THE ALKALOIDS Chemistry and Pharmacology VOLUME 36
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THE ALKALOIDS Chemistry and Pharmacology Edited by Arnold Brossi Narionul Instirures of Health Bethesda, Maryland
VOLUME 36
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I’KINItil) IN 11111 LINl’ltU S T A I E S 01- AMI;KICA XY
‘If1
‘)I
92
~H~:N?OI338
184- I86
S. drcrrssura (Pappe) Gilg.
Af
48
C,9H?oN:O: 304 C I ~ H ~ P N ? O308 Z
141- 143 221-223
S. hirsrrra Spruce ex Benth. S. hirsutu Spruce ex Benth.
Am Am
50 50
(continued)
TABLE I1 (Continued) Alkaloid
Decuwne
Structure
R = H
Molecular formula
MW
mp ( T I
203-205
[alD
Specie5
S.dale de Wild. S.decusmu (Pappe) Gilg. S. elueocurpu Gilg. ex
L
0
K
IO-Hydroxy-3.14dihydrodecussine
= H, 3.14-dihvdro
R = OH. 3.14-dihydro
78-82
Leewenberg S. floribunda Gilg. S. dulr de Wild. S. deciissuru (Pappel Gilg. S. elueocurpu Gilg. ex Leewenberg S. decussoru (Pappe) Gilg.
Location
Ref.
Af Af Af
Sl 5 1 . /43 51
Af Af Af Af
43, 44 51 51 51
Af
51
2
M
-
6
d
0
C
N
.
L
10
Q Q
300
9-Methoxycanthin-6-one (19)
C i5H ioNzOz
175-176
10-Hydroxycanthin-6-one (20)
CiiH&"02
288-293 (dec.)
10-Methoxycanthin-6-one (21) 11-Hydroxycanthin-6-one (amarorine)
C I ~ H I O N Z O ~175-178 323-325 CI4HgN2O2
(22)
11-Methoxycanthin-6-one (amoridine) (2.3) I -H ydrox y- 1 1-methox ycanthin-6-one (24)
C15HION202 237-238 CISHIoN~O,
-
Source
Family
Ref.
Peniaceras australis Zanihoxylum carbibaeum Zanihoxylum elephaniiasis Ailanthus excelsa Burcea aniidysenierica Odyendea gabonensis Picrasma excelsa Picrasma quassioides Ailanthus uliissimu Ailanihus excelsa Odyendea gabonensis Pierreodendron kersiingii Simaba muliipora Simaba cuspidaia Simaba multipora Eurycoma longifolia Simaba muliipora Simaba mulripora Amaroria soulameoides (syn Soulamea soulameoides) Brucea anridysenierica Quassia kersiingii Amaroria soulameoides
Rutaceae Rutaceae Rutaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae
35 88 72 25 47 37 53,82 84 36 25 37,38 39 40 2 41 43 42 42 44,45
Simaroubaceae Simaroubaceae Simaroubaceae
46 29 44
Brucea antidysenrerica
Simaroubaceae
26,47
Brucea antidysenterica
Simarou baceae
26
Soulamea pancheri Brucea antidysenterica Picrasma quassioides
Simaroubaceae Simaroubaceae Simaroubaceae
28 46 48
Simaroubaceae Simaroubaceae
53 49-52,54
Simaroubaceae Simaroubaceae
37 55
258 (dec.)
Picrasma excelsa Picrasma quassioides (syn. P. ailanthoides) Odyendea gabonensis Quassia africana (syn. Simaba africana) Ailanthus altissima cell culture Simaba multijora Simaba cuspidata Simaba multij7ora Samadera indica
Simaroubaceae Simaroubaceae Simaroubaceae Simaroubaceae
13,15 42 2 42 56
280-290 (dec.)
Simaba mulfijlora
Simaroubaceae
42
>330
274 (dec.)
Picrasma quassioides Quassia amara Picrasma quassioides
Simaroubaceae Simaroubaceae Simaroubaceae
3,57,58 59 48
294-295 (dec.) 171-172 (dec.) 199-200 (dec.) >350
Picrasma quassioides Picrasma quassioides Picrasma quassioides Cortinarius infractus
Simaroubaceae Simaroubaceae Simaroubaceae Corinariaceae
3 48 61 4
1 I-Hydroxy-I-methoxycanthin-6-one
(25) 1 , I I-Dimethoxycanthin-6-one(26)
4-Hydroxy-5-rnethoxycanthin-6-one (picrasidine Q ) (27) 5-Hydroxy-4-methoxycanthin-6-one (nigakinone) (28)
-
217-220 (dec.) 286-289 224-225
4,5-Dimethoxycanthin-6-one (8)
145-146
4,5-Dihydrocanthin-6-one (29) Canthin-2,6-dione (31) 3-Methoxycanthin-2,6-dione (2)
128 290-305 (dec.) >330
f 5-Methoxycanthin-2,6-dione (indacanthinone) (30) lO-Hydroxy-3-rnethoxycanthin-2,6dione (32) 3-Methylcanthin-5,6-dione (picrasidine L) (3) 3-Methyl-4-methoxycanthin-5,6-dione (picrasidine 0) (33) Picrasidine M (4) Picrasidine N (34) Picrasidine U (35) Infractopicrin ( 5 )
142
TAICHI OHMOTO AND KAZUO KOIKE
canthind-one (l),4,5-dimethoxycanthin-6-one(8), and pyridine, it was found that the N-oxides showed an approximately 0.5-ppm high-field shift relative to 1,8, and pyridine. On this basis, compound 9 was assumed to be a 3-oxide. Haynes et a/. ( I )obtained 9 by oxidizing 1 with hydrogen peroxide. Ohmoto et a / . (18) obtained 9 through oxidizing 1 with m-chloroperbenzoic acid and further converted 9 to 1through hydrogenation with palladium on carbon. On the basis of these results, structure 9 was confirmed.
I-Hydroxycanthin-6-one (10) 1-Hydroxycanthin-6-one (10) has been isolated from the root bark (20) and cell cultures (13,m 6 ) of Ailanthus altissima and from the heartwood (21) of A. giraldii. By comparison of the mass spectra and splitting patterns in the 'H-NMR spectra of 5-hydroxycanthin-6-one (16) and 8-hydroxycanthin-6-one (U), 10 was estimated to be the I-hydroxyl derivative. Through direct comparison between methylated 10 and I methoxycanthin-6-one (ll),structure 10 was confirmed. I-Methoxycanthin-6-one (11) I-Methoxycanthin-6-one (11) has been isolated from the root bark (19,221, wood (18), stem ( 2 3 , and leaves (24) of A. altissima, its cell culture (14-16), the root bark of A. excelsa (25), the ground wood of Brucea antidysenterica (26), the root of Hannona klaineana (27), and Soulamea pancheri (28). Based on results of comparisons between 4-methoxy- I -methoxycarbonyl-P-carboline obtained by oxidizing 11 (18)
4iT&hoxy-1iW.hoxycarbony 1-o-carboline
3. CANTHIN-6-ONE ALKALOIDS
I43
with KMn04 and I methoxycarbonyl-P-carboline together with comparison of the chemical shifts of H-3 in the 'H-NMR spectra of I-ethyl4-methoxy-p-carboline and I -ethyl-P-carboline, structure 11 was determined. 1-Methoxycanthin-&one N-Oxide (12)
1-Methoxycanthin-6-one N-oxide (12) has been isolated from the root bark of A. aftissima (19) and its cell culture (15). High-resolution MS proved that the number of oxygens in 12 was larger than that of 11 by one. Also, through a comparison of the 'H-NMR spectra of 11 and 12, Ohmoto et a / . assumed that this compound would be the N-oxide derivative. Ohmoto et a / . (18) then made a direct comparison of natural product 12 with a synthetic compound obtained by oxidizing 11 with m-chloroperbenzoic acid and determined structure 12 for I-methoxycanthin-6-one N-oxide.
2-Hydroxycanthin-6-one (13) and 4-Hydroxycanthin-6-one (14)
Both 2-hydroxy- (13) and 4-hydroxycanthin-6-one (14) have been isolated from cell cultures of A. altissima (13,f5),and 13 has also been isolated from the stem bark of Quassia kerstingii (29). From the facts that the mass spectra of both 13 and 14 show an mlz 236 ion and that both compounds show the same fragmentation pattern, formed by losing CO then HCN from the molecular ion, it was supposed that they are hydroxycanthin-6-ones. Crespi-Perellino et al. (13,151 compared the 'H-NMR spectrum of 13 with those of 1 and 10 and supposed 13 to be the
Ri
Fh
(13)
OH
H
(14)
H
OH
144
TAICHI OHMOTO A N D KAZUO KOIKE
1- or 2-hydroxyl derivative. In addition, acetylated 13 did not agree with l-acetoxycanthin-6-one, and structure 13 was determined. As 'H-NMR spectral data for the indole ring and positions 1 and 2 of 14 did not differ from those of 1, the substituent effect on the chemical shift of H-5 was in accordance with the known substituent effect on acetylated 14 (30),and, according to Nelson and Price (31), a hydroxyl derivative obtained from 4-methylthiocanthin-6-one (6) with alcoholic alkali agreed with 14. Based on these findings, structure 14 was determined. 4-Methoxycanthin-6-one (15)
4-Methoxycanthin-6-one (15) has been isolated from the bark of both the stem and root of Charpentiera obouata (Amaranthaceae) (32) and the aerial parts of Drymariu cordata (Caryophyllaceae) (33).This is one of the compounds not found in the families Rutaceae and Sirnaroubaceae. Its UV and IR spectra proved that it was a canthin-6-one alkaloid, and the 'H-NMR spectrum confirmed the methoxyl function. As natural 15 was identical with an authentic compound synthesized by Nelson and Price (31), structure 15 was determined.
4-Merhylthiocanthin-6-one (6) 4-Methylthiocanthin-6-one (6) is one of the first canthin-6-one alkaloids isolated from the bark (stem, root, branch, and sapling) and wood of Pentaceras australis (31)together with 1 and 7 (5).Readers are referred to Volume 3 (p. 249) of this treatise for its structure (5). 5-Hydroxycanthin-6-one (16)
5-Hydroxycanthin-6-one (16) has been isolated from the root bark of Simarouba umara (34) and from cell cultures of A . alrissima (12,15). Since its UV spectrum was similar to that of 5-methoxycanthin-6-one (25), Lassak et a f .(34)assumed it to be 4- or 5-hydroxycanthin-6-one. The prominent peak at M+-56 (mlz 180, Mf-2CO) in the mass spectrum supported structure 16 having an OH adjacent to the carbonyl group. The final confirmation of structure 16 was made through direct comparison of methylated 16 with authentic 7.
3. CANTHIN-6-ONE ALKALOIDS
I45
5-Methoxycanthind-one (7)
5-Methoxycanthin-6-one (7)has been found in the bark (stem, root, branch, and sapling) and leaves of Pentaceras australis (35). This compound has also been found in three species of Rutaceae including P . australis, five species of Simaroubaceae, and one species of Caryophyllaceae. The number of species of plants containing 7 is second only to those containing 1. Readers are referred to Volume 3 (p. 249) of this treatise for its structure (5). 5-Hydroxymethylcanthin-6-one( I 7)
5-Hydroxymethylcanthin-6-one (17)has been isolated from the root bark of A . altissima (36).This is the only canthin-6-one alkaloid which has a hydroxymethyl function. In the 'H-NMR spectrum the signal arising from the methylene protons (6 4.60, 2H, dd, J = 5.7 and 1.2 Hz) in C&OH showed long-range coupling with an aromatic proton (6 8.02, l H , t, J = 1.2 Hz) at position 4 o r 5 . It also showed coupling with a hydroxyl proton (6 5.56, l H , t, J = 5.7 Hz). On this basis, 17 was supposed to be a 4- or 5-hydroxymethyl alkaloid. Through comparisons of the 'H-NMR spectrum of 17 with that of 1 and that of acetylated 17, structure 17 was determined.
8-Hydroxycanthind-one (18)
8-Hydroxycanthin-6-one (18)has been isolated from the root bark of Ailanthus excelsa (25), the trunk (37) and stem bark (38) of Odyendea gabonensis, the stem bark of Pierreodendron kerstingii (39), and the wood of Simaba multifora (40).The bathochromic shift caused by alkali addition shown in the UV spectrum together with MS data suggested that
146
TAlCHl OHMOTO A N D KAZUO K O l K E
18 would be a hydroxycanthin-6-one alkaloid. Since a doublet I6 8.08 and 8.78 (each d, J = 4.9 Hz) and 6 6.97 and 8.12 (each d, J = 10.6 Hz)] arising from two isolated pairs of vicinal protons at H-l and H-2 as well as H-4 and H-5 is observed in the 'H-NMR spectrum, the hydroxy group should be located in ring A of the benzenoid nucleus. Moreover, since continuous three aromatic protons of ABC-type exist, the hydroxy group should be located at position 8 or 1 I . Comparison of the 'H-NMR spectra of 1 and 18 confirmed structure 18. 9-Methoxycanthin-6-one (19) 9-Methoxycanthin-6-one (19) has been isolated from the bark of Simabu cuspidata (2) and the stem bark of Simaba mufrijlora (41). From IR, U V , high-resolution MS (M+ at mlz 250.0735), and 'H-NMR (6 3.99, 3H, s), it was supported that 19 was a methoxycanthin-6-one. From the fact that there are two isolated pairs of vicinal protons (H-l and H-2; H-4 and H-5) in the 'H-NMR spectrum, it is obvious that the methoxyl group does not exist in ring C or D. In addition, the lower field proton H-8 (6 8.13, I N , d , J = 2 Hz) in ring A does not show ortho coupling. These findings led to determination of structure 19.
10-Hydroxycanthin-6-one (20) and 10-Methoxycanthin-6-one (21) Both I0-hydroxy- (20) and 10-methoxycanthin-6-one (21) have been isolated from the wood of S. muftijlora (42),and 20 has also been isolated from the root of Eurycoma longifolia (43). The U V spectrum of 20 shows a large bathochromic shift (76 nm) on alkali addition. Both the UV and IR spectra of 20 resemble those of 18. Acetylation and methylation of 20 produce the monoacetate and monomethyl ether, respectively. In the 'H-NMR spectrum of 20, two isolated pairs of vicinal protons in rings C and D and 1 , 2, 4-trisubstituted aromatic protons are observed. From these observations Arisawa et a f . (42) concluded that the hydroxyl function should associate with position 9 or 10. The position was fixed by the interlocking evidence, and structure 20 was determined. There was an increase in the molecular ion of 14 mass units in the mass spectrum of 21 as compared to 20 and the appearance of a new signal (6 3.91, 3H, s)
3. CANTHIN-6-ONE ALKALOIDS
147
R
(203 R=H (21) R = C k
arising from the methoxyl function in the 'H-NMR spectrum of 21, but otherwise both the UV and 'H-NMR spectra of 20 and 21 were alike. Thus, 21 was assumed to be the monomethyl ether of 20. Direct comparison of methylated 20 and naturally produced 21 supported structure 21. 11-Hydroxycanthin-6-one (Amarorine, 22) and 11 -Methoxycanrhin-6-one (Amaroridine, 23)
Both 1l-hydroxy- (22) and 1 I-methoxycanthin-6-one (23) have been isolated from the bark and wood of Amaroria soulameoides (44,45), and 22 has also been isolated from the stem of Brucea antidysenterica (46)and the stem bark of Quassia kerstingii (29). Since the UV spectrum of 22 showed a bathochromic shift on alkali addition, it was supposed that 22 had a phenolic hydroxyl group, and, together with findings from the studies on the IR and 'H-NMR spectra, Clarke et al. (44) assumed 22 to be a hydroxycanthin-bone derivative. When 22 was methylated, 23 was produced. Since the 'H-NMR spectra of both 22 and 23 showed pairs of aromatic vicinal protons at H-1 and H-2 as well as H-4 and H-5 and three coupled aromatic protons of the ABC type on ring A, it was concluded that the functional group should be attached to position 8 or 1 1. Through X-ray crystallographic studies on the monohydrate of 22, Clarke et af. assigned structure 22 to the compound having a hydroxyl function and structure 23 to the compound having a methoxyl function (44). Incidentally, 22 obtained from B . antidysenterica formed 11-O-bromobenzoylcanthin-6-one, X-ray analysis of which supported structure 22 (46).
(22) R=H (23) R=C%
148
TAICHI OHMOTO AND KAZUO KOIKE
I-Hydroxy-1I-methoxycanthin-6-one (24) and I 1 -Hydroxy-l-methoxycanthin-6-one (25)
Both I-hydroxy-1 l-methoxy- (24)and 1I-hydroxy-l-methoxycanthin6-one (25) have been isolated from the wood of B. antidysenterica (26, 47). In addition, 24 has been isolated from its root bark (47) and 25 from Soulamea pancheri (28). The UV spectrum of 24 shows a bathochromic shift on addition of alkali, and its mass spectrum shows the molecular ion at mlz 266 (M+, 100%). These facts support the supposition that 24 is a canthin-6-one alkaloid which has a hydroxyl and a methoxyl substituent. Since the 'H-NMR spectrum shows aromatic proton signals, a pair of vicinal protons at H-4 and H-5, a singlet at H-2, and three protons from H-8 through H-10, it is assumed that this compound is either 24 or 25. The 'H-NMR spectrum of 24 is not the same as that of 25, and irradiation of 11-OCH3induced an NOE (20%) of the H-10 signal but did not affect the H-2 signal. Based on these findings, structure 24 was determined. Structure 25 was determined after comparison of the 'H-NMR spectral data of 25 and those of 24 and from the NOE observed (H-2, 20%) by irradiation of l-OCH3 of acetylated 25.
Ri (24) (25)
Rz
H Cl-b Cl-b H
I , I I-Dimethoxycanthin-&one (26)
1,11-Dimethoxycanthin-6-one (26) has been isolated from the stem of B. antidysenterica (46). The IR and UV spectra indicate a typical canthin-6-one structure, the 'H-NMR spectrum shows two methoxyl signals [a 4.00 and 4.16 (each 3H, s)], and the high-resolution mass spectrum also suggests a dimethoxycanthin-6-one alkaloid. Since the 'H-NMR spectrum shows the presence of aromatic protons at H-4 and H-5, three protons on ring A, and also a IH singlet, it was considered that one of the two methoxyl groups is attached to either C-1 or C-2 and the other methoxyl to C-8 or C-1 I . NOE experiments on 26 revealed that irradiation of l-OCH3 and lI-OCH3 induced 12 and 13% enchancement at H-2 and H-10, respectively. From these facts, structure 26 was determined.
149
3. CANTHIN-&ONE ALKALOIDS
4-Hydroxy-5-methoxycanthin-6-one (Picrasidine Q , 27) Picrasidine Q (27) has been isolated from the root wood of Picrasmu quassioides (48).The IR spectrum of 27 shows a hydroxyl group, and its UV spectrum shows the typical absorption arising from the canthin-6-one chromophore. Its 'H-NMR spectrum shows a methoxyl group, a pair of ortho-coupled signals at H-1 and H-2, and signals of four continuous aromatic protons at H-8 through H-11. It is understood, therefore, that the hydroxyl and methoxyl substituents are located at positions 4 and 5, respectively. Methylated 27 is identical with 8 but is not identical with authentic 28. On the basis of these facts, structure 27 was determined.
cw 0
OH Cl-b
m
5-Hydroxy-4-methoxycanthin-6-one (nigakinone, 28) and 4,5Dimethoxycanthin-&one (8) Both nigakinone (28) and 4,5-dimethoxycanthin-6-one(8) have been isolated from the heartwood (49,501, stem (51), and wood (52) of P. quassioides and from the wood of P . excelsa (53). In addition, 8 has been isolated from the wood of P . quassioides (syn. P . ailanrhoides; 54), the trunk bark of Odyendea gabonensis (371, and the root bark of Quassia
4,5-dihydroxycanthind-one
0ocI-b l-cnethoxycarbony 1-o-carboline
150
TAICHI OHMOTO AND KAZUO KOIKE
africana (55). Acetylation of 28 gave the monoacetate, and direct comparison between methylated 28 and authentic 8 proved that they were fully identical. In addition, the CO absorption shown in the IR spectrum of 28 differs from that of 8 in wave number, that is, 28 and 8 show absorptions at 1632 and 1663 cm-', respectively. KMn04 oxidation of 28 produces 1-methoxycarbonyl-P-carboline.From these facts, structure 28 was determined. Incidentally, there is a report (17) which states that 28 is contained in 17 of 33 species of the genus Rhododendron of the family Ericaceae grown in China and 8 in 28 species of this genus.
4,5-Dihydrocanthin-6-one(29)
4,5-Dihydrocanthin-6-one(29) has been isolated only from cell cultures of A. altissima (13,15).Its mass spectrum shows the molecular ion at mlz 222, and the fragmentation pattern is very similar to that of canthin-6-one, with some differences; e.g., [M - HIf and [M - H - CO]' have higher intensity than the fragments obtained from 1, and 29 is larger than 1 by two mass units. The 'H-NMR spectrum of 29 shows the same data as that of 1 at the indole ring and positions 1 and 2, but aromatic protons at positions 4 and 5 have been substituted with two multiplets [6 3.22 and 3.49(each 2H)] which correspond to the methylene group, and hence structure 29 was determined. Further evidence was obtained by Haynes et al. when they synthesized 29 through reduction (HJRaney Ni or Zn plus AcOH) of 1(I).
S-Methoxycanthin-2,6-dione (Indacanthinone, 30), 3-Methoxycanthin-2,6-dione (2), Canthin-2,6-dione (31), (32) and IO-Hydroxy-3-methoxycanthin-2,6-dione Indacanthinone (30) was the first compound isolated as a canthin2,6-dione alkaloid from the wood of Samadera indica (56). 3Methoxycanthin-2,6-dione(2), canthin-2,6-dione (31), and lO-hydroxy-3methoxycanthin-2,6-dione (32) have been isolated from the wood of Simaba multiJora (42), and 2 has been also isolated from the bark of S. cuspidata ( 2 ) .Compounds 30, 2, 31, and 32 give intense yellowish green fluorescence in solution with organic solvents. Their UV spectra strongly
3. CANTHIN-6-ONE ALKALOIDS
I51
fluorescence in solution with organic solvents. Their UV spectra strongly resemble those of canthin-6-one alkaloids. The mass spectrum of 30 proves the molecular formula C ~ S H ~ O NIt~has O ~been . made clear from the 'H-NMR spectrum and measurement of methoxyl groups by Zeisel's method that one of the three oxygens in the formula belongs to a methoxyl group. The IR and mass spectra of 30 suggest it has two CO groups. One of them shows a -NHCO- function and weak basicity. By allowing SO2 gas to pass through the orange-red solution of 30 prepared in an organic solvent, the solution is decolorized, regaining the orange-red color on aeration. Reactions between 30 and POC13 result in substitution of the enol O H with Cl to form the chlorine-containing compound (CI5H9N2O2CI). When the cool chloroform solution of 30 absorbs bromine, the bromine-containing compound (ClsH9N202Br),which shows the same UV spectrum as that of 30, is produced. These facts strongly support structure 30. The UV spectra of 2, 31, and 32 are similar to that of 30. MS of these three compounds shows parent ions at mlz 266 (CI5Hl0N2O3),mlz 236 ( C I S H ~ N ~ Oand ~ ) , mlz 282 (CI5HION2O4), respectively. The 'H-NMR spectrum of 2 shows, in addition to a methoxyl signal (6 4.20, 3H, s), a pair of doublets which are assigned to H-4 and H-5 and a proton signal (6 7.26, IH, s) which is assigned to H-1. In addition it indicates the existence of four aromatic protons on ring A. On the basis of these findings, structure 2 was determined. Compound 31 is produced through reduction of 2 with sodium hydrogen sulfide. Direct comparison indicates that this synthetic product is identical with naturally produced 31. Thus, structure 31 was determined. On the other hand, Giesbrecht et al. (2) made comparisons among the signals arising from N-OCH3 of 2 (6 64.8). C-OCH3 of 2-methoxypyridine (6 53.1), and N-OCH3 of pyridone (6 64.7)
152
TAlCHl OHMOTO A N D KAZUO KOIKE
and determined structure 2. The 1R spectrum of 32 indicates the existence of a hydroxyl group (3440 cm-I), and acetylation of 32 produces the monoacetate. By comparing the 'H-NMR spectra of the monoacetate and lO-acetoxycanthin-6-0ne, structure 32 was determined.
