Novel acyl carbamates and acyl / diacyl ureas show in vitro efficacy against Toxoplasma gondii and Cryptosporidium parvum

Abstract

Toxoplasma gondii and Cryptosporidium parvum are protozoan parasites that are highly prevalent and opportunistically infect humans worldwide, but for which completely effective and safe medications are lacking. Herein, we synthesized a series of novel small molecules bearing the diacyl urea scaffold and related structures, and screened them for in vitro cytotoxicity and antiparasitic activity against T. gondii and C. parvum. We identified one compound (GMG-1-09), and four compounds (JS-1-09, JS-2-20, JS-2-35 and JS-2-49) with efficacy against C. parvum and T. gondii, respectively, at low micromolar concentrations and showed appreciable selectivity in human host cells. Among the four compounds with efficacy against T. gondii, JS-1-09 representing the diacyl urea scaffold was the most effective, with an anti-Toxoplasma IC₅₀ concentration (1.21 μM) that was nearly 53-fold lower than its cytotoxicity IC₅₀ concentration, indicating that this compound has a good selectivity index. The other three compounds (JS-2-20, JS-2-35 and JS-2-49) were structurally more divergent from JS-1-09 as they represent the acyl urea and acyl carbamate scaffold. This appeared to correlate with their anti-Toxoplasma activity, suggesting that these compounds' potency can likely be enhanced by selective structural modifications. One compound, GMG-1-09 representing acyl carbamate scaffold, depicted in vitro efficacy against C. parvum with an IC₅₀ concentration (32.24 μM) that was 14-fold lower than its cytotoxicity IC₅₀ concentration in a human intestinal cell line. Together, our studies unveil a series of novel synthetic acyl/diacyl urea and acyl carbamate scaffold-based small molecule compounds with micromolar activity against T. gondii and C. parvum that can be explored further for the development of the much-needed novel anti-protozoal drugs.

Keywords: Acyl carbamate; Acyl urea; Cryptosporidium parvum; Diacyl urea; Drug discovery; Toxoplasma gondii.

Graphical abstract

Scheme 1

Elaboration of acyl isocyanates (II) obtained from primary amides (I). Reagents and conditions: (a) i. 1.0 mmol of I, dry CH₂Cl₂, RT, 5 min, ii. 3.0 eq of (COCl)₂, reflux 2.5–3 h, iii. RT, in vacuo solvent evaporation, (b) i. 1.1–1.25 eq of the appropriate nucleophile (IIIa-c), dry PhMe, reflux, 2.5–3 h, ii. RT, solvent evaporation.

Fig. 1

Analysis of the effects of test compounds on the growth of Cryptosporidium parvum in human ileocecal adenocarcinoma (HCT-8) cells in vitro. Equal amounts of freshly excysted sporozoites of C. parvum were inoculated into HCT-8 cells in culture and immediately treated with 20 μM of compound JS-1-07, JS-1-09, JS-1-11, JS-1-37, JS-2-20, JS-2-35, JS-2-49, GMG-1-08 or GMG-1-09. Infected cells without compound-treatment, but with equivalent amount of DMSO to that used in compound-treated cells (1% v/v) were set as negative controls. Positive control cultures were treated with paromomycin (800 μM). After 72 h the cultures were analyzed for parasite proliferation by immunofluorescence assays. The fluorescence generated by C. parvum was quantified and is shown on the Y-axis representing the parasite load. The data shown represent the means from three independent experiments with standard error bars, and levels of statistical significance depicted by asterisks (*, P < 0.05).

Fig. 2

Analysis of the effects of test compounds on the growth of T. gondii in vitro. Equal amounts (500) of T. gondii tachyzoites constitutively expressing yellow fluorescent protein (YFP) were used to infect confluent monolayers of human foreskin fibroblasts (HFF) and immediately treated with 20 μM of compound JS-1-07, JS-1-09, JS-1-11, JS-1-37, JS-2-20, JS-2-35, JS-2-49, GMG-1-08 or GMG-1-09. Infected cells without compound-treatment, but with DMSO equivalent to volume used in compound-treated cells (1% v/v) were set as negative controls. Positive control cultures were treated with atovaquone (0.5 μM). After 48 h, the cultures were analyzed for parasite proliferation by measuring the parasite YFP fluorescence by fluorescence microscopy. The fluorescence generated by T. gondii was quantified and is shown on the Y-axis representing the parasite load. The data shown represent the means from three independent experiments with standard error bars, and levels of statistical significance depicted by asterisks (*, P < 0.05).

Fig. 3

Effect of varying concentrations of compound GMG-1-09 on the growth of Cryptosporidium parvum in HCT-8 cells. Equal amounts of freshly excysted sporozoites of C. parvum were inoculated into HCT-8 cells in culture and varying concentrations of GMG-1-09 added at the time of infection (solid line) or at 2 h post-infection (p.i.) (dashed line). Control infected cells (dotted line) were treated immediately p.i. with volumes of DMSO equivalent to those used in the compound-treated cultures. The cells were analyzed for parasite infectivity and proliferation by an immunofluorescence assay after 72 h of culture. The fluorescence generated by intracellular C. parvum merozoites was quantified and is shown on the Y-axis representing the parasite load. The data shown represent means of three independent experiments with standard error bars and levels of statistical significance between groups indicated by asterisk (*, P < 0.05).

Fig. 4

Effect of varying concentrations of compounds on the growth of Toxoplasma gondii in human foreskin fibroblasts (HFF) cells. Equal amounts (500) of T. gondii tachyzoites constitutively expressing yellow fluorescent protein (YFP) were used to infect confluent monolayers of HFF cells and immediately (solid lines) or 2 h (dashed lines) after infection treated with varying concentrations of compound JS-1-09 (A), JS-2-49 (B), JS-2-35 (C), or JS-2-20 (D). Infected cells without compound-treatment, but with DMSO equivalent to volumes used in compound-treated cells were set as negative controls (DMSO h p.i.). After 48 h, the cultures were analyzed for parasite proliferation by measuring the parasite YFP fluorescence by fluorescence microscopy. The fluorescence generated by T. gondii was quantified and is shown on the Y-axis representing the parasite load. The data shown represent the means from three independent experiments with standard error bars, and levels of statistical significance depicted by asterisks (*, P < 0.05).

Fig. 5

Structures of the potent antitubercular agents: AU1235 and SQ109.