Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity

Abstract

The bifunctional enzyme N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 μM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 μM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP(+) and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.

Figure 1

Proposed reaction mechanism of TbFolD.

Figure 2

(A) Structures of 1 and 2; (B) Molecular formula of one symmetry-independent molecule of intermediate 3 and of the DMSO unit showing the atom numbering scheme.

Scheme 1. Synthesis of Compounds 2–5

Scheme 2. Synthesis of Compounds 16–20

Figure 3

Compound 2 bound in the active site of TbFolD. The polypeptide is depicted as off-white ribbon, the interacting residues, and the nicotinate moiety of the cofactor, which was modeled, are represented with C atoms as orange sticks, the ligand as cyan sticks. All O, N, and H atom positions are red, blue, and white, respectively. Blue dashed lines represent potential hydrogen bonding interactions.

Figure 4

(a) Two possible binding modes of 16 within TbFolD as resulted from docking studies. FolD is shown using the same color scheme employed in Figure 3. The ligand C atoms are represented as white sticks. (b) Binding modes of 18 within TbFolD.

Figure 5

Superimposition of the human FolD (PDB code 1DIG) and TbFolD/2 (PDB 4LRR) complexes, represented as green and orange ribbons and sticks, respectively. Nonconserved residues between the two species are labeled. The NADP⁺ cofactor and water molecules were removed for clarity. This picture was obtained using Chimera software (UCSF).