Praveen K. Shukla

Synthesis and antifungal activity of 1,2,3-triazole containing fluconazole analogues

Fluconazole based novel mimics containing 1,2,3-triazole were designed and synthesized as antifungal agents. Their antifungal activities were evaluated in vitro by measuring the minimal inhibitory concentrations (MICs). Compounds 12, 15, and 16 were found to be more potent against Candida fungal pathogens than control drugs fluconazole and amphotericin B. The studies presented here provide structural modification of fluconazole to give 1,2,3-trazole containing molecules. Furthermore, these molecules were evaluated in vivo against Candida albicans intravenous challenge in Swiss mice and antiproliferative activities were tested against human hepatocellular carcinoma Hep3B and human epithelial carcinoma A431. It was found that compound 12 resulted in 97.4% reduction in fungal load in mice and did not show any profound proliferative effect at lower dose (0.001 mg/ml).

Mucoadhesive nanoparticles for prolonged ocular delivery of natamycin: In vitro and pharmacokinetics studies

The aim of this study was to prepare natamycin encapsulated lecithin/chitosan mucoadhesive nanoparticles (NPs) for prolonged ocular application. These NPs were characterized by their mean particle size 213nm, encapsulation efficiency 73.57%, with a theoretical drug loading 5.09% and zeta potential +43. In vitro release exhibited a biphasic drug release profile with initial burst followed by a very slow drug release. The MIC(90) and zone of inhibition of NPs showed similar antifungal activity as compared to marketed suspension and free natamycin against Candida albicans and Aspergillus fumigates. The ocular pharmacokinetics of NPs and marketed formulation were evaluated in NZ rabbits. The NPs exhibit significant mucin adhesion. The AUC((0-∞)) was increased up to 1.47 fold and clearance was decreased up to 7.4-fold as compared to marketed suspension. The PK-PD and pharmacokinetic simulation was carried out to estimate optimum dosing regimen for good efficacy. Thus, lecithin/chitosan NPs could be considered useful approach aiming to prolong ocular residence and reduce dosing frequency.

Mycotic keratitis: an overview of diagnosis and therapy

The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus‐positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. The yield of clinical examination in the diagnosis of keratomycosis is 63–83% and KOH mount is 91%. This still highlights the limitation of routine clinical examination and smear examination, which is not performing 100% efficiently. It is for these 37%, 17% and 9% of cases, every day advanced technologies are called for. Those who deal with patient care are aware of certainties and uncertainties of results of clinical examination. The best reported figures at specialized centres might not translate into clinical practice. Another factor to be kept in mind is that many patients who come after secondary and tertiary referrals are already treated with antibiotics, antivirals, steroids and sometimes even antifungals that distort the clinical picture completely. Further, one has to consider as well the cases caused by yeast‐like fungi, which resemble bacterial keratitis. Confirmation of diagnosis, not only in case of mycotic keratitis but also for other diseases, to initiate prompt and accurate therapy would avoid unnecessary and indiscriminate use of steroids/antibacterials/antivirals and antifungals.

An eco-friendly synthesis and antimicrobial activities of dihydro-2H-benzo- and naphtho-1,3-oxazine derivatives

A series of 3,4-dihydro-2H-benzo[e]-, 2,3-dihydro-1H-naphtho[1,2-e]-, 3,4-dihydro-2H-naphtho[2,1-e][1,3]oxazine and 1,2-bis(3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-yl)ethane derivatives was obtained through an eco-friendly Mannich type condensation-cyclization reaction of phenols or naphthols with formaldehyde and primary amines in water at ambient temperature. Preliminary in vitro antimicrobial activity of the synthesized compounds was assessed against six pathogenic fungi, two Gram-negative and two Gram-positive bacteria. Some of the screened compounds have shown significant in vitro antimicrobial effect. Cytotoxic activities of the lead compounds (2m, 2n, 3c and 3d) against mouse fibroblast cell line (L929) were determined by MTT method. The assay results revealed that these molecules offered remarkable viability (>90%) of L929 cells at concentration of 25 microg/mL.

