Potent Thymidylate Synthase Inhibitor Research Articles

Involvement of Concentrative Nucleoside Transporter 1 in Intestinal Absorption of Trifluorothymidine, a Novel Antitumor Nucleoside, in Rats

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

In Silico Screening of the Library of Pyrimidine Derivatives as Thymidylate Synthase Inhibitors for Anticancer Activity

We here report the virtual screening of several series of pyrimidine derivatives for in silico Thymidylate Synthase (TS) inhibition to arrive at possible potential inhibitors of TS with acceptable pharmacokinetic or ADME (Absorption, Distribution, Metabolism and Excretion) properties. Library of the molecules was constructed based upon structural modifications of pyrimidines nucleus. Structural modifications in descending order were performed for the series of pyrimidines, viz from pyrimidines with five membered heterocyclic ring to pyrimidines with four membered heterocyclic ring to simple pyrimindine carboxylates in an order to arrive at pyrimidines with better inhibition scores (G‐Scores) as compared with Raltitrexed (RTX) and active metabolite of 5‐Fluorouracil (5‐FUMP). The molecules with better G‐Scores were subjected to predict pharmacokinetic or ADME properties. The molecules with acceptable ADME properties and better G‐Scores were prioritized for synthesis and anticancer evaluation. Three molecules from pyrimidine carboxylate series PIC1-31 were found acceptable with G‐Scores and pharmacokinetic properties. Thus a library of pyrimidine derivatives was constructed based upon the feasibility of synthesis and in silico screened to prioritize the molecules and to obtain potential lead molecules as TS inhibitors.

Design, synthesis and evaluation of potent thymidylate synthase X inhibitors

Three synthesized series of compounds based on a thiazolidine core allowed identification of potent inhibitors of thymidylate synthase X. The evaluation of the catalytic activity of the enzyme in the presence of these molecules revealed two distinct classes of compounds that inhibit ThyX with submicromolar concentrations, which could lead, after optimization, to effective inhibitors with potential biomedical interest.

Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo

Murine L1210 leukaemia cells expressing either the reduced folate carrier (RFC) or the membrane folate receptor (MFR) were studied in vitro and in vivo to assess the dynamics of membrane transport of two categories antifolates; folate-based inhibitors of dihydrofolate reductase (methotrexate, edatrexate, aminopterin, PT523, and PT644) and thymidylate synthase (TS) [CB3717, raltitrexed, plevitrexed (BGC9331), pemetrexed and GW1843]. The potency of in situ inhibition of TS was used as an endpoint to analyze the in vitro dynamics of RFC/MFR-membrane transport of these antifolates. Both for L1210-RFC and L1210-MFR cells, the potency of in situ TS inhibition was closely correlated with increasing affinities of these transporters for the antifolates (r = 0.64, P < 0.05 and r = -0.65, P < 0.05, respectively). Within the group of antifolates for which MFR had a low binding affinity, those that had the ability to become polyglutamylated, were more potent inhibitors of TS in situ activity than non-polyglutamatable antifolates. In vivo activity of methotrexate, edatrexate, raltitrexed and pemetrexed was assessed in L1210-RFC and L1210-MFR bearing mice that were fed either a standard or a folate-deficient chow. Dietary folate depletion significantly reduced the MTD for methotrexate (sevenfold), edatrexate (sevenfold), raltitrexed (50-fold) and pemetrexed (150-fold). Based on increased life spans, antitumor effects of methotrexate and edatrexate were markedly better in L1210-RFC bearing mice on the folate-deficient chow (ILS: 455 and 544%, respectively) than on standard chow (ILS: 213 and 263%, respectively). No therapeutic effects of methotrexate and edatrexate were observed for L1210-MFR bearing mice on either chow condition, which may be consistent with the low binding affinity for MFR. Irrespective of the folate diet status, pemetrexed and raltitrexed were inactive against both L1210-RFC and L1210-MFR bearing mice, which may be due to high circulating plasma thymidine levels. Collectively, this study underscores that modulation of dietary folate status can provide a basis within which the therapeutic effect of antifolates may be further improved.