3-Mefhylcanfhin-5,6-dione(Picrasidine L , 3) Picrasidine L (3) has been isolated from the root bark ( 3 ) ,root (57), and wood (58) of P . quassioides and from the wood of Quassia umara (59). The compound reported as 3-methylcanthin-2,6-dione was identified as 3 by Ohmoto and Koike (3).The compound isolated from the wood of Q . amara (59) was also corrected from 3-methylcanthin-2,6-dioneto 3 (60). The I3C-NMR spectrum of 3 indicates the existence of two carbonyl carbons (6 156.34 and 169.79). The UV spectrum of 3 resembles that of 2; however, while addition of acid or base produces no change in the UV spectrum of 2, addition of acid causes a large hypochromic shift in U V spectrum of 3, though base induced no subsequent change. 'H-NMR spectral data of 3 reveal four vicinal protons which are positioned at H-8 through H-1 1 of ring A , a pair of ortho-coupled signals at H-1 and H-2, a methyl proton signal (6 3.98, 3H, s), and an olefinic proton signal (H-4, 6 5.98, l H , s); hence, structure 3 was proposed. The synthetic product obtained by methylation of 16 completely agrees with natural 3, and thereby structure 3 was confirmed.
3-Methyl-4-methoxycanthin-5,6-dione (Picrasidine 0 , 33)
Picrasidine 0 (33) has been isolated from the root wood of P . quassioides (48).The UV spectrum shows, like 3, a hypochromic shift on addition of acid. The 'H-NMR spectrum shows signals arising from a methyl (6 3.82, 3H, s) and a methoxyl (6 4.26, 3H, s) but lacks the aromatic proton at H-4, and hence structure 33 was proposed. Since the synthetic compound obtained through methylation of 28 agrees with naturally produced 33, structure 33 was confirmed.
3. CANTHIN-&ONE ALKALOIDS
153
Picrasidines M (4), N (34), and U (35)
Picrasidine M (4) has been isolated from the root bark of P . quassioides (3) and picrasidines N (34) and U (35) from the root wood of the same plant (48,61).All three compounds emit the strong yellowish fluorescence specific to canthin-5,bdiones in organic solvents. From elemental analyses of 4 and 34 their molecular formulas were obtained: 4, C29HZZN404; 34, C30H24N404. While the IR spectrum of 4 shows an absorption band arising from an amino group at 3420 cm-', 34 does not show this absorption. In the 'H-NMR spectrum of 4, signals of the AzBztype [6 3.72 ( 2 H , t, J = 7.1 Hz) and 4.77 (2H, t, J = 7. I Hz)] are observed, and hence a -CH2CH2-unit should be present. Two methoxyl signals [6 3.97 (3H, s) and 4.13 (3H, s)] are observed, and aromatic proton signals are also observed at H-3' (6 7.99, l H , s), H-8 through H-1 1 , and H-5' through H-7'; hence, it has been suggested that 4 is a compound in which the canthin-5,6-dione and I-ethyl-4,8-dimethoxy-P-carbolinestructures are found together between N-3 and C-1'. Compound 4 gives 5acetoxycanthin-bone and 4,8-dimethoxy-I-vinyl-~-carbolinewhen treated with acetic anhydride. Methylation of 5-acetoxycanthin-6-one produces 7. From direct comparison among synthesized 5-
154
TAICHI OHMOTO AND KAZUO KOlKE
methoxycanthin-6-one and 7 and synthesized 4,8-dimethoxy-l-vinyl-Pcarboline and respective authentic samples, structure 4 was determined. The IR and 'H-NMR spectra of compound 34 lack signals of an NH proton of the indole moiety in the p-carboline structure, but otherwise they are very much similar to those of 4. Compound 34 produces when 5-acetoxycanthin-6-one and 4,9-dimethoxy-I-vinyl-~-carboline treated with acetic anhydride, and hence structure 34 was determined. Comparison between the 'H-NMR spectrum of 35 and that of 4 shows a lack of an H-4 signal in the former but an increase in the methoxyl signal by one. Otherwise the patterns of both spectra are the same. Cleavage of 35 with acetic anhydride produces 5-acetoxy-4-methoxycanthin-6-one and 4,8-dimethoxy-I-vinyl-P-carboline, and hence structure 35 was determined.
Infractopicrin ( 5 )
Infractopicrin (5) has been isolated as a bitter substance from the fruiting bodies of Cortinurius infractus ( 4 ) . The IR and U V spectra suggest that 5 is a canthin-6-one alkaloid. Its 'H-NMR spectrum shows the existence of a -CH2CH2CH2-group [6 2.47 (2H, m), 3.22 (2H, t t , J = 6 and 0.6 Hz), and 4.96 ( 2 H , t , J = 6 Hz)]. Its mass spectrum shows mlz 36 and 38 ions, whose existence was confirmed by chlorination. Thus, structure 5 was determined.
IV. 13C-NMR Spectroscopy Over 30 canthin-6-one alkaloids have already been isolated, and their structures have been determined. There are not many reports, however, which mention that "C-NMR spectroscopy was used for structural
3. CANTHIN-bONE ALKALOIDS
155
determination (2, 3, 48, 89, 90). The reason for this seems to be that, although canthin-6-one alkaloids have low molecular weights, they have many tertiary carbons, and this, together with two nitrogen atoms in the molecule, makes it difficult to assign the carbon atoms causing chemical shifts in I3C-NMR spectrum. Koike and Ohmoto (90) measured I3C-IH shift correlation two-dimensional NMR (91) for the first time and clearly assigned proton-bearing carbons (Fig. I). Koike and Ohmoto then assigned the chemical shifts in the "C-NMR spectra of canthin-6-one alkaloids using spin-spin coupling between 13C and 'H. They obtained a spin-spin network (Fig. 2) between I3C and 'H and coupling constants ( ' J , Table 11; 'f and ' J , Table 111) using protoncoupled "C NMR and the long-range selective decoupling (LSPD) method (92). Thus, assignment of all carbon atoms in canthin-6-one alkaloids have been established (Table IV). By comparing ring C of 1 with its model compound, pyridine, it is seen that spin-spin coupling constants of the two are very similar (Fig. 2). Both carbonyl carbons at position 6 of 1 and in acrylic acid, which is the model compound of 1, have very large 3J values for vicinal trans coupling (Fig. 2), and this fact also strongly supports the assignment of carbon atoms in canthin-6-one alkaloids. Investigation of substituent effects of 11,28, and 8 revealed that C-2 in the ortho position and C-16 in the para position in 11, which possesses a substituted methoxyl group at position 1 , showed about 14 ppm and 7 ppm high-field shifts, respectively, as compared with 1. Substituent effects owing to oxygen functional groups at positions 4 and 5 were shifted about 6 ppm upfield for C-15 in 28 and about 3 ppm upfield for C-15 in 8. Another observation disclosed that C-4 in 8, which is produced by substituting a methoxyl for a hydroxyl group at position 5 in 28, showed an approximately 10 ppm down-field shift (90).
V. Synthesis
A. SYNTHESIS OF CANTHIN-6-ONES Cook et al. (93,94) obtained lactam 37a, in 82% yield, by refluxing Nb-benzyltryptamine (36a) and 2-ketoglutaric acid in toluene in the presence of p-toluenesulfonic acid. Then they oxidized 37a with SeOz in dioxane. As a result, loss of the Nb-benzyl group and aromatization of ring C proceeded simultaneously, and canthin-6-one (1) was successfully synthesized in 33% yield. Under similar conditions, using Nb-benzyl tryptophan methyl ester (36b), 2-methoxycarbonylcanthin-6-one (38) was
N
Y
m
w
a d
-LT
I'
7
R
-f
a
m
157
3. CANTHIN-6-ONE ALKALOIDS
Hy7 JH f 0
14.1
FIG. 2. Spin network of 1, pyridine, and acrylic acid ( J , Hz).
synthesized in 66% yield, forming 37b on its way. Rosenkranz ef ul. (95) synthesized Nb-succinyltryptophan (39a) from tryptophan and succinic acid. They 39a was added to polyphosphoric acid, V205, and P0Cl3, and the mixture was heated at 115°C. Thus, 1was synthesized in one step at 75% yield. 4-Ethylcanthin-6-one (40) was synthesized by the same method (Scheme I). In addition, there are reports of the following syntheses: 4,5dimethoxycanthin-6-one (8) from 1-methoxymethyl-P-carbolineand succhic acid anhydride (96), 1 using 4-oxo-l,4,6,7,12,12b-hexahydroindo
TABLE I1 ONE-BOND "C-'H COUPLING CONSTANTS (Hz) FOR COMPOUNDS 1, 11, 28, A N D 8 Carbon 1 2 4 5 8 9 10
II I-OCHI 4-OCH3 5-OCH,
1
11
28
8
165. I 179.7 165.1 168.0 168.5 162.1 162.1 162.1
178.6 164.0 168.0 168.7 160.0 160.0 160.0 145.5 -
164.0 179.0 -
164.0 178.6
-
168.0 160.0 160.0 160.0 146.7 145.7
168.0 160.0 160.0 160.0 146.5
u
W
m
9 10
II
12
dd 'J(C-9, H-1 I ) dd 'J(C-lo, H-8) ddd *J(C-11, H-10) 'J(C-11, H-9)
13
t
14
'J(C-13. H-9) 'J(C-13, H-11) dd ?J(C-14, H-1) 'J(C-14, H-2)
15
8.8 8.8
'J(C-13, H-9) 'J(C-13, H-11) d 'J(C-14, H-2)
8.0
8.0 1.5
8.0
t
6.0 6.0
t
3.7 8.1 8.1 8.1
t
'J(C-16, H-2) 'J(C-16. H-5) 1-OCHS 4-OCH' 5-OCH'
'J(C-12, H-10) 'J(C-12, H-8)
1.5
t
'J(C-15, H-I) 'J(C-1.5, H-4) 16
6.0 6.0
8.1
t
'J(C-12, H-10) ~J(c-12, H-8)
-$
8.1
dd )J(C-9, H-11) dd 'J(C-lO, H-8) ddd *J(C-Il, H-10) 'J(C-11, H-9)
8.1
1 I .4
11.4
d 'J(C-15, H-4)
t 'J(C-16, H-2) 'J(C-16, H-5) q
8.8 8.8 8.0
8.0
11.0 11.0
dd 'J(C-9, H-l I ) dd 'J(C-lo, H-8) ddd 'J(C-11, H-10) 'J(C-11, H-9) t ' ~ ~ ( c - 1 H-10) 2, 'J(C-12, H-8) t 'J(C-13, H-9) 'J(C-13, H-11) dd *J(C-14, H-I) 'J(C-14, H-2) d ?I(C-15, H-I) d 'J(C-16, H-2)
1.5 8.0
dd 'J(C-9, H-11) dd 'J(C-lo, H-8) ddd 'J(C-11, H-10) 'J(C-ll, H-9)
6.0 6.0
'J(C-12, H-10) 'J(C-12, H-8)
8.0 8.0
'J(C-13, 'J(C-13, dd 'J(C-14, 'J(C-14, d 'J(C-15,
8.0 8.0
8.0 8.0 1.5
8.0
t
6.0 6.0
t
3.5 8.0 8.0
12.5
H-9) H-11)
8.0 8.0
H-I) H-2)
3.5 8.0
H-I)
8.0
d 'J(C-16, H-2)
12.8
I60
TAICHI OHMOTO A N D KAZUO KOIKE
TABLE IV "C-NMR SPECTRAL DATA(ppm) FOR COMPOUNDS 1, 11, 28, A N D 8 Carbon
1
11
28
8
I 2 4 5 6 8 9 10 I1 12 13 14 15 16 I-OCH3 4-OCHj 5-OCHz
115.37 144.84 138.57 127.98 158.21 116.29 129.84 124.69 121.61 123.33 138.24 128.99 130.91 135.23 -
152.23 130.69 138.69 124.61 160.21 116.73 129.59 125.66 124.37 123.88 138.17 130.18 130.83 128.70 56.70 -
113.70 144.11 142.68 139.60 156.27 115.22 129.62 124.65 122.39 124.20 137.38 127.96 124.82 133.46 60.51 -
114.89 144.48 152.05 139.35 157.43 116.24 130.01 124.59 121.78 123.96 138.22 129.15 127.61 132.71 61.17 61.04
-
pCH3 1
4.4
O+OC&
[2,3-a]chinolizine and NaNOz (97), and 1 using naturally produced eburnamonine and Se (98). Moreover, Hagen and Cook (99) synthesized 3-0x0-9-methoxycarbonyl indolizino[8,7-b]indole (41) from tryptamine hydrochloride and dimethyl a-ketoglutamate in refluxing methanol. As
161
3. CANTHIN-bONE ALKALOIDS
tryptamine : W tryptophan : Rco(x1
3 6 3 : RH 331: Fi4JX-b
7 days
37a : RH 3ib: m
1: W
38:
rn
SCHEME I
illustrated in Scheme 2, they obtained 1-methoxycanthin-6-one by treatment of 41 with dichlorodicyanobenzoquinone (DDQ) and followed by hydrolysis, methylation, and cyclization.
2) WQ dioxane,
A 11 SCHEME2
162
T A l C H l OHMOTO AND KAZUO KOIKE
P
I
UJ
2
1
I
2
FIG. 3. UV spectra of 3 and 16. ~, EtOH; ...... 16 in EtOH + CHI.
B.
3 in Et0H;---,
3 in EtOH
+ HCI; -.-.-,16
in
S Y N T H E S I S OF C A N T H I N - 5 , 6 - D I O N E S
UV spectra of canthin-5,6-dione alkaloids 3 and 33, which were isolated by Ohmoto and Koike (3,48) from Picrasma quassioides, showed a characteristic absorption between 400 and 500 nm. This absorption was hypochromically shifted under acidic conditions. The UV spectrum of 3 in acidic solvents resembles that of 5-hydroxycanthin-6-one (16) (Fig. 3). This fact, that is, that 3 undergoes chemical shift in acidic solvents, indicates that the carbonyl at position 5 of the canthin-5,ddione skeleton in 16 is protonated, producing 42a. On the basis of the above observations, Ohmoto and Koike refluxed 16 and 33 with dimethyl sulfate in acetone and synthesized 3 and 28 (Scheme 3).
I63
3. CANTHIN-6-ONE ALKALOIDS
'H
+
d
7
'ab
w
56?
acetone,
w
M
0 3 : RSI 28: rn
16: RH
33: F#x)b
42a: Mi
a :F#x)b
SCHEME 3
Similarly, Matus and Fischer (100) heated I-alkyl-p-carboline with an excess of dialkyl oxalate and synthesized canthin-5,6-dione derivatives in 20-65% yield (Scheme 4). Catalytic hydrogenation of Nb-benzylcanthin5,ddione (43a) produces 16. Based on these reactions, the structures of canthin-5,bdione derivatives were elucidated (Scheme 5 ) . YOOR2
R,J
q
q
N CbR
+
R 43aH b
H
c H dCH3 e C& f C &
g
H
COO&
Fb
RI H
M
H
C& C&
47
C-kFl-l
64
H n %
OcH3CA-k SCHEME 4
o=f&& Ph
=AD
0
42
44 20 39
C&
H H H
yield (%)
29
0j-q
0
OH
16
43a SCHEME 5
164
TAICHI OHMOTO AND KAZUO KOIKE
VI. Biosynthesis It was hypothesized that the biosynthetic pathway to canthin-6-one alkaloids started from tryptophan as a precursor and produced tryptamine on the way to canthin-6-one (1) (101) (Scheme 6). Anderson et al. (103) established, for the first time, the method of tissue cultivation of Ailanthus altissima using cell and cell suspension cultures. It was recognized that yield of 1 from tissue cultures was 100 to 1000 times higher than the content of 1 in the plant body. From feeding experiments using cell suspension cultures and providing ~ - [ m e t h y l e n e ' ~ C ] tryptophan as the precursor, production of radioactive 1, 10, and 11 was confirmed. The rate of production gradually increased in accordance with the length of feeding time (16,102).
H
H
tryptophan
tryptamine
0canthin-ne
SCHEME 6
Similarly, Crespi-Perellino et al. (13,15), using cell cultures of A. altissima and providing L-, D-, and D,L-[methylene-'4C]tryptophanas the precursor, carried out tracer experiments and proved the biosynthetic pathway to canthin-6-one alkaloids to be as follows (Scheme 7): tryptophan + P-carboline-I-propionic acid + 4,5-dihydrocanthin-6-one(29) + canthin-6-one (1) + I-hydroxycanthin-6-one (10)+ I-methoxycanthin-6one (11) + I-methoxycanthin-6-one 3-oxide (12).In the biosynthetic pathway to canthin-6-one alkaloids, oxidation proceeds stepwise. The hydroxyl group at position 1 of canthin-6-one is methylated, and 11 is readily formed; this formation is considered to be a transmethylation promoted by a specific enzyme. Anderson et al. (103) carried out feeding experiments with cell cultures and obtained radioacof A. altissima and ~-[methylene-'~CImethionine tive 14. They supposed, therefore, that L-methionine would become S-adenosylmethionine and associate with transmethylation of 10 to produce 11. In accordance with growth of the cell culture, radioactive 11 gradually increases; hence, they stated that the 0-methyltransferase acts at the last stage of synthesis.
3. C A N T H I N - b O N E ALKALOIDS
165
SCHEME 7
VII. Bioassay and Pharmacology Over twenty canthin-6-one alkaloids have been bioassayed in the following areas. The antimicrobial activities of 1and 11against 1 I kinds of bacteria were almost negligible compared to that of streptomycin (70,87). Activities of 1 on various bacterial and fungal strains were investigated, and it was found that in general the activity was strong toward fungi relative to bacteria (66,86,104). When the hydroxyl group of the canthin-6-one nucleus was changed to a methoxyl group, the antibacterial activity decreased (70). Both 8 and 28 inhibited growth of Staphylococcus aureus and its drug-resistant strains (50). As it is well known that Ailanthus altissima (23) and Eurycoma longfolia (43) are used as febrifuges and antimalarial agents among folk medicines in Southeast Asian countries, their antimalarial and amebicidal activites were measured. Several quassinoids in such plants showed activity but 1,11,18,21, and 31 did not (23,43).The amebicidal activity of quassin and 1 was determined by bioassay using axenic Entamoeba histolytica. While the EDSovalue of quassin was 0.5 pg/ml, that of 1was as large as 23 pg/ml (82,105).
166
TAICHI OHMOTO AND KAZUO KOIKE
The antiherpes activity of four kinds of canthin-6-one alkaloids was assayed biologically together with 10 P-carboline derivatives. Among these compounds, 8 and 28 had activity on a level with that of acyclovir, the control. It was noticed, however, that the therapeutic ratio was small (106).
As part of a search for antitumor agents in plants, testing of cytotoxic activity has been caried out in uitro with three kinds of cell systems. In the guinea pig keratinocyte (GPK) system, 1, 7, 9, and 11 did not show statistically significant cytotoxic activity (14) in GPK epithelial cells as judged from inhibition of DNA synthesis. In the P388 lymphocytic leukemia system, both 22 and 26 had cytotoxic activity (45,46). In the nasopharynx (KB) system, none of the compounds 1, 7, 11, and 18 showed full cytotoxic activity (25). Compounds 20,21, and 22 had weak activity (42,45). The activities of 16 canthin-6-one alkaloids, including 1, 7, and 11, which were already reported, and 13 other synthetic compounds were assayed biologically. The results indicated that 22 and 26 had the strongest activity (26). When the structure-cytotoxicity relationship was investigated, it was found that compounds such as 22 and 26 which were oxidized at position C-10 or C-11 of ring A showed strong cytotoxic activities. It was also noted that activity was reduced by replacing the hydroxy group in 11-hydroxy derivatives with methyl ether or ester (26,45). Compounds 1 and 2 manifested phototoxicity on bacteria and fungi on irradiation with near-UV light (320-400 nm). Both compounds inhibited mitosis of Chinese hamster ovary cells and induced chromosomal changes. It was apparent that the two compounds could be photosensitizers, though their activities were weaker than 8-methoxypsoralen, the control (107). With the inhibitory activity against cyclic adenosine monophosphate phosphodiesterase as an index, in uitro bioassay of the activity of 21 canthin-6-one alkaloids was carried out. The strongest inhibitory activities were detected with 4, 17, and 27 among the compounds tested. The activities shown by 10,28, and 34 were the same, twice as strong, and 15 times as strong, respectively, as the activity of papaverine, the control. Acetylation and methylation of the hydroxy derivatives of canthin-6-one decreased activity (108,109). The rate of blood flow in the stomach and intestine of rabbits was assayed in uiuo by the hydrogen clearance method. Compounds 1,28, and 29 increased the blood flow rate in the intestine by 15, 25, and 35%, respectively. In the stomach, however, 1 increased the rate by only lo%, and 28 decreased it (110).
3. CANTHIN-6-ONE ALKALOIDS
I67
REFERENCES
1 . H. F. Haynes, E. R. Nelson, and J. R. Price, Ausr. J. Sci. Res., Ser. A 5, 387 (1952). 2. A. M. Giesbrecht, H. E. Gottlieb, 0. R. Gottlieb, M. 0. F. Goulart, R. A. De Lima, and A. E. G. Sant’ana, Phyrochemisrry 19, 313 (1980). 3. T. Ohmoto and K. Koike, Chem. Pharm. Bull. 33, 3847 (1985). 4. W. Steglich, L. Kopanski, M. Wolf, M. Moser, and G. Tegtmeyer, Terrahedron L e f t . 25, 2341 (1984). 5 . W. I. Taylor, in “The Alkaloids” (R. H. F. Manske, ed.), Vol. 8, p. 249. Academic Press, New York, 1965. 6. G. A. Cordell, “Introduction to Alkaloids: A Biogenetic Approach,” p. 619. Wiley, New York, 1981. 7. J. A. Joule, in “The Alkaloids” (J. E. Saxton, ed.), Vol. I , p. 156. Royal Society of Chemistry, London, 1971. 8. J. E. Saxton, in “The Alkaloids” (M. F. Grundon, ed.), Vol. 7, p. 183. Royal Society of Chemistry, London, 1977. 9. J. E. Saxton, in “The Alkaloids” (M. F. Grundon, ed.), Vol. 8, p. 154. Royal Society of Chemsitry, London, 1978. 10. J. E. Saxton, in “The Alkaloids” (M. F. Grundon, ed), Vol. 9, p. 152. Royal Society of Chemistry, London, 1979. 1 1 . J. E. Saxton, in “The Alkaloids” (M. F. Grundon, ed.), Vol. 11, p. 145. Royal Society of Chemistry, London, 1981. 12. J. E. Saxton, Nat. Prod. Rep., 591 (1987). 13. N. Crespi-Perellino, A. Guicciardi, G. Malyszko, and A. Minghetti, J . Nar. Prod. 49, 814 (1986). 14. L . A. Anderson, A. Harris, and J. D. Phillipson, J. Nar. Prod. 46, 374 (1983). 15. N. Crespi-Perellino, A. Guicciardi. G. Malyszko, E. Arlandini, M. Ballabio, and A. Minghetti, J. Nar. Prod. 49, 1010 (1986). 16. L. A. Anderson, C. A. Hay, M. F. Roberts, and J . D. Phillipson. Planr Cell Rep. 5,387 (1986). 17. Laboratory of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, Acra Eor. Sin. 19, 257 (1977). 18. T. Ohmoto, K. Tanaka, and T. Nikaido, Chem. Pharm. EiiII. 24, 1532 (1976). 19. T. Ohmoto, K. Koike, and Y. Sakamoto, Chern. Pharm. EIIII. 29, 390 (1981). 20. E. Varga, K. Szendrei, J. Reisch, and G. Maroti, Fitoterapia 52, 183 (1982). 21. S . A. Khan and K. M. Shamsuddin, Phytochemisfry 20, 2062 (1981). 22. E. Varga, K . Szendrei, J. Reisch, and G. Maroti, PIanra Med. 40, 337 (1980). 23. D. H . Bray, P. Boardman, M. J. O’Neill, K . L . Chan, J . D. Phillipson, D. C. Warhurst, and M. Suffness, Phytorher. Res. 1, 22 (1987). 24. C. Souleles and R. Waigh, J. Nar. Prod. 47, 741 (1984). 25. G. A. Cordell, M. Ogura, and N. R. Farnsworth, Lloydia 41, 166 (1978). 26. N. Fukamiya, M. Okano, T. Aratani, K. Negoro, Y. M. Lin. and K. H. Lee, Planfa Med. 53, 140 (1987). 27. L . Lumonadio and M. Vanhaelen, Phyrochemisrry 23, 453 (1984). 28. B. Viala, Thesis, Universite de Paris-Sud, Centre d’Orsay, France (1971). 29. G. R. Pettit, S. B. Singh, A. Goswami, and R. A. Nieman, Tetrahedron 44,3349 (1988). 30. L. M. Jackman and S. Sternhell, “Application of NMR Spectroscopy to Organic Chemistry,” 2nd ed., p. 202. Pergamon, New York, 1969.
