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CYP5122A1, an enzyme involved in sterol biosynthesis in Leishmania, was recently characterized as a sterol C4-methyl oxidase. Screening of a library of compounds against CYP5122A1 and CYP51 from Leishmania resulted in the identification of two structurally related classes of inhibitors of these enzymes. Analogs of screening hit N-(3,5-dimethylphenyl)-4-(pyridin-4-ylmethyl)piperazine-1-carboxamide (4a) were generally strong inhibitors of CYP51 but were less potent against CYP5122A1 and typically displayed weak inhibition of L. donovani promastigote growth. Analogs of screening hit N-(4-(benzyloxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18a) were stronger inhibitors of both CYP5122A1 and L. donovani promastigote proliferation but also remained selective for inhibition of CYP51. Two compounds in this series, N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18e) and N-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18i) showed modest selectivity for inhibiting L. donovani promastigote proliferation compared to J774 macrophages and were effective against intracellular L. donovani with EC 50 values in the low micromolar range. Replacement of the 4-pyridyl ring present in 18e with imidazole resulted in a compound (4-(2-(1H-imidazol-1-yl)ethyl)-N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)piperazine-1-carboxamide, 18p) with approximately fourfold selectivity for CYP5122A1 over CYP51 that inhibited both enzymes with IC 50 values ≤ 1 µM, although selective potency against L. donovani promastigotes was lost. Compound 18p also inhibited the proliferation of L. major promastigotes and caused the accumulation of 4-methylated sterols in L. major membranes, indicating that this compound blocks sterol demethylation at the 4-position in Leishmania parasites. The molecules described here may therefore be useful for the future identification of dual inhibitors of CYP51 and CYP5122A1 as potential antileishmanial drug candidates and as probes to shed further light on sterol biosynthesis in Leishmania and related parasites., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Karl Werbovetz has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Pankaj Sharma has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Chris La Rosa has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Michael Zhuo Wang has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Yiru Jin has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes., (© 2024. The Author(s).)

Visceral leishmaniasis, caused by Leishmania donovani , is a life-threatening parasitic disease, but current antileishmanial drugs are limited and have severe drawbacks. There have been efforts to repurpose antifungal azole drugs for the treatment of Leishmania infection. Antifungal azoles are known to potently inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania . However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets for these compounds. Our previous study reveals that, in Leishmania , lanosterol undergoes parallel C4- and C14-demethylation reactions to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. Overexpression of CYP5122A1 results in growth delay, differentiation defects, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro . Dual inhibitors of CYP51 and CYP5122A1, e.g., clotrimazole and posaconazole, possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors, e.g., fluconazole and voriconazole, have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes., Competing Interests: Competing Interest Authors declare no competing interests.

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect., Competing Interests: Declaration of competing interest Authors declare no competing interests or conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nitrogen-containing heterocyclic rings are common structural components of marketed drugs. Among these heterocycles, imidazole/fused imidazole rings are present in a wide range of bioactive compounds. The unique properties of such structures, including high polarity and the ability to participate in hydrogen bonding and coordination chemistry, allow them to interact with a wide range of biomolecules, and imidazole-/fused imidazole-containing compounds are reported to have a broad spectrum of biological activities. This review summarizes recent reports of imidazole/fused imidazole derivatives as anticancer agents appearing in the peer-reviewed literature from 2018 through 2020. Such molecules have been shown to modulate various targets, including microtubules, tyrosine and serine-threonine kinases, histone deacetylases, p53-Murine Double Minute 2 (MDM2) protein, poly (ADP-ribose) polymerase (PARP), G-quadraplexes, and other targets. Imidazole-containing compounds that display anticancer activity by unknown/undefined mechanisms are also described, as well as key features of structure-activity relationships. This review is intended to provide an overview of recent advances in imidazole-based anticancer drug discovery and development, as well as inspire the design and synthesis of new anticancer molecules.

Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis. Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c ( N -(4-((8-(1 H -imidazol-1-yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC 50 value of 0.53 μM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong inhibitors of recombinant CYP51 from L. donovani , as assessed by a fluorescence-based assay, additional targets are likely to play an important role in the antileishmanial action of these compounds.

