Bioactive Natural Products (Part I)

studies in Natural Products Chemistry Volume 28 Bioactive Natural Products (Part I)

studies in Natural Products Chemistry edited by Atta-ur-Rahman

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Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidaton (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure and Chemistry (Part B) Stereoselective Synthesis (Part F) Stereoselective Synthesis (Part G) Stereoselective Synthesis (Part H) Bioactive Natural Products (Part A) Stereoselective Synthesis (Part I) Structure and Chemistry (Part C) Stereoselective Synthesis (Part J) Structure and Chemistry (Part D) Stereoselective Synthesis (Part K) Structure and Chemistry (Part E) Structure and Chemistry (Part F) Bioactive Natural Products (Part B) Bioactive Natural Products (Part C) Bioactive Natural Products (Part D) Bioactive Natural Products (Part E) Bioactive Natural Products (Part F) Bioactive Natural Products (Part G) Bioactive Natural Products (Part H) Bioactive Natural Products (Part I )

studies in natural Products Chemistry Volume 28 Bioactive natural Products (FM I)

Edited by

Atta-ur-Rahman H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistm

2003

ELSEVIER Amsterdam - Boston - Heidelberg - London - New York - Oxford - Paris San Diego - San Francisco - Singapore - Sydney - TolRahinan (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.

BIOACTIVE COMPOUNDS FROM THE GENUS BROUSSONETIA DONGHO LEE^ and A. DOUGLAS KINGHORN* Program for Collaborative Research in the Pharmaceutical Sciences and Department ofMedicinal Chemistry and Pharmacognosy, College of Pharmacy, University ofIllinois at Chicago, Chicago, Illinois 60612, USA, ABSTRACT: The genus Broussonetia of the Moraceae (mulberry family) is of both ethnomedical and industrial interest. Of the approximately 30 species in this genus, only three have been subjected to previous phytochemical investigation, namely, B. kazinoki, B. papyriferay and B, zeylanica. From over 100 compounds isolated from these species, the major secondary metabolites reported thus far are alkaloids of the pyrrolidine type and several types of flavonoids. Some of these compounds have exhibited various biological activities, such antioxidative, aromatase inhibitory, cytotoxic, glycosidase inhibitory, and platelet aggregation inhibitory effects. The biologically active constituents of the species in the genus Broussonetia are discussed in detail.

INTRODUCTION The genus Broussonetia L*Her. ex Vent, of the Moraceae (mulberry family) is represented by lactiferous trees or shrubs. Broussonetia comprises about 30 species and is distributed throughout various regions of the w^orld including Africa, East Asia, and North America [1,2]. Thus far, only three species of the genus Broussonetia have been studied for their secondary metabolites, namely, B, kazinoki, B. papyrifera, and B. zeylanica. Broussonetia kazinoki Siebold & Zucc. is a deciduous tree growing to 4.5 m that flowers in August. It occurs in mainland China, Japan, and Korea [1]. The plant requires well-drained soil but can grow in Address correspondence to this author at Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, U.S.A. E-mail: [email protected]. ^Current address: Chemistry and Life Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709, U.S.A.

nutritionally poor soil [3]. Preparations made from B. kazinoki have been used as a tonic to increase vision and sexual potency, and to treat boils, eczema, infant colic, and leukorrhea [4,5]. Various extracts of 5. kazinoki have exhibited antifungal, antiinflammatory, antioxidant, and antispasmodic activities [6-10]. Broussonetia papyrifera (L.) L'Her. ex Vent, is a deciduous tree growing up to 15 m that is commonly called the paper mulberry. It is native to East Asia, then later introduced and naturalized in the United States. It flowers from August to September, and the seeds ripen from September to November [1,11]. The plant prefers light and well-drained soil and' is easily cultivated in a warm sunny position in any soil of reasonable quality [3]. Fibers from the bark are used in making paper, cloth, and rope. These fibers can be produced by beating strips of bark on a flat surface with a wooden mallet [12]. 5. papyrifera has been used for cancer, dyspepsia, and pregnancy [13]. In mainland China, the fruits of 5. papyrifera have been employed for impotency and ophthalmic disorders [4,14], Also, the leaf juice of 5. papyrifera is diaphoretic and laxative and the stembark is hemostatic [4]. Antifungal and antioxidant activities of the extracts ofB. papyrifera were reported [6,7,9]. Broussonetia zeylanica (Thwait.) Comer is endemic to Sri Lanka and its tough bark-fibers were used to make string [15]. Several types of bioactive compounds have been reported from the genus Broussonetia including glycosidase inhibitory alkaloids and aromatase inhibitory or cytotoxic flavonoids. This chapter reviews the biologically active constituents from the genus Broussonetia reported by the end of 2001. BIOACTIVE COMPOUNDS FROM BROUSSONETIA KAZINOKI The bioactive secondary metabolites reported from Broussonetia kazinoki can be classified into major two groups, alkaloids and flavonoids (Table 1), Fig. (1). The Kusano group at Osaka University of Pharmaceutical Sciences in Japan reported over 20 pyrrolidine alkaloids, broussonetines A-H, K-M, 0-T, V-X, and Mi, and broussonetinines A and B, four pyrrolidinyl piperidine alkaloids, broussonetines I, J, Ji, and J2, two pyrroline alkaloids, broussonetines U and Ui, and one pyrrolizidine alkaloid, broussonetine N, from hot water extracts of 5. kazinoki [16-24]. As shown in Table 1, some of these alkaloids exhibited strong

Table 1. Bioactive Compounds from Broussonetia kazinoki Compound type/name

Activity

Reference

ALKALOIDS Pyrrolidines Broussonetine C (1)

Inhibition of glycosidases*

[16]

Broussonetine D (2)

Inhibition of glycosidases*

[16]

Broussonetine E (3)

Inhibition of glycosidases"

[17]

Broussonetine F (4)

Inhibition of glycosidases*

[17]

Broussonetine G (5)

Inhibition of glycosidases*

[18]

Broussonetine H (6)

Inhibition of glycosidases*

[18]

Broussonetine K (7)

Inhibition of glycosidases*

[20]

Broussonetine L (8)

Inhibition of glycosidases*

[20]

Broussonetine M (9)

Inhibition of glycosidases*

[21]

Broussonetine 0 (10)

Inhibition of glycosidases*

[21]

Broussonetine P (11)

Inhibition of glycosidases*

[21]

Broussonetine Q (12)

Inhibition of glycosidases*

[21]

Broussonetinine A (13)

Inhibition of glycosidases*

Broussonetinine B (14)

Inhibition of glycosidases*

[17]

Inhibition of glycosidases*

[22]

KazinolD(16)

Cytotoxicity against human tumor cell lines'*

[25]

Ka2inolK(17)

Cytotoxicity against human tumor cell lines**

[25]

I

[17]

Pyrrolizidine Broussonetine N (15) FLAVONOIDS Diphenylpropanes

Table 1. Bioactive Compounds from Broussonetia kazinoki (continued) Compound type/name

Activity

Reference

Flavans 7,4'-Dihydroxyflavan (18)

Cytotoxicity against human tumor cell lines'*

[25]

KazinolA(19)

Antioxidant activity*" Inhibition of tyrosinase**

[26] [26]

KazinolE(20)

Antioxidant activity*^ Inhibition of tyrosinase'

[26] [26]

KazinolQ(21)

Cytotoxicity against human tumor cell lines'*

[25]

KazinolR(22)

Cytotoxicity against human tumor cell lines'*

[25]

Broussonol A (23)

Cytotoxicity against human tumor cell lines'*

[27]

Broussonol B (24)

Cytotoxicity against human tumor cell lines'*

[27]

Broussonol C (25)

Cytotoxicity against human tumor cell lines'*

[27]

Broussonol D (26)

Cytotoxicity against human tumor cell lines'*

[27]

Flavonols

1

"Glycosidase inhibitory activity expressed as ICso value (jiM); 1: p-Gal = 0.036, p-Man = 0.32; 2: P-Gal = 0.029, P-Man = 0.34; 3: a-Glc = 3.3, P-Glc == 0.055, P-Gal = 0.002, p-Man = 0.023; 4: a-Glc = 1.5, p-Glc = 0.01, P-Gal = 0.004, P-Man = 0.028; 5: P-Glc = 0.024, P-Gal = 0.003, P-Man = 0.76; 6: P-Glc = 0.036, p-Gal = 0.002, P-Man = 0.32; 7: P-Glc = 0.026, P-Gal = 0.005, P-Man = 0.3; 8: P-Glc = 0.017, P-Gal = 0.004, p-Man = 0.2; 9: P-Gal = 8.1; 10: P-Glc = 1.4, P-Gal = 0.17, P-Man = 8.2; 11: P-Glc = 2.4, P-Gal = 0.2, P-Man = 7.6; 12: P-Glc = 1.4, P-Gal = 0.6, P-Man = 20.0; 13: P-Gal = 0.016, a-Man = 0.3; 14: P-Gal = 0.01, a-Man = 0.29; 15: P-Glc = 6.7, p-Gal = 2.9, P-Man = 3.3 (P-Gal = p-Galactosidase; a-Glc = a-Glucosidase; P-Glc = pGlucosidase; a-Man = a-Mannosidase; P-Man = P-Mannosidase). '*Cytotoxicity expressed as ED50 value (^ig/mL); 16: PLC/PRF/5 = 3.3, 212 = 7.0, HT3 = 3.6; 17: HT3 = 8.6: 18: HT3 = 11.6, SiHa = 8.9, CaSki = 17.4; 21: PLC/PRF/5 = 3.5, T24 = 2.3, 212 = 3.8, HT3 = 4.3, SiHa - 4.7; 22: HT3 = 9.3, SiHa = 9.3, CaSki = 8.2; 23: A546 = 8.7, HCT-8 = 9.1; 24: A546 = 5.52, HCT-8 = 8.8; 25: A546 = 7.8, HCT-8 = 9.6; 26: KB = 4.5 (key to cell lines; 212 = inducible Ha-ras oncogene transformed NIH/3T3; A549 = human lung carcinoma; CaSki = human cervical carcinoma; HCT-8 = human ileocecal carcinoma; HT3 = human cervical carcinoma; KB = human epidermoid carcinoma; PLC/PRF/5 = human hepatoma; SiHa = human cervical carcinoma; T24 = human hepatoma). '^Antioxidant activity shown by l,l-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity (IC50 pM); 19:41.4,20:33.4. ''Activity not specified. nCso 241.3 nM.

H CH2OR2

H(?

OH 1 R,=H,R2 = H 3 R, = OH, R2 = H 7 R, = 0H,R2 = Glc 10 R, = H,R2 = H,A-3',4'

H HOHjC^i^^V-*'^

H(f

CH2OR2

OH

2 R, = H,R2 = H 4 R, = 0H,R2 = H 8 R, = 0H,R2 = Glc 11 Ri=H,R2 = H,A-3',4'

HOH2C*^^\.-*'^

Ha

OH

H

OH ^O

H0H2CM^^'\.»^'^

H(f

O

OH

CH2OH

Fig. (1). Continued H CH2OR

HO

OR

12 R = Glc 13 R==H

CH2OH

HOF^C^^'^V.^^^'

HC)

OH

14

HOH2C

I?

CH2OH

OH

16 17 A^3,4

HO.

TCO' 19

18

Fig. (1). Continued

OH

OH

O 24

Fig. (1). Structures of bioactive constituents of Broussonetia kazinoki.

glycosidase inhibitory activity with IC50 values ranging from 0.002 to 8.2 [xM. Selective inhibition of glycosidase enzymes has a number of potential therapeutic uses, including the treatment of cancer, diabetes, and HIV-AIDS [28-32]. Also, the prenylated flavonoid derivatives, kazinols D (16) and K (17) (diphenylpropanes), 7,4'-dihydroxyflavan (18),

10

kazinols Q (21), and R (22) (flavans), and broussonols A-D (23-26) (flavonols), were isolated as moderate to weak cytotoxic principles against several human cancer cell lines with ED50 values ranging from 2.3 to 17.4 ^ig/mL [25,27]. Two flavans, kazinols A (19) and E (20), were reported as antioxidative principles using the l,l-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay (IC50 41.4 and 33.4 |iM, respectively) [26]. These compounds (19 and 20) also exhibited inhibitory activity against tyrosinase, which is a key enzyme in melanin biosynthesis and plays a role in the conversion of tyrosine to DOPA and DOFA to dopaquinone [26,33]. An antioxidative effect and the suppression of melanin biosynthesis are useful for cosmetic products in relation to hyperpigmentation [34]. Broussonetine C (1), a monocyclic polyhydroxy pyrrolidine alkaloid, showed a yellow spot on TLC when sprayed with ninhydrin reagent and heated (ninhydrin reaction), and its molecular formula was determined by a positive high-resolution mass spectrometry (C18H36NO5, [M + H]^, m/z 346.2579). The IR spectrum displayed a hydroxy band at 3370 cm'^ and a carbonyl band at 1706 c m \ The ^H- and '^^C-NMR signals were assigned using the ^H-^H correlated spectroscopy (^H-^H COSY), heteronuclear single quantum coherence (HSQC), and distortionless enhancement by polarization transfer (DEFT) pulse sequences. The position of the carbonyl carbon and the linkage of the pyrrolidine ring and the aliphatic side chain were determined using the heteronuclear multiple bond coherence (HMBC) NMR technique [HMBC correlations were observed for the carbonyl carbon signal (5c 210.8) with the proton signals at 6H 2.71 (H-11') and 6H 2.12 (H-120, and for the C-5 carbon signal (5c 62.9) of the pyrrolidine ring with the proton signals at 5H 4.44 (H-4) and 5H 2.04 (H-l'), respectively] [16]. The relative stereochemistry of the pyrrolidine ring of broussonetine C (1) was determined from its coupling constants (vicinal coupling, ^2,3 = •/3,4 = •/4,5 = 6.4 Hz) and nuclear Overhauser enhancement effects (H-2/H-4 and H-3/H-5). The absolute stereostructure was disclosed as (2i?,3/?,4i?,5/?) using the benzoate chirality method [35]. A diacetylacetoamide was prepared from broussonetine C (1) by treatment with acetic anhydride in pyridine at room temperature, and then a dibenzoate (la) was obtained by benzoylation of the diacetylacetoamide. The circular dichroism (CD) curve of l a displayed a negative Cotton

11

effect (A8237 -15.9) and a positive effect (AS223 +16.4), which indicated a negative chirality as shown in Fig. (2) [16]. C0CH3

BzO

OBz la

Ae(nin): +16.4(223) -15.9(237)

Fig. (2). Determination of the absolute stereostnicture of broussonetine C (1) by the benzoate chirality method.

Broussonetine L (8) showed similar physical and spectroscopic properties to those of broussonetine C (1) [16], except for proton signals of a p-glucose (anomeric proton, 8H4.78, 1H, doublet, J- 7.8 Hz) moiety in the H-NMR spectrum. Hydrolysis of broussonetine L (8) with 1 N HCl provided broussonetine F (4) [17] and D-glucose ([a]D +40.6°). Therefore, the structure of broussonetine L (8) was determined to be 13'O-p-D-glucopyranosylbroussonetine F due to the glycosylation shift of C13' (6c 69.3) of broussonetine L (8) (broussonetine F, 4, 6c-i3' 61.6) and HMBC long-range correlations observed between H-13' (6H 3.69 and 4.09) and an anomeric carbon (5c 104.4), and between an anomeric proton and C-13'[20]. The absolute stereochemistry of broussonetine L (8) was determined by the combination of the benzoate chirality method and the Mosher's method [35-37]. A carbamate (8a) was prepared from broussonetine F (4) by reaction with phenyl chloroformate in tetrahydrofuran-H20 (7:3), and a diacetate (8b) was prepared from 8a with acetic anhydride in pyridine. Finally, a dibenzoate (8c) was obtained by benzoylation of 8b. The CD curve of 8c showed a negative Cotton effect (AE237 -30.9) and a positive effect (Ae223 +15.9) to confirm a counter-clockwise chirality between two benzoyl groups. Fig. (3) [20].

12

OH

IN.

nd

CH2OGIC

OH

H

OH CH2OH

HOHjC*^'^.*^'^

Hcf

O—CO

OH

PhOCOCl NaHCOj

QH CH2OH

CH2OAC

HO

OH PhCOCl pyridine

O—CO

OAc CH2OAC

Bz(f

8c

OBz OBz

BzQ

A8(nm): +15.9(223) -30.9 (237)

Fig. (3). Determination of the absolute stereostructure of the pyrrolidine ring of broussonetine L (8) by the benzoate chirality method.

13

The absolute configuration of C-l' of 8 was then investigated by the Mosher's method. The di- (R)- and (S)-2-methoxy-2-phenyl-2(trifluoromethyl)-acetic acid (MTPA) esters (8dR and 8d5) and tri- (R)and (iS)-MTPA esters (SeR and SeS) prepared from 8a, were analyzed by ' H - ' H C O S Y N M R (500 MHz) and A6 values (SS-SR) were measured. These values established the R configuration of C-l' of 8 by comparison of the di-MTPA esters (8d/? and 8d5) and the tri-MTPA esters (SeR and 8fty),Fig.(4)[20]. O—CO

OR4 CH2OR1

R-,(f

OR, 8dif, 8d5: Ri = R2 = MTPA, R3 - R4 = H 8ei?, ScJ: R, = R2 = R4 = MTPA. R3 = H

A^(^S-^R)

3

4

5

r

+0.100

-0.039

-0.019

0.000

+0.020

-0.332

-0.161

-0.037

-0.060

+0.013

1"

2

8d

+0.030

1 8e

-0.154

r 0.000 1 +0.050

Fig. (4). Determination of the absolute configuration of C-l' of broussonetine L (8) by the Mosher's method.

Also, the absolute stereochemistry of the pyrrolizidine ring and Cr of broussonetine N (15) was established by the Mosher's method. The tri- (Ry and (5)-MTPA esters (15a/? and 15a5) and penta- (Ry and (5)MTPA esters (15bif and IShS) were prepared from 15 and A6 values (658R) were measured. Accordingly, the R configuration of C-l of the pyrrolizidine ring from 15a and the R configuration of C-l' from 15b were determined, respectively, Fig. (5) [22]. A biosynthetic study of the 18-carbon chain skeleton of broussonetines was reported [38]. To verify the biosynthetic route of these alkaloids, the plant was grown on an aseptic medium and the enriched ^^C of the isolated alkaloids was analyzed by NMR after feeding with [l-^^C]glucose. The labeling pattem of broussonetine J (27) obtained

14

MTPAO -Z. -0.006 "L

!?-^^^_,,, H r

C-0.029

MTPAOH2C

+0.013 +0.001

c V l

CHoOMTPA

H

+0.061

OH

MTPAO -0.075'^

''-^\,ri^ -

-^-^'^

CH2OMTPA OMTPA

15b

Fig. (5). Determination of the absolute configuration of broussonetine N (15) by the Mosher's method.

from the feeding experiment indicated that C-4 through C-18 were formed via palmitoyl CoA through the acetate-malonate pathway, whereas C-1 through C-3 were derived via serine from 3-phosphoglyceric acid. Therefore, the 18-carbon chain of broussonetine J (27) was assumed to be formed initially by condensation of serine and palmitoyl CoA [38], As shown in Fig. (6), the absolute stereochemistry of the pyrrolidine rings of the broussonetines is related to o-serine and that of broussonetine U (28) is related to L-serine. Out of a series of over 30 alkaloids obtained from B, kazinoki, some of them showed potent glycosidase inhibitory activity as shovm in Table 1. Interestingly, only broussonetines E and F (3 and 4), which have a hydroxyl group on C-T, demonstrated potent inhibitory activity against a-glucosidase [17]. However, broussonetines G and H (5 and 6), which also have a hydroxyl group on C-l', did not inhibit a-glucosidase [18]. These results suggested that the inhibition of a-glucosidase might be attributed to the hydroxyl groups on both C-T and C-13' and the keto groups of C-9' or C-10' [17,18]. However, additional studies seem to be required to verify this suggestion [24].

15

CHjOH -O.

COOH

OH

OH

OH

OH OH D-[l-^^C]glucose

O

II

^

.COOH

HO SCoA

NH2 D-serine

NH2 L-serine

CoAS

OH

HO HO

OH

Fig. (6). Biosynthesis of broussonetines J and U (27 and 28).

COOH H O - ^ '

16

BIOACTIVE PAPYRIFERA

COMPOUNDS

FROM

BROUSSONETIA

The major types of bioactive constituents reported from Broussonetia papyrifera are the prenylated flavonoids, which include compoxmds of the diphenylpropane, chalcone, flavan, flavanone, flavone, flavonol, and aurone classes (Table 2), Fig. (7). An early study on B, papyrifera resulted in the isolation of two diphenylpropanes, broussonins A (29) and B (30), and a coumarin, marmesin (52), with antifungal activity [39]. Also, a diprenylated diphenylpropane derivative, kazinol F (31) [40], was reported as an antioxidant and tyrosinase inhibitory constituent [34]. Table 2. Bioactive Compounds from Broussonetia papyrifera Activity

Compound type/name

Reference(s)

FLAVONOroS Diphenylpropanes Broussonin A (29)

Antifungal activity* Inhibition of aromatase**

[39]

Broussonin B (30)

Antifungal activity*

[39]

Kazinol F (31)

Antioxidant activity (scavenging free radicals)^ Inhibition of tyrosinase**

[34]

[34]

1

Antioxidant activity (inhibition of lipid peroxidation)^ Inhibition of cyclooxygenase* Inhibition of nitric oxide production'^ Inhibition of respiratory burst in neutrophils* Platelet aggregation inhibitory activity**

[42]

1

Inhibition of aromatase**

[41]

Isogemichalcone C (34)

Inhibition of aromatase*'

[41]

1 2,4,2',4'-Tetrahydroxy-3'1 prenylchalcone (35)

Inhibition of aromatase**

[41]

1

[41]

1

Chalcones

Broussochalcone A (32)

1 3'-[y-Hydroxymethyl-(£)-ymethylallyl]-2,4,2',4'. tetrahydroxychalcone 1 r - 0 coumarate (33)

[43] [42] [44] [431

Flavans Broussoflavan A (36)

Antioxidant activity (inhibition of lipid peroxidation)*^ 1 Platelet aggregation inhibitory activity**

1 1

[43] [45]

1

17

Table 2. Bioactive Compounds from Broussonetia papyrifera Compound type/name KazinolA(19) KazinolB(37)

(continued)

Activity

RefereDce(s)

Antioxidant activity' Inhibition of tyrosinase' Platelet aggregation inhibitory activity** Inhibition of cyclooxygenase* Platelet aggregation inhibitory activity**

1

[26] [26] [43]

[43] [43]

1 1

Flavanones (25)-Abyssinone II (38)

Inhibition of aromatase**

[41]

(2.S)-2',4'-Dihydroxy-2"-(lhydroxy-1-methylethyl)dihydrofuro[2,3-Alflavanone (39)

Inhibition of aromatase*'

[41]

(2iS)-Euchrenone a? (40)

Inhibition of aromatase*'

[41]

(2.S)-Naringenin (41)

Inhibition of aromatase**

[41]

Inhibition of aromatase**

[41]

Inhibition of aromatase**

[41]

Platelet aggregation inhibitory activity*"

[43]

Antioxidant activity (inhibition of lipid peroxidation)' Antiproliferative activity* Inhibition of aromatase** Inhibition of cyclooxygenase* Platelet aggregation inhibitory activity*" Antioxidant activity (inhibition of lipid peroxidation)*^ Antiproliferative activity*

[45] [45] [41] [43] [43]

Inhibition of aromatase*'

[41]

(25)-5,7,2',4'1 Tetrahydroxyflavanone (42) Flavone 1 5,7,2',4'-Tetrahydroxy-31 geranylflavone (43) Flavonols Broussoflavonol £ (44)

Broussoflavonol F (45)

Broussoflavonol G (46) Isolicoflavonol (47)

[45]

'

Aurone Broussoaurone A (48)

[45]

Antioxidant activity (inhibition of lipid peroxidation)' Inhibition of cyclooxygenase* Platelet aggregation inhibitory activity*"

[45] [43]

L. _ [43I_.

MISCELLANEOUS AlbanoIA(49)

Inhibition of aromatase**

[41]

Betulinic acid (50)

Selective cytotoxic activity against melanoma cell lines'"

[46]

1

18

Table 2. Bioactive Compounds from Broussonetia papyrifera (continued) Compound type/name

Activity

Reference(s)

Demethylmoracin I (51)

Inhibition of aromatase**

[41]

Marmesin(52)

Antifungal activity*

[39]

MoracinN(53)

Inhibition of aromatase**

[41]

Ursolic acid (54)

Inhibition of HIV-1 protease dimerization''

[47]

"Antifungal activity (presented as a range) expressed as the minimum concentration (mM) required for complete inhibition of fungal growth including Fusarium roseum, F. lateritium, F. solani, Diaporthe nomuraU Stigmina mori, Sclerotinia sclerotiorum, Bipolaris leersiae, and Rosellinia necatrix\ 29: 0.2-0.9, 30: 0.05-0.9, 52: 0.9-4.0. ''Aromatase inhibitory activity determined as IC50 value (^iM); 29: 30.0, 33: 0.5, 34: 7.1, 35: 4.6, 38: 0.4, 39: 0.1,40: 3.4,41: 17.0,42: 2.2,43: 24.0,45: 9.7,47: 0.1,49: 7.5,51: 31.1,53: 31.1. ^Antioxidant activity expressed as IC50 value (pM); 31: 6.7 (jig/mL); 32:0.63,36: 2.1,45:2.7,46:1.0,48: 1.2. ''The tyrosinase inhibitory activity of 31 was IC50 0.39 |ig/mL. *Cyclooxygenase inhibitory effect determined as IC50 value (ng/mL); 32: 19.4,37: 155.3,45: 17.5,48: 22.7. ^Inhibitory effect (IC50) of 32 on nitric oxide production was 11.3 ^iM. ^Compound 32 inhibited O2 consumption in formylmethionyl-leucyl-phenylalanine- and phoibol 12-myristate 13-acetate-stimulated rat neutrophils with IC50 values of 70.3 and 63.9 ^M, respectively. •^Antiplatelet activity induced by arachidonic acid was expressed by IC50 value (^M); 19: 11.4, 32: 6.8, 36: 86.7,37: 32.6,44: 39.9,45:16.9,48: 15.4. 'Activity found as a constituent of Broussonetia kazinofd. ^Antiproliferation activity shown by the inhibition of ['H]thymidine incorporation into DNA in the proliferation of rat vascular smooth muscle cells. The effect was expressed as % of control; 45: 0-7.8,46: 0-0.4. ''Activity found as a constituent of a plant other than a Broussonetia species.

Broussochalcone A (32) [48], a prenylated chalcone, is one of the most completely studied constituents of B. papyrifera biologically. Broussochalcone A (32) inhibited platelet aggregation induced by arachidonic acid with an IC50 value of 6.8 |aM as well as induction by adrenaline in human platelet-rich plasma. The antiplatelet effect of 32 was partially due to an inhibitory effect on cyclooxygenase activity and by reducing thromboxane fomiation [43]. Also, broussochalcone A (32) inhibited O2 consiraiption in fomiylmethionyl-leucyl-phenylalanine- and phorbol 12-myristate 13-acetate-stimulated rat neutrophils with IC50 values of 70.3 and 63.9 jiM, respectively. This inhibitory effect of 32 on respiratory burst in neutrophils was not mediated by the reduction of phospholipase C activity, but was mediated by the suppression of protein kinase C activity through interference with the catalytic region and by the

19

RiO

29Ri = CH3,R2 = H 30Rj = H,R2 = CH3

31

33R = H 34R = OCH3

OH

36

20

Fig. (7). Continued

OH

OH

37

OH

OH

41R = H 42R = OH

21

Fig. (7). Continued

HO,

48

49

22

Fig. (7). Continued

COOH

50

HO-

51

o ^ ^ ^ -o- - o 52

CCX)H

54

Fig. (7). Structures of bioactive constituents of Broussonetia papyrifera.

attenuation of O2*" generation from the NADPH oxidase complex, which might inhibit the generation of toxic oxygen radicals and terminate the tissue damage [43]. Furthermore, broussochalcone A (32) showed antioxidant activity in iron-induced lipid peroxidation in a rat brain

23

homogenate model with an IC50 value of 0.63 |iM as well as in the DPPH system, and exhibited an inhibitory effect on nitric oxide (NO) production with an IC50 value of 11.3 jaM. This potent inhibitory effect on NO production was mediated by suppression of nuclear factor (NF)-KB activation, phosphorylation and degradation of iKBa (an inhibitory protein of NF-KB), and inducible NO synthesis expression, which have been associated with autoimmune or inflammatory diseases [42]. In an effort to investigate antioxidant constituents with antiproliferative effects in rat vascular smooth muscle cells (VSMC), broussoflavan A (36) [49], broussoflavonols F (45) [50] and G (46) [51], and broussoaurone A (48) [49] were found to inhibit the Fe^^-induced thiobarbituric acid-reactive substance formation in rat brain homogenate. Furthermore, broussoflavonols F (45) and G (46) inhibited fetal calf serum-, 5-hydroxytryptamine-, or ADP-induced [^H]thymidine incorporation into rat VSMC [45]. Antioxidant activities and inliibitory effects on proliferation of rat VSMC with potent antiplatelet activities of 45 and 46 may be useful for vascular diseases and atherosclerosis [43,45]. The concept of cancer chemoprevention is becoming wellestablished and refers to the pharmacological intervention to arrest or reverse the process of carcinogenesis, and thus prevent cancer [52,53]. It has become evident that various phytochemical components of the diet are able to prevent cancer formation in full-term carcinogenesis inhibition studies in animal models [54]. As part of a U.S. National Cancer Institutefunded program project conducted at the University of Illinois at Chicago [55-57], an ethyl acetate extract of the whole plants ofB, papyrifera was found to significantly inhibit aromatase activity in an in vitro assay [58,59] (74% inhibition at 80 |ig/mL) [41]. This was only one of a handful of extracts found to significantly inhibit aromatase activity with the bioassay protocol used, out of over 1,000 extracts screened [60]. This target was chosen for investigation, because aromatase catalyzes the final, rate-limiting step in estrogen biosynthesis [61], and is regarded as a target relevant to the treatment or prevention of breast and prostate cancers [62]. Several synthetic aromatase-inhibitory drugs have been developed, including aminoglutethimide, substrate androstenedione derivatives, imidazoles, and triazoles [63-65]. From the active extract of B, papyrifera were isolated several aromatase inhibitors with IC50 values in the range 0.1-31.1 ^M, inclusive of broussonin A (29) [66], 3'-[Y-hydroxymethyl-(£)-y-methylallyl]-

24

2,4,2',4'-tetrahydroxychalcone 11 '-O-coumarate (33) [41], isogemichalcone C (34) [41], 2,4,2',4'-tetrahydroxy-3'-prenylchalcone (35) [67], (25)-abyssinone II (38) [68], (25)-2',4'.dihycIroxy-2"-(lhydroxy-1 -methylethyl)-dihydrofuro[2,3-A]flavanone (39) [41], (25)euchrenone a7 (40) [69], (25)-naringenin (41) [70], (25)-5,7,2',4'tetrahydroxyflavanone (42) [71], 5,7,2',4'-tetrahydroxy-3-geranylflavone (43) [41], broussoflavonol F (45) [50], isolicoflavonol (47) [72], albanol A (49) [73], demethylmoracin I (51) [41], moracin N (53) [74]. Of these aromatase inhibitors, five of the compounds were new (33, 34, 39, 43, 51), and details of structure elucidation of 33, 34, and 43 are presented as examples in the following two paragraphs. The isolates 3'-[y-hydroxymethyl-(£^-Y-methylallyl]-2,4,2',4'. tetrahydroxychalcone 11'-O-coumarate (33) and 3'-[Y-hydroxymethyl(£)-y-methylallyl]-2,4,2',4'-tetrahydroxychalcone 11 '-O-ferulate (isogemichalcone C, 34) were obtained as orange powders and were shown by positive HRFABMS to possess molecular formulas of C29H26O8 (m/z [M + Na]^ 525.1884) and C30H28O9 (m/z [M + N a ] \ 555.1577), respectively. The ^H- and ^^C-NMR spectra of 33 and 34 exhibited characteristic chalcone signals, and signals for a coumarate group for 33 at 6H 7.54 (2H, 7 = 8.6 Hz, H-2" and H-6"), 6H 6.87 (2H, J = 8.5 Hz, H3" and H.5''), 5H 7.59 (IH, 7 = 16.0 Hz, H-T'), and 5H 6.35 (IH, J = 16.0 Hz, H-8") and signals for a ferulate group for 34 at 6H 7.34 (IH, 7 = 1.6 Hz, H-2"), 6H 6.85 (IH, / = 8.1 Hz, H.5"), 6H 7.12 (IH, 7 = 1.7 and 8.2 Hz, H-6"), 5H 7.57 (IH, J = 16.0 Hz, H.7"), 5H 6.40 (IH, J= 15.9 Hz, H8"), and 6H 3.91 (3H, singlet, OCH3). Based on these observations, the structures of 33 and 34 were concluded to be prenylated chalcones with a coumarate and a ferulate unit attached, respectively, which were confirmed by 2D-NMR techniques. Fig. (8). In case of isogemichalcone C (34), it was concluded to be a regioisomer of gemichalcone C by comparing its spectra with those of the latter compound [75]. This was confirmed using a NOESY NMR experiment. Thus, the NOE correlations between H-7' and H-10', and H-8' and H-IT clearly indicated E stereochemistry of the prenyl group. Moreover, the chemical shift differences at positions C-10' and C-1T of the E and Z isomers supported the stereochemistry proposed. Fig. (8) [41,75,76]. 5,7,2',4'-Tetrahydroxy-3-geranylflavone (43) exhibited a molecular ion [M]"^ at m/z 422.1719 by HREIMS, consistent with an

25

Carbon

6c 33

34

Gemichalcone C [75]

10'

14.2

14.2

64.2

ir

70.2

70.2

22.8

'

Fig. (8). Selected HMBC (->) and NOE (

COOEt Bu.Sn^

^COOEt

Fig (3). Treatment of 2,2,6-trimethylcyclohexanone with lithium diisopropylamide (LDA) followed by phenyltriflimide (7V-phenylbis (trifluoromethanesulphonimide) gave the corresponding triflate [24]. The

73

best coupling reaction could be achieved with Farina's 'soft' palladium (Pd2(dba)3) with AsPhs as ligand and DMPU, Fig (4) [25]. OTf

a)LDA

^COOEt

b) Bu3Sn"

phenyltriflimide

Pd2(dba)3, NMP, DMPU, AsPh3

^^COOU

COOEt c) K:OH, EtOH, H2O

Fig. (4). Dominguez, Iglesias, and De Lera (1998, 2001). As an extension of this procedure, they synthesized the side chain of 9Z-retinoate stereoselectively and attached it to the hydrophobic ring by a high yielding thallium accelerated Suzuki cross-coupling reaction, Fig. (5) [26]. The tetraenylstannate used for the coupling reaction was obtained by Mn02 oxidation of the known stannyldienol [27], to the corresponding aldehyde (86%), followed by condensation with the phosphonate (52%) and reaction of the tetraenylstannate with a solution of iodine. The product was immediately added to the organoborane, in the presence of Pd(PPh3)4 then TIOH was added, to provide the 9Zretinoate in 84% yield. The organoborane was freshly prepared from the cyclohexanone, via its hydrazone, which was transformed into the iodide. COOEt

a) BuLi,

1 BuSn

BuSnH, CuCN OH4

BuSn

^

^

b) Mn02 K2C03^

[ ""OH

-^:^ ^

BuSn''

OEt ^j^Q

c) BuLi, DMPU

•

COOEt

COOEt

I J) /BuLi, B(0Me)3

e) H2N>fH2 I2, Et3N, DBN

"

Pd(PPh3)4

*"

Fig. (5). Pazos, and De Lera (1999).

B(0H)2 J ^ ^ ^

kjl\

COOEt

74

In this exhaustive work De Lera et al described the syntheses of the retinoid skeleton via the Stille coupUng for the formation of side-chain single bonds [28]. C(7)-C(8) strategies: A stereoselective synthesis of all E retinal, via a condensation of a Cio chloroacetal with p-cyclogeranylsulfone was described by Julia et al. [29]. The chloroacetal was reacted with the silylenol ether, using TiCl4/Ti(OMe)4, to give in 63% yield, the chloromethoxyacetal derivative as a mixture of ElZ isomers (80/20). The aldehyde was converted in 97% yield into the corresponding acetal with HC(0Me)3 and camphorsulfonic acid in methanol, Fig. (6).

OMe OMe

OMe

OMe

a) Ti(0Me)4 TiCl4

OMe

b) HC(0Me)3 CHO

OMe CI

01

CI

Fig. (6). This building block was condensed with the anion of (icyclogeranylsulfone. During flash-chromatography the intermediate was hydrolyzed to the sulfone-aldehyde, as a mixture of three isomers in 95% yield. Retinal was obtained from this sulfone by treatment with MeONa, for 10 days, in the dark (90%), Fig. (7).

OMe I

OMe OMe

SO2

I

OMe I

CHO

Fig. (7). Chemla, Julia, and Ugen (1993).

75

Chabardes developed a process for the preparation of vitamin A and its intermediates, from cyclogeranylsulfone and Cio aldehyde-acetals [30], For example, chlorocitral reacted with ethylene glycol, HC(0Me)3 and pyridinium tosylate to provide the chloroacetal (40%), as a mixture of two isomers. Reaction of this allylchloride with A^-methylmorpholine oxide (NMO) and Nal furnished the aldehyde, as a mixture of four isomers. These compounds underwent condensation with pcyclogeranylsulfone. Further chlorination of the sulfone-alkoxide salts, led to a mixture of sulfone-chloride acetals and their products of hydrolysis in 45-50% yield. Double elimination of the chloride and the sulfone, followed by hydrolysis with pyridinium tosylate (PPTS) gave retinal, as a mixture of all E and 13Z isomers (78/22). The overall yield from the chloroacetal was 18%. In another 'one-pot' example, retinal was obtained in 52% yield from the aldehyde, and was then isomerised and reduced to retinol (all E: 95.5, 13Z: 4, 9Z: 0.5) Fig. (8).

s

a) NMO CI

I

Nal, DMF

I

O-^ ^) L A

^2

/PrMgCl c) SOCI2, pyridine

d) MeOK

j^^^^'-'^^::^.-'^^

e) PPTS

Fig. (8). Chabardes (1994). Honda et al described a highly Z stereoselective [2,3]-sigmatropic rearrangement that provided trisubstituted E,Z synthons, starting from A^tiglyl-p-methallyldimethylammonium salts [31]. The application of this key triene synthon to the stereoselective synthesis of 13Z-retinol was reported from a trieneester. Thus, prenylbenzyl ether was converted via ene-type chlorination followed by amination into internal allylamine. This was reacted with ethyl 3-bromotyglate in acetonitrile to give the

76

quaternary salt. Treatment of the latter with EtOK in ethanol resulted in the formation of an ylide. This latter underwent [2,3]-sigmatropic rearrangement to furnish the diene that possessed a newly formed Z and tiglyl-origin E stereochemistry, Fig. (9). a) Br

COOEt

OSi/BuMeo

OSi/BuMeo

h) EtOK

NMeo

"^ EtOOC 0SirBuMe2 COOEt

Fig. (9). Treatment of this synthon with peracetic acid resulted in the formation of a A^-oxide intermediate. A Cope elimination gave the triene, Fig. (10).

c) AcOOH

EtOOC

EtOOC 0Si/BuMe2

0Si/BuMe2 ^0°C

EtOOC 0Si/BuMe2

Fig. (10). ?BuMe2Si was then replaced by rBuPh2Si and the transformation of the ester group to formyl group was carried out by treatment with aluminium hydride (AIH3), followed by manganese dioxide oxidation. This triene aldehyde was reacted with the anion of p-cyclogeranylsulfone and quenched with AC2O. Desilylation to the acetoxysulfone (80%), and

77

reductive cleavage with sodium amalgam gave the desired 13Z-retinol (63%), Fig. (11). e)Bu4NF,y)TBDPSCl

»• OHC

EtOOC

^

g) AlCl3/LiAlH4, h) Mn02 OSi/BuMeo

OSi/BuPh,

OSi/BuPho

Fig. (11). Honda, Yoshii, and Inoue (1996). A one-pot procedure was developed by Otera et al from pcyclogeranylsulfone [32]. Its lithium salt reacted with 3,7-dimethyl-8oxo-2,6-octadienyl acetate to the sulfone-alcohol. The hydroxyl group was protected to the MOM ether with MeOCH2Cl. Double elimination could be achieved with potassium MeOK to provide vitamin A in 50% yield. Fig. (12).

.o 9 o.

OHC

^ ^ so. OAc

OAc OH

a) Nal, BuLi

b)MOM-ci

y^^^Yi^^^^ ^

\ X \

c) MeOK

OMOM

Fig. (12). Orita, Yamashita, Toh, and Otera (1997).

^^^^^^^^^^^V^^OAc

78

A similar synthesis was patented by Odera [33]. Two patents by Takahashi et al reported the synthesis of vitamin A via a Cio dihalogeno derivative [34,35]. In one procedure the halogenodiene was prepared by bromination of 3,7-dimethyl-2,5,7octatrien-1-yl acetate. Addition of the latter and /BuOK in DMF to the Cio sulfone provided the retinol sulfone (34%). Again, double elimination (MeOK), gave vitamin A acetate, Fig. (13).

a) Br2

Brv

OAc b) /BuOK, DMF

PY

SOo

I

Br

c) MeOK

-^::s^^^^

OAc

Fig. (13). Takahashi, Furutani, and Seko (2000). They also developed a second process via other dihalo-compounds [36]. Treatment of the 1,2-bromo-hydroxy chain with TiCU in DME, gave mainly the l-bromo-4-chloro unit. Condensation with the Cio sulfone in DMF, in the presence of /BuOK gave the retinylsulfoneacetate. Elimination of the tolylsulfmate with KOH in DMF produced vitamin A acetate in 87% yield. Fig. (14).

Fig. (14). Takahashi, and Seko (2001).

79

In a similar route, Takahashi et al made use of non-halogenated sulfones [37]. Similar processes were related. TiCU was added to a solution of the diol to give a crude mixture of isomers in which the 5-chlorosulfone was the main compound in 95% yield. The mixture was treated with MeOK to produce crude retinol. Acetylation with acetic anhydride (AC2O) in pyridine, in the presence of DMAP, provided the retinyl acetate in 70% from the diol [38,39], Fig. (15).

OH

c) AC2O, DMAP

Fig. (15). Takahashi, Furutani, and Seko (2000). C(io)-C(ii) strategies: Mestres et al [40] published a regioselective addition of a lithium trienediolate (generated from hexa-2,4-dienoic acid or dihydropyran-2one) to p-ionone. Dehydration of the hydroxyacid, afforded a mixture of 9EIZ, 13£'/Zretinoic acids which, isomerised in the presence of I2, led to all E retinoic acid in 35% and 30% yield, starting from dienic acid and pyranone, respectively, Fig. (16).

COOH

COOH

Fig. (16). Aurell, Parra, Tortajada, Gil, and Mestres (1990); Aurell, Came, Clar, Gil, Mestres, Parra, and Tortajada (1993); Aurell, Ceita, Mestres, Parra, and Tortajada (1995).

80

A concise preparation of retinoids via new enaminodiesters synthons was described by Valla et al [41]. For example, all £-retinoic acid was synthesized within one day by a 'one-pot' process. The enaminodiester synthon was prepared from methyl isopropylidenemalonate and dimethylformamide dimethylacetal (DMF-DMA) and then condensed with the lithium enolate of p-ionone. A Grignard reaction with the obtained ketodiester led to the retro carbomethoxyretinoate. Saponification and concomitant decarboxylation, provided mainly all E retinoic acid {all E/UZ: 90/10, 72% from (J-ionone), Fig. (17).

^v

COOMe

a)LDA

l^^^J^

b) MeMgBr

COOMe

COOMe COOMe COOMe

COOH

c)K0H,Me0H,H20

^ COOMe

^HCllM

Fig. (17). Valla, Cartier, Labia, and Potier (2001). A short synthesis of retinal was described by Taylor et al. [42] based on the addition of a Cn vinylalane to a methylpyrylium salt. The 13Zretinal (48%) was isomerised to all E retinal by a previous procedure [43]. p-Ionone was first converted into the alkyne and then into the vinylalane, using the Negishi methodology [44]. Addition of an excess of this alane to 4-methylpyrilium tetrafluoroborate [45] gave 13Z-retinal, being isomerized to the all E isomer (I2 in benzene/ether). Fig. (18). AlMejBuLi

a) MejAl, ZrClz

^ (TI-C5H5)2, BuLi

c)l2 CHO

Fig. (18). Hemming, De Meideros, and Taylor, (1994); Taylor, Hemming, and De Meideros (1995).

CHO

81

Through two successive Stille reactions, Parrain et al [44] realized a stereo selective synthesis of all E, 13Zand 9-A2or-retinoic acids. First, the coupling of £'-l,2-bis(tributylstannyl)ethene and Z- or E-tributylstannyl3-iodoalk-2-enoates was performed, followed by iododestannylation. The second step involved another vinyltin which was synthesized by stannylation of the Negishi dienyne, derived from p-ionone [47]. To obtain the substituted vinylstannate, the dienyne was treated with lithium butyltributylstannylcyanocuprate (Lipshutz reagent) [48] to yield the intermediate vinylcuprate, which was trapped with an excess of Mel in the presence of hexamethylphosphoramide (HMPA). The reaction occurred to the advantage of the terminal vinylstannate (up to 92%). The coupling partner was obtained from tetrolic acid, which was converted into E vinyliodide by stannylcupration of the generated stannate [49]. The Z vinyliodide was more classically obtained by hydroiodination [50]. Stille coupling of the P-iodovinylic acids (protected as the corresponding tributyltin esters) with £'-l,2-bis(tributylstannyl)ethene, catalyzed by dichlorobis(acetonitrile)palladium provided dienyltins with retention of the configuration of the two double bonds in fair yields. Iododestannylation yielded quantitatively the dienic acids, Fig. (19). a) Bu3SnBuCuLi, yr-——

^^

j T-

C00HBu3Sn^

^COOH

^

d) BuSnOMe, PdClzCMeCN); e)l2./)KF,HC\

V c)HI

j

I

SnBu3 g)PdCl2(MeCN)2,DMF

COOH

I

I

^ O ^ : ^ ^ ^ ^

^ \^-!v

COOH

Fig. (19). Thibonnet, Abarbri, Duchene, and Parrain (1999). The first palladium-catalyzed cross-coupling reaction used in the synthesis of retinoids was described by Negishi and Owczarczyk from a Ci4 alkenylzinc [51]. The synthesis was carried out via a Pd(PPh3)4

82

catalyzed coupling of the C14 alkenylzinc (obtained from the iodide) with the Ce iodide (derived from 3-methyl-2£'-penten-4-yn-l-ol), followed by further deprotection with BU4NF. Vitamin A was obtained in 38% yield based on p-ionone, with complete control of stereo- and regiochemistry, Fig. (20).

Q

a) LDA, Cl-P(0)(0Et)2, LDA b) MesAl, Cl2ZrCp2,12,

c) DIBAL-H, I2 ClSiPh2/Bu, EtsN, DMAP

d) /BuLi, ZnBr2 P(i(PPh3)4 e) BU4NF

Fig. (20). Negishi, and Owczarczyk (1991). A highly stereoselective synthesis of retinol vz^ a CM + C6 route was depicted by De Lera et al [52]. A Suzuki reaction of a C14 alkenyliodide with a C6 alkenylboronic acid afforded retinol in 83% yield, with retention of the geometries of the coupling partners. The alkenyliodide was obtained by a zirconium-mediated methylalumination and a subsequent Al/I exchange by slow addition of ICN. Coupling with the C6 boronic acid (12 hrs to reach completion), afforded retinol in 83% yield [53], Fig. (21).

Fig. (21). Torrado, Iglesias, Lopez, and De Lera (1995).

83

C(ii)-C(i2) strategies: Stereoselective syntheses of all E, 9Z-retinoic-acids and llZ-retinal were developed from p-ionone-tricarbonyliron complex [12]. Treatment of the complex (prepared from p-ionone and dodecacarbonyliron, (Fe3(CO)i2)), with the lithium salt of acetonitrile, Wada et al obtained the nitrile, in 88% yield, Fig. (22). CHO

O

a) LDA, MeCN ^ Fe(C0)3 Q (EtO)2P''''~V^COOEt

^ c) BuLi

COOEt

COOH

,)j^30H MeOH, HjG

Fig. (22). Contrarily, the reaction of the lithium enolate of ethyl acetate with subsequent dehydration gave predominantly the ethyl 9Zionylideneacetate in 89% yield, Fig. (23).

O

a) LDA, MeCOOEt ^

Fe(C0)3

/'-p^

CHO ^)BuUTHF

(C0)3 g) NaOH, ^

^ COOEt

MeOH, H2O COOH

COOEt Fig. (23). Wada, Hiraishi, Takamura, Date, Aoe, and Ito (1997); Wada, (2000).

84

These compounds were converted to the corresponding all E and 9Zretinoic acids via P-ionylideneacetaldehydes. Thus, the reaction with the Uthium sah of (EtO)2P(0)CH2C(Me)=CHCOOEt in THF made possible the C20 ester-complex. The complex was removed by CUCI2 in EtOH (98%) and saponification of the ethyl retinoate, the retinoic acids could be obtained {all E: 89%, 13Z: 8% and 9Z: 59%, 9Z,13Z: 12%, respectively). The Peterson reaction of the chlorovinyl-complex with ethyl trimethylsilylacetate provided the HZ isomer preferentially (77%), and the 1 IJE" isomer as a secondary product (15%). The ester was transformed into the Cig ketone (Ph3SnCH2l, BuLi, Et20, 79%). Reaction with (/PrO)2P(0)CH2CN afforded the llZ-retinonitrile in 73% yield. The complex was removed by CuCb (72%) and DIBAL-H reduction led quantitatively to llZ-retinal, Fig. (24). EtOOC

Fig. (24). Wada, Hiraishi, Takamura, Date, Aoe, and Ito (1997); Wada (2000). Wada et al. [13] have previously reported similar syntheses of all E, 9Z-retinoic acids and 1 IZ-retinal. A short access to retinal was reported by Duhamel et al. [54,55] via the enolate of prenal, prepared from the corresponding silyl enol ether or enol acetate. The diene reacted with p-ionylideneacetaldehyde to give the dihydropyranol as the single reaction product. The dihydropyranol was

85

easily converted into retinal (43% yield) by dehydration, ring opening and further dehydration in the presence of a catalytic amount of pyridinium chloride or boric acid, Fig. (25).

OAc

a) MeLi

J^„." t ^

OSiMe,

pyridinium chloride, DMF

Fig. (25). Duhamel, Guillemont, and Poirier (1991); Cahard, Duhamel, Lecomte, and Poirier (1998). These authors also described a three-step synthesis of 13Z-retinoic acid [56]. The obtained hydroxydihydropyrane (66%) was oxidized either by Jones's reagent (CrOs, water, H2SO4, 90%) or Corey's reagent (pyridinium chlorochromate (PCC), 65%). Finally, the dihydropyranone was transformed into retinoic acid (as a mixture of9E, 13Z, and 9Z,13Z), by /BuOK, according to a known procedure [57], Fig. (26).

or PCC

GOGH

Fig. (26). Cahard, Mammeri, Poirier, and Duhamel (2000); Cahard, Duhamel, Lecomte, and Poirier (1998). This French group patented a process for the preparation of vitamin A from vinyl-P-ionol, by BF3-Et20 catalyzed condensation with a C5 sulphide (50% yield) [58]. The phenylthioretinal was reduced with NaBH4 to give the corresponding alcohol (99.5%), which was acetylated (AC2O, -100%).

