Structure-based discovery of pyrazolamides as novel ERRγ inverse agonists.

Estrogen-related receptor-gamma (ERRγ) is an orphan nuclear receptor with high structural similarity to estrogen receptors (ERα and ß). The endogenous ligand of the receptor has yet to be identified. Only two classes of molecules-stilbene (diethylstilbestrol, 4-hydroxytamoxifen, and GSK5182) and flavonol (kaempferol) have been known to modulate the transcriptional activity of the receptor to date. Further, these agents lack selectivity to ERRγ suggesting the need for a new inverse agonist. Thus, virtual screening was used to identify pyrazolamide 7 as a novel ERRγ inverse agonist. Structure-based diversification and optimization of the compound further led to the identification of derivative 19 as a potent inverse agonist of ERRγ with selectivity over other nuclear receptors including those of ERR family. Pyrazolamide 19 exhibits strong affinity towards ERRγ and inhibits the expression of hepcidin, fibrinogen and gluconeogenic genes, which suggests that these compounds may have antimicrobial, anti-coagulant and antidiabetic activities.

Synthesis of Aryl Trimethylstannane via BF3·OEt2-Mediated Cross-Coupling of Hexaalkyl Distannane Reagent with Aryl Triazene at Room Temperature.

BF3·OEt2-mediated cross-coupling of (SnMe3)2 with aryl triazene offers a new strategy for the synthesis of aryl stannane. A variety of synthetically useful aryl trimethylstannanes were produced in moderate to good yields with this metal-free approach. One-pot sequential Stille cross-coupling with different aryl bromides provides a short entry to both symmetrical and unsymmetrical biaryl compounds.

Design, synthesis, and systematic evaluation of 4-arylpiperazine- and 4-benzylpiperidine napthyl ethers as inhibitors of monoamine neurotransmitters reuptake.

Two series of 4-arylpiperazine- and 4-benzylpiperidine naphthyl ethers were designed based on structure-activity relationship (SAR) and docking model of reported monoamine neurotransmitters reuptake inhibitors. The compounds were synthesized in 3-simple steps and their biological activities were evaluated. Several compounds were proven to be potent inhibitors of serotonin and norepinephrine reuptake. Computer docking was performed to study the interaction of the most potent compound 35 with human serotonin transporter. The results of the analyses suggest that 4-arylpiperazine- and 4-benzylpiperidine naphthyl ethers might be promising antidepressants worthy of further studies.

Design, synthesis and docking study of 4-arylpiperazine carboxamides as monoamine neurotransmitters reuptake inhibitors.

Rational drug design method has been used to generate 4-arylpiperazine carboxamides in an effort to develop safer, more potent and effective monoamine neurotransmitters reuptake inhibitors. Out of twenty-seven synthesized compounds, compound 9 displayed potent monoamine neurotransmitter reuptake inhibitory activity against HEK cells transfected with hSERT or hNET. A Surflex-Dock docking model of 9 was also studied.

Design, synthesis, and biological evaluation of 1,3-diarylisoquinolines as novel topoisomerase I catalytic inhibitors.

With a goal of identifying potent topoisomerase (topo) inhibitor, the C4-aromatic ring of the anticancer agent, 3,4-diarylisoquinolone, was strategically shifted to design 1,3-diarylisoquinoline. Twenty-two target compounds were synthesized in three simple and efficient steps. The 1,3-diarylisoquinolines exhibited potent anti-proliferative effects on cancer cells but few compounds spared non-cancerous cells. Inhibition of topo I/IIα-mediated DNA relaxation by several derivatives was greater than that by camptothecin (CPT)/etoposide even at low concentration (20 µM). In addition, these compounds had little or no effect on polymerization of tubulin. A series of biological evaluations performed with the most potent derivative 4cc revealed that the compound is a non-intercalative topo I catalytic inhibitor interacting with free topo I. Collectively, the potent cytotoxic effect on cancer cells including the drug resistance ones, absence of lethal effect on normal cells, and different mechanism of action than topo I poisons suggest that the 1,3-diarylisoquinolines might be a promising class of anticancer agents worthy of further pursuit.

Triple reuptake inhibitors: Design, synthesis and structure-activity relationship of benzylpiperidine-tetrazoles.

Monoamine transporters are important targets in the treatment of various central nervous disorders. Several limitations of traditional reuptake inhibitors, like delayed onset of action, insomnia, and sexual dysfunction, have compelled the search for safer, more effective compounds. In this study, we have sought to identify novel monoamine reuptake inhibitors. Based upon the docking study of compounds that we had reported previously, aromatic rings (A1) were modified to generate a novel series of benzylpiperidine-tetrazoles. Thirty-one compounds were synthesized and evaluated for their triple reuptake inhibition of serotonin, norepinephrine and dopamine. Triple reuptake inhibitor, compound 2q, in particular, showed potent serotonin reuptake inhibition, validating our design approach.

