Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti- Trypanosoma brucei Agents

Abstract

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (K_i = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Keywords: Leishmania infantum; Trypanosoma brucei; antiparasitic agents; dihydrofolate reductase inhibitors; pteridine reductase inhibitors; triazino and guanidino benzimidazoles.

Figure 1

(a) Antiprotozoal drugs Cyc and Pyr, as template molecules targeting TbDHFR and TbPTR1; (b) previously studied TbPTR1 inhibitors characterized by a key substitution on the core structure of Cyc or by a pyrimidine-based tricyclic scaffold (PI).

Figure 2

Active site view of the complexes (A) TbDHFR-NADP(H) (light green cartoon and carbons; cofactor in sticks, light pink carbons) and Pyr (in sticks, pink carbons), PDB id 3QFX; (B) TbPTR1-NADP(H) (light cyan cartoon and carbons; cofactor in sticks, cyan carbons) and Pyr (in sticks, pink carbons), PDB id 7OPJ. In both panels, oxygen atoms are colored red, nitrogen blue, sulfur yellow, and phosphorus magenta. H-bonds are shown as tan dashed lines.

Scheme 1. Reagents and Conditions: (i) HCl Conc. (1 Eq), H₂O, Reflux 6 h

Scheme 2. Reagents and Conditions: (i) 0.5 Eq of Piperidine, Reflux 7 h

Figure 3

Crystal structure of TbDHFR (light green cartoon and carbons) in the ternary complex with the cofactor NADP(H) (in sticks, turquoise carbons) and 1g (in sticks, magenta carbons). (A) Overall fold of TbDHFR (α-helices and β-strands are colored yellow-green and dark green, respectively). (B) View of the cofactor biding site. (C) Active site view, showing binding of 1g; the inhibitor surrounded by the omit map (green mesh contoured at the 2.5σ level) is displayed in the inset. (D) Active site view of the superimposition between the complexes TbDHFR-NADP(H)-1g and TbDHFR-NADP(H)-Pyr (cofactor and Pyr in sticks, pink carbons; PDB id 3QFX). In all panels, oxygen atoms are colored red, nitrogen blue, sulfur yellow, and phosphorus magenta. H-bonds are shown as tan dashed lines.

Figure 4

Active site view of TbPTR1 (light cyan car A–E): Active site view of TbPTR1 (light cyan cartoon and carbons) in the ternary complex with the cofactor NADP(H) (in sticks, turquoise carbons) and (A) 1g (in sticks, magenta carbons), (B) 1f (in sticks, light yellow carbons), (C) 2f (in sticks, green carbons), (D) 2d (in sticks, orange carbons), and (E) 2c (in sticks, purple carbons). The inhibitor surrounded by the omit map (green mesh contoured at the 2.5σ level) is displayed in the inset of each panel. (F) Structural comparison among the binding modes of compounds 1g (in sticks, magenta carbons), 1f (in sticks, light yellow carbons), 2f (in sticks, green carbons), 2d (in sticks, orange carbons), and 2c (in sticks, purple carbons) within the TbPTR1 active site (light cyan cartoon and carbons). The comparison highlights the slight opening of Phe97 in the complexes with the tricyclic compounds 1g and 1f (in yellow). This rotation is required to accommodate the bulkier substituents on their dihydrotriazine rings, distorting the peculiar π-sandwich with the cofactor nicotinamide. On the other hand, in the complexes with 2c, 2d, and 2f, Phe97 (in purple) is parallel to the guanidino benzimidazole moiety of the inhibitors and the NADP(H) nicotinamide. In all panels, oxygen atoms are colored red, nitrogen blue, sulfur yellow and phosphorus magenta. Water molecules are shown as red spheres (arbitrary radius) and H-bonds as tan dashed lines.

Figure 5

Active site view of the superimposition between the structure of TbPTR1 (light cyan cartoon and carbons) in complex with NADP(H) (in sticks, turquoise carbons) and (A) 1g (in sticks, magenta carbons) and Cyc (in sticks, gold carbons; PDB id 6HNC(25)); (B) 2c (in sticks, purple carbons) and Pyr (in sticks, pink carbons; PDB id 7OPJ(27)); (C) 1g (in sticks, magenta carbons) and the tricyclic inhibitor PI (1, in sticks, gray carbons; PDB id 6TBX(21)); (D) 2c (in sticks, purple carbons) and the tricyclic inhibitor PI (1, in sticks, gray carbons; PDB id 6TBX(21)). In all panels, oxygen atoms are colored red, nitrogen blue, sulfur yellow, and phosphorus magenta.