168
TAICHI OHMOTO A N D KAZUO KOIKE
E. R. Nelson and J. R. Price. ANSI.J. Sci. Res. Ser. A 5, 768 (1952). P. J. Scheuer and T. R. Pattabhiraman, Lloydia 28, 95 (1965). W. S. Chen, Acta D o t . Sin. 28, 450 (1986). E. V. Lassak, J . Polonsky, and H. Jacquemin, Phytochemistry 16, 1126 (1977). E. R. Nelson and J. R. Price, Aust. J. Sci. R e s . , Ser. A 5, 563 (1952). T. Ohmoto and K. Koike, Chem. Pharm. Bull. 32, 170 (1984). P. Forgacs, J. Provost, and A. Touche, Planta Med. 46, 187 (1982). P. G. Waterman and S. A. Ampofo, Plurrttr M i d . 50, 261 (1984). S. A. Ampofo and P. G. Waterman, J . Nut. Prod. 48, 863 (1985). M. Arisawa, A . Fujita, N . Morita, A. D. Kinghorn, G. A. Cordell, and N. R. Farnsworth. Planta Med. 51, 348 (1985). 41. J. Polonsky, J. Gallas, J. Varenne, J. Prance, C. Pascard, H. Jacquemin, and C. Moretti, Tetrahedron Leu. 23, 869 (1982). 42. M. Arisawa, A. D. Kinghorn, G. A. Cordell, and N. R. Farnsworth, J. Nut. Prod. 46, 222 (1983). 43. K. L. Chan, M. J. O'Neill, J. D. Phillipson, and D. C. Warhurst, Planta Med. 52, 105 (1986). 44. P. J. Clarke, K. Jewers. and H. F. Jones, J . Chem. Soc. Perkin Trans. I , 1614 (1980). 45. S. S. Handa. A. D. Kinghorn. G. A . Cordell. and N. R. Fransworth, J. Nut. Prod. 46, 359 (1983). 46. N. Fukamiya, M. Okanao. T. Aratani, K. Negoro, A. T. McPhail, M. Ju-ichi, and K. H. Lee, J. Nut. Prod. 49, 428 (1986). 47. A. Harris. L. A. Anderson, J. D. Phillipson, and R. T. Brown, Planta Med., 51, 151 (1985). 48. T. Ohmoto and K. Koike, Chem. Pharm. Bull. 33, 4901 (1985). 49. Y. Kimura, M. Takido, and S. Koizumi, Yakugaku Zosshi 87, 1371 (1967). 50. J. S. Yang, S. R. Luo. X. L. Shen, and Y. X. Li, Acta Pharm. Sin. 14, 167 (1979). 51. Y. Kondo and T. Takemoto, Chem. Pharm. Bull. 21, 837 (1973). 52. T. Ohmoto and K . Koike, Chem. Pharm. Bull. 32, 3579 (1984). 54. N. Inamoto, S. Masuda, 0. Simamura. and T. Tsuyuki, Bull. Chem. Soc. J p n . 34,888 ( 1961). 55. L. Lumonadio and M. Vanhaelen, J. Nut. Prod. 49, 940 (1986). 56. V. S. lyer and S. Rangaswami, Curr. Sci. 41, 140 (1972). 57. T. Ohmoto and K. Koike, Chem. Pharm. Bull. 30, 1204 (1982). 58. J. S. Yang and D. Gong, Acfu Chim. Sin. 42, 679 (1984). 59. P. Barbetti. G. Grandolini, G. Fardella, and I. Chiappini, Planta Med. 53, 289 (1987). 60. G. G r a n d o h i , private communication. 61. K. Koike and T. Ohmoto, Phytochemistry 27, 3029 (1988). 62. I. A. Benages, M. E. A. De Juarez, S. M. Albonico, A. Urzua, and B. K. Cassels, fhvtochemistry 13, 2891 (1974). 63. E. M. Assem, 1. A. Benages, and S. M. Albonico, Planta Med. 48, 77 (1983). 64. P. R. Torres and B. K. Cassels, A n . Asoc. Quirn. Argent. 63, 187 (1975). 65. F. Fish and P. G . Waterman, Phytochemistry 10, 3325 (1971). 66. 0. 0. Odebiyi and E. A . Sofowora. Planto Med. 36, 204 (1979). 67. S. Najiar. G. A. Cordell. and N. R. Farnsworth, Phytochemistry 14, 2309 (1975). 68. C. M. Kim and 1. 0. Huh, Korean J . Phurmucogn. 12, 5 (1981). 69. F. Fish, A. I. Gray, and P. G. Waterman, Phytochemistry 14, 2073 (1975). 70. L. A . Mitscher, H. D. H. Showalter, M. T. Shipchandler, R. P. Leu and J. L . Beal, Lloydiu 35, 177 (1972). 71. F. Fish. A. I. Gray, and P. G. Waterman, 1.Phurm. Pharmcol. 28, Suppl., 69p (1975). 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
3. CANTHIN-&ONE ALKALOIDS
169
A. T. Awad, J. L. Beal, S. K. Talapatra, and M. P. Cava, J. Pharm. Sci.56,279 (1967). P. G. Waterman, Phytochemistry 15, 578 (1976). S. K. Talapatra, S. Dutta, and B. Talapatra, Phytochemistry 12, 729 (1973). H. K. Desai, D. H. Gawad, T. R. Govindachari, B. S. Joshi, P. C. Parthasarathy. K. S . Ramachandran, K. R. Ravindranath, A. R. Sidhaye. and N. Viswanathan, Indian J. Chem. Sect. B 14B, 473 (1976). 76. G. B. Guise, E. Ritchie, R. G. Senior, and W. C. Taylor, Aust. J. Chem. 20, 2429 (1967). 77. J. R. Cannon, G . K. Hughes, E. Ritchie, and W. C. Taylor, Aust. J. Chem. 6, 86 (1953). 78. J. Vaquette, A. Cave, and P. G . Waterman, Plunta Med. 35, 42 (1979). 79. K. Szendrei, T. Korbely, H. Krenzien. J. Reisch. and I. Novak, Herha Hung. 16, 15 (1977). 80. A Harris, L . A. Anderson, and J. D. Phillipson, J. Pharm. Pharmacol. 33, 17p (1981). 81. L. Lumonadio and M. Vanhaelen, Phytochemistry 23, 2121 (1984). 82. A. H a m s and J. D. Phillipson. J. Pharm. Pharmacol. 34, 43p (1982). 83. T. Ohmoto, K. Koike, and K. Kageil, Shoyakugaku Zasshi 41, 338 (1987). 84. T . Ohmoto and K. Koike, Chem. Pharm. Bull. 31, 3198 (1983). 85. M. Arisawa, A. Fujita, N. Morita, P. J. Cox, R. A. Howie, and G. A. Cordell, Phytochemistry 26, 3301 (1987). 86. M. Yokota, H. Zenda, T. Kosuge, and T. Yamamoto. Yakugakrr Zusshi 98, 1508 (1978). 87. L. Lumonadio, M. Vanhaelen, and M. J. Devleeschouwer, Fitotempiu 57,291 (1986). 88. D. D. Casa and M. Sojo, J. Chem. Soc. C , 2155 (1967). 89. N. Fukamiya, M. Okano, T. Aratani, K. Negoro, A. T. McPhail. M. Ju-ichi, and K. H. Lee, J . Nat. Prod. 49, 4281 (1986). 90. K. Koike and T. Ohmoto, Chem. Pharm. Bull. 33, 5239 (1985). 91. A. Bax, “Two Dimensional Nuclear Magnetic Resonance in Liquids.” Delft Univ. Press. Reidel, Dordrecht, 1982. 92. H. Seto, T. Sasaki, H. Yonehara, and J. Uzawa, Tetrahedron Lett. 18, 923 (1978). 93. 0. Campos, M. DiPierro. M. Cain. R. Mantei, A. Gawish, and J. M. Cook, Heterocycles 14, 975 (1980). 94. M. Cain, 0. Campos, F. Guzman, and J. M. Cook, J . A m . Chem. Soc. 105,907 (1983). 95. H. J . Rosenkranz, G. Botyos, and H. Schmid. Justus Liehigs Ann. Chem. 691, 159 (1966). 96. L. A. Mitscher, M. Shipchandler, H . D. H . Showalter. and M. S. Bathala, Heterocycles 3, 7 (1975). 97. R. Oehl, G. Lenzer, and P. Rosenmund, Chem. Ber. 109, 705 (1976). 98. M. F. Bartlett and W. 1. Taylor, J . A m . Chem. Soc. 82, 5941 (1960). 99. T. J . Hagen and J . M. Cook, Tetrahedron Lett. 29, 2421 (1988). 100. I. Matus and J. Fishcer, Tetrahedron Lett. 26, 385 (1985). 101. R. Hegnauer, in “Chemical Plant Taxonomy“ (T. Swain, ed.), p. 410. Academic Press, New York, 1963. 102. L. A. Anderson, M. F. Roberts, and J. D. Phillipson, Plant Cell Rep. 6, 239 (1987). 103. L. A. Anderson, C. A. Hay, J. D. Phillipson, and M. F. Roberts, Plunt Cell Rep. 6,242 (1987). 104. T. Ohmoto and Y. 1. Sung, Shoyakugaku Zasshi 36, 307 (1982). 105. A. T. Keene, A. Horris, J. D. Phillipson, and D. C. Warhurst. Planta Mad. 52, 278 (1986). 106. T. Ohmoto and K. Koike, Shoyakugaku Za.tshi 42, 160 (1988). 72. 73. 74. 75.
170
TAICHI OHMOTO AND KAZUO KOIKE
107. G. H. N . Towers and Z. Abramowski. J . Nur. Prod. 46, 576 (1983). 108. Y. I. Sung, K. Koike, T. Nikaido, T. Ohmoto, and U. Sankawa. Chem. Pharm. Bull. 32, 1872 (1984). 109. T. Ohmoto, T . Nakaido, K. Koike, K. Kohda, and U. Sankawa, Chem. Pharm. Bull. 36, 4588 (1988). 110. T. Ohmoto, Y. I. Sung, K. Koike, and T. Nikaido, Shoyakuguku Zasshi 39,28 (1985).
- Chapter 4 PHENETHYLISOQUINOLINE ALKALOIDS TETSUJ I KAMETANI~ Institute of Medicinal Chemistry Hoshi Universiry Tokyo. Japan
MASUOKOIZUMI Fujigoremha Research Lahorarories Chugai Pharmaceuriccil C o . . Ltd. Shizuoka, Japun I. Introduction ......... ........
.................................
i72
11. Structural Elucidation, Chemical Reaction, and Stereochemistry of
Phenethylisoquinoline Alkaloids .......................................................... .................. A. Simple Phenethylisoquinolines. .......
173 173
C. Bisphenethylisoquinolines ............................................... D. Homoproaporphines .................................................. E. Homoaporphines.... ............... ....................................................... ................. F. Homoerythrina Alkaloids ............. G. Dibenz[dflazecines ................................................................ H. Miscellaneous ............................................... Ill. Biosynthesis ......................... .......... .............. A. Androcymbines ... ............ ............................................................ B. Homoaporphines............. ........................... s ..............................
191 191
189
194
195 197 198
200 200 200 20 1 20 1 202 IV. Synthesis .................................................... 202 A. Phenol Oxidation ......................................................................... 206 B. Nonphenolic Oxidation .... .............. 208 C. Anodic Oxidation ................................................. D. Lead Tetraacetate Oxidation via Quinol Acetates ................... ........... 208 209 E. Photolytic Cyclodehydrobromination ........... ............ F. Asymmetric Synthesis .............. ................ ............., ........... ........... 213 214 G. Miscellaneous Methods. ........... V. Pharmacology ..................................... ...... ................. ....................... 219 220 References .....................................
' Deceased. 171
THE ALKALOIDS, VOI.. 36 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
172
TETSUJI KAMETANI A N D MASUO KOIZUMI
I. Introduction
The chapter on phenethylisoquinoline alkaloids that appeared in Volume 14 of this treatise reviewed the literature up to the middle of 1972 ( I ) . In the succeeding years covered in this review (1972-1988) significant advances have been made in all aspects of study regarding this family of alkaloids. Phenethylisoquinoline alkaloids are classified into seven major alkaloid groups based on structural differences: simple I-phenethylisoquinolines (l),homomorphinanedienones (2), bisphenethylisoquinolines (3), homoproaporphines (4), homoaporphines (9, homoerythrines (6), and dibenz[dflazecines (7)which are related to benzylisoquinoline alkaloids. Although tropolone and Cephalotaxus alkaloids also belong to the
0
R1
2
1
Me-N
3
8
4
7
5
R3 a4 6 10
2
1
173
4. PHEN ETHY LISOQUINOLIN E ALKALOIDS
phenethylisoquinoline alkaloid group, these alkaloids are not included in this chapter since they were reviewed earlier (2). This chapter is organized in the same manner as the preceeding reviews, listing the alkaloids according to structure. Some sections that were prominent in the last review are absent or received scantily, because little new information has appeared, whereas new sections have been added or others expanded when warranted. Synthesis of the alkaloids is discussed in a separate section; this arrangement seemed desirable since some of the synthetic approaches are applicable to more than one ring system. Nearly 40 new phenethylisoquinoline alkaloids have been isolated and characterized since the last review in Volume 14 of this treatise (I). These alkaloids are listed in Table I together with their botanical sources and physical properties.
11. Structural Elucidation, Chemical Reaction, and Stereochemistry of
Phenethylisoquinoline Alkaloids A. SIMPLE PHENETHYLISOQUINOLINE ALKALOIDS Of the simple phenethylisoquinoline alkaloids dysoxyline (8), (S)-( +)homolaudanosine (9), and (-)-isoautumnaline (lo), 8 and 9 were isolated from Dysoxyfum lenticeflare Gillespie (3):Alkaloid 10 was isolated from Colchicum ritchii R.B. ( 4 ) . Alkaloids 8 and 9 were identified as simple I-phenethylisoquinolinesby means of their mass spectra. The parent ions of 8, mlz 355 (CzlHz5NO4), and 9 mlz 371 (C22H24N04), differ by 16. Both compounds show a base peak at mlz 206 resulting from the loss of a phenethyl radical from the parent ion. A similar loss of the C-1 benzyl radical produces the base peak
8 ’ 7 7 ’
0@R5 R3
0
R4
g : 9 : 10: 11:
R1 Me Me H Me
R2 Me Me Me
H
R3
R4
-cHzMe Me
Me Me MO Me
R5 R6 H BOH H IIH OH OH MH
I74
LT.
r"
I
I75
'D
TABLE I (Continued) UV Compound
Colchiritchine (14). CmH21N04
CC-20 11.5). C ~ I H ? I N O S
rnp ("C)
Amorphous
2 10-2 12
[aid")"
t207 10.I5,MeOH)
Amax,
nm
(log d
213 l4.4ld 241 (4.08) 277 (3.701
243 (4.27) 280 13.781
IR ymrr. c m ~ l 16fdY
1630 1610
1665 1635 1605
'H NMR 6. pprn I .4M IH.m.H-I2Pl' I .7M 1 H.m.H-6P)
2.341 IH.m.H-5P) 2.42( 1H.m.H-6a) 2.821 IH.m.H-5al 2.85(2H,m.H-I3uP) 3.?7(IH.m.H-l2a) 3.67(3H,s.OMel 4.08(3H.s.OMe) 4.1 1IIH.m.H-71 5.93 5.97l?H.d,Jl.4.OCHzOl 6.27( 1H.s.H-4) 6.3 I ( 1 H.s.H-81 6.81lIH.r.H-I I 1
2.9M3H.r.NMe)' 3.79(3H.r.OMel 4.14(3H.s.OMe) 5.95(2H,r.OCHzO) 6.261IH.s.Ar-H) 6.48(IH.s.olefinic H ) 6.75( 1H.r.olefinic H)
Source Colchirum rirrhii R.B.
Ref. 4
h
cml r
Lo
0
PI ci
N
I
*
N n
I78
a
N
I
N
?
179
0 I
t
%
TABLE I (Conrinued)
Source
Compound lolantamine 1271. C I P H ~ ~ N O ~
215-216
-
+ II 2 (CHCIII
3350 1650 1630
Ref.
2.38(3H.s.NMe)' 3.78(3H,s,OMe) 6.451 IH.s.Ar-HI
Merendpro jolonrue
I5
2.37OH.r.NMe)' 3.5 II3H.s.OMel 3.78(3H.s.OMe) 3.79( IH,c,H-I2) 6.46(1H.s.Ar-H1
Colrhiciim Iurerrm
I6
lhon
231-232
-96 (MeOHl
m
228 (3.891'' 272 (4.051
0
Me0
Jolanline (31). C20H27N04
269-270
-
2lod 2x5
Me0
3535 1677 1667 1617 1600
3400 1650 1630 I600 1460
Baker
Merendero jolonrae
17
+
0
N P
N I
N
181
WX N19 0 -
I
N ICi I
0
r? N
... "I
I82
E m
m
Merenderine (491, C ~ ~ H Z J N O S
CC-24 (501. CiiH25NOr
Colrhirunr szouirsii
Amorphous
245-249
249 (3.901 281 (3.601
342W
276(3.28Ih 284 (3.301
3320h
Colchrrum
23
5.24
cornigerum
Hornoerythrina alkaloids
Holidinine (52). CloH27NOd
164-165
t Y 1 (1.03. CHCI31
1580
1480
3.25(3H,a.OMel' 3.76(3H.a.OMe) 3.93(3H.s.OMel 5.5h(lH.H-I1 5.581 IH.s.OH1 6.631IH.s,Ar-Hl
Phdline 5p. afT.
25
HO
Meo% Me0 P' (continued)
TABLE 1 (Continued)
Compound Comosidine 671, C ~ U H ~ N O ?
mp 1°C)
143-145
[alDI")"
+72 (0.17. CHCIi)
Uv A,,,. nm (log el
1R ymrx. cm
236 (3.%lh 283 (3.511
1610h 1580
'H NMR
1515 1469
-
2.7-Dihydrohomoerysotrine 1.541. CroHyNOi
Amorphous
-
- I18 (0.58. CHChl
e
3-Epi-2.7-d1hydrohomoerysotrine (53).
CmW?7NOi
Amorphou,
+ I ? ? (0.50. CHCIjl
282 (3.481h 259 (3.021
-
6 . ppm
Source
3.7646H.s.OMel' 3.84(3H.s.OMel 6 . W IH.s.Ar-HI 6.07(IH,s.Ar-Hl
Phellinr sp. aff. Phpllirle wmim
IRCM IH.q.H-4,,1' ?.68(3H,a,OMeI 3.76(3H.s.OMel 3.8213H.s.OMeI 5.241IH.m.H-II 6.561 1H.s.H-18) 6.89( IH.>.H-I5)
CeptluloroxllJ hurringrotiiu
I .58( IH.I.H-4,, )' ?.78( IH.q.H-4,,1 3.19(3H.r.OMe) 3.7713H.r.OMel 3.83I3H.b.OMel 5.25f IH.m.H-I I 6 . 6 3 1H.s.H-18) 6.74( 1H.s.H-151
Ref. 25.33
Labill.
3'
260 (hydrochloride)
+75 ( I . ? , EtOHl
242 (3.681 291 (3.65)
1615h 1495
I480 I44?
I .55(preudo-t.J4,,.4,q IZ,J3,4,, I 1 .H-4,,IC 2.53( IH.q,J?H-4,,,) 3.?7(s.OMe) ?.76( IH.d.Jl0.H-I) 5.86(?H.OCHzOl 6.021 1H.m.H-2) 6.?6( IH,b.Ar-H)
[email protected])
Phelline
?.85(3H,s,OMe)' 3.3X 1H.rn.H-3) 5. I512H.d.JI .4.OCH?O) 5.30(q1J 10.1.8 S.hl(d.JI0) 6.27( 1H.s.H-18) 7.lO(lHs.H-lY
Phelline
33
comu.w
Labill.
MeO" Alkaloid 6 (59). C I V H ? I N O ~
I26
t 6 3 (1.8. EtOH)
243 (3.691 292 (3.67)
1620h 1500
1489 1465
Wilconine (601. C20HzcN04
150-151
-51.4 (0.55, CHCI,)
281 13.41)h 258 (2.75) 233 (3.901
1610" 1?80
1460 1120
'
I .67( IH,q.H-4,,)' 2.96OH.s.OMe) 3.1 I(IH.m,H-4,,,) 3.76(6H.s.OMe) 3.84( IH.rn.H-3,, 1 5.864 IH.d.Jl0.5.H-Il 6.CfAIH.rn.H-2) 6.?3( IH.s.Ar-H) 6.82(lH.s.Ar-H)
33
comoscr
Labill.
Crphrrloroxrrs
34
wlsoniunu
(continued)
TABLE 1 (Continued) UV Am,,.
nm
Compound
mp ("C)
Lalol")"
(log C)
IR ymrr.cm
'
'H NMR 6 . PPm
Epiwilsonine (61). CmH25N04
103-104
t60.7 (0.S5,CHCI?)
281 13.44)h 2sn 12.77) 233 13.92)
1.701IH.q.H-4,,)' 3.1411 H . m .H-4,, ) 3.2913H.s.OMe) 3.3MIH.m.H-3,,) 3.7913H.s.OMe) 3.8013H.s.OMe) ?.77IlH.q.H-I) 6.041 1H.m.H-2) 6.611 IH.s.Ar-H) 6.981 IH.s.Ar-H)
Dibeoz[dJlszednes Dysazecine 166). C ~ I H ~ S N O ~
217-219 (picrate)
t 8 3 10.22. EtOH)
230 (4.18Ih 291 (3.861
2.1013H.s.NMe)' A ?.MlIH.td.JI1.3) 3.8213H.s.OMe) 3.92(3H.s.OMe) 5.961IH.d.JI.O 5.981IH.d.JI . 5 ) 6.521IH.a.Ar-H) b.S3( 1H.s.Ar-H) 6.76OH.s.Ar-H)
r
O
Me0 '@-Me
0 Me
Source Crpholoroxrrs nil5uniuno
Ref. 34
Miscellaneous alkaloids Holidine (67). CigH24N203
Amorphous
+I75 (1.0. CHCI71
233 (3.931b 272 0.631
1714h 1580
1.66(IH.dd.ll?.H-4,,)' 2.70( 1H.dd ,J 12.3.H-4,, ) 3.14(IH,m.H-3) 3.22(3H.s.OMe) 3.99(3H.s.COOMel 5.70(1H.H-I) 7 . W I H.s,Ar-H) 8.50( IH,s,Ar-Hl
Phelline sp.
232 (3.99)h 272 (3.651
3350h 1680
I .68(lH.dd.llZ.H-4,,)' 2.68(IH.dd.J12,3.H-4.,) 3.13(IH.m.H-3) 3.23(3H.s.OMe) 5.69(2H.s.NHz) 5.79( I H.H-I 1 7.94( IH.s.Ar-H) 8.34( IH,s,Ar-HI
P h e l l i n ~sp.
MeOOC
25
aff.
MeO" Phellinarnide (68).C I R H ~ ~ N K ~
206
+I80 (1.0. CHCI3)
25
aff.
(continued)
TABLE I (Continued)
Source
Compound I1 I4
Phellibilidine (69). C I ~ H ~ ~ N O ~
- 1 1 (1.0. CHCI,)
213 (4.02) 298 (3.11)
3570 3400 2930 1715
1610 1450
1.4M 1H,dd.J12H-4,,)'~m 3.36(3H,s.OMe) 3.58(I H,s.OH) 3.61(IH.m,H-3) 4.34.4. IZ(ZH.Zd.Jl2.H-181 5 . 5 3 1H.rn.H- I ) 5.70(IH.s.H-l5)
Helline
1.5MIH.dd.J12.H-4,,)'J 1.97( IH.m.Hb) 2.56(1H.dd,J12.3.5,H-4,q) 2.W 1H.d.J16.Ha) 3.4 I(3H.s.OMel 3 . W 1H Im.H-3,") 3.82(IH.d.J12.H-18) 3.96(1H,d.J12.H-18) 5.54(1H.rn.H-I) 5.6M 1H.s.H- 15)
Hrfline billiardierr
Ref. 26
billrardreri
% O MeO" lsophellibilidine (70). C,,H23NO4
(Concentration. solvent).
132
EtOH. ' CDCll.
+204 (1.2, CHCI,i
MeOH. ' D20.
' CF,COOH.
213 (3.98$' 280 13.34)
KBr.
-
CHCI,. ' CD6. 400 MHz.
' 270 MHz. ' 100 MHz.
