Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers,as potent JAK3 kinase inhibitors

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.     

Lead identification and characterization of hTrkA type 2 inhibitors

Virtual in silico structure-guided modeling, followed by in vitro biochemical screening of a subset of commercially purchasable compound collection resulted in the identification of several human tropomyosin receptor kinase A (hTrkA) inhibitors that bind the orthosteric ATP site and exhibit binding preference for the inactive kinase conformation. The type 2 binding mode with the DFG-out and αC-helix out hTrkA kinase domain conformation was confirmed from X-ray crystallographic solution of a representative inhibitor analog, 1b. Additional hTrkA and hTrkB (selectivity) assays in recombinant cells, neurite outgrowth inhibition using rat PC12 cells, early ADME profiling, and preliminary pharmacokinetic evaluation in rodents guided the lead inhibitor progression in the discovery screening funnel.     

An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.     

Discovery of noscapine derivatives as potential β-tubulin inhibitors

Twenty novel 1,2,3-triazole noscapine derivatives were synthesized starting from noscapine by consecutive N-demethylation, reduction of lactone ring, N-propargylation and Huisgen 1,3-dipolar cycloaddition reaction. In order to select the most promising molecules to subject to further biophysical and biological evaluation, a molecular docking analysis round was performed using noscapine as reference compound. The molecules featuring docking predicted binding affinity better than that of noscapine were then subjected to MTT assay against MCF7 cell line. The obtained results disclosed that all the selected triazole derivatives exhibited a remarkably lower cell viability compared to noscapine in the range of 20 μM in 48 h. In an attempt to correlate the biological activity with the ability to bind tubulin, the surface plasmon resonance (SPR) assay was employed. Compounds 8a, 8h, 9c, 9f and 9j were able to bind tubulin with affinity constant values in the nanomolar range and higher if compared to noscapine. Integrating computational predictions and experimental evaluation, two promising compounds (8h and 9c) were identified, whose relevant cytotoxicity was supposed to be correlated with tubulin binding affinity. These findings shed lights onto structural modifications of noscapine toward the identification of more potent cytotoxic agents targeting tubulin.     

Synthesis of β-ionone derived chalcones as potent antimicrobial agents

A series of chalcones (3a–v) have been synthesized by condensation of β-ionone (1) with a variety of aldehydes (2a–v). The synthesized compounds have been screened for their in vitro antimicrobial activity against five bacterial and five fungal strains, using disc diffusion assay. The evaluated compounds display a wide range of activities, from completely inactive to the highly active compounds. Some of the compounds are also active against methicillin resistant staphylococcus aureus (MRSA).     

Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir1^27-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1^27-149 in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.     

Synthesis of novel hetero ring fused pyridine derivatives; Their anticancer activity,CoMFA and CoMSIA studies

A series of novel furo[2,3-b]pyridine-2-carboxamide 4a–h/pyrido[3′,2′:4,5]furo[3,2-d] pyrimidin-4(3H)-one derivatives 5a–p were prepared from pyridin 2(1H) one 1 via selective O-alkylation with α-bromoethylester followed by cyclization, then reaction with different aliphatic primary amines to obtain 4 and further reaction with triethyl orthoacetate/triethyl orthoformate. Also prepared novel furo[2,3-b]pyridine-2-carbohydrazide Schiff’s bases 7a–h and pyrido [3′,2′:4,5]furo[3,2-d]pyrimidin-4(3H)-one derivatives 8a–h starting from furo[2,3-b]pyridine carboxylate derivatives 3 by reaction with hydrazine hydrate to form 6 and reaction with diverse substituted aldehydes and cyclization. Products 4a–h, 5a–p, 7a–h and 8a–h were screened against four human cancer cell lines (HeLa, COLO205, Hep G2 and MCF 7) and one normal cell line (HEK 293). Compounds 4e, 4f, 4g, 5h, 7c, 7d, 7e and 7f showed significant anticancer activity against all the cell lines at micro molar concentration and found to be non-toxic to normal cell line. Studies for HeLa, COLO205 and MCF-7 using CoMFA and CoMSIA. Models from 3D-QSAR provided a strong basis for future rational design of more active and selective HeLa, COLO205 and MCF-7 cell line inhibitors.     

