In silico assessment and sonochemical synthesis of 2-alkynyl 3-chloropyrazines as prospective ligands for SARS-CoV-2.

The recent global pandemic caused by COVID-19 has triggered an intense effort worldwide towards the development of an effective cure for this disease. In our effort we have explored the 2-alkynyl substituted 3-chloropyrazine framework as a potential template for the design of molecules for this purpose. Our strategy was supported by the in silico studies of representative compounds to assess their binding affinities via docking into the N-terminal RNA-binding domain (NTD) of N-protein of SARS-CoV-2. Thus we created a small library of molecules based on the aforementioned template via an environmentally safer method that involved the rapid synthesis of 2-alkynyl 3-chloropyrazine derivatives under Cu-catalysis assisted by ultrasound. The reactions proceeded via the coupling of 2,3-dichloropyrazine with commercially available terminal alkynes in the presence of CuI, PPh3 and K2CO3 in PEG-400. Further molecular modelling studies helped in establishing a virtual SAR (Structure Activity Relationship) within the series and identification of three potential hits. The desirable ADME was also predicted for these three molecules suggesting their prospective medicinal value.

Pyrrolo[2,3-b]quinoxalines in attenuating cytokine storm in COVID-19: their sonochemical synthesis and in silico / in vitro assessment.

In view of the recent global pandemic caused by COVID-19 intense efforts have been devoted worldwide towards the development of an effective treatment for this disease. Recently, PDE4 inhibitors have been suggested to attenuate the cytokine storm in COVID-19 especially tumour necrosis factor alpha (TNF-α). In our effort we have explored the 2-substituted pyrrolo[2,3-b]quinoxalines for this purpose because of their potential inhibitory properties of PDE-4 / TNF-α. Moreover, several of these compounds appeared to be promising in silico when assessed for their binding affinities via docking into the N-terminal RNA-binding domain (NTD) of N-protein of SARS-CoV-2. A rapid and one-pot synthesis of this class of molecules was achieved via the Cu-catalyzed coupling-cyclization-desulfinylation of 3-alkynyl-2-chloroquinoxalines with t-butyl sulfinamide as the ammonia surrogate under ultrasound irradiation. Most of these compounds showed good to significant inhibition of TNF-α in vitro establishing a SAR (Structure Activity Relationship) within the series. One compound e.g. 3i was identified as a promising hit for which the desirable ADME and acceptable toxicity profile was predicted in silico.

Ultrasound assisted synthesis of 3-alkynyl substituted 2-chloroquinoxaline derivatives: Their in silico assessment as potential ligands for N-protein of SARS-CoV-2.

In view of recent global pandemic the 3-alkynyl substituted 2-chloroquinoxaline framework has been explored as a potential template for the design of molecules targeting COVID-19. Initial in silico studies of representative compounds to assess their binding affinities via docking into the N-terminal RNA-binding domain (NTD) of N-protein of SARS-CoV-2 prompted further study of these molecules. Thus building of a small library of molecules based on the said template became essential for this purpose. Accordingly, a convenient and environmentally safer method has been developed for the rapid synthesis of 3-alkynyl substituted 2-chloroquinoxaline derivatives under Cu-catalysis assisted by ultrasound. This simple and straightforward method involved the coupling of 2,3-dichloroquinoxaline with commercially available terminal alkynes in the presence of CuI, PPh3 and K2CO3 in PEG-400. Further in silico studies revealed some remarkable observations and established a virtual SAR (Structure Activity Relationship) within the series. Three compounds appeared as potential agents for further studies.

Ameliorative effects of curcumin towards cyclosporine-induced genotoxic potential: an in vitro and in silico study.

Several studies documented the ameliorative effects of curcumin which plays a pivotal role in radical scavenging activities. It also participates in various cellular pathways and interacts with multiple targets. In the present study, we investigated the ameliorative effect of curcumin upon chromosomal genotoxicity induced by cyclosporine, an immunosuppressant, using in vitro approaches. A plausible mechanism of how curcumin mitigates the genotoxic implications of cyclosporine was ascertained using in silico tools. We observed that the curcumin reduces the genotoxic consequences made by cyclosporine upon cell cycle checkpoints and associated chromosomal/DNA manifestations. In addition, we presented the mechanistic details of curcumin interaction with various biomacromolecule types using docking experiments which showed that the possible radical scavenging activities can only be emerged by inducing the expression of antioxidant enzymes, supported by available experimental evidences. We anticipate that the induction of antioxidant enzymes by curcumin would activate Nrf2-Keap1 pathway as the plausible mechanism to exert anti-inflammatory response as demonstrated in renal epithelial cells.

Ameliorative effects of curcumin against lead induced toxicity in human peripheral blood lymphocytes culture.

Lead, a heavy metal and multifaceted toxicant, is well studied for its distribution and toxicity in ecosystem, yet there is no consensus on its amelioration by any synthetic or phytochemical compounds. Curcumin, a known antioxidant and dietary element, is a well-known herb, for its therapeutic uses and having a wide spectrum of its beneficial properties against several adverse effects. Hence, the current study was taken into consideration to evaluate the ameliorative effects of curcumin (3.87 µM, i.e. 1.43 µg/ml) against lead acetate (doses: 10-6 M, i.e. 0.379 µg/ml and 10-4 M, i.e. 37.9 µg/ml, durations: 24 h and 69 h) induced genotoxicity and oxidative stress in human peripheral blood lymphocyte cultures (PBLC). On one hand, antigenotoxic and antioxidative potentials of curcumin against lead were simultaneously evaluated by the array of genotoxicity and oxidative stress indices. The result postulated that lead acetate showed dose- and duration-dependent increase in both genotoxicity and oxidative stress whereas curcumin, when added along with lead acetate, showed the significant amelioration in all genotoxic and oxidative stress-related indices. The study indicated that, due to alteration in antioxidant defense system, there is an adverse genotoxic effect of lead. On the other hand, curcumin, a potent antidote, can protect chromatin material against lead -mediated genotoxicity by balancing the activity of antioxidant defense system.

Antigenotoxic effect of melatonin against mercuric chloride in human peripheral blood lymphocytes.

Melatonin (MLT) is an extraordinary antioxidant, which plays an important role in reducing reactive oxygen species (ROS) by scavenging them directly or indirectly. Mercury (Hg) is a heavy metal, which induces cytogenetic alterations via various mechanisms, leading to genotoxicity. It induces genotoxicity by enhancing the ROS chiefly. In the present study, the antigenotoxic effect of MLT was evaluated against mercuric chloride (HgCl2). All experiments were conducted in vitro in peripheral blood lymphocytes. Blood cultures were exposed to various concentrations of HgCl2 (2.63, 6.57, and 10.52 microM) for 24 h to study a range of genotoxic parameters. MLT (0.2 mM) supplementation, alone and in combination with the high concentration of Hg, was administered to blood cultures for 24 h. Genotoxic parameters, such as chromosomal aberrations (CAs; structural aberrations (chromatid gaps and breaks, chromosomal gaps and breaks) and numerical aberrations), micronuclei (MNs), and comet assay, were evaluated and analyzed using suitable statistical analysis. Hg treatment revealed a significant increase in CAs, MNs, and comet length. Co-supplementation of MLT along with Hg showed marked protection of these genotoxic end points in treated cultures. In conclusion, our findings suggest that MLT protects against Hg-induced augmentation in genotoxic indices because of its antioxidant property.

The genotoxic effects of anti-cancer drug gossypol on human lymphocytes and its mitigation by melatonin.

PURPOSE OF STUDY: To determine melatonin as a potential natural antioxidant to mitigate the genotoxic effects of promising anti-cancer drug gossypol in human lymphocytes. INTRODUCTION: Gossypol, is a polyphenolic compound naturally occurring in cotton seed, was originally identified as a male contraceptive but it has several proposed clinical applications. Gossypol has anti-proliferative effects on cancer cell lines. However, its genotoxic effects on normal cells are not much studied. Hence, there is a paucity of data available. Hence, the study was conducted to investigate gossypol-induced genotoxic effects on lymphocytes. METHODS: Peripheral blood lymphocyte cultures (PBLC) were done and exposed by two different doses of an anti-cancer drug, gossypol (0.274 mM, 1.645 mM) to check genotoxic effects. Melatonin (0.2 mM) is used as an antioxidant. Genotoxic indices such as sister chromatid exchanges (SCEs), cell cycle proliferative index (CCPI), average generation time (AGT), population doubling time (PDT) were assayed in the cultures. RESULT: Gossypol-treated groups indicated significant increases in frequency of SCEs calculated for SCE/plate and SCE/chromosome. Furthermore, CCPI showed a remarkable reduction and increased AGT and PDT levels were found in exposed cultures. When the higher dose of gossypol cultures was treated along with melatonin, these indices were found to be declined and comparable to control. CONCLUSION: Gossypol, an anti-cancer drug, induces genotoxicity on lymphocyte cells and co-supplementation of melatonin antioxidant ameliorates these toxic effects of gossypol.

Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.

Lead (Pb) which plays a significant role in modern industry is related to a broad range of physiological, biochemical, behavioural and genetical dysfunctions. Its exposure leads to an increased frequency of genetic aberrations in humans. Hence, this study was designed to assess the genotoxic effect of lead acetate at three dosage levels (10, 25 and 50 µg/mL) by employing: the Cytokinesis Block Micronucleus (CBMN) assay and the Comet assay in Peripheral Blood Lymphocyte Cultures. The results of this study revealed an increased level of DNA damage among treated groups. A significant increase in the tail length of comets and other indices was observed at 25 and 50 µg/mL concentrations comparatively. Thus, lead acetate induced single-strand breaks (SSB) and double strand breaks (DSB) in DNA, alkali-labile sites (ALS), oxidative DNA damage as well as DNA-DNA/DNA-protein/DNA-metal cross linking as evidenced by the Comet assay. The chromosome breakage, DNA misrepair, chromosome loss and telomere end fusion were determined by the Micronucleus assay. Micronucleus frequency in treated lymphocytes was significantly higher as compared to controls. Nucleoplasmic bridges increased significantly and Nuclear buds increased at higher two doses only in exposed cultures. Thus, these assays are better indices for lead induced genotoxicity and metal-nucleus interactions.

Identification of putative SNPs in progressive retinal atrophy affected Canis lupus familiaris using exome sequencing.

Progressive retinal atrophy (PRA) is one of the major causes of retinal photoreceptor cell degeneration in canines. The inheritance pattern of PRA is autosomal recessive and genetically heterogeneous. Here, using targeted sequencing technology, we have performed exome sequencing of 10 PRA-affected (Spitz=7, Cocker Spaniel=1, Lhasa Aphso=1 and Spitz-Labrador cross breed=1) and 6 normal (Spitz=5, Cocker Spaniel=1) dogs. The high-throughput sequencing using 454-Roche Titanium sequencer generated about 2.16 Giga bases of raw data. Initially, we have successfully identified 25,619 single nucleotide polymorphisms (SNPs) that passed the stringent SNP calling parameters. Further, we performed association study on the cohort, and the highly significant (0.001) associations were short-listed and investigated in-depth. Out of the 171 significant SNPs, 113 were previously unreported. Interestingly, six among them were non-synonymous coding (NSC) SNPs, which includes CPPED1 A>G (p.M307V), PITRM1 T>G (p.S715A), APP G>A (p.T266M), RNF213 A>G (p.V1482A), C>A (p.V1456L), and SLC46A3 G>A (p.R168Q). On the other hand, 35 out of 113 unreported SNPs were falling in regulatory regions such as 3'-UTR, 5'-UTR, etc. In-depth bioinformatics analysis revealed that majority of NSC SNPs have damaging effect and alter protein stability. This study highlighted the genetic markers associated with PRA, which will help to develop genetic assay-based screening in effective breeding.

