Cancer chemotherapy

Cancer Chemotherapy In Cancer Chemotherapy; Sartorelli, A.; ACS Symposium Series; American Chemical Society: Washington...

Author: Alan C. Sartorelli

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Cancer Chemotherapy

In Cancer Chemotherapy; Sartorelli, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


Cancer Chemotherapy A l a n C . Sartorelli,

EDITOR

Yale University

A symposium sponsored by the Division of Medicinal Chemistry at the 169th Meeting of the American Chemical Society, Philadelphia, Pa., April 7, 1975.

30

ACS SYMPOSIUM SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1976

American Chemical Society Library 1155 16th st. N . W. In Cancer Chemotherapy; Sartorelli, A.; ACS Symposium Series; American Chemical Washington, DC, 1976. Washington, D. C.Society: 20036

Library of Congress CIP Data Cancer chemotherapy (ACS symposium series; 30 ISSN 0097-6156) Includes biographical references and index. 1. Cancer—chemotherapy. I. Sartorelli, Alan Clayton, 1931II. Series: American Chemical Society. ACS symposium series; 30. D N L M : 1. Antineoplastic agents—Therapeutic use— Congresses. 2. Neoplasms—Drug therapy—Congresses. QZ267 RC271.C5C292 ISBN 0-8412-0336-9

C215 1975 616.9'94'061 76-24825 ACSMC8 30 1-126 (1976)

Copyright © 1976 American Chemical Society A l l Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E U N I T E D STATES O F AMERICA


ACS Symposium Series Robert F . G o u l d , Editor

Advisory Board Kenneth B. Bischoff Jeremiah P. Freeman E. Desmond Goddard Jesse C. H . Hwa Philip C. Kearney Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Aaron Wold


FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishin format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.


PREFACE linical results of the past decade have demonstrated that the use of ^ chemical agents, largely in various combinations, in the treatment of disseminated cancer of man has resulted in a significant cure rate of various neoplasms, particularly the rapidly proliferating cancers of childhood. Thus, diseases such as acute lymphocytic leukemia, Ewing's sarcoma, Hodgkin's disease, choriocarcinoma, embryonal rhabdomyosarcoma, Burkitt's lymphoma, Wilms' tumor, and testicular carcinoma have responded dramatically to chemotherapy. Progress has been less spectacular with the more slowly growing neoplasms, e.g., carcinoma of the breast, lung, and colon, whic lems created by cancer. To combat these forms of malignancy, considerable hope is placed upon the use of mixed modality therapy; the conceptual approach of such therapy is to lessen the neoplastic cell burden of the host by use of surgery and/or irradiation followed by chemotherapy designed to attack metastatic neoplastic cells. Although gains are to be expected from such therapeutic onslaughts, it seems reasonable to predict that addition of new agents to our therapeutic armamentarium will be of value. Thus, agents with biochemical mechanisms of action different from those currently used in the clinic are to be sought, as well as molecular modifications of existing agents designed to increase the therapeutic differential between the cancer and the host. In addition, the medicinal chemist may be required to design drugs with new actions. For example, potent antiviral agents may ultimately be necessary as part of the treatment of diseases such as acute leukemia. Therapeutic approaches leading to extensive prolongation of survival or cure, which rely on the extensive use of irradiation, alkylating agents, and/or other mutagens will be expected to induce new cancers; therefore, effort will necessarily be required to develop drugs capable of preventing carcinogenesis. Finally, noncytotoxic therapies must be devised; here, concepts such as differentiation therapy designed to convert neoplastic cells to less or noncancerous cells should be considered. The proceedings of this symposium represent a sampling of a segment of the current approaches to the development of new and more efficacious antitumor drugs. The first chapter represents a class of agents, a-(N)-heterocyclic carboxaldehyde thiosemicarbazones, which in theory should provide a useful derivative for the treatment of cancer, since the ix


molecular target for these agents, the enzyme ribonucleoside diphosphate reductase, is of great importance to the synthesis of D N A and cell replication. Adriamycin, perhaps the most exciting drug to appear in cancer therapy recently, has a wide spectrum of anticancer activity, particularly affecting several solid tumors. The rate-limiting cardiac toxicity of this material presents a challenge to medicinal chemistry, for it seems quite reasonable to assume that the molecular determinants of this toxicity to the heart are different than those responsible for cytotoxicity to tumor tissue. Thus, structural modification to minimize or eliminate cardiac toxicity should enhance considerably its clinical usefulness. For this reason, two chapters are included on this important antibiotic. The alkylating agents represent an old class of highly active agents in which many derivatives have been synthesized and tested. Should additional compounds with this type of reactivity be designed and syn thesized? The answer i alkylating moiety, which direct alkylating potential largely to different cellular targets, are employed, or if other concepts are used to enhance anticancer specificity. The next chapter addresses itself to this latter possibility, attempting in its consideration of design principle, to take advantage of the anticipated higher reducing potential of hypoxic cells of solid tumors. The last chapter represents an important discovery of recent years, the nitrosoureas, a class of agents which possesses both alkylating and carbamoylating activity. These materials represent a particular chal lenge in drug design, since a large number of these compounds are clinically active, and selection of additional members of this class for trial in man has become particularly difficult. Thus, as an aid to such a selection, it is extremely important to understand the relative importance of both alkylating and carbamoylating activities to anticancer potency, as well as to formulate an understanding of the intracellular molecular target(s) of these materials and the structural features which dictate tissue specificity. It is to be expected that the tools of medicinal chemistry, particularly when guided by those of biochemistry and pharmacology, will provide new and better anticancer agents, and that the use of these materials with other drugs and treatment modalities will ultimately allow an expansion of the cure rate of various kinds of malignancy. November 5, 1975

A L A N C. SARTORELLI

χ


1 Development of

(N)-Heterocyclic Carboxaldehyde

Thiosemicarbazones with Clinical Potential as Antineoplastic Agents ALAN C. SARTORELLI and KRISHNA C. AGRAWAL Department of Pharmacology and Section of Developmental Therapeutics, Division of Oncology, Yale University School of Medicine, New Haven, Conn. 06510 The initial r e p o r t f the a n t i n e o p l a s t i activity f ( N ) - h e t e r o c y c l i c carboxaldehyd 1956 by Brockman and h i (1) reporte mylpyridine thiosemicarbazone (PT) produced an increase i n the life span of mice bearing the L1210 leukemia; the f u r t h e r devel opment o f t h i s compound as a p o t e n t i a l cancer chemotherapeutic agent was c u r t a i l e d , however, because o f its relatively low ther apeutic index. Several years l a t e r , French and Blanz (2,3) des c r i b e d the synthesis o f 1-formylisoquinoline thiosemicarbazone (IQ-1) and a v a r i e t y o f other ( N - h e t e r o c y c l i c carboxaldehyde thiosemicarbazones. These i n v e s t i g a t i o n s demonstrated that s e v e r a l h e t e r o c y c l i c r i n g systems, i n c l u d i n g p y r i d i n e , isoquinol i n e , q u i n a z o l i n e , phthalazine, pyrazine, p y r i d a z i n e , and purine possessed s i g n i f i c a n t a n t i n e o p l a s t i c activity when the carbonyl attachment o f the s i d e chain was l o c a t e d at a p o s i t i o n to the to the r i n g n i t r o g e n atom; attachment of the side chain βor h e t e r o c y c l i c Ν atom r e s u l t e d i n i n a c t i v e antitumor agents. Members of t h i s c l a s s have shown a n t i c a n c e r activity against a wide spectrum o f t r a n s p l a n t e d rodent neoplasms, i n c l u d i n g Sar coma 180, E h r l i c h carcinoma, Leukemia L1210, Lewis lung c a r c i noma, Hepatoma 129, Hepatoma 134, Adenocarcinoma 755, and B16 melanoma. In a d d i t i o n , spontaneous lymphomas o f dogs have shown ( N ) - h e t e r o c y c l i c carboxaldehyde thiosemicar s u s c e p t i b i l i t y to bazones. Such broad spectrum activity denotes great clinical p o t e n t i a l and suggests that a drug o f t h i s c l a s s may w e l l have utility i n cancer therapy. Extensive m o d i f i c a t i o n o f the formyl thiosemicarbazone side chain o f IQ-1 was c a r r i e d out by Agrawal and Sartorelli (4) to a s c e r t a i n the importance o f t h i s p a r t of the molecule f o r a n t i cancer a c t i v i t y . A v a r i e t y o f the s u b s t i t u t i o n s and a l t e r a t i o n s of the various p o s i t i o n s o f the side chain that were made are shown i n Figure 1; these changes uniformly l e d to complete l o s s or marked decrease i n t u m o r - i n h i b i t o r y potency. In a d d i t i o n , replacement of the h e t e r o c y c l i c r i n g Ν with C a l s o r e s u l t e d in a biologically i n a c t i v e compound. These f i n d i n g s i n d i c a t e d the

1


2

CANCER

Figure 1.

Some modifications of the side chain of thiosemicarbazone

CHEMOTHERAPY

1-formylisoquinoline


1.

SARTORELLI AND AGRAWAL

Antineoplastic

Agents

3

e s s e n t i a l i t y o f t h i s p o r t i o n o f the molecule and supported the i n i t i a l suggestion o f French and Blanz (3) that a conjugate N*-N*-S* t r i d e n t a t e l i g a n d system was a r e q u i s i t e f o r tumor i n hibitory activity. Extensive s u b s t i t u t i o n o f the i s o q u i n o l i n e and p y r i d i n e r i n g systems o f ( N ) - h e t e r o c y c l i c carboxaldehyde thiosemicarbazones has been c a r r i e d out by our l a b o r a t o r y (5-10) and by Blanz et a l . (11,12). A d e t a i l e d summary o f the e f f e c t s o f some o f these subs t i t u e n t s on a n t i n e o p l a s t i c e f f i c a c y has been reported (13,14). Unfortunately, these i n v e s t i g a t i o n s have not allowed d e t a i l e d p r e d i c t i o n s to be made concerning the importance o f these m o d i f i c a t i o n s to the a c t i v i t y o f t h i s c l a s s o f agents against neoplas tic cells. The 100

83

90

> 1000

57

13 (25)

0. 8

0.9

(12.5)

112 (1)

a

S e e Tables I I I and IV f o r d e t a i l s .

b

P388 mouse lymphocytic leukemia, QD1-9 schedule, i p tumor and drug.


36

CANCER

CHEMOTHERAPY

the f u l l and i r r e v e r s i b l e p o s i t i v e charge present i n the drug molecules i s a probable explanation f o r the i n v i t r o e f f e c t because p h y s i c a l data on the daunomycin d e r i v a t i v e i n d i c a t e l i t t l e change i n the b i n d i n g p r o p e r t i e s with DNA. For example, the A T for 31 i s 16.3° vs 16.8° f o r daunomycin. P h y s i c a l data on the adriamycin d e r i v a t i v e are not y e t a v a i l a b l e but both 30 and 31 can be accommodated by the model receptor complex. S u r p r i s i n g l y the adriamycin quaternary r e t a i n e d a p p r e c i a b l e i n v i v o a c t i v i t y while the daunomycin d e r i v a t i v e was i n a c t i v e . However q u a t e r n i z a t i o n i s c l e a r l y d e t r i m e n t a l to i n v i v o a c t i v i t y i n both cases. Before l e a v i n g the subject of d e r i v a t i v e s the work of Trouet et a l . on DNA complexes of daunomycin and adriamycin should be mentioned although these m a t e r i a l s a r e not d e r i v a t i v e s i n the chemical sense used e a r l i e r i n t h i s s e c t i o n . Because tumor c e l l s have a higher p i n o c y t o s i s r a t e than normal c e l l s , these i n v e s t i gators reasoned that complexin c a r r i e r that could ente i n s e l e c t i v e drug uptake by tumor c e l l s . Inside the c e l l sub sequent lysosomal d i g e s t i o n of the c a r r i e r would occur and the r e s u l t i n g drug r e l e a s e would cause c e l l death. They a p p l i e d t h i s p r i n c i p l e , termed lysosomotropic drug a c t i o n , to daunomycin and adriamycin v i a the drug-DNA complexes because of the high s t a b i l i ty of these complexes. Although the o r i g i n a l work (111) used daunomycin, adriamycin-DNA complex a l s o proved advantageous over the f r e e drug. Table V I I I presents comparative i n v i v o a n t i tumor t e s t data f o r adriamycin and i t s DNA complex from recent work by A t a s s i and Tagnon (112). The complex i s c l e a r l y s u p e r i o r to the f r e e drug at a l l dose l e v e l s and induced one long-term s u r v i v o r out of ten t r e a t e d animals at the highest dose. The m

Table V I I I .

A c t i v i t y of Adriamycin and Adriamycin-DNA Complex Against Leukemia L1210 i n DBA/2 M i c e a

Dose (mg/kg)

S u r v i v a l time i n c r e a s e (%) Adriamycin Adr iamy c in-DNA

6

83

216 (1/10)

5

141

216

3

92

125

a

D a t a from r e f . 112.

b

Dose schedule QD1-5, i v . Dose of adriamycin-DNA complex r e f e r s to wt. of adriamycin i n complex.


2.

HENRY

Adriamycin

37

t o x i c i t y of the complex, based on drug content, i s reduced substantially. T h i s type of r e s u l t (113) l e d to s u c c e s s f u l c l i n i c a l use of daunomycin-DNA complex i n the m a j o r i t y of p a t i e n t s i n a group of 25 with acute leukemias (114,115). A f a v o r a b l e r e s u l t with the one p a t i e n t t r e a t e d w i t h adriamycin-DNA complex was a l s o reported (114). Reduced t o x i c i t y was the major advantage c i t e d f o r the complexes over the f r e e drugs. Additional c l i n i c a l t r i a l s with both of these promising m a t e r i a l s are c u r r e n t l y underway (40). The p o s s i b i l i t y that the r o l e of the DNA i n the adriamycinDNA complex i s not to c a r r y the drug i n t o c e l l s by endocytosis has been r a i s e d by the work of Marks et a l . They found that DNA i n j e c t e d s e p a r a t e l y b e f o r e and a f t e r adriamycin, and a l s o without adriamycin, i n c r e a s e d s u r v i v a l time i n tumor b e a r i n g mice (116). Sgmij^ Within the d e f i n i t i o n used i n logs are p o s s i b l e , thos a daunosamine s u b s t i t u t e and those c o n s i s t i n g of an aglycone s u r rogate coupled to daunosamine. As the f i r s t examples of the former type Penco (117) coupled daunomycinone w i t h glucose and glucosamine v i a a Koenigs-Knorr sequence to g i v e compounds 32 and 33, r e s p e c t i v e l y . No r e p o r t s on the b i o l o g i c a l p r o p e r t i e s of 32

32

R = OH

33

R = NH

2

are known to the author but the glucosamine analog has been s t u d i e d e x t e n s i v e l y . I t binds l e s s s t r o n g l y to DNA than daunomycin (60), i s l e s s potent or i n a c t i v e i n a v a r i e t y of i n v i t r o assays (77,118), and i s i n a c t i v e a g a i n s t the a s c i t i c sarcoma 180 tumor i n mice (77). The pronounced d e l e t e r i o u s e f f e c t of r e p l a c i n g daunosamine with glucosamine i s not e n t i r e l y c o n s i s t e n t with the molecular model of F i g u r e 4 because an e q u a t o r i a l


CANCER

38

CHEMOTHERAPY

f

2 -ammonium group can bond w i t h the adjacent phosphate anion about as w e l l as a 3'-ammonium. However, the 2 -ammonium s t e r i c a l l y i n t e r a c t s with the 8-methylene of the Α-ring and a conformational change i n the r e l a t i o n s h i p of the sugar to the Α-ring could r e a d i l y account f o r a poor f i t i n the h y p o t h e t i c a l receptor complex. Very r e c e n t l y the F a r m i t a l i a group (119) reported semi s y n t h e t i c analogs of daunomycin and adriamycin i n which the 4 hydroxyl i s i n v e r t e d with respect to the parent a n t i b i o t i c s (34, 35). These compounds were prepared by coupling the corresponding Ν,Ο-trifluoroacetyl-blocked c h l o r o sugar (obtained from dauno samine) with daunomycinone and with adriamycinone, blocked at the 14-hydroxy group by a k e t a l . In a d d i t i o n to the n a t u r a l a-anomers these syntheses simultaneously provided 3-anomers 36 and 37 i n l e s s e r y i e l d s . The 4 - e p i m e r i c a n t i b i o t i c s r e t a i n e d roughly com parable a c t i v i t y to the parents as i n h i b i t o r s of Murine Sarcoma 1

1

f

v i r u s (Maloney) f o c i formation and mouse embryo f i b r o b l a s t p r o l i f e r a t i o n but appeared somewhat l e s s potent as i n h i b i t o r s of HeLa c e l l colony formation. In v i v o 34 and 35 r e t a i n e d s u b s t a n t i a l a c t i v i t y against a s c i t i c Sarcoma 180 and Gross leukemia i n the mouse but e f f i c a c y was moderately reduced r e l a t i v e to the parents. 4 - E p i - a d r i a m y c i n was e q u a l l y e f f i c a c i o u s but s l i g h t l y l e s s potent than adriamycin i n a Sarcoma 180 s o l i d tumor t e s t as w e l l . I n t e r e s t i n g l y the 3-anomer of 4 -epi-daunomycin (36) r e t a i n e d s i g n i f i cant a c t i v i t y i n the v a r i o u s i n v i t r o t e s t s and against a s c i t i c Sarcoma 180 i n v i v o , d e s p i t e i t s marked configurâtional d i f f e r e n c e from daunomycin; potency i n every t e s t was reduced s u b s t a n t i a l l y i n comparison with daunomycin or 34. 1

1

A very s i g n i f i c a n t f i n d i n g i n t h i s study was the l a c k of e f f e c t of 35 on the beating r a t e of c u l t u r e d mouse embryonic heart c e l l s at 1 pg/ml when adriamycin showed a t o x i c e f f e c t at 0.1 yg/ml.


2.

HENRY

Adriamycin

39

T h i s c l e a r l y demonstrates that small s t r u c t u r a l m o d i f i c a t i o n s can f a v o r a b l y a f f e c t c a r d i o t o x i c p r o p e r t i e s without d e s t r o y i n g a n t i tumor a c t i v i t y . Daunomycin analogs 34 and 36 were a l s o r e c e n t l y prepared at Stanford Research I n s t i t u t e by somewhat d i f f e r e n t methods (120, 121). Not unexpectedly we found that 4 -epi-daunomycin was e s s e n t i a l l y i d e n t i c a l to daunomycin as an i n h i b i t o r of n u c l e i c a c i d s y n t h e s i s and a l s o gave a AT value n e a r l y as high as the parent (Table IX). The 3-anomer (36) r e t a i n e d about one-tenth of T

m

Table IX.

Compound

In v i t r o Comparison of Daunomycin and Two Configurâtional Isomers.

DN

34

0.7

36

6.7

2.9

5.2

daunomycin

0.80

0.32

16.8

a

S e e Table IV f o r d e t a i l s .

b

S e e Table VI f o r d e t a i l s .

of the potency of daunomycin i n suppressing n u c l e i c a c i d s y n t h e s i s and i t s AT value was depressed by 11.5° below that of daunomycin. P r e l i m i n a r y i n v i v o NCI t e s t data i n d i c a t e that both 34 and 36 are a c t i v e i n the L1210 system. 4 -Epi-daunomycin i s r e a d i l y accommodated by the proposed receptor model of F i g u r e 4 with p o s s i b l e l o s s of the 4 -hydroxy1 hydrogen bond as the only change. Because of i t s molecular shape the 3-anomer (36) cannot be f i t t e d n e a r l y as r e a d i l y as can 34. The aglycone r e s i d u e of 36 has become e q u a t o r i a l to the pyranose r i n g , r a t h e r than a x i a l as i n the α-anomers. However a reasonable conformation f o r 36 that permits aglycone i n t e r c a l a t i o n and forma t i o n of the i o n i c bond and the 9-hydroxy hydrogen bond can be obtained by r o t a t i o n about the two carbon-oxygen bonds of the g l y c o s i d i c l i n k , thus q u a l i t a t i v e l y r a t i o n a l i z i n g the decreased potency and AT . S t r u c t u r e 38 i s another semisynthetic analog d e r i v e d from daunomycinone (122). In t h i s compound the daunosamine moiety i s replaced by the b a s i c but much simpler 3 - a l a n i n e e s t e r f u n c t i o n . Because of the f l e x i b i l i t y of the a c y c l i c 3 - a l a n i n e s i d e chain t h i s compound can r e a d i l y be accommodated by the receptor model. Compared to daunomycin 38 i n h i b i t s DNA and RNA s y n t h e s i s i n c u l t u r e d L1210 c e l l s at about t e n - f o l d higher l e v e l s ( E D = 8.2 and 5.7 μΜ, r e s p e c t i v e l y ) . In v i v o antitumor a c t i v i t y was a l s o m

1

1

m

50


CANCER

40

CHEMOTHERAPY

aa found with t h i s compound, g i v i n g increases i n s u r v i v a l time i n two t e s t s of 60 and 26% at 12.5 mg/kg and 69 and 26% at 6.25 mg/kg (P388, QD1-9 i p regimen). The A T value f o r 38 under our s t a n dard c o n d i t i o n s (Table VI) was reduced but s i g n i f i c a n t at 6.3°. Although the i n v i v o a c t i v i t y of 38 i s unremarkable compared to adriamycin and daunomycin i t r e i n f o r c e s the c o n c l u s i o n suggested p r e v i o u s l y by the p r o p e r t i e nothing i r r e p l a c e a b l e i simply be a c a r r i e r f o r a potency-enhancing ammonium moiety. T h i s point w i l l be considered f u r t h e r i n f o l l o w i n g d i s c u s s i o n s . Several e s t e r s of daunomycinone analogous to 38 but derived from amine-bearing cyclohexane c a r b o x y l i c a c i d s have been patented and claimed as e f f e c t i v e against L1210 lymphocytic leukemia i n the mouse (123). Moving t o the second type of semisynthetic analog, Table X presents a s e r i e s of g l y c o s i d e s of daunosamine with a v a r i e t y of s y n t h e t i c aglycones (124). These compounds a r e part of a s e r i e s designed to determine what f e a t u r e s of the n a t u r a l aglycones a r e e s s e n t i a l f o r b i o l o g i c a l a c t i v i t y . D e l e t i n g a l l aromatic charac ter and r e t a i n i n g only r i n g A of daunomycinone provided 39. V i r t u a l l y a l l a c t i v i t y as an i n h i b i t o r of DNA and RNA s y n t h e s i s i n c e l l c u l t u r e i s l o s t by t h i s s i m p l i f i c a t i o n . The A T value f o r 39 has not been determined but a low value would be expected because i n t e r c a l a t i o n i s impossible with no aromatic group. Coupling of daunosamine to α-tetralol, thus r e i n s t a t i n g a l i t t l e aromatic character i n the aglycone, y i e l d e d the diastereomeric p a i r , 40 and 41. Neither compound d i s p l a y e d s i g n i f i c a n t DNA/RNA synthesis i n h i b i t i o n nor provided A T values a p p r e c i a b l y above b a s e l i n e ( i . e . , no b e t t e r than daunosamine). Extension of the aromatic area of the s e r i e s by use of b i p h e n y l - 4 - c a r b i n o l as aglycone provided compound 42. A n e a r l y t e n - f o l d enhancement of n u c l e i c a c i d s y n t h e s i s i n h i b i t i o n r e s u l t s from t h i s change but the a b i l i t y of the compound to thermally s t a b i l i z e DNA remains i n s i g n i f i c a n t . Moving to anthraquinone-2-carbinol as aglycone, thus adding a t h i r d aromatic r i n g and quinonoid character to the system, provided analog 43. A l l a c t i v i t y parameters improved; DNA and RNA synthesis i n h i b i t i o n values f e l l below 10 μΜ and the ATm rose to the low but s i g n i f i c a n t value of 2.2°. Coupling daunosamine to a s i m p l i f i e d tetrahydronaphthacene quinone analog of daunomycinone that was prepared p r e v i o u s l y i n our l a b o r a t o r i e s (37,125) y i e l d e d m

m

m


HENRY

2.

