Potent Thymidylate Synthase Inhibitor Research Articles

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid.

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).

ChemInform Abstract: Synthesis of Novel, Nonclassical 2‐Amino‐4‐oxo‐6‐(arylthio)ethylpyrrolo[2,3‐d]pyrimidines as Potential Inhibitors of Thymidylate Synthase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of novel, nonclassical 2‐amino‐4‐oxo‐6‐(arylthio)ethylpyrrolo[2,3‐d] pyrimidines as potential inhibitors of thymidylate synthase

AbstractA novel series of 14 nonclassical 6‐substituted pyrrolo[2,3‐d]pyrimidines 2a ‐ 2n were designed as potential inhibitors of thymidylate synthase, based on previously reported 2‐amino‐4‐oxopyrrolo[2,3‐d]‐pyrimidines 1a and 1b. The synthesis of the target compounds 2a‐2n was accomplished by nucleophilic displacement of the mesylate 11 with appropriately substituted aromatic thiols. Most of the target compounds did not show inhibition of either Escherichia coli thymidylate synthase or recombinant human thymidylate synthase at the concentrations tested. However, compounds 2h (2,4‐dichloro), 2j (3,4‐dichloro) and 2m (4‐nitro) did show 25%, 40% and 35% inhibition of human thymidylate synthase at 23 μM, 23 μM and 24 μM, respectively. These observations are in accordance with previous reports, which suggest that strong electron withdrawing substituents on the side chain aromatic ring are conducive to inhibition of thymidylate synthase.

Theoretical Studies Suggest a New Antifolate as a More Potent Inhibitor of Thymidylate Synthase

A set of free energy calculations were performed for selected antifolate compounds as inhibitors of thymidylate synthase (TS). These calculations reproduced the nonadditive behavior of different su...

ChemInform Abstract: Nonclassical 5‐Substituted Tetrahydroquinazolines as Potential Inhibitors of Thymidylate Synthase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Synthesis of Novel Quinazoline‐Based Antifolates with Modified Glutamate Side Chains as Potential Inhibitors of Thymidylate Synthase and Antitumor Agents.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Raltitrexed. A review of its pharmacological properties and clinical efficacy in the management of advanced colorectal cancer.

Raltitrexed (ZD-1694) is a quinazoline-based folate analogue that exerts its cytotoxic activity by the specific inhibition of thymidylate synthase. In vitro studies show that raltitrexed is actively transported into cells and is then rapidly and extensively metabolised to a series of polyglutamates. These metabolites are significantly more potent inhibitors of thymidylate synthase than the parent drug and are retained intracellularly, producing prolonged cytotoxic effects without the need for continuous drug exposure. Phase III clinical trials in patients with advanced colorectal cancer evaluated raltitrexed 3 mg/m2 administered as a 15-minute intravenous infusion once every 3 weeks. This schedule produced objective response rates of 14.3 to 19.3%, which were similar to those in patients treated with fluorouracil plus leucovorin (15.2 to 18.1%). Median survival durations ranged from 9.7 to 10.9 months with raltitrexed treatment and from 10.2 to 12.7 months with fluorouracil plus leucovorin. The major toxicities associated with raltitrexed involve the haematological and gastrointestinal systems, although severe asthenia also occurred in 6 to 18% of patients receiving the drug. Grade 3 or 4 nausea or vomiting occurred in up to 13% of raltitrexed recipients and grade 3 or 4 diarrhoea in up to 14%. Similar incidences of grade 3 or 4 nausea or vomiting and diarrhoea were seen with fluorouracil plus leucovorin treatment. Raltitrexed generally showed significant advantages over fluorouracil plus leucovorin with respect to the incidence of leucopenia and mucositis. A greater proportion of raltitrexed than fluorouracil plus leucovorin recipients were able to receive the scheduled dosage. Thus, with its similar efficacy to fluorouracil-based regimens, convenient administration schedule and favourable tolerability profile, raltitrexed provides clinicians with a worthwhile alternative to fluorouracil-based treatment for patients with advanced colorectal cancer.

