Synthesis and biological evaluation of 1-phenyl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-one/thiones as anticancer agents.

Cancer is associated with uncontrolled cell proliferation invading adjoining tissues and organs. Despite the availability of several chemotherapeutic agents, the constant search for newer approaches and drugs is necessitated owing to the ever-growing challenge of resistance. Over the years, DNA has emerged as an important druggable therapeutic drug due to its role in critical cellular processes such as cell division and maintenance. Further, evading apoptosis stands out as a hallmark of cancer. Hence, designing new compounds that would target DNA and induce apoptosis plays an important role in cancer therapy. In the current work, we carried out the synthesis and anticancer evaluation of 1-aryl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-ones/thiones (26 compounds) against selected human cancer cell lines. Among these, compounds 8ae, 8ad, 8cf, 10ad and Kenpaullone have shown good inhibitory properties against HeLa cells (IC50 < 2 µM) with good selectivity over the non-cancerous human embryonic kidney (Hek293T) cells. In cell cycle analysis, the compounds 8ad and 8cf have exhibited G2/M cell cycle arrest in HeLa cells. In addition, the compounds 8ad and 8cf induced apoptosis in a dose-dependent manner in the Annexin-V FITC staining assay. The DAPI staining clearly demonstrated the condensed and fragmented nuclei in 8ad, 8cf, 8ae and Kenpaullone-treated HeLa cells. In addition, these compounds strongly suppressed the healing after 48 h in in vitro cell migration assay. The DNA binding experiments indicated that compounds 8ae, 8cf, and 8ad as well as Kenpaullone interact with double-stranded DNA by binding in grooves which may interrupt the DNA replication and kill fast-growing cells. Molecular docking studies revealed the binding pose of 8ad and Kenpaullone at HT1 binding pocket of double-stranded DNA. Compounds 8ad and 8cf demonstrated moderate topo II inhibition which could be a possible reason for their anticancer properties. Compounds 8ad and 8cf may cause the topo II and DNA covalent complex, which leads to the inhibition of DNA replication and transcription. This eventually increases the DNA damage in cells and promotes cell apoptosis. With the above interesting biological profile, the new 1-aryl-2,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(4H)-one/thione derivatives have emerged as promising leads for the discovery of new anticancer agents.

Cinnamide derived pyrimidine-benzimidazole hybrids as tubulin inhibitors: Synthesis, in silico and cell growth inhibition studies.

An approach in modern medicinal chemistry to discover novel bioactive compounds is by mimicking diverse complementary pharmacophores. In extension of this strategy, a new class of piperazine-linked cinnamide derivatives of benzimidazole-pyrimidine hybrids have been designed and synthesized. Their in vitro cytotoxicity profiles were explored on selected human cancer cell lines. Specifically, structural comparison of target hybrids with tubulin-DAMA-colchicine and tubulin-nocodazole complexes has exposed a deep position of benzimidazole ring into the αT5 loop. All the synthesized compounds were demonstrated modest to interesting cytotoxicity against different cancer cell lines. The utmost cytotoxicity has shown with an amine linker of benzimidazole-pyrimidine series, with specificity toward A549 (lung cancer) cell line. The most potent compound in this series was 18i, which inhibited cancer cell growth at micromolar concentrations ranging 2.21-7.29 µM. Flow cytometry studies disclosed that 18i inhibited the cells in G2/M phase of cell cycle. The potent antitumor activity of 18i resulted from enhanced microtubule disruption at a similar level as nocodazole on ß-tubulin antibody, explored using immunofluorescence staining. The most active compound 18i also inhibited tubulin polymerization with an IC50 of 5.72 ± 0.51 µM. In vitro biological analysis of 18i presented apoptosis induction on A549 cells with triggering of ROS generation and loss of mitochondrial membrane potential, resulting in DNA injury. In addition, 18i displayed impairment in cellular migration and inhibited the colony formation. Notably, the safety profile of most potent compound 18i was revealed by screening against normal human pulmonary epithelial cells (L132: IC50: 69.25 ± 5.95 µM). The detailed binding interactions of 18i with tubulin was investigated by employing molecular docking, superimposition and free energy analyses. Thus remarks made in this study established that pyrimidine-benzimidazole hybrids as a new class of tubulin polymerization inhibitors with significant anticancer activity.

