Synthesis, molecular modelling and QSAR study of new N-phenylacetamide-2-oxoindole benzensulfonamide conjugates as carbonic anhydrase inhibitors with antiproliferative activity.

In continuation of our previous studies to optimise potent carbonic anhydrase inhibitors, two new series of isatin N-phenylacetamide based sulphonamides were synthesised and screened for their human (h) carbonic anhydrase (EC 4.2.1.1) inhibitory activities against four isoforms hCA I, hCA II, hCA IX and hCA XII. The indole-2,3-dione derivative 2h showed the most effective inhibition profile against hCAI and hCA II (KI = 45.10, 5.87 nM) compared to acetazolamide (AAZ) as standard inhibitor. Moreover, 2h showed appreciable inhibition activity against the tumour-associated hCA XII, similar to AAZ showing KI of 7.91 and 5.70 nM, respectively. The analogs 3c and 3d showed good cytotoxicity effects, and 3c revealed promising selectivity towards lung cell line A549. Molecular docking was carried out for 2h and 3c to predict their binding conformations and affinities towards the hCA I, II, IX and XII isoforms.

Discovery of a Potent Glutathione Peroxidase 4 Inhibitor as a Selective Ferroptosis Inducer.

Potent and selective ferroptosis regulators promote an intensive understanding of the regulation and mechanisms underlying ferroptosis, which is highly associated with various diseases. In this study, through a stepwise structure optimization, a potent and selective ferroptosis inducer was developed targeting to inhibit glutathione peroxidase 4 (GPX4), and the structure-activity relationship (SAR) of these compounds was uncovered. Compound 26a exhibited outstanding GPX4 inhibitory activity with a percent inhibition up to 71.7% at 1.0 µM compared to 45.9% of RSL-3. At the cellular level, 26a could significantly induce lipid peroxide (LPO) increase and effectively induce ferroptosis with satisfactory selectivity (the value of 31.5). The morphological analysis confirmed the ferroptosis induced by 26a. Furthermore, 26a significantly restrained tumor growth in a mouse 4T1 xenograft model without obvious toxicity.

Discovery of antiproliferative and anti-FAK inhibitory activity of 1,2,4-triazole derivatives containing acetamido carboxylic acid skeleton.

Small molecule inhibitors of the focal adhesion kinase are regarded as promising tools in our armamentarium for treating cancer. Here, we identified four 1,2,4-triazole derivatives that inhibit FAK kinase significantly and evaluated their therapeutic potential. Most tested compounds revealed potent antiproliferative activity in HepG2 and Hep3B liver cancer cells, in which 3c and 3d were the most potent (IC50 range; 2.88 ~ 4.83 µM). Compound 3d possessed significant FAK inhibitory activity with IC50 value of 18.10 nM better than the reference GSK-2256098 (IC50 = 22.14 nM). The preliminary mechanism investigation by Western blot analysis showed that both 3c and 3d repressed FAK phosphorylation comparable to GSK-2256098 in HepG2 cells. As a result of FAK inhibition, 3c and 3d inhibited the pro-survival pathways by decreasing the phosphorylation levels of PI3K, Akt, JNK, and STAT3 proteins. This effect led to apoptosis induction and cell cycle arrest. Taken together, these results indicate that 3d could serve as a potent preclinical candidate for the treatment of cancers.

Phosphonate as a Stable Zinc-Binding Group for "Pathoblocker" Inhibitors of Clostridial Collagenase H (ColH).

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.

Probing phenylcarbamoylazinane-1,2,4-triazole amides derivatives as lipoxygenase inhibitors along with cytotoxic, ADME and molecular docking studies.

