Synthesis and Biological Studies of Dodecameric Cationic Antimicrobial Peptides Containing Tetrahydrofuran Amino Acids.

We report here a concise route to synthesize various stereoisomers of tetrahydrofuran amino acids (TAAs) and the synthesis of TAA-containing linear cationic dodecapeptides. Some of these linear peptides show slightly better antimicrobial activities than their tetra- and octameric congeners, but no activity against Mycobacterium tuberculosis, for which octapeptides exhibited by far the best results; this implies that antibacterial activity is dependent on the length of these linear peptides. All the dodecapeptides described here were found to be toxic in nature against Vero cells. The study helps to delineate the optimal length of this series of linear peptides and select potential leads in the development of novel cationic peptide-based antibiotics.

Design, synthesis, in vitro and in silico studies of 2, 3-diaryl benzofuran derivatives as antitubercular agents.

As a part of our drug discovery program for anti-tubercular agents, a total of seventeen 2, 3-diaryl benzofuran hybrids were designed, synthesized and screened for their anti-tubercular potential against Mycobacterium tuberculosis H37Ra avirulent strain. Out of seventeen, four derivatives showed significant activity against M. tuberculosis H37Ra avirulent strain (ATCC 25177) with MIC value ranging from 12.5 to 50 µg/mL but out of four, one derivative (9E) was significantly active (MIC 12.5 µg/mL), which was further supported by the molecular docking energy (-8.4 kcal/mol) with respect to the first line anti-tubercular drug, isoniazid (-6.2 kcal/mol) on the target Polyketide Synthase-13. All the derivatives were also evaluated for their cytotoxicity against the normal lung cell line L-132 by the MTT assay and no toxicity was observed up to 27.4 µg/mL concentration. This report on the antitubercular potential of benzofuran derivatives may be of great help in anti-tubercular drug development.

A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts.

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C-terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail-forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane-defined vacuoles. Functional complementation studies indicated that the C-terminus, but not the N-terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non-pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.

Synthesis and biological evaluation of novel 1,2,3-triazole derivatives as anti-tubercular agents.

A library of seventeen novel 1,2,3-triazole derivatives were efficiently synthesized in excellent yields by the popular 'click chemistry' approach and evaluated in vitro for their anti-tubercular activity against Mycobacterium tuberculosis H37Ra (ATCC 25177 strain). Among the series, six compounds exhibited significant activity with minimum inhibitory concentration (MIC) values ranging from 3.12 to 0.78µg/mL and along with no significant cytotoxicity against MBMDMQs (mouse bone marrow derived macrophages). Molecular docking of the target compounds into the active site of DprE1 (Decaprenylphosphoryl-ß-d-ribose-2'-epimerase) enzyme revealed noteworthy information on the plausible binding interactions.

New class of methyl tetrazole based hybrid of (Z)-5-benzylidene-2-(piperazin-1-yl)thiazol-4(%H)-one as potent antitubercular agents.

In search of potential therapeutics for tuberculosis, we describe here the synthesis and in vitro antitubercular activity of a novel series of thiazolone piperazine tetrazole derivatives. Among all the synthesized derivatives, four compounds (10, 14, 20 and 33) exhibited more potent activity (MIC=3.08, 3.01, 2.62 and 2.51 µM) than ethambutol (MIC=9.78 µM) and pyrazinamide (MIC=101.53 µM) against Mycobacterium tuberculosis. Furthermore, they displayed no toxicity against Vero cells (C1008) and mouse bone marrow derived macrophages (MBMDMϕ). These investigated analogues have emerged as possible lead molecule to enlarge the scope of the study.

Interactive effect of oral anti-hyperglycaemic or anti-hypertensive drugs on the inhibitory and bactericidal activity of first line anti-TB drugs against M. tuberculosis.

Co-existence of life style disorders, like, Diabetes or Hypertension, increases risk of, treatment failure, deaths and developing drug-resistant TB. Concomitant administration of drugs to treat dual/multi-morbidities may alter their effectiveness, in additive/synergistic or adverse/antagonistic manner. We evaluated interactive effect of 7 anti-hyperglycaemic (HG) and 6 anti-hypertensive (HT) drugs on the inhibitory (MICs) and bactericidal (% killing of intracellular bacilli) activities of anti-TB drugs, Isoniazid (INH), Rifampicin (RFM), Ethambutol (EMB) and Streptomycin (STR) against M. tuberculosis. Five anti-HG drugs, namely, Acarbose, Acetohexamide, Glyburide, Repaglinide and Sitagliptin imparted either 'additive' or 'no effect' on the activities (inhibition or % killing) of all the four anti-TB drugs, as evident by their lower FICs (Fractional Inhibitory concentrations) and higher bacterial killing in combination. Metformin and Rosiglitazone, however, exerted adverse effect on the Ethambutol (FICs >2.0). All the six anti-HT drugs, namely, Atenolol, Hydrochlorothiazide, Ramipril, Valsartan, Nifedipine and Verapamil exerted either 'additive'/'synergistic' or 'no effect' on the activities of anti-TB drugs. These findings may help clinicians to select safe and helpful anti-HG or anti-HT drugs for TB patients, if, suffering with diabetes or hypertension like co-morbidities and receiving DOTs (a set regimen for the treatment of TB based on the WHO guidelines).

