Synthesis, biological, and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.     

New fluoro derivatives of the pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide system: evaluation of fluorine binding properties in the benzodiazepine site on γ-aminobutyrric acid type A (GABA(A)) receptor. Design, synthesis, biological, and molecular modeling investigation

In the search for potent ligands at the benzodiazepine site on the GABA(A) receptor, new fluoro derivatives of the pyrazolo[5,1-c][1,2,4]benzotriazine system were synthesized to evaluate the importance of the introduction of a fluorine atom in this system. Biological and pharmacological studies indicate that the substitution at position 8 with a trifluoromethyl group confers pharmacological activity due to potential metabolic stability in comparison to inactive 8-methyl substituted analogues. In particular, the compound 3-(2-methoxybenzyloxycarbonyl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (21) emerges because of its selective anxiolytic profile without side effects. An analysis of all the newly synthesized compounds in our pharmacophoric map confirms the essential interaction points for binding recognition and the important areas for affinity modulation. The fluorine atom was able to form a hydrogen bond interaction only when it is not in position 3.