Hunting small molecules as anti-inflammatory agents/drugs is an expanding and successful approach to treat several inflammatory diseases such as cancer, asthma, arthritis, and psoriasis. Besides other methods, inflammatory diseases can be treated by lipoxygenase inhibitors, which have a profound influence on the development and progression of inflammation. In the present study, a series of new N-alkyl/aralky/aryl derivatives (7a-o) of 2-(4-phenyl-5-(1-phenylcarbamoyl)piperidine-4H-1,2,4-triazol-3-ylthio)acetamide was synthesized and screened for their inhibitory potential against the enzyme 15-lipoxygenase. The simple precursor ethyl piperidine-4-carboxylate (a) was successively converted into phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide (3) and N-phenylated 5-(1-phenylcarbamoyl)piperidine-1,2,4-triazole (4), then in combination with electrophiles (6a-o) through further multistep synthesis, final products (7a-o) were generated. All the synthesized compounds were characterized by FTIR, 1H, 13C NMR spectroscopy, EIMS, and HREIMS spectrometry. Almost all the synthesized compounds showed excellent inhibitory potential against the tested enzyme. Compounds 7c, 7f, 7d, and 7g displayed potent inhibitory potential (IC50 9.25 ± 0.26 to 21.82 ± 0.35 µM), followed by the compounds 7n, 7h, 7e, 7a, 7b, 7l, and 7o with IC50 values in the range of 24.56 ± 0.45 to 46.91 ± 0.57 µM. Compounds 7c, 7f, 7d exhibited 71.5 to 83.5% cellular viability by MTT assay compared with standard curcumin (76.9%) when assayed at 0.125 mM concentration. In silico ADME studies supported the drug-likeness of most of the molecules. In vitro inhibition studies were substantiated by molecular docking wherein the phenyl group attached to the triazole ring was making a π-δ interaction with Leu607. This work reveals the possibility of a synthetic approach of compounds in relation to lipoxygenase inhibition as potential lead compounds in drug discovery.

Bimodal Molecule as NIR-CT Contrast Agent for Hepatoma Specific Imaging.

With currently available molecular imaging techniques, hepatocellular carcinoma (HCC), a liver cancer with high mortality rates and poor treatment responses, is mostly diagnosed at its late stage. This is largely due to the lack of highly sensitive contrast agents with high liver specificity. Herein, we report a novel bimodal contrast agent molecule CNCI-1 for the effective detection of HCC at its early stage both in vitro and in vivo. The agent has high liver specificity with effective X-ray computed tomography (CT)/near-infrared (NIR) imaging functions. It has been successfully applied to in vivo NIR imaging with high sensitivity and high selectivity to the HCC region of the HepG2 tumor-xenografted mice model and LM3 orthotopic hepatoma mice model. Moreover, the agent was found to be noninvasive and hepatocarcinoma cells preferential. Furthermore, it also enhanced the tumor imaging by revealing the blood vessels nearby for the CT image acquisition in the VX2 orthotopic hepatoma rabbit model. Our design strategy provides a new avenue to develop the medical relevant bimodal contrast agents for diagnosis of HCC at its early stage.

Optical Control of the GTP Affinity of K-Ras(G12C) by a Photoswitchable Inhibitor.

Photocontrol of protein activity is an emerging field in biomedicine. For optical control of a mutant small GTPase K-Ras(G12C), we developed small-molecule inhibitors with photoswitchable efficacy, where one configuration binds the target protein and exert different pharmacological effects upon light irradiation. The compound design was based on the structure feature of a previously identified allosteric pocket of K-Ras(G12C) and the chemical structure of covalent inhibitors, and resulted in the synthesis and characterization of two representative azobenzene-containing compounds. Nucleotide exchange assays demonstrated the different efficacy to control the GTP affinity by photoswitching of one potent compound PS-C2, which would be a useful tool to probe the conformation of mutational K-Ras. Our study demonstrated the feasibility of designing photoswitchable modulators from allosteric covalent inhibitor of small GTPases.

Discovery and Characterization of XY101, a Potent, Selective, and Orally Bioavailable RORγ Inverse Agonist for Treatment of Castration-Resistant Prostate Cancer.

We report the design, optimization, and biological evaluation of nuclear receptor RORγ inverse agonists as therapeutic agents for prostate cancer treatment. The most potent compound 27 (designated as XY101) exhibited cellular activity with an IC50 value of 30 nM in a cell-based reporter gene assay with good selectivity against other nuclear receptor subtypes. The cocrystal structure of 27 in complex with the RORγ ligand binding domain provided a solid structural basis for its antagonistic mechanism. 27 potently inhibited cell growth, colony formation, and the expression of AR, AR-V7, and PSA. 27 also exhibited good metabolic stability and a pharmacokinetic profile with oral bioavailability of 59% and a half-life of 7.3 h. Notably, 27 demonstrated promising therapeutic effects with significant tumor growth inhibition in a prostate cancer xenograft model in mice. The potent, selective, metabolically stable, and orally available RORγ inverse agonists represent a new class of compounds as potential therapeutics against prostate cancer.

Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor.

Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC50 value of 32 µM in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC50 value (37 µM). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.

Potent, Selective, and Cell Active Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor Developed by Structure-Based Virtual Screening and Hit Optimization.

PRMT5 plays important roles in diverse cellular processes and is upregulated in several human malignancies. Besides, PRMT5 has been validated as an anticancer target in mantle cell lymphoma. In this study, we found a potent and selective PRMT5 inhibitor by performing structure-based virtual screening and hit optimization. The identified compound 17 (IC50 = 0.33 µM) exhibited a broad selectivity against a panel of other methyltransferases. The direct binding of 17 to PRMT5 was validated by surface plasmon resonance experiments, with a Kd of 0.987 µM. Kinetic experiments indicated that 17 was a SAM competitive inhibitor other than the substrate. In addition, 17 showed selective antiproliferative effects against MV4-11 cells, and further studies indicated that the mechanism of cellular antitumor activity was due to the inhibition of PRMT5 mediated SmD3 methylation. 17 may represent a promising lead compound to understand more about PRMT5 and potentially assist the development of treatments for leukemia indications.

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth.

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

Design, Synthesis and Structure-Activity Relationship Studies of Novel 4 (1-adamantyl) Phenyl Analogues as HIF-1α Inhibitors.

Hypoxia inducible factor-1 (HIF-1) is a key mediator during cancer cells to adapt tumor hypoxic condition. In this study, a series of adamantane-based compounds were synthesized and evaluated as potential inhibitors of HIF-1α. Examination of their structure-activity relationship (SAR) identified the adamantane-containing indole derivative 20a as a potent inhibitor of HIF-1α in Hep3B cell lines under tumor hypoxia (IC50 = 0.02 µM). The study herein may provide valuable information for the development of novel therapeutics against cancer and tumor angiogenesis.

Pyrimidine sulfonylacetanilides with improved potency against key mutant viruses of HIV-1 by specific targeting of a highly conserved residue.

Based on molecular simulation, the etravirine-VRX-480773 hybrids previously disclosed by our group were optimized to yield novel pyrimidine sulfonylacetanilides 8 with improved activity against a panel of seven clinically relevant single and double mutant strains of HIV-1. The improvement in potency in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits the reverse transcriptase (RT), and represents a remarkable step forward in the development of anti-HIV drugs.

ortho-Carboranylphenoxyacetanilides as inhibitors of hypoxia-inducible factor (HIF)-1 transcriptional activity and heat shock protein (HSP) 60 chaperon activity.

ortho-Carboranylphenoxy derivatives were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter gene assay. Among the compounds synthesized, compound 1d showed the most significant inhibition of hypoxia-induced HIF-1 transcriptional activity with the IC50 of 0.53µM. Furthermore, compound 1h was found to possess the most significant inhibition of heat shock protein (HSP) 60 chaperon activity among the reported inhibitors: the IC50 toward the porcine heart malate dehydrogenase (MDH) refolding assay was 0.35µM.

Synthesis, in vitro evaluation and cocrystal structure of 4-oxo-[1]benzopyrano[4,3-c]pyrazole Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) inhibitors.

Cryptosporidium inosine 5'-monophosphate dehydrogenase (CpIMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]benzopyrano[4,3-c]pyrazole derivative was identified as a moderately potent (IC50 = 1.5 µM) inhibitor of CpIMPDH. We report a SAR study for this compound series resulting in 8k (IC50 = 20 ± 4 nM). In addition, an X-ray crystal structure of CpIMPDH·IMP·8k is also presented.

Design and development of oxazol-5-ones as potential partial PPAR-γ agonist against cancer cell lines.

Recent era aims at developing safer partial Peroxisome proliferator-activated receptor-γ (PPAR- γ) agonists in order to dodge the toxicity issues related to full agonists. With a view to develop non-thiazolidinediones as partial PPAR-γ agonists, novel analogues of oxazol-5-ones (3a-3q) were designed and virtually analyzed for their molecular and drug like properties. The newly synthesized compounds were further evaluated for their preliminary cytotoxicity in a panel of eight cancer cell lines using four concentrations at 10- fold dilutions. Sulforhodamine B (SRB) protein assay was used to estimate cell stability or growth. All the compounds demonstrated distinct effect in the extent of cytotoxicity in the breast cancer cell line MCF-7 with 3g specifically exhibiting partial PPAR-γ agonist activity and adipogenesis stimulating ability.

