No abstract

3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. The aim of the design program was to identify TS inhibitors with different pharmacological characteristics from classical folate analogs and, most notably, to develop non-glutamate-containing molecules which would not require facilitated transport for uptake and would not undergo intracellular polyglutamylation. One molecule which resulted from this program, AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention. In vivo, AG337 was highly active against the thymidine kinase-deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress.

The X-ray crystal-structure-based design, synthesis, and biological activity of a novel family of benz[cd]indole-containing inhibitors of thymidylate synthase (TS) are described. The structure-activity of the lead compound was studied by conceptually dividing the molecule into four regions and independently optimizing the substituents for each region. Combination of favored substituents for each region led to inhibitors with Ki's against the human enzyme in the range of 10-20 nM. Thymidine shift experiments suggested that the cytotoxic properties of the best enzyme inhibitors were due to TS targeting in cells. The inhibitors were synthesized from substituted 6-aminobenz[cd]indol-2(1H)-ones by alkylation with both a simple alkyl group and a substituted benzylic portion. The 2,6-diaminobenz[cd]indoles were prepared from the corresponding lactams by conversion to the thiolactam, alkylation to the methylated thiolactam, and then displacement with a substituted or unsubstituted amine.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART) are described. The compounds were designed using the X-ray crystal structure of human GART. The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-2, 6-diamino-4(3H)-pyrimidinone, 20. The bicyclic compounds were prepared in both racemic and diastereomerically pure forms using two distinct synthetic routes. The compounds were found to have human GART KiS ranging from 30 microM to 2 nM. The compounds inhibited the growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar range and were found to be selective for the de novo purine biosynthesis pathway. The most potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety. Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds 10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity.