Synthesis of rigid trichostatin A analogs as HDAC inhibitors

New inhibitors of histone deacetylase (HDAC) have been synthesized and evaluated for their activity toward non small lung cancer cell line H661. Their design is based on indanone (or tetralone) systems leading to trichostatin A (TSA) analogs with limited conformational mobility. Molecular modelization at the AM1 level revealed that the conformations of indane-based analogs and TSA bound to HDAC like protein are similar. The synthesis of these new analogs was achieved by alkylation of an appropriate indanone (or tetralone) to introduce the side chain bearing a terminal ester group, the latter being a precursor of hydroxamic acid and aminobenzamide derivatives. Hydroxamic acids with the TSA side chain were found to be the most active compounds and the presence of the dimethylamino group on the phenyl ring turned out to be essential to achieve low micromolar activities against H661 cancer cells.     

5-Aryl indanones and derivatives as non-steroidal progesterone receptor modulators

Novel 5-aryl indanones, inden-1-one oximes, and inden-1-ols were synthesized and evaluated as progesterone receptor (PR) modulators using the T47D cell alkaline phosphatase assay. Both PR agonists and antagonists were achieved with appropriate 3- and 5-substitution from indanones and inden-1-ols while inden-1-one oximes provided only PR antagonists. Several compounds such as 10 and 11 demonstrated potent in vitro PR agonist potency similar to that of steroidal progesterone (1). In addition, a number of compounds (e.g., 12, 13, 17, 18) showed potent PR antagonist activity indicating the indanones and derivatives are promising PR modulator templates.     

Design, synthesis and biological evaluation of 1,4-benzodiazepine-2,5-dione-based HDAC inhibitors

New histone deacetylase inhibitors have been synthesized and evaluated for their activity against non-small lung cancer cell line H661. These compounds have been designed with diversely substituted 1,4-benzodiazepine-2,5-dione moieties as cyclic peptide mimic cap structures, and a hydroxamate side chain. Biological evaluations demonstrated that benzodiazepine-based HDACi bearing an aromatic substituent at the N1 position exhibited promising antiproliferative and HDAC-inhibitory activities.     

Functional interplay between histone demethylase and deacetylase enzymes

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.     

Synthesis and anticancer activity of 2-benzylidene indanones through inhibiting tubulin polymerization

In an attempt to discover a potent and selective anticancer agent, gallic acid has been modified to benzylidene indanones as tubulin polymerization inhibitors. These compounds were evaluated against several human cancer cell lines and also evaluated for inhibition of tubulin polymerase in in vitro assays. Three of the analogues exhibited strong cytotoxicity against human cancer cell lines IC(50)=10-880 nM and also showed tubulin polymerization inhibition (IC(50)=0.62-2.04 μM). Compound 9j, the best candidate of the series was found to be non-toxic in acute oral toxicity in Swiss-albino mice up to 1000 mg/kg dose.     

The concise synthesis of chalcone, indanone and indenone analogues of combretastatin A4

A series of aryl- and aroyl-substituted chalcone analogues of the tubulin binding agent combretastatin A4 (1) were prepared, using a recently introduced one-pot palladium-mediated hydrostannylation-coupling reaction sequence. These chalcones were converted to indanones by Nazarov cyclisation, followed by oxidation to give the corresponding indenones. Indenones were also prepared using a palladium-mediated formal [3+2]-cycloaddition process between ortho-halobenzaldehydes and diarylpropynones. All compounds were assessed as inhibitors of tubulin polymerisation, but only E-31 had activity similar to that of 1. However, compound E-31 did not exhibit antiproliferative activity against the MCF-7 cell line.     

Gallic acid-based indanone derivatives as anticancer agents

Gallic acid-based indanone derivatives have been synthesised. Some of the indanones showed very good anticancer activity in MTT assay. Compounds 10, 11, 12 and 14 possessed potent anticancer activity against various human cancer cell lines. The most potent indanone (10, IC₅₀ = 2.2 μM), against MCF-7, that is, hormone-dependent breast cancer cell line, showed no toxicity to human erythrocytes even at higher concentrations (100 μg/ml, 258 μM). While, indanones 11, 12 and 14 showed toxicities to erythrocytes at higher concentrations.     

Metronidazole acid acyl sulfonamide: a novel class of anticancer agents and potential EGFR tyrosine kinase inhibitors

A series of novel metronidazole derivatives were recently reported as potent anticancer agents targeting EGFR and HER-2 by our group [Qian, Y.; Zhang, H. J.; Zhang, H.; Xu, C.; Zhao, J.; Zhu, H. L. Bioorg. Med. Chem.2010, 18, 4991]. Based on the previous results, we designed and synthesized a new series of metronidazole acid acyl sulfonamide derivatives and a new series of phenylacetyl benzenesulfonamide derivatives and their anticancer activities were evaluated as potential EGFR and HER-2 kinase inhibitors. Among all the compounds, compound 12 displayed the most potent inhibitory activity EGFR and HER-2 (IC(50)=0.39 μM for EGFR and IC(50)=1.53 μM for HER-2) and it also showed the most potent growth inhibitory activity against A549 and B16-F10 cancer cell line in vitro, with an IC(50) value of 1.26 μg/mL for A549 and 0.35 μg/mL for B16-F10. Docking simulation was further performed to position compound 12 into the EGFR active site to determine the probable binding model.     

Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II

A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.     

Synthesis and antiproliferative evaluation of amide-containing flavone and isoflavone derivatives

Certain amide-containing flavone and isoflavone derivatives were synthesized and evaluated for their antiproliferative activities. These compounds were synthesized via alkylation of hydroxyl precursors followed by the reaction with H(2)SO(4) and NaN(3) (Schmidt reaction). The preliminary assays indicated that the inhibitory activity against the growth of NCI-H661 decreased in an order of linked chromophore flavone-6-yl 16a-d>flavone-7-yl 17a-d>flavone-3-yl 15a-d and isoflavone-7-yl 18a-d. Among these flavone-6-yl derivatives, N-(4-methoxyphenyl)-2-(4-oxo-2-phenyl-4H-chromen-6-yloxy)acetamide (16c) was the most potent with a GI(50) value of 0.84 microM. The inhibitory activity against the growth of NPC-TW01 decreased in an order of linked chromophore flavone-6-yl 16a-d>isoflavone-7-yl 18a-d>flavone-7-yl 17a-d>flavone-3-yl 15a-d. Flavone-6-yl derivatives 16a-d demonstrated significant inhibitory activities against the growth of NPC-TW01 cell with an average GI(50) value of 0.84 microM. The oxime derivatives 1 and 2 caused accumulation of NPC-TW01 cell in G(2)/M phase which were distinct from that of their amide isomers 16b and 16c, respectively, which induced cell-cycle arrest in G(0)/G(1) phase followed by apoptosis. Therefore, the antiproliferative mechanism of flavone derivatives was affected not only by the phenyl benzopyran-4-one pharmacophore but also by the peripheral substituents.     

Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6

A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.     

1-Chloro-2-formyl indenes and tetralenes as antitubercular agents

1-Chloro-2-formyl indenes and tetralenes have been synthesized using Vilsmeier-Haack-Arnold reaction onto indanones and tetralones. Most of these analogues exhibited antitubercular activity against Mycobacterium tuberculosis H37Rv strain with MICs ranging from 30 to 500 μg/mL. Analogue 13 was further modified to some derivatives. The most active analogue 23 showing MIC at 30 μg/mL was further evaluated for acute oral toxicity in Swiss albino mice and was found to be safe up to 300 mg/kg dose.     

Structural insights into the inhibitor binding and new inhibitor design to Polo-like kinase-1 Polo-box domain using computational studies

Polo box domain (PBD) from Polo-Like Kinase-1 (PLK-1) a cell cycle regulator is one of the important non-kinase targets implicated in various cancers. The crystal structure of PLK-1 PBD bound to phosphopeptide inhibitor is available and acylthiourea derivatives have been reported as potent PBD inhibitors. In this work, structure and ligand-based pharmacophore methods have been used to identify new PBD inhibitors. The binding of acylthiourea analogs and new inhibitors to PBD were assessed using molecular docking and molecular dynamics simulations to understand their binding interactions, investigate the complex stability and reveal the molecular basis for inhibition. This study provides the binding free energies and residue-wise contributions to decipher the essential interactions in the protein-inhibitor complementarity for complex formation and the design of new PBD inhibitors with better binding.

Communicated by Ramaswamy H. Sarma     

Chemical synthesis and in silico molecular modeling of novel pyrrolyl benzohydrazide derivatives: Their biological evaluation against enoyl ACP reductase (InhA) and Mycobacterium tuberculosis

In efforts to develop new antitubercular agents, we report here the synthesis of a series of novel pyrrole hydrazine derivatives. The molecules were evaluated against inhibitors of InhA, which is one of the key enzymes involved in type II fatty acid biosynthetic pathway of the mycobacterial cell wall as well as inhibitors of Mycobacterium tuberculosis H37Rv. The binding mode of compounds at the active site of enoyl-ACP reductase was explored using the surflex-docking method. The model suggests one or two H-bonding interactions between the compounds and the InhA enzyme. Some compounds exhibited good activities against InhA in addition to promising activities against M. tuberculosis.     