Synthesis and Structural Activity Relationship Study of Ursolic Acid Derivatives as Antitubercular Agent.

OBJECTIVE: The chemical transformation of ursolic acid (UA) into novel C-3 aryl ester derivatives and in vitro and silico assessment of their antitubercular potential. BACKGROUND: UA is a natural pentacyclic triterpenoid with many pharmacological properties. Semisynthetic UA analogs have demonstrated enhanced anticancer, antimalarial, and antifilarial properties in our previous studies. METHOD: The C-30 carboxylic group of previously isolated UA was protected, and various C-3 aryl ester derivatives were semi-synthesized. The agar dilution method was used to evaluate the in vitro antitubercular efficacy of Mycobacterium tuberculosis (Mtb) H37Ra. In silico docking studies of the active derivative were carried out against Mtb targets, catalase peroxidase (PDB: 1SJ2), dihydrofolate reductase (PDB: 4M2X), enoyl-ACP reductase (PDB: 4TRO), and cytochrome bc1 oxidase (PDB: 7E1V). RESULTS: The derivative 3-O-(2-amino,3-methyl benzoic acid)-ethyl ursolate (UA-1H) was the most active among the eight derivatives (MIC1 2.5 µg/mL) against Mtb H37Ra. Also, UA-1H demonstrated significant binding affinity in the range of 10.8-11.4 kcal/mol against the antiTb target proteins, which was far better than the positive control Isoniazid, Ethambutol, and co-crystallized ligand (HEM). Moreover, the predicted hit UA-1H showed no inhibition of Cytochrome P450 2D6 (CYP2D6), suggesting its potential for favorable metabolism in Phase I clinical studies. CONCLUSION: The ursolic acid derivative UA-1H possesses significant in vitro antitubercular potential with favorable in silico pharmacokinetics. Hence, further in vivo assessments are suggested for UA-1H for its possible development into a secure and efficient antitubercular drug.

Influence of vehicles used for oral dosing of test molecules on the progression of Mycobacterium tuberculosis infection in mice.

Preclinical evaluation of drug-like molecules requires their oral administration to experimental animals using suitable vehicles. We studied the effect of oral dosing with corn oil, carboxymethyl cellulose, dimethyl sulfoxide, and polysorbate-80 on the progression of Mycobacterium tuberculosis infection in mice. Infection was monitored by physical (survival time and body weight) and bacteriological (viable counts in lungs) parameters. Compared with water, corn oil significantly improved both sets of parameters, whereas the other vehicles affected only physical parameters.

Bioenhancing and antimycobacterial agents from Ammannia multiflora.

The methanolic extract of Ammannia multiflora (Lythraceae) showed significant bioenhancing activity with the antibiotic nalidixic acid. Bioassay-guided fractionation of MeOH extract resulted in the isolation of a novel compound, 2,5-bis-(3,3'-hydroxyaryl)tetrahydrofuran, named as ammaniol (5), along with 9 other known compounds (1-4, 6-10). Furthermore, compound 4-hydroxy- α-tetralone (1) was converted into five semisynthetic acyl derivatives, 1A-1E, which were evaluated along with compounds 1, 5, 6, 9, and 10 for their bioenhancing activity in combination with nalidixic acid against the two strains, CA8000 and DH5 α, of Escherichia coli. The results showed that the methanolic extract of A. multiflora and compounds 1 and 9 possessed significant bioenhancing activity and reduced the dose of nalidixic acid fourfold while compounds 5, 6, 10 and semisynthetic derivatives 1A- 1E reduced the dose of nalidixic acid twofold. Compound 5 was also tested for antimycobacterial activity against Mycobacterium H37Rv and was found to show moderate activity (MIC 25 µg/mL) against this pathogen.

Design and Synthesis of Quinazolinone-Triazole Hybrids as Potent Anti-Tubercular Agents.

