Vol. 2, 227-243, February 1996 Clinical Cancer Research 227 Minireview Thymidylate Synthase Inhibitors Nikolaos Touroutoglou and Richard Pazdur 1 Introduction Division of Medicine, The University of Texas M. D. Anderson TS 2 inhibitors are a new class of compounds that target TS, Cancer Center, Houston, Texas 77030 an enzyme in the folate metabolic pathways necessary for thy- midylate synthesis, specifically. By inhibiting TS, these agents decrease de novo thymidylate synthesis, which is necessary for Abstract DNA synthesis and repair. Contrary to prior empiric approaches Folate analogues that inhibit thymidylate synthase (TS) to anticancer drug discovery, the development of new TS inhib- selectively were developed based on TS and folate molecular itors was based on the targeted and specific design of new structures and properties. The structure-activity relation- molecules and relied on extensive knowledge of the structure, ship, preclinical and clinical development, and issues of conformation, and properties of TS and the mode of action of potential importance in the future success of these TS inhib- folate analogues. These compounds are being tested currently in itors are reviewed herein. Properties of these new com- preclinical and clinical settings, and their role in clinical practice pounds depend mainly on the use of the reduced folate as single agents or in combination regimens remains to be carrier (RFC) proteins for cellular entry and on intracellu- defined. This review will provide a background of the mecha- lar polyglutamation, which augments TS inhibitory function nism of TS inhibition, the development and current status of and cellular retention. CB3717 [Zeneca (formerly ICI Phar- folate analogues that inhibit TS selectively, the mechanisms of maceuticals), Macclesfield, United Kingdom], the first selec- resistance to TS inhibition, and the current approaches to aug- tive TS inhibitor developed, demonstrated antineoplastic ment the efficacy of these new compounds. activity in Phase I trials, but its development was abandoned due to nephrotoxicity. ZD1694 (Tomudex; Zeneca), a water- TS soluble, nonnephrotoxic quinazoline, demonstrated activity A critical step in the de novo pathway of DNA synthesis is in colorectal, breast, and pancreatic cancer. Phase III trials the production of the pyrimidine nucleotide TMP from dUMP. of Tomudex in advanced colorectal cancer were completed This reaction is catalyzed by the enzyme TS using the folate recently. LY231514 (Eli Lilly Research Labs, Indianapolis, cosubstrate CH2THF (Fig. 1) and is the only source of de novo cellular thymidylate. IN) uses the RFC and polyglutamation to exert its selective TS (Fig. 1) is a two-substrate enzyme that accepts both TS inhibitory action. Phase I trials of this compound have dUMP and CHzTHF to form a ternary complex through which been completed. ZD9331 (Zeneca), currently in preclinical reductive methylation of dUMP to TMP occurs. In this process, studies, was designed to obviate the use of the RFC for tetrahydrofolate is oxidized to dihydrofolate. This reaction pro- cellular entry. 1843U89 (Glaxo-Wellcome, Research Trian- vides the de novo cellular thymidylate nucleotides necessary for gle Park, NC), currently in Phase I trial, does not require the DNA synthesis and repair. TS inhibition results in a thymine- RFC; polyglutamation of this potent TS inhibitor leads to its less state, which is toxic to actively dividing cells. This cyto- higher cellular retention without augmenting its TS inhibi- toxicity is effected likely by DNA fragmentation that results tory activity. Currently in clinical trials, AG337 (p.o. and i.v. from dTTP depletion, which increases misincorporation of forms) and AG331 (both by Agouron, La Jolla, CA) are dUTP (1). Cells containing the salvage enzyme TK, which uses lipophilic potent TS inhibitors with action independent of circulating thymidine, can circumvent this cytotoxicity. How- the RFC and polyglutamation. Multiple mechanisms ever, in normal tissues and in some tumor cells, the circulating through which cells may overcome TS inhibition have been thymidine concentrations may not be sufficient to achieve a described. Combining TS inhibitors with other agents that significant effect (2). affect TS, interfere with TS gene and mRNA regulation, or The enzyme involved in regeneration of tetrahydrofolate inhibit salvage mechanisms of thymidylate depletion may from dihydrofolate is DHFR (Fig. 1). Folate analogues (antifo- potentially enable greater clinical utility of this class of lates), such as MTX, inhibit DHFR, deplete tetrahydrofolate, compounds. and cause a marked decrease in pyrimidine and purine synthesis (3-5). Mechanisms of