Cancer Chemotherapy - ACS Publications - American Chemical Society

2 Adriamycin DAVID W. HENRY

Downloaded by CORNELL UNIV on July 19, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0030.ch002

Stanford Research Institute, Menlo Park, Calif. 94025

I n t r o d u c t i o n . Adriamycin (1, a l s o c a l l e d doxorubicin) is an a n t h r a c y c l i n e a n t i b i o t i c and w a s " f i r s t i s o l a t e d i n the l a t e 1960s by Arcamone and colleagues i n the F a r m i t a l i a research l a b o r a t o r i e s (1,2). I t is produced by a mutant s t r a i n of Streptomyces peucetius, the microorganism that produces the c l o s e l y r e l a t e d a n t i b i o t i c daunomycin (2). Daunomycin (also known as rubidomycin, daunorubicin and rubomycin) was independently discovered i n three l a b o r a t o r i e s s e v e r a l years e a r l i e r than adriamycin (3-5) and has

0

OH

0

OH

HO 1 2

R = OH adriamycin R = H daunomycin

achieved a s i g n i f i c a n t place i n the treatment of acute lymphocytic and myelogenous leukemias (6,7). Adriamycin i s a l s o an a c t i v e antileukemic drug but is of much greater i n t e r e s t because of its activity against a broad spectrum of s o l i d tumors (8-10). I t i s the o b j e c t i v e of t h i s paper to briefly summarize s i g n i f i c a n t background data on the chemical, biochemical and clinical p r o p e r t i e s of adriamycin and daunomycin and subsequently to d e s c r i b e more fully the status of s t r u c t u r e - a c t i v i t y s t u d i e s stemming from the

15

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


16

CANCER

CHEMOTHERAPY

parent a n t i b i o t i c s . I t is appropriate and necessary to i n c l u d e considerable information on daunomycin because of the c l o s e r e l a t i o n s h i p between the chemical and biochemical p r o p e r t i e s of the two a n t i b i o t i c s and because daunomycin has been a v a i l a b l e f o r study longer than adriamycin. 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 are produced by the s t r e p o mycetes and are c h a r a c t e r i z e d by the presence of a t e t r a h y d r o naphthacene quinone moiety. They bear s e v e r a l a d d i t i o n a l oxygen f u n c t i o n s i n the aromatic and saturated p o r t i o n s of the aglycone and v a r i a t i o n i n the number and p o s i t i o n of these s u b s t i t u e n t s ( u s u a l l y hydroxyls) d i s t i n g u i s h i n d i v i d u a l compounds. Many members of t h i s group are known, l a r g e l y through the work of Brockmann (11,12). S i g n i f i c a n t c y t o t o x i c a c t i v i t y i s almost always a s s o c i a t e d with b a s i c g l y c o s i d e s of the parent nucleus, r a t h e r than the aglycones themselves, and complete s t r u c t u r a l information i s o f t e n l a c k i n g f o r these sugar conjugates. A second group of a n t h r a c y c l i n e s with i n v i t r o and/or i n v i v o c y t o t o x i c character are given i n Figure 1. With the exception of carminomycin, these compounds have been studied comparatively l i t t l e and they w i l l not be f u r t h e r discussed i n any d e t a i l . Carminomycin (3) has r e c e i v e d much a t t e n t i o n i n the USSR (13-21) s i n c e f i r s t reported by Gauze et a l . i n 1973 and data on i t w i l l be c i t e d where p e r t i n e n t . Chemistry. The s t r u c t u r a l assignment of adriamycin r e s t s h e a v i l y on a c o r r e l a t i o n with that of daunomycin (22). M i l d a c i d i c h y d r o l y s i s of adriamycin y i e l d s the aglycone, a d r i a mycinone (8, F i g u r e 2), and the amino sugar, daunosamine (10)· The u l t r a v i o l e t and v i s i b l e s p e c t r a of 8 were i d e n t i c a l to those of the parent and daunomycin, thus e s t a b l i s h i n g the chromophore s t r u c t u r e . D e t a i l e d s p e c t r a l analyses of 8, 7-deoxy-adriamycinone (9), bisanhydroadriamycinone (11), and t h e i r a c e t y l a t i o n products f u r t h e r e s t a b l i s h e d the c l o s e r e l a t i o n s h i p to daunomycin and f i x e d the p o s i t i o n of the a d d i t i o n a l hydroxyl group i n the a c e t y l side c h a i n . The s t r u c t u r e of daunomycin was e s t a b l i s h e d i n two stages. The o r i g i n a l chemical p u b l i c a t i o n s by Arcamone et a l . (23,24) described the h y d r o l y s i s to the aglycone, daunomycinone, and the amino sugar, daunosamine. The major s t r u c t u r a l f e a t u r e s of daunomycinone were determined by a combination of s p e c t r a l analyses and chemical degradation, l e a v i n g the stereochemistry of the tetrahydro r i n g and the l o c a t i o n s of the methoxy and g l y c o s i d i c groups u n s p e c i f i e d . Several years l a t e r (25,26) the I t a l i a n group completed the s t r u c t u r a l assignments of daunomycinone by a f u r t h e r degradative sequence y i e l d i n g 1,6,10,11-tetrahydroxynaphthacene-5,12-dione d e r i v a t i v e s (e.g., 12) whose s p e c t r a l p r o p e r t i e s s p e c i f i e d the c o r r e c t h y d r o x y l a t i o n p a t t e r n . Simultaneously they succeeded i n f i x i n g the g l y c o s i d i c l i n k at the 7hydroxy by c a r e f u l nmr a n a l y s i s of daunomycin pentaacetate and by c a t a l y t i c r e d u c t i o n of daunomycin to 7-deoxydaunomycinone.


2.

HENRY

HO

17

Adriamycin

0

OH

0

OH

Ô

MeO

0

OH


NH 3

carminomycin

4

2

dihydrodaunomycin (duborimycine)

OH 5

ό

0

OH

β-rhodomycin I (rhodomycin B)

6

cinerubin A

NMe

2

7

ό

pyrromycin Figure 1.

Some anihracycline antibiotics with cytotoxic activity


18

CANCER

OH


0

CHEMOTHERAPY

14 15 Figure 2.

R=H R = Me

Degradation products used for structure proof of adriamycin and daunomycin


2.