Cyanine compounds have previously shown excellent in vitro and promising in vivo antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22 analogs of thiazole orange ((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy. Cyanines possessing substitutions on the quinolinium ring system displayed potency against Leishmania donovani axenic amastigotes that differed little from the parent compound (IC 50 12-42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA melting temperature were modest when synthetic oligonucleotides were incubated with selected analogs (ΔTm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter. Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat SAR suggests that further information regarding the target(s) of these molecules is needed to aid their development as antileishmanials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Current therapeutic options against cutaneous leishmaniasis are plagued by several weaknesses. The effective topical delivery of an antileishmanial drug would be useful in treating some forms of cutaneous leishmaniasis. Toward this end, a microneedle based delivery approach for the antileishmanial drug amphotericin B was investigated in murine models of both New World (Leishmania mexicana) and Old World (Leishmania major) infection. In the L. mexicana model, ten days of treatment began on day 35 post infection, when the area of nodules averaged 9-15 mm 2 . By the end of the experiment, a significant difference in nodule area was observed for all groups receiving topical amphotericin B at 25 mg/kg/day after application of microneedle arrays of 500, 750, and 1000 μM in nominal length compared to the group that received this dose of topical amphotericin B alone. In the L. major model, ten days of treatment began on day 21 post infection when nodule area averaged 51-65 mm 2 in the groups. By the end of the experiment, there was no difference in nodule area between the group receiving 25 mg/kg of topical amphotericin B after microneedle application and any of the non-AmBisome groups. These results show the promise of topical delivery of amphotericin B via microneedles in treating relatively small nodules caused by L. mexicana. These data also show the limitations of the approach against a disseminated L. major infection. Further optimization of microneedle delivery is needed to fully exploit this strategy for cutaneous leishmaniasis treatment.

Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani -infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis., (Copyright © 2017 American Society for Microbiology.)

Arylimidamide (AIA) compounds containing two pyridylimidamide terminal groups (bis-AIAs) possess outstanding in vitro antileishmanial activity, and the frontrunner bis-AIA DB766 (2,5-bis[2-(2-isopropoxy)-4-(2-pyridylimino)aminophenyl]furan) is active in visceral leishmaniasis models when given orally. Eighteen compounds containing a single pyridylimidamide terminal group (mono-AIAs) were synthesized and evaluated for their antileishmanial potential. Six of these compounds exhibited sub-micromolar potency against both intracellular Leishmania donovani and Leishmania amazonensis amastigotes, and three of these compounds also displayed selectivity indexes of 25 or greater for the parasites compared to a J774 macrophage cell line. When given orally at a dose of 100mg/kg/day for five days, compound 1b (N-(3-isopropoxy-4-(5-phenylfuran-2-yl)phenyl)picolinimidamide methanesulfonate) reduced liver parasitemia by 46% in L. donovani-infected mice. Mono-AIAs are thus a new class of candidate molecules for antileishmanial drug development., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Two new compounds, namely, a para-benzoquinone ring-containing abietane (1) and a para-benzoquinone ring-containing 7,8-seco-abietane (2), and 14 other known highly oxidized abietane diterpenoids (3-16) were isolated from an extract prepared from the cones of Taxodium distichum, collected in central Ohio. The active subfraction from which all compounds isolated in this study were purified was tested in vivo using Leishmania donovani-infected mice and was found to dose-dependently reduce the parasite burden in the murine livers after iv administration of this crude mixture at 5.6 and 11.1 mg/kg. The structures of 1 and 2 were established by detailed 1D- and 2D-NMR experiments, HRESIMS data, and electronic circular dichroism studies. Compounds 3 and 4 were each fully characterized spectroscopically and also isolated from a natural source for the first time. Compounds 2-16 were tested in vitro against L. donovani promastigotes and L. amazonensis intracellular amastigotes. Compound 2 was the most active against L. amazonensis amastigotes (IC50 = 1.4 μM), and 10 was the most potent against L. donovani promastigotes (IC50 = 1.6 μM). These compounds may be suggested for further studies such as in vivo experimentation either alone or in combination with other Taxodium isolates.