Quinolidene based monocarbonyl curcumin analogues as promising antimycobacterial agents: Synthesis and molecular docking study

A series of quinoline incorporated monocarbonyl curcumin analogues was efficiently synthesized using [HDBU][HSO₄] as catalyst via Knoevenagel type condensation and evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Ra (MTB) and Mycobacterium bovis BCG in dormant state. The analogues 3e, 3h, 4a and 4e exhibited very good antitubercular activity. The antiproliferative activity of the analogues against MCF-7, A549 and HCT-116 cell lines was evaluated using modified MTT assay and these compounds were found to be non-cytotoxic. Molecular docking study has been carried out against M. tuberculosis pantothenate synthetase (MTB PS) enzyme in an effort to enhance the understanding of their action as antitubercular agents. The potency, low cytotoxicity and selectivity of these analogues support them as valid leads for further optimization.     

Synthesis,antimicrobial, and mosquito larvicidal activity of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones

A series of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones (6a–h) have been synthesized by cyclization of ethyl-3-aryl-4-(2-chlorophenyl)-6-methyl-1-(5-methylisoxazol-3-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 4a–h with 3-amino-5-methylisoxazole 5. Compounds 4a–h were obtained by Biginelli reaction, by condensation of aromatic aldehyde 1, ethyl acetoacetate 2, and isoxazolyl thioureas 3 in a one-pot reaction catalyzed by ceric ammonium nitrite (CAN). Compounds 6a–h were tested for their antibacterial and antifungal activities against various bacterial and fungal strains. The results showed that these compounds exhibited good antibacterial and antifungal activity compared with that of standard antibiotics. Mosquito larvicidal activity of the newly synthesized compounds 6a–h is also studied against fourth instar larvae Culex quinquefasciatus. Some of the compounds are proved to be lethal for mosquito larvae.     

An assay specific for the active form of liver phosphorylase kinase

An assay specific for the active form of liver phosphorylase kinase (EC 2.7.1.38) has been developed utilizing inhibition of the inactive form of phosphorylase kinase by β-glycerophos, phate and ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid. Following in vitro activation the results compared favorably with those obtained using a less specific assay previously available. (J. R. Vandenheede, S. Keppens, and H. DeWulf, 1977, Biochim. Biophys. Acta.481, 463–470;D. D. Doorneweerd, A. W. H. Tan, and F. Q. Nuttall, 1978, Diabetes27, 474). The in vitro activation of phosphorylase kinase was not associated with the formation of a small-molecular-weight form of the enzyme. The utility of the assay in monitoring in vivo interconversion reactions in response to various physiological stimuli was demonstrated.     

Stereoisomeric guaiacylglycerol-β-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells

Two 8-O-4′-type neolignan epimers erythro-guaiacylglycerol-β-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-β-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC₅₀ = 45.56 μM) and 2b (IC₅₀ = 39.02 μM) had more cytotoxic effect than its enantiomers 1a (IC₅₀ = 82.66 μM) and 2a (IC₅₀ = 67.97 μM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 μM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-β-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.     

Synthesis and biological evaluation of novel tricyclic oxazine and oxazepine fused quinazolines. Part 1: Erlotinib analogs

Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthesized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and Calu-3 cell lines. Compared to erlotinib and gefitinib, compounds 1a–1h were found to demonstrate more potent antitumor activities. Several derivatives could counteract EGF-induced phosphorylation of EGFR in cells, and their potency was comparable to the reference compounds. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib.     

Structure-based design,synthesis and biological evaluation of a newer series of pyrazolo[1,5-a]pyrimidine analogues as potential anti-tubercular agents

In-depth study of structure-based drug designing can provide vital leads for the development of novel, clinically active molecules. In this present study, twenty six novel pyrazolo[1,5-a]pyrimidine analogues (6a-6z) were designed using molecular docking studies. The designed molecules were synthesized in good yields. Structural elucidation of the synthesized molecules was carried out using IR, MS, ¹H NMR and ¹³C NMR spectroscopy. All the synthesized compounds were evaluated for their in-vitro anti-tubercular activity against H37Rv strain by Alamar Blue assay method. Most of the synthesized compounds displayed potent anti-tubercular activities. Amongst all the tested compounds 6p, 6g, 6n and 6h exhibited promising anti-tubercular activity. Further, these potent compounds were gauged for MDR-TB, XDR-TB and cytotoxic study. None of these compounds exhibited potent cytotoxicity. Stability of protein ligand complex was further evaluated by molecular dynamics simulation for 10 ns. All these results indicate that the synthesized compounds could be potential leads for further development of new potent anti-tubercular agents.     

