Vibha Tandon

A Copper-Catalyzed Tandem Synthesis of Indolo- and Pyrrolo[2,1-a]isoquinolines†

Transition-metal-catalyzed tandem reactions have emerged as a useful tool for the synthesis of multiring heterocyclic compounds because of the intriguing selectivity, atom economy, and exceptional ability to activate p systems, especially alkynes, towards intermolecular and intramolecular nucleophilic attack. Among the transition-metal-catalyzed reactions, palladium is extensively used because of its tolerance of many functional groups and its low toxicity. However, in recent years copper-catalyzed reactions have received considerable attention because of their efficiency and low costs. 5] The reported annulation chemistry for the synthesis of heterocycles from alkynes proceeds through p complexation of the alkyne and subsequent attack of the resulting h-metal complex onto the appropriate adjacent functionalized arene. 6] However, the synthesis of polyheterocycles by the nucleophilic addition of N heterocycles onto alkynes and subsequent in situ ring closure by C C bond formation is still unknown. Indolo[2,1-a]isoquinolines and pyrrolo[2,1-a]isoquinolines have unique nitrogen-containing tetracyclic and tricyclic structures, and their reduced and oxidized forms occur widely among natural products, biologically active pharmaceuticals, and p-conjugated functional materials, such as organic semiconductors and luminescent materials. The reported methods for the syntheses of indoloand pyrrolo[2,1-a]isoquinolines, typically require multistep syntheses and expensive reagents. Methods for the construction of these structures include well known benzyne reactions or the oxidative couplings of 1-benzylisoquinoline. F rstner and co-workers reported the synthesis of analogous isoquinolines by the cycloisomerization of biaryl alkynes using PtCl2, AuCl, AuCl3, GaCl3, or InCl3. [2a] Herein, we report the first coppercatalyzed synthesis of this class of heterocycles by the tandem addition of N heterocycles onto alkynes and subsequent intramolecular cyclization of the in situ generated enamine by C2 arylation. In continuation of recently developed methods for the copper-catalyzed N-arylation using benzotriazole as a ligand and the electrophilic cyclization of alkynes, 15] we hypothesized that the direct synthesis of polycyclic heteroaromatic compound 6 could occur in a one-pot reaction of N heterocycle 1 with ortho-haloarylalkyne 2 by sequential N C and C C bond formation under the proper conditions such that intermediate 5 would not have to be isolated (Scheme 1, route A). We also anticipated the possible formation of regioisomer 3 by initial arylation of N heterocycle 1 at the C2position by the ortho-haloarylalkyne 2 and subsequent intramolecular attack of the N heterocycle onto the carbon– carbon triple bond of the in situ generated intermediate 4 (Scheme 1, route B). This designed tandem reaction features the use of benzotriazole (L1) and benzotriazol-1-ylmethanol (L2) as novel and inexpensive ligands in copper-catalyzed reactions. To identify the optimal reaction conditions for the reaction, a number of copper catalysts, including CuI, CuCl, CuBr, Cu2O, and Cu(OAc)2, and several different organic solvents and ligands were examined in the reaction of 3methylindole (1a) with 2-bromophenyl-4-methoxyphenylethyne (2a ; Table 1). Interesting observations emerge from the data in Table 1. We first reacted 1 a (0.5 mmol) with 1.1 equivalents of 2a, 10 mol% of CuI, and 1.4 equivalents of KOtBu in 1.0 mL of DMF at 110 8C for 24 hours—the desired coupling product 3a was not observed (Table 1, entry 1). However, the addition of 20 mol% of ligand L1 to the reaction afforded the desired product 3a in a 65 % yield (Table 1, entry 2). The designed ligand L2, was subsequently found to be more effective than ligand L1 (Table 1, entry 3), and from entries 4 and 5 in Table 1 it is apparent that the solvent has a significant influence on the reaction. DMSO was found to be quite successful for the transformation as compound 3a was obtained in an 82 % yield when DMSO was used as the solvent instead of DMF (Table 1, entry 4). When we used toluene as the solvent, the desired product 3a was obtained in only a 38% yield (Table 1, entry 5). Different bases were tested in this reaction system, but KOtBu proved to be most effective (Table 1, entries 4, 6, and 7). The yield of [*] Dr. A. K. Verma, J. Singh, Dr. V. Tandon Dr. B. R. Ambedkar Center for Biomedical Research University of Delhi, Delhi 110007 (India) E-mail: averma@acbr.du.ac.in