60 MHz.
26
189
4. PHENETHYLISOQUINOLINE ALKALOIDS
in the mass spectra of benzylisoquinolines. The difference of 16 amu between 8 and 9 is explained by the presence of a methylenedioxy group in 8 and two methoxy groups in 9. The difference is confirmed by the presence of ions derived from the cleavage of the C-7‘-C-8’ bond in 8 at mlz 135 and in 9 at mlz 151. The ‘H-NMR spectra are in full agreement with structures 8 and 9. The circular dichroism (CD) spectra obtained from 8 and 9 each showed positive Cotton effects near 280 and 240 nm. Since the CD spectrum of (S)-( +)-laudanosine contains bands of a similar sign at these wavelengths, the absolute configuration for alkaloids 8 and 9 must be ( S ) (3). The diphenolic isoquinoline (lo), CZIH27N05,was obtained together with the known isomeric base (-)-autumnaline (11). The ‘H-NMR spectrum of 10 differed from that of 11 only by a slight shift of the H-5 and H-8 absorptions. This indicated that the isomerism resided in ring A, more specifically in the relative positions of the methoxy and hydroxy substituents. Complete NOE studies of 10 and 11 conclusively established the substitution pattern in rings A and C in each case (4).
B. HOMOMORPHINANEDIENONES A N D ANALOGS I . Androcymbine-Type Alkaloids Three alkaloids, namely alkaloids CC-10 (12) and CC-20 (15) and collutine (13), were isolated from Colchicum cornigerum (5) and Colchicum luteum ( 6 ) , respectively. Recently, a fourth alkaloid, colchiritchine (14), was isolated from Colchicum ritchii ( 4 ) .
RS
12: 13: 14 : 15 : 16 : 11: 18:
R1 H
R2 Me Me Me -CH,-CH2Me H Me Me Ma Me
R3 Me H Me Me Me Me Me
R4 I ~ 4H “H
R5 HMe Me H ~ f i HMe 4I-I Me 4 4 Me IIH Me
The IR spectra of these alkaloids showed the characteristic absorptions of a cross-conjugated dienone system, and their U V spectra were similar to that of androcymbine (16). Their similarity to 16 was also evident from the mass spectra of CC-10 and collutine which established the molecular formula CZIHZ5NO5. Alkaloid CC-10 is thus isomeric with androcymbine (16) and with collutine (13), but it was chromatographically different from both. Since methylation of 12 and 13 with diazomethane yielded 18, an
190
TETSLJJI KAMETANI A N D MASUO KOIZUMI
enantiomer, 0-methylandrocymbine (171, was obtained by similar methylation of 16. The mass spectrum of CC-20 confirmed the structure and also established the molecular formula CzlHzlNOs that is two hydrogens less than that of 12 and 13. This is due to the presence of a methylenedioxy group in CC-20, also confirmed in the 'H-NMR spectrum which showed signals for one aromatic methoxy, one vinylic methoxy, and an N-methyl group. It must be stressed that the 'H-NMR spectrum of CC-20 was virtually identical with that of 16, apart from obvious differences in the methoxy and methylenedioxy regions. The combined data lead to structure 15 for alkaloid CC-20. The spectral data of colchiritchine (14) was identical with that of CC-20, apart from the absence of an N-methyl group ( 4 ) . 2. Alkaloid CC-2 and Szovitsidinr Alkaloid CC-2 (19) and szovitsidine (21) were isolated from Colrhicum cornigerum (5) and Colchicum szovitsii (7), respectively. The molecular formula of alkaloid CC-2 is C21H27N05, corresponding to four hydrogens more than present in CC-20. Structure 19 was determined for CC-2 in the following way. The absorption at 3536 cm-' in its IR spectrum was assigned to an alcoholic hydroxy group because the U V spectrum was unchanged on addition of strong base. The 'H-NMR spectrum showed signals corresponding to one N-methyl, one aromatic methoxy, one methoxy attached to saturated carbon, one methylenedioxy group, and one aromatic proton. The coupled signals present correspond to HC (6 4.5, d, J = 4 Hz), H B (6 5.9, t, J = 4 Hz), and HA (6 6.4, m); this set of signals is closely similar to the three protons (HA, HB, HC) in kreysiginine (20).Irradiation of HC in the spectrum of CC-2 caused the HB signal to collapse to a doublet (J = 4 Hz), indicating a cis relationship between HA and HB.
19:R= H
20
21
Mild oxidation of CC-2 with manganese dioxide afforded a conjugated enone, C21H2SNOS,showing the allylic nature of the original hydroxy group. Furthermore, the 'H-NMR spectrum of the enone was identical with that of the oxidation product of 19; in particular, the signal for HA
4. PHENETHYLISOQUINOLINE ALKALOIDS
191
now appeared as a triplet (6 4.12, J = 8 Hz), which supported the presence of the methylene group at the carbon adjacent to the OMe group. The foregoing information is best accommodated by structure 19 for alkaloid CC-2. Rigorous structural proof came from X-ray crystallographic studies ( 2 7 ) . The data reported on szovitsidine (21) are not sufficiently complete to unambiguously assign its structure. It may be assumed that szovitsidine is a reduced derivative of 0-methylandrocymbine (17) whose structure, including absolute stereochemistry, was previously elucidated together with that of androcymbine (16) (28). C. BISPHENETHYLISOQUINOLINES The new bisphenethylisoquinoline alkaloid is jolantinine 22, which was isolated from Merederu joluntue (8).The structure of (22) was determined by IR, 'H-NMR, and mass spectroscopy.
22
D. HOMOPROAPORPHINES Colchicum kesselringii Rgl. gave seven alkaloids, kesselridine (23) ( l o ) , regelamine (24) (1I ) , kesselringine (25) (12). regeline (26) ( 1 3 , regelinone (32) (18), isoregelinone (33) (20), and jolantimine (34) ( 1 9 ) , whereas Colchicum luteum afforded t h e alkaloids luteidine (28) ( 1 6 ) and luteicine (29) ( 9 ) . Jolantamine (27) (15) and jolantine (31) ( 1 7 ) were isolated from Merendsru joluntue, and trigamine (30) was obtained from Merendera triginu (Adams) (14).This group comprises several types of compounds. They differ in the nature of ring D and consequently in their U V , IR, and NMR spectroscopic properties. When there is a keto group at C-12 or C-13 of ring D, it may form a cyclic half-acetal bridge with the phenolic
192
TETSUJI KAMETANI A N D MASUO KOIZUMI
I2 S4 : Me H R1
Me H R2
26: M e
Me
M
s
R2 0
HO
12
'
M
e
R 27: H 28: ome
0
0
M$HO ?-Me
-
g 8
R'P
Ho'
M
MeO H
Ho 29
30
31
R OH 33 : - O H
32:
group at C-1. The half-acetal hydroxyl can be free or etherified with a methyl group. Moreover, the double bond in ring D can be in the cis or trans configuration to the hydrogen at C-6A. Reduction of regelamine (24) in ethanol containing sodium gave the ring-cleavage products 35 and. 36. Sodium borohydride reduction of jolantamine (27) and subsequent hydrogenation in the presence of Raney nickel gave 36 (29,301. Treatment of kesselringine (25) and regeline (26) with acetic anhydride containing sodium acetate gave the acetates 39 (12) and 40 (13),respectively. On the other hand, treatment of 26 with acetic anhydride containing sulfuric acid gave 37 (12). Methylation of 26 with dimethyl sulfate gave 43 (13). Furthermore, methylation of 25 with diazomethane afforded 26, which was hydrolyzed with acid to 24. Treatment of 25 with butanol and hydrochloric acid furnished 38. Acetylation of 26 with acetic anhydride gave 40, while treatment of 24 or 26 with acetyl chloride or acetic anydride gave 42. Treatment of 25 with benzyl chloride gave 41 (31). Reduction of luteidine (29) by sodium borohydride or hydrogenation in the presence of Raney nickel gave tetrahydroluteidine (44), whereas Wolff-Kishner reduction of 28 gave the cyclopropane 45. Cyclization of 28 in acetic acid containing hydrogen chloride gas gave the acetal46 (16).
35: R = O H 36:R=H
R1 31: Ac 38: H
R2 Me Bu
R3 Ac H
R1
39: Ac 4 0 : Me 41 : Bz 42: M e
R2 R3 M e Ac Me Ac M e Bz Ac Ac
R4 Me Me Ph
Me
43
194
TETSUJI KAMETANI A N D MASUO KOIZUMI 28
46
The structure of all the alkaloids were established spectroscopically, and typical examples are described here. The elemental analysis and the mass spectrum of kesselringine (25) established the molecular formula C19H25N04.The UV spectrum showed that kesselringine contains one aromatic ring. In the UV spectrum measured in alkaline ethanol, the bands at 292 and 231 nm underwent a bathochromic shift by approximately 10 nm and showed hypochromicity, indicating the presence of a free phenolic group. According to the IR spectrum, the alkaloid possesses a hydroxy group but no 0x0 group. The 'H-NMR spectrum of kesselringine exhibits one N-methyl group, one aliphatic methoxy group, a multiplet arising from one proton of the OCH type, and a singlet arising from the isolated aromatic proton. Consequently, kesselringine is a homoproaporphine alkaloid with a phenolic group at C-2. A phenolic hydroxy group at C-1 forms the ketal group. Since the ketal group is located at C-12 on ring D, the secondary hydroxy group must be in the axial position, as shown in the 'H-NMR analysis, and, consequently, in the quasi-cis position to the vicinal methoxy group in the ketal. Kesselringine shows negative Cotton effects in its CD spectrum at 250 and 300 nm and a positive Cotton effect at 220 nm. By analogy with proaporphine alkaloids, it is assumed that kesselringine has the (6aR,8aS,1 IS, 12R) configuration. E. HOMOAPORPHINES
The alkaloids 0-methylkreysigine (47) (21),szovitsamine (48) (22),and merenderine (49) (23) were isolated from Colchicum szouitsii. A fourth alkaloid, CC-24 (50), was isolated from Colchicum cornigerum (5,24).The chemical behavior of 50 has not been described. All four alkaloids were
'
47: 48: 49: 50:
R2 0
I95
4. PHENETHYLISOQUINOLINE ALKALOIDS
R1 Me Me
RZ
Me H
H H
Me
51:H
Me
H
R3 R1 OMe I I H OMe I I H OH IIH OMe ' I H OH - H
M R3
presented as optical antipodes of known alkaloids; in particular, the ( R ) form of merenderine (49) was identical with the (S)form of floramultine (51).
F. HOMOERYTHRINA ALKALOIDS
Cephalotaxus harringtonia (32) gave two alkaloids, 3-epi-2,7dihydrohomoerysotrine (53) and 2,7-dihydrohomoerysotrine (54), whereas Cephaloraxus wilsoniana (34) afforded wilsonine (60) and epiwilsonine (61). Alkaloids 57, 58, and 59 were isolated from Phelline comosa Labill. (3.9, and holidinine (52) and comosidine (57) were isolated from Phelline sp. aff. (25). The spectroscopic properties of the homoerythrina skeleton parallel those of the Erythrina group. The IR and 'H-NMR spectral characteristics are similar, particularly in rings A and B. The stereochemistry at C-3 may be assigned from chemical shift and coupling constants. Elemental analysis and mass spectroscopy of two isomeric alkaloids, 3-epi-2,7-dihydrohomoerysotrine (53) and 2,7-dihydrohomoerysotrine (54) established the molecular formula CzaHz7N03.Their spectroscopic properties closely resembled schelhammericine (55) except that the 'H-NMR spectra revealed the presence of two aromatic methoxy groups instead of a methylenedioxy group as in 55. Distinction between the two epimers on the basis of 'H-NMR data was made as follows. In the (3s) methoxy group of 53, the methoxy resonance occurs at 6 2.68, with a quartet for the axial C-4 proton near 6 1.80. In the (3s) methoxy group of 54, the methoxy resonance occurs at 6 3.19 with an apparent triplet for the axial C-4 proton at 6 1.58. The configuration at C-5 is considered the same as that in 55, since their optical rotations are of the same magnitude (32). In the same way, the physical and spectral data of holidinine (52) indicate that one of the methoxy groups of comosivine (56) is replaced by a hydroxy group (25). Alkaloid 57 (C20H27N03) was found to differ from 6,7-dihydrohomoerythramine (58) in that it contained two aromatic methoxy groups in place
196
TETSUJI KAMETANI A N D MASUO KOIZUMI
52: 53: 54: 55: 56:
R1 R2 R3 OMeMe H H Me Me H Me Me H -C&OMeMe Me
R4 R5 H OMe OMeH H OMe OMeH H OMe
R1 R2 5 7 : Me Me 58 : -CH~-
R2
R4
of the methylenedioxy group. The C-3 proton was, from 'H-NMR coupling constants, found to be axial, and structure 57 was consistent with the data. The configuration at C-5 is assumed, although its CD curve is the inverse of that of 58 in the 235 nm region. Structure 58 corresponds to 6,7-dihydrohomoerysotrine (33). The epoxy alkaloids 59 (CI9HZINO4) and 61 (C20H2SN04)exhibited similar physical and spectral characteristics. Both contain an allylic methoxy group, a disubstituted double bond, and para-oriented aromatic protons; however, the former alkaloid contains a methylenedioxy group and the latter two aromatic methoxy groups. Their IR spectra show no hydroxy or carbonyl group, suggesting that the fourth oxygen atom is contained in a ring. The mass fragmentation patterns of the two alkaloids are almost identical, showing that they differ only in the aromatic substituents. Structures 59 and 61 were assigned to the two alkaloids on the basis of spectroscopic evidence and chemical transformations.
4. PHENETHYLISOQUINOLINE ALKALOIDS
197
R20
OH MeO"
2 R1 R2 6 2 : Me Me 6 3 : -CH2-
R1 R2 64:Me Me 65: -CH2-
Reduction of 61 with lithium aluminum hydride gave the alcohol 62 with preservation of the double bond. The positions of both the double bond and the hydroxy group were evident from the 'H-NMR and mass spectral data of 62. The downfield shift experienced by the proton at C-14 (6 1.36) could be accounted for if the hydroxy group were at C-6 near the aromatic proton at C-14. Similar reduction of 59 gave the corresponding alcohol 63. Catalytic reduction of 61 led to alcohol 64, and spectral data revealed that isomerization of the double bond had occurred. Similar reduction of 59 gave the alcohol 65, which exhibited spectroscopic properties similar to those of 64. That the original alkaloids 59 and 61 contained a 6,7-epoxy group was concluded from the reduction experiments, but the stereochemistry of the epoxide remains uncertain. Alkaloid 61 was later isolated from a different plant along with its C-3 epimer 60. The name wilsonine was given to 60, and 3-epiwilsonine to 61.
G.
DIBENZ[df]AZECINES
A new dibenz[df]azecine is dysazecine (66), which was isolated from Dysoxylum lenticellare (3). The mass spectrum of 66 established the molecular formula C21H25N04 and suggested the presence of a nitrogen atom in the large heterocyclic ring. The 'H-NMR spectrum of 66 revealed four noncoupled aromatic protons, one methylenedioxy group, two methoxy groups, and an N-methyl group in a uniquely shielded position (6 2.10). The I3C-NMR spectrum shows the presence of four oxygenated
198
TETSUJI KAMETANI AND MASUO KOIZUMI
10
Me0 0-
Me 66
quaternary aromatic carbons (6 144.7- 148.3), four quarternary aromatic carbons (6 133.0-135.4), four protonated aromatic carbons ortho to oxygens (6 107.5-1 12.8), and five aliphatic methylene groups (two dishielded by the nitrogen at 6. 49.6 and 59.0, and three resonating between 6 27.8 and 30.5). The narrow ranges of the chemical shifts within some groups precludes individual assignments in the absence of model compounds. By analogy to dysoxyline (8) and schelhammericine (55), dysazecine is a dibenz[d,flazecine with a three-carbon bridge between nitrogen and the methylenedioxy phenyl ring. Spectral data do not distinguish between this molecule and that in which the third methylene group connects the dimethoxyphenyl with the nitrogen, and further work is required to settle this point. The CD spectrum of 66 is dominated by strong (0 > lo4) Cotton effects at 295 nm (positive) and 232 nm (negative). Since 66 contains no chiral carbon, its optical activity arises solely from the inherently nonsymmetric diphenyl ring system held in one chiral conformation. The literature contains a report on the transformation of schelhammeridine to optically active, bridged biphenyls which differ from 66 by the absence of both methoxy groups and the presence of a chiral C-7 hydroxy function (35). The ( R ) chirality for the biphenyl system was associated with a positive Cotton effect at 290 nm in the ORD spectrum of these compounds (35). This assignment seems in agreement with the signs of the Cotton effect generally observed in the CD spectra of optically active biphenyls. The combined data lead to structure 66 for dysazecine.
H. MISCELLANEOUS 1 . Holidine and Phellinamide
Two new alkaloids, holidine (67) and phellinamide (68), were isolated from Phelline sp. aff. P . lrrcida along with several other homoerythrinan
4. PHENETHYLISOQUINOLINE ALKALOIDS
199
and related alkaloids (25).The mass spectra of holidine and phellinamide and C18H23N302, established their molecular formulas as C19H24N203 respectively. Their spectroscopic properties closely resembled each other. The 'H-NMR spectrum of 68 revealed the presence of an amide group in place of a methyl ester group in 67. Hydrolysis of the amide group of 68 with 1 N hydrochloric acid followed by esterification with diazomethane afforded a compound identical in all respects to 67. This chemical correlation confirms that the configurations at C-3 and C-5 are the same in both alkaloids. These data lead to structures 67 and 68 for holidine and phellinamide, respectively.
6 7 : R=OMe
68: R= NH,
H
MeO" 69
70
2. Phellihilidine and Isophellibilidine Helline hilliardieri afforded two new alkaloids, phellibilidine (69) and isophellibilidine (70), respectively (26). The mass spectrum of 70 shows it to be isomeric with 69 (CI7HZ3NO4). Moreover, several common peaks were found in the spectra of 69 and 70, indicating the same partial structure. The IR absorption at 3380 cm-' for 70 agrees with the presence of a hydroxy group; the absorption at 1745 cm-' can be attributed not to an unconjugated ester or &lactone but rather to an a,p-unsaturated
200
TETSUJI KAMETANI A N D MASUO KOIZUMI
lactone, which is also compatible with the U V and 'H-NMR data. The 'H-NMR spectrum of 70 discloses the presence of two trisubstituted double bonds in addition to a methoxy group and two protons of an AB system. Reduction of 69 and 70 with lithium aluminum hydride did not lead to the same compound: 69 gave rise to diol71 while 70 afforded trio1 72. The above data for phellibilidine and isophellibilidine lead to proposed structures 69 and 70 and to assignment of the pseudoequatorial orientation to the methoxy group at C-3.
72
71
111. Biosynthesis
Although biosynthesis of the phenethylisoquinoline alkaloids has not yet been studied in full, that of androcymbines, homoaporphines, and homoerythrinans has been examined by work with radioactive tracers. In this section tracer experiments as well as hypothetical biogenetic routes in the synthesis of the phenethylisoquinoline alkaloids are discussed. A. ANDROCYMB~NES Extensive tracer experiments show that tropolone alkaloids of the species Colchicum are derived from the I-phenethylisoquinoline system (73) by way of the dienone O-methylandrocymbine (16) (36). These findings, when combined with results of the earlier work (37), support the sequence shown.
B. HOMOAPORPHINES Specifically I4C-labeled I-phenethylisoquinolineswere administered to Kreysigia rnultijlora plants, and the alkaloids were isolated and degraded to unambiguous sequences. The results show that the C-homoaporphine
20 1
4. PHENETHYLISOQUINOLINE ALKALOIDS
-I-
-
16
73
no
::qo NHCOMe
skeleton of 50 originates from autumnaline (73), probably by ortho-para phenol coupling. Taxonomic interest in these findings was related to the biosynthesis of colchicine (74) in Cofchicurn autumnale (38).
C. HOMOERYTHRINA A N D DIBENZ[~,JAZECINE ALKALOIDS Tracer experiments suggest that schelhammeridine (78) of the species Schelhammera is derived from the 1-phenethylisoquinoline 75 by way of
the dibenz[df]azecine 76a and dienone 77 (39).
Me 71
76a
77
70
D. HOLIDINE A N D PHELLINAMIDE Holidine (67) and phellinamide (68) probably originate from the reaction of ammonia with aldehydes 80 and 81 formed by cleavage of the aromatic
202
TETSUJI KAMETANI A N D MASUO KOIZUMI
67 : R=OMe 68: R=NH,
81
80
79
ring of homoerythrinan precursors such as 79 (25).The hypothesis shown was proposed by Barton (26).
IV. Synthesis This section describes various synthetic methods, each of which gives rise to a different type of phenethylisoquinoline alkaloid, depending on reactivity and reaction conditions. A. PHENOLOXIDATION One-electron withdrawing inorganic reagents have been used to perform biomimetic syntheses of phenolic phenethylisoquinoline alkaloids. In order to obtain androcymbine compounds of type 85, the diphenolic isoquinoline 82a was subjected to phenol oxidation with manganese dioxide. The homoaporphine 83a coupled at the ortho-ortho position to the hydroxy group was the only product formed under these reaction
R2 82
a
b c d e
83
Rl
R2
R3
OH H OMe H H
Me Me Me H M e
Me Me Me Me H
R2 OH Me H Me OMeMe H Me OMeH on ~e R1
a
b C
d e
t
85
84
R3
OMe OMe OMe OH OMe
n
R1
R3
b
H OMe OMeOMe
c
OMeH
a
a~
b
R1 R2 H OMeMe
4. PHENETHYLISOQUINOLINE ALKALOIDS
203
conditions (40).Although diphenolic oxidative coupling reactions play an important role in the biosynthesis of alkaloids (41), the synthetic utility of the above reaction has been limited owing to low yield. Therefore, attention has been directed toward utilization of monophenolic substrates in an attempt to develop effective intramolecular coupling methods for use in alkaloid synthesis. Efficient syntheses of different alkaloids have resulted from intramolecular oxidative coupling of monophenolic isoquinolines using vanadium oxytrifluoride (VOF3)in trifluoroacetic acid (TFA). Thus, treatment of 82b and 82c with VOF3-TFA gave homoaporphines 83b and 83c and homoproaporphines 84b and 84c, with dienone 84b undergoing a dienone-phenol rearrangement in concentrated sulfuric acid to give the homoaporphine 83e (42).As model enzymatic reaction, phenol oxidation of the hydrochloride of 82d with cuprous chloride and oxygen in pyridine gave (+)-kreysiginone (84c) and isomer 84a, while 82e-hydrochloride provided ortho-ortho (830, ortho-para (83d), and para-para (85a) coupled products (43,44). The colchicine precursor 0-methylandrocymbine (85b) seemed a particularly challenging synthetic goal for applying the new procedure, since previous attempts to prepare the alkaloid by oxidative coupling of diphenol 82a had met with failure (45).Treatment of phenethylisoquinoline 82c with diborane in T H F , followed by two-electron oxidation using thallium(II1) trifluoroacetate gave, after removal of the blocking group with anhydrous sodium carbonate in refluxing methanol, (+)-0methylandrocymbine (85b) in 20% overall yield (46). The existence of homoerythrina alkaloids has been anticipated from biosynthetic considerations. Homoerythrina dienone 77 was synthesized in the following way. Oxidation of the diphenolic isoquinoline 86 with vanadium oxytrichloride in methylene chloride afforded the expected prohomoerythrinadienone 87 (47),which was transformed to the imine 88 in quantitative yield by 1 N sodium hydroxide at 0°C. Sodium borohydride reduction of the iminium chloride of 88 gave 76. Oxidative phenolic coupling of 76 with potassium hexacyanoferrate in methylene chloride afforded homoerythrina dienone 77 in 45% yield and homoerysodienone 89a in 15% yield (48). Moreover, the lactam dienone 91 was prepared in excellent yield by oxidation of the N-acyltetrahydroquinoline 90 with potassium ferricyanide (49). Reduction of the lactam carbonyl group of 91 would afford the required homoerythrina dienone 89a, but this could not be achieved in the presence of interfering functional groups. Consequently, lactam 91 was protected by benzylation, and reduction of the product with sodium borohydride gave a mixture of epimeric lactam dienols 92a in 70% overall
IL-
0
T
T
T
\ /
Or'
p L
W
m
204
t
E
b
m
m m
k0 \ /
gb n q
::a+
j
Me0
+ Z
0
OMe 90
a: X=O b:X=H,
P
MeO
\
R1 0
91
0 R4 76
a b c d e
R1 H
R2
02
Me Me Me Me
Me
H
H 02
R3 Me Me Me Me H
0
89 a : R=H b : R=0z
92 M
MR Me0
0 H
R4 H H Bz 0z Me
X HI 0 2
0 HZ H2
206
TETSUJI KAMETANI A N D MASUO KOlZUMl
yield. Further reduction of 92a with lithium aluminum hydride afforded the corresponding base 92b. Jones oxidation of 92b gave dienone 89b, which was treated with aqueous TFA to give the required phenolic dienone 89a in virtually quantitative yield (50). On the other hand, reductive cleavage of the dienone lactam 91 with chromium(I1) chloride gave the dibenz[df]azecine 76b in 87% yield. Protection of 76b by benzylation gave lactam 76c, which was reduced to amine 76d with lithium aluminum hydride. Deprotection of 76d by hydrogenolysis afforded the diphenolic dibenz[d,Aazecine 76a, a likely biosynthetic precursor of the Schelhammera alkaloids. Oxidation of the diphenol76a by potassium ferricyanide in the two-phase system gave the expected 5,7-fused dienone 77 in 61% yield (50). Reaction of the N-oxide 93a with cuprous chloride in methanol gave the homoaporphine 83d. Compound 93b provided (?)-kreysiginone (84a) under similar reaction conditions (51).