Visceral leishmaniasis is a neglected parasitic disease that has a high fatality rate in the absence of treatment. New drugs that are inexpensive, orally active, and effective could be useful tools in the fight against this disease. We previously showed that N(2),N(4)-disubstituted quinazoline-2,4-diamines displayed low- to sub-micromolar potency against intracellular Leishmania, and lead compound N(4)-(furan-2-ylmethyl)-N(2)-isopropyl-7-methylquinazoline-2,4-diamine (4) exhibited modest efficacy in an acute murine model of visceral leishmaniasis. In the present work, thirty-one N(2),N(4)-disubstituted quinazoline-2,4-diamines that had not previously been examined for their antileishmanial activity were evaluated for their potency and selectivity against Leishmania donovani, the causative parasite of visceral leishmaniasis. Quinazoline-2,4-diamines with aromatic substituents at both N(2) and N(4) exhibited potent in vitro antileishmanial activity but relatively low selectivity, while compounds substituted with small alkyl groups at either N(2) or N(4) generally showed lower antileishmanial potency but were less toxic to a murine macrophage cell line. Based on their in vitro antileishmanial potency, N(4)-benzyl-N(2)-(4-chlorobenzyl)quinazoline-2,4-diamine (15) and N(2)-benzyl-N(4)-isopropylquinazoline-2,4-diamine (40) were selected for in vivo evaluation of their pharmacokinetic and antileishmanial properties. While 15 displayed a longer plasma half-life and a greater area under the curve than 40, both compounds showed low efficacy in an acute murine visceral leishmaniasis model. Although the present study did not identify new quinazoline-2,4-diamines with promising in vivo efficacy, the reduced in vitro toxicity of derivatives bearing small alkyl groups at either N(2) or N(4) may provide clues for the design of safe and effective antileishmanial quinazolines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

N1-Benzylated dihydroquinolin-6-ols and their corresponding esters display exceptional activity against African trypanosomes in vitro, and administration of members of this class of compounds to trypanosome-infected mice results in cures in a first-stage African trypanosomiasis model. Since a quinone imine intermediate has been implicated in the antiparasitic mechanism of action of these compounds, evaluation of the hepatotoxic, mutagenic, and methemoglobin-promoting effects of these agents was performed. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate showed outstanding in vitro selectivity for Trypanosoma brucei compared to the HepG2, Hep3B, Huh7, and PLC5 hepatocyte cell lines. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were not mutagenic when screened in the Ames assay, with or without metabolic activation. The latter 2 compounds promoted time- and dose-dependent formation of methemoglobin when incubated in whole human blood, but such levels were below those typically required to produce symptoms of methemoglobinemia in humans. Although compounds capable of quinone imine formation require careful evaluation, these in vitro studies indicate that antitrypanosomal dihydroquinolines merit further study as drug candidates against the neglected tropical disease human African trypanosomiasis., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2014.)

A series of N(2),N(4)-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 N(2),N(4)-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents.

Sixty-two cationic benzyl phenyl ether derivatives (36 amidines and 26 prodrugs) were prepared and assayed for activities in vitro and in vivo against Trypanosoma brucei rhodesiense (STIB900), and in vitro against Plasmodium falciparum (K1) and Leishmania donovani axenic amastigotes. 3-Amidinobenzyl 4-amidino-2-iodo-6-methoxyphenyl ether dihydrochloride (55, IC50 = 3.0 nM) and seven other compounds exhibited IC50 values below 10 nM against T. b. rhodesiense in vitro. The 2-bromo-4,4'-diamidino analogue 19 (IC50 = 4.0 nM) and 12 other analogues were more potent than pentamidine (IC50 = 46 nM) against P. falciparum. The 3',4-diamidino-2,6-diiodo analogue 49 (IC50 = 1.4 μM) and two other compounds were more effective than pentamidine (IC50 = 1.8 μM) against L. donovani. A prodrug, 3',4-bis(N″-methoxy)amidino-2-bromo derivative 38, was the most efficacious against trypanosome infected mice, attaining 4/4 cures in four daily 25 mg/kg oral doses, and the 2-chloro-4,4'-diamidine 18 cured 3/4 mice in four daily 5 mg/kg intraperitoneal doses., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

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.