Antimetabolites and cancer: emerging data with a focus on antifolates

Antimetabolites, especially antifolates, play an important role in the treatment of a variety of both malignant, and non-malignant diseases, such as rheumatoid arthritis, and bacterial and parasitic infections. Recently, new antimetabolites have become an area for anticancer drug expansion. Gemcitabine has emerged as an important new agent in several tumour types, including non-small cell lung cancer, pancreatic, 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 been developed; raltitrexed and pemetrexed are now commercially available for the treatment of mesothelioma, non-small cell lung cancer and other solid cancer types. This review will describe the most recent findings and their potential clinical applications.

Novel 2‐Amino‐4‐oxo‐5‐arylthio‐substituted‐pyrrolo[2,3‐d]pyrimidines as Nonclassical Antifolate Inhibitors of Thymidylate Synthase.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Structures of Leishmania major Pteridine Reductase Complexes Reveal the Active Site Features Important for Ligand Binding and to Guide Inhibitor Design

Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductase found in parasitic trypanosomatid protozoans. The enzyme participates in the salvage of pterins and represents a target for the development of improved therapies for infections caused by these parasites. A series of crystallographic analyses of Leishmania major PTR1 are reported. Structures of the enzyme in a binary complex with the cofactor NADPH, and ternary complexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8-tetrahydrobiopterin reveal that PTR1 does not undergo any major conformational changes to accomplish binding and processing of substrates, and confirm that these molecules bind in a single orientation at the catalytic center suitable for two distinct reductions. Ternary complexes with cofactor and CB3717 and trimethoprim (TOP), potent inhibitors of thymidylate synthase and dihydrofolate reductase, respectively, have been characterized. The structure with CB3717 reveals that the quinazoline moiety binds in similar fashion to the pterin substrates/products and dominates interactions with the enzyme. In the complex with TOP, steric restrictions enforced on the trimethoxyphenyl substituent prevent the 2,4-diaminopyrimidine moiety from adopting the pterin mode of binding observed in dihydrofolate reductase, and explain the inhibition properties of a range of pyrimidine derivates. The molecular detail provided by these complex structures identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value.

Synthesis of 2‐Amino‐4‐oxo‐5‐substitutedbenzylthiopyrrolo[2,3‐d]pyrimidines as Potential Inhibitors of Thymidylate Synthase.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of Classical and Nonclassical 2‐Amino‐4‐oxo‐6‐benzylthieno[2,3‐d]pyrimidines as Potential Thymidylate Synthase Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

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

A series of 17 novel 2-amino-4-oxo-5-[(substituted phenyl)thio]pyrrolo[2,3- d]pyrimidines were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor agents. The analogues contain a variety of electron withdrawing substituents on the phenyl ring of the side chain and were evaluated as inhibitors of human TS (hTS) and Escherichia coli TS and of human and E. coli dihydrofolate reductase (DHFR). The analogues 14, 17, and 18 were potent inhibitors of hTS with IC 50 values of 0.28, 0.21, and 0.22 μM, respectively, and were more potent than the clinically used ZD1694, 2 and LY231514, 3 against human TS.

Loss of Reduced Folate Carrier Function and Folate Depletion Result in Enhanced Pemetrexed Inhibition of Purine Synthesis

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 micromol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC(50), in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.

Synthesis of 2-amino-4-oxo-5-substitutedbenzylthiopyrrolo[2,3-d]-pyrimidines as potential inhibitors of thymidylate synthase

A series of ten novel 2-amino-4-oxo-5-[(substitutedbenzyl)thio]pyrrolo[2,3-d]pyrimidines 2-11 were synthesized as potential inhibitors of thymidylate synthase and as antitumor agents. The analogues contain various electron withdrawing and electron donating substituents on the benzylsulfanyl ring of the side chains and were synthesized from the key intermediate 2-amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine, 14. Appropriately substituted benzyl mercaptans were appended to the 5-position of 14 via an oxidative addition reaction using iodine, ethanol and water. The compounds were evaluated against human, Escherichia coli and Toxoplasma gondii thymidylate synthase and against human, Escherichia coli and Toxoplasma gondii dihydrofolate reductase. The most potent inhibitor, (6) which has a 4′-methoxy substituent on the side chain, has an IC50=25 μM against human thymidylate synthase. Contrary to analogues of general structure 1, electron donating or electron withdrawing substituents on the side chain of 2-11 had little or no influence on the human thymidylate synthase inhibitory activity.

