In Vitro and In Silico Studies of Glycyrrhetinic Acid Derivatives as Anti- Filarial Agents.

BACKGROUND: Lymphatic filariasis is one of the chronic diseases in many parts of the tropics and sub-tropics of the world despite the use of standard drugs diethylcarbamazine and ivermectin because they kill microfilaries and not the adult parasites. Therefore, new leads with activity on adult parasites are highly desirable. OBJECTIVE: Anti-filarial lead optimization by semi-synthetic modification of glycyrrhetinic acid (GA). METHODS: The GA was first converted into 3-O-acyl derivative, which was further converted into 12 amide derivatives. All these derivatives were assessed for their antifilarial potential by parasite motility assay. The binding affinity of active GA derivatives on trehalose-6-phosphate phosphatase (Bm-TPP) was assessed by molecular docking studies. RESULTS: Among 15 GA derivatives, GAD-2, GAD-3, and GAD-4 were found more potent than the GA and standard drug DEC. These derivatives reduced the motility of Brugia malayi adult worms by up to 74% while the GA and DEC reduced only up to 49%. Further, GA and most of its derivatives exhibited two times more reduction in MTT assay when compared to the standard drug DEC. These derivatives also showed 100% reduction of microfilariae and good interactions with Bm-TPP protein. CONCLUSION: The present study suggests that 3-O-acyl and linear chain amide derivatives of glycyrrhetinic acid may be potent leads against B. malayi microfilariae and adult worms. These results might be helpful in developing QSAR model for optimizing a new class of antifilarial lead from a very common, inexpensive, and non toxic natural product.

Sulfonamide chalcones: Synthesis and in vitro exploration for therapeutic potential against Brugia malayi.

Keeping in mind the immense biological potential of chalcones and sulfonamide scaffolds, a library of sulfonamide chalcones has been synthesized and evaluated for in vitro antifilarial assay against human lymphatic filarial parasite Brugia malayi. Experimental evidence showcased for the first time the potential of some sulfonamide chalcones as effective and safe antifilarial lead molecules against human lymphatic filarial parasite B. malayi. Sulfonamide chalcones 4d, 4p, 4q, 4t and 4aa displayed the significantly wide therapeutic window. Particularly chalcones with halogen substitution in aromatic ring proved to be potent antifilarial agents against Brugia malayi. Sulphonamide chalcones with lipophilic methyl moiety (4q and 4aa) at para position of terminal phenyl rings of compounds were found to have remarkable antifilarial activities with therapeutic efficacy. Observed preliminary evidence of apoptosis by effective chalcone derivatives envisaged its fair possibility to inhibit folate pathway with consequent defect in DNA synthesis.

Synthesis and biological evaluation of 4-oxycoumarin derivatives as a new class of antifilarial agents.

A series of 4-oxycoumarin derivatives was synthesized, characterized and evaluated in vitro and in vivo for antifilarial activity against the human lymphatic filarial parasite, Brugia malayi. A majority of the compounds studied showed potent in vitro activity with low IC50 values in the micro molar (µM) range (0.014-1.73 and 0.0056-0.43) against adult worms and microfilariae, respectively. Compounds 8 and 9 were identified to be the most promising antifilarial candidate molecules exhibiting activity in the nanomolar (nM) range. The IC50 values for compound 8 were 14 nM and 5.6 nM while for compound 9 were 94 nM and 13 nM, respectively, for adult worm and microfilaria. These two compounds also displayed promising adulticidal activity (74.9 ± 4.8% and 69.4 ± 2.8%, respectively) in the primary rodent (jird) screen. This study also serves as a starting point for investigating structure-activity relationship with different amino substituents.

Synthesis and antifilarial activity of chalcone-thiazole derivatives against a human lymphatic filarial parasite, Brugia malayi.

Here we report the synthesis of novel chalcone-thiazole compounds and their antifilarial activity. The antifilarial properties of these hybrids were assessed against microfilariae as well as adult worms of Brugia malayi. Among all the synthesized compounds, only two compounds, namely 4g and 4n were identified to be promising in vitro. These active compounds were tested in B. malayi-jird (Meriones unguiculatus) and B. malayi-Mastomys coucha models. Compound 4n showed 100% embryostatic effect and 49% macrofilaricidal in jirds and M. coucha models, respectively. This study provides a new structural clue for the development of novel antifilarial lead molecules.

New chemotypes for wALADin1-like inhibitors of delta-aminolevulinic acid dehydratase from Wolbachia endobacteria.

Substituted benzimidazoles of the wALADin1-family have recently been identified as a new class of species-selective inhibitors of delta-aminolevulinic acid dehydratase (ALAD) from Wolbachia endobacteria of parasitic filarial worms. Due to its Wolbachia-dependent antifilarial activity, wALADin1 is a starting point for the development of new drugs against filarial nematodes. We now present several other chemotypes of ALAD inhibitors that have been identified based upon their molecular similarity to wALADin1. A tricyclic quinoline derivative (wALADin2) with a different inhibitory mechanism and improved inhibitory potency and selectivity may represent an improved drug lead candidate.

In vitro and in vivo antifilarial activity evaluation of 3,6-epoxy [1,5]dioxocines: a new class of antifilarial agents.

A series of 3,6-epoxy [1,5]dioxocines were synthesized and evaluated for their antifilarial activity against adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro. Out of these, six compounds (4a-f) possessed improved in vitro anti-filarial activity and examples 4d and 4f were also found to be active in the in vivo experiments. These results demonstrate that 3,6-epoxy [1,5]dioxocines exhibits potent antifilarial activity and might be developed into a new class of antifilarial drug.

Design, synthesis, and biological activities of closantel analogues: structural promiscuity and its impact on Onchocerca volvulus.

Onchocerciasis, or river blindness, is a neglected tropical disease that affects more than 37 million people worldwide, primarily in Africa and Central and South America. We have disclosed evidence that the larval-stage-specific chitinase, OvCHT1, may be a potential biological target for affecting nematode development. On the basis of screening efforts, closantel, a known anthelmintic drug, was discovered as a potent and highly specific OvCHT1 inhibitor. Originally, closantel's anthelmintic mode of action was believed to rely solely on its role as a proton ionophore; thus, the impact of each of its biological activities on O. volvulus L3 molting was investigated. Structure-activity relationship studies on an active closantel fragment are detailed, and remarkably, by use of a simple salicylanilide scaffold, compounds acting only as protonophores or chitinase inhibitors were identified. From these data, unexpected synergistic protonophore and chitinase inhibition activities have also been found to be critical for molting in O. volvulus L3 larvae.

Towards novel antifilarial drugs: challenges and recent developments.

Filariasis is caused by thread-like nematode worms, classified according to their presence in the vertebrate host. The cutaneous group includes Onchocerca volvulus, Loa loa and Mansonella streptocerca; the lymphatic group includes Wuchereria bancrofti, Brugia malayi and Brugia timori and the body cavity group includes Mansonella perstans and Mansonella ozzardi. Lymphatic filariasis, a mosquito-borne disease, is one of the most prevalent diseases in tropical and subtropical countries and is accompanied by a number of pathological conditions. In recent years, there has been rapid progress in filariasis research, which has provided new insights into the pathogenesis of filarial disease, diagnosis, chemotherapy, the host-parasite relationship and the genomics of the parasite. Together, these insights are assisting the identification of novel drug targets and the discovery of antifilarial agents and candidate vaccine molecules. This review discusses the antifilarial activity of various chemical entities, the merits and demerits of antifilarial drugs currently in use, their mechanisms of action, in addition to antifilarial drug targets and their validation.

Synthesis and screening of 1-methyl-4-substituted benzoyl piperazides against adult Setaria digitata for antifilarial activity.

There is no safe and effective drug for killing the adult worms for the elimination/control of lymphatic filariasis and research is very much warranted towards the development of a macrofilaricidal drug. Therefore, the synthesis and evaluation of 1-N-methyl-substituted benzoyl/phenyl acetyl piperazides for macrofilaricidal activity were carried out. The title compounds, 1-N-methyl-substituted benzoyl/phenyl acetyl piperazides were synthesized and purified by slightly modifying the reported procedure and characterized by FT-IR, NMR and mass spectral data. The compounds were screened for macrofilaricidal activity against adult Setaria digitata, the bovine filarial worm for motility and MTT reduction assays using the reported procedures. All the compounds synthesized were characterized by spectral data. Out of 26 compounds synthesized and screened for macrofilaricidal activity, six compounds exhibited moderate antifilarial activity. The structure-activity relationships are discussed. In the case of benzoyl piperazides chloro-substitution in the para position enhanced the activity compared to its substitution in meta and ortho positions. In the case of phenyl acetyl piperazides when methyl group was in the meta position it was more active than when it was in para position. In dimethyl substituted compounds, substitutions at 3, 5-positions were more effective than 2, 3 and 3, 4 substitutions.

Synthesis of 4-amino-5-cyano-2, 6-disubstituted pyrimidines as a potential antifilarial DNA topoisomerase II inhibitors.

A novel series of 4-amino-5-cyano-2, 6-disubstituted pyrimidines have been synthesized and evaluated for their in vitro antifilarial DNA topoisomerase II activity against filarial parasite Setaria Cervi. In particular compounds bearing 4-chloro-phenyl substitutent at position-6, exhibited strong inhibition at 40 microg/mL and 5 microg/mL concentration. The present study based on the biological results obtained, suggests that the nature of substitutent at position-4 in the phenyl ring directly affects DNA topoisomerase II inhibitory activity. Most of the compounds have shown better topoisomerase II inhibitory activity than the standard antifilarial drug (DEC) and the topoisomerase II inhibitors (Novobiocin, Nalidixic acid).

Synthesis of 2-sulfanyl-6-methyl-1,4-dihydropyrimidines as a new class of antifilarial agents.

A series of 2-sulfanyl-6-methyl-1,4-dihydropyrimidines (8-21) were synthesized in good yields by alkylation of 5-methyl-6-phenyl-2-thioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid ethyl esters (2-7) with different alkyl or aralkyl halides in the presence of a combination of anhydrous K(2)CO(3) and catalytic amount of tetrabutyl ammonium bromide. The title compounds were evaluated for their antifilarial activity against adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro and in vivo at various concentrations. One of the compounds (18) showed promising antifilarial activity.

Glutathione metabolism of filarial worms: A vulnerable target for the design and synthesis of new antifilarial agents.

The main problem regarding the chemotherapy of filariasis is that no safe and effective drug is available yet to combat the adult human filarial worms. One of the main reasons is the prolonged existence, i.e. survival of filarial worms in mammalian hosts for many years, and having a very strong antioxidant system. Glutathione (GSH) has been identified as an important part of the antioxidant system of many, if not all, living cells and, together with glutathione reductase (GR), it maintains the correct intracellular redox balance. It protects the cell against oxidative damage by non-enzymatic scavenging of free radicals and by enzymatic neutralization of toxic hydrogen peroxide, lipid hydroperoxides, and derivatives by glutathione-dependent peroxidases (GPXs) and glutathione-S-transferases (GSTs). Work in this direction reveals that filarial worms can synthesize and recycle GSH, and its depletion may be useful in chemotherapeutic situations in which the cells to be killed and the cells to be spared have substantially different quantitative requirements for GSH. All normal mammalian cells have a considerable amount of GSH, whereas filarial worms may have GSH concentrations close to that required for their survival and, therefore, a little manipulation of the glutathione metabolism of filarial worms may have drastic consequences. The present review details the application of the glutathione metabolism of filarial worms as a target for the design and synthesis of new antifilarial agents.

Synthesis and activity of substituted anthraquinones against a human filarial parasite, Brugia malayi.

Lymphatic filariasis (elephantiasis) is a global public health problem caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. We have previously reported anthraquinones from daylily roots with potent activity against pathogenic trematode Schistosoma mansoni. Here we report the synthesis of novel anthraquinones A-S and their antifilrarial activity. Anthraquinones A-S were synthesized by a single-step Friedel-Crafts acylation reaction between phthalic anhydrides and substituted benzenes. The antifilarial properties of these synthetic anthraquinones were tested against microfilaria as well as adult male and female worms of B. malayi. The most active anthraquinone was K, which showed 100% mortality within 1, 5, and 3 days, respectively, against microfilaria and adult male and female worms at 5 ppm concentration. Albendazole, an oral drug currently used to treat parasitic infections, was used as a positive control. Methylated products of anthraquinones did not affect the microfilaria. Histological examination of treated adult female parasites showed most of the anthraquinones caused marked effects on intrauterine embryos.

Syntheses of 2,4,6-trisubstituted pyrimidine derivatives as a new class of antifilarial topoisomerase II inhibitors.

A series of 21 compounds of trisubstituted pyrimidine derivatives have been synthesized and evaluated for their in vitro topoisomerase II inhibitory activity against filarial parasite Setaria cervi. Out of these, seven compounds (8, 11-14, 25 and 28) have shown 60-80% inhibition at 40 and 20 microg/mL concentration. Five compounds (12, 13, 14, 25 and 28) exhibited 70-80% inhibition at 10 microg/mL concentration and three compounds (13, 14 and 28) have shown 40-60% inhibition at 5 microg/mL concentration. All the above mentioned compounds have shown better topo II inhibitory activity than standard antifilarial drug (DEC) and enzyme topo II inhibitors (Novobiocin, Nalidixic acid).

7-O-[4-methyl piperazine-1-(2-acetyl)]-2H-1-benzopyran-2-one: a novel antifilarial lead compound.

In preliminary studies we found that benzopyrones (coumarins), which are known to exert many biological activities including anti-inflammatory effect, possess promising macrofilaricidal action as well. In order to explore the possibility of combining such a macrofilaricidal activity with the microfilaricidal potential of the known piperazine pharmacophore, we synthesized a series of compounds and evaluated their antifilarial effect. In the present study, one of these compounds, 7-O-[4-methyl piperazine-1-(2-acetyl)]-2H-1-benzopyran-2-one (2), which has shown promising macrofilaricidal action against rodent filariid Litomosoides carinii in cotton rats, was evaluated against infection with Brugia malayi in Mastomys coucha and jird (Meriones unguiculatus). In the B. malayi-M. coucha system, the compound at a dose of 300 mg/kg, oral (p.o.) x5 days showed 53.6% adulticidal and 46.0% microfilaricidal activity along with 46.3% sterilization effect on the female worms. In addition, the compound interfered with the establishment of infective larvae (L(3))-induced infection to an extent of 50% at the same dose level. At 1 microM concentration it inhibited protease activity of B. malayi to 82%. The compound thus provides a novel lead for further synthesis and development of antifilarial agents with macrofilaricidal, microfilaricidal, female-sterilizing and possible larvicidal efficacy.

Synthesis and antifilarial evaluation of 7-O-acetamidyl-4-alkyl-2H-1-benzopyran-2-ones.

A series of 7-O-acetamidyl-4-alkyl-2H-1-benzopyran-2-ones (5-23) has been synthesized by amidation of 7-O-(carbethoxymethyl)-4-alkyl-2H-1-benzopyran-2-ones (2a, 2b) with different primary and secondary amines in fair to good yield. The resulting compounds were screened for their filarial DNA topoisomerase inhibitory activity under in vivo condition in Setaria cervi. The compounds were tested in vitro against Brugia malayi. A few of the compounds possess promising antifilarial activity.

Potential transition state phosphoramidate inhibitors of beta-tubulin as antifilarial agents.

Transition state phosphoramidate inhibitors of beta-tubulin were designed as potential antifilarial agents. The reaction of 2-aminobenzimidazole with diisopropyl phosphite and carbon tetrachloride at a low temperature gave the unexpected 1-diisopropoxyphosphoryl-2-aminobenzimidazole, which on heating gave the novel benzimidazole derivative, 2-(diisopropoxyphosphoryl)aminobenzimidazole. Both products were fully characterized and the synthetic procedure to both compounds was optimized. The procedure was used to prepare the related 5-benzoyl-2-(diisopropoxyphosphoryl)aminobenzimidazole and 5-benzoyl-2-(diethoxyphosphoryl)aminobenzimidazole (1d). In a preliminary trial against Brugia pahangi compound 1d was found to have no antifilarial activity. This lack of activity may be attributed to its extreme insolubility and thus low bioavailability. The synthesis of analogous, more soluble, phosphorothioate-substituted benzimidazoles using the same methods may yield compounds with greater antifilarial activity.

Beta-carbonyl substituted glutathione conjugates as inhibitors Of O. volvulus GST2.

A series of beta-carbonyl substituted glutathione conjugates were prepared and evaluated as inhibitors of OvGST2. Their specificity for the parasite derived protein was assessed through comparison with their inhibition of human piGST. Inhibition of OvGST2 has been demonstrated at low micromolar concentrations for these conjugates and selectivity for OvGST2 over human pi-GST of greater than 10-fold has been achieved.

Solid phase synthesis of quinolones.

Solid phase syntheses of ethyl 6-carboxyquinol-4(1H-)-one-3-carboxylate (5) and N-substituted 6-carboxyquinol-4(1H)-one-3-carboxamides 7a-d have been described. Antifilarial in vitro activities of 5,7a-d against Brugia malayi have also been delineated.

Potent 1,3-disubstituted-9H-pyrido[3,4-b]indoles as new lead compounds in antifilarial chemotherapy.

Substituted 9H-pyrido[3,4-b]indoles (beta-carbolines) identified in our laboratory as potential pharmacophore for designing macrofilaricidal agents, have been explored further for identifying the pharmacophore responsible for high order of adulticidal activity. This has led to syntheses and macrofilaricidal evaluations of a number of 1-aryl-9H-pyrido[3,4-b]indole-3-carboxylate derivatives (3-7). The macrofilarical activity was initially evaluated in vivo against Acanthoeilonema viteae. Amongst all the synthesized compounds, only twelve compounds namely 3a, 3c, 3d, 3f, 4c, 4d, 4f, 5a, 6f, 6h, 6i and 7h have exhibited either > 90% micro- or macrofilaricidal activity or sterilization of female worms. These compounds have also been screened against Litomosoides carinii and of these only 3f and 5a have also been found to be active. Finally these two compounds have been evaluated against Brugia malayi. The structure activity relationship (SAR) associated with position-1 and 3 substituents in beta-carbolines have been discussed. It has been observed that the presence of carbomethoxy at position-3 and an aryl substituent at position- in beta-carbolines effectively enhance antifilarial activity particularly against A. viteae. Amongst the various compounds screened, methyl 1-(4-methylphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4c) has shown highest adulticidal activity and methyl 1-(4-chlorophenyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxyla te (3a) has shown highest microfilaricidal action against A. viteae at 50 mg/ kg x 5 days (i.p.). Another derivative of this compound namely 1-(4-chlorophenyl)-3-hydroxymethyl-9H-pyrido[3,4-b]indole (5a) exhibited highest activity against L. carinii at 30 mg/kg x 5 days (i.p.) and against B. malayi at 50 mg/kg x 5 days (i.p.) or at 200 mg/kg x 5 days (p.o.).