Hemozoin inhibiting 2-phenylbenzimidazoles active against malaria parasites

Abstract

The 2-phenylbenzimidazole scaffold has recently been discovered to inhibit β-hematin (synthetic hemozoin) formation by high throughput screening. Here, a library of 325,728 N-4-(1H-benzo[d]imidazol-2-yl)aryl)benzamides was enumerated, and Bayesian statistics used to predict β-hematin and Plasmodium falciparum growth inhibition. Filtering predicted inactives and compounds with negligible aqueous solubility reduced the library to 35,124. Further narrowing to compounds with terminal aryl ring substituents only, reduced the library to 18, 83% of which were found to inhibit β-hematin formation <100 μM and 50% parasite growth <2 μM. Four compounds showed nanomolar parasite growth inhibition activities, no cross-resistance in a chloroquine resistant strain and low cytotoxicity. QSAR analysis showed a strong association of parasite growth inhibition with inhibition of β-hematin formation and the most active compound inhibited hemozoin formation in P. falciparum, with consequent increasing exchangeable heme. Pioneering use of molecular docking for this system demonstrated predictive ability and could rationalize observed structure activity trends.

Keywords: Bayesian statistics; Benzimidazoles; Docking; Hemozoin; Malaria; QSAR.

Figure 1.

Strategy for selection of compounds for investigation. Leading structural fingerprints previously identified using Bayesian statistics for both β-hematin inhibition and in vitro P. falciparum growth inhibition encompassed the benzimidazole scaffold (A) [24]. Disconnection strategy used for synthesis and enumeration of all possible compounds based on available starting materials (B). Strategy for filtering enumerated structures based on predicted β-hematin inhibition, parasite growth inhibition, solubility and generalizations taken from the predicted active list used to simplify the final list of target compounds (C). Suitable aryl groups are discussed in the main text.

Figure 2.

Summary of SAR trends in the R group found for β-hematin inhibition activity (larger = more active). ERG = electron-releasing group, EWG = electron-withdrawing group.

Figure 3.

Summary of SAR trends in the R group found for parasite growth inhibition activity (larger = more active). ERG = electron-releasing group, EWG = electron-withdrawing group.

Figure 4.

Correlation of (A) parasite IC₅₀ and β-hematin inhibition activity and (B) observed pIC₅₀ plotted against predicted pIC₅₀ for inhibition of P. falciparum growth based on a fitted multiple correlation equation. In (A) R² = 0.59 and P = 0.0002. In (B) pIC₅₀ = 28.7(1/IC₅₀ β-hematin) + 3.71(HBD) – 1.43(molecular depth) – 8.90. F = 9.81 > F_crit^0.01 = 5.56, r² = 0.68, P < 0.0001. t for the individual parameters > t_crit^0.10 = 1.761 (5.11, 1.88 and 2.56 respectively for the first, second and third coefficients).

Figure 5.

Deconstruction of compound 5. All the compounds representing fragments of 5 (20 – 25) exhibited no significant β-hematin or parasite growth inhibition activity.

Figure 6.

Quantities of undigested hemoglobin (A), haemozoin (B) and freely exchangeable heme (C) in the NF54 strain of P. falciparum cultured in vivo after 32 h incubation with compound 5. Parasites were synchronized, and drug added to early ring stage parasites. Amounts are expressed in term of fg heme iron per cell, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus control.

Figure 7.

A representation of the number of π-π interactions with the (011) and (001) faces of the β-hematin crystal found by molecular docking and inhibitory activity for compounds 2 - 19 (A). In this representation + refers to IC₅₀ values below 80 μM and sum refers to the number of π-stacking interactions on both the (001) and (011) faces with a distance less than 3.8 Å. In the case of the (011) face, a direct proportionality was found between adsorption energy (E_ads⁰¹¹) and IC₅₀ for inhibition of β-hematin formation; r² = 0.70, P = 0.0004 (B).

Figure 8.

Molecular docking of compound 5 to the (011) face (A) and (001) face (B) of hemozoin. Interactions 1 and 2 represent π-stacking and 3 hydrogen bonding. A structure activity relationship hypothesis for this pharmacophore is presented in C. Modification of the imidazole NH group in compound 26 had relatively little influence on activity (D).

Scheme 1.

(i) PPA, 220 °C, 5 h; (ii) pyridine, THF or DMF, −40 °C, 2 – 3 h.