A straightforward and convenient methodology has been developed for the reaction of 2-aminobenzamide and carbonyls affording 2,3-dihydroquinazolin-4(1H)-ones using aqueous solution of [C12Py][FeCl3Br]. The developed methodology was applied for the synthesis of 25 quinazolinone-triazole hybrids followed by evaluation of their in vitro anti-tubercular (TB) activity. The results revealed that 8 quinazolinone-triazole hybrids displayed promising activity having MIC values of 0.78-12.5 µg/mL. The compound 3if with MIC 0.78 µg/mL was found to be the lead nominee among the series, better than Ethambutol, a first line anti-TB drug and comparable with Rifampicin. The active compounds with MIC values ≤ 6.25 µg/mL were subjected to in vitro cytotoxicity and found nontoxic. In drug-drug interaction, compounds 3ia and 3ii interacted synergistically with all the three anti-TB drugs, INH, RFM, and EMB. Other 3 compounds interacted either in synergistic or additive manners. Important information on the binding interaction of the target compounds with the active sites of 1DQY Antigen 85C from Mycobacterium tuberculosis and Enoyl acyl carrier protein reductase (InhA) enzymes was obtained from molecular docking studies. Screening of the drug-likeness properties and bioactivity score indicates that synthesized molecules could be projected as potential drug candidates. Based on the current study, quinazolinone-triazole hybrids framework can be useful in drug development for TB.

Synthesis and biological evaluation of substituted 4-arylthiazol-2-amino derivatives as potent growth inhibitors of replicating Mycobacterium tuberculosis H37Rv.

In search of potential therapeutics for tuberculosis, we describe herein synthesis and biological evaluation of some substituted 4-arylthiazol-2-amino derivatives as modified analogues of the antiprotozoal drug Nitazoxanide (NTZ), which has recently been reported as potent inhibitor of Mtb H(37)Rv (Mtb MIC=52.12 µM) with an excellent ability to evade resistance. Among the synthesized derivatives, the two compounds 7a (MIC=15.28 µM) and 7c (MIC=17.03 µM) have exhibited about three times better Mtb growth inhibitory activity over NTZ and are free from any cytotoxicity (Vero CC(50) of 244 and 300 µM respectively). These two compounds represent promising leads for further optimization.

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents.

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H(37)Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 µg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 µg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.

Brevifoliol and its Analogs: A New Class of Anti-tubercular Agents.

Brevifoliol is an abeo-taxane isolated from the Taxus wallichiana needles; eighteen semisynthetic esters derivatives of brevifoliol were prepared by Steglich esterification and screened for their anti-tubercular potential against Mycobacterium tuberculosis H37Ra avirulent strain. The 3- [chloro (7)] and 3, 5-[dinitro (8)] benzoic acid ester derivatives were most active (MIC 25 ug/ml) against the pathogen. Further, in silico docking studies of the active derivative 7 with mycobacterium enzyme inhA (enoyl-ACP reductase) gave the LibDock score of 152.68 and binding energy of -208.62 and formed three hydrogen bonds with SER94, MET98, and SER94. Similarly, when derivative 8 docked with inhA, it gave the LibDock score of 113.55 and binding energy of -175.46 and formed a single hydrogen bond with GLN100 and Pi-interaction with PHE97. On the other hand, the known standard drug isoniazid (INH) gave the LibDock score of 61.63, binding energy of -81.25 and formed one hydrogen bond with ASP148. These molecular docking results and the way of binding pattern indicated that compounds 7 and 8 bound well within the binding pocket of inhA and showed a higher binding affinity than the known drug isoniazid. Additionally, both the derivatives (7 and 8) showed no cytotoxicity, with CC50 195.10 and 111.36, respectively towards the mouse bone marrow-derived macrophages.

Substituted hydrazinecarbothioamide as potent antitubercular agents: synthesis and quantitative structure-activity relationship (QSAR).

A series of novel substituted hydrazinecarbothioamides was synthesized and evaluated for anti-TB activity. Three most active compounds viz. 1, 6 and 12 were found to exhibit minimum inhibitory concentration (MIC) of 0.4 microg/mL, whereas four compounds viz. 3, 5, 10 and 11 showed comparatively lesser activity with MIC value of 0.8 microg/mL against Mycobacterium tuberculosis strain. A highly significant QSAR equation explaining 81.8% variance is described.

Synthesis and bio-evaluation of alkylaminoaryl phenyl cyclopropyl methanones as antitubercular and antimalarial agents.

A series of 4-alkylaminoaryl phenyl cyclopropyl methanones (6a-6u and 8a-8c) were synthesized from 4-fluorochalcones (3a and 3b) by cyclopropanation of double bond followed by nucleophilic substitution of F with different amines. The compounds were screened for their antitubercular and antimalarial activities against Mycobacterium tuberculosis H37Rv and Plasmodium falciparum 3D7 strains in vitro respectively. Several compounds (6a, 6d-6h, 6p, 6q and 8a-8c) exhibited good in vitro antitubercular activities with MIC values 3.12-12.5µg/mL and preferentially inhibited the growth of P. falciparum in vitro (4a, 4c, 6a-6d, 6f, 6s, 8a and 8c) with IC50 as low as 0.080 and 0.035µg/mL and SI values 4975 and 6948, respectively. Molecular docking studies and in vitro evaluation against FAS-II enzymes using reporter gene assays were carried out to elucidate the mode of action of these molecules. Two compounds 4a and 6g showed significant inhibition at 25µM concentration of the compound.