Antidiabetic activity of 50% ethanolic extract of Ricinus communis and its purified fractions.

We investigated the antidiabetic activity of 50% ethanolic extract of roots of Ricinus communis (RCRE) along with its bioassay-guided purification. Five-hundred milligram per kilogram body weight appeared to be the effective dose as it caused the maximum lowering of the fasting blood glucose, both in normal as well as type 1 diabetic animals. The maximum hypoglycemic effect was always observed at the 8th h up to which the study has been conducted. Administration of the effective dose of RCRE to the diabetic rats for 20 days showed favorable effects not only on fasting blood glucose, but also on total lipid profile and liver and kidney functions on 10th and 20th day. RCRE was purified using silica gel column chromatography. Out of several different fractions tested, only one fraction (R-18) showed significant antihyperglycemic activity. RCRE seemed to have a high margin of safety as no mortality and no statistically significant difference in alkaline phosphatase, serum bilirubin, creatinine, serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase and total protein was observed even after the administration of the extract at a dose of 10 g/kg b.wt. Thus R. communis seems to have a promising value for the development of a potent phytomedicine for diabetes.

Insulinotropic effect of cinnamaldehyde on transcriptional regulation of pyruvate kinase, phosphoenolpyruvate carboxykinase, and GLUT4 translocation in experimental diabetic rats.

Diabetes mellitus is a chronic metabolic disorder affecting about 6% of population worldwide with its complications and is rapidly reaching epidemic scale. Cinnamomum zeylanicum is widely used in alternative system of medicine for treatment of diabetes. In the present study, we have performed bioassay guided fractionation of chloroform extract of C. zeylaniucm and identified cinnamaldehyde (CND) as an active principle against diabetes. In continuation to it, a detailed study was undertaken to elucidate its mode of antidiabetic action in STZ induced diabetic rats. Oral administration of CND (20 mg/kg bw) to diabetic rats for 2 months showed significant improvement (p<0.001) in muscle and hepatic glycogen content. In vitro incubation of pancreatic islets with CND enhanced the insulin release compared to glibenclamide. The insulinotropic effect of CND was found to increase the glucose uptake through glucose transporter (GLUT4) translocation in peripheral tissues. The treatment also showed a significant improvement in altered enzyme activities of pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and their mRNA expression levels. Furthermore, the median lethal dose (LD(50)) of CND could not be obtained even at 20 times (0.4 g/kg bw) of its effective dose. With the high margin of safety of CND, it can be developed as a potential therapeutic candidate for the treatment of diabetes.

Nutritional and Hypoglycemic Effect of Fruit Pulp of Annona squamosa in Normal Healthy and Alloxan-Induced Diabetic Rabbits