86

The resulting sulphide-acetate was oxidized with w-chloroperbenzoic acid (MCPBA) and the sulfoxide was eliminated by heating in CCI4 to supply vitamin A acetate in 76% yield. Fig. (27). SPh OH

OMe

^^O 6)NaBH4

a) BF3-Et20

c) Bi^N, AC2O

•Qll

^Y^vA.^^

SPh

d) MCPBA

^Y^^^A-^^^^

ecu ^

kA.

reflux

SOPh

OAc

Fig. (27). Ancel, Bienayme, Duhamel, and Duhamel (1992). Another work of Duhamel and Ancel [59] related this synthesis of retinal via p-ionylideneacetaldehyde. Condensation of methallylmagnesium chloride with diethyl phenyl orthoformate (Et02CH0Ph) led after bromination of the ene-acetal, deshydrohalogenation (NaOH 50%), ethanol elimination with hexamethyldisilazane (HMDS) and ISiMes, to the bromo-dienol ether. This latter was submitted to bromine lithium exchange and the lithio enol ether was then condensed with pionylideneacetaldehyde to give retinal. Fig. (28). GEt MgCl (Et0)2CH0Ph

GEt Br,

GEt

GEt

GEt NaGH

GEt Br

/BuLi

HMDS Ov-^CHG

Fig. (28). Duhamel, and Ancel (1992). In a similar approach, Duhamel et al [60] studied the catalyzed condensation (BF3-Et20 or ZnCb) of vinyl-P-ionol with a chloroenolether. The intermediary aldehyde {all EI9Z\ 65/35) had been

87

dehydrohalogenated (l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 86% or LiCl, 75%), to a mixture of retinals. This mixture had been isomerized to all E retinal, according to literature procedures, [61] Fig. (29).

a) BF3-Et20 or ZnCl2^

CHO orLiCl, DMF

Fig. (29). Duhamel, Duhamel, and Ancel (1994). In connection with a work related to the syntheses of C5 building blocks, Quintard et al [62] described a synthesis of retinal from Pcyclocitral. This aldehyde was condensed with the vinyl lithium salt of the C5 acetal. The lithiated compound was obtained via the vinyltin derivative which was first converted into the vinyl iodide before doing the halogen-metal exchange. Fig. (30 and 31). OEt J^^^^

OEt

.;Bu3SnMgMe,CuCN

i ^ J . ^ ^ ^ ^

c)BuUort^.U

u^J.^^^ OEt

b)\2

Fig. (30). In an iterative fashion, the hydroxyacetal (intermediately formed by condensation of vinyllithium salt with p-cyclocitral) was dehydrated with aqueous HBr. This allowed the simultaneous hydrolysis into pionylideneacetaldehyde, as a mixture of7E,9E (80%) and 7£,9Z (20%). The reaction had been repeated with the same C5 unit and finally, retinal could be obtained as a mixture of isomers, containing 68% of all E isomer (47%) yield from P-cyclocitral), Fig. (31).

>CcCHO

Fig. (31). Beaudet, Launay, Parrain, and Quintard, (1995); Launay, Beaudet, and Quintard (1997). Bienayme and Yezeguelian [63] described a new synthesis of retinal via a Heck vinylation of a C15 tertiary allylic alcohol with a C5 iodoacetal. Thus, the bromo acetal was prepared by a known procedure [64], by a bromination-dehydrobromination reaction sequence (E and Z isomers: 40/60). The iodo acetal could be easily obtained (as a mixture of E and Z isomers, 40/60), by a nickel catalyzed iodine-bromine exchange. This synthon reacted smoothly with the C15 tertiary allylic alcohol in the presence of a catalytic amount of palladium acetate and a stoechiometric amount of either a silver or a thallium salt. The C20 hydroxy-acetal was obtained in 38% yield, as a mixture of E and Z isomers (48/52). Finally retinal was obtained by treatment with dilute HBr in refluxing acetone, as a mixture of £" and Z isomers (C(9)=C(io) and C(i3)=C(i4)), Fig. (32).

I

GEt ""^^ 1 ^ 3

I

OEt

I ' c)lK,NiBrJn

^

OEt ^^^

I

OH

^Pd(OAc),

Fig. (32). Bienayme, and Yezeguelian (1994) In another study, Bienayme [65] obtained retinal in three steps from pionone, involving a Pd-catalyzed rearrangement of a mixed carbonate, derived from ethynyl-retro-ionol.

89

Thus, the P-ionone was smoothly deconjugated and ethynylated to give ethynyl-retro-ionol as a mixture of ElZ stereoisomers. Formation of the carbonate and its Pd-catalyzed rearrangement produced straightforward a mixture of aldehydes and a allene compound. After silica-gel chromatography, the allenic-aldehyde was conjugated with a catalytic amount of HBr in acetone. Retinal was obtained as a mixture of E and Z isomers (75/25), which could be converted into the all E isomer by simple equilibration. Fig. (33).

uC^^ /^^^N.x^^

T^

...^^

c) Pd(0Ac)2

^.

fl)MeONa,NMP

?$^ ^ " ^

^ QC

r^y^^^^'''''^^

—"

^^ ^E~MgCl

o 6)Pd(dba)3,P(napht)3

Y^

CHO

^HBr

CHO

Pd(dba)3, P(napht)3

Fig. (33). Bienayme (1994, 1995). A similar route was patented by Ancel and Meilland [66]. The ethynyl-retro-ionol was acetylated (Ac20-DMAP-Et3N) and this propargylic acetate was reacted with methyl butadiene acetate in the presence of BF3-Et20. The allenic-retinal, obtained in 61% yield was isomerised in retinal by HBr in acetone (yield: 50%), Fig. (34).

CHO

Fig. (34). Ancel, and Meilland (2000).

90

Salman et al [67] described a process for the preparation of 13Zretinoic acid (isotretinoin) in a single step from piony lideneacetaldehy de. Thus, isotretinoin was obtained by treating methyl-3,3dimethylacrylate with LDA, followed by addition of pionylideneacetaldehyde and further hydrolysis with 10% sulphuric acid. The pH had to be adjusted to 2.8 ±0.5, Fig. (35).

'

b)

MeO a) LDA

c)H2S04,pH=2.8

\ X \

COOH

Fig. (35). Salman, Kaul, Babu, and Kumar (2001). Recently Valla et al showed that new 'P-methylenealdehydes' synthons could be substituted to 7jE',9£'-ionylideneacetaldehydes (derived from a and P-ionones) in a Stobbe reaction [68,69]. Regioselective isomerization of these P-methylenaldehydes in Et2NH produce the compound {EIZ\ 97/3). These synthons were synthesized by formylation of ionones and concomitant acetalysation of the sodium salts of the hydroxymethylenic compounds. Wittig reaction and acidic hydrolysis of the p-methyleneacetals produced the pmethy lenealdehydes. Hence, Stobbe-like condensation with dimethyl-isopropylidene malonate and saponification of malonic acid, half-esters afforded the corresponding 14-carboxyretinoic acids, as a mixture of all E and 9Z isomers (80/20). The all E diacid was easily removed by crystallization from MeCN or ether, Fig. (36). A stereospecific decarboxylation in 2,6dimethylpyridine led to isotretinoin.

91

O >Q

a) MeONa, HCOOMe

OMe "OMe

c) ?h^?CH2

b) H2SO4, MeOH COOMe

OMe OMe

d) HCOOH

COOMe y)NaOH COOH

'

COOH

g) ether

COOH

h) 2,6.dimethyl pyridine

COOH

Fig. (36). Valla, Andriamialisoa, Prat, Giraud, Laurent, and Potier (1999); Giraud, Potier, Andriamialisoa, and Valla (1999). A related stereoselective synthesis of all E retinoic acid was also performed by Valla et al [70] from the 14-carboxyretinoic acid, derived from p-ionone, using pyridine (2 eq.) at room temperature for 20 hrs. The crude retinoic acid mixture {all E/13Z: 97/3) was crystallized in MeCN or AcOEt to provide pure all E retinoic acid. Fig. (37). COOH

COOH

a) pyridine ^

COOH

Fig. (37). Valla, Andriamialisoa, Prat, Laurent, Giraud, and Potier (2000). A new preparation of the Cig ketone, an important synthon for the synthesis of vitamin A had also been published by Valla et al [71]. Hence P-ionone and acetonitrile were condensed in the presence of KOH, to afford the nitrile (80%, ElZ isomers: 80/20). A Reformatsky reaction of ethyl bromoacetate with the nitrile provided the ethyl Pionylideneacetoacetate in 70% yield. Subsequent reduction with NaBH4, followed by esterification (MeS02Cl) and desulfonation of the unstable

92

ester, led to the acid {ElZ isomers, 80/20) in 80% yield. Reaction of the latter with MeLi afforded the Cig ketone in 70% yield, as a mixture of 9£/Z isomers (80/20), Fig. (38).

^V-'^^O

CN

ci) MeCN

b) Zn, BrCHsCOOEt

KOH

V^^^^^^/J^^^^

c) NaBH4

COOH

d) MeS02Cl-Me3N

Fig. (38). Andriamialisoa, Valla, Zenache, Giraud, and Potier (1993). In addition, these researchers described a series of 9- and 13methylene analogues. The synthesis of 9 and 13-methylene isomers of retinal has also been reported [72]. Hence, the above described Pmethylenealdehyde was condensed with the carbanion of diethyl 2oxopropylphosphonate, to give the methylene ketone in 51% yield. Condensation of the ketone with A^-ethylidenecyclohexylamine afforded the 9-methylene isomer of retinal, as a 13£/13Z mixture (80/20), Fig. (39).

CHO

a) (EtO)2POCH2COMe

^)MeCH=NC6Hii c) (C00H)2

Fig. (39). Laurent, Prat, Valla, Andriamialisoa, Giraud, Labia, and Potier (2000).

93

The synthesis of the 13-methylene isomer was performed from Pionylideneacetaldehyde {ElZ: 80/20). Condensation with acetone provided the conjugated ketone which, after formylation (MeONa/HCOOEt) and ketalisation (H2SO4/CH3OH), produced the pketoacetal {9EIZ\ 80/20). A Wittig reaction with methyltriphenyl phosphorane (/BuOK/PhaP^CHs, Br") followed by hydrolysis of the pmethyleneketal, produced the 13-methylene isomer of retinal, as a 9E and 9Z mixture (80/20), Fig. (40). O P>.

i.

|c)DIBAL-H

r^^^'^Y^''^^:-^^

y)Mn02

.COOEt

CHO

v^^^^^CHO O

^

(EtO)2P'xA^CN x ^ e) DIBAL-H CN

Fig. (41). Valla, Prat, Laurent, Andriamialisoa, Giraud, Labia, and Potier (2001). These French chemists described a synthesis of ethyl 9-methylene13£ and 13Z-retinoates via the Julia strategy [74]. The required new C15 sulfone was prepared by O-silylation of p-ionone, followed by catalytic condensation (ZnBr2) of the enol with PhSCH2Cl. A Peterson olefmation of the ketosulphide led to the methylenic sulphide. Oxidation (using bis(trimethylsilyl) peroxide [75]), gave the Ci5 9-methylenesulphone, without any detectable oxidation of the double bonds. Thus, condensation with ethyl 4-bromo-3-methyl-2-butenoate (2£/2Z: 50/50) provided the sulphone-ester, as a mixture of isomers (13£'/13Z: 50/50). Elimination to the ethyl 9-methylene-retinoate (2£/2Z: 50/50) was done by treating the crude mixture with EtONa in cyclohexane. Fig. (42).

95

OSiMe Q

b) PhSCHsCl, ZnBr2

a) LDA, Me3SiCl

O l^'^^^Y'^^

^) Me3SiCH2MgCl peroxyde

X^^^^^^A^ I

H

Br-

COOEt

SOoPh

r^^^Y^^^/^^^

COOEt

e) BuLi

Fig. (42). Valla, Laurent, Prat, Andriamialisoa, Cartier, Giraud, Labia, and Potier (2001). These researchers also described further syntheses of modified retinoids such as: 9-demethyl-14-carboxyretinoic acid [76], 9-methylene13-demethyl analogues of natural retinoids [77], aromatic 9-methylene and 13-demethyl-retinol, retinal, and ethyl 13-demethyl-9-methylene retinoate [78], Fig. (43). COOH COOH

R = CH20H;CH0; COOEt

Fig (43). Giraud, Andriamialisoa, Valla, Zennache, and Potier (1994); Valla, Prat, Laurent, Andriamialisoa, Cartier, Labia, and Potier (2001).

96

The Wittig reaction of lithium a-(dimethylamino)-alkoxydes and a Ci5 alkyltriphenylphosphonium salt was used by Wang et al to elaborate the ethylenic linkage of retinol [79]. This in situ method offers the unique advantage in its application to labile aldehydes, which otherwise would become isomerised or self-condensed, Fig. (44).

P"Ph3,Br- ^> /Bir^^'^0 /BuLi, /BuOK

Fig. (44). Wang, Wei, and Schlosser, (1999). Three analogous processes involved the reaction of the C15 phosphonium salt with the 5-hydroxy-4-methyl-2(5/i/)-furanone, in the presence of a base, as described below. To generate the phosphorane, Magnone [80,81], Wang et al [82] and John and Paust [83] used respectively sodium methoxide, triethylamine/MgCb in A^,A^-dimethylacetamide and LiOH in A^,A^dimethylformamide. For the isomerization step, the two first authors emploied rose Bengal as photosensitizer and the latter Erythrosine B, to give isotretinoin. Fig. (45). a)

BrMg"^

b) PhsP, HCl, EtOH OH

*^

c) NaOMe or Et3N, MgClj, AcNMe2 or LiOH, DMF

e) KOH, rose Bengal or Erythrosine B

Fig. (45). Magnone (1996,1999); Wang, Bhatia, Hossain, and Towne (1999); John, and Paust (1994).

97

White et al. developed a stereospecific synthesis of Z-olefins, including isotretinoin [84]. Thus, isotretinoin was obtained by a Reformatsky reaction of p-cyclocitral with the C5 bromoester, followed by DIBAL-H lactone reduction, lactol ring opening, selective olefin bond formation with ethyl 4-diethoxyphosphoryl-3-methyl-2-butenoate and further saponification, Fig. (46). OH

0

\

/

II

t^

\ /

\ >

>C^"« .)EtO^^^^

Uk

'A

1

zii

>

^

b) DIBAL-H

^

" L0

°

d) KOH, EtOH, H2O

JC"

I

•rS r^

r^^

COOH

Fig. (46). White, Hwang, and Winn (1996). Tanaka et al reported a synthesis of vitamin A derivatives from C15 phosphonates [85]. Vitamin A acetate was prepared in 92% yield by reaction of the C15 phosphonate with 2-methyl-4-acetoxy-2-butenal, Fig. (47).

a) /BuONa, DMF, PhMe

Fig. (47). Tanaka, Hanakoa, and Takanohashi (1994). Babler and Schlidt [86] described a route to a versatile C15 phosphonate, used for a stereoselective synthesis of all E retinoic acid and p-carotene. Base-catalyzed isomerization of the vinyl-phosphonate afforded the corresponding allyl-phosphonate as the sole product. Horner-Emmons olefination with ethyl 3-methyl-4-oxo-2-butenoate concluded the facile synthesis of all E ethyl retinoate. The C15 phosphonate was synthesized starting from the epoxide of p-ionone. Subsequent isomerization with MgBr2, afforded the C14 aldehyde in 93%

98

from p-ionone. A modified Homer-Emmons olefmation with tetraethyl methylenediphosphonate led to the vinyl phosphonate in 93% yield. Isomerization to the allylic phosphonate was perfomied with /BuOK. The synthesis of ethyl retinoate was carried out via Homer-Emmons olefination with ethyl 3-methyl-4-oxo-2£-butenoate (61%), Fig. (48).

^v

>Q fl)Me2S=CH2

c)CH2(P(OEt)2)2

Z>)MgBr2

/ ^ ^ ^ ^ C H O

r^V^V-^^^- 80 _g/ml) against human pathogenic fungi such as Candida albicans and Aspergillus fumigatus, and in this respect does not share the activity of certain other tetramic acid metabolites such as the aurantosides that are active against C albicans. Interestingly, P. oryzae is the most sensitive pathogen to cryptocin. This fungus, which causes rice blast and is responsible for significant crop losses, is one of the five targeted diseases in the development of fungicides [70]. Cryptocin is also active against R. solani, a representative of the basidiomycetes that cause cankers, heart and stem rots, root rots, and blights of woody and viney plants. A metabolite (CJ-17,572) from a strain of the fungus Pezicula sp appears to be identical to cryptocin, although the possible identity of the two was not mooted [71]. The lack of reported details, NMR parameters for

124

cryptocin and m.p. for the Pezicula metabolite, makes comparison difficult. Of some interest is the observation that attempted acetylation of the Pezicula metabolite yielded a derivative (37) in which the secondary alcohol had been eliminated and the enol oxygen at C4 acetylated. The metabolite (CJ-17,572) inhibited the growth of multi-drug resistant strains of Staphyllococcus aureus (MIC 10 |ig/ml) and Enterococcus faecalis (MIC 20 |ig/ml) and exhibited cytotoxicity against HeLa cells (ICg^ 7.1 Jig/ml).

NH2

39

40

Yet another analogue (CJ-21,058) (38) of equisetin was isolated from an unidentified soil fungus found at Nagasaki, Japan [72]. It showed marginally greater activity than CJ-17,572 against S. aureus (MIC 5 |ig/ml) and E. faecalis (MIC 5 ^g/ml). Interestingly, CJ-21,058 was discovered using an assay for SecA inhibiting activity. Sec A is a dimer of 102 kDa subunits found in the cytoplasm and bound to the inner membrane and is the peripheral domain of a core containing an integral domain comprising SecY, SecE and SecG proteins. SecA couples the energy from ATP binding and hydrolysis to protein translocation through repeated cycles of insertion and deinsertion of SecA. Compounds that inhibit association of the enzyme complex or of ATPase activity of SecA could provide a new class of antibiotics. CJ-21,058 showed an IC50 of 15 |ig/ml. Other examples in which the decalin system has been modified have been described. The epoxide (39) (PF1052) has been reported as a metabolite from an isolate of a Phoma sp. It showed good activity against Staphylococcus aureus (MIC 3.13 |ig/ml). Streptococcus parvulus (0.78 |Lig/ml) and Clostridium perfringens (0.39 |Lig/ml) [73]. A Microtetraspora sp isolate recovered at Andhra Pradesh in India, produced a metabolite BU-4514N assigned structure (40) from NMR data

125

[74]. It has been claimed to be active against Gram-positive bacteria and to be effective as a nerve growth factor (NGF) mimic. NGF is a protein known to be essential for the development and maintenance of certain sympathetic and sensory neurons in the peripheral nervous system. NGF appears to have functions in the cholinergic neurons in the basal forebrain. BU-4514N is useful for treating neurodegenerative disorders such as Alzheimer_s disease by mimicking the effect of NGF. Cultures of PCI2 rat pheochromocytoma cells respond to NGF by differentiating into sympathetic neuron-like cells. The cells stop dividing, produce nuritelike structures and produce increased levels of neurotransmitters and neurotransmitter receptors [75].

N—r^^ CO^Hs

o»»'

42

Vermisporin (41) is produced by the fungus Ophiobolus vermisporis [76]. Its structure was determined by chemical degradation to the derivative (42) which was studied by X-ray crystallography and provided the absolute configuration [77]. Vermisporin exhibits antimicrobial activity towards Bacteroides spp (0.25-2 |ig/ml), Clostridium perfringens (0.25-2 fig/ml) and methicillin-resistant Staphylococcus aureus (0.12-0.5 |Lig/ml). A metabolite of Ophiobolus rubellus produces the tetramic acid (43) that has been claimed to be an inhibitor of proline hydroxylase (IC5019|LiM) [78]. Three related tetramic acids have been reported from Chaetomium globosum. Two (44, 45) differ in the stereochemistry of the amino acid component, and the third is the methyl ester of 44 [79]. It is claimed that these compounds are chemokine receptor antagonists and can be used to treat HIV-1 infections.

126

44Ri = C02H;R2=OH 4 5 R i = OH;

R2 = C02F

An isolate of Streptomyces lydicus gave lydicamycin (46), a metabolite that showed activity against gram-positive bacteria, Bacillus subtilis (MIC quercetin > kaempferol > luteolin. As for biflavones, the best radical scavengo* is amentoflavone, followed by bilobetin, ginkgetin, isoginkgetin, and sdadopitysin [137]. Recently, free radical scavenging activities of terpene-free EGb and quercetin w^e revealed by means of an in vitro electro-spin resonance assay [138]. Additionally, the in vivo experiments showed that terpene-free EGb inhibits cutaneous blood flux, whidi reflects the skin inflammatory level [138]. In regard to ginkgo terpenes, it has been revealed by means of electron paramagnetic resonance and U\7VIS spectroscopy that ginkgolides B, C, J and M, as well as bilobalide but not ginkgolide A, scavenge superoxide and hydroperoxyl radicals in dimethyl sulfoxide as an aprotic solvent [139]. Akiba et d. showed that EGb prevents the platelet aggregation induced by a combination of 100 f4M terr-butyl hydroperoxide and Fe^*. However, ginkgolides A, B and C, which are known to be PAF-antagonists, have no influence on this aggregation. Therefore, it was suggested that free radicals, but not FAF, might be involved in platelet aggregatk)n induced by oxidative stress [140]. Serotonin (5-HT) produces a rapid elevation of superoxide that stimulates the mitogenesis of bovine pulmonary artery smooth muscle ceUs (SMCs). EGb scavenges superoxide elevated by 5-HT, hence preventing 5-HT-induced mitogenesis on both SMCs and Chinese hamster lung fibroblasts. These results indicate that EGb inhibits the cellular transduction signaling process that leads to mitogenesis, as a result of its antioxidant activity [141]. In addition to radical scavenging properties, it has been reported that EGb reacts with nitric oxide (NO) in in vitro systems [136], and inhibits NO production induced by lipopolysaccharide plus intoferon-Y in maaophage cell Une RAW 264.7 [142]. Fre-treatment with oral administration of EGb reduced nitric oxide overproduction after transient brain ischemia in the MongoHan gerbil [143]. Further experiments showed that EGb inhibits NO production by attenuating the level of iNOS mRNA in a human endothelial cell line (ECV304) [144], also inhibits the activation of protein kinase C (PKC) induced by sodium nitroprusside (SNP), NO generator, and that its flavonoid constituents have protective properties against toxicity induced by SNP on cells of the hippocampus [145]. Recently, it was shown that ginkgolide A, ginkgolide B and bilobalide inhibit NO production in macrophages derived from a human monocytic cell line through attenuation of iNOS mRNA expression. However, these components have no effect on the eNOS-mediated NO production in endothelial ceUs [146].

181

Influences on the Neurotransmitters Numerous studies have demonstrated age-related changes in levels of neurotransmitters and their recq>tors in certain areas of the brain. There is a decrease in the levels of acetylcholine and in the numbers of muscarinic receptors and 6-adrenocqptors in the c^ebral cortex and hippocampus of the brain in patients suffering from Alzheimer's disease and in the brains of aging rodents, diaracterized behaviorally by a sevore impairment ia cognitive functions [147, 148, 149]. Numbers of 5-HT recq)tors and levels of dopamine and noradrcnalin and 5-HT have also shown age-related diminution [150, 151, 152], and are known to be involved in the regulation of mood [153]. Furthermore, it has been demonstrated that the activity of monoanune oxidase (NfAO), which r^ulates the brain concentrations of 5-HX norq)inephrine and other biogenic amines, inaeases with advancing age [154]. Hius, the inhibition of NfAO has been shown to produce antidepressant or anxiolytic responses in animal models and in man [155]. Brain Levels of Biogenic Monoamines Nforier-Teissier et d. [156] determined that administration of EGb alters the levels of catecholamines, indolamines and their metabolites in some brain areas of young rats and mice. Marked changes in the EGb-treated brain were found for norepinephrine, 5-HT, and its metabotite, 5-hydroxyindole-3-acetic add, whereas it was less effective for dopamine and its m^abolite 3,4-dihydroxy-phenylacetic add. EGb-induced changes depend on the route of administration (p. o. or L p.), dose and duration of treatment (acute or dironic). In old rats (26 months old), oral administration of EGb (10 mg/kg and 30 mg/kg, for 7 days) produces elevations of 5-HT in the frontal cortex, hippocampus, striatum and hypothalamus, and of dopamine levels in the hippocampus and hypothalamus compared with controls. On the other hand, EGb decreases the 5-HT level in the pons, and those of norepinephrine in the hippocampus and hypothalamus [157]. In this connection, Racagni et al, [158] showed that the O-methylated amine metaboUte of norepinephnne, normetanq)hrine, was markedly elevated (+500%) in the cerdjral cortex by du:onic oral administration of EGb (100 mg/kg, for 14 days), suggesting an increase of norq)inephrine turnover. In additbn, treatment with EGb (50 or 100 mg/kg/day, for 20 days) diminished the inareased plasma levels of epiDq)hrine, norepinephrine, and corticosterone induced by acute auditory stress in young and old rats [113]. GABA is the major inhibitory neurotransmitter in the CNS and acts to counter glutamateinduced exdtatk)n. Bilobalide (30 mg/kg/day, p.o., for 4 days) elevates GABA levels in the hippocampus and cerebral cortex in mice. These effects of bilobalide are due to a potentiation in glutamic add decarboxylase activity and an enhancement in the protein amount of 67 kDa glutamate decarboxylase. Furthermore, isoniazid and 4-O-methylpyridoxine, pot^t convulsants, induce reductk)ns in brain GABA levels, whereas bilobalide counteracts these effects. These results indicate that potentiation of GABAergic transmission induced by bilobalide might explain its anticonvulsant activity against isoniazid and 4-Omethylpyridoxine [159,160]. Monoamine Oxidase Activity White et al, [161] explored in rat brain mitodiondrial extracts the effect of EGb on MAO activity in vitro. MAOA and MAOB activities wore assayed using [^H]5-HT and [^*C]B-

182

phenetfaylamme as substrates, respectively. EGb inhibited both NfAOAand MAOB activities of rats and mice in vitro to similar extents. These results have suggested that the inhibition of MAO may be a mechanism underlying antidq)ressant or anxiolytk: responses of this extract obtained in animal models and man. Similar observations using a fluorimetric method were shown for EGb, but not for ginkgolide A and ginkgolide B [162]. Sloley et d. showed that kaempferol is a primal in vivo, but not ex vivo, rat brain MAQ-inhibitor in EGb [163]. Besides, the effects of long-term treatment with EGb (500 mg^g/day, for 7 months) on c^ebral MAO activity were investigated in mice subjected to a chronic mild stress. EGb induced reductions in basal MAO activity in 18-month-old mice. Hie age-related inaease in brain MAO activity was lower in die untreated mice subjected to stress and EGb potentiated this effect [164]. Recently, the effects of EGb on aggression woe investigated using MAO-A knockout nuce. EGb reduced their aggressive behavior in resident-intruder confrontations to levels seen in wild types, and decreased their [^H]ketanserin binding to 5-HT2A reoq)tors in the frontal cortex [165]. On the other hand. Fowler et d, recently measured MAO-A and MAO-B activities in the human brain using positron emission tomography and ["C]dorgyline and ["C]Ir dopamine > 5-HT [173]. Similar results were obtained by Ramassamy er d, [174]. These workers showed that EGb deaeased the specific uptakes of [^H]dopamine, [^H]5-HT and [^H]choline by synaptosomes prepared from tiie striatum of mice in a concentration-dependent manner. Tlie IQ^ values were 637 figfiol for [^H]dopamine uptake, 803 /ig/ml for [^H]5-HT uptake, >2000 //gAnl for [^H]choline uptake. However, they conduded that the inhibition of amine uptake caused by EGb appears to be non-specific, since EGb also prevents the specific binding of the dopamine uptake inhibitor [^H]GBR12783 to membranes prq)ared from striatum. EGb in vitro modifies the [^H]5-HT uptake by synaptosomes prepared firom nuce cerebral cortex in a biphasic manner. As mentbned above, the uptake of [^H]5-HT is inhibited by a high concentration of EGb [174]. On the other hand, low concentrations of EGb (4-16 //g/ml) A similar inaease was also obtained when significantly inaease [^H]5-HT uptake. synaptosomes were prepared from the cortk:es of mice treated orally with EGb, either acutely (100 mg/kg, 14 hours and 2 hours before death) or semi-dironically (2 x 100 mg/kg/day, for 4 days). Furthermore, such an inaement in the [^H]5-HT uptake is attributed to the flavonoid constituents of EGb [175], and may be associated with the mechanism of its antidq)ressant activity.

5'HT Receptors Adeaeased (22%) number of 5-HTi^ recq)tor binding sites labeled by [^H]8-hydroxy-2(di-/i-

184

propylainino)tetralin (pHJS-OH-DPAT), a S-HTi^ receptor agonist, in cerebral cortex membranes of Wistar rats was observed in aged (24 months old) rats as compared with young (4 months old) animals. Chronic treatment with EGb (5 mg/kg/day, for 21 days) did not alter the B ^ value in young rats, whereas it significantly inaeased it in aged rats (33%) [176]. On the other hand, Bolanos-Jim^iez et d, showed that chronic treatment with EGb (50 mg/kg/day, 14 days) produced a relatively small diminution in pHJS-OH-DPAT binding to hippocampal 5-HTi;^ receptors in 18-month-old rats [177]. There is at the moment no dear explanation for this disaepancy. An inhibitory effect of S-OH-DPAT on forskolin-stimulated adenylyl cyclase activity is observed in hippocampal membranes of the guinea pig and rat, and has been used as an index of the functional activities of S-HTj^ receptors [178]. Q)ld stress induces a reduction of the inhibitory effect of S-OH-DPAT in the hippocampus isolated from 18-month-old rats, although it has no influence on either the affinity or number of [^H]8-0H-DPAr binding sites. The administration of EGb (50 mg/kg p.o. for 14 days) prevents the cold stress-induced reduction in the inhibitory effect of 8-(Xl-DPAr on forskolin-stimulated adenylyl cyclase activity in old rats. These results indicate that EGb prevents the stress-induced desensitization of hippocampal 5-HTu^ receptors; thus, its effects might explain anti-stress and antidepressant properties of EGb [177].

NMDA Receptor Taylor [173] showed that EGb acts in vitro as an inhibitor of radioligand binding to the competitive and non-competitive sites of ^-methyl-i>-aspartate (NMDA) receptors. In addition, the most potent inhibition (K^ = 0.5 mg/ml) is observed for non-competitive NMDAsites labeled by ['H]MK-801. MPTP-Induced Dopaminergic Neurotoxicity It is known that l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) selectively causes degeneration of the nigrostriatal dopaminergic neuronal pathway in several animal species, which is considered an animal model of Parkinson's disease [179]. As shown in Figure (2), MPTP administered systematically crosses the blood brain barrier, and is oxidized by MAOB into MPP*. This metaboUte is concentrated into dopaminergic neurons and consequently destroys such neurons by generation of ifree radicals [180, 181]. In mice implanted subcutaneously with osmotic minipumps releasing MPTP for 7 days (105 /^g/h/mouse) (approximately 100 mg/kg/day), a decrease in [^H]dopamine uptake by a synaptosomal fraction prepared from striatum was observed. This neurotoxic effect was prevented by the chronic injection of EGb (approximately 100 mg/kg/day in drinking tap water) for 17 days. Such a protective activity of EGb against MPTP neurotoxicity is unlikely to depend on inhibition of the MPTP uptake by dopamine neurons, since the concentration at which EGb prevents [^H]dopamine uptake is too high to reach the brain under in vivo experimental conditions [174, 182]. Therefore, it appears likely that a possible explanation lies in thefree-radicalscavenging property of EGb, which neutralizes free radicals generated from MPP* in the dopaminergic neurons. In this regard, another study showed that protective and curative treatments with EGb prevented the reduction of striatal dopamine levels induced by MPTP. MPTP (30 mg/kg/day.

185

BBB I i^Astrocyte

Nigrostriatal dopamine neuron

MPTP • ' - > MPTP injection

Fig. (2). Hypothesized mechanisms of neurotoxicity of MPTP. After injection of MPTP, its native fomi crosses the blood brain barrier (BBB) and is oxidized by monoamine oxidase B (MAO-B) into MPP^. This metabolite is transported and concentrated into nigrostriatal dopamine and exerts a neurotoxic effect

i.p. for 6 days) sigtdficantly reduced striatal dopamine levels in C57 mice. On the other hand, when C57 mice were pretreated with EGb (20, 50, 100 mg/kg/day, Lp.) for 7 days and then treated with the same extract 30 min before MPTP injection for 6 days, the neurotoxic effect of MPTP was antagonized in a dose-dependent manner. Moreover, in mice treated with EGb (50 mg/kg/day, Lp.) for 2 weeks after MPTP-lesion, the recovery of striatal dopamine levels was accelerated. MPTP is oxidized by MAO-B into MPP^ a positively charged species, whereas EGb, but not ginkgolides A and B, inhibits MAO-B activity. Therefore, another possible explanation for this protectk)n might lie in the inhibition of MAO activity caused by EGb to prevent the oxidization of MPTP into MPP* [162]. Effect of EGb on the Neuroendocrine System It is well known that neuro^docrine dianges with advancing age provide information about CNS functions [183]. The serum prolactin (PRL) level inaeases in old rats (26 months old) compared with young rats (3 months old). Administration of EGb (10 mg/kg/day, p.o., for 7 days) deaeases the blood PRL level, while greatly inaeasing adrenocorticotrophic hormone (ACTH) in old rats compared with age-matched controls. On the other hand, EGb, at a dose of 30 mg/kg, deaeases the serum level of growth hormone (GH) and ACTH in young rats compared with age-matdied controls [157]. Glucocorticoids are also essential for many aspects of normal brain development; however, hyperseaetion induces pathological states such as damage to the hippocampus [184]. Treatment with EGb (100 mg/kg/day, for 8 days) causes a 50% reduction of the plasma corticosterone level This reduction is probably due to deaeases in the number (B,^), mRNA

186

expression and protein levels of adrenal mitochondrial peripheral-type benzodiazepine receptors (PBR), whidi are a key element in the r^ulation of cholesterol transport Similar results have been observed in the chronic administration of ginkgolides A and B (2 mg/kg/day, i.p. for 8 days)[185]. Further study demonstrated that EGb and ginkgolide B also decreased PBR expression and cell proliferatk)n in the highly aggressive human breast cancer cell line MDA-231, which is ridi in PBR [186]. With regard to steroidogenesis, in addition to PBR, an ACTH-dependent process is also responsible for its regulatfon. Using cultured adrenocortical cells, Amri et al, [187] have shown that ex vivo treatment with EGb (100 mg/kg/day, p.o., for 8 days) and ginkgolide B (2 mg/kg/day, i p . , for 8 days) reduces ACTHstimulated cortkx)Sterone production by 50% and 80%, respectively. Moreover, Mardlhac et al. [188] showed that administration of EGb (50 or 100 mg/kg p.o., for 14 days) reduces basal cortkx)sterone seaetion and the subsequent inaease in oortkx>tropin-releasing hormone (CRH) and arginine vasopressin {/^/?) gene expression. Ginkgolide B (2 mg/kg/day i.p., for 14 days) reduces basal corticosterone seaetion without alteration in the subsequent CRH and /WF inorease. However, the stimulation of CRH gene expression by insulin-induced hypoglycemia is attenuated by ginkgolide B. These results indkrate that EGb and ginkgolide B are also able to affect the hypothalamic-pituitary-adrenal axis at the hypothalamic level Corticosteroids play a pivotal role for the development of behavbral sensitization to an^hetamine through the type II glucocorticoid receptor [189], Trovero et d. [190] showed that EGb reduces D-amphetamine (0.5 mg/kg, i.p., for 12-24 days)-induced behavioral sensitization as estimated by increasing values of locomotor activity, although EGb itself has no locomotor effect Furthermore, chronic administration of D-amphetamine reduces the density of [^H]dexamethasone binding sites of type II glucocorticoid recq)tors in the dentate gyrus and the CAl hippocampal regions of D-amphetamine-treated animals, indkating down-regulation of type II glucocorticoid receptors. On the other hand, pretreatment with EGb (50 or 100 mg/kg/day, p.o., for 20-24 days) prevents this down-regulation of type II glucocorticoid receptors, suggesting that EGb restores the density of these receptors. Taken together, these studies indicate that EGb is able to modukte both stress-induced and age-related behavioral sensitizatk>n by regulating alterations of the neuroendocrine system. Effect of EGb on the Phospholipid Metabolism Effects of EGb on ischemia-induced principal changes in the cerebral lipid metabolism were reviewed by Robin et al. [191]. Figure (3) shows diagram of cerebral lipid metabolism following ischemia. Pretreatment witii EGb could normalize the increased mitodiondrial lipid peroxide content and cytosolic lactase dehydrogoiase activity and the deaeased mitochondrial phospholipid contents and superoxide dismutase activity in rat brain after occlusion of common carotid arteries [192]. Rogue et d. [193] have shown that EGb is a potent inhibitor of phospholipase C (PKQ in vitro (ICJQ: 82 //g/ml). Furthermore, in isdiemic rats pretreated with a single injection of EGb (100 mg/kg, i.p.), a deaease in PKC activity is observed as compared with untreated ischemic animals. Impairment of membrane-bound Na,K-ArPase, whidi is responsible for maintaining and restoring membrane potential, and an increased level of malondialdehyde (MDA), which is a known as an index of lipid peroxidation, are seen aft^ unilateral focal cerebral ischemia in the mouse. Pretreatment witii EGb (100 mg/kg/day, p.o. for 10 days) preserves the Na,K-ArPase activity during cerebral ischemia and prev«its the kiaeased MDA levels caused by cerebral

187

Hydroperoxide Prostaglandin Leukotriene

Fig. (3). Diagram of cerebral lipid metabolism following ischemia. During ischemia, AIT-synthesis is disturbed by deficiencies in oxygen and glucose supplies. The subsequent energy failure stimulates phospholipase C (PLC) and Aj/Aj (PIAj/Ai), and thereby leads to formation of diacylglycerol (DAG), which is converted to free fatty adds (FFA) by Upasc, and leads to accumulation FFA and lysophospholipids. Among FFA, especially, the peroxidation of arachidonic add (AA) initiates a cascade leading to lq)oxygenase and cydooxygenase metabolites (prostaglandins and leukotrienes) and hydroperoxide, which are augmented during reperfusion following ischemia. Aoetylation of lyso-platelet-activating factor (lyso-PAF) leads to PAF, a mediator of inflammation.

ischemia [194]. Similar inhibitory effects of EGb on MDA production induced by hydrogen peroxide have been shown in erythrocyte membranes [195, 196]. Electroconvulsive shock (ECS), as well as ischemia, induces inaeases in free fatty add (FFA) and diacylglycerol (DAG) in the rat brain, probably due to the breakdown of membrane phospholipids through the activation of phospholipases (PLC, PLAj/Aj). EGb treatment (100 mg/kg/day, p.o. for 14 days) selectively decreases endogenous FFA levels and increases endogenous DAG levels in the hippocampus. Therefore, ECS-induced accumulation of FFAis prevented in the hippocampus of EGb-treated rats during clonic seizures (30 sec to 2 min after

188

ECS). Furthermore, the inaeased DAG levels induced by ECS are delayed by EGb treatment and the subsequent decrease in DAG levels is accelerated by EGb treatment in both the hippocampus and cortex [197]. Hypoxic or ischemic conditions led to an immediate release of free dioline via the breakdown of choline-containing phospholipids in rat hippocampus slices. Klein et d. [198] showed that bilobalide inhibited the hypoxia-induced dioline release in a dose-dependent manner both in vitro (ECjo*. 0.38 /^M) and ex vivo (2-20 mgAcg, p.o.). Asimilar reduction of dioline release was confirmed after administration of EGb (200 mg/kg, p.o.). Bilobalide also inhibits the ^-methyl-D-aspartate-induced, PLAj-dependent release of dioline from hippocampal phosphol4)ids both in vitro (10-100;^M) and in vivo (20 mg/kg, Lp.) [199]. Rabin et d. [200] investigated the effect of EGb on the rate of FFAreincorporation into brain phospholipids during reperfiision following ischemia in the gerbil brain by means of quantitative autoradiography and biodiemical analysis. Isdiemia-reperfusk>n selectively reincorporated arachidonic add (AA) into brain phospholipids. Pretreatmait with EGb (50 or 150 mg/kg/day, for 14 days) accelerated AA reincorporation following isdiemia, suggesting that EGb ameliorates the neurotoxic reaction caused by prolonged ^posure of the brain to high concentrations of AAand its metabolites, and stabilizes the membrane bilayer. Taken together, these results showed that EGb can prevent isdiemia-induced Na,K-ArPase injury, and suppress hypoxia- and ECS-induced membrane phospholipid breakdown in the brain, and bilobalide might be associated with its protective action. In addition, EGb reduces AA-induced neuronal damage as a consequence of the increase in reincorporation of A A Therefore, these medianisms might provide a possible explanation for neuroprotective properties of EGb and bilobalide against oxidative damage. Anti-PAF Platelet-activating factor (PAF), a potent phospholipid inflammatory mediator, enhances glutamatergic exdtatory synaptic transmission in the h^>pocampus [201]. Braquet et d, [202, 203] showed that ginkgolides, mainly ginkgolide B, act as potent antagonists of PAF in various cell types. Pretreatment of ginkgolides (10 mg/kg/day, p.o., for 7 days) ameliorated behavioral impairments assessed by the MacGraw stroke index, and inq)roved the mitodiondrial respiration evaluated by the respiratory control ratio (RCR) following c^ebral ischemia obtained by bilat^al ligature of the common carotid arteries in Mongolian g^bils [204]. The order of these effects in cerebral isdiemia was ginkgolide B > ginkgolide A > ginkgolide C > ginkgolide J, and was correlated with that of their PAF antagonistic properties described by Braquet et d, [203]. Consistent with this finding, liu et d. showed that both preand post-hypoxic treatment with ginkgolide B (25 mg/kg/dose, two serial doses) decreased the inddence of cerebral infarction in hypoxic ischemic brain injury of immature rats [205]. Moreover, Akisu et d, [206] found that endogenous PAF concentrations in brain tissue markedly inaeased in the hypoxic-ischemic brain in immature rats. Pretreatment with EGb reduces endogenous PAF concentrations in cerebral hypoxic-ischemic brain injury of immature rats as compared with controls. These results indicate that the PAF-antagonistic activity of the ginkgolides contributes to the neuroprotective effect against brain injury associated with an episode of the post-isdiemic phase. This idea was supported further by the observation that in primary neuronal cultures isolated from onbryonic rat cerebral cortex, ginkgolide B demonstrates protective effects against glutamate neurotoxidty involving PAF [207].

189

It has been demonstrated that ginkgolide B prevents bng-tenn potentiation (LIP) induced by PAF in the h^}pocampus [208] and in the voitral part of the medial vestibular nudei [209]. These results suggest that PAF might act as a retrograde messenger in ITP, which activates the presynaptic mechanisms enhancing the glutamate release. Izqui^do et d, found that pre- or immediate post-training intrahippocampai or intraamygdala infusion of the PAF antagonist, ginkgolide B, produces amnesia for avoidance tasks. These results support the idea that PAF may play a role in memory formatfon [210]. Amine Uptake and Membrane Fluidity Protonged incubatbn of synaptosomes prqiared from the striatum in the presence of ascorbk: add (10^ M) decreases the ability of synaptosomes to take up [^H]dopamiae. Similar inhibition is also observed in the ability of cortical synaptosomes to take up [^H]5-HT. Furthermore, this decrease is potaitiated by addition of Fe^* tons. EGb (4-16 /igAni), in particular its flavonoid fraction, prevents the reduction in the ability of synaptosomes to take up either 5-HT or dopamine [211]. Moreover, EGb (10 //g/ml) prevents a decrease in binding of [^H]GBR12783, the dopamine uptake inhibitor, to dopamine recq)tors in the presence of the combination of ascorbic add/Fe^* ions. These results suggest that EGb-induced prevention of the impainnent of the ability of synaptosomes to take up [^H]amine by the ascorbic add/Fe^* ions is related its inhibitory properties in generation of free radicals. However, EGb does not modify the inaeased [^H]dopamine release that is triggered by high potassium concentrations in the presence of the combination of ascorbate/Fe^*, suggesting that the vesicular exocytotic dopamine release does not seem to depend upon peroxidation [212]. Furthermore, Ramassamy et d. [213] showed that the combination of ascorbic acid/Fe^* ions could deaease synaptosomal membrane fluidity measured by fluorescence polarization using 1,6-diphenyl 1,3,5-hexatriene in a concentration-dq)endent manner. Free radical generation by ascorbic add/Fe^* results in a decrease of membrane fluidity through the peroxidation of neuronal membrane Upids. These membrane altCTations were prevented by either EGb (2-16 ;-Methylglabridin (123) Hispaglabridin A (3'-prenylglabridin) Glabrol (25) 3-Hydroxyglabrol (26)* Glabrone (DMP;4',3']-2',7-dihydroxyisoflavone) Medicarpin (3-hydroxy-9-niethoxypterocarpan) Shinpterocarpin (DMP;3,4]-9-hydroxypterocarpan) Euchrenone as (DMP;4',3']-7-hydroxy-8-prenylflavanone) Glyinflanin K (2DMP;7,8, ;2',3']-isoflavan) Glyinflanin G (2DMP;4,5, ;4',3']-2',3-dihydroxychalcone) Kanzonol U (DMP;2',3']-4',6-dihydroxy-2-arylbenzofuran] Kanzonol V (DMP;2',3']-4',6-dihydroxy5-prenyl-2-arylbenzofuran) Kanzonol W (DMP;7,8]-2',4'-dihydroxy-3-arylcoumarin) Kanzonol X (3',8-diprenyl-2',4',7-trihydroxyisoflavan) Kanzonol Y (3,5'-diprenyl-a,2',4,4'-tetrahydroxy-dihydrochalcone) Kanzonol Z (DMP;7,8]-3,4'-dihydroxy-3'-prenylflavanone) 3-Hydroxyparatocarpin C**

+-H-¥ +++ ++ ++ +++ + ++ ++ + ++ +++ ++ ++ -

European [50,51 ] -

+++ ++++ ++ ++-H+++ +++ ++ ++++ +++ ++ ++ ++ ++ +++ +++ +++ +++ +++ +++

Yields from dried licorice roots: -H-i-H=more than 0.01%; +-H-=between 0.01 and 0.001%; ++=0.001-0.0001%; +=1-0.1 ppm. • The compound was obtained from the stolons. ° 2,2-dimethylpyrano[b=DMP. ** Tentative name used here (DMP;4,5]-3'-prenyl-2',3,4'-trihydroxychalcone).

The difference of the substituents at C-5 is expected that European and Chinese licorices exhibit different actions in therapeutically use. For example, 5,6-disubstituted isoflavans do not showed a potency of

207

anti-HIV activity in vitro, but two isoflavans with no substituent at both 5- and 6-positions obtained from Erythrina lysistemon (Leguminosae) have the activity as described later [52]. As described the above, moraceous plants and Glycyrrhiza species are rich sources of isoprenylated phenolic compounds. The phenolic nuclei having the isoprenoid-derived substituents, e.g., simple isoprene or a monoterpenoid, vary over a wide range from a simple phenol to complicated ones. Some of the moraceous plants studied by our group have been used as traditional herbal medicines in the native countries. It is interesting to clarify the relationship between the usage and biological activities of the isoprenylated phenolic compounds. So we studied some of the biological activities of these compounds. This article reviews the biological activities of the isoprenylated flavonoids isolated from the moraceous plants and isoprenoid-substituted phenols (flavonoids, xanthones, dihydrostilbenes, and dihydrophenanthrenes) from Glycyrrhiza species by our group and other several groups. 11. HYPOTENSIVE ACTIVITY OF ISOPRENYLATED FLAVONOIDS FROM THE ROOT BARK OF MORUS SPECIES The first report for the hypotensive effect of the mulberry tree was presented by Fukutome in 1938, who asserted that oral administration of the hot water extract of the mulberry tree showed a remarkable hypotensive effect in rabbits [53]. Ohishi reported the hypotensive effect of the ethanol extract of mulberry root bark [54]. Suzuki and Sakuma reported that the hypotensive activity seemed to be due to phenolic substances, and that the effect disappeared on acetylation [55]. Later, Katayanagi, et aL reported that the ether extract of the root bark gives to rabbit (6 mg/kg, i.v.) showed a marked hypotensive effect and that the active constituents seemed to be a mixture of unstable phenolic compounds [56]. Tanemura ascribed the activity of mulberry root bark to acetylcholine and its analogous presumably contained in the alcohol soluble fraction, and that the hypotensive constituents produced a yellowish-brown precipitate on treatment with Dragendorff reagent [57]. Yamatake, et al reported that n-butanol- and water-soluble fractions of mulberry root bark had similar effect except for those on the cardiovascular system. Both fractions showed cathartic, analgesic, diuretic, antitussive, anti-edema, sedative, anticonvulsant, and hypotensive actions in mice, rats, guinea pigs and dogs [7]. On the beginning of our study of mulberry tree, the hypotensive constituents had not been identified. In view of the reports, we assumed that the hypotensive compounds of the plant would be a mixture of unstable

208

phenolic compounds and therefore undertook a study of the phenolic constituents of the root bark of the cultivated mulberry tree. The root bark of the cultivated mulberry tree was extracted successively with n-hexane, benzene, and methanol. The methanol extract, 1-20 mg, showed a dose-dependent decrease in arterial blood pressure in pentobarbital-anesthetized rabbit, Fig. (4). The extract was fractionated successively by silica gel column chromatography (C.C.), polyamide C.C, silica gel preparative (p.) TLC, and p. HPLC leading to isolated of kuwanons G (1, 0.2% yield) [9] and H (2, 0.13% yield) [10]. The root bark of Moms alba n-Hexane Residue Benzene

Extract

Residue I Methanol Residue

Extract -T extract Ethyl acetate soluble portion I C.C, p. TLC, p. HPLC

C.C, p. TLC Morusin (3), kuwanons C (42), D, E (43), F, oxydihydromorusin (46), mulberroftiran A (47)

Kbwanons G (1), H (2), L (44), M (35), albanol B (97) mulberrofurans C (28), F (29), and G (30) Fig. (4). Isolation procedure of flavonoids from the root bark of Morus alba.

mmHg

PN n > < l i < M H H H H M M ( H i t i i kuwanon G 1 mg/kg i.v. mmHg

tsmmm |ioo 50

iilSliSirJ'^^

kuwanon H I mg/kg i.v.

'

10 s

Fig. (5). Effects of kuwanon G (1) and kuwanon H (2) on blood pressure. Electrocardiogram (ECG), phrenic nerve discharge (PN), and electroencephalogram (EEG) in a gallamine-immobilized rabbit.

209

Both compounds (1 and 2) almost equally caused decrease of arterial blood pressure in a dose dependent and reversible manner at the dose of between 0.1 and 3 mg/kg, i.v. in pentobarbital-anesthetized as well as in un-anesthetized, gallamine-immobilized rabbits. Fig. (5), [58]. These hypotensive actions of kuwanons G (1) and H (2) were not modified by atropine or eserine, suggesting the non-cholinergic nature origin. Furthermore, neither propranol nor diphenhydramine affected their actions on the arterial blood pressure. Although they produced no significant change in both electrocardiogram (ECG) and respiration when administered intravenously in rabbits. The hypotensive effects of kuwanon G (1) and H (2) did not accompany with heart rate change [58]. In pentobarbital-anesthetized pithed dogs, kuwanons G (1) and H (2) also significantly decrees of femoral arterial blood pressure. These effects suggested that mechanism of hypotensive effects of kuwanons G (1) and H (2) mediated through peripheral system. Mulberrofurans C (28) [59], F (29) [60], and G (30) [60], Fig. (6), were also isolated as hypotensive components from the mulberry tree. Mulberrofuran C (28) is considered to be formed by a Diels-Alder type of enzymatic reaction process of a chalcone derivative and dehydromoracin C (31) or its equivalent. Furthermore, mulberrofurans F (29) and G (30) seems to be Diels-Alder type adducts derived from chalcomoracin (32) and mulberrofuran C (28), respectively, by the intra-molecular ketalization reaction of the carbonyl group with the two adjoining hydroxyl groups, 3'(5')-OH and 2"-0H. Intravenous injection of mulberrofuran C (28, 1 mg/kg) produced a significant hypotension (37 mmHg fall) in rabbit (male, 3.3 kg) anesthetized with pentabarbital sodium (30 mg/kg). Single intravenous injection of mulberrofurans F (29) and G (30) (both 0.1 mg/kg) caused a marked depressor effect in rabbit by 26 mm Hg and 16 mm Hg, respectively. On the other hand, in Japan, "Sang-Bai-Pi" (the root bark of Chinese mulberry tree) imported from China has been used as an herbal medicine, hence a study of the components of this crude drug purchased in the Japanese market was undertaken. Its phenolic components are different from those of Japanese mulberry tree. For example, morusin (3) and kuwanon G (1) are the main phenolic components of Japanese mulberry tree, in the case of "Sang-Bai-Pi", these components are minor ones, while sanggenons A (4) [16], C (5) [17], and D (33) [61] are the main components [24]. Sanggenons C (5) and D (33) showed the hypotensive effects as follows: Sanggenon C (5) caused transient decrease in arterial blood pressure at the doses of 1 mg/kg in pentobarbital-anesthetized rabbit by 15 mm Hg, while at the doses of 5 mg/kg the compound (5) caused a transient decrease by 100 mm Hg, which continued for more

210

mulberrofuran C (28): R = H chalcomoracin (32): R = CH2CH=CMe2

mulberrofuran F (29): R = CH2CH=CMe2 mulberrofuran G (30): R = H

kuwanon E (43) sanggenon B (45)

Fig. (6).

Structures of flavonoids (28 - 44) from moraceous plants.