Adriamycin

41

the d i a s t e r i o m e r i c p a i r of analogs 44 and 45. Compound 44 now resembles the parent a n t i b i o t i c s q u i t e c l o s e l y , possessing a chromophore w i t h very s i m i l a r e l e c t r o n i c c h a r a c t e r , c o r r e c t stereochemistry a t the 7 - p o s i t i o n and only l a c k i n g the 9 - p o s i t i o n s u b s t i t u e n t s and the 4-methoxy group. These seeming advances toward the parent a n t i b i o t i c s improved the DNA/RNA i n h i b i t o r y c h a r a c t e r o f the drug somewhat but by a f a c t o r of l e s s than two over anthraquinone 43. On the other hand the A T value f o r 44 rose q u i t e s i g n i f i c a n t l y to 12.6°. Perhaps most importantly, however, the diastereomer of unnatural R c o n f i g u r a t i o n a t the 7p o s i t i o n (45) was as good an i n h i b i t o r o f n u c l e i c a c i d s y n t h e s i s as was 44 d e s p i t e t h e i r marked stereochemical d i f f e r e n c e . The AT of 45 a l s o improved over t h e simpler analogs but much l e s s so than that of 44. The A T data a r e c o n s i s t e n t w i t h the r e c e p t o r s i t e model given i n F i g u r e 4. Fo should possess an aromati with the surrounding base p a i r s . T h i s c o n d i t i o n i s met w i t h the analogs showing s i g n i f i c a n t A T v a l u e s . In a d d i t i o n , the c o r r e c t c o n f i g u r a t i o n a t the 7 - p o s i t i o n combined w i t h the l a r g e s t aromat i c moiety (compound 44) y i e l d e d the h i g h e s t DNA s t a b i l i z a t i o n seen i n the s e r i e s . The n u c l e i c a c i d s y n t h e s i s i n h i b i t i o n values a l s o c o r r e l a t e g e n e r a l l y with what the model would p r e d i c t : s t r u c t u r a l f e a t u r e s expected t o g i v e a more s t a b l e complex a r e associated with greater i n h i b i t o r y e f f e c t s . However compound 45 i s an important exception to t h i s g e n e r a l i z a t i o n . The i n v e r s i o n of the b e n z y l i c carbon (equivalent to C-7 of adriamycin) y i e l d s a molecule that e s s e n t i a l l y cannot p a r t i c i p a t e i n the h y p o t h e t i c a l receptor s i t e . The molecular shape of 45 d i f f e r s s h a r p l y from that of 44; t h e l a t t e r f i t s i n t o the s i t e as e a s i l y as adriamycin ( l e s s the 9- and 14-hydroxy hydrogen bonds) while 45 can be made to f i t only p o o r l y by assuming improbable conformations. Although i t i s p o s s i b l e according to model s t u d i e s f o r analog 45 to form i o n i c a l l y bound i n t e r c a l a t i v e complexes w i t h DNA that d i f f e r subs t a n t i a l l y from that of F i g u r e 4, adriamycin and daunomycin cannot be accommodated. Thus the s i m i l a r E D 5 0 v a l u e s f o r 44 and 45 could be caused f o r t u i t o u s l y by a unique DNA complex assumed by 45, but not by the a n t i b i o t i c s . However the data seem b e t t e r r a t i o n a l i z e d by the p r o p o s i t i o n that adriamycin, daunomycin, 45 and a l l of the i n v i t r o - a c t i v e analogs can a f f e c t n u c l e i c a c i d s y n t h e s i s by an a l t e r n a t e and comparatively n o n s t e r e o s p e c i f i c mechanism i n a d d i t i o n t o the s i n g l e s p e c i f i c mechanism i m p l i e d by the molecular complex p i c t u r e d i n F i g u r e 4. A l l analogs i n Table X except 39 have been evaluated i n v i v o by NCI i n the P388 system. A l l were found i n a c t i v e a t non-toxic doses. The l a c k o f i n v i v o a c t i v i t y from any of the analogs, e s p e c i a l l y 44, i m p l i e s that one o r more of the 4- and 9 - p o s i t i o n s u b s t i t u e n t s of adriamycin and daunomycin a r e necessary f o r a u s e f u l drug. The DNA/RNA s y n t h e s i s i n h i b i t i o n shown by 44, while not as potent as that of the parents, i s w e l l w i t h i n the range m

m

m

m


42

CANCER

Table X.

Glycosides of Daunosamine. Inhibition of N u c l e i c A c i d Synthesis i n Cultured L1210 C e l l s and E f f e c t on Thermal Denaturation of C a l f Thymus DNA.

NH

2

ΕΡ

No.

CHEMOTHERAPY

R

ς η

(yM)

a

DNA

RNA

210

210

AT

m

(°C)

OCH 39

OH

(diastereomeric mixture)

0.5

0.9

1.1


HENRY

2.

43

Adriamycin

Table X

(Concluded)

ED ς n ( y M )

(°Q

2.2

5.6

4.7

12.6

5.4

4.5

6.8

adriamycin

0.8

0.9

17.8

daunomycin

0.3

0.3

16.8

OH

o l To 0

OH

o-

45 0

OH

0-

daunosamine

See Table

m

7.4

0 44

AT

RNA

9.0

43

3

c

DNA

No.

>

100

>

100

IV f o r d e t a i l s .

See Table VI f o r s p e c i f i c c o n d i t i o n s .


1.0

44

CANCER

CHEMOTHERAPY

where u s e f u l i n v i v o - a c t i v e d e r i v a t i v e s appear (see Tables I I I and IV). T h i s suggests that the need f o r these a d d i t i o n a l sub s t i t u e n t s may l i e i n the realm of host e f f e c t s r a t h e r than i n t h e i r importance f o r i n c r e a s i n g i n t r i n s i c t u m o r - c e l l k i l l i n g character. T o t a l l y S y n t h e t i c Analogs of Adriamycin. Analogs of t h i s group are a s s o c i a t e d with the a n t h r a c y c l i n e n a t u r a l products p r i m a r i l y by possessing two s t r u c t u r a l f e a t u r e s i n common, a q u i n i n o i d aromatic u n i t and a b a s i c s i d e c h a i n . Analogs of t h i s type were f i r s t described by M u l l e r e t a l . (126) who prepared racemic 46 and s e v e r a l other r e l a t e d compounds as analogs of rhodomycin Β and daunomycin. No data on the b i o l o g i c a l or b i o p h y s i c a l p r o p e r t i e s of these compounds have y e t been published however. Very r e c e n t l y Doubl f i v e anthraquinones bearin (e.g., 47-49) as models of s e v e r a l i n t e r c a l a t i n g drugs i n c l u d i n g

0

CHo I NHCH(CH ) NEt

0

R»

&® R

T

2

3

2

47

R = Η, R

=0H

48

CH I R = H, R = NHCH(CH ) NEt

49

CHo I R = NHCH (CH ) N E t , R = Η

3

f

2

3

2

T

2

3

2

adriamycin. Spectrophotometric DNA-binding s t u d i e s determined that compounds 47 and 48 had b i n d i n g constants s l i g h t l y greater than adriamycin but that the number of receptor s i t e s on the DNA was reduced by 25-50%. Compound 49 possessed a b i n d i n g constant about one-half that of adriamycin^but the apparent number of receptor s i t e s was e q u i v a l e n t to the a n t i b i o t i c . No b i o l o g i c a l data on these compounds have been published but i t i s s i g n i f i c a n t that such r e l a t i v e l y a c c e s s i b l e and simple analogs e x h i b i t DNA b i n d i n g c h a r a c t e r i s t i c s very s i m i l a r t o those of the much more complex parent a n t i b i o t i c s .


2.

HENRY

45

Adriamycin

Racemic 3-alanine e s t e r 50 was prepared by Yamamoto et a l . (128) and i s a c l o s e r e l a t i v e of corresponding daunomycinone e s t e r 38. T h i s compound i n h i b i t s DNA and RNA s y n t h e s i s i n c u l tured L1210 c e l l s with ED q v a l u e s of 2.6 and 2.8 μΜ, r e s p e c t i v e ly. These values are only t h r e e - f o l d higher than adriamycin i n 5

0

OH

50

the same t e s t and s e v e r a l f o l d l e s s than those of J8. The A T value f o r 50 i s 5.2° compared to 17.8° f o r adriamycin and 6.3° f o r 38 under s i m i l a r c o n d i t i o n s (see Table V I ) . Analog 50 was i n a c t i v e i n v i v o i n the standard L1210, P388 and B16 t e s t s . The h i g h i n v i t r o potency of 50, d e s p i t e i t s c o n s i d e r a b l e s t r u c t u r a l d e v i a t i o n from adriamycin and daunomycin, provides a d d i t i o n a l encouragement that s i m p l i f i e d analogs can possess the necessary cytotoxicity. The l a c k of i n v i v o a c t i v i t y of 50 when compared to 38 emphasizes again the apparent importance of one or more of the 4- and 9-substituents i n attempting to reproduce the t h e r a p e u t i c c h a r a c t e r i s t i c s of the n a t u r a l products i n l e s s complex molecules. Table XI presents three f u l l y aromatic dihydroxynaphthacenequinones bearing b a s i c s u b s t i t u e n t s that may be regarded as ana logs of adriamycin. Compound 51 was reported by F i n k e l s t e i n and Romano (129) to have i n v i v o a c t i v i t y against Sarcoma 180 and s o l i d E h r l i c h carcinoma tumors but no a c t i v i t y was seen i n the L1210 and P388 systems of the NCI. B e l i e v i n g that a longer, more b a s i c s i d e chain would confer g r e a t e r s i m i l a r i t y to daunomycin, b a s i c amides 52 and 53 were prepared from 51 (130). The N,Nd i e t h y l g l y c y l s i d e chain of 52 provided no improvement over 51 as an i n h i b i t o r of n u c l e i c a c i d s y n t h e s i s or i n v i v o . The longer, N , N - d i e t h y l - 3 - a l a n y l , homolog (53) was d e t e c t a b l y a c t i v e i n the i n v i t r o t e s t but s i g n i f i c a n t a c t i v i t y a g a i n s t L1210 and P388 i n v i v o was not seen. DNA m e l t i n g temperature measurements on 51-53 were not p o s s i b l e because of water i n s o l u b i l i t y . m


46

CANCER

Table X I .

CHEMOTHERAPY

5,12-Dihydroxynaphthacene-6,11-diones as I n h i b i t o r s of N u c l e i c A c i d Synthesis.

EDgp (μΜ) Structure

No. 0

DNA

RNA

OH

51

> 100

> 100

52

> 100

> 100

53

49

39

adriamycin

0.8

0.9

See Table IV f o r d e t a i l s .

In Table XII are presented two N-substituted-l-amino-4hydroxyanthraquinone that may be regarded as t r i c y c l i c analogs of adriamycin (131). Both d i s p l a y moderate l e v e l s of n u c l e i c a c i d s y n t h e s i s i n h i b i t i o n and s u b s t a n t i a l thermal s t a b i l i z a t i o n of DNA. Compound 54 i s e s s e n t i a l l y a lower homolog of 47, one o f the com pounds d e s c r i b e d by Double and Brown (127) as b i n d i n g to DNA with a constant equal to adriamycin. Compound 54 has shown marginal but confirmed a c t i v i t y against P388 lymphocytic leukemia i n the mouse i n the QD1-9 schedule (ca 25% i n c r e a s e i n s u r v i v a l time at


HENRY

2.

47

Adriamycin

Table XII.

l-Amino-4-hydroxyanthraquinones. I n h i b i t i o n of Nucleic Acid Synthesis in Cultured L1210 C e l l s and E f f e c t on Thermal Denaturation of C a l f Thymus DNA.

ED No.

54

Structure

Ο

Π 0

55

a

RNA

AT

(°C)

m

6.7

12.4

OH 19

NHÔ^CH NMe 2

adriamycin a

(yM)

8.2

LOI^^ 0

S0

DNA

15

10

6

·

2

0.8

0.9

17.8

S e e Table IV f o r d e t a i l s . See Table VI f o r s p e c i f i c

conditions.

50-100 mg/kg) but was i n a c t i v e against L1210. In v i v o assays on 55 have not been obtained. The l a s t group (132) of t o t a l l y s y n t h e t i c analogs, l i s t e d i n Table X I I I , i s c h a r a c t e r i z e d by a v a r i e t y of b a s i c side chains i n the 2 - p o s i t i o n of the aromatic nucleus. The compounds with 1,4-dihydroxyanthraquinone n u c l e i and a l i p h a t i c s i d e chains (56, 57) were s i g n i f i c a n t l y i n h i b i t o r y i n the i n v i t r o t e s t but the presence of a phenyl r i n g i n the s i d e chain destroyed a l l a c t i v i t y (58, 59). The presence of a phenyl i n a b a s i c side chain on the 1 - p o s i t i o n (55, Table XII) was much l e s s detrimental to i n v i t r o a c t i v i t y . The l a s t three compounds of Table X I I I , a l l i n c o r p o r a t i n g naphthoquinone r i n g s , are the most remote from the a n t h r a c y c l i n e a n t i b i o t i c s of a l l analogs prepared. Appreciable DNA/RNA syn t h e s i s i n h i b i t i o n was nevertheless encountered, e s p e c i a l l y with 62. The p o s s i b i l i t y must be considered that these h i g h l y

American Chemical Society Library 1155 16th St. N. w. In Cancer Chemotherapy; Washington, D. C. Sartorelli, 20036 A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

CANCER

48

Table X I I I .

CHEMOTHERAPY

2 - S u b s t i t u t e d A n t h r a q u i n o n e s and N a p h t h o q u i n o n e s as I n h i b i t o r s o f Nucleic Acid Synthesis i n Cultured L1210 C e l l s . a

ED s η

No.

Structure

adriamycin a

(μΜ)

DNA

0.8

S e e T a b l e IV f o r d e t a i l s .


RNA

0.9

2.

HENRY

Adriamycin

49

s i m p l i f i e d compounds are a c t i n g through a t o t a l l y d i f f e r e n t mechanism s i n c e the i n t e r c a l a t i n g p o t e n t i a l of the naphthalene r i n g i s decreased because of i t s smaller s i z e . A T data are not a v a i l a b l e on a l l compounds but 60 and 61 provided v a l u e s of only 1.5° under our standard c o n d i t i o n s . A l l analogs i n Table X I I I were i n a c t i v e at n o n - t o x i c doses i n the P388 and L1210 systems. Naphthoquinone 62 was notable f o r i t s h i g h t o x i c i t y , causing a h i g h p r o p o r t i o n of e a r l y deaths among t r e a t e d mice at doses above 1.5 mg/kg on the QD1-9 schedule. A l l analogs i n Tables XI-XIII are s t r u c t u r a l l y capable of i n t e r c a l a t i n g i n t o DNA with the b a s i c s i d e chains forming i o n i c bonds w i t h adjacent phosphates. However the l a c k of s i d e c h a i n and aglycone o p t i c a l asymmetry and the conformational f l e x i b i l i t y of the s i d e chains make i t d i f f i c u l t to draw u s e f u l c o n c l u s i o n s r e l a t i v e to the proposed adriamycin-DNA r e c e p t o r complex d i s c u s s e d earlier. These molecule b l e b i n d i n g modes accordin obvious advantage can be assigned to any one of them. I t i s s i g n i f i c a n t that DNA b i n d i n g parameters comparable to those of adriamycin and daunomycin are found with these muchs i m p l i f i e d analogs and that s u b s t a n t i a l DNA and RNA s y n t h e s i s i n h i b i t o r y c h a r a c t e r f r e q u e n t l y p a r a l l e l s t h i s behaviour. While data are inadequate to f i r m l y a s s o c i a t e t h e i r mechanism of a c t i o n with that of adriamycin, t h e i r comparatively ready s y n t h e t i c a c c e s s i b i l i t y coupled with t h e i r f r e q u e n t l y i n t e r e s t i n g e f f e c t s on n u c l e i c a c i d s y n t h e s i s make them a c l a s s of compounds w i t h good prospects f o r y i e l d i n g a u s e f u l antitumor drug. The i n v i v o a c t i v i t y found f o r 54 f u r t h e r supports t h i s suggestion. m

Conclusions. The most obvious c o n c l u s i o n to be drawn from the f o r e g o i n g d i s c u s s i o n i s t h a t i t i s d i f f i c u l t to improve on Nature. In only a few i n s t a n c e s have s t r u c t u r a l manipulations w i t h adriamycin or daunomycin provided s u p e r i o r e f f i c a c y , and i n no case has s u p e r i o r potency r e s u l t e d . Yet, improved p r o p e r t i e s appear to have o c c a s i o n a l l y r e s u l t e d from s t r u c t u r e s such as rubidazone (18), AD32 (Figure 6 ) , 4'-epi-adriamycin (35), and the a n t i b i o t i c - D N A complexes (Table V I I I ) . Further research at the p r e c l i n i c a l and c l i n i c a l l e v e l w i l l be necessary to f u l l y r e v e a l the v a l u e of these m a t e r i a l s f o r human therapy. To date i n v i v o antitumor a c t i v i t y has been a s s o c i a t e d almost e x c l u s i v e l y w i t h d e r i v a t i v e s and analogs that have carbon s k e l e tons i d e n t i c a l to those of adriamycin and daunomycin. The most e f f i c a c i o u s m o d i f i c a t i o n s have been obtained by d e r i v a t i z a t i o n of the parent a n t i b i o t i c s (e.g., Table I I I ) or by a s m a l l s t e r e o chemical change as i n the 4 - e p i m e r i c a n t i b i o t i c s (34 and 35)· The work with the daunosamine g l y c o s i d e s (Table X) and the βa l a n y l e s t e r s (38 and 50) suggest that optimum i n v i v o a c t i v i t y i s r e l a t e d to the presence of one or more of the 4- and 9s u b s t i t u e n t s . Yet a p p r e c i a b l e , i f not remarkable, i n v i v o a c t i v i t y has been d i s p l a y e d by compounds w i t h s k e l e t o n s d e v i a t i n g ,


50

CANCER

CHEMOTHERAPY

s i g n i f i c a n t l y from those of the parents (28, 29, 38), thus p r o v i d i n g encouragement that there i s no absolute requirement f o r the n a t i v e skeleton and other s k e l e t a l v a r i a n t s could o f f e r b e t t e r antitumor p r o p e r t i e s and decreased t o x i c i t y . The number of p o s s i b l e s t r u c t u r a l parameters of adriamycin p o t e n t i a l l y sub j e c t to i n v e s t i g a t i o n i s very l a r g e and only a few have been approached, due p r i m a r i l y to the d i f f i c u l t chemistry of the anthracyclines. T h i s s i t u a t i o n w i l l , unquestionably, change i n the f u t u r e because of the a c c e l e r a t i n g therapeutic i n t e r e s t i n adriamycin. The breadth of s t r u c t u r a l types showing s i g n i f i c a n t i n h i b i t o r y e f f e c t s on n u c l e i c a c i d s y n t h e s i s i n c e l l c u l t u r e i s much greater than that showing i n v i v o antitumor a c t i o n . While i t i s w e l l known that i n v i t r o systems c h a r a c t e r i s t i c a l l y provide p o s i t i v e r e s u l t s more f r e q u e n t l y than whole-animal t e s t s , the s t r u c t u r a l spectrum y i e l d i n very broad. Whether th (e.g., 62) are t r u l y r e l a t e d to adriamycin, or s e r e n d i p i t o u s l y act by d i f f e r e n t mechanisms, the p o t e n t i a l f o r developing i n v i v o antitumor a c t i v i t y from them seems high because they are r e a d i l y approached s y n t h e t i c a l l y and s t r u c t u r a l parameters can be e a s i l y altered. Perhaps the most v a l u a b l e r e s u l t from the d e r i v a t i v e and analog work reviewed here w i l l be separation of antitumor a c t i v i ty from c a r d i o t o x i c i t y . U n t i l r e c e n t l y very l i t t l e was known about the r e l a t i o n of chemical s t r u c t u r e to c a r d i o t o x i c i t y (133, 134) because no animal models f o r t h i s syndrome were a v a i l a b l e . Several t e s t systems have been reported r e c e n t l y (135-138) and c a r d i o t o x i c i t y e v a l u a t i o n s may be expected to become an e s s e n t i a l part of analog e v a l u a t i o n . The f a v o r a b l y a l t e r e d e f f e c t s of 4 epi-adriamycin (35) when compared to adriamycin was p r e v i o u s l y noted (119). ~~ The DNA binding s i t e hypothesis c i t e d throughout the d i s cussion has been a f a s c i n a t i n g t o o l f o r the study of adriamycin analogs. I t has provided a conceptual base on which to evaluate the e f f e c t s of s t r u c t u r a l changes on b i o p h y s i c a l and b i o l o g i c a l p r o p e r t i e s of drug candidates but has not yet provided any unique i n s i g h t s that have l e d to design of improved drugs. The l a t t e r p o i n t i s not too s u r p r i s i n g i n view of our incomplete knowledge on the s t a t e of DNA i n the nucleus of l i v i n g c e l l s ; many f a c t o r s i n a d d i t i o n to receptor f i t must govern the e f f e c t of a drug on the complexities of DNA f u n c t i o n . On the other hand, the p h y s i c a l data that were obtained by D i Marco and colleagues (51) on the binding of daunomycin d e r i v a t i v e s and analogs to i s o l a t e d DNA, as w e l l as our AT measurements, are c o n s i s t e n t with the model. S t r u c t u r a l changes that d e l e t e or decrease the v a r i o u s b i n d i n g f o r c e s maintaining the complex r e s u l t i n d e s t a b i l i z a t i o n . The divergence of the b i o l o g i c a l p r o p e r t i e s of analogs such as 45 from t h e i r DNA b i n d i n g p r o p e r t i e s can thus be i n t e r p r e t e d as e v i dence that the mechanism of a c t i o n of the a n t h r a c y c l i n e 1

m


2.