Identification and characterization of in vitro and in vivo fidarestat metabolites: Toxicity and efficacy evaluation of metabolites.

The progression of diabetic complications can be prevented by inhibition of aldose reductase and fidarestat considered to be highly potent. To date, metabolites of the fidarestat, toxicity, and efficacy are unknown. Therefore, the present study on characterization of hitherto unknown in vitro and in vivo metabolites of fidarestat using liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) is undertaken. In vitro and in vivo metabolites of fidarestat have been identified and characterized by using LC/ESI/MS/MS and accurate mass measurements. To identify in vivo metabolites, plasma, urine, and feces samples were collected after oral administration of fidarestat to Sprague-Dawley rats, whereas for in vitro metabolites, fidarestat was incubated in human S9 fraction, human liver microsomes, and rat liver microsomes. Furthermore, in silico toxicity and efficacy of the identified metabolites were evaluated. Eighteen metabolites have been identified. The main in vitro phase I metabolites of fidarestat are oxidative deamination, oxidative deamination and hydroxylation, reductive defluroniation, and trihydroxylation. Phase II metabolites are methylation, acetylation, glycosylation, cysteamination, and glucuronidation. Docking studies suggest that oxidative deaminated metabolite has better docking energy and conformation that keeps consensus with fidarestat whereas the rest of the metabolites do not give satisfactory results. Aldose reductase activity has been determined for oxidative deaminated metabolite (F-1), and it shows an IC50 value of 0.44 µM. The major metabolite, oxidative deaminated, did not show any cytotoxicity in H9C2, HEK, HEPG2, and Panc1 cell lines. However, in silico toxicity, the predication result showed toxicity in skin irritation and ocular irritancy SEV/MOD versus MLD/NON (v5.1) model for fidarestat and its all metabolites. In drug discovery and development research, it is distinctly the case that the potential for pharmacologically active metabolites must be considered. Thus, the active metabolites of fidarestat may have an advantage as drug candidates as many drugs were initially observed as metabolites.

Exploration of carbamide derived pyrimidine-thioindole conjugates as potential VEGFR-2 inhibitors with anti-angiogenesis effect.

The development of new small molecules from known structural motifs through molecular hybridization is one of the trends in drug discovery. In this connection, we have combined the two pharmacophoric units (pyrimidine and thioindole) in a single entity via molecular hybridization strategy along with introduction of urea functionality at C2 position of pyrimidine to increase the efficiency of H-bonding interactions. Among the synthesized conjugates 12a-aa, compound 12k was found to exhibit significant IC50 values 5.85, 7.87, 6.41 and 10.43 µM against MDA-MB-231 (breast), HepG2 (liver), A549 (lung) and PC-3 (prostate) cancer cell lines, respectively. All these compounds were further evaluated for their inhibitory activities against VEGFR-2 protein. The results specified that among the tested compounds, 12d, 12e, 12k, 12l, 12p, 12q, 12t and 12u prominently suppressed VEGFR-2, with IC50 values of 310-920 nM in association to the positive control (210 nM). Angiogenesis inhibition was evident by tube formation assay in HUVECs and cell-invasion by transwell assay. The mechanism of cellular toxicity on MDA-MB-231 was found through depolarisation of mitochondrial membrane potential, increased ROS production and subsequent DNA damage resulting in apoptosis induction. Moreover, clonogenic and wound healing assays designated the inhibition of colony formation and cell migration by 12k in a dose-dependent manner. Molecular docking studies also shown that compound 12k capably intermingled with catalytically active residues GLU-885, ASP-1046 of the VEGFR-2 through hydrogen-bonding interactions.

Synthesis of Combretastatin-A4 Carboxamidest that Mimic Sulfonyl Piperazines by a Molecular Hybridization Approach: in†vitro Cytotoxicity Evaluation and Inhibition of Tubulin Polymerization.

