The Need for Additional Alkylating Agents and Antimetabolites

(CANCER RESEARCH 29, 2435-2442, December 1969] The Need for Additional Alkylating Agents and Antimetabolites

Charles Heidelberger1

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

The title that was assigned to me is a clear invitation to XCH2CH2CI HN CHSN pontificate. Instead, I will share with you a few modest and XCH2CH2CI VCH2CH2CI XCH2CH2CI not very original thoughts and speculations. These are based on a large literature and a small experience of tilling the soil I E HI ,CH2CH2CI among certain obscure and unnatural pyrimidines. This engage CH3N ment with the rationality of antimetabolites followed an 'l CICH,CH2X ignoble exodus from the quagmire surrounding an even more 0 obscure alkylating agent. Thus, my bias is already exposed. 0 >C ... /CH2-0XMCH2 It is not my purpose to cover the enormous literature in ^i-'N f XXCH2CH2CI P-lN these two fields of alkylating agents and antimetabolites. ^^rr CH,-N'H3ZECH2-CH2XCH2CH2CI1XSCH2CH2CI Rather, I shall refer to reviews whenever possible. Nor do I H intend to explore systematically structure-activity relation m ships of every series of compounds, detailed screening results, comparative toxicology, clinical pharmacology, and mechanisms of action. I will try merely to focus on certain o facts, trends, and promising leads that happen to interest me CICH2CH2NH C- NCH2CH2CI and suggest points of departure for future synthetic chemical NO CH2-N^N^Nsl forays. I shall emphasize drug design at the expense of ignoring i^ the exciting advances in cell-cycle kinetics and clinical com â€¢yirr bination chemotherapy that have been emphasized elsewhere IS. CH2-CH2 in this Symposium. I shall forbear from intoning the custom ary litany of our ignorance of the essential molecular lesions of 0 0 malignancy and intend to proceed with what is known. But I CH^SOCH2CH2CH2CH2OSCH2 had better proceed. 0 0 CH2-CH2

ALKYLATING AGENTS X The alkylating agents as a group are among the clinically Chart 1. I, sulfur mustard; II, noi-HN2; III, nitrogen mustard, HN2; most useful compounds in cancer chemotherapy. Their chem IV, Nitromin; V, phenylalanine mustard (SarcolyÃ¡n, Melphalan); VI, istry and attempts to design agents showing selectivity of uracil mustard; VII, cyclophosphamide (Cytoxan, Endoxan); VIII, l,3-bis(2-chloroethyl)-l-nitrosourea, BCNU; IX, triethylenemelamine action have been reviewed by Ross (46). Hirschberg (24) has (TEM); X, Thio-TEPA; XI, Myleran. made a very comprehensive compilation of the earlier informa tion about their chemotherapeutic properties, and this has been expanded and brought up to date by Ochoa and with their characteristic 0-chloroethyl groups. Two examples Hirschberg (39). Various aspects of the mechanism of action of alkylating agents in many systems have been reviewed by of ethyleneimines (aziridines) are IX and X, and the remaining Brookes and Lawley (7), Lawley (32), Wheeler (57, 58), and important class of methane sulfonates is illustrated by Myleran Warwick (56). Oliverio and Zubrod (40) have discussed their (XI). Literally thousands of these compounds have been synthesized, in which the reactive 0-chloroethyl, ethylene- clinical pharmacology. imine, or methane sulfonate groups have been hung onto The alkylating agents with chemotherapeutic activity are innumerable "carriers," which some mystics endow with chemically reactive and at least difunctional. The structures of a few of the important compounds are shown in Chart 1. remarkable biologic specificity. Thus, we have phenylalanine Compound I is sulfur mustard, the original prototype, and mustard (V, Sarcolysin, Melphalan), uracil mustard (VI), II-VIII are members of the nitrogen mustard (HN2) family, antimalarial mustards, steroidal mustards, aromatic mustards, quinonoid mustards, etc. Do these "carriers" in fact impart any biologic specificity to the reactive groups that they embrace? In order to answer this question it is necessary to have reliable comparative data on 'American Cancer Society Professor of Oncology. the activities of a large number of compounds. Fortunately,