Use of PCR Targeting of Internal Transcribed Spacer Regions and Single-Stranded Conformation Polymorphism Analysis of Sequence Variation in Different Regions of rRNA Genes in Fungi for Rapid Diagnosis of Mycotic Keratitis

ABSTRACT The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. Immunological methods of diagnosis have not been found to be satisfactory, and recent research has been diverted to the use of PCR for the sensitive and early diagnosis at the molecular level. In the present study we targeted different regions of the rRNA gene to diagnose cases of mycotic keratitis and identify the causal agents. Six fungus-specific primers (primers ITS1, ITS2, ITS3, ITS4, invSR1R, and LR12R) were used, and the amplified products were analyzed by single-stranded conformation polymorphism (SSCP) analysis. Dendrograms of these SSCP patterns, prepared on the basis of Jaccards coefficient, indicated that the PCR products obtained with primer pair ITS1 and ITS2 were the best for the identification of fungi. The results were confirmed by sequencing of the PCR products, and the approach was successfully tested experimentally for the detection of mycotic keratitis caused by Aspergillus fumigatus and was used for the diagnosis of fungal corneal ulcers in patients.

2,3-Disubstituted-1,4-naphthoquinones, 12H-benzo[b]phenothiazine-6,11-diones and related compounds: Synthesis and Biological evaluation as potential antiproliferative and antifungal agents

A series of 2-chloro-3-arylsulfanyl-[1,4]naphthoquinones (2), 2,3-bis-arylsulfanyl-[1,4]naphthoquinones (3) and 12H-benzo[b]phenothiazine-6,11-diones and their analogs 6-8 were synthesized and evaluated for their antiproliferative activity against human cervical cancer (HeLa) cells. Compounds 3a and 3b were found to possess most potent antiproliferative and cell killing ability. Compounds 1-8 were also evaluated for antifungal activities. The structure-activity relationship of these compounds was studied and the results show that compound 2a (MIC(50)=1.56 microg/mL) exhibited in vitro potent antifungal activity compared to the clinically useful antifungal drug Fluconazole (MIC(50)=2.0 microg/mL) against Sporothrix. schenckii. Compound 2a (MIC(50)=1.56 microg/mL) also exhibited same antifungal activity compared to clinically useful drug Amphotericin-B (MIC(50)=1.56 microg/mL) against Trichophyton. mentagraphytes.

Chloropyrimidines as a new class of antimicrobial agents.

In the course of our investigations of pyrimidines as antimycotic agents, we have identified a sub-class, with significant in vitro activity against mycobacteria. The salient feature of these pyrimidine derivatives (3a-o and 7a,b) is their appended aryl, heteroaryl and alkylthio substituent at position 6 and also alkylthio substituent at position 2. The rational design, synthesis, and evaluation of the in vitro antibacterial activity against six pathogenic bacteria including virulent and non-virulent strains of Mycobacterium tuberculosis is described. Some of the synthesized compounds (3c, 3h, 3i, 3o) have displayed only potent in vitro antimycobacterial activity with MIC of 0.75 microg/mL except 3i which also demonstrated activity against Escherichia coli at 12.5 microg/mL concentration. Only two compounds, 3a and 3b, demonstrated antibacterial activity against Pseudomonas aeruginosa and E. coli with MIC 12.5 microg/mL. All the synthesized compounds were also evaluated for their antimycotic activity against five pathogenic fungi but only some of them 3j-n and 7a,b were found most potent against Aspergillus fumigatus and Trichophyton mentagrophytes.

Monoclonal Immunoglobulin G1 Directed against Aspergillus fumigatus Cell Wall Glycoprotein Protects against Experimental Murine Aspergillosis

ABSTRACT Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log10 units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 × 105 CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.

Tetrahydronaphthyl azole oxime ethers: The conformationally rigid analogues of oxiconazole as antibacterials

A series of novel (Z)- and (E)-2-imidazolo-/triazolo-methyl tetrahydronaphthyl oxime ethers (7-28) were synthesized as conformationally constrained analogues of oxiconazole and evaluated for antifungal and antibacterial activities. Many of these derivatives exhibited potent antibacterial activity and surprisingly none of them was active against fungal strains. The SAR studies showed that imidazole oxime ethers were more active than the corresponding triazole oxime ethers. Imidazole derivatives 8, 11, 12, 15, 18, 19, 21 and 23 exhibited high inhibitory activity with 1.56-0.39 microg/mL MIC values against Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus. These compounds represent new structure scaffolds that can be further optimized to give new antibacterial agents with structures significantly different from those of existing classes of antibiotics.

Antifungal anthraquinones from Saprosma fragrans.

Abstract A new 3,4-dihydroxy-1-methoxy anthraquinone-2-corboxaldehyde (1) together with a known anthraquinone, damnacanthal (2), were isolated from the chloroform fraction of the aerial part (whole plant without root) of Saprosma fragrans. The isolated anthraquinones (1) and (2) were found to exhibit antifungal activity against Trichophyton mentagrophytes and Sporitrichum schenckii. Their structures were established by chemical and spectral analysis.