Design,synthesis, and biological evaluation of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs in MCF-7 and MDA-MB-468 breast cancer cell lines

A series of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs were designed and synthesized for developing pyrazinoindolone scaffolds as anti-breast cancer agents. Compounds 1h and 1i, having a furan-2-yl-methylamide and benzylamide group, respectively, exhibited more potent cytotoxicity in MDA-MB-468 triple-negative breast cancer (TNBC) cells than compounds possessing aliphatic groups. Compounds 2a and 2b, as (R)-enantiomers of 1h and 1i, also had inhibitory activity against MDA-MB-468 cells. Moreover, analogs (3a–b and 3d–e) bearing a benzyl group at the N-2 position showed more potent activity than gefitinib, as a potent EFGR-TK inhibitor. Especially, compound 3a exhibited selective cytotoxic activity against MDA-MB-468 cells; it also had a synergistic effect in combination with gefitinib against MDA-MB-468 cells. In addition, we confirmed that compounds 3a and 3d inhibit phosphorylation of Akt in MDA-MB-468 cells using western blot analysis. Therefore, these 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs may be helpful for investigating new anti-TNBC agents.     

New amide linked dimeric 1,2,3-triazoles bearing aryloxy scaffolds as a potent antiproliferative agents and EGFR tyrosine kinase phosphorylation inhibitors

A search for potent antiproliferative agents has prompted to design and synthesize aryloxy bridged and amide linked dimeric 1,2,3-triazoles (7a–j) by using 1,3-dipolar cycloaddition reaction between 2-azido-N-phenylacetamides (4a–e) and bis(prop-2-yn-1-yloxy)benzenes (6a–b) via copper (I)-catalyzed click chemistry approach with good to excellent yields. All the newly synthesized compounds have been screened for their in vitro antiproliferative activities against two human cancer cell lines. The compounds 7d, 7e, 7h, 7i and 7j have revealed promising antiproliferative activity against human breast cancer cell line (MCF-7), whereas, the compounds 7a, 7b, 7c, 7i and 7j were observed as potent antiproliferative agents against human lung cancer cell line (A-549). The active compounds against MCF-7 have been also analysed for their mechanism of action by the enzymatic study, which shows that the compounds 7d, 7h and 7j were acts as active EGFR tyrosine kinase phosphorylation inhibitors. In support to this biological study, the molecular docking as well as in silico ADME properties of all the newly synthesized hybrids were predicted.     

Synthesis,antiplatelet and in silico evaluations of novel N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides

This paper describes the synthesis, antiplatelet and theoretical evaluations of 10 N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides (2a–j). These compounds were synthesized, characterized and screened for their in vitro antiplatelet profile against human platelet aggregation using arachidonic acid, adrenaline and ADP as agonists. Among NAH derivatives 2a–j, the compounds 2a, 2c, 2e, 2g and 2h were the most promising molecules with significant antiplatelet activity.     

Synthesis and docking studies of novel antitumor benzimidazoles

In this work, the benzimidazole-pyrrole conjugates 6a–h and benzimidazole-tetracycles conjugates 12–14 were prepared. The cytotoxicity of the compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 was tested against lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural product (UK-1), the standard. The tested 4g,h, 6a–h, 10 and 12–14 exhibited remarkable cytotoxicity activity against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized compounds 4g,h, 6a–h, 10 and 12–14 has been estimated by studying their possible inhibitory effects on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the Epstein–Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells and their effects being stronger than that of a representative control, oleanolic acid.Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has been in correlation with the antitumor activity. All synthesized compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thiophene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giving the best docking results, were further studied to estimate their effect on the level of uPA using AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting the expression of uPA.     

