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.).     

Glycyrrhetinic acid and its analogs: A new class of antifilarial agents

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5 μM; IC₅₀: 1.20 μM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a–7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50 μM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC₅₀ values were found to be 8.8 and 2.2 μM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100 mg/kg, s.c. ×5 days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.     

Synthesis and antibacterial activity of new polyfluoro-1,3,5-oxadiazines]

A number of polyfluro-3,6-dihydro-2H-1,3,5-oxadiazines and polyfluoro-4H-1,3,5-oxadiazines was designed. Relationship between their chemical structure and in vitro activity was investigated. The highest activity against grampositive microorganisms was shown by N1,N6-bis-[2,2,6,6-tetrakis(trifluoromethyl)-3,6-dihydro-2H-1,3,5-oxadiazine-4-yl]-1,6-hexane diamine.     

Dipetalonema viteae and Brugia pahangi transplant infections in gerbils for use in antifilarial screening

Transplanted infections of Dipetalonema viteae and Brugia pahangi have been evaluated as tools for experimental chemotherapy. Attempts were made to establish these filariae in similar pharmacokinetic sites within the same host, so that direct comparisons of in vivo drug susceptibilities could be made. Unfortunately, it was not possible to establish B. pahangi in the subcutaneous tissues, the preferred site of D. viteae. Therefore, intraperitoneal B. pahangi and subcutaneously implanted D. viteae in gerbils were used for the study. D. viteae infections were significantly enhanced by concomitant infections with B. pahangi, while B. pahangi infection rates were unaffected by the presence of D. viteae. Experiments with amoscanate, CGP6140 and Mel W demonstrated the importance of employing both B. pahangi and D. viteae for antifilarial discovery work and the fundamental effect of parasite location on drug efficacy. D. viteae rapidly migrate from the peritoneal cavity of gerbils following implantation; twenty one hours after infection 73% of transplanted worms were found in the subcutaneous tissues. It was shown that the migration response could be used as a stringent parameter for demonstrating antifilarial activity. D. viteae were exposed to antifilarial drugs for 24 hours in vitro, washed and implanted into the peritoneal cavity of gerbils. At autopsy, 5 days later, 10(-8)M ivermectin and milbemycin D had prevented migration; CGP6140, amoscanate, suramin, flubendazole and furapyrimidone were also detected at less than 10(-6)M using this parameter. In all cases the migration response was more sensitive to drugs than parasite kill. Ivermectin's ability to inhibit worm migration through the tissues is discussed, with respect to the role of itinerant males in the reproductive cycle of Onchocerca volvulus.     

Synthesis and analgesic/anti-inflammatory evaluation of fused heterocyclic ring systems incorporating phenylsulfonyl moiety

A series of pyrazolo[1,5-a]pyrimidine, triazolo[1,5-a]pyrimidine, and pyrimido[1,2-a]benzimidazole ring systems incorporating phenylsulfonyl moiety were synthesized via the reaction of 3-(N,N-dimethylamino)-1-aryl-2-(phenylsulfonyl)prop-2-en-1-one derivatives 2a,b with appropriate nitrogen nucleophiles. The analgesic and anti-inflammatory activities of the newly synthesized compound were investigated in vivo. 3-Bromo-2-phenyl-6-(phenylsulfonyl)-7-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5e) was found to have an excellent analgesic activity in comparison with indomethacin as a reference drug, while the highest anti-inflammatory effect was observed in the case of 2-(4-bromophenyl)-6-(phenylsulfonyl)-5-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5d). From the structure-activity relationship (SAR) point of view, the analgesic/anti-inflammatory activity of pyrazolo[1,5-a]pyrimidine derivatives was found to be much higher than triazolo[1,5-a]pyrimidine and pyrimido[1,2-a]benzimidazole derivatives.     

Syntheses and antifilarial profile of 7-chloro-4-(substituted amino) quinolines: a new class of antifilarial agents

The syntheses of 7-chloro-4-(substituted amino) quinolines (2-22) and their antifilarial activities are delineated. Some of the screened compounds have shown promising filarial response and sterilization effect on female Acanthocheilonema viteae in rodents.     

Synthesis and initial SAR studies of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines: a new class of KDR kinase inhibitors

We have synthesized and evaluated the activity of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines as a new class of KDR kinase inhibitors. Starting with screening lead 1, potency against isolated KDR was fully optimized with 3-thienyl and 4-methoxyphenyl substituents at the 6- and 3-positions (3g, KDR IC(50)=19 nM), respectively. The synthesis and SAR of these compounds are described.     