HENRY

Adriamycin

51

A n t i b i o t i c s does not i n v o l v e a s i n g l e s t e r e o s p e c i f i c r e c e p t o r . Future s t r u c t u r e - a c t i v i t y work w i l l undoubtedly shed more l i g h t on t h i s p o i n t . Some of the concepts d e s c r i b e d i n t h i s paper were b r i e f l y discussed i n an e a r l i e r symposium report (139). Acknowledgment. The author g r a t e f u l l y acknowledges the work of Dorrïs"Îayïorând Brenda Williamson who ably performed the n u c l e i c a c i d s y n t h e s i s i n h i b i t i o n assays and DNA b i n d i n g s t u d i e s . Thanks a r e due a l s o to Dr. Harry B. Wood, J r . of the D i v i s i o n of Cancer Treatment, NCI, f o r suggestions and h e l p f u l d i s c u s s i o n s . Supported by Contract No. l-CM-33742 from the D i v i s i o n of Cancer Treatment, NCI, NIH, USPHS. Literature Cited 1.

2. 3. 4.

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2.

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51. 52. 53. 54. 55. 56. 57. 58.

59. 60.

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94. 95. 96. 97. 98. 99.

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109.

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55

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56

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120. 121. 122. 123. 124. 125.

126. 127. 128. 129. 130. 131. 132. 133. 134. 135.

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Tong, George and Henry, D. W. In p r e p a r a t i o n . Trouet, Andre, Deprez-de Campeneere, D., and de Duve, C. Nature New B i o l . (1972), 239, 110. A t a s s i , Ghanem, and Tagnon, H. J . Europ. J . Cancer (1974), 10, 399. Trouet, Α., Duprez-de Campeneere, D., de Smedt-Malengreaux, Μ., and A t a s s i , G. Europ. J . Cancer (1974), 10, 405. Sokal, G., Trouet, Α., Michaux, J.-L., and Cornu, G. Europ. J . Cancer (1973), 9, 391. Cornu, G., Michaux, J.-L., Sokal, G., and Trouet, A. Europ. J . Cancer (1974), 10, 695. Marks, Thomas, K l i n e , I r a , and V e n d i t t i , J . M. Proc. Am. Asso. Cancer Res. (1974), 16, 92. Penco, S. Chim. Ind., M i l a n (1968), 50, 908. D i Marco, Α., Zunino, F., Silvestrini, R., Gambarucci, C., and Gambetta, R. Arcamone, F., Penco F r a n c h i , G., D i Marco, Α., Casazza, Α., Dasdia, T., F o r m e l l i , F., Necco, Α., and Soranzo, C. J . Med. Chem. (1975), 18, 703. Fujiwara, Α. Ν., Wu, Η., Lee, W. W., and Henry, D. In preparation. Lee, W. W., Wu, H. Y., C h r i s t e n s e n , J . Ε., Goodman, Leon, and Henry, D. W. J . Med. Chem. (1975), 18, 768.. Mosher, C. W. and Henry, D. W. In p r e p a r a t i o n . French Patent 1,593,555 (Rhone-Poulenc, J u l y 10, 1970). Fujiwara, A. N., Stankorb, J . , Acton, Ε. Μ., Lee, W. W., and Henry, D. W. In p r e p a r a t i o n . Lee, W. W., Martinez, A. P., and Henry, D. W. A b s t r . 167th Nat. Meeting of the Am. Chem. Soc., Los Angeles, April 1974. Abstr. No. MED1-58. M u l l e r , Werner, F l u g e l , R o l f , and S t e i n , C a r l . Liebigs Ann. Chim. (1971), 754, 15. Double, J . C. and Brown, J . R. J . Pharm. Pharmacol. (1975), 27, 502. Yamamoto, Κ., Acton, Ε. Μ., and Henry, D. W. In prepara tion. F i n k e l s t e i n , J . and Romano, J . A. J . Med. Chem. (1970), 13, 568. Fujiwara, A. N., Acton, Ε. Μ., and Henry, D. W. In prepara tion. B i c k n e l l , R. B. and Henry, D. W. In p r e p a r a t i o n . Grange, E. W., Lee, W. W., and Henry, D. W. In p r e p a r a t i o n . Herman, E., Mhatre, R., Lee, I. P., V i c k , J . , and Waravdekar, V. S. Pharmacology (1971), 6, 230. Mhatre, R., Herman, E., Huidobro, Α., and Waravdekar, V. J . Pharmacol E x p t l . Therapeut. (1971), 178, 216. C a r g i l l , C., Bachmann, Ε., and Zbinden, G. J . Nat. Cancer Inst. (1974), 53, 481.


2.

HENRY

136. 137. 138. 139.

Adriamycin

57

Jaenke, R. S. Lab. I n v e s t . (1974), 30, 292. Young, D. M. and F i o r a v a n t i , J . L. Proc. Am. Asso. Cancer Res. (1974), 17, 73. Necco, A. and Dasdia, T. IRCS L i b r . Compend. (1974), 2, 1293. Henry, D. W. Cancer Chemo. Rpts., P a r t 2 (1974), 4, 5.


3 Biochemical Pharmacology of the Anthracycline Antibiotics N I C H O L A S R.

BACHUR

Biochemistry Section, Baltimore Cancer Research Center, National Cancer Institute, 3100 Wyman Park Dr., Baltimore, M d . 21211

Medical s c i e n t i s t s have a cumulative eighteen year clinical experience w i t h the a n t h r a c y c l i n daunorubicin. Daunorubici in I t a l y by F a r m i t a l i a and i n France by Rhône-Poulenc in 1964. A few years l a t e r , adriamycin was announced by F a r m i t a l i a and started into clinical trials. These trials i n d i c a t e d t h a t daunor u b i c i n was an impressive agent f o r remission i n d u c t i o n i n acute leukemia whereas adriamycin had a wider spectrum of a c t i v i t y a g a i n s t s o l i d tumors as w e l l as leukemias (1,2,3,4,5). There i s little doubt t h a t the a n t h r a c y c l i n e a n t i b i o t i c s i s o l a t e d from Streptomyces have had a major impact on the prospects of cancer chemotherapy, because o f t h e i r degree o f activity f o r i n d u c i n g remission or stopping p r o g r e s s i o n o f malignant growth and t h e i r wide ranging activity a g a i n s t malignancies. However, complicating t h e i r u s e f u l n e s s are the t o x i c s i d e e f f e c t s a s s o c i a t e d with t h e i r a d m i n i s t r a t i o n : myelosuppression, s t o m a t i t i s , nausea, vomiting, a l o p e c i a , e l e c t r o c a r d i o g r a p h i c changes, and a s e r i o u s cumulative dosage r e l a t e d myocardiopathy (6). Although the agents are n e a r l y i d e n t i c a l s t r u c t u r a l l y ( F i g . 1), adriamycin's potency i s about one and one-half times g r e a t e r than daunorubicin in both pharmacologic e f f e c t and i n t o x i c i t y . The only d i f f e r e n c e between the complex molecules is a t the number fourteen carbon p o s i t i o n where adriamycin has an a d d i t i o n a l hydroxyl. Although many s t r u c t u r e s were reviewed e x t e n s i v e l y i n the preceeding d i s c u s s i o n by Dr. Henry, I t h i n k we can reexamine the fundamental s t r u c t u r e from a biochemical view p o i n t . The a n t i b i o t i c molecule is double headed with a hydrophobic end and a h y d r o p h i l i c end. The A, B, and C r e s o n a t i n g r i n g system comprises the hydrophobic end; and the D r i n g , with attached amino sugar are the h y d r o p h i l i c head o f the molecule. In a d d i t i o n to t h i s dual p h y s i c a l c h a r a c t e r i s t i c , the molecules are amphoteric with a b a s i c amino group and the a c i d i c p h e n o l i c hydroxyls of the a n t h r a c y c l i n e r i n g . These p h y s i c a l p r o p e r t i e s p l u s abundance of r e a c t i v e s i t e s o f f e r the p o t e n t i a l f o r numerous i n t e r a c t i o n s with cellular components such as n u c l e i c a c i d s , p r o t e i n s , and lipids.

58


3.

BACHUR

Anthracycline

Antibiotics

ADRIAMYCI1M

OH

DAUNORUBICIN

H

Figure 1.

Structures of adriamycin and daunorubicin


CANCER

60

CHEMOTHERAPY

Figure 2. Chromosomal damage in human cells induced by daunorubicin (10)

Figure 3.

Kinetics of adriamycin and daunorubicin uptake at 37°C leukemia cells in vitro

in L1210 murine


3.

BACHUR

Anthracycline

Antibiotics

61

The a v a i l a b i l i t y o f b i o t r a n s f o r m a t i o n groups i n c r e a s e s even f u r t h e r the c o m p l i c a t i o n s o f drug d i s p o s i t i o n i n mammals. A c e n t r a l s t r u c t u r a l component of both molecules i s the g l y c o s i d i c bond j o i n i n g the sugar, daunosamine, and the anthrac y c l i n e nucleus. T h i s bond i s very l a b i l e to chemical and enzymatic cleavage; and the s c i s s i o n of the bond i n a c t i v a t e s the compound. For t h i s reason n e i t h e r adriamycin nor daunorubicin are g i v e n o r a l l y s i n c e the g l y c o s i d i c bond i s s p l i t i n the gastrointestinal tract. Both adriamycin and daunorubicin must be g i v e n p a r e n t e r a l l y to be e f f e c t i v e . When adriamycin o r daunorubicin are administered i n t r a venously to animals o r i n humans, the compounds are r a p i d l y absorbed i n t o c e l l s and l o c a l i z e d p r i m a r i l y i n the c e l l nucleus (7,8,9). There i s good anatomical and chemical evidence to i n d i c a t e t h a t the compounds both l o c a l i z e i n the c e l l nucleus and a l s o i n t e r a c t w i t h the nuclear m a t e r i a l Chromosomal prepara t i o n s from human lymphocyte r u b i c i n treatment ( F i g t i o n , r i n g formation, s p l i t t i n g , s e p a r a t i o n , and other forms of damage. Since both adriamycin and daunorubicin have a unique f l u o r e s c e n c e , they can be d e t e c t e d by f l u o r e s c e n c e microscopy i n cells. With t h i s technique the drug f l u o r e s c e n c e i s l o c a l i z e d a t the n u c l e a r s t r u c t u r e s (8). I t i s remarkable t h a t the seemingly i n s i g n i f i c a n t e x t r a hydroxyl o f adriamycin makes not only a more potent compound, but a l s o a drug w i t h wider spectrum of a c t i v i t y a g a i n s t malignancy. For t h i s reason the drugs have been s t u d i e d and compared. Since the whole animal s t u d i e s are q u i t e complex, we o r i g i n a l l y compared adriamycin and daunorubicin i n mammalian c e l l c u l t u r e c o n t a i n i n g L1210 murine leukemia c e l l s . L1210 c e l l s and other mammalian c e l l l i n e s are i n h i b i t e d more by daunorubicin than by adriamycin. Daunorubicin has a g r e a t e r a c t i v i t y f o r i n h i b i t i n g both DNA and RNA metabolism as seen i n our l a b o r a t o r y and i n others (Table 1) (11,12,13). T h i s , o f course, i s the opposite o f the c l i n i c a l f i n d i n g s and o f f i n d i n g s i n v i v o and was a p e r p l e x i n g o b s e r v a t i o n . The p o s s i b i l i t y remained t h a t the two drugs may be e n t e r i n g the c e l l s at d i f f e r e n t r a t e s , so we compared the accumulation of adriamycin and daunorubicin i n the L1210 c e l l s . Daunorubicin i s taken i n t o the c e l l much more r a p i d l y and to a higher degree than i s adriamycin ( F i g . 3) (11). Therefore the e f f e c t i v e l e v e l of drug i n the c e l l i s much h i g h e r i n the case of daunorubicin. T h i s r e s u l t s i n an apparent s u p e r i o r i t y of daunorubicin over adriamycin a t i n h i b i t i n g n u c l e i c a c i d s y n t h e s i s . However, the s p e c i f i c a c t i v i t i e s o f the amount o f drug i n the c e l l s compared to the amount o f i n h i b i t i o n produced by the drug i n d i c a t e s t h a t adriamycin has a g r e a t e r s p e c i f i c a c t i v i t y than does daunorubicin (11) . A l t e r a t i o n s on the s i d e chain a t the 9 p o s i t i o n have profound e f f e c t s on the p o l a r i t y and s o l u b i l i t y o f the a n t h r a c y c l i n e a n t i biotics. T h i s apparently d i r e c t l y e f f e c t s a b s o r p t i o n of the drug



3

HeLa C e l l

HeLa C e l l

3.

4.

survival

survival

c

L1210 incorporation Uridine-2-14 i n t o RNA

L1210 incorporation Thymidine-methyl H

2.

1.

System

(13)

Kim & Kim

0.03

0.2

Meriwether & Bachur (11)

Meriwether & Bachur (11)

Ref.

DiMarco e t a l (12)

(yM)

0,9

ID 50 μΜ

29 60 73

27 55 74

Drug cone

1.9

12 36 57

16 32 59

Adriamycin Daunorubicin Percent I n h i b i t i o n

In V i t r o I n h i b i t i o n of C e l l A c t i v i t i e s by Adriamycin and Daunorubicin

Table 1


2.

Malony sarcoma v i r u s F r i e n d Leukemia v i r u s Rausher Sarcoma v i r u s

RNA Tumor V i r u s DNA Polymerase

4

E. C o l i - T / L 1 4 1 phage DNA polymerase

67 56 64

22 58 89 98

67 64 66

15 39 72 95

Adr. Daun. % Inhibition

0.066 0,066 0.066

0.011 0.028 0.057 0.114

Drug Cone. (mM)

Chandra e t a l (15)

Goodman e t a l (14)

Ref.

Adriamycin and Daunorubicin I n h i b i t i o n o f DNA Polymerases

Table 2

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i n t o the c e l l . We have compared metabolites and chemical d e r i v a t i v e s o f adriamycin and daunorubicin with changes only on the C9 side chain. Compounds with the lowest " p o l a r i t y " as measured by p a r t i t i o n c o e f f i c i e n t have the h i g h e s t uptake i n t o L1210 c e l l s . There i s a near l i n e a r r e l a t i o n s h i p of p o l a r i t y to drug uptake (Fig. 4 ) . In order to avoid the dynamics of the c e l l membrane, we i n v e s t i g a t e d drug e f f e c t s d i r e c t l y in_ v i t r o on a group of p u r i f i e d DNA polymerases s u p p l i e d by Dr. Maurice Bessman. The polymerases were T4 bacteriophage induced i n E. C o l i . I n h i b i t i o n o f the DNA polymerases was s l i g h t l y g r e a t e r with adriamycin than with daunor u b i c i n (Table 2) (14). From our data and from the data of others (Table 2) (15), i t i s d i f f i c u l t to e x p l a i n why adriamycin has a higher potency i n mammals. The potency o f the two agents i s nearly i d e n t i c a l i n these i n v i t r o DNA polymerase assays. In a d d i t i o n , t h e i r b i n d i n g to DNA i s q u i t e s i m i l a r and the unwinding angle t h a t they produce e x p l a i n the c l i n i c a l d i f f e r e n c e rubicin. Studies on p u r i f i e d T4 phage induced polymerases uncovered another unpredicted a c t i o n o f the a n t h r a c y c l i n e s which may have s i g n i f i c a n c e i n t h e i r s e l e c t i v e a c t i o n a g a i n s t malignant c e l l s . The p u r i f i e d phage polymerases f a l l i n t o three phenotypic groups, mutagenic, w i l d type, and antimutagenic. The mutagenic s t r a i n s have a high mutation frequency, the antimutagenic s t r a i n s have a low mutation frequency, and the w i l d type f a l l s between with a normal mutation r a t e . We observed that at low drug concentrations the mutagenic polymerases were not i n h i b i t e d but were stimulated (ex., L56) ( F i g . 5) whereas the antimutagenic polymerases (L141) were uniformly i n h i b i t e d as p r e d i c t e d (14). Simultaneously, a 5' exonuclease which i s a p a r t of each enzyme and i s t h e o r i z e d to be the e d i t i n g or e r r o r c o r r e c t i n g system f o r the phage DNA s y n t h e s i s i s uniformly i n h i b i t e d a t a l l concentrations of the drugs. This means t h a t at low drug l e v e l s the mutagenic DNA polymerase i s made even more mutagenic s i n c e the low f i d e l i t y polymerase i s s t i m u l a t e d while the a s s o c i a t e d c o r r e c t i o n system i s i n h i b i t e d . T h i s does not occur i n the antimutagenic enzymes which are uniformly i n h i b i t e d . T h i s may be a clue to how these drugs are s e l e c t i v e f o r leukemic c e l l s . Since the DNA polymerase i n acute leukemia c e l l s i s b e l i e v e d to be mutagenic (16), the drugs may cause the leukemic c e l l s to i n c o r p o r a t e too many e r r o r s i n t o t h e i r DNA to be compatible with s u r v i v a l . As p r e v i o u s l y s t a t e d , a l l of the s t u d i e s i n v e s t i g a t i n g the p h y s i c a l - c h e m i c a l i n t e r a c t i o n s of adriamycin and daunorubicin and the comparative e f f e c t s on c e l l and t i s s u e c u l t u r e i n h i b i t i o n , on enzymes such as DNA polymerase and RNA polymerase, or on b i n d i n g to DNA have shown l i t t l e i f any d i f f e r e n c e between the two compounds. I t i s only i n whole animal s t u d i e s that s i g n i f i c a n t d i f f e r e n c e s between adriamycin and daunorubicin e f f e c t s are e a s i l y d i s c e r n a b l e . T h i s i n d i c a t e s that other u n r e a l i z e d mechanisms are


3.

BACHUR

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65

1.6

Daunorubicin

1.4-

Ia> ο

1.2 -

to

Ο

Rubidazone Δ/ ο Ε S

0.8

LU

g

Adriamycin 0.6

D CD ZD

rr 0.4 û 0.2 • Adriamycinol 1.0

2.0

3.0

4.0

PARTITION COEFFICIENT Figure 4.

Relationship of drug uptake into L1210 cells and n-butanol-water partition coefficient of several anthracycline derivatives


66

CANCER

CHEMOTHERAPY

r e s p o n s i b l e . From t h a t p o i n t of view we can examine other aspects of the biochemical pharmacology of these compounds i n the mammal such as t h e i r metabolic d i s p o s i t i o n . P a t i e n t s t r e a t e d with a d r i a mycin or daunorubicin excrete s i g n i f i c a n t q u a n t i t i e s of the drugs and the metabolites i n the u r i n e (17,18). From these metabolites we have been able t o determine q u a l i t a t i v e l y and q u a n t i t a t i v e l y the metabolic sequences and pathways which the drugs t r a v e r s e (19) . The major metabolic step f o r both adriamycin and daunorubicin i n mammals i s v i a the keto r e d u c t i o n r e a c t i o n (20,21,22) ( F i g . 6 ) . T h i s r e a c t i o n i s c a t a l y z e d by a s o l u b l e aldo-keto reductase found i n a l l c e l l s analyzed. The enzyme r e q u i r e s NADPH as a c o f a c t o r and produces the p h a r m a c o l o g i c a l l y a c t i v e products, d a u n o r u b i c i n o l and adriamycinol r e s p e c t i v e l y from daunorubicin and adriamycin. A f t e r animals are administered adriamycin, the parent drug i s the predominant m a t e r i a l found i t i s s u e t e i g h t hours but a d r i a mycinol i s a s i g n i f i c a n administration, daunorubicino t i s s u e s and excreted (22). Since the aldo-keto reductase produces p h a r m a c o l o g i c a l l y a c t i v e m e t a b o l i t e s which d i f f e r from the parent i n p h y s i c a l - c h e m i c a l c h a r a c t e r i s t i c s , t h i s enzyme and i t s l e v e l i n tumor and t i s s u e may help determine the pharmacodynamic e f f e c t s o f these drugs. In studying the t i s s u e d i s t r i b u t i o n o f aldo-keto reductase, we examined human t i s s u e s obtained a t autopsy. A l l human t i s s u e s examined c o n t a i n a c t i v e l e v e l s o f aldo-keto reductase (23). Daunorubicin i s a b e t t e r substrate f o r the a l d o keto reductases i n most animal t i s s u e s and with p u r i f i e d aldo-keto reductase p r e p a r a t i o n s (21,22,23). Both subsequent t o o r concurrent with carbonyl r e d u c t i o n , the a n t h r a c y c l i n e g l y c o s i d e s are metabolized by g l y c o s i d a s e s i n most t i s s u e s (20,24,25). These microsomal enzymes s p l i t the drug or reduced metabolites i n t o aglycones and f r e e amino sugar ( F i g . 7) ( r e a c t i o n s : I -> IV, I I I I I , I I -*• V) . The major g l y c o s i d a s e (I •> IV, II V) i s r e d u c t i v e i n i t s mechanism, r e q u i r e s NADPH f o r a c t i v i t y , produces deoxyaglycones (IV,V) and i s i n h i b i t e d by oxygen (24). As i n the carbonyl r e d u c t i o n , daunorubicin i s the b e t t e r substrate f o r the g l y c o s i d a s e s than i s adriamycin. The aglycone products o f these r e a c t i o n s apparently have no a n t i c a n c e r a c t i v i t y . Since they are l i b e r a t e d i n t r a c e l l u l a r l y , however, these aglycones may have a c t i v i t i e s which are not r e a d i l y apparent and more r e s e a r c h i s necessary to r e s o l v e t h i s q u e s t i o n . The aglycones because o f t h e i r high l i p i d , low water s o l u b i l i t y , are e x c r e t e d p r i n c i p a l l y i n the b i l e ; but must be conjugated f i r s t to i n c r e a s e water s o l u b i l i t y (22,25,26,27,28). P r i o r to conjugation, i n order to o f f e r a more appropriate conjugation s i t e , there i s a 4-0 demethylation o f both daunorubic i n and adriamycin to y i e l d the demethylated aglycone (V + VI) (19). Demethylated aglycones serve as substrate f o r O - s u l f a t i o n and O-beta-glucuronidation y i e l d i n g the 4-0-sulfate conjugate and 4-0-beta glucuronide conjugate (VI VI, VI -> VIII) to be


Figure 6.

Aldo-keto reductase reaction

co

5

Ci"

1

«s.

S

3*

3

Ci

Χ

td > ο

δ.


S3 Pathways for human metabolism of adriamycin (19) Figure 7.


3.