Combined Therapeutic Efficacy of the Thymidylate Synthase Inhibitor ZD1694 (TOMUDEX) and the Immunotoxin B43(Anti-CD19)-PAP in a SCID Mouse Model of Human B-Lineage Acute Lymphoblastic Leukemia

The quinazoline antifolate N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (ZD1694; Tomudex) is a potent inhibitor of thymidylate synthase and causes cell death through disruption of DNA synthesis and repair by blocking the obligatory thymidine nucleotide synthesis. B43(anti-CD19)-PAP immunotoxin is a potent inhibitor of protein synthesis in CD19+ B-lineage acute lymphoblastic leukemia (ALL) cells and causes apoptosis. In this model, 100% of SCID mice challenged with 1 × 106 human NALM-6 B-lineage ALL cells develop overt and invariably fatal leukemia. All of the 22 control SCID mice treated with phosphate-buffered saline died of disseminated human leukemia between 31 and 61 days with a median survival of 41.2 days. Treatment with ZD1694 resulted in improved leukemia-free survival with a median survival of 69.2 days (P < 0.001, log-rank test). B43-PAP treatment was more effective than ZD1694 (P = 0.026) and resulted in 51.0 % long-term leukemia-free survival with a median survival of 187.5 days (P < 0.0001. log-rank test). The combination of ZD1694 and B43-PAP was more effective than either agent alone and resulted in 100% long-term leukemia-free survival. To our knowledge, this preclinical study is the first to demonstrate the feasibility and therapeutic advantage of combining an anti-leukemia immunotoxin with a thymidylate synthase inhibitor.

The role of thymidine kinase activity in murine colon tumours treated with 5-fluorouracil.

5-Fluorouracil (5FU) is widely used in the treatment of colon cancer. To exert its antitumour effect 5FU has to be converted into toxic metabolites: FUTP, FdUTP and FdUMP. FUTP and FdUTP are incorporated into RNA and DNA, respectively. FdUMP provides a potent inhibitor of thymidylate synthase (TS) and thereby inhibits de novo formation of dTMP and DNA synthesis. The latter is considered the main mechanism of anti-tumour activity.1,2

Increased cell killing and mutagenecity by DNA alkylating agents in cells with decreased TTP pools.

Numerous studies have demonstrated that an imbalance of deoxyribonucleoside triphosphates (dNTPs) pools is mutagenic and can alter the lethality and the mutagenicity of DNA-damaging agents [1]. In order to examine the effect(s) of the changes in dNTP pools on DNA fidelity, various drugs, including methotrexate (MTX), fluorodeoxyurid-ine (FdUrd) and fluorouracil (FUra), have been used. The attempts to utilize these drugs as inhibitors of dNTP synthesis, however, have been hampered because they have several sites of action. For the purpose of investigating whether the reduced TTP pool affected the lethality and the mutagenesis induced by DNA-alkylating agents, we have employed two strategies. First, a thymidylate synthase (TS) inhibitor, ICI D l694 [2], was used. ICI D l694 is a specific and potent TS inhibitor, which exerts its action mainly through metabolizing to its polyglutamate derivatives. Although the monoglutamate of ICI D l694 has similar inhibitory activity against TS and dihydrofolate reductase, the polyglutamate forms are considered to be much more specific for TS [3]. Therefore, treatment of cells with ICI D l694 is expected specifically to decrease intracellular TTP levels. Second is to introduce a mutational loss of thymidine kinase (TK), which is an important salvage enzyme for maintaining TTP pools. In a human promyelocytic leukemia cell line, HL-60, TK deficient mutant had only one fifth of TTP pools of the wild type cells [4]. In the present study, we try to determine the effect of the diminished TTP pool on the killing or mutagenicity induced by DNA-alkylating agents using these strategies.KeywordsThymidine KinaseHypoxanthine Phosphoribosyl TransferasedNTP PoolHPRT LocusHuman Promyelocytic Leukemia Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

New antimetabolites in cancer chemotherapy and their clinical impact.

It is almost 50 years since antimetabolites were first found to have clinical antitumour activity, with Farber's discovery that aminopterin could cause remission in acute leukaemia. In the following 10 years, methotrexate, 6-mercaptopurine and 5-fluorouracil (5-FU) found their way into clinical practice. Subsequently, cytosine arabinoside was found to have activity in acute leukaemia, but, until recently, other significant developments have involved optimizing the efficacy of existing antimetabolites, including the use of leucovorin with methotrexate or 5-FU. Recently, new antimetabolites have become a fertile area for anti-cancer drug research. Gemcitabine (GEMZAR) has emerged as an important new agent in several tumour types, including pancreatic, non-small-cell lung, bladder, breast and ovarian cancers. Capecitabine is an intriguing new prodrug, offering tumour selectivity and prolonged tumour exposure to 5-FU. More potent thymidylate synthase inhibitors have also emerged; raltitrexed is now commercially available for the treatment of colorectal cancer. Others under development include LY231514, which has other sites of action, hence the acronym MTA (multi-targeted antifolate). A novel target is glycinamide ribonucleotide formyltransferase (GARFT) and LY309887 and AG2034 are undergoing clinical investigation as GARFT inhibitors. A critical element with LY309887 appears to be co-administration of folate. It seems entirely possible that several novel antimetabolites will establish themselves in clinical practice in future for the treatment of solid tumours.