211

than one hour by 15 mm Hg [17,62]. Sanggenon D (33) caused a transient decrease at the dose of 1 mg/kg in pentobarbital and urethane anesthetized male Wister strain rat by 35 mm Hg, while the compound (33) caused a decrease by 80 mmHg at the doses of 1 mg^g in spontaneously hypertensive rat [61,63]. III. ANTI-TUMOR PROMOTING ACTIVITY OF MORUSIN (3) Cancer chemoprevention is the most important subjects in cancer research at present and is a new medical strategy for cancer prevention, which was established by recent understanding of molecular multistage carcinogenesis in humans. To find nontoxic cancer preventive agents, Fujiki and his coworker studied natural products derived from marine and plant sources [64,65]. In 1987, Yoshizawa, et aL reported that (-)epigallocatechin gallate (EGCG), which is a main constituent of green tea, inhibited tumor promotion by teleocidin in mouse skin [66]. In 1988, Fujita, et aL reported the inhibitory effect of EGCG on carcinogenesis with 7V-ethyl-A^-nitro-A^-nitrosoguanidine in mouse duodenum [67]. On the other hand, in the course of our examination the constituents of the Morus root bark, we found the following novel photo-oxidative cyclization. When a solution of morusin (3) in chloroform (CHCI3) was irradiated using high-pressure mercury lamp, morusin hydroperoxide (34), Fig. (6), was obtained in ca, 80% yield [68]. The reaction did not occur in the dark and was depend on the solvent; the reaction occurred in low polar or nonpolar solvent such as CHCI3 and benzene, but not in protic solvent. The reaction mechanism was suggested as follows [69]: morusin (3) in the ground state interacts with an oxygen molecular to form a contact charge transfer complex [3 O2] (CCTC). On irradiation, the CCTC gives an excited charge transfer state that presumably leads to reactive species such as free radicals as described in Fig. (7). Recently, the proof of presence of the CCTC was provided by laser desorption/ionization time-of-flight mass spectrometry of 3 [70]. The hydroperoxide (34) was also obtained with the oxidation of morusin (3) with singlet oxygen or radical initiator [71]. HO^^s^^OH hv 34 •OOH

Fig. (7). Reaction mechanism of photo-oxidative cyclization of morusin (3).

212

This photoreaction and the relative reaction of morusin (3) along with the anti-tumor promoting activity of EGCG encouraged us to examine the anti-tumor promoting activities of a series of isoprenylated flavonoids isolated from Morus species. First we examined the inhibition against three biochemical effects; the specific binding of ^H-12-O-tetradecanolylphorbol-13-acetate (TPA) to mouse particulate fraction, the activation of Ca^'^-activated phospholipid-dependent protein kinase (protein kinase C) with teleocidin, and induction of ornithine decarboxylase (ODC) with teleocidin in mouse skin [72]. Interestingly, of the eight isoprenylated flavonoids, morusin (3), kuwanons G (1) and M (35), mulberroforan G (30), and sanggenon D (33) gave similar results in these biochemical tests as described in Table 2. Table 2.

Effects oi Morus flavonoids on biological and biochemical activities Inhibiting of specific [^H]TPA binding (ED50 jimol/L)

57 99 100 85 34 62 48 60

Morusin (3) Kuwanon G (1) Kuwanon H (2) Kuwanon M (35) Mulberrofuran G (30) Sanggenon A (4) Sanggenon C (5) Sanggenon D (33)

Inhibition of activation of protein kinase C (ED50 fimol/L)

80 40 80 22 46 80 46 42

Inhibition of ODC induction

(%) 43 34 -35 25 10 -62 -17 17

100

^

o

o

Concentration (mol/L) of morusin (3) Fig. (8). Effects of morusin (3) on specific binding of [^H]TPA to a mouse skin particulate fi-action. Various concentrations of morusin (•) or TPA (o) were incubated with a particulate fi-action of mouse skin in the presence of 4 nmol/L [^H]TPA for 2 h at 4°C, and the assay mixture was filtered on glass filter membrane with acetone cooled in a dry ice-ethanol bath. Non-specific bindings were measured in the presence of 500-fold excess of unlabelled TPA.

213

Of these five compounds, morusin (3) is the least toxic and can be isolated as one of the main phenolic compounds from the root bark. The more detailed data for the above these biochemical tests of morusin (3) were as follows [73]. As shown in Fig. (8), morusin (3) caused dose-dependent inhibition of the specific binding pHJTPA to a mouse skin particulate fraction. The concentration of morusin (3) for 50% inhibition (ED50) was 57 |amol/L, whereas that of unlabelled TPA was 4 nmol/L. As morusin (3) was assumed to interact with the phorbol ester receptor, we examined whether it inhibited the activation of protein kinase C by teleocidin in vitro [73]. Fig. (9) shows that morusin (3) inhibited the phosphorylation of histone type III-S by protein kinase C dose-dependent and that 80 |imol/L morusin caused 50% inhibition. 100

o

VA

50

a

0.

VA

10-

\o-

10-*

Concentration (mol/L) of morusin (3) Fig. (9). Inhibition by morusin (3) of activation of protein kinase C by teleocidin in vitro. The assay mixture (0.25 mL) contained 20 jimol/L CaCh, 7.5 |ag of phosphatidylserine, 2.3 (^mol/L teleocidin, and various concentrations of morusin (3) with 0.05 units of partially purified enzyme. Enzyme activity was measured as the incorporation of ^^P from [7-^^P]ATP into histone type III-S during incubation for 3 min. at 30^.

Furthermore, we examined the inhibition of the induction of ODC induction by teleocidin in mouse skin. Application of 11.4 nmol morusin (3) caused 43% inhibition of the induction of ODC by 11.4 nmol teleocidin [73]. From the results of these three tests, morusin (3) might inhibit the tumor-promoting activity of teleocidin on mouse skin. As shown in Figs. (10) and (11), the percentage of tumor bearing mice in the group treated with 7,12-dimethylbenz[a]anthracene (DMBA) plus teleocidin reached 100% by week 15, o in Fig. (10). In contrast, the onset of tumor formation was delayed 5 weeks by treatment with morusin (3), • in Fig. (10), and the percentage of tumor-bearing mice in the group treated with DMBA plus teleocidin and morusin (3) was 60% at week 20. The average number of tumors per mouse in week 20 was also reduced from 5.3, o in Fig. (11), to 1.1, • in Fig. (11), by morusin (3) treatment.

214

On the other hand, morusin (3) itself did not show a tumor promoting activity on mouse skin, x in Figs. (10) and (11). From these results, morusin (3) is an anti-tumor promoter judging from its ability to inhibit the short-term effects induced by tumor promoters.

100

to

10

20

Weeks of promotion

10

20

Weeks of promotion

Figs. (10) and (11). Inhibition by morusin (3) of tumor promotion by teleocidin in a two-stage carcinogenesis experiment on mouse skin. Inhibition was achieved by a single application of 100 ^g of DMBA, and teleocidin (2.5 }ig) and morusin (1 mg) were applied twice a week throughout the experiments.

As mentioned the above, morusin (3), kuwanon G (1), kuwanon M (35), mulberroforan G (30), and sanggenon D (33) showed inhibitory effects in the three biochemical tests. The anti-tumor promoting activities of later four flavonoids with one or two isoprenoid groups have not been tested in a two-stage carcinogenesis experiments, due to limitations of their amounts available, but their inhibitory potencies to the three biochemical tests were almost similar to that of morusin (3). Furthermore, the twelve isoprenylated flavonoids from the moraceous plants and two flavonol glycosides (48 and 49) from Epimedium species (Berberidacaceae) [74] along with quercetin (50) were tested for inhibitory effects on carcinogenesis by a test for inhibition of specific binding of [^H]TPA to a mouse skin particulate fraction. While the other biochemical tests and the inhibition of tumor promotion of teleocidin in a two-stage carcinogenesis experiment have not been carried out, due to limitation in their amounts available, some of isoprenylated flavonoids from the moraceous plants showed the similar inhibitory potencies to those of morusin (3) and the related compounds, Figs. (6) and (12), as shown in Table 3. On the other hand, EGCG and green tea extract are acknowledged cancer-preventive agents in Japan [75,76]. Natural products with antitumor promotion activity isolated from foodstuff and medicinal plants have been summarized by Konoshima and his co-worker and Akihisa and

215

his co-worker [77,78]. Considering these results as well as the results of biochemical tests and anti-tumor promoting activity of the isoprenylated flavonoids from the moraceous plants in a two-stage carcinogenesis experiment with teleocidin, the isoprenylated poly-phenolic compound seems to be interesting compounds for finding cancer preventive agents and the more detailed experiments should be carried out. Table 3.

Effects of the isoprenylated flavonoids on inhibition of specific [^H]TPA binding (ID50, ^mol/L)

Kazinol C (36) Kazinol E (37) Kazinol F (38) Kazinol J (39) Kazinol M (40) Kazinol N (41) Kuwanon C (42) Kuwanon E (43)

Kuwanon L (44) Sanggenon B(45) Oxydihydromorusin (46) Mulberroftiran A (47) Ikarisoside A (48) Ikarisoside B (49) Quercetin (50)

80 70 98 90 100 >100 80 83

80 95 95 >100 >100 >100 >100

OMe oxydihydromorusin (46) mulberrofuran A (47)

ikarisoside A (48): R = Rha ikarisoside B (49): R = Glu(1 ^ 2)Rha

OCH3 OH

O

quercetin (50): Ri = OH,R2=R3 = H cirsilioi (51): Ri = H, R2 = 0Me, R3 = Me

antiarone L (57) artonin H (56)

Fig. (12). Structures of flavonoids (46 - 57) from moraceous plants, Epimedium species, and test reagents (50 and 51).

IV. INHIBITION OF ARTONIN E (7) AND RELATED COMPOUNDS ON 5-LIPOXYGENASE Previously, we reported the effects of Morus flavonoids on arachidonate metabolism in rat platelet homogenates, such as inhibition of 12-hydroxy5,8,10-heptadecatrienoic acid (HHT), thromboxane B2, and 12-hydroxy5,8,10,14-eicosatetraenoic acid (12-HETE) [79,80]. As described in the

216

introduction, Artocarpus plants (Moraceae) have been used as traditional medicine in Indonesia for swelling and malarial fever. This usage seems to be expecting for effect of anti inflammation. As leukotrienes are known to be chemical mediators of anaphylaxis and inflammation, a number of compounds have been studied and developed as selective inhibitors of 5-lipoxygenase, the enzyme initiating leukotriene biosynthesis from arachidonic acid. So the inhibitory effect of the Artocarpus flavonoids against arachidonate 5-lipoxygenase was examined [81]. Yamamoto, et aL screened various flavonoids, and found that cirsiliol (51), Fig. (12), potently inhibited 5-lipoxygenase and proposed two structural factors of the flavonoids for the specific inhibitory activity, one is catechol type of the B ring and the other is the presence of an alkyl-like side chain at the C-3 position [82,83]. We had interesting for the inhibitory effects of a series of Artocarpus flavones on the 5-lipoxygenase activity. Seven Artocarpus flavonoids and morusin (3) were tested for their inhibitory actions on arachidonate-5-lipoxygenase purified from porcine leukocyte [84]. As shown in Fig. (13), the IC50 values varied depending on the structural modification of the compound. The compounds having three hydroxyl groups at positions 2\ 4\ and 5' on the B ring (compounds 7, 8, 52 and 55) were more potent inhibitors. Thus, the vicinal diol partial structure was important for 5-lipoxygenase inhibition.

OH o heterophyllin (52)

OH

0

artonin A (54)

cycloheterophyllin (53)

Inhibitory effects (IC50 ± SD, N=3, ^imol/L) on arachidonate 5-lipoxygenase activity

OH

0

artonin B (55)

Morusin (3) Artonin E (7) Artobiloxanthone (8) j Cycloartobiloxanthone (9) Heterophyllin (52) Cycloheterophyllin (53) Artonin A (54) Artonin B (55)

2.9 ± 0.4 0.36 db 0.03 0.55 db 0.20 1.3 ±0.2 0.73 ±0.21 1.6±1.0 4.3 ± 0.5 1.0 ±0.1

1

'

Fig. (13). The inhibitory effect (IC50 ± SD) on arachidonate 5-lipoxygenase activity.

As shown in Fig. (14), 5-lipoxygenase was inhibited depending on the concentration of artonin E (7), which gave the lowest IC50 (0.36 |Limol/L) of all the eight compounds. On the other hand, morusin (3), which

217

lacked the 5'-hydroxyl group of artonin E (7), was a less potent 5lipoxygenase inhibitor (IC5o=2.9 |Limol/L). Artonin E (7) was significantly more potent than cirsiliol (51, Fig. (12), IC5o=1.3 |Limol/L), which was reported as a 5-lipoxygenase inhibitor. This finding was consistent with the report that the inhibitory activity of cirsiliol (51) with 5-lipoxygenase was enhanced by introducing a lipophilic alkyl group at the C-3 position of theflavoneskeleton. Inhibitory actions of artonin E (7) and morusin (3) on other mammalian arachidonate oxygenases were examined. Artonin E (7) inhibited two 12-lipoxygenase from porcine leukocytes and human platelets, 15-lipoxygenase from rabbit reticulocytes, and fatty acid cyclooxygenase from bovine vesicular glands (IC5o=2.3, 11, 5.2, and 2.5 |amol/L, respectively). However, IC50 values for these oxygenases were higher by one order of magnitude than that for 5-lipoxygenase. Morusin (3) also inhibited these enzymes (except for human platelet 12lipoxygenase) with IC50 values of micro molar order as follows: two 12lipoxygenase from porcine leukocytes and human platelets, 15lipoxygenase from rabbit reticulocytes, and fatty acid cyclooxygenase from bovine vesicular glands; IC5o=3.4, > 30, 3.3 and 1.6 |imol/l, respectively. These results indicated that artonin E (7) was a relatively specific inhibitor for 5-lipoxygenase. Thus, which the selectivity for 5-lipoxygenase was not observed with morusin (3). Significant differences of IC50 values of artonin E (7) and morusin (3) between porcine leukocyte 12-lipoxygenase and the human platelet 12-lipoxygenase should be noted since the leukocyte and platelet 12-lipoxygenase were distinct both catalytically and immunologically.

Concentration (|imol/L) Fig. (14). Dose-dependent inhibition of 5-lipoxygenase by artonin E (7, • ) , morusin (3, o), and cirsiliol (51, A).

V. INHIBITION OF ARTONIN E (7) AND RELATED COMPOUNDS ON MOUSE TNF-a RELEASE AND THEIR CYTOTOXIC ACTIVITIES

218

As described in Chapter III, morusin (3) has been found to be anti-tumor promoter in a two-stage carcinogenesis experiment with teleocidin. Considering the similarity of the structures between morusin (3) and artonin E (7), artonin E (7) was expected to be an anti-tumor promoter. Furthermore we found a novel photo-oxidative cyclization of artonin E (7) as follow: photo-reaction of artonin E (7) in CHCI3 containing 4% ethanol solution with high-pressure mercury lamp produced artobiloxanthone (8) and cycloartobiloxanthone (9), and the treatment of artonin E (7) with radical reagent (2,2-diphenyl-l-picrylhydrazyl: DPPH) resulted in the same products, Fig. (15), [84].

(±)-artobiloxanthone (8)

artonin E (7)

hv, 24 h. CHCI3 DPPH, 24 h, CHCI3 (in the dark)

Fig. (15).

8

9

34% 70%

3% 4%

OH 0 (±) -cycloartobitoxanthone (9)

Photoreaction of artonin E (7) and the reaction with radical reagent.

As described in Chapter III, we have reported the photo-oxidative cyclization on morusin (3). These results suggested that the photo-

OH 0 (±) -cycloartobiloxanthone (9)

OH 0 (±)-artobiloxanthone (8)

Fig. (16). Plausible mechanism for the formation of artobiloxanthone (8) and cycloartobiloxanthone (9) from artonin E (7).

219

oxidative cyciization of artonin E (7) may proceed through phenol oxidation via the semiquinone radicals described in Fig. (16). This chemical reactivity and the similarity of the structures between morusin (3) and artonin E (7) encourage us to examine the anti-tumor promoting activity of artonin E (7). Recently, Fujiki, et al. proposed a new tumor promotion mechanism applicable to human cancer development on the basis of experiment with okadaic acid. They described that tumor necrosis factor-a (TNF-a) induced by okadaic acid acts as a mediator of human carcinogenesis [65]. As briefly summarized in Fig. (17), okadaic acid inhibits the action of protein phosphatase type 1 and 2A, resulting in the accumulation of phosphorylated protein. Fujiki's group has shown that TNF-a acts as a timior promoter in BALB/3T3 cell transformation in vitro. The results of the studies on the okadaic acid class tumor promoters suggest that inflammatory stimuli or chemical tumor promoters induce TNF-a release from target tissues, and TNF-a gene expression in the initiated cells. This released TNF-a acts as a tumor promoter in the autocrine and paracrine system. According to the assumption that TNF-a is an endogenous tumor promoter associated with inflammatory potential, many historical puzzles of tumor promotion, such as its relationship to inflammation, can be solved. Based on this new tumor-promotion pathway, inhibition of TNF-a production leads to inhibition of tumor promotion. Furthermore, recent investigation has revealed that TNF-a is involved in various diseased, such as rheumatoid arthritis, Crohn's disease, multiple sclerosis, graft-versus-host disease, HIV, malaria, sepsis, and cachexia associated with cancer [85-90]. So, specific inhibitions of TNF-a production will almost certainly be effective not only in cancer prevention but also in the therapy and prevention of these other diseases.

—1 protein i j — ' phosphatase 1

okadac acid

^ phosphorylated proteins

t 1

{jene expression

- c-fos ojun

NF-KB

TNF-a - •

p

phosporylated proteins

~3

V

t ' _

ODC TNF-a

— •

_

Fig. (17). Mechanism of tumor promotion with okadaic acid.

Based on the above descriptions, we examined the inhibitory effect of the Artocarpus flavonoids on TNF-a release stimulated by okadaic acid using BALB/3T3 cells. This experiment was carried out in co-operation with Dr. Fujiki's group (Saitama Cancer Center Research Institute, Japan). All the compounds tested inhibit the TNF-a release stimulated by

220

okadaic acid at suitable lower concentration. This result suggests that several Artocarpus flavonoids act as anti-tumor promoter against to the okadaic acid type promotion. However, the detail mechanism is not clear at present, Fig. (17). The comparison of the inhibitory effects of the Artocarpus flavonoids against the TNF-a release (Table 4) and arachidonate 5-lipoxygenase, Fig. (13), was carried out. Artonin E (7) was the most potent inhibitor on both tests and the other compounds, artobiloxanthone (8) and heterophyllin (52), inhibited stronger than cycloartobiloxanthone (9), cycloheterophyllin (53), and morusin (3). The compounds showing stronger activity, all have three hydroxyl groups in the B ring. This characteristic feature might be important factor for both biological activities [91,92]. It is also noteworthy that the bioactivities of these flavonoids may reflect the use of Artocarpus species to the treatment for inflammation and malarial fever in Jamu medicines as is stated above. Table 4. Inhibitory effects (IC50, Mmol/L) of six flavonoids for the release of TNF-a from BALB/3T3 cells by treatment of okadaic acid Morusin (3) Artobiloxanthone (8) Heterophyllin (52)

1.76 0.94 0.48

Artonin E (7) Cycloartobiloxanthone (9) Cycloheterophyllin (53)

0.43 1.94 7.8

We also examined the cytotoxic activities of the Artocarpus flavonoids, artonins A (54), B (55), E (7), H (56), heterophyllin (52), and cycloheterophyllin (53), against cancer cells, mouse L-1210 and colon 38. All compounds tested showed the cytotoxic activities against both cancer cells (Table 5) [93]. Among them, cytotoxicity of heterophyllin (52), artonins B (55) and E (7) w^ere stronger than critical drug, l-(2-tetrahydrofuryl)-5-fluorouracil (TFFU). While we examined the cytotoxic activities of three dihydrochalcone derivatives isolated from Antiaris toxicaria (Moraceae), antiarones J (19), K (20), and L (57), against the two cancer cells [94]. All the compounds showed the weak cytotoxic activities against both cancer cells. Artonin E (7) also exhibited Table 5. Cytotoxic activities (IC50, |ig/mL) of Artocarpus and Antiaris flavonoids against L-12i0 and Colon 38 cells

Artonin A (54) Artonin B (55) Artonin E (7) Artonin H (56) Heterophyllin (52) ' Positive control.

L-1210

Colon 38

8.8 23 2.2 8.8 2.3

14.3

1.4 1.9 3.5 1.3

L-1210 Cycloheterophyllin (53) Antiarone J (19) Antiarone K (20) Antiarone L (57) TFFU*

Colon 38

4.7

4.6

77.0 81.3 80.4

70.4 46.3 >100

2.9

3.9

221

cytotoxic activities against human oral cells and MT4-cells as shown in Chapter VII (Table 7). VI.

BOMBESIN RECEPTOR ANTAGONISTS, KUWANONS G (1) AND H (2), ISOLATED FROM MORUS SPECIES

Bombesin and its mammalian counterparts, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to have a wide range of physiological and pharmacological functions [95]. Ligand-binding and molecular cloning studies have revealed two pharmacologically distinct G-protein-coupled receptor subtypes for mammalian bombesinlike peptides; a GRP-preferring (GRP-R) and an NMB-preferring bombesin receptor (NMB-R) [96]. A series of observations indicates that the mammalian bombesin-like peptides may act autocrine growth factors in human small cell lung carcinoma (SCLC) and other cancers. First, many human SCLC cell lines have been shown to express bombesin-like peptides [97]. Second, peptide bombesin receptor antagonists or anti-bombesin antibodies inhibit SCLC cell growth in vitro and in vivo [98,99]. These data suggested that the bombesin receptor antagonists might be useful for the treatment of some kinds of SCLC and other cancers. Because most antagonists reported thus far are peptides except for CP-70,030 and CP-75,998 (first synthetic non-peptide antagonists) [100-102], so, Fujimoto's group (Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan) screened the four hundred plant extract samples to search for non-peptide bombesin receptor antagonists. The methanol extract of the underground part of cultivated mulberry tree, Morus bombycis, was found to potently inhibit [^^^I]GRP binding to Swiss 3T3 cells. Bioassay-directed fractionation led to the isolation of two known flavone derivatives, kuwanons G (1) and H (2), which were identified by direct comparison with the authentic samples [103]. The antagonistic profiles of kuwanons G (1) and H (2) were characterized from the following results [103]. Kuwanon H (2) inhibited specific binding of [^^^I]GRP to GRP-referring receptors in murine Swiss 3T3 fibroblasts with K{ value of 290±50 nmol/L, which is more potent than that of kuwanon G (1), K\ value=470±60 nmol/L. The Ki value of 2 was about one order of magnitude more potent than those of CP-70,030 and CP-75,998, but had no effect on endothelin-1 or neuropeptide Y binding. While kuwanon H (2) inhibited specific binding of [^^^I]bombesin to rat esophagus membranes, the Ki value was about one order of magnitude less potent, Ki value of 2=6,500±2,000, than that of [^^^I]GRP toSwiss 3T3 cells. While bombesin (10 ^ mol/L) increased intracellular Ca^"^ levels in Swiss 3T3 cells, kuwanon H (2, 500 nmol/L) attenuated the bombesin-

222

induced increase in cytosolic free Ca^"^ concentration ([Ca^"^]!) by 60%, but not bradykinin- or endothelin-1-induced increase in [Ca^"^]}, Fig. (18).

r\

r

808

215

t\

< \

f t V

BOM

t

t

S

BOM

t

BK

t

S

t

BK

A^ Y1 t t t t V

ET-1

S

ET-l

Fig. (18). Effect of kuwanon H (2) on agonist-induced increases in [Ca^^\ in Swiss 3T3 cells. Cells were stimulated by 10"* mol/L bombesin (BOM), 10"* mol/L endothelin-1 (ET) or 10"* mol/L bradykinin (BK). Kuwanon H (S, 500 nmol/L at the final concentration) or dimethyl sulfoxide (V) was added 1 min before stimulation.

In Swiss 3T3 cells, GRP stimulates ["^H]thymidine incorporation in a concentration-dependent manner. Kuwanon H (2) inhibited GRPinduced DNA synthesis in Swiss 3T3 cells. The IC50 value was around 100 nmol/L, close to its K, value for [^^^I]GRP binding to Swiss 3T3 cells, Fig. (19). Kuwanon H (2) demonstrated selectivity toward GRP, as concentration of 10"^ mol/L uninfluenced basal and 5% serum-induced [ HJthymidine incorporation. From above results, kuwanon H (2) appears to be a selective antagonist for GRP-R.

B o.

B o

35000

25000

15000

-log (2) mol/L Fig. (19). Dose-dependent effects of kuwanon H (2) on basal (o) and GRP (10" mol/L)-induced DNA syntheses in Swiss 3T3 cells (•). Values are the mean ± S.E. for four determinations.

As bombesin family peptides are thought to be autocrine growth factors for SCLC, the results described above suggested that kuwanon H

223

(2) might be useful against SCLC. Unfortunately, however, kuwanon H (2) had no effect on the growth of two human SCLC lines, Lu-134 and NCI-HI 28. At the time, kuwanon H (2) was the most potent of non-peptide bombesin receptor antagonists (NPBRA) that had been reported. Its affinity might be too low to determine whether the non-peptide antagonist is effective against human lung cancers. However, kuwanon H (2), and possibly kuwanon G (1) also, can serve as lead compounds for more rational drug design in the synthesis of more potent antagonists. Furthermore, these compounds may be useful tools on the study of GRP-R. Recently, it was reported that NPBRA, PD 176252, with high binding affinity which was developed via the application of a peptoid drug design strategy [104]. VIL

EFFECTS OF PHENOLS AGAINST BACILLUS SUBTILIS (M45) (REC-ASSAY), HUMAN ORAL CELLS, AND HIV-INFECTED MT-4 CELLS

Rec-assay was developed by Kada et al. for screenings chemical and enveloped mutagens. Recombination less mutant strain of Bacillus subtilis (M45) is more sensitive to the cell-killing action of chemical mutagens, e.g., mytomycin C, A^-nitroso-A/-methylurethane, etc., than the wild-type bacteria (HI7) [105]. This assay was also useful for prescreening of anticancer drugs, such as enediyne-family antibiotics [106]. For the constituents of plants, the assay was modified and used exclusively for the detection of anti-mutagen compounds [107]. Since the sensitivity of the rec-assay to chemicals having induction activity of DNA damage is higher than from other screening technique, such as Ames test, this method may be useful for pre-screening of anticancer agents in crude drugs. Furthermore, the antibacterial compounds against the wild-type strain (HI7) may be expected that these antibacterial compounds have another bioactive potency. We tried the application of the rec-assay (unmodified) for the detection of bioactive phenolic compounds obtained from Glycyrrhiza species [51], and spore rec-assay [108,109] was used for moraceous flavonoids as shown in Table 7. Sixty-nine Glycyrrhiza phenols out of a total 108 compounds showed inhibitory activity against the growth of both HI7 and M45 strains. Cytotoxic activities of these antibacterial compounds {Glycyrrhiza phenols and moraceous phenols) against human oral squamous cell carcinoma (HSC-2) and human T-lymphoblastoid cell line MT-4 cells were also shown in Table 7 [110-113] along with other biological activities reported until the middle of 2002. In the Table, relatively strong-cytotoxic compounds against HSC-2 (CC5o50 55 22 6 18 2 2 10 ND >100

-H-

±

12

10

Glabridin (23)

+++

±

13

4

Glabrol (25) Glycycoumarin Glycyrin Glycyrol (76) (neoglycyrol) Glyasperin A (77) Glyasperin B Glyasperin C (61) Glyasperin D (62) Glyasperin J Glyasperin K Glisoflavanone Glyinflanin A (glycyrdione A) Glyinflanin B Glyinflanin C (glycyrdione C) Hispaglabridin A (124) 3-Hydroxyglabrol (26) 3-Hydroxyparatocharpin C^ Isoderrone Isoglycyrol(117) Isoliquiritigenin (70)

4-+ -f+

±

18 32 14 100 13 11 14

-

-

AFE, FDC

ABM ABM, EBV, LAT ABE ABH, APA, EBV, ODD ALR, 5LG ABM ABH, ABM

ALR, ICO, 5LG, NKA, IC0,5LG,NKA ALR, IOC, 5LG, NKA AIA, IPP ABE, ABH, ABM, AFE, AMA, AOA, EAA ABE, ABH, ABM, AFE, AOA, EAA, EAB, ETA, ICO, IMI, PSO, SAE ABE ABC, ABE, AOA, CPH ABC, ABE, ABH ABC, 5LG

-

10 ND ND 46 ND

++ ++

-

31 19

27 8

+

-

14 31

>100 12

ABE, AOA, MOS ABE

-

38 ND 16 22

12 ND >100 14

ALA

+ -H-H-H-

++ + ++ ++

-H-

+ ++

-

++

-

± ±

++

ABM ABM, ABH

ABC, ALR ABE, ATP, EAA, MCC, MGA, TFV, UFI

229

Table 7 (continued) Kanzonol B (81) Kanzonol G Kanzonol H Kanzonol P Kanzonol R Kanzonol S Kanzonol U (glabrocoumarone A) Kanzonol V Kanzonol W Kanzonol X (tenuifolin B) Kanzonol Y Kumatakenin (73)

+ + + + ++

—

Licochalcone A (59) Licochalcone B (82) Licoflavonol Licoisoflavanone (66) Licoisoflavone A (phaseoluteone) Licoisoflavone B (67) Licoricidin (63) Licoricone (120) Licorisoflavan A Medicarpin 1-Methoxyphaseollidin (125) 1 -Methoxyficifolinol 3-(9-Methylgancaonin P 4'-(9-Methylglabridin (123) Naringenin Paratocarpin L (macarangaflavanone B) Pinocembrin 6-PrenyleriodictyoF (71) S-PrenyleriodictyoF (72) 6-Prenylnaringenin (90) Semilicoisoflavone B (68) Shinpterocarpin Sigmoidin A Sigmoidin B (99) Topazolin Wighteone (84) (erythrinin B)

±

-

ND ND 46 ND ND ND ND

ND ND >10 ND ND ND ND

-

ND ND ND

ND ND ND

—

ND 375

-H-

—

20

ND 51 TCD 15

-

-

4 22 72 55

16 13 40 21

43 8

7 15

45 14 45 28 11 ND ND ND 24

64 47 53 12 8 ND ND ND 14

105 ND 35 29 78 ND 20 43 19 20

>100 ND 60 32 22 ND 29 26 4 12

+

17 100 11 >100 11 ND ND

-hH-

±

100 >100 50 25 >100 >100 50 50 >50 >50 >100 12.5 12.5 12.5 12.5 12.5 25 25 >50 >50 12.5 12.5 6.25 6.25 >50 >50 >100 >100 0.05 0.025

ATCC 43526 >100 >100 50 25 >100 >100 50 50 >50 >50 >100 12.5 12.5 12.5 12.5 12.5 25 25 >50 >50 12.5 12.5 6.25 6.25 >50 >50 >100 >100 0.05 0.025

ZLM 1007

GP98

>100 >100 >100 >100 50 50 25 25 >100 >100 >100 >100 50 50 50 50 >50 >50 >50 >50 12.5 >100 12.5 12.5 12.5 25 12.5 12.5 12.5 12.5 12.5 12.5 25 25 12.5 12.5 >50 >50 >50 >50 12.5 6.25 6.25 6.25 6.25 6.25 3.13 6.25 >50 >50 >50 >50 >100 >100 >100 >100 0.20 0.05 0.025; 0.10

(cfii)'* (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b)

source

G. glabra G. glabra G. inflata G. inflata G. uralensis G. uralensis G. uralensis G. uralensis

* (a): 2x10^ colony forming units (=cfti), (b): 2x10^ cfii. ** Positive control; amoxicillin (=AMOX). ATCC 43504, ATCC 43526, and ZLM 1007 are CLAR-sensitive strains.

Next, we attempted to isolate further flavonoids exhibiting anti-^. pylori activity from the extract of G. uralensis. In 1967, Takagi and Ishii reported that one of the flavonoid-rich fractions of G. uralensis (FMIOO), which also included about 15% glycyrrhizic acid (110), is effective in prevention of digestive gastric ulcer by suppressing gastric secretion [258,259]. The fraction was developed as an anti-ulcer drug and ten similar medicines containing licorice extract have been also supplied as prescribed drugs for treatment of gastric ulcer, duodenal ulcer, and gastritis [255]. Our study of FMIOO showed that the medicine exhibited anti-/f. pylori activity but did not contain licoricidin (63), which is the main isoprenoid-substituted flavonoid in G. uralensis [259,260] and exhibited anti-//. pylori activity as described above. The other antibacterial agent 67 was not detected in FMIOO on TLC analysis. The above investigations indicated strongly that licorice extract contains some anti-//. pylori flavonoids.

242

H3CO.

3-0-methylglycyrol (118)

OH glycyrin (121)

6,8-diprenylorobol (124)

0

isdicofiavonol (122)

1-methoxyphaseollidin (125) HO^ ^..^ ^ 0 ^

OH O

0CH3 gancaonin I (126)

gancaond C (127)

_ . ^ ^

dihydroisoflavone A (128)

OCH3 4'-0-methylglabriclin (129)

hispagiabridin A (130)

shinflavanone(131)

Fig. (31). Structures of compounds 117-128 isolated from the active fractions of the methanol extract of G. uralensis and compounds 129 -131 from the dichloromethane extract of G. glabra (Russian licorice).

The isolation of flavonoids from the methanol extract of G. uralensis was carried out under non-basic conditions, because some flavonoids isomerize under basic conditions, e.g. racemization of flavanones and isoflavanones, ring-open reaction of flavanones etc. Bioactive fractions were separated by some chromatographic methods and each step was monitored with anti-//. pylori activity with the paper disk method. Eighteen compounds were isolated from these bioactive fractions and

243

their anti-H. pylori activities were shown in Table 10. The MICs of the growth of H. pylori of vestitol (119), licoricone (120), 1-methoxyphaseollidin (125), and gancaonol C (127), Fig. (31), were similar to that of licoricidin (63). The activities of the other flavonoids were weak and similar to those of glycyrrhetic acid (111) and liquiritigenin (101). All the compounds investigated here had weaker anti-//. pylori activity; however, these compounds may be chemopreventive agent agents the H. pylori infection. Furthermore, these compounds may be bacteriostatic agents for the bacteria in the stomach and prevent peptic ulcer or gastric cancer disease in H. pylori-mfQCtcd people. However, further pharmacological and clinical studies including the antibacterial effect in liquid medium are required for confirmation of this hypothesis. Imakiire et al. also reported antibacterial activities of compound 23, 4'-0-methylglabridin (129), hispaglabridin A (130), glabrol (25) and shinflavanone (131), Fig. (31), from the lipophilic extract of Russian licorice, G. glabra; Maruzen P-TH® that is a material of medicines and cosmetics [126,127]. Table 10. Anti-Helicobacter pylori activities (MIC, |ig/mL) of the flavonoids from Glycyrrhiza uralensis ATCC 43504 ATCC 43526 ZLM 1007 Glyasperin D (62) 3-0-Methylglycyrol (118) Vestitol (119) Licoricone (120) Glycyrin (121) Isolicoflavonol (122) Gancaonol B (123) 6,8-Diprenylorobol (124) l-MethoxyphaseoUidin (125) Gancaonin I (126) Gancaonol C (127) DihydrolicoisoflavoneA(128)^ CLAR** AMOX**

25 25 >16 >16 12.5 12.5 12.5 12.5 50 50 50 25 >32 32 >50 50 16 16 50 50 16 16 >25 >25 0.025 0.0125 0.05 0.025

25 25 >16 >16 12.5 12.5 12.5 12.5 50 50 25 25 32 16 >50 50 16 8 50 50 16 8 >25 >25 0.0125 < 0.0063 0.05 0.025

12.5 12.5 >16 >16 12.5 12.5 12.5 12.5 50 25 25 25 32 32 50 50 16 16 50 50 32 16 25 25 < 0.0063 < 0.0063 0.05 0.025

* (a): 2x10^ cfu, (b): 2x10^ cfu. ^ Tentative name used here. ** Positive control; clarithromycin (=CLAR) and amoxicillin (=AMOX).

ZLM 1200 25 25 >16 >16 12.5 12.5 25 12.5 50 50 50 25 32 16 >50 50 16 16 >50 50 32 16 >25 >25 0.0125 < 0.0063 0.025 0.125

GP98

(cfu)*

12.5 6.25 >16 >16 12.5 6.25 12.5 12.5 25 25 25 12.5 16 16 50 50 16 8 50 50 16 16 25 25 50 12.5 0.2 0.1

(a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) (a) (b)

244

Protonpump inhibitor-based triple therapy is now the most commonly accepted eradication regimen for peptic ulcer patients with H. pylori infection. However, CLAR resistance is an increasing problem as its use has become more common in recent years [234,261]. It is interesting that licorice flavonoids exhibited anti-//. pylori activity against not only CLAR and AMOX-sensitive strains but also CLAR and AMOX-resistant strain CP98: Although licorice has been used as a crude drug in Japan from more than 1200 years [262], these strains have not developed resistance to the licorice flavonoids. These compounds may be useful as lead compounds in the development of a new class of anti-//. pylori agents. X. EFFECTS OF ISOPRENYLATED FLAVONOIDS FROM MORUS SPECIES ON TESTOSTERONE 5a-REDUCTASE In Japan, the extracts of mulberry tree have been used for promotion of hair growth and prevention of baldness [263]. Testosterone 5a-reductase catalyses the reduction of testosterone to its active form, 5a-dihydrotestosterone (5a-DHT). 5a-DHT has been implicated in certain androgen-dependent conditions such as benign prostatic hyperplasia, acne, and male pattern boldness [264]. And 5a-reductase activity is high in situ. Inhibitions of 5a-reductase may be usefuU for the treatment of these diseases. Therefore we studied on 5a-reductase inhibitory activity of some isoprenylated flavonoids isolated from the root bark of Japanese mulberry tree [265]. Table 11 shows the 5a-reductase inhibitory activity of flavonoids isolated from the root bark of Morus species. Most of the flavonoids had inhibitory activities against 5a-redactase, and showed the activity in the range of 10^ - lO"'' mol/L. Kuwanon E (43) had the most potent activity of these compounds and its IC50 value is 6.9x10"^ mol/L, while kuwanon G (1) had no effect at 10"^ mol/L. Fig. (32) shows the effects of kuwanon E (43) on the Lineweaver-Burk plots of rat prostate 5a-reductase activity using testosterone as a substrate. The addition of 3x10"^ mol/L kuwanon E (43) produced a parallel shift indicating un-competitive inhibitor. And the apparent K\ value is 7.6x 10~^ mol/L. Enzyme kinetic studies of inhibitor are very important for considering as a therapeutic agent. It is interesting to note that isoprenoid-substituted flavonoids having non-steroidal structures are potent un-competitive inhibitors of 5a-reductase. So, it would be expected that the isoprenoid-substituted flavonoid derivertive would be an interesting lead compounds for testosterone 5a-reductase inhibitor.

245

Table 11. Effects of Morus flavonoids on testosterone 5a-reductase Inhibition (%)* 59.6 35.6 63.0 94.0

Morusin (3) Oxydihydromorusin (46) Kuwanon C (42) Kuwanon E (43) Kuwanon G (1) Kuwanon H (2) Kuwanon L (44) Mulberrofiiran A (47) Mulberrofuran G (30)

0 100 53.0 24.2 37.0

IC50 (mol/L)

8.2x10"^ 6.9x10"' 1.8x10"^ 4.4x10"^

' Final concentration at 100 |imol/L.

lA'estosterone (1/10^ mol/L) Fig. (32). Lineweaver-Burk plots of inhibition of prostatic 5a-reductase by kuwanon E (43). The assay was carried out at varied concentration of [4-*'*C]testosterone in the absence (o) or in the presence of 0.3 Hmol/L kuwanon E (•).

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[63] Zenyaku Kogyo Co., Ltd., Jpn. Kokai Tokkyo Koho, 1983, JP58041894; Chem. Abstr,, 99, 93725. [64] Fujiki, H.; Suganuma, M.; J. Toxicol Toxin Rev., 1996, 75, 129 - 156. [65] Fujiki, H.; Suganuma, M.; Okabe, S.; Sueoka, E.; Suga, K.; Imai, K.; Nakachi, K.; Cancer Detection Prevention, 2000,2^, 91 - 99. [66] Yoshizawa, S.; Horiuchi, T.; Fujiki, H.; Yoshida, T.; Okuda, T.; Sugimura, T.; Phytother. Res., 1987,1,44-47. [67] Fujita, Y.; Yamane, T.; Tanaka, M.; Kuwata, K.; Okuzumi, J.; Takahashi, T.; Fujiki, H.; Okuda, T.; Jpn. J. Cancer Res., 1989, 80, 503 - 505. [68] Nomura, T.; Fukai, T.; Yamada, S.; Katayanagi, M.; Chem. Pharm. Bull, 1978, 26, 1431-1436. [69] Nomura, T.; Fukai, T.; Heterocycles, 1978, 9, 635 - 646. [70] Fukai, T.; Nomura, T.; Rapid Commun. Mass Spectrom., 1998,12, 1945 - 1951. [71] Nomura, T.; Fukai, T.; Matsumoto, J.; J. Heterocyclic Chem., 1980, 77, 641 646. [72] Nomura, T.; Fukai, T.; Hano, Y.; Yoshizawa, S.; Suganuma, M.; Fujiki, H. In Progress in Clinical and Biological Research', Cody, V.; Middleton, E., Jr.; Harbome, J.B.; Beretz, A., Eds.; Alan R. Liss, Inc.: New York, 1988; Vol. 280, pp. 2 6 7 - 2 8 1 . [73] Yoshizawa, S.; Suganuma, M.; Fujiki, H.; Fukai, T.; Nomura, T.; Sugimura, T.; Phytother. Res., 1989, i, 193 - 195. [74] Fukai, T.; Nomura, T.; Phytochemistry, 1988, 27,259 - 266. [75] Okabe, S.; Ochiai, Y.; Aida, M.; Park, K.; Kim, S.-J.; Nomura, T.; Suganuma, M.; Fujiki, H.; Jpn. J Cancer Res., 1999, 90,133 - 739. [76] Sueoka, N.; Suganuma, M.; Sueoka, E.; Okabe, S.; Matsuyama, S.; Imai, K.; Nakachi, K.; Fujiki, H.; Ann. N. Y. Acad Sci., 2001, 928, 21A - 280. [77] Konoshima, T.; Takasaki, M. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier Science B.V.: Amsterdam, 2000; Vol. 24, pp. 215 -267. [78] Akihisa, T.; Yasukawa, K. In Studies in Natural Products Chemistry, Atta-ur-Rahman, Ed.; Elsevier Science B.V.: Amsterdam, 2001; Vol. 25, pp. 3 42. [79] Kimura, Y.; Okuda, H.; Nomura, T.; Fukai, T.; Arichi, S.; Chem. Pharm. Bull., 1986, 34, 1223 - 1227. [80] Kimura, Y.; Okuda, H.; Nomura, T.; Fukai, T.; Arichi, S.; J Nat. Prod., 1986, 49, 639-644. [81] Reddy, G.R.; Ueda, N.; Hada, T.; Sackeyfio, A.C.; Yamamoto, S.; Hano, Y.; Aida, M.; Nomura, T.; Biochem. Pharm., 1991, 41, 115-119. [82] Yamamoto, T.; Furukawa, M.; Yamamoto, S.; Horie, T.; Wakanabe-Kohno, S.; Biochem. Biophys. Res. Commun., 1983, 776, 612-618. [83] Yamamoto, S.; Ueda, N.; Hada, T.; Horie, Y.; In Flavonoids in Biology and Medicine: Current Issues in Flavonoids Research', Das, N.P., Ed.; National University of Singapore: Singapore, 1990, Vol. 3, pp. 435-446. [84] Aida, M.; Yamagami, Y.; Hano, Y.; Nomura, T.; Heterocycles, 1996, 43, 2361 2566. [85] Gorman, J.D.; Sack, K.E.; Davis, J.C, Jr.; New Engl. J Med., 2002, 346, 1349 1356. [86] KeatingG.M.; Perry, CM.; BioDrugs, 2002. 16,\\\148.

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[87] Ghezzi, P.; Mennini, T.; Neuroimmunomodulation, 2001, P, 178-182. [88] Fowler, D.E.; Wang, P.; Int. J. Mol Med.; 2002, P, 443 - 449. [89] Siddiqi, N.J,; Alhomida, A.S.; Dutta, G.P.; Pandev, V.C; In Vivo, 2002, 16, 67 70. [90] Trotti, R.; Rondanelli, M.; Anesi, A.; Gabanti, E.; Brustia, R.; Minoli, L.; J. Hematother. Stem Cell Res.; 2002, / / , 369 - 375. [91] Aida, M.; Nomura, T.; Abstract Papers of The I15th Annual Meeting of Pharmaceutical Society of Japan; Sendai, 1995, p. 205. [92] Aida, M.; Hano, Y.; Fujiki, H.; Nomura, T.; Abstract Papers of 7PP5 International Chemical Congress of Pacific Basin Societies, Honolulu, 1995, Vol. 2, p. 873. [93] Hano, Y.; Aida, M.; Nomura, T.; Kozasa, M.; Fujimoto, M.; Abstract Papers of The Illth Annual Meeting of Pharmaceutical Society of Japan; Tokyo, 1991, Vol. 2, p. 229. [94] Uno, Y.; Mitsui, P.; Nomura, T.; Jpn. Kokai Tokkyo Koho, 1992, JP 04169548; Chem. Abstr.,ni,21>9U9. [95] Tache, Y.; Melchiorri, P.; Negri, L., Eds., Bombesin-Like Peptides in Health and Disease, In Ann. N.Y. Acad Sci., 1988, 547, 1-541. [96] Battey, J.; Wada, E.; Trends Neurosci., 1991,14, 524 - 528. [97] Moody,T.W.;Cuttitta,R.;Z//e5c/., 1993,52, 1161-1173. [98] Trepel, J.B.; Moyer, J.D.; Cuttitta, F.; Frucht, H.; Coy, D.H.; Natale, R.B.; Mulshine, J.L.; Jensen, R.T.; Sausville, E.A.; Biochem. Biophys. Res. Commun., 1988,/5ECg>EGC [185]. In the same study was also demonstrated that pyrogallol and gallic acid exert inhibitory activity and a mixture of these two compounds inhibited histamine release as strongly as EGCg. 7. Hepatoprotective activity Polyphenols are also endowed to have hepatoprotective effects. For example, quercetin reduces liver oxidative damage, ductural proliferation and fibrosis in biliary-ostructed rats, suggesting that it may

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be a useful liver protective agent in patients with biliary obstruction [186]. 8. Antiviral and antimicrobial activity Polyphenols may act as antimicrobial and antiviral agents as demonstrated by several studies in vitro. Polyphenol-rich extracts from various plants, such as Betula pubescens^ Epilobium angustifolium, Perillafrutescens, Pinus sylvestris, Rubus chamaemorus, Rubus idaeus. Solarium tuberosum, propolis and pure compounds, were tested to evaluate their antimicrobial activity against different bacteria and yeasts species, such as Bacillus subtilis, Escherichia coli, Mycobacterium tuberculosis H37Rv, Pseudomonas aeruginosa. Salmonella spp, Staphylococcus aureus. Streptococcus piogenes, Aspergillus niger, Candida albicans, Saccharomyces cerevisiae and showed growth inhibitory and bactericidal effect at different concentrations [187-192]. Naturally occurring flavonoids with antiviral activity have been recognized since the 1940s [193]. Quercetin, morin, rutin, taxifolin, dihydrofisetin, leucocyanidin, pelargonidin chloride, apigenin, catechin, hesperidin, and naringin have been reported to possess antiviral activity against some of 11 types of viruses [193]. (-)-Epigallocatechin gallate and theaflavin digallate inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney cells in vitro [194]. 9. Oestrogenic activity Plant-derived oestrogens may exert both oestrogenic and antioestrogenic effects, depending on several factors, including their concentration, the concentrations of endogenous oestrogens, and individual characteristics, such as gender and menopausal status [195,196]. The anti-oestrogenic activity of phytoestrogens may be partially explained by their competition with endogenous 17p-estradiol for oestrogen receptors [197]. Many of the potential health benefits of phytoestrogens may be attributable to features that do not involve oestrogen receptors, such as their influence on enzymes, protein synthesis, cell proliferation, angiogenesis, calcium transport, Na"^/K"*" adenosine triphosphatase, growth factor action, vascular smooth muscle cells, lipid oxidation, and cell differentiation. Phytoestrogens may have

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favorable effects on the risk of cardiovascular disease and are thought to be hypocholesterolemic, anticarcinogenic, antiproliferative, antiosteoporotic, and hormone altering [195,196,198,199]. Finally, flavonoids can bind to structural proteins and this feature could explain their ability to enhance the integrity of connective tissue. EPIDEMIOLOGIC EVIDENCE HEALTH BENEFITS

OF PLANT POLYPHENOL

1. Risk of CHD diseases Several epidemiological studies have reported inverse relation between intakes of flavonols and flavones and cardiovascular heart diseases (CHD). In a prospective study of 3454 men and women (age 55 years and older), a significant inverse association between the intake of catechinrich tea and radiographically quantified aortic atherosclerosis was found [200]. Similarly, inverse association between the consumption of red wine and CHD mortality (French paradox) have been suggested [201]. This beneficial effect of red wine may be due to the antioxidant ability of the wine phenolics to inhibit the oxidation of LDL to an atherogenic form [202], In the Zupthen Elderly Study [203] flavonol and flavone intake at baseline in 1985 of approximately 800 men (aged 65-85 years) was determined using the cross-check dietary history method. Men were divided into tertiles of flavonol and flavone intake. After five years of follow-up 43 men died from heart disease in this period. Flavonol and flavone intake, expressed as tertiles, was inversely associated with mortality from coronary heart disease and to a lesser extent with the incidence of first myocardial infarction. Furthermore, the association between long-term flavonol and flavone intake and risk of stroke in a cohort of 552 middle-aged Dutch men free fi"om history of stroke at baseline was also investigated within this study. Men were divided into quartiles of flavonol and flavone intake, and followed for 15 years. During this period 42 men had a first stroke event. Flavonol and flavone intake was strongly inversely associated with stroke risk. In both studies, the men in the highest category of flavonol and flavone intake (>30mg/day) had about one-third the risk of getting the disease compared

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with men in the lowest category. The major sources of dietary quercetin and other flavonols were revealed as tea and onions (fruits and vegetables had minor importance). The same authors [204] confirmed these results in the Seven Country Study. The contribution of flavonols and flavones in explaining the variance in coronary heart disease mortaUty rates across 16 cohorts from seven countries was studied. Flavonol and flavone intake was inversely correlated with mortality from coronary heart disease. Thesefindingare in line with the results of a cohort study in Finnland [205], where a significant inverse gradient was observed between dietary intake of flavonoids and total and coronary mortality. A modest but not significant inverse correlation between the intake of flavonols and flavones and subsequent mortality rates was found in a prospective cohort study of US Health Professionals by Rimm et al [206]. The authors do not exclude thatflavonoidshave a protective effect in men with established coronary heart disease although strong evidence was missing. Also other studies failed to demonstrate a significant statistical association between the intake of polyphenols and CHD. In Great Britain for instance coronary and total mortality even rose with the intake of the majorflavonolsource, tea [207]. The most likely explanation for the latter observation is that in this study tea consumption merely acted as a marker for a lifestyle that favours the development of cardiovascular disease. Indeed, men with the highest intake of tea and flavonols tended to be manual workers, and they smoked more and ate more fat [208]. 2. Risk of cancer The epidemiological evidence for a beneficial support of polyphenols in cancer disease is contradictory and less clear than its role in CHD. The Zupthen Elderly Study found a weak inverse association between flavonoid intake from fruit and vegetables sources and cancer of the alimentary and respiratory tracts combined [209]. The same authors observed no independent association with mortality from other causes between flavonoid intake and cancer mortality in the Seven Country Study [204]. ICnekt et al [210] studied the relation between the intake of flavonoids and subsequent cancer among 9959 finnish men and women during a follow-up in 1967-1991, An inverse association was observed between the intake offlavonoidsand incidence of all sites of cancer combined. Of

302

the major flavonoid sources, the consumption of apples showed an inverse association with lung cancer incidence. The cancer protective effects of black and green tea consiraiption, important sources of flavonol in specific countries, have been investigated mainly in case-control studies. Kohlmeier et al [211] evaluated the epidemiologic literature about tea and cancer prevention, concluding that cohort studies do not suggest a protective role for tea drinking in the total risk of cancer. Site-specific studies give a more complex picture. For example, a protective effect of green tea on the development of colon cancer is suggested. On the other hand, evidence for black tea is less clear, with some indication of a risk of colon or rectal cancer associated with regular use of black tea. In another cohort study of a Japanese population, researcher surveyed more than 8000 individuals over 40 years of age on their living habits, including daily consumption of green tea. Results found a negative association between green tea consumption and cancer incidence, especially among females drinking more than 10 cups per day [212]. 3, Vasoprotective effects (Hypertension) Experimental studies have shown that the administration of green teaenriched water to laboratory animals is associated with a reduction in blood pressure [213]. Different epidemiologic studies have suggested that drinking either green or black tea may lower cholesterol concentration and blood pressure [214,215]. In a epidemiological study of Japanese women, a history of stroke was less common among those who drank more green tea. There was no statistically significant reduction in blood pressure alone among those women who drank more tea [206]. 4. Oestrogenic effects Phytoestrogens represent a family of plant compounds that have been shown to have both oestrogenic and anti-oestrogenic properties. Accumulating evidence from molecular and cellular biology experiments, animal studies and, to a limited extent, human clinical trials suggests that phytoestrogens may potentially confer health benefits related to

303

cardiovascular diseases, cancer, osteoporosis, and menopausal symptoms. These potential health benefits are consistent with the epidemiological evidence that the risk of heart disease, various cancers, osteoporotic fractures, and menopausal symptoms is lower among populations that consume plant-based diets, particularly among cultures with diets that are traditionally high in soy products. One study over 9 months noted a significant reduction in total cholesterol in premenopausal women when they consumed soy products with 45 mg conjugated isoflavones/day in comparison to levels during a control period when they were fed isoflavone-free soy products. The treatment group difference was significant despite the small sample size and the selection of healthy, normocholesterolemic women who had limited room for detectable improvements [216]. The pattem of soy intake and its association with blood lipid concentrations in the Hong Kong Chinese population was studied in a total of 500 men and 510 women with an age range of 24-74 years by Ho et al [217]. In men, soy intake and total plasma cholesterol were negatively correlated (r = 20.09, P = 0.04), as were soy intake and LDL cholesterol fr = 20.11, P = 0.02). The respective values in women tors. Crocin did not affect the inhibiticm of non-NMDA response by 100 mM ethanol, but significantly blocked the inhibition of NMDA response by 10-50 mM etiianol. We perfcnmed whole-cell patch receding with primary cultured rat hippocampal neurons, and confirmed tiiat crocin blocked etfianol inhibition of inward currents evoked by the application of NMDA. We also demonstrated that crocin suppresses the effect of tumor necrosis factor (TNF)-a on neuronally differentiated PC-12 cells. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-a~induced release of cytochrome c from the mitochondoria. Crocin also blocked the cyotochrome c-induced activation of caspase-3. We found that crocin inhibited the effect of daunorubicin as well. The present paper focuses on the pharmacological actions of crocin on the central nervous system and reviews briefly the findings of such studies on the prevention of neuronal progranmaed cell death (apoptosis).

