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

Abstract

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 1

Classical (methotrexate) and non-classical (trimetrexate) antifolates

Figure 2

a) Conserved hydrogen bonds with the enzyme and pyrimidine ring b) the active site of human DHFR bound to compound 35.

Figure 3

Docked poses of compounds 25, 29 and 35 bound to the active site of human DHFR. Interactions with residues Phe 31 and Phe 34 are shown.

Figure 4

a) The value of the propargyl linker and substitution at the C6 position is evident in a comparison with trimethoprim b) Surface rendering of the active site of human DHFR bound to TMP (white, from the crystal structure 2W3A), docked compounds 25 (purple) and 35 (tan).