Ameliorative effect of certain antioxidants against mercury induced genotoxicity in peripheral blood lymphocytes.

Various antioxidants play an important role in reducing the reactive oxygen species (ROS) by scavenging them directly or indirectly. Mercury (Hg) is one of the known hazardous genotoxicant, induces the genotoxicity by enhancing the ROS. In the present study, three structurally different bioactive compounds such as melatonin (0.2 mM), curcumin (3.87 µM) and andrographolide (0.4 µM) were evaluated against the genotoxic effect of mercury. All the experiments were conducted using the peripheral blood lymphocytes In Vitro. The cultures were exposed to different doses (2.63 µM; 6.57 µM; 10.52 µM) of mercury salt (HgCl2) for studying various genotoxic indices. All three antioxidant compounds, alone and in combination with high dose of mercury, were added to the cultures with controls. For ascertaining genotoxicity, sister chromatid exchanges (SCEs), cell cycle proliferative index/replicative index (CCPI/RI), average generation time (AGT), population doubling time (PDT), %M1, %M2 and %M3 were assessed and analyzed using suitable statistical analysis. The results revealed a dose dependent increase in SCEs, AGT and PDT, with a concomitant reduction in CCPI values after treatment of mercury. Supplementation of these three antioxidant compounds effectively negated these genotoxic endpoints in treated cultures with improvement in the cell cycle kinetics i.e. CCPI. The antimutagenic activity of these compounds on mercury induced genotoxicity was in the following order: melatonin > curcumin > andrographolide. In conclusion, these compounds have ameliorated mercury induced increase in genotoxic indices due to their excellent antioxidant properties and the combination seems to be effective.

Synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole moiety: their in vitro evaluation against PDE4B.

A number of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties were synthesized by using Pd/C-Cu mediated coupling-cyclization strategy as a key step. The o-iodoanilides or o-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one using 10%Pd/C-CuI-PPh3 as a catalyst system and Et3N as a base to give the target compounds. All the synthesized compounds were tested for their PDE4B inhibitory potential in vitro using a cell based cAMP reporter assay. Some of them showed fold increase of the cAMP level when tested at 30 µM. A representative compound showed encouraging PDE4B inhibitory properties that were supported by its docking results.

Mitigative role of melatonin and α-tocopherol against mercury-induced genotoxicity.

The present study was conducted to elucidate the protective effect of melatonin (MLT) and α-tocopherol on mercury-induced genotoxicity in human blood cultures. The in vitro effects of inorganic mercury added to human lymphocytes on the cell-cycle proliferative index (CCPI)/proliferation replicative index (PRI) and sister chromatid exchange (SCE) using fluorescence plus Giemsa staining were examined. A significant increase occurred in SCE per metaphase (SCE/chromosome and SCE/cell) and inhibition of proliferative kinetics, which resulted in a decline of the replicative index, in comparison to the controls. Treated lymphocyte cultures also exhibited a reduction in %M1 and %M2 metaphase plates, but an increase in %M3 metaphase plates was noticed. Adding α-tocopherol and MLT individually and in combination indicated a mitigative effect by reducing the genotoxic potential of treated cultures. The percent amelioration for all the three parameters, namely, frequency of SCE, SCE/plate and SCE/chromosome as well as CCPI, was comparatively high with MLT and α-tocopherol in combination than MLT followed by α-tocopherol. The percent mitigation was better in combined antioxidant additions to toxicant-exposed cultures, compared to MLT, whereas the percent mitigation by α-tocopherol alone was less for average generation time and population doubling time, respectively.

Evaluation of in vitro anti-genotoxic potential of melatonin against arsenic and fluoride in human blood cultures.