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1969 American Association for Cancer Research. CharlesHeidelberger these data have been provided by a truly heroic and Table 2 monumental study by Schmidt et al. (50). These investigators TumorSarcoma D11!111.5.0.0.6.5.71.02.51.02.51.7L/DÃ¨11112.5.0.0.6.6 studied 51 ÃŸ-chloroethylamines, 25 aziridines, 39 methane sulfonates, and 12 nonalkylating compounds for their toxicity subcutaneousAdenocarcinoma180, to mice, rats, dogs, and monkeys and their activities against 18 subcutaneousL1210,755, transplanted rat tumors and 7 transplanted mouse tumors. subcutaneousL1210, ascitesEhrlich This tour de force was published in a monograph of 1528 ascitesAdenocarcinoma pages (50). SAH-I-I, subcutaneousL1.52.5.1.04.04.53.5DL1.02.51.02.53.02.0L/DLa It has been alleged by many that the proof of the chemotherapeutic specificity of the "carrier" rests on the Effects of optical isomers of phenylalanine mustard on transplantable "fact" that the L-phenylalanine mustard has greater tumor- rat tumors (50). LDi0/ED90 orLD10/ED60. Â°L/DL,mean of 6 tumors, 1.38 Â±0.11. inhibitory effectiveness than the D isomer or the DL mixture , mean of 5 tumors, 1.54 Â±0.26. (V). Let us examine the most reliable information on this subject as presented by Schmidt et al. (50). Their data on the rat tumors are given in Table 1. At the bottom of the table is methane sulfonate series, there was no indication that specific the mean for 10 tumors of the ratio of the LDi0/ED90 (or classes of alkylating agents or members within a class were ED60), the therapeutic index, for the L isomer over the D endowed with unique antitumor activity. Apart from these isomer, which is 1.05 Â±0.19. Thus, there is no difference in the exceptions, cyclophosphamide (VII) and the isomerie phenyl therapeutic index of the two isomers although, admittedly, the alanine mustards (V) were the outstanding representatives in L isomer is more toxic and is more active against the tumors virtually every test system examined whether the neoplasm than the D isomer. On the other hand, when the ratio of the L was highly sensitive or relatively insensitive to alkylating agents." isomer to the DL mixture is taken for 16 tumors, it is 0.76 Â± 0.08. This finding that the DL mixture has a better Let us now examine cyclophosphamide (VII, Cytoxan, therapeutic index than the L isomer resists rational Endoxan) a little more closely. This compound "was designed explanation. However, the same comparison in mouse tumors in accordance with the principle of transversion of an inactive (Table 2) shows that the L isomer is better than the D and DL, transport form to an active form at special sites in the body" but not strikingly so. If the ratios of all the rat and mouse tumors (5). It was thought that tumors are rich in phosphoamidases, combined are taken, the mean L/DL ratio of the therapeutic and hence that the P-N bond would be selectively cleaved in index for 22 tumors is 0.92 Â±0.09, and the L/D ratio for 16 the tumor to activate the drug. However, it was soon found tumors is 1.20 Â±0.15. Neither of these ratios is significantly that cyclophosphamide was not active against the growth of different from 1. In addition to demonstrating biologic tumor cells in culture but was metabolized to an active form in variability, these data show clearly that if these optical isomers the liver (15). Hence, the properties of the compound were as represent the proof for the chemotherapeutic specificity of the designed, except that the activation takes place in the liver "carrier," then the case is lost, even though there are rather than in the tumor. Since that time a great deal of work differences in overall toxicity. Schmidt et al. (50) have has been done, particularly by Brock (5) and Friedman (17) to concluded that "with possibly two exceptions, both in the elucidate the nature of this activation process. It was originally believed that after a series of hydrolyses, nor-HN2 (II) was liberated and was the therapeutically active compound. Table 1 However, this does not seem to be so, and the present state of Tumor L DL L/DI/7 D L/D6 this exceptionally complicated situation appears to be that the metabolism of cyclophosphamide in vivo does not parallel its WalkersubcutaneousWalker 256, CH, pulmonaryWalker256, CH, chemical behavior. Considerably more work is needed before ascitesWalker256, CH, an understanding is reached of the almost unique tumor- subcutaneousWalker256, CB, inhibitory activity of cyclophosphamide. pulmonaryWalker256, CB, Another compound of particular interest is 1,3-bis(2-chlo- subcutaneousWalker256, SK, roethyl)-l-nitrosourea (BCNU) (VIII), which is unusual in that pulmonaryYoshida256, SK, hepatomaYoshidaascites it crosses the blood-brain barrier and is active against sarcomaDunningascites intracerebral L1210 leukemia (49). Although it contains subcutaneousDunningIRC-741 leukemia, ÃŸ-chloroethyl groups, there is some doubt as to whether it is ascitesLymphomaIRC-741 leukemia, really an alkylating agent, since it reacts to only a slight extent subcutaneousAdenocarcinoma8, subcutaneousAdenocarcinomaR-35, with 4-(p-nitrobenzyl)-pyridine (59). It inhibits DNA and pulmonaryMurphy-SturmR-35, RNA synthesis in LI210 leukemia cells in vivo and in vitro lymphosarcoma,subcutaneousNovikoff (60), and its effect on a DNA polymerase system is comparable to that of /3-chloroethylisocyanate ; it appears to hepatoma, ascites178338.5331220332812.5112.51583121101012445425.5142.515.51.131.001.060.410.330.331.000.450.611.001.461.000.620.401.000.46102121101415310112.51.701.570.410.331.422.200.670.801.001.000.46react with the enzyme rather than with the primer (61). Effects of optical isomers of phenylalanine mustard on It is clear that there is very little relationship between the transplantable rat tumors (50). LDi 0 'I DC,,,or LD10/EDoo- distribution, excretion, and metabolism of the alkylating Â°L/DL:mean of 16 tumors, 0.76 Â±0.08. agents and their mechanism of action (39,40). Although they bL/D: mean of 10 tumors, 1.05 Â±0.19. are often considered to be "radiomimetic" compounds, this

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analogy is probably overdone (56). Studies on the mechanism chemical type will be found which will affect some neoplasms of resistance of experimental tumors to alkylating agents have in a unique way ...." Therefore, I feel that continuation of a been critically reviewed by Wheeler (58). The main fact is that vast effort of synthesis aimed at the random attachment of there is considerable cross-resistance among these structurally alkylating groups to a fanciful collection of "carriers" can no diverse compounds, but Wheeler (58) cautions against assum longer be justified. However, there appear to me to be three ing from that fact that the alkylating agents act by a common areas in which specific and rational syntheses could be justified mechanism; he considered altered permeability as an impor at this time: (a) preparation of possible metabolites of tant factor in resistance, and the repair of alkylated DNA (see cyclophosphamide and its derivatives, (b) synthesis of further below) was unknown at that time. derivatives of BCNU, and (c) once we have a much more What then is the mechanism of action of the alkylating agents, sophisticated knowledge of the parameters regulating the if, indeed, a single mechanism exists? It appears from an specificity of the attack on DNA, this information might be overwhelming body of data that the primary action of these put to use in the area of drug design. compounds is a direct attack on DNA. The chemistry of this has been worked out in a brilliant series of investigations by ANTIMETABOLITES Brookes and Lawley, which has been reviewed by Lawley (32). They showed that sulfur mustard (I) exerts a nucleophilic Let us herewith define a metabolite as some naturally attack on the N-7 of guanine residues in the DNA, which occurring compound produced during metabolism and an labilizes the glycosidic bond and causes the release of guanine antimetabolite as a compound related structurally to the from the DNA, which can thus give rise to mutations. They metabolite which prevents its further utilization by competing also showed that sulfur mustard could react with two guanines with it for an enzyme. This definition and field came out of to produce a cross-linking, which they postulate to be the the pioneering efforts of D. D. Woods (64), who in 1940 primary cytotoxic action, and which would prevent DNA recognized the metabolite-antimetabolite relationship of replication (7). They have demonstrated with sensitive and p-aminobenzoic acid and sulfanilamide. The role of these resistant strains of bacteria that in the latter there is an active compounds in pharmacology and biochemistry was explained repair mechanism that excises the cross-linked guanines from when the structure and function of folie acid were elucidated. the DNA (33). They also found in bacteria that similar Reviews of various aspects of cancer chemotherapy with cross-linking was obtained with triethylenemelamine (TEM) antimetabolites of many sorts have been provided by Stock (IX) and butadiene diepoxide, and produced additional evi (53), Langen (30), Baker (2), and Timmis (54). dence that the cross-linking is interstrand (34). Crathorn and Antifolics Roberts extended this work to mammalian cells (HeLa) and have demonstrated that with sulfur mustard there is a direct The antifolics in cancer chemotherapy have three distinc relationship between lethality and attack on DNA; they also tions: (a) they were the first compounds to be effective against found repair-excision of alkylated guanines from the DNA acute leukemia in children; (b) they cure a high percentage of (11). Ruddon has reported that DNA reacted with HN2 (HI) female patients with choriocarcinoma; and (c) they are now has a decreased template activity for RNA polymerase, and to where the action is. a lesser extent for DNA polymerase (48), which is opposite to Folie acid (XII) is reduced to tetrahydrofolate by the the inhibitory effects of nucleic acid biosynthesis found in enzyme dihydrofoÃate reducÃase,which is powerfully inhibited intact cells. by antifolates such as Amethopterin (XIII, Methotrexate). However, the view that the primary chemotherapeutic effect Tetrahydrofolate reacts with formaldehyde (or other com of alkylating agents results from their attack on DNA is not pounds at the same oxidation level) to give isomerie formyl or universally accepted, largely upon the basis of work done in mÃ©thylÃ¨netetrahydrofolates, which are the coenzymes of all mice with tumors. Golder et al. (19) found that the extent of one-carbon metabolism. Consequently, these coenzymes are alkylation by HN2 was very slight and that there was some required for two steps of purine synthesis and for thymidylate evidence of crosslinking DNA to protein. Wheeler and Alex synthetase. A great deal of work has been done on the ander (59) studied the in vivo alkylation of sensitive and mechanism of resistance to antifolics, and suffice it to say that resistant tumors implanted bilaterally into the same mouse, this is not fully understood at present. Although Amethop and found no correlation between the extent of alkylation of terin is a very powerful inhibitor of dihydrofolate reductase, the DNA and chemotherapeutic effect. However, it is evident there is still a need to obtain more selective and specific to all those who are concerned with the binding of carcinogens compounds. to DNA that there is considerable specificity in binding, and in A simple modification, by the addition of a mÃ©thylÃ¨negroup fact Doskocil and Sormova (13) have found that not all to give homofolic acid (XIV), has been studied by Friedkin et guanines in DNA are equally reactive to sulfur mustard. Until al. (43). They have found some antimalarial activity with this the nature of this specificity is understood, the mechanism of compound and have further demonstrated that tetrahydro- action of the alkylating agents will remain somewhat obscure. homofolates are inhibitors of thymidylate synthetase. Very What then is the need for additional alkylating agents? recently, Hutchison (27) has reported that a series of Schmidt et al. (50) have stated, "In one sense the lack of quinazoline analogs, of which XV is the most active, exert specificity of the various alkylating agents in both the greater inhibitions of bacteria and tumor cell growth than does therapeutic activity and toxicity spheres is disappointing. On Amethopterin. Obviously, further work along these two lines the surface at least, it offers little hope that any agent of this is indicated.

