Exploration of Benzo[b]carbazole-6,11-diones as anticancer agents: Synthesis and studies of hTopoIIα inhibition and apoptotic effects.

Two series of (hetero)arylamino-naphthoquinones and benzo-fused carbazolequinones were considered for study with the rationale that related structural motifs are present in numerous drugs, clinical trial agents, natural products and hTopoIIα inhibitors. Total 42 compounds were synthesized by reactions including dehydrogenative CN and Pd-catalyzed CC bond forming transformations. These compounds were screened against numerous cancer cells including highly metastatic one (MCF-7, MDA-MB-231, H-357 and HEK293T), and normal cells (MCF 10A). Some of the active compounds were evaluated for clonogenic cell survival and apoptotic effects in cancer cells (DAPI nuclear staining, Comet assay, Annexin-V-FITC/PI dual staining, flow cytometry, and western blot analysis with relevant proteins). All compounds were tested for hTopoIIα inhibitory activity. The investigated series compounds showed important properties like significant apoptotic antiproliferation in cancer cells with cell cycle arrest at S-phase and downregulation of NF- κß signaling cascade, relatively less cytotoxicity to normal cells, and hTopoIIα inhibition with more efficiency compared to an anticancer drug etoposide.

Co-administration of zinc phthalocyanine and quercetin via hybrid nanoparticles for augmented photodynamic therapy.

The photodynamic anticancer activity of a photosensitizer can be further increased by co-administration of a flavonoid. However, this requires that both molecules must be effectively accumulated at the tumor site. Hence, in order to enhance the activity of zinc phthalocyanine (ZnPc, photosensitizer), it was co-encapsulated with quercetin (QC, flavonoid) in lipid polymer hybrid nanoparticles (LPNs) developed using biodegradable & biocompatible materials and prepared using a single-step nanoprecipitation technique. High stability and cellular uptake, sustained release, inherent fluorescence, of ZnPC were observed after encapsulation in the LPNs, which also showed a higher cytotoxic effect in breast carcinoma cells (MCF-7) compared to photodynamic therapy (PDT) alone. In vivo studies in tumor-bearing Sprague Dawley rats demonstrated that the LPNs were able to deliver ZnPc and QC to the tumor site with minimal systemic toxicity and increased antitumor effect. Overall, the photodynamic effect of ZnPc was synergized by QC. This strategy could be highly beneficial for cancer management in the future while nullifying the side effects of chemotherapy.

E-pharmacophore guided discovery of pyrazolo[1,5-c]quinazolines as dual inhibitors of topoisomerase-I and histone deacetylase.

In the quest to ameliorate the camptothecin (CPT) downsides, we expedite to search for stable non-CPT analogues among 11 motifs of pyrazoloquinazolines reported. E-pharmacophore drug design approach helped filtering out pyrazolo[1,5-c]quinazolines as Topoisomerase I (TopoI) 'interfacial' inhibitors. Three compounds, 3c, 3e, and 3l were shown to be potent non-intercalating inhibitors of TopoI specifically and showed cancer cell-specific cytotoxicity in lung, breast and colon cancer cell lines. The compounds induced cell cycle arrest at S-phase, mitochondrial cell death pathway and modulated oxidative stress in cancer cells. Furthermore, a preliminary study was conducted to explore the feasibility of these compounds to be developed as dual TopoI-HDAC1 (histone deacetylase 1) inhibitors (4a) to combat resistance. Compound 4a was found to possess dual inhibitory capabilities in-vitro. Cytotoxic potential of 4a was found to be significantly higher than parent compound in 2D as well as 3D cancer cell models. Probable binding modes of 4a with TopoI and HDAC1 active sites were examined by molecular modelling.

Exploration of the expeditious potential of Pseudomonas fluorescens lipase in the kinetic resolution of racemic intermediates and its validation through molecular docking.

A profoundly time-efficient chemoenzymatic method for the synthesis of (S)-3-(4-chlorophenoxy)propan-1,2-diol and (S)-1-chloro-3-(2,5-dichlorophenoxy)propan-2-ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic alcohols, respectively. Docking of the R- and S-enantiomers of the intermediates demonstrated stronger H-bond interaction between the hydroxyl group of the R-enantiomer and the key binding residues of the catalytic site of the lipase, while the S-enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost-effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs.

Anticancer activity of dihydropyrazolo[1,5-c]quinazolines against rat C6 glioma cells via inhibition of topoisomerase II.

The design and synthesis of dihydropyrazolo[1,5-c]quinazolines (1a-h) as human topoisomerase II (TopoII) catalytic inhibitors are reported. The compounds were investigated for their antiproliferative activity against the C6 rat glial cell line. Two compounds, 1b and 1h, were found to be potent cytotoxic agents against glioma cells and exerted selective TopoII inhibitory activity. Furthermore, the compounds induced alterations in reactive oxygen species levels as measured by DCFDA assay and were found to induce cell cycle arrest at the G1 phase at lower concentrations and profound apoptosis at higher concentrations. The interaction of selected investigational molecules with TopoII was further corroborated by molecular modeling.

Design, Sustainable Synthesis, and Programmed Reactions of Templated N-Heteroaryl-Fused Vinyl Sultams.

A de novo design and synthesis of N-heteroaryl-fused vinyl sultams as templates for programming chemical reactions on vinyl sultam periphery or (hetero)aryl ring is described. The key features include rational designing and sustainable synthesis of the template, customized reactions of vinyl sultams at C═C bond or involving N-S bond cleavage, and reactions on the periphery of the heteroaryl ring for late-stage diversification. The simple, easy access to the template coupled with opportunities for the synthesis of diversely functionalized heterocyles from a single template constitutes a rare study in contemporary organic synthesis.

Imine/amide-imidazole conjugates derived from 5-amino-4-cyano-N1-substituted benzyl imidazole: Microwave-assisted synthesis and anticancer activity via selective topoisomerase-II-α inhibition.

Microwave-accelerated synthesis and anticancer activity of novel imine/amide-imidazole conjugates derived from 5-amino-4-cyano-N1-substituted benzyl imidazole against a panel of seven cancer cell lines are reported for the first time. Compounds ARK-4, 10 and 12 in the series show promising in vitro anti proliferative activity with low micromolar IC50 values against A-459 (lung), Hep-G2 (liver) and H-460 (liver) cancer cell lines. Compounds caused the increase in ROS levels as well as mitochondrial membrane depolarization, which might induce apoptosis. Further, mechanistic interventions on biological and molecular modeling data supported that compounds inhibited topoisomerase-II selectively.

Indenoindolone derivatives as topoisomerase II-inhibiting anticancer agents.

Based on known heterocyclic topoisomerase II inhibitors and anticancer agents, various indenoindolone derivatives were predicted as potential topoisomerase II-inhibiting anticancer agents. They are hydrazones, (thio)semicarbazones, and oximes of indenoindolones, and indenoindolols. These derivatives with suitable substitutions exhibited potent specific inhibition of human DNA TopoIIα while not showing inhibition of topoisomerase I and DNA intercalation, despite the fact that parent indenoindolones are known poor/moderate inhibitors of topoisomerase II. The potent topoisomerase II inhibitor indenoindolone derivatives exhibited good anticancer activities compared to etoposide and 5-fluorouracil, and relatively low toxicity to normal cells. These derivatizations of indenoindolones were found to result in enhancement of anticancer activities.

Synthesis of imine-pyrazolopyrimidinones and their mechanistic interventions on anticancer activity.

Design, synthesis and anticancer activity of a series of imine-pyrazolopyrimidinones is reported for the first time. Compounds 9d, 9n and 9o in the series show encouraging in vitro anticancer activity with low micromolar IC50 values against prostate (PC3) and breast (MCF7) cancer cell lines. Some notions about structure-activity relationships and plausible mechanism of biological activity are presented.

Antibacterial Potential of Gold Nanoparticles Synthesized From Leaf Extract of Syzygium cumini Against Multidrug-Resistant Urinary Tract Pathogens.

BACKGROUND: Urinary tract infection (UTI) is one of the most commonly encountered bacterial infections. Due to the misuse or excessive use of antibiotics, the upsurge of multidrug-resistance cases in UTIs has now become a global threat to public health. Exploring a newer or safer treatment using green synthesized nanoparticles (NPs) is another substitute for eliminating multidrug-resistant pathogens. METHODOLOGY: Leaf extract of Syzygium cumini was used for green synthesis of gold NPs. Synthesis of Syzygium cumini gold nanoparticles (ScAu-NPs) was achieved by optimizing various reaction parameters. These ScAu-NPs were characterized through UV-visible spectroscopy, transmission electron microscope, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction. ScAu-NPs were then processed for antibacterial activity against clinically isolated multidrug-resistant pathogens like Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus faecalis. RESULTS: Characterization of NPs revealed that biosynthesized NPs were spherical in shape. FTIR spectroscopy showed the presence of phenolics and aromatic compounds. Biosynthesized NPs exhibit good antibacterial activity with a significant bacterial reduction seen against all bacterial isolates compared to the controls. CONCLUSION: From the results of the present study, the formulation of biosynthesized ScAu-NPs can be utilized in drug development for eliminating infections caused by multidrug-resistant pathogens.

N-(1,3-Diaryl-3-oxopropyl)amides as a new template for xanthine oxidase inhibitors.

A series of forty two N-(1,3-diaryl-3-oxopropyl)amides were synthesized via an efficient, modified Dakin-West reaction and were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure-activity relationship analyses have been presented. Selected active xanthine oxidase inhibitors (3r, 3s, and 3zh) were assessed in vivo to study their anti-hyperuricemic effect in potassium oxonate induced hyperuricemic mice model. Compound 3s emerged as the most potent xanthine oxidase inhibitor (IC(50)=2.45 µM) as well as the most potent anti-hyperuricemic agent. The basis of significant inhibition of xanthine oxidase by 3s was rationalized by its molecular docking into catalytic site of xanthine oxidase.

A rational approach for the design and synthesis of 1-acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles as a new class of potential non-purine xanthine oxidase inhibitors.

Xanthine oxidase is a complex molybdoflavoprotein that catalyses the hydroxylation of xanthine to uric acid. Fifty three analogues of 1-acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles were rationally designed and synthesized and evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Some notions about structure activity relationships are presented. Six compounds 41, 42, 44, 46, 55 and 59 were found to be most active against XO with IC(50) ranging from 5.3 µM to 15.2 µM. The compound 59 emerged as the most potent XO inhibitor (IC(50)=5.3 µM). Some of the important interactions of 59 with the amino acid residues of active site of XO have been figured out by molecular modeling.

Response surface methodology of nitrilase production by recombinant Escherichia coli.

Growth and nitrilase production by recombinant Escherichia coli cells harbouring pET 21 (b) plasmid, for the expression of Pseudomonas putida nitrilase were improved using response surface methodology. Central composite design was used for obtaining ideal concentration of critical medium components which include fructose, tryptone, yeast extract and lactose. The optimal values for the concentration of fructose, tryptone, yeast extract and lactose were found to be 1.13, 2.26, 3.25 and 0.9 % (w/v), respectively. Here, fructose served as carbon source for the growth while lactose was preferably used as inducer for the expression of foreign protein. Yeast extract in the medium was used as a growth promoter while tryptone was added as a major nitrogen source. Using this optimized medium, an experimental growth of 6.67 (OD at 600 nm) and nitrilase activity of 27.13 U/ml was achieved. This approach for medium development led to an enhancement of the growth and enzyme activity by 1.4 and 2.2 times, respectively, as compared to the un-optimized medium.

Light-assisted anticancer photodynamic therapy using porphyrin-doped nanoencapsulates.

Light activatable porphyrinic photosensitizers (PSs) are essential components of anticancer and antimicrobial therapy and diagnostic imaging. However, their biological applications are quite challenging due to the lack of hydrophilicity and biocompatibility. To overcome such drawbacks, photosensitizers can be doped into a biocompatible polymer such as gelatin and further can be used for biomedical applications. Herein, first, a novel A4 type porphyrin PS [5,10,15,20-tetrakis(4-pyridylamidephenyl)porphyrin; TPyAPP] was synthesized via a rational route with good yield. Further, this porphyrin was encapsulated into the gelatin nanoparticles (GNPs) to develop hydrophilic phototherapeutic nanoagents (PTNAs, A4por-GNPs). Notably, the synthesis of such porphyrin-doped GNPs avoids the use of any toxic chemicals or solvents. The nanoprobes have also shown good fluorescence quantum yield demonstrating their applicability in bioimaging. Further, the mechanistic aspects of the anticancer and antimicrobial efficacy of the developed A4por-GNPs were evaluated via singlet oxygen generation studies. Overall, our results indicated porphyrin-doped biodegradable polymeric nanoparticles act as effective phototherapeutic agents against a broad range of cancer cell lines and microbes upon activation by the low-cost LED light.