HENRY

Adriamycin

19


The absolute stereochemistry of daunomycin followed from the i s o l a t i o n of carbon atoms 6a, 7, 8 and 9 as S(-)-methoxy-succinic a c i d (13) and the s p e c i f i c a t i o n of a c i s o r i e n t a t i o n f o r the 7and 9-hydroxyls v i a acetonide 14. As daunosamine had been f u l l y i d e n t i f i e d i n the e a r l i e r work (24) and pmr a n a l y s i s of dauno mycin pentaacetate r e q u i r e d the α-glycosidic c o n f i g u r a t i o n , these r e s u l t s f u l l y c h a r a c t e r i z e d a l l chemical and s t e r i c f e a tures of the molecule. Almost simultaneously Iwamoto et a l . (27) a r r i v e d at s i m i l a r c o n c l u s i o n s regarding the b a s i c s t r u c t u r e and r e l a t i v e stereochemistry of daunomycin through 220 Mc pmr spec t r a l a n a l y s i s of the parent a n t i b i o t i c and by p r e p a r a t i o n of acetonide 15. An X-ray c r y s t a l l o g r a p h i c a n a l y s i s of N-bromoacetyldaunomycin confirmed the assigned s t r u c t u r e (28). In a formal sense a t o t a l s y n t h e s i s of adriamycin has been completed i f the work of s e v e r a l groups of i n v e s t i g a t o r s are combined. Arcamone and h i s a s s o c i a t e s succeeded i n c o n v e r t i n g daunomycin to adriamycin i n a seven-step process i n 1969 (29), thus p e r m i t t i n g the i n c o r p o r a t i o n of a daunomycin s y n t h e s i s i n t o an adriamycin s y n t h e s i s . Daunosamine was f i r s t prepared from L-rhamnose i n twelve steps by Marsh et a l . i n 1967 (30). A l e n g t h i e r s y n t h e s i s from glucose was r e p o r t e d by Yamaguchi and Kojima s e v e r a l years l a t e r (31). Very r e c e n t l y Horton and Weckerle d e s c r i b e d a h i g h - y i e l d process f o r transforming D-mannose to daunosamine (32). Daunomycinone was prepared i n 1973 by Wong et a l . (33). S e v e r a l r e l a t e d s t u d i e s accompanied t h i s major accomplishment (34-37), e s p e c i a l l y noteworthy being the recent r e g i o s p e c i f i c s y n t h e s i s of 9-deoxydaunomycinone by Kende et a l . (38). The f i n a l segment i n the t o t a l s y n t h e s i s of a d r i a mycin was provided by Acton et a l . who e f f e c t e d c o u p l i n g of daunosamine with daunomycinone to g i v e daunomycin (39). The syn t h e s i s of adriamycin provided by combining these s y n t h e t i c u n i t s t o t a l s w e l l over 40 steps; o b v i o u s l y there i s ample opportunity for an improved r o u t e . C l i n i c j l ^J l e ^ ^ . Although a f u l l d i s c u s s i o n of c l i n i c a l r e s u l t s i s beyond the scope of t h i s paper i t i s a p p r o p r i a t e to b r i e f l y note the reasons f o r the c u r r e n t i n t e n s e i n t e r e s t i n adriamycin as an antitumor drug. I t i s not only a c t i v e against some s o l i d tumors i n man but a g a i n s t a wide spectrum of these tumors, many of which are p o o r l y or nonresponsive to other drugs. As a c l a s s s o l i d tumors are most r e s i s t a n t to chemotherapy and past progress has been g r e a t e s t i n the disseminated malignancies. Table I provides s e l e c t e d data from a recent review by C a r t e r and Blum on the use of adriamycin a g a i n s t human cancer (8). A response was d e f i n e d i n t h i s study as a g r e a t e r than 50% r e d u c t i o n i n tumor mass except i n the case of acute leukemia. While none of these response r a t e s approach 100%, i t i s important to note that the r e s u l t s have been obtained i n many cases from p a t i e n t s i n which other treatments ( f r e q u e n t l y i n c l u d i n g chemotherapy) have p r e v i o u s l y f a i l e d . Regarding


20

CANCER

Table I.

CHEMOTHERAPY

Some Human Tumors Responding to A d r i a m y c i n . a

Breast cancer Sarcomas Lung cancer Malignant lymphomas Acute leukemia


a

36% 26% 19% 41% 24%

(44/121) (46/176) (44/229) (61/147) (47/195)

D a t a from r e f . 8. Numbers i n parentheses are responding p a t i e n t s over évaluable patients.

adriamycin's broad spectrum of a c t i v i t y Carter r e c e n t l y reported that adriamycin i s c l e a r l y a c t i v e against 9 of 19 human tumors on which treatment s t a t i s t i c s are s a t i s f a c t o r y and c l e a r l y i n a c t i v e i n only three of them (40). Another s i g n i f i c a n t p o i n t regarding the data i n Table I i s that they are based upon s i n g l e drug treatment. Many of the most e f f e c t i v e chemotherapeutic regimens f o r cancer are now based upon combinations of drugs, not the use of s i n g l e e n t i t i e s (41). Consequently adriamycin i s c u r r e n t l y being studied i n a broad a r r a y of combination chemotherapy t r i a l s (42,43). Among e a r l y r e s u l t s of these s t u d i e s Jones et a l . report an 80% response r a t e i n a group of 50 breast cancer p a t i e n t s using a cyclophosphamide-adriamycin combinat i o n (44). G o t t l i e b et a l . have shown i n a spectrum of sarcomas that adriamycin plus 4(5)-dimethyltriazeno i m i d a z o l e - 5 ( 4 ) carboxamide (DIC), with and without v i n c r i s t i n e , provides a 50% response r a t e , as opposed to a 30% response with s i n g l e drugs (45). Combined modality approaches to cancer therapy are i n an e a r l y s t a t e of development but e a r l y r e s u l t s here are a l s o very promi s i n g . Watring et a l . r e c e n t l y reported s u b s t a n t i a l b e n e f i t s by simultaneous use of adriamycin with X-ray treatment i n a small group of p a t i e n t s with advanced g y n e c o l o g i c a l cancers (46). Adriamycin i s subject to the t o x i c side e f f e c t s common to many antitumor drugs. Table I I summarizes the incidence of the most common t o x i c r e a c t i o n s , as reviewed by C a r t e r and Blum (47). Although of high frequency these e f f e c t s are g e n e r a l l y r e v e r s i b l e and manageable except the myocardiopathy. T h i s i s expressed most o f t e n as a r a p i d l y p r o g r e s s i n g syndrome of congestive heart f a i l u r e and c a r d i o r e s p i r a t o r y decompensation. I f detected e a r l y t h i s c o n d i t i o n may be r e v e r s i b l e (48) but f r e q u e n t l y has not responded w e l l to treatment (49). The c a r d i o - t o x i c e f f e c t s are dose r e l a t e d and a cumulative dose below 500 mg/M y i e l d s a very low i n c i d e n c e of myocardiopathy (49). In p a t i e n t s r e c e i v i n g a greater t o t a l dose the i n c i d e n c e climbs r a p i d l y and reached 30% i n one r e t r o s p e c t i v e a n a l y s i s (49). C a r d i o t o x i c i t y i s the s i d e e f f e c t most l i m i t i n g to the use of adriamycin at the present time because treatment must be stopped while the tumor i s s t i l l 2


2.

HENRY

Adriamycin

Table

21

II.

Incidence of Common Toxic E f f e c t s of A d r i a m y c i n . a

Alopecia Nausea/vomit ing Stomatitis Leukopenia

100% 20-55% 80% 60-75%

Cardiac i r r e g u l a r i t i e s EKG Myocardiopathy

6-30% 0.4-1.2%


âta

from r e f .

responding to the drug. i s a l s o w e l l documented

47.

The c a r d i o t o x i c character of daunomycin (50).

^ S h m i s m ^ ^ c j ^ A c t i o n . D i Marco and h i s a s s o c i a t e s have done the m a j o r i t y of mechanistic s t u d i e s on 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 and Arcamone and D i Marco have r e c e n t l y reviewed t h i s subject (51). The m a j o r i t y of i n v e s t i g a t i o n s i n the area have used daunomycin as the subject but many s t u d i e s i n d i c a t e that adriamycin a c t s very s i m i l a r l y at the molecular l e v e l . Both a n t i b i o t i c s r a p i d l y i n h i b i t n u c l e i c acid synthesis i n various cultured c e l l l i n e s (e.g., 52-56) and i n c e l l s of tumor bearing a n i mals (52,57). T h i s phenomenon i s thought to be the primary b i o chemical l e s i o n caused by the drugs and to be due to s e l e c t i v e b i n d i n g of drug to nuclear DNA. A l a r g e body of b i o p h y s i c a l and biochemical data support t h i s c o n c l u s i o n . Autoradiographic experiments c l e a r l y show, f o r example, that daunomycin concen t r a t e s i n the n u c l e i of tumor (KB) and normal ( r a t l i v e r ) c e l l s i n c u l t u r e , and f l u o r e s c e n c e quenching i n drug-treated c e l l n u c l e i a l s o supports t h i s contention (58). In v i t r o b i o p h y s i c a l s t u d i e s have e s t a b l i s h e d that daunomycin and adriamycin form s t a b l e complexes with n a t i v e DNA and that the aglycone p o r t i o n of the drugs i n t e r c a l a t e s between base p a i r s of the DNA h e l i x i n the complex (51). C h a r a c t e r i s t i c a l t e r a t i o n s i n the u l t r a v i o l e t and the v i s i b l e spectrum of the drug (bathochromic s h i f t and decreased absorption) demonstrate a s i g n i f i c a n t drug-DNA a s s o c i a t i o n , as does a pronounced decrease of daunomycin f l u o r e s cence i n the presence of DNA. DNA a l t e r s drug chemistry as w e l l , i n h i b i t i n g i o n i z a t i o n of the p h e n o l i c hydroxyls at h i g h pH and v i r t u a l l y e l i m i n a t i n g s u s c e p t i b i l i t y to p o l a r o g r a p h i c reduc t i o n (59,60). Apparent b i n d i n g constants f o r t h i s process (2.3-3.3 χ 10 M" ) are of the same magnitude as f o r the a c t i n o mycins, another s t r o n g l y i n t e r a c t i n g group of DNA i n t e r c a l a t i n g drugs (61). T h i s strong b i n d i n g process s a t u r a t e s at about one drug molecule per f i v e n u c l e o t i d e s f o r adriamycin and about s i x 6

1


22

CANCER

CHEMOTHERAPY

n u c l e o t i d e s f o r daunomycin (60). At high drug:DNA r a t i o s f u r t h e r drug b i n d i n g occurs by weaker processes. Daunomycin binding to denatured DNA i s reduced by a f a c t o r of 20 but the number of s i t e s remains the same. The p r o p e r t i e s of DNA are a l t e r e d by complexation with daunomycin and adriamycin. As expected f o r i n t e r c a l a t i v e b i n d i n g (62, 63) the complex sediments more slowly and has a lower buoyant d e n s i t y than the DNA i t s e l f . The v i s c o s i t y of s o l u t i o n s of a n t i b i o t i c complexes increases with i n c r e a s i n g drug:DNA r a t i o s , a l s o d i a g n o s t i c f o r i n t e r c a l a t i v e b i n d i n g . In a d d i t i o n the drugs s t a b i l i z e h e l i c a l DNA to thermal dénaturâtion, increases i n melting temperature (AT ) of 14.8° and 13.4° being observed f o r a d r i a mycin and daunomycin, r e s p e c t i v e l y , i n 0.01 M pH 7.0 T r i s b u f f e r at a drug:DNA r a t i o of 1:10 (60). More d i r e c t evidence f o r the i n t e r c a l a t i v e binding mechanism has a l s o been found. Dall'acqua et a l . determined by flow d i chroism experiments that the chromophores of bound daunomycin and adriamycin are p e r p e n d i c u l a r to the DNA h e l i x a x i s (64). Waring demonstrated that i n c r e a s i n g c o n c e n t r a t i o n s of daunomycin e f f e c t r e v e r s a l of the s u p e r c o i l s of 0X174 closed c i r c u l a r DNA, a phenomenon considered h i g h l y d i a g n o s t i c f o r i n t e r c a l a t i o n (65). Pigram et a l . studied f i b e r s of DNA-daunomycin complex by X-ray d i f f r a c t i o n and concluded that the p a t t e r n s obtained were c o n s i s t e n t only w i t h i n t e r c a l a t i v e b i n d i n g (66). These l a t t e r authors a l s o proposed a s p e c i f i c molecular model f o r the complex i n which the aglycone was l a r g e l y overlapped by adjacent base p a i r s and the daunosamine s i d e chain of the drug p r o j e c t e d i n t o the major groove of the h e l i x with the ammonium moiety i n t e r a c t i n g i o n i c a l l y with a phosphate one base p a i r away from the i n t e r c a l a t i o n s i t e . The p o s s i b i l i t y of a hydrogen bond between the 9-hydroxy and an a d j a cent phosphate was a l s o suggested. I t i s of i n t e r e s t that daunomycin binds only weakly to double stranded RNA and probably by a mechanism i n v o l v i n g only e l e c t r o s t a t i c i n t e r a c t i o n s (67). Consistent with the DNA complexing p r o p e r t i e s of these a n t i b i o t i c s s e v e r a l i n v e s t i g a t o r s have reported that DNA and RNA synthesis i n v a r i o u s i s o l a t e d polymerase systems (mammalian, b a c t e r i a l , v i r a l ) i s i n h i b i t e d by daunomycin and adriamycin and that t h i s i s due to drug-template b i n d i n g r a t h e r than d i r e c t i n h i b i t i o n of the enzyme (56,68-72). Recent evidence suggests that adenine-thymine templates are most s e n s i t i v e to t r a n s c r i p t i o n i n h i b i t i o n by daunomycin but t h i s p o i n t i s not f i r m l y establ i s h e d (70). I n h i b i t i o n of v i r a l polymerase systems, both RNA-directed (69, 72-75) and DNA-directed (76), has been i n v e s t i g a t e d e x t e n s i v e l y . In one comparative study of adriamycin and daunomycin i n h i b i t i o n of DNA polymerases from murine sarcoma v i r u s , r a t l i v e r and bact e r i a , the v i r a l enzyme was the most s e n s i t i v e (69). The a n t i b i o t i c s have been shown e f f e c t i v e i n v i v o against tumors induced by murine sarcoma v i r u s (77), F r i e n d leukemia v i r u s and Rous sarcoma v i r u s (74,75).


m


2.