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impermeable to the compound (3). Hopefully, this difficulty may not be insurmountable. If so, we can look forward to A Vc-NHCHCH2CH2COOH inhibitors of dihydrofolate reductase with unsurpassed COOH specificity to cancer tissue. Here, then, is a supreme example xn of carrying out enzymatic structure activity studies combined with skillful and imaginative chemistry. These latter studies of Hitchings (25) and Baker (3) gave rise ^-C-NHCHCH2CH2COOH to the third category of distinction listed above. COOH XIII NH OH OH

_O~VcONHCHCH2CH2COOH

COOH

XVIII ' 0 SH SH k ^-C-NHCHCH2COOH

COOH zz k CH3 xn XXtt XXTT1 oc><5>NHo NH2 NH2 , XVII HO Chart 2. XII, folie acid; XIII, amethopterin (Methotrexate); XIV, homofolic acid; XV, quinazoline analog; XVI, pyrimethamine (Daraprim); XVII, Bakei and Meyer's analog. "XXVI ~ "~" CH NHCH2CH=CC >!>

A classical study of dihydrofolate reducÃase inhibition by another series of inhibitors, the 2,4-diaminopyrimidines, illus trated by XVI, Daraprim, an active antimateria!, has been carried out by Hitchings (25). He has studied the kinetics of interaction of a series of these compounds with dihydrofolate reductases isolated from bacteria and various protozoa and has found HO OH striking differences. This makes it possible to achieve a remarkable specificity in the chemotherapy of infectious disease by taking advantage of these species differences in Chart 3. XVIII, adenine; XIX, guanine; XX, 8-azaguanine; XXI, enzymes. 6-mercaptopurine; XXII, thioguanine; XXIII, dimethyltriazenoirnid- Baker has for many years been working on "active-site- azole carboxamide; XXIV, psicofuranine; XXV, arabinosyladenine; directed irreversible inhibitors" of various enzymes, including XXVI, Cordycepin; XXVII, R = H, Tubercidin, R = CM, Toyocamycin, dihydrofolate reducÃase. This productive research has been R = CONH2, Sangivamycin; XXVIII, Formycin; XXIX, isopentenyl- described in his book (2) and in innumerable papers since. By adenosine. studying the kinetics and nature of the inhibition of this enzyme with a large number of structurally related com Furine Ant Â¡metabolites pounds, he has "mapped" the region of the active site in terms of ionic and hydrophobic regions. As the near-culmination of Various aspects relating to these compounds have been this approach, Baker and Meyer (3) have recently reported reviewed by Brockman (6), Cohen (10), Fox (16), and that compound XVII has the fantastic specificity of being a Heidelberger (23). The naturally occurring purine bases, powerful irreversible inhibitor of the enzyme isolated from adenine (XVIII) and guanine (XIX), have a large number of LI210 cells, but not from mouse liver, spleen, and intestine! analogs related to them in various ways. A number of these This finding has remarkable connotations as to the nature of represent isosteric replacements in the imidazole ring: 8-aza the carcinogenic change, but it cannot yet be interpreted. guanine (XX), Tubercidin (XXVII), and Formycin (XXVIII). Surely, this compound must be the "magic bullet" that we Two very important purine base analogs are 6-mercaptopurine have all been searching for, with apparent specificity for (XXI) and thioguanine (XXII), which are effective in cancer irreversible inhibition of only the tumor enzyme. Unfor chemotherapy. Their nucleosides are known and have been tunately, however, this compound is inactive in vitro at studied extensively, and it is of great interest that the a-anomer of 2'-deoxythioguanosine, in contrast to all others inhibiting the growth of LI210 cells which are presumably