Tailoring a stable and recyclable nanobiocatalyst by immobilization of surfactant treated Burkholderia cepacia lipase on polyaniline nanofibers for biocatalytic application.

Polyaniline nanofibers were synthesized by the oxidative polymerization of aniline. Surfactant treated lipase from Burkholdaria cepacia was immobilized on these polyaniline nanofibers by adsorption. The activity of immobilized preparation was six times higher than that of free lipase with an enhanced dispersion in organic solvents. Five-level-four-factor central composite design was applied for the optimization of immobilization parameters (viz. reaction time, pH, stirring rate and enzyme-support ratio) which were evaluated on the basis of lipase loading and activity. The thermal stability of the lipase in the nanobioconjugate, demonstrated in terms of the half-life at 80 °C was almost sixteen-fold higher than in the free form. The reusability data revealed the utility of the nanoconjugate for seven consecutive cycles with a slow and gradual decline in the activity. However, the nanoconjugate retained almost 30% of their initial activity after seven cycles of reuse revealing its utility of in industrial applications. The nanoconjugate was used in the kinetic resolution of (RS)-1-(7-(3-chloro-2-hydroxypropoxy)benzofuran-2-yl) ethanone, racemic intermediate of an important ß-blocker (Befunolol), with a high conversion rate of 48.2%, 98% ee-value and enantioselectivity (E) of 188, which signify its importance as a nanobiocatalyst.

Burkholderia cenocepacia: a new biocatalyst for efficient bioreduction of ezetimibe intermediate.

Ezetimibe is a selective acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor used in hypercholesterolemia. Synthesis of ezetimibe requires enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one (FOP alcohol) as a crucial intermediate which is produced by reduction of the corresponding prochiral ketone (FOP dione). A new biocatalyst from acclimatized soil was screened for bioreduction of the above prochiral ketone. The microorganism was identified by 16S mRNA sequencing, as Burkholderia cenocepacia. Various physicochemical conditions were optimized to increase cellmass and enzyme activity. The overall increase in cellmass concentration and enzyme activity was 2.06 and 1.85-fold, respectively. Various reaction conditions, for example pH, temperature, agitation, and cellmass concentration, were optimized for maximum product yield (chiral alcohol) with excellent enantioselectivity. Best reduction was achieved in phosphate buffer (50 mM, pH 8.0) at 40 degrees C (200 rpm) and the yield of enantiopure alcohol from the corresponding prochiral ketone was 54%. This biocatalyst was also used for the reduction of various other prochiral ketones.

Tailoring a robust and recyclable nanobiocatalyst by immobilization of Pseudomonas fluorescens lipase on carbon nanofiber and its application in synthesis of enantiopure carboetomidate analogue.

Pseudomonas fluorescens lipase (PFL) was covalently immobilized on carbon nanofiber (CNF) using 1­ethyl­3­[3­dimethylaminopropyl] carbodiimide (EDC)/N­hydroxysuccinimide (NHS). Surface functionalization of carbon nanofiber augments dispersibility as well as efficiency of covalent immobilization. Crucial parameters for immobilization such as pH, enzyme-support ratio, reaction time and mixing rate were optimized using one factor at a time (OFAT) approach. The nanobiocatalyst prepared under optimized conditions demonstrated a ten-fold increase in enzyme activity and the advantage of high thermal stability (up to 85 °C) along with 10 cycles of reusability. Subsequently practical application of the nanobiocatalyst was explored in the kinetic resolution of racemic 1­phenylethanol into (S)­1­phenylethanol [C = 49.1%, eep = 99.5%, ees = 98.5% and E value = 151.4] followed by Mitsunobu reaction with a substituted pyrrole, giving an enantiopure (R)-carboetomidate analogue (yield = 83%).

Facile development of biodegradable polymer-based nanotheranostics: Hydrophobic photosensitizers delivery, fluorescence imaging and photodynamic therapy.