HENRY

Adriamycin

23

The c e l l c y c l e phase s p e c i f i c i t i e s of daunomycin and a d r i a mycin have been i n v e s t i g a t e d by s e v e r a l groups but general c o n c l u sions cannot yet be drawn. Tobey (78) and Kim et a l . (79,80) report both a n t i b i o t i c s to be S-phase s p e c i f i c i n Chinese hamster and HeLa c e l l s , r e s p e c t i v e l y . Bhuyan and F r a s e r concluded s i m i l a r l y f o r adriamycin i n Chinese hamster f i b r o b l a s t s (81). However a v e r y d e t a i l e d study by S i l v e s t r i n i et a l . i n d i c a t e d that daunomycin caused d e t e c t a b l e i n h i b i t i o n of DNA and/or RNA synthesis i n the G}, S and G phases of the c e l l c y c l e i n r a t f i b r o b l a s t s (82). Mizuno et a l . (71) report no phase s p e c i f i c i t y f o r t r i t i a t e d daunomycin uptake by L - c e l l s i n c o n t r a s t to the r e s u l t s of S i l v e s t r i n i et a l . (83) who found uptake maximal i n the l a t e S-phase. Mizuno et a l . noted maximal c y t o t o x i c i t y by daunomycin i n the l a t e S, G and M phases but an e f f e c t was evident at a l l p o i n t s i n the c e l l c y c l e (71). 2


2

In view of the v e r y minor s t r u c t u r a l d i f f e r e n c e between adriamycin and daunomycin, numerous i n v e s t i g a t o r s have sought to determine the b a s i s f o r the chemotherapeutic s u p e r i o r i t y u s u a l l y seen with the former (2,84). At the enzymic l e v e l Tatsumi et a l . found the two drugs to be e s s e n t i a l l y equivalent as i n h i b i t o r s of L1210-cell DNA polymerase (56) but Zunino and c o l l a b o r a t o r s (68) found adriamycin a somewhat more potent i n h i b i t o r than daunomycin i n s e v e r a l c e l l - f r e e DNA and RNA polymerase systems. Slightly s u p e r i o r potency f o r adriamycin over daunomycin was reported e a r l i e r i n v i r a l DNA polymerase systems (69,76) but the d i f f e r ences are small and may vary w i t h the template employed (74). At the c e l l u l a r l e v e l adriamycin has u s u a l l y been found somewhat l e s s potent than daunomycin when judged by c r i t e r i a such as c y t o t o x i c i t y , a n t i m i t o t i c index and i n h i b i t i o n of DNA s y n t h e s i s , but the d i f f e r e n c e i s u s u a l l y q u i t e small (54,55,79,85,86) and not always evident (57) . Daunomycin i s i n i t i a l l y taken up by c e l l s more r a p i d l y than adriamycin however (54,56,57,87) and t h i s absorption d i f f e r e n c e i s c i t e d by Zunino et a l . (68) as the probable reason f o r the apparent s u p e r i o r i t y of daunomycin i n L1210 c e l l systems. S i l v e s t r i n i et a l . noted that the h a l f - l i f e of adriamycin i n Sarcoma 180 c e l l s i n mice was s u b s t a n t i a l l y greater than daunomycin (12 hr vs 4 hr) and that adriamycin was i n h i b i t o r y to RNA s y n t h e s i s i n these c e l l s at l e v e l s where daunomycin was i n e f f e c t i v e (57). In a d d i t i o n these i n v e s t i g a t o r s found that daunomycin k i l l e d c e l l s more q u i c k l y than adriamycin but that those c e l l s s u r v i v i n g the daunomycin were capable of r a p i d l y resuming p r o l i f e r a t i o n . In c o n t r a s t adriamycin had a slower c y t o t o x i c onset but fewer c e l l s were v i a b l e on attempted c l o n i n g a f t e r drug exposure. Razek et a l . (86) a l s o noted s i g n i f i c a n t d i f f e r ences between the two drugs. They found AKR leukemia c e l l s to be more s e n s i t i v e to both drugs than normal hematopoietic stem c e l l s from the mouse. However the d i f f e r e n t i a l t o x i c i t y between the two c e l l types ( T D 5 0 normal c e l l s / T D s o AKR c e l l s ) f o r adriamycin was n e a r l y twice as l a r g e as that f o r daunomycin (6.3 vs 3.6).



24

CANCER

CHEMOTHERAPY

One of the most 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 daunomycin l i e s i n the area of host immunosuppression. Casazza et a l . reported that adriamycin i n t e r f e r e d l e s s with the host-mediated spontaneous r e g r e s s i o n of murine sarcoma v i r u s (Maloney) induced tumors than d i d daunomycin (88). Schwartz and Grindey found that the s u b s t a n t i a l therapeutic advantage of a d r i a mycin and daunomycin i n t r e a t i n g P288 lymphocytic leukemia i n mice i s l o s t when the host i s immunosuppressed by whole-body i r r a d i a t i o n or cyclophosphamide treatment p r i o r to tumor implantation (87, 89). They a l s o found that daunomycin uptake by spleen was twice that of adriamycin whereas l i v e r , thymus and tumor c e l l s absorbed the two drugs e q u a l l y . I t i s c l e a r that adriamycin and daunomycin have profound d e l e t e r i o u s e f f e c t s on n u c l e i c a c i d synthesis and t h i s property must be involved i n t h e i r mechanism of a c t i o n . However, not a l l DNA-related b i o l o g i c a l e f f e c t s can be d i r e c t l y a t t r i b u t e d to synt h e s i s i n h i b i t i o n . For example, S i l v e s t r i n i et a l . noted that a n t i m i t o t i c e f f e c t s were evident i n c u l t u r e d mouse c e l l s at daunomycin l e v e l s much too low to a f f e c t n u c l e i c a c i d synthes i s (82). They a l s o demonstrated that the drug abruptly blocked m i t o s i s when given only a few minutes before prophase. Similarly Schwartz r e c e n t l y noted that severe chromosome breakage was e v i dent i n the tumor c e l l s of daunomycin/adriamycin treated P288bearing mice w i t h i n 1-3 hr a f t e r treatment (90). Such breakage i s not an i n t r i n s i c property of the drug-DNA i n t e r a c t i o n however as i s o l a t e d DNA i s not degraded when incubated with daunomycin (71). Several p o s s i b l e mechanisms of a c t i o n f o r the a n t h r a c y c l i n e s have been suggested that are apparently unrelated to DNA metabol i s m . Gosalvez et a l . i n v e s t i g a t e d the e f f e c t s of adriamycin and daunomycin on the r e s p i r a t i o n of i s o l a t e d mitochondria and of i n t a c t normal and tumor c e l l s and found s i g n i f i c a n t i n h i b i t i o n (91). S i m i l a r l y F o l k e r s and co-workers determined i n v i t r o that a d r i a mycin, carminomycin, daunomycin and adriamycin-14-0-octanoate i n h i b i t e d succinoxidase and NADH-oxidase, r e s p i r a t o r y chain enzymes that r e q u i r e coenzyme Qiq as c o f a c t o r (92). Comparatively high drug l e v e l s were r e q u i r e d to e l i c i t these e f f e c t s i n both s t u d i e s . A l t e r a t i o n of c e l l surface a r c h i t e c t u r e i s a new e f f e c t of adriamycin r e c e n t l y reported by Murphree et a l . (93). Using a new assay technique (94) they found that concanavalin A-induced a g g l u t i n a t i o n of S180 c e l l s was enhanced s e v e r a l f o l d f o l l o w i n g drug exposure. As the amount of l e c t i n bound was unchanged t h i s suggests that adriamycin a f f e c t s the c l u s t e r i n g of concanavalin A binding s i t e s . The p o s s i b i l i t y that adriamycin and daunomycin act by i n t e r ference with microtubule f u n c t i o n during m i t o s i s has been r a i s e d by the work of Dano (95,96). Induced r e s i s t a n c e to v i n c r i s t i n e , v i n b l a s t i n e or the two a n t h r a c y c l i n e s i n E h r l i c h a s c i t e s tumors was accompanied by c r o s s - r e s i s t a n c e to each of the others; v i n c r i s t i n e and v i n b l a s t i n e are thought to act p r i m a r i l y on microtubules.