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1969 American Association for Cancer Research. Additional Alkylating Agents and Antimetabolites studied, is biologically active and is incorporated into DNA and is incorporated into DNA instead of thymine, but it has (36). It has also been found that 6-methylmercaptopurine been disappointing in cancer chemotherapy (cf. 23). An ribonucleoside has quite a different mechanism of action than interesting approach to increasing the chemotherapeutic effect 6-mercaptopurine (6-MP) and its ribonucleoside (4), and that of a close relative, 5-iodo-2'-deoxycytidine (ICDR), was made the periodate oxidation product of its ribonucleoside inhibits by Woodman (63), who complexed the 5 -phosphate of ICDR tumor growth and has other interesting biologic actions (28). with various polycations and found that it was incorporated Another interesting compound is 4(5X3,3-dimethyl- into mouse tumor DNA in vivo to a considerably greater triazene)-imidazole-5(4)-carboxamide (XXIII), which is an extent than was the nucleoside. Such an approach may find analog of an intermediate in purine biosynthesis and has application to other nucleotide analogs. considerable clinical activity against melonomas (51). Its biochemical mode of action is not yet known (to this author), but it represents an important new series of compounds that is worthy of further chemical and biochemical exploration. Arabinosyladenine (XXV) has considerable activity against some tumors, and studies of its mechanism of action have been carried out (cf. 10, 23). Several purine nucleoside antibiotics with interesting structures and tumor-inhibiting activity have been isolated, characterized, and studied. These include Cordycepin (XXVI), Psicofuranine (XXIV), Tubercidin, Toyo- camycin, Sangivamycin (XXVII), and Formycin (XXVIII) (cf. 16). This latter compound is of particular interest because a number of its biologic properties have been explained by Ward and Reich (55) as being due to the fact that, in polynucleotide form, its individual residues exist in the syn, rather than in the common anti, conformation. This poses interesting possibili ties for other C-nucleosides. Isopentenyl adenosine (XXIX) was isolated from transfer RNA and its structure determined by Hall et al. (20). It was found to be a powerful cytokinin in plants and also had inhibitory activity against tumor cells and possibly against acute leukemia in children. Because of this interesting activity, a number of related compounds have been synthesized and tested by Fleysher et al. (14). Although this presentation has been necessarily brief and superficial, it is clear that there are a number of interesting structural modifications of purine nucleosides with biologic activity. There are also several other different types of structural modifications that I have not listed here. Therefore, it is quite clear that further synthetic work with purine and purine nucleoside analogs should be biologically, and probably chemotherapeutically, rewarding. Chart 4. XXX, uracU; XXXI, thymine, XXXII, cytosine; XXXIII, 5-fluorouracil; XXXIV, 5-fluoro-2'-deoxyuridine, FUDR; XXXV, Pyrimidine Antimetabolites trifluorothymidine; XXXVI, 5-iodo-2'-deoxyuridine, IUDR; XXXVII, arabinosyl cytosine; XXXVIII, 6-azauridine; XXXIX, 5-azauridine; XL, Some general reviews of this subject are those of Stock (53), 3-deazacytidine; XLI, S'-fluorothymidine; XLII, Showdomycin. Langen (30), Baker (2), Timmis (54), and Brockman (6). The pyrimidine bases that occur in the nucleic acids are uracil (XXX), thymine (XXXI), and cytosine (XXXII). The Arabinosylcytosine (ara-C) (XXXVII) is a compound that is only pyrimidine antimetabolite that is chemotherapeutically very active against tumors, human acute leukemias, and DNA active as the free