Manoj Kushwaha

Metal-Free Oxidative Amidation of 2-Oxoaldehydes: A Facile Access to α-Ketoamides

A novel and efficient method for the synthesis of α-ketoamides, employing a dimethyl sulfoxide (DMSO)-promoted oxidative amidation reaction between 2-oxoaldehydes and amines under metal-free conditions is presented. Furthermore, mechanistic studies supported an iminium ion-based intermediate as a central feature of reaction wherein C1-oxygen atom of α-ketoamides is finally derived from DMSO.

Capsaicin production by Alternaria alternata, an endophytic fungus from Capsicum annum; LC-ESI-MS/MS analysis

Alternaria alternata, an endophytic fungus capable of producing capsaicin (1) was isolated from Capsicum annum. The endophyte was found to produce capsaicin upto three generations. Upscaling of the fermentation broth led to the isolation of one known and one compound characterized as 2,4-di-tert-butyl phenol (2) and alternariol-10-methyl ether (3) respectively. Compound 1 and 3 were identified and quantified using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system through multiple reaction monitoring (MRM). Furthermore, compound 3 displayed a range of cytotoxicity against a panel of human cancer cell lines and was found to induce apoptosis evidenced by Hoechst staining and loss of mitochondrial-membrane potential in HL-60 cells.

Biodegradable polymeric system for cisplatin delivery: Development, in vitro characterization and investigation of toxicity profile ☆ ☆☆

Cisplatin is one of the most potent anticancer agent used in the treatment of various solid tumors, however, its clinical use is limited due to severe adverse effects including nephrotoxicity. In this investigation cisplatin loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles were developed and characterized for various in vitro characteristics including size distribution, zeta potential, drug loading and release profile. PLGA nanoparticles were successfully developed as investigated using scanning electron microscopy and exhibited average particles size and zeta potential as 284.8 nm and -15.8 mV, respectively. Fourier transform infrared spectroscopy and differential scanning calorimetry indicated an absence of any polymer-drug interactions. Cisplatin nanoparticles exhibited in vitro anticancer activity against A549 cells comparable to that of cisplatin solution. The biodistribution study in mice indicated that the kidney cisplatin level was significantly (p<0.01) lower with cisplatin nanoparticles than cisplatin solution. Following two cycles of cisplatin treatment, a week apart, blood urea nitrogen level was found to be higher in case of cisplatin solution as compared to cisplatin nanoparticles. Further, there was a significant (p<0.01) increase in plasma creatinine level in case of cisplatin solution as compared to cisplatin nanoparticles. Histopathological examination of kidney from cisplatin nanoparticles treated group revealed no kidney damage, however, a sign of nephrotoxicity was observed in the case of cisplatin solution. The results suggest that PLGA nanoparticle based formulation could be a potential option for cisplatin delivery.

Isolation and characterization of bioactive metabolites from Xylaria psidii, an endophytic fungus of the medicinal plant Aegle marmelos and their role in mitochondrial dependent apoptosis against pancreatic cancer cells

BACKGROUND The genus Xylaria has been reported as a rich source of biologically active secondary metabolites. In the present study, an endophytic fungus Xylaria psidii has been isolated from the leaf sample of Aegle marmelos (L.) Corr., characterized on the basis of its morphological features and sequence data for the ITS region (KU291350) of the nuclear ribosomal DNA. Biological screening of ethyl acetate extract of Xylaria psidii displayed a potential therapeutic effect on pancreatic cancer cells. HYPOTHESIS This study was designed systematically to explore Xylaria psidii, an endophytic fungus for the identification of biologically active secondary metabolites against pancreatic cancer cells. METHODS While exploring the bioactive secondary metabolites, a sensitive and reliable LC-MS based dereplication approach was applied to identify four compounds A-D from fungal extract. Further bioactivity guided isolation of fungal extract yielded two major metabolites 1 and 2. The structures of 1 and 2 have been determined by detailed spectroscopic analysis including MS, NMR, IR and UV data and similarity with published data. Xylarione A (1) is new whereas (-) 5-methylmellein (2) is reported for the first time from X. psidii. Both the isolated compounds were screened for their effect on the viability and proliferation against a panel of cancer cell lines (MCF-7, MIA-Pa-Ca-2, NCI-H226, HepG2 and DU145) of different tissue origin. RESULTS Compounds 1 and 2 exhibited cytotoxicity against pancreatic cancer (MIA-Pa-Ca-2) cells with IC50 values of 16.0 and 19.0 µm, respectively. The cell cycle distribution in MIA-Pa-Ca-2 cells, confirmed a cell cycle arrest at the sub-G1 phase. Cell death induced by 1 and 2 displayed features characteristic of apoptosis. Flow cytometry based analysis of 1 and 2 using Rhodamine-123 displayed substantial loss of mitochondrial membrane potential in a concentration dependent manner by both the compounds. CONCLUSION Results conclude that the isolated compounds 1 and 2 are responsible for the activity shown by crude ethyl acetate extract and may act as potential leads for medicinal chemists for designing more potent analogs.

