N9-substituted 2,4-diaminoquinazolines: synthesis and biological evaluation of lipophilic inhibitors of pneumocystis carinii and toxoplasma gondii dihydrofolate reductase.

N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C2H5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C2H5 substituted compound 8 was highly potent, with IC50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.

Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

Design, synthesis, and computational affinity prediction of ester soft drugs as inhibitors of dihydrofolate reductase from Pneumocystis carinii.

A series of dihydrofolate reductase (DHFR) inhibitors, where the methylenamino-bridge of non-classical inhibitors was replaced with an ester function, have been prepared as potential soft drugs intended for inhalation against Pneumocystis carinii pneumonia (PCP). Several of the new ester-based inhibitors that should serve as good substrates for the ubiquitous esterases and possibly constitute safer alternatives to metabolically stable DHFR inhibitors administered orally, were found to be potent inhibitors of P. carinii DHFR (pcDHFR). Although the objectives of the present program is to achieve a favorable toxicity profile by applying the soft drug concept, a high preference for inhibition of the fungal DHFR versus the mammalian DHFR is still desirable to suppress host toxicity at the site of administration. Compounds with a slight preference for the fungal enzyme were identified. The selection of the target compounds for synthesis was partly guided by an automated docking and scoring procedure as well as molecular dynamics simulations. The modest selectivity of the synthesized inhibitors was reasonably well predicted, although a correct ranking of the relative affinities was not successful in all cases.