base is 5-fluorouracil (FU) (XXXIII), which viruses (cf. 10, 23). Its mechanism of action is also under was originally synthesized on the basis of a very definite intensive study. It is rapidly deactivated by deamination to rationale. With all other pyrimidine analogs, the nucleosides arabinosyluracil (ara-U), and interesting research has been are required for biologic activity. A derivative more active than carried out to find inhibitors of this deaminase in the hope of FU is 5-fluoro-2'-deoxyuridine (FUDR) (XXXIV); and trifluo- increasing the chemotherapeutic effects of ara-C (8). rothymidine (XXXV), in addition to being a potent tumor Isosteric replacements have also been fruitful with pyrimi inhibitor, is also the most active compound known to inhibit dine nucleosides. 6-Azauridine (XXXVIII) and 5-azauridine the replication of DNA viruses. These compounds have been (XXXIX) have shown moderate activity against various thoroughly reviewed (22, 23). 5-Iodo-2 '-deoxyuridine (IUDR), tumors, and this field has been reviewed by Skoda (52). It is of (XXXVI) is clinically active against herpes simplex keratitis interest that although trifluorothymidine and 6-azathymidine

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1969 American Association for Cancer Research. CharlesHeidelberger have biologic activity, a compound, 5-trifluoromethyl-6-aza- demonstrated that double-stranded poly-inosinic:poly- 2'-deoxyundine, that combines both substitutions, was cytidylic acid preparation (poly-IC) is capable of preventing biologically inert (12). Another compound with an isosteric or inhibiting viral replication in vitro and in vivo by stimula replacement that has activity against bacteria and tumor cells ting interferon production. Very recently, Levy et al. (37) and in culture is 3-deazacytidine (XL) as very recently reported by Homan et al. (26) have reported that poly-IC inhibits the Robins et al. (45). In our laboratory we are synthesizing growth of several tumors in mice, and they have done some comparable compounds in the deoxyribonucleoside series, as pharamacologic studies with the polymer. This opens up an exciting new field of macromolecular chemotherapy. Particu- well as other pyrimidine nucleoside analogs with isosteric replacements. Langen and Kowollik (31) have recently pre lary when more is known about the mechanisms of these pared 5 '-fluorothymidine (XLI), which is active against thy- effects, it appears possible that major advances in specific midylate kinase, and hence is a nucleotide, rather than a cancer chemotherapy may be in the offing. In these days of nucleoside, analog. wild speculations about genetic engineering, it may not be A compound that appears superficially to resemble a totally inappropriate to prophesy that one day cancer may be pyrimidine nucleoside in the antibiotic Showdomycin (XLII) reversed by appropriate polynucleotide chemistry [for a (cf. 16). However, biochemical work by Roy-Burman et al. review of the chemistry of polynucleotides, see Michelson et (47) indicates that it does not act as a pyrimidine analog, but al. (38)]. For example, if as proposed by Pitot and rather it inhibits various enzymes as a consequence of the Heidelberger (42), carcinogenesis is the result of the alkylating properties of the maleiimide moiety. perpetuated deletion of a represser, then if the messenger As in the case of the purine antimetabolites, the pyrimidine RNA that codes for this represser were synthesized, it could nucleosides are continuing to provide interesting new phar cause the revision of that cancer to normal. Impossible? Who macologically active compounds, including many that are not knows? specifically mentioned here. Further chemical efforts in this field seem to be fully justified. Other Miscellaneous Antimetabolites