BACHUR

Anthracycline

Antibiotics

69

e x c r e t e d . A l l o f these m a t e r i a l s are found i n human b i l e and human u r i n e (26,27,28). Since the reduced compounds, adriamycinol and d a u n o r u b i c i n o l , show s u b s t a n t i a l a n t i c a n c e r a c t i v i t y , i t i s p o s s i b l e t h a t other m e t a b o l i t e s may have o t h e r types of a c t i v i t y . There are numerous m e t a b o l i t e s w i t h the p o t e n t i a l f o r these substances to be b i o l o g i c a l l y as w e l l as p h a r m a c o l o g i c a l l y a c t i v e . The p o t e n t i a l s are being examined a t the p r e s e n t time. S t u d i e s to date s t i l l have not e x p l a i n e d f u l l y the pharmaco l o g i c d i f f e r e n c e s between adriamycin and d a u n o r u b i c i n . There are major d i f f e r e n c e s i n the d i s p o s i t i o n and metabolism of the agents i n v i v o and i n the p e n e t r a t i o n through c e l l membranes as we have shown. A l s o d i f f e r e n c e s i n t h e i r e f f e c t s on immunosuppression have been r e p o r t e d (29,30). The evidence i s accumulating t o d e f i n e the p r e c i s e mechanisms of a c t i o n o f these drugs. The a n t h r a c y c l i n e a n t i b i o t i c s show promise as cancer chemo t h e r a p e u t i c agents. I daunorubicin have comple f e e l t h a t we can look forward to encouraging progress i f we l e a r n and understand more about the b i o l o g i c a l i n t e r a c t i o n s of these agents. Then we may be b e t t e r prepared to design analogs t h a t are s a f e r and more e f f e c t i v e i n t h e i r purpose. LITERATURE CITED 1. B o i r o n , Μ., J a c q u i l l a t , C., W e i l , M., Tanzer, J . , Levy, D., S u l t a n , C., and Bernard, J . : Lancet (1969) 1, 330-333. 2. Wiernik, Ρ.Η. and S e r p i c k , A.A.: Cancer Res. (1972) 32, 2023-2026. 3. Tan, C., Tasaka, Η., Yu, K.P., Murphy, M.L., and Karnovsky, D. Cancer (1967) 20, 333. 4. Bonadonna, G., M o n f a r d i n i , S., Delena, M., F o s s a t i - B e l l a n i , F. and B e r e t t a , G.: Cancer Res. (1970) 30, 2572-2582. 5. Middleman, E., Luce, J . , and F r e i , E.: Cancer (1970) 28, 837-843. 6. Blum, R.H. and C a r t e r , S.K.: Ann. I n t . Med. (1974) 80, 249-259. 7. Bachur, N.R., E g o r i n , M.J., and H i l d e b r a n d , R.C.: Biochem. Med. (1973) 8, 352-361. 8. E g o r i n , M.J., Hildebrand, R.C., Cimino, E.F., and Bachur, N.R.: Cancer Res. (1974) 34, 2243-2245. 9. Silvestrini, R., Gambarucci, C., and Dasdia, T.: Tumori (1970) 56, 137-148. 10. Whang Peng, J . , L e v e n t h a l , B.G., Adamson, J.W., and Perry, S. Cancer (1969) 23, 113-121. 11. Meriwether, W.D. and Bachur, N.R.: Cancer Res.(1972) 32, 1137-1142. 12. DiMarco, Α., Zunino, F., Silvestrini, R., Gambarucci, C., and Gambetta, R.A.: Biochem. Pharmacol. (1971) 20, 1323-1328.


70

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

CANCER C H E M O T H E R A P Y

Kim, S.H. and Kim, J.H.: Cancer Res. (1972) 32, 323-325. Goodman, M.F., Bessman, M.J., and Bachur, N.R.: P r o c . Nat. Acad. S c i . , USA (1974) 71, 1193-1196. Chandra, P., Zunino, F., Gotz, D., Gericke, R., Thorbeck, R., and DiMarco, Α.: FEBS L e t . (1972) 21, 264-268. Springgate, C.F. and Loeb, L.A.: Proc. Nat. Acad. S c i . , USA (1973) 70, 245-249. Huffman, D., Benjamin, R.S., and Bachur, N.R.: Clin. Pharmacol. Therap. (1972) 13, 895-905. Benjamin, R.S., Riggs, C.E., J r . , and Bachur, N.R.: Clin. Pharmacol. Therap. (1973) 14, 592-600. Takanashi, S. and Bachur, N.R.: Proc. Amer. Assoc. Cancer Res. (1974) 15, 76. Bachur, N.R. and Gee, M.: J . Pharmacol. Exp. Ther. (1971) 177, 567-572. F e l s t e d , R.L., Gee Μ. d Bachur N.R. J Biol. Chem (1974) 249, 3672-3679 Bachur, N.R., Hildebrand, , , Pharmacol. Exp. Ther. (1974) 191, 331-340. Bachur, N.R., Takanashi, S., Arena, E.: Proc. XI I n t e r . Cancer Congress, F l o r e n c e , 1974. Bachur, N.R. and Gee, M.: Fed. Proc. (1972) 31, 835. B u l l o c k , F . J . , Bruni, R.J., and A s b e l l , Α.: J . Pharmacol. Exp. Ther. (1972) 182, 70-76. Cradock, J.C., E g o r i n , M.J., and Bachur, N.R.: Arch. I n t e r . Pharmacodyn. (1973) 202, 48-61. Bachur, N.R., E g o r i n , M.J., Hildebrand, R.C., and Takanashi, S.: Proc. Amer. Assoc. Cancer Res. (1973) 14, 14. Benjamin, R.S., Riggs, C.E., Jr., Serpick, Α.Α., and Bachur, N.R.: C l i n . Res. (1974) 22, 483. Schwartz, H.S. and Grindey, G.B.: Cancer Res. (1973) 33, 1837-1844. Casazza, A.M.: Adriamycin 2nd I n t . Sympos. B r u s s e l s , 1974.


4 Potential Bioreductive Alkylating Agents AI JENG LIN, L U C I L L E A. COSBY, and ALAN C. SARTORELLI Department of Pharmacology and Section of Developmental Therapeutics, Division of Oncology, Yale University School of Medicine, New Haven, Conn. 06510

Clinical t e s t s of the past years have repeatedly demonstrated that a l k y l a t i n g agent therapies against variou encourage the expenditure o f continuous e f f o r t to develop new types of a l k y l a t i n g agents with v a r i e d c a r r i e r groups designed to o r i e n t the a l k y l a t i n g p o r t i o n o f the molecule to d i f f e r e n t i n t r a cellular s i t e s (1), as well as of d e r i v a t i v e s which r e q u i r e unique modes o f a c t i v a t i o n . Thus, a l k y l a t i n g agents, such as the n i t r o s o u r e a s (2-4), cyclophosphamide (5,6), and the t r i a z i n e d e r i v a t i v e s (7-10), which r e q u i r e e i t h e r enzymatic or chemical a c t i v a t i o n p r i o r to a l k y l a t i o n , are examples of e f f i c a c i o u s m a t e r i a l s of t h i s c l a s s with d i f f e r e n t n e o p l a s t i c specificities. These a l k y l a t i n g agents may be c l a s s i f i e d as compounds with l a t e n t activity (11). The use o f a l a t e n i z a t i o n p r i n c i p l e allows the design of compounds which may e x p l o i t biochemical d i f f e r e n c e s between the most s u s c e p t i b l e normal t i s s u e s and n e o p l a s t i c c e l l s . The e f f e c t i v e employment of such a p r i n c i p l e , u l t i m a t e l y , requires the biochemical monitoring of i n d i v i d u a l cancers to s e l e c t those most prone to a c t i v a t e the l a t e n t a l k y l a t i n g p o t e n t i a l . Our i n t e r e s t i n the development of p o t e n t i a l b i o r e d u c t i v e a l k y l a t i n g agents, a r e l a t i v e l y new c l a s s o f drugs which requires r e d u c t i v e a c t i v a t i o n p r i o r to maximum e x e r t i o n of a l k y l a t i n g potential, i s based upon (a) the assumption that i n the hypoxic n e o p l a s t i c c e l l s of s o l i d tumors d i s t a l to blood v e s s e l s , which t r a d i t i o n a l l y are extremely r e s i s t a n t to chemotherapy, the decreased oxygen t e n s i o n creates c o n d i t i o n s conducive to reduction; such c e l l s should t h e o r e t i c a l l y be p a r t i c u l a r l y s e n s i t i v e to q u i nones which r e q u i r e b i o r e d u c t i o n p r i o r to e x e r t i o n o f t h e i r g r o w t h - i n h i b i t o r y p o t e n t i a l , and (b) studies of the biochemical mechanism of a c t i o n of mitomycin C, an a n t i n e o p l a s t i c agent which has a c t i v i t y against s o l i d tumors o f both animals and man (12-15). Thus, compounds o f t h i s type may be p a r t i c u l a r l y u s e f u l against c e r t a i n s o l i d tumors. Iyer and Szybalsky (16) have presented evidence to i n d i c a t e that the mitomycins act as b i f u n c t i o n a l a l k y l a t i n g agents which 71


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72

add across both strands o f the DNA double h e l i x to cause crossl i n k i n g . Furthermore, i t has been demonstrated that (a) the r e duction of the benzoquinone r i n g o f the mitomycin molecule to dihydrobenzoquinone was an e s s e n t i a l step f o r b i o l o g i c a l a c t i v i t y (17) and (b) an NADPH-dependent microsomal system was i n v o l v e d i n the r e d u c t i v e a c t i v a t i o n step (18,19). K i n o s h i t a and h i s co workers (20,21) have reported a p o s i t i v e c o r r e l a t i o n between both the a n t i n e o p l a s t i c and a n t i m i c r o b i a l a c t i v i t i e s o f a s e r i e s o f mitomycin d e r i v a t i v e s and t h e i r reduction p o t e n t i a l s . These i n v e s t i g a t o r s have also provided evidence that the carbamyl group and the a z i r i d i n e r i n g o f the mitomycins were not e s s e n t i a l f o r b i o l o g i c a l a c t i v i t y , proposing that the e s s e n t i a l p o r t i o n s of the mitomycin molecule were the s t r u c t u r e s shown i n formulas I and II (Scheme I ) . I t i s conceivable that charge d e r e a l i z a t i o n of the dihydroquinone hydroxyl groups of II r e s u l t s i n o-quinone

Scheme I

χI

methide ( I I I ) - l i k e intermediates, IV and V, which are the forms that act to a l k y l a t e DNA (Scheme I I ) .

Scheme II o-Quinone methides (III) have been reported to be a c t i v e intermediates i n several chemical r e a c t i o n s ; i n d i c a t i o n s f o r t h e i r p o s s i b l e involvement i n a number of biochemical processes have also been published (22). In view of the s i m i l a r i t i e s i n s t r u c t u r e and i n p o s s i b l e chemical r e a c t i v i t y between the pre sumed a c t i v e form of the mitomycin analogs (V) and quinone


LIN ET A L .

4.

R

Bioreductive

Alkylating

Agents

73

l 2 III

VII

VIII

methides ( I I I ) , i t was a n t i c i p a t e d that bis(ο-quinone methides) (VII), generated i n c e l l s , would have the p o t e n t i a l to a l k y l a t e DNA, as w e l l as other macromolecules o f b i o l o g i c a l importance, and thereby perhaps be e f f e c t i v e t u m o r - i n h i b i t o r y agents. Based upon t h i s concept, a s e r i e s o f benzo- and naphthoquinones ( V I I I ) , possessing one or two s i d e chains with the p o t e n t i a l to a l k y l a t e f o l l o w i n g r e d u c t i o n , wer (23-25) Since the NADPH-dependen mitomycins In v i v o apparently has l i t t l e s p e c i f i c i t y , conceivably the same quinone reductase system w i l l convert the quinones o f the b i o r e d u c t i v e a l k y l a t i n g s e r i e s to t h e i r corresponding dihydroquinones, a r e a c t i o n e s s e n t i a l f o r the expression o f a l k y l a t i n g potential. Furthermore, s i n c e Cater and P h i l l i p s (26) reported a s i g n i f i c a n t l y lower o x i d a t i o n - r e d u c t i o n p o t e n t i a l f o r tumor t i s s u e , r e l a t i v e to most normal t i s s u e s , i t i s conceivable that a t h e r a p e u t i c d i f f e r e n t i a l w i l l e x i s t between normal t i s s u e s and some cancers f o r compounds r e q u i r i n g b i o r e d u c t i v e a c t i v a t i o n . Antineoplastic Effects. The naphthoquinone d e r i v a t i v e s (Table I) o f t h i s s e r i e s produce s i g n i f i c a n t p r o l o n g a t i o n o f the l i f e span o f mice bearing e i t h e r Adenocarcinoma 755 or Sarcoma 180 a s c i t e s c e l l s , with con s i d e r a b l y greater potency being e x h i b i t e d i n the Adenocarcinoma 755 t e s t system (24). Benzoquinone d e r i v a t i v e s , however, were, i n general, only a c t i v e against Adenocarcinoma 755 (25); an ex c e p t i o n was 2,3-dimethyl-5,6-bis(chloromethyl)-1,4-benzoquinone, which was a l s o moderately a c t i v e against Sarcoma 180. Among the naphthoquinones t e s t e d (Table I ) , d i f f e r e n c e s i n maximal a c t i v i t y between compounds i n a given tumor system were r e l a t i v e l y s m a l l . D e r i v a t i v e s with two groups capable o f a l k y l a t i o n a f t e r b i o r e d u c t i o n appeared to be equal i n antitumor a c t i v i t y to agents possessing only one arm with a l k y l a t i n g p o t e n t i a l . Furthermore, s i m i l a r i t i e s were observed i n the a n t i n e o p l a s t i c potencies o f chloromethyl, bromomethyl, and acetoxymethyl d e r i v a t i v e s o f naphthoquinones, implying that the type o f l e a v i n g group present i n the molecule was not c r i t i c a l f o r tumor i n h i b i t o r y potency. The mechanism o f a c t i o n o f t h i s c l a s s o f compounds (Scheme III) has been hypothesized (23,27) to i n v o l v e b i o r e d u c t i o n i n v i v o , i n a manner analogous t o mitomycin C, presumably by an NADPH-dependent quinone reductase enzyme system, although other


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74

CHEMOTHERAPY

Table I. A n t i n e o p l a s t i c A c t i v i t i e s o f Benzo- and Naphthoquinone D e r i v a t i v e s Against Sarcoma 180 and Adenocarcinoma 755 Ascites C e l l s . a

Inhibitor

Ri

None

*2

—

-CH C1 -CH C1 -CH C1 -CH Br -CH B -CH -CH -CH

-H -CH -CH C1 -CH H

-CH C1 -CH 0Ac -CH 0Ac

-CH C1 -CH 0Ac -H

2

0 u

1

ρ 1

r

2

2

2

3

2

3

CH

3

ΛL i Ύ

Λ

2

2

-R

2

2

Sarcoma 180°

13.3

11.8

47.6 39.8 38.6 45.4 47.6

23.4 19.2 22.6 25.0 24.6

2

2

3

Adenocarcinoma 755b

10.4

-H

2

2

28.7 37.0

18.5 14.0 12.4

0

CH o

-CH90AC

9.4

3

-CH 0Ac 2

a

D

-H

39.8

11.2

A d m i n i s t e r e d once d a i l y f o r 6 consecutive days, beginning 24 h r a f t e r tumor i m p l a n t a t i o n . Average s u r v i v a l time (days) o f tumor-bearing mice at the optimal dosage schedule.

p o s s i b i l i t i e s cannot be discounted, t o form corresponding dihydroquinones (X). The dihydroquinones are unstable and spontaneously


4.

LIN ET AL.

Bioreductive

Alkylating

Agents

75

decompose t o form the a n t i c i p a t e d r e a c t i v e intermediates, oquinone methides (VII). Such r e a c t i v e species presumably then f u n c t i o n as i n h i b i t o r s o f n e o p l a s t i c growth by a l k y l a t i o n o f UNA, RNA, and/or other b i o l o g i c a l m a t e r i a l s i n a manner s i m i l a r to that o f the mitomycins.

Scheme I I I Although no biochemical evidence i s c u r r e n t l y a v a i l a b l e to support the e x i s t e n c e o f an o-quinone methide i n v i v o , chemical evidence (28) has been obtained t o s u b s t a n t i a t e the formation

XVII

w i n Scheme IV


76

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CHEMOTHERAPY

of t h i s intermediate i n the r e d u c t i v e amination of 2,3-dimethyl5,6-bis(acetoxymethyl)-1,4-benzoquinone (XII) by a n i l i n e and morp h o l i n e . This was demonstrated by reducing compound XII with 1 molar equivalent of NaBrfy i n methanol at i c e - c o l d temperature (Scheme IV). Two major yellow products were obtained a f t e r column chromatography on s i l i c a g e l . These products were i d e n t i f i e d as duroquinone (XIII) and the s p i r o dimer, 3 ,4 -dihydro-3,4,6 ,7 tetramethyl-6-methylenespiro[3-cyclohexene-l, 2 (1 H)-naphthalene]2,5,5 ,8*-tetradone (XIV). The mechanism i n v o l v e d i n the format i o n o f products XIII and XIV can best be explained by the i n i t i a l r e d u c t i o n of compound XII by NaBrfy to the corresponding dihydrobenzoquinone XV, which decomposes to generate XVI. Further r e duction or d i m e r i z a t i o n o f XVI produces the observed products XIII and XIV. To provide a d d i t i o n a l evidence f o r the existence of XVI, the reduction o f XII by NaBrty was c a r r i e d out i n the presence o f morpholine and a n i l i n e Th expected adduct XVII and XVIII were obtaine mediate XVI to a l k y l a t morpholin suggeste p o t e n t i a l to c o v a l e n t l y b i n d to b i o l o g i c a l materials i n v i v o , i f intermediate XVI were generated e n z y m a t i c a l l y i n v i v o . The concept o f b i o r e d u c t i v e a c t i v a t i o n of these quinones r e quires s t r i c t s t r u c t u r a l c o n s t r a i n t s which allow the generation of an o-quinone methide, as w e l l as a redox p o t e n t i a l f o r the quinone r i n g that i s compatible with b i o l o g i c a l a c t i v a t i o n . Studies o f the r e l a t i o n s h i p s between s t r u c t u r e and a c t i v i t y with a s e r i e s o f benzo- and naphthoquinone d e r i v a t i v e s of t h i s c l a s s have demonstrated that e s s e n t i a l l y a l l compounds possessing the quinone r i n g and an appropriate s i d e chain(s) have antitumor act i v i t y (23,24); whereas, compounds with a s i d e chain(s) u l t i m a t e l y capable o f a l k y l a t i o n , but without the quinone nucleus or v i c e versa (Table I I ) , were t o t a l l y devoid of a n t i n e o p l a s t i c potency. These f i n d i n g s were i n t e r p r e t e d to i n d i c a t e that these molecules were unable to generate the required o-quinone methide intermediate. f

f

1

1

1

f

Table I I .

Some Compounds Devoid of A n t i n e o p l a s t i c A c t i v i t y Against Sarcoma 180 Which Demonstrate S t r u c t u r a l Requirements f o r Bioreductive A l k y l a t i n g Agents

2,5-Dimethoxy-3,4-dimethyl-l-chloromethylbenzene 2,5-Dimethoxy-3,4-dimethyl-l-acetoxymethylbenzene 2,5-Dimethoxy-3,4-dimethyl-l,2-bis(acetoxymethyl)benzene 2-Methyl-1,4-naphthoquinone 2-Acetoxyethyl-l,4-naphthoquinone 2,3,5,6-Tetramethy1-1,4-benzoquinone (duroquinone)


f

4.

LIN ET A L .

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Alkylating

Agents

77

P r e l i m i n a r y s t u d i e s (29), on the r e l a t i o n s h i p between the half-wave p o t e n t i a l s ( E 1 / 2 ) o f a s e r i e s o f benzo- and naphthoquinone d e r i v a t i v e s o f t h i s c l a s s and t h e i r antitumor e f f e c t s , i n d i c a t e d that compounds with r e l a t i v e l y low redox p o t e n t i a l s g e n e r a l l y possessed the most e f f i c a c i o u s antitumor a c t i v i t i e s , with the exception o f 2,3-dimethyl-5,6-bis(chloromethyl)-1,4benzoquinone which has moderate antitumor a c t i v i t y , even though i t s redox p o t e n t i a l was i n the range o f Sarcoma 180 i n a c t i v e m a t e r i a l s (Table III)· I f the c o r r e l a t i o n between redox Table I I I .

Inhibitor

Half-Wave P o t e n t i a l (£1/2) and A n t i n e o p l a s t i c A c t i v i t y Against Sarcoma 180 o f Benzo- and Naphthoquinones Rl

R2

Antineoplastic Activity

E

l/2 (volt)

-CH -CH2C

-CH C1 -CH Br -CH 0Ac 2

-H -CH Br -CH 0Ac

-0.24 -0.21 -0.21

-CH C1 -CH 0Ac -CH 0Ac 2

-CH C1 -CH 0Ac -H

-0.05 -0.11 -0.11

-CH 0Ac -CH 0Ac

-CH 0Ac -H

-0.05 -0.06

2

2

2

2

2

2

2

2

CH3O 2

2

C H

2

0

3 '

p o t e n t i a l and antitumor a c t i v i t y i s an important feature f o r compounds o f t h i s c l a s s , i t would be expected that m a t e r i a l s with even lower redox p o t e n t i a l s might e x h i b i t more potent a n t i n e o p l a s t i c properties. In an e f f o r t t o a l t e r the redox p o t e n t i a l s i g n i f i c a n t l y , an e l e c t r o n donating (methyl) and an e l e c t r o n withdrawi n g (chloro) group were introduced i n t o the benzenoid r i n g o f 2,3-bis(chloromethyl)-1,4-naphthoquinone. The r e s u l t s obtained (Table IV) i n d i c a t e that the i n t r o d u c t i o n o f a methyl- or c h l o r o f u n c t i o n i n t o the 5- or 6- p o s i t i o n o f the parent compound does not s i g n i f i c a n t l y a f f e c t e i t h e r the redox p o t e n t i a l or the a n t i tumor a c t i v i t y o f the parent compound. A s i m i l a r o b s e r v a t i o n ,


78

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Table IV. Half-Wave P o t e n t i a l ( E 1 / 2 ) and A n t i n e o p l a s t i c A c t i v i t y Against Sarcoma 180 o f Some 5- or 6-Substituted Naphthoquinones a

R

u

Λ γ ^

1

N^CH C1 2

E

R2

Ri

l

1 / 2

(volt)

Av. s u r v i v a l time (days)b

Control CI

H

-0.26

25.0

H

Cl

-0.28

21.8

H

-0.28

25.8

-0.31

19.4

CH H

a

D

3

CH

3

A d m i n i s t e r e d once d a i l y f o r 6 consecutive days, beginning 24 h r a f t e r tumor implantation. Average s u r v i v a l time o f tumor-bearing mice at the optimal dosage schedule.

that s u b s t i t u e n t s on the benzenoid r i n g o f the naphthoquinone nu cleus have l i t t l e e f f e c t on the redox p o t e n t i a l o f the quinonoid r i n g , has been reported (30). Furthermore, i t has been shown that s u b s t i t u e n t s on the quinonoid r i n g o f the naphthoquinone moiety exert stronger e f f e c t s on the redox p o t e n t i a l o f the r i n g than s u b s t i t u e n t s on the benzenoid r i n g (30). In a d d i t i o n , e a r l i e r s t u d i e s on the r e l a t i o n s h i p between s t r u c t u r e and a c t i v i t y i n a s e r i e s o f naphthoquinone d e r i v a t i v e s o f t h i s c l a s s i n d i c a t e d that compounds with one side chain capable o f a l k y l a t i o n f o l l o w i n g r e duction are as a c t i v e as agents with two a l k y l a t i n g side chains (23,24). Based upon these c o n s i d e r a t i o n s , another s e r i e s o f 2chloromethyl- and 2-bromomethy1-1,4-naphthoquinone d e r i v a t i v e s with various s u b s t i t u e n t s a t the 3 - p o s i t i o n was prepared and t h e i r redox p o t e n t i a l s and antitumor e f f e c t s against Sarcoma 180 were measured (Table V) (31). These d e r i v a t i v e s were found to be r e l a t i v e l y potent i n h i b i t o r s o f the growth o f Sarcoma 180 a s c i t e s c e l l s , with the exception o f 2-chloromethyl-3-benzamido-l,4naphthoquinone, which was i n a c t i v e against t h i s tumor c e l l l i n e .