A549肺癌细胞株中癌睾丸抗原的表达及生物学意义研究

目的：癌睾丸抗原（Gancer．testisantigen,CTA）是一类主要表达于睾丸组织和癌组织,并具有较强诱导体液和细胞免疫应答能力的抗原,可以作为抗肿瘤的理想靶分子。本研究探讨了A549肺癌细胞系中CTA的表达情况及其生物学意义。方法：本研究中选取了A549肺癌细胞株,运用RT-PCR的方法检测了其中九种常见CTA的表达情况,进一步通过AV—PI双染法观察了表柔比星,紫杉醇,卡铂,伊立替康,依托泊苷五种化疗药物对A549细胞的凋亡作用及其调控CTA表达的情况。另外用转化生长因子β1（TGF-β1）处理A549细胞,验证了TGF-β1促进细胞生长的作用,进一步检测此时CTA的表达调控。结果：结果显示MAGEAl。MAGEA3,MAGECl,HCA661在A549细胞系中稳定表达,表柔比星能够强烈诱导细胞发生凋亡,其诱导作用岁浓度增高而增强。表柔比星下调了MAGEAl,MAGEA3,MAGECl的表达,对HCA661的表达无影响。TGF-β1增强了肿瘤细胞增殖能力,与此同时下调HCA661的表达,对MAGEAI,MAGEA3,MAGECI的表达无影响。结论：MAGEAl,MAGEA3,MAGECl,HCA661在肺癌细胞系A549中稳定表达。TGF-β1作用48小时,使HCA661的表达下调,MAGEAl,MAGEA3,MAGECl的表达无变化。表柔比星作用48小时,使MAGEAl,MAGEA3,MAGECl的表达下调,HCA661表达无影响,卡铂,紫杉醇,伊立替康,依托泊苷对CTA的表达无调控,为进一步明确CTA的表达调控机制奠定了研究基础,同时为将HCA661作为临床中诊断与治疗的新靶点提供了可行性。     

The synthesis of L-carvone and limonene derivatives with increased antiproliferative effect and activation of ERK pathway in prostate cancer cells

Thirty-one novel derivatives of carvone, carveol, and limonene were designed and synthesized using L-carvone as a starting material via chlorination, nucleophilic substitution, and reduction. The structures of these derivatives were characterized by MS and 1H NMR. The antiproliferative effect was evaluated in human prostate cancer LNCaP cells. L-carvone, L-carveol, and L-limonene were weak cell growth inhibitors and introduction of 4-(2-methoxyphenyl)piperazine to carvone, carveol or limonene significantly increased their antiproliferative effect. The antiproliferative effect was correlated with ERK activation and p21(waf1) induction.     

Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor

Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a approximately 32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The approximately 32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR(+)/trkA(-) 661w cells. Our study shows that a approximately 32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.     

Redox survival signalling in retina-derived 661W cells

Reactive oxygen species have been implicated in processes involving cellular damage and subsequent cell death, especially in organs such as the eye that are constantly exposed to excitatory signals. However, recent studies have shown that oxidant species can also act as intracellular signalling molecules promoting cell survival, but little is known about this mechanism in the retina. The present study demonstrates for the first time that hydrogen peroxide (H2O2) is generated rapidly and acts as a pro-survival signal in response to a variety of apoptotic stimuli in retina-derived 661W cells and in the retinal ganglion cell line RGC-5. Focussing on 661Ws and serum deprivation, we systematically investigated pro-survival and pro-death pathways and discovered that the rapid and transient burst of H2O2 activates the AKT survival pathway. Activation of the apoptotic machinery takes place following the decline of H2O2 to basal levels. To substantiate this proposed pro-survival role of H2O2, we inhibited the oxidant burst, which exacerbated cell death. Conversely, maintenance of the oxidant signal using exogenous H2O2 enhanced cell survival. Overall, the results presented in this study provide evidence for a novel role of H2O2 in mediating survival of retinal cells in response to apoptotic stimuli.     

Synthesis, antiproliferative, and antiplatelet activities of oxime- and amide-containing quinolin-2(1H)-one derivatives

Certain oxime- and amide-containing quinolin-2(1H)-one derivatives were synthesized and evaluated for their antiproliferative and antiplatelet activities. These compounds were synthesized via alkylation of hydroxyl precursors followed by the reaction with NH(2)OH or NaN(3) (Schmidt reaction). The preliminary assays indicated that amide derivatives are either weakly active or inactive while the oxime counterparts exhibited potent inhibitory activities against platelet aggregation induced by collagen, AA (arachidonic acid), and U46619 (the stable thromboxan A(2) receptor agonist). Among them, (Z)-6-[2-(4-methoxyphenyl)-2-hydroxyiminoethoxy]quinolin-2(1H)-one (7c) was the most active against AA induced platelet aggregation with an IC(50) of 0.58microM and was inactive against cell proliferation. For the inhibition of U46619 induced aggregation, 7a and 8a-c exhibited very potent activities with IC(50) values in a range between 0.54 and 0.74microM. For the antiproliferative evaluation, N-(biphenyl-4-yl)-2-(2-oxo-1,2-dihydroquinolin-7-yloxy)acetamide (11d) was the most potent with GI(50) values of <10, 10.8, and <10microM against the growth of MT-2, NCI-H661, and NPC-Tw01, respectively, and possessed only a weak antiplatelet activity. Further evaluation of 11d as a potential anticancer agent is on-going.