A new BET inhibitor, 171, inhibits tumor growth through cell proliferation inhibition more than apoptosis induction.

The bromodomain and extra-terminal domain (BET) family of proteins, especially bromodomain-containing protein 4 (BRD4), has emerged as exciting anti-tumor targets due to their important roles in epigenetic regulation. Therefore, the discovery of BET inhibitors with promising anti-tumor efficacy will provide a novel approach to epigenetic anticancer therapy. Recently, we discovered the new BET inhibitor compound 171, which is derived from a polo-like kinase 1 (PLK1)-BRD4 dual inhibitor based on our previous research. Compound 171 was found to maintain BET inhibition ability without PLK1 inhibition, and there was no selectivity among BET family members. The in vitro and in vivo results both indicated that the overall anti-tumor activity of compound 171 was improved compared with the (+)-JQ-1 or OTX-015 BET inhibitors. Furthermore, we found that compound 171 could regulate the expression of cell cycle-regulating proteins including c-Myc and p21 and induce cell cycle arrest in the G0/G1 phase. However, compound 171 only has a quite limited effect on apoptosis, in considering that apoptosis was only observed at doses greater than 50 µM. To determine the mechanisms underlying cell death, proliferation activity assay was conducted. The results showed that compound 171 induced clear anti-proliferative effects at doses that no obvious apoptosis was induced, which indicated that the cell cycle arresting effect contributed mostly to its anti-tumor activity. The result of this study revealed the anti-tumor mechanism of compound 171, and laid a foundation for the combination therapy in clinical practice, if compound 171 or its series compounds become drug candidates in the future.

Tranylcypromine and 6-trifluoroethyl thienopyrimidine hybrid as LSD1 inhibitor.

Tranylcypromine moiety extracted from LSD1 inhibitors and 6-trifluoroethyl thienopyrimidine moiety from menin-MLL1 PPI inhibitors were merged to give new chemotypes for medicinal chemistry study. Among 15 new compounds prepared in this work, some exhibited nanomolar LSD1 activity and good selectivity over MAO-A/B, low micromolar menin-MLL1 PPI inhibitory activity, as well as submicromolar MV4-11 antiprofilative activities. Intracellular LSD1 engagement of compounds with higher enzymatic and antiproliferative activities was confirmed by CD86 mRNA up-regulation experiments.

A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC50] = 2.6 µM) and RNH functions (IC50 = 0.65 µM) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.

Preparation of 5'-deoxy-5'-amino-5'-C-methyl adenosine derivatives and their activity against DOT1L.

From a readily available 5-C-Me ribofuranoside, we have realized a reliable route to valuable 5'-deoxy-5'-amino-5'-C-methyl adenosine derivatives at gram scale with confirmed stereochemistry. These adenosine derivatives are useful starting materials for the preparation of 5'-deoxy-5'-amino-5'-C-methyl adenosine derivatives with higher complexity. From one of the new adenosine derivatives, some 5'-deoxy-5'-amino-5'-C-methyl adenosine DOT1L inhibitors were prepared in several steps. Data from DOT1L assay indicated that additional 5'-C-Me group improved the enzyme inhibitory activity.

Design and synthesis of benzylpiperidine inhibitors targeting the menin-MLL1 interface.

Menin is an essential oncogenic cofactor for mixed lineage leukemia (MLL)-mediated leukemogenesis, functioning through its direct interaction with MLL1 protein. Therefore, targeting the menin-MLL1 protein-protein interface represents a promising strategy to block MLL-mediated leukemogenesis. On the basis of co-crystal structure analysis, starting from thienopyrimidine chemotype, we have investigated the detailed structure-activity relationship of the piperazinyl-dihydrothiazole moiety. Several compounds were found with potent inhibitory activity against menin and better activities in cell-based experiments than MI-2-2. Molecular docking analysis revealed a less explored subpocket, which could be used for the design of new menin-MLL1 inhibitors.

Multi-substituted 8-aminoimidazo[1,2-a]pyrazines by Groebke-Blackburn-Bienaymé reaction and their Hsp90 inhibitory activity.

Using a 2,3-diamino pyrazine substrate and yttrium triflate catalyst, various 2-alkyl and aryl substituted 3,8-diaminoimidazo[1,2-a]pyrazines were efficiently prepared through Groebke-Blackburn-Bienaymé MCR. In particular, a novel 2-piperonyl 3,8-diaminoimidazo[1,2-a]pyrazine structure was prepared exclusively with this new method and was found to have moderate Hsp90 inhibitory activity. A crystalline complex with N-terminus ATP domain of Hsp90 and one of the new Hsp90 inhibitors was also obtained to elucidate the origin of activity of 2-piperonyl 3,8-diaminoimidazo[1,2-a]pyrazines.

Direct glycosylation of bioactive small molecules with glycosyl iodide and strained olefin as acid scavenger.

A new strategy for diversity-oriented direct glycosylation of bioactive small molecules was developed. This reaction features (−)-ß-pinene as acid scavenger and work with glycosyl iodides under mild conditions. With the aid of RP-HPLC and chiral SFC separation techniques, the new direct glycosylation proved effective at gram scale on bioactive small molecules including AZD6244, podophyllotoxin, paclitaxel, and docetaxel. Interesting glycoside derivatives were efficiently created with good yields and 1,2-cis selectivity.

Strained olefin enables triflic anhydride mediated direct dehydrative glycosylation.

For the first time, we demonstrated that Tf2O mediated direct dehydrative glycosylation was possible simply with strained olefins, and other typical bases were inhibitors of this reaction. We optimized the glycosylation conditions and found that typical benzyl protected 1-OH pyranosyl donors and certain alcohol acceptors were suitable for our glycosylation system. Furthermore, we found that complete 1,2-trans selectivity and a wider acceptor scope could be achieved with 2-O-Bz 3,4,6-tri-O-Bn pyranosyl donors.

Synthesis and bioassay of ß-(1,4)-D-mannans as potential agents against Alzheimer's disease.

AIM: Oligomannurarate 971 derived from a marine plant has shown neuroprotective effects. In this study we synthesized a series of truncated derivatives of the oligosaccharide, and investigated the effect of these derivatives against Aß peptide toxicity in vitro. METHODS: The sulfoxide method was applied to synthesize the derivatives. SH-SY5Y human neuroblastoma cells were treated with Aß1-40 (2 µmol/L), and the cell viability was detected using a CCK8 assay. RESULTS: A series of ß-(1,4)-D-mannosyl oligosaccharide, ranging from the disaccharide to the hexasaccharide, were synthesized. Addition of 10 µmol/L ß-(1,4)-D-mannobiose 6, ß-(1,4)-D-mannotriose 9 or ß-(1,4)-D-mannotetraose 12 in SH-SY5Y cells significantly attenuated Aß1-40-induced toxicity. The efficacies were similar to those caused by 10 µmol/L oligomannurarate 971 or alzhemed. Other oligosaccharides including oligomaltoses and oligocelluloses were less active. CONCLUSION: Synthetic homogeneous short chain ß-(1,4)-D-mannans shows neuroprotective effect against Aß peptide toxicity similar to that of heterogeneous oligomannurarate 971 and alzhemed.

Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations.

AIM: To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. METHODS: Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. RESULTS: Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π-π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. CONCLUSION: The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met.

Preparation of bicyclo[1.1.1]pentane-derived nucleoside analogues.

Nucleoside analogues are prevalent in drug design and call for more diversified structures. Bicyclo[1.1.1]pentane (BCP) structure has recently found wide applications in drug discovery. However, incorporation of BCP fragment into nucleoside analogues is hitherto unknown. Thus, from readily available BCP-containing building blocks, six new compounds, including pyrimidine nucleoside analogues, purine nucleoside analogues, and C-nucleoside analogues were prepared in 1-4 steps, generally with good yields.

Sorafenib inhibits transforming growth factor ß1-mediated epithelial-mesenchymal transition and apoptosis in mouse hepatocytes.

Epithelial-mesenchymal transition (EMT) is a physiological process that has been recognized to occur during the progression of an increasingly large number of human diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. The activation of transforming growth factor ß (TGF-ß) signaling is considered a critical event during EMT, and efforts have been made to screen small molecules that interfere with the TGF-ß signaling pathway during EMT. Here we report the identification of sorafenib, a clinical agent that inhibits TGF-ß signaling. When applied to AML12 cells and primary hepatocytes, sorafenib strikingly suppressed TGF-ß1-induced EMT and apoptosis. Additionally, sorafenib inhibited TGF-ß1-induced signal transducer and activator of transcription 3 phosphorylation. We further present in vitro evidence that sorafenib ameliorates the proapoptotic and profibrotic effects of TGF-ß1 in mouse primary hepatocytes, suggesting that this drug exerts a protective effect on hepatocytes and has therapeutic potential for the treatment of liver fibrosis.

C-6 aryl substituted 4-quinolone-3-carboxylic acids as inhibitors of hepatitis C virus.

Quinolone-3-carboxylic acid represents a highly privileged chemotype in medicinal chemistry and has been extensively explored as antibiotics and antivirals targeting human immunodeficiency virus (HIV) integrase (IN). Herein we describe the synthesis and anti-hepatitis C virus (HCV) profile of a series of C-6 aryl substituted 4-quinlone-3-carboxylic acid analogues. Significant inhibition was observed with a few analogues at low micromolar range against HCV replicon in cell culture and a reduction in replicon RNA was confirmed through an RT-qPCR assay. Interestingly, evaluation of analogues as inhibitors of NS5B in a biochemical assay yielded only modest inhibitory activities, suggesting that a different mechanism of action could operate in cell culture.

The design, synthesis and biological evaluations of C-6 or C-7 substituted 2-hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus.

C7-Substituted 2-hydroxyisoquinoline-1,3-diones inhibit the strand transfer of HIV integrase (IN) and the reverse-transcriptase-associated ribonuclease H (RNH). Hepatitis C virus (HCV) NS5B polymerase shares a similar active site fold to RNH and IN, suggesting that N-hydroxyimides could be useful inhibitor scaffolds of HCV via targeting the NS5B. Herein we describe the design, chemical synthesis, replicon and biochemical assays, and molecular docking of C-6 or C-7 aryl substituted 2-hydroxyisoquinoline-1,3-diones as novel HCV inhibitors. The synthesis involved an improved and clean cyclization method, which allowed the convenient preparation of various analogs. Biological studies revealed that the C-6 analogs, a previously unknown chemotype, consistently inhibit both HCV replicon and recombinant NS5B at low micromolar range. Molecular modeling studies suggest that these inhibitors may bind to the NS5B active site.

1-(N,N-diisopropylcarbamoyloxy)-1-tosyl-1-alkenes--a2d1 synthons via tandem umpolung.

1-(N,N-diisopropylcarbamoyloxy)-1-tosyl-alkenes have been developed as an a(2)d(1) synthon via tandem umpolung. Upon addition of Grignard reagents and further quench by carbonyl compounds, this synthon produces alpha,alpha'-branched-alpha'-oxygenated ketones. Strategically, this widely applicable method installs respectively a carbanion unit and a carbonyl unit formally onto the alpha-carbon and the carbonyl center of an aldehyde in one-pot.

A 17 gene panel for non-small-cell lung cancer prognosis identified through integrative epigenomic-transcriptomic analyses of hypoxia-induced epithelial-mesenchymal transition.

As a critical feature of the tumor microenvironment, hypoxia is known to be a potent inducer of tumor metastasis, and it has been proposed that the initial steps in metastasis involve epithelial-mesenchymal transition (EMT). The strong correlation among hypoxia, EMT, and metastasis suggests that integrative assessment of gene expression and the DNA modification program of hypoxia-induced EMT via high-throughput sequencing technologies may increase our understanding of the molecular basis of tumor invasion and metastasis. Here, we present the genomewide transcriptional and epigenetic profiles of non-small-cell lung cancer (NSCLC) cells under normoxic and hypoxic conditions. We demonstrate that hypoxia induces EMT along with dynamic alterations of transcriptional expression and epigenetic modifications in both A549 and HCC827 cells. After training using a dataset from patients with invasive and noninvasive lung adenocarcinomas with an artificial neural network algorithm, a characteristic 17-gene panel was identified, consisting of genes involved in EMT, hypoxia response, glycometabolism, and epigenetic modifications. This 17-gene signature clearly stratified NSCLC patients with significant differences in overall survival across three independent datasets. Our study may be suitable as a basis for further selection of gene signatures to potentially guide prognostic stratification in patients with NSCLC.

Ilex latifolia Thunb protects mice from HFD-induced body weight gain.

Kuding tea is implicated in alleviating metabolic disorders in traditional Chinese medicine. However, the role of Ilex latifolia Thunb (kuding tea), one of the large leaf kuding tea species, in the prevention of the development of obesity remains to be determined. We show here that 7-week-old male mice treated with an Ilex latifolia Thunb supplement for 14 weeks were resistant to HFD-induced body weight gain and hepatic steatosis, accompanied by improved insulin sensitivity. Ilex latifolia Thunb supplementation dramatically reduced the systemic and tissue inflammation levels of mice via reducing pro-inflammatory cytokine levels, increasing anti-inflammatory cytokine levels in the circulation and inhibiting p38 MAPK and p65 NF-κB signaling in adipose tissue. Together, these results indicate that Ilex latifolia Thunb protects mice from the development of obesity and is a potential compound pool for the development of novel anti-obesity drugs.

ω-3 free fatty acids and all-trans retinoic acid synergistically induce growth inhibition of three subtypes of breast cancer cell lines.

All-trans retinoic acid (ATRA), one of vitamin A derivatives, shows greater growth inhibition of breast cancer cell for ER-positive than ER-negative cells, while triple negative breast cancer cell such as MDA-MB-231 cell is poorly responsive to ATRA treatment. In this study, we found that combination of ω-3 free fatty acids (ω-3 FFAs) and ATRA exhibited synergistic inhibition of cell growth in three subtypes (ER+ MCF7, HER2+ SK-BR-3, Triple negative HCC1806 and MDA-MB-231 cells) of human breast cancer cell lines. The combined treatment of ω-3 FFAs and ATRA resulted in cell cycle arrest. ω-3 FFAs combined with ATRA synergistically provoked cell apoptosis via the caspase signals but not p53. These findings suggest that combined chemotherapy of ω-3 FFAs with ATRA is beneficial for improvement of ATRA sensitivity in breast cancer cells.

Discovery of a series of dihydroquinoxalin-2(1H)-ones as selective BET inhibitors from a dual PLK1-BRD4 inhibitor.

Recent years have seen much effort to discover new chemotypes of BRD4 inhibitors. Interestingly, some kinase inhibitors have been demonstrated to be potent bromodomain inhibitors, especially the PLK1 inhibitor BI-2536 and the JAK2 inhibitor TG101209, which can bind to BRD4 with IC50 values of 0.025 µM and 0.13 µM, respectively. Although the concept of dual inhibition is intriguing, selective BRD4 inhibitors are preferred as they may diminish off-target effects and provide more flexibility in anticancer drug combination therapy. Inspired by BI-2536, we designed and prepared a series of dihydroquinoxalin-2(1H)-one derivatives as selective bromodomain inhibitors. We found compound 54 had slightly higher activity than (+)-JQ1 in the fluorescence anisotropy assay and potent antiproliferative cellular activity in the MM.1S cell line. We have successfully solved the cocrystal structure of 52 in complex with BRD4-BD1, providing a solid structural basis for the binding mode of compounds of this series. Compound 54 exhibited high selectivity over most non-BET subfamily members and did not show bioactivity towards the PLK1 kinase at 10 or 1 µM. From in vivo studies, compound 54 demonstrated a good PK profile, and the results from in vivo pharmacological studies clearly showed the efficacy of 54 in the mouse MM.1S xenograft model.