Melatonin has been known for its anti-oxidant potential but not much studied for the anti-genotoxic potential. The current study elucidate the role of melatonin against in vitro genotoxicity by arsenic (As) and fluoride (F). Human peripheral blood cultures were exposed to As (1.4 microM) and F (34 microM), alone and in combination, with and without melatonin (0.2 mM). Ethyl methane sulphonate (EMS; 1.9 mM) was selected as the positive control to analyze the genotoxic parameters like sister chromatid exchanges (SCEs), cell cycle proliferative index (CCPI) and primary DNA damage. The frequency of SCE/cell, SCE/chromosome and primary DNA damage reduced significantly (p<0.001) accompanied with a marked increase in CCPI upon addition of melatonin. Similarly marked recovery was attained by melatonin from As and F induced primary DNA damage as studied using comet assay. The results clearly indicate towards the protection of lymphocytes from toxic effects af As and F by melatonin in vitro.

Melatonin protection on mercury-exerted brain toxicity in the rat.

The effect of melatonin on the neurotoxicity induced by mercuric chloride was studied. Adult rats were fed orally with two different doses of mercuric chloride (2 mg; 4 mg/kg body weight) to evaluate brain toxicity with respect to cerebral hemisphere, cerebellum, and medulla oblongata regions for 60 days with or without supplementation with melatonin (5 mg/kg body weight) intraperitoneally. The results suggest that the graded doses of mercury elicit the depletion of enzymatic activities, such as adenosine triphosphatase, succinate dehydrogenase, phosphorylase, alkaline phosphatase, acid phosphatase, altered glycogen, total protein, and lipid peroxidation levels in the cerebral hemisphere, cerebellum, and medulla oblongata of the brain, thereby affecting their respective functions. Blood glucose and mercury levels increased, followed by a reduction in body and organ weights. All these effects seemed to be severe in the cerebral hemisphere of the brain. Further affected indices were, to some extent, maintained in the brain of animals cotreated with melatonin, showing its protective role against mercury-exerted neurotoxicity.

Stereoselective total synthesis of Patulolide C.

Stereoselective total synthesis of Patulolide C has been accomplished from easily available and inexpensive (S)-chiral epoxide. The key steps involved in the concise synthesis of Patulolide C utilizes ring opening of chiral epoxide, cleavage of 1,2-diol, deprotection of benzyl ether of hydroxyl acid and Yamaguchi macrolactonisation dilution conditions as key steps. The advantage of this method include inexpensive starting material, mild reaction conditions and high purity of products.

Synthesis of 2-substituted Furo[3,2-b]pyridines Under Pd/C-Cu Catalysis Assisted by Ultrasound: Their Evaluation as Potential Cytotoxic Agents.

BACKGROUND: Compounds containing furo[3,2-b]pyridine framework have shown interesting pharmacological properties, including anticancer activities. Though these compounds are generally synthesized via the heteroannulation processes involving acetylenic derivatives, some of them are complex. OBJECTIVE: The study aimed to explore a series of 2-substituted furo[3,2-b]pyridines for their cytotoxic properties against cancer cell lines in vitro. METHODS: We developed a convenient synthesis of 2-substituted furo[3,2-b]pyridines via sequential (i) C-C coupling followed by (ii) C-O bond-forming reactions in a single pot. The reactions were performed under ultrasound irradiation in the presence of Pd/C as an inexpensive, stable and widely used catalyst. A range of 2- substituted furo[3,2-b]pyridines were synthesized via coupling of 3-chloro-2-hydroxy pyridine with terminal alkynes in the presence of 10% Pd/C-CuI-PPh3-Et3N in EtOH. The in vitro evaluation of all these compounds was carried out against MDA-MB-231 and MCF-7 cell lines and subsequently against SIRT1. RESULTS: The furo[3,2-b]pyridine derivative 3b showed encouraging growth inhibition of both MDAMB-231 and MCF-7 cell lines and inhibition of SIRT1. The compound 3b also showed apoptosis-inducing potential when tested against MCF-7 cells. CONCLUSION: The Pd/C-Cu catalysis under ultrasound accomplished a one-pot and direct access to 2-substituted furo[3,2-b]pyridine derivatives, some of which showed anticancer properties.

Ultrasound Assisted Synthesis of 2-Substituted Benzofurans via One-Pot and Sequential Method: Their In Vitro Evaluation.