Berberine bioisostere Q8 compound stimulates osteoblast differentiation and function in vitro.

The Q8 compound is a unique derivative of berberine. The present study investigated the functional role of Q8 to evaluate its potential for use in bone regeneration, especially in osteoblast differentiation. The safe concentration of Q8 increased BMP4-induced alkaline phosphatase (ALP) activity, and induced RNA expression of ALP, bone sialoprotein (BSP), and osteocalcin (OC). The activities of ALP-, BSP- and OC-luciferase reporters were also increased by Q8. During osteoblast differentiation, Q8 stabilized the Runx2 and Osterix protein abundance by blocking the ubiquitin-proteasome pathway, which in turn promoted Runx2 and Osterix induced transcriptional activity and subsequently increased the osteoblast differentiation. Meanwhile, depletion of Runx2 and Osterix markedly abolished the bone anabolic effect of Q8 on osteoblast differentiation. To evaluate the signal transduction pathway involved in the Q8-mediated regulation of Runx2 and Osterix, we examined the reporter assay using various kinase inhibitors. Treatment with a protein kinase A (PKA) inhibitor, H89 inhibited the Q8-mediated regulation of Runx2 and Osterix. Based on these findings, this study demonstrates that Q8 promotes the osteoblast differentiation by stabilization of Runx2/Osterix through the increased activation of PKA signaling. The enhancement of osteoblast function by Q8 may contribute to the prevention for osteoporosis.

Discovery of Isoquinolinoquinazolinones as a Novel Class of Potent PPARγ Antagonists with Anti-adipogenic Effects.

Conformational change in helix 12 can alter ligand-induced PPARγ activity; based on this reason, isoquinolinoquinazolinones, structural homologs of berberine, were designed and synthesized as PPARγ antagonists. Computational docking and mutational study indicated that isoquinolinoquinazolinones form hydrogen bonds with the Cys285 and Arg288 residues of PPARγ. Furthermore, SPR results demonstrated strong binding affinity of isoquinolinoquinazolinones towards PPARγ. Additionally, biological assays showed that this new series of PPARγ antagonists more strongly inhibit adipocyte differentiation and PPARγ2-induced transcriptional activity than GW9662.

Insights into the Structural Features Essential for JAK2 Inhibition and Selectivity.

The Janus kinase 2 (JAK2)-mediated signaling pathway plays an important role in controlling cell survival, proliferation, and differentiation. A mutation of JAK2 (V617F in specific) that results in constitutive activation of the enzyme is found in patients with myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. The genetic, biological, and physiological evidence available to date has established JAK2 inhibitors as effective chemotherapeutic agents for the treatment of MPNs as well as solid tumors, hepatitis C virus (HCV) infection, Alzheimer's disease, and Parkinson's disease. Important features essential for JAK2 inhibitors are potent enzymatic inhibition and a high degree of selectivity among other isoforms of JAK. The extent of the potency and selectivity of JAK2 inhibitors is dependent upon receptor-ligand interactions and structural difference between isoenzymes. Thus, detailed knowledge regarding structural characteristics and the binding mode between JAK2 and its inhibitors is necessary. Accordingly, we compiled in this review a comprehensive summary of the three dimensional (3D) structural features of reported JAK2-ligand complexes and the structureactivity relationship (SAR) of JAK2 inhibitors, with particular focus on potent JAK2 inhibition and specificity.

Design and synthesis of novel androgen receptor antagonists via molecular modeling.

Several androgen receptor (AR) antagonists are clinically prescribed to treat prostate cancer. Unfortunately, many patients become resistant to the existing AR antagonists. To overcome this, a novel AR antagonist candidate called DIMN was discovered by our research group in 2013. In order to develop compounds with improved potency, we designed novel DIMN derivatives based on a docking study and substituted carbons with heteroatom moieties. Encouraging in vitro results for compounds 1b, 1c, 1e, 3c, and 4c proved that the new design was successful. Among the newly synthesized compounds, 1e exhibited the strongest inhibitory effect on LNCaP cell growth (IC50=0.35µM) and also acted as a competitive AR antagonist with selectivity over the estrogen receptor (ER) and the glucocorticoid receptor (GR). A docking study of compound 1e fully supported these biological results. Compound 1e is considered to be a novel, potent and AR-specific antagonist for treating prostate cancer. Thus, our study successfully applied molecular modeling and bioisosteric replacement for hit optimization. The methods here provide a guide for future development of drug candidates through structure-based drug discovery and chemical modifications.

Substituted 2-arylquinazolinones: Design, synthesis, and evaluation of cytotoxicity and inhibition of topoisomerases.