Poly imdeot Â¡des Again, the listing of these compounds will not be complete, but are selected arbitrarily according to my o Â».ncurrent There is now abundant evidence that mammalian cells can interests. Glutamine (XLIII) is an important biosynthetic take up intact polynucleotides. This has been reviewed for intermediate, and diazo-oxo-norleucine (DON, XLIV) is its DNA by Ledoux (35), and Click has shown that DNA antimetabolite. In view of the interesting therapeutic effects of obtained from mouse thymus can actually inhibit the growth L-asparaginase, Handschumacher et al. (21) designed an of L1210 tumors in vivo (18). In our own studies, we made analogous inhibitor of the substrate asparagine (XLV), namely oligonucleotides of FUDR-5'-phosphate (FUDRP) in 1961 diazo-oxo-nor-L-valine (XLVI), which they showed to have (44), but these did not show any greater activity than FUDR enzymatic specificity. Such an approach may be applied to at inhibiting the incorporation of formate into the DNA other amino acids, should other enzymes of amino acid thymine of Ehrlich ascites cells in vitro. metabolism be found with tumor-inhibitory activities. An important recent discovery by Park and Baron (41) Hydroxyurea (XLVII) has been shown at high concentrations to be a reversible inhibitor of DNA synthesis (65), and it has some cancer chemotherapeutic activity which has stimulated NEN-CH2CO CH2CH2CH-COOH H2 N C CH2CH2Ã‡H COOH the synthesis of related analogs. It has been shown by Krakoff NH, NH2 et al. (29) to be an inhibitor of ribonucleoside diphosphate

XL-Ill XLIV reducÃase. Lastly, I would like to consider three aldehydes. Very recently Apple and Greenberg (1) reported that the combina HgH-C-CH2CH COOH N5NCH2CO CH2CH COOH tion of two metabolites in minor branches of the 3-carbon NH2 NH2 stage of glycolysis, 2-oxopropanal (XLVIII) and 2,3-dihy-

TTCV XLVI droxypropanal (XLIX), cured some mice with transplanted tumors. It has been demonstrated by Ciaranfi et al. (9) that L-eryrfiro-2,3-dihydroxybutyraldehyde, an inhibitor of pro HgN-C-NHOH CH3C-CHO CH2-CH-CHO tein synthesis and not of glycolysis, has considerable O OH Ã’H antitumor activity. Thus, it appears that more synthesis of yo/ir XLVIM XLIX aldehydes may be in order. CH,CH-CH CHO 3 i i OH OH CONCLUSIONS

Chart 5. XLIII,glutamine; XLIV, diazo-oxo-norleucine (DON); XLV, What is the need for additional alkylating agents and asparagine; XLVI, diazo-oxo-norvaline; XLVII, hydroxyurea; XLVIII, antimetabolites? In my opinion there is no need for additional 2-oxopropanal; XLIX, 2,3-dihydroxypropanal; L, L-erythro-2,3-duiy- alkylating agents dreamed up ad hoc, but possibly there is a droxybutyraldehy de. need for compounds specifically designed relative to the

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Charles Heidelberger

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