BACKGROUND: The 2-substituted benzofuran framework has attracted enormous attention due to its presence in a range of bioactive compounds and natural products. While various methods for the synthesis of 2- substituted benzofuran derivatives are known, several of them suffer from certain drawbacks. OBJECTIVE: The main objective of this work was to explore a series of 2-(het)aryl substituted benzofurans derivatives for their cytotoxic properties against cancer cell lines in vitro. METHODS: In our efforts, we have developed a one-pot synthesis of this class of compounds via sequential C-C coupling followed by C-Si bond cleavage and subsequent tandem C-C/C-O bond-forming reaction under ultrasound irradiation. The methodology involved coupling of (trimethylsilyl)acetylene with iodoarenes in the presence of 10% Pd/C-CuI-PPh3-Et3N in MeOH followed by treating the reaction mixture with K2CO3 in aqueous MeOH and finally coupling with 2-iodophenol. A variety of 2-substituted benzofurans were synthesized using this methodology in good yield. All the synthesized compounds were tested in vitro against two cancer cell lines, e.g. MDAMB-231 and MCF-7 cell lines subsequently against SIRT1. RESULTS: The benzofuran derivative 3m showed encouraging growth inhibition of both MDAMB-231 and MCF- 7 cell lines and significant inhibition of SIRT1. The compound 3m also showed a concentration-dependent increase in the acetylation of p53. CONCLUSION: Our efforts not only accomplished a one-pot and direct access to 2-(het)aryl substituted benzofurans but also revealed that the benzofuran framework presented here could be a potential template for the identification of potent inhibitors of SIRT1.

Ultrasound Assisted Synthesis of 3-(het)aryl Isocoumarin Derivatives and their in vitro Pharmacological Evaluation.

BACKGROUND: In view of numerous biological activities of 3-substituted isocoumarins a number of analogues based on this scaffold were synthesized for their in vitro pharmacological evaluation. METHODS: The syntheses of 3-substituted isocoumarins were carried out via a Pd/C-catalyzed Suzuki- Miyaura coupling of 3-chloroisochromen-1-one with a range of boronic acid derivatives. This C-C bond forming reaction was facilitated by ultrasound irradiation to afford the desired products in good yields. A number of 3-(het)aryl isocoumarin derivatives were prepared by using this methodology and subsequently tested for their TNF-α inhibitory properties in vitro followed by cytotoxicities via the MTT assay. RESULTS: Several compounds showed inhibition of TNF-α with one compound showing an IC50 value of 9.01±1.25 µM. Three compounds also showed promising cytotoxic properties against two cancer cell lines with IC50 ~ 0.9-2.7 µM. CONCLUSION: The isocoumarin framework could be an effective template for the design and discovery of new inhibitor of TNF-α for the potential treatment of related diseases.

Lemon Juice Mediated Reaction under Ultrasound Irradiation: Synthesis of Indolofuroquinoxalines as Potential Anticancer Agents.

BACKGROUND: A non-hazardous synthetic methodology has been developed for the preparation of compounds based on indolofuroquinoxaline framework. Lemon juice that is known to play the role of a biocatalyst in various organic reactions was used for this purpose. METHOD: A number of indolofuroquinoxaline derivatives were prepared via the lemon juice mediated condensation of methyl 2-(2-chloro-1H-indol-3-yl)-2-oxoacetate or its N-alkyl derivatives with 1,2- diamines under ultrasound irradiation. All the synthesized compounds were screened via an MTT assay for their potential anticancer properties in vitro using a number of cancer cell lines including MDA-MB 231, and MCF7, K562, Colo-205 and IMR-32 and the non-cancerous HEK293 cell line. Compounds 3a, 3b and 3c showed promising growth inhibition against K562, MDA-MB 231 and MCF7 cell lines but no significant effects on HEK293 cell line suggesting their selectivity towards cancer cells. RESULTS AND CONCLUSION: Moreover, according to their IC50 values, all these compounds appeared to be relatively more potent towards K562 cell line over MDA-MB 231 and MCF7 cell lines indicating their potential against leukemia.