C.N. Khobragade

Synthesis and biological evaluation of a novel series of pyrazole chalcones as anti-inflammatory, antioxidant and antimicrobial agents

A novel series of 1-(2,4-dimethoxy-phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-propenone (3) have been prepared by the Claisen-Schmidt condensation of 1-(2,4-dimethoxy-phenyl)-ethanone (1) and substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2). Substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2) were prepared by Vilsmeir-Haack reaction on acetophenonephenylhydrazones to offer the target compounds. The structures of the compounds were established by IR, (1)H NMR and mass spectral analysis. All the compounds were evaluated for their anti-inflammatory (TNF-alpha and IL-6 inhibitory assays), antioxidant (DPPH free radical scavenging assay) and antimicrobial activities (agar diffusion method) against some pathogenic bacteria and fungi. Of 10 compounds screened, compounds 3a, 3c and 3g exhibited promising IL-6 inhibitory (35-70% inhibition, 10 microM), free radical scavenging (25-35% DPPH activity) and antimicrobial activities (MIC 100 microg/mL and 250 microg/mL) at varied concentrations. The structure-activity relationship (SAR) and in silico drug relevant properties (HBD, HBA, PSA, cLogP, molecular weight, E(HOMO) and E(LUMO)) further confirmed that the compounds are potential lead compounds for future drug discovery study. Toxicity of the compounds was evaluated theoretically and experimentally and revealed to be nontoxic except 3d and 3j.

Synthesis and biological evaluation of a novel series of 2,2-bisaminomethylated aurone analogues as anti-inflammatory and antimicrobial agents

This is the first report on aurones as a new class of drugs with anti-inflammatory and antimicrobial agents. A series of 2,2-bisaminomethylated aurone analogues (4a-j) were synthesized by Mannich reaction from 1,3,5-trimethoxybenzene in three steps. The structures of the newly synthesized compounds were confirmed by IR, (1)H NMR and mass spectral analysis. All the synthesized compounds were screened against the pro-inflammatory cytokines (TNF-alpha, IL-6) and antimicrobial (antibacterial and antifungal) activity. Compounds 4a, 4b, 4c, 4d, and 4e showed promising results against IL-6 at 10 microM concentration (74-100%). Compounds 4a, 4b and 4c were found to be active against TNF-alpha (76-100%) at 10 microM. Interestingly, all compounds have shown good antimicrobial activity. Compounds 4d, 4e and 4f showed excellent antimicrobial activity as compared with standard drugs.

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9

An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55oC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/minrespectively.

Synthesis of novel 3,5-diaryl pyrazole derivatives using combinatorial chemistry as inhibitors of tyrosinase as well as potent anticancer, anti-inflammatory agents

In the present article, we have synthesized a combinatorial library of 3,5-diaryl pyrazole derivatives using 8-(2-(hydroxymethyl)-1-methylpyrrolidin-3-yl)-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (1) and hydrazine hydrate in absolute ethyl alcohol under the refluxed conditions. The structures of the compounds were established by IR, (1)H NMR and mass spectral analysis. All the synthesized compounds were evaluated for their anticancer activity against five cell lines (breast cancer cell line, prostate cancer cell line, promyelocytic leukemia cell line, lung cancer cell line, colon cancer cell line) and anti-inflammatory activity against TNF-alpha and IL-6. Out of 15 compounds screened, 2a and 2d exhibited promising anticancer activity (61-73% at 10 microM concentration) against all selected cell lines and IL-6 inhibition (47% and 42% at 10 microM concentration) as in comparison to standard flavopiridol (72-87% inhibition at 0.5 microM) and dexamethasone (85% inhibition at 1 microM concentration), respectively. Cytotoxicity of the compounds checked using CCK-8 cell lines and found to be nontoxic to slightly toxic. Out of 15, four 3,5-diaryl pyrazole derivatives exhibiting potent inhibitory activities against both the monophenolase and diphenolase actions of tyrosinase. The IC(50) values of compounds (2a, 2d, 2h and 2l) for monophenolase inhibition were determined to range between 1.5 and 30 microM. Compounds 2a, 2d, 2h and 2l also inhibited diphenolase significantly with IC(50) values of 29.4, 21.5, 2.84 and 19.6 microM, respectively. All four 3,5-diaryl pyrazole derivatives were active as tyrosinase inhibitors (2a, 2d, 2h and 2l), and belonging to competitive inhibitors. Interestingly, they all manifested simple reversible slow-binding inhibition against diphenolase.