I2/Aqueous TBHP-Catalyzed Coupling of Amides with Methylarenes/Aldehydes/Alcohols: Metal-Free Synthesis of Imides

We present a metal-free method for the synthesis of imides by the direct coupling of NH-amides with methylarenes under iodine/aqueous TBHP conditions. The optimized conditions worked very well with benzaldehydes and benzyl alcohol and furnished the corresponding imides in good to excellent yields. A series of control and radical scavenger experiments were also performed, which suggested the involvement of radical pathways. The labeling experiment in the presence of (18)O-labeled H2O suggested water as a source of oxygen in the imides.

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases

Evolutionary and functional divergence of gene paralogs expressing polyketide synthases from Rheum emodi play into secondary metabolism and substrate selectivities. Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.

Chrysomycins A–C, antileukemic naphthocoumarins from Streptomyces sporoverrucosus

Two known naphthocoumarins, chrysomycins A (1) and B (2), along with one new naphthocoumarin chrysomycin C (3) were isolated from the antimicrobial strain of Streptomyces sporoverrucosus (MTCC11715) (isolated from soil samples from the Jammu hills) and characterized. The structure of the new compound 3 was established from 2D-NMR data. Chrysomycins A (1) and B (2) were identified using a strategic HPLC–PDA/LCMS and Dictionary of Natural Products (DNP) based fast dereplication. Additionally, two new naphthocoumarins, chrysomycins D and E were identified using LCMS, UV and DNP information. Chrysomycins A–C (1–3) were isolated for the first time from Streptomyces sporoverrucosus and were screened for cytotoxicity against a panel of cancer cell lines (A549, Colo205, PC-3, MIAPaCa-2, and HL-60), amongst which the most potent activity was observed against human leukemia HL-60 cells with IC50 values of 0.9, 0.95 and 11 μM, respectively. The mechanistic studies indicated that chrysomycins A (1) and B (2), at 1 μM concentration, distorted the cellular and nuclear morphology with significant DNA damage and apoptosis in HL-60 cells.

Encapsulation: entrapping essential oil/flavors/aromas in food

Imparting flavors to foods can greatly enhance the finished product quality, cost, and consumer satisfaction. The food industry is continuously in search of unique flavors with high acceptability ingredients, processing methods, and packaging materials. The industry devotes lot of money to research and development for the enhancement and preservation of flavors, as which are delicate and volatile. Most available aromatic compounds are either extracted from natural sources or synthesized. Many factors linked to aroma affect the overall quality of the food. Since manufacturing, storage, packaging, and ingredients in food often cause modifications in overall flavor by reducing their intensity or producing off-flavor components, the stability of the matrices is an important criterion to preserve the properties of the flavored materials. To limit aroma degradation or loss during processing and storage, it is beneficial to encapsulate volatile ingredients prior to their use in foods or beverages. Encapsulation is the technique by which one material or a mixture of materials is coated with or entrapped within another material or system. This chapter presents an overview of the encapsulation of essential oil/flavor or aroma compounds in food and its importance in the food industry.

Trifluoroacetic acid catalyzed thiophenylmethylation and thioalkylmethylation of lactams and phenols via domino three-component reaction in water

An efficient one-pot trifluoroacetic acid catalyzed thiophenylmethylation and thioalkylmethylation of lactams, isatins and phenols via domino three-component coupling (3CC) with thiols and formaldehyde in water has been described. The developed protocol has wide substrate-scope for a variety of thiols, lactams and isatins. Utility of the protocol for ortho-/para-thiophenylmethylation of phenols indicated that reaction proceeds through in situ formation of a thiophenylmethylium cation intermediate. LC-ESIMS-based mechanistic investigation further confirmed formation of this intermediate. For isatins, the N- versus O-thiophenylmethylation was confirmed by recording the X-ray crystal structure of compound 4e. Thionaphthyl analog 3e exhibited significant antiproliferative activity in MCF-7 cells (IC50 8 μM) via apoptosis-induction.