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The Evolution of a Cardioprotective Antimetastatic and Antitumor Drug: An International Adventure Raghunathan V. Nair and Donald T. Wiiak Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, OH 43210 The medicinal chemical research leading to the discovery of the antitumor, antimetastatic, and cardioprotective properties of a group of compounds known generally as the bis(dioxopiperazines) began in the laboratories of the Imperial Cancer Research Fund (ICRF) in London in the late 1960's ( I , . The three important biological properties (antitumor, antimetastatic, and cardioprotective) of his(dioxupiperazines) are likely independent of one another, but may in part be related to chelatiunof biomrtals. Metastasis is the spread of cancer cells from its primary site to a distant location, and antimetastatic drugs block this spread. Although antitumor and antimetastatic activities are particularly important, these compounds also possess the unique ability to protect vital organs such as the heart from toxic effects of other antitumor drugs such as the anthracyclines. Based upon the hypothesis (2) that many antitumor drugs work because they are chelators of metal ions, Creighton and co-workers a t the ICRF investigated the antitumor properties of the potent chelating agent ethylenediaminetetraacetic acid (EDTA) (1) and several of its derivatives (Fig. 1, structures 1-9). EDTA (1) itself had previously been reported to exhihi t no significant antitumor properties (3).These investigators suspected thatthis may have been due to the inability of EDTA to penetrate cellular membranes. If this were true,
1, R = H, EDTA 2, R = CHB 3, R = C2H5
5, R = H, ICRF-154 6, R = CH3, ICRF-159 6a, (+) isomer of ICRF-159 (ICRF-187) 6b, (-) isomer of ICRF-159 (ICRF-186) 7, R = C2Hs, ICRF-192
4
8, meso 9, dl
Figure 1. EDTA and less polar derivatives tested for antitumor properties.
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Journal of Chemical Education
then i t would follow that the less polar derivatives should show improved bioavailability. In addition to bioavailability and selective tumor-killing ability, cancer researchers desire drugs that have low toxicity to normal tissues, good water solubility to facilitate ease of administration, shelf life stability, and antimetastatic properties limiting spread of the disease. Structure-Activity Relationships
The initially prepared compounds were evaluated for antitumor properties using Sarcoma (S180) and the Leukemia (L1210) cell lines in mice (4,5). Simple esters of EDTA (2, 3) as well as the amide-acid (4) also did not show any antitumor properties. The his(imide) of EDTA known as ICRF-154 (5) was the first to show the anticipated activity. I t was obtained as the product of reaction between EDTA and formamide. Administration of this bis(dioxopiperazine) (5) produced a 91% inhibition (5 doses at 30 mglkg) in the growth of S180 tumor and a 67% (9 doses a t 30 mglkg) increase in survival time of L1210 inoculated mice (4). These doses are high in comparison to the potent clinically important anthracyclines, which are active a t about 10 mglkg. Subsequently, several structurally related bis(dioxopiperazines) were prepared and a structure-activity profile was formulated. Alterations in the terminal dioxopiperazine rings afforded ineffective compounds (5).Introduction of a methyl group into the ethylene chain of ICRF-154 yielded bidimide) (6) (ICRF-159), which exhihited improved antitumor properties. Bis(imide) (6) produced a 93% (5 doses at 30 mglkg) inhibition of Sl8O tumor in mice and 137% (13 doses at 30 mglkg) increase in survival time over controls in the L1210 leukemia model (4). Additional structure-activity comparisons among the his(dioxopiperazine) derivatives were conducted in vitro (6).The relative concentrations of several related bis(imides) required for a 50% inhibition of colony formation (IDsa) of mouse L cells were determined (6). Whereas ICRF-154 (5) had an IDboof 7.3 pM concentration in this assay, ICRF-159 (6) exhihited an IDSOof 3.0 pM. Substitution of the methyl group in ICRF-159 (6) with an ethyl group resulted in an ineffective (ID50 720 pM) compound (7) (ICRF-192). Methyl substitution on both ethylene carbons of ICRF-154 was useful only when the molecule had the meso conformation. Thus, bis(dioxopiperazine) (8) exhibited an IDso of 0.09 pM, whereas the