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The effects of reducing the molecular weight of the antileishmanial compound DB766 on DNA binding affinity, antileishmanial activity and cytotoxicity are reported. The bis-arylimidamides were prepared by the coupling of aryl S-(2-naphthylmethyl)thioimidates with the corresponding amines. Specifically, we have prepared new series of bis-arylimidamides which include 3a, 3b, 6, 9a, 9b, 9c, 13, and 18. Three compounds 9a, 9c, and 18 bind to DNA with similar or moderately lower affinity to that of DB766, the rest of these compounds either show quite weak binding or no binding at all to DNA. Compounds 9a, 9c, and 13 were the most active against Leishmania amazonensis showing IC(50) values of less than 1 μM, so they were screened against intracellular Leishmania donovani, showing outstanding activity with IC(50) values of 25-79 nM. Despite exhibiting little in vitro cytotoxicity these three compounds were quite toxic to mice., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels.

Dihydroquinoline derivative OSU-40 (1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate) is selectively potent against Trypanosma brucei rhodesiense in vitro (50% inhibitory concentration [IC(50)], 14 nM; selectivity index, 1,700) and has been proposed to cause the formation of reactive oxygen species (ROS) in African trypanosomes (J. Fotie et al., J. Med. Chem. 53:966-982, 2010). In the present study, we sought to provide further support for the hypothesis that OSU-40 kills trypanosomes through oxidative stress. Inducible RNA interference (RNAi) was applied to downregulate key enzymes in parasite antioxidant defense, including T. brucei trypanothione synthetase (TbTryS) and superoxide dismutase B (TbSODB). Both TbTryS RNAi-induced and TbSODB RNAi-induced cells showed impaired growth and increased sensitivity toward OSU-40 by 2.4-fold and 3.4-fold, respectively. Decreased expression of key parasite antioxidant enzymes was thus associated with increased sensitivity to OSU-40, consistent with the hypothesis that OSU-40 acts through oxidative stress. Finally, the dose-dependent formation of free radicals was observed after incubation of T. brucei with OSU-40 utilizing electron spin resonance (ESR) spectroscopy. These data support the notion that the mode of antitrypanosomal action for this class of compounds is to induce oxidative stress.

Bioactivity-directed fractionation of the MeOH fraction of the extract of Mimulus bigelovii by means of an axenic Leishmania amastigote assay and chromatographic techniques resulted in the isolation of four C-geranyl flavanones, diplacone (1), 3'-O-methyldiplacone (2), 4'-O-methyldiplacone (3), 3'-O-methyldiplacol (4), together with a geranylated flavone, cannflavin A (5). These compounds were separated from M. bigelovii for the first time. All compounds showed moderate antileishmanial activity against axenic Leishmania donovani amastigotes with IC(50) values ranging from 4.8 to 14.6 μg/mL. The compounds were also tested against the related kinetoplastid parasite Trypanosoma brucei brucei and they showed activity with IC(50) values ranging from 1.4 to 7.2 μg/mL., (Copyright © 2011 John Wiley & Sons, Ltd.)

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., (Copyright © 2011. Published by Elsevier Ltd.)

Purpose: To develop an HPLC-UV method for determination of a novel antitrypanosomal compound (OSU-36) and its ester prodrug (OSU-40) in rat plasma and to apply the method for pharmacokinetic evaluation of both compounds in rats., Methods: Since an attempt to assay for OSU-36 and OSU-40 in non-stabilized plasma resulted in highly non-linear calibration curves and poor sensitivity due to instability of the compounds, the plasma was stabilized using paraoxon and ascorbic acid. The sample treatment included protein precipitation by acetonitrile; evaporation; reconstitution with acetonitrile and filtration. The chromatography conditions included Xterra RP18 3.5 µm 4.6X100 mm column and gradient mobile phase system of acetonitrile-water., Results: The limits of quantification (LOQ) were 50 ng/mL and 40 ng/mL for OSU-36 and OSU-40, respectively. The intra- and interday precision and accuracies were below 13% for low, medium and high quality control samples for both compounds. While OSU-40 has been stable in all tested handling conditions, OSU-36 was unstable in plasma after 20 days storage in -80 °C or 4h 28 °C storage. The developed method has been applied for a pharmacokinetic study in rats which revealed that an ester prodrug OSU-40 is rapidly converted to OSU-36 within the plasma compartment by plasma esterases. OSU-36, in turn, relatively quickly undergoes oxidative metabolism, including within the plasma compartment., Conclusions: A supplementation of rat plasma with an esterase inhibitor to prevent degradation of ester prodrug (OSU-40), and with antioxidant to prevent oxidation of OSU-36, is necessary for reliable determination of both compounds. Due to limited stability of OSU-36 in stabilized rat plasma, long-term storage of samples or prolonged handling in room temperature conditions is not recommended.

Analogs of the trypanocidal lead compound 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were prepared to extend the structure-activity relationship in this series of molecules, improve the in vivo antitrypanosomal activity of the lead, and determine whether ester prodrugs are needed to overcome the instability of the dihydroquinolin-6-ols. Two of the most active compounds identified in this study were 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxy)benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride. These stable solids possessed low nanomolar IC(50) values against Trypanosoma brucei rhodesiense STIB900 in vitro and provided cures in an early treatment acute mouse model of African trypanosomiasis when given ip at 50mg/kg/day for four consecutive days., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin, and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii, and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provide additional support for the proposed dinitroaniline binding site on α-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.

The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracellular parasite Toxoplasma gondii is reported. Using in vitro cultures of parasites in human fibroblasts, we determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several displayed sub-micromolar potency against the parasite. The most potent compound was N(1),N(1)-dipropyl-2,6-dinitro-4-(trifluoromethyl)-1,3-benzenediamine (18b), which displayed an IC(50) value of 36 nM against intracellular T. gondii. Based on these data and another recent report [Ma, C.; Tran, J.; Gu, F.; Ochoa, R.; Li, C.; Sept, D.; Werbovetz, K.; Morrissette, N. Antimicrob. Agents Chemother. 2010, 54, 1453], an antimitotic structure-activity relationship for dinitroanilines versus Toxoplasma is presented., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Although miltefosine and paromomycin were registered as clinical agents against visceral leishmaniasis in the last decade, the antileishmanial drug arsenal still requires improvement, particularly in the area of oral antileishmanial drugs for both visceral and cutaneous diseases. Several new compounds and formulations have displayed promising efficacy in animal models of leishmaniasis, including the 8-aminoquinoline NPC1161, a series of bis-quinolines, DB766, rhodacyanine dyes, amiodarone, and an oral formulation of amphotericin B. Herein we provide a review of those molecules whose antileishmanial properties have been described over the past few years and a brief assessment of the studies required to identify new preclinical antileishmanial candidates., (2010 Elsevier Ltd. All rights reserved.)

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC(50)], <1 microM) 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 (IC(50) < or = 0.12 microM) 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.

Objectives: To assess the impact of visceral leishmaniasis (VL) on the concentration of amphotericin B (AmB) recovered in the liver and spleen following either intravenous (AmBisome) or oral (iCo-009) AmB administration to mice., Methods: Livers and spleens previously obtained from VL-infected BALB/c mice (following intravenous AmBisome or oral AmB treatments) were analysed for AmB concentrations. Then, non-infected BALB/c mice were divided into three treatment groups: a single dose of intravenous AmBisome (2 mg/kg, n = 5); and oral AmB every 12 h for 5 days (10 mg/kg, n = 6 and 20 mg/kg, n = 6). The animals were sacrificed 7 days after the initiation of the treatment and the livers and spleens were harvested for drug analysis by HPLC., Results: The single intravenous injection of AmBisome resulted in a 77-fold lower concentration of AmB in infected compared with non-infected liver tissue, while the difference in AmB concentration in the spleen was only 5-fold. The multiple dose oral administration of AmB resulted in a 3-fold lower concentration of AmB in infected compared with non-infected livers for both oral doses, while the differences in AmB concentrations in the spleen were not statistically different for the oral treatment groups., Conclusions: VL significantly lowered the concentration of AmB in the liver and the spleen when compared with uninfected animals. This effect seems to correlate with the degree of infection of the tissue. In the case of the intravenous liposomal formulation (AmBisome), the differences between the infected and non-infected tissues are of a higher magnitude than in the case of orally administered AmB (iCo-009).