LIN ET AL.

4.

Table V.

Bioreductive

Alkylating

79

Agents

Half-Wave P o t e n t i a l (Εχ/2) and Antitumor E f f e c t s Against Sarcoma 180 o f 2-Halomethyl-l,4-Naphthoquinones a

R

X

E

l / 2(

v o l t

)

Av. s u r v i v a l time (days) Ρ

CI

-0.28

29.7

NHCC.H. fl

CI

-0.23

13.0

SC H

CI

-0.27

30.4

CI

-0.23

23.2

Br

Br

-0.24

29.8

CI

Br

-0.25

29.8

-0.44

40.9

C H 6

5

6

2

S C

5

5

H

6 5

Mitomycin C

Control 12.4 Administered once d a i l y f o r 6 consecutive days, beginning 24 h r a f t e r tumor implantation. Average s u r v i v a l o f tumor-bearing mice at the optimal dosage schedule. a

D

Although the optimal d a i l y dosage l e v e l o f 15 mg/kg o f 3-phenyl2-chloromethyl-l,4-naphthoquinone was about equal i n maximal ac t i v i t y t o that o f 3-chloro- o r 3-bromo-2-bromomethylnaphthoquinone, the 3-phenyl d e r i v a t i v e produced s a t i s f a c t o r y antitumor a c t i v i t y over a wider range o f dose l e v e l s , suggesting that t h i s agent had the h i g h e s t t h e r a p e u t i c index. However, the redox p o t e n t i a l s and the antitumor e f f e c t s o f these compounds were found t o be s i m i l a r to those o f the parent compound, i n d i c a t i n g that the f u n c t i o n a l groups introduced i n t o the molecule had minimal e f f e c t on the r e dox p o t e n t i a l o f the quinonoid r i n g , corresponding t o the r e l a t i v e l y minor e f f e c t s o f these m a t e r i a l s on antitumor a c t i v i t y r e l a t i v e t o the parent compound. Although hydroxyl and amino functions were reported to decrease the redox p o t e n t i a l when i n troduced onto the quinonoid r i n g o f the naphthoquinones (30), s u b s t i t u t i o n o f these f u n c t i o n a l groups onto the 3 - p o s i t i o n o f 2-chloromethyl-l,4-naphthoquinone would be expected to r e s u l t i n an unstable molecule due t o an e l e c t r o n i c e f f e c t .


CANCER

80 Table VI.

CHEMOTHERAPY

A n t i n e o p l a s t i c A c t i v i t y Against Sarcoma 180 of Quinoline-5,8-dione and Naphthazarin D e r i v a t i v e s a

Av. Inhibitor None 0 ^ . 1 1

survival (days)b 12.4

time

Br

CH

18.6

ι^ N - if^ y ^ιΓC H

Br

0 HO

0 CH Br 2

f | 1

HO

19.6 !

^Br

0

0 11.2

14.4 0CH 0 3

OH

0 10.4

a

D

A d m i n i s t e r e d once d a i l y f o r 6 consecutive days, beginning 24 hr a f t e r tumor i m p l a n t a t i o n . Average s u r v i v a l time o f tumor-bearing mice at the optimal dosage schedule.


4.

LIN ET

AL.

Bioreductive

Alkylating

Agents

81

It i s i n t e r e s t i n g to note that the half-wave p o t e n t i a l f o r mitomycin C under the same experimental c o n d i t i o n s was found to be -0.44 v o l t s , a value c o n s i d e r a b l y lower than any o f the compounds o f t h i s s e r i e s ; furthermore, i t s antitumor a c t i v i t y i n t h i s system was a l s o c o n s i d e r a b l y g r e a t e r . These r e s u l t s encourage the f u r t h e r design and s y n t h e s i s o f compounds of the b i o r e d u c t i v e a l k y l a t i n g type with redox p o t e n t i a l s lower than those already available. Both benzo- and naphthoquinone d e r i v a t i v e s o f t h i s s e r i e s g e n e r a l l y are p o o r l y water s o l u b l e . T h i s f a c t o r adds to the comp l i c a t i o n o f the u l t i m a t e p r e p a r a t i o n o f a s u i t a b l e p a r e n t e r a l dosage form. As p a r t o f a study to develop new a n t i n e o p l a s t i c agents o f t h i s c l a s s with (a) lower redox p o t e n t i a l s and thus p o s s i b l y g r e a t e r t h e r a p e u t i c potency, and (b) b e t t e r water s o l u b i l i t y ( i n s a l t form), the s y n t h e s i s o f a number of 2-chloromethyl- and 2-bromomethyl d e r i v a t i v e f naphthazarin d 2,3 bis(bromomethyl)quinoline-5,8-dion compounds (Table VI) wei posses t i v i t y , p r o l o n g i n g the l i f e span o f tumor-bearing mice from 12 to 13 days f o r untreated tumor-bearing c o n t r o l animals to 19 to 20 days. However, these r e s u l t s were obtained at the expense o f subs t a n t i a l host t o x i c i t y , as measured by body weight l o s s during the drug treatments. 2-Bromomethyl-3-bromo-6,7-dimethylnaphthazarin was found to be i n a c t i v e against Sarcoma 180 at dosage l e v e l s up to 40 mg/kg per day, and as expected, 6,7-dimethylquinoline-5,8dione and 2,3-dibromo-l,2,3,4-tetrahydro-5,8-dimethoxy-2,3methylene-l,4-dioxonaphthalene, p r e c u r s o r s o f the f i n a l product, were found to be i n a c t i v e as a n t i n e o p l a s t i c agents. These r e s u l t s f u r t h e r s u b s t a n t i a t e d the v a l i d i t y o f the proposed model compound (VIII). Biochemical Studies. The involvement of coenzyme Q (CoQ) i n mitochondrial e l e c t r o n t r a n s p o r t has been w e l l recognized i n b i o l o g i c a l systems. Mammal i a n succinoxidase and NADH-oxidase systems have been e x t e n s i v e l y s t u d i e d and may be considered r e p r e s e n t a t i v e of CoQ e l e c t r o n t r a n s p o r t sequences. A r e l a t i o n s h i p has been documented between a n t i m a l a r i a l potency o f benzoquinone and naphthoquinone d e r i v a t i v e s and the degree o f i n h i b i t i o n o f m i t o c h o n d r i a l succinoxidase a c t i v i t y (32-34), a f i n d i n g i n d i c a t i v e o f the importance of CoQ to t h i s p a r a s i t e (35); however, no comparable evidence i s a v a i l able to i n d i c a t e a s i m i l a r r e l a t i o n s h i p between a n t i n e o p l a s t i c a c t i v i t y o f quinone d e r i v a t i v e s and i n h i b i t i o n o f e l e c t r o n t r a n s p o r t . Profound e f f e c t s o f ethyleneiminoquinones, which are r e l a t i v e l y potent a n t i n e o p l a s t i c agents, on the r e s p i r a t i o n of c a r c i noma c e l l s have been reported e a r l i e r (36,37). These c o n s i d e r a t i o n s prompted an i n v e s t i g a t i o n of the e f f e c t s of the b i o r e d u c t i v e a l k y l a t i n g agents s y n t h e s i z e d i n t h i s laborat o r y on beef heart m i t o c h o n d r i a l NADH-oxidase and succinoxidase



H

CH OC(0)CH

3

3

3

CH Br

CH OC(0)CH

CH 0C(0)CH

CH 0C(0)CH

2

2

2

2

2

2

H

CH

3

2

3

2

31.0+4.4

42.9+5.0

23.6+_.8.5

69.5+15.3

37.4+7.1

74.5+19.1

81.7+19.8

46.1+11.6

53.4+16.3

87.6+12.7

51.7+_14.7

60.3+_ 7.2

+_9.8

4.3+5.2

71.3+10.9

24.5+5.8

24.1

16.8+7.7

10.9+6.1

20.2+3.1

24.8+5.0

16.0+3.8

Succinoxidase, % 3.3xlO-4Mb 1.7xlO-4Mb

a

95.6+5.4

91.2+4.6

96.3+17.6

24.1+3.8

73.2+8.7

55.3+23.4

95.4+5.2

70.8+19.0

95.4+9.7

3.3x10-^

P e r cent o f u n i n h i b i t e d c o n t r o l s +_ standard d e v i a t i o n . bConcentration o f i n h i b i t o r employed. [From: J . Med. Chem., _16, 1268, 1973; courtesy o f the American Chemical S o c i e t y ] .

a

CH

CH Br

3

29.2+3.3

CH Br

CH Br

2

20.0+4.3

23.7+_3.8

H

CH C1

47.1+J.7

51.5+9.9

3

CH

CH C1

2

1

NADH-oxidase, %a 3.3xlO"5Mb 1.7xlO-4Mb

36.2+8.6

2

3.3xlO" M

b

CH C1

2

R2

4

0

I n h i b i t i o n o f M i t o c h o n d r i a l Succinoxidase and NADH-oxidase by Naphthoquinone D e r i v a t i v e s

CH C1

Ri

Table VII.

LIN ET

4.

Bioreductive

AL.

Alkylating

Agents

83

activities. The r e s u l t s (Table VII) i n d i c a t e d that at concentra t i o n s o f 1.7 to 3.3 χ 10-4M, a l l quinones t e s t e d depressed mito c h o n d r i a l succinoxidase and NADH-oxidase a c t i v i t i e s to about 50% or l e s s of the u n i n h i b i t e d c o n t r o l s , except f o r 2-methyl-3acetoxymethylnaphthoquinone. At a lower c o n c e n t r a t i o n (3.3 χ 10~ M), only bromomethylnaphthoquinone (R^ = -CH2Br; R = H) s t r o n g l y i n h i b i t e d succinoxidase a c t i v i t y ( i . e . , to below 30% of the u n i n h i b i t e d c o n t r o l ) . I n t e r e s t i n g l y , at the same concentra t i o n (3.3 χ 10~ M), bromomethylnaphthoquinone only weakly de creased NADH-oxidase a c t i v i t y . This f i n d i n g suggests a s i t e of a c t i o n by t h i s compound at Complex II ( i . e . , succinate-CoQ reduc tase). In c o n t r a s t , the three compounds with a 2-methyl group (R = CH3) t e s t e d , s e l e c t i v e l y i n h i b i t e d NADH-oxidase a c t i v i t y at 3.3 χ 10-5M, to at l e a s t 50% o f c o n t r o l a c t i v i t y . This suggests a p r e f e r e n t i a l s i t e of a c t i o n at Complex I ( i . e . , NADH-CoQ reduc t a s e ) . Among these three methyl compounds the chloromethyl (Rj = CH C1; R2 = CH3) and acetoxymethy analogs showed an equa t i v i t y at the two concentrations t e s t e d . Introduction of a l i p o i d a l s i d e chain (pentadecyl) i n t o the acetoxymethyl or c h l o r o methylbenzoquinone or naphthoquinone analogs r e s u l t e d i n a reduc t i o n of enzyme i n h i b i t o r y a c t i v i t y (Table VIII) and complete e l i mination o f antitumor a c t i v i t y (38). These r e s u l t s suggest that the l i p o i d a l s i d e chain may cause s t e r i c i n t e r f e r e n c e with the r e a c t i v e a l k y l a t i n g s i d e chain o f the i n h i b i t o r molecule or that l i p o p h i l i c i t y p l a y s a negative r o l e i n the b i o l o g i c a l a c t i o n o f these compounds. The g r e a t e r potency o f the benzoquinones r e l a t i v e t o naphthoquinones as i n h i b i t o r s o f CoQ-mediated enzyme systems does not correspond to t h e i r l e s s e r a c t i v i t i e s when com pared to naphthoquinone d e r i v a t i v e s as a n t i n e o p l a s t i c agents, suggesting that the s u s c e p t i b i l i t y o f mitochondrial e l e c t r o n t r a n s p o r t i s not the major or s o l e determinant i n the biochemical mechanism o f a c t i o n o f t h i s c l a s s o f compounds. A c c o r d i n g l y , other p o t e n t i a l metabolic s i t e s o f a c t i o n have been sought. 2,3Bis(chloromethyl)-1,4-naphthoquinone, a member o f t h i s s e r i e s which i s a r e l a t i v e l y potent i n h i b i t o r o f the growth of both Adenocarcinoma 755 and Sarcoma 180, has been found to cause greater i n h i b i t i o n o f the s y n t h e s i s o f DNA i n v i v o than of the formation of e i t h e r RNA or p r o t e i n (39). For example, a s i n g l e dose o f 30 mg/kg o f t h i s compound produced 80% i n h i b i t i o n of the i n c o r p o r a t i o n o f thymidine-H3 i n t o DNA, with i n h i b i t i o n p e r s i s t i n g f o r up to 24 hours a f t e r drug treatment. R a d i o a c t i v i t y from C l 4 l a b e l e d 2,3-bis(chloromethyl)-1,4-naphthoquinone was found to b i n d t i g h t l y to DNA, RNA, and p r o t e i n i s o l a t e d from Sarcoma 180 a s c i t e s c e l l s exposed to t h i s agent, suggesting the p o s s i b l e a l k y l a t i o n o f these c e l l u l a r macromolecules. This compound a l s o changed the sedimentation p a t t e r n i n a l k a l i n e sucrose g r a d i e n t s o f DNA from drug-treated c e l l s , i n d i c a t i n g i n t r o d u c t i o n of s i n g l e strand breaks i n these molecules. 5

2

5

2

2



3

3

R

1

2

2

4

1 4

3

3

4

1 4

1 -H -H -(CH )l CH -(CH ) CH

2

3

2

-H -H -(CH )i CH -(CH ) CH

31.0 47.4 35.0 91.9

34.7 39.7 22.4 17.4

+

5.2

+ + + +

4.4 7.7 3.9 4.1

+ 17.2 + 14.4 + 0.4

69.5 87.6 80.2 113.8

39.2 103.9 93.9 89.9

+ + + +

0

15.3 12.7 17.6 13.3

II

2

CH X

+ 15.6 + 21.1 + 24.1 + 3.6

a

b

NADH-oxidase (% c o n t r o l a c t . ) 3.3 χ 10-4M& 3.3 χ 10^M

4.3 24.1 44.2 86.6

9.1 13.8 54.9 50.3

+ + + +

5.2 9.8 8.8 2.4

+ 0.8 + 3.6 + 8.6 + 5.6

Succinoxidase 3.3 χ 10-4Mb

a

95.6 95.4 89.9 104.4

39.3 63.5 89.2 91.9

+ + + +

5.4 5.2 9.7 7.3

+ 18.1 + 26.4 + 14.9 + 8.0

(% c o n t r o l a c t . ) 3.3 χ 10-i>Mb

I n h i b i t i o n o f Beef Heart M i t o c h o n d r i a l NADH-Oxidase and Succinoxidase by Some Benzoquinone and Naphthoquinone B i o r e d u c t i v e A l k y l a t i n g Agents

P e r c e n t o f u n i n h i b i t e d c o n t r o l s +_ standard d e v i a t i o n . bConcenration o f i n h i b i t o r employed. [From: J . Med. Chem. Γ7, 668, 1974; courtesy o f the American Chemical Society.]

a

-CI

-02CCH3

-CI

-O2CCH3

X

-OCH3

3

3

-CH -OCH -CH

Table V I I I .

4.

LIN ET A L .

Bioreductive

Alkylating

Agents

85

The biochemical data a v a i l a b l e t o date i n d i c a t e that t h i s c l a s s o f agents produces a v a r i e t y o f metabolic l e s i o n s i n sus c e p t i b l e n e o p l a s t i c c e l l s , a f i n d i n g presumably i n d i c a t i v e o f their alkylating potential. Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

S c h n i t z e r , R. J., and Hawking, F., Experimental Chemother., Volume V, pp. 1-94, Academic Press, New York, 1967. Johnston, T.P., McCaleb, G. S., and Montgomery, J . Α., J . Med. Chem., (1963) 6, 669-681. Schabel, F. M., J r . , Johnston, T. P., McCaleb, G. S., Montgomery, J . Α., L a s t e r , W. R., and Skipper, Η. E., Cancer Res., (1963) 23, 725-733. Nies, Β. Α., Thomas, L. Β., and F r e i r e i c h , Ε. J., Cancer, (1965) 18, 546-553 Arnold, H., Bourseaux (1958) 45, 64-66. Arnold, Η., Bourseaux, F., and Brock, N., Nature (London) (1958) 181, 931. Shealy, Y. F., Montgomery, J . Α., and Laster, W. R., Jr., Biochem. Pharmacol., (1962) 11, 674-675. Gerulath, Α. Η., and Loo, T. L., Biochem. Pharmacol., (1972) 21, 2335-2343. Luce, J . K., Thurman, W. R., Isaacs, B. L., and T a l l e y , R. W., Cancer Chemother. Rep., (1970) 54, 119-124. Kingra, G. S., Comis, R., Olson, Κ. B., and Horton, J . , Cancer Chemother. Rep., (1971) 55, 281-283. Ross, W. C. J., B i o l o g i c a l A l k y l a t i n g Agents, pp. 177-180, Butterworths, London, 1962. L i v i n g s t o n , R. Β., and C a r t e r , S. K., S i n g l e Agents in Can cer Chemotherapy, pp. 385-388, IFI/Plenum, New York, 1970. Frank, W., and Osterberg, A. E., Cancer Chemother. Rep., (1960) 9, 114-119. Sugiura, K., Cancer Res., (1959) 19, 438-445. M i l l e r , E., S u l l i v a n , R. D., and Chryssochoos, T., Cancer Chemother. Rep., (1962) 21, 129-135. Iyer, V. N., and S z y b a l s k i , W., Proc. Nat. Acad. S c i . U.S., (1963) 50, 355-362. Schwartz, H. S., Sodergren,J. E., and P h i l i p s , F. S., Science, (1963) 142, 1181-1183. Schwartz, H. S., J. Pharmacol. Exp. Ther., (1962) 136, 250258. Iyer, V. N., and S z y b a l s k i , W., Science (1964), 145, 55-58. K i n o s h i t a , S., Uzu, Κ., Nakano, Κ., Shimizu, Μ., Takahashi, T., and Matsui, M., J. Med. Chem., (1971) 14, 103-109. K i n o s h i t a , S., Uzu, K., Nakano, K., and Takanashi, T., J. Med. Chem., (1971) 14, 109-112. Turner, A. B., Quart. Rev. Chem. Soc., (1964) 18, 347-360.


86

23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

33. 34.

35.

36. 37. 38. 39.

CANCER

CHEMOTHERAPY

L i n , A. J., Cosby, L. Α., Shansky, C. W., and Sartorelli, A. C., J. Med. Chem. (1972) 15, 1247-1252. L i n , A. J., P a r d i n i , R. S., Cosby, L. Α., and Sartorelli, A. C., J. Med. Chem., (1973) 16, 1268-1271. L i n , A. J., Shansky, C. W., and Sartorelli, A. C., J. Med. Chem., (1974) 17, 558-561. Cater, D. Β., and Phillips, A. F., Nature (London), (1954) 174, 121-123. L i n , A. J., Cosby, L. Α., and Sartorelli, A. C., Cancer Chemother. Rep., (1974) 4, 23-25. L i n , A. J., and Sartorelli, A. C., J. Org. Chem., (1973) 38, 813-815. L i n , A. J., and Sartorelli, A. C., Biochem. Pharmacol., (1975), in p r e s s . F i e s e r , L. F., and F i e s e r , M., J. Amer. Chem. Soc., (1935) 57, 491-494. L i n , A. J., Lillis, Chem., (1975), in p r e s s S c h n e l l , J. V., S i d d i q u i , W. Α., Geiman, Q. M., Skelton, F. S., Lunan, K. D., and F o l k e r s , K., J. Med. Chem., (1971) 14, 1026-1029. Skelton, F. S., P a r d i n i , R. S., Heidker, J . C., and F o l k e r s , K., J. Amer. Chem. Soc., (1968) 90, 5334-5336. C a t l i n , J . C., P a r d i n i , R. S., Daves, G. D., Jr., Heidker, J . C., and F o l k e r s , K., J. Amer. Chem. Soc., (1968) 90, 3572-3574. Skelton, F. S., Lunan, K. D., F o l k e r s , Κ., S c h n e l l , J. V., S i d d i q u i , W. Α., and Geiman, Q. M., Biochemistry, (1969) 8, 1284-1287. Hayashi, S., Ueki, Η., and Ueki, Y., Gann (1963) 54, 381390. Hayashi, S., Ueki, Η., and Ueki, Y., Gann (1964) 55, 1-8. L i n , A. J . , P a r d i n i , R. S., Lillis, B. J., and Sartorelli, A. C., J. Med. Chem., (1974) 17, 668-672. Cosby, L. Α., L i n , A. J., and Sartorelli, A. C., unpublished data.