corresponding dl stereoisomer (9), with an IDso of 150 pM, was much less effective. Although bis(imide) (8) was more potent than ICRF-159 (6) in this assay, it had a lower therapeutic index in vivo (4, 6). The calculated ratio of maximum tolerated dose to the dose required for 90% inhibition of 5180 tumor growth was 6.7 for ICRF-193 (8). This is less desirable than the value of 9.8 for ICRF-159 (6). Thus, bis(imide) (8) had the higher toxicity. The reasons for such activity differences among these closely related molecules were not clear. In general, compounds having five or less carbons between the two dioxopiperazine rings were relatively more potent. Subsequent re-
search in this area has focused on ICRF-154 (5), ICRF-159 (6) and derivatives of these bis(dioxopiperazines). Cllnlcal Testing The first clinical results with ICRF-154 (5) and ICRF-159 (6) were reported by Hellman and co-workers a t the ICRF (7). The drugs were evaluated in six patients with acute leukemia and three with lymphosarcoma. ICRF-154 (5) was ineffective on acute lymphoblastic, myeloid, or monocytic leukemia and was not investigated further in the clinic. ICRF-159 (6), in contrast, showed considerable leukopenic activity in nearly all patients. Subsequently, ICRF-159 (6) has undergone extensive clinical investigations. Treatment with this compound has shown partial remission in patients having acute leukemia and lymphosarcoma (7), non-Hodgkins lymphoma (8),Kaposi's sarcoma (9),and psoriasis (10). This drug was not particularly useful in the treatment of advanced cancer (11). -~~~- ~ - ~ broncho~enic ~" . ., malienant melanoma (12),carcinoma of the breast (13,14), squamous cell carcinoma of the head and neck (15).advanced eastric cancer (16). and pediatric solid tumor; (ii). The initial renorta on the bioloeical of biddiox" -vronerties opiperazines) led to the participation of several researchers from the United States, Canada, and China into this area of research. Racemic ICRF-159 (6) was named razoxane by the British Pharmaco~oeiacommission. The aqueous solubility of this material was approximately 3 mg/mL a t 25 "C. PO& solubility in water presented a problem in the administration of this drug. In 1976, Repta and co-workers (18) in the United States reported the use of pure enantiomers of ICRF-159 (6) , ~as , a solution to this nroblem. Each enantiomer of 6having approximately 15 m g / i solubility ~ in water at 25 OC was equally potent as the racemic material (18). Thus, owing to ease of formulation, pure enantiomers, especially the (+) isomer (ICRF-187) have been preferred in later clinical investigations. X-ray crystal structure determinations in Canada (19) revealed that the two dioxopiperazine rings in racemic ICRF-159 (6) adopted a cis arrangement. This is in contrast with the ooticallv pure ICRF-187, whose two rings existedanti to e a c h b t h e r . ~ h edifferences in water solubility between these crystal forms and the racemic mixture have been attributed (19) to the greater number of intermolecular interactions that exist in the racemic material. ~
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Antirnetastatlc ProDertles Inspiteofthe inadequaciesobserved with ICRF-159 (6) as aclinicallv useful antitumor drue, the finding: that this compound iniibited cancer metastazs was a breakthrough. Metastasis is a major problem in the treatment of cancer. The selective antimetastatic property of ICRF-159 (6) was first noted in the Lewis lung carcinoma (3LL) model (20). Whereas all the control miceshowed multiple metastatic lung nodules 21 days after tumor implantation, only five out of the 27 animals treated with ICRF-159 (6) (30 mglkglday for 10 days) showed a few small nodules. There was no significant effect on the weight of the primary tumor. Further studies led these English investigators to suggest that ICRF-159 (6) was involved in normalizine the develonine tumor blood vessels (angiometamorphic Lffect) and therefore prevented the release of malienant cells into the circulation (21-23). A dose-response study for inhibition of XLI, metastasis in mice showed significant reduction in metastasis at a dose of 12.5 mglkg of ~ C R F - (6) I ~for ~ 7 days (24). Stereochemical studies designed to assess the preferred relative conformation of the dioxopiperazine moieties in ICRF-154 (5) and ICRF-159 (6) for optimal antimetastatic effects were pursued by investigators in the United States (see Fig. 2). The trans-10 and cis-11 cyclopropyl analogues of ICRF-159 (6) were prepared in the late 1970's (25, 26). Com~ounds10 and 11differ from ICRF-159 (6) by only two hydrogen atoms in their molecular weight. The dioxopiperazine rings in these molecules, however, are held anti (10) or
