Carbamoyl- N-aryl-imine-urea: a new framework to obtain a putative leishmanicidal drug-candidate

Abstract

Leishmaniasis is a neglected parasitic disease, and current treatment includes limitations of toxicity, variable efficacy, high costs and inconvenient doses and treatment schedules. Therefore, new leishmanicidal drugs are still an unquestionable medical need. In this paper we described the design conception of a new framework, the carbamoyl-N-aryl-imine-urea, to obtain putative leishmanicidal drug-candidates. Compounds 9a-e and 10a-e were designed and synthesized and their leishmanicidal activity was studied in comparison to pentamidine, miltefosine and meglumine antimoniate. The conformational profile of the new carbamoyl-N-aryl-imine-urea framework was investigated by X-ray diffraction studies, using compound 9a as a model. The plasma stability of this putative peptide mimetic subunit was studied for compound 10e (LASSBio-1736). Among the congeneric series, LASSBio-1736 was identified as a new antileishmanial drug-candidate, displaying plasma stability, cytotoxicity against amastigote forms of L. amazonensis and L. braziliensis, and leishmanicidal activity in a cutaneous leishmaniasis murine model, without preliminary evidence of hepatic or renal toxicity.

Fig. 1. Examples of cysteine proteases inhibitors bearing a peptide mimetic framework.

Fig. 2. Design conception of compounds 9a–e and 10a–e bearing a new peptide mimetic framework – the carbamoyl-N-aryl-imine-urea.

Fig. 3. Synthetic route to obtain the target compounds 9a–e and 10a–e.

Fig. 4. Asymmetric unit of compound 9a (LASSBio-1491) elucidated in this study. Non-hydrogen atoms are represented as 30% probability ellipsoids and randomly labeled, while hydrogens are shown as arbitrary radius spheres. Cyan dashed lines draw hydrogen bonds.

Fig. 5. A molecular superposition of conformers A (blue) and B (green) of compound 9a (LASSBio-1491). Hydrogen atoms were omitted for clarity. Some key atoms were labeled to identify molecular fragments. The two molecular planes crossing through both conformers were split over two frames (left and right).

Fig. 6. Effect of pentamidine and carbamoyl-N-aryl-imine-urea derivatives (9a–e and 10a–e) against intracellular amastigote forms of L. amazonensis at concentration of 30 μM.

Fig. 7. Plasma stability of 10e (LASSBio-1736) ▲ and standard benzocaine ■. Data were obtained through the compound recovery percentage analyzed by UPLC-MS/MS.

Fig. 8. In vivo efficacy of meglumine antimoniate, miltefosine and 10e (LASSBio-1736) treatments (30 μmol per kg per day × 28 days) in BALB/c mice infected with L. amazonensis. Lesion sizes were monitored weekly. Values are the mean of lesion sizes in five mice in each group and bars represent the standard error of the mean.

Fig. 9. Images of lesions after of treatment of BALB/c mice infected with L. amazonensis untreated and treated with meglumine antimoniate, miltefosine and 10e (LASSBio-1736) at dose 30 μmol per kg per day × 28 days. Photographs were taken on the first day after end of treatment. In the control groups, infected control, and meglumine antimoniate, the lesions showed an intense swelling and ulcerated by after treatment cessation. In groups treated with miltefosine and 10e (LASSBio-1736), the photographs revealed a complete healing of the nodules and ulcers.

Fig. 10. Parasite burden throughout the course of miltefosine (30 μmol per kg per day × 28 days, p.o.), meglumine antimoniate and LASSBio-1736 (10e) (30 μmol per kg per day × 28 days, i.p.) treatments in BALB/c mice infected with L. amazonensis. (A) log₁₀ of parasites loads of the infected ear. (B) log₁₀ of parasites loads of the draining lymph node. The parasite loads of infected ears and draining lymph nodes were determined using a quantitative limiting-dilution assay. Values are the mean of parasites loads in five mice in each group and bars represent the standard error of the mean. ***P < 0.001 vs. control.

Fig. 11. Survival of BALB/c mice infected with L. amazonensis untreated and treated with meglumine antimoniate, miltefosine, and LASSBio-1736 (10e) at dose 30 μmol per kg per day × 28 days. Data are from representative experiments, n = 5 mice per group. Treatment with meglumine antimoniate induced 20% of mortality in the group and other treatments presented survival rate of 100%.

Fig. 12. In vivo effect of miltefosine (30 μmol per kg per day × 28 days, p.o.), meglumine antimoniate and LASSBio-1736 (10e) (30 μmol per kg per day × 28 days, i.p.) in spleen weight of BALB/c mice infected with L. amazonensis. Spleen weight was verified in last day of treatment. Values are the mean of the spleen weight in five mice in each group and bars represent the standard error of the mean. *P < 0.05 vs. control.

Fig. 13. In vivo effect of miltefosine (30 μmol per kg per day × 28 days, p.o.), meglumine antimoniate and LASSBio-1736 (10e) (30 μmol per kg per day × 28 days, i.p.) treatments on serum ALT (A), AST (B), creatinine (C) and urea (D) levels of BALB/c mice infected with L. amazonensis. Values are the mean of the plasma levels of biochemical parameters in five mice in each group and bars represent the standard error of the mean. *P < 0.05, **P < 0.01 vs. control.