2.

HENRY

Adriamycin

25


A l l of the above i n v e s t i g a t i o n s on non-DNA-related e f f e c t s of adriamycin and daunomycin are at p r e l i m i n a r y stages but the p o s s i b i l i t y that these drugs a c t by m u l t i p l e mechanisms must now be s e r i o u s l y considered. Molecular Models. Based upon the evidence reviewed above (and ô^T^soê^^dliîa^nÎt d e s c r i b e d here) Arcamone and D i Marco (51) concluded that DNA-adriamycin complex i n v o l v e s three types of b i n d i n g : hydrophobic i n t e r a c t i o n due to the i n t e r c a l a t e d a g l y cone, e l e c t r o s t a t i c a t t r a c t i o n between the protonated 3'-amino group of the daunosamine and phosphate groups of the h e l i x , and hydrogen bonds of u n s p e c i f i e d c h a r a c t e r . The molecular model proposed by Pigram et a l . (66) f o r the daunomycin-DNA complex i s c o n s i s t e n t w i t h these c o n c l u s i o n s . The l a t t e r authors employed a conformation f o r the daunomycin A r i n g s i m i l a r to that found i n the c r y s t a l s t r u c t u r e of N-bromoacetyldaunomycin (Figure 3a) (28).

Figure 3.

Possible conformations of ring A of daunomycin

I f another conformation of r i n g A i s p o s t u l a t e d (Figure 3b) a model i s p o s s i b l e which s i m i l a r l y permits the chromophore to i n t e r c a l a t e between base p a i r s and the protonated amino sugar i n the major groove to form an e l e c t r o s t a t i c bond w i t h a DNA phosphate l o c a t e d one n u c l e o t i d e away from the i n t e r c a l a t i o n s i t e . However the l a t t e r conformation a l s o c l e a r l y allows the 9-hydroxy to hydrogen bond f i r m l y t o the phosphate adjacent to the i n t e r c a l a t i o n gap and the 4'-hydroxy to probably form a hydrogen bond to the phosphate two n u c l e o t i d e s away from the i n t e r c a l a t i o n s i t e . A t h i r d hydrogen bond i s p o s s i b l e between the 14-hydroxyl and N-7 of a p u r i n e base i n the adjacent base p a i r . T h i s a l t e r n a t e model complex, i l l u s t r a t e d i n F i g u r e 4, i s c o n s i s t e n t w i t h a l l of the b i o p h y s i c a l data and e s p e c i a l l y emphasizes the importance of the hydrogen bonding f o r c e s that are known to c o n t r i b u t e s i g n i f i c a n t l y to the s t a b i l i t y of the complex (97).



26

CANCER

CHEMOTHERAPY

Figure 4. Proposed intercalative complex of adriamycin and helical B-form DNA as viewed from the major groove. Much DNA structure has been omitted to clarify specific bonding points. Four consecutive phosphate groups from one polydeoxyribosephosphate chain are shown extending from the upper right to the lower middle positions of the drawing. The phosphate ester links in this chain are in the 3 ' —» 5 ' direction starting from the upper right. Part of a thymine-adenine base pair is shown immediately above the aglycone. The drawing is based on a CPK molecular model.



2.

HENRY

Adriamycin

27

The drug f i t i n t h i s model suggests a preference f o r an adenine-thymine base p a i r on the same s i d e of the i n t e r c a l a t e d aglycone as the sugar r e s i d u e , with the thymine l o c a t e d adjacent to the methoxy-bearing D - r i n g . However a l l combinations of adenine-thymine and guanine-cytosine bases around the aglycone can be accommodated except those that p l a c e the thymine toward r i n g A and on the same s i d e of the molecule as the sugar r e s i d u e . In these cases the thymine methyl s t e r i c a l l y prevents formation of the i o n i c and hydrogen bonds a s s o c i a t e d with that r e g i o n of the complex. The a l t e r n a t e conformation p o s t u l a t e d f o r r i n g A (Figure 3b) i n the model of F i g u r e 4 p l a c e s the 9 - a c e t y l group i n an a x i a l p o s i t i o n and the 7- and 9-oxygen f u n c t i o n s e q u a t o r i a l . T h i s i s c o n t r a r y to the c r y s t a l l o g r a p h i c a l l y determined conformation of N-bromoacetyldaunomycin (28) and the conformation u s u a l l y found i n other a n t h r a c y c l i n o n e A r i n g s (38) ( i . e . , F i g u r e 3a), thus r a i s i n g the q u e s t i o n of i t s p r o b a b i l i t y i n a DNA complex. No f i r m energ e t i c j u s t i f i c a t i o n has been made but the 3a ·*• 3b conformational change p l a c e s the l a r g e sugar moiety e q u a t o r i a l , p o s s i b l y a n o t too-unequal exchange f o r the a x i a l placement of the a c e t y l . In a d d i t i o n the s i g n i f i c a n t energy g a i n i n forming the complex might be expected to more than o f f s e t any energy r e q u i r e d to achieve the new conformation. O v e r a l l , the proposed r e c e p t o r complex p i c t u r e d i n F i g u r e 4 seems s a t i s f a c t o r y because a maximum number of drug f u n c t i o n a l groups have been used i n the b i n d i n g process and there are no unf i l l e d c a v i t i e s between drug and DNA or dangling p a r t s of the drug l e f t unused. The model o f f e r s something r a r e i n m e d i c i n a l chemist r y , the o p p o r t u n i t y to work with a reasonably p o s s i b l e , t h r e e dimensional, l a r g e l y c h e m i c a l l y - d e f i n e d drug-receptor s t r u c t u r e . The concept of the s p e c i f i c r e c e p t o r s i t e has permeated much of drug r e s e a r c h i n recent years but f u l l use of the idea has r a r e l y been p o s s i b l e because the d e t a i l e d chemical s t r u c t u r e of the r e c e p t o r s are not known. H e l i c a l DNA i s one of the very few b i o l o g i c a l macromolecules of known importance as a drug r e c e p t o r that can be d e s c r i b e d i n chemical and three-dimensional terms. In the f o l l o w i n g d i s c o u r s e the model w i l l be d i s c u s s e d i n terms of i t s a b i l i t y to r a t i o n a l i z e t e s t data among a n t h r a c y c l i n e d e r i v a t i v e s and analogs. Daunomycin and Âîamycin Dérivât The c a r b o n y l group at C-ÎTôT^dajji^ selectively derivat i z e d by standard carbonyl reagents and, as would be expected, the amino group i s a l s o s e l e c t i v e l y attacked by a c y l a t i n g agents. For t h i s reason d e r i v a t i v e s on these f u n c t i o n a l groups r e c e i v e d e a r l y a t t e n t i o n i n s t r u c t u r e - a c t i v i t y s t u d i e s (51,98). Most i n v e s t i g a t i o n s on these c l a s s e s of d e r i v a t i v e s used daunomycin as s u b s t r a t e but the l i m i t e d work on s i m i l a r adriamycin d e r i v a t i v e s suggests comparable e f f e c t s . An important part of the data used by Arcamone and D i Marco to reach t h e i r c o n c l u s i o n s r e g a r d i n g the


