2,4-Diamino-5-substituted-quinazolines as Inhibitors of a Human Dihydrofolate Reductase with a Site-Directed Mutation at Position 22 and of the Dihydrofolate Reductases from Pneumocystis carinii and Toxoplasma gondii

Abstract

2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a classical (p-aminobenzoyl)-L-glutamate side chain, were synthesized as potentially selective inhibitors of a site-directed mutant of human dihydrofolate reductase (DHFR) containing phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross resistance to the nonclassical antifolates piritrexim (PTX) and trimetrexate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild-type enzyme. The potency of the analogue with a classical (p-aminobenzoyl)-L-glutamate side chain was similarly diminished in comparison with MTX, but the difference in its binding affinity to the two DHFR species was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to mutational alterations in the active site of the target enzyme. Also, to test the hypothesis that DHFR from Pneumocystis carinii and Toxoplasma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the lipophilic analogues in the series were carried out against the P. carinii and T. gondii reductases in comparison with the enzyme from rat liver. In contrast to their preferential binding to mutant versus wild-type human DHFR, binding of these analogues to the P. carinii and T. gondii enzymes was weaker than binding to rat enzyme. It thus appears that, if the active site of the DHFR from these parasites is less sterically restrictive than the active site of the mammalian enzyme, this difference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.

Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations

“…Quinazolines IV.1 to IV.3 were obtained from the corresponding anthranilonitriles and chloroformamidine hydrochloride (62). Quinazolines IV.4 to IV.7 were obtained from 2,4-diamino-5-iodoquinazoline and an alkene or alkyne via a palladiumcatalyzed Heck reaction, followed by catalytic hydrogenation in the case of IV.6 and IV.7 (65). N 10 -unsubstituted quinazolines IV.8 to IV.12 were obtained from 2,4-diaminoquinazoline-5-carbonitrile and the appropriate substituted anilines by reductive coupling in the presence of Raney nickel (65).…”

Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae

“…These lipophilic DHFR inhibitors are assumed to cross the plasma membrane by passive or facilitated diffusion and, therefore, to obviate drug susceptibility and resistance problems associated with carrier-or receptor-mediated folate transport mechanisms (7). Many of these compounds have previously been assayed against P. carinii DHFR and T. gondii DHFR-TS in vitro (48,49,(51)(52)(53)(54)(55)(56). For example, the IC 50 of PY875 against the P. carinii DHFR enzyme in vitro was ϳ7 M (51), suggesting that PY875 might also inhibit the growth of Pc-yeast; Table 6 shows this to indeed be the case.…”

“…The activities of many of these drugs against these parasites or their purified DHFR enzymes was known already. For example, members of the PY series had been tested in vitro to measure inhibition of the P. carinii or T. gondii enzyme (48,49,(52)(53)(54)56). We knew at the outset which compounds were effective in vitro against the DHFRs of other pathogens, which allowed us to quickly identify drugs that did not effectively penetrate the yeast host.…”

Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of Their Effects on β‐Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry

“…Previously 2,4-diamino-5-substituted quinazolines have been shown to act as inhibitors of human and bacterial DHFR. [43,44] This may explain the observed toxicity at concentrations greater than 30 μM. The fact that substituents on the 6- and 7-positions of 2,4-diaminoquinazoline result in decreased GCase inhibition and an increase in DHFR inhibition suggests that selectivity of 1 congeners for GCase over DHFR could be increased by modifying the substituents on the 5-position of quinazoline.…”