Multinuclear NMR spectroscopy and antitumor activity of novel platinum(II) complexes with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines

Novel platinum(II) complexes with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines have been synthesized and characterized by infrared and multinuclear magnetic resonance spectroscopic techniques (1H, 13C, 15N, 195Pt). The complexes are of two types: [PtCl2(L)2] and [PtCl2(NH3)(L)], where L=5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp) and 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp). Significant 15N NMR upfield shifts (92-95 ppm) were observed for N(3) atom indicating this nitrogen atom as a coordination site. The molecular structure suggest that Pt(II) ion has the square planar geometry with N(3) bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines, N-bonded second ligand (NH3 for cis-[PtCl2(NH3)(L)] or, respectively, 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines for cis-[PtCl2L2]) and two cis chloride anions. The antiproliferative activity in vitro of complexes (1-4) have been tested against the cells of four human cell lines: SW707 rectal adenocarcinoma, A549 non-small cell lung carcinoma, T47D breast cancer and HCV29T bladder cancer. The results indicate a moderate antiproliferative activity of (4) against the cells of rectal, breast and bladder cancer and a marked and selective cytotoxic effect of (1-3) against the cells of all studied human cancer lines.     

Synthesis and BZR affinity of pyrazolo[1,5-a]pyrimidine derivatives. Part 1: study of the structural features for BZR recognition

Examination of the pharmacophoric points of the pyrazolo[1,5-a]pyrimidine derivatives, ligands for BZR, previously published led us to the design of a novel class of 3,6-diaryl-4,7-dihydro-pyrazolo[1,5-a]pyrimidin-7-ones and to determine the groups involved in the BZR recognition.     

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).     

Pyrazolopyridines with potent activity against herpesviruses: effects of C5 substituents on antiviral activity

Synthesis of a series of 5-substituted as well as 5,7-disubstituted 3-[2-(cyclopentylamino)-4-pyrimidinyl]-2-phenylpyrazolo[1,5-a]pyridin-7-amines with potent activity against herpes simplex viruses is described. Synthetic approaches allowing for variation of the substitution pattern are outlined and resulting changes in antiviral activity are highlighted. Several compounds with in vitro antiviral activity similar to or better than acyclovir are described.     

Determination of antifilarial compound UMF-078 and its metabolites in plasma by high-performance liquid chromatography

UMF-078, methyl (±)-[5-(α-amino-4-fluorobenzyl)benzimidazol-2-yl]carbamate, is a new antifilarial compound being developed by the World Health Organization. In the present study, a HPLC method for the simultaneous estimation of UMF-078 and its metabolites (flubendazole, decarbamoylated flubendazole, UMF060 and decarbamoylated UMF-060) in plasma was developed, validated and applied to pharmacokinetic studies. Linearity was observed between 20 and 1000 ng/ml for decarbamoylated UMF-060 and between 10 and 500 ng/ml for other analytes. Recoveries were consistent over the concentration ranges studied for all the analytes. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of ±20% at the lowest limit of quantitation, whereas at higher concentrations it was ±15%. The analytes showed stability up to two freeze–thaw cycles in plasma. No degradation was observed for any of the analytes even after 72 h of storing the dry plasma extracts at −30°C. The assay method was employed to study the pharmacokinetics of hydrochloride salt of UMF-078 in rats. The parent compound and its metabolites viz: decarbamoylated UMF-060, UMF-060 and flubendazole were quantitated in serum and the compounds could be monitored up to 168 h post-dose.     

Diagnostic value of IgG isotype responses against Brugia malayi antifilarial antibodies in the clinical spectrum of brugian filariasis

To study the diagnostic significance of antifilarial IgG subclasses in the clinical spectrum of brugian filariasis, IgG1, IgG3 and IgG4 antifilarial antibodies were determined in an exposed population comprising 74 asymptomatic amicrofilaraemics, 30 microfilaraemics, 20 lymphangitis and 16 elephantiasis patients resident in Narathiwart province, an area endemic for Brugia malayi lymphatic filariasis in southern Thailand. The dominant isotype of antifilarial antibody was IgG4. A significantly higher percentage of individuals were positive for IgG1 in the microfilaraemic and lymphangitis groups compared with the elephantiasis and endemic normal patients, while a significantly higher positive rate of IgG3 was found in those with lymphangitis. The possible role of these isotypes for diagnostic purposes and the pattern of antibody response in various clinically manifesting groups are discussed.     

Synthesis and biological evaluation of novel steroidal[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines

The preparation of steroidal[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines and their biological evaluation as potential anticancer agents are herein reported. These novel heterosteroids (2, 4) were prepared through the condensation reaction of 3-amino-1,2,4-triazole with 16-arylidene-17-ketosteroids (1, 3). All the synthesized compounds were evaluated for their anticancer activity in vitro against PC-3 (human prostatic carcinoma), MCF-7 (human breast carcinoma) and EC9706 (human esophageal carcinoma) cell lines. Among the screened compounds, 2i, 2n and 4f showed significant inhibitory activity against all the three human cell lines.     