Microbial and xanthine dehydrogenase inhibitory activity of some flavones

Xanthine dehydrogenase (XDH) is responsible for the pathological condition called Gout. In the present study different flavones synthesized from chalcone were evaluated in vitro for their inhibitory activity. Inhibitory activity of flavones on XDH was determined in terms of inhibition of uric acid synthesis from Xanthine. The enzymatic activity was found maximum at pH 7.5 and temperature 40°C. The flavones 6-chloro-2-[3-(4–hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F1) and 6-chloro-7methyl-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F2),were noncompetitive and competitive inhibitor with Ki values 1.1 and 0.22 respectively. The flavones (F1), (F2), 6-chloro-2-[3-(4-chloro-phenyl)-1phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F3), 8-bromo-6-chloro-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F4), 2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F5) and 6-methyl-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F6) were also screened for their antimicrobial activity, measured in terms of zone of inhibition. A broad spectrum antifungal activity was obtained against Trichoderma viridae, Candida albicans, Microsporum cannis, Penicillium chrysogenum and Fusarium moniliformae. In case of Aspergillus niger and Aspergillus flavous only spore formation was affected, while antibacterial activity was observed against Staphylococcus aureus, Bacillus subtilis and Serratia marsecens only. The flavones were further analyzed for quantitative structural activity relationship study (QSAR) by using PASS, online software to determine their Pa value. Toxicity and drug relevant properties were revealed by PALLAS software in terms of their molecular weight. Log P values were also studied. The result showed both the F1 and F2 flavones as antigout and therefore supports the development of novel drugs for the treatment of gout.

Synthesis of new heterocyclic hybrids based on pyrazole and thiazolidinone scaffolds as potent inhibitors of tyrosinase

As a part of ongoing studies in developing new Tyrosinase inhibitors, a class of structurally novel 2-(2,4-dimethoxy phenylamino)-5 methylene-4-thiazolinone derivatives were synthesized by incorporating 2-(2,4-dimethoxy-phenylamino)-thiazol-4-one with various 1-(1-methyl-buta-1,3-dienyl)-3-phenyl-1H-pyrazole-4-carbaldehyde. The results showed that some of the synthesized compounds exhibited significant inhibitory activities. Especially, 5-[3-(2-chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-dimethoxy-phenylamino)-thiazol-4-one (5h) and 5-[3-(3-chloro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-2-(2,4-dimethoxy-phenylamino)-thiazol-4-one (5g) possessing 2-chloro-phenyl and 3-chloro-phenyl group exhibited the most potent tyrosinase inhibitory activity with an IC(50) value of 34.12 and 52.62 μM, respectively. The inhibition mechanism analysis of 5h and 5g thiazolidinone derivatives demonstrated that the inhibitory effects of the compounds on tyrosinase were reversible and competitive. Preliminary structure-activity relationships (SAR) analysis suggested that further development of such compounds might be of interest, as it manifests simple reversible slow binding inhibition against monophenolase and diphenolase.

Homology modeling and docking study of xanthine oxidase of Arthrobacter sp. XL26

Hyperuricemia is a condition of defective purine metabolism characterized with elevated serum uric acid (UA) level that further leads to gout and gouty nephrolithiasis disorders. Gout is a world wide distributed rheumatic disease comprises 1% of the total population and still is in increasing state. One of the factors contributing to overproduction of UA is the hydroxylation of xanthine catalyzed by xanthine oxidase (XO). In the present study, 3D modeling of Arthrobacter sp. XL26 (xodB) protein was performed by comparative modeling approach using Rhodobacter capsulatus XDH (PDB ID: 2W3sF) as template in SWISS-MODEL, Geno3D and MODELLER program server. The best model was selected based on overall stereochemical quality (Procheck, PROSA, GenThreader), energy minimized, refined and used for active site characterization in BioMed CAChe workspace. The enzyme-inhibitor interaction was studied by docking to screen the possible inhibitors and application of model in design and development of anti-gout agents.

Exploring glycolate oxidase (GOX) as an antiurolithic drug target: molecular modeling and in vitro inhibitor study.