INTRODUCTION Saffron {Crocus sativus L. ; Iridaceae)findsuse in medicine as well as a flavoring and coloring agent. It has three main chemical compounds. The bright red coloring carotenoids; a bitter taste, picrocrocin; and a spicy

314

aroma, safranal. The carotenoid pigments consist of oooelin (JKP4>^Jiioo^^ estei; crooetin

CH, Y "^

\^'\^^'f\}^^'

(50%

I.OSO4/NMO 2. Nal04 3. NaBH4

^ (50%)

6

(PHB)

Less active than MFA Fig. (2). Conversion of MFA to PHB

As mentioned earlier, the only structural difference between PHA and MFA resides in ring G, and therefore our analog program centered on this ring. An added reason was that the envisaged synthetic pathway could also provide PHA, a compound not in our possession at the time, and essential for comparative assays. Removal of the methyl and hydroxyl groups at C14 in PHA would yield paraherquamide B (PHB), a compound identical to MFA except for the size of ring G (PHB five versus MFA six). The planned synthetic sequence required the opening of ring G and an

334

oxidative removal of one carbon atom, to be followed by ring closure as shown in Fig. (2). Treatment of MFA (1) with cyanogen bromide [6] opened ring G to yield the bromo derivative 3 [7]. Attempts to dehydrobrominate 3 in one step via a base-catalyzed elimination with DBU/CH3CN, KOH/MeOH, or r^rr-BuOK/DMSO were unsuccessful. However, the required methylene entity could be introduced by converting 3 first to a selenide, then oxidation with periodate, followed by thermolysis in benzene to provide compound 4. Hydrolysis of the cyano group with NaOH in ethylene glycol [8] produced 5 (50% yield). Osmium catalyzed oxidation of 5 in the presence of 4-methylmorpholine A^-oxide (NMO) gave a diol, which was cleaved to an aldehyde upon treatment with periodate. Treatment of the aldehyde with sodium cyanoborohydride resulted in an intramolecular reductive amination to yield the desired product PHB (6). The seven step conversion of PHB to PHA is shown in Fig. (3). Oxidation of PHB (6) in THF/H2O with iodine in the presence of bicarbonate [9] gave 16-oxo-paraherquamide B (7, 40%). Treatment of 7 with LDA and phenylselenyl chloride followed by oxidation of the resulting selenide with H2O2 gave the a,p-unsaturated lactam 8. Attempted epoxidation of 8 with H202/NaOH, m-CPBA, isovaleraldehydeA^O(acac)2, or n-BuLi/H202 failed to give the epoxide 9. However, when 8 in THF was treated with r^rf-butylperoxide [10] in the presence of triton B, the epoxide 9 (58%) was obtained. It was assumed that attack on the double bond by peroxide would occur from the least hindered side to yield an a-epoxide; proof was obtained from the stereochemistry of alcohol 11 (vide infra). In any case, the stereochemistry of these chiral centers is of little consequence, since they are removed at a later stage of the synthesis. While standard methods utilizing NaBHj, BH3-THF, or superhydride failed to open the epoxide ring, samarium iodide [11] provided the required 14a-hydroxy compound 10 in a good yield (85%). Although this single electron transfer reagent has been shown to cleave epoxy-ketones, to our knowledge this is the first instance of its application to amides. Reduction of 10 with LAH/AICI3 gave 11 in a modest yield (24%). The stereochemistry of 11 was established by comparison of its ^H NMR spectrum with that reported by Blizzard et al. in their synthesis of 11 by an altemative synthetic route [5j]. Swem oxidation of 11 produced ketone 12 (71%). Reaction of 12 with methyl magnesium bromide in THF gave PHA in a 50% yield (based on recovered starting material) with the

335

formation of only a trace of the related a-methyl epimer. Semisynthetic PHA proved to be identical by TLC, ^H NMR and HRMS to the natural product kindly provided by Professor Yamazaki. We were able to then confirm that the nematocidal activity of PHA is superior to MFA, which in turn is superior to that of PHB.

1.LDA/PhSeCI

,

2. H2O2 (58%) 6

(PHB)

Y-

OH

triton B (58%)

LAH/AICI3 (24%)

Swern Oxidation

MeiVlgBr 1

•

HO

(50%)

(71%)

CH3

Hr,CCH,,^v04-^H3 2 H3C fif °

\ Q CH3'-'

Fig. (3). Conversion of PHB to PHA

(PHA)

More active than l\^FA

336

Hydroxylation of MFA at C14, C15, C16 via a Novel Cyanogen Iodide Reaction While the reaction of MFA with cyanogen bromide (BrCN) in refluxing chloroform caused ring fission to yield 3 [Fig. (4)], under the same conditions cyanogen iodide did not provide the iodo analog 13. CH^

J

14B reflux 1 h

Fig. (4). Reaction of MFA with CNI

15A

15B

Cyanogen Iodide (ICN) has been used extensively for the cyanation of alkenes and aromatic compounds [12], iodination of aromatic compounds [13], formation of disulfide bonds in peptides [14], conversion of dithioacetals to cyanothioacetals [15], formation of rran^-olefins from dialkylvinylboranes [16], lactonization of alkene esters [17], formation of guanidines [18], lactamization [19], formation of a-thioethter nitriles [20], iodocyanation of alkenes [21], conversion of alkynes to alkyl-iodo alkenes [22], cyanation/iodination of p-diketones [23], and formation of alkynyl iodides [24]. The products obtained from the reaction of ICN with MFA in refluxing chloroform were fran^-16-iodo-17-cyanomarcfortine A (14)

337

and 17-cyanomarcfoitine A (15) in 90% and 10% yields, respectively [25]. The likely mechanism of the formation of these products is shown in Fig. (5). CH3 HoC C H o x ; ^ ^ 0 4 - - C K

Fig. (5). A plausible mechanism of the reaction of MFA with CNI

The iminium ion intemiediate 16 is generated by a free radical oxidation of 1 and is in equilibrium with the enamine intermediate 17; cyanide ion addition to 16 gives a low yield of compound 15 while the favored trans addition provides the main product 14. Although generation of an iminium intermediate with chlorine dioxide [26] or bromine [27] has been reported, these reagents did not produce compounds such as 14, suggesting that equilibration with the enamine did not occur. The Polonovski-Potier reaction applied to aspidospermane generated an enamine intermediate, which upon treatment with CNBr gave a product similar to 14 in three steps [28]. The trans adduct 14 upon treatment with 45% aqueous KOH in MeOH for 3 h at room temperature gave the 16,17-dehydro derivative 18 in 90% yield. The utility of this novel cyanogen iodide reaction was further demonstrated on MFA by affording a procedure for the regiospecific introduction of a single hydroxyl group at C14, C15, and C16. These analogs were required to determine the significance of the hydroxyl group

338

on anthelmintic activity. Compound 18 thus became the common intermediate for the synthesis of 14a-hydroxymarcfortine A (23), 15ahydroxymarcfortine A (27), and 16a-hydroxymarcfortine A (29).

I.LDA/PhSeCI 2. HgOg/NaOH (65%)

More active than MFA

Fig. (6). Synthesis of 14a-hydroxymarcfortine A from 18

Preparation of 23 from 18 was achieved in five steps [Fig. (6)]. Hydrolysis of 18 with a catalytic amount of p-toluenesulfonic acid in 95% methanol at room temperature for 1 h gave 19 (90%). The C15-C16 double bond was then introduced by selenation [Lithium diisoproylamide (LDA) and phenylselenenyl chloride] at C16 followed by hydrogen peroxide oxidation and subsequent elimination of phenylselenenic acid by an aqueous alkaline workup ( I N NaOH) to give the C15-C16 dehydro analog 20 (65%). Compound 20 underwent allylic oxidation with Se02in refluxing dioxane (1.3 equiv, 1 h) to provide in a modest yield (35%) the desired a-hydroxyl derivative 21. Reduction of the double bond with lithium triethylborohydride (7 equiv/THF, 0 ""C, 0.5 h) gave 22 (86%). The regiospecific reduction of the C17 amide in 22 was achieved by treatment with BH3-DMS (10 equiv/THF) to provide 14ahydroxymarcfortine A (23, 75%). The couphng constant between the C14 hydrogen and the CI5 hydrogen is 2 Hz, indicating the hydroxyl group is axial.

339

3

1.LDA

18

Inactive

18 NMO (80%) inactive

Fig. (7). Synthesis of 15 a- and 16a-hydroxyniarcfortine A from 18

The preparation of 15a-hydroxymarcfortine A (27) and 16ahydroxymarcfortine A (29) is shown in Fig. (7). Treatment of 18 with LDA (4 equiv/THF, -78 ""C) followed by 1.5 equiv of Davis's reagent 24 [(2-benzenesulfonyl)-3-phenyloxaziridine] [29] gave the desired C15hydroxlated material 25 (y-hydroxylation, 30%) along with the 17-oxo derivative 19 (a-hydroxylation, 10%). ^H NMR of 25 indicated a single stereoisomer in which the C15 hydrogen has two pseudoaxial and two pseudoequatorial couplings. Reaction of 25 with 2.6 equiv of Se02 in aqueous ethanol at ambient temperature for 16 h furnished 26 (50%), which was reduced with LAH (2.75 equiv/THF, 0 ""C, 0.5 h) to yield 27 (25%). Treatment of 18 with a catalytic amount of OSO4 and 4.2 equiv of NMO gave 28 (80%); the carbonyl at C17 was reduced with BH3-DMS (6

340

equiv/THF, 0 ""C, 1 h) to yield 16a-hydroxy-MFA 29 (60% yield based on recovered starting material). While compounds 27 and 29 lack nematocidal activity, we found 23 to be more active than MFA. Synthesis of 14p-hydroxy-MFA (31) and 14p-methyl-14a-hydroxyMFA (32)

CH^ HoC CHq

^O-^UcHg /)

Swern Oxidation

CHg^

30

MeMgBr NaBH. CH^ HX

32

as active as PHA

^-.^04-CH3

31 (50%) + 23 (3%)

Fig. (8). Synthesis of 14P-hydroxy-MFA and 32

Because of the enhanced biological activity of 14a-hydroxy-MFA (23), the synthesis of its antipode was carried out as shown in Fig. (8). Swern oxidation (oxalyl chloride, DMSO, NEts, -78 ''C, 1 h) of 23 provided 14-oxo-MFA (30, 74%). Reduction of 30 with NaBHU (6 equiv/THF, 0 ''C, 1 h) gave M^-hydroxy-MFA 31, 50%) and 14ahydroxy-MFA (23, 3%). Compound 31 was inactive, thus demonstrating the need for correct stereochemistry of the hydroxyl group at C14. To assess the effect of the C14 methyl group on biological activity, 30 was reacted with methylmagnesium bromide to yield 32 (50% based on recovered starting material), the six membered homologue of PHA. The

341

related a-methyl epimer was present only in trace amounts. Compound 32 is the first MFA analog with nematocidal activity comparable to that of PHA. An Improved and Practical Synthesis of 14a-hydroxy-MFA (23). SPh

1.LDA 2. PhS-SPh

\

(60%)

4 V-N

33

CHo

1.,x;s^COOOMg

CI,

"COQ-

(76%)

2. heat

COOOMg f^'^o

23 (70 g)

Fig. (9). An Improved and Practical Synthesis of 14a-hydroxy-MFA

To provide additional material for clinical trials, and to eliminate the use of hazardous reagents such as ICN, PhSeCl, and Se02, an improved synthesis of 23 was devised [Fig. (9)]. Treatment of MFA 1 with sodium bicarbonate and iodine [30] in refluxing aqueous THF produced 19 in one step (76%, 350 g scale), eliminating the use of ICN and Se02. Compound 19 was reacted with LDA and phenyl disulfide to give 33 (65%). Oxidation with the magnesium salt of perphthalic acid followed by refluxing in toluene provided 34 (76%). Repeated reaction with the magnesium salt of perphthalic acid and diethylamine gave the rearranged product 22 (61%) [31]. The carbonyl group at C17 in compound 22 was reduced with BH3DMS (10 equiv/THF) to provide 14a-hydroxy-MFA 23 (70 g, 50%). The stereospecific rearrangement of 34 was previously described [32].

342

Synthesis of 15a-Methyl-14a-Hydroxy-MFA (36) and ISp-Methyl14a-Hydroxy-MFA (41) Due to the enhanced nematocidal activity observed following the introduction of a methyl group at C14 [32, Fig. (8)], its effect on C15 was next investigated.

Me2CuLI (60 %)

11 Q

BH3-DMS (50 %)

HoC--/

9-BBN NaOH/HgOg

1.MsCI/NEt3 2. DBN (70%)

45

Inactive

Fig. (11). Synthesis of 45, a C14,15-fused ring analog.

While compound 36 demonstrated excellent nematocidal activity, the epimer 41 at the same concentration was totally inactive. Perhaps the conformation of the G-ring relative to the hydroxyl group is more important than the chirality of the methyl group at CI5. To further

344

investigate this hypothesis, compound 45, having a C14-C15 fused ring, was synthesized. In addition, 45 could also define the importance of hydrogen bonding at C14 [Fig. (11)]. Synthesis of 45 , a C14,15-Fused Ring Analog Compound 21 [Fig. (11)] was reacted with vinyl magnesium bromide and copper iodide in THF at 0 ^C to give the 1,4 addition product 42 in 48% yield [35]. Hydroboration with 9-BBN followed by NaOH and H2O2 workup provided a mixture of the desired alcohol 43 (20%), recovered starting material 42 (20%), and the C17 reduced alcohol 44 (5%). Further reduction of 43 with the BH3-DMS complex gave 44 in 35% yield. The two combined samples of 44 were mesylated (CH3S02Cl/NEt3) and then treated with excess DBN to give the furan-containing MFA analog 45 in 70% yield. Compound 45 is inactive, thus demonstrating that hydrogen bonding of the C14 hydroxyl group is indeed important for anthelmintic activity. The significance of the geometry of ring G on anthelmintic activity was next investigated. Fission of ring-G: The Synthesis of Analog 48 CH,

O4-CH3 // QJJ ^^" O'

C;H3

COCI2 Pyridine /toluene

U ^

J

O H^C-

bH O

CH, LiBEtgH

HaCs^aC^ H3C CH3 ^^^YO-lr-CHa

HO-4—(•-v4J> Inactive

Fig. (12). Synthesis of 48.

-NH 48

^^^^

H3C

NaBHgCN H O 80%

H,

46 40%

H H3C CH3 ^?v^o4-CH3

345

Compound 2 [Fig. (12)] in pyridine was treated with phosgene in toluene [5j] to give the carbamate 46, which on reduction with lithium triethyl borohydride gave 47 in a 40% yield. Subsequently, compound 47 was treated with formaldehyde and sodium cyanoborohydride to yield 48 (80%). Compound 48 is biologically inactive, and in our computer model (minimum energy conformation) it resembles 14p-hydroxy-MFA 31, which is also inactive. It can be concluded that the naturally occurring conformation of ring G is essential for biological activity. Economic Viability The biological profile of compounds 32 and 36 points towards their use in large food animals. The cost of these compounds, however, is prohibitive if synthesized by the procedures outlined in Fig. (8) and Fig. (9). Therefore, an alternative scheme had to be developed, as outlined in Fig. (13). The envisaged route required the selective introduction of the crucial 14-hydroxyl group into MFA via a biotransformation procedure. The resulting 23 would then be a common intermediate for the synthesis of 32 (two chemical steps) and 36 (three chemical steps).

BJotransformation Primary Fermentation

- • IVIFA

. m Q^ VN/ O

Three Chemical Steps

23 Tvw) Chemical Steps

HoC

36 Fig. (13). Economic viability

32

CH,

346

Microbial Hydroxylation (Biotransformation) at Eight Individual Carbon Atoms of MFA Many reviews and hundreds of papers have been published on the use of mono-oxygenases for the introduction of oxygen atoms onto various substrates [36]. We were especially interested in nonactivated stereospecific carbon atom hydroxylation. Therefore, a large number of biotransformation experiments were carried out utilizing cultures reported in the literature, and random samples from the Pharmacia culture collection. Screening was performed by adding a solution of 1 (10 mg) [37] in DMF (0.4 mL) to vigorously growing culture fermentations in 500 mL wide-mouth flasks. Incubation was conducted at 28 °C, and shaking was continued for 1-3 days depending on the culture. The mixture was thoroughly extracted with chloroform, centrifuged, and the solvent removed at 35 °C under reduced pressure. The residue was analyzed by TLC (three solvent systems) and HPLC. Promising samples were scaled up 10- to 100-fold and refermented in a Labraferm fermentation tank. Purification was achieved by various chromatographic techniques, and the structure determined with the aid of NMR and MS. Whenever semisynthetic samples were available, a side by side comparison was also performed.

27

CH3

l>^0-4-CH.

o I to N-Demethylatlon/^ ^ " s

Arrows indicate position of hydroxylation Fig. (14). Biotransformation products

The great majority of cultures either totally metabolized 1 or left it unchanged. However, a few cultures did provide hyroxylated products.

347

Extensive efforts were undertaken to increase the yields of biotransformation products by changing the media, temperature, time of fermentation, etc. The highest yields obtained were for compounds 23, 27 and 29, which on scale-up gave a 10-15% yield, in addition to recovered starting material (30%). In summary, utilizing biotransformation techniques, eight out of the 28 carbon atoms in MFA were successfully hydroxylated. In Fig. (14), arrows indicate the sites of hydroxylation. It is noteworthy that despite the hindered nature of C14, cultures UC 5059, UC 11141, and UC 11144 were able to introduce a hydroxyl to give 23 in a 10-15% yield. Discovery of 2-Desoxo-15a-Methyl-14a-Hydroxy-MFA (49) Scale up studies of a second potential candidate yielded the desired 36 and a small amount of a less polar compound, characterized as the 2desoxo derivative 49 [Fig. (15)]. The excellent nematocidal activity of 49 encouraged us to attempt the selective reduction of the C-ring carbonyl in MFA (1), to yield 52. Reduction of 1 with three or ten equivalents of LAH yielded 50 (carbonyl reduced in ring F) and 51 (carbonyls reduced in rings C and F) respectively, both lacking nematocidal activity; we were unable to synthesize 52. Discovery of 2-Desoxo-PHA (PNU-141962,53) To follow up the excellent nematocidal activity of 49, PHA was selectively reduced with the alane-NMe2Et complex to furnish 53, albeit in only a 5% yield [Fig. (16)]. Attempts to improve the yield of this one step procedure with various reducing agents, such as LAH, LAH/AICI3, NaBEU/Acetic acid, NaBIVCFsCOOH, Red-Al, Super-hydride, Li-9-BBN-hydride, BH3-THF, Li-tri-r-butoxyaluminum hydride and LiBHU, were unsatisfactory. The highest yield obtained was with LiBEU (10-20%). A lengthier but higher yielding synthesis of 53 was developed (4 steps 60-70%) by using our previously described process [38]. Compound 2 was reacted with 9fluorenylmethyl chloroformate (Fmoc-Cl, 1.5 equiv) in the presence of NaH (3 equiv) at 0 °C to give a quantitative yield of 54 [39]. Reduction of 54 with NaBHU in MeOH at 0 °C gave 55, which was deprotected with piperidine in THF to give the imine intermediate 56.

348

HO

;HN '/>^NH

35 (20 grams)

^"3

BH3-DMS CH3

CH3

04-CHo

O-4-CH3

oi + 49 (10 miligrams) as active as 36

36 (10 grams) CH3

04-CH, -//LAH(3or10equiv)

0

CK 52

(3 equiv)

O-UCH,

5Q

Inactive

51

Inactive

Fig. (15). Synthesis of 49,50, and 51

This was further reduced with NaBKU in MeOH at 0 °C to give 53. Compound 53 displayed excellent activity in our jird and sheep models. Indeed, in our hands, this compound was two to four times more potent in sheep than the parent compound (PHA) against the important

349

gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus colubriformis.

^

O

CH 53

PNU-141962

CHo

CH3

0-4-CHo

O-V-CH3

K)—>"

J, IN-Fmoc

H3C Jl'X ^

CHq

^'^

NaBH. ^

Overall yield 60-70%

Hqi 3

O

CH3 OH

^

CH,

56

Fig. (16). Discovery of 2-Desoxo-PHA (PNU-141962,53)

It should be noted that workers at Merck found PHA to be considerably more potent than we did [40a], a difference which may be due to the use of different vehicles. While this was an exciting development, we were concerned about the toxicity of this PHA analog, because Merck workers reported [40b] that PHA is quite toxic to mice, with an estimated LD50 of < 15 mg/kg. In dogs, the toxicity is even greater, with death seen at doses as small as 0.5 mg/kg [40b], reducing chances for commercialization. Interestingly, PHA is relatively safe in jirds, sheep and rats. Structure activity relationship studies performed by Merck and Pfizer workers did not yield analogs with lower toxicity. In contrast, compound 53 was not toxic to mice at doses up to 50 mg/kg. Furthermore, dogs treated with 53 at 20 mg/kg experienced no toxic effects besides mild and reversible

350

mydriasis. The exceptional improvement in selective biological activity due to the removal of a single oxygen atom in ring C is noteworthy indeed. In conclusion, rational drug design led to the synthesis of two highly active compounds, 32 and 36. Scale up studies of 36 yielded a minor product, the 2-desoxo-MFA derivative 49, which in turn led to the discovery of 2-desoxo-PHA (PNU-141962), a compound that is currently under development. Efficacy of MFA, PHA and PNU-141962 Drug evaluations were conducted in Mongolian gerbils (jirds) concurrently infected with the gastrointestinal nematodes H, contortus and T, colubriformiSr or monospecifically infected with O. ostertagU using established techniques [41a,b]. This model permits assessment of the activity of experimental compounds directly against target parasites in vivo, using very little drug. Treatments were given on day 10 or day 6 post-inoculation in the K contortus and T. colubriformis or O. ostertagi models, respectively. Table 1. Efficacies of MFA, PHA and PNU-141962 against gastrointestinal nematodes in experimentally infected jirds following oral dosing.

95% Effective Dose (mg/jird)

Compound H. contortus

T. colubriformis

O. ostertagi

MFA

0.33

0.11

4.0

PHA

0.33

0.11

0.5

PNU-141962

0.33

0.11

1.0

Drugs were administered orally in 0.2 ml vehicle (17% DMSO: 83% vehicle #98). Animals were examined for worm burdens on day 13 {H. contortus and T. colubriformis) or day 8 (O. ostertagi) post-inoculation (3 days post-treatment). Approximate ED95 values for MFA, PHA and PNU-141962 against 3 nematode species in jirds are shown in Table 1. Against H, contortus, the approximate ED95 for MFA, PHA and PNU-141962 was 0.33 mg/jird

351

(roughly 10 mg/kg). These 3 compounds were also approximately equipotent against T. colubriformis in the jird model, with an EDc^^ of 0.11 mg/jird. Against O. ostertagU the approximate ED95 for each compound was higher than that observed for the other nematodes. Against this species, the ED95 values for PHA and PNU-141962 were 0.5 and 1.0 mg/jird, respectively; these values were 4- to 8-fold lower than those for MFA (-4 mg/jird).

Table 2. Efficacy of Marcfortines, PHA and PNU-141962 in sheep experimentally infected with H. contortus following oral dosing of conqiounds in 60:40 propyleneglycol/glycerol formal vehicle.

95 % Effective Dose (mg/kg) H. contortus Compound

MFA

12.5

23

7

32

2

PHA

1-2

PNU-141962

0.5-1

36

4-5

,

The compounds were also tested in sheep experimentally infected with Haemonchus contortus. The treatments were given orally in propylene glycol/glycerol formal (60:40 v:v) vehicle on day 35 post-inoculation. Animals were necropsied 7 days post-treatment and examined for worm burdens. Approximate ED95 for the marcfortine compounds, PHA and PNU-141962 are shown in Table 2. Against K contortus, the approximate 95% effective dose for PNU-141962 was 0.5-1.0 mg/kg; ED95 for the marcfortines ranged from 1-12.5 mg/kg and for PHA 1-2 mg/kg. It should be noted that formulation has not been optimized for ruminants, and subsequent preliminary pharmacokinetic studies have shown that the bioavailability of PNU-141962 in sheep is low (10-15%) in the vehicle used, when dosed orally or subcutaneously (unpublished observations).

352

Mode of Action of PNU-141962, MFA and PHA Similarities in structure and anthelmintic spectra of the paraherquamides and marcfortines suggest that these compounds share an anthelmintic mechanism. This concept is supported by observations that PHA and PNU-141962 displaced [^H]marcfortine A in competition binding assays using membranes prepared from adult H, contortus; competition was also observed in binding assays using membranes prepared from the free-living nematode Panagrellus redivivus and [^H]PNU-141962 as ligand (our unpublished observations). PHA, PNU-141962, and related compounds rapidly induce flaccid paralysis of parasitic nematodes in vitro, without affecting ATP levels [42]. While the mechanism of action of these new anthelmintic agents was initially poorly understood, they appear to share a binding site with phenothiazines in membranes prepared from C elegans [43]. Recent observations in insects suggest that PHA binds to invertebrate nicotinic acetylcholine receptors (nAChR) and is an antagonist of acetylcholine (ACh) at these receptors [44]. To determine if a similar mechanism of action is found for these compounds in nematodes, we investigated the mechanism of action of this anthelmintic class using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum [45a]. Our findings can be summarized as follows. None of the compounds significantly altered A. suum muscle tension or membrane potential when given alone. However, paraherquamides blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by ACh and nicotinic agonists, including the anthelmintics levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic action of PNU-141962 and related paraherquamides and marcfortines is due to blockade of cholinergic neuromuscular transmission. To further test that concept, we examined the effects of these compounds on three subtypes of human nAChR. In these studies, a Ca^"^ flux assay was used to measure the function of nAChR expressed in cultured mammalian cells. PNU-141962 blocked nicotinic stimulation of cells expressing a3 ganglionic (IC50 = 6 \\M) and muscle-type (IC50 = 3 |LIM) receptors, but was inactive at 100 |xM vs the a7 CNS subtype. This compound also paralyzed the parasite H. contortus in culture with an IC50 value of approx. 0.1 uM, thus demonstrating the basis for host vs. parasite selectivity. It is noteworthy that PHA is more effective in blocking mammalian nAChR than is PNU141962, perhaps explaining the greater mammalian toxicity observed with the prototype drug.

353

Isotopic Labeling of PNU-141962 (53) with Deuterium [45b]

2. NaBHaCN (preferred)

(500/^

Fig. (17). Isotopic Ubeling of PNU-141962 (53) with Deuterium

Modifications of MFA and PHA led to the discovery of 2desoxoparaherquamide A (PNU-141962, 53) which is as active as PHA and has an improved safety profile. In order to do preclinical studies, we

354

wished to synthesize radio-labeled PNU-141962. In this reason, we prepared [CD3]-2-desoxoparaherquamide A (62). Although the synthesis of [C24"^H]-PHA has been reported [45c], the labile nature of position-24 with respect to acid hydrolysis rendered such labeling unsuitable for our preclinical studies. Using a deuterium labeled reagent, we have developed a synthetic strategy that is suitable for the introduction of '"C and ^H into the 14-methyl group of PNU-141962 through the appropriate choice of labeling in the reagent [Fig. (17)]. The dehydration of PNU-141962 to exo-olefin 57 through the use of DAST was readily accomplished. A method has been reported for the conversion of an exo-olefin derivative of PHA to its ketone by sequential reactions involving bromination, ozonolysis and debromination with zinc [5j]. However, these procedures were long, low yielding and, worse, the benzene ring of PHA was also brominated. In our hands, glycolation of the exo-olefin with osmium tetroxide followed by oxidation of the glycol to the ketone with sodium periodate proved more suitable to our purposes. Earlier, we reported a method for the stereospecific addition of MeMgl to 14-oxoparaherquamide B [7]. Using this methodology we successfully methylated the carbonyl at position-14 of ketones 60 and 61 in a highly stereoselective manner. Treatment of 53 with DAST [(diethylamino)sulfur trifluoride] in methylene chloride provided 57 in 50% yield. Compound 57 was treated with osmium tetroxide at 5 °C in the presence of NMO (4methylmopholine iV-oxide) for 18 h to provide 58 and 59, which were separated by silica-gel chromatography. Compounds 58 and 59 were treated with sodium periodate at 5 °C for 18 h to provide 60 and 61 respectively. Compounds 60 and 61 were treated with CDsMgl followed by NaBHsCN to give 62 in 20% and 50% yields, respectively. In conclusion, isotopic labeling of 2-desoxoparaherquamide A (PNU141962) with deuterium was achieved from PNU-141962 in four steps in anticipation of using its method of synthesis for the preparation of the corresponding ^^C and ^H labeled products.

Semi-Synthesis of 3-Epi-paraherquamide A (65) To investigate the significance of the chirality of the C3 position on anthelmintic activity, we prepared 3-epi-PHA [65, Fig. (18)].

355

CH« 0-4-CH3

H3C CH3 ^

= \=. C o^ y" 3 CHo

^ 0 bH,

O

CH,

63

56 f-BuOCI NEtg CH2CI2,0 °C

HgCf^a

rt 16h

-3 :T CH3

AcOH

o '^=\P^3 ^y

^-kteCXJ "°-. V-v^, H3C Jt-^^

65

0 CH3

CI 64

Fig. (18). Semi-Synthesis of 3-Epi-paraherquamide A (65)

Compound 56 [39] was heated under refluxing in xylene to give rearranged product 63 in good yield. Compound 63 was subjected to conditions described by Williams [46] to provide 65 in low yield. Compound 65 did not show ant anthelmintic activity . €26 Dialkyl and Spiroalkyl Analogs of MFA

HCOOH MFA

•

Fig. (19). Outlined synthesis of 67

356

To investigate the effect of changes at the C26 position of MFA on anthelmintic activity, several of C26-dialkyl and spiroalkyl analogs were prepared [47]. The analogs were synthesized in four steps starting with cathecol 66 [Fig. (19)], which was prepared from MFA in 80% yield by stirring in formic acid for 16 h.

Br

"V

\—'——^

R

K2CO3 Kl

R

66

m-CPBA

68a: R, R == cyclobutyl 68b: R, R == cyclohexyl 68c: R, R == diethyl 68d: R. R =: ethyl-methyl 68e: R, R =: dimethyl

C[

^O-^

r\

R

SnCU

•

MTPI

Fig. (20). Synthesis of 67

The general route outlined below, follows the modified method of Williams [48] used his preparation of the g^m-dimethyl dioxepin ring of PHB. By this method we were able to prepare the four dioxepin-ring anaologs in which the geminal methyl groups at C26 of MFA were replaced by a cyclobutyl 67a, cyclohexyl 67b, diethyl 67c, and ethylmethyl 67d groups [Fig. (20)].

357

The catechol 66 was coupled with the appropriate bromo reagent 68a-d in the presence of K2CO3 and KI in acetone/water to give mono-alkylated products 69a-d in 29-82% yield.Epoxidation with m-CPBA in CH2CI2 followed by workup with sodium bisulfite [49] (to remove the N-oxide) gave epoxides 70a-d in 40-100% yield. Ring closure using SnCUin THF provides alcohols 71a-d (50-85% yield) which were dehydrated with methyltriphenoxyphosphonium iodide (MTPI) in THF/DMF to provided the final products 67a-d in 20-30% yield. To verify the regiochemistry of the alkylation described above we reduced MFA with borane-methyl sulfide complex to provide 72 in 40% yield [Fig. (21)]. This compound was identical to the one prepared from the catechol 66 and 4-bromo-2-methyl-2-butene (73) using the chemistry reported in step 1 of Fig. (20), thereby conforming the assigned regiochemistry of compounds 68a-d. The biological activity of compounds 67a-d was evaluated in our standard anthelmintic assay which uses immunosuppressed Mongolian gerbils inoculated with Haemonchus contortus and Trichostrongylus colubriformis [41]. The compounds were administrated orally at a dosage rate of 0.33 mg/gerbil. Unlike MFA, none of these compounds gave the 95% clearance of helminthes we use as a criteria for determining activity, and were deemed inactive.

MFA

BH3-DMS /—\

•(

^aS. P^;

N-^K^ Ji^jJ^

I ^

^

K2CO3/KI

66

Acetone/HgO

Fig. (21). Reaction of MFA with BH3-DMS

C24 and C25 Substituted MFA Derivatives To investigate the effect on anthelmintic activity by changing the substituent pattern at C24/C25, we synthesized a number of analogs [Fig. (22)] [50].

358

Swem oxidation of 71e provided the ketone 74, which was subjected to Wittig olefination with methyltriphenylphosphonium bromide and n-BuLi to give the exocyclic methylene containing compound 75 in 50% yield. The versatile ketone 74 was also epoxidized with trimethylsulfoxium iodide and potassium r-butoxide in DMSO to give 76 in 35% yield, whereas Grignard chemistry (MeMgBr) gave a quantitative yield of 77. Treatment of 77 with DAST afforded a 35% yield of the desired analog 78.

V-OH

^

Fig. (22). C24 and C25 Substituted MFA Derivatives

We reasoned apriori that both compounds 75 and 78 would have anthelmintic activity since they contained the crucial C26 dimehtyl moiety and likewise retained the proper geometry of the A ring found in the parent compound MFA based on modeling studies. Furthermore, these structures should be less hydrolyzed under acidic conditions.

359

With the exception of compound 75 none of these compounds were active. Since the exocyclic methylene analog 75 had activity we chose to prepare a simplified analog [Fig. (23)]. Thus, the catechol 66 was reacted with 79, K2CO3 and Nal in DMF for 16 h to give the exocyclic methylene compound 80 which lacked the C26 dimethyls. Compound 80 was inactive, which further emphasizes the importance of C26 dimethyls. KgCOg/Nal CI 79

^'

Fig. (23). Synthesis of 80

STEREOCONTROLLED TOTAL SYNTHESIS OF (+)-PHB As part of on going efforts of Williams and his coworkers [46] to elucidate the biosynthesis of the core bicyclo[2.2.2] ring system of the related alkaloids the brevianamides [51], they have applied methodology originally developed for the stereocontrolled total synthesis of (-)brevianamide B [52] to complete the first stereocontrolled total synthesis of(+)-PHB. Retrosynthetic analysis As outlined in Fig. (24), a convergent synthesis of the enantiomer of the natural PHB was envisioned to contain four key carbon-carbon bondforming reactions. The first task would involve the construction of a suitably a-alkylated proline derivative [52]. The second important coupling would be the Somei/Kaetani-type alkylation [53] of the suitably protected gramine derivative 86 and the requisite piperazinedione 85. The third and most crucial C-C bond-forming reaction was a stereofacially controlled intramolecular SN2' cyclization reaction and concomitantly installs the isopropenyl group that will be utilized in the fourth C-C bond-forming reaction. Standard procedures to effect this transformation involve strong protic acids [52], and there was reason for concern about the reactivity of

360

the more highly oxygenated indole 83 as a practical synthetic precursor to 82.

^i"

84

(R)N MeOgC—'N^COOEt

Q:

(60-80%)

Boc I ^N

O

Boc I cOOEt

^N

U^-^Y^OTBS

OH

113

^"

112

111

cOOEt

(86%)

^Y^NH V - - ^ v ^ ^ OTBS

(93%)

^ . . ^ - ^ ^ OTBS

c.d.e

f,g (79%)

^N

cOOEt OTBS OMOM 114

Reagents: (a) Baker's yeast; (b) LDA, THF, HMPA, (E)-ICH2CH=C(Me)CH20TBS; (c) 5.7 equiv MOMCl, (/Pr)2NEt, CH2CI2; (d) 2.7 equiv ZnBr2, CH2CI2; 0 °C (e) K2CO3. 2 equiv BrCHiCOBr, CH2CI2; (f) NH3 in MeOH (5.7 M), 25 °C; (g) 3 equiv NaH, toluene, HMPA, 25 °C; (h) 1.3 equiv n-BuU, THF, 11.1 equiv ClCOOMe, - 78 °C; then 4 equiv ClCOOMe, 5 equiv LiN(TMS)2. - 78 °C. Fig. (31). Synthesis of 116

Protection of the secondary alcohol as the corresponding methoxy methyl (MOM) ether, followed by removal of the Boc group with ZnBr2 in dichloromethane and acylation of the incipient secondary amine with bromoacetyl bromide in the presence of K2CO3 afforded the bromoacetamide 114 in 86% yield from 113. Treatment of 114 with methanolic ammonia afforded the corresponding glycinamide which was directly subjected to cyclization in the presence of NaH in toluene/HMPA to afford the bicyclic compound 115 in 79% overall yield from 114. Next, a one-pot double carbomethoxylation reaction was performed by the sequencial addition of n-BuLi in THF followed by addition of methylchloroformate, that carbomethoxylated the amide nitrogen atom. Subsequent addition of four equiv of methyl chloroformate followed by the addition of 5 equiv of LiN(TMS)2 afforded 116 as a mixture of diastereomers in 93% yield that were taken on directly without separation.

369

Construction of the Tryptophan Derivative 120 Somei-Kamatani coupling of 116 [Fig. (32)] with the gramine derivative 94 in the presence of tri(n-butyl)phosphine gave the tryptophan derivative 117 as a 3:1 mixture of diastereomers in 70% yield. TBS* n

O

br"*"*^' R' = COOMe,R" = MOM :MOM

94

(70%)

OTBS OMOM

OTBS

116 R' = COOMe

(68 -71%)

•

/--p^N V - N , ^

120

OTBS

Boc

Boc

Reagents: (a) 0.7 equiv («-Bu)3P, MeCN; (b) 5 equiv LiCl, H2O, HMPA, 105 °C, 5 h; (c) 2.5 equiv Me30BF4, CS2CO3. CH2CI2.25 °C; (d) DMAP, 3 equiv (Boc2)0, CH2CI2; 0 °C (e) 3.3 equiv TBAF, THE, 25 °C; (f) 1.1 equiv Msci coUidine, CH2CI2, 0 °C; (g) 3.3 equiv TBSOTf, 2,6-lutidine, CH2CI2,0 °C. Fig. (32). Synthesis of 120

Decarboxylation of 117 was effected by treatment of 117 with LiCl in hot, aqueous HMPA at 105 °C providing 118 as a mixture of diastereomers that were separated and carried forward individually. Protection of the secondary amide group as the corresponding methyl lactim ether was accomplished by treating 118 with trimethyloxonium tetrafluoroborate in dichloromethane that contained cesium carbonate. Next, the indole nitrogen atom was protected as the corresponding Boc derivative by treatment with dicarbonic acid bis(f^rr-butyl)ester in the presence of DMAP and the silyl ether was removed with tetrabutylammonium fluoride to provide diol 119 in 52-78% overall yield from 118. Selective conversion of the allylic alcohol to the corresponding

370

allylic chloride was accomplished by mesylation in the presence of coUidine. Silylation of the secondary alcohol with rerr-butyldimethylsilyl triflate in the presence of 2,6-lutidine afforded the key allylic chloride 120 in 68-71% yield over the two steps. Construction of the Bicyclo[2.2.2] Ring System and the Final C-C Bond-Forming Reaction on the Indole

OMOM, h (85%), i (97%) OH

Reagents: (a) 20 equiv NaH, TOP, reflux 30 h; (b) 3.1 equiv AgODp4, 4.68 equlv PdCh. MeCN, propylene oxide; then NaBH4, EtOH; (c) 0.1 M HCl, THF; (d) 2-hydroxypyridine, toluene, 120 °C, 2 h; (e) 5 equiv (iBu)2AlH, CH2CI2. 0 °C; (f) NaH, Mel, DMF, 0 °C; (g) 6 equiv B-bromocatecholborane, CH2CI2, 0 °C; (h) Sequiv l,l,l-triacetoxy-l,l-dihydro-l,2-benziodoxol-3(7f/)-one (Dess-Martin periodinane), CH2CI2, 25 °C; (i) TFA, CH2CI2, 25 °C.

Fig. (33). Synthesis of 124

The stage now set to effect the SN2' reaction. [Fig. (33)] Compound 120 was refluxed in THF with 20 equiv of NaH, resulting in a very clean

371

and high-yielding cyclization reaction furnishing the desired product 121, and the undesired anr/-diastereomer was not detected. Closure of the seventh ring was effected by treatment of 121 with 4.68 equiv of PdCl2 and 3.1 equiv of AgBF4 [58] in acetonitrile containing propylene oxide as an acid scavenger. The incipient heptacyclic apallladium adduct was worked up immediately by the addition of the ethanol and NaBH4to afford the desired indole 122. Cleavage of the lactim ether 122 was effected with 0.1 M HCl to give the corresponding ring-opened amine methyl ester that was recyclized by treatment of this material with 2-hydroxypyridine in hot toluene. Chemoselective reduction of the secondary amide was effected by treatment of of the product obtained from the previous step with excess diisobutylaluminum hydride in CH2CI2 to furnish 123 (50-72% yield) [64]. Methylation of the secondary amide 123 proceeded in 96% yield. Cleavage of the MOM ether with bromocatecholborane [65] (91% yield) followed by oxidation of the secondary alcohol with Dess-Martin periodinane [66] (85% yield) and cleavage of the Boc group and TBS ether with TFA (97% yield) gave the ketone 124. Formation of the Spiro Oxindole The final, critical oxidative spirocyclization of the 2,3-disubstituted indole to the spiro oxindole was effected by treatment of 124 with tertbutyl hypochlorite in pyridine to provide the labile 125 [Fig. (34)]. The Pinacol-type rearrangement was conducted by treating compound 125 with p-toluenesulfonic acid in THF/water. It is assumed that the chlorination of 124 proceeds from the least hindered face of the indole, to give the a-chloroindolene 125. The hydration of the imine functionality must also occur from the same a-face that is syn to the relatively large chlorine atom furnishing the ^yn-chlorohydrin 126, that subsequently rearranges stereospecifically to the desired spiro oxindole 127. The dioxepin ring was then formed by dehydration of the secondary alcohol 127 with MTPI in DMPU to afford 14-oxo-PHB 12 [7]. This material has been previously described by a Pharmacia group (obtained semi-synthetically from MFA) and comparison of the authentic and synthetic materials [^H and ^^C NMR, IR, exact mass, mobility on thinlayer chromatography (TLC)] conformed the identity of this substance. Treatment of the synthetic ketone with MeMgBr gave (-)-PHA (2) in 42% yield that was identical in all respects [^H and ^^C NMR, IR, exact mass,

372

mobility on TLC, m.p. (250 °C (dec)), [ajo = -22 (C= 0.2 MeOH)] to the natural PHA [4]. OH TsOH

f-BuOCI

O

THF/HgO

OH

CH3

^*^LNx^xk^O

• (55%) O 54 % from 125

3

CH3O 127

MeMgBr (42%)

12

Fig. (34). Formation of the spiro oxindole

CONVERSION OF MFA TO PARAHERQUAMIDES VIA A NOVEL PLATINUM-OXYGEN-MEDIATED RING CONTRACTING REACTION [33] Our earlier conversion of MFA to PHA required 13 steps [7]. By employing a ring contracting reaction utilizing platinum and oxygen (Pt/Oi) at a key point in the synthesis, we were able to directly convert marcfortine A to the intermediate 16-oxoparaherquamide B (7), thereby eliminating six steps in our earlier synthesis.

373

H3C Ch CH«

A

Pt/C dioxane/water

o 7

CH,

(14-23%)

o p \J/

H3C CH,

06^

CH^ 128

(2-3%)

129

130

(9-19%)

(14-19%)

MCPBA 130

7

(80%)

1

4

MCPBA

H

o

p^JiiU/M^c CH3

P

p<j^^H^C

CH3

CH-, Fig. (35). Reaction of MFA with oxygen

Although the Pt/02 reaction has been used for oxidation of primary and secondary alcohols [67], hydroxylation of 12a-deoxytetracyclines [68], oxygenation of cholesterol [69], and oxidation of tertiary amines [5f], this

374

reaction has not been used extensively compared to singlet oxygen reactions [70]. When marcfortine A was treated with Pt on carbon (10%) in dioxane/water under an oxygen atmosphere (balloon pressure, room temperature, 2-4 days), four products (7, 128-130) were isolated [Fig. (35)]. The reaction was not accelerated by heating. Performing the reaction under oxygen at 1000 psi resulted in acceleration with very little effect on the product ratios. Yields varied depending on the activity of the platinum.

o p

^3^. p^3

%J/

Pt/Og followed by MCPBA

H

HO

23

10 (43%)

HQC

CH-N

°

>-N 131

HO-f-^4v> H3C

H3C CHo

V ^

o

133 (10%)

H3C CH3 ,

,,^

M:^40<J (3%)

H3C

Y\

CH^

132

Fig. (36). Reaction of 23,32 with oxygen

PHA AIHg-NMegEt

375

Dioxo compound 130 was converted to 7 in 80% yield by treatment with m-chloroperbenzoic acid (m-CPBA). According to the literature, sixmembered rings containing a 1,2-dicarbonyl moiety are converted to fivemembered ring hydroxy acids only in the presence of a strong base [71]. By contrast, our method is performed under neutral conditions and is more efficient. Subsequently, we examined several MFA derivatives as substrates for the Pt/02 reaction. When 32 was subjected to Pt/Oa chemistry, three products (132-134) were isolated in poor yield [Fig. (36)]. In the case of 23, the Pt/02 reaction mixture was treated straightaway with m-CPBA without isolation of any intermediate products, giving the desired compound 10 (43% yield). Compound 10 can be converted to PHA (2) in three steps. Compound 132 was converted to PHA (2) by treatment with alane-dimethylethyl amine complex in THF (30% yield). HqC CHo

Pt/0«

HO^

O

36 135

Fig. (37). Reaction of 36 with oxygen

(6%)

376

When 36 was subjected to Pt/02 chemistry [Fig. (37)], four products (35, 135-137) were isolated. Although the Pt/Oi chemistry performed on 36 does give 137 (the desired product) in trace amounts, it can be more readily prepared by oxidation of 136 with m-CPBA (80% yield). Subsequently, 137 was reduced with LAH/AICI3 in THF to give 138 (40% yield).

CH«

Pt/C

0-0

\

[O] B

\ Partial aiiyiiic oxidation o

CH3

C: X = H,H D: X = H,OHorO [0]

130

[H2O2]

*•

7

oo [H2O2]

[0]

128+ 129

o CH3 E: X = H,OHorO >

Fig. (38). A plausible mechanism for the Pt/O^ meddiated ring contracting reaction

377

A plausible mechanism for the Pt/Oi-mediated ring contracting reaction is shown in [Fig. (38)]. In this mechanism, A exists in equilibrium with B (one can speculate about the formation of A). Intermediate B undergoes molecular oxidation accompanied by partial allylic hydroxylation to give intermediates C and D, Each of these is further oxidized to give putative intermediate E and product 130. A final oxidative step, probably involving hydrogen peroxide generated in situ and proceeding by a mechanism paralleling that described for the m-CPBA reaction, leads to products 7, 128, and 129.

ACKNOWLEDGEMENTS We are grateful to Professor Yamazaki at Chiba University for furnishing a sample of natural paraherquamide A.

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[55] [56] [57]

[58] [59]

(a) Conder, G.A.; Johnson, S.S.; Guimond, P.M.; Cox, D.L.; Lee, B.L. J. Parasitol 1991, 77, 621; (b) Conder, G.A., Johnson, S.S. J. Parasitol 1996, 82, 100. Thompson, D.P.; Klein, R.D.; Geary, T.G. Parasitology 1996, 775, S217. Schaeffer, J. M.; BHzzard, T.A.; Ondeyka, J.; Goegelman, R.; Sinclair, P. J.; Mrozik, H. Biochem. Pharmacol 1992,43, 679-684. Nauen, R.; Ebbinghaus, U.; Tietjen, K. Pestic. Sci. 1999,55,608-610. (a) Zinser, E.W; Wolfe, M.L.; Alexander-Bowman, S.J.; Thomas, E.M.; Groppi, V.E.; Davis, J.P.; Thompson, D.P.; Geary, T.G. J. Vet. Pharmacol Ther., submitted, (b) Lee, B. H. / Labelled Compd Radiopharm 2002, 45, 0000. (c) Blizzard TA, Rosegay A, Mrozik H, Fisher MH. / Labelled Compd Radiopharm 1990,2S, 461-464. (a) Gushing, T. D.; Sanz-Cervera, J. F.; Williams, R. M. J. Am. Chem. Soc. 1996, 77S, 557. (b) Gushing, T. D.; Sanz-Cervera, J. F.; Williams, R. M. J. Am. Chem. Soc. 1993, 775, 9323. Lee, B. H.; Clothier, M. F. Bioorg. Med Chem. Lett, 1997, 7, 1261. Gushing, T. D.; Williams, R. M. Tetrahedron Lett. 1990, 31, 6325. McWhorter, W. W.; Gleave, D. M.; Savall, B. M. Syn. Comm. 1997,27, 2425. Lee, B. H.; Clothier, M. F. Bioorg. Med Chem. Lett, 1998, 5, 3415. Sanz-Cervera, J. F.; Glinka, T.; Williams, R. M. J. Am. Chem. Soc. 1993, 775, 347. Williams, R. M.; Glinka, T.; Kwast, E.; Coffman, H.; Stille, J. K. J. Am. Chem. Soc. 1990, 772, 808. (a) Somei, M.; Karasawa, Y.; Kaneko, C. Heterocycles 1981, 16, 941. (b) Kametani, T.; Kanaya, N.; Ihara, M. / Am. Chem. Soc. 1980, 702, 3974. (a) Beer, R. J. S.; Clarke, K.; Davenport, H. F.; Robertson, A. /. Chem. Soc. 1951, 2029. (b) Bennington, F.; Morin, R. D.; Clark, L. C , Jr. J. Org. Chem. 1959,2^,917. Kosuge, T.; Ishida, H.; Inabe, A.; Nukaya, H. Chem. Pharm. Bull 1985, 33, 1414. (a) Magid, R. M.; Fruchey, O. S.; Johnson, W. L.; Allen, T. G. J. Org. Chem. 1979,44, 359.(b) Magid, R. A. Tetrahedron 1980,36, 1901. The idea that the stereochemical outcome of an intramolecular enolate alkylation is determined by chelation in the transition state was recently demonstrated by Denmark and Henke, who observed a marked preference for a "closed" transition state (coordination of the cationic counterion to an enolate and the developing alcohol) resulting in a syn product. For example, the highest syn.anti ratio (89:11) was obtained in toluene and the lowest syn:anti ratio (2:98) was obtained with a crown ether. These observations parallel the facial selectivities described herein and in ref 11 on the intramolecular SN2'reaction; see: (a) Denmark, S. A.; Henke, B. R. / Am. Chem. Soc. 1991, 775, 2177. (b) Denmark, S. A.; Henke, B. R. /. Am. Chem. Soc. 1989, 777, 8022. Trost, B. M.; Fortunak, J. M. D. Organometallics 1982, 7, 7. In a recently reported synthesis of gelsemine, a tertiary lactam was reduced in the presence of a secondary lactam with DIB AH. However, this reagent failed on substrates 101; see: Dutton, J. K.; Steel, R. W.; Tasker, A. S.; Popsavin, V.; Johnson, A. P. J. Chem. Soc, Chem. Commun. 1994, 765.