A series of 2-arylquinazolinones with structural homology to known 3-arylisoquinolines were designed and synthesized in order to develop safe, effective, and selective cytotoxic agents targeting topoisomerases (topos). 2-Arylquinzolinones with various substitutions on the aromatic rings were obtained by thermal cyclodehydration/dehydrogenation on reacting anthranilamides and benzaldehydes. The compounds had superior topo I-inhibitory activities but were generally inactive against topo IIα. Among the 6-methyl-, 6-amino-, and 7-methylquinazolinones, 6-amino-substituted derivatives displayed potent cytotoxicity at submicromolar to nanomolar concentrations against human colorectal adenocarcinoma cells (HCT-15), human ductal breast epithelial tumor cells (T47D), and cervical cancer cells (HeLa). There was a good correlation between topo I inhibition and the cytotoxic effects of 6-aminoquinazolinones. Docking models demonstrated that topo I inhibition by these compounds is owing to intercalation and H-bond interactions with the DNA bases and amino acid residues at the enzymatic site.

Computer-aided discovery of aminopyridines as novel JAK2 inhibitors.

The Janus kinase 2 (JAK2)-mediated signaling pathway plays an important role in controlling cell survival, proliferation, and differentiation. In recent years, genetic, biological, and physiological evidence has established JAK2 inhibitors as effective chemotherapeutic agents for the treatment of many different cancers. For this reason, we sought to identify novel small molecule inhibitors of JAK2. Using Surflex-Dock software, we tested 3010 compounds with known chemical structures in silico for their ability to interact with the JAK2 ATP-binding pocket. We selected the 10 highest-scoring compounds and tested their abilities to inhibit JAK2 activity in vitro. Compound 1a (ethyl 1-(5-((3-methoxyphenyl)carbamoyl)-3-nitropyridin-2-yl)piperidine-4-carboxylate) was identified. Optimization of 1a using docking studies led to the discovery of compounds 1b and 1d, potent JAK2 inhibitors. Furthermore, as V-shaped kinase inhibitors can curve around the protein backbone and access deep into the pocket, we developed a new series of compounds with a non-linear sulfonamide bond. Nine compounds were prepared and evaluated for JAK2 inhibitory effects. Compounds 7e (IC50=6.9µM) and 7h (IC50=12.2µM) showed better JAK2 inhibition, validating our design approach. This study successfully applied virtual screening for hit discovery, and a docking study for hit optimization. In addition, a novel approach to drug discovery, combining structure- and shape-based drug design, facilitated the design of more potent JAK2 inhibitors. The methods provide a guide for future development of inhibitors targeting JAK2 and other kinases.

Modification of 3-arylisoquinolines into 3,4-diarylisoquinolines and assessment of their cytotoxicity and topoisomerase inhibition.

Inspired by the initial success of the monoarylisoquinolines and the quest to identify more potent and selective anticancer agents with topoisomerase (topo) inhibitory activity, series of diarylisoquinolines (3,4-diarylisoquinolones and 3,4-diarylisoquinolinamines) were designed and synthesized. Synthesis of these compounds primarily involved lithiated toluamide-benzonitrile cycloaddition, Suzuki coupling, and nucleophilic aromatic substitution reactions. Eight of the derivatives were selectively toxic against human ductal breast epithelial tumor cells (T47D), human prostate cancer cells (DU145), and human colorectal adenocarcinoma cells (HCT-15), but had no effect on normal human breast epithelial cells (MCF10A). The topo inhibitory activities of the diarylisoquinoline compounds were relatively dependent upon their chemical structure. 3,4-Diarylisoquinolones generally did not inhibit topo I and only showed moderate inhibition of topo II. In contrast, several 3,4-diarylisoquinolinamines showed superior topo I inhibitory activity. Isoquinolinamine derivatives had greater affinity for topo I than for topo II. Topo inhibition by 3,4-diarylisoquinolines was further supported by docking models showing intercalative and/or H-bond interactions between these compounds and the DNA/topo(s). An analysis of the correlation between the cytotoxicity and topo inhibition of these compounds indicated that the primary biological target of derivatives with potent cytotoxicity was topo, which in turn establishes diaryl-substituted isoquinolines as a novel class of potential anticancer drugs.

Design, synthesis and systematic evaluation of cytotoxic 3-heteroarylisoquinolinamines as topoisomerases inhibitors.

A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity, topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor (T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145) cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15) cells than camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent cytotoxicity with topos and cell cycle inhibitory activities.

SAR based design of nicotinamides as a novel class of androgen receptor antagonists for prostate cancer.

Molecular knowledge of pure antagonism and systematic SAR study offered a direction for structural optimization of DIMN to provide nicotinamides as a novel series of AR antagonists. Nicotinamides with extended linear scaffold bearing sterically bulky alkoxy groups on isoquinoline end were synthesized for H12 displacement. AR binding affinity and molecular basis of antiandrogenic effect establish the optimized derivatives, 7au and 7bb, as promising candidates of second generation AR antagonists for advanced prostate cancer.