CANCER

28

CHEMOTHERAPY


DNA binding mode and mechanism of a c t i o n of the a n t h r a c y c l i n e s was derived from t h i s type of d e r i v a t i v e (51). In general, formation of an N-acyl d e r i v a t i v e (amide, urea, thiourea) of daunomycin r e s u l t s i n a marked decrease or t o t a l l o s s of i n v i v o antitumor a c t i v i t y i n experimental animal screens. Potency i s always decreased markedly and u s u a l l y e f f i c a c y as w e l l . That t h i s i s not u n i v e r s a l l y so, however, i s i l l u s t r a t e d by data on N - a c e t y l daunomycin (16) i n Table I I I . A c t i v i t y against L1210 i s v i r t u a l l y l o s t i n t h i s " d e r i v a t i v e but s l i g h t l y s u p e r i o r e f f i c a c y r e s u l t s i n the P388 system at a higher dose. The r e s u l t with the p - f l u o r o p h e n y l t h i o u r e a of adriamycin (17) i n the P388 system i s more t y p i c a l with poorer e f f i c a c y at a h i g h dose l e v e l being found. I t i s s i g n i f i c a n t however that a p p r e c i a b l e i n v i v o Table I I I .

In v i v o Antitumor A c t i v i t y of Adriamycin and Daunomycin D e r i v a t i v e s i n Three Experimental Systems . a

No.

Compound

L1210

10

dauno-N-COCH

17 (20)

17

adria-N-CSNHC R\F-p

18

dauno-13=NNHC0C H

19

dauno-13=NNHC0CH 0H

20

adria-13=NNHC0C H

3

6

6

2

6

adriamycin daunomycin

a

5

5

% Increased s u r v i v a l time at optimal dose (mg/kg) B16 P388

-

50 (25)

-

118

34 (7)

-

43 ( 1 . 7 )

-

75 (2)

(5.9)

63 ( 1 . 7 )

91 (12.5) 2/26C

(2)

100

(3) l / 1 0 C

275

(6) 3/6C

152 (6) 2/6C 101 (8) 1/6C c

c

112 >140 (0.5) 3/6C 70 (0.5) 108 (0.25)

b

—

94 ( 0 . 5 )

b

D

188

(1) 4/10C

D a t a from standard NCI mouse t e s t systems using QD1-9 dose schedule (99). F r a c t i o n s f o l l o w i n g e n t r i e s i n d i c a t e long term s u r v i v o r s ("cures") over t o t a l animals i n t e s t group. Underl i n e d f i g u r e s are averages of two or more v a l u e s .

b

D a t a from r e f . 100. c Data from r e f . 84.


b


2.

HENRY

Adriamycin

29

a c t i v i t y i s r e t a i n e d a f t e r i n s e r t i o n of such a l a r g e and comp a r a t i v e l y nonpolar group. Carbonyl d e r i v a t i z a t i o n at C-13 a l s o causes a decrease i n potency i n animal t e s t s but the e f f e c t i s l e s s than with Na c y l a t i o n and the e f f i c a c y of the d e r i v a t i v e i s o c c a s i o n a l l y b e t t e r than the parent a n t i b i o t i c . Table I I I l i s t s three c a r b o n y l derivatives. Daunomycin benzhydrazone (rubidazone, s t r u c t u r e 18) i s equal or s l i g h t l y s u p e r i o r to daunomycin i n e f f i c a c y i n the P388 and B16 systems but 3-6 times as much drug i s r e q u i r e d f o r optimum a c t i v i t y . In the L1210 system rubidazone appears s l i g h t l y poorer than daunomycin but i t should be p o i n t e d out that both rubidazone (and adriamycin) are schedule s e n s i t i v e and the QD1-9 regimen i s not optimum a g a i n s t L1210. With dosing every three hours on day one a f t e r tumor i m p l a n t a t i o n , f o r example, rubidazone g i v e s an i n c r e a s e i n s u r v i v a l time of 61% vs 33% f o r daunomycin at t o t a l doses of 32 and 4 mg/kg, r e s p e c t i v e l y . On t h i s schedule adriamycin p r o v i d e s a 156% i n c r e a s e i n s u r v i v a l time and two out of e i g h t long-term s u r v i v o r s at 8 mg/kg (100). Daunomycin g l y colohydrazone (19) i s c l e a r l y s u p e r i o r to daunomycin i n the P388 and B16 systems according to the l i m i t e d data a v a i l a b l e . Adriamycin benzhydrazone (20) i s c l e a r l y l e s s potent than the parent i n the one t e s t system f o r which data are a v a i l a b l e but e f f i c a c y i s not s e r i o u s l y reduced. The data i n Table I I I are provided to give a general comparison of d e r i v a t i v e s with the parent a n t i biotics. D e t a i l e d comparisons should be regarded w i t h c a u t i o n because the data are not drawn from s i n g l e experiments that d i r e c t l y compared a l l compounds a g a i n s t each tumor. Rubidazone (18), o r i g i n a l l y r e p o r t e d by Maral et a l . (101), has a g r e a t e r t h e r a p e u t i c index than daunomycin (about two-fold)

