2,4-Diamino-5-(2′-arylpropargyl)pyrimidine derivatives as new nonclassical antifolates for human dihydrofolate reductase inhibition

Dihydrofolate reductase (DHFR) has been a well-recognized target for the development of therapeutics for human cancers for several decades. Classical inhibitors of DHFR use an active transport mechanism to gain access to the cell; disabling this mechanism creates a pathway for resistance. In response, recent research focuses on nonclassical lipid-soluble DHFR inhibitors that are designed to passively diffuse through the membrane. Here, a new series of propargyl-linked antifolates are investigated as potential nonclassical human DHFR inhibitors. Several of these compounds exhibit potent enzyme inhibition with 50% inhibition concentration values under 500 nM. Molecular docking investigations show that the compounds maintain conserved hydrogen bonds between the pyrimidine ring and the enzyme as well as form van der Waals interactions with critical residues in the active site. Interestingly, the most potent compound, 2,4-diamino-5-(3-(3,4,5-trimethoxyphenyl)prop-1-ynyl)-6-ethylpyrimidine (compound 35), is 3500-fold more potent than trimethoprim, a potent inhibitor of bacterial DHFR but weak inhibitor of human DHFR. The two structural differences between compound 35 and trimethoprim show that the propargyl linkage and the substitution at C6 of the pyrimidine ring are critical to the formation of contacts with Thr 56, Ser 59, Ile 60, Leu 22, Phe 31 and Phe 34 and hence, to enhancing potency. The propargyl-linked antifolates are efficient ligands with a high ratio of potency to the number of non-hydrogen atoms and represent a potentially fruitful avenue for future development of antineoplastic agents.

Figure optionsDownload high-quality image (191 K)Download as PowerPoint slideResearch highlights▶ Dihydrofolate reductase (DHFR) is a target for the development of new antineoplastic agents. ▶ A new series of nonclassical antifolates, the propargyl-linked inhibitors, are explored for their ability to inhibit human DHFR. ▶ Several of these compounds exhibit IC50 values under 500 nM. ▶ Molecular docking experiments show that interactions between the C6 substituent on the pyrimidine ring and residues Phe 31 and Phe 34 in the active site are important for activity, as are interactions between the aryl ring and Thr 56, Ser 59 and Ile 60. ▶ The most potent of these compounds (compound 35) has two critical substitutions relative to trimethoprim, a known weak inhibitor of human DHFR but potent inhibitor of bacterial species of DHFR. ▶ Docked complexes of compound 35 exhibit more extensive interactions with Thr 56, Ser 59, Ile 60, Leu 22, Phe 31 and Phe 34 compared to trimethoprim, most likely explaining its greater potency. ▶ The new series of propargyl-linked antifolates are highly efficient ligands and represent a new avenue for the development of antineoplastic agents.