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[60] [61]

[62] [63] [64] [65] [66] [67] [68] [69] [70] [71]

Martin, S. F.; Benage, B.; Geraci, L. S.; Hunter, J. E.; Mortimore, M. J. Am. Chem.Soc.l991J 13, 6161. (a) Yoon, N. M.; Brown, H. C. J. Am. Chem. Soc. 1968, 90, 2927. (b) Marlett, E. M.; Park, W. S.; /. Org. Chem. 1990, 55, 2968. (c) Jorgenson, M. J. Tetrahedron Lett. 1962, 559. (d) Another very recent synthesis of gelsemine reported the reduction of the same gelsemine precursor (as in ref 59) with AIH3. Newcombe, N. J.; Fang, Y.; Vijn, R. J.; Hiemstra, H.; Speckamp, W. N. /. Chem. Soc, Chem. Commun. 1994, 767 Williams, R. IVl.; CaO, J.; Tsujishima, H. Angew. Chem. Int. Ed. 2000,39, 2540. (a) Williams, R. M.; CaO, J. Tetrahedron Utt. 1996, 37, 5441. (b) Cooper, J.; Gallgher, P. T. ; Knight, D. W. J. Chem. Soc, Perkin Trans 1 1993, 1313. Fukuyama, T.; Liu, G. Pure Appl Chem. 1997,69,501. Boeckman, R. K.; Potenza, J. C. Tetrahedron Utt. 1985,26,1411. (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155; (b) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc 1991,113, 7277. Sneeden, R. P. A.; Turner, R. B. J. Am. Chem. Soc 1955, 77, 190. Muxfeldt, H.; Buhr, G.; Bangert, R. Angew. Chem., Int. Ed. Engl. 1962,1, 157. Sakamaki, H.; Take, M.; Akihisa, T.; Matsumoto, T.; Ichinohe, Y. Bull. Chem. Soc Jpn. 1988,61,3023. Wasserman, H. H.; Ives, J. L. Tetrahedron 1981, 37, 1825. (a) Bhatt, M. V. Tetrahedron 1964, 20, 803. (b) Aoyage, P.; Moriyama, Y.; Tsuyuki, T.; Takahashi, T. Bull. Chem. Soc Jpn. 1973,46, 569.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.

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ACARICIDES OF NATURAL ORIGIN, PERSONAL EXPERIENCES AND REVIEW OF LITERATURE (1990-2001)* GUIDO FLAMINI Dipartimento di Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy ABSTRACT: Acari are responsible for millions of dollars worth of damages each year as a result of infestations in animals, plants and man. They affect our health directly and prosperity as animal and plant parasites, vectors of disease, and producers of allergens. The indiscriminate use of pesticides has destroyed many of their natural enemies of hitherto harmless species and quickly induced resistance in many parasites. At present, the control of acarid parasitic diseases in agriculture, human and veterinary medicine is mainly based on the use of drugs; for this reason the lack of effective drugs often prevents the control of some parasitic diseases, making them more serious and important. The use of commercial drugs involves many problems; besides the drugresistance shown by the most important parasites, the environmental damage and the toxicity of many synthetic drugs, represent the main problems that strongly limit their use. In addition, drug residues in plant and animal food products are important reasons forfiirthereconomic losses for farmers and must be regarded as potentially hazardous to man and envh-onment. Plant-derived compounds are generally more easily degradable and could show a smaller negative environmental impact with respect to synthetic drugs. For these reasons, the evaluation of the antiacarid activity of plant extracts is increasingly being investigated in order to obtain new leads, as demonstrated by recent studies that have evaluated and confirmed the effectiveness of many plant compounds on bacteria,fimgi,protozoa, hehnints and arthropods. This review will be limited to the class Arachnida, sub-class Acaridi, particularly to their control in agriculture, veterinary and human medicine with natural methods.

INTRODUCTION Mites and ticks, collectively known as the Acari, constitute the second most diverse group of animals on the planet today and are of interest to humans for a variety of reasons. They affect our health and weel being directly as plant, animal and human parasites, vectors of disease, and producers of allergens. They are responsible for millions of dollars worth

Dedicated to the memory of Prof Serena Catalano

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of economic losses each year as a result of infestations of animals, man and plants. The acari are ubiquitous, being members of every major ecosystem on earth, and their specific habitats run the gamut from the familiar to the truly bizarre. From our backyards to the geothermal springs of the Yellowstone caldera and from the subcutaneous tissue of turtles to our very own hair follicles, mites carry out a nearly invisible existence. While awareness of the acari dates back to ancient Egypt (1550 BC) and was further demonstrated through the writings of the major Greek scholars, the science of acarology originated in 18th century in Europe. Linnaeus described the first mite species, Acarus siro, in 1758, thus laying the groundwork for the field of systematic acarology. Today, over 48000 described species of mites and ticks have been described worldwide, while current estimates place total diversity at over a halfmillion species. The Class Arachnida, to which the order Acari belongs, together with the Class Insecta, the Class Crustacea and others, constitute the Phylum Arthropoda. All the classes contain species useful to man, but also many pests that can cause economic losses and/or diseases. This review will be limited to the order Acari, particularly to their control with natural methods in agriculture, veterinary and human medicine. The indiscriminate use of inorganic pesticides, destroyed many harmless species including natural enemies of these mites and ticks [1]. From 1940 onwards, organochlorine and organophosphate pesticides were introduced, but tresistance was quickly acquired in many arthropod parasites, including acari; fortunately many useful predatory mites became resistant too. The emergence of resistance to parasiticides is one of the most serious challenges faced by modem farmers. Perhaps it is the simplicity of treating parasite attacks with very effective drugs or pesticides on a routine basis and the proven cost-effective gains in productivity that will accrue in the short term, that has led to the predominance of synthetic pesticides [2]. Broadly speaking, resistance is the ability of the parasites to survive doses of drugs that would normally kill parasites of the same species and stage. It is inherited and selected for because the survivors of the pesticide treatment pass genes for resistance on to their offspring. These genes are initially rare in the population or arise as rare mutations in genes but as selection continues, the proportion of resistance genes in the population increases and so does the proportion

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of resistant parasites. Drug susceptibility is a resource that needs to be preserved using appropriate techniques of parasite management. In many cases susceptibility to certain pesticides in some parasites has been lost forever. One approach to this problem is to develop new susceptibilities by using novel drugs. However, the application of synthetic chemical substances is still the common method to control or eradicate parasites of plant and animals. Unfortunately, many acaricides have non-specific properties, affecting other organisms (crops, non-vertebrates and vertebrates) to varying degrees. The damage to these non-target organisms may be reflected by different effects, like direct mortality, long term population reductions, bioaccumulation within the food web. The indiscriminate dispersion of these substances must be regarded as potentially hazardous to man and environment. Plants are the richest source of organic compounds on Earth. Many of these natural chemicals are endowed with pesticide properties. It is well known that some pesticides of plant origin have been in use before the time of the Romans (i.e. pyrethrum, obtained from the flower heads of Chrysanthemum cinerarifolium). In 1959, the term Integrated Pest Management (IPM) was coined [3]. IPM consist in the application of the best mix of control tactics for a given pest problem in comparison with the yield, profit and safety of the alternative mixes [4]; these control methods ought to be environmentally acceptable and sustainable [5]. However, many of these studies have little bearing on what is actually happening in the field. In veterinary medicine, the control of ectoparasites is of great importance due to their effects on livestock profitability and the health status of animals. Infestations on livestock can cause intense irritation leading to poor condition, weight loss, reduced milk yield, and hide or fleece damage. Furthermore, many species of acari are responsible for transmission of diseases to the host animals themselves or act as vectors of a number of diseases to humans [6]. APICULTURE During the 90s, several cases of resistance to common acaricides employed in beekeeping by Varroa mites (Acari: Varroidae) were reported from different countries [7-10]. In Italy, the consequences of the resistance were disastrous colony losses. Available statistics show that in

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certain districts, losses often exceeded 70% and in some locations even reached 90% [11]. Varroa mites originated in southeast Asia, where they parasitized the Asian honeybee. Gradually they have spread and now^ afflict most of the European honeybees. Though the Asian honeybee has been able to tolerate the mite: it was a pest, but not a fatal one, European honeybees have almost no resistance, and the mite has proved fatal to millions of the colonies in Europe and America. Varroa mites suck the body fluids from adults and brood, prefering the latter, especially drone brood. The problem of developing suitable treatments was difficult in the case of the Varroa mites because most substances effective against the parasites have unacceptable side-effects on bees. Towards the end of the 80s products based on pyrethroids were used. These substances were very effective on mites, and without any appreciable effects on bees. Pyrethroids are synthetic analogs of pyrethrins, secondary metabolites obtained from the flower heads of Chrysanthemum cinerarifolium. Synthetic pyrethroids replaced the natural ones because they apparently exhibited no negative effect on plants, animals and humans. Unfortunately, the limits of this "magic bullet" soon became evident, with serious impact on beekeeping because of the resistance developed by Varroa. Since the creation of EU Varroa experts' group, several lines of research in alternative control measures have been explored: apicultural techniques for reducing the number of mites, increasing bee resistance, and searching for acaricidal products that are generally recognized as safe for humans, such as some natural derivatives [12]. Among these compounds many simple carboxylic acids have been used, such as formic, oxalic and lactic acids. The first report on the use of formic acid as miticide was published in 1980 [13]. Five years later Wachendorfer et al. [14] developed and carried out field trials on the "lUertisser Milbenplatte" (=mite plate), an adsorbent cardboard soaked in formic acid on which the bees alighted before entering in the hive. Other methods of treatment included dispensation of formic acid by means of evaporators, wood shavings, or use of liquid reservoirs with wicks [15-27]. The mortality rate of Varroa mites was however quite variable, with percentages ranging from 12% [24] to 98.8% [21], with a prevalence of intermediate values (47%-56%) [18,21,26]. A strong dependence upon the administration method was observed: in western Switzerland the percentage of mites killed was 80% when formic acid was applied on the plates, but 90%

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when applied on viscose cloth [16]. Many of the above techniques involve removal of one or more frames from the hive, and most involve handling of liquid acid by the beekeeper, with the risks associated with dispersion and the corrosive nature of this substance. To resolve these problems, recently a gel formulation of formic acid has been developed. Kochansky and Shimanuki [28] experimented with various gelling agents, obtaining the desired rheological characteristics and releasing behavior of the active principle, with fumed silicas and Carbopols 934 and 941. The fumed silicas gave the best results, with optimum evaporation speed for application to bee hives (2-3 weeks). A gel formulation tested in Argentina under autumnal climatic conditions showed a 92% mortality in Varroa jacohsoni with a low variability, indicating that this kind of formulation could be the best one [29]. Formic acid was evaluated for its effect on the honey bee colony; Westcott and Winston [30] examined various parameters such as colony weight gain, brood survival, sealedbrood area, emerged-bee weight, number of retumed foragers, pollen-load weight and worker longevity in treated and control colonies. Only sealedbrood area was lower in formic acid treated colonies; furthermore, also the honey production was lower in treated colonies, but this difference from control was not statistically significant. Another study confirmed that the use of formic acid can be considered safe, in fact the average length of life of the treated bees was 24.4 days compared with 23.8 days of controls [31]. Also during heavy Varroa Jacobsoni infestations and hives weakened by the dry summer conditions, it was demonstrated that 8 consecutive treatments with formic acid during the month of August gave satisfactory mite control without harming or irritating the bees [15]. However, these conditions are not sufficient for a proper use of formic acid in apiculture, but it is necessary to evaluate whether this substance can accumulate in honey, wax and propolis. Various HPLC methods have been developed [32,33], but only one [34] compared treated honey samples and controls, conclusively showing that the former contained higher levels of formic acid (25-51 mg/kg) than the latter (20 mg/kg). Recently, the influence of formic acid on honey taste has been evaluated by means of an expert test panel [35]. The acid was artificially added to different honeys and the testing persons remarked an increased acid taste in the honeys adulterated with acid quantities above the taste threshold, that is the lowest correctly distinguishable concentration of an additive in

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honey. The taste threshold for acacia honey was about half of the one for honeydew honey, because the latter has a much stronger aroma and can whitstand more acid than acacia honey, which has only a weak aroma. The taste thresholds of formic acid were 150-300 mg/kg in acacia honey and 300-600 mg/kg in honeydew honey; in pure water it was 10 mg/kg, about 20 to 50 times below that in honey. Similar thresholds were also found in Italy [36]; Italian authors also noted that after application in autumn at the end of the bee season, the formic acid content increases significantly and may exceed the natural content. Later on, the formic acid content decreases slowly due to evaporation, to reach the original level in the following spring. Therefore, formic acid treatments should be performed in autumn to avoid adverse consequences to the taste of honey, harvested the following spring. Further to formic acid treatment, oxalic acid [21,21,37-40] and lactic acid [17,18,20,21,41-46] have been evaluated for their activity against Varroa jacobsoni and V. destructor. The application of oxalic acid has been studied both in different periods and by means of different methods. It seems that the best period is autumn-winter, the broodless period (average efficacy 99.44% vs. 52.25% in the presence of brood) [40]. These data have been confirmed in Italy, where a spray treatment of oxalic acid in water was compared with a topical application of the acid in a sucrose-water solution; the best result was obtained using the former method. In general, for both treatments, the highest mite mortality coincided with the treatment applied with no sealed brood [38]. With regard to the toxicity of oxalic acid, two contrasting studies are present in literature. The first one claims that bee mortality in treated hives was not significantly different from the bee mortality in controls [37], while the other paper suggests negative long-term effects, with a statistically significant negative effect on brood development and the death of some queens [39]. On the presence of oxalic acid in honey, it seems that its content in samples taken from the nest after treatment did not differ significantly between treated and control ones [37,38]. If the application is performed in autumn, the honey content of oxalic acid is comparable to its natural amount, that is below its taste threshold, determined in 300400 mg/kg for acacia honey and 700-900 mg/kg for honeydew honey [35]. Lactic acid was less active than the previous organic acids [18,21] and again it showed a greater effectiveness during late summer-winter

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treatments [18]. When its activity was compared with some commercial acaricides, results show it is less effective than coumaphos, a synthetic coumarin (umbelliferone) derivative, but also less toxic [42,45]. Rademacher [47] prepared a new formulation containing a synthetic acaricide, cymiazole hydrochloride, and lactic acid as active ingredients; furthermore, the content of cymiazole was 50% less than in a commercial preparation. The mixture resulted highly toxic to Varroajacobsoni, with the tolerance of honey bees a 100 times greater than that of the mites; field tests have shown that the mixture killed 83% and over 90% of mites in colonies with and without brood, respectively. Some authors reported an increased bee mortality after treatments with lactic acid [18,41]. Immediately after applications in autumn, the lactic acid content of honey increased up to 1500 mg/kg, but four weeks later it decreased to about 500 mg/kg: this was below the taste threshold of 800-1600 mg/kg in rape honey [35]. Formic acid was also found effective against another bee parasite, Tropilaelaps clareae (Acari: Laelapidae). This mite develops in brood cells and causes disturbances in metamorphosis, resulting in abnormal, incompletely developed individuals; in the case of a severe attack, bee larvae are killed. It caused substantial losses of Apis mellifera colonies in Pakistan, but a single application of formic acid (20 ml) to infested colonies gave complete control of the mite population, however, it also destroyed all the brood and some adult bees. Two treatments of 15 ml also gave complete control of the mite population, but caused only minimal destruction of brood [48]. The same findings were obtained in another study [49]. A following study confirmed these results and also demonstrated the effectiveness of sulfur (450 mg/frame) for T, clareae control [50]. Furthermore, sulfur was not toxic for bees [31]. Tracheal mites were also killed by formic acid [51]. This parasite, Acarapis woodi (Acari: Tarsonemidae), lives in the tracheal tubes of adult honey bees. The bees die because of the disruption to respiration caused by the mites clogging the tracheae. In summary, although these organic acids are natural honey constituents, the international food legislation prohibits honey additives adulterating its taste. Therefore, the residues of these substances in honey have to remain below their taste threshold [35]. Another natural substance tested against bee mites was the oil obtained

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from neem tree, Azadirachta indica, kernels. Its first use in beekeeping as acaricide dates back to 1995, when it was tested against the tracheal mite Acarapis woodi [52]. A commercial preparation of neem oil containing 3000 ppm was used to prepare a syrup containing 3 or 6 ml/1 of the drug. The colonies of Apis mellifera were treated in mid-May, and in August no mites were found in the colonies treated with 3 ml/1, while they were still high in controls. Furthermore, the treatment not only killed adult mites but also reduced the number of mite eggs. Treated colonies collected more pollen and produced more honey. Topical applications of neem oil in laboratory to infested bee were highly effective against both Varroa jacobsoni dead Acarapis woodi [53]. Approximately 50-90% V.jacobsoni mortality was observed 48 h after treatment with neem oil, with associated bee mortality lower than 10%. Although topically applied neem oil did not result in direct A. woodi mortality, it offered significant protection of bees from reinfestation. Significantly, azadirachtin-rich extract was ineffective at controlling both the mites, suggesting that azadirachtin. Fig. (1), one of the main insecticide compounds of neem tree, has no significant miticidal properties; in fact, honey bees were also deterred from feeding on a sucrose syrup containing more than 0.01 mg/ml of azadirachtin-rich extract. MeOO£. 1 ^OH

MeOOC MeOOC

^i

o

Fig. (1). Structure of azadirachtin

The same research group evaluated neem oil in the field [54]. They sprayed a 5% solution of the oil on infested honey bee colonies, killing about 90% Varroa mites but obtaining only a slight but not statistically significant decrease in tracheal mite infestation levels. Unfortunately this treatment caused 50% queen loss and the treated colonies showed onethird as many adult bees and one-sixth as much brood as untreated

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colonies. The authors hope that these negative effects on bees could be remedied by changes in the formulation, the application technology, and the season of application. In another trial the same researchers compared the spray applications of neem oil with feeding oil or azadirachtin-rich extract in a sucrose syrup [55]. They concluded that spray application was more effective than the other methods only on Varroa. They also reported that spray treatments had no effect on adult honey bee populations, however treatments reduced the amount of sealed brood in colonies by 50% and caused queen loss at higher doses. Besides neem oil, Majeed also tested Azadirachta indica dust obtaining an effective level of control and increased honey production with both the formulations [56]. Also pure azadirachtin, both commercially formulated and purified, was tested for its effects on Varroa mites, infested bees and healthy bees [57]. Azadirachtin was administered in 50% sucrose syrup at various concentrations, ranging from 6 to 162 |i87.6%), and probably contributes significantly to the acaricidal activity of the oil. The paper demonstrated the importance of a further factor affecting the variability in the effectiveness of extracts obtained from the same species, that is the technique used for the extraction. Once again, I would like to underline the significance of knowing the composition of the essential oils used in the tests; unfortunately, in the case of the three absinth oils, authors were not able to identify its major constituent; moreover, another sesquiterpene, present in the DSD oil and absent in the other two, that could be responsible of the greater toxicity of the former oil, was also unidentified. The toxicity of vapours of the essential oils obtained from four plant species, seeds of Cuminum cyminum and Pimpinella anisum (Apiaceae), leaves of Origanum syriacum var. bevanii (Lamiaceae) and fruits of Eucalyptus camaldulensis (Myrtaceae) against another species of the same genus, the carmine spider mite, T. cinnabarinus, was investigated in Turkey [262]. This mite is a major greenhouse pest in this country and throughout the world. It attacks a large range of 100 cultivated crops and weeds. It is a serious pest on beans, eggplant, pepper, tomatoes, cucurbits, and many other vegetables. It is also a pest of papaya, passion fruit, and many other fruits. The carmine spider mite also attacks many flowers and ornamental

428

plants such as carnation, chrysanthemum, cymbidium, gladiolus, marigold, pikake, and rose. The oils were assayed at 0.25, 0.50, 1.00 and 2.00 |il/l of air. The phytotoxicity of the vapours of these essential oils was estimated by exposing tomato, bean and cucumber seedlings to the highest dose for 96 hours. All essential oils, except that of eucalyptus, caused 100% mortality of the carmine spider mite at or below the maximum dose after 2-3 days of exposure. When the essential oils were compared on the basis of their LT50 and LT99 values, the order of toxicity was oregano>cumin>anise>eucalyptus. Phytotoxicity to seedlings was manifested as discoloration and eventual drying of the first two leaves. Cumin and anise oils were toxic to all plants tested, oregano was toxic only to tomato, whereas eucalyptus to none. Some terpenes, commonly found in essential oils, are pheromones of some arthropods species. Pheromones are volatile chemicals used for communication within individuals of the same species and, occasionally, between different species (usually the latter are known as allomones) [263]. Two examples are represented by famesol and nerolidol. Fig. (7), two highly attractive compounds to Tetranychus mites. These compounds have been added to common synthetic acaricides to improve their effectiveness against moving stages of mites [264,265].

nerolidol

CH.OH famesol Fig. (7). Nerolidol and famesol, two sesquiterpene pheromones

Also many non-volatile compounds have been found effective against Tetranychus mites. Among these chemicals, particularly interesting as a new class of acaricides, are naphthoquinones, in particular two derivatives isolated from Calceolaria andina (Scrophulariaceae), protected by patent application, and designated as BTG 505 and BTG 504, Fig, (4) [171,266,267]. These chemicals were found to exhibit high activity, even against strains of T, urticae that are most resistant to a wide range of

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commercial acaricides. Furtheraiore, the levels of activity were low on many beneficial species, both insects and acari {Phytoseiulus persimilis and Typhlodromus piri). Naphthoquinones have long been known to inhibit mitochondrial respiration, however the primary site of action may vary, depending upon the nature of substituents. Complex III was found to be the primary site of action for the two 2-hydroxy-l,4naphthoquinones isolated from C. andina. Complex III (ubiquinol:cytochrome c oxidoreductase), is found in mitochondria, photosynthetic bacteria and other prokaryotes; the general function of the complex is electron transfer between two mobile redox carriers, ubiquinol (QH2) and cytochrome c. Electron transfer is coupled with the translocation of protons across the membrane thus generating an electrochemical proton potential that can drive ATP synthesis by ATP synthase. Since the primary loss mechanism of applied naphthoquinones from leaf surfaces was proved to be volatilization (though some degradation also occurred leading to a half life of about 20 hours), authors have shown that different types of formulations (not reported, patent pending) can increase the efficacy against parasites and reduce phytotoxicity. Concerning the commercial potential of these products, the authors observed that naphthoquinones can occur in concentrations up to 5% w/w in dried aerial parts of C andina, but this herb would be a poor source for commercial production of the natural products. However, this genus is amenable to hybridization, and from preliminary studies it seems that it could be possible to produce more vigorous and highyielding cultivars. Furthermore, these compounds can be synthesized in 2-3 steps. Other synthetic (and natural, see below) acaricides, i.e. tebufenpyrad, pyridaben, fenazaquin, have been shown to be active by inhibition of another electron transport system of the mitochondrial respiratory chain, Complex I (NADH:ubiquinone oxidoreductase). Because of their high activity against various mite species, these METI (mitochondrial electron transport inhibitors) acaricides are in widespread use, but some strains of T. urticae from different parts of the world have been reported to exhibit resistance to these substances. Very recently, a strain of T. urticae from hops in UK was confirmed to have crossresistance to all the METI acaricides, despite having only been exposed to a single compound. Naphthoquinones inhibit Complex III in the mitochondrial respiratory chain, a system distinct from the Complex I,

430

but given the unpredictability of cross-resistance patterns and the fact that metabolic resistance can confer resistance between chemical groups, it was important to evaluate whether METI resistance also protected mites against the naphthoquinones [268]. Experiments with METI acaricide resistant strains and the standard reference susceptible strain, ascertained that the activity of naphthoquinones against T. urticae remained uncompromised.

•Mil

Fig. (8). Acetogenins from Annona glabra

From the seeds of another species, Abrus precatorius (Fabaceae), other classes of compounds have shown promising effects against T. urticae. From the non-saponified fraction of a crude petroleum ether extract, coumarin, p-amyrin and a mixture of sterols were isolated and tested against females and eggs in laboratory conditions. P-amyrin was the most effective compound against both stages. Spraying females with sub-lethal doses of p-amyrin caused a significant reduction in fecundity and the viability of resulting eggs [269]. From the seeds of Annona glabra (Annonaceae), three acetogenins, squamocin. Fig. (6), desacetyluvaricin and asimicin, Fig. (8), have been extracted and their toxicity against insects and mites evaluated; they have shown good insecticidal activity, but no acaricidal effect against T. urticae [270] while, recently, squamocin was found effective against the house dust mite Dermatophagoides pteronyssinus [226]. The genus Pimpinella (Apiaceae) produces rare phenylpropanoids with an unusual substitution pattern at the phenyl ring: the (l^*)propenyl-2-hydroxy-5-methoxybenzene skeleton of these compounds

431

has been named pseudoisoeugenol [271], Fig (9). Also derivatives of (1£)propenyl-4-hydroxybenzene have been isolated in some Pimpinella species. The activity in the contact assay of 100 ppm of eight Pimpinella phenylpropanoids against the red spider mite, T. telarius, has been evaluated [272]. Epoxy-anoltiglate was the most effective compound, killing 100% of the mites, while four other substances, epoxypseudoisoeugenolisobutyrate, epoxy-pseudoisoeugenoltiglate, pseudoisoeugenolisobutyrate and isoeugenolisobutyrate showed lesser effectiveness, killing 80-90% of the mites.

X) HoCO'

H3CO' Epoxy-anoltiglate

epoxy-pseudoisoeugenolisobutyrate

isoeugenolisobutyrate

^ O H3CO"

"^^

epoxy-pseudoisoeugenoltiglate

H3CO" pseudoisoeugenolisobutyrate

Fig. (9). Phenylpropanoids from Pimpinella species

The other phenylpropanoids tested, anoltiglate, isoeugenolisobutyrate and isoeugenol were completely ineffective. Other uncommon natural derivatives are 2-acylcyclohexane-l,3-diones or p-triketones, typical of hops {Humulus lupulus, Cannabinaceae). The p-acids from hop belong to this class of chemicals and are by-products of hop processing for brewing. The fraction containing these products has been examined in a choice bioassay for its effect on the feeding behavior

432

of T. urticae [273]. The results showed that both the highest concentrations of culupulone, Fig. (10), the chiefs-acid component of the fraction, and the whole p-acid fraction repelled T. urticae and also affected its survival. The greatest difference between the pure compound and the crude fraction treatments was seen in the oviposition of the mites: significantly fewer eggs were found in the whole p-acid fraction. This suggested that culupulone was not the only active component in the pacid fraction. Some secondary metabolites, epitaondiol diacetate, stypetriol triacetate, epitaondiol monoacetate and epitaondiol. Fig. (10), isolated from the brown alga Stypopodium flabelliforme (Dictyotaceae) have been tested on adults of T. urticae [274]. Only epitaondiol showed little acaricidal activity at 500 ppm (14% mortality), while the other compounds resulted inactive at 1000 ppm.

epitaondiol culupulone

ajoene stypetriol Fig. (10). Structure of hop and algal metabolites

Ajoene, Fig. (10), an unsaturated sulfoxide disulfide, is the principal chemical responsible for garlic's anticoagulant properties. It has been also investigated for its acaricidal activity on T. urticae [275]. Complete mortality (100%) by ajoene was observed at 0.075% after 14 h of treatment, a dose comparable with other synthetic acaricides used in the experiment. At lower concentrations (0.05%), it affected female fecundity

433

and only 31.5% of the juvenile stages. These results suggested that ajoene, besides having direct acaricidal effect, could also control resurgence of the pest. Glandular trichomes of wild tomato, Lycopersicon hirsutum f. glabratum (Solanaceae), yielded two methyl ketones: 2tridecanone and 2-undecanone [276]. They are known to cause mortality in several herbivorous insect species; it seems that this kind of chemicals could be considered defensive substances against pollen-feeding animals, as confirmed also by their abundance in wind-pollinated plants [277]. Dutch researchers investigated the effects of these compounds on two strains of T. urticae, collected from tomato and cucumber crops in greenhouses [276]. The two ketones were tested separately, in combination in the ratio found in L hirsutum f glabratum and in several other ratios to detect any synergistic interaction between them. Synergistic effects were not detected. They measured both the direct contact and residual toxicity, as well as the viability of the eggs produced by ketone-treated females. Both compounds showed LC50 values comparable to the formulated acaricide amitraz; 2-tridecanone was slightly more toxic than 2-undecanone, but only against the tomato strain. In the bioassays for the residual effects, no significant mortality occurred, however the mites avoided feeding on the treated surface and the eggs were laid almost exclusively on the untreated area. Furthermore, there was no significant egg viability for most of the treatments. A new plant species. Quassia sp. aff. bidwillii (Simaroubaceae) was discovered in Australia and the MeOH extract of its aerial parts was tested against T, urticae [278]. Because of its effectiveness, subsequent fractionation by RP-chromatography gave the pure active quassinoid derivative chapparinone, which showed a LC5o=47 ppm. Neem extracts, pure constituents (i.e. azadirachtin) and formulated products showed positive results against Tetranichus mites [279-283]. Less polar extracts were considerably more toxic than polar ones or coldpressed neem oil or commercial neem oil, and reduced the fecundity of the mites on treated plants and the survival of nymphs hatched from treated eggs; application of pentane extract or neem oil in sublethal concentrations, caused growth disrupting effects on the nymphal stages and ovicidal effects. Quantification of the insecticidal substance azadirachtin in the extracts revealed that this compound was not the most active principle against the mites [284].

434

Other promising control agents are microbial metabolites. Abamectin was found much less toxic to the useful predatory mite Phytoseiulus persimilis than to the parasite mite T, urticae [285]. A strain of the soil bacterium Streptomyces platensis yielded three new substances having a marked acaricidal activity against T. urticae, AB3217 A, B and C, Fig. (11) [286,287], while another strain of Streptomyces, NKl 1687, produced gualamycin. Fig. (11), that was able to kill 100% of dicofol-sensitive and resistant mites (adults and larvae) at 250 |ig/ml [288].

AB3217A:R=H O

^

^^

11

I

AB3217 B:R= —c—(CH2)4—C-(CH3)2

(f

AB3217C:R= —C—(CH2)2—CH-(CH3)2 ^

H N ^ ^

gualamycin

Fig. (11). Acaricidal agents of bacterial origin

Finally, as altematives to chemical compounds or as part of integrated pest management programs, predatory arthropods or fungal pathogens have been used. Among predators, the most used are phytoseiid mites such as Phytoseiulus persimilis and Neoseiulus fallacis [289-293], while the fungus Neozygites adjarica was tested as pathogen agent [294,295]. The acaricidal properties of some of the previous compounds were

435

attributed to the interference with mitochondrial electron transport due to inhibition of Complex III. Some structurally diverse miticides are able to inhibit Complex I (NADHiubiquinone oxidoreductase), a system distinct from Complex III. Complex I is the first electron transport complex of the mitochondrial respiratory chain. It oxidizes NADH and transfers the electrons via a flavin mononucleotide cofactor and several iron-sulfur clusters to ubiquinone (Q). So, Complex I contributes to the protonmotive force that drives ATP synthesis. Besides many synthetic products, some secondary products from microbial and plant sources exhibit biological activity against agricultural pests because of their action on Complex I. Rotenone and piericidin A, Fig. (12), were known for a long time as high-affinity inhibitors of proton-translocating NADHiQ oxidoreductase [296]. Rotenone is the most widely used inhibitor of Complex I because of its high inhibitory potency and commercial availability. It is the most potent member of the rotenoids, a family of isoflavonoids extracted from Fabaceae plants. All known natural rotenoids have been isolated in the thermodynamically stable cis-B/C ring fusion; synthetic analogues in which B and C-rings planes are almost coplanar were about 100-fold less active than natural rotenone, indicating that the bent form is essential for the activity [297]. This conclusion is supported by the observation that rotenol, in which the whole conformation is not fixed due to opening of the C-ring, is about 200-fold less active than rotenone. Another important feature for the activity is the configuration of the isopropenyl group linked to the E-ring. Cube resin, the roots extract of Lonchocarpus utilis and L. urucu (Fabaceae), is an important acaricide. The four principal active constituents are rotenone, rotenolone, deguelin and tephrosin. Fang and Casida identified further 25 minor rotenoids having variations in the B, D and E-rings, thereby providing a new and unique set of compounds to elucidate structureactivity relationships for the activity on Complex I [298]. The rotenone series and the deguelin series, with modifications in the B, C and D-rings, followed similar overall substituent effects on activity. In particular, the parent compounds rotenone and deguelin were more potent than any of their derivatives. Hydroxylation or methoxylation in the A-D ring system considerably reduced the potency. The trans isomers were 7-100 fold less active than the corresponding cis isomers. Authors affirmed that, considering the potency and the amounts, the four

436

major rotenoids accounted for more than 95% and probably almost all of the biological activity of the cube resin as inhibitor of Complex I. Many kinds of Streptomyces strains produce piericidin homologues. Piericidin A is a very potent inhibitor of Complex I. The natural side chain of piericidin A is not essential for the activity since piericidin B, C and D analogues, in which the region from C-5 to C-13 differs, exhibit activity as high as or only slightly less than piericidin A. On the basis of studies with synthetic analogues, it was concluded that a branched methyl group at C-3 and unsaturation between C-2 and C-3 are important for potent activity [297].

OCHo

OCH OCHo

OCHo

DeguelinR=H Tephrosin R=OH

Rotenone R=H Rotenolone R=OH

HoC

H3CO'

Piericidin A

HO*

Capsaicin

Fig. (12). Complex I inhibitors

Capsaicin, Fig. (12), the pungent principle of Capsicum species

437

(Solanaceae), acts as competitive inhibitor for ubiquinone in Complex I. Methyl capsaicin is more potent than capsaicin, indicating that the phenolic OH is not essential for the activity [297]. Other natural inhibitors of Complex I are annonaceous acetogenins. These compounds belong to a wide group of natural products isolated from several species of the Annonaceae family, which include more than 250 molecules with diverse chemical structures. Among the various classes, it seems that monotetrahydrofuranic derivatives are less potent than other acetogenins [296,299]. CONCLUSIONS The control of parasitic diseases is mainly based on the use of effective drugs, both in agriculture or human and veterinary medicine; for this reason the lack of effective drugs often prevents the control of some parasitic diseases, making them more serious and important. At present, however, the use of commercial drugs involves many problems that strongly limit their use: foremost the drug-resistance problem shown by the most important parasites, the environmental damage and the toxicity of many synthetic drugs. In addition, drug residues in plant and animal food products are important reasons of considerable economic losses for farmers. The European Community's recent law (EC n. 1804/99) regarding biological animal farming, limits the use of synthetic drugs, while the use of homeopathic remedies and phytotherapies is allowed. All these problems are stimulating the search for new and alternative control methods, including the search of effective compounds characterized by smaller environmental impacts in terms of residues and toxicity. Since plant-derived compounds are generally more easily degradable and could show a reduced environmental damage with respect to synthetic drugs, at present the evaluation of the antiparasite activity of plant extracts is being increasingly investigated, as demonstrated by the recent studies that have evaluated and confirmed the effectiveness of many plant compounds on bacteria, fungi, protozoa, helmints and arthropods. Much of present day antiparasite chemotherapy is derived from practices and advances made in the 19-20th Centuries, during which the antiparasite activity of some plants has been scientifically confirmed. Nowadays higher plants are still important sources of new active principles, among which the antimalarial compound artemisinin is one of the most recently introduced.

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Even so, the pharmacological control of some parasitic diseases is still very difficult; among them we can find arthropod-related diseases. Perhaps human and veterinary medicine are the most suitable fields for a real application of natural drugs, in fact the treatment of these pathologies is mostly topical, and particular drug-formulations are not required. Furthermore, generally only a few treatments are necessary to kill all the parasites. In agriculture, in spite of the studies performed to date, these substances are perhaps still far from their effective use: their main usefiil feature, that is their biodegradability, is also their weakness. Often, many products are not able to persist in the environment for a period of time sufficient for pests control. Further studies are necessary to prepare better formulations that allow us to solve this problem. Other important future research topics should concentrate on the evaluation of the toxicity of these compounds, an unknovm feature for many natural compounds. REFERENCES [I]

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.

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PODOLACTONES: A GROUP OF BIOLOGICALLY ACTIVE NORDITERPENOIDS ALEJANDRO F. BARRERO, JOSE F. QUILEZ DEL MORAL and M. MARHERRADOR Department of Organic Chemistry, Institute ofBiotechnology, University of Granada, Avda. Fuentenueva, 18071, Granada, Spain ABSTRACT: More than seventy podolactones have been isolated mainly from Podocarpus species. Some of these structures have also been found in filamentous fungi. The different biosynthetic origin of these molecules considering their vegetal or fungic source is discussed in this review. These molecules present a wide range of biological activities: anti-tumor activity, anti-inflammatory activity, fungicide activity, insecticidal activity and plant growth regulatory activity. These biological properties are analyzed in detail, and in the light of their results, structure/activity relationships are discussed. Finally, chemical reactivity, including interconversion reactions, and synthetic approaches to these compounds are summarized.

INTRODUCTION Podolactones are considered to be a group of natural products whose basic skeleton contains a y-lactone between carbons 19-6 and a 6-lactone between carbons 12-14, which are their characteristic functions Fig. (1) [1]. The numbering of the podolactone skeleton has been assigned on the basis of the totarane skeleton from which most of podolactones have been proposed to be derived.

Fig.{l)

The podolactones with a nor- or bisnorditerpenoid structure are mainly found in different species of the Podocarpus plant type [2-3]. Apart from

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the Podocarpus species, five podolactones have also been isolated from a New Zealand mistletoe, Ileostylus micranthus, which was parasiting P. totara, from which it has been suggested that podolactones were assimilated [4]. A small number of tetranorditerpenoid dilactones isolated from the filamentous fungi Oidiodendron truncatum [5-6], O. griseum [7], Aspergillus wentii [8] and a non-identified species of the Acrostalagmus genus [9] have also been considered as fomiing a part of this group. These natural substances are of great interest due to both their unusual structures and the potent wide-ranging spectrum of biological activities that they possess: antitumor activity [10] in vitro and/or in vivo, antiinflammatory activity [7], fungicidal activity [11], herbivorous manmialian antifeedant activity [12], insecticide activity against house-fly larvae and other insects [13] and, lastly, a potent plant growth regulatory activity, both as inhibitors and as stimulants [14-15]. BIOSYNTHETIC ORIGIN All natural podolactones have been isolated from two types of natural sources, plants related to the genus Podocarpus, from which the majority of podolactones have been described, and the filamentous fungi. This different natural source also possesses a different biosynthetic origin. The first noticeable evidence supporting this difference is that, while podolactones isolated from plants are nor- orfcwnorditerpenoids(i.e. nagilactone C and nagilactone E), the dilactones isolated from fungi have lost four carbons from a diterpene precursor (i.e. oidilactone C).

Nagilactone C

Nagilactone E

Oidiolactone 0

Appearance of podolactones in the extracts of Podocarpus species along with various derivatives with a totarane skeleton (totarol, 12-

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hydroxytotarol, 19-hydroxytotarol, totaral and 4p-carboxy-19-nortotarol) [16] led the Hayashi's team to postulate the following biosynthetic scheme for these substances (Scheme 1)* The pathway starts with 12hydroxytotarol, which suffers a meta-pyrochatecase-type fission to give a hydroxy-acid, intemiediate which decarbonylates leading to the corresponding a-pyrone, transformation already described in other catechols [17],

Scheme 1. Proposed biogenetic pathway for podoactones isolated from plants.

On the other hand, the C-16 terpenoid dilactones isolated from fiingi have been postulated to have a biosynthetic origin different from that reported for podolactones from plants. Two possible biosynthetic ways were envisaged, one supposing an oxidative loss of four carbons from a diterpene precursor, and secondly, the addition of a C-1 unit to a sesquiterpenoid [18]. The results obtained by administering isotopically labelled acetic and mevalonic acids to an Acrostalamus fungi, together with the isolation from this same fungus of acrostalidic acid, acrostalic acid and isoacrostalidic acid let us to conclude an attribution of a diterpenoid origin for these lactones with the following biogenetic pathway proposal[19] (Scheme 2).

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.CO2H

-^

CO2H

*

ciS' and frans-communic acid

COgH

CO2H

aerostatic acid

CO2H

'OMe

0

^

"" CO2H III

acrostaiidic acid

^^ 'CO2H isoacrostalidic acid

Scheme 2. Proposed biogenetic pathway for podoactones isolated from fungi.

The route could start from the mixture of cis- and rrans-coimnunic acids (or also from other diterpenes, such as isocupresic acid). The isolation of hydroxyacid I as a natural product from Acrostalagmus reinforces the possiblity of the existence of diene II as a key precursor, not only of isoacrostalidic acid and acrostaiidic acid, but also of dilactone III. The straightforward chemical conversion of I into III in a recent publication by Barrero et al. [20] supports this hypothesis.

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STRUCTURES OF NATURAL PODOLACTONES Natural podolactones can be classified into three major stractural types depending on the nature of the conjugated lactone system in the B/C ring moiety [21], Fig. (2). Type A: a-pirone [8(14),9(ll)-dienolide], Type B: 7a,8a-epoxy-9(ll)-enolide, Type C: 7,9(1 l)-dienolide.

Type A

TypeB

TypeC

Fig. (2).

Below we can see all the podolactones described up to date, distributed according to the aforementioned classification with an indication of the species from which they were identifed and their bibliographic reference. First, the podolactones isolated from Podocarpus species are presented and, later, the podolactones found in fungi will be listed.

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Podolactones from plants Type A

nagilactone A 1 P. nagi, P. macrophyllus P. philippinensis, P. polystachyus [M, 22-22]

0

nagilactone B 2

P.nagi[\7\ 0

nagilactone C 3 P. nagi, P. nivalis, P. halii, P. macrophyllus, P. purdeanus, lleostylus micranthus [4,17,25-27] O

inumakilactone E 6 P. macrophyllus, P. polystachyus [24, 26] O

C02Me

hallactone A

7 P. ham [30]

1 -deoxy-2a-hydroxynagiiactone A 8 P./lag/[31]

15-methoxycarbonylnagilactone D

9 P. nag/ [31]

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10 P. nagi [32]

O 1-deoxy-2p,3pepoxynagilactone A 13 P. nagi [33]

15-hydroxynagitactone D

3p-hydroxynagiiactone A 11 P. nagi [32]

urbalactone 14 P. urbanii [34]

12 P. nag/ [32]

O

R=p-D-glc nagilactoside A 15 P. nagi [35]

O

3-deoxynagilactone C 16 lleostylus micranthus [4]

3-ephnagilactone C 17

P. na^/ [36]

1-deoxynagiiactone A 18 P. nag/[16]

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2,3-dehydro1-deoxynagilactone A

2,3-dehydronagilactone A

20 P. na^/[16]

19

P. nag/[16]

R=p-D-glc nagilactoside B 21 P. nagi [37]

R*,

epi-sellowin C 22 P. nagi [37]

R=p-D-glc-(1-^6)p-D-glc-nagilactoside E 25 P. nagi [38]

R=p-D-glc-(1 -•Sj-p-D-glcnagilactoside C 23 P. nagi [38]

R=p-D-glc-(1-*-3)p-D-glc-(1-^ 6)-p-D-glcnagilactoside F 26 P. nagi [39]

R=p-D-glc-(1^^6)p-D-glc-nagilactoside D 24 P. nagi [38]

R=:p-D-glc-{1-#-6)p-D-glc-(1-^ 3)-p-D-glcnagilactoside G

27 P. nag/ [39]

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TypeB

OH

0 inumakilactone A 28 P. macrophyllus, P. philippinensis [40-41,22] O

O

podolactone A 29 P. neriifolius [42-43]

O podolactone B 30 P. neriifolius [42]

SOMe

0

inumakilactone B 31 P. polystachyus, P. macrophyllus P. neriifolius [23,44-45]

^

O

podolactone C 32 P. neriifolius P. milanjianus [45-48] O

podolactone D 33 P. neriifolius [45-47] 0

'%^

O

sellowin B 35 P. sellowii [29,47]

nagilactone E 36 P. nap/ [49]

462

SOaMe

::o I

0Glu(0H)4

inumakiiactone A giucoside 37 P. macrophylus P, philippinensis [40-41,22]

O

nagilactone Q 40 P. sellowii P. milanjianus [50]

2p,3p-epoxypodolide 43 P. nagi [52]

o;

hallactone B 38 P. hallii, P. sellowii P. polystachyus [24,30,47]

podolide

39 P. gracilor [9]

O

16-hydroxypodollde (salignone H) 41 P. saligna [51]

milanjilactone A 44 P. milanjianus [53]

O

2,3-dihydro16-hydroxypodolide 42 P. nagi [52]

salignone I 45 P. saligna [54]

463

K^

O 3-deoxy-2a-hydroxynagiiactone E 46

O

salignone M 47 P. saligna [55]

P. nagi lleostylus micranthus [4,36]

16-hydroxynagiiactone E 48

P. nagi [56]

TypeC

Gluc-0'

O

ponaiactone A

49

P. na/fa// [57]

-O d ' ip-hydroxynagilactone F 52 P. nagi

O ponaiactone A glucoside 50 P. r?a/fa// [57]

nubilactone A 53 P. nubigena [58]

podolactone E 51 lleostylus micranthus P. neriifolius [4,45]

O

3p-hydroxynagilactone F

54 P. naflf/ [33]

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nagilactone F 55 P. nagi, P. milanjianus, P. sellowii P. macrophilus [49-50,25]

2,3-dehydro-16-hldroxinagilactone F

miianjilactone B 56

57 P. nagi [56]

P. milanjianus [53]

Ha,

nagilactone I 58 P. nagi [66]

2a-hydroxynagilactone F

59 P. nagi lleostylus micranthus [4,36]

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Podolactones from fungi

-O PR 1388 60 Oidiodendron truncatum Oidiodendron griseum [6,7]

0^ Oidiolactone C (Oidiodendroiide C) 61 Oidiodendron truncatum Oidiodendron griseum [7,11] O

oidiolactone D (oidiodendroiide A) 62 Oidiodendron truncatum Oidiodendron griseum [7,11]

'''OMe

O

LL-21271a 0 LL-Z1271Y 64 63 Acrostalagmus Acrostalagmus Oidiodendron griseum [7,9,59] Oidiodendron griseum [7,9,59]

wentilactone A 65 Aspergillus wentii [8]

0

O wentilactone B 66 Aspergillus wentii [8,20]

oidiodendroiide B 67 Oidiodendron truncatum [11]

Oidiodendron griseum [7]

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Miscellaneous

OH '''OH

HO'

inumakilactone C 69 P. macrophyllus [44]

inumakilactone D 70 P. macrophyllus [60]

saljgnone A 71 P.sa//flfna[51,61]

OH

"'^^x^OH H / —0

C02Me

0^

V ^ ^ x * \ ^

COaMe

saiignone J 73 P. sa//flfna [61]

saiignone B 72 P. saligna [61]

saiignone K 74 P. saligna [55]

C02Me

saiignone L 75 P. sa//p/7a [55]

nagilactone J 76 P. /lagf/ [62]

O dlhydrodeoxynubilactone A 77 P. saligna [63]

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BIOLOGICAL ACTIVITY OF PODOLACTONES Anti-tumoral Activity. a) Yoshida Sarcoma. The in vitro bioactivitivity of 29 podolactones, 15 of them natural products, against cultured Yoshida Sarcoma cells [64-65] was investigated by Hayashi's group during the period between 1975 and 1979.

H2O3PO'

468

Table 1 summarizes the obtained results. Podolactones were grouped on the basis of the previously reported structural subgroups in which these natural compounds had been classified. Table 1. Citotoxidty of natural and synthetic podolactones against Yoshida Sarcoma Type A Lactones Nagilactone A (1) Nagilaaone B (2) Nagilactone C (3) Nagilactone D (4) 1 -deoxy-2a-hydroxynagilactone A(8) 3p-hydroxynagilactone A (11) 15-methoxycarbonyl nagilactone D (9) 10 78 79 80 81

TypeB Lactones ICso (x 10-^ M) 3l0 17.2 22.5 3.32 16.4 487.0 21.5 305.0 1460.0 1000.0 138.0 119.0

Nagilactone E (36) NagUactone G (40) 82 23-ciihydro-16hydroxypodolide (42) and 16-hydroxy podolide (41) Inumakilactone A( 28) Inumakilactone B (31) 83 84 85 86 87 88

TypeC ICso (xlO^M) 336 1.48 3.72 14.8 10.4 4.11 18.3 87.0 607.0 4,19 20.6 110.0

Lactones Nagilactone F (55) 89 90 91

ICso (XIQ-^ M)

o

12.2 16.1 18.9

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Synthetic derivatives 92 and 93, which can not be not included in any of the three A-C subgroups, turned out to be inactive against this cell line.

On the basis of the reported data, the following structure-activity relationships were proposed: i) The dilactones with few or no polar substituents showed strong activity, a factor that could be related to the permeability of these substances through the cell membrane. Thus, the most active compounds (ICso -- 0.015 jig/mL), nagilactone F (55) and 7,8-epoxy-nagilactone F (91), do not have any hydroxyl group. The monohydroxylated dilactones nagilactone D (4), inumakilactone B (29) and nagilactone E (34) are slightly less active, but still maintain a substantial activity (IC5o=0.11-0.14 M-g/mL), while the dihydroxylated lactones nagilactone A (1), nagilactone C (3) and inumakilactone A (26), and the trihydroxylated 10 and 3p-hydroxynagilactone A (11) are, respectively, 10 and 100 times less active than the non-hydroxylated ones. ii) The dienic system conjugated with the y-lactone is one of the most important functional groups for anti-tumor activity. ///) The y-lactone group in positions 4p and 6P is important for the activity, but not essential. iv) The acetylation of hydroxyl groups reduces the activity by 10-50 times, probably due to steric effects. v) The 7,8-epoxy group of the type-B dilactone is crucial, since the hydrogenolysis product of the epoxide ring is completely inactive. vi) Activity decreases when the configuration of the substituent is changed on Ci? from a to p. vii) Oxidation of the hydroxyl groups to ketones or the introduction of a p epoxide in the A ring do not affect activity, but the introduction of an a epoxide in position 2,3 reduces activity up to ten times.

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b) P-388 Mouse Linfome Podolide was the first dilactone reported to have antitumor activity in vivo against P-388 leukemia in mice and citotoxycity in vitro towards cells derived from P-388 murine leukemia [9]. More detailed reports on the antileukemic activity of nagilactone C (3) and nagilactone E (36) was given by Hayashi in 1975 [64]. Both lactones were effective with a dose of 20 mg/kg/day (T/C 125%). Podolactone C also showed antineoplasic activity against P 388 cells in vivo at 20 mg/kg/day (151% T/C) [48]. Finally, Bloor and Molly reported the cytotoxic activity of five podolactones isolated from a New Zealand mistletoe. The more active lactones were 3-deoxy-2a-hydroxynagilactone E (46), 2ahydroxynagilactone F (59) and podolactone E (51) showing IC50 (jxg/mL) values of 0.06, 0.06 and ) on distinct cell typesfrominnate immunity (monocytes/macrophages, NK cells, PMNs, dendritic cells) and adaptive immunity (lymphocytes). • ) other cells or directly the tumor, These cells produce cytokines or NO ( C 3 ) which target ( resulting in toxicity ( • « ^ ) . Lipid A can also inhibit angiogenesis, bloodflowin the tumor and the secretion of immunosuppressive TGF-p by the tumor cells.