Molecular hybridization approach is a promising structural modification tool to design new chemical entities (NCEs) by mimicking two different pharmacophoric units into one scaffold to enhance the biological properties. With this aim, combretastatin-A4 acids were integrated with sulfonyl piperazine scaffolds as a one molecular platform and evaluated for their in vitro antiproliferative activity against a panel of human cancer lines cell lines namely, lung (A549), mouse melanoma (B16F10), breast (MDA MB-231and MCF-7) and colon (HCT-15) by MTT assay. Amongst which the compound (E)-3-(4-Chlorophenyl)-1-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5 ab) displayed significant IC50 values in the range of 0.36 to 7.08 µm against the selected cancer cell lines. Moreover, 5 ab was found to be the most potent member of this series with IC50 0.36±0.02 µm. Further investigations revealed that the compound 5 ab displayed significant inhibition of tubulin assembly with IC50 5.24±0.06 µm and molecular docking studies also disclosed the binding of 5 ab effectively in CA4 binding space at the colchicine binding site. The flow cytometric analysis demonstrated that the compound 5 ab caused cell cycle arrest at G2/M phase in A549 cells. Compound 5 ab induced apoptosis in A549 cells which was further evaluated by different staining assays such as DAPI and AO which undoubtedly speculated, the induction of apoptosis. To study the anti-migration with 5 ab, cell migration/scratch wound assay was performed and the extent of apoptosis was studied by Annexin-V, including mitochondrial potential by JC-1 staining.

Pyrazolo-benzothiazole hybrids: Synthesis, anticancer properties and evaluation of antiangiogenic activity using in vitro VEGFR-2 kinase and in vivo transgenic zebrafish model.

A series of new pyrazolo-benzothiazole hybrids (7-26) were synthesised and screened for their cytotoxic activity towards several cancer cell lines [colon (HT-29), prostate (PC-3), lung (A549), glioblastoma (U87MG)] and normal human embryonic kidney cell line (Hek-293T). Compounds 8, 9, 13, 14, 18, 19, 23, and 24 displayed significant activity, with compound 14 being particularly potent towards all the tested cancer cell lines with IC50 values in the range 3.17-6.77 µM, even better than reference drug axitinib (4.88-21.7 µM). Compound 14 also showed the strongest growth inhibition in 3D multicellular spheroids of PC-3 and U87MG cells. The mechanism of cellular toxicity in PC-3 cells was found to be cell cycle arrest and apoptosis induction through depolarisation of mitochondrial membrane potential, increased ROS production and subsequent DNA damage. Further, compound 14 displayed significant in vitro (VEGFR-2 inhibition) and in vivo [transgenic zebrafish Tg(flila:EGFP) model] antiangiogenic properties. Overall, these results provide strong evidence that compound 14 could be considered for a lead candidate in anticancer and antiangiogenic drug discovery.

Synthesis and in vitro cytotoxicity evaluation of ß-carboline-combretastatin carboxamides as apoptosis inducing agents: DNA intercalation and topoisomerase-II inhibition.

To explore a new set of cytotoxic agents, ß-carboline-combretastatin carboxamide conjugates were designed, synthesized and evaluated for their in vitro cytotoxicity potential, DNA binding affinity and Topoisomerase-II (topo-II) inhibition activity. Among the designed hybrids, 10v and 10af have shown significant cytotoxic effect against A549 (lung cancer) cell line having IC50 value 1.01 µM and 1.17 µM respectively. Further, it was speculated that treatment with compound 10v may induce apoptosis among A549 cells, which was supported by Hoechst staining, DCFDA, Annexin V-FITC and morphological assays. Flow cytometric analysis revealed that the hybrid 10v arrests A549 cells in G2/M phase of cell cycle in a dose dependent manner. Amongst the active hybrids, most potent hybrid 10v was tested for DNA topo-II inhibition activity. Moreover, to further support the biological activity and to infer the mode of interaction between ligands and DNA, spectroscopy and molecular docking studies were carried out. The docking and spectroscopy results showed that the ligands exhibited an intercalative mode of binding with DNA and could efficiently bind to DNA and form topo-II ternary complex. Based on these experiments, the hybrids 10v and 10af were identified as proficient new scaffolds which need to be developed as hit molecules for therapeutic interest.