The effect of caffeic acid phenethyl ester analogues in a modified resistant hepatocyte model.

We present a study of the chemoprotective effects of two caffeic acid phenethyl ester (CAPE)-related structures: LQM717 and LQM706. The modified resistant hepatocyte model in rats was used to study the chemoprevention of these CAPE analogues, which are inexpensive and easily obtained. In the liver cancer model used, we detected extensive necrosis and lipid peroxidation after 24 h, many altered hepatic foci, putatively preneoplastic lesions with γ-glutamyl transpeptidase staining after 30 days, and liver tumors at 12 months. We tested the effect of the CAPE analogues on necrosis, lipid peroxidation, proliferation, p65 activation, altered hepatic foci, and tumors. Both compounds exerted protective effects on lipid peroxidation, necrosis, cell proliferation, p65 activation, and preneoplastic lesions. Rats under a carcinogenic protocol showed a 52, 71.74, and 51.6% decrease in the number of preneoplastic nodules when pretreated with CAPE, LQM706, and LQM717, respectively. At 12 months after carcinogenic treatment, eight of eight rats developed liver cancer, whereas in the group of rats that received pretreatment with CAPE, LQM706, or LQM717, 62.5, 83.3, or 42.85%, respectively, had tumors. In conclusion, LQM717 has the potential to enhance chemoprotection activity much better than CAPE by markedly reducing the formation of liver cancers in this model, and this is a compound that is easy to obtain.

Inhibitor of ovarian cancer cells growth by virtual screening: a new thiazole derivative targeting human thymidylate synthase.

Human thymidylate synthase (hTS) was targeted through a virtual screening approach. The most optimal inhibitor identified, 2-{4-hydroxy-2-[(2-hydroxybenzylidene)hydrazono]-2,5-dihydrothiazol-5-yl}-N-(3-trifluoromethylphenyl)acetamide (5), showed a mixed-type inhibition pattern, with a K(i) of 1.3 µM and activity against ovarian cancer cell lines with the same potency as cisplatin. X-ray studies revealed that it binds the inactive enzyme conformation. This study is the first example of a nonpeptidic inhibitor that binds the inactive hTS and exhibits anticancer activity against ovarian cancer cells.

Discovery of highly potent and selective pan-Aurora kinase inhibitors with enhanced in vivo antitumor therapeutic index.

Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis. Currently a number of Aurora kinase inhibitors with different isoform selectivities are being evaluated in the clinic. Herein we report the discovery and characterization of 21c (AC014) and 21i (AC081), two structurally novel, potent, kinome-selective pan-Aurora inhibitors. In the human colon cancer cell line HCT-116, both compounds potently inhibit histone H3 phosphorylation and cell proliferation while inducing 8N polyploidy. Both compounds administered intravenously on intermittent schedules displayed potent and durable antitumor activity in a nude rat HCT-116 tumor xenograft model and exhibited good in vivo tolerability. Taken together, these data support further development of both 21c and 21i as potential therapeutic agents for the treatment of solid tumors and hematological malignancies.

Synthesis and biological evaluation of 2-(3-fluoro-4-nitro phenoxy)-n-phenylacetamide derivatives as novel potential affordable antitubercular agents.

A novel series of 2-(3-fluoro-4-nitrophenoxy)-N-phenylacetamide compounds were designed, synthesized and in vitro assessed for their antitubercular activities by a microdilution method. All the novel derivatives exerted potent or moderate active against M. tuberculosis H37Rv, with MIC values ranging from 4 to 64 µg/mL. The most potent derivative 3m showed an identical MIC value of 4 µg/mL for both M. tuberculosis H37Rv and rifampin-resistant M. tuberculosis 261. It demonstrated no inhibitory effects against six different tumor cell lines by a MTT assay and had a good safety profile in a vero cell line, providing a good lead for subsequent optimization in search of novel affordable antitubercular agents.