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Treatments in animal models LPS and lipids A treatments

LPS treatments In animals, the first in vivo experiments were performed with bacterial extracts in a guinea pig sarcoma model by Gratia and Linz [88], and with LPS in mouse primary subcutaneous tumors by Shear and Turner [4]. The antitumoral effect of LPS on the growth of subcutaneous or intramuscular tumors has been extensively investigated [61,147-153]. On ascitic tumors, treatment with LPS was shown to be efficient in some cases [153-155] while failing in others [61,156]. We tested the effect of LPS in a model of peritoneal carcinomatosis (solid tumor) induced by PROb colon cancer cells in syngeneic BDIX rats. We showed that i.p. injections of LPS from E. coli can cure 20 % of the rats [157]. Comparing the effect of LPS from different strains in this model, we found that the efficacy depends on the bacterial strain and on the structure of the lipid A used. Whatever the lipid A used, we have shown a correlation with in vitro macrophage secretion of IL-ip but not with NO, TNF-a or IL-6 [83]. Lipids A treatments Here, we will emphasize on treatments with lipid A, considering only curative treatments beginning after tumor cell injection. After a review of the literature, we will detail the effects and mechanisms of DT-5461, ONO-4007 and OM-174, the three lipids A which have been mostly documented. Parr et al. [61] showed that lipid A has the same antitumoral effect as whole endotoxin preparations on murine L5178Y lymphoma. The effects of LPS and synthetic lipid A treatments were compared by Shimizu et al. [158-161] on Meth A fibrosarcoma in BALB/c mouse. The antitumoral activity of different lipids A has also been investigated. Ribi et al. [162] used an extract from S, typhimurium containing lipid A, which when injected directly into hepatocarcinoma line 10 tumors in guinea pigs shows an antitumoral effect. This activity is attributed to a monophosphoryl diglucosamine derivative of lipid A [163]. Synthetic lipid A analogs also proved to be active in this system [164], as well as

534

when injected i.p. in Meth A fibrosarcoma-bearing BALB/c mice presensitized with Propionibacterium acnes. The i.v. injection of the monoglucosamine GLA-27 slows the growth rate of RL-1 lymphoma and Meth-A sarcoma in BALB/c mice [165]. Shimizu et al. [160] compared several lipid A analogs with regards to their antitumoral activity using Meth A tumors in BALB/c mice. Antitumoral activity is not correlated with mitogenicity of C3H/He mice splenocytes and NO production in Swiss mice macrophages, but is correlated with macrophage TNF-a production. A synthetic lipid A was shovm to inhibit the growth of tumors induced in nude mice by the injection of ML\ paca-2 or Panc-1 human pancreatic tumor cells, likely through TNF-a secretion by macrophages [134]. Association of lipids A with other immunomodulators Treatments with lipids A were tested in association with diverse immunomodulators. Intravenous injections of GLA-60 in association with IFN-Y were found to reduce B16 melanoma lung metastases in C57BL/6 mice [114]. DT-5461 injected i.v. in association with indomethacin increases the survival of BALB/c mice bearing peritoneal, liver and lung C26 colon carcinoma [166] through the inhibition of angiogenesis. MDP (muramyl dipeptide), a Mycobacteria derivative, potentiates the antitumoral effect on Meth A fibrosarcoma in BALB/c mice, of several lipids A (A-171, A-172, 56, A-606, A-607, A-608) injected i.v., but the associations were less efficient than LPS alone [160,167]. MDP also increases the efficacy of DT 5461 in the same model [158]. The increase was correlated with an in vitro mitogenic effect of DT 5461 on spleen cells, as well as the production of NO and TNF-a by macrophages. In 1996, the same team tested several lipid A analogues, finding that the association with MDP showed no better efficacy than LPS on Meth A sarcoma in BALB/c mice. In these mice, cyclophosphamide injected 7 days prior an ONO-4007 treatment in order to inhibit the immunosuppressive response, enhanced the efficacy of the lipid A [108]. Treatment with the lipid A DT 5461 The synthetic diglucosamine compound DT-5461 has been reported to reduce the weight of various tumors including murine Meth A

535

fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis Lung carcinoma, and C38 colon carcinoma, but not that of C26 colon carcinoma [168] or L5178Y lymphoma [169]. The tumor size was reduced by a necrotic process. DT-5461 i.v. injections induced TNF-a secretion in subcutaneous Meth A tumors in BALB/c mice [112] as well as in B16-BL6 tumors in C57BL/6 mice, decreased angiogenesis, and reduced the number of spontaneous metastases [94]. The effect of DT5461 was accompanied by a reduced blood flow in the tumor which could be reversed by antisera directed against TNF-a, IFN-oc/|3, and IFNY [105,112]. No effect on the life span of animals bearing ascitic tumors was observed. The antitumoral effect of i.v. injections of DT-5461 in a rabbit hepatic carcinoma [113] was associated with blood flow reduction in the tumor area. In vitro, it was shown that in the murine macrophage cell line J774, DT-5461 enhanced cytokine production using LPS receptors [30], and that in the murine macrophage cell line RAW 264, signal transduction involved phosphorylation of MAP kinases [170]. Treatment with the lipid A ONO-4007 The monoglucosamine compound ONO-4007, injected i.v. slowed the growth of subcutaneous MM46 murine mammary carcinoma in C3H/He mice, increasing TNF-a secretion by intratumoral macrophages [80]. It induced TNF-a production by spleen cells from BALB/c mice bearing intradermic murine Meth-A fibrosarcoma, as well as their proliferation in vitro [135]. Intravenous injections increased the survival of WKAH rats bearing KDH-8 hepatocarcinoma subcutaneous tumors, but had no effect on rats bearing KMT-17 fibrosarcoma, or SST-2 mammary adenocarcinoma [171]. In WKAH rats, ONO-4007 acted by inducing the production of TNF-a [99], as was confirmed in C3H/He mice bearing MM46 mammary carcinoma or MH134 hepatoma [144]. The efficacy of this lipid A may be limited to TNF-a-sensitive tumors in WKAH rats [172]. While 3 i.v. injections every 7 days inhibited TNF-a production in liver and blood, no tolerization was found in tumors [99]. A similar effect was seen in hamsters bearing pancreatic carcinoma [173]. The prolongation of survival of WKAH rats bearing c-WRT-7 myelomonocytic leukemia by i.v. injections of ONO-4007 can be explained by a differentiating effect [174] that could not be reproduced

536

by a cytokine (BL-la, IL-6 or TNF-a) treatment. Recently, Mizushima et al. [175] showed that subcutaneous 13762NF mammary tumors, but not peritoneal or lung tumors, were cured by i.v. injections of ONO-4007 in F-344 rats. The efficacy of ONO-4007 was enhanced in mice bearing Meth A fibrosarcoma when cyclophosphamide was injected 7 days prior treatment in order to inhibit an immunosuppressive response [106]. Subcutaneous injections of ONO-4007 increased vascular permeability in the skin of normal mice by increasing the production of TNF-a and ILip [176]. In the same conditions, i.v. injections induced NO production in the lungs [177] but these effects were not studied in the context of tumor growth. Treatment with the lipid A OM-174 We investigated the antitumoral activity of OM-174 in a model of peritoneal carcinomatosis induced by PROb colon cancer cells in syngeneic BDIX rats. These cells are chemoresistant [178], NK-resistant [179] and TNF-a-resistant [81]. Without treatment, all rats die of their tumors. Treatment of peritoneal carcinomatosis (solid tumors) always started after the formation of macroscopic nodules up to 3 mm. The cumulative volume of these numerous nodules corresponds to the volume of a large tumor. An equivalent stage of tumors in himians cannot be resected and have always a fatal evolution. OM-174, which is a triacylated diglucosamine, has a partial structure ofE, coli lipid A [180]. Repeated i.v. injections of OM-174, every 2 days, cured 90 - 100 % of the rats. Such a success has never been obtained with a treatment of this kind. Tumor disappeared via the apoptotic pathway without an inflammatory reaction. OM-174 is not toxic to tumor cells in vitro^ therefore it does not induce tumor cell apoptosis directly. The establishment of a specific immune response was evidenced with a Winn-type assay, e.g. the protection of naive rats against a tumor by the injection of spleen cell from rats cured of the same tumor. Treatment efficacy depended on the number and frequency of injections which indeed induced the tolerance of macrophages to OM-174 decreasing their TNF-a production [81]. After a peak following the 2 first injections, TNF-a in tumors returned to basal levels. Moreover, PROb cells are resistant to TNF-a in vitro, in consequence in our model, TNF-a is probably not involved in tumor regression. On the contrary, the efficacy

537

of both DT-5461 in CDFl mice [169], and ONO-4007 in BALB/c mice [99], depended on TNF-a. In our model, during lipid A-induced txmior regression, NOS II mRNA and protein levels were induced in tumor cells with the concomitant production of NO [104]. Neither OM-174 nor TNFa induced NO production by tumor cells in vitro, whereas NOSII expression is induced by IFN-y and IL-ip in these cells [122]. Therefore, the in vivo NOS II induction may be indirectly due to the presence of IFN-y and IL-ip in the tumors of treated animals [96]. Accordingly, we determined that the treatment with OM-174 causes IFN-y and IL-ip accumulation in tumors, at the mRNA and proteins levels (unpublished results). The NO thus produced is autotoxic for tumor cells provoking their apoptosis. Moreover, treatment with OM-174 inhibited the synthesis of TGF-pi by PROb tumor cells [107], therefore abrogating its immunosupressive role [181]. Furthermore the inhibition of TGF-pl enhanced the synthesis of NOSII [107], thus increasing the autotoxic effect of NO on tumor cells. Therapeutic vaccination

Lipids A are also used in therapeutic cancer vaccination to cure tumors. In this case, lipids A are used as adjuvants, e.g. administered simultaneously with tumor extracts or tumor antigens, to increase the immunogenicity of the vaccine or to inhibit the tumor-induced tolerance. Therapeutic vaccines were tested in BALB/c mice bearing TA3-Ha mammary carcinoma. The treatment consisted of 4 subcutaneous injections, at 3-6 days intervals, of Detox [a commercial preparation of cell wall skeletons from Mycobacterium phlei and non-toxic monophosphoryl lipid A from Salmonella minnesota (S. minnesota) in squalane oil and Tween 80 from Ribi Immunochemical research, Montana, USA] mixed with Thomsen-Friedenreich (TF) antigen coupled with KLH (Keyhole Limpet Hemocyanin) performed 5 days after the tumor cell injection. This vaccination achieved the survival of 25 % of the mice. Pretreatment of mice with cyclophosphamide in order to inhibit any suppressive response, increased survival to 50 % when the treatment began 5 days after tumor cell injection, and to 90 % when the treatment began 2 days after tumor cell injection. Both antibody as well as delayed-

538

type hypersensivity (DTH) responses were obtained. Moreover, lymph node cells were protective in a Winn-type assay [182], In C3H/HeN mice bearing MH134 hepatoma, monophosphoryl lipids A from P. gingivalis or S, minnesota Re 595 increased the survival of mice when administered in combination with timior cell lysates and Freund*s incomplete adjuvant [31]. Conciusion In conclusion, various lipid A have been tested as treatments for tumorbearing animals, using different routes (intratumoral, intraperitoneal, intravenous, intradermic). While the intradermic route permits the use of greater doses without toxicity [76,77], most of the studies were performed using several i.v. injections of lipid A. Optimum doses range from 1 to 5 mg/kg for the 3 lipids A DT 5461, ONO 4007 and OM-174 in rats and mice. In our model of colon carcinoma in rats, we showed that the i.v. treatment is more efficient than an intraperitoneal one [81]. In general, most studies showed that the treatments increase survival, or slow the growth of established tumors in mice [80,93,94,99,105,112,135,144,160,168], rats [12,81,96,107,171174,181], and rabbits [113]. To our knowledge, only two laboratories reported the total cure of established tumors. Mizushima et al. [175] showed that ONO-4007 cures subcutaneous tumors but not intraperitoneal or lung ones. Onier et al. [81] reported that OM-174 cures 90-100 % of rats bearing peritoneal carcinomatosis consisting of a large number of nodules between 1 and 3 mm, while all untreated rats die of their cancer. Lipids A are generally considered to act through TNF-a secretion [112]. For instance ONO-4007 was shown to be efficient only on TNF-asensitive tumors [171,172], therefore, only well vascularized timiors can be affected. However, in our model, we showed that TNF-a is probably not involved since this cytokine peakes after the first two injections and then retums to basal levels before tumor regression. The efficacy of OM174 relies on an indirect induction of an autotoxic production of NO by the tumor cells [104]. Perhaps a more important aspect is the immunogenicity of tumor cells. After apoptosis or necrosis of tumor

539

cells, apoptotic bodies or debris can be phagocytosed by macrophages and dendritic cells. These cells will then present the immunogenic peptides to helper T lymphocytes which will induce a specific immune response. CLINICAL STUDIES The aim of phase I trials is to determine the toxicity of potential drugs. The following phase II trials are designed to study the pharmacological properties and the potential effectiveness of a drug. The aim of a phase III trial is to study the efficacy and safety of a particular protocol. Treatments with LPS Several phase I trials have been performed with LPS from Salmonella abortus equi administered i.v. in patients who suffered from disseminated cancer. White blood cell number decreased after each injection and retumed to basal level by 24 hours. There were no changes in coagulation parameters, and no disseminated intravascular coagulation was observed. After the first injection of LPS, increases in TNF-a concentration and IL-6 activity in serum were detected. However LPS tolerance which is accompanied by a decrease in TNF-a and IL-6 production depended on the intervals between repeated injections, but it was not determined whether it was a benefit or a draw-back. Injections of IFN-y prevented this decrease in TNF-a and IL-6, and ibuprofen attenuated LPS toxicity [183,186]. In a phase II trial, patients with colorectal cancer or non-small cell lung cancer, LPS showed a low grade toxicity and induced a reduction of TNF-a concentration in serum. One complete remission, stable for 36 months, was achieved [187]. In another phase I trial, i.d. injections of LPS from Pantoea agglomerans were given to patients who suffered from disseminated cancer and received cyclophosphamide, and ibuprofen, to attenuate fever. Increases in the serum concentrations of TNF-a, IL-6 and G-CSF were observed, without tolerance [188].

540

The low response of cancer patients to LPS treatments may be due to low maximal tolerated dose (MTD), 4 ng/kg. In order to avoid this problem, several trials were performed with lipids A. Treatments with lipids A A phase I trial was conducted with monophosphoryl lipid A (MPLA) prepared from S, typhimurium or S, minnesota injected i.v. in patients with disseminated cancer. Fever, chills andfetiguewae Ihe most OMnmoti side eflFects and the dose of 250 \x^jr^ i.e. (250x1.7): 65=6 jugl^ was estimated acceptable [189]. Another trial used i.v. injections of the synthetic lipid A SDZ MRL 953 in patients with disseminated cancer who received ibuprofen. The most common toxicity was fever, and the MTD was not reached. The lipid A had no significant effect on the serum concentrations of TNF-a, EL-ip, IL-8, G-CSF and IL-6. White cell number increased within 12 hours after the first injection, mainly due to PMNs, and then retumed to normal after 48 hours [37]. In a recent phase I trial the synthetic lipid A analog ONO-4007 was given by i.v. injections to patients with cancer unresponsive to the standard therapy. The limited systemic toxicity disappeared within 24 hours. The MTD was defined as 125 mg/patient [e.g. (125:65=2 mg/kg]. The lipid A increased serum concentrations of TNF-a and IL-6, without affecting the concentrations of GM-CSF, IFN-y and neopterin. There was a significant drop in lymphocyte counts after injections, but no effect on clotting parameters [190]. The results of phase I trials of LPS and lipids A in cancer patients show that the tested lipids A are approximately 30,000 to 500,000 fold less toxic than the LPS (table 1). The MTD in humans ranging from 6 |Lig/kg to 2 mg/kg, is lower than or close to the optimal doses of lipids A observed in rodents. Humans are more sensitive to lipid A than rodents, therefore it is possible that similarly to the toxic dose, the effective dose for humans is lower than for rodents. To this day the very few existing phase n trials cannot answer this question. These trials are summed up in Table 1.

541

Table 1. List of tfie clinical tiiab performed with LPS or lipids A injected to cancer patients.

Phase Vosika et al. Cancer Immunol Immunother 1984 Engelhardt et al JBiolRespModif 1990 1 Engelhardt et al. Cancer Res 1991 1 Mackensen et al. Blood 1991 1 Mackensen et al. Eur Cytokine Netw 1992 Ottoetal. Eur J Cancer 1996 1 Goto et al. Cancer Immunol Immunother 1996 1 Kiani et al. Blood 1997 1 DeBonoetal, Clin Cancer Res

1

2000

Product

Tumor

Route

MTD

Tested 1 Parameters CC

I

Lipid A Diverse (Salmonella)

i.v.

250 jig/m^ 6^g/kg

I

LPS (Salmonella)

Diverse

i.v.

4ng/kg

CC, CK

Diverse LPS (Salmonella)

i.v.

8ng/mg

CC, CK

LPS (Salmonella)

Diverse

i.v.

CK

LPS Diverse (Salmonella)

i.v.

CC,CK

CC, CK

I

II

LPS (Salmonella)

Diverse

i.v.

I

LPS (Pantoea)

Diverse

s.c.

I

Diverse Lipid A SDZMRL 953 1 Diverse Lipid A ONO-4007

I.v.

>1800ng/kg

CK

> 39.6 ng/kg

CC, CK

125 mg/patient 2mg/kg

1

CK

'

CC = cell count, CK = cytokines, i.v. = intravenously, s.c. = subcutaneously, MTD = maximum tolerated dose

Therapeutic vaccines Several trials of therapeutic vaccination used the adjuvant property of lipid A to enhance the vaccination efficacy against human tumors, which are often considered as weakly immunogenic, or even tolerogenic. The first trials of therapeutic vaccination against cancer using lipid A as adjuvant were performed on melanoma patients with 0.25 ml Detox. The composition of commercial vaccines used as therapeutic vaccines in humans is given in Table 2. Some trials used a pretreatment vsdth 300

542

mg/m of cyclophosphamide to inhibit an eventual suppressive response to the vaccine. Table 2 . Composition of commercial vaccines used as therapeutic vaccines in cancer patients.

DETOX (Ribi Immunochem Research, Inc., Hamilton, Montana, USA) 0.25 ml: 250 mg cell wall skeleton from Mycobacterium phlei, 25 mg monophosphoiyl lipid A from Salmonella minnesota R595 prepared as an oil-in-water emulsion with 2% squalane and 0.4% Tween 80 in 2X noraaal saline. Melacine (Ribi Immunochem Research, Inc., Hamilton, Montana, USA) Homogenates of melanoma tumor cell lines mixed with Detox. THERATOPE sTn-KLH (Biomira Inc., Edmonton,AB, Canada) 100 mg sTn-KLH emulsified in 0.25 ml Detox. (sTn = sialyl-Tn = DAcNeu2-6aGalNAc-0-Ser/Thr). OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA, USA) 1.2 ml: 100 mg/ml recombinant PSA + liposomes of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol + 200 mg /ml monophosphoiyl lipid A from Salmonella minnesota R595.

Melacine

The first phase I trial, performed by Mitchell et al. [191] and several further phase I and II trials by the same group, studying different parameters [192,193] are summed up by Mitchell et al. [194]. Homogenates of 2 melanoma cell lines, mixed with Detox were injected s.c. to melanoma patients. Toxicity was minimal and local. There was no correlation between the antibody response against melanoma determinants and the clinical response. The remissions were correlated with the presence of cytotoxic T lymphocyte (CTL) precursors. Most of the CD8+ and CD4+ isolated clones lysed MHC-matched melanoma target cells. Histological studies of regressing lesions showed the presence of CD3+ lymphocytes, mostly CD4H-, perivascularly and at the periphery of the tumor as well as the presence of macrophages throughout the lesions. Cyclophosphamide did not improve the number of responding patients, however lESf-a given to non-responding patients led to a clinical response. The vaccine, referred to as Melacine (Ribi Immunochemical Research, Inc.), contains homogenates of melanoma cells mixed with 0.25 ml Detox. In a multicenter phase II trial, with patients with

543

disseminated melanoma, the toxicity was moderate, mostly local. PreCTL number increased, and the expansion of CD8+ T cells was correlated with increased survival Clinical responses (remissions and disease stabilizations) were obtained [195]. The efficacy of Detox was contrasted with that of other adjuvants. Helling et al. [196], treated melanoma patients with cyclophosphamide, comparing Detox, BCG and the saponin QS-21 in a vaccine containing the ganglioside GM2, conjugated with KLH. Detox toxicity was only local. The antibody response was not increased by Detox, in contrast to the other adjuvants. Schultz et al. [197] used vaccines made of materials shed from melanoma cell lines, mixed with Detox or alum. Local side effects occurred in the Detox group. In this trial, an antibody response was present and more frequent in the Detox group than in the alum group while there was no difference in DTH. However, the disease-free survival was lower in the Detox groups than the alum group. Eton et al. [198] mixed irradiated melanoma cells with Detox. Toxicity was only local. Peripheral blood mononuclear cell cytotoxicity against autologous melanoma cells was correlated with survival, but no NK cell cytotoxicity occured. Two major responses were obtained, not correlated with DTH response. Jheratope

Detox was also used in immunotherapy directed against other types of cancers. Vaccines generally used sialylated (s)Tn, which are mucin epitopes expressed on epithelial tumors, conjugated with KLH. This vaccine was commercialized as Theratope (Biomira Inc., Edmonton, Canada). In a phase I study O'Boyle et al. [199] injected the vaccine to colorectal cancer patients. Toxicity was only local, and an antibody response was observed. Several trials were performed on breast cancer patients treated with cyclophosphamide. Little local toxicity was found, and an antibody response was evidenced. Partial clinical responses and disease stabilizations were obtained. [200-202]. Adluri et al. [203] compared vaccines using Detox or QS-21 in an adjuvant therapy for colorectal cancer patients. Toxicity was mostly local. An antibody response was

544

induced against synthetic epitopes but not against natural antigens. No DTH response was detected. A phase II trial was performed on metastatic breast cancer patients with or without cyclophosphamide [204]. The results showed a local toxicity. Cyclophosphamide was not efficient. An antibody response was evidenced. No complete remission was obtained. On the other hand, a randomized trial with breast, ovarian and colorectal cancer patients, showed an increase in the antibody response by cyclophosphamide. This antibody response was correlated with increased survival, when antibody levels to mucins were low before immunotherapy. The beneficial role of this response might be due to a blockage of immunosuppressive mucins [205-207]. In another study involving patients with metastatic breast, colorectal and ovarian cancer, increased anti-sTn titers were correlated with better survival. Even if before treatment, elevated titers of antibodies against the mucin MUCl, were correlated with a poor response to immunotherapy, CTL precursors to the MUCl were detected in carcinoma patients [208]. A vaccine using 10 |Lig/ml MUCl-KLH mixed with Detox was injected s.c. in breast cancer patients treated with cyclophosphamide. A weak antibody response, and an ex vivo CTL response against HLA-matched adenocarcinoma cell lines were seen. No correlation with the clinical outcome was available [209]. Theratope was given to patients with breast or ovarian cancer who received peripheral blood stem cell rescue after chemotherapy. Toxicity was mostly local. In vitro, NK activity which was low before immunization returned to normal values, toxicity against cells bearing sTn antigen appeared, and lymphocytes responded to sTn, by proliferation and IFN-y production. Antibodies against sTn were detected in 16 patients, while the anti-MUC-1 antibody titer decreased [210]. The remissions were longer in treated patients and there was a tendency to a decreased risk of relapse [211]. Another vaccine, formulated by mixing irradiated cells from colon carcinoma cell lines with Detox was injected to patients with colorectal metastatic adenocarcinoma, with or without DL-la [212]. The vaccine induced local toxicity, and fatigue which was increased in the group treated with IL-la. DTH occurred in both groups. No clinical response was available.

545

Since point mutations of the ras proto-oncogene are often found in cancer, a vaccine was made with mutated ras peptides mixed with Detox. In a phase I study, CD4+ proliferation and CD8+ cytotoxicity specific to the mutated peptide, were observed. The side effects were minimal and one patient showed a stabilisation of the disease [213]. OncoVax

Trials of therapeutic vaccination against prostate cancer used OncoVax-P (Jenner Biotherapies, Inc, San Ramon, California). OncoVax-P consists of 200 ng monophosphoryl lipid A (similar to that used in Detox) added to 1 ml liposomes and 100 ^g PSA (prostate-specific antigen). Patients received injections by different routes (intramuscular, intravenous or subcutaneous) according to the trial, with or without GM-CSF, IL-2 or BCG and cyclophosphamide pretreatment. No serious side effects were seen. DTH and antibody responses were achieved. Vaccination increased the PSA-reactive T cell frequency as determined by IFN-y secretion, but no toxicity against PSA-expressing target cells was detected. The most effective strategy could not be determined, and no conclusion about the clinical efficacy of the treatment was possible [214,215]. Conclusion

The phase I and phase II trials of therapeutic vaccines (Table 3.) show a weak toxicity of lipids A, furthermore they show a stimulation of the acquired antitumoral immune response. CTL responses correlate better than antibody responses to clinical outcomes, in agreement with current concepts of antitumor immunity. Phase HI trials are now necessary to determine the effective protocol.

546

Table 3.

List of clmical trials performed with lipids A as adjuvant of therapeutic vaccines administered to cancer patients.

Phase 1 Adjuvant 1 Mitchell et al. Cancer Res, 1988 Mitchell etal., AfmNYAcadSci,\99^

1

Schultzetal. Vaccine, 1995 | Elhot et al. Semin Surg Oncol, 1993 1 Eton et al. Clin Cancer Res, 1998 Longenecker et al. Ann NY Acad Sci, 1993 Adluri et al. Cancer Immunol Immunother, 1995 1 Miles et al. Brit J Cancer, 1996 Reddish et al. Cancer Immunol Immunother, 1996 MacLean et al. J Immunother, 1996 MacLean et al. J Immunother, 1996 1 MacLean et al. J Immunother, 1997 Sandmaier et al. J Immunother,\999 Holmberg et al. Bone Marrow transplant, 2000 1 Reddish et al. Int J Cancer, 1998 Kleif et al. J Immunother, 1999 Woodlock et al. J Immunother, 1999 1 Harris et al. 1 Semin Oncol, \999

Antigen

Tumor

I

Detox

Cell material 1

Melanoma

1

Detox

Cell material

Melanoma

II I

Detox

Cell material 1

Detox 1 Cell material

Melanoma Melanoma

II

Detox

Cell material]

Melanoma

I

Detox

Cell material

Melanoma

CTL,DHT

Breast

AR

AR,CTL AR,DTH

Detox

sTn-KLH j

I

Detox

sTn-KLH

Colorectal

AR,DTH

II

Detox

sTn-KLH

Breast

AR

Detox

sTn-KLH

AR

Detox

sTn-KLH

Theratope

sTn-KLH

Breast, ovarian, colorectal Breast, ovarian, colorectal Breast

Theratope J sTn-KLH

Breast, ovarian

AR

sTn-KLH

Breast, ovarian

I

Theratope

AR AR

1 sTn-KLH

Theratope

MUCl-KLH 1

Detox I

Detox

I

Detox

m\

QncoVax-P

1 Breast, ovarian Breast

Ras

Colorectal, pancreas, lung Cell material 1 Colorectal PSA

1

Prostate

1

AR = antibody response, CTL = cytotoxic T lymphocytes, DTH = delayed type hypersensitivity, PR = proliferative response

Tested i parameters AR,DTH, CTL AR,CTL

AR,CTL, PR CTL AR, CTL CTL, PR DTH AR,DTH

547

CONCLUSION LPS immunotherapy was the first immunotherapy for cancers assayed in patients in spite of its toxicity. The standardisation of animal models of cancer, the discovery of the LPS composition and of lipid A activity, the discovery of lipid A structure leading to its chemical synthesis, and the synthesis of lipid A derivatives far less toxic than the natural lipids A, restarted research in this field. At the same time, advances in immunology allowed a better understanding of the mechanisms of action of LPS and lipids A in whole organisms. Most of the articles report a significant enhancement of the life span of treated animals. However recent results show that it is now possible to definitely cure animals bearing large tumors while the untreated counterparts die of their cancer. The most effective structure so far consists of diglucosamine acylated by 3 long chain fatty acids, and the substitution of the diglucosamine backbone is now under investigation. The best treatments consist of repeated i.v. injections, where frequency is an important parameter, and the optimal dose is not necessarily the maximal one. With the same lipid A, the best treatment schedule changes from one animal model to the other. Comparative studies have not been performed to elucidate if species and tumor origin and/or the immunogenicity of tumor cells and their immunosuppressive effect are important parameters at the origin of these differences. Three lipids A have been more intensively studied in animal models, all of them having indirect effects, mediated in vivo by the immune system. For two of them, DT-5461 and ONO-4007, TNF-a is an important mediator acting at the vascular level that provokes tumor necrosis. For the third one, OM-174, the treatment induces the accumulation of IFN-y and DL-lp in tumors, which activate NOS II transcription in tumor cells that produce autotoxic NO, which then provokes the apoptosis of tumor cells. At the same time this treatment inhibits the production of TGF-pl by tumor cells which reduces the TGFpi induced immunosuppression and enhances NO production. Acquired immune response, probably completes the tumor regression started by the apoptosis process and, most probably induces specific memory. Important questions have to be answered to facilitate the definition of protocols for humans. For example, is the lipid A tolerance of

548

macrophages an important parameter for treatment effectiveness ? The answer will influence the choice of doses and frequency of injections, in order to determine whether to increase progressively the dose of lipid A injected to each patient or not. Several lipids A have been tested in cancer patients: MPLA, SDZ MRL 953, and ONO-4007 were injected i.v. in phase I trials. The maximal tolerated dose found is lower than or close to the optimal dose defined in animals. Humans are more sensitive to lipid A than rodents so it is possible that similarly to the toxic dose, the effective dose is lower in humans than in animals. Because it requires small amounts of lipid A generally injected s.c, the adjuvant effect of lipid A has been largely investigated in cancer patients, but only with MPLA. Phase I and phase II trials show weak toxicity of different vaccines with MPLA and the development of an immune response. Phase III are now necessary to find an effective protocol. Therefore it is now to soon to know or to predict whether the lipids A will become an antitimioral medicine. A great deal of data are now available, which justify and impose the necessity of phase III trials. New efforts have to be made quickly because of the thousands of patients who will die in the near future. ABBREVIATIONS APC = antigen-presenting cells; BPI = bactericidal permeabilityincreasing protein; CSF = colony stimulating factor; CTL: cytotoxic T lymphocytes; DG = diacyl-glycerol; DTH = delayed-type hypersensivity; ECSIT = Evolutionarily-Conserved Signaling Intermediate in Toll pathway; FADD = Fas associated death domain; G-CSF = granulocyte colony stimulating factor; GPI = glycosylphosphatidylinositol; HDL = high density lipoprotein; ICAM = intercellular adhesion molecule; i.d.: intradermal; IKK == IkB-inducing kinase; IL-lp = interleukine-lp; IFN-y = interferon-y; IP3 = inositol triphosphate; IL-IR = IL-1 receptor; IRAK = IL-lR-associated kinase; i.p. = intraperitoneal; i.v. = intravenous; KLH = Keyhole Limpet Hemocyanin; LAK = Lymphokine-activated killer; LBP = LPS binding protein; LDL = low density lipoprotein; LPS ==

549

lipopolysaccharide; MCP = monocyte chemoattractant protein; MDP = muramyl dipeptide; MIP = macrophage inflammatory protein; MHC = major histocompatibility complex; MPLA = monophosphorylated lipid A; MTD = maximum tolerated dose; NIK = K-inducing kinase; NFKB = nuclear factor KB; NK = natural killer; NO == nitric oxide; NOS == nitric oxide synthase; PAF = platelet-activating factor; PGE2 = prostaglandin E2; PKC = protein kinase C; PMN = polymorphonuclear neutrophils; PSA = prostate-specific antigen; RSLA = Rhodobacter sphaeroides lipid A; s.c. = subcutaneous; SCID == severe combined immuno deficiency; SLPI = secretory leukocyte protease inhibitor; TF = ThomsenFriedenreich; TLR = Toll Like Receptor; TNF-a = Tumor necrosis factor-a.

ACKNOWLEDGEMENTS The authors thank Conseil Regional de Bourgogne, association pour la Recherche contre le Cancer (ARC), Ligues contre le Cancer de Bourgogne et de Haute-Mame and Fondation pour la Recherche Medicale for theirfinancialsupport.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistryy Vol. 28 © 2003 Elsevier Science B.V. All rights reserved.

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Prevention of Cancer Chemotherapy Drug-Induced Adverse Reaction, Antitumor and Antimetastatic Activities by Natural Products YOSHIYUKI KIMURA Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan. ABSTRACT: Although it has recently been thought that a number of medicinal plants and foodstuffs have antitumor and antimetastatic activities, the basis for this hearsay is unclear. Therefore, to clarify whether natural products have antitumor and antimtastatic actions, I have been using biochemical and pharmacological approaches to study the natural products isolated from various medicinal plants and foodstuffs. In the review, we will introduce the biological and pharmacological actions of various components isolated from some medicinal plants and foodstuffs on tumor growth and metastasis in tumor-bearing mice. Chitosan and fish oils prevented the adverse reactions such as gastrointestinal toxicity and myelotoxicity caused by cancer chemotherapy drugs without interfering the antitumor activity of chemotherapy drugs. Stilbenes derivatives isolated from Cassia or Polygonum species inhibited the tumor growth and metastasis to the lung in highly metastaic tumor-bearing mice. Furthermore, I found that stilbenes inhibited the angiogenesis in in vivo and in vitro models.

INTRODUCTION Cancer is the largest single cause of death in both men and women, claiming over 6 million lives each year v^orldwide. Cancer chemotherapy drugs such as 5-fluorouracil (5-FU) derivatives, cisplatin (CDDP), mitomycin, doxorubicin, taxisol, etc. have been used extensively for the treatment of certain types of cancer. However, with these treatments, severe gastrointestinal toxicity with diarrhea and mucosis, and hematologic toxicity with leukopenia and immunosuppression, appear to be dose-limiting factors. Furthermore, the removal of malignant tumor by surgical operation, radiation therapy and/or adjuvant therapy with cancer chemotherapy drugs may be curative. However, the removal of certain cancers, for example, breast carcinoma, colon carcinoma and osteogenic sarcoma, may be followed by the rapid growth of distant metastases to lung, liver etc. Therefore, efforts are underway to develop new modulators that inhibit the adverse reactions without loss of antitumor activity and new drugs having antitumor and antimetastatic activities without adverse

560

reactions.

In this review, I describe the following articles.

1) Prevention by Natural Products of Adverse Reactions Induced by Cancer Chemotherapy Drug without Loss of Antitumor Activity. a) Prevention by Chitosan of Myelotoxicity, Gastrointestinal Toxicity and Immunocompentent Organic Toxicity Induced by S-Fluorouracil (5-FU) [1] or Doxorubicin [2] without Loss of Antitumor Activity in Tumor-Bearing Mice, Chitin and chitosan are polymers with molecular weight of about 1000 kDa, and contain more than 5000 acetylglucosamine and glucosamine units, respectively. Chitin is widely distributed in natural products such as the protective cuticles of crustaceans and insects, as well as being found in the cell walls of some fungi and microorganisms, and is usually prepared from the shells of crabs and shrimps. Chitin is converted to chitosan by deacetylation with 45% NaOH at lOO'^C for 2 h. Though chitosan is reported to augment the natural killer activity, the antitumor activity of chitosan is not clear yet. First, I examined the antitumor effects of chitosan, but it had no effect. Gastrointestinal toxicity and myelotoxicity are caused by the 5-FU after the phosphorylation in the digestive tract and bone marrow tissue. To clarify whether chitosan enhances the antitumor activity of 5-FU or doxorubicin and prevents the adverse reactions induced by 5-FU or doxorubicin, I examined the antitumor activity and adverse reactions, such as myelotoxicity, immunocompetent organ toxicity, and gastrointestinal toxicity of combined treatment with chitosan and 5-FU or doxorubicin in sarcoma 180-bearing mice. 5-FU (12.5 mg/kg twice daily) plus chitosan (150, 375 and 750 mg/kg twice daily) inhibited the tumor growth as well as 5-FU alone. Chitosan (150 and 750 mg/kg twice daily) blocked the reduction of blood leukocyte number caused by 5-FU administration, and it prevented the injury of the small intestinal mucosa membrane and delayed the onset of diarrhea induced by 5-FU "Fig. (1), Fig. (2) and Fig. (3)". Furthermore, chitosan (750 mg/kg twice daily) prevented the reduction of spleen weight induced by 5-FU in sarcoma 180-bearing mice "Fig. (4)", and the reduction of lymphocyte, CDS^ and NKl.l.^ T cell numbers induced by 5-FU "Fig. (5)". Intraperitoneal doxorubicin (5 mg/kg on days 1 and 8 after inoculation of tumor cells) significantly inhibited tumor volume and tumor weight, compared with sarcoma 180-bearing mice. Similarly, doxorubicin plus chitosan (200 and 800 mg/kg twice daily) also inhibited the tumor growth, compared with tumor-bearing mice "Fig. (6)". On the other hand, a remarkable reduction in body weight of mice after 8 days was observed in the mice receiving intraperitoneal doxorubicin compared with tumor-bearing mice. Oral administration of chitosan (400 and 800 mg/kg

561

I I

I sarcoma 180-bearing mice

H

^-^

B

I sarcoma ISO-bearing mice SFU

f^J

Chitosan

E 2 I Chitosan

i E E o E 51

5

2-D-glucoside or piceid was administered orally twice daily for 32 consecutive days, starting 12 h after tumor implantation. Results are expressed as means ± S.E. of 4-5 mice in each group. *P2)]-Xyl; A" 3c Holotoxio A [21] R = [3-0-Me-Glc^l->3)-Gic-(l-^)]-[3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui2)]-Xyl; A^ 3d Holotoxin Bi [20] R = [Glc3)-X)1-(l-^)-Qai-(l->2)]-Xyl; A^ 3e Holotoxin B [21] R = [G^c3)-G^c-(l->4>Qui-(1^2)^Xyi; A" 3f Neothynidioside [22] R = 3-0-Me-Glc-(l-y3)-Xyl-4)-Qui-2)^W)S03Na-Xyl; A^' 3g Psolusoside A [23] R = 6-OS03Na-3-^-Me-Glo3)-€-OS03Na-Glc4)-Qui-(l->2)-Xyl; A^' 3h Qadoloside A [24] R = 3-0-Me-Glc-(l-^3)-Xyi-(1^4)-Qui-(l->2)-Xyi; A" 3i Cladoloside B [24] R = [Glc-(l->4)]-[3-0-Me-Glc-2)-4-OS03Na-Xyi; A^

Fig. (4). Structure of 3p,12a-dihydro3Qiiolost-9(l l)-ene aglyoone based glycosides

Some glycosides contain two hydroxyl groups at positions 12a and 17a of the holostanol skeketon, Fig. (5):

HO O

5a Echinoside B [28] R = Qui-(1^2)-4-OS03Na.Xyl; R' = H 5b Echinoside A [28] R = 3-0-Me-Glc3>Glo4>Qiii-(l->2>4-OS03Na-Xyl; R* = H 5c 22-Acetoxy-echinoside A [29] R = 3-0-Me-Glc3)-Glc2)^M3S03Na-Xyl; R* = OAc 5d Holothurin A, [30] R = 3-2>4-0S03Na-Xyl; R* = C« 5e 24-Dehydroechinoside B [31] R = Qui-(l->2)^K)SOjNa-XyI; R* = H; A^ 5f 24-Dehydroechiiioside A [31] R = 3-0-MeGlc4)-Qui-2)-4-0S03Na.Xyl; R^ = H; A^ 5g 22-Hydroxy-24-dehydroechiiioside A [29] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-2)-4-0S03NarX>i; R' = OH;A^

Fig. (5), Stiucture of 3p,12a, 17a-trihydrox54iolost-9(l l)-«ie aglycone based glycosides

Glycosides 5c, 5d and 5g together with glycosides 6, Fig. (6) and 7, Fig. (7) are characterized by additional acetoxy or hydroxy groups in the side chain.

592

OH

R

O'

6 24(5)-hydroxy-25-dehydroechinoside A [29] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-(l-^2)-4-OS03Na^Xyl Fig. (6). Structure of a sul&ted tetraglycoside isolatedfixMnthe sea oxccashei Actinopygaflammea

HO O^

^O

OAc

7a Holothurinosidc B [32] R = [3-0-Me-Glc-(l->3)-CHc-(l->4)-Qui-2)]-[CHc-(l->4)]-Xyl; R ' = OH; A^ 7b Pervicoside A (Neothyosidc A) [27] R = 3-C>-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l->2)-4-OS03Na-Xyl; R^ = H 7c Neothyoside B [33] R = Qui-(l->2)-4-OS03Na-Xyl; R ' = H Fig. (7). Structure of 25-acetoxi-3p,12a-dihydroxjiiolost-9(l l)-€ne aglycone based glycosides

Holothurins A (8a) and B (8b) isolated from the sea cucumber Holothuria leucospilota [34] as well as Desholothurin A (8d), and Holothurinosides A (8c), C (8e) and D (8f), Fig. (8)fromHolothuria forskali [32] are the only examples of glycosides containing the side chaininafiiranform. Compounds 3a, 3g-31 and 7c are the only A^'^ ^-glycosides isolated from sea cucumbers belonging to the order Dendrochirotida. In general, 3|3-hydroxyholost-9(ll)-ene based aglycones were characterized in holothurins isolated from animals of the order Aspidochirota.

593

8a Holothurm B [34] R = Qui-(1^2)-4-OS03Na-Xyl; R ' = OH 8b Holothurm A [34] R = 3-0-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l-^2)-4-OS03Na-X)4; R^ = OH 8c Holothurinoside A [32] R = [Glc-(1^4)]-[3-0-Me-Glc-(l-^3)-Glc-(l->4)-Qiii-(l->2)]-Xyi; R^ = OH 8d Desholothurm A [32] R = 3-0-Me-CHc-(l->3)-Glc-(l->4)-Qui-(l->2)-Xyl; R ' = OH 8e Holothurinoside C [32] R = 3-0-Me-Glc-(l->3)-Glc-(l-^)-Qui-(l->2)-Xyi; R ' = H 8f Holothurinoside D [32] R = Qui-(1^2)-Xyi; R ' = H Fig. (8). Structures of glycosides isolatedfromtbe sea cucumbers Holothuria leucospilota and Holothuria forskalii

3P-HydroxyhoIost-7-ene aglycones Frondoside B (9a), Cucumariosides A2-4 (9b) and A7-3 (9c), Fig. (9) as well as several triterpene glycosides isolated from the sea cucumbers Stichopus chloronotus (lOa-lOh) and Thelenota ananas (lOi, lOj), Fig. (10) contain the simple 3P-hydroxyholost-7-ene as the aglycone. An additional acetoxyl group in the side chain is present in compounds 10alOj.

9a Frondoside B [35] R = [3-0-Me-Glc-(l-^3)-6-OS03Na-Glc-(l->4)]-pCyl-2)-4-OS03Na.Xyl; A'; A^ 9b Cucumarioside K2-A [36] R = [3-0-Me-Glo3)-Glc-(l->4)]-pCyl-(l->2)]-Qui-2)-40S03Na-Xyl;A';A^ Fig. (9). Structures of glycosides isolatedfromthe sea cucumbers Cuctanariafrondosa and Cucumaria japonica

594

Glycosides lOa-lOj were isolated from Stichopus chloronotus and Thelenota ananas, two sea cucumbers belonging to the order Aspidochirota [37].

lOa Stichloroside C, (Stichoposide C) [37] R = [3-0-Me-Glo3)-Glc-(l->4)]-[3-0-Me-ac3)-Xyi-(l-»4)Qum-(l-^2)]-Xyi 10b Stichloroside B, (Stichoposide D) [37] R = [3-0-Me-Glc-(l->3)-Glc-(l-^)]-[3-0-Me-Glc-2)]-X>4; A^' lOg Stichloroside B2 [37] R = [3-0-Me-Glc-(l->3)-Glc-(l->4)]-[3-0-Me-Glo3)-Xyi-(l-M)-Glc-(l-^2)]-Xyi; A^^ lOh Stichloroside A2 [37] R = [3-0-Me-Glc-{l->3)-Glc-3)-CHc-2)]-Xyl; A^ lOi Thelenotoside A [37] R = 3-0-Me-Glc-(l->3)-Xyi-(l-^)-Qui-(1^2)-Xyl lOj Thelenotoside B [37] R = 3-3)-X>i-(l->4)-Glc-(l->2)-Xyl Fig. (10). Structures o f glycosides isolated fixjm the sea cucunibers Stichopus chloronotus and Thelenota ananas

3p-Hydroxyholost-7-ene aglycones with a carbonyl group at C-16 have been isolated exclusively from the sea cucumber Cucumaria japonica, Fig. (11).

R—a

l l a Cucumarioside A2-3 [36] R = [3-0-Me-Glc-(l-^3)-Glc-2)-Xyi

595 l i b Cucumarioside Ar-2 [36] R = [6-OS03Na-3-0-Me-Glc-(l->3)-6-OS03Na-Glc-(1^4)]-[X>d-(l->2)]-Qui-(l->2)4-OS03N2hXyl l i e Cucumarioside Ao-3 [38] R = [3-0-Me-Glc-(l->3)-Xyi-2)]-Qui-(l-^2)-4-OS03Na.X)d; A" U d Cucumarioside A,-2 [38] R = [6-OAc^c-(l->3)-Glc-(l->4)]-pCyi-(l->2)]-Qui-(l->2H-OS03Na-Xyl; A^' l i e Cucumarioside A2-2 [36] R = [3-0-Me-CHc-(l->3)-Glc-(l-^)]-pCyi-(l->2)]-Qui-(1^2)-XyI; A" l l f Cucumarioside A7-I [36] R = [6-OS03Na-3-0-Me-Glc-(l-^3)-6-OS03Na-Glc-(l->4)]-[X>d-(l->2)]-Qui-(l->2)4-OS03Na-Xyl;A^' l l g Cucumarioside A3 [39] R = [3-(9-Me-ac4)]-[X54-(l->2)]-Qui-(l->2)-4-OS03NaXyi;A" l l h Cucumarioside A6-2 [39] R = [6-OS03Na-3-0-Me-Gac-(l->3)-Glc-(l->4)]-pCyi-(l->2)]-Qui-(l->2)-4-OS03NaXyl;A" Hi Cucumarioside A4-2 [36] R = [GIc-(l->3)-ac-2)-4-OS03Na-Xyl 12b Frondoside Ai [41] R = 3-0-Me-Glc-(l->3)-X>i-(l->4)-Qui-(l->2)-4-OS03Na-Xyl 12c Liouvilloside B [42] R = 6-OS03Na-3-0-Me-Glc-(l->3)-6-OSOjNa-Glc-(l->4)-Qui-(l->2)-4-OS03Na-Xyi 12d Cucumarioside Ao-2 [38] R = [3-0-Me-Glc-(l-^3)-Xyi-(l-^)]-pCyl-(l-^2)]-Qui-(l-^2)-4-OS03Na-Xyl; A^^ 12e Neothyonidioside C [43] R = 6-OS03Na-3-6>-Me-Glc-(1^3)-X5d-(l->4)-Qui-(l->2)-4-OS03Na-Xyi; A^ 12f Cucumarioside G, [44] R = 3-0-Me-Xyi-(l->3)-GJc-(l->4)-Qui-(l->2)-4-OS03N»-Xy!; A^ 12g Liouvilloside A [42] R = 6-OS03Na-3-0-Me-Glc-3)-6-OS03Na-Glc-(1^4)-Qui2)-4-OS03Na-Xyi; A^ 12h Cucumarioside C^ [45] R = [3-0-Me-Xyl-(l->3)-Glc4)]-[Xyi-(l->2)]-Qui-(l->2)-Xyi; HE, A^'* 12i Cucumarioside H [46] R = 3-0-Me-Xyl-3)-2)-4-OS03Na-X>4; 22£'; A^ 12k Cucumarioside Cj [45] R = [3-0-Me-X)d-(l->3)-Glc-2)]-Qui-(l->2)-Xyl; 22Z; A^ 121 Cucumarioside G3 [47] R = 3-0-Me-ac-(l-^3)-Cac4)-Qui-(l-^2)-4-OS03Na.Xyl; 22Z; A^ Fig. (12). structure of 16p-acetoj^-3p4iydroxjiiolost-7-eoe aglycone based glycosides

Some of the glycosides containing a 16p-acetoxy group also present an allylic hydroxyl group at C-25, Fig. (13).

596

OH

13a Cucumarioside G4 [47] R = 3-(9-Me-X)4-(l->3)-Glc-{l->4)-Qui-(1^2)^U)S03Na.Xyl 13b Eximisoside A [48] R = 3-0-Me-Glc-(l-^3)-Xyl-(l->4)-Glc-(l->2)-X5d 13c Caldgeroside E [49] R = [6-OS03Na.3-0-Me-Glc3)-a(Kl->4)]-[Glc-(l-^2)]-Qui-{l->2)-4-OS03Na-Xyl Fig. (13). Structure of 16p-acetoxy-3p,25-dihydrox>iiolosta-7,22-diene aglyccaie based glycosides

Four glycosides isolated from the sea cucumber Cucumaria lefevrei [50] are the only examples of holothurins with a 16a-acetoxy group in their aglycones, Fig. (14). Lefevreiosides A2 (14b), B (14c) and C (14d) show the same monosulfated tetrasaccharide chain and differ in the degree of unsaturation or the position of the double bond in their side chains. Lefevreioside Ai (14a) is the desulfated analog of glycoside 14b.

14a Lefevreioside A, [50] R = 3-0-Me-Glc-(l->3)-Glc-(l->4)-Qui-(l-^2)-Xyl 14b Lefevreioside A2 [50] R = 3-0-Me-Glc-(l-^3)-Cac-(l->4)-Qui-2)-4-OS03Na-X>d 14c Lefevreioside B [50] R = 3-0-Me-Glc-(l-^3)-Glc-(l->4)-Qiii-(l->2)-4-OS03Na-Xyi; A^ 14d Lefevreioside C [50] R = 3-0-Me-Glc-(l->3)-G!c-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; A^^ Fig. (14). Structures o f glycosides isolatedfixatnthe sea cucumber Cucumaria

lefevrei

Several triterpene glycosides isolated from the sea cucumbers Cucumaria echinata and Pentamera calcigera contain a carbonyl group at C-23 in the side chain, Fig. (15). This structural feature is absent in 3phydroxyholost-9(ll)-ene aglycones.

597

15a Cucumechinoside C [51] R = 3-(9-Me-Glc-(l->3)-6-OS03Na-ac-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; R^ = H 15b Cucumedimoside F [51] R = 6-OS03Na-3-0-Me-Glc-(l->3)-6-OS03Na^c-(l->4)-Qui-(1^2)-4-OS03Na-Xyl; R' = H 15c Caldgeroside Cj [52] R = [3-0-Me-XyKl->3)-Glc-(l->4)]-[CHc-(l->2)]-Qui-(l->2)-4-OSOjNa-Xyl; R^ = H 15d Caldgeroside D2 [49] R = [3-C>-Me-XyH1^3)-6-OS03Na.Glc-(l->4)]4Glc2)]-Qm-3)-6-OS03Na-Glc-(1^4)-Qui-(l->2)-4-OS03Na-Xyi; R* = O 15f Cucumedimoside B [51] R = 3-O-Me-Glc-(l->3)-2-OSO3N2hXyi-(l->4)-Qui-(l->2)-4-OS03Na-Xyl; R' = O 15g Cucumechinoside D [51] R = 6-OS03Na-3-0-Me-d; R^ = O 15i Cucumarioside Ao-1 [38] R= [3-O-Me-Glc-(l->3)-X>i-(l-^)]-[Xyi-2)]-Qui-(l->2)-4-OS03Na-Xyi; R^ = P-OAc Fig. (15). Structures of glycosides isolated fix>tn the sea cucumbers Cucumaria echinata and Pentamera calcigera

Recently, we have isolated an antifungal holothurin from the sea cucumber Psolus patagonicus [53]. Patagonicoside A (16), Fig. (16) is the &st example of a 3p-hydroxyholost-7-ene aglycone substituted with 12a- and 17a-hydroxy groups. H%o-Me-Glc-(l->3)-6-OS03NarGlc-4)-Qui-(l->2)-4-OS03N»-Xyl Fig. (16). Structure of patagonicoside A, an antifungal oligoglycoside isolated fixxn the sea cucumber Psolus patagonicus

Non-holostane aglycones

598

Recently, some examples of holothurins having uncommon nonholostane aglycones have appeared in the literature. These glycosides have been isolated from seven species of sea cucimibers belonging to the order Dendrochirota. All are sulfated compounds, the majority monosulfated at the glucose or xylose units. Five glycosides contain aglycones with an 18(16)-lactone and a A^unsaturation, Fig. (17) and (18). OAc

17 Psolusoside B [54] R = [6-OSO,Na-Gl(Kl->4)]-[Glc-(l->4)-Glo(l-^2)]-X>i Fig. (17). Structure of Psolusoside B, isolatedfixmithe sea cucumber Psoltds fabricii

^N^^ •iiiH

18a Cucumarioside G^ [55] R = 3-0-Me-Xyl-(l-^3)-Glc(l->4)-Qui-(l-^2)-4-OS03Na.Xyl 18b Caldgeroside B [52] R= [3-0-Me-X>i-(l->3)-Glc(l->4)]-[Qw-2)]-Qiii-(l-^2)-4-OS03Na-Xyi 18c Caldgeroside C, [52]R = [3-0-Me-X>d3)-CHc(l->4)]-[Glc-(l-^2)]-Qui-(l->2)-4-OS03Na.X>1 18d Caldgeroside D, [49] R = [3-0-Me-Xyl-(l->3)-6-OS03Na-Glc(l-^)]-[CHcji-(l->2)-4-OS03Na-X^ Fig. (18). structures of noo4iolostane glycosides isolated fixxn the sea cucumbers Et^ntacta fraudatrix and Pentamera cahigera

Avilov et al. [56,57] reported three holothurins that are devoid of a lactonefiinctionand have a shortened side chain. Kurilosides A (19a) and

599

C (19b) contain a 9(ll)-double bond aglycone moiety and 16a-acetoxy group, Fig. (19).