0
R2
93 R1
R2
a
H
Me
b
Me
H
B. NONPHENOLIC OXIDATION The use of phenolic oxidative coupling for in uitro synthesis of isoquinoline alkaloids has as a whole proved disappointing, although it still is considered to be a key step in the biosynthesis of these compounds. In recent years, it has been shown that phenolic ethers may efficiently be coupled by reagents such as vanadium oxytrifluoride (VOF3), thallium tristrifluoroacetate (TTFA), and ruthenium(1V) tetrakis(trifluor0acetate) (RUTFA), thus providing more rewarding routes to phenethylisoquinoline alkaloids.
207
4. PHENETHYLISOQUINOLINE ALKALOIDS
Treatment of N-trifluoroacetylisoquinoline 94a with VOF3 in trifluoroacetic acid gave the homoerythrina dienone 96a in 64% yield along with homoaporphine 97. Similarly 94b gave 95a in 50% yield and 96b in 42% yield, and 94c gave 95c in 3% yield along with 96a (52). Similar oxidation of 94d with VOF3 gave 96d in 65% yield, which when treated with 1 M sodium hydroxide in methanol yielded imine 98. Reduction of the imine hydrochloride with sodium borohydride in ethanol gave the dibenz[d,f]azecine 76e (53).
94
9s a , c
Ei
R2 me Me
R3 me Me
me
EZ
me
me
me
EZ
R1 me
w
a,b.d
97
Me
?
96d
6
Me 98
Nonphenolic is quinolines 99a,b were subject d to oxidatia with TTFA (54) and with RUTFA (55) to give the homoaporphines 100a,b. Moreover, oxidation of 99c with TTFA in trifluoroacetic acid gave lOOc, while 99d provided 101 under the same conditions. Treatment of 101 with sulfuric acid resulted in smooth dienone-phenol rearrangement with concomitant debenzylation to give an 81% yield of (2)-multifloramine (83e) (56).
208
TETSUJI K A M E T A N I A N D MASUO KOIZUMI
R30
0 Me
0 R3
99 a b C
d
100 a , b , c
R1
R2
R3 Me
Me Me Bz Bz
H OMe Me OMe Me OMe Bz
Me0 0
99d
M
~
o
$
?
-
~
~
Me 0 101
C. ANODIC OXIDATION Although diphenols have not yet been coupled electrochemically, their methyl ethers have recently been coupled with considerable success. Yields have been high, and the reactions seem remarkably clean. Oxidation of the hydrochloride of 102 was carried out on a graphite anode in water using tetraethylammonium perchlorate as the electrolyte. Potentials were controlled at 0.7 V. The dienone 103 was obtained in 23% yield (57). Similar anodic oxidation of 94a gave the homoaporphine lOOa (58).
D. LEADTETRAACETATE OXIDATION V I A QUINOL ACETATES Treatment of the quinol acetates derived from 5-, 6-, or 7-hydroxytetrahydroisoquinolines with lead tetraacetate (LTA) in acid gives different types of alkaloids. LTA oxidation of 7-hydroxy bases 82b,c,f,g in acetic
209
4. PHENETHYLISOQUINOLINE ALKALOIDS
102
103
acid gave the p-quinol acetates 104,which were treated with concentrated sulfuric acid-acetic anhydride to give O-acetylhomoaporphines 105a,b,c,d (59-61). On the other hand, treatment of p-quinol acetates 104b,c,g with trifluoroacetic acid gave the homomorphinandienones 106b,c,gand the hornoproaporphines 84a,b,calong with homoaporphines (62,63). Oxidation of 5-hydroxyisoquinolines 107a,b,c with LTA in dichloromethane gave the o-quinol acetate 108 which was converted with trifluoroacetic acid to the 3-hydroxyhomoaporphine 109 (64,65).Treatment of o-quinol acetate 111 prepared from 6-hydroxyisoquinoline 110 with acetic anhydride in the presence of an acid (concentrated sulfuric acid, boron trifluoride, or trifluoroacetic acid) gave the 2-hydroxyhomoaporphine 112 (66).
E. PHOTOLYTIC CYCLODEHYDROBROMINATION Since the total synthesis of O-methylandrocymbine (17)was accomplished via a photolytic cyclodehydrobromination reaction of a 1 4 2 bromophenethyl)-7-hydroxyisoquinoline(67), many phenethylisoquinoline alkaloids have been synthesized by this reaction. Irradiation of the bromoisoquinoline 113a,b with a Hanovia 450-W mercury lamp, using a Pyrex filter, in the presence of an excess of sodium hydroxide and sodium hydrogen sulfite gave alkaloid CC-24 (83a)(68) and the homoaporphine 114 (69), respectively. The first total synthesis of dysazecine (66) was accomplished in the following way (70). Irradiation of bromoamide 115 in methanol in the presence of sodium hydroxide with a 1 0 0 - W high-pressure mercury lamp gave cyclicamide 116a, which was reduced with diborane to the amine 116c. Conversion of 116a to dysazecine was achieved by allowing 116a to react with methyl iodide and potassium carbonate in ethanol to give the O-rnethyl derivative 116b. Reduction of 116b with sodium borohydride
M He
_j
R1
'
OR3
0
R2
H
-CH2-
g
O M e Bz
'
0
OR3
R4
R2
82 R1
Rl
0
R2
f
-
% M:e-
104
105
R3
R2
Me
+
106
R3
R4
a b c d
Me Me OMeMe Me H -CH2O M e Bz M e
R1 '
0 P
H
-
0 0 R3 04
M
e
21 I
4. PHENETHYLISOQUINOLINE ALKALOIDS
107 R1
a
b C
R2
108
109
R3
H H
Me Me -CH2OMe Me Me
O
\
AcO Me%-Me
MOO OMe OMe
110
112
111
0 R4
OMe 113 Rl
a
b
R2
Me H -CHz-
R3
R4
OH Me H H
R1
R2
83a : Me H 114 : -CHz-
R3
OH H
R4 Me
H
212
TETSUJI KAMETANI A N D MASUO KOIZUMI
115
116
a
b c
R H Me Me
X 0 0 H,
and boron trifluoride-etherate in tetrahydrofuran afforded the expected amine 116c. N-Methylation of 116c with formaldehyde and sodium borohydride gave (+)-dysazecine (66). An application of the photolytic cyclodehydrobromination reaction to bromoamide 117 gave the 1 1-membered ring lactams 118 and 119, which could be useful compounds for the synthesis of homoaporphines (71). Cyclization of 119 with phosphorous oxychloride in acetonitrile afforded the expected homoaporphine 120 in excellent yield (72).
117 Rl a
on
b c d e
OH Owe
on
119
R4 RS OMeOMe
n -ocn,o-
H ti OMeOMe O m e o n OMeOMe Omen
OH H H
118
R2 R3 OMe n
H
RS 120
213
4 . PHENETHYLISOQUINOLINE ALKALOIDS
The dibenzazecine 122a was readily prepared by a photocyclization reaction of bromophenol 121. Reduction of 122a with diborane gave the secondary amine 122b, which was converted to the dienone 123 by Birch reduction. Cyclization of 123 on heating in 5% hydrochloric acid afforded the desired compound 124a, and subsequent 0-methylation of 124a with an excess of diazomethane gave 124b (73).
122
121
a
b
123
X=O X=H,
124
a
R=H
b
R=Me
F. ASYMMETRIC SYNTHESIS Two asymmetric syntheses of phenethylisoquinolines have been reported. Simple exchange between isoquinoline 125 and imine 126 gave the chiral formamidine 127. Methylation of 127 with tert-butyllithium gave the lithiated formamidine, which was alkylated with 3,4-dimethoxyphenethyl iodide and hydrazinolyzed to give the (S)-( -)-isoquinoline 128 in 95% enantiomeric excess (e.e.) (74). The e.e. of 128 was determined by chiral-column HPLC analysis, as developed by Pirkle and applied to chiral N-heterocycles and other amines (75). Reaction of aldehyde 129 and ylide 130,derived from 3,4,5-trimethoxybenzyltriphenylphosphonium chloride obtained by treatment of the salt in T H F with n-butyllithium, afforded the trans-alkene 131 which was
214
TETSUJI KAMETANI A N D MASUO KOlZUMl
J
L -0uo
OMe 125
126
127
128
catalytically reduced to urethane 132a. When 132a was treated with an excess of methyllithium in THF, the reaction proceeded in acceptable chemical yield and without loss of optical purity to give base 132b (76). Conversion of 132b to homoprotoberberine 133 was accomplished by treatment of the hydrochloride of 132b with formaldehyde using an established procedure (77). The spectroscopic properties of 133 were the same as those already reported (77), and the optical purity was 81.9% based on published data. Compound 132c, prepared by reduction of 132a with lithium aluminum hydride, was treated with thallium(II1) trifluoroacetate to give (S)-O-methylkreysigine (51) in 27% chemical yield and 84% e.e.
G . MISCELLANEOUS METHODS The adduct 135 was formed in high yield when dichlorocarbene was generated by phase-transfer catalyzed decomposition of chloroform in the presence of the oxyberberine 634. Reduction of 135 with lithium aluminum hydride in hot THF lead to an enlargement of ring C with formation of the vinylic chloride 136 (78). On the other hand, N carboethoxydehydroaporphine 137 was reacted with dichlorocarbene under the same conditions to give adduct 138. Its reduction with lithium aluminum hydride followed by catalytic hydrogenation gave homodicentrine (139) in 63% overall yield from 137 (79). The benzyne reaction of the bromoisoquinoline 140 was examined by using sodium methylsulfinylmethanide, and dibenz[b,g]azecine 141 was obtained (80,81). An attractive modification appeared to be the expansion of the central ring of (+)-homoargemonine (143), since it seemed possible that 143 could be a representative of a new, as yet undiscovered alkaloid class originating from a I-phenethyltetrahydroisoquinolineprecursor. The synthesis of 143 was accomplished by reaction of the 1,2dihydroisoquinoline 142 and formic acid-phosphoric acid (82). Stereo-controlled total synthesis of homoerythrina alkaloids was accomplished by the new method and proved a useful tool for the synthesis
8
+
OMe
OMe OMe
134
137
135
138
136
139
217
4. PHENETHYLISOQUINOLINE ALKALOIDS
Me0 " O
W
N
-
B
Me
-
OH
CH,SCH,
bOMe
h
OMe
140
Me0
0
M
e
141
A
0
e
7
Me
M
e
Me0
0
g
) 143
&
y
)
M
e OMe
MeO OMe 142
of these alkaloids. When enone 144 was reduced with tetra-n-butylammonium borohydride, alcohol 145 was produced stereoselectively (a: b, 6 : I ) in 80% yield. On the other hand, reduction of 144 with sodium borohydride-cerium(II1) chloride gave the alcohol 145 as a major product (a : b, 1 : 5) in 81% yield. Methylation of 145a with methyl iodide afforded 0-methyl ether 14% in 44% yield. The isomeric alcohol 145b similarly gave the O-methyl ether 145d in 73% yield. Reduction of 145c with lithium aluminum hydride-aluminum chloride ( 1 : 1) in T H F gave 55, which was identical with schelhammericine. Similar reduction of the isomeric 0methyl ether 145d afforded 3-epischelhammericine (146)(83). Reduction of the enone 147 with sodium borohydride-cerium(II1) chloride in methanol gave a 2 : 1 mixture of the unsaturated alcohols,
2
A
fP
A
0
A0
0
A0
0
ZZ==
0000
1010
99
A
A0 0
0
?!P @
A
219
4. PHENETHYLISOQUINOLINE ALKALOIDS
which were separated after methylation to the O-methyl derivatives 148a (54% yield) and 148b (25% yield). Reduction of 148a with aluminum hydride gave amine 149, whose spectral data were identical to those of alkaloid A. Similarly 148b gave 6,7-dihydrohomoerythraline(58) (84).
V. Pharmacology Simple I-phenethylisoquinolineswere studied extensively by Brossi et af. at Hoffmann-La Roche. The results of their studies led to the discovery of (+)-1-(4’-chlorophenethyl)-6,7-dimethoxy-l,2,3,4-tetrahydroisoquinoline (150a), whose hydrochloride was clinically evaluated as an analgesic under the generic name methopholine. The compound was found to be a clinically effective analgesic with a potency similar to that of codeine. Although the general toxicity of methopholine was excellent (851, the compound was never marketed owing to the formation of cataracts in dogs during chronic toxicity studies, later found not to be drug related. The chemistry and pharmacology of methopholine was summarized (86) and chemical details reported (87).
Meo2-M
Me0
/
.Me
Cl
Cl
150a
1 Sob
It was later found that the analgesic effect of methopholine rested entirely with the (R)-(-) antipode (150b), in retrospect a better drug (88). The story of methopholine is a classic example of how structure-activity relationships should be resolved early, and the enantiomers studied, before a decision on which compound to be developed is reached. Chlorinated analogs of methopholine were found to have antitussive activity (89),also resting with the ( R ) isomer.
220
TETSUJI KAMETANI AND MASUO KOIZUMI
Recently, dysoxyline (8) and (S)-( +)-homolaudanosine (9)were demonstrated to have significant cardiac effects as assayed using isolated atrial muscles of the rat by Aladesanmi and Ilesanmi (90).
Acknowledgments
The author (M.K.) is most grateful to Dr. A. Brossi, Department of Health and Human Services, National Institutes of Health, who carefully read and made critical comments on various portions of the manuscript. Thanks are also due Professor Dr. Toshio Honda, Institute of Medicinal Chemistry, Hoshi University, Tokyo, for critical reading of the text. The author is extremely grateful for family support and especially acknowledges Miss Namie Koizumi for preparing all the drawings and typing the manuscript.
REFERENCES
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4. PHENETHYLISOQUINOLINE ALKALOIDS
22 1
16. N. L . Mukhamedyarova, M. K. Yusupov, and M. G . Levkovich, Khim. Prir. Soedin. 354 (1976). 17. Kh. Turdikulov, V. D. Nguyon, and M. K. Yusupov, Khim. Prir. Soedin. 555 (1976). 18. M. K . Yusupov, A. M. Usmanov, A. K . Kasimov, and Kh. Turdikulov, Khim. Prir. Soedin. 867 (1977). 19. D. A. Abdullaeva, M. K. Yusunov, A. K. Kasyrnov, N. VanDau, and K. A. Aslanov, Khim. Prir. Soedin. 12 (1976). 20. A. M. Usmanov and M. K. Yusupov, Khim. Prir. Soedin., 195 (1981). 21. M. K . Yusupov, B. N . DinhThi, and Kh. A. Aslanov, Khim. Prir. Soedin. 11, 526 (1975). 22. M. K. Yusupov, N . DinhThi, Kh. A. Aslanov, and A. S. Sadykov, Khim. Prir. Soedin. 11, 109 (1975). 23. A. A. Trozyan, M. K . Yusupov, and Kh. A. Aslanov, Khim. Prir. Soedin. 11, 527 (1975). 24. A. K . Kasimov, E. Kh. Timbekov, M. K. Yusupov. and Kh. A. Aslanov. Khim. Prir. Soedin. 11, 230 (1977). 25. N. Langlois, J. Razafimbeld. R. Z. Andriamialisoa, J . Pusset. and G. Chauviere, Heterocycles 22, 2453 (1984). 26. D. H. R . Barton, R. D. Bracho, C. J . Potter, and D. A. Widdowson, J . Cliem. Soc.. Perkin Trans. I , 2278 (1974). 27. A. F. Cameron and C. Hannaway, J . Chem. Soc., Perkin Trans. 2 , 1002 (1973). 28. A. R. Battersby, R. B. Herbert, L. Pijewska, F. Santavy, and P. Sedmera, J. Chem. Soc., Perkin Trans. I , 1736 (1972). 29. M. K . Yusupov, D. A. Abdullaeva, F. G . Kamaev, and A. S. Sadykov, Dokl. Akad. Nauk Uzb. SSR, 51 (1976). 30. E. Kh. Timbekov, A. K . Kasimov, D. A. Abdullaeva, M. K . Yusupov, and Kh. A. Aslanov, Khim. Prir. Soedin. 328 (1976). 31. M. K. Yusupov, N . L. Mukhamedyarova, A. S. Sadykov, L. Dolejs, P. Sedmera, and F. Santavy, Collect. Czech. Chem. Comnrun. 42, 1518 (1977). 32. R. G. Powell, Phytochemistry 11, 1467 (1972). 33. N. Langlois, B. C. Das, P. Potier, and L. Lacombe, Bull. Soc. Chim. Fr. 3535 (1970). 34. R. G. Powell, K . L. Mikolajczak, D. Weisleder, and C. R. Smith, Jr., Phytochemistry 11, 3317 (1972). 35. S . R. Johns, J . A. Lamberton, A. A. Sioumis, and H. Suares, Aust. J. Chem. 22, 2203 ( 1969). 36. A. R. Battersby, R. B. Herbert, E. McDonald, R. Rarnage, and J . H. Clements, J. Chem. Soc. Perkin Trans. I , 1741 (1972). 37. A. R . Battersby, R. B. Herbert, E. McDonald, R. Ramage. and J . H . Clements, Chem. Cornmun. 603 (1966). 38. A. R . Battersby, P. Bohler, M. H. G . Munro, and R. Ramage. J. Chem. Soc., Perkin Trans. I , 1399 (1974). 39. A. R. Battersby, E. McDonald, J . A. Milner, S. R. Johns, J . A. Lamberton, and A. A. Sioumis, Tetrahedron Lett. 3419 (1975). 40. T. V. P. Rao, Curr. Sci. 45, 453 (1976). 41. W. 1. Taylor and A. R. Battersby, eds., “Oxidative Coupling of Phenols.” Dekker. New York. 42. S . M. Kupchan. 0. P. Dhingra, and C.-K. Kim, J . Org. Chem. 41, 4049 (1976); 43,4076 (1978). 43. T. Karnetani, Y. Satoh. M. Takemura, Y. Ohta, M. Ihara. and K. Fukumoto, Heterocycles 5, 175 (1976).
222
TETSUJI KAMETANI AND MASUO KOIZUMI
44. T. Kametani, M. lhara, M. Takemura, Y. Satoh, H. Terasawa, Y. Ohta. and K. Fukumoto, J. A m . Chem. Soc. 99, 3805 (1977). T. Kametani, H. Yagi, F. Satoh. and K. Fukurnoto, J. Chem. Soc. C. 271 (1968). M. A. Schwartz, B. F. Rose, and B. Vishrnuvajiala, J. A m . Chem. Soc. 95,612 (1973). J . P. Marino and J . M. Sarnanen. Tetruhedron Letr. 45.53 (1976). J. P. Marino and J . M. Samanen, J. Org. Chem. 41, 179 (1976). E. McDonald and A. Suksamrarn. Tetrcihedron Letr. 4421 (1975); J . Chem. Soc.. Perkin Truns. I , 440 (1978). 50. E. McDonald and A. Suksamrarn, J. Chem. Soc.. Perkin Trcrns. 1 , 434 (1978). 51. T. Kametani. M. Ihara, M. Takemura, and Y. Satoh, Heterocycles 14, 817 (1980). 52. S. M. Kupchan, 0. P. Dhingra. and C.-K. Kim, J. Org. Chern. 41, 4047 (1976). 53. S. M. Kupchan, 0. P. Dhingra. and C.-K. Kim, J . Chrrn. Soc,., Chem. Commun. 847 (1977); J . Org. Chem. 43, 4464 (1978). 54. F. R. Hewgill and H. C. Pass, Aitst. J . Chem. 38, 555 (1985). 55. Y. Landais, D. Rambault, and J . P. Robin, Tetruhedron Letr. 28, 543 (1987). 56. E. C . Taylor, J. G. Andrade. G. J. H . Rall, and A. Mckillop, J. A m . Chem. Soc. 102, 6513 (1980). 57. J. M. Bobbitt, I. Noguchi, R. S. Ware. K. N. Chiong, and S. J . Huang, J. Org. Chem. 40, 2924 (1975). 58. S. M. Kupchan, 0. P. Dhingra, C.-K. Kim. and V. Kameswaran, J. Org. Chem. 43, 2521 (1978). 59. 0. Hoshino, T. Toshioka, and B. Umerawa, J. Chem. Soc. Chem. Commun. 740 (1972); Chem. Phurm. Bull. 22, 1307 (1974). 60. 0. Hoshino, H. Hara, N . Serizawa, and B. Umezawa, Chem. Phurm. Bull. 23, 2048 (1975). 61. H. Hara, 0. Hoshino, and B. Umezawa, Heterocycles 5 , 213 (1976). 62. H. Hara, 0. Hoshino, and B. Umezawa, J. Chem. Soc. Perkin Truns. 1 . 2657 (1979). 63. H. Hara, 0. Hoshino, B. Umezawa, and Y. litaka, Heterocycles 7 , 307 (1977). 64. H. Hara, H. Shinoki, 0. Hoshino, and B. Umezawa, H e t e r o c y l e s 20, 2155 (1983). 65. H. Hara, H. Shinoki, T. Komatsu, 0. Hoshino. and B. Urnezawa, Chem. Pharm. Bull. 34, 1924 ( 1986). 66. 0. Hoshino, K. Kikuchi, H. Ogose, B. Umezawa, and Y. litaka, Chem. Pharm. Bull. 35, 3666 (1987). 67. T. Kametani, Y. Satoh, S. Shibuya, M. Koizumi. and K. Fukumoto, J. Org. Chem. 36, 3733 (1971). 68. T. Kametani, Y. Satoh. and K. Fukurnoto, Tetrcrhedron 29, 2027 (1973). 69. T. Govindachari. K. Nagarajan, S. Rajeswari. H. Suguna, and B. R. Pai, H e l u . Chim. Actu 60, 2138 (1977). 70. H. Tanaka. Y. Takamura. K. Ito, K. Ohira. and M. Shibata, Chc,m. Phcmii. B d . 32, 2063 (1984). 71. 0. Hoshino. H. Ogasawara. A. Takahashi, and B. Umezawa, Heteroc.ycles 23, 1943 (1984). 72. 0. Hoshino, H. Ogasawara. A. Takahashi, and B. Urnezawa. Heteroc,ycles 25, 15.5 (1987). 73. H. Tanaka. Y. Takamura, and M. Shibata. Chem. Phorm. Bull. 34, 24 (1986). 74. A. I. Meyers. M. Boes. and D. A. Dickrnan, Angew. Chem.. I n ! . Ed. Engl. 23, 458 (1984). 75. W. H. Pirkle and C. I. Welsh, J . Org. Chon. 49, 13X (1984). 76. Z. Czanocki. D. B. MacLean. and W . A. Szarek. J . Chem. Soc.. Chem. Commrrn. 493 (1984): Can. J . Chem. 65, 23.56 (1987). 45. 46. 47. 48. 49.
4. PHENETHYLISOQUINOLINE ALKALOIDS
223
77. A. Brossi and S. Teitel, Hclu. Chiin. Actu 52, 1228 (1969). 78. G. Manikumar and M. Shamma. J . Org. Chem. 46, 386 (1981). 79. J . L. Castro, L. Catedo, and R. Riguera. T ( > t r t i h d r o nLett. 1561 (1985); J . Org. Chc>in. 52 3579 (1987). 80. S. Kano, T. Ogawa, T . Yokomatsu. E. Komiyama, and S. Shibuya, Telrcihedron Lett. 1063 (1974). 81. S. Kano, E. Komiyama, T. Ogawa. Y. Takahashi. T . Yokomatsu. and S. Shibuya. Chem. Pharm. Bull. 23, 2058 (1975). 82. F. R. Stermitz and D. K. Williams, J. O r g . Chem. 58, 2099 (1978). 83. Y. Tsuda, S. Hosoi. T . Ohshima. S. Kaneuchi. M. Murata. F. Kiuchi. J. Toda. and T . Sano. Chem. Phurm. B1~11.33, 3574 (1985). 84. Y. Tsuda and M. Murata, Tetruhedron Lett. 27, 3385 (1986). 85. H. Besendorf. B. Pellmont, H. P. Bachtold, and A. Studer. Experientiu 18, 446 (1962). 86. A. Brossi, H. Besendolf, L. A. Pirk. and A. Reiner, in “Medical Chemistry” (J. de Stevens, ed.), Vol. 5, pp. 281-330. Academic Press. New York, 1965. 87. A. Brossi. H. Besendorf, B. Pellmont. M. Walter, and 0 . Schnider. H d u . Chim. Actu 43, 1459 (1960). 88. A. Brossi and F. Burkhardt, Helu. Chitn. Actti 44, 1558 (1961). 89. A. Rheiner, Jr. and A. Brossi. E.rpc.ric,ntiu 20, 488 (1964). 90. A. J . Aladesanmi and 0 . R. Ilesanmi. J . Ncit. Prod. 50, 1041 (1987).