Structure-based virtual screening and identification of a novel androgen receptor antagonist.

Hormonal therapies, mainly combinations of anti-androgens and androgen deprivation, have been the mainstay treatment for advanced prostate cancer because the androgen-androgen receptor (AR) system plays a pivotal role in the development and progression of prostate cancers. However, the emergence of androgen resistance, largely due to inefficient anti-hormone action, limits the therapeutic usefulness of these therapies. Here, we report that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) acts as a novel anti-androgenic compound that may be effective in the treatment of both androgen-dependent and androgen-independent prostate cancers. Through AR structure-based virtual screening using the FlexX docking model, fifty-four compounds were selected and further screened for AR antagonism via cell-based tests. One compound, DIMN, showed an antagonistic effect specific to AR with comparable potency to that of the classical AR antagonists, hydroxyflutamide and bicalutamide. Consistent with their anti-androgenic activity, DIMN inhibited the growth of androgen-dependent LNCaP prostate cancer cells. Interestingly, the compound also suppressed the growth of androgen-independent C4-2 and CWR22rv prostate cancer cells, which express a functional AR, but did not suppress the growth of the AR-negative prostate cancer cells PPC-1, DU145, and R3327-AT3.1. Taken together, the results suggest that the synthetic compound DIMN is a novel anti-androgen and strong candidate for useful therapeutic agent against early stage to advanced prostate cancer.

Application of ring-closing metathesis for the synthesis of benzo[3,4]azepino[1,2-b]isoquinolin-9-ones.

Cycloaddition reaction between toluamides and benzonitriles was applied to prepare the 3-arylisoquinolines, and their chemical transformation to the dienes 4 was performed. The ring-closing metathesis (RCM) reaction afforded the desired heterocyclic compounds, benzo[3,4]azepino[1,2-b]isoquinolinones 5 in good yield.

Synthesis of benzo[3,4]azepino[1,2-b]isoquinolin-9-ones from 3-arylisoquinolines via ring closing metathesis and evaluation of topoisomerase I inhibitory activity, cytotoxicity and docking study.

Benzo[3,4]azepino[1,2-b]isoquinolinones were designed and developed as constraint forms of 3-arylisquinolines with an aim to inhibit topoisomerase I (topo I). Ring closing metathesis (RCM) of 3-arylisoquinolines with suitable diene moiety provided seven membered azepine rings of benzoazepinoisoquinolinones. Spectral analyses of these heterocyclic compounds demonstrated that the methylene protons of the azepine rings are nonequivalent. The shielding environment experienced by these geminal hydrogens differs unusually by 2.21ppm. As expected, benzoazepinoisoquinolinones displayed potent cytotoxicity. However, cytotoxic effects of the compounds were not related to topo I inhibition which is explained by non-planar conformation of the rigid compounds incapable of intercalating between DNA base pairs. In contrast, flexible 3-arylisoquinoline 8d attains active conformation at drug target site to exhibit topo I inhibition identical to cytotoxic alkaloid, camptothecin (CPT).

Design and synthesis of 4-amino-2-phenylquinazolines as novel topoisomerase I inhibitors with molecular modeling.

4-Amino-2-phenylquinazolines 7 were designed as bioisosteres of 3-arylisoquinolinamines 6 that were energy minimized to provide stable conformers. Interestingly, the 2-phenyl ring of 4-amino-2-phenylquinazolines was parallel to the quinazoline ring and improved their DNA intercalation ability in the DNA-topo I complex. Among the synthesized 4-amino group-substituted analogs, 4-cyclohexylamino-2-phenylquinazoline 7h exhibited potent topo I inhibitory activity and strong cytotoxicity. Interestingly, consistency was observed between the cytotoxicities and topo I activities in these quinazoline analogs, suggesting that the target of 4-amino-2-phenylquinazolines is limited to topo I. Molecular docking studies were performed with the Surflex-Dock program to afford the ideal interaction mode of the compound into the binding site of the DNA-topo I complex in order to clarify the topo I activity of 7h.

Design, synthesis and docking study of 5-amino substituted indeno[1,2-c]isoquinolines as novel topoisomerase I inhibitors.

Various 5-amino group-substituted indeno[1,2-c]isoquinolines 7a-f were synthesized based on the previous QSAR study as rigid structures of 3-arylisoquinolines. Amino group-substituted compounds, especially 5-piperazinyl indeno[1,2-c]isoquinoline 7f, displayed potent topoisomerase I inhibitory activity as well as cytotoxicities against five different tumor cell lines. A Surflex-Dock docking model of 7f was also studied.