OAc

R-

19« Kuriloside A [56] R = [3-O.Me-Glc-(1^3)-6-OS03N»4)-Qiii-(l->2)]-Xyl 19b Kuriloside C [56] R = [3-(9-Me-Glc-(l->3)-6-OS03Na^c-(l->4)]-[Qui-2)]-Xyl Fig. (19). Structures of ^yootsides isolatedfixxnthe sea cucumber Duasmodactyla kurilensis

Koreoside A (20) isolated from Cucumaria koraiensis is one of the two examples of non-holostane glycosides with three sulfete groups in the oligosaccharide chain, Fig. (20). COCH, IIH

20 Koreoside A [57] R = [6-OS03Na-3-C>-Me-3)-6-OS03Na-Glc-(l->4)]-PCyl-(l->2)]-Qui-(l->2)-4OSO^a-Xyi Fig. (20). Glycoside isolatedfixxntbe sea cucumber Cucumaria koraiensis

Ds-Penaustrosides A (21a) and B (21b), as well as Frondoside C (21c), also lack the lactone function and have an additional hydroxyl group at C20, Fig. (21).

600

21a Ds-Penaustroside A [19] R = [3-0-Me-Xyi4)]-[Qui-(l-^2)]-Qui-(1^2)-4-OS03Na-Xyl; R^ = H 21b Ds-Penaustroside B [19] R = [3-0-Me-Xyi-2)]-Qui-(l->2)-4-OS03Na-X>4; R^ = H; 21c Frondoside C [58] R = [3-0-Me-Xyi4)]-[Qiii-(l->2)]-Qui-2)-4-OS03Na-Xyl; R' = OAc; A^ Fig. (21). Structures of tiOQ4K>lostane glyoosides isolated from the sea cucumbers Pentacta australis and Cucumariafrondosa

Most of sea cucumber triterpene glycosides are tetra- or pentaglycosides. The few disaccharides that have been isolated show a Qui-(1^2)-4-OS03Na-Xyl chain attached to C-3 of the triterpenoid aglycone [28, 31, 33, 34, 37]. Bivittoside A (4a) and Holothurinoside D (8f) show no sulfate group while Stichoposide B (lOe) is the only example of a disaccharide with a glucose unit attached to C-2 of the xylose unit. Some hexasaccharides have been isolated from sea cucumbers of the order Aspidochirota: Stichopus japonica [21], Stichopus chloronotus [37], Parastichopus californius [20] and Bohadschia bivittata [18]. They are non-sulfated glycosides with a linear 3-0-Me-Glc-(l->3)Glc-(l->4)-Xyl chain and a branching of a linear trisaccharide at C-2 of the xylose unit. The only example with a glucose unit instead of the terminal 3-0-Me-glucose is Holotoxin Bi (3d). Most tetrasaccharides show a linear chain with the most common 3-0In some Me-Glc-(l->3)-Glc-(l->4)-Qui-(l->2)-Xyl structure. tetrasaccharides the glucose imit is replaced by a xylose [22, 24, 37, 38, 40, 43, 51] while Cucumariosides Gi (12f) and G4 (13a) show a terminal 3-0-Me-xylose unit. Thelenotoside B (lOj) and Eximioside A (13b) show a different tetrasaccharide chain: 3-0-Me-Glc-(l-^3)-Xyl-(l->4)-Glc(1~>2)-Xyl with no quinovose unit. Non-holostane triterpenoids, such as Psolusoside B (17), Kuriloside C (19b) and Bivittoside B (4b) are the only examples of tetrasaccharides with a non-linear chain. Most tetrasaccharides are sulfated at C-4 of the xylose unit. Additional sulfete

601

groups at C-6 of the 3-0-Me-glucose unit and at C-6 of the glucose unit have been found in trisulfeted tetraglycosides. Pentaglycosides isolated from sea cucumbers show a variety of carbohydrate chains, Fig. (22). Most glycosides contain chains I-IV. Chain IV is typical for glycosides isolated from the sea cucumber Pentamera calcigera: Calcigerosides Ci (18c), C2 (15c), Di (18d), D2 (15d) and E (13c). Cucumarioside Ai-2 (lid) is the only example of a triterpene glycoside containing an acetate group at C-6 of the terminal glucose unit (chain XII). Pentasaccharide chains with glucose as the terminal sugar are uncommon and were found in a few glycosides, such as Cucumarioside A4-2 (Hi) (chain VII), Cladoloside B (3i) (chain X) and Holothurinoside A (8c) (chain XT). [3-^-M&2)]9

66.6

78.1

64.2

73.4

6"'^^

62.2

17.9

•lnPy^3-D20(5:l) **InPy^5 ' Py-rfj-DzO (8:2) *toesulfeted analog of 18b "InPy^5P20(4:l)

610

One common structural feature is the presence of a xylose unit attached to C-3 of the aglycone and substituted at C-2' with a quinovose unit. As shown in Table 2, carbons involved in the interglycosidic linkages show chemical shifts at 6 ca. 82-88 ppm, shifted downfield from those expected for the corresponding methyl glycopyranosides. Glycosides containing a terminal glucose (12a, 8c, 9a) or xylose (Des18b) substituted with a methoxyl group at C-3 show an additional signal at 5 ca. 86-88 ppm due to the substitution at this carbon. Frondosides A (12a) and B (9a) and the desulfated derivative of Calcigeroside B (Des18b) present a branched 2,4-disubstituted quinovose residue with a xylose unit attached to C-2" in 9a and 12a and a quinovose unit in Des-18b. This substitution pattern is deduced from the downfield shifts of C-2" and C-4" of the 2,4-disubstituted quinovose in comparison with those carbon resonances in a terminal quinovose vmit (Des-18b). On the other hand, Holothurinoside A (8c) with a 2,4-disubstituted xylose unit attached at C-3 of the aglycone shows signals at 6 83.3 ppm (C-2') and 77.9 ppm (C-4') for the carbon atoms involved in the glycosidic bonds. As shown in Table 3, due to the proximity of carbon resonances of the different sugar units in the oligosaccharide chain, it is diflBcult to assign unambiguously each signal on the only basis of comparison with published data, sometimes performed in different solvents or solvent mixtures. Recent application of two-dimensional NMR techniques (^H-^H COSY, relay COSY, HETCOR, COLOC, HMBC and HMQC) to the structural elucidation of holothurins [32, 35, 39, 40, 48, 52, 57] has allowed the unambiguous assignment of all ^H and ^^C resonances of the oligosaccharide chain. The NOESY spectrum of Patagonicoside A clearly showed the correlations between the protons (Fig. (23)) of the oligosaccharide chain. These correlations confirmed the iaterglycosidic linkages deduced previouslyfi-omanalysis of ^H-^H COSY and HETCOR spectra as well as the site of attachment of the sul&ted xylose unit to the C-3 of the aglycone [53].

611

Fig. (23). NOESY oorrelatiocis of the oligosaccharide moiety ofpatagonicoide A

Another common structural feature in holothurins is the presence of one, two or three sulfate units attached to the sugar residues of the oligosaccharide chain. The location of these groups has been determined by comparison of ^^C-NMR data of the native glycosides and the corresponding desulfated derivatives. Desulfetion of the native holothurins is easily achieved by hydrolysis in a mixture of pyridine and dioxane at 120X and further purification of the desulfated derivatives by HPLC [53]. Those holothurins containing an acetoxyl group at C-16, as Liouvilloside A (12g), are desulfeted by acid hydrolysis in HCl-MeOH in order to prevent hydrolysis of the acetate group [42]. Table 4 shows the ^H- and ^^C-NMR data for two glycosides, Patagonicoside A (16) and Hemoiedemoside A (3k), containing the same disulfeted tetrasaccharide chain and the trisulfated Liouvilloside A (12g), that differsfi^om16 and 3k in the presence of an additional sulfete group at C-6 of the terminal 3-0-Me-glucose unit. The three glycosides differ in their aglycone structures. As observed in Table 4 the esterified carbons with a sulfete group show downfield shifts of ca, 4-6 ppm with respect to their desulfeted derivatives, while upfield shifts of ca. 2-3 ppm are observed for the vicinal carbons. The chemical shifts of these carbons vary with the solvent used for performing the spectra. For example, the xylose unit with a sulfate group at C-4', common to all sulfeted holothurins, shows a characteristic signal for C-4' at 5 77.1 ppm in CD3OD, while the same carbon resonance is shifted downfield to 5 75.8 and 74.4 ppm in CsDsN.DaO (5:1) and DMSO-e/6, respectively. Sulfate groups at C'6 of glucose or a 3-0-Me-glucose residue show typical signals for C-6 at 5 65.6-68.5 ppm in these deuterated solvents.

612

Table 4. *H- and "C-IVMR data for the Sugar Moieties of Patagonicoside A (16), Hemoiedemoside A (3k) and LiouviUoside A (12g).

16

c r T 3' 4' 5'

3k

12g

Sc*-'

SH'(«/mHz)

5c ^'^

5H"(./inHz)

Sc^-^

SH'(>/mHz)

105.6 82.7

4.46 d (7.9) 3.55 m 3.78 m 4.23 m 3.37 m;

104.9 82.4

4.69 d (7.1) 3.69 m 4.27 dd (9, 8.7) 5,11m 3.72 m

104.2 82.0 14.1 (+2) 74.4 (-4.8) 63.2 (+2.3)

4.32 d (7.3) 3.36 m 3.54 m 3.97 m 3.19 m

103.8 74.9 74.2 86.2

4.49 d (8) 3.10 3.32 m 3.03

70.4 17.4

3.34 m 1.25 d (5.3)

103.1 72.6 85.9

4.40 d (7.8) 3.25 m 3.49 m

68.7 74.2 (+2.1) 65.9 (-4.9)

3.20 m 3.53 m 3.78 m, 4.04 dd

75.1 (+2.3) 77.1 (-6) 63.8 (+2.6)

74.8 (+2.6) 75.8 (-5.5) 63.9 (+2.3)

4.17 m

1" 2" 3" 4"

104.8 76.3 75.6

5" 6"

115

r"

4.75 m 4.92d/7.7)

4.61 d (7.6) 3.36 m 3.55 m 3.23 m

104.6 75.5 75.6 87.8

18.0

3.49 m 1.35 d (6.1)

71.3 17.8

3.88 dd 3.97 m 3.44 dd (8.7, 8.9) 3.66 m 1.63 d (6.1)

873

104.8

4.45 (6.9)

104.6

4.76 d (7.7)

ij'i'tt

74.3

3'"

87A

74.3 86.5

4»»»

10.2 75.2 (+2.7) 68.5 (-6.1)

3.41m 3.60 m 3.42 m 3.69 m 4.12 m;

3.95 4.25 3.79 4.21 4.68 m, 5.14 dd (2,10.7)

5'" 6'"

69.9 74.7 (+2.7) 67.5 (-5.7)

4.38 m 1»»9

105.2 75.4 87.6 71.1

4.57 d (7.9) 3.31m 3.10 m 3.34 m

104.9 74.5 87.4 70.3

78.1 62.5

OCH3

61.1

3.32 m 3.65 m; 3.85 bd (10.5) 3.62 s

113

6""

2"" 3"" 4"" «9»»

61.8 60.6

5.29 d (7.8) 3.96 m 3.71m 4.02 dd (8.9, 9.3) 3.95 m 4.19 m, 4.43 dd (2,11.9) 3.85 s

(18,10) 103.8 73.6 85.8 69.3

4.47 d (7.9) 3.15 m 3.00

75.1 (+1.8) 65.6 (-4.6)

3.34 m 3.83m,4.04dd (18,10) 3.49 s

60.1

3.21m

' Reoofxied at 125 MHz in Me^hanoW4; ^ Italics = interglycx)sidic positions, bold = sul&te positions; (Ac = 6c - ScdesDtfated andog); *" Rfiootded at 500 MHz in Metbanol-rozdova, O.A.; Kalinovsky, A.I.; Stonik, V.A.; Gudimova, E.N.; Chem. Nat. Comp., 1993,29,216-218. Maier, MS.; Roccatagliata, A.J.; Kuriss, A.; Chludil, HD.; Seldes, A.M.; Pujol, C.A.; Damonte, E.B.; J. Nat. Prod., 2001, 64^ 732-736. Avilov, S.A.; Kalinovsky, A.I.; Stonik, V.A.; Chem. Nat. Comp. 1990,26,42-45. Afiyatullov, S.S.; Tishchenko, L.Y.; Stonik, V.A.; Kalinovsky, A.I.; Elyakov, G.B.; Khim. Prirodn. Soedin, 1985, 2, 244-248. Afiyatullov, Sh.Sh.; Kalinovsky, A.I.; Stonik, V.A.; Khim. Prirodn. Soedin, 1987, 6, 831-837. Kalinin, V.I.; Afiyatullov, Sh.Sh.; Kalinovsky, A.I.; Khim. Prirodn. Soedin, 1988, 2,221-226. Kalinin, V.I.; Avilov, S.A.; Kalinovsky, A.I.; Stonik, V.A.; Mlgrom, Y.M. Rashkes, Y.N.; Khim. Prirodn. Soedin, 1992, 6, 691-694.. Kalinin, V.I.; Avilov, S.A.; Kalinina, E.Y; Korolkova, O.G.; Kalinovsky, A.I. Stonik, V.A.; Riguera, R.; Jimenez, C; J. Nat. Prod., 1997, 60, 817-819. Avilov, S.A.; Antonov, A.S.; I>rozdova, O.A.; Kalinin, V.I.; Kalinovsl^^, A.I. Riguera, R; Lenis, L.A.; Jimenez, C; J. Nat. Prod., 2000, 63, 1349-1355. Rodriguez, J.; Riguera, R; J. Chem. Res., 1989, 2620-2636. Myamoto, T.; Togawa, K.; Hguchi, R; Komori, T.; Sasaki, T.; Liebigs Ann. Chem., 1990,453-460. Avilov, S.A.; Antonov, A.S.; Drozdova, O.A.; Kalinin, V.I.; Kalinovsky, A.L; Stonik, V.A.; Riguera, R; Lenis, L.A.; Jimenez, C; J. Nat. Prod, 2000, 63, 6571. Murray, A.R; Muniain, C.C; Seldes, A.M.; Maier, M.S.; Tetrahedron, 2001,57, 9563-9568. Kalinin, V.I.; Kalinovsky, A.I.; Stonik, V.A.; Dmitrenok, RS.; Elkin, I.N.; Chem. Nat. Comp., 1989, 25, 311-317.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 28 © 2003 Elsevier Science B.V. All rights reserved.

617

SULFUR-CONTAINING NATURAL PRODUCTS FROM MARINE INVERTEBRATES MICHELER.PRINSEP Department of Chemistry, University ofWaikato, Private Bag 3105, Hamilton, New Zealand. ABSTRACT: An overview of sulfur-containing natural products isolated from the marine invertebrate phyla that are commonly studied by natural products chemists, is provided. The material is arranged by phyla and sulfated compounds are included, except for the Echinodermata where sulfated saponins and steroids are specifically excluded. A total of 638 compounds and 530 references are recorded. The review covers the published literature up until the end of 2001. References to reported syntheses and comments on biological activities of metabolites are included.

INTRODUCTION Marine invertebrates such as sponges, bryozoans and tunicates are well known sources of a wide variety of secondary metabolites or natural products. Many of these compounds possess novel structures with unprecedented ring systems or unusual combinations of functional groups. There is a high incidence of biological activity among the secondary metabolites of marine invertebrates, which is not surprising, given the environment that the organisms live in. Marine invertebrates are either completely sessile, or if able to move, such as is the case for example, for nudibranchs (sea slugs) and ophistobranchs (sea hares), they do so only very slowly. Most marine invertebrates lack physical protection in the form of spines, a sting or a shell, so would seem an ideal meal for a passing predator. However, these invertebrates flourish and the fact that they do so, is thought to be due to chemical protection from predation, overgrowth and infection that their secondary metabolites convey. For this reason, many research groups worldwide investigate the natural products of marine invertebrates, primarily concentrating on their biological activity. A myriad of structural types are found amongst the secondary metabolites of marine invertebrates. Halogenation is typical of marine natural products, which is not unexpected, given that seawater contains

618

very high concentrations of chloride, bromide and iodide ions (559 mM, 0.86 niM and 0.45 |iM respectively) [1]. Sulfur is the fourth most common element in seawater after chlorine, sodium and magnesium and the sulfate anion is the second most abundant after chloride [2]. There are many excellent reviews available on marine natural products in general. The annual reviews by Faulkner [3-20] cover all secondary metabolites from the major marine phyla. Very few reviews however, deal solely with sulfur-containing marine metabolites. The first of these to be published arranged compounds according to structural types [21]. Another review covers sulfated marine compounds only and these are arranged by phylum [2]. The most recent review deals with marine sulfur-containing natural products excluding sulfates and metabolites are organised according to the sulfur functional groups that they contain [22]. This review will discuss all sulfur-containing natural products from the main marine invertebrate phyla studied by natural product chemists: Bryozoa, Chordata, Cnidaria, Mollusca, Porifera and a selection of those from the Echinodermata. The vast majority of sulfurcontaining metabolites that have been isolated from echinoderms are sterol sulfates and saponins and this review will not include coverage of these but will only deal with other types of sulfur-containing metabolites from echinoderms. For discussions of the sterol sulfates and saponins of echinoderms, the reader is referred to other sources [2,3-20]. The current review concentrates on isolation and biological activity of sulfurcontaining metabolites and covers the literature up to the end of 2001. As pointed out by Christophersen [21], the distinction between primary and secondary metabolites is somewhat blurred, therefore, some compounds that could perhaps be classified most readily as primary metabolites are included, but macromolecules are specifically excluded. Every endeavour has been made to be comprehensive, but inevitably some metabolites may have been overlooked. Metabolites are organised by phylum and within phyla, compounds are arranged in families, or by structural type.

Bryozoans A bryozoan or moss animal is a sedentary colony of minute filterfeeding individuals called zooids [23]. They are widely distributed

619

throughout the marine environment. There are about 4,000 living species and over 10,000 as preserved fossils. Apart from a very fev^ freshw^ater species, all living bryozoans are marine dwelling [24]. The zooids that comprise a bryozoan colony each consist of a body wall (the box or tube) and a polypide. The polypide is made up of a simple U-shaped gut and an apparatus of tentacles called a lophophore [25]. The inner faces of the tentacles are covered with cilia that beat to generate a current of water towards the mouth. The supportive outer body wall is often reinforced by either chitin or calcium carbonate or both. Zooids can be specialised and take on specific roles within a colony, such as feeding, brood or funicular (transport or communication) zooids [25]. Contact with the bryozoan Alcyonidium gelatinosum gives rise to "Dogger Bank itch", an allergic contact dermatitis. The causative agent is (2-hydroxyethyl)dimethylsulfoxonium ion (1). Synthesis of 1 was achieved by base-catalysed condensation of trimethylsulfoxonium chloride and formaldehyde [26].

OH X"

The sulfur-containing p-carboline alkaloid, l-ethyl-4-methylsulfone-p-carboline (2) was isolated from the New Zealand bryozoan Cribricellina cribraria, along with several other p-carboline alkaloids. l-Ethyl-4-methyl-sulfone-P-carboline (2) exhibited only modest antimicrobial activity, especially compared to some of the other alkaloids isolated, which were both cytotoxic and antimicrobial [27].

Two sulfur-containing isoquinoline alkaloids, 2-methyl-6,7di(methylthio)-2//-isoquinoline-3,5,8-trione (3) and 2-methyl-6methylthio-2H-isoquinoline-3,5,8-trione (4) were isolated from a

620

Tasmanian collection of the Australian bryozoan Biflustra perfragilis. The crystal structure of compound 3 was determined and it exhibited activity in the brine shrimp assay and against cultures of marine bacteria but was inactive against human bacterial pathogens [28]. Simultaneously, 7-amino-2-methyl-6-methylthio-"2//-isoquinoline-3,5,8trione (5) and compound 3 were reported from a South Australian collection of the same bryozoan by another research group and named perfragilins A and B respectively. The bryozoan was referred to as Membranipora perfragilis but is in fact the same species as B. perfragilis [28]. Both perfragilins showed activity against the P388 murine leukaemia cell line, with perfragilin B (3) being about one order of magnitude more active than perfragilin A (5) [29]. Single crystal Xray structures of perfragilins A (5) and B (3) were determined [30] and perfragilin B (3) was later synthesised [31]. These compounds are all structurally very similar to mimosamycin, which has been isolated from a terrestrial bacterium and from two marine sponges [28]. This suggests that the perfragilins are of bacterial origin.

Me-S

The Japanese fouling bryozoan, Dakaira subovoidea was the source of two 6//-anthra[l,9-Z?c]thiophene derivatives (6) and (7). The structure of compound 6 was determined by X-ray crystallography [32]. Both compounds were found to act as antioxidants and were patented for use as hypolipemics, but no other biological activity was reported [33]. Compound 7 has also been synthesised [34]. Our own studies of the bryozoan Watersipora subtorquata [35] led to the isolation of the closely related anthraquinone 5,7-dihydroxy-6-oxo-6//-anthra[l,9-Z?c] thiophene-l-carboxyhc acid (8), which was identified by nuclear magnetic resonance (NMR) and mass spectral analysis [36]. Compound 8 exhibited significant cytotoxicity against the African green monkey kidney cell line, BSC-1 [36].

621

6 R = CH2OH 7 R = C02Me 8 R = CO2H

Two ceramide 1-sulfates (9-10) have been obtained from the Japanese bryozoan Watersipora cucullata. These compounds are potent deoxyribonucleic acid (DNA) topoisomerase I inhibitors [37].

HO3SO,

C9H19 HO3SO.

C7H 7"15

Three new alkaloids, euthyroideones A, B and C (11-13) were isolated from the New Zealand bryozoan Euthyroides episcopalis [38]. All three compounds contain the unique heterocyclic pyrido(4,3-h)-l,4benzothiazine skeleton. The structure of euthyroideone A (11) was determined by X-ray crystallography, NMR spectroscopy and mass spectrometry. Euthyroideone B (12) exhibited modest cytotoxicity against the BSC-1 cell line [38].

11

12

13

Tunicates (Ascidians) Tunicates or ascidians belong to the phylum Chordata. Ascidiacea is a class of the subphylum Urochordata (Tunicata) and members of this

622

class are often referred to as tunicates or sea squirts, because their body is covered with a sack or tunic and many species expel water through a siphon when disturbed [39]. There are approximately 2,000 living species of tunicate and of these, ascidians are the most abundant. They may be solitary or colonial and are sessile, filter-feeding organisms [39]. Biologically active metabolites are quite conmionly found in ascidians and many of these compounds are derived from amino acids. Ascidiacyclamide (14), a cytotoxic, cyclic peptide, was isolated from an unidentified species of ascidian [40]. The absolute configuration was determined and the structure confirmed by total synthesis [41]. An Xray crystal structure was carried out [42] and further X-ray crystallographic studies determined the conformation of the molecule in the solid- and solution-states [43].

VN H

V

o=< \

y/^

NH

HN

\

14

Lissoclinum species of tunicates produce a range of cyclic peptides, which contain thiazole, thiazoline and oxazoline rings. Most belong to the general families patellamides (octapeptides), lissoclinamides (heptapeptides) or bistratamides (hexapeptides) [22]. The heptapeptide ulicyclamide (15) and the octapeptide ulithiacyclamide (16) were the first representatives of a series of cyclic peptides to be isolated from Lissoclinum patella. Their structures were elucidated by interpretation of spectral data [44]. A revised structure was later put forward for ulicyclamide (15) as a result of a detailed analysis of the fast atom bombardment (FAB) mass spectrum. The same paper reported the isolation of two more polar cyclic peptides and another, which was present as a minor component. These heptapeptides were called lissoclinamides 1-3 (17-19) [45]. An unidentified tunicate from the Great Barrier Reef contained ulithiacyclamide (16) and ascidiacyclamide (14) [46]. Two syntheses of ulithiacyclamide (16)

623

have been reported [47,48]. Ulicyclamide (15) was later also synthesised in high yield by solid phase synthesis [49]. The conformational properties of ulithiacyclamide (16) were probed using NMR spectroscopy and molecular mechanics calculations [50]. Ulithiacyclamide B (20) was isolated from L. patella from Pohnpei and was cytotoxic against the human oral carcinoma cell line, KB [51]. o

R

'•\. K'\ O

Ph O 15

\ ^

O

16 R = CH2CHMe2 20 R = CH2Ph

Y

^

17

18 Rj = Me, R2 = H 19Ri=H,R2 = Me

Three cyclic octapeptides, patellamides A-C (21-23) were isolated from L. patella and cytotoxicity data for these compounds and for ulicyclamide (15) and ulithiacyclamide (16) against L1210 murine leukaemia cells and the human acute lymphoblastic leukaemia (ALL) cell line CEM were reported [52]. The structures of the patellamides were later reassigned on the basis of synthetic studies. The proposed structures of patellamides B (22) and C (23) were synthesised and the products were shown to differ from the natural products. This led to new structures being proposed [53,54]. Separate syntheses of

624

patellamide A (21) [55], patellamide B (22) [56] and of patellamides B and C (22-23) [57] produced compounds that were identical to the natural products.

yr^

oK

21

o=(

y-T

22 R = CH2CHMe2 23 R = CH(Me)Et

The spectral data of the patellamides was also reasssigned and the new assignments used for elucidation of the structures of three new metabolites, the octapeptide prepatellamide B formate (24) and the heptapeptides, prelissoclinamide 2 (25), and preulicyclamide (26) [58]. The molecular conformation of patellamide A (21) was determined by X-ray crystallography [59,60] and the solution conformations of patellamides B (22) and C (23) were determined by NMR spectroscopy and molecular dynamics [61]. The octapeptide preulithiacyclamide (27) is a potent inhibitor of Macrophage Scavenger Receptor and was isolated from L. patella from Palau along with other known cyclic peptides [62].

\=M

H

^U

>=0

o\"r-NH HN-< \ P - -

O 59

^^ Ph 60

631

°y:A: ^"xrx VrT 61

62

63 R = Me 64R = H

A Didemnum sp. from Palau was the source of didemnaketal C (65), which contains a sulfonic acid group [95]. The in vitro anti-human immuodeficiency virus (anti-HTV) activity of D. molle from Pohnpei is associated with the sulfated mannose polysaccharide kakelokelose (66) [96].

o o o" o Me02C>,,^s!>s^AA,^^.->^^

-^Ov.^ OSOjNa HO-^JUQ;^

OSOsNa

NaOsSO-^^"^"^^

66

Lamellarin T-V and Y sulfates (67-70) were isolated from an unidentified ascidian from the Arabian Sea coast of India [97]. Four additional lamellarin sulfates, the 20-sulfates of lamellarins B, C and L and lamellarin G 8-sulfate (71-74) were isolated from Didemnum chartaceum from the Great Barrier Reef [98]. Unusually long relaxation times were observed for certain signals in the ^H NMR spectra of these compounds. Lamellarin a 20-sulfate (75) was isolated from an unidentified ascidian from India and was an inhibitor of human immunodeficiency virus type 1 (HTV-l) integrase [99].

632

.O^^O

R,0. R7O

OH

R.O

o R i OMe

OR4

OMeORs

67 Ri Ell = Me, R2 = OMe, R3 = H

71 ^5, R^ = SOsNa, R2 = Me, R3 = H, R4 = Me, R5 = Me, R^ = OMe,

68 Ri = Me, R2 = H, R = H

72 RJ = SOaNa, R2 = Me, R3 = H, R4 = Me, R5 = Me, Re = OMe

69 Ri = Me, R2 = OMe, R3= OH

73 R^ = S03Na, R2 = Me, R3 = Me, R4 = H, R5 = H, R^ = H

70 Ri = H, R2 = H, R3= H

74 Ri = Me, R2 = H, R3 = Me, R4 = H, R5 = S03Na, Re = H 75 Ri = S03Na, R2 = Me, R3 = H, R4 = Me, R5 = Me, Rg = H

Didemnum rodriguesi from New Caledonia contained the unusual peptidyl alkaloid caledonin (76), that formed a complex with Zn^"^ and Cu^ ions between thiol and primary amine groups [100]. The minalemines D-F (77-79) are peptide guanidine derivatives isolated from a Caribbean collection of D. rodriguesi and contain a sulfamic acid group [101]. The stereochemistry of cyclodidemniserinol trisulfate (80) from a Palauan specimen of Didemnum guttatum was partially determined [102]. f^

N

o NH2

. N O "

H2N

H N^ NH

H

»03?

9

O R

f "

NH

H O

'N H

77 R = C7H15 78R = C8Hn 79R = C9Hi9

OSOaNa NHS03Na

NaOSOj^ 80

U

NH2 ^

633

The structure of the cytotoxic metabolite dendrodoine (81) from the tunicate Dendrodoa grossularia was determined by X-ray analysis [103] and it was later synthesised by a convergent route [104].

cxA:^ V

N

.Me

Me

81

The eudistomins are p-carboline alkaloids isolated from Eudistoma olivaceum. Eudistomins C, E, F, K and L (82-86) all contain a novel oxathiazepine ring [105]. It was later proposed that the stereochemistry of eudistomins C, E, F, K and L (82-86) should be revised on the basis of a nuclear Overhauser enhancement difference spectroscopy (NOEDS) study of eudistomin K (85) from Ritterella sigillinoides [106]. Eudistomin K sulfoxide (87), an antiviral agent from /?. sigillinoides [107] was synthesised from eudistomin K (85), and the structure and absolute configuration of compound 85 were determined by X-ray analysis [108]. R. sigillinoides also contains debromoeudistomin K (88), in addition to known eudistomins [109]. A^(10)-Methyleudistomin E (89) was isolated from E, olivaceum from the Caribbean [110].

82 Ri = H, R2 = OH, R3 = Br, R4 = H

87

89

83 R, = Br, R2 = OH, R3 = H, R4 = H 84 Ri = H, R2 = OH, R3 = Br, R4 = C2H3O2 85 R, = H, R2 = H, R3 = Br, R4 = H 86 Ri = H, R2 = Br, R3 = H, R4 = H 88 Rj = H, R2 = H, R3 = H, R4 = H

Syntheses of both (-)-eudistomin F (84) [111] and of (-)-eudistomin L (86) [112] have been reported, while (-)-eudistomins C, E, F, K and L (82-86) were later also synthesised from the corresponding A^hydroxytryptamines and D-cysteinal [113].

634

Eudistomidin C (90) was one of three antileukaemic p-carboline alkaloids isolated from an Okinawan sample of Eudistoma glaucus. It was identified by spectral methods and synthesis of a derivative [114]. Eudistomidins E (91) and F (92) were isolated from E, glaucus from Okinawa and identified by spectroscopic techniques [115]. Eudistomidins C (90) and F (92) contain a methyl sulfide group while eudistomidin E (91) contains a methyl sulfoxide. 14Methyleudistomidin C, (93) was isolated from Eudistoma gilboverde along with known eudistomins [116]. 14-Methyleudistomidin C (93) exhibited potent cytotoxic activity with IC50 values less than 1 |Lig/mL against four human tumour cell lines.

SMe

Citorellamine (94) is an indole disulfide dihydrochloride from the Fijian tunicate Polycitorella mariae. It exhibits potent antimicrobial and insecticidal activity in addition to cytotoxicity [117]. The structure was later revised and syntheses of the proposed and true structures carried out [118].

N H 94

'NH k / S f .2HC1

A Didemnum sp. from Rota in the northern Mariana Islands contained four new p-carboline alkaloids, didemnolines A-D (95-98), together with known metabolites [119]. The didemnolines are characterised by substitution at N9 as opposed to CI. A straightforward synthesis of the didemnolines was reported [120].

635

MeS^N

1

Me

H

95 R = Br

97 R = Br

96R = H

98 R = H

Two antineoplastic 24-membered macrolide sulfates, iejimalides C (99) and D (100) were isolated from Eudistoma cf. rigida and identified by interpretation of spectral data [121]. o Me0s.^x^^x::^>s^^^^0^.Av^'10 |Lig/mL for kuanoniamine B (134), 5 [Xg/mL for kuanoniamine D (136), to 1 |Lig/mL for kuanoniamine A (133) [145]. Kuanoniamine A (133) has also been synthesised [146,147].

133

640

NHR 134 R = COCH2CHMe2 135 R = COEt 136 R = Ac

Polycarpamines A-E (137-141) are unusual sulfur-containing antifungal agents from Polycarpa auzata from the Philippines. The structures were elucidated by interpretation of spectral data [148]. Polycarpine (142), a cytotoxic, dimeric, disulfide alkaloid, the corresponding dihydrochloride (143) and two sulfur-containing related monomers (144-145) were isolated from Polycarpa clavata from Western Australia [149]. Polycarpine (142) was also isolated with two monomers (144,146) from P. aurata from Chuuk [150] and later it was synthesised in three steps from p-methoxyphenacyl bromide [151]. NMeo

NMe2

Y\S

MeO.

MCO^'YTC^ MeSS

R OMe

NMe7

MeO.

HO,

MeSS

MeOS

R

137 R = H

138 R = O

140 R = COMe

139 R = S

OMe

141

NH2 OMe

01U'

MeO

Me^^^Y

NH2

c

Me

VN

jr^'^" ..o^^

142

144 R = OMe

143 = .2HC1

145 R = OH

146

The in vivo antitumour activity of extracts of the tunicate Ecteinascidia turbinata was noted in the late 1960s [152] but the active metabolites were only isolated and identified much later by two research groups. These complex alkaloids were termed the ecteinascidins and are

641

abbreviated as Et with a number representing the value of the highest mass ion observed in the positive ion FAB mass spectrum. The Harbor Branch group [153] identified two compounds that were identical to ecteinascidins 729 (147) and 743 (148), identified at Illinois where compounds 745 (149), 759A (150), 759B (151) and 770 (152) were also reported [154]. The stereochemical representations at the 11,13 bridgehead differ between the two groups. Ecteinascidins 759A (150) and 759B (151) were tentatively assigned as A^-oxides of ecteinascidin 743 (148) [154]. X-ray crystal structures of the N12-formyl derivative of ecteinascidin 729 and of the natural N12-oxide (153) of ecteinascidin 743 (apparently different from compounds 150 and 151) were determined [155]. An enantioselective total synthesis of ecteinascidin 743 (148), which entered phase I clinical trials as an anticancer agent, has been reported [156] and synthesis of 148 from the fermentation product cyanosafracin B can provide sufficient quantity for clinical trials [157]. OMe HO^ Js^^Me

OMe H0,^Js^Me

MeO. J ^

147 Ri = H, R2 = OH

-NH

153

148 Rj = Me, R2 = OH 149Ri=Me, R2 = H 150 Ri = Me, R2 = OH, TV-oxide 151 Ri = Me, R2 = OH, N-oxide 152Ri=Me,R2 = CN

Ecteinascidins 597 (154), 583 (155), 594 (158) and 596 (158) are putative biosynthetic precursors of ecteinascidins and were isolated from £". turbinata from the Caribbean [158]. A recent review on the chemistry and pharmacology of the ecteinascidins has been published [159].

642

OMe

OMe

OMe

MeO

Four simple sulfates (158-161) were identified as antimicrobial constituents of Halocynthia roretzi from Japan [160]. Sodium (or potassium) 2,6-dimethylheptyl sulfate (161) was also found in Polycitor adriaticus from Croatia [161]. The absolute configuration of 2,6dimethylheptyl sulfate (161), which has also been found in other Mediterranean ascidians, has been determined using Mosher's method [162].

The Mediterranean ascidian Halocynthia papillosa contained two cytotoxic sulfates, 6-methylheptyl sulfate (162) and (F)-oct-5-enyl sulfate (163) [163]. ^OSOjNa

^OSOjNa 162

163

Ascidia mentula from the Mediterranean was the source of two antiproliferative alkyl sulfates, sodium salts of 3,7,11,15tetramethylhexadecane-l,19-diyl disulfate (164) and heneicosane-1,21diyl disulfate (165) respectively [164]. Microcosmus vulgaris, also

643

collected in the Mediterranean, was the source of the sodium (or potassium) salt of (3Z)-4,8-dimethylnon-3-en-l-yl sulfate (166) [165]. ^OSOgNa OSO.Na 164 NaO^; 165

166

The Mediterranean tunicate Sidnyum turbinatum contained four alkyl sulfates, 1-heptadecanyl sulfate (167), 1-octadecanyl sulfate (168), sodium (25)-2,6,10,14-tetramethylpentadeca-l,18-diyl sulfate (169) and 1-hexyl sulfate (170). The structures were determined by spectroscopic and chemical methods. All exhibited antiproliferative activity in vitro against the murine fibrosarcoma cell line, WEHI 164 [166]. NaO.SO

NaOiSO 168

167 ^0S03Na NaOaSO

169

Na03S0 170

The structure of polyclinal (171), an aromatic sulfate from a Califomian specimen of Polyclinum planum, was determined by X-ray crystallography [167]. OH ^CHO ^OSOsNa OH 171

644

Uoamines A (172) and B (173) are piperidine alkaloids, isolated from Aplidium uouo from Maui, Hawaii. They differ only in the geometry of the 3-thiomethylacrylate ester group [168]. OH

A^A^ O

SMe

H 172

173

Tasmanian collections of Clavelina cylindrica yielded the alkaloids cylindricines F (174) and G (175), the first thiocyanates isolated from an ascidian [169]. Cylindricines H-J (176-178) were later isolated from the same species [170].

,0Ac

,0Ac SCN

NCS"

RH2C

174R = (CH2)4Me

176 R = SCN

175 R = (CH2)2Me

177 R = NCS

A Micronesian ascidian, Nephteis fasicularis, was the source of fasicularin (179), a tricyclic, thiocyanate-containing alkaloid that was active in a DNA damaging assay [171]. The structure was confirmed by total synthesis [172]. C^Hv NCS.

The virenamides A-C (180-182), thiazole-containing cytotoxic linear peptides, were isolated from the colonial ascidian Diplosoma virens collected on the Great Barrier Reef, Australia. Their structures were

645

deduced from NMR spectral data and confirmed using Marfey's procedure [173]. Virenamides D (183) and E (184) were also obtained from D. virens from the Great Barrier Reef [174] and virenamide B (181) has been synthesised [175].

^ . \

,,

180

181 R = CHMej 182 R = CHjPh

183

184

An enediyne antitumour antibiotic, namenamicin (185) was isolated from Polysyncraton lithostrotum from Fiji [176].

NHC02Me

HO-^'

wo

o^

Y

OH

HN.,

185

646

Cnidaria (Coelenterates) The Cnidaria comprise about 8,000 living species and include jellyfish, corals, soft corals or gorgonians, sea anemones and hydrozoans. They are the lowest members of the animal kingdom with cells organised into specialised organs [177]. Cnidarians have a single internal cavity, which acts as a stomach and a single opening above, which is encircled by tentacles and through which food enters and waste escapes [178]. Some Cnidaria are solitary and consist of a single polyp such as sea anemones and others are colonial such as corals but all Cnidaria are radially symmetrical [178]. Many have nematocysts or stinging cells but these organisms are less likely to contain secondary metabolites for use in chemical defence, as they are not really required. Terpenoids are very commonly isolated from this phylum but very few sulfur-containing compounds have been found in Cnidarians. The marine hydroid Tridentata marginata contained the aromatic alkaloids tridentatols A-C (186-188). Tridentatol A (186) inhibited feeding by the planehead filefish. The structure of tridentatol C (188) was elucidated by a single crystal X-ray diffraction study [179]. ^QXJ

SMe

^QXJ

NySMe

kjk ^

SMe 186

1 >-SMe

187

188

^

A zoanthid from the Indian Coast, Zoanthus sp., contained the sulfated sphingolipid hariamide (189) [180]. o C9H19

Two new ultraviolet (UV) absorbing compounds, palythrinethreonine-sulfate (190) and palythrine-serine-sulfate (191) were isolated from the reef-building coral Stylophora pistillata [181].

647

jj

R

OMe

OH yC HO ^OSOjH 190R=Me 191 R = H

The sea anemone Anthopleura elegantissima was the source of the sulfonic acid-containing compound mycosporine-taurine (192) [182], o HOH2CJ X

^

SOH

H 192

Molluscs The phylum MoUusca comprises approximately 100,000 species, making it one of the largest animal phyla [177]. The name mollusc means "softbodied". Molluscs are non-segmented, have a head with tentacles and move by crawling on a foot. For bivalves such as mussels and oysters, the foot is a digging tool and for cephalopods such as squid and octopuses, it is formed into tentacles. The outer body covering is termed the mantle and usually secretes a shell to protect the body [183]. The shell-less molluscs such as the carnivorous nudibranchs (sea slugs) and herbivorous ophistobranchs (sea hares), are well known sources of bioactive secondary metabolites but in many cases the mollusc itself does not produce the compound but sequesters it from its diet. Similarly, filter-feeding bivalves have been the sources of large toxic compounds but the actual producers of these compounds are thought to be microorganisms. Adenichrome is an Fe (Ill)-containing pigment from bronchial heart of Octopus vulgaris. It consists of a mixture of closely related peptides derived from glycine and the isomeric amino acids adenochromines A, B and C (193-195) [184,185].

648

.CO2H

193 Ri= ^-S^

NH

. R,= f - S

R4N^N

194Ri = H.

R2= ^-S^ =(

^pNH2

h

R3 = H

R4 = H or Me .CO2H R3= ^ NH2 R4N^N

R4 = H or Me 195 R

NH9

R4N^N

R4 = H or Me CO2H

ro^H /-


256

255

254

The structure of 5-isothiocyanatopupukeanane (257), a sesquiterpene isothiocyanate from an Axinyssa species from Guam, was determined by X-ray analysis [260]. Two isomeric sesquiterpene thiocyanates, 2thiocyanatoneopupukeanane (258) and 4-thiocyanatoneopupukeanane (259) were isolated from an unidentified sponge from Pohnpei and from Phycopsis terpnis from Okinawa [261]. A sample of Axinyssa (= Trachyopsis) aplysinoides from Palau yielded a rare thiocyanate, 2thiocyanatopupukeanane (260), while two specimens from Pohnpei yielded 13-isothiocyanatocubebane (261), 1-isothiocyanatoaromadendrane (262) and 2-thiocyanatoneopupukeanane (258) [262]. This last compound had previously been assigned different stereochemistry at C2 [261]. (-)-4Thiocyanatoneopupukeanane has been synthesised in an enantiospecific manner (259) [263]. Both enantiomers of 2-thiocyanatoneopupukeanane (258) have been synthesised from (/?)-carvone [264].

SCN\

NCS

,

. H '

258 Ri = SCN, R2 = H ^^'

259Ri=H,R2 = SCN

^60

261

262

A sesquiterpene isothiocyanate, halipanicine (263) has been isolated from an Okinawan specimen of Halichondria panicea [265]. The relative stereochemistry of halipanicine (263) was established by synthesis [266] and later, a total synthesis was achieved [267].

SCN

CO w 263

662

Three new antiparasitic sesquiterpene isothiocyanates, 4isothiocyanato-9-amorphene (264), 10-isothiocyanato-4,6-amorphadiene (265) and 10-isothiocyanato-5-amorphen-4-ol (266) were isolated from a Fijian specimen of Axinyssa fenestratus. The compounds were identified by spectral data interpretation [268]. Two isomeric isothiocyanates (267268) were isolated from Acanthella klethra from the Great Barrier Reef and their structures were determined by X-ray crystallography and spectral data examination [269]. . .Ncs SCN»

A sesquiterpene thiocyanate, cavemothiocyanate (269) was isolated from Acanthella cf. cavernosa and the structure was elucidated on the basis of spectral data. The nudibranch Phyllidia ocellata also contained cavemothiocyanate [270]. Acanthene B (270) is a sesquiterpene isothiocyanate isolated from a British Columbian Acanthella sp. [271]. The sesquiterpene thiol, T-cadinthiol (271) was isolated from Cymbastela hooperi from Kelso Reef on the Great Barrier Reef [272]. A sesquiterpene isothiocyanate that displayed modest in vitro antimalarial activity, (-)-9-isothiocyanatopupukeanane (272) was isolated from an Axinyssa sp. from the Great Barrier Reef [273]. Great Barrier Reef samples of A. cavernosa contained lO-isothiocyanatocadin-4-ene (273) [274]. H\^SH

269

^^^

270

271

^^^^f^

272

HTNCS

273

Two isothiocyanates, epipolasins A and B and the corresponding (3phenylethylamine adducts, epipolasinthioureas A (274) and B (275) were isolated from the sponge, Epipolasis kushimotoensis. The structures of the epipolasins were determined by chemical degradation to known compounds [275]. The structure of epipolasin A is identical to that

663

previously assigned to a metabolite of the nudibranch Cadlina luteomarginata (240) [249] and the physical data is also similar except for the sign and magnitude of the optical rotation. The structure of epipolasin B is identical to that previously assigned to axisothiocyanate 2 (232) [239]. Synthesis of (-)-(10/?)-10-isothiocyanoaromadendrane indicated that it was the enantiomer of epipolasin B, previously isolated from E, kushimotoensis [276]. .-'^"S"'^

w)

CQ ^K!^

^

240 R = NCS

232 R = NCS

274 R = NHC(S)NHCH2CH2Ph

275 R = NHC(S)NHCH2CH2Ph

The structure of a diterpenoid isothiocyanate (276) extracted from a Halichondria sponge, was determined from chemical and spectral data [277].

NCS 276

Kalihinols G (277) and H (278) were trace components of a species of Acanthella from Guam and kalihinol X (279) was isolated from a Fijian species of Acanthella, All inhibited growth of Bacillus subtilis. Staphylococcus aureus and Candida albicans [278]. 10-Epi-isokalihinol H (280) and 15-isothiocyanato-l-epi-kalihinene (281) were isolated from Acanthella cavernosa from the Seychelles [279]. A Japanese specimen of A. cavernosa contained a sesquiterpene isothiocyanate (282) and lOPformamido-5P-isothiocyanatokalihinol A (283). Structures were assigned by spectral data interpretation [280]. Phakellia pulcherrima from the Philippines contained the minor diterpene isothiocyanates kalihinol L (284), 10-isothiocyanatokalihinol G (285), 10-epi-kalihinol H (286) and 10-isothiocyanatokalihinol C (287) [281]. 10-Epi-kalihinol I (288) and 5,10-bisisothiocyanatokalihinol G (289) were isolated from an Acanthella sp. from Okinawa [282].

664

HOJ

HOj

1 HJ

1 HJ THJ[>

C N ^

A^^

NCS

H CI

279

277 Ri = NC, R2 = NCS

280

281

278 Ri = NCS, R2 = NC

NCS

H\.NHCHO

^K

- ^

282

283

SCN ^yJC P

SCN V C

CN V Q

CI

NCS

NCS

NCS

NCS

HO-

289

A Japanese sponge of the Adociidae family contained 10isothiocyanatobiflora-4,15-diene (290), which was identified by spectral analysis [283]. A. cavernosa from Fiji yielded a diterpene isothiocyanate (291) [284]. NHCHO

NCS

NCS 291

665

Cymbastela hooperi from the Great Barrier Reef contained four diterpene isothiocyanates (292-295) amongst other diterpenes [285]. An amphilectene isonitrile (296) was isolated from a Caribbean Cribochalina sp. [286]. NCS

NCS

A series of eighteen long chain, aUphatic a,a)-bis-isothiocyanates (297-314) and three a-isothiocyano-c?-formyl analogues (315-317) was isolated from a Fijian species of Pseudaxinyssa [287]. The major constituents (297), (305) and (315) all have the same length of aliphatic chain (CI8). Unlike terpenoid isothiocyanates, this series was not accompanied by the corresponding isocyanides or formamides. SCNr ^ ( C H 2 ) n / ^ ^ I NCS

SCN-(CH2)n

297n=14 301n = l l

3 0 5 n = 1 6 310n = 13

315 n = 15

298n = 8

302n = 12

306n = 9

316 n = 9

299n = 9

303n=13

3 0 7 n = 1 0 312n = 15

300n=10

304n=14

308n=ll

313n=17

309n=12

314n=18

NCS

311n=14

SCN

(CH2)n-CH0

317n=16

Dysidea herbacea contains linear polychlorinated peptides with a thiazole residue. The metabolites can be divided into the dysidenin, the isodysidenin and the dysideathiazole series of compounds [22]. Dysidenin (318) was isolated from D. herbacea from Cooktown, Australia without stereochemical assignments [288]. The structure of isodysidenin (319), isolated from a sample of D, herbacea from Papua New Guinea, was determined by X-ray diffraction analysis [289]. It was proposed that the two compounds differ in stereochemistry at C5 [290]. The absolute configurations were later revised [291].

666

Me

^H JL

Me

Q H

318 R = Me 325 R = H

*^

-^H J ^

Q H '^

319 R = Me, X = CI, Y = CI 322 R = H, X = CI, Y = CI 323R = H,X = C1,Y = H 324R = H,X = H,Y = C1

Two thioacetates, thiofurodysin acetate (320) and thiofurodysinin acetate (209) were isolated from a Dysidea species from Sydney, Australia. They were converted by treatment with Raney nickel to a mixture containing furodysin and furodysinin respectively [214]. These were the first thiol acetates isolated from natural sources. The absolute configurations of (-)-(6/?,ll/?)-thiofurodysinin acetate (209), (-)-(6/?,ll/?)furodysinin disulfide (208) and (+)-(6/?,ll/?)-methoxythiofurodysinin acetate lactone (321), isolated from a Fijian specimen of D. herbacea were determined by chemical interconversion [292]. H

Acs

HA 320

^o

^""YT^^) ^""^^

OMe O

^ 209 R = SAc

321

208 R = -S-S- (dimer)

A collection of D. herbacea from near Bowen, Australia yielded 13demethylisodysidenin (322), 11 -monodechloro-13-demethylisodysidenin (323) and 9-monodechloro-l3-demethylisodysidenin (324), all derivatives of isodysidenin, and a dysidenin derivative, 13-demethyldysidenin (325). 13-Demethylisodysidenin (322) and 13-demethyldysidenin (325) were epimeric at C5 by direct comparison of the dechlorinated derivatives of each [293]. Syntheses of both (+)-13-demethyldysidenin (325) and (-)-13demethylisodysidenin (322) have been described [294]. The results of this synthetic study imply that absolute configurations at C2 and C7 in all of the natural materials are 5, opposite to those assigned by X-ray crystallography to isodysidenin (319). In an Australian specimen of D, herbacea, 13-demethylisodysidenin (322) was found to be localised in cells of the cyanobacterium Oscillatoria spongeliae, while two sesquiterpenes were associated with the sponge cells [295].

667

Thiofurodysinin (326), a furanosesquiterpene from Dysidea avara from Australia, was the first report of a sesquiterpene mercaptan from a sponge [296]. HS^V^r^^x 326

A Palauan species of Dysidea contained 15-acetylthioxyfurodysinin lactone (327), that binds to human leukotriene B4 (LTB4) receptor. The structure was determined by spectral data analysis and confirmed by synthesis involving photo-oxidation of 15-acetylthioxyfurodysinin (328), which co-occurs with it in the sponge [297,298]. An Australian species of Euryspongia also contained 15-acetylthioxyfurodysin (329) and 15acetylthioxyfurodysinin (328) [299]. A sample of Z). herbacea from the Great Barrier Reef contained (-)-neodysidenin (330) and the absolute configuration was determined by capillary electrophoresis of Marfey's derivatives [300]. H OH AcS''"V**'"'^f'^^^^>^Q

Acs

H/ 327

H

329

OH

W

NH

330

The dysideathiazoles A-E (331-335) are a series of polychlorinated amino acid derivatives from Pacific Island collections of D. herbacea. The structures were determined by X-ray analyses and the absolute configurations were determined by X-ray crystallography of a brominated derivative [301]. Herbamide A (336), a chlorinated amide was isolated from a Papua New Guinean sample of D. herbacea as a minor component [302]. D. herbacea from the southern Great Barrier Reef contained a thiazole (337) amongst other known metabolites [303]. A Dysidea sp.

668

from Okinawa contained the benzothiazole S1319 (338), as a Padrenoreceptor agonist [304]. XCI2C.

CCI2Y

331 R = H, X = CI, Y = CI

336

332 R = Me, X = CI, Y = CI 333R=:Me,X = Cl,Y = H 334 R = H, X = CI, Y = H 335 R = Me, X = H, Y = H Me ^ C ^ N ^ , CHCI2

NFP 337

NHMe

y

338

Dysidea avara from the Solomon Islands contained the melemeleones A (339) and B (340), which were identified by spectroscopic analyses [305]. They consist of a sesquiterpene linked to a quinone with an attached taurine residue [22].

SOaH

340

Dysidea sp. from Bararin Island in the Philippines, has yielded the dysideaprolines A-F (341-346), proline-derived analogues of dysidenin (318). The barbaleucamides A (347) and B (348), which are structural analogues of the cyanobacterial metabolite barbamide, were also isolated. The structures were elucidated by NMR spectroscopic analysis. It is most probable that all of these compounds are derived from a symbiotic cyanobacterium found in close association with the Dysidea sp. [306].