Glycolate oxidase (GOX) is one of the principal enzymes involved in the pathway of oxalate synthesis. It converts glycolate to glyoxylate by oxidation and then glyoxylate is finally converted to oxalate. Therapeutic intervention of GOX in this consequence thus found potential in the treatment of calcium oxalate urolithiasis. In present investigation, we explored GOX in search of potential leads from traditional resources. Molecular modeling of the identified leads, quercetin and kaempherol, was performed by employing Glide 5.5.211 (SchrodingerTM suite). In the absence of pure human glycolate oxidase (hGOX) preparation, in vitro experiments were performed on spinach glycolate oxidase (sGOX) as both enzymes possess 57% identity and 76% similarity along with several conserved active site residues in common. We aimed to identify a possible mechanism of action for the anti-GOX leads from Tribuls terrestris, which can be attributed to anti-urolithic drug development. This study found promising in development of future GOX inhibitory leads.

Comparative structural modeling and docking studies of oxalate oxidase: Possible implication in enzyme supplementation therapy for urolithiasis

In humans oxalate is end product of protein metabolism, with no enzyme present to act on it. In conditions of its enhanced endogenous synthesis or increased absorption from the diet, oxalate accumulation leads to hyperoxaluria which can further lead to a number of pathological conditions including urolithiasis. Urolithiasis has been a perplexing problem due to its high incidence and rate of recurrence after treatment like Extracorporeal-shock wave lithotripsy (ESWL). Hence other prophylactic treatment becomes necessary. One of the newer approaches of curing such metabolic disorders is the enzyme supplementation therapy. Oxalate oxidase (OxOx) is a commonly occurring enzyme in plants, bacteria and fungi that catalyses oxidative cleavage of oxalate to CO(2) with reduction of dioxygen to H(2)O(2). Present study, used Hordeum vulgare OxOx crystal structure (PDB ID 2ET1A) as a template for constructing 3D models of OxOx from Triticum aestivum, Arabidopsis thaliana, Sclerotiana sclerotiarum. Similarly Homology models for isoforms Ceriporiopsis subvermispora 336, C. subvermispora 422 were constructed by using template Bacillus subtilis oxalate decarboxylase (Oxdc) (PDB ID 2UY8A) by comparative modeling approach in SWISS MODEL, MODELLER, 3D JIGSAW and GENO 3D program server. Based on overall stereochemical quality (PROCHECK, PROSA, VARIFY 3D), best models were selected, energy minimized, refined and characterized for active site in BioMed CaChe V 6.1 workspace. Selected models were further studied for structure function relationship with substrate (oxalate) and its analogue (glycolate) by using docking approach. Calculated interaction energy between the oxalate and constructed enzyme indicated that homology models for OxOx of T. aestivum, A. thaliana and S. sclerotiarum, can account for better regio-specificity of this enzyme towards oxalate. That supports the interested metabolism and thus may further implement in enzyme supplementation therapy for urolithiasis.

Comparative structural modeling and docking studies of uricase: Possible implication in enzyme supplementation therapy for hyperuricemic disorders

Uricase (EC 1.7.3.3, UC) catalyzes the oxidation of uric acid (UA) to more soluble allantoin thereby lowering plasma UA levels. In humans, when concentration of UA exceeds >7mg/dl, it leads to hyperuricemia, gout, nephrolithiasis and urolithiasis. A new remedy to cure such metabolic diseases is the enzyme supplementation therapy by UC but with high degree of antigenic independence. Therefore screening of new uricase sources to expand its usefulness and reduced antigenecity is needed. Present study employed cheminformatics approach to construct models of reported UC from different sources viz. Bacillus megaterium, Streptomyces bingchenggensis BCW-1, Paenibacillus sp, Solibacter usitatus Ellin6076, Truepera radiovictrix DSM 17093 and Ktedonobacter racemifer DSM 4496 in order to study their structure-function relationship for enzyme mass production and modification for improved characteristics. BioMed CAChe version 6.1 was further used to study enzyme-substrate interactions of models with uric acid using docking approach. Results indicated that models for UC of Streptomyces bingchenggensis BCW-1 accounted for better regio-specificity towards UA, supporting the interested metabolism and thus may further be implicated in enzyme supplementation therapy for hyperuricemic associated disorders.