ag-331 and raltitrexed

Antifolate drugs, as a class, have broad-spectrum activity against both hematologic and solid human malignancies. The pharmacokinetics of the classical antifolate methotrexate have been well-defined and pharmacokinetic data can be exploited to reduce the toxicity and enhance the activity of the drug. Methotrexate remains the only anticancer drug for which plasma drug level monitoring is used in routine clinical practice. Recently, novel classical and nonclassical antifolates have been developed that target either specific folate-dependent enzymes (e.g., thymidylate synthase [CB3717, raltitrexed, ZD9331, 1843U89, nolatrexed, AG331], glycinamide ribonucleotide transformylase [lometrexol, LY309887, AG2034] or multiple folate-dependent enzymes (e.g., MTA/LY231514). In the early clinical trials of these agents, a number of pharmacokinetic-pharmacodynamic relationships were identified and it is highly likely that the full therapeutic potential of these new drugs will also require the exploitation of pharmacokinetic data.

Topics: Antimetabolites, Antineoplastic; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Humans; Indoles; Isoindoles; Methotrexate; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase

Thymidylate synthase (TS) is a critical enzyme for DNA replication and cell growth because it is the only de novo source of thymine nucleotide precursors for DNA synthesis. TS is the primary target of 5-fluorouracil (5-FU), which has been used for cancer treatment for more than 40 years. However, dissatisfaction with the overall activity of 5-FU against the major cancers, and the recognition that TS still remains an attractive target for anticancer drugs because of its central position in the pathway of DNA synthesis, led to a search for new inhibitors of TS structurally analogous to 5,10-methylenetetrahydrofolate, the second substrate of TS. TS inhibitory antifolates developed to date that are in various stages of clinical evaluation are ZD 1694 and ZD9331 (Astra-Zeneca, London, UK), (Eli Lilly, Indianapolis, IN), LY231514 (BW1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), and AG337 and AG331 (Agouron, La Jolla, CA). Although each of these compounds has TS as its major intracellular site of action, they differ in propensity for polyglutamylation and for transport by the reduced folate carrier. LY231514 also has secondary target enzymes. As a result, each compound is likely to have a different spectrum of antitumor activity and toxicity. This review will summarize the development and properties of this new class of TS inhibitors.

Topics: Animals; Antimetabolites, Antineoplastic; Colorectal Neoplasms; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase

The enzyme, thymidylate synthase (TS) is considered an important target for the development of new anticancer agents. Moreover, the folate-binding site in TS is believed to offer better opportunities for the design of highly specific inhibitors than the pyrimidine (dUMP) binding site. This belief led to the design of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline-based drug which had antitumour activity in clinical studies. Occasional, but serious nephrotoxicity led to the withdrawal of CB3717 from further clinical study. More water-soluble and non-nephrotoxic analogues were developed with an interesting diversity in biochemical profile, particularly with respect to interactions with the reduced-folate cell membrane carrier (RFC) and folylpolyglutamate synthetase (FPGS). An example of a compound that uses both of these processes well is the quinazoline, ZD1694 (Tomudex), a drug which is about to complete phase III evaluation for colorectal cancer. High chain length polyglutamates are formed that are up to 70-fold more potent TS inhibitors than the parent drug (Ki tetraglutamate = 1 nM). Furthermore they are retained in cells/tissues for a prolonged period. A number of other novel folate-based TS inhibitors are currently in pre-clinical or clinical study. For example, LY231514 is a pyrrolopyrimidine analogue in phase I study and, although less potent as a TS inhibitor, has biochemical properties similar to ZD1694. Another compound in phase I study is the benzoquinazoline, BW1843U89 which has somewhat different properties. It is a very potent TS inhibitor (Ki = 0.09 nM) and an excellent substrate for the RFC (human) and FPGS, but polyglutamation proceeds to diglutamate only and is not accompanied by increased TS inhibition. Another highly water-soluble compound in pre-clinical development is ZD9331 which was specifically designed to use the RFC but not be a substrate for FPGS. Potent TS inhibition (Ki = 0.4 nM) was achieved through a rational programme of computerised molecular modelling of the active site of TS and a large database of structure-activity relationships. Two lipophilic compounds were designed to be devoid of interactions with either the RFC or FPGS. High resolutions crystal complexes of E. coli TS were central to obtaining potent TS inhibitors and both AG337 (Ki human recombinant TS = 16 nM) and AG331 (Ki = 12 nM) are in clinical studies. This portfolio of novel compounds therefore comprehensively addresses the potential of TS as

Topics: Antineoplastic Agents; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase

The patterns of DNA fragmentation were evaluated following a brief exposure (2 h) of the human ileocecal adenocarcinoma cell line, HCT-8, to several specific thymidylate synthase inhibitors, a quinazoline (ZD1694) and benz[cd]indole-containing molecule (AG-331). The magnitude and size of DNA fragmentation induced by the two agents were assessed by alkaline elution for DNA single-strand breaks (ssbs), and by pulsed- and constant-field gel electrophoresis for DNA double-strand breaks (dsbs). Both agents induced dose-dependent DNA dsbs. While AG-331 induced ssbs and dsbs only in nascent DNA, ZD1694 affected both genomic and nascent DNA. The fragments of newly synthesized and genomic DNA, estimated by pulsed-field gel electrophoresis assay, were associated with the bands in the range of 0.05 to 1.1 and 1.1 to 5.7 megabases, respectively. 5-fluoro-2'-deoxyuridine (FdUrd), like ZD1694, produced both mature and nascent DNA fragmentation, whereas only nascent DNA breakage induced by 5-fluorouracil (FUra) was detected, similar to AG-331. The induction of both mature and nascent DNA fragmentation by ZD1694 and FdUrd appears to correlate with the higher, but similar, potency of these agents. Aphidicolin, a DNA polymerase inhibitor, protects from DNA dsbs and cytotoxicity by ZD1694 and AG-331. These observations suggest that replicative DNA synthesis is an important factor in ZD1694- and AG-331-induced DNA fragmentation and, subsequently, cell growth arrest. The results indicate that although the new antimetabolites investigated herein were developed and extensively evaluated as specific and potent thymidylate synthase inhibitors, DNA damage appears to be an important additional determinant of drug effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antimetabolites, Antineoplastic; Aphidicolin; Cell Division; DNA Damage; DNA, Neoplasm; Enzyme Inhibitors; Floxuridine; Fluorouracil; Genome; Humans; Indoles; Quinazolines; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

The effects of de novo dTMP inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2- thenoyl)-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6-thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC90 were 6.0 and 9.0 nM, respectively and IC50 and IC90 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 microM for 24-h exposures, and IC50 and IC90 for TS inhibition were 0.7 and 3.0 microM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5-amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 microM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.

Topics: Carrier Proteins; Humans; Indoles; Membrane Proteins; Nucleoside Transport Proteins; Quinazolines; Thiophenes; Thymidine Kinase; Thymidylate Synthase; Tumor Cells, Cultured; Urinary Bladder Neoplasms