669

OMe

^^A, X2HC' " ^ ^N R2

Cl^C

R .^N,^-^CCl3

O

341 Ri = H, R2 = Me, X = CI, R3 = CHCI2

347 R = H

342 Ri = Me, R2 = Me, X = CI, R3 = CHCI2

348 R = Me

343 Ri = H, R2 = H, X = CI, R3 = CHCI2 344 Ri = H, R2 = Me, X = H, R3 = CHCI2 345 Ri = H, R2 = Me, X = CI, R3 = CH3

346 Ri = H, R2 = Me, X = CI, R3 = CH2CI

The burrowing sponge Siphonodictyon coralliphagum and other species of the same genus, contain a series of sesquiterpene hydroquinones. Siphonodictyal D (349), and siphonodictyols G (350) and H (351) occur as sodium sulfates and the structure of siphonodictyal D (349) was determined by X-ray crystallography [307]. A deep water collection of S. coralliphagum contained bis(sulfato)cyclosiphonodictyol A (352) which inhibits binding of LTB4 to human neutrophils [308]. Na03S0,^^^CH0

349

OH

350

Na03S0^^

Na03S0-f3-0S03Na "CH2OH

351

Na03S0

670

Agelas nakamurai from Japan produced the sesquiterpene sulfone, agelasidine A (353), which possessed antispasmodic activity. The structure was deduced from spectral data [309]. A simple synthesis of agelasidine A (353) utilised a hetero-Claisen rearrangement [310]. A biomimetic synthesis of 353 was also reported [311] and another synthesis of agelasidine A (353) was carried out in three steps from famesol [312].

353

Two diterpene derivatives of hypotaurocyamine, agelasidines B (354) and C (355) were also isolated from Agelas nakamurai The structures were determined by interpretation of spectral data. Both are antimicrobial, inhibit smooth muscle contraction and enzyme activity of Na"*'/K"^transporting adenosine triphosphate (ATP)ase [313]. Agelasidine C (355) has been synthesised [314]. (-)-Agelasidine C (356) and (-)-agelasidine D (357) were isolated from the Caribbean sea sponge Agelas clathrodes. The structures were confirmed by interpretation of the spectral data and by comparison of this data with those of the known antipode (+)-agelasidine C (355) [315]. NH

o \ 354

355

O'^O

356

H .N>^NH2 NH

NH

357

Suvanine (358), an acetyl cholinesterase inhibitor was first isolated from species of Ircinia [316] and then later from a Coscinoderma species from Fiji and Palau when the structure was revised [317].

671

Me,

+ NHo

Me

NHo

A Califomian sponge of the Halichondriidae family contained a sulfated sesterterpene hydroquinone and five sulfated sesterterpenes. The structures of the halisulfates 1-5 (359-363) were determined by interpretation of spectral data and a structure was proposed for halisulfate 6 (364). The halisulfates are antimicrobial and antiinflanmiatory [318]. The absolute configuration of halisulfate 3 (361), which was also isolated from Ircinia sp. from the Philippines, has been determined by application of the chiral amide method and by chemical degradation techniques [319]. Halisulfate 7 (365) is a sesterterpene sulfate from a Coscinoderma sp. from Yap, Micronesia [320].

359

360 NaOgSO-

CH20S03Na-0 361

362

672

NaOaSO'

Bioassay directed isolation of serine protease inhibitors from Coscinoderma mathewsi yielded the 1-methylherbipoline salts (366-367) of known sesterterpenes halisulfate-1 (359) and suvanine (358) [321]. Coscinosulfate 1 (368), a sesquiterpene sulfate, was isolated from a New Caledonian collection of C mathewsi. It displayed significant activity as an inhibitor of the protein phosphatase Cdc25 [322]. A total synthesis starting from (+)-sclareolide was described [323].

OSOa

^O

Me

367

OS03Na

Sulfircin (369), an antifungal sesterterpene sulfate was isolated as the N,A^-dimethylguanidinium salt from a deepwater Ircinia species and its structure was determined by X-ray analysis [324]. Two sesterterpene sulfates, hipposulfates A (370) and B (371), were isolated from Hippospongia cf. metachromia from Okinawa and their structures were elucidated by interpretation of spectroscopic data. Both compounds possess an enolsulfate functionality [325].

673

oso

NaO^SO^

370R = H 371 R = OH

Akaterpin (372) is an inhibitor of phosphatidylinositol-specific phospholipase C from a Callyspongia sp. [326]. The relative stereochemistry of the ring junction in the upper decalin moiety of akaterpin was shown to be cis by synthesis of model compounds [327]. NaOsSO-f

VoSOgNa

Four unstable sulfate esters (373-376) of known furanosesterterpenes were obtained from Ircinia variabilis and from /. oros from the northern Adriatic Sea [328]. The 22-(9-sulfates of palinurin (377) and fasiculatin (378) were isolated from /. variabilis and from /. fasiculata respectively. Both were toxic to brine shrimp [329]. Ircinianin sulfate (379) was isolated from /. (Psammocinia) wistarii from the Great Barrier Reef as a very unstable metabolite [330]. PSO3K

674

379

Adociasulfates 1-6 (380-385) were isolated from a Haliclona (aka Adocia) sp. from Palau and were all inhibitors of kinesin motor proteins [331]. Adociasulfate 2 (381) had earlier been shown to inhibit the activity of the motor protein kinesin by interference with its binding to microtubules [332]. An Adocia sp. from the Great Barrier Reef contained adociasulfates 1 (380), 7 (386) and 8 (387), which inhibit vacuolar YCATPase [333]. Adociasulfates 5 (384) and 9 (388) were obtained from Adocia aculeata from the Great Barrier Reef [334]. The structure of adociasulfate 1 (380) was confirmed by an enantioselective total synthesis [335]. Adociasulfate 10 (389) from Haliclona sp. from Palau also inhibits the kinesin motor proteins [336].

NaOaSO-ZT^OSOsNa

HO^: 380Ri = SO3Na,R2 = SO3Na

381R = S03Na

384 Ri = SOsNa, R2 = H

385 R = H

386 Ri = H, R2 = SOsNa

382

675

H0-/^-0S03Na

OSOaNa

387

383

NaOaSO.

COoH

OSOaNa

389

388

Shaagrockols B (390) and C (391) from the Red Sea sponge Toxiclona toxius, are antifungal hexaprenylhydroquinone disulfates and were identified by spectral data interpretation [337]. Toxicols A (392), C (393) and toxiusol (394) are hexaprenoid hydroquinones that were also isolated from Toxiclona toxius. The structures were determined by spectral data examination [338].

390

OSOaNa

GSOsNa

0S03Na

OSO^Na

676

OSOsNa

OSOgNa

392 R = SOgNa

394

393 R = H

A hexaprenyl-hydroquinone sulfate (395) was identified as an H^/K"^ATPase inhibitor from a Japanese species of Dysidea [339]. Sarcotragus spinulosus from deep water contained the Na"^/K"^-ATPase inhibitors sarcochromenol sulfates A-C (396-398) and sarcohydroquinone sulfates A-C (399-401) [340]. The structures were determined by spectral data analysis of the natural products and of derivatives.

.rd"*^"

NaOaSO'

396 n = 5 397 n = 6 398 n = 7

OSOaNa 399 R = H, n = 6 400R = SO3Na,n = 7 401 R = H, n = 8

A heptaprenylhydroquinone derivative (402) was isolated from an Indian sample of Ircinia fasciculata [341]. Ircinia spinulosa from the Adriatic contained three sulfated 2-prenylhydroquinones (403-405) that are toxic to brine shrimp [342]. An Ircinia sp. from New Caledonia contained a sulfated 2-prenylhydroquinone (406) and a sulfated furanoterpene (407) [343]. An Australian Sarcotragus sp. contained octaprenylhydroquinone sulfate (408) and nonaprenylhydroquinone sulfate (409) as inhibitors of al,3-fucosyltransferase VII [344].

677

OSO^Na

402

ry^^^yiry^

0S03Na OSO^Na

407

The yellow pigment halenaquinol sulfate (410) has been isolated from the Okinawan sponge Xestospongia sapra and is a pentacyclic hydroquinone [345]. The absolute stereochemistry was determined to be 65 by comparing the CD spectrum of a derivative with a theoretically calculated spectrum [346]. Theoretical calculation of CD spectra of halenaquinol sulfate (410) isolated from X exigua and X sapra determined that the absolute stereostructure was 12b5' [347]. The pentacyclic compound, xestoquinol sulfate (411) has been isolated from an Okinawan collection of X. sapra and its structure was elucidated on the basis of spectroscopic data and a chemical conversion [348]. NaOjSO. OSOaNa

Xestoquinolide B (412) was obtained from Xestospongia cf. carbonaria and the protein kinase activity of it and related compounds reported. The structure of this merosesquiterpenoid was elucidated by spectral data interpretation [349]. A Xestospongia species from the

678

Philippines contained the topoisomerase n inhibitors, secoadociaquinones A (413) and B (414) [350]. HN"^

0 ' "SOjNa NH

HNO'ITV ^ , ^ C O N H

426

680

Halicylindramides A-C (427-429) are antifungal and cytotoxic depsipeptides from Halichondria cylindrata from Japan [358]. Two additional peptides, halicylindramides D (430) and E (431) of which the former is antifungal and cytotoxic, were also isolated from a Japanese collection of//, cylindrata [359]. H V.

.

O

/

***, „ ' O

^ ^ ° / O rf^ H

Ph

CONH2 427 Ri = H, R2 = Me 428 Ri = Me, R2 = H 429 Ri = Me, R2 = Me j ^

O^^NHCHO

r S

?ONH2

Ph O H2N

N "

H2NOC

u ^NH O

-^

430

Br-f

V-,

O. NHCHO A-
-(L-proline-L-thioproline) (498) was isolated from Tedania ignis but a bacterial origin for the metabolite was suggested [423]. Cyd(7-(L-proline-L-methionine) (499) was isolated from Pseudomonas aeruginosa associated with the Antarctic sponge, Isodictya setifera. The structure was elucidated by spectroscopic methods and confirmed through synthesis [424]. o

o

498

499

o

693

There have been three reports of the same dimeric disulfide. It was first isolated from an unidentified sponge from Guam and the structure elucidated by analysis of spectral data. The (E,E) stereochemistry of the disulfide (500) was defined by comparing the ^^C NMR spectroscopic data with those of the (E,Z)Asomer (501) that was obtained as an unstable minor product [425]. Compound 500 was isolated from a species of Psammaplysilla and was called psammaplin A [426]. It was also isolated from Thorectopsamma xana, collected from the same location in Guam, together with a minor dimeric metabolite bisaprasin (502). Both compounds inhibited growth of Staphylococcus aureus and Bacillus subtilis [427]. Psammaplin A (bisprasin) (500) was later isolated from a Dysidea species of sponge and shown to act on Ca^^-induced Ca^"^ release channels of skeletal muscle [428].

500n= !(£,£) 501 n = 1 (E,Z) 502n = 2

Four minor metabolites, psammaplins B-D (503-505) and presammaplin A (506) were isolated from Psammaplysilla purpurea, in addition to psammaplin A (500). Psammaplin B (503) is a thiocyanate bromotyrosine derivative, while psammaplin C (502) is a sulfanamide. Psanmiaplin D (505) displayed antimicrobial activity and mild tyrosine kinase inhibition [429]. The psammaplins Ai (507) and A2 (508) and aplysinellins A (509) and B (510) were isolated from Aplysinella rhax from both Pohnpei and Palau. These compounds inhibit famesyl protein transferase and leucine aminopeptidase [430]. Another sample of A. rhax from the Great Barrier Reef, Australia contained psammaplin A 11'sulfate (511) and bisaprasin 11'-sulfate (512), both of which inhibited [ H]-l,3-dipropyl-8-cyclopentylxanthine binding to rat brain adenosine Ai receptors [431].

694

O

H

T

H

0

HO' 506

503 R = SCN 504 R = SO2NH2 505 R=

—S-S^^^^^

o N H

A

OMe

507 Ri = H, R2 = SO3', n = 1 508 Ri = SO3-, R2 = 503', n = 2 511 Ri = H, R2 = S03Na, n = 0

^^H

OH Br Br^

HO2C

695

34-Sulfatobastadin 13 (513) is an inhibitor of endothelin A receptor from lanthella sp. from the Great Barrier Reef [432]. Three new bastadin analogues including 15,34-0-disulfatobastadin 7 (514) and lO-Osulfatobastadin 3 (515) were isolated from lanthella basta from Exmouth Gulf, Western Australia. They showed moderate differential activity as sarcoplasmic reticulum-Ca^'*"-channel agonists of the skeletal muscle receptor-protein complex, RylR FKBP12 [433]. NOH

ON,5j^^Na03SO ^>Ss^Br

NOH NOH

0--V^Br 0S03Na

NaOjSO^ H(

OlP

v^

Br' NOH 514

NOH 515

lantherans A (516) and B (517) are dimeric tetrabrominated benzofuran derivatives that were isolated from an Australian lanthella species. The structures were determined by spectroscopic and chemical methods. lantheran A (516) includes a (Z,Z)-1,3-butadiene moiety, whereas iantheran B (517) is the geometric isomer possessing a (Z,£:)-1,3butadiene moiety. Both compounds were Na"^/K'^-ATPase inhibitors [434,435]. lanthesines C (518) and D (519) showed potent NaVK^-

696

ATPase activity and are additional dibromotyrosine derivatives from an Australian lanthella sp. [436].

516

517 R = S03Na

MeO

.SOgNa H

N^xv^O

OMe 518

Br,

i)H

MeOBr HN^^^^^Oyk

Hj^^S03Na

B,A:A^C02H 519

A two Sponge association of a thin crust of Haliclona sp. overlaying an unidentified choristid (probably not Jaspis) sponge contained two enol sulfates, presumed to be from the choristid sponge [437]. These enol sulfates were also found as the sodium salts jaspisin (520) [438] and isojaspisin (521) [439] and (£)- and (Z)-narains (522-523) [440] from

697

Japanese specimens of Jaspis sp. The jaspisins (520-521) inhibited hatching of sea urchin embryos and the narains (522-523) induced metamorphosis in ascidian larvae. Three 3,4-dihydroxystyrene sulfate dimers (524-526) were also isolated from the same Jaspis species [441]. H0^^^5yx%^OS03-R

H0.,^ - !NH2 ^ - - NT

ROsSO' 6SO3R 533

A Sterol disulfate (534) and a sterol trisulfate (535), both closely related to halistanol, have been found in a species of Halichondria [447] and in Trachyopsis halichondrioides [448] respectively.

699

.-kAJ

H4N"'03SO'

OSOsNa 534

535

A Japanese species of Epipolasis contained five sterol sulfates named halistanol sulfates A-E (536-540), which differ from the original halistanol sulfate (532) from Halichondria moorei [449]. Structures were elucidated by spectroscopic and chemical techniques. Halistanol sulfates F-H (541543) are three additional sterol sulfates from Pseudaxinyssa digitata that inhibit HIV in vitro [450].

>s.-4Ct/

Na03S04 3380

H • 0S03Na

b=

536 Ri = a, R2 = H 537 Ri = b, R2 = H

c=

538 Ri = c,R2 = H 539 Ri = d, R2 = H

d=

540 Ri = e, R2 = O H

The sterol sulfate, halistanol disulfate B (544) was isolated from a South African Pachastrella sp. The structure and stereochemistry of compound 544 were established mainly by interpretation of spectral data. Halistanol disulfate B (544) was active in the endothelin converting enzyme (ECE) assay at a micromolar concentration [451]. Three sterol trisulfates (545-547) have been isolated from the sponges Trachyopsis halichondrioides and Cymbastela coralliophila [452].

700

R Na03Sa

I

I J H

NaOjSO' OSOgNa 544

545 R = ^^.^.^-^^ 546 R = ^^Y^ 'VV,

547 R =

f

The structure of sokotrasterol sulfate (548), isolated from sponges of the family Halichondriidae was determined by X-ray analysis [453-455]. The steroid, 26-norsokotrasterol sulfate (549), was isolated from the marine sponge Trachyopsis halichondrioides and was identified by NMR spectroscopic analysis [456].

HOaSQ HO3SO' OSOjNa 548

Ibisterol sulfate (550) is a sulfated sterol from a deepwater Topsentia sp. that was cytoprotective against HIV-1 in the NCI primary screen [457].

0S03Na 550

701

A Topsentia sp. from Okinawa contained five antimicrobial 14-methyl sterol sulfates, topsentiasterol sulfates A-E (551-555) [458]. Ophirapstanol trisulfate (556) from deepwater Topsentia ophiraphidites showed inhibition in the guanosine diphosphate/G-protein RAS exchange assay [459].

Na03S04

I^T'TI''^

NaOjSO'' HO ^ OSOjNa 551 R =

^ "r"^^ '^ o V-OH 552 R = ^^t^^r=\ -OH HO^O

553 R =

o-^^o 554 R =

il \ O

555 R =

OSOaNa 556

702

An unusual 6a-sterol sulfate (557) was isolated from Dysideafragilis, from the Venetian lagoon and displayed cytotoxicity against two different tumour cell lines in vitro [460]. Tamosterone sulfates (558-559) are a C14 epimeric pair of polyhydroxylated sterols isolated from a new species of Oceanapia [461]. The Japanese marine sponge Epipolasis sp. contained the steroid polasterol B sulfate (560) along with the known compound halistanol sulfate (532). The structure of compound 560 was determined on the basis of spectroscopic evidence and a chemical conversion [462].

NaOjSQ

HO Y Y ^OH GSOsNa

OH OH 558 R = a-H 559 R = P-H

557

NaOaSO^i 4 y . ^ L i x ^ Na03S0' OSOjNa 560

An Acanthodendrilla sp. from Japan contained ten steroidal sulfates, acanthosterol sulfates A-J (561-570). Acanthosterol sulfates I (569) and J (570) showed antifungal activity against Saccharomyces cervisiae and its mutants [463]. Clathsterol (571), was isolated from the Red Sea sponge Clathria sp. The structure was established mainly by interpretation of spectral data and a chemical transformation. Clathsterol (571) was active against HIV-1 reverse transcriptase (RT) at a concentration of 10 |LiM [464]. Toxadocia zumi contains three sterol sulfates (572-574) that are antimicrobial, cytotoxic, ichthyotoxic and larvicidal [465].

703

RjO. R20^ 0S03Na 561

R,0,

^ T1 l Tii OSOaNa

OSOsNa 562Ri=H,R2 = Ac

564 Ri = H, R2 = Ac

563Ri = H,R2 = H

566 Ri = H, R2 = H

568Ri=Ac,R2 = H

569 Ri = Ac,R2 = H

NaOjSOi HI H NaO^SO' " ^ ^^

v^

OH' OAc

«^jdb

I JL J NaOaSO^^-^^^

0S03Na 565 Ri = H, R2 = Ac

571

572 R =

567 Rj = H, R2 = H

573 R =

570Ri=:Ac,R2 = H 574R= .

The sterol sulfates haplosamates A (575) and B (576) are inhibitors of HFV-l integrase from two Philippines Haplosclerid sponges and were reported to be the first naturally occurring sulfamates [466] but the structures were revised after re-examination of spectral data [467].

-O •bP02H0Me

NaOsSO' Y Y ^OR OH OH 575 R = H 576R = P03H2

704

A sterol sulfate, 3P,4P-dihydroxypregn-5-en-20-one 3-sulfate (577), was isolated from Stylopus australis from New Zealand and was the first known sterol sulfate with a 5-pregnene skeleton [468].

HO3SO'

The Pacific deepwater sponge Poecillastra laminaris contained annasterol sulfate (578), which had glucanase inhibitory activity [469].

'^OAc

NaOaSa 578

Polymastiamide A (579), an antimicrobial steroid with an unusual side chain modification involving an amide bond to a non-protein amino acid, was isolated from the Norwegian marine sponge Polymastia boletiformis. The structure of polymastiamide A (579) was elucidated by analysis of spectroscopic data and chemical interconversions [470]. Polymastiamides B-F (580-584), additional amino acid conjugates of steroids, were later isolated from the same sponge [471]. O

'CO2H NaOsSO'^ >

MeO 579

NaOjSa 580Ri = H,R2 = OMe 581Ri=Me,R2 = H

CO2H

705

O

NaOsSO^^^V""^"--^

CO2H

582 Ri = Me, R2 = OMe 583Ri = H,R2 = OMe 584Ri=Me,R2 = H

Echinoclasterol sulfate phenethylammonium salt (585), an antifungal and cytotoxic steroid, was isolated from the South Australian sponge Echinoclathria subhispida [472].

^x:!;55^^\^NH3

0380^

585

Three antiviral sterol disulfate orthoesters, orthoesterol disulfates A-C (586-588) were isolated from Petrosia weinbergi and their structures were determined by spectral data elucidation [473].

NaOaSa

I

I J H

NaOjSO'

586R= / .

587

R=c5^0C

588R:

c^^

706

Weinbersterol disulfates A (589) and B (590) are also antiviral metabolites from P. weinbergi [474].

589Ri = H,R2 = OH 590Ri = OH,R2 = H

Haliclostanone sulfate (591) is an unusual polyhydroxylated sterol sulfate from Haliclona sp. from Malaysia [475].

HO' Y ' y ^OH O l f OH 591

Crellastatin A (592) is the first of a series of cytotoxic bis-steroidal sulfates isolated from a Crella sp. from Vanuatu [476].

,0S03Na

592 Ri = OH, R2 = OH 593 Ri = H, R2 = OH 594 Rj = OH, R2 = H 595 Ri = H, R2 = H

707

Crellastatins B-M (593-604) are twelve additional cytotoxic, dimeric 4,4'-dimethylsterols from the same Crella sp. [477,478].

,0S03Na

596

.OSOaNa

597 R = OH 598 R = H

.OSOjNa

OSOjNa

600

708

^OSOjNa

^OH

r^^. 602

,0S03Na

OSO^Na

^OH OSOjNa

Benzylthiocrellidone (605) was isolated from Crella spinulata and the structure was confirmed by synthesis [479]. It is the first reported example of a natural product containing a dimedone unit [22].

709

Ph O

S^

OH

00 605

Pateamine (606), a potent cytotoxin containing a dilactone functionality, was isolated from a New Zealand species of Mycale and identified by analysis of spectral data [480]. Total synthesis of pateamine A (606) involved a P-lactam based macrocyclisation [481,482], while another total synthesis of pateamine employed a concise and convergent route [483].

A sulfated galactolipid, M-6 (607) was isolated from Phyllospongia foliascens. M-6 (607) consisted of an inseparable mixture of compounds with variations occurring in the carboxylic acid portion of the molecule. Compound 607 has resistant activity against complement fixation in serological reactions [484]. CH20S03"Na'"

OH 607

hOR ^O"^

R = (a:b=l:2) a =C0(CH2)6CH=CHC7Hi5 b = C0Ci5H3i

710

The cytotoxic polyether acanthifolicin (608), which is structurally very similar to okadaic acid, was isolated from Pandaros acanthifolium. It is unique in having an episulfide group on a long-chain polyether (C38) backbone. The structure and absolute configuration were determined by X-ray analysis [485]. Acanthifolicin (608) is thought to be a product of a microbial or microalgal symbiont of the sponge. Desulfurisation of acanthifolicin with a Zn/Cu couple yielded okadaic acid [486]. HQH

Mycothiazole (609) is a novel thiazole-containing lipid with anthelmintic properties isolated from Spongia mycofijiensis. The structure was established by analysis of spectral data [487]. A total synthesis of (-)mycothiazole (609) utilised a convergent strategy. The optical rotation of the product was the same sign as that of the natural material but significantly larger [488].

NHC02Me 609

The theonezolides are 37-membered macrocycles, consisting of fatty acid chains with attached functionalities such as a sulfate ester and a thiazole [22]. Theonezolide A (610) is a cytotoxic metabolite of Theonella sp. from Okinawa. The structure was reported without stereochemical details [489], The structures of theonezolides B (611) and C (612) from a Japanese Theonella sp. were determined by spectroscopic methods but without stereochemistry, except at one centre [490].

711

Toxadocials A-C (613-615) and toxadocic acid (616) are sulfated long chain alcohols isolated from Toxadocia cylindrica that inhibit thrombin. Their structures were determined by chemical and spectral means [491,492]. NaOgSO

GSOsNa

NaOsSO 613 R = CHO 616 R = CO2H

0S03Na

712

NaOsSO

OSOgNa

CHO

NaOaSO

OSO^Na

614 OHC NaOaSO NaOsSO

0S03Na

OSOaNa 615

Callyspongins A-B (617-618) are sulfated compounds from a Japanese sample of Callyspongia truncata. They inhibit fertilisation of starfish (Asterias amurensis) gametes [493].

"OSO^Na 617 R = SGjNa 618 R = H

Mycale sp. from Japan contained thiomycalolides A (619) and B (620) as minor metabolites. They are highly cytotoxic glutathione adducts of the known metabolites mycalolides A and B [494]. OHC

^o HO2C

NH

HO2C o'^-'^s^ o

I O

VQ OMe

619R = 0 620 R = H, 0C0CH(0Me)CH20Me

713

Penares sp. from Japan contained penarolide sulfates Ai (621) and A2 (622), which were a-glucosidase inhibitors [495]. O

•3"7 o c.H

C4H9

An Oceanapia sp. collected off the northern Rottnest Shelf, Australia, has yielded three novel dithiocyanates, thiocyanatins A-C (623-625). The structures were determined by spectroscopic analysis and confirmed by total synthesis. The thiocyanatins contain an unprecedented dithiocyanate functionality and an unusual 1,16-difunctionalised n-hexadecane carbon skeleton. They possess nematocidal activity [496]. NCS^

^SCN

SCN 624

623 "'SCN 625

Three sulfated ceramides, calyceramides A-C (626-628) were isolated as inhibitors of neuraminidase from the marine sponge Discodermia calyx. Their structures were determined by spectroscopic and chemical methods [497].

NaOjSO.

NaOjSi

714

NaOaSO.

Irciniasulfonic acid (629) was obtained from Ircinia sp. from Japanese waters. Spectroscopic and chemical analyses revealed it to consist of three different kinds of acids; common fatty acids, a novel unsaturated branched CIO fatty acid and an isethionic acid. Irciniasulfonic acid (629) reverses multidrug resistance in human carcinoma cells caused by overexpression of membrane glycoprotein [498].

R=

629

Bastaxanthins B, C, D, E, and F (630-634) are novel carotenoid sulfates from the marine sponge lanthella basta from the Great Barrier Reef, Australia [499]. The stereostructure of bastaxanthin C (631) was determined on the basis of infrared (IR), ^H and ^^C NMR, and CD spectra, and by chemical transformations [500]. Bastaxanthins were also isolated from /. flabelliformis from the Great Barrier Reef including bastaxanthin C (631) (major), B (630), D (632), and F (634) and bastaxanthin G (635) [501]. Bastaxanthin G (635) was not fully characterised but was the most polar of the carotenoids isolated and was tentatively described as a disulfate [501],

715

630 R = CH2OH

Na03S0'

631 R = CHO 633 R = CO2H

632 R = CH2OH

NaOgSa

634 R = CO2H

A sulfone (636) is a minor constituent of the Mediterranean sponge Anchinoe tenacior [502]. Sulfolane (637), a familiar industrial chemical, was isolated from a mixture of the sponge Batzella sp. and a Lissoclinum tunicate from Victoria, Australia [503]. It is possibly an absorbed compound rather than a natural product [12].

(!) 637

636

5-Thio-D-mannose (638), the first example of a naturally occurring 5thio-sugar has been isolated from Clathria pyramida [504] and was later synthesised in 12 steps from D-mannose [505]. HOpH

HOAOH

638

716

(2-Hydroxyethyl)dimethylsulfoxonium chloride (1), the causative agent of Dogger Bank Itch which has previously been isolated from the marine bryozoan Alcyonidium gelatinosum [26], has now been isolated as a cytotoxic component of the marine sponge Theonella aff. mirabilis [506].

Echinoderms The phylum Echinodermata comprises about 7000 living species [177]. Echinoderm means "spiny-skinned" and these organisms are characterised by the tube feet, which they use to move about. These have suction discs on the ends, which operate by an internal bulb pumping water in and out of the foot, causing expansion and contraction. The phylum is sub-divided into five classes; the asteroids (sea stars), the holothurians (sea cucumbers), the crinoids (sea lilies), the echinoids (sea urchins) and the ophiuroids (brittle stars) [178]. As stated in the introduction to this review, sulfated sterols and saponins, which comprise the majority of echinoderm metabolites containing sulfur, are not included here. The histidine derivatives, l-methyl-5-thiolhistidine (639) and its disulfide (640) were isolated from unfertilised eggs of the sea urchin Paracentrotus lividus [507] and from those of other echinoderms [508]. Their structures were revised after an unambiguous synthesis [509]. LOvothiol A disulfide (640) was also shown to be the egg release pheromone of the marine polychaete worm Platynereis dumerilii [510]. Me CO2H NNf--^NH2 /—< S-S NH2 H2N Y\ N^N-Me H02C^^-^N

HS

639

640 ^^

The ovothiols, a family of mercaptohistidine compounds, have been isolated from marine invertebrate eggs. Ovothiol B (641) from the scallop

717

Chlamys hastata, ovothiol C from the sea urchin Strongylocentrotus purpuratus (642) and ovothiol A from the starfish Evasterias troschelli were isolated from eggs of ovarian tissue [511,512]. The structure of ovothiol A is identical to that of l-methyl-5-thiolhistidine (639). CO2H

641 R = H 642 R = Me

The ovaries of the Japanese sea urchin Hemicentrotus pulcherrimus contained a bitter tasting amino acid, pulcherrimine (643) [513]. A sulfonoglycolipid isolated from the shell of the sea urchin Anthocidarias crassispina was a 96:4 mixture of r-0-palmitoyl-3'-0-(6-sulfo-a-Dquinovopyranosyl)glycerol (644) and the myristoyl counterpart (645) [514]. o

OH

^^ 643

OH^ 644R = Ci5H3i 645R = Ci3H27

Many starfish cause an escape response in usually sessile marine invertebrates [7]. The starfish Dermasterias imbricata causes the sea anemone Stomphia coccinea to release its basal disc from the substratum and swim away on contact. Bioassay-directed fractionation of the starfish extract led to the isolation of the compound found to elicit this response, the benzyltetrahydroisoquinoline alkaloid imbricatine (646). The structure of compound 646 was elucidated by spectral data interpretation. The amino acid residue in imbricatine is related to the thiol containing amino acids ovothiols A-C. Imbricatine (646) is active in both L1210 and P388

718

assays [515,516]. The structural elucidation and partial synthesis of imbricatine (646) were later reported fully [517].

Forbesin (647), a sulfated glycolipid and a disodium salt, eicosane1,16-disulfate (648) were isolated from the sea star Asterias forbesi, Forbesin was also isolated from A. vulgaris [518]. NaOsSO

647

HOMO

NaOaSO OSOgNa

648

The sea star Henricia laeviuscula contained the anthraquinone sodium isorhodoptilometrin-2'-sulfate (649) [519].

OSOjNa

719

The naphthopyrone, comantherin sulfate (650) was isolated from the crinoid Comantheria perplexa [520] and three naphthopyrones (651-653) were isolated from Comanthus parcivirrus timorensis. Both species were collected off Australia [521].

NaOsSO' 651 Ri = OH, R2 = H

650

652 Ri = OH, R2 = OMe 653Ri = OMe,R2 = OMe

The crinoid Comatula pectinata contained three anthraquinones (654656) [522] while two further anthraquinones (657-658) were isolated from the crinoid Ptilometra sp. l,8-Dihydroxy-3-propyl-9,10-anthraquinone-60-sodium sulfate (657) and l,8-dihydroxy-3-(r-hydroxypropyl)-9,10anthraquinone-6-O-sodium sulfate (658) have not previously been isolated from a natural source. l,8-Dihydroxy-3-(l-hydroxypropyl)-9,10anthraquinone-6-O-sodium sulfate (658) was cytotoxic [523]. OH O NaOgSO.

.OMe NaOsSO' OR2 0

ORi

0

654 Ri = H, R2 = H

657R = H

655 Rj = Me, R2 = H

658R = OH

656 Ri = Me, R2 = Me

The deepwater stalked crinoid Gymnocrinus richeri contained the gymnochromes C (659) and D (660) and isogymnochrome D (661). These compounds have a helical chirality and chiral atoms in the sidechains give rise to isomers [524].

720

HO

0

OH

fWS

Brv

HO" HO.

HO -%H

Wy^ OH 0 OH 659

riT

HOs ^B,6S03H

rl

\J

Br^

OH

0

if' ^ II ""^

HO"JLX

TI T11 TL

Br''

Brv

HO ,Br

O

OH

9SO3H

II

1

11^

^

JS ^

PSO3H 'Br

OH 0 OH 660

PSO3H OH O OH 661

The holothurian Cucumaria frondosa contains 2,6-dimethylnonane-lsodium sulfate (662) and 2,4,6-trimethyl-nonane-l-sodium sulfate (663). The structures were proposed without stereochemical detail [525].

LAjJx.'

OSOsNa

662 R = H 663 R = Me

The Japanese sea cucumber Cucumaria echinata contained a ganglioside CG-1 (664) with neuritogenic activity toward the rat pheochromocytoma PC-12 cell line [526]. Similar activity was reported for the ganglioside HPG-8 (665) isolated from the sea cucumber Holothuria pervicax from Japan [527]. 0SO3H HO^^^A^OH HN^3H ..OH HO' ^ *OHHO^ OH 664

721

OH H

loX>^2C0^0.

H03SO'

HO' ^ 'OHHO^ OH 665

A carotenoid sulfate, ophioxanthin (666), was isolated from the ophiuroid Ophioderma longicaudum from the Mediterranean Sea and shown to be 5,6,5',6-tetrahydro-P,P-carotene-3,4,3',4-tetraol 4,4'-disulfate [528]. The carotenoid, dehydroophioxanthin (667) was isolated from the ophiuroid Ophiocomina nigra off Spain and the structure was determined by spectral data analysis [529].

(3Z)-4,8-Dimethylnon-3-en-l-yl sodium sulfate (166), which is also found in the ascidian Microcosmus vulgaris [166], is a sulfated alkene that was isolated from the ophiuroid Ophiocoma echinata from Colombia [530].

722

ABBREVIATIONS ALL

= acute lymphoblastic leukaemia

Anti-fflV

= anti-human immunodeficiency virus

ATP

= adenosine triphosphate

CD

= circular dichroism

DNA

= deoxyribonucleic acid

DSP

= diarrhetic shellfish poisoning

EC50

= effective concentration needed to reduce cell growth by 50%

ECE

= endothelin converting enzyme

ED50

= effective dose needed to reduce cell growth by 50%

FAB

= fast atom bombardment

fflV-1

= human immunodeficiency virus type 1

IC50

= inhibitory concentration needed to reduce cell growth by 50%

IR

= infrared

mg

= milligram

mL

= millilitre

mM

= millimolar

[iM

= micromolar

nM

= nanomolar

NCI

= National Cancer Institute

NMR

= nuclear magnetic resonance

NOEDS

= nuclear Overhauser enhancement difference spectroscopy

723

NSP

= neurotoxic shellfish poisoning

RT

= reverse transcriptase

SPLA2

= secreted phospholipase A2

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753

SUBJECT INDEX (Vol. 28) Abamectin 434,406 eigmnst Hyalomma spp, 407 against Phytoseiulus persimilis 434 against Rhipicephalus spp. 407 against Tetranichus urticae 434 Absolute stereostructure 11 of broussonetine C 11 ofbroussonetineL 12 Aburatubolactam A 139 f^om Streptomycessp. 139 Aburatubolactam C 139 fvom Streptomycessp. 139 (2S)-Abyssinone II 17 as aromatase inhibitor 17 Acacia honey 386 aroma of 386 Acanthella 663 kalihinol G of 663 kalihinol H of 663 Acanthella cavernosa 663 10-^/?/-isokalihinolHfrom 663 15-isothiocyanato-1 -epi-kalihinene from 663 Acanthella klethra 662 isothiocyanates from 662 A canthella pulcherhma 660 isothiocyanates from 660 sesquiterpenes from 660 Acanthifolicin 710 as cytotoxic agent 710 episulfide group of 710 from Pandaros acanthifolium 110 similarity to okadaic acid 710 Acanthodendrilla sp. 702 acanthosterol sulfate A of 702 acanthosterol sulfate B of 702 acanthosterol sulfate C of 702 acanthosterol sulfate D of 702 acanthosterol sulfate F of 702 acanthosterol sulfate G of 702 acanthosterol sulfate H of 702 acanthosterol sulfate I of 702 acanthosterol sulfate J of 702 against Saccharomyces cerevisiae

702 antifimgal activity of 702 A carapis woodi 387,390 in honey bees' tracheal tubes 387 infestations in Minnesota 390 Acaricidal activities 403,406,412,422 against Dermatophagoides pteronyssinus 422 against Psoroptes cuniculi 412 against Rhipicephalus appendiculatus 406 for killing adult ticks 403 of caffeine 422 of Cuminum cyminum 427 of essential oils 412,427 of Eucalyptus camaldulensis All of eugenol 403 of extract prepared by microwave assisted process (MAP) 427 of Lavandula angustifolia 412 of linalool 412 of Margaritaria discoidea 406 of Origanum syriacum var. bevanii All of phenylpropanoid derivatives 403 of Pimenta dioica 403 of Pimpinella anisum All of Tanacetum vulgare 's extracts 427 of P-thujone 427 Acaricidal properties 400,406 of benzaldehyde 406 of carvacrol 406 of cedrene 406 of a-cyclocitral 406 of P-cyclocitral 406 of Euphorbia obovalifolia 's latex 400 of Ficus brachypoda's htex 400 of geraniol 406 of (£)-geranylacetone 406 of a-ionone 406 of linalool 406 of w-cymene 406 of methyl salicylate 406 ofnerol 406

754

of nerolidol 406 of nonanal 406 of P-ocimene 406 of phenylacetaldehyde 406 of phenylacetonitrile 406 of a-terpineol 406 Acaricide 381,429,435 cross-resistance of 429 deguelinas 435 effectiveness of 404 fenazaquinas 429 from Annona squamosa 404 from Azadirachta indica 404 literature about 381 Lonchocarpus urucu as 435 of natural origin 381 pyridabenas 429 rotenoloneas 435 rotenoneas 435 tebufenpyrad as 429 tephrosinas 435 A cams siro 382 as mite species 382 Acrostalamus fungi 455 acrostalidic acid from 455 acrostalic acid from 455 isoacrostalidic acid from 455 3-Acyl tetramic acid 111,112,114 from Alternaha alternata 114 from Alternaha longipes 114 from Alternaha tenuis 114 biosynthetic pathways of 111 from Pyricularia oryzae 114 tautomeric forms of 112 Adenichrome 647 Fe(III)-containing pigment as 647 from Octopus vulgaris 647 Adociasp. 674 adociaquinone A from 678 adociasulfate from 674 from Great Barrier Reef 674 structure of 674 Adociasulfates 1-6 674 as kinesin motor proteins inhibitors 674 from Haliclona (aka Adocia) sp. 674

Adociidae family 664 10-isothiocyanatobiflora-4,15diene of 664 spectral analysis of 664 P-Adrenoceptors 183 with [^H]dihydroalprenolol 183 AflastatinA 127,128 as aflatoxin inhibitor 128 from Streptomyces griseochromogenes 127 structure of 128 AflastatinB 127 as aflatoxin inhibitor 128 from Streptomyces griseochromogenes 127 Anatoxins 128 from Aspergillus flavus 128 from Aspergillus nomius 128 from Aspergillus parasiticus 128 from Aspergillus tamarii 128 African tick species 396 Amblyomma hebraeum as 396 Boophilus decoloratus as 396 Hyalomma sp. as 396 Rhipicephalus appendiculatus as 396 Rhipicephalus evertsi evertsi as 396 Agelas dispar 692 from Bahamas 692 pyridinebetaine B of 692 Agelas nakamurai 670 agelasidine A from 670 agelasidine B from 670 antispasmodic activity of 670 Na^/K^-transporting adenosine triphosphate (ATP)ase inhibitor from 670 spectral data of 670 structure of 670 synthesis of 670 Agricultural pests 423 of forage crops 423 of fruits 423 of ornamentals 423 of timber 423 of vegetables 423

755

Ajoene 432 acaricidal activity of 432 against Tetranyehus urticae 432 anticoagulant properties of 432 Akaterpin 673 as phosphatidylinositolphospholipase C inhibitor 673 from Callyspongia sp. 673 stereochemistry of 673 AlbanolA 17 as aromatase inhibitor 17 Albanol B 234 from Morus uralensis 234 Alcyonidium gelatinosum sp. 619 (2-hydroxyethyl) dimethylsulfoxonium ion from 619 Dogger Bank itch by 619 Aldose reductase inhibitor 691 from Dictyodendhlla sp. 691 a-Alkyl-p-hydroxyproline moiety 367 construction of 367 Allelopathic activity 483 of natural podolactones 484 of synthetic podolactones 484 Allium sativum (LilidicediQ) 415 Alphitolic acid 40 from Licania heteromorpha var. heteromorpha 40 Althiomycin 143 from Cystobacter fuscus 144 frovci Myxococcus xanthus 144 from Streptomyces althioticus 143 from Streptomyces matensis 143 Amblyomma 394,397 by Beauveria bassiana 397 by hyperparasitic fimgi 397 by Metarhizium anisopliae 397 control of 397 in goats 394 in sheeps 394 Amblyomma variegatum 395 repellent properties of 395 [^H]Amine uptake 183 of Ginkgo biloba L. 183 Ancorinoside A 120 fvom Ancorinasp. 120

Ancorinoside A Mg salt 120 ficom Ancorinasp. 120 Ancorinoside B 120 ficom Ancorinasp. 120 Ancorinoside C 120 from Ancorina sp. 120 Ancorinoside D 120 from Ancorinasp. 120 Annona glabra seeds 430 acetogenins from 430 against Dermatophagoides pteronyssinus 430 against Typhlodromus urticae 430 asimicinfrom 430 desacetyluvaricin from 430 squamocinfrom 430 Annona squamosa 415 extract of 415 Anthelmintics 331,332 broad spectrum activity of 332 diminished activity of 331 new class of 332 Anthocyanidins 275 cyanidinas 275 delphinidin as 275 malvidinas 275 pelargonidin as 275 structure of as 275 Anthocyanins 275,276,277,292 cyanidin-3-glucoside as 275 delphinidin-3-glucoside as 275 for coronary heart disease 292 from black grapes 276 in blackberry 277 in blueberry 277 in cabbage, red 277 in cherry 277 inchokeberry 277 in cranberry 277 in currant (black) 277 in food plants 276 in grape (red) 277 in onion 277 in organe, blood O'uice) 277 in raspberry, red 277 in strawberry 277 in wines, porto 277 in wines, red 277

756

malvidin-3-glucoside as 275 pelargonidin-3-glucoside as 275 Anthopleura elegantissima 647 mycosporine-taurine from 647 Anti-apoptotic effects 175 Antiahs toxicaria 203 antiarone A from 203 antiarone B from 203 antiarone E from 204 antiarone J from 203 antiarone K from 203 ficusins A from 204 uses for arrow poison 203 Anti-atherosclerotic activity 257,293 of polyphenols 257,293 Anti-bacterial activity 140 of ikarugamycin 140 Anti-carcinogenic activity 257,293 of polyphenols 257,293 Anti-con vulsant activity 176 ofbilobalide 176 Anti-depressant effects 177 of Ginkgo biloba L. 177 Anti-feedant activity 480 against mammals 480 of l-deoxy-2P,3p-epoxynagilactoneA 480 of nagilactone A 480 of nagilactone C 480 Anti-ftingal activity 66,473,475 of2-hydroxynagilactoneF 475 of intrapetacin A 66 of intrapetacin B 66 of LL-Z1271a 473 of nagilactone C 475 of nagilactone E 475 of oidiodendrolideB 475 ofoidiolactoneD 475 Anti-fungal holothurin 597 from Psolus patagonicus 597 Anti'Helicobacter pylori activities 234,241,243 of 6,8-diprenylorobol 243 of dihydrolicoisoflavone A 243 of formononetin 241 of gancaonini 243 ofgancaonol B 243

of gancaonol C 243 ofglabrene 241 ofglabridin 241 ofglyasperinD 243 ofglycyrin 243 ofglycyrol 241 ofglycyrrheticacid 241 ofglycyrrhizicacid 241 of isoglycyrol 241 of isolicoflavonol 243 oflicochalcone A 241 oflicochalconeB 241 oflicoisoflavoneB 241 of licorice flavonoids 234 of licorice-saponin 241 oflicoricidin 241 oflicoricone 243 ofliquiritigenin 241 ofliquiritin 241 of l-methoxyphaseoUidin 243 of3-(9-methylglycyrol 243 ofvestitol 243 Anti-Human inmiunodeficiency virus (HIV) activity 225,226 of antiarone I 226 of broussoflavonol B 226 of broussoflavonol C 226 ofgancaoninR 226 ofglyasperin A 226 ofglycyrol 226 ofkazinolB 226 of kumatakenin 226 ofkuwanonH 225 oflicochalconeB 226 ofmoracinC 226 of morusin 225 of mulberry tree 225 ofnorartocarpetin 226 ofprenylflavones 225 of wighteone 226 Anti-HIV flavonoids 226 2-arylbenzofiiran as 226 from Glycyrrhiza species 226 from moraceous plants 226 Anti-inflammatory activity 200,257,293 of genus Morw^ 200 of polyphenols 257,293

757

Anti-metastatic activity 559 of natural products 559 Anti-microbial activity 62,224,257,293 ofalphitolicacid 62 of AMOX 224 ofbetulinicacid 62 of formononetin 224 ofgancaonini 224 ofgancaonolB 224 of glabrene 224 of glabridin 224 ofglyasperinD 224 of glycyrin 224 of glycyrol 224 of isoglycyrol 224 of isolicoflavonol 224 of Licania heteromorpha var. heteromorpha 62 of licochalcone A 224 of licochalconeB 224 of licoisoflavoneB 224 of licorice flavonoids 224 of licoricidine 224 of licoricone 224 of liquiritigenin 224 of liquiritin 224 of 3-0-methylglycyrol 224 of 3|3-0-d5-/?-coumaroyl maslinic acid 62 of 3P-0-cw-/?-coumaroyl alphitolic acid 62 of 3 ^'O'tranS'P'C0\xmdC[0y\ alphitolic acid 62 of 3 p-(9-^rv^Wea species 666 (I5*,45*,65*,7/?*)-4-Thiocyanato-9cadinene 660 from Trachyopsis aplysinoides 660 X-ray analysis of 660 5-Thio-D-mannose 715 as naturally occurring 5-thiosugar 715 from Clathria pyramida 115 Thiofiirodysinin 667 asfiiranosesquiterpene667 from Dysidea avara 667 Thymol 391 employed as Frakno thymol frame 391 Thymovar 391 employed as Frakno thymol frame 391 Thymus vulgaris 391,415 against Knemidocoptes pilae 415 essential oils from 391,415 Tick-borne diseases 394 in livestock 394

Ticks toxicity 403 byeugenol 403 byisoeugenol 403 by methyleugenol 403 bysafrole 403 Tirandamycin A 131 biological activity of 131 from Streptomyces tirandis 131 Topsentiasp. 701 in guanosine diphosphate/G-protein RAS exchange assay 701 sulfates of 701 topsentiasterol sulfate A from 701 topsentiasterol sulfate B from 701 topsentiasterol sulfate C from 701 topsentiasterol sulfate D from 701 topsentiasterol sulfate E from 701 Tormentic acid 40 from Licania licaniaeflora 40 from Licania pyrifolia 40 Total synthesis 502 of nagilactone F 502 ToxadocialA 711 as sulfated long chain alcohols 711 from Toxadocia cylindrica 711 thrombin inhibition by 711 ToxadocialB 711 as sulfated long chain alcohols 711 from Toxadocia cylindrica 711 thrombin inhibition by 711 ToxadocialC 711 from Toxadocia cylindrica 111 thrombin inhibition by 711 Toxic essential oils 393 from Apis mellifera 393 from Varroajacobsoni 393 Toxiclona toxius 675 toxicol A from 675 toxiusol from 675 Trachyopsis halichondrioides 700 26-norsokotrasterol sulfate from 700 Tracheal mites 389 effects ofvegetable oils on 389 Triandamycin B 131 from Streptomyces flaveolus 131 Trichostrongylus colubriformis 342

796

Tridacna maxima 652 arsenic-containing sugar sulfate from 652 Tridentata marginata 646 tridentatol A from 646 tridentatol B from 646 tridentatol C from 646 Trididemnum sp. 638 from Guam 638 shermilamine A from 638 P-Triketone 117 from Apiosordaria effusa 117 Triterpene glycoside 596,587,589 antifiingal activity of 589 cytotoxic activity of 589 cytostatic activity of 589 from Cucumaria echinata 596 from Pentamera calcigera 596 from sea cucumbers 587 hemolytic activity of 589 immunomodulatory activity of 589 Tropical ixodid ticks 404 Hyalomma genera as 404 Rhipicephalus genera as 404 Tryptophan derivative 369 construction of 369 Tumor 532 effectof lipid A on 532 in host response to LPS 531 Tumor growth in LLC-bearing mice 581 effects of 2,3,5,4'-tetrahydroxystilbene-2-O-D-glucoside on 581 effects of piceid on 581 Tumor necrosis factor-a (TNF-a) 219 as tumor promoter 219 by okadaic acid 219 Tunicates (Ascidians) 621 active metabolites of 622 ascidiacyclamide from 622 bistratamides from 622 from Phylum chordata 621 lissoclinamides from 622 patellamides from 622 Tyrindoxyl sulfate 652 as Tyrian purple dye 652 from Murex truncatus 652

Tyrophagus putrescentiae 421 against 1,8-cineole 421 against fenchone 421 against isomers of caryophyllene 421 against linalool 421 against linalyl 421 against menthone 421 against myrtanol 421 against Pinus halepensis 420 digdxnsX Pinus nigra 420 against Pinus pinaster 420 against Pinus pinea 420 against pinene 421 against pulegone 421 against a-terpinene 421 against y-terpinene 421 against terpineol 421 against valencene 421 UoamineA 644 from Aplidium uouo 644 UoamineB 644 of Aplidium uouo 644 3-thiomethylacrylate ester group of 644 Urbalactone 459 from Podocarpus urbanii 459 Ursolic acid 18,40 from Licania carii 40 from Licania licaniaeflora 40 from Licania pittieri 40 from Licania pyrifolia 40 inhibition of HI V-1 protease dimerization by 18 Uvaria pauciovulata ATI effects on Dermatophagoides pteronyssinus All squamocin from 422 structure of 422 Vancoresmycin 150 activity against gram-positive bacteria 150 from Amycolatopsin sp. 150

797

Varroajacobsoni 387 honey bees tolerant to 387 toxicity of 387 VarroaxmiQS 383,384,392 as honey bee parasites 384 biological activity of 392 Vasoprotective effects 302 of green tea 302 Vermisporin 125 antimicrobial activity of 125 from Ophiobolus vermisporis 125 Vernonia amygdalina 400 tick toxicity of 400 Veterinary medicine 383 for ectoparasites control 383 Vetiver grass 399 for controlling ticks 399 Phetchabunas 399 'Si Sa Kef as 399 'Uthai Thani' as 399 VirenamideA 644 as cytotoxic linear peptides 644 from Diplosoma virens 644 Virenamide B 644 as cytotoxic linear peptides 644 from Diplosoma virens 644 Virenamide C 644 as cytotoxic linear peptides 644 from Diplosoma virens 644,645 Vitamin A synthesis 71 by Baadische Anilin by 71 by Hoffrnann-LaRoche 71 by Rhone-Poulenc 71 SodaFabrik 71 Waiakeamide 684 as cyclic hexapeptide 684 from Ircinia dendroides 684 Watersipora subtorquata 620 5,7-dihydroxy-6-oxo-6//anthra[ 1,9-6c]thiophene-1 carboxylic acid from 620 Wentilactone A 465 from Aspergillus wentii 465 Wentilactone B 465 from Aspergillus wentii 465

Wheat flour 259 vanillic acid in 259 syringic acid in 259 Xanthobaccin A 140 asfiingitoxicmetabolite Xanthobaccin B 140 asftmgitoxicmetabolite Xanthobaccin C 140 asfiingitoxicmetabolite Xestoquinolide B 677 from Xestospongia cf. carbonaria 611 protein kinase activity of spectral data of 677

140 140 140

677

Yessotoxin 653 analogues of 653 from Patinopecten yessoensis 653 in diarrhetic shellfish poisoning (DSP) 653 Zoanthus sp. 646 sphingolipid hariamide from 646 zoanthid A from 646 Zyzzya cf marsailis 686 discorhabdin A from 686 makaluvamine F from 686 total synthesis of 686

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