4-Aminopyridyl-based CYP51 inhibitors as anti-Trypanosoma cruzi drug leads with improved pharmacokinetic profile and in vivo potency

Abstract

CYP51 is a P450 enzyme involved in the biosynthesis of the sterol components of eukaryotic cell membranes. CYP51 inhibitors have been developed to treat infections caused by fungi, and more recently the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. To specifically optimize drug candidates for T. cruzi CYP51 (TcCYP51), we explored the structure-activity relationship (SAR) of a N-indolyl-oxopyridinyl-4-aminopropanyl-based scaffold originally identified in a target-based screen. This scaffold evolved via medicinal chemistry to yield orally bioavailable leads with potent anti-T. cruzi activity in vivo. Using an animal model of infection with a transgenic T. cruzi Y luc strain expressing firefly luciferase, we prioritized the biaryl and N-arylpiperazine analogues by oral bioavailability and potency. The drug-target complexes for both scaffold variants were characterized by X-ray structure analysis. Optimization of both binding mode and pharmacokinetic properties of these compounds led to potent inhibitors against experimental T. cruzi infection.

Scheme 1. Syntheses of Compounds 1–12

Reagents and conditions: (a) arylboronic acid, 5 mol % Pd₂(dba)₃, 10 mol % PCy₃, 2M K₃PO₄, dioxane, 100 °C (microwave), 1 h, ca. 90%; (b) H₂SO₄/MeOH (1/10), 70 °C, 24 h, 91%; (c) H₂ (balloon), Pd/C, MeOH–acetone, 23 °C, 24 h, 92%; (d) 4-fluorobenzyl bromide, K₂CO₃, acetone, 70 °C, 5 h, 95%; (e) 10% NaOH (aq), MeOH/THF (1/1), 60 °C, 3 h, ca. 95%; (f) acetic anhydride, Et₃N, CH₂Cl₂, 0–23 °C, 1 h, 84%; (g) 10% NaOH (aq), MeOH/THF (1/1), 23 °C, 2 h, 36%; (h) 1-(aryl)piperazine, Pd(OAc)₂, P(o-tolyl)₃, Cs₂CO₃, toluene, 60 °C, 48 h, ca. 70%; (i) 13, 14, 15, 16, 17, 18, 19, 22b, 24b, 26b, 27b, 28b, or 29b (as appropriate), PyBOP, HOBt, Et₃N, CH₂Cl₂, 23 °C, 1 h, ca. 70%.

Figure 1

Animal model of T. cruzi Y luc infection. (A) Development of parasitemia in the untreated mice over the course of 21 days postinfection with T. cruzi Y luc parasites. Parasite count in fresh blood samples (red) was paralleled by the luminescence reading of whole amimals (blue). Each measurement is an average of five mice. (B) Evolution of the parasitemia in a single experimental animal by luminescence.

Figure 2

Efficacy of compounds upon ip administration. In two independent experiments, (A) and (B), compounds were administered at 40 mg/kg, ip, bid. Luminescence in mice was read upon luciferin injection on day 3 postinfection and prior to treatment (black bars) and on day 7 postinfection and after four days of treatment (gray bars). Each data point is an average of five mice; dpi-days postinfection. Posaconazole (Pos) served as a positive control. Percent inhibition for each compound is calculated relative to the untreated control on day 7 postinfection. *Values significantly different than vehicle-treated controls (p ≤ 0.05), except for LP10.

Figure 3

Tissue tropism of compounds. Tissue distribution of selected inhibitors administered orally as a single 50 mg/kg dose in 20% HPβCD (A) or 20% Kolliphor (B). Compound concentration detected in a tissue after 2 (blue) or 8 (orange) hours of exposure is plotted in μM. Tabulated data are presented in Supporting Information, Tables S1 and S2.

Figure 4

Anti-T. cruzi efficacy of compounds in four-day mouse model of infection. (A) Dose–response in Kolliphor (KOL) versus HPβCD (CD) administration for compounds 5 (empty bars) and 12 (filled bars). Compound 5 in Kolliphor was more active than in HPβCD (p < 0.05) versus compound 12, which had comparable activity in both vehicles (P < 0.05). (B) Dose–response for Kolliphor administration of 5, 6, 7, and 12. Benznidazole (BNZ) served as a positive control.

Figure 5

Inhibitors in the active site of TcCYP51. (A,D) Slice through the binding site shows bound inhibitors (yellow spheres) and the protein surface colored by hydrophobicity, hydrophobic areas are in orange and hydrophilic areas are in blue. Heme is in dark-red spheres. (B) Piperazine group separating two phenyl rings in the 12 (yellow sticks) allows smooth bending of the long substituent along the β-sheet saddle (magenta). A fragment of the electron density map (blue mesh) contoured at 1.2 σ delineates position of 12 at 2.04 Å resolution. Protein is in ribbon, heme is in spheres. (C,F) Residues within 5 Å from the inhibitor (yellow sticks) are highlighted in blue, heme is in gray sticks. (E) Binding mode of 1 resembles that of 12 (B), with fewer contacts for the long substituent at the chiral carbon center. Electron density map at 2.84 Å is contoured at 0.8 σ. Images here and otherwise were generated using CHIMERA or PYMOL

Figure 6

Interactions of the terminal phenyl ring in the N-arylpiperazine scaffold. Fluoro-substituted edges of the terminal phenyl ring in compounds 12 (A) and 11 (B) face residues I45, F48, F55, and I70. Inhibitors are shown in van der Waals spheres highlighted in yellow. van der Waals radii of the amino acid residues (blue sticks) are marked by blue dots. Heme is in stick mode. Heteroatoms are colored by type: oxygen in red, nitrogen in blue, fluorine in cyan, iron in ochre.

Figure 7

Pyridine-based CYP51 inhibitors. (A) N-Indolyl-oxopyridinyl-4-aminopropanyl-based analogues. (B) Fenarimol analogues. Drug–heme van der Waals interactions are shown as resolved in the X-ray structures of the corresponding drug–target complexes (PDB ID codes are in parentheses). Heme is in gray van der Waals spheres; inhibitors colored by atom types with carbon highlighted in yellow are labeled by the small-molecule codes. The branching points in chemical structures are highlighted in green.