Thenutritive value of the pulp of the edible fruit of Annona squamosa and its effect on various biochemical parameters has been assessed in normal and alloxan-induced diabetic rats. Different doses (2.5, 5.0, 10.0 g/kg b.w.) of fresh fruit pulp of A. squamosa were given to the three groups each of normal healthy and diabetic rabbits orally daily for 1 month. There was a control group of normal as well as diabetic animals which did not receive fruit pulp. Protein efficiency ratio (PER), digestibility coefficient (DC), biological value (BV) and net protein utilization (NPU) were calculated and improvement in the nutritional level was studied by comparing with the control group. Effect of the fruit pulp was also studied on various biochemical parameters, namely fasting blood glucose (FBG), total cholesterol (TCH), HDL-cholesterol, triglyceride (TG), total protein (TPR), alkaline phosphatase (ALKP), serum glutamate oxaloacetate and pyruvate transaminases (SGOT and SGPT), serumcreatinine (CRTN) and serum bilirubin (BIL). Protein andglucose in urine were also estimated. Total hemoglobin and glycohemoglobin (HbAc) were estimated in blood before and after 1 month of feeding fruit pulp. Fruit pulp increased the net protein utilization by 29.3 in normal healthy rabbits with 10 g/kg b.w. and 34.1 in induced diabetic (induced by alloxan) animals with 5 g/kg b.w. of the fruit pulp feeding when compared with the control group of rabbits (p < 0.001). Feeding fruit pulp with the same amount increased the total hemoglobin content by 21.0% in normal rabbits and 10.8% in diabetic rabbits. Fruit pulp also reduced the total cholesterol level by 45–46% in normal and 32.4% in diabetic animals with increased HDL-cholesterol. Feeding pulp improved the liver function in normal as well as diabetic rabbit as shown by reduction in the serum SGOT, SGPT, ALKP and bilirubin levels. The optimal improvement in nutritive value of normal animals was found with 5.0–10.0 g/kg b.w. of the fruit pulp feeding, while in diabetic animals it was 2.5–5.0 g/kg b.w. In the diabetic animals pulp feeding between 2.5 and 5.0 g/kg b.w. showed improvement in the glucose tolerance. Further, 5 g/kg b.w. of fruit pulp brought down urine sugar, urine protein and glycohemoglobin in diabetic rabbits. Feeding pulp had increased utilization of dietary protein, body weight as well as the ratio of gain in body weight per gram of protein consumed. It had a protective effect on liver and heart as indicated by reduction in the SGOT, SGPT, ALKP and serum bilirubin levels.

Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkhead-associated domains (FHA), the phosphorylation-responsive modules, have emerged as prominent players in STPK mediated signaling. In this study, we demonstrate the association of the previously uncharacterized FHA domain-containing protein Rv0019c with cognate STPK PknB. The consequent phosphorylation of Rv0019c is shown to be dependent on the conserved residues in the Rv0019c FHA domain and activation loop of PknB. Furthermore, by creating deletion mutants we identify Thr36 as the primary phosphorylation site in Rv0019c. During purification of Rv0019c from Escherichia coli, the E. coli protein chloramphenicol acetyltransferase (CAT) specifically and reproducibly copurifies with Rv0019c in a FHA domain-dependent manner. On the basis of structural similarity of E. coli CAT with M. tuberculosis PapA5, a protein involved in phthiocerol dimycocerosate biosynthesis, PapA5 is identified as an interaction partner of Rv0019c. The interaction studies on PapA5, purified as an unphosphorylated protein from E. coli, with Rv0019c deletion mutants reveal that the residues N-terminal to the functional FHA domain of Rv0019c are critical for formation of the Rv0019c-PapA5 complex and thus constitute a previously unidentified phosphoindependent binding motif. Finally, PapA5 is shown to be phosphorylated on threonine residue(s) by PknB, whereas serine/threonine phosphatase Mstp completely reverses the phosphorylation. Thus, our data provides initial clues for a possible regulation of PapA5 and hence the phthiocerol dimycocerosate biosynthesis by PknB, either by direct phosphorylation of PapA5 or indirectly through Rv0019c.