A Simple Work-Up-free, Solvent-free Approach to Novel Amino Acid Linked 1,4-Disubstituted 1,2,3-Triazoles as Potent Antituberculosis Agents.

An efficient, green strategy for synthesis of 1,4-disubstituted-1,2,3-triazole has been developed using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) acetate ionic liquid (200 µL) under a solvent- and external base-free condition. This protocol is further applied for the synthesis of novel amino acid containing 1,2,3-triazole molecules, which were then evaluated for potential antitubercular and antibacterial activities. Cytotoxicity assay of the compounds was also performed. In silico analysis of the promising compounds selected through experimental analysis was thereafter performed for visualizing molecular interactions and predicting binding affinities between our synthesized molecules, which exhibited good activity in experimental studies and the DprE1 target protein of Mycobacterium tuberculosis. Durg-likeness studies also show potential of the synthesized molecules as drug candidates.

Solution-phase synthesis of a library of carbapeptide analogues based on glycosylamino acid scaffolds and their in silico screening and antimicrobial evaluation.

A well-organized and efficient approach toward the solution phase synthesis of a library of carbapeptide analogues based on glycosyl amino ester scaffold is described. The reported synthetic route involves a five step preparation of heptofuranuronamides 6a-h and octopyranuronamide 7e from glycosyl amino esters 1 and 7, respectively. Coupling of glycosyl amino esters 1 or 7 with three different N-Fmoc protected amino acids afford the N-Fmoc protected intermediates 2a-c and 7a. Deprotection of Fmoc group in 2a-c and 7a with piperidine gave respective compounds 3a-c and 7b with free amine. Subsequent coupling of 3a-c and 7b with different aromatic acids furnishes respective heptofuranuronates 4a-h and octopyranuronate 7c in good yields. The latter, on ester hydrolysis by LiOH gave the corresponding glycopeptide analogues 5a-h and 7d with terminal carboxyl group. The carboxyl group in these compounds was amidated with oxalyl chloride/ NH(4)OH to afford heptofuranuronamides 6a-h and octopyranuronamides 7e. In vitro screening of all compounds displayed moderate antifungal, antitubercular, and general antibacterial activities. Reverse docking calculations involving over 841 protein drug targets have identified two potential targets for these compounds. These results will form the basis for synthesizing second-generation antimicrobial compounds.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents.

INTRODUCTION: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. MATERIALS AND METHODS: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A - DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. RESULTS: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. CONCLUSION: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

Dihydroartemisinin and its Analogs: A New Class of Antitubercular Agents.

BACKGROUND: Tuberculosis is one of the leading causes of mortality worldwide. Resistance against the frontline anti-tubercular drugs has worsened the already alarming situation, which requires intensive drug discovery to develop new, more effective, affordable and accessible anti-tubercular agents possessing novel modes of action. OBJECTIVE: Chemical transformation of dihydroartemisinin for anti-tubercular lead optimization. METHODS: Dihydroartemisinin, a metabolite of artemisinin was chemically converted into eight acyl derivatives and were evaluated for anti-tubercular potential against H37Rv virulent strain of Mycobacterium tuberculosis by agar-based proportion assay. Further, synergistic activity of 12-O-m-anisoyl dihydroartemisinin was also studied with the front-line anti-TB drugs, isoniazid and rifampicin. RESULTS: The results showed that all the derivatives were active but out of eight, 12-O-m-anisoyl dihydroartemisinin and 12-O-p-anisoyl dihydroartemisinin were significantly active (MIC 25.0 µg/mL). In synergistic activity evaluation, the 12-O-m-anisoyl dihydroartemisinin derivative showed reduction in MIC (by 1/8th, i.e. 3.12 µg/mL and that of rifampicin by »th, i.e. 0.05 µg/mL) with the front-line anti-TB drug, rifampicin. The sumfractional inhibitory concentration (Σ FIC) was 0.375. CONCLUSION: These results suggested a synergistic effect of the 12-O-m-anisoyl dihydroartemisinin with rifampicin and established its base for the development of anti-tubercular agents from an in-expensive and non-toxic natural product. To the best of our knowledge this is the first ever report on the anti-tubercular potential of dihydroartemisinin and its derivatives.

Thiophene containing triarylmethanes as antitubercular agents.

A new series of thiophene containing triarylmethane derivatives were synthesized from the Friedel-Crafts alkylation of diarylcarbinols followed by incorporation of amino alkyl chains. These were evaluated against Mycobacterium tuberculosis H37R(v) and showed the activity in the range of 3.12-12.5 microg/mL in vitro.