MeO

0

OH

NH 18

6

2

rubidazone



30

CANCER

CHEMOTHERAPY

and was evaluated a g a i n s t acute m y e l o b l a s t i c leukemia by J a c q u i l l a t et a l . (102). While a 45% complete remission r a t e was achieved i n t h i s p r e l i m i n a r y c l i n i c a l study and hematological t o x i c i t y was lessened and b e t t e r c o n t r o l l e d w i t h rubidazone, c l e a r s u p e r i o r i t y over daunomycin was not e s t a b l i s h e d . Against s o l i d tumors r e s u l t s were l e s s promising p r i m a r i l y due to unacceptable s i d e e f f e c t s (103). Resistance to rubidazone can be induced i n the E h r l i c h a s c i t e s tumor i n v i v o and r e c i p r o c a l c r o s s - r e s i s t a n c e i s found with daunomycin, adriamycin, v i n c r i s t i n e and v i n b l a s t i n e (104). In v i t r o 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 by amino and carbonyl d e r i v a t i v e s g e n e r a l l y p a r a l l e l t h e i r i n v i v o antitumor e f f e c t s , i . e . , potency i s almost always decreased i n comparison to the parent a n t i b i o t i c s . Table IV l i s t s E D 5 0 values f o r DNA and RNA s y n t h e s i s i n h i b i t i o n i n c u l t u r e d L1210 c e l l s f o r a s e r i e s of d e r i v a t i v e s of daunomycin and adriamycin. Similar data f o r s e v e r a l other unmodified a n t h r a c y c l i n e a n t i b i o t i c s are a l s o presented f o r comparison. N-Acyl d e r i v a t i v e s u s u a l l y s u f f e r a greater l o s s of potency r e l a t i v e to the parent than do the C-13 carbonyl d e r i v a t i v e s . The f i r s t f i v e compounds i n Table IV were c i t e d i n Table I I I f o r t h e i r s i g n i f i c a n t i n v i v o antitumor a c t i v i t y . A l l of t h i s group are i n h i b i t o r y f o r both DNA and RNA below 10 μΜ but i n almost every case have l o s t a p p r e c i a b l e potency r e l a t i v e to adriamycin and daunomycin. D e r i v a t i v e s 21-27 of Table IV i l l u s t r a t e a d i f f e r e n t e f f e c t on DNA and RNA s y n t h e s i s that f r e q u e n t l y r e s u l t s upon dérivâtization. The e f f e c t on DNA synthesis i s greater than i t i s on RNA s y n t h e s i s by a f a c t o r of three or more f o r each of these compounds. Daunomycin and a d r i a mycin cause equal e f f e c t s on the two types of n u c l e i c a c i d under the c o n d i t i o n s of t h i s assay. Since the DNA and RNA t e s t s are performed under i d e n t i c a l c o n d i t i o n s the s e l e c t i v i t y a f f o r d e d by these compounds suggests that some aspect of t h e i r mechanism of a c t i o n has been changed by d e r i v a t i z a t i o n and that t h i s e f f e c t could be e x p l o i t e d to o b t a i n drugs with f a v o r a b l y a l t e r e d t o x i c i t y or tumor spectrum. Three of t h i s group of d e r i v a t i v e s (21, 23, 25) are i n a c t i v e i n v i v o i n the L1210 or P388 systems but 22, 24, 2&, and 27 r e t a i n s i g n i f i c a n t a c t i v i t y . I t i s of i n t e r e s t that carminomycin i s the only compound encountered that showed p r e f e r e n t i a l i n h i b i t i o n of DNA s y n t h e s i s over RNA s y n t h e s i s i n t h i s system. A similar d i f f e r e n t i a l effect was a l s o noted i n a bacterium (17). Rhodomycin, nogalamycin, and the c i n e r u b i n s s t r o n g l y favor i n h i b i t i o n of RNA s y n t h e s i s . The high i n v i t r o potency of carminomycin i s r e f l e c t e d i n i t s i n v i v o activity. Shorin et a l . (15) report somewhat s u p e r i o r e f f i c a c y with carminomycin against L1210 leukemia at doses about oneseventh those of daunomycin. These authors a l s o r e p o r t s i g n i f i cant carminomycin a c t i v i t y i n s e v e r a l other experimental tumor systems. An i n t e r e s t i n g c l a s s of 14-0-acyl d e r i v a t i v e s of adriamycin (Figure 5) was reported r e c e n t l y from the F a r m i t a l i a


2.

HENRY

Adriamycin

31

Table IV.


No.

I n h i b i t i o n of N u c l e i c A c i d Synthesis i n Cultured L1210 C e l l s by A n t h r a c y c l i n e A n t i b i o t i c s and D e r i v a t i v e s

3

ΕΡςη (μΜ) RNA DNA

Compound

16

dauno-N-COCH

7.5

6.8

17

adria-N-CSNHCgH^F-p

1.9

0.9

18

dauno-13 = NNHCOC H

2.7

2.0

19

dauno-13 = NNHC0CH 0H

3.5

2.6

20

adria-13 = NNHCOC H

5

6.5

3.0

21

dauno-N-COCH CH CH

3

22

dauno-N-CSNHCH

23

dauno-N-CSNH(CH ) CH

24

dauno-N-CHO

3

6

5

2

6

2

2

3

2

3

3

66

21

> 100

15

32

7.1

25

8.7

2.1

0.7

25

dauno-13 = N0CH

26

dauno-13 = NNHCO(CH ) CH

27

dauno-13 = NNHCOCgHÊt-p

6.0

1.0

1

adriamycin

0.8

0.9

2

daunomycin

0.3

0.3

3

carminomycin

0.04

0.2

4

d ihydrο daunomyc i n

1.2

2.1

5

6

-

3

2

6

3

21

6.5

2.1

0.1

0

4

0.4

c i n e r u b i n A*

5

0.3

0.03

b

1.5

0.2

rhodomycin B nogalamycin

cinerubin B

b

3

Determined by drug e f f e c t on i n c o r p o r a t i o n of H - l a b e l e d thymidine and u r i d i n e i n t o DNA and RNA, r e s p e c t i v e l y . E D 5 0 i s the drug concentration e f f e c t i n g a 50% r e d u c t i o n of l a b e l i n g i n i s o l a t e d DNA and RNA. See r e f . 105 f o r d e t a i l e d methodology. ' A n t i b i o t i c s k i n d l y provided

by Dr. Martin Apple, UCSF.


CANCER

32

CHEMOTHERAPY


L a b o r a t o r i e s (106,107). These compounds, prepared from 14-bromodaunomycin by displacement with carboxylate s a l t s , f r e q u e n t l y

Figure 5. Adriamycin-14-O-carboxylates prepared by Arcamone et al (106). R = CH , C H, n-C H , C H , CH C H,, 3-pyridyl, CH (l-naphthyl). 3

2

y

7

15

ft

5

2

6

2

d i s p l a y e d i n v i t r o and i n v i v o c y t o t o x i c e f f e c t s comparable to adriamycin. In some cases, notably the octanoate e s t e r , c l e a r l y s u p e r i o r antitumor a c t i v i t y was found against Gross leukemia and transplanted mammary carcinoma i n the mouse. Pharmacokinetic s t u d i e s with the octanoate demonstrate that i t has a l t e r e d prope r t i e s r e l a t i v e to adriamycin; i n p a r t i c u l a r the d e r i v a t i v e accumulates i n heart t i s s u e to a smaller extent. Preliminary data suggest that the octanoate ester i s cleaved i n t r a c e l l u l a r l y by n o n - s p e c i f i c esterases (107). Another s i g n i f i c a n t group of adriamycin-14-0-carboxylates was r e c e n t l y reported by I s r a e l et a l . These i n v e s t i g a t o r s prepared a s e r i e s of N - t r i f l u o r o a c e t y l a t e d e s t e r s (Figure 6) that showed c y t o t o x i c i t y i n v i t r o (108) and y i e l d e d one compound, Nt r i f l u o r o a c e t y l a d r i a m y c i n - 1 4 - 0 - v a l e r a t e (code named AD32), that provided marked improvement i n e f f i c a c y i n t h e i r L1210 and P388 mouse leukemia systems (109). As shown i n Table V, against P388 adriamycin at 4 mg/kg gave about one-third of the s u r v i v a l time increase of AD32 at 40 mg/kg and no 60-day s u r v i v o r s . The


2.