Studies on filarial GST as a target for antifilarial drug development-in silico and in vitro inhibition of filarial GST by substituted 1,4-naphthoquinones

Eleven 1,4-naphthoquinone analogues with different amino substitutions at position 3 of the quinone ring earlier reported for macrofilaricidal activity were selected and screened against purified cytosolic GST isolated from the bovine filarial worm Setaria digitata and IC₅₀ values were determined. Of the 11 compounds tested, 8 showed good inhibition against S. digitata GST. The IC₅₀ values of the most effective macrofilaricidal compounds—11 [2-(4-methylpiperazin-1-yl)naphthalene-1,4-dione] and 9 {2-[(1,3-dimethylbutyl)amino]naphthalene -1,4-dione}—were 0.872 and 0.994 mM, respectively. Compounds 9 and 11 were further studied for type of enzyme inhibition and found to exhibit competitive and uncompetitive inhibition kinetics, respectively, with respect to substrate GSH. All 11 compounds were in agreement with Lipinski’s rule of five and passed through the FAFDrugs ADME/tox filter. Molecular docking was carried out using the modeled 3D structure of wbGST PDB ID:1SFM as receptor and substituted naphthoquinones as ligands using AutoDock 4.0. The binding energy of nine compounds varied from −9.15 to −6.58 Kcal mol⁻¹, whereas compounds 8 and 10 did not show any binding to the receptor. Among the compounds studied, compound 7 {2-[3-(diethylamino) propyl]aminonaphthalene-1,4-dione} showed maximum affinity towards wbGST as it exhibited the lowest binding energy, followed by compounds 11 and 9. However compound 7 was not macrofilaricidal while 11 and 9 exhibited macrofilaricidal activity. The results of in silico and in vitro studies with the synthesized 1,4 -naphthoquinone analogues on filarial GST and in vitro macrofilaricidal activity against adult bovine filarial worm S. digitata open up a promising biochemical target for antifilarial drug development.     

2-cyanoaminopyrimidines as a class of antitumor agents that promote tubulin polymerization

A series of 4-chloro-2-cyanoamino-6-fluoroalkylamino-5-phenylpyrimidines was prepared as a result of our efforts to modify a series of [1,2,4]triazolo[1,5-a]pyrimidines that proved to be potent anticancer agents with a unique mechanism of tubulin inhibition. On the cyanoamino nitrogen, a methyl group is optimal for activity among alkyl groups introduced. The structure-activity relationship for the rest of the molecule resembles that of [1,2,4]triazolo[1,5-a]pyrimidines. A lead compound (5) retained in vitro potency compared with TTI-237. In the mechanism of action studies, it behaved in the same manner as TTI-237. In addition, it is also capable of overcoming multidrug resistance due to P-gp. These findings strongly suggest that this series of 2-cyanoaminopyrimidines binds at the same site and in the same binding mode as TTI-237. Further modifications of the 2-cyanoamino group are underway.     

Synthesis and SAR of substituted pyrazolo[1,5-a]quinazolines as dual mGlu2/mGlu3 NAMs

Herein we report the design and synthesis of a series of substituted pyrazolo[1,5-a]quinazolin-5(4H)-ones as negative allosteric modulators of metabotropic glutamate receptors 2 and 3 (mGlu₂ and mGlu₃, respectively). Development of this series was initiated by reports that pyrazolo[1,5-a]quinazoline-derived scaffolds can yield compounds with activity at group II mGlu receptors which are prone to molecular switching following small structural changes. Several potent analogues, including 4-methyl-2-phenyl-8-(pyrimidin-5-yl)pyrazolo[1,5-a]quinazolin-5(4H)-one (10b), were discovered with potent in vitro activity as dual mGlu₂/mGlu₃ NAMs, with excellent selectivity versus the other mGluRs.     

Phosphodiesterase inhibitors. Part 4: Design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-4,4-dimethylpyrazolones

(-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. Here we show that a 4,4-dimethylpyrazolone subunit serves as an effective surrogate for the 5-methyl-4,5-dihydropyridazin-3(2H)-one ring of KCA-1490 whilst lacking a stereogenic centre. The 2- and 7-substituents in the pyrazolo[1,5-a]pyridine subunit markedly influence the PDE-inhibitory profile and can be adjusted to afford either potent PDE4-selective inhibitors or dual PDE3/4 inhibitors. A survey of bicyclic heteroaromatic replacements for the pyrazolo[1,5-a]pyridine allowed further refinement of the inhibitory profile and identified 3-(8-methoxy-2-(trifluoromethyl)imidazo[1,2-a]pyridin-5-yl)-4,4-dimethyl-1H-pyrazol-5(4H)-one as an orally active, achiral KCA-1490 analog with well-balanced dual PDE3/4-inhibitory activity.     

Comparison of different heterocyclic scaffolds as substrate analog PDE5 inhibitors

Several different heterocyclic systems were compared as PDE5 inhibitor scaffolds. In addition to the known 3H-imidazo[5,1-f][1,2,4]triazin-4-ones and pyrazolopyrimidinones, isomeric imidazo[1,5-a][1,3,5]triazin-4(3H)-ones were also shown to be potent and selective PDE inhibitor scaffolds with in vivo activity. SAR trends were elucidated for sulfonamide derivatives with generality across different scaffolds.