5 A Review of Studies on the Mechanism of Action of Nitrosoureas G L Y N N P. W H E E L E R Southern Research Institute, 2000 Ninth Ave. S., Birmingham, Ala. 35205

This review supplements and updates several previous reviews (1,2,3,4), and some of the material covered in those reviews will be included here to provide background for more recent developments and to present an overview of this subject. However, some of the details and literature references that were given previously will not be repeated. Historical Background In 1959 the routine screening of compounds for therapeutic activity against murine leukemia L1210, under the auspices of the Cancer Chemotherapy National Service Center, showed that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was somewhat active. This finding stimulated the initiation of an investigation of compounds that might be progenitors of diazomethane, with nitrosoguanidines being studied at Stanford Research Institute (5,6) and the nitrosoureas being studied at Southern Research Institute. It was soon found that replacement of the methyl group on the nitrosated nitrogen atom of each series of compounds by a 2-haloethyl group gave improved anticancer activity and also that the nitrosoureas were more active agents than the nitrosoguanidines. A n additional stimulus for pursuing the study of nitrosoureas at Southern Research Institute was the observation by Skipper, Schabel, T r a d e r , and Thomson (7) that in contrast to methotrexate, 6-mercaptopurine, cyclophosphamide, azaserine, 5-fluorouracil, mitomycin C, and M N N G , intraperitoneally administered N-methyl-N-nitrosourea (MNU) was active against intracerebrally inoculated L1210 cells. In the continuing program of preparation and evaluation of nitrosoureas by Johnston, McCaleb, Montgomery, and collaborators, early results (8) showed that N , N'-disubstituted-N-nitrosoureas 87


CANCER

88

CHEMOTHERAPY

were active compounds. Subsequent tests (9,_1Q) led to the conclusion that greater activity was obtained when the substituent on N - l was a 2-chloroethyl or a 2-fluoroethyl group and the substituent on N-3 was a 2-chloroethyl, 2-fluoroethyl, cycloaliphatic, or heteroalicyclic group. Therefore, most of the compounds that have been prepared and tested bear a 2-chloroethyl group on N - l and miscellaneous groups on N - 3 . The three disubstituted nitrosoureas shown below are undergoing extensive clinical t r i a l s . They are N , N -bis( 2-chloroethyl )-N-nitrosourea ( B C N U ; NSC 409962), N-( 2-chloroethyl)N - c y c l o h e x y l - N - n i t r o s o u r e a ( C C N U ; NSC 79037), and N - ( 2 chloroethyl) - N -(trans-4-methylcyclohexyl)-N-nitrosourea ( M e C C N U ; NSC 95441). T

f

T

î C1CH CH 2

NO B C N U (NSC 409962)

C1CH CH - N-C-ΝΉ 2

2

Ί ο

Π

C C N U (NSC 79037)

Ο C 1 C H C H - N - ë - ΝΉ 2

2

M e C C N U (NSC 95441 J * * 0

3

Mode of Decomposition It was hypothesized early that the observed biological effects of MNNG ( 5 ) and of M N U (11) were due to their decom position at physiological conditions to yield diazomethane, which would then alkylate biological materials. Subsequent studies have shown that alkylation of biological materials does in fact occur, but experiments with M N N G (12), M N U (13,14), and N-ethyl-N-nitrosourea (1J5) that were labeled with C , 3H, or H in the methyl or ethyl group showed that methyl or ethyl 1 4

2


5.

WHEELER

Action

of

Nitrosoureas

89

groups of the alkylation products contained the same ratios of isotopes as the parent compounds, and therefore a diazoalkane could not be an intermediate. This and other evidence is con sistent with the intermediate formation (as shown below) of methanediazohydroxide, which then decomposes to generate a methyl cation. Ο CEL-N-C-NH 3

,

CH N=NOH

2

3

+

OCNH

2

NO CH3N=N+ CH3+

HOH

+ OH"" + N.

HOH

ι

[HOOCNH ]

C0

2

2

+ NH3

CH OH 3

H+ By analogy with the decomposition of M N U , it would be expected that the decomposition of N-( 2-chloroethyl)-N - s u b stituted-N-nitrosoureas would yield 2-chloroethanediazohydroxide and the corresponding isocyanate. The 2-chloroethanediazohydroxide would give rise to a 2-chloroethyl cation, which could combine with a hydroxide ion or water to f o r m 2-chloroethanol. In 1967 Montgomery and co-workers (16.) reported that decomposition of B C N U and of C C N U in water yielded p r i m a r i l y acetaldehyde rather than 2-chloroethanol. This suggested the intermediate generation of a vinyl cation rather than a 2-chloroethyl cation, and they suggested the following mechanistic sequence. By this scheme the loss of a proton and a chloride ion gives rise to an unstable oxazolidine intermediate, which then breaks down into ethylenediazohydroxide and an i s o cyanate. In contrast to B C N U , B F N U ( N , N -bis( 2-fluoroethyl)N-nitrosourea) decomposed in the normal manner to yield 2-fluoroethanol. T

f


CANCER

90 O©

Ο C 1 C H C H - N - C - •NHR 2

CHEMOTHERAPY

2

NO

C1CH CH - - N - C : = N R I 2

2

NO

CH,

f ICCHH VI

ιΘ C=NR + CI N J

ι '

C H = C H N = N O H + OCNR 2

C H = C H N = N © + OH® 9

CH3CHO —

[CH =CHOH] 2

+

H.

2.

C H =CH® ^ c r i 8

+ N

2

In 1974 Colvin, Cowens, Brundrett, K r a m e r , and L u d l u m ( Γ 7 ) studied the decomposition of C - l a b e l e d B C N U in phosphate buffer at pH 7. 4 and found that contrary to the results of Montgomery et al. 63% of the identified volatile material was 2-chloroethanol and 31% was acetaldehyde; other volatile compounds included dichloroethane and vinyl chloride. They also obtained evidence (18) for the formation of a 2-chloro ethyl cation f r o m C C N U . Reed et al. ( 19 ) reported evidence supporting the formation of the 2-chloroethyl cation and of 2-chloroethanol when C C N U or M e C C N U decomposes in buffered aqueous solutions, and they proposed the following mechanism, which involves base catalysis. Montgomery and co-workers presented (20) the results of additional studies of the decom position of six N-( 2-chloroethyl)-N -substituted-N-nitrosoureas (including B C N U and C C N U ) in both water and buffered solutions, and they concluded that it is likely that at physiological con ditions both of the above modes of decomposition occur. The relative extents of the two modes depend upon whether or not the solution is buffered at or near physiological pH. In unbuffered solutions the major volatile product was acetaldehyde, and only very small quantities of 2-chloroethanol were 14

T


5.

WHEELER

Action of Nitrosoureas

91

ClCH CH NCONH-R I 2

2

NO OHO |! C 1 C H C H N - Cs- N - R 2

M

2

V

Ν

Η

r

C 1 C H2 ,CH

N-OH C1CH=CH

. " ^| -H 0 N z

2

2

ClCH CH -^NÔH 2

2

I

C 1 C H C H © + N + OH"" 2

2

OH" ClCH CH OH 2

2

formed. In buffered solutions much less acetaldehyde and much more 2-chloroethanol were produced. The 2-chloroethyl cation can be a precursor of acetaldehyde (20), so part of the acetaldehyde might be formed by this pathway in addition to its formation via the vinyl cation. The isocyanates that are generated upon the decomposi tion of the nitrosoureas can react with water to form carbamic acids, which in turn decompose to yield carbon dioxide and the corresponding amines. If the concentration and the pH of the mixture are suitable, the amine may react with the isocyanate to yield the symmetrical urea.


CANCER

92

H 0 R N C O — - — ^ [RNHCOOH]

CHEMOTHERAPY

2

V

-RNH

2

+

C0

2

J

^

RNH-C-NHR

II

Ο If nucleophiles (including biological materials) other than hydroxyl ion are present during the decomposition of the nitrosourea the carbonium ions might alkylate them. A l s o if compounds containing active hydrogen atoms are present they might react with the isocyanate by a carbamoylation reaction. Either alkylation or carbamoylation (or both) of biological materials might cause the observed physiological effects of these agents. In the special cases where the substituent upon N-3 is a 2-haloethyl grou acid would be a 2-haloethylamine as an alkylating agent; this would be the case for B C N U . Products of Alkylation Utilizing C C N U labeled with C in the 2-chloroethyl group, Cheng, Fujimura, Grunberger, and Weinstein ( 21 ) observed the binding of the isotope to poly U , poly A , poly ( i 3 ) > poly G , poly C , tRNA, D N A , albumin, histone, ribonuclease A , and cytochrome C upon incubating mixtures in buffered solutions, pH 7. 2 at 3 7 ° . They also observed that the C was bound to the R N A , D N A , and protein of leukemia L1210 cells following incubation of the C C N U with cells in vitro, and Connors and Hare (ji2) s i m i l a r l y observed the binding of C to R N A , D N A , and protein upon incubation of this labeled agent with murine T L X 5 ascites cells. Following the adminis tration of the C C N U to mice bearing the ascitic f o r m of the leukemia L1210, C was associated with the three types of macromolecules. K r a m e r , Fenselau, and Ludlum (.23) have identified an alkylated base that was formed upon exposure of a polynucleotide to a N-( 2-chloroethyl)-N-nitrosourea. These investigators incubated Γ 2-chloroethyl- C ] B C N U with poly C , and after acid hydrolysis of the polymer they isolated two products which they identified as 3- ( 2-hydroxy ethyl) C M P (I) and 3, N - e t h a n o - C M P (II). Both of these compounds upon decomposition yielded 3- ( 2-hydroxyethyl) - U M P ( III). They suggest that a 3- ( 2-chloroethyl) cytosine moiety may have been an intermediate product in the formation of the isolated products. 1 4

G

U

1 4

1 4

1 4

14

4


5.

WHEELER

Action

of

Nitrosoureas

93

B e c a u s e of the w e l l - k n o w n m u t a g e n i c and c a r c i n o g e n i c a c t i v i t i e s of M N U and N - e t h y l - N - n i t r o s o u r e a ( E N U ) (see b e l o w ) the a l k y l a t i o n of n u c l e i c a c i d s by t h e s e agents h a s b e e n s t u d i e d by a n u m b e r of i n v e s t i g a t o r s . T a b l e I l i s t s the a l k y l a t e d b a s e s that hav the t r e a t m e n t of d e o x y g u a n o s i n t R N A ( 3 0 ) , T M V - R N A ( 3 1 ) , and b a c t e r i o p h a g e R17 ( 3 2 ) . The p r o d u c t s that h a v e b e e n i s o l a t e d a f t e r i n c u b a t i n g i n t a c t c e l l s with M N U o r E N U a r e g i v e n i n T a b l e II. In m o s t i n s t a n c e s , 7 - M e G u a was f o r m e d i n m u c h g r e a t e r q u a n t i t y than the o t h e r methylated bases. T h e e v i d e n c e (26, 33) i n d i c a t e s that the extent of f o r m a t i o n of p h o s p h o t r i e s t e r s i s g r e a t e r t h a n that of m o s t of the a l k y l a t e d b a s e s but s t i l l m u c h l e s s , with two e x c e p t i o n s ( 29, 31 ), t h a n that of 7-MeGua. T a b l e III l i s t s the a l k y l a t e d b a s e s that have b e e n i s o l a t e d f r o m n u c l e i c a c i d s of v a r i o u s t i s s u e s f o l l o w i n g the a d m i n i s t r a t i o n of M N U or E N U to e x p e r i m e n t a l a n i m a l s .



Q6-MedGuo Οβ-EtdGuo

3-Et Thy Phosphotriester ( Indirect evidence)

3-MedThd O-MedThd

Xp(Me)Y

1-EtAde 3-EtAde 7-EtAde 3-EtCyt

1-MeAde 3-MeAde 7-MeAde

1-MedAdo 3-MeAde 7-MeAde

0 6 - EtGua 7- EtGua 3-EtGua

3-MedCyd

QS-MeGua 7-MeGua

Q6-MedGuo 7-MeGua 3-MeGua

3-MeCyt

1-MeAde 3-MeAde 7-MeAde

Q6-MeGua 7-MeGua

Phosphotriester ( Indirect evidence)

3-EtCyt

1-EtAde 3-EtAde 7-EtAde

œ-EtGua 7-EtGua 3-EtGua

Phosphotriester (Indirect evidence)

3-MeCyt

1-MeAde 3-MeAde

Oe-MeGua 7-MeGua 3-MeGua

Table I Products Isolated after Incubation of Various Materials with M N U or E N U DNA Deoxyguanosine DNA DNA t-RNA T M V - R N A Bacteriophage (24) (25.26,27) (28) (29) (30) (31) B17 (32)

5.

WHEELER

Action

of

95

Nitrosoureas

Table II Products Isolated after Incubating C e l l s with M N U or E N U HeLa Cells L-Cells E. Coli (33) (15) (29) Ce-EtGua 7-EtGua 3-EtGua 1-EtAde 3-EtAde 7-EtAde

C^-MeGua 7-MeGua

00-EtGua 7-EtGua

3-MeAde

3-EtAde

3-EtCyt 3-EtThy Phosphotriester ( Indirect evidence)

Phosphotriester ( Indirect evidence)

Table III Products Isolated f r o m Tissues of Experimental Animals Following Administration of M N U or E N U (13,34-M) 06- MeGua O^-EtGua 7- MeGua 7-EtGua 3-EtGua 3-MeAde 3-EtAde 7-EtAde Experiments have shown that the relative extents of alkylation of the various positions on the various bases of DNA and R N A differ for several biological alkylating agents. C o m parison of the sites and extents of alkylation of DNA by methyl methane sulfonate, ethyl methane sulfonate, isopropyl methanesulfonate, and M N U (27) and by diethyl sulfate, ethyl methanesulfonate, and E N U (29) showed that M N U and E N U caused relatively more alkylation of the O -position of guanine than the other agents. Similar results were obtained in experiments with R N A and: diethyl sulfate, ethyl methane sulfonate, and E N U (31); dimethyl sulfate, methyl methane sulfonate, M N U , and MNNG (32); and dimethyl sulfate, M N U , and MNNG (45); M N U and MNNG yielded similar results. The nitrosoureas also caused the formation of larger quantities of phosphotriesters than the other agents (29, 31, 32). There is also evidence that ethyl methane sulfonate alkylates the Opposition of guanine 6


CANCER CHEMOTHERAPY

96

moieties more extensively than methyl methane sulfonate and that E N U alkylates this position more than M N U (24» 27, 46). Lawley (27, 46) has pointed out that these results are consistent with the Swain-Scott factors for the alkylating agents and the nucleophilicities of the sites on the bases. The relationships of these differences to biological effects will be mentioned below. Consideration of the multiplicity of alkylation products listed above for M N U and E N U makes it evident that much work remains to be done to isolate and identify alkylation products obtained with N-( 2-chloroethyl )-N-nitrosoureas. The task may be more difficult, if the alkylation products are 2-chloroethyl amines or sulfides, because these compounds themselves might be chemically active as alkylating agents. Products of Carbamoylation Soon after the initiation of studies with nitrosoureas it was recognized that carbamoylation of biological materials might play a role in causing the physiological effects of the agents (16,47). In 1968 we reported (48) that 2-chloroethyl isocyanate was as effective as B C N U in decreasing the DNA nucleotidyltransferase activity of crude preparations f r o m leukemia L1210 cells, and we obtained evidence (49) that carbamoylation of the e-amino group of lysine moieties occurred when B C N U was incubated with histone. Oliverio and co-workers reported (50, 51) that following the administration of C C N U to dogs and monkeys the cyclohexyl portion, but not the 2-chloroethyl portion, of the parent compound was bound to the plasma protein. Weinstein and collaborators (21, 52) observed that the cyclohexyl portion of C C N U was extensively bound to globulin, ribonuclease A , cytochrome C , histone, albumin, and polylysine (in increasing order) and to proteins of L I 210 cells, and hydrolysis of the latter four treated mater i a l s yielded NS-cyclohexylcarbamoyllysine. We have recently observed (J53) that at physiological conditions the carbamoylation of the N of lysine occurs more extensively than c a r bamoylation of N . Table IV shows the relative amounts of products obtained when a mixture of equimolar quantities of C C N U and lysine was incubated in 0.1 M phosphate buffer at 37°. 2

6

Table IV Relative Quantities of Products F o r m e d Upon Incubating a Mixture of L - [ U - ^ C ] Lysine and C C N U Relative Product Quantity N - ( Cyclohexylcarbamoyl) lysine N - ( Cyclohexylcarbamoyl ) lysine N , r ^ - D i i cyclohexylcarbamoyl)lysine 2

6

2

1.00 0. 38 0. 09


WHEELER

5.

Action

of

97

Nitrosoureas

Experiments with other a -amino acids and with dipeptides also demonstrated the carbamoylation of N of amino acids and of terminal amino groups of peptides. Incubation of a mixture of insulin and C C N U , and hydrolysis of the reaction product yielded N-cyclohexylcarbamoylglycine and N-cyclohexylcarbamoylphenylalanine, which shows that carbamoylation of the amino terminal moieties occurred. We did not detect any N^cyclohexylcarbamoyllysine, which would have been indicative of carbamoylation of the single lysine moiety present in the molecule, but it is emphasized that we did not diligently seek N - cyclohexylcarbamoyllysine. We have observed (J53) that even at room temperature cyclization of the 2-chloroethylcarbamoylamino groups can occur to f o r m oxazolinyl groups as shown for N - ( 2-chloroethylcarbamoyl)lysine. Similar cyclization occurs if the 2

6

6

Ο II

ClCH CH NHCNHCH CH CH CHCOO2

2

2

2

2

NH + 3

H

2

CNHCH CH CH CH CHCOO^η + 2

H c_rf 2

2

2

2

3

2-chloroethylcarbamoyl group is on N of an amino acid. The oxazolinyl group is more basic than the 2-chloroethylcarbamo ylamino group, and the oxazolinyl compound migrates electrophoretically s i m i l a r l y to the parent amino acid. T h i s c y c l i zation is analogous to that which occurs when 1, 3-bis( 2-chloro ethyl) urea (54) or chloroethylbiurets (55.) are heated in boiling water. Under physiological conditions that permit cyclization of 2-chloroethylcarbamoyl groups, there is no evidence of alteration of cyclohexylcarbamoyl groups. Therefore, proteins carbamoylated upon treatment with B C N U might have different biochemical properties f r o m those carbamoylated upon treat ment with C C N U and other nitrosoureas. There is evidence that carbamoylation of nucleic acids might occur to a small extent. Utilizing [ cyclohexyl- C1 C C N U (21) and [ c a r b o n y l - C 1 C C N U (52) in experiments with isolated nucleic acids and with intact L1210 cells the Columbia University group detected a relatively minute quantity of C associated with R N A and with DNA in comparison to the quantity associated with proteins under the same conditions. Serebryanyi and co-workers (56) incubated a mixture of Γ c a r b o n v l - C ]MNU with D N A and observed that the * C became bound to the DNA. 2

14

14

1 4

14

4


CANCER

98

CHEMOTHERAPY

Upon consideration of the relative nucleophilicities of phosphate, hydroxyl, and amino groups at pH 7, they suggested that c a r bamoylation would occur chiefly with the phosphate groups but carbamoylation of the bases might also occur. Upon conversion of the carbamoylated D N A to the corresponding apurinic acid a small amount of C was retained. No carbamoylation product was identified. More recently Serebryanyi and Mnatsakanyan (57) have incubated adenosine or cytidine with M N U at physio logical conditions and have obtained chromatographic and ultra violet spectral data that are consistent with the formation of NS-carbamoyladenosine and N -carbamoylcytidine. 1 4

4

Metabolism of Nitrosoureas There i s evidence that certain nitrosoureas are altered by enzymatic metabolism to yield the products shown in Table V .

Agent

Table V Products of Metabolism of Nitrosoureas System Products

BCNU

Microsomal

BCNU CCNU

L i v e r cytosol Microsomal and in vivo

N, N - B i s ( 2-chloroethyl)urea Unidentified Ring-hydroxylated C C N U C i s - 2 and / or trans-2 Trans.-3 Cis-3 Trans-4 Cis-4

Reference (58)

f

(58) (60,61,62)

H i l l et al. (58) observed that B C N U is converted to 1, 3-bis(2-chloroethyl)urea by microsomal mixed-function oxidase that requires ΤΡΝΉ and oxygen. When the microsome preparation was replaced with a l i v e r cytosol preparation, a product that has not been identified was obtained. May, Boose, and Reed (59) and H i l l , K i r k , and Struck (58) presented evidence that hydroxylation of the cyclohexyl ring occurs very rapidly when C C N U is incubated with a l i v e r m i c r o s o m a l preparation in the presence of oxygen and T P N H . Reed and May (60, 61) identified five metabolites, which they obtained in vitro and in vivo and which are listed in the table. They did not specify the configuration of the 2-hydroxy deriva tive, but in a personal communication D r . Reed stated that it is the c i s - 2 - i s o m e r . Hilton and Walker (62) have independently identified the two pairs of 3- and 4 - i s o m e r s and the trans-2isomer as products of in vitro incubation and also found them in the plasma of rats. Only the c i s - 4 - i s o m e r and the trans-4isomer were found in human plasma following the intravenous


5.

WHEELER

Action

of

Nitrosoureas

99

administration of C C N U ; these isomers were present in approximately equal quantities (63). The data of all of these groups of investigators indicate that the rate of metabolic hydroxylation exceeds the rate of chemical breakdown of C C N U , and therefore, it is likely that the hydroxylated metabolites are intermediate precursors of the therapeutically active moieties. There is also evidence (58, 59) that hydroxylation of M e C C N U occurs. Because of these facts the various hydroxylated compounds are being synthesized (60, 64) and evaluated in chemotherapeutic trials. The ç i s - 4 - and t r a n s - 4 - i s o m e r s were more active against leukemia L I 210 and more toxic than C C N U (64). Cowens, Brundrett, and Col vin (65) observed that Ν - ( 2chloroethyl)-N , N-dimethyl-N-nitrosourea is stable in aqueous solution and is inactive against L I 210 leukemia in vitro but is active against this leukemia in vivo. This suggests that meta bolic alteration gives rise to a more labile nitrosourea. The types and extent ureas bearing other types of substituents on N-3 are not known, and the relevance of the known alterations to therapeutic effec tiveness is not presently known. It can be expected, however, that metabolism would alter the physical and chemical properties of the parent compounds, and therefore one must exercise caution in inferring the transposition of results obtained in vitro to an in vivo situation. Reed and May (60) also observed that a major urinary metabolite of C C N U in mice is thiodiacetic acid, which is evi dence of thiol alkylation. They suggest that the alkylation might involve a 2-chloroethyl cation or 2-chloroacetaldehyde, which could be formed upon enzymatic oxidation of 2-chloroethanol. T

Relationships of Phvsicochemical and Chemical Properties to Therapeutic Usefulness One of the advantages that the most effective nitrosoureas have over several other types of anticancer agents is the fact that their degree of lipoid solubility permits them ( or perhaps their metabolically altered derivatives) to cross such interfaces as the blood-brain b a r r i e r " and thus k i l l neoplastic cells present in the brain (7). Hansch, Smith, Engle, and Wood (66) used the octanol-water partition coefficients to study the relationship of lipophilicity to activity against intracerebrally inoculated LI210 cells, and Montgomery, Mayo, and Hansch (67) c a r r i e d out a s i m i l a r study with subcutaneous Lewis lung carcinoma. They found that there is an optimum range for the partition coefficient. While lipophilicity is surely an important factor in determining the biological activity of the nitrosoureas, it seems likely that ultimately the biological effects will depend upon the alkylating and carbamoylating activities of the agents or their metabolic derivatives. Therefore we attempted (68) to relate tT


100

CANCER

CHEMOTHERAPY

Table VI Ο II

R-N-C-NH-R' I

NO R' C1CH CH 9

T

o,5

(min)

0. 520

53

9

^540

dpm 42.000

C1CH CH -

16,438

C1CH CH

9

30,218

C1CH CH

9

9

9

9

9

C1CH CH 9

9

C1CH CH 9

9

M a r r o w toxicity High (fifi)

44

1. 133

21.076

High

(70)

49

1. 106

42.789

High

(70)

21

2. 35

824

Low

(71)

16

2. 74

41,197

Low

(69)

HO

C1CH CH 9

9

N

AcO —î

H a l f - l i f e in ethanol/phosphate buffer, ( 1 / 5 0 ) , p H 7 . 4 , 3 7 ° . It was n e c e s s a r y to initially dissolve some of the compounds in acetone, and in such instances equal volumes of acetone were added to the blanks. b

C

A is a m e a s u r e of the concentration of alkylated 4-(jg-nitrobenzyl)pyridine in an ethyl acetate extract of a mixture of the nitrosourea and 4- (jD-nitrobenzyl) pyridine in acetate buffer, pH 6. 0, that had been incubated at 3 7 ° for 2 hr. 5 4 0

T h e dpm is a measure of the radioactivity present in unidentified reaction products obtained upon incubating the nitrosourea with l y s i n e - C in phojphate buffer, pH 7. 4, at 3 7 ° for 6 hr. l 4


5.