HENRY

Adriamycin

33


NHCOCF3 Figure 6. N-Trifluoroacetyhted adriamycin-14-O-carboxyhtes prepared by Israel et al (108). R = CH , C H CH(CH ) , (CH ) CH , C(CH ) , (CH ),CH (CH ) CH , (CH ) CH . 3

2

3

3

3

3

2

3y

2

6

2

5

3

3

2

8

2

3

advantage of AD32 over adriamycin was even more pronounced a g a i n s t L1210. T h i s tumor i s i n t r i n s i c a l l y much l e s s s e n s i t i v e to a n t h r a c y c l i n e s and t h i s i s shown by the low s u r v i v a l time i n c r e a s e of

Table V.

Experimental Antitumor A c t i v i t y o f AD32 a

Optimal Dose (mg/kg)

% Increase S u r v i v a l Time

60-day Survivors

Tumor

Drug

P388

adria AD32

4.0 40

132 429

0/6 3/5

L1210

adria AD32

4.0 50

42 > 400

0/7 5/7

a

Schedule:

QD1-4, i p .

Data from r e f . 109.

42% and l a c k of long-term s u r v i v o r s given by adriamycin. In c o n t r a s t AD32 gave f i v e out of seven 60-day s u r v i v o r s , an imp r e s s i v e r e s u l t as long-term s u r v i v o r s w i t h any a n t h r a c y c l i n e a r e not common i n the L1210 system. As expected with an N-acylated d e r i v a t i v e optimum dose l e v e l s f o r AD32 a r e at l e a s t t e n - f o l d g r e a t e r than f o r adriamycin. Before l e a v i n g t h i s group of d e r i v a t i v e s i t should be noted that t h e i r b i o l o g i c a l a c t i v i t i e s a r e g e n e r a l l y i n q u a l i t a t i v e agreement with the r e c e p t o r s i t e hypothesis d i s c u s s e d above.



34

CANCER

CHEMOTHERAPY

Amino, C-13 carbonyl and 14-hydroxy d e r i v a t i v e s are a l l s t e r i c a l l y acceptable according to molecular model s t u d i e s . The amine and a c e t y l moieties are r e l a t i v e l y uncrowded i n the complex and can accept bulky m o d i f i c a t i o n s without s e r i o u s l y d i s t u r b i n g c r i t i c a l b i n d i n g p o i n t s . The markedly decreased potencies u s u a l l y found with N-acyl d e r i v a t i v e s are r a t i o n a l i z e d by the drug's l o s s of b a s i c c h a r a c t e r and consequent l o s s of the e l e c t r o s t a t i c b i n d ing component. The DNA b i n d i n g constant of N-acetyldaunomycin f o r example i s about 200-fold l e s s than that of daunomycin but the number of b i n d i n g s i t e s i s reduced only by one t h i r d (60). Table VI presents two compounds that have r e c e n t l y emerged from the Stanford Research I n s t i t u t e program (105). Periodate treatment of adriamycin provides c a r b o x y l i c a c i d 28 i n high y i e l d by o x i d a t i v e removal of the 14-carbon. The a c i d i s q u i t e poor as a 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 o r i n v i t r o but the e s t e r (29) i s Table VI.

No.

ΕΡςη DNA

R

28

OH

29

0CH

> 100 3

adriamycin

3

B i o l o g i c a l A c t i v i t y of Periodate-modified D e r i v a t i v e s of Adriamycin

(μΜ) RNA

% increase i n s u r v i v a l time (mg/kg)

65

b

AT

63 (25)

2.0

0.9

46

0.8

0.9

112

m

(°C) 2.6

(50)

12.6

(1)

17.8

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

P388 mouse lymphocytic leukemia, QD1-9 and drug.

schedule, i p tumor

°Sonicated c a l f thymus DNA i n O.OlO M, pH 6.0 P 0 drug:DNA molar r a t i o of 1:10.

4

b u f f e r at a


C

2.

HENRY

Adriamycin

35

n e a r l y as potent as adriamycin. Both compounds proved a c t i v e i n v i v o i n the P388 system although potency and e f f i c a c y are g r e a t l y reduced r e l a t i v e to adriamycin. T h i s r e s u l t i s of i n t e r e s t none-the-less because these compounds are the f i r s t i n which the carbon s k e l e t o n of the parent has been a l t e r e d with r e t e n t i o n of i n v i v o a c t i v i t y . There are no s t e r i c c o n s t r a i n t s to the f i t o f e i t h e r 28 or 29 i n t o the proposed r e c e p t o r complex of F i g u r e 4. However 28 i s probably z w i t t e r i o n i c at p h y s i o l o g i c a l pH and the carboxylate anion would undoubtedly introduce a strong e l e c t r o s t a t i c r e p u l s i o n t o the DNA phosphates and thereby i n h i b i t b i n d i n g . T h i s e f f e c t i s r e f l e c t e d i n the r e l a t i v e a b i l i t i e s of 28 and 29 to s t a b i l i z e h e l i c a l DNA. The A T o f 28 i s only 2.6 ~vs 12.6° f o r e s t e r 29 and 17.8° f o r adriamycin under the standard c o n d i t i o n s noted i n Table V I . In f u r t h e r new work treatment of adriamycin and daunomycin with excess methyl i o d i d e , followed by i o n exchange, provided quaternary ammonium c h l o r i d e s (30, 31) given i n Table V I I (110). DNA and RNA s y n t h e s i s i n h i b i t i o n i s g r e a t l y depressed by q u a t e r n i z a t i o n of both a n t i b i o t i c s . Reduced c e l l p e n e t r a t i o n because of m


2r

Table V I I .

a

B i o l o g i c a l A c t i v i t y of Quaternary Ammonium D e r i v a t i v e s of Adriamycin and Daunomycin.

MeO

0

OH

^

NMe +

3

No.

_R_

30

OH

31

H adriamycin

ED .ς η (μΜ) RNA DNA

CI"

% increase i n ^ s u r v i v a l time (mg/kg)

> 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 complexing a drug with a macromolecular c a r r i e r that could enter c e l l s only by p i n o c y t o s i s would r e s u l t 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 subsequent 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 t h i s paper two types of s e m i s y n t h e t i c analogs are p o s s i b l e , those c o n t a i n i n g a n a t u r a l aglycone coupled to 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.

DNA

RNA

34

0.79

0.24

15.0

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 .

AT

m

(°C)

b

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 s of 34-36 that daunosamine provides nothing i r r e p l a c e a b l e i n 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 and may 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. For maximum s t a b i l i t y o f the complex the drug should possess an aromatic component that permits maximum overlap 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.

R

OCH 39

CHEMOTHERAPY

ς η

(yM)

a

DNA

RNA

210

210

AT

m

(°C)

3

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 Double and Brown (127) prepared a s e r i e s of f i v e anthraquinones bearing one or two aminoalkylamino s i d e chains (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. Sartorelli; Cancer Chemotherapy Washington, D. C. 20036 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

Structure

(μΜ)

DNA

RNA


No.

adriamycin a

0.8

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


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 molecules can p a r t i c i p a t e i n a spectrum of p o s s i b l e b i n d i n g modes according to molecular model s t u d i e s and no 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 g ED50 values below 10 μΜ i s nevertheless very broad. Whether the most s t r u c t u r a l l y remote compounds (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.

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110. 111. 112. 113. 114. 115.


116. 117. 118. 119.

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2.

HENRY

136. 137. 138.

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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.


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