Synthesis of classical and nonclassical 2‐amino‐4‐oxo‐6‐benzylthieno‐[2,3‐d]pyrimidines as potential thymidylate synthase inhibitors

AbstractA series of seven nonclassical 2‐amino‐4‐oxo‐6‐substituted thieno[2,3‐d]pyrimidines 2‐8 and one classical N‐[4‐(2‐amino‐4‐oxo‐3,4‐dihydrothieno[2,3‐d]pyrimidin‐6‐ylmethyl)benzoyl]‐L‐glutamic acid 9 (Table I) were designed as the first in a series of 6‐substituted 6‐5 fused ring analogs as potential thymidylate synthase (TS) inhibitors and as antitumor agents. The target compounds were synthesized via a Heck coupling of appropriately substituted iodobenzenes and allyl alcohol followed by cyclization using cyanoacetate and sulfur powder to afford substituted thiophenes. The resulting thiophenes were then cyclocondensed with chloroformamidine hydrochloride to afford 2‐amino‐4‐oxo‐6‐substituted thieno[2,3‐d]pyrimidines 2‐8 and 26. Hydrolysis of 26 followed by coupling with diethyl L‐glutamate afforded 28. The classical analog 9 was obtained by hydrolysis of 28. None of the target compounds inhibited human recombinant thymidylate synthase at 23 μm except 9 for which the IC50 value was 100 μm.

Thymidylate synthase: a critical target in cancer therapy?

For the last four decades, synthesis and testing of potentially active drugs (e.g., antimetabolites) have focused on structural modification of existing metabolites as precursors of DNA and RNA synthesis. In recent years, the focus has shifted to synthesis of target-specific agents. Thus, the current emphasis of drug development is directed at inhibiting specific target(s) expressed preferentially, if not exclusively, in tumor tissues, with the ultimate goal of improving the therapeutic efficacy and selectivity of these new agents. Preclinically, proof-of-principle studies were carried out in tumors with specific expression of the intended target. With the hope of translating preclinical findings to the design of implementation of clinical trials. Thymidylate synthase (TS) continues to be a critical target for 5-fluorouracil (5-FU) and its prodrugs, UFT/LV (Orzel), capecitabine (Xeloda), and S-1, primarily because this enzyme is essential for the synthesis of 2-deoxythymidine-5-monophosphate, a precursor for DNA synthesis. While fluoropyrimidine antimetabolites have other sites of action, antifolates ZD1694 (raltitrexed, Tomudex) and AG337 (Thymitag) are more specific and potent TS inhibitors. Thus, it is hoped that pronounced and sustained inhibition of this enzyme could result in downstream regulation of molecular markers associated with sensitivity and resistance to these agents. It is also critical to recognize that the degree and duration of inhibition of the target enzyme may depend on the expression level of the target enzyme, thymidylate synthase. Correlative studies in preclinical and clinical systems demonstrated a close relationship between the enzyme level (mRNA and protein) and response to therapy of colorectal cancer patients treated with fluoropyrimidine or Tomudex. However, significant overlap was demonstrated between responders and non-responders. These data are consistent with the hypothesis that prediction of response to anticancer drugs is multifactorial, and TS is one target. Clinically, although overall response of colorectal cancer patients to a variety of TS inhibitors is similar, toxicity profiles are different. The availability of the 5-FU prodrugs offers the possibility of greater therapeutic selectivity based on the demonstration that thymidine phosphorylase, the activating enzyme for 5-FU, is expressed at a higher level in tumor tissue compared with normal tissue counterparts. It is likely that successful application of TS inhibitors will not only be based on measurement of the TS level in tumors vs. normal tissues, but on the delineation of the consequences of this inhibition on molecular markers associated with cellular proliferation, apoptosis and cell cycle regulation.

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2 S)-2-{ O-fluoro- p-[ N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)- N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15 min to a slower, constant rate over the next 45 min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50 nM [ 3 H ]pemetrexed, 25 nM [ 3 H ]5-formylTHF, or 50 nM [ 3 H ]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (∼25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.

Antifolate chemistry: synthesis of 4-[N-[(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino]benzoic acid via a (propargyl)Co2(CO)6+ complex.