The current chemotherapy for second stage human African trypanosomiasis is unsatisfactory. A synthetic optimization study based on the lead antitrypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate (TDR20364, 1a) was undertaken in an attempt to discover new trypanocides with potent in vivo activity. While 6-ether derivatives were less active than the lead compound, several N1-substituted derivatives displayed nanomolar IC(50) values against T. b. rhodesiense STIB900 in vitro, with selectivity indexes up to >18000. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (10a) displayed an IC(50) value of 0.014 microM against these parasites and a selectivity index of 1700. Intraperitoneal administration of 10a at 50 (mg/kg)/day for 4 days caused a promising prolongation of lifespan in T. b. brucei STIB795-infected mice (>14 days vs 7.75 days for untreated controls). Reactive oxygen species were produced when T. b. brucei were exposed to 10a in vitro, implicating oxidative stress in the trypanocidal mode of action of these 1,2-dihydroquinoline derivatives.

Novel dicationic triazoles 1-60 were synthesized by the Pinner method from the corresponding dinitriles, prepared via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The type and the placement of cationic moieties as well as the nature of aromatic substituents influenced in vitro antiprotozoal activities of compounds 1-60 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and their cytotoxicity for mammalian cells. Eight congeners displayed antitrypanosomal IC(50) values below 10 nM. Thirty-nine dications were more potent against P. falciparum than pentamidine (IC(50) = 58 nM), and eight analogues were more active than artemisinin (IC(50) = 6 nM). Diimidazoline 60 exhibited antiplasmodial IC(50) value of 0.6 nM. Seven congeners administered at 4 x 5 mg/kg by the intraperitoneal route cured at least three out of four animals in the acute mouse model of African trypanosomiasis. At 4 x 1 mg/kg, diamidine 46 displayed better antitrypanosomal efficacy than melarsoprol, curing all infected mice.

Tubulin is a highly conserved, negatively charged protein that is found in essentially all eukaryotic cells. These properties ensure that isolation protocols successful in one system will likely work, with a few modifications, in most systems. Tubulin has been isolated most frequently from mammalian brain, and the main difference encountered in other systems versus brain is that tubulin is much less abundant in nearly all other sources than it is in brain. This means that attempting to purify tubulin by direct polymerization from a homogenate will often fail or be quite inefficient. However, the conservation of negative charge on tubulin means that an initial ion exchange step can be used to both purify and concentrate the protein from most systems. Polymerization-competent tubulin can usually be obtained by inducing polymerization in the salt eluate from the ion exchange step. We describe protocols for this procedure and describe its application to a number of vertebrate, fungal, protozoal, and plant sources., (Copyright 2010 Elsevier Inc. All rights reserved.)

Forty-eight cationically substituted pentamidine congeners possessing benzofuran rings were synthesized by a copper mediated heteroannulation of substituted o-iodophenols with phenyl acetylenes. Activities of compounds 1-48 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and cytotoxicities for mammalian cells were influenced by the nature of cationic substituents, placement of the benzofuran fragment, and the length of the carbon linker between aromatic moieties. Several dications exhibited superior antiplasmodial and antileishmanial potencies compared to pentamidine.

