Trupti Pandharkar

Novel arylimidamides for treatment of visceral leishmaniasis.

ABSTRACT Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC50], <1 μM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC50 ≤ 0.12 μM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.

Potent antiprotozoal activity of a novel semi-synthetic berberine derivative

Treatment of diseases such as African sleeping sickness and leishmaniasis often depends on relatively expensive or toxic drugs, and resistance to current chemotherapeutics is an issue in treating these diseases and malaria. In this study, a new semi-synthetic berberine analogue, 5,6-didehydro-8,8-diethyl-13-oxodihydroberberine chloride (1), showed nanomolar level potency against in vitro models of leishmaniasis, malaria, and trypanosomiasis as well as activity in an in vivo visceral leishmaniasis model. Since the synthetic starting material, berberine hemisulfate, is inexpensive, 8,8-dialkyl-substituted analogues of berberine may lead to a new class of affordable antiprotozoal compounds.

Antileishmanial Activity of a Series of N2,N4-Disubstituted Quinazoline-2,4-diamines

A series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against Leishmania donovani and L. amazonensis intracellular amastigotes. A structure–activity and structure–property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC50 values in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 23 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg kg–1 day–1 for 5 consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N2,N4-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents.

Synthesis and antiprotozoal activity of dicationic m-terphenyl and 1,3-dipyridylbenzene derivatives

4,4″-Diamidino-m-terphenyl (1) and 36 analogues were prepared and assayed in vitro against T rypanosoma brucei rhodesiense , Trypanosoma cruzi , Plasmodium falciparum , and Leishmania amazonensis . Twenty-three compounds were highly active against T. b. rhodesiense or P. falciparum. Most noteworthy were amidines 1, 10, and 11 with IC50 of 4 nM against T. b. rhodesiense, and dimethyltetrahydropyrimidinyl analogues 4 and 9 with IC50 values of ≤ 3 nM against P. falciparum. Bis-pyridylimidamide derivative 31 was 25 times more potent than benznidazole against T. cruzi and slightly more potent than amphotericin B against L. amazonensis. Terphenyldiamidine 1 and dipyridylbenzene analogues 23 and 25 each cured 4/4 mice infected with T. b. rhodesiense STIB900 with four daily 5 mg/kg intraperitoneal doses, as well as with single doses of ≤ 10 mg/kg. Derivatives 5 and 28 (prodrugs of 1 and 25) each cured 3/4 mice with four daily 25 mg/kg oral doses.

Identification of New Antileishmanial Leads from Hits Obtained by High-Throughput Screening

ABSTRACT A previous screen of ∼200,000 compounds from the PubChem database identified 70 compounds possessing 50% effective concentrations (EC50s) below 1 μM against Leishmania major promastigotes that were not toxic to mammalian epithelial cancer cells at this concentration (E. Sharlow et al., PLoS Negl. Trop. Dis. 3:e540, 2009). Based on availability and chemical exclusion criteria, 31 of these compounds were purchased from commercial suppliers and evaluated for in vitro activity against intracellular L. donovani and L. amazonensis parasites. Benzothiazole cyanine compounds (PubChem 16196319 and 16196223) displayed potent activity against intracellular amastigotes, prompting a search for commercially available compounds that were structurally related. Pubchem 123859 (the cyanine dye thiazole orange) showed exceptionally potent activity against intracellular L. donovani in vitro (50% inhibitory concentration [IC50] = 21 ± 12 nM) and low cytotoxicity against Vero cells (IC50 = 7,800 ± 200 nM). Administration of 123859 and 16196319 at a dose of 1 mg/kg of body weight intraperitoneally (i.p.) daily for 5 days resulted in 44% ± 4% and 42% ± 3% suppression of liver parasitemia in L. donovani-infected BALB/c mice, respectively, compared to the untreated control group (the reductions in liver parasitemia were 30% ± 5% and 27% ± 4%, respectively, compared to the (2-hydroxypropyl)-β-cyclodextrin solution (HPβCD) vehicle control, which itself displayed some antileishmanial activity). Benzothiazole-containing cyanine dyes are thus potential lead compounds for the discovery of novel antileishmanial agents.

Antileishmanial bis-arylimidamides: DB766 analogs modified in the linker region and bis-arylimidamide structure-activity relationships.

Analogs of the lead antileishmanial bis-arylimidamide DB766 were prepared that possess unsymmetrical substitutions on the diphenylfuran linker, and an additional compound was synthesized that contains isopropoxy groups meta to the central furan. These agents all displayed nanomolar in vitro potency against intracellular Leishmania with selectivity indexes >100 compared to J774 macrophages. While the unsymmetrical analogs were toxic to mice when given ip at 30 mg/kg/day, the compound bearing the meta isopropoxy groups was well tolerated by mice and showed activity in a murine model of visceral leishmaniasis when administered ip at 30 mg/kg/day for five days.

Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

ABSTRACT Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 half-knockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

8,8-dialkyldihydroberberines with potent antiprotozoal activity.

Semisynthetic 8,8-dialkyldihydroberberines (8,8-DDBs) were found to possess mid- to low-nanomolar potency against Plasmodium falciparum blood-stage parasites, Leishmania donovani intracellular amastigotes, and Trypanosoma brucei brucei bloodstream forms. For example, 8,8-diethyldihydroberberine chloride (5b) exhibited in vitro IC50 values of 77, 100, and 5.3 nM against these three parasites, respectively. In turn, two 8,8-dialkylcanadines, obtained by reduction of the corresponding 8,8-DDBs, were much less potent against these parasites in vitro. While the natural product berberine is a weak DNA binder, the 8,8-DDBs displayed no affinity for DNA, as assessed by changes in the melting temperature of poly(dA·dT) DNA. Selected 8,8-DDBs showed efficacy in mouse models of visceral leishmaniasis and African trypanosomiasis, with 8,8-dimethyldihydroberberine chloride (5a) reducing liver parasitemia by 46% in L. donovani-infected BALB/c mice when given at an intraperitoneal dose of 10 mg/kg/day for five days. The 8,8-DDBs may thus serve as leads for discovering new antimalarial, antileishmanial, and antitrypanosomal drug candidates.

Synthesis and pharmacological evaluation of mono-arylimidamides as antileishmanial agents

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Correction to Antileishmanial Activity of a Series of N2,N4-Disubstituted Quinazoline-2,4-diamines

Pages 5143 and 5144. In the footnote to Tables 1 and 2, the EC50 of podophyllotoxin against J774A.1 macrophages should be “25 ± 5 nM” instead of “250 ± 10 nM”. Page 5154. In the right-hand column, second and third lines from the top, the description of the J774A.1 cytotoxicity assays should read that the assays were conducted as mentioned previously “... except that 10 cells/well were incubated with compounds for 72 h.” rather than “... except that cells were incubated with compounds for 72 h.” Addition/Correction