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- Chapter 5 ALKALOIDS OF THE CALABAR BEAN SEIICHI TAKANO A N D KUNIO OGASAWARA Phurmuceuticul Instilute Tohoku Universitv Aohayumu, Sendui 980. Japan
I . Introduction .......
.................. ................................................................. HI. Synthesis of the Alkaloids .................................................................. ........... A. Brief Outline of Syntheses Established prior to 1970 B. Syntheses after 1971 ..................................................................... IV. Pharmacology.. ................................................................... .......................................... References 11. Structures of the
225 225 226 226 226 247 249
I. Introduction
The alkaloids of the Calabar bean (Physostigmu uenenosum) were reviewed in Volumes 2 ( I ) , 8 (2), 10 ( 3 ) ,and 13 (4) of this treatise, covering the period up to 1970. In the intervening years no new alkaloids have been discovered. However, considerable advances have been made in both the synthesis and pharmacology of the alkaloids. A number of syntheses including entirely new approaches and an enantiocontrolled route as well as the first total synthesis of racemic geneserine have been accomplished. In addition, the remarkable enantiospecificity in pharmacological activities such as antiacetylcholinesterase and analgesic activities has been recognized. This chapter outlines investigations reported during the period from 1971 to the end of 1988, focusing mostly on synthesis.
11. Structures of the Alkaloids
Isolation of seven alkaloids from the Calabar bean is reported to date (Fig. I). Of these, the structures of (-)-calabatine (6) (C17H~5N~03) 225
THE ALKALOIDS. VOL 76 Copyright 0 1989 hy Academic !‘re\\. Inc All right\ of reproduction in any form renerved
226
SEIICHI T A K A N O A N D K U N l O OGASAWARA
(-)-physostigmine (1)
MeycoQ&J HO
(-)-norphysostigrnine (2)
M HeO N
C
o
e
,
I H Me
N 0'
(-)-eseramine (3)
(-)-calabatine (6);C17H,503N, Me (-)-calabacine ( 7 ) ;C17H,503N,
Me
(-)-physovenine (4)
(-)-geneserine (5)
FIG. I . Alkaloids isolated from the Calabar bean.
and (-)-calabacine (7) (C17H25NZ03) have not been determined since their isolation was reported in 1963 ( 5 ) . 111. Synthesis of the Alkaloids
A. BRIEFOUTLINE OF SYNTHESES ESTABLISHED PRIOR TO 1970 Of the five alkaloids with known structures, physostigmine (l),eseramine (3), and physovenine (4) have been synthesized (1-4). Since the conversion of physostigmine (l),a principal alkaloid, to physovenine (4) (6) and geneserine ( 5 ) (7,8) has also been established, synthesis of the former implies acquisition of the latter two alkaloids in a formal sense. Up to 1970, the synthesis of geneserine ( 5 ) was not reported because its structure had been considered to be the N-oxide of physostigmine (1)until 1969 (9-11) since its first isolation in 1915 (12). The four approaches to the synthesis of physostigmine (1) may be classified into four types based on the key step employed: (i) the Fischer indolization route, (ii) the indole alkylation route, (iii) the oxindole alkylation route [including synthesis of physovenine (411, and (iv) the oxidative indolization route (1-4) (Scheme 1).
B.
S Y N T H E S E S A F T E R 1971
There have been more than 10 syntheses of physostigmine (1) and related alkaloids reported since 1971. They include entirely new ap-
227
5. ALKALOIDS OF THE CALAEAR BEAN
(i) Fischer indolization route
1
EtMgBr Me1
L+
rNHNH2
R
O
e
N
p
(ii) lndole alkylation route
h
-
t
(iii) Oxindole alkylation route
Hlo
I
1
h
9
EtONa CICHzCN
,
Me Me
Me
physostigmine (1)
13
(iv) Oxidative indolization route
\
aq. K3Fe(CN)6 I H I
14
Me Me 15
SCHEME I . Outline of syntheses established prior to 1970.
proaches to construct the alkaloid framework, the first enantiocontrolled synthesis of natural (-)-physostigmine (l), and the first total synthesis of racemic geneserine ( 5 ) . It should be pointed out that the same intermediate 27 of geneserine (9,which could be obtained by (v) the isochromanone route or by (vi) the radical cyclization route, may be used not only for geneserine (5) but also for physostigmine (1)and physovenine (4) (Scheme 2). Improved syntheses based on classical routes such as the Fischer indolization route and the oxindole alkylation route have also been reported. The former could provide substantial amounts of the racemic alkaloids, while the latter made possible the practical production of both the natural and the unnatural enantiomers of the alkaloids with the development of a highly efficient method for resolving the racemic intermediate. The latter may be particularly interesting from the pharma-
228
SEIICHI TAKANO A N D KUNIO OGASAWARA
(iii) Enantiocontrolled route
Y
M e o w22c N
lhv Me
H2
1) LDA 2) hydrolysis 3) alkylation
I
C02Et 20
17
18
Me physostigrnine (1)
I
I
Me Me 25
k 27
4
I
F-
I
Me 29
t
BuaSnH
26 (v) lsochrornanone route
I
I
Me Me TfO24 (iv) 1,3-Dipolar addition route
I
Me 28 (vi) Radical cyclization route
SCHEME 2. Outline of syntheses established after 1971.
5. ALKALOIDS OF THE CALABAR BEAN
229
cological point of view, since the enantiospecificity of biological activities has been recognized in recent pharmacological investigations of physostigmine (1) and related alkaloids. The syntheses established after 1971 are outlined chronologically in the order in which they were developed.
I . Synthesis of Physostigrnine a. Synthesis of Racemic Physostigrnine via the Photochemical Route. The photochemical valence isomerization of 1,2-dihydronaphthaIenes to l,la,2,6b-tetrahydrocycloprop[b]indenes (e.g., 30 + 31) is well established (13). Ikeda and co-workers applied this photochemical rearrangement to 1,2-dihydroquinoline derivatives and observed that the same type of reaction took place in these heterocycles to give rise to cycloprop[blindoles in moderate yields (e.g., 32 + 33) (14). This finding was immediately exploited in a synthesis of racemic physostigmine (1)by the same authors (15). Unfortunately, the key reaction did not proceed in good yield with the appropriate substrate for construction of the natural product; nevertheless, an entirely new approach to the alkaloid was established.
30
C02Et
32
31
CO,Et 33
Photolysis of 1,2-dihydroquinoline 19, obtained in 47% yield from 6-methoxy-4-methylquinoline 34 by the Reissert reaction (16), in ethanol in a Pyrex tube afforded the endo-cyanocycloprop[b]indole 20 in 10% yield as a single product. On alkaline hydrolysis 20 furnished the furo[2,3-b]indole 38 in 69% yield. Formation of 38 was assumed to occur by sequential hydrolysis to the anion 35, ring opening to the indolenine 36, hydrolysis of the cyano group, and recyclization as shown in Scheme 3 . After N-methylation of 38 with methyl iodide in a sealed tube, the resulting 39 was heated with methylamine to give the lactam 40 which was reduced with LiAIH4 to give racemic esermethole (41) in 25% overall yield from 38. Conversion to 41 to racemic physostigmine (1) in two steps had already been established (1,2).
230
SEllCHI TAKANO AND KUNIO OGASAWARA
Me
hv,
CN
KCN, 47%
EtOH, Pyrex tube 2"C.g h
I
34
CO,Et
I
CO,Et
19 10% KOH
M
aq. EtOH 120-130 "C
35
20
e
o
e
C
N
36
Meocfb Me1
YHo H
37
38
Me 39
25%
69%
acetone 60 "C sealed tube 4.5 h
Me Me 40
100%
(f)-eserrnethole (41) 100%
SCHEME 3. Synthesis of racemic physostigmine. The lkeda approach
b. Synthesis of Racemic Physostigmine via the Acyliminium Route. In 1978, Wijnberg and Speckamp (17) disclosed a new approach to racemic physostigmine (1)employing acyliminium cyclization (18)as the key step, a route which they developed by themselves. The synthesis devised by these authors did not start from an indole derivative but from the succinimide 47 corresponding to the A-C framework of the alkaloid. In order to avoid the difficulties encountered in the preparation of the nitrated imide 48 starting from the nitrated precursor, the introduction of the nitro group was performed at a later stage. Thus, 3-ethoxybenzaldehyde (42) was first converted to the imide 47 in 60% yield via a sequence
-[
j
23 1
5. ALKALOIDS OF THE CALAEAR BEAN
E t O D C H O 8). These melanins are described by the base used in the synthetic procedure, such as NaOH-melanin, ammonia-melanin, diethylamine-melanin (212), to characterize eventual structural differences. In a typical experiment (253) air was bubbled for 3 days through a solution of d,I-dopa (10 g) in deionized water (2 liters) adjusted with concentrated ammonia to pH 8; 3.5 g of a precipitate was formed after acidification to pH 2, which was then washed with 10 mM hydrochloric acid and deionized water. Melanogenesis by autooxidation of 5,6-dihydroxyindole proceeds much more rapidly than that of dopa and is further accelerated (reaction time of a few minutes) by a number of heavy metal ions such as Cu(II), Zn(II), or Fe(III), which commonly occur in pigmented tissues (194). Metalcatalyzed autooxidation of 5,6-dihydroxy-2-carboxylic acid (DICA) using Co(II1) at slightly alkaline pH proceeds rapidly to give a dark brown melanin (192). C. ELECTROCHEMICAL SYNTHESIS In the 1980s electrochemical studies provided a fundamentally new mechanistic insight into the early stages of the melanization processes. Cyclic voltammetry of several catecholamines identified and clarified the cascade of chemical steps that precede the final polymerization of the respective 5,6-indolequinones. These studies allowed the identification of each electron-transfer process and determination of the rate constants of the coupled chemical (nonoxidative) reactions. Furthermore, the voltammetric data established a background for the quantitative determination of catecholamines using a selective amperometric detector in combination with liquid chromatography (LCEC technique) (254). The tendency of this class of compounds to absorb at electrode surfaces (such as platinum) allowed the determination of
6 . CHEMISTRY OF MELANINS
273
catecholamine concentrations at the 5 x lo-' M or 10 ppb level (255). Relevant electroanalytical data that serve in the detection and determination of important catecholamines and related compounds (see Appendix for clarification of abbreviations) are presented in Table 111. Several of the references provide information on the mechanism of the anodic electrode processes. Several publications on electrochemical mechanistic studies of the oxidative transformations of catecholamines followed the contribution by R. N . Adam's group (256)and involved a-methyldoparnine, a-methylnoradrenaline, dopamine (257), a-rnethyldopa, 5,6-dihydroxy-2-methylindole (258), and dopa (259). These studies (257) (Scheme 5), which confirmed the validity of the melanization scheme by Mason and Raper (Ref. 7, p. 50), explored the pH effect on the sequence of events that characterize the electrooxidation of catecholamines. Thus, the cyclic voltammogram in 1 M HC104(pH 0.6) shows only peaks corresponding to the catechol-quinone redox couple as the protonation of the amino group prevents the cyclization step. At pH 6.36, however, the cyclization products appear as a new redox couple that corresponds to the respective dihydroindole product. This process is of particular biological significance since the rate of cyclization of the oxidized form of catecholamines is a major factor in determination of catecholamine toxicity. Such toxicity results from competitive reactions of the oxidized quinonoid form of the catecholamines with sulfhydry1 groups of some essential enzymes. Thus, the fast cyclizing N-methylsubstituted catecholamines are less toxic than the unsubstituted ones that cyclize more slowly. Moreover, E l l ? potentials (rotating carbon electrode) of catecholamines were successfully correlated with their cytotoxicity (279),justifying the importance of the electron-transfer step. At an even higher pH (>7.68) the absence of a cathodic peak estimates the half-life of the corresponding quinone to be of the order of tens of milliseconds (257). The darkening around the anode is considered evidence for an electrochemically accomplished rnelanogenesis. The electrocatalytic effect (oxidation of NADH) observed with a glassy carbon electrode coated with polymer containing dopamine (covalently attached to a polyrnethyl methacrylic matrix) is analogous to bulk reactions of melanins (see Section V). The overall electrochemical behavior, however, indicates a very slow reaction involving only a few monolayers (280). No direct electron transfer between melanin particles suspended in aqueous buffers and electrodes has been observed. This allowed the use of the polarographic method in monitoring the concentration changes of TIt3and Fe+3mediators reacting with D,L-dopa melanin (210).Moreover,
E J
l . P
TABLE Ill ELECTROANALYTICAL OXIDATION DATAOF VARIOUSCATECHOLAMINES A N D RELATED COMPOUNDS Compounds DA, dopa DA, NADR, ADR, A-MNADR, IPNADR A-MDA, A-MNADR, DA A-MD, 5,6-DHMI Dopa ADR, NADR ADR, DA, NADR, SER. A-MDA, dopa NADR, ADR, DA ADR, NADR, DA, DHBA, dopa ADR, NADR NADR, dopa, DA, MTAM, VMA, ADR, HMVA, SER, 5HI-3AA
Isolation~electrochemicaldetection
Ref.
M or 10 ppb Polarographic three-electrode cell system; detection limit 5 x Cyclic voltammetry; planar carbon paste electrode Cyclic voltammetry; carbon paste electrode in I M HCIO, Cyclic voltammetry; carbon paste electrode in I M HCIO, Cyclic voltammetry; carbon paste electrode Polarography after KIOl oxidation HPLC; carbon paste electrode; detection limit 0.4-0.6 ng/ml HPLC with electrochemical detector: detection limits (in fmol): NE, 80; E. 180; DA, 200 HPLC with amperometric detector Polarography after air oxidation Polarography on carbon fibers, graphite powder, polyester resin, and glass tubes; SCE and Ag/AgCI used as reference electrodes; range of 200-570 mV
255
256 25 7 258 25 9 260 26 I 262 263 264 265
ADR, NADR, IPNADR, A-MD DA, NADR Dopa, DA, ADR, NADR ADR, NADR, DA, DOE, dopa, DHEPH NADR. DA, ADR, DHBA NADR, ADR, DA UA Dopa, UA, AA UA SHI-3AA, dopa, 6-HDA, 6-ADA, DA, NADR, AA NADR, dopa, DA, ADR, DHBA, SER, DHPAA, VMA, MN, NMN, CRT, N ;;1 HMVA,A-MD DA, SAL, ADR, NADR -
Differential pulse polarography using glassy carbon, SCE, and auxilliary Pt electrodes Double pulse voltammetry using Pt surfaces HPLC with electrochemical detection Three-electrode polarography system; El:?range 0.33-0.22 V/ECS HPLC with amperometric detector; wax impregnated carbon paste detector electrode; E +0.5 V vs. Ag/AgCI Reversed-phase liquid chromatography with thin-layer amperometric detector operated at +0.720 V vs. AgiAgCl HPLC with direct electrochemical oxidation; detection limit I pg HPLC with sandwich-type thin-layer cell and carbon paste, graphite, glassy carbon, gold, platinum, and mercury as working electrodes HPLC with electrochemical detection Liquid chromatography with sandwich-type thin-layer cell
266 267 268 269 270
HPLC with electrochemical detector; detection limits 0.05-0.20 ng
2 76
HPLC with electrochemical detector HPLC with amperomrtric detector (review)
277 2 78
271 2 72 273 2 74 2 75
276
RAIMONDO CRIPPA ET A L
2H'
+
2e-
1
R" H
HO H
melanoid pigment
SCHEME 5. Electrochemical oxidation of catecholamines to melanoid pigments (257).
the formation of hydrogen peroxide during the enzymatic and autooxidative melanogeneses (in I M KOH) was also monitored with dc polarography (264). This study suggests the following reaction scheme:
277
6. CHEMISTRY OF MELANINS
The charge-transfer processes between chlorpromazine cation radicals (CPZ'.) and catecholamines were studied spectrochemically in order to determine the biological function of chlorpromazine (281). Electrochemical oxidation of the neurotransmitter serotonin (SER, which carries a single phenolic group) produced polyhydroxylated compounds and the corresponding quinones (282) which are the most potent neurotoxins known.
D. PHOTOCHEMICAL SYNTHESIS Catecholamines are thermodynamically and photochemically unstable compounds that yield aminochromes and melanins on photooxidation (283-285) (Scheme 6 ) . Thus, irradiation (254 nm) of oxygen-saturated dilute solutions of adrenaline, isoprenaline, and noradrenaline produced the corresponding aminochromes in 65, 56, and 35% yield, respectively (285). Longer irradiation produced melanins, thus providing evidence for the photolabile character of aminochrome (284). Studies of the action spectrum confirmed the excited state of the catecholamine as the primary
catecholamine
catecholamine-quinone
indoline
Tested compounds NADR
R1
R2 = H
R3
ADR
R1 = O H
R2
R3 = CH)
DA
R1 = H
R2 = H
R3 = H
Dopa
R1
=
R2 = COOH
R3
=
H
IPNADR
R1
= OH
R2
=
H
R3
=
(CH3)zCH
EP
R1 = H
R2
=
H
R3 = CHJ
= OH
H
=
H
=
H
R3 aminochrome
J melanin
+
0 O
m
.
"
:
R3
+
H o Q ) 7 J :
HO
R'
indole-quinone
SCHEME6 . Thermochemical decomposition of catecholamines (284).
278
RAIMONDO CRIPPA E T A L
factor in the transformation processes. N-Substituted catecholamines were found to react more rapidly than the corresponding N-unsubstituted ones (284). A method was established (286) for detecting the presence of radicals during the protolysis of catecholamines and for assigning the hyperfine structures of the corresponding o-semiquinone anion radicals. An investigation of the oxidation of melanin precursors in the presence of azide radicals using pulse radiolysis has been reported (219). Thus, dopa and cysteinyldopa yielded first the unstable semiquinones that disproportionated to a quinone-quinol complex. The quinones decayed to more stable products; dopaquinone produced dopachrome while cysteinyldopa-quinones rearranged to benzothiazine isomers. Photooxidation of various melanin precursors, e.g., DI, has been studied in connection with investigation of the mechanism of the immediate pigment darkening, i.e., natural skin tanning (287). The experiments were performed both under physiological conditions (phosphate buffer, pH 7) and in organic solvents (methanol). These studies can generally be characterized as preliminary, and only a few conclusions can be drawn. Experiments in aqueous media showed significant competition between the primary photochemical and autooxidative processes. Irradiation of all
cH3c00fx2
CHICOO
\
CH,COO
16
17
OCOCH, CH,COO CHJCoo
CHICOO
I&/
CH3COO
CHICOO
\
CH,COO
2
\j
c
H
CH, 20
3
6. CHEMISTRY OF MELANINS
279
investigated compounds (DI, M-DI, and their 0,O-diacetyl- and 0.0dimethyl derivatives) in methanol produced different colors and yielded complex mixtures of unidentified products (288). The one identified process was a photo-Fries rearrangement of the 5,6-diacetoxyindole to 5-acetoxy-7-acetylindole (289). U V irradiation of N-methyl-DI using a Pyrex glass filter yielded a mixture of products with low conversion (290). Acetylation and chromatographic separation on silica gel TLC plates produced two isomeric triacetoxy-I-methylindoles (16 and 17), two pentaacetoxybisindolyls (19 and 20), as well as 5,6-diacetoxyindole (18) identified by NMR and mass spectroscopy. The structures of the photooxygenation products reveal the marked tendency of the indole moiety to undergo light-catalyzed oxygenation at the 2 , 4 , and 7 positions. The reaction conceivably involves interaction of the semiquinone radicals with triplet oxygen to give peroxide products which, together with the semiquinones, take part in the highly complex polymerization processes.
IV. Isolation, Purification, and Characterization
A. ISOLATION A N D PURIFICATION Selection of the procedure that will lead to the isolation and purification of natural melanins depends on the source material. Generally melanins are minor components of tissues and rarely exist in the free state, as in sepia ink (granules with dimensions of -0.2 pm (Ref. 7, p. 60). According to a widely used procedure, the tissue is homogenized in a blender and the protein components solubilized by extensive hydrolytic treatment with mineral acids, such as concentrated HCI at room temperature for 7 days [sepiomelanin, melanoma (Ref. 7, p. 92) and eye melanin (Ref. 7, p. I O l ) ] or boiling 6 N HCI. Such drastic conditions (204,291)lead to considerable alteration of the pigment as evidenced by the evolution of carbon dioxide. Thus, permanganate oxidation of acid-treated eumelanins, either natural or synthetic, gives much lower yields of pyrrole-2,3,5-tricarboxylicacid (PTCA) than the corresponding untreated pigments (II I). This suggests that the COz liberated during acid treatment arises from loss of the carboxyl group at position 2 of the indole or pyrrole rings (204,292).Thus, development of milder procedures is desirable. In the favorable case of cephalopod ink, minimal damage can be achieved by mechanical separation of the pigment granules, followed by a short treatment with 0.5 N MCl at room temperature and extensive
280
RAIMONDO CRIPPA E T A L
sonication in deionized water (291). Alternatively, insoluble melanin granules have been disaggregated using various solubilization processes (293) which provide a method for the separation of proteins and other extraneous material from melanin particles. Pigmented epidermal appendages such as hair, wool, and feathers have been widely used in the harvesting of melanins and are solubilized via various techniques such as acid hydrolysis (102,f 1 2 ) , alkaline degradation (294), or phenolthioglycollic acid extraction (295). The extent of structural modification that results from these rather drastic procedures should be taken into consideration when such samples are used in further studies. Recently, however, much milder isolation techniques (114,296)based on enzymatic digestion of keratin at ambient temperature and neutral pH have been attempted. Gel permeation chromatography has been used in studies of watersoluble melanins. Several fractions were separated from allomelanin from Aspergillus niger on Sephadex G gels (Ref. 7 , p. 131). Similarly, humic acids were separated into three fractions on Sephadex G-75 (297).
B. SOLUBILITY A N D SOLUBILIZATION The relatively poor solubility of natural and synthetic eumelanins (Table IV) is a considerable obstacle in structural determination. The rate of particle sedimentation in aqueous suspensions of synthetic melanins is
TABLE IV SOLUBILITY OF NATIVE MELANINS" Solvent
Squid
octopus
Dog
Man
Concentrated sulfuric acid Liquid ammonia Phenol 15% Sodium hydroxide Formic acid Dimethylformamide Dimethyl sulfoxide Ethylene chlorohydrin Basic sodium borohydride Dilute hydrogen peroxide/ammonia Solulene
5 5 5 4-5 5 5 5 5 4
4 5 5 4-5 5 5 5 4 4 I
4 5 5 4-5 4 5 4 4 4
I
4 5 5 4-5 4 5 4 4 4 1
1
1
I
I
1
" Solubilities range from 5 (totally insoluble) to I (totally soluble). Data are experimental observations by L. J . Wolfram and M . A. Berthiaume.