WHEELER

Action

of

Nitrosoureas

101

each of these three parameters, namely, partition coefficient, alkylating activity, and carbamoylating activity, and the summation of them to the therapeutic activity against i . p. L1210. Upon the basis of mathematical correlation we concluded that all three parameters are important. We suggested that the lipophilicity was a major factor because it determined the extent of delivery of the agent to the desired site, that a dominant influence of the carbamoylating activity might be associated with the whole animal toxicity of the agent, and that the alkylating activity is important in determining the therapeutic index. We stress that these assignments are merely suggestions, because it is quite difficult to obtain and properly evaluate data for a multifactor system such as this. The possible relevance of carbamoylating activity to whole animal toxicity is suggested by a consideration of the data in Table V I , which lists the chemical h a l f - l i v e s , the alkylating activities, and the carbamoylatin determined for several nitrosoureas, and the relative marrow toxicities that others have reported. C C N U , which has a high in vitro carbamoylating activity, has a high marrow toxicity (69), and therefore, by inference, one would assume that at least certain of the hydroxylated metabolic derivatives of C C N U would also be myelosuppressive. The data show that the c a r bamoylating activities of the c i s - 4 and trans-4-hvdroxv compounds have lower, but still moderately high, carbamoylating activity, as do also the acetvlated c i s - 2 and trans-2-hydroxy compounds. Although the relevance of the in vitro data to the in vivo situation might be questionable, since it is quite possible that the acetyl groups are removed in vivo, these two compounds did have high marrow toxicity in vivo. Chlorozotocin [ 2- [3- ( 2-chloroethyl)-3-nitrosoureido] - 2 - d e o x y - D - g l u c o pyranose] (12), which has a high alkylating activity and a low carbamoylating activity, has a low marrow toxicity (71.). The low marrow toxicity of the chlorozotocin tetraacetate along with the high in vitro carbamoylating activity is perhaps due to removal of the acetyl groups in vivo. In the N - m e t h y l - N nitrosourea series of compounds (73) the decrease in marrow toxicity parallels the decrease in carbamoylating activity (Table VII). Comparison such as this is being extended to analogs of chlorozotocin as they become available. Schein et al. (74) observed that chlorozotocin was much less inhibitory than B C N U for DNA synthesis by human marrow in vitro which correlated with the differences in carbamoylating activity.



Q

0

5

b

48

0.190

0.016

5 4 0

486

A 0.356

a

7

(min) c

1,629

19,777

10,892

dpm (73)

Low (73)

High

Marrow toxicity

H a l f - l i f e in ethanol/phosphate buffer, (1 /50), pH 7. 4, 3 7 ° . It was necessary to initially dissolve some of the compounds in acetone, and in such instances equal volumes of acetone were added to the blanks.

HO

/

_H

T

14

The dpm is a measure of the radioactivity present in unidentified reaction products obtained upon incubating the nitrosourea with l y s i n e - C in phosphate buffer, pH 7. 4, at 3 7 ° for 6 hr.

5 4 0

Â is a measure of the concentration of alkylated 4- (p-nitrobenzyl) pyridine in an ethyl acetate extract of a mixture of the nitrosourea and 4- (]D-nitrobenzyl) pyridine in acetate buffer, pH 6. 0, that had been incubated at 3 7 ° for 2 hr.

a

CH -

CH,

CH,-

R'

R-N-ë-NH-R' I NO

Ο

Table VII

5.

WHEELER

Action

of

Nitrosoureas

103

Table VIII lists the half-lives, the alkylating activities, and the carbamoylating activities of a number of nitrosoureas that have been synthesized by Johnston and co-workers at Southern Research Institute (8,9,10). These properties were determined as described previously (68). Although the activi ties of a number of these compounds against L1210 leukemia ( 8 , 9 , 1 0 ) , have been determined, the marrow toxicities of most of them have not yet been examined. It is obvious that the substituents on both Ν and N have great influences upon the properties of the compounds and that to a considerable degree one can obtain compounds with specified properties by the proper selection of substituents. T

Biochemical Effects A number of studies (reviewed in j$) have shown that BCNU, CCNU, MNU, synthesis of D N A , R N A , and protein in vitro and in vivo. At lower concentrations or lower doses the inhibition of the syn thesis of DNA was usually greater than the inhibition of synthesis of R N A and of protein. At still lower doses, and even at early times after the administration of the higher doses, stimulation of macromolecular synthesis often occurred. Similar inhibitions of the synthesis of D N A , R N A , and protein have been reported for streptozotocin (75), and inhibition of synthesis of DNA by chlorozotocin (71) and by chlorozotocin tetraacetate (69) was observed. Several studies to investigate the effects of nitrosoureas upon specific steps of macromolecular synthesis have been carried out. Incubation of crude cell-free preparations from L1210 cells with B C N U or C C N U caused decreases in the DNA nucleotidyltransferase activity of the preparations, but N - ( 2 chloroethyl)N-nitrosourea and M N U had much less effect (48). 2-Chloroethyl isocyanate caused as much decrease as B C N U or C C N U , and it was suggested that the apparent deactivation of the enzyme by B C N U and C C N U was due chiefly to the reactions of the isocyanates generated f r o m them. In experiments with purified D N A polymerases I and II isolated f r o m rat l i v e r and hepatoma (76), B C N U , C C N U , M e C C N U , 2-chloroethyl isocya nate, and cyclohexyl isocyanate inhibited DNA polymerase II but did not affect the activity of DNA polymerase I. D N A polymerase II is also sensitive to thiol-blocking agents, but DNA polymerase I is not (77). Auxiliary data (77) led to the suggestion that enzyme I is a repair enzyme and enzyme II is a replicative enzyme. Preincubation of D N A with the nitrosoureas or isocya nates did not significantly decrease its template activity in the assay system (48,76). Thus, it appears that the inhibition of replicative DNA synthesis might be due at least partially to carbamoylation of the replicative polymerase. In studies of the


In Cancer Chemotherapy; Sartorelli, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976. 1. 3

- H

CH3

7.0

- H

2

C1CH CH -

ClCrLCH -

2

FCH CH -

129967

129966

2

2

FCH CH -

125649

2

2

2

C1CH CH -

95441

2

2

2

FCH CH -

2

87974

2

C1CH CH -

C H , -

2

79037

79653

2

2

<X

Ό H

3

-0

2

-CH CH F

68

FCH CH -

91728

2

43

2

-CH CH C1

2

C1CH CH -

409962

2

254

-CILCrLCl

CrLj-

409935

2

CH3-

23909

R

R-N-C-NH-R' I NO

47547

NSC No.

T A B L E VIII

0.1

4

252

520

4

-

4

3.3

-

0.

4.4

0.

568

0.420

610

0.520

-

0.

-

-

0.

0. 8

3

640

016

0.

46

12

257

1.

-

4

31789

0.022

49031

38160

49113

30206

017

071

070

42000

2

1

3

1

3

( Arbitrary reference

1

19777

1 3

1 28716 19777

1 8851

1

η

8558

10892

dpm

Carbamoylating Activity

074

0.

0.

4

12

0.

12

0.

0.

28

7

004

0.

2

091

093

0.

3

15

0.

1.

—

382

010

0.

081

2

0.

0.020

>3.

-

4

SD

2

356

η

0

0.

η

Δ ·ίΐιι

Alkylating A c t i v i t y

—

21

0.8

9

12

SD

η

PROPERTIES O F NITROSOUREAS

WHEELER

:

Action

of

Nitrosoureas

105

I ι

I

I

af υ

υ

X

υ

in

ι υ y υ

u υ

ι

aT υ

υ y G


I

υ


C1CH CH -

C1CH CH -

FCrLCH,-

204817

88106

93492

129968

61.3

C1CFLCH,-

110800

4

4

2

47.9

2

C1CH CH -

132921

4

—

43.7

4

48

0>

6

48.5

4

C1CH,CH,-

2

1(g)

16.1

4

2

2

CH OAc

2.5

3.5

—

3.0

1.3

0.9

0.8

1.2

6

21.1

SD

2.3

0. 5

45.9

1

(continued)

55.7

2

2

2

C1CH CH -

204818

2

C1CH,CH -

114460

2

ClCrLjCr^-

2

178248

3

CH CH -

174793

T A B L E VIII

1.339

0.633

0.547

4

4

12

2

2

0.647

0.853

4

4

4

4

1.133

1.106

2.740

2. 350

0.110

0.034

0.171

0.068

0.016

0.072

0.062

0.048

0.326

15730

34968

18625

21076

42789

41197

824

dpm


FCH CH -

C1CH CH -

FCH CH -

C1CH CH -

132085

128303

136900

136902

80590

C1CH CH -

105763

2

2

2

2

2

ο

2

2

FCHgCî^—

106767

2

2

2

FCH CH -

2

2

2

114459

2

C1CH CH -

2

103534

2

C1CH CH —

95466

Ό

Ο

2

•Ό

^-So

- Ο

- Ο

Ό

5.6

.3

9.1

4

4

6

0.2

1.9

1.1

>3.0

1.818

33

12

12

12

2

2

4

2.097

>3.0

1.351

1.142

1.854

34

0.3

—

_

1.3

1.

5

4

—

_

5

5

2.369

22.4

19

_

49.1

26.

0.286

0.215

0.117

-

—

0.093

0.320

13566

11476

10866

—

10065


3

2

FCrLjCr^-

95459

2

2

2

2

I NO

NO

2

CrLCl

CH^rLjF

Q .N H - C - N - C H

-(CH ) CN

-H

- O

3

VIII

8.1

4

—

—

4

4

4

3

4

3

4

4

n

31.9

6.6

3.0

52. 8

286

41. 3

202

14.7

0

T .5

(continued)

540

0.120

—

0. 846

0.040

0. 064

0.161

0.013

0

0.040

0

0.301

0

A

2. 2

0. 2

0. 3

2.1

7

1.7

5

0.6

1. 8

SD

-

—

-

—

1

2

0. 024

1 1 -

c

T h e dpm i s a m e a s u r e of the radioactivity present in unidentified reaction products obtained upon incubating the n i t r o s o u r e a with l y s i n e - " C i n phosphate buffer, pH 7. 4, at 37° for 6 h r .

5 4 0

^Α i s a m e a s u r e of the concentration of alkylated 4 - ( p - n i t r o b e n z y l ) pyridine in an ethyl acetate extract of a mixture of the nitrosourea and 4 - ( p - n i t r o b e n z y l ) p y r i d i n e in acetate buffer, pH 6. 0, that had been incubated at 37° for 2 h r .

—

7006

2308

6474

26272

28391

10287

13733

25143

9776

—

-

dpm

SD

1 -

1

1

1

1

'

1

n

H a l f - l i f e in ethanol / phosphate buffer, (1 / 50), pH 7. 4, 37°. It was n e c e s s a r y to initially dissolve some of the compounds in acetone, and in such instances equal volumes of acetone were added to the blanks.

2

C1CH CH2-

2

93372

2

Ω

Ο

5.

WHEELER

Action

of

Nitrosoureas

109

repair of D N A that had been irradiated with X - r a y s (78) or ultraviolet light (79) it was observed that B C N U and 2-chloroethyl isocyanate interfered with the rejoining of broken strands but not with the repair synthesis, which implies inhibition of the ligase as a result of carbamoylation of it. These facts suggest that the carbonium ions derived from the nitrosoureas might alkylate D N A and the isocyanates derived f r o m them might interfere with the repair of the damaged D N A . One might speculate that if nitrosoureas were used in combination with other alkylating agents, both agents might cause alkylation of D N A in an additive manner, and the generated isocyanate might inhibit repair. Incubation of DNA-dependent R N A polymerase f r o m E h r l i c h ascites cells with M N U or N - p r o p y l - N - n i t r o s o u r e a resulted in inhibition of the enzyme (80). Injection of these agents into mice bearing E h r l i c h ascites cells caused an activation of the Mg+ 2-dependen nuclei f r o m the ascites cells and at higher concentrations an inhibition of the Mn+2/(NH ) S0 -dependent R N A synthesis by these nuclei (81). In studies with cultured leukemia L1210 cells it was found that B C N U inhibited the "processing" of 45S nucleolar R N A more readily than the synthesis of R N A , and it also inhibited the "processing" or exit of high-molecular weight R N A f r o m the nucleoplasmic fraction (82). "processing" of nucleolar R N A was also inhibited by C C N U , N - ( 2- fluoroethyl ) - N - cyclohexyl- N-nitrosourea, 2- chloroethyl isocyanate, and cyclohexyl isocyanate, but not by M N U , N - ( 2 chloroethyl)-N-nitrosourea, or streptozotocin. It was concluded that a carbamoylation reaction was responsible for interference with the "processing". A comparison (83) of the effects of nitrogen mustard (HN2), B C N U , and C C N U upon the synthesis and "processing" of R N A by cultured H e L a cells yielded the following results: BCNU CCNU HN2 Inhibits formation of 45-S nRNA No* Yes Yes Inhibits "processing" of 45-S nRNA Yes No Yes Produces shortened HnRNA No No Yes Inhibits appearance of p o l y ( A ) containing cytoplasmic R N A No Yes Yes 4

2

4

T

h

e

f

•inhibition occurs after extended exposure. Since each of these agents is an active anticancer agent, it is obvious that bifunctionality, carbamoylation, and interference with the formation and "processing" of R N A are not requisites for anticancer activity. A study (84) ° f the manner in which M N U interferes with protein synthesis led to the conclusion that this agent can damage polyribosomes and protein-soluble factors of the cell


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sap, which leads to inhibition of polypeptide initiation and elongation. Other data (8»5 ) were interpreted to indicate that changes in the polyribosome profile induced by M N U reflects the mechanism of inhibition of protein synthesis rather than being a direct consequence of methylation of polysomal m R N A . M N U , E N U , and N-butyl-N-nitrosourea caused a significant stimulation of aminoacyl-tRNA complex formation but had no effect on the binding of nRNA to ribosomes (86). The pharmacology of the nitrosoureas has been reviewed recently (4) and will not be discussed here, except to say that the half-lives of the compounds in the blood are very short, that the agents are widely distributed throughout the body without evidence of concentration in neoplastic tissues, and that the agents cross the "blood-brain b a r r i e r " and thus reach the brain and brain tumors. A s is the case with most anticancer agents, the reason(s) for the preferential cytotoxicit plastic tissues is not known. When rates of synthesis of D N A (87» 88» 89» 90) and of protein (84) are used as indicators of the "state of health" of various host and neoplastic tissues, it appears that both host and neoplastic tissues are damaged, but the host tissues recover the ability to synthesize these macromolecules more readily than the neoplastic tissues. The mechanisms of this recovery are not known, but they may include the repair of macromolecules and/or the repopulation of the tissues with viable cells with the accompanying loss of dead cells. It has been observed that deletion of methylated purines occurs following the treatment of E s c h e r i c h i coli with E N U (15), of L - c e l l s with M N U (33), and of mice with M N U (40,42). Following the administration of [ C H ] M N U to mice bearing hepatomas, deletion of radioactivity from the D N A , R N A , protein, and lipids occurred more rapidly for spleen and l i v e r than for the hepatoma (91). The relevance of these deletions to survival of the cells and tissues is considered below. At minimal concentrations of streptozotocin and streptozotocin tetraacetate that were cytotoxic to cultured leukemia L1210 cells these agents did not significantly lower the levels of activity of thymidine kinase, thymidylate synthetase, and D N A polymerase (92). O* the other hand, streptozotocin, M N U , and B C N U caused decreases in the observed activity of NAD glycohydrolase and the level of NAD in a variety of biological systems (reviewed in 3). 1 4

3

1

Biological Effects Many published articles report that a number of nitrosoureas are active as carcinogenic, mutagenic, and teratogenic agents in a number of biological systems, and it is not deemed necessary to refer to each of those articles here. F o r the


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purpose of this review it is sufficient to state the nitrosoureas are quite potent in causing each of these biological effects. On the basis of the presently held dogmas of the role of nucleic acids in determining heritable traits in biological systems one might speculate that these effects derive f r o m alteration of the nucleic acids of the cells that were exposed to the agents. The contents of Tables I, II, and III show that many alterations of the nucleic acids occur upon exposure to M N U and E N U , and one might ask whether the extent(s) of occurrence of one or more of these alterations correlates with the causation of one or more of the effects. A number of these investigators(13, 35, 36» 37».38» 39) isolated and quantitatively determined only the 7-alkylguanines, and several of them (ljj, 36, 38, 39) concluded that there was no correlation between the extent of formation of 7-alkylguanine and carcinogenicity. F r e i (34) detected and determined C^-MeGua and 3-MeAde in addition to 7 - M e G u a and suggested that thi to relate the formation of alkylated bases to carcinogenicity. This possibility is of interest, because it has been suggested by Loveless (24) that mutagenesis by these agents might be related to the formation of C^-alkylguanine moieties. Walker and Ewart (33) observed that following treatment of L - c e l l s with M N U the deletion of 3-MeAde and 7-MeGua was more rapid than that of Οβ-MeGua. Goth and Rajewsky (44) found that after the treatment of rats with E N U , the deletion of 3-EtAde and 7-EtGua f r o m l i v e r and f r o m brains occurred relatively rapidly, as did also the deletion of C^-EtGua f r o m l i v e r , but the deletion of Œ - E t G u a from brain occurred slowly. Kleihues and Margison (42) obtained analogous results with M N U for l i v e r and brain and found that the rate of deletion of Οβ-MeGua f r o m the D N A of kidney was intermediate between the rates for l i v e r and brain. F r e i and Lawley (40) compared the rates of deletion of alkylated purines f r o m the D N A of bone marrow, small bowel, kidneys, l i v e r , lungs, spleen, and thymus, but not the brain, of mice following the adminis tration of M N U and found that 3-MeAde and 7-MeAde were deleted more rapidly than 3-MeGua, 7-MeGua, and O^-MeGua; there was little difference in the rates of deletion of 7-MeGua and C^-MeGua during the brief period (18 hours) of observa tion. Margison and Kleihues (43) observed that, when M N U was administered to rats in repeated small weekly doses that rather specifically gave rise to tumors of the nervous system, C^-MeGua accumulated in the D N A of the brain to a much greater extent than in the kidney, spleen, small intestine, and l i v e r . Since the nervous system is the primary site of tumor formation following the administration of M N U and E N U (11,93) and the kidney (93 ) is another frequent site, these results imply that the retention of CP-alkylguanine in the DNA might be con ducive to carcinogenesis.


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The cause of the cytotoxicity of the nitrosoureas i s not known, and it seems likely that it might result from a combination of several mechanisms. V e r l y (94) and Lawley et al. (95) have suggested that the delayed inactivation of bacteriophage by monofunctional alkylating agents i s related to the presence of apurinic sites in the DNA following the elimination of alkylated purines, and Lawley and Brooks (26) and V e r l y and co-workers (97) have observed that the rate of elimination of 3-alkyladenine is much greater than that of 7-alkylguanine. The formation of and the subsequent hydrolysis of phosphotriesters might also contribute to single-strand breaks of D N A (94) and chain breaks of phage R N A (.32,98) with resultant phage deactivation. These same mechanisms may contribute to cytotoxicity in cells and tissues, but alkylation or carbamoylation of other c r i t i c a l cellular constituents might also be contributing factors. Experiments hav to sensitivity of mammalian cells in the various phases of the cell cycle and to determine the effects of treatment with these agents upon progression through the cell cycle. The literature prior to 1972 was reviewed (j$), and it will not be repeated here. Several studies performed subsequent to that review have confirmed and extended the previous conclusions. Tobey et a l . (99,100) compared the effects of B C N U , C C N U , M e C C N U , chlorozotocin, and streptozotocin in a single experimental system and found the first four of these compounds had the following s i m i l a r effects: (a) had little effect upon the progression of cells through G ; (b) caused a prolongation of S; (c) caused cells to accumulate in late S or early G ; (d) caused mitotic non-disjunction and polyploidy (pell enlargement); (e) were more cytotoxic to non-cycling cells than to cycling cells; and (f) caused an altered clonal morphology and growth pattern in surviving cells. Streptozotocin differed from the other four compounds by causing less prolongation of S and less accumulation of cells in late S and G and by being more cytotoxic to cycling cells than to non-cycling cells at low concentrations. Based upon another study Tobey (101 ) concluded that chlorozotocin arrested cells in early G , streptozotocin arrested them about one-third of the way through G , and B C N U and C C N U arrested them about two-thirds of the way through G . Other investigators (102,103) also observed that B C N U , C C N U , and M e C C N U were more cytotoxic to plateau-phase or nondividing cells than to exponentially growing cells. Hahn et a l . (104) suggested that the differential sensitivity of non-cycling and cycling cells might partially be due to the anomolous binding of B C N U to serum protein, but this is probably not the total cause, because in the experiments of Tobey et al. serum was present in the medium for both the cycling and the non-cycling cells. Contrary to the above observation for streptozotocin, 1

2

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M N U prolonged the S phase without killing the cells (105). These observations of the greater cytotoxic sensitivity of plateau-phase cells and non-cycling cells to the nitrosoureas differ f r o m the observations with hematopoietic cells in vivo (106.107) in which the rapidly proliferating cells were more sensitive than the normal bone marrow. However, non-pro liferating cells were sensitive to B C N U (108). Although cultured cells are sensitive to the cytotoxic effects of the nitro soureas in a l l phases of the c e l l cycle (75,109-113), cells are reported to be most sensitive in certain phases. The most sensitive phases have been reported variously for different cell lines and different agents to be the Gj^ IS boundary (111 ), mid-S (75,112), and G (113). It was reported in 1963 (114) that streptozotocin is diabetogenic, and many studies related to this property of the compound and the disease it initiates have been performed (115). There appears to be a ficity for elliciting diabetes, because although the ethyl analog of streptozocin i s diabetogenic, the 2-chloroethyl analog (chlorozotocin) and chlorozotocin tetraacetate are not ( 116). A l s o , M N U (117) and several analogs of streptozotocin in which the glucosyl portion of the molecule i s altered (118 ) are also non-diabetogenic. Streptozotocin causes a decrease in the NAD content of pancreatic islets (119,120.121 ) perhaps by reducing the tissue uptake of precursors and decreasing the synthesis of NAD (119 ) and by inducing NAD degradation, which can be prevented by inhibitors of NAD-glycohydrolases (120). The diabetogenic action is probably related to this decrease in NAD concentration, since this action can be pre vented by the administration of NAD (122 ). M N U also decreases the NAD content of pancreatic islets but to a lesser extent than streptozotocin (121 ) , and other nitrosoureas can reduce the NAD content of various tissues (reviewed in j$). Therefore the glucose residue (121 ) in conjunction with the presence of the methyl or ethyl group on the nitrosated nitrogen (116) must cause additional specific cytotoxicity for the β - c e l l s of the pancreas. Another observed effect of the nitrosoureas that might be of potential practical usefulness is the prevention of the in vitro sickling of r e d cells containing hemoglobin S (123) by M N U , B C N U , and C C N U . It appears likely that this prevention results f r o m the carbamoylation of the hemoglobin S, since prevention also results when the cells are treated with 2-chloro ethyl isocyanate or cyclohexyl isocyanate and the treatment of the cells or of mice with B C N U alters the electrophoretic properties of the hemoglobin. This reaction i s probably analo gous to the reaction of cyanate, which has undergone extensive testing for this purpose, but the use of nitrosoureas as pro genitors of isocyanates has the potential for accomplishing some specificity of carbamoylation. 2


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Conclusions It i s always difficult to specifically define a chemical and / or biochemical mechanism by which a drug elicits a thera peutic response, and the difficulty is greater if the agent has a multiplicity of chemical activities and biochemical and physio logical effects. Nevertheless, knowledge of the possible factors that may be involved in bringing about such a response can aid in rationally planning therapeutic regimens for use of the agents. It i s hoped that the facts that have been presented in this review will be useful for such planning. It is believed that the nitrosoureas have an established place in the cancer chemotherapy armamentarium. A n ex panding knowledge of the various aspects of their mechanisms of action should make possible a more effective use of the compounds already being used and serve as a guide in the preparation of other nitrosourea peutic indexes and more desirable physical and chemical properties. Literature Cited 1.