Photodynamic therapy (PDT) is reported to be a promising technique to eradicate various cancers. As most of the photosensitizers (PSs) are hydrophobic in nature, thus, the effective delivery of PSs at the targeted site is the main hurdle associated with PDT. Zinc phthalocyanine and Zinc naphthalocyanine are reported as good PSs, however, highly hydrophobic characteristics restrict their use for clinical applications. To circumvent this limitation here we developed the advanced polymer-based nano-delivery system having polyethylene glycol (PEG) coated polymeric core with ~90% PS encapsulation. The PEG coating was responsible for the stabilization of probe in the physiological environment and storage conditions. The developed theranostic probes showed efficient in vitro fluorescence and singlet oxygen quantum yields upon irradiation with 620-750 nm (30 mW/cm2) light. The clathrin-mediated endocytosis (CME) based mechanism of cellular internalization was evaluated. The fluorescence of treated MCF-7 cells showed the ability of the probes as imaging agents. Moreover, up to 65% cell inhibition showed their cytotoxic efficiency. Further, comparatively higher tumor-accumulation of PSs without significant hepato/nephro-toxicity shown in vivo experimentation using breast tumor-bearing female Sprague Dawley (SD) rats suggested the featured passive targeting ability of preparations and clinically safe to be used. The study explored the exceptional delivery system for hydrophobic PSs with commendable theranostic applications.

Self-Assembled Gold Nanoparticle-Lipid Nanocomposites for On-Demand Delivery, Tumor Accumulation, and Combined Photothermal-Photodynamic Therapy.

In this study, a distinct photoamenable nanoparticle-based drug delivery system was developed for highly efficient targeted on-demand delivery, fluorescence imaging, and therapy by incorporating zinc phthalocyanine (ZnPc) and gold nanoparticles (AuNPs) into liposomes. The hyperthermia, produced by AuNPs under LED light irradiation, enhanced the liquidity of liposomal membrane and promoted the instantaneous release of ZnPc from the carriers realizing the concept of on-demand release. In addition, the local hyperthermia also resulted in thermal damage of cancer cells along with photodynamic effect and achieved a synergetic effect of photodynamic and photothermal therapy. The developed probes showed a high breast cancer carcinoma cells (MCF-7 cell line) inhibition up to 86.7% under red light irradiation. Further, in vivo experiments suggested the high tumor accumulation as well as the antitumor effect in breast tumor-bearing female Sprague-Dawley (SD) rats. The outcomes demonstrate the capability of these probes as a novel drug delivery system to codeliver therapeutic agents with photothermal agents and will have an enormous potential for future diagnosis and therapy.

Porphyrin Functionalized Gelatin Nanoparticle-Based Biodegradable Phototheranostics: Potential Tools for Antimicrobial Photodynamic Therapy.

Photomedicine-based antimicrobial therapy has emerged as an alternative treatment for antibiotic-resistant microbial infections. Although various photosensitizers (PSs) have been reported as efficient antimicrobial agents, their efficient delivery to the specific target area requires further investigation. In the current study, development of a biodegradable phototheranostic nanoagent (PTNA) by incorporation of a PS (trans-AB-porphyrin) and gelatin nanomatrix is described. The antimicrobial efficacy of the PTNA against Gram-positive bacteria, Gram-negative bacteria, and yeast strains, along with other properties including hydrophilicity, biocompatibility, and targeting ability, is evaluated. Unlike the commonly used membrane permeabilizing chemicals that are toxic, the delivery vehicle gelatin used in this study is biocompatible and biodegradable. Here, the method offers a sustainable synthesis of gelatin-based stable formulation of nanotheranostic agents with high loading (>85%). The study revealed that the reactive oxygen species (ROS), generated in situ by the PTNAs, are primarily responsible for microbial cell death. The developed PTNAs described herein featured "nano size (<200 nm), have high fluorescence and singlet oxygen quantum yields, retain photophysical properties of PS after incorporation into the gelatin matrix, could be activated by a cost-effective light irradiation, and have efficient antimicrobial photodynamic activity." This antimicrobial photodynamic therapy using the newly synthesized phototheranostic nanoagent has manifested its competence, therapeutic modality of general acceptance, and wide-spectrum antimicrobial action.