resistance to MTX include reduced drug transport across cell membranes (6-8), reduced drug polyglu- 2 The abbreviations used are: TS, thymidylate synthase; CH2THF, 5,10- methylene tetrahydrofolate; DHFR, dihydrofolate reductase; MTX, methotrexate; 5-FU, 5-fluorouracil; FdUrd, fluorodeoxyuridine; IdUrd, Received 5/31/95; revised 9/11/95; accepted 9/27/95. iododeoxyuridine; RFC, reduced folate carrier; FPGS, folylpolygluta- 1 To whom requests for reprints should be addressed, at Division of mate synthase; FdUMP, fluoro-dUMP; TK, thymidine kinase; DLT, Medicine, Box 92, The University of Texas M. D. Anderson Cancer dose-limiting toxicity; MTD, maximum tolerated dose; PR, partial re- Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) sponse; LV, leucovorin; ICso, 50% inhibitory concentration; AZT, 792-3634; Fax: (713) 745-1827. zidovudine. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. 228 Thymidylate Synthase Inhibitors MTX MTX (Glun) I=,,, MTX I "~ " ,~ I ........ t u 'I I Thymidylate Synthase I methylene I,DMFRI FH4 Glun) N5-'1° FH2 (Glun) FH4 (Glun) FH4 (Glun) A ! t- I °~ 1 dUMP dTMP i de novo Pyrimidine DNA Synthesis Synthesis ......L..2... ~ ~ ~-iv ~ _ ..:L ~ :..~~ V N 5"I°methenyl _.., IcYCLO I N 10 formyl I G.APJAICAR I "= ~ FH4 (Glun) FH4 (Glun) -'" "'- FH4 (Glun) -__....__"-- E o Purines -r T N5 meth'¢l FH4 -'~ N5 formyl FH4 Fig. 1 Folate metabolic pathways. Inner square, role of TS in DNA synthesis. Dashed arrows, inhibitory effect. FH4, tetrahydrofolate; FH2, dihydrofolate; (Glu.), polyglutamate; MS, methionine synthase; CYCLO, cyclo,5,10,methenyl-FH4-cyclohydroxylase; GAR, glycinamide ribotide transformylase; A1CAR, aminoimidazole carboxamide ribotide transformylase. tamation (polyglutamation confers intracellular drug retention), the intracellular level of CHaTHF, resulting in the stabilization gene mutation resulting in altered forms of DHFR to which of the ternary complex and thus a higher degree and duration of MTX cannot bind effectively, and an increase in cellular DHFR TS inhibition (13, 14). via DHFR gene amplification (9). The development of antifo- Selective TS inhibition is of interest in drug development lates that inhibit TS (rather than DHFR) selectively may cir- for a variety of reasons. Immunological quantitation using cumvent MTX tumor resistance, especially that resulting from monoclonal antibodies against TS (15, 16) revealed an inverse increased levels of DHFR. association between the treatment response to 5-FU and the TS inhibition can be achieved with either fluoropyrimi- cellular expression of TS; thus, TS overexpression of tumor dines or folate analogue compounds. The first compounds to cells can be a marker and prognostic indicator for 5-FU resis- have clinically significant TS-inhibiting activity were the flu- tance. An association between the degree of TS inhibition and oropyrimidines 5-FU and FdUrd; these are metabolized to the clinical response to chemotherapy in colorectal and breast 5-fluoro-2-deoxyuridylate (the most efficacious analogue of de- cancers has been noted (17, 18). Moreover, expression of TS oxyuridylate) and form a ternary complex together with TS and was found to predict for disease-free survival and survival in CH2THF, resulting in potent TS inhibition. These drugs also patients with rectal cancer independently (19). Folate analogues may become incorporated into RNA or DNA via fluoro-UTP are less susceptible to metabolic degradation than fluoropyrimi- (10, 11) or 5-fluoro-dUTP (12), respectively. These events may dines and are not incorporated into RNA or DNA (3). Finally, obscure quantitation of TS inhibition. Despite this, potentiation tetrahydrofolate is a relatively large molecule providing a vari- of the cytotoxic effects of 5-FU and FdUrd was predicted and ety of sites amenable to manipulation in the design of novel achieved successfully by the addition of folinic acid to increase TS-specific inhibitors. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. Clinical Cancer Research 229 HN--N I II Ptefldlne Ring p-AmlnobenzolcAcid GlutamylResidues O COOH 0 10 II I . .NH__~ ,~--C- NH-- CH N CONH H 3 N I 1 COOH ZD9331 " " Tetrahydrofolate 0 COOH ?. .-(. .,,,"~ CHe~"~,. '~ l/ I H." .'T -"X ~", ]", / II I c CH H,N/~N~ IcflH CB 3717 c~oo. NH~ ~ i(~ CIOOH COOH I - /NH"('. ,)-C- NH-- CH o / s \~ I HN~CHr~% /~'C -NH-iH CH' I II I c,, H.N,,~N,,~ ZD 1694 CH. Memotrexam COOH COOH /NH2 NH COOH --c~ so~--. i"oo. j 1843U89 CH2 SO,- N O H~C \ / CH~ AG-331 Fig. 2 Chemical structures of tetrahydrofolate, MTX, 1843U89, ZD9331, CB3717, ZDl694, AG337, and AG331. To reach and inhibit target enzymes such as DHFR or TS, tiation of TS inhibition that may exceed by 100-fold that of the antifolates enter