6 . CHEMISTRY OF MELANINS
28 I
generally accelerated by lowering the pH. This effect results from decreased solvation of the hydrophilic groups and formation of large agglomerates resulting from hydrophobic interactions of the individual indole units. Detailed studies using static and dynamic light scattering methods revealed the existence of two distinct pH ranges: one between pH 3.4 and 7.0 where the aggregation was slow (20 hr and fractal dimensions of 2.23) and another below pH 3.4 where the aggregation was fast (30 min and fractal dimensions 1.8). The fractal nature of the aggregates accounts for the relative stability of melanin suspensions (298). The different hydration and ionization states were correlated with the dielectric property of melanins (299).The dielectric constants and specific conductivities of melanin suspensions followed the sequence acidic > neutral > basic pH and showed dependence on the time of hydration. The solubilization of eumelanins has been attempted under a variety of conditions. Table IV summarizes the results obtained with some native melanins (300). A melanin is considered completely solubilized if the solution does not scatter light. So far only two approaches have been successful, one of which is based on treating the pigment with Solulene 100 (0.1 M solution of dimethyl-n-dodecyl-n-undecyl ammonium hydroxide in toluene; incubation for 2.5 hr at 75°C). Such solutions were used in the characterization and quantitative determination of melanins (124,301). The extinction coefficient (absorption at 400 nm) for hair melanin was of the order of 3000 M - ' cm-' per indole unit, while that of melanoma melanin was only about 70% that of synthetic dopa melanin (124). The mechanism of the Solulene solubilization process is unknown, and degradation of the pigment cannot be excluded. Interestingly, full neutralization of the melanin solution in Solulene with acetic acid did not precipitate the pigment (302). The second approach to solubilization involves treatment of natural melanosomes and synthetic melanins with a dilute solution of hydrogen peroxide at pH 9-10 (303). The solubilized melanin precipitates under acidic conditions and is readily redissolved in basic media. There is only a slight increase in the carboxyl content, suggesting only limited degradation of the pigment. The fact that melanins can be solubilized in both polar and nonpolar media is a clear manifestation of the ability of the melanin structure to accommodate highly diverse demands on its solvation characteristics. Melanin solubilization provides a unique opportunity for determination of molecular weights. Three approaches using various melanin preparations have been attempted (300,303,304).These included viscosity, gel permeation chromatography, and vapor pressure osmometry. Surprisingly the molecular weights were lower than expected, ranging between
282
RAIMONDO CRIPPA E T A L
1100 and 6000 irrespective of melanin origin (sepia, dopa-tyrosinase, or autooxidative 5,6-dihydroxyindole). The molecular weights of melanin samples solubilized by the oxidative method were not much different from those of Solulene-solubilized ones. Thus, the melanin prepared oxidatively from human hair had a molecular weight of about 10,000 (vapor phase osrnornetry) (303) and is generally unaffected by the length of oxidation time, between 10 and 1440 min (302).
C. ANALYSIS A N D STANDARDIZATION The semiquantitative and quantitative methods discussed in this section are based on the optical properties of melanins in both the transmission and reflection mode, and they may require solubilization and/or partial degradation of the samples (305).The melanin content in tissues has been determined visually (306) following treatment with Fe" and potassium ferricyanide (307) and by reflectance (308)and remittance (143) methods. For fluorimetric determination of melanins (melanoma cells), the sample is solubilized with alkaline hydrogen peroxide (pH 7.8, 100°C, 30 min); the excitation wavelength is 410 nm, emission 500 nm (148). Fairly good chemical stability of melanins has been determined in gravimetric determinations after separation from all other constituents of melanosomes with acid digestion (6 N HCI, I O O T , 72 hr) (110). Methods based on quantitative markers combined with TLC and HPLC (using an electrochemical detector) have been developed both for eu- and phaeomelanins. Thus, for eumelanins the marker is pyrrole-2,3,5-
TABLE V MOLECULAR WEIGHTSOF SOLUBILIZED MELANINS" Solubilization time (min)
MNh
MW
10 30 60 120 240 480 1,440
2,100 2,700 2,200 3,900 2,020 2,530 1,930
3,100 5.400 6,200 (14,700) 6,340 6,100 4.500
" L. J . Wolfram and M. A. Berthiaume. unpublirhed experimental data. Number average molecular weight. ' Weight average molecular weight.
6. CHEMISTRY OF M E L A N I N S
283
tricarboxylic acid produced by permanganate oxidation, for phaeomelanins the aminohydroxyphenylalanine produced by hydrolysis with hydroiodic acid (149). Yields of analytical markers vary significantly for melanins of different origin and are generally low. They are particularly useful, however, in estimating the relative ratios of eumelanins to phaeomelanins in mixed o r hybrid pigments (150). The free radical properties of melanins suggest an obvious marker, and ESR signals have been used for both identification and characterization of melanins in tissues and body fluids (309).
V. Structure and Chemical Properties The understanding of melanin structure has been attempted via analytical and biosynthetic approaches. The analytical one originally explored by Nicolaus (3,7,310) has led to the development of a number of useful methods for characterizing natural and synthetic melanins in terms of elemental composition. functional groups, and structural features of the pigment backbone. These methods helped in the elucidation of the partial polymeric structure of the eumelanin sepiomelanin (3) seen below.
COOH
0
284
RAIMONDO CRIPPA ET A L
Some properties of both eu- and phaeomelanins such as insolubility, heterogeneity, and unusuai spectral properties have been an obstacle in obtaining information on both structures and chemical properties. The biosynthetic approach, which originated with Raper’s pioneering studies in the 1920s (154), has provided information on the ultimate monomeric precursors of eu- and phaeomelanins. Thus, eumelanins are considered as polymers or copolymers resulting from the oxidative coupling of 5,6-dihydroxyindole (DI) and 5,6-dihydroxyindole-2carboxylic acid (DICA), while phaeomelanins are derived from the oxidative cyclization of cysteinyldopa adducts via the intermediate 1 ,Cbenzothiazines. Since the melanin precursors are known and since the mode of interactions to form the pigment is not unlimited, one might expect that the chemical reactivity pattern of melanin should reflect that of its precursors. The results of investigations suggest that this is indeed the case, and, thus, the long-held view of the chemical inertness of this material is being rapidly abandoned. A. ELEMENTAL COMPOSITION The content and relative ratios of heteroelements have been used as criteria in the differentiation of melanin families. Even in the same family, however, the content of heteroatom(s) depends on the origin (156). Thus, the nitrogen content for eumelanins ranges from 5.18% for one synthetic dopa-melanin to 12.13% for melanoma melanin (Ref. 7, p. 97) (9.42% N calculated for 5,6-indolequinone homopolymer). For phaeomelanins the sulfur and nitrogen content varies between 10 and 12% and between 7 and 9%, respectively (Ref. 7, p. 116) (10.26% S and 8.97% N calculated for the cysteinyldopa homopolymer). The empirical formula and particularly the carbon/sulfur (US) ratio have been useful in determining the degree of heterogeneity of hybrid melanins, i.e., the ratio of dihydroxyindole versus cysteinyldopa units. Thus, in one investigation (118) the C/S ratio for black hair melanin was found to be 40, while for red hair melanin the C/S ratio was 7. These data suggest the presence of some cysteinyldopa units even in black hair melanins. Furthermore, the higher content of oxygen in a synthetic melanin compared to the expected C8H5N02for poly(DI), [C9H5N04for poly(DICA)] suggests the presence of more hydroxyls or carboxyls in the melanin structure. The minimum sulfur content in skin was found to vary with hair color, owing to dissimilation of sulfur in the skin during hair growth (123).
6. CHEMISTRY OF MELANINS
285
The presence of proteins is one of the major factors contributing to pigment heterogeneity in native melanins. Depending on the source of the pigment and the method of melanin isolation, the quantity of melanoproteins vary widely from as little as 4.3% (113) to as high as 55.8% (118). No difference between the amino acid compositions of the hydrolysates from black and red hair melanoproteins was detected. The content of melanoproteins varies according to the method of isolation, but the degree of subsequent removal by acid hydrolysis has not, in our view, been satisfactorily validated. The heteroatom count of melanin preparations is affected to a much lesser extent by the bound metals such as Na, K , Ca, Mg, Fe, Zn, Cu, Cr, Pb, Mn, Cd, and Sr determined in human hair and skin (119). Neutron activation analysis of melanins isolated from dark human hair and banana peels gave evidence for the presence of Au, Br, Sb, Ag, Fe, Zn, Co, Cr, Ni, and Hg (311). B. DEGRADATION The conventional spectrophotometric techniques (UV-visible, IR, NMR) are of limited use in structural determination of melanins. Consequently , an array of degradation techniques that yield easily identifiable, low molecular weight fragments has been developed. Many of these methods were developed in the 1950s and 1960s and are documented by Nicolaus (7). The degradation methods are classified as reductive, oxidative, pyrolytic, and photochemical, and recent findings are described below.
I . Reductive Methods Melanins have been degraded reductively via catalytic hydrogenation, as well as with hydriodic acid and sodium borohydride. Thus, sepiomelanin at 150°C with hydrogen and palladium in ethanol produced 5,6-dihydroxyindole (Ref. 7, p. 81). On the other hand, under surprisingly mild conditions (0. I N NaOH/NaBH4) sepiomelanin and biosynthetic eumelanins gave 5,6-dihydroxyindole-2-carboxylicacid (14/). Degradation with hydriodic acid was found to be a specific method in the identification of phaeomelanins (117); aminohydroxyphenylalanine, the degradation product identified by HPLC, is characteristic for melanins derived from 5-S-cysteinyldopa. Owing to the chemical nature of the reagent this degradation involves both reductive and hydrolytic processes. No effect on the number of 5,6-dihydroxyindole units in the melanin polymer was observed on reduction with ascorbic acid or sodium dithionite (222).
286
RAIMONDO CRIPPA ET A1
2. Oxidative Methods Melanins have been aerobically degraded to a number of pyrrolecarboxylic acids with alkali hydroxides either by high-temperature fusion (above 200°C) (Ref. 7, p. 80) or in boiling dilute aqueous solution (e.g., 4% NaOH) (Ref. 7, p. 81). This finding may support the hypothesis that carboxyl-substituted pyrrole moieties (e.g., 2,3,5-pyrroletricarboxylic acid) represent one constituent of the melanin structure (Ref. 7, p. 85). It is much more likely, however, that the majority of the pyrrolecarboxylic acids result from oxidative-hydrolytic degradation of the 5,6dihydroxyindole moieties of the melanin. This process parallels the oxidative degradation of 5,6-indole-2-carboxylic acid with peracetic acid (Ref. 7, p. 80) which also leads to pyrrole-2,3,5-tricarboxylicacid. Alkali fusion (308°C) of several eumelanins and allomelanins isolated from animals and plants (312) produced 5,6-dihydroxyindole and 3,4dihydroxybenzoic acid. A more detailed study using sodium hydroxide degradation of both natural and synthetic melanins revealed the formation of two different components: one (the more stable under the reaction conditions used) which absorbs in the visible region and a second absorbing in the U V region. It was speculated that the former is composed of stacks of planar monomer units and that the latter represents the “core” of the polymer providing the protective function against the harmful U V radiation (313). A number of studies have been devoted to the truly oxidative degradation of all types of melanins. Generally, eumelanins undergo oxidative degradation in several stages for which various reagents, such as hydrogen peroxide and potassium permanganate, have been utilized. Hydrogen peroxide oxidation in mild alkaline (pH 9-10) media first solubilizes melanin with no obvious structural change (see Section IV). It is the second stage, the bleaching process, which is most probably associated with the oxidative breakdown of the polymer structure. Complete bleaching of melanin in specimens embedded in paraffin or polystyrene is possible in 1-3 hr at 37°C in a mixture of benzyl alcohol (20 ml), acetone (10 ml), 10% hydrogen peroxide (5 ml), and 25% ammonia (4 drops). Results are identical to those obtained after 24-48 hr of oxidation in 10% hydrogen peroxide (314). Oxidative degradation can be terminated at the solubilization stage by decomposition of the excess hydrogen peroxide (Pt-black, catalase). Acidification (pH > 2,3 = 2,3,4,5. The same samples after decarboxylation at 200°C followed the sequence 2,3,5 > 2,3 > 2,5 = 2,4 = 2,3,4,5. The decrease in 2,3,5 triacids and the increase in 2,3 diacids are attributed to the loss of carboxyl groups owing to the thermal treatment (7). Resistance to further oxidative degradation uhder specific experimental conditions may substantially influence the ratio of the individual pyrrolecarboxylic acids formed (315). 3. Other Degradation Methods
Photooxidation of adrenochrome melanin under oxygen at high pressure led to its degradation and formation of low molecular weight products (316). Natural black (human hair, bovine eyes) and synthetic (tyrosine, dopa, and dopamine) melanins were investigated by Curie point pyrolysis-gas chromatography-mass spectrometry (86,96).The pigments were characterized by different ratios of degradation products identified as aromatic hydrocarbons, phenols, catechols, pyrroles, and indoles. The amount of ash in karakul lamb wool was correlated to its color, with black producing the most (3.9%) and white the least (1.2%). Similar studies showed a correlation with the calcium content (317.3f8).
c. NONDEGRADATIVE METHODS I . Redox System One of the most characteristic functional properties of melanins is their ability to exchange electrons with reducing and oxidizing agents; this accounts for their existence in both the oxidized quinone and reduced
288
RAIMONDO CRIPPA ET A L
quinol forms, respectively. Unlike oxidative and reductive degradations, these processes merely involve a reversible exchange of two electrons and two protons. Typically, Ti3+,Fe3+,ascorbic acid (2101, Fe(CN):-, sodium hydrosulfite, NADH and NADPH, cytochrome c, dichlorophenolindophenol ( 2 2 3 , nitroxide free radicals ( 3 / 9 ) ,and Nitro Blue Tetrazolium (320)undergo exchange of oxidation states with aqueous melanin suspensions. Eumelanins can act either as electron acceptors or electron donors in a fashion similar to that of a large number of electronexchanging synthetic polymers characterized by the quinone-quinol functionality (322). ESR is the method used extensively to characterize directly changes in the oxidation state of melanins (75). Spectrophotometric or electrochemical methods have been useful in monitoring concentration changes of the reagents-mediators (oxidants, reductants). The electrochemical method allows the monitoring of nontransparent suspensions without separating the melanin that does not exchange electrons with the electrode. Owing to the presence of acidic groups in melanins (carboxyls, phenolic groups) positively charged reagents react faster than anions or neutral species, especially in basic media. Thus, cationic nitroxides react much faster than anionic ones, and the reaction is twofold faster at pH 10 than pH 5 . The slow reaction with Nitro Blue Tetrazolium is dramatically accelerated in the presence of a cationic detergent (92). Generally reduction of both natural black wool and synthetic L-dopa and tyrosine melanins results in a lighter color and changes in the ESR spectra (/53).Relatively minor changes are observed on treatment with mild reducing agents [ascorbic acid/water, sodium borohydride/ aluminum chloride/diglyme, sodium borohydride/ferric chloride/diglyme, homogeneous high-pressure catalytic hydrogenation using tris(tripheny1phosphine)chlororhodium in chloroform]. Much more significant changes are observed under the drastic condition4 of Birch reduction (sodium in liquid ammonia). Interestingly, products of the reduction in nonaqueous media show an increased free radical content, while the reverse is observed when aqueous media are used. Mechanistically, the quinone-quinol forms in melanins are coupled via the relationship Melred
Mel,,
+ 2n e - + 2n H'
where n is an integer. Experimentally, however, this relationship has never been examined quantitatively in order to determine coupled irreversible chemical processes such as cross-linking or carbon-carbon bond cleavage.
6. CHEMISTRY OF MELANINS
289
The populations and role of semiquinone states assumed to be responsible for the characteristic ESR signal have been extensively studied by ESR spectrometry for all types of melanins (75). The increase in the free radical content after reduction of melanins in nonaqueous media may indicate an increased population of semiquinones (153) and/or quinhydrone-type complexes. In such a case a maximum intensity signal should be observed with half-oxidized-half-reduced melanin. Both reduction and oxidation processes have been found to be biphasic. Thus, in kinetic studies of the reduction of synthetic d,l-dopa melanin with Ti3+ and oxidation with Fe3+, respectively, a fast first electron-exchange step was followed by a slow second step (210). Whereas the quinone-quinol relationship involves an exchange of two electrons, only 0.5 electrons were accounted for the fast reaction step between d,l-dopa melanin and Ti3+; similarly, only 0.02 electrons per indole unit was exchanged with Fe3+(210). From the 25: 1 ratio for the fast reduction versus oxidation steps, it was concluded that melanin in an air atmosphere exists predominantly in the quinonoid form. This finding was further supported by an experiment in which reduced d,l-dopa melanin was partially reoxidized by air. The biphasic character of the electron-exchange processes was interpreted as the difference in reaction mechanisms involving the surface and the core of the melanin granules. Using the oxidation-reduction capacities obtained for the fast electron-exchange processes, one-fourth of the indole units were found at the particle surface. Assuming the same fast rate of electron exchange in both the oxidation and reduction, respectively, the slow diffusion of the reagent (Ti3+,Fe” , and H+)in and out of the melanin particle is believed to control the rate in the second phase. Alternatively, the slow step may represent an electron transfer between the outside indole units exchanging electrons with the reagent and the indole units of the particle interior, combined with a diffusion of protons. This mechanism resembles processes which characterize electron transfer in redox-conducting polymeric films of similar chemical structure deposited at solid electrodes (322). Whereas only 0.02 electron per indole unit was exchanged in the fast Fe3+ oxidation process, long exposure of d,I-dopa melanin resulted in total consumption of two electrons. This observation was associated with an oxidative cross-linking step involving two hydrogen atoms (210). Unlike the Fe3+-oxalate oxidation, the potassium ferricyanide one in pH 7.2 buffer afforded 0.25 electrons per indole unit. When the reaction with potassium ferricyanide was allowed to proceed to completion (time not specified) about 0.75 electrons per indole unit were exchanged, again suggesting deeper structural changes.
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RAIMONDO CRIPPA ET A L
The electron-exchange properties of melanins have been studied with a number of special reagents in order to elucidate the electron exchange mechanism itself and the role of the melanin redox properties in biological systems. It was thus found that nitroxide radicals were reversibly reduced by melanins in the dark (319)and that the redox equilibria were altered on irradiation (see Section VI). Moreover, the reduction of nitroxides (R2N0.)was inhibited by oxygen. The equilibrium
K
=
[Mel,,] [R2NOH]/[Mel,,~][R2N0.]
and the reaction rates were determined quantitatively. Melanins such as d,I-dopa melanin, phaeomelanin, and retinal pigment slowly reduced Nitro Blue Tetrazolium in aqueous dimethylformamide (aeorobic conditions, pH 7.4) (320). The reaction was strongly accelerated by cationic detergents (e.g., cetyltrimethyl ammonium bromide) with no significant photoeffect (92). Hydrogen peroxide, which oxidatively degrades eumelanins, undergoes disproportionation with catechol melanin to produce oxygen and water (205). Of particular significance to biological systems is the reaction of melanins with oxygen. The effect of external factors on this reaction, e.g., pH, illumination with visible light, temperature, and catalase, has been studied in detail (323). Melanins (d,I-dopa and bovine eye melanin) were studied in their native, reduced (sodium borohydride in 12% sodium hydroxide), oxidized (potassium ferricyanide in pH 6.8 buffer), and methylated (first reduced with sodium borohydride, then reacted with dimethyl sulfate) forms, and the reaction was monitored via ESR. The rates of oxygen uptake were, generally, higher with illumination. Over the pH range 5.5-1 1.9 the rates increased more than three orders of magnitude, while the free radical intensity fourfold. The sodium-reduced d,l-dopa melanin reacted faster (up to two orders of magnitude at low pH) while the methylated substrates slower (one order of magnitude). Activation energies for reaction with oxygen determined for the dark and photoactivated processes were 10 and 5 kcal/mol, respectively. However, only a negligible difference in the oxygen consumption rates for untreated and ferricyanide-oxidized melanin has been found. Results of the study of the effect of hydrogen peroxide and catalase suggest processes leading to hydroxylated melanins via a hydroperoxide intermediate rather than a quinole to quinone oxidation:
The studies aimed toward the examination of the role of melanins in
6 . CHEMISTRY OF MELANINS
29 I
living systems (especially the processes involving NADH, NADPH, and cytochrome c ) (223) are directly linked to their redox properties. Generally, the chemical changes of melanins, both natural and synthetic, were monitored via ESR, while concentration changes of the reactants were determined spectrophotometrically. In a way similar to reactions reported earlier, the electron-transfer processes were found to be strongly irradiation dependent (both by visible and U V light). The following equations characterize the mechanism of NADH oxidation with melanin (324):
+ NADH + H ' C
MelFed+ NAD' Melred+ 0: C Mel,,, + HzOz NAD' + 2 HzO NADH + H' + HzOZ
Mel,,
In this system the rate of NADH oxidation was increased by eliminating H202 using catalase. In addition to direct electron exchange, melanins exhibit interesting properties characteristic of electron-transfer agents (223).Thus, synthetic dopa, dopamine, adrenaline, adrenochrome, and hydroquinone melanins accelerated the oxidation of NADH with Fe(CN):-. (optimum pH 5.5-8.5) and 2,6-dichlorophenolindophenol-Cu?'. The rate with all three components present was higher than the combined rates of oxidation of NADH with either reagent alone (68,195). Interestingly, the 1 : 2 molar ratio of NADH oxidized and Fe(CN)63- reduced was approximately the same irrespective of the amount of the melanin used. The reversible character of the entire system was documented by the rate decrease after addition of any of the reaction products [Fe(CN):- and NAD']. The use of various reagents as cooxidants (e.g., KMn04, benzoquinone, iodine, and ferric chloride) enhanced the oxidation of NADH and decreased the reduction of ferricyanide (325).
2. Acid Functional Groups Information on the acidic functional groups of melanins was obtained by acid o r base titrations (326).Melanins were prepared by autooxidation of the precursors in the presence of bases (such as sodium hydroxide, ammonia, diethylamine, and glutathione). Since incorporation of highly nucleophilic bases in the polymeric matrix is quite likely, it is not surprising that the resulting titration curves showed large differences for samples of different origin. In addition, the results were also influenced by the titration procedure itself (e.g., the waiting time) most probably because of the biphasic mechanism. The titration curves were characteristic for both reversible and irreversible processes; the latter involved reactions other than proton exchange, such as the loss of bases attached by coulombic forces and of absorbed carbon dioxide following treatment
292
RAIMONDO CRIPPA ET A L
with acids. The titration curves are unique for each melanin type and, therefore, are well-suited for characterization and differentiation purposes. The amino acid composition of pigmented wool melanins was determined, and the effect of cationic surfactants on the reduction of bleeding of wool in alkaline solution was explained on the basis of neutralization of the carboxyl groups in melanins by the cationic surfactants (327). 3 . Derivatization Melanins have been derivatized with various reagents in processes involving both phenolic and carboxyl functional groups. The native phenolic functional groups of melanins have been methylated with diazomethane directly. Samples with higher numbers of methoxy groups were prepared by reducing the quinone functionalities with sodium borohydride prior to methylation with dimethyl sulfate. Such derivatized melanins underwent oxidation by oxygen (in the dark, pH 10.5) 10 times slower compared to the native sample (Ref. 7, p. 80; 323) A similar effect was observed on methylation of the phenolic groups in humic acids (327). The reverse trend was observed, however, with a methylated melanin on illumination (pH > Li’, Ba” >> Mg”). However, the exceedingly high affinity found for Pb’+ when compared with similar
296
RAIMONDO CRIPPA ET A L
divalent ions suggests the possible contribution of other factors. The equal affinity found for native and synthetic melanins indicates that the proteins present in native melanins play a minor role in the binding of metals (95). Detailed study of the affinity of Mn’+ was prompted by the fact that occupational exposure to manganese affects the nervous system and, in particular, nerve cells in substantia nigra. Using S4Mnand autoradiographic techniques, the highest binding was found for bovine eye melanin (1.33 pmol/mg; corresponding to one Mn atom per 4.8 indole units), the lowest for synthetic DA melanin (0.15 pmol/mg) (87). Complexation of grape pomace melanin with metals (Co”, Mn”) enhanced its effectiveness in carrot and onion germination (108). Binding studies combined with ESR spectroscopy provided deep mechanistic insight into the nature of the interaction of the metal ions with melanin. This technique allowed the identification of chelation of di- and trivalent diamagnetic metal ions by the o-semiquinone radical centers (253);this interaction often results in an increase of the total free radical concentration. Studies carried out over a broad pH range demonstrated different binding mechanism of ions below and above pH 7. At lower pH binding involved primarily carboxyl groups or complexation with a bidentate nitrogen-carboxyl ligand. At higher pH binding involved mainly phenolic hydroxyls. The binding capacity varied for melanins of different origin: the number of reactive sites in a bovine eye melanin was less than that in synthetic melanins (203). Organic compounds used in binding studies with melanins were mostly bases, often positively charged (quarternary ammonium cations). Paraquat and diquat studied both in uitro and in uiuo were found to bind strongly to eye melanin, and the cation-exchange mechanism was fully identified (344). A systematic structure-affinity study was reported for a series of heterocyclic compounds and synthetic d,I-dopa melanin (345).The structural variables of the substrate molecules were basicity, extent of the 7~ system, and planarity of the molecule (346).Relative affinities determined from adsorption in pH 7 phosphate buffer followed the sequences pyridine 9-methyliminostilbene > 9,lOdimethyliminostilbene Thus, the extent of the r-electron system in series I, the 7~ system and basicity in series 11, and the steric factors and degree of buckling of the central ring in series 111 are the determining factors for the affinity toward
6 . CHEMISTRY OF MELANINS
297
melanin. These results are consistent with the expected stabilities of the charge-transfer complexes between the respective heterocycle, the .rr-donor, and the oxidized melanin, a .rr-acceptor. The practical consequences of this structure-affinity relationship suggest applications in the development of drugs which may selectively target melanocytes (such as melanoma cells) o r drugs with low toxicity that are not accumulated in melanin-containing tissues, such as eyes. Analysis of experimental data from binding studies of chloroquine, chlorpromazine, paraquat, and Nil’ using Scatchard plots support the concept of more than one binding site participating in these processes (343). VI. Spectroscopic Characterization
A. ULTRAVIOLET-VISIBLE A N D INFRARED SPECTROSCOPY The history of spectroscopic investigations of melanins attests to many attempts to obtain UV-visible and IR spectra with sufficient resolution to allow structural determinations. In the UV-visible range, the insolubility of natural eumelanins and the scarce solubility (at high pH) of artificial ones produces problems of scattered light, which prevent structural spectrophotometric determinations by traditional means. Typical spectra of both eu- and pheomelanins in the range 180-700 nm are characterized by a monotonic increase of the absorbance with decreasing wavelength coupled with one or more barely detectable shoulders that possibly reflect relative amounts of the various monomers present in the pigment. Solid films of eumelanins show spectra even less resolved (347). Despite the poor resolution, such spectra can provide comparative parameters in terms of optical absorbance ratios at selected wavelengths attempts to characterize melanins of different origin. An original approach to the absorbing and scattering properties of melanin granules, leading to a light-trap role in uiuo, was suggested by Wolbarsht r t ul. (328). Their hypothesis takes into account the effects arising from Rayleigh scattering (by the molecules) and Mie scattering (by the melanosomes) and, through a semiquantitative treatment, provides a model of the overall optical properties based on multiple scattering and multiple absorption with consequent high optical density. The proposed absorption mechanism of melanin as an amorphous semiconductor (315) with phonon coupling to excited electronic states helps to explain the efficient absorption of internally scattered light. A strong dependence of this effect on the hydration state further improves the description of this
298
RAIMONDO CRIPPA ET At-.
peculiar optical behavior (348). A more detailed discussion of the interaction of melanins with light is presented in Section V . The infrared characterization of melanins in the fingerprint region gives fairly good results when performed using very “dilute” KBr pellets (99). The sensitivity of the method allowed the study of protonation and deprotonation of titrable groups at different pH, thus monitoring the binding of iron to various chelating functional groups and allowing the comparison of natural and various synthetic melanins (99,349,350).Table V lists the IR vibration bands for various synthetic and natural melanins. 1R analysis of hydration in melanins was performed on samples dried at different temperatures (99). Spectra of samples of synthetic L-dopa melanin heated under reduced pressure (2 X lo-’ torr) at 400 K and 670 K show a decrease of the bands in the water absorption regions (3400, 1600, and 600 cm-’) and a concomitant increase of the background (mainly at shorter wavelengths) attributed to light scattering. Simple analysis of the transmittance T over a path of length x in a medium containing only spherical scatterers of radius r gives T =
e-yx
where y = n i k is the scattering coefficient. The scattering area ratio k is a function of the ratio rlA. The theory gives, for small particles, y a K4 (Rayleigh scattering) and, for larger particles, y A-$ (Mie scattering) when $ approaches zero. For L-dopa melanin $ equals 1.26. This value cannot be explained by the use of simplified approximations, thus reflecting the complex distribution of shapes and sizes of the pigment granules. Such studies on natural pigments are rather limited owing to the possible interference of proteins and other strongly bound cellular components to the IR spectra of melanins.