Novel 5,6-disubstituted pyrrolo[2,3-d]pyrimidine derivatives as broad spectrum antiproliferative agents: Synthesis,cell based assays,kinase profile and molecular docking study

Two new series of 5-subtituted and 5,6-disubstituted pyrrolo[2,3-d]pyrimidine octamides (4a–o and 6a–g) and their corresponding free amines 5a–m and 7a–g have been synthesized and biologically evaluated for their antiproliferative activity against three human cancer cell lines. The 5,6-disubstituted octamides 6d–g as well as the amine derivative 7b have shown the best anticancer activity with single digit micromolar GI₅₀ values over the tested cancer cells, and low cytotoxic effects (GI₅₀?>?10.0?µM) against HFF-1 normal cell. A structure activity relationship (SAR) study has been established and disclosed that terminal octamide moiety at C2 as well as disubstitution with fluorobenzyl piperazines at C5 and C6 of pyrrolo[2,3-d]pyrimidine are the key structural features prerequisite for best antiproliferative activity. Moreover, the most active member 6f was tested for its antiproliferative activity over a panel of 60 cancer cell lines at NCI, and exhibited distinct broad spectrum anticancer activity with submicromolar GI₅₀ and TGI values over multiple cancer cells. Kinase profile of compound 6f over 53 oncogenic kinases at 10?µM concentration showed its highly selective inhibitory activity towards FGFR4, Tie2 and TrkA kinases. The observed activity of 6f against TrkA (IC₅₀?=?2.25?µM), FGFR4 (IC₅₀?=?6.71?µM) and Tie2 (IC₅₀?=?6.84?µM) was explained by molecular docking study, which also proposed that 6f may be a type III kinase inhibitor, binding to an allosteric site rather than kinase hinge region. Overall, compound 6f may serve as a promising anticancer lead compound that could be further optimized for development of potent anticancer agents.     

Synthesis,anti-inflammatory,analgesic, COX-1/2 inhibition activities and molecular docking study of pyrazoline derivatives

Design, synthesis and pharmacological activities of a group of 1,3,5-trisubstituted pyrazolines were reported. The chemical structures of the synthesized compounds have been assigned on the basis of IR, MS, ¹H NMR, and ¹³C NMR spectral analyses. The synthesized 1,3,5-trisubstituted pyrazoline derivatives were evaluated in vivo for anti-inflammatory, analgesic activities and in vitro for COX-1/2 inhibition assay. Among the tested compounds, derivatives 4h, 6e, 7a, 7e, and 9 showed more potent anti-inflammatory and analgesic activities than the reference drug celecoxib. On the basis of their higher activities in the in vivo studies compared with celecoxib, the five compounds 4h, 6e, 7a, 7e and 9 were selected to test their inhibitory activities against ovine COX-1/2 using an in vitro cyclooxygenase inhibition assay. Docking study of compounds 7a, 7e and 9 into the COX-2 binding site revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

2-(chromon-3-yl)imidazole derivatives as potential antimicrobial agents: synthesis,biological evaluation and molecular docking studies

A series of novel 2-(chromon-3-yl)-4,5-diphenyl-1H-imidazoles (4a-h) were synthesized by one pot condensation of substituted 3-formylchromones (1a-h), benzil (2) and ammonium acetate (3) in refluxing acetic acid at 110 °C under N2 atmosphere. Allylation of compounds 4a-h with allyl bromide in the presence of fused K₂CO₃ furnished N-allyl-2-(chromon-3-yl)-4,5-diphenyl-1H-imidazoles (6a-h). The synthesized compounds were characterized spectroscopically and evaluated for in vitro antimicrobial activity against various pathogenic bacterial and fungal strains by disc diffusion method. Compounds bearing electron withdrawing substituents such as bromo (4f) showed significant inhibitory activity against S. cerevisiae (MIC 1.4 μg/ml) and 4g containing chloro substituent, displayed more inhibitory potential against C. albicans (MIC 1.5), as compared to the standard drugs. Compounds 6a and 4c exhibit remarkable inhibitory potential against B. subtilis with MIC 0.98 and 1.23, respectively. The time kill assay for active compound 6a was performed by viable cell count (VCC) method to elucidate the microbicidal nature of 2-(chromon-3-yl)imidazoles. A molecular docking study of most active compounds with target ‘lanosterol 14α-demethylase’ (CYP51) was performed to unravel the mode of antifungal action.