A new route to compound 3 (4-[N-(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino]benzoic acid), a crucial intermediate for the synthesis of potent inhibitors of thymidylate synthase (TS), is described. In this sequence the C6-N10 bond was constructed first, by the reductive amination of 5-acetamido-6-bromoindan-1-one 6 with tert-butyl 4-aminobenzoate, then the cyclopenta[g]quinazolinone ring was formed and the propargyl group was introduced on the N10-position using the (propargyl)Co2(CO)6+ complex as the electrophilic propargyl reagent.

2‐Amino‐4‐oxo‐6‐substituted‐pyrrolo[2,3‐d]pyrimidines as Potential Inhibitors of Thymidylate Synthase.

AbstractFor Abstract see ChemInform Abstract in Full Text.

2‐amino‐4‐oxo‐6‐substituted‐pyrrolo[2,3‐d]pynmidines as potential inhibitors of thymidylate synthase

AbstractClassical, antifolate inhibitors of thymidylate synthase often suffer from a number of potential disadvantages when used as antitumor agents. These include impaired uptake due to an alteration of the active transport system required for cellular uptake, as well as the formation of long acting, non‐effluxing polygluta‐mates via folypolyglutamate synthetase, which are responsible for toxicity to normal cells. To overcome some of the disadvantages of classical thymidylate synthase inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical inhibitors, which could enter cells via passive diffusion and are not substrates for folypolyglutamate synthetase. A series of eight nonclassical 6‐substituted 2‐amino‐4‐oxo‐pyrrolo[2,3‐d]pyrimidines 2a‐2h were designed as potential inhibitors of thymidylate synthase. The synthesis of the target compounds 2a‐2h was achieved via regioselective iodination at the 6‐position of 5, palladium‐catalyzed coupling with the appropriate phenylacetylenes, reduction of the C8‐C9 triple bond followed by saponification. Preliminary biological results indicated that none of the target compounds showed inhibitory activities against thymidylate synthase from Escherichia coli, Lactobacillus casei, rat or human thymidylate synthase at the concentrations tested. None of the target compounds showed inhibitory activity against dihydrofolate reductase from Escherichia coli, Lactobacillus casei, rat or human at 3.0 × 10−5 M. However, 50% inhibition of dihydrofolate reductase from Pneumocystis carinii and from Toxoplasma gondii was achieved with compound 2d and with compound 2g at 3.0 × 10−5 M.

Effects of impaired renal function on the pharmacokinetics and toxicity of i.v. ZD9331, a novel non-polyglutamated thymidylate synthase inhibitor, in adult patients with solid tumors

ZD9331 is a potent thymidylate synthase inhibitor. Renal and hepatic clearances were found to be important routes of elimination. The objectives of this pharmacologic trial were to investigate the effect of renal impairment on the pharmacokinetics of ZD9331, to study the toxicity profile and to document any antitumor effects of ZD9331 when administered i.v. to patients with different degrees of renal impairment. Patients were treated with ZD9331 130 mg/m2 given as an i.v. infusion on day 1 of a 4-week cycle to allow full pharmacokinetic assessment. Subsequent cycles involved the administration of ZD9331 on days 1 and 8, every 3 weeks. Patients were stratified according to their renal function assessed by the creatinine clearance: normal renal function (creatinine clearance > or =60 ml/min), mildly impaired renal function (creatinine clearance > or =40 to <60 ml/min) and moderately impaired renal function (creatinine clearance >25 to <40 ml/min). For pharmacokinetic analysis plasma sampling was performed during the first course and assayed using a validated liquid chromatographic tandem mass spectrometry assay. Twenty-three patients were entered on the study, of whom 21 received 130 mg/m2 ZD9331 in the first treatment cycle. No relationship was seen between renal impairment and plasma clearance nor with the area under the concentration-time curve of free ZD9331. Increasing renal impairment was associated with a greater incidence of myelosuppression. No predictive relationship between the clearance of free ZD9331 and the degree of renal impairment as determined by creatinine clearance could be assessed. However, data from this trial indicate that increased renal impairment may be associated with greater ZD9331-induced toxicity, particularly myelosuppression, although this cannot be attributed to any alteration in the plasma pharmacokinetics of ZD9331. Therefore, it may be necessary to administer a reduced dose of ZD9331 to patients with impaired renal function.

Sulfamide antifolates inhibiting thymidylate synthase: synthesis, enzyme inhibition and cytotoxicity.

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.