C a r t e r , S. K., Schabel, F . Μ . , Jr., Broder, L . Ε., and Johnston, T . P. Adv. Cancer Res. (1972) 16, 273-332. 2. Wheeler, G . P. Biochem. Pharmacol. (1974) 23, Supplement No. 2, 225-232. 3. Wheeler, G . P. Handb. E x p . Pharmacol. (1975) 38/2, 65-84. 4. Oliverio, V . T., Kohn, K. W., and Wooley, P. V . Proc. of 11th Inter. Cancer C o n g . , Florence, 1974 (1975) 5, 238-247. 5. Skinner, W. A., G r a m , H . F., Greene, Μ. Ο., Greenberg, J., and Baker, B . R. J . Med. Pharm. Chem. (1960) 2, 299-333. 6. Hyde, Κ. A., Acton, Ε., Skinner, W. A., Goodman, L., Greenberg, J., and Baker, B. R. J . Med. Pharm. Chem. (1962) 5, 1-14. 7. Skipper, Η. Ε., Schabel, F . M., Jr., T r a d e r , M . W., and Thomson, J . R. Cancer Res. (1961) 21, 1154-1164. 8. Johnston, Τ . P., McCaleb, G . S . , and Montgomery, J . A . J . Med. Chem. (1963) 6, 669-681. 9. Johnston, Τ . P., McCaleb, G . S . , Opliger, P. S . , and Montgomery, J . A . J . Med. Chem. (1966) 9, 892-911. 10. Johnston, Τ . P., McCaleb, G . S . , Opliger, P. S . , Laster, W. R . , and Montgomery, J . A . J . Med. Chem. (1971) 14, 600-614. 11. Druckrey, H., Ivankovid, S . , and Preussmann, R. Z. Krebsforsch. (1965) 66, 389-408.


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Wunderlich, V., and Tetzlaff, I. A r c h . Geschwulstforsch. (1970) 35, 251-258. 39. Goth, R . , and Rajewsky, M . F. Cancer Res. (1972) 32, 1501-1505. 40. F r e i , J . V., and Lawley, P. D . Chem. - B i o l . Interact. (1975) 10, 413-427. 41. Kleihues, P., Stavrou, D., Scharrer, Ε., and Bucheler, J . Z . Krebsforsch. Klin. Onkol. (1973) 80, 317-322. 42. Kleihues, P., and Margison, G . P. J . Natl. Cancer Inst. (1974) 53, 1839-1841. 43. Margison, G . P., and Kleihues, P. Biochem. J . (1975) 148. 521-525. 44. Goth, R., and Rajewsky, M . F. Proc. Natl. Acad. Sci. USA (1974) 71, 639-643. 45. Lawley, P. D., and Shah, S. A . Biochem. J . (1972) 128. 117-132. 46. Lawley, P. D . Mutat 47. Wheeler, G . P. Cancer Res. (1962) 22, 651-688. 48. Wheeler, G . P., and Bowdon, B. J . Cancer Res. (1968) 28, 52-59. 49. Bowdon, B. J., and Wheeler, G . P. Proc. A m . Assoc. Cancer Res. (1971) 12, 67. 50. Oliverio, V . T., Vietzke, W. Μ., Williams, Μ. Κ., and Adamson, R. H . Proc. A m . A s s o c . Cancer Res. (1968) 9, 56. 51. O l i v e r i o , V . Τ., Vietzke, W. Μ., Williams, Μ. Κ., and Adamson, R. H . Cancer Res. (1970) 30, 1330-1337. 52. Schmall, Β . , Cheng, C . J., F u j i m u r a , S . , Gersten, Ν . , Grunberger, D., and Weinstein, I. B. Cancer Res. (1973) 33, 1921-1924. 53. Wheeler, G . P., Bowdon, B. J., and Struck, R. F . Cancer Res. (In p r e s s ) . 54. Kreling, Μ. -Ε., and McKay, A . F . Can. J. Chem. (1959) 37, 504-505. 55. Johnston, T . P., and Opliger, P. S. J . Med. Chem. (1967) 10, 675-681. 56. Serebryanyi, Α. Μ., Smotryaeva, Μ. A., Kruglyakova, Κ. Ε., and Kostyanovskii, R. G . Dokl. A k a d . , Nauk SSSR (1969) 185, 847-849. 57. Serebryanyi, Α. Μ., and Mnatsakanyan, R. M . F E B S Lett. (1972) 28, 191-194. 58. H i l l , D . L., K i r k , M . C., and Struck, R. F . Cancer Res. (1975) 35, 296-301. 59. May, Η. Ε., Boose, R . , and Reed, D. J . Biochem. B i o phys. Res. Commun. (1974) 57, 426-433. 60. Reed, D. J., and May, H . E. Life Sciences (1975) 16, 1263-1270. 61. Reed, D . J . Proc. A m . A s s o c . Cancer Res. (1975) 16, 92.


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CANCER

CHEMOTHERAPY

Wheeler, G . P., and Alexander, J . A . Cancer Res. (1974) 34, 1957-1964. Rosenoff, S. H., Bostick, F . W . , DeVita, V . Τ., and Young, R. C . Proc. A m . A s s o c . Cancer Res. (1973) 14, 77. Young, R. C . C e l l Tissue Kinet. (1973) 6, 35-43. Brereton, H . D., Bryant, T . L., and Young, R, C . Cancer Res. (1975) 35, 2420-2425. L e r m a n , M . I., Abakumova, O . Yu., Kucenco, N . G., Gorbacheva, L . Β., Kukushkina, G . V., and Serebryanyi, A . M . Cancer Res. (1974) 34, 1536-1541. Bhuyan, Β . K. Cancer Res. (1970) 30, 2017-2023. Druckrey, Η., Schagen, Β., and Ivankovic, S. Z . K r e b s forsch. (1970) 74, 141-161. V e r l y , W. G . Biochem. Pharmacol. (1974) 23, 3-8. Lawley, P. D., Lethbridge, J . Η., Edwards, P. A., and Shooter, Κ. V . J Lawley, P. D., and Brookes, P. Biochem. J . (1963) 89, 127-138. V e r l y , W. G., Barbason, Η . , Dusart, J., and PetitpasDewandre, A . Biochim. Biophys. Acta (1967) 145, 752762. Shooter, Κ. V., Howse, R . , and M e r r i f i e l d , R. K. Biochem. J . (1974) 137, 313-317. Tobey, R. Α., and C r i s s m a n , H . A . Cancer Res. (1975) 35, 460-470. Tobey, R. A., Oka, M . S., and C r i s s m a n , H . A . E u r . J. Cancer (1975) 11, 433-441. Tobey, R. A . Nature (1975) 254, 245-247. Barranco, S. C., Novak, J . Κ . , and Humphrey, R. M . Cancer Res. (1973) 33, 691-694. Barranco, S. C., Novak, J . Κ . , and Humphrey, R. M . Cancer Res. (1975) 35, 1194-1204. Hahn, G . Μ., Gordon, L . F., and Kurkjian, S. D. Cancer Res. (1974) 34, 2373-2377. F r e i , J . V., and Oliver, J . Exp. C e l l Res. (1972) 70, 49-56. Bruce, W. R., and Valeriote, F . A . " T h e Proliferation and Spread of Neoplastic C e l l s , " pp 409-420. The Williams and Wilkins Company, Baltimore, 1968. van Putten, L . Μ . , Lelieveld, P . , and Kram-Idsenga, L . K. J . Cancer Chemother. Rept. Part 1 (1972) 56, 691-700. Wilkoff, L . J., Dulmadge, Ε . A., and L l o y d , Η. H . J. Natl. Cancer Inst. (1972) 48, 685-695. Wilkoff, L . J., Dixon, G . J., Dulmadge, Ε . A., and Schabel, F . Μ . , J r . Cancer Chemother. Rept. Part 1 (1967) 51, 7-18.


5.

WHEELER

110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 121. 123.

Action

of

Nitrosoureas

Kelly, F., and Legator, M . Mutat. Res. (1971) 12, 183-190. Bhuyan, Β . Κ . , Scheldt, L . G., and F r a s e r , T . J . Cancer Res. (1972) 32, 398-407. Barranco, S. C., and Humphrey, R. M . Cancer Res. (1971) 31, 191-195. Drewinko, Β., Brown, B. W . , and Gottlieb, J . A . Cancer Res. (1973) 33, 2732-2736. Rakieten, N., Rakieten, M . L., and Nadkarni, M . V . Cancer Chemother. Rept. Part 1 (1963) 29, 91-98. Rudas, B. A r z n e i m . - F o r s c h . (1972) 22, 830-861. Schein, P . , McMenamin, Μ . , and Anderson, T . Int. Cancer Congr. A b s . 11th 1974 (No. 3) 437-438. Schein, P. S. Cancer R e s . (1969) 29, 1226-1232. Bannister, B. J . Antibiot. (1972) 25, 377-386. Schein, P. S . , Cooney, D. A., McMenamin, M . G., and Anderson, T 2625-2631. Hinz, M., Katsilambros, N., M a i e r , V., Schatz, Η., and Pfeiffer, E . F . F E B S Lett. (1973) 30, 225-228. Gunnarsson, R . , Berne, C., and Hellerstroöm, C . Biochem. J . (1974) 140, 487-494. Schein, P. S., Cooney, D . Α., and Vernon, M . L. Cancer Res. (1967) 27, 2324-2332. Wheeler, G . P., Bowdon, B. J., and Hammack, W. J . Biochem. Biophys. Res. Commun. (1973) 54, 10241029.


INDEX A Acetonide 18,19 N-Acetyl daunomycin 28 Actinomycins 21 Acute leukemia, use of adriamycin against 20,58 Adenocarcinoma 755 73,74, 83 Adriamycin 16 acidic hydrolysis of 16,18 N-acyl derivatives of 27 14-O-acyl derivatives of 30 antimitotic effects 24 antitumor activity of derivative carbonyl derivatives of 2 cell cycle phase specificities 23 in chemotherapy trials 20 clinical results 19 conversion from daunomycin 19 degradation products 18 D N A complexes of 36 formation with native D N A 21 and helical B—from D N A 26 host immunosuppression 24 human tumors responding to 20 keto reduction reaction 66 periodate treatment of 34 quaternary ammonium derivatives of 35 racemic β-alanine ester of 45 in Sarcoma 180 cells 23 semisynthetic analogs of 37 structure of 15 synthesis of 19 synthetic analogs of 44 toxic side effects 20,21 tricyclic analogs of 46 N-trifluoroacetylated esters 32 Adriamycin benzhydrazone 29 Adriamycin-14-O-octanoate 24 Adriamycinol 66 Adriamycinone 16,18 Aglycone 16,18 Aldo-keto reductase 66 Alkylating agents 71 antineoplastic effects 73 biochemistry of 81 3- ( m-Aminophenyl ) pyridine thiosemicarbazone 10 4- ( m-Aminophenyl ) pyridine thiosemicarbazone 10 5- ( m-Aminophenyl ) pyridine thiosemicarbazone 10 l-Amino-4-hydroxyanthraquinones 47

5-Amino-IQ-l Anthracycline antibiotics effects of respiration of mitochondria inhibition of nucleic acid synthesis interaction with nucleic acids mechanism of action metabolic disposition structural differences between structure of toxic side effects Anthracycline derivatives

11 16, 61 24 31, 61 21 21 66 23 58 58 31

Antineoplastic activity of benzoquinone derivatives of naphthazarin derivatives of naphthoquinone derivatives of quinoline-5,8-dione of 5- or 6-substituted naphthoquinones Antitumor activity of mitomycin C ....

73 80 73 80 78 81

Β Benzoquinones 73 antineoplastic activity 77 half-wave potential 77 Bisanhydroadriamycinone 16,18 2,3-Bis ( bromomethyl ) quinoline5,8-dione 81 2,3-Bis ( chloromethyl ) -1,4naphthoquinone 83 N,IV'-Bis ( 2-chloroethyl ) -N-nitro sourea ( B C N U ) structure 88 B C N U , decomposition in phosphate buffer 90 B C N U , inhibition of nucleic acid synthesis 103, 109 Ν,Ν'-Bis ( 2-fluoroethyl ) -Nnitrosourea 89 Bis ( isoquinoline-1-carboxaldehyde thiosemicarbazanato ) -nickel ( II ) monohydrate 3 Bis ( O-quinone methides) 73 Blood-brain barrier 99, 110 Breast cancer, use of adriamycin against 20 N-Bromoacetyldaunomycin 19,25 2-Bromomethyl-3-bromo-6,7dimethylnaphthazarin 81 Bromomethylnaphthoquinone 83 2-Bromomethyl-1,4-naphthoquinone derivatives 78

123


CANCER

124

c Carcinomycin 16, 17, 24, 30 3-Chloro-2-bromomethylnaphthoquinone 79 N - ( 2-chloroethyl ) -N'-cyclohexylN-nitrosourea ( C C N U ) 88, 92, 109 C C N U inhibition of nucleic acid synthesis 103 2-Chloroethyl isocyanate 103 N - ( 2-Chloroethyl-N',N-dimethylN-nitrosourea 99 N-(2-Chloroethyl)-N-nitrosourea 103 N - ( 2-Chloroethyl ) -N-substitutedN-nitrosoureas 89 N - ( 2-Chloroethyl ) - N ' - ( frarw-4-methylcyclohexyl)-N-nitrosourea 88 2-Chloromethy 1-3-benzamido-1,4naphthoquinone 78 2-Chloromethyl-l,4-naphthoquinon derivatives 7 Chlorotocin 101, 103 Chlorotocin tetraacetate 103 Cinerubin A 17 Cinerubins 30 Concanavalin A 24 N - ( Cyclohexylcarbamoyl ) lysine 96 N -(Cyclohexylcarbamoyl)lysine 96 Cyclophosphamide 71 2

6

Deoxythymidine triphosphate (dTTP) 5 Diabetogenic action 113 2,3-Dibromo-l,2,3,4-tetrahydro-5,8dimethoxy-2,3-methylene1,4-dioxonaphthalene 81 N , N - D i ( cyclohexylcarbamoyl ) lysine 96 Diethyl sulfate, alkylation of nucleic acid by 95 Dihydrodaunomycin 17 Dihydroquinones 75 Dihydroxynaphthacenequinones 45 N,N'-Disubstituted-N-nitrosoureas .... 87 2,3-Dimethyl-5,6-bis ( chloromethyl ) 1,4-benzoquinone 73, 76, 77 6,7-Dimethylquinoline-5,8-dione 81 DNA -adriamycin binding 25 alteration of properties 22 2

6

inhibition site inhibition of biosynthesis inhibition of polymerases Doxorubicin Drug receptor dTTP Duborimycine Duroquinone

5 5 63 15 27 5 17 76

Ε

D Daunomycin N-acyl derivatives of antimitotic effects autoradiographic experiments carbonyl derivatives of cell cycle phase specificities conformations of ring A of conversion to adriamycin degradation products - D N A complex host immunosuppression quaternary ammonium derivatives of in Sarcoma 180 cells semisynthetic analogs of Daunomycin benzhydrazone Daunomycin glycolohydrazone Daunomycinone, preparation of Daunomycinone, structural assignments of Daunorubicin inhibition of cell activities inhibition of D N A polymerases keto reduction reaction Daunorubicinol Daunosamine glycosides of structure of synthesis of 7-Deoxyadriamycinone

CHEMOTHERAPY

15,16 27 24 21 29 23 25 19 18 36,37 24 35 23 37 29 29 19 16 15 62 63 66 66 40 16,18 19 16,18

Ehrlich ascites cells Ehrlich ascites tumors Ehrlich carcinoma 4'-Epi-adriamycin 4'-Epi-daunomycin Escherici colt Ethyl methane sulfonate N-Ethyl-N-nitrosourea ( E N U ) alkylation of nucleic acid by alkylation products alterations of nucleic acids 5'-Exonuclease

109 24 1, 11, 45 38 39 110 95 88,93 95 94,95 94,95 64

F 2-Formyl-4- ( m-amino ) phenylpyridine thiosemicarbazones 9 2-Formylpyridine thiosemicarbazone (PT) ι administration to mice 3 enzyme-inhibitory activity 9 metal ion interactions 3 tumor inhibitor activity 9 1-Formylquinoline thiosemicarbazone (IQ-1) 1-3,5 Friend leukemia virus 22,63 G O-Glucuronide Glycosidase


8 66

INDEX

125

Gross leukemia Guanazole Gynecological cancers

32,38 7 20

H Half-wave potential of 2-halomethyl-l,4naphthoquinones 79 of mitomycin C 81 of some 5- or 6-substituted naphthoquinones 78 2- Halomethyl-l,4-naphthoquinones .. 79 Hepatoma 129 1,11 a- ( Ν ) -Heterocyclic carboxaldehyde thiosemicarbazones affinity for iron 3 antineoplastic activity of 1 biochemical mechanism 3 blockage of ribonucleotide reductase 5 clinical trials 7 as coordinating agents for transition metals 3 inhibition of ribonucleoside diphos phate reductase by 6 3- Hydroxy-2-formylpyridine thiosemicarbazone 6 5-Hydroxy-2-formylpyridine thiosemicarbazone ( 5-HP ) inhibition of ribonucleotide reductase 6 as inhibitor of ribonucleoside diphosphate reductase 7 as inhibitor of tumor growth 8 structure of 3 toxicity 7 treatment of cancer patients 3,7 Hydroxyurea 3,7 I Intercalative binding Isopropyl methanesulfonate

22 95

Κ Keto reduction reaction

66

L Leucine-C 5 Leukemia L1210 anthracycline antibiotics used against 23,28-33,36,39, 40,45,46,49,61, 64 D N A biosynthesis in 5 α- ( Ν ) -heterocyclic carboxaldehyde thiosemicarbazones used against 1, 5, 11 14

Leukemia L1210 (continued) nitrosoureas used against 87, 92, 96, 97, 99, 103,109,110 Leukemia P388 11 anthracycline antibiotics used against 28-33, 35, 41, 45,46,49 Lewis lung carcinoma 1 Lipophilicity 99 Lung cancer, use of adriamycin against 20 Lymphocytic leukemia 15, 24 Lysosomotropic drug action 36

M Malignant lymphomas Malony sarcoma virus

20 63

S( — )-Methoxysuccinic acid 18,19 2-Methyl-3-acetoxymethylnaphthoquinone 83 4-Methyl-5-amino IQ-1 11 Methyl methanesulfonate 95 N-Methyl-iV'-nitro-iV-nitrosoguanidine ( M N N G ) 87 alkylation of nucleic acid by 95 N-Methyl-N-nitrosourea ( M N U ) alkylation of nucleic acid by 95 alkylation products 94,95 alterations of nucleic acids 94,95 inhibition of nucleic acid synthesis 103 structure 87 Mitomycins 71, 81 Murine sarcoma virus, use of anthra cycline antibiotics against 22, 24, 38 Myelogenous leukemias, use of daunomycin against 15 Myocardiopathy 20 Ν NADH-oxidase, inhibition of 24, 82, 84 Naphthazarin derivatives 80 Naphthoquinones 73 antineoplastic activity 77 derivatives 82 half-wave potential 77 Nitrogen mustard 109 Nitrosoureas 71 alkylation products 92 alkylating activities 100, 104 biochemical effects of 103 biological effects 110 carbamoylating activities 100, 104 carbamoylation products 96 cause of cytotoxicity 112 chemical half-lives 100 decomposition modes 88 effects on macromolecular synthesis 103


CANCER

CHEMOTHERAPY

Ribonucleotide reductase Rous sarcoma virus Rubidazone Rubidomycin Rubomycin

5, 6 22 29 15 15

126 Nitrosoureas (continued) half-lives 104 marrow toxicity 100 metabolism of 98 pharmacology of 110 prevention of in vitro red cell sickling 113 properties of 104 Nogalamycin 30 Novikoff hepatoma 11 Nucleic acid(s) carbamoylation of 97 inhibition of synthesis 30, 31, 46, 47, 61,103 interaction of anthracycline antibiotics with 21 synthesis 22

Partition coefficient T4 Phage-induced polymerases 3-Phenyl-2-chloromethyl-l,4naphthoquinone N-Propyl-N-nitrosourea Pyrromycin

S Sarcoma 180 anthracycline antibiotics used against 23, 38, 45 D N A biosynthesis in 5 a- ( Ν ) -heterocyclic carboxaldehyde thiosemicarbazones used against 1, 5, 7-9, 11 naphthoquinone derivatives used against 73-81, 83 Sarcomas, adriamycin used against .... 20 Streptozotocin 103 Succinoxidase 24, 82, 84

10 64 79 103 17

Τ Triazine derivatives 1,6,10,11-Tetrahydr oxynaphthacene5,12-dione Thymidine-H Thymidine kinase Thymidylate synthetase N-Trifluoroacetyladriamycin14-O-valerate N-Trifluoroacetylated adriamycin14-O-carboxylates 3

Quinoline-5,8-dione O-Quinone methides

80 72

R Rausher sarcoma virus Rhodomycin β-Rhodomycin I Ribonucleoside diphosphate reductase

63 30 17 6,11

71 16,18 5 5 5 33 33

U Uridine-H

3


5

Cancer chemotherapy

Cancer chemotherapy

Handbook of Cancer Chemotherapy

Fischer Cancer Chemotherapy Handbook