A series of 37 dicationically substituted bis(phenoxymethyl)benzene bis(phenoxymethyl)naphthalene, and bis(benzyloxy)naphthalene analogues of pentamidine was prepared and evaluated for antiprotozoal activities and cytotoxicity in in vitro. 1,3-Bis(4-amidinophenoxymethyl)benzene (1) was the most active against Trypanosoma brucei rhodesiense (IC(50)=2.1 nM). 1,3-Bis[4-(N-isopropylamidino)phenoxymethyl]benzene (2) was most active against Plasmodium falciparum (IC(50)=3.6 nM) and displayed a selectivity index more than 50 times greater than that of pentamidine. Several other compounds displayed lower antiplasmodial IC(50) values and higher selectivity indices relative to pentamidine. 1,4-Bis(4-amidinophenoxymethyl)benzene (14) was the most active against Leishmania donovani (IC(50)=1.3 microM). Compound 2 displayed the greatest activity against T. b. rhodesiense in vivo, curing three of four infected mice dosed intraperitoneally at 5 mg/kg x 4 days.

A series of novel pyridyl analogues 1-18 of antiprotozoal drug 1,5-bis(4-amidinophenoxy)pentane (pentamidine) has been synthesized and tested for in vitro activities against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, and for cytotoxicity against mammalian cells. Antiprotozoal properties of compounds 1-18 depended on the placement of cationic moieties on the pyridine rings as well as the nature of substituents on the amidine groups. Diamidine 6 with cationic moieties adjacent to pyridine nitrogen atoms was the most promising compound in the series showing superior in vitro activities against T. brucei rhodesiense, P. falciparum, and L. donovani compared to pentamidine. An oral prodrug of diamidine 6, diamidoxime 9, administered at 25 mg/kg daily for 4 days, exhibited excellent antitrypanosomal efficacy in vivo curing all infected animals in the STIB900 acute mouse model of trypanosomiasis.

Visceral leishmaniasis is a deadly parasitic disease caused by obligate intramacrophage protozoans of the Leishmania genus. The World Health Organization estimates the annual death toll to be 50,000, with 500,000 new cases each year. Without treatment, visceral leishmaniasis is inevitably fatal. For the last 70 years, the first line of defense has been pentavalent antimonials; however, increased resistance has brought amphotericin B to the forefront of treatment options. Unfortunately, the difficult route of drug administration, toxicity issues, and cost prevent amphotericin B from reaching the infected population, and mortality continues to rise. Our reformulation of amphotericin B for oral administration has resulted in a highly efficacious antileishmanial treatment that significantly reduces or eradicates liver parasitemia in a murine model of visceral leishmaniasis. This formulation has overcome amphotericin B's significant physicochemical barriers to absorption and holds promise for the development of a self-administered oral therapy for the treatment of visceral leishmaniasis.

Diamidine 1 (pentamidine) and 65 analogues (2-66) have been tested for in vitro antiprotozoal activities against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, and for cytotoxicity against mammalian cells. Dications 32, 64, and 66 exhibited antitrypanosomal potencies equal or greater than melarsoprol (IC(50) = 4 nM). Nine congeners (2-4, 12, 27, 30, and 64-66) were more active against P. falciparum than artemisinin (IC(50) = 6 nM). Eight compounds (12, 32, 33, 44, 59, 62, 64, and 66) exhibited equal or better antileishmanial activities than 1 (IC(50) = 1.8 microM). Several congeners were more active than 1 in vivo, curing at least 2/4 infected animals in the acute mouse model of trypanosomiasis. The diimidazoline 66 was the most promising compound in the series, showing excellent in vitro activities and high selectivities against T. b. rhodesiense, P. falciparum, and L. donovani combined with high antitrypanosomal efficacy in vivo.

BTB 06237 (2-[(2,4-dichloro-5-methylphenyl)sulfanyl]-1,3-dinitro-5-(trifluoromethyl) benzene), a compound previously identified through QSAR pharmacophore development and a virtual screen of the Maybridge database, possesses potent and selective activity against Leishmania parasites. In the present study, several analogs of BTB 06237 were synthesized and analyzed for activity against Leishmania axenic amastigotes, their ability to reduce the level of parasitemia in peritoneal macrophages, and their ability to generate reactive oxygen species (ROS) in L. donovani promastigotes. It was found that an aromatic ring must be present in the position occupied by the 2,4-dichloro-5-methylphenyl group in the lead compound, but changing the functional groups generally has little effect on the antileishmanial potency. Alterations to the 1,3-dinitro-5-(trifluoromethyl)benzene ring have more influence on antiparasitic activity with two aromatic nitro groups and a third electron-withdrawing group being required. This structural requirement corresponds with redox potential, the ability to generate ROS in the parasites, and dissipation of the mitochondrial membrane potential. Finally, we used this collection of data to design a new antileishmanial compound with strong activity in vitro and improved properties as an antileishmanial candidate.