syn (11) to each other. Bis(dioxopiperazines) 10 and 11 were examined for antimetastatic effects in the bronchoaenic adenocarcinoma LG1002 in Syrian golden hamsters 6 6 ) . At 15 mglkg, intraperitoneal administration of either ICRF-159 (6) or cis-11 reduced the metastatic growth of LG1002 and showed no effect on the growth of the primary tumor. In contrast, the trans compound (101.itimulated both the lung metastasis and the primary tumor growth. Similar stereoselective effects of isomers 10 and 11 were also observed on lung metastasis of B16-F10 melanoma in mice (27). Pretreatment of B16-F10 cell lines for 24 h with trans-10 a t 2 rM and 20pM concentrations resulted in an increase in lung melanoma colony formation. Such pretreatment with the cis isomer (11) reduced the metastasis to the lung. ICRF-159 (6). like the cis isomer (11). was alao antimetastatic at these husesin this assay. Results in vitro werecomparable to those in vivo for ICHF-159 ( 6 ) and trans-10. cis-ll was found to stimulate colony formation in vitru. I.itrle or no effect was ohsewed with thesynthetic intermediates (12, 13,and their methyl ester hydrochlorides) u, these his(dioxopiperazinesr. suggesting that the dioxopiperazine rings are necessary for ~nttmetastaticeffects. Results uf iniectine oretreated cells into mice indicated that the antimetastatrckffect of ICRF159 (6) . . and cis-11 mav be inde~endentof anv. aneiometamorphicmechanism that would have to arise by the action in vivo of the drue on the vasculature of the ~ r i m a r vtumor. No of tumor primary tumoiwas established after cells in these experiments. Lntraperitoneal injections (30 mglkg) of either trans-10 or cis-11 into tumor-bearing mice accelerated the tumor growth. Tumors were detected six days after implantation and grew to a size of 100-250 mm2 after 22 days. In animals treated with similar dose of ICRF-159 (6), tumors did not appear until 17 days and were only 30 mm2 in size after 22 days. In control animals, tumors appeared 12-15 days after implantation and reached a size of 60-80 mm2 by day 22. These results seemed to correlate with the stimulation in colony formation in vitro observed with these cyclopropyl bis(dioxoninerazine) stereoisomers. .. ~om~arativeevaluation oitronc-cyclopropyl bis(dioxopiperazine) (10) and tetraacid (121with ICHF-159 (6) and its
13 14 Figure 2. Stereoisomers studied to determinethe preferred spatial wienmtion of funclional groups for biological activity. Volume 65
Number 6 June 1988
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precursor tetraacid (13) in V-79A fibroblasts in tissue culture showed ICRF-159 (6) to he most cytotoxic and mutagenic (25). Conformationally restricted trans-10 was least cytotoxic and mutagenic in this assay. The nontoxic nature of trans-10 and previously reported (5) inactivities of dl-9 and trans-cyclohutyl his(dioxopiperazine) (14) in the sarcoma (S180), leukemia (L1210), and [3H]-thymidine assays were suggested to be related to their conformational similarities. Among the tetraacid precursors to the bidirnides), conformationally rigid 12 was more toxic a t all concentrations and eenerallv more mutagenic than the acvclic counthe differences in terpart (l$. ~heseinvesti~atordsu~~ested cytotoxicities between 12 and 13 to be an indication of a mechanism involving asymmetric enzymes since similar activity profiles were expected if chelation of cations were resp-oniihle for their effect. Tetraacid (12) effectively blocked scheduled DNA synthesis a t a concentration of 1 0 - W hut weaklv induced unscheduled DNA synthesis. ~is(dioxopiperazines)(6), t r a m 10 as well as the tetraacid (13) were less effective in inhihiting scheduled DNA synthesis hut induced dose-dependent unscheduled DNA synthesis. Most compounds that inhibit scheduled DNA synthesis also inhibit unscheduled DNA svnthesis (28). The differential effect exhibited bv 12 a t a iigh concentration M) was suggested to he a reflection of inhibition of asymmetric enzymes involved in scheduled as well as unscheduled DNA synthesis. Damaged DNA thus enters DNA replication subsequently activating the errorprone postrepl