B. X-RAYDIFFRACTION A N D RAYLEIGH SCATTERING OF MOSSBAUER RADIATION STUDIES Early X-ray diffraction studies on melanins gave evidence of only a short range order in the arrangement of the indole rings with the appearance of a lamellar structure with an average interlayer spacing of about 3.4 A (351). Reinvestigation of this subject was recently made possible through the introduction of a new technique, Rayleigh scattering of Mossbauer radiation (RSMR) (352). This diffraction technique has an extremely high energy resolution (AEIE and provides both detailed structural and dynamic information. The spectra show a broad structured peak centered at Q = 1.78 A-‘ arising mainly from interlayer distances
TABLE V VIBRATION BANDSA N D CHARACTERISTIC IR ABSORPTION REGIONS FOR SYNTHETIC A N D NATURAL MELANINS ~~
~
L-Dopa melanin
Dopamine melanin
Sepia melanin
~
~~
Eye melanin
V
Vibrational band
(cm-')
N - H - NH2 symmetrical and asymmetrical stretching OH - H bonded stretching ( H 2 0 ,carboxylic, phenolic) N - H - NH3' stretching Aliphatic C - H stretching
3400-3500 3440 3200 2930 2860 2700-2500 I700 I600 I600 1400 1400 1300-1200 900-730 600
Carboxylic H-bonded OH stretching C = 0 COOH stretching OH bending (HzO) Carboxylate ion asymmetrical stretching Carboxylate ion symmetrical stretching Carboxylic C - 0 stretching or OH bending Aromatic C - H bending OH librations (HzO)
pH2
+ + + + +
pH 10
pH2
+
+
+
pH 10
+
+ +
+ + +
+ +
+
+
+
t
+ +
+
+ +
+ +
+ +
+
+ +
pH2
+ +
+
pH2
pH 10
+
+
+ +
+
+
+ +
+
+
+ +
+
pH 10
+ +
+
+ +
+ + + +
+ +
+ +
+
+ + + +
+ +
+ +
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RAIMONDO CRIPPA E T A L
and a broad second peak at Q = 5.4 k 'corresponding to intermolecular distances. The total curve is affected by the contribution of water coordinated to the melanin, which is responsible for the inelastic part of the spectrum. Consequently, the net radial distribution function deduced from the elastic part reflects the atomic distribution of the melanin structure alone. The main peaks correspond to the average bond lengths (C-N, C=O, C-C) in the melanin monomer (1.45 A), to distances between next-nearest neighbors (2.4 A), to the perpendicular interlayer spacing between indole planes (3.4 A), and to distances between atoms in adjacent layers occupying different positions in each monomer unit (4.4 The assignment of currently unidentified peaks may provide additional valuable structural information. The dynamics of the system are typical of a layered structure characterized by large anisotropies in the bonding forces. The mean square displacements measured along the direction of interlayer bonds < u: >, is one order of magnitude greater than that measured for bond distances in the monomer plane, < u i >. This result is confirmed by the large difference between the Debye temperatures 13, and 811 for motions perpendicular and parallei to the monomer planes (0, = 109.5 K , 1911 = 456 K), reflecting the strong anisotropy of the thermal vibrations. The X-ray diffraction curve for lyophilized melanosomes gives a radial distribution function similar to the curve obtained for a synthetic melanin (90). Thus, a peak at 3.4 A corresponds to the distance between indole planes, and two peaks are assigned to distances between first and second neighboring atoms, respectively. Other peaks, not observed in pure melanin, are assigned to distances relative to the protein matrix. Smallangle X-ray investigations confirmed the presence of periodicity in melanoproteic organelles (353). The results of recent X-ray studies of melanin films prepared from 5,6-dihydroxyindole (DI) are consistent with a pentameric structure with the DI units being linked at the 7 and 4 positions and twisted in a helix with a 180" repeat at each end (354).
A).
C. MOSSBAUER SPECTROSCOPY The ion-exchange capability of various types of melanins allows binding of the 57Feisotope, the most common probe used in Mossbauer spectroscopy. This method has proved to be a useful and accurate technique in the investigation of molecular and supramolecular structures of melanins. Both natural sepia and bovine eye melanins, as well as synthetic d,l-dopa melanin, were subjected to such studies (39,349,355).
6. CHEMISTRY OF MELANINS
30 1
Generally, the Mossbauer spectra show characteristics consisting of two components: two Zeeman sextets and a central quadrupole doublet. Studies performed at variable temperatures report a redistribution of the intensities between the components with a temperature-dependent line broadening. The results suggest that, in all samples, melanins occur in the form of very small paracrystalline particles with a broad size range and showing a superparamagnetic behavior. D. NMR SPECTROSCOPY The ability of NMR spectrometers to operate in the cross-polarization/ magic angle spinning mode is a powerful tool for structural elucidation of insoluble materials (356). Natural abundance solid-phase I3C-NMR spectra could be obtained for synthetic L-dopa eumelanin. The inordinate number of resonance signals, however, prevented definitive assignments of the peaks to specific carbons. Subsequently Chedekel et al. (357)used this technique to study the conversion of specifically labeled L-dopa and 5,6-dihydroxyindole to melanin. In the enzymatically produced melanin the I3C-NMR spectrum identified unequivocally the benzylic carbon of I-dopa as the C-3 carbon in the DI (or its carboxyl derivative) repeating unit. In the melanin formed by autooxidation, however, the C-3 carbon was in the form of both a pyrrolelike and a carbonyl carbon. Eumelanins produced in a similar way from DI showed no presence of carbonylcarrying structural units. These results also strongly suggest that the polymerization step involves predominantly the 4 and 7 positions of the indole. Recent work by Aime and Crippa (358) shows that spectral features differentiate samples according to their various sources and that different functionalities present in eumelanins can be identified. E. ELECTRONIC STRUCTURE Despite the lack of knowledge of the precise molecular structure of melanins, many of their physical properties can be understood in terms of the electronic structure. Thus, the optical and electrical behaviors of melanins and melanosomes, which are consistent with the role observed or hypothesized in uiuo, can be interpreted in terms of the formalism of the solid-state theory. Pullman and Pullman were the first to perform a calculation of the band structure for an idealized indole-5,6-quinone polymer, and their results enabled the prediction of an exceptional electron-accepting ability arising from extension of the lowest empty band in the bonding energy region
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RAIMONDO CRIPPA E T A L
(359). The possibility that melanins are intrinsic semiconductors was investigated experimentally in order to explain their properties, such as conductivity, photoconductivity, as well as the light and temperature dependence of the paramagnetism. Unfortunately, inconsistencies emerged between the models and experimental data. An analysis of this problem led McGinness (360) to suggest that such discrepancies could be worked out by appiication of theories on the electronic structure of amorphous materials proposed by Mott (361). The application of these new ideas yielded a model of melanin granules as hypothetical solid-state devices that might assume many physiological roles. The quantum mechanical solution of the random-square well model for the single particle wave function yields a set of energy levels different from those of crystals. For amorphous materials, the density of states is gaussian and the states under the peak are extended (i.e., the electron has the same probability of being found anywhere in the solid). On the contrary, the states under the tails are localized, and the electrons are restricted to a local volume. In this picture the mobility of the electrons in localized states depends on tunneling or phonon-assisted hopping. The resulting conductivity is not dependent on a gap in the density of states, as in the case of crystals, but is based on the mobility of electrons in localized states. For this reason the concept of “mobility gaps” substitutes the usual term “band gaps.” Many simplified reviews on this topic have been published, and readers are referred to the detailed treatment of the theory by Cohen (362).A short account of the concept is presented by Davis and Mott (363). Thus, one can test the validity of the model and calculate the most important parameters of the energy band structure of melanins. Experiments using a different approach gave somewhat different results. Working with melanin suspensions in 0.1 M hydrochloric acid, Strzelecka obtained an Eo value 1.4 eV and was able to reveal the presence of a band of states at the Fermi level (364). Despite the discrepancies, attributed to differences in the nature of the samples (melanins are very sensitive to degree of hydration and pH) the experiments confirm the consistency of the proposed model and provide useful data for further interpretation of the physical properties of melanins. Typical figures are (+ = 10-’2-10-’’ R-’ cm-’; (EF - Ev)= 1 eV, and Eo = 1.4-3.4 eV for synthetic melanins from L-dopa and hydroquinone (365) and for natural melanins extracted from bovine eyes, hair, and banana peels (82). The threshold switching in melanins and melanosomes, a rather exotic property of amorphous semiconductors, was studied by McGuiness et al.,
6. CHEMISTRY OF MELANINS
303
who demonstrated that it can happen at biologically attainable electrical field strengths (366-368). Study of the alteration of both the conductivity and threshold switching characteristics after doping of melanins with other molecules of biological importance and coupled with lowtemperature specific heat determinations (369) suggested the doping molecules as the carriers contributing to the conduction states. These findings support the hypothesis of a relation between electronic properties and cellular functions of inelanosomes as nonlinear energy transduction devices operating by phonon-electron coupling mechanisms (3f5).This biophysical model justifies the observed transition from a cytoprotective state at low energy input rates to a cytotoxic state at the high energy input rates. Strictly connected with these ideas are experiments on the absorption and dispersion of sound waves in melanins (370). A resonance absorption was found at I MHz, and a rather sophisticated theoretical interpretation allowed correlation of the shear spectrum with the presence of partially ordered structures. Particularly interesting is the observation that hydrated melanins and melanosomes are exceptionally “black” materials with respect to ultrasound absorption. More strictly related to the organization of pigmented tissues are studies on the electrical charge and/or polarization storage in melanins (which can consequently be classified as bioelectrets). This effect was discovered in synthetic melanins (365) but was also found in pigment epithelium-choroid complexes (371). Experiments performed via the thermally stimulated depolarization current (TSDC) technique showed a large depolarization current in the physiological temperature range in fresh pigmented eye tissue. This result can be explained only by preferential displacement of opposite charges or by natural orientation of electric dipoles in melanin molecules. The biological relevance of this peculiar histological feature is still not fully understood. It should be noted that the role of occular pigmentation is possibly more complex than believed on the basis of simple light absorption mechanisms. A fast photostable electrical response of the eye caused by melanin was identified (372), but its significance for vision processes is still doubtful. The possible biophysical consequences of the introduction of new physical models of melanins are stimulating and puzzling at the same time. New fields of investigation are open to test these models, in particular, the role of melanins in the inner ear and the functional significance of neuromelanin in the brain. Theoretical hypotheses on this last topic (373,374) are based on the electronic structure and physicochemical behavior of such pigments.
304
RAIMONDO CRIPPA E T A I
F. ESR SPECTROSCOPY Historically melanins were among the first biological molecules submitted to ESR investigations (375). The origin of their paramagnetism was debated for several years, but the presence of stable free radicals was almost always considered as the probable cause for the ESR signal. The classic publication by Blois et al. (376) provided definitive data on the spectral characteristics (including the g value, linewidth, temperature dependence, and magnetic interactions with Cu2+and confirmed both the presence of trapped free radicals and the close similarity between natural squid melanin and various synthetic preparations. More recently, the problem received great attention in an attempt to correlate the free radical properties of melanins to their chemical structure, biosynthesis, and possible physiological role in cells and tissues (75). 1 . Characteristics of ESR Spectra
The small differences in the ESR spectra of a large number of natural and synthetic eumelanins studied under various physical conditions were attributed to preparation and experimental conditions. Common parameters of the spectra are a single, slightly asymmetrical line that is nonhomogeneously broadened, without hyperfine coupling; g -2.004 and AH4-10 G,concentration 4-10 x lo” spins/g, corresponding to 1 radical per 3000 monomers of an average molecular weight of 200. The presence of residual protein moieties or metal ions (e.g., Zn, Cu, Fe in concentrations of 25-950 pg/g in samples extracted from bovine eyes) in natural eumelanins does not influence the ESR lineshape or intensity. Saturation recovery measurements gave values of relaxation times TI ranging between 10 and 20 sec, depending on the type of melanin and experimental conditions (377). All these experiments were performed on dried eye melanin or frozen suspensions. At present, no firmly based experimental data exist on TI and T2 for melanin under physiological conditions, primarily because of the strong dependence of the microwave saturation on the oxygen pressure. The effect of temperature on the paramagnetism (378), which was doubtful earlier, was recently definitely established both with regard to the spin concentration and linewidth on samples of suspended material. It was, moreover, correlated to temperaturedependent equilibria between diamagnetic groups (quinone, hydroquinone, or their donor-acceptor complexes) and their paramagnetic counterparts (biradicals and semiquinone radicals). All the spectral characteristics point to the presence of immobilized semiquinones as the simple radicals that originate the paramagnetism in melanins (S = l/2).This view is also supported by many experiments
305
6. CHEMISTRY OF MELANINS
involving oxidizing and reducing agents and the pH dependence of free radical concentration. The redox properties of many melanins were tested with various reactants, but their consequences on ESR spectral intensities are not unequivocal (75,324). However, it is still reasonable to attribute to melanins a scavenging ability for OH., H-, e-aq and other radicals, owing to the presence of various electron-exchange groups (379). The influence of pH on free radical concentrations is, on the other hand, well established and is due to the equilibrium (380) MQ
+ MQH2 $ 2
MQ-.
+ 2 H‘
As an example, the ratios of radical concentrations in aqueous suspensions of natural melanins at pH 1,7, and 14 are 0.5, 1 , and 7 respectively (309). Moreover differences in g values and linewidth were also found at different pH values, indicating the presence of various ionizable forms of the free radicals. However, at present, exact determinations of the pK, for melanins are subject to experimental difficulties owing to the appearance of irreversible changes during the titration with H+ and to the pH-dependent shift of the oxidation state (326). Equilibrium among the various forms of ionizable groups on melanins can also explain the well-known effects of metal ions on ESR spectral features. Diamagnetic ions generally enhance the ESR spectrum by a factor varying between 1.2 and 9. The equilibrium MQ
+
MQHz
2 H’
+ 2 MQ
2 1”’ ‘
2 MQ . I“+
(where I indicates a metal ion) can justify the finding that the extent of radical formation depends on the complexation ability of a particular ion. A concomitant broadening of the spectral line was noted with ions that possess a nuclear moment. The decrease of the ESR intensity observed with paramagnetic ions, originally reported by Blois et al. (376) with Cu” and also studied with synthetic and natural melanins ( 2 0 3 , has a different physical explanation. The formation of complex(es) places the ion in close proximity to the free radical, and the consequent strong magnetic interaction quenches the signal drastically. Details on the theoretical treatment of this phenomenon in melanins have been reported by Sarna et al. (381). Characteristics of the ESR spectra of phaeomelanins are quite different and deserve some comment. The usual spectrum is composed of a triplet with g = 2.0052, a value typical of immobilized radicals with hyperfine splitting due to nitrogen ( I = 1). Accurate measurements were accomplished on synthetic cysteinyldopa melanins (382) at various pH in DzO
306
RAIMONDO CRIPPA ET A L .
and in the presence of metal ions. All results point to the presence of semiquinonimine radicals. The most important consequence of such detailed studies is the definitive assignment of the ESR spectral characteristics of natural melanins, considered copolymers of dopa- and cysteinyldopa-derived monomers, as suggested by Prota (34).In a comparative study of synthetic melanins, prepared with different ratios of dopa and cysteinyldopa as starting materials, and natural malanins, it was possible to demonstrate the presence of both 0 , O - and p-N,Osemiquinones and semiquinonimine free radicals (382) in ratios directly related to the chemical composition.
2. Efiect of Light on Free Radicals Studies of the absorption of electromagnetic radiation in both the UV and visible regions by melanins resulted in an enhanced population of radicals being characterized by ESR. The absorption of light by melanin suspensions induces transient free radicals at wavelengths throughout the visible and UV region. They differ slightly from intrinsic radicals, showing a more complex ESR spectrum, with a higher g value, broader linewidth, and, possibly, a shorter T I .This induced population consists of two components, one characterized by a low yield (of the order of 1-2%) and a decay time of a few seconds and the other with a decay time of a few milliseconds that accounts for about 50% of the signal (252).The complex kinetics, temperature independent for the fast decay component, probably involve physical effects such as electron tunneling mechanisms and is further complicated by pH and oxygen pressure dependence, as suggested by accurate measurements with spin traps and superoxide dismutase (383, indicating the formation of 02--in transient equilibria under light. The transient nature of the light-induced radicals with the first half-life (time resolution 0.1 sec) of around 1 sec (384-386) was confirmed in more detailed studies (252). Using a pulse photolysis system (time resolution 0.2 msec), the existence of a slow (half-life 5 sec, second-order kinetics, large temperature dependence) and fast (chemical lifetime 50 msec, no temperature dependence) decaying spectral component was revealed. During continuous irradiation the contribution of the fast decaying component is dominant (50- 100 times), and the process shows characteristics of a singlet-triplet intersystem crossing mechanism. The entire photoexcitation process, which is considered by some to have relevance to the photoprotective action of melanins, is formulated as follows: Q
+ QH* 2
singlet
+ triplet -+
QH.
+ QH. + Q + QH?
where Q, QH2, and QH. represent quinone, quinol, and semiquinone units of the polymer, respectively.
307
6. CHEMISTRY OF MELANINS
During photolysis of phaeomelanins biologically active OH. and 0 2 - . are produced in concentrations about 100 times higher than in eumelanins. Nanosecond laser-flash photolysis indicates a photoionization of the excited state of the molecule producing a phaeomelanin radical action and hydrated electrons (387) that are responsible for the reduction of molecular oxygen to 0 2 - .The . biological and pathological implications of the deexcitation pathways of melanins are discussed in a review by Chedekel (388) with particular emphasis on the consequences of the formation of HPETEs during the photodegradation of phaeomelanins in the presence of arachidonic acid. These reactions open new areas in the study of the physiopathology of human skin cancer under sunlight irradiation. The Appendix lists the abbreviations that were used throughout the chapter.
Appendix
Abbreviation AA
Compound name
Structure CH,OH
Ascorbic acid
I
HO
6-ADA
6-Aminodopamine
ADR
Adrenaline (epinephrine)
OH
CHCHZNHCH3
I
OH
A-MD
y z
a-Methyldopa
H ~ ~ H ~ - : - C O O H
HO
\
CH3
(conrinued)
308
RAIMONDO CRIPPA E T A L .
Appendix (Continued)
Abbreviation A-MDA
Compound name
Structure
a-Methyldopamine
CH,CHNH,
I
CH 3
A-MNADR
a-Methylnoradrenaline
/
CHCHNH,
I I
HO CH,
BZQ
Benzoquinone
BZQ-2M-31
2-(2-Methyl-3-indolyI)benzoquinone
CAT
Catechol
CPZ
Chlorpromazine
CRD
Carbidopa
CRT
Creatinine
309
6 . CHEMISTRY OF MELANINS
Appendix (Continued)
Abbreviation DA
Compound name Dopamine (3-hydroxytyramine)
Structure H 0 ~ C H z C H z N H z HO
DHBA
3,4-Dihydroxybenzylamine
DHEPH
3.4-Dihydroxyephedrine
DI
5.6-Dihydroxyindole HO H
DICA
5.6-Dihydroxyindole-2-carboxylicacid COOH
H
3,4-DHMA
p
OH
3.4-Dihydroxymandelic acid
OH
CHCOOH
I
OH
5.6-DH MI
5.6-Dihydroxy-2-methylindole
DHPAA
3,4-Dihydroxyphenylacetic acid H o ~ C H z C o o H HO
DOE
Dioxethedrine
OH
OH
CHCHNHCH~CH,
I
I
OHCH,
3 10
RAIMONDO CRIPPA ET A L
Appendix (Continued)
Abbreviation Dopa
Compound name Dopa (3.4-dihydroxyphenylalanine)
Structure HODHz;".f"oH HO
EPH
Ephedrine
EPI
Epinine (deoxyepinephrine)
OH
OH
0 ~H,CH ,NHCH,
6-HDA
p
OH
6-H ydrox ydopamine
OH
CH ,CH,NHCH,
5HI-3AA
5-Hydroxyindole-3-acetic acid H
HMVA
Homovannilic acid
,COOH
CH H J p,01
HO
HQ
IND
Indole
IPADR
N-lsopropyladrenaline
OH
OH
@
CH CH ,NCH
I
OH
I
CH(CH,),
31 1
6. CHEMISTRY OF MELANINS
Appendix (Continued)
Abbreviation IPNADR
Compound name
N-Isopropylnoradrenaline(isoproterenol. isoprenaline)
Structure OH
OH
>-=
LHCH,NHCH(CH,), I OH
M-DI
HO
MN
Metanephrine
6"
CH,O
CHCHZNHCH,
I
OH MTAM
3-Methox ytyramine
CH30
OH
@
CH,CH,NH,
NMN
Normetanephrine (3-0-methylnoradrenaline)
CH30
$H CHCH ZNH 2
I
OH NADR
Noradrenaline (norepinephrine)
SAL
Salsoline
(continued)
312
RAIMONDO CRIPPA ET A L
Appendix (Continued)
Abbreviation
Compound name
SALOL
Salsolinol
SER
Serotonin
TRP
Tryptophan
TYR
Tyrosine
VMA
Vanillylmandellic acid
Structure
CH COOH
I
OH UA
Uric acid
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