Nonclassical 5‐substituted tetrahydroquinazolines as potential inhibitors of thymidylate synthase

AbstractClassical inhibitors of thymidylate synthase such as Nl0‐propargyl‐5,8‐dideazafolic acid (1), N‐(5‐[N‐(3,4‐dihydro‐2‐methyl‐4‐oxoquinazolin‐6‐ylmethyl)‐N‐methylamino]‐2‐thenoyl)‐L‐glutamic acid (ZD1694, 2) and N‐[2‐amino‐4‐oxo‐3,4‐dihydro(pyrrolo[2,3‐d]pyrintidin‐5‐yl)ethylbenzoyl]‐L‐glutamic acid (LY231514, 3) while potent, suffer from a number of potential disadvantages, such as impaired uptake due to an alteration of the active transport system required for their cellular uptake, as well as formation of long acting, non‐effluxing polyglutamates via the action of folylpolyglutamate synthetase, which are responsible for toxicity. To overcome some of the disadvantages of classical inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical thymidylate synthase inhibitors, which could enter cells via passive diffusion. In an attempt to elucidate the role of saturation of the B‐ring of non‐classical, quinazoline antifolate inhibitors of thymidylate synthase, analogues 7‐17 were designed. Analogues 13‐17 which contain a methyl group at the 7‐position, were synthesized in an attempt to align the methyl group in an orientation which allows interaction with tryptophan‐80 in the active site of thymidylate synthase. The synthesis of these analogues was achieved via the reaction of guanidine with the appropriately substituted cyclohexanone‐ketoester. These ketoesters were in turn synthesized via a Michael addition of the appropriate thiophenol with 2‐carbethoxycyclohexen‐1‐one or 5‐methyl‐2‐carbethoxycyclo‐hexen‐1‐one to afford a mixture of diastereomers. The most inhibitory compound was the 3,4‐dichloro, 7‐methyl derivative 17 which inhibited the Escherichia coli and Pneumocystis carinii thymidylate syntheses 50% at 5 × 105 M. Our results confirm the importance of the 7‐CH3 group and electron withdrawing groups on the aromatic side chain for thymidylate synthase inhibition.

Chemotherapy for the treatment of patients with metastatic colorectal cancer: an overview.

Approximately 50% of patients with colorectal cancer develop locally recurrent or distant metastatic disease during the course of their illness and eventually die. Since the 1950s the mainstay of treatment for patients in need of palliative therapy has been and continues to be the fluoropyrimidines. When 5-fluorouracil (5-FU) was first introduced into the clinic it was used as a single agent given by rapid intravenous injection. Results with this drug have been disappointing, with response rates consistently low, usually of brief duration, and with little or no impact on survival. During the 1970s and 1980s, multidrug regimens were evaluated with little or no improvement in outcome. More recently, our understanding of the metabolism, pharmacology, and the mechanisms of action as well as the potential mechanisms of resistance to 5-FU has led to its more rational use. This knowledge has resulted in the design of treatment programs with improved therapeutic effects by changing its route of administration, combining it with biochemical modulators and using it in conjunction with other chemotherapeutic agents. These strategies have created new optimism for improved results with less toxicity. More potent inhibitors of thymidylate synthase (TS) such as tomudex and trimetrexate have been developed and are currently being evaluated in the clinic either alone or in combination with 5-FU. Semisynthetic topoisomerase inhibitors such as irinotecan have shown encouraging results as first-line therapy, in combination with 5-FU or as salvage therapy.

Synthesis of novel quinazoline-based antifolates with modified glutamate side chains as potential inhibitors of thymidylate synthase and antitumour agents

Several novel antifolates, derivatives of 2-desamino-2-methyl- N 10-propargyl-5,8-dideazafolic acid, were synthesised as inhibitors of thymidylate synthase (TS) and antitumour agents. This was accomplished by first developing routes to the key intermediates Glu-Ome-γ-ψ[CSNH]Glu(OEt)-OEt ( 8), Glu-OBu t-γ-ψ[CH 2NH]Glu(OBu t)-OBu t ( 16), Glu-OMe-γ-ψ[CN 4]Gly-OMe ( 23) and its 2,5-disubstituted regioisomer ( 22), followed by DEPC coupling to 4-[ N-[3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)-methyl]- N-prop-2-ynylamino]benzoic acid ( 9) or 4-[ N-[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)-methyl]- N-prop-2-ynylamino]-2-fluorobenzoic acid ( 24), and finally removal of the protecting groups. The resulting quinazoline-based antifolates with modified glutamate side chains, and in particular, the tetrazole derivatives 26 and 29 displayed potent TS and L1210 cell growth inhibitory activities ( e.g., for 26: TS IC 50 = 2.4 nM, L1210 IC 50 = 1.3 μM).