In vivo evaluation of anti-oxidant and anti-lipidimic potential of Annona squamosa aqueous extract in Type 2 diabetic models

AIM OF THE STUDY Diabetes is known to involve oxidative stress and changes in lipid metabolism. Many secondary plant metabolites have been shown to possess antioxidant activities, improving the effects of oxidative stress due to diabetes. The present study was aimed to evaluate the effect of water extract of Annona squamosa leaves on antioxidant enzymes and lipid profile of animal models of type 2, non-insulin dependent diabetes mellitus (NIDDM). MATERIAL AND METHODS The plant material was extracted with boiling water for 2 h. Albino Wistar rats (n=24) were divided into four groups. Diabetes was induced by streptozotocin injection (ip) at a dose of 50 mg/kg. Animals of treated groups were given the dose of 350 mg/kg of the extract. The excised rat tissues were rinsed in ice-cold saline, blotted dry and weighed. RESULTS AND CONCLUSIONS The results clearly suggest that the water extract of Annona squamosa leaves possessed antioxidant activity as shown by increased activities of scavenging enzymes, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione reductase (GR) and glutathione-s-transferase (GST) and decrease in malondialdehyde levels present in various tissues. Administration of the extract also improved the lipid profile of the treated groups indicating thereby that the high levels of triglyceride and total cholesterol associated with diabetes can also be significantly managed with the extract.

Preliminary studies on activity of Ocimum sanctum, Drynaria quercifolia, and Annona squamosa against Neisseria gonorrhoeae.

Background: Despite the progressive increase of antimicrobial resistance of Neisseria gonorrhoeae worldwide, there are limited reports of alternative remedies from plants. Goal: The aim of the current study was to screen 3 plants, Ocimum sanctum, Drynaria quercifolia, and Annona squamosa, for activity against Neisseria gonorrhoeae. Study: By disc diffusion method, extracts of these 3 plants were screened for activity against Neisseria gonorrhoeae; their antimicrobial activity was calculated as percentage inhibition and compared with penicillin and ciprofloxacin. Results: The extracts of all 3 plants caused inhibition of Neisseria gonorrhoeae clinical isolates and World Health Organization (WHO) strains, more so than the multidrug resistant Neisseria gonorrhoeae. Conclusion: Neisseria gonorrhoeae clinical isolates and WHO strains were sensitive to extracts of Ocimum sanctum, Drynaria quercifolia, and Annona squamosa. This motivates us to isolate the active component/second from the 3 plants.

Synthesis and biological activity of novel inhibitors of topoisomerase I: 2-Aryl-substituted 2-bis-1H-benzimidazoles

Inhibitors of topoisomerase I constitute a novel family of antitumor agents. The class of benzimidazole derivatives contains compounds possessing affinity to DNA. For example, fluorescent stains Hoechst 33342 and Hoechst 33258 interact with DNA as ligand and produce nonspecific inhibition of the catalytic activity of many enzymes involved in DNA synthesis, including DNA topoisomerase and DNA helicase. Several 2-aryl-5-substituted-2,5-bisbenzimidazole derivatives were synthesized and ability of these derivatives to induce DNA cleavage in the presence of topoisomerase I was evaluated in vitro. These analogs were also assayed for their cytotoxicity against U87, MCF7 and HeLa human tumor cells. All the four compounds showed a potent growth inhibitory effect on all the cell lines, with IC50 in the μM range.

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.

In vitro activity of eugenol, an active component from Ocimum sanctum, against multiresistant and susceptible strains of Neisseria gonorrhoeae

In view of the widespread emergence of resistant isolates, an attempt was made to isolate and characterise the component(s) of Ocimum sanctum with activity against Neisseria gonorrhoeae. Bioassay-guided purification of the hexane extract of leaves of O. sanctum was carried out, which yielded H12c as the active compound. H12c was characterised and was determined to be eugenol, with a minimum inhibitory concentration of 85-256 mg/L. The antigonorrhoeal efficacy of H12c was better against multiresistant strains. The 50% lethal dose (LD50) of H12c was found to be 2g/kg body weight in rats. In view of its efficacy and lower toxicity, eugenol may be a potentially suitable molecule to be developed clinically in response to emerging resistant isolates of N. gonorrhoeae.