Cruciferous vegetables contain significant amounts of ascorbigen and related substances with known molecular structures. This study tested the hypothesis that ascorbigen demonstrates antioxidant properties and protects human umbilical cord endothelial cells against hyperglycemic toxicity in vitro. It was observed that ascorbigen, in micromolar concentrations, protected against endothelial cell death from glucose toxicity. Additionally, ascorbigen at 3.0 mm shifted the concentration response curve of l-phenylephrine to the right, with a reduction in the maximal contractile effects of the agonist. This action was not related to alpha-adrenoceptor blockade. Ascorbigen also relaxed the vascular tone induced by l-phenylephrine, which is not mediated by an endothelial cell nitric oxide-dependent mechanism. On the guinea-pig ileum, the spasmogenic effects of carbachol, histamine and serotonin were reduced in the presence of 3 mM ascorbigen. Spasm of the gut induced by the acetylcholinesterase inhibitor, physostigmine, was antagonized by ascorbigen with an IC50 of 286 microM. This natural product also has a weak antiparasitic activity. The cytoprotective effects of ascorbigen may be highly relevant in the optimum physiological regulation of the function and the therapeutic value of this substance in disease settings needs to be further investigated., ((c) 2008 John Wiley & Sons, Ltd.)

Tubulin is the proposed target for drugs against cancer and helminths and is also a validated target in kinetoplastid parasites. With the aim of identifying new lead compounds against Leishmania sp., tubulin isolated from L. tarentolae was used to screen a 10 000 compound library. One compound, Chembridge No. 7992831 (5), displayed an IC(50) of 13 microm against Leishmania tubulin in an in vitro assembly assay and showed a greater than threefold selectivity over mammalian tubulin. Another compound, Chembridge No. 9067250 (8), exhibited good activity against mammalian tubulin (IC(50) = 5.0 microm). This compound was also toxic to several cancer cell lines with IC(50) values in the region of 1 microm. Subsequent testing of analogues of 8 contained within the library identified two compounds with greater potency against mammalian tubulin (IC(50) values of 1.1 and 2.8 microm). The more potent antitubulin agent also showed promising activity against cancer cell lines in vitro, with IC(50) values ranging from 0.18 to 0.73 microm.

A series of cationically substituted 2-phenylbenzofurans 1- 49 have been synthesized, and their in vitro antiprotozoal properties against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, as well as cytotoxicity against mammalian cells, have been evaluated. Eight dications exhibited antitrypanosomal activities comparable to that of pentamidine and melarsoprol. Twenty-six compounds were more active than pentamidine, and seven dications demonstrated increased activities against P. falciparum than artemisinin. Five congeners were more active against L. donovani than pentamidine. Introduction of methoxy or hydroxy groups in the 7- and/or 2'-position afforded derivatives that were highly selective against T. b. rhodesiense, P. falciparum, and L. donovani. Fourteen 2-phenylbenzofurans displayed excellent in vivo efficacies in the acute mouse model of trypanosomiasis, curing 3/4 or 4/4 animals at 4 x 5 mg/kg. Diamidine 1 and di( N-isopropyl)amidine 45, administered at 4 x 1 mg/kg, exhibited potency comparable to that of melarsoprol, providing 3/4 and 2/4 cures, respectively.

Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.

Kinetoplastid parasites are responsible for the potentially fatal diseases leishmaniasis, African sleeping sickness and Chagas disease. The current treatments for these diseases are far from ideal and new compounds are needed as antiparasitic drug candidates. Tubulin is the accepted target for treatments against cancer and helminths, suggesting that kinetoplastid tubulin is also a suitable target for antiprotozoal compounds. Selective lead compounds against kinetoplastid tubulin have been identified that could represent a starting point for the development of new drug candidates against these parasites.