Folate-based inhibitors of thymidylate synthase: synthesis and antitumor activity of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

In an effort to synthesize inhibitors of thymidylate synthase (TS) that do not undergo polyglutamation, a series of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) was prepared. A methyl, ethyl, or propargyl group was incorporated into the gamma-glutamyl amide bond of gamma-linked L,L dipeptide derivatives of ICI 198583, such as ICI 198583-gamma-L-Glu. In addition, steric bulk was introduced on either side of the gamma-glutamyl bond of ICI 198583-gamma-L-Glu or ICI 198583-gamma-L-Ala. The resulting dipeptide analogues, e.g., ICI 198583-gamma-MeGlu and ICI 198583-gamma-Aib, were apparently stable to in vivo hydrolysis but poorer inhibitors of TS and L1210 cell growth. However, introduction of 7-Me, 2'-F substitution into the quinazoline nucleus gave significant improvement in the inhibitory activity against thymidylate synthase. Compounds 28-30, the 7-Me, 2'-F derivatives of ICI 198583-gamma-MeGlu, ICI 198583-gamma-EtGlu, and ICI 198583-gamma-PgGlu, respectively, were potent inhibitors of TS (K(iapp) = 0.21-1.1 nM) and L1210 cell growth (IC50 = 0.05-0.34 microM) and were similar to that seen with the most potent gamma-linked L,D dipeptide derivatives of ICI 198583 previously synthesized. Furthermore, the low cross-resistance ratios for the L1210:R(D1694)/L1210 cell line indicated that 28-30 do not undergo polyglutamation.

The role of thymidine kinase activity in murine colon tumours treated with 5-fluorouracil

5-Fluorouracil (5FU) is widely used in the treatment of colon cancer. To exert its antitumour effect 5FU has to be converted into toxic metabolites: FUTP, FdUTP and FdUMP. FUTP and FdUTP are incorporated into RNA and DNA, respectively. FdUMP provides a potent inhibitor of thymidylate synthase (TS) and thereby inhibits de novo formation of dTMP and DNA synthesis. The latter is considered the main mechanism of anti-tumour activity.1,2

Folate-based thymidylate synthase inhibitors in cancer chemotherapy.

Understanding the relationship between chemical structure and biological properties of folate analogs, particularly their interactions with the target enzymes, transport proteins and folate-metabolizing enzyme, folylpolyglutamate synthetase (FPGS), has enabled the rational design and development of the selective thymidylate synthase (TS) inhibitors with folate-based structures for clinical uses. These compounds specifically inhibit TS devoid of concomitant effects at other loci, unlike 5-fluorouracil (5-FU). ZD1694 ('Tomudex') was designed as a non-nephrotoxic and highly active analog of N10-propargyl-5,8-dideazafolic acid (CB3717), which is a potent TS inhibitor but had unacceptable nephrotoxicity caused by its poor water solubility. The potent cytotoxic activity of ZD1694 is dependent upon active uptake into cells via the reduced folate carrier (RFC), and subsequent rapid and extensive metabolism to polyglutamate forms inside cells. Marked enhancement of the TS inhibitory activity has been noted as the glutamate chain is elongated. Polyglutamation is critical to the biological activity of ZD1694 against tumor and normal proliferating tissues. The retentive property of ZD1694 polyglutamates inside cells led to a single, infrequent administration schedule in clinical studies. ZD1694 has completed phase I and phase II evaluation with activity observed in several tumor types, particularly in colorectal cancer with a 26% objective response rate. A recent European phase III study of ZD1694, randomized against a 5-FU plus leucovorin regimen, demonstrated an equivalent response rate for advanced colorectal cancer (complete or partial responses; 20 versus 17%) and less toxicity than seen with the latter regimen. The newer selective TS inhibitors, which retain potency for TS inhibition but are not substrates for RFC and/or FPGS, are currently under clinical evaluation. These classes of compound may have benefits for circumvention of resistance by virtue of alterations in these protein functions and for the management of toxicity.

Resistance to Tomudex (ZD1694): Multifactorial in human breast and colon carcinoma cell lines

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 μM, resulted in stably resistant cell lines (MCF TDX and H630 TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCF TDX and a 52-fold increase in TS levels in H630 TDXcell lines. Despite continued exposure of these cells to 2.0 μM TDX, TS protein and TS mRNA expression decreased to parental levels in H630 TDX cells, whereas in MCF TDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCF TDXcell line. TDX uptake was 2-fold higher in resistant MCF TDX cells than in parental MCF-7 cells, whereas in H630 TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630 TDX cells was noted when compared with the H630 parental cells. In H630 TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630 TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCF TDX and H630 TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.

2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase.

Six novel 2-amino-4-oxo-5-[(substituted phenyl)sulfanyl]pyrrolo[2,3-d]pyrimidines 7-12 were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor and/or antibacterial agents. The analogues contain a 5-thio substituent with a phenyl, 4'-chlorophenyl, 3',4'-dichlorophenyl, 4'-nitrophenyl, 3',4'-dimethoxyphenyl, and 2'-naphthyl on the sulfur, and were synthesized from the key intermediate 2-(pivaloylamino)-4-oxo-6-methylpyrrolo[2,3-d]-pyrimidine, 17. Appropriately substituted aryl thiols were appended to the 5-position of 17 via an oxidative addition reaction using iodine, ethanol, and water under conditions which also resulted in the deprotection of the 2-amino group. The compounds were evaluated against human, Lactobacillus casei, Escherichia coli, Streptococcus faecium, and Pneumocystis carinii (pc) TSs and against human, rat liver (rl), pc, and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues with the 3',4'-dichloro and the 4'-nitro substituents in the side chain (9 and 10) were more potent than N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop- 2-ynylamino]benzoyl]-L-glutamic acid (PDDF, 1) and N-[5-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- methylamino]-2-thenoyl]-L-glutamic acid (ZD1694,2) against human TS. Analogues with the 4'-chloro, 3',4'-dimethoxy, and naphthyl side chains (8, 11 and 12) were more potent than the unsubstituted phenyl analogue (7) but less than 2, 9, and 10 by 1 order of magnitude. They were all poor inhibitors of human, rl, and pc DHFRs (IC50 = 10(-5) M) but moderate inhibitors (IC50 = 10(-6) M) of tg DHFR. The 4-nitro analogue, 10 (EC50 1.5 microM), was comparable to PDDF in its potency as an inhibitor of the growth of the FaDu human squamous cell carcinoma cell line.

Relationships between resistance to cisplatin and antifolates in sensitive and resistant tumour cell lines

Possible relationships between tumour resistance to cisplatin and the folate-based thymidylate synthase (TS) inhibitors, CB3717 and ZD1694 (tomudex), have been investigated in vitro using a panel of tumour cell lines (predominantly human ovarian), either parental or possessing acquired resistance to cisplatin or ZD1694. Across eight parent human tumour cell lines, ZD1694 was the most potent drug (mean ic 50 of 1.9 × 10 −8 M), being over 250 times as potent as its prototype CB3717 (mean ic 50 of 4.8 × 10 −6 M). In five pairs of acquired cisplatin-resistant human tumour cell lines (three ovarian, one cervical and one testicular) which encompass all of the main known mechanisms of platinum drug resistance, ZD1694, CB3717 and the DHFR inhibitor, methotrexate, all exhibited non-cross-resistance. The cervical line, HX/155cisR, showed collateral sensitivity to ZD1694, CB3717, 5-fluorouracil (FUra) and fluorodeoxyuridine (FdUrd). One cell line, A2780cisR, showed a low level of cross-resistance to FUra (resistance factor, RF, of 1.5) and FdUrd (RF of 3.8). A2780cisR, in common with two other cisplatin-resistant lines, did not possess elevated TS activity compared with its parent. Cisplatin retained activity in four acquired ZD1694-resistant cell lines (encompassing reduced folate transport, elevated TS and defective polyglutamation mechanisms of resistance). Furthermore, combinations of ZD1694 with each of the platinum-based drugs, cisplatin, carboplatin and the recently introduced orally administrable, JM216, all showed additive growth inhibitory effects by median effect analysis. These data suggest that the tumour inhibitory properties of the recently introduced highly potent TS inhibitor, ZD1694, and cisplatin, and, moreover, their respective mechanisms of resistance, do not overlap. Therefore, these drugs may be considered for combination in the clinic.