Elucidation of pharmacokinetics of novel DNA ligase I inhibitor, S012-1332 in rats: Integration of in vitro and in vivo findings.

S012-1332 is the first DNA ligase I inhibitor that demonstrated in vivo anti-breast cancer activity. The present study aimed to assess the in vivo pharmacokinetics of S012-1332 in rats and interpret them with in vitro findings. A sensitive and selective liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated to determine S012-1332. Following oral administration, the absolute bioavailability was 7.04%. The absorption was prolonged which can be explained by low solubility in simulated gastric fluid and several folds higher solubility in simulated intestinal fluid. The effective permeability across the intestinal membrane in in situ single pass perfusion study for S012-1332 was 5.58 ± 1.83 * 10-5 cm/sec compared to 5.99 ± 0.65 * 10-5 cm/sec for carbamazepine, with no significant difference, indicating S012-1332 has high permeability. It was rapidly partitioning into plasma in blood, where it was stable. Plasma protein binding was moderate which may have attributed to the rapid distribution out of the vascular compartment. The pharmacokinetics of S012-1332 was characterized by extensive clearance as seen with rat liver and intestinal microsomes. In vitro results elucidate the in vivo pharmacokinetic data. These findings provide crucial information for further development of S012-1332 as anti-breast cancer agent.

A Novel Benzocoumarin-Stilbene Hybrid as a DNA ligase I inhibitor with in vitro and in vivo anti-tumor activity in breast cancer models.

Existing cancer therapies are often associated with drug resistance and toxicity, which results in poor prognosis and recurrence of cancer. This necessitates the identification and development of novel therapeutics against existing as well as novel cellular targets. In this study, a novel class of Benzocoumarin-Stilbene hybrid molecules were synthesized and evaluated for their antiproliferative activity against various cancer cell lines followed by in vivo antitumor activity in a mouse model of cancer. The most promising molecule among the series, i.e. compound (E)-4-(3,5-dimethoxystyryl)-2H-benzo[h]chromen-2-one (19) showed maximum antiproliferative activity in breast cancer cell lines (MDA-MB-231 and 4T1) and decreased the tumor size in the in-vivo 4T1 cell-induced orthotopic syngeneic mouse breast cancer model. The mechanistic studies of compound 19 by various biochemical, cell biology and biophysical approaches suggest that the compound binds to and inhibits the human DNA ligase I enzyme activity that might be the cause for significant reduction in tumor growth and may constitute a promising next-generation therapy against breast cancers.

The regulation of Hh/Gli1 signaling cascade involves Gsk3ß- mediated mechanism in estrogen-derived endometrial hyperplasia.

The present study was undertaken to explore the functional involvement of Hh signaling and its regulatory mechanism in endometrial hyperplasia. Differential expression of Hh signaling molecules i.e., Ihh, Shh, Gli1 or Gsk3ß was observed in endometrial hyperplasial (EH) cells as compared to normal endometrial cells. Estradiol induced the expression of Hh signaling molecules and attenuated the expression of Gsk3ß whereas anti-estrogen (K1) or progestin (MPA) suppressed these effects in EH cells. Cyclopamine treatment or Gli1 siRNA knockdown suppressed the growth of EH cells and reduced the expression of proliferative markers. Estradiol also induced the nuclear translocation of Gli1 which was suppressed by both MPA and K1 in EH cells. While exploring non-canonical mechanism, LY-294002 (Gsk3ß activator) caused a decrease in Gli1 expression indicating the involvement of Gsk3ß in Gli1 regulation. Further, Gsk3ß silencing promoted the expression and nuclear translocation of Gli1 demonstrating that Gsk3ß serves as a negative kinase regulator of Gli1 in EH cells. Similar attenuation of Hh signaling molecules was observed in rats with uterine hyperplasia undergoing anti-estrogen treatment. The study suggested that Hh/Gli1 cascade (canonical pathway) as well as Gsk3ß-Gli1 crosstalk (non-canonical pathway) play crucial role in estrogen-dependent cell proliferation in endometrial hyperplasia.

Palladium-Catalyzed Synthesis of Phenanthridine/Benzoxazine-Fused Quinazolinones by Intramolecular C-H Bond Activation.

A highly efficient synthesis of phenanthridine/benzoxazine-fused quinazolinones by ligand-free palladium-catalyzed intramolecular C-H bond activation under mild conditions has been developed. The C-C coupling provides the corresponding N-fused polycyclic heterocycles in good to excellent yields and with wide functional group tolerance.

Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA ligases and can distinguish them from human DNA ligase I.

DNA ligases are critical components for DNA metabolism in all organisms. NAD(+)-dependent DNA ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA ligase or Mycobacterium tuberculosis NAD(+)-dependent DNA ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA ligase I & T4 DNA ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.

Tandem C-2 functionalization-intramolecular azide-alkyne 1,3-dipolar cycloaddition reaction: a convenient route to highly diversified 9H-benzo[b]pyrrolo[1,2-g][1,2,3]triazolo[1,5-d][1,4]diazepines.

An efficient diversity-oriented synthetic approach to annulated 9H-benzo[b]pyrrolo[1,2-g][1,2,3]triazolo[1,5-d][1,4]diazepines has been developed using a Sc(OTf)3-catalyzed two-component tandem C-2 functionalization-intramolecular azide-alkyne 1,3-dipolar cycloaddition reaction. The reaction shows high substrate tolerance and provides a library of fused heterocycles that may lead to novel biologically active compounds or drug lead molecules.

Chemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K/Akt/FOXO and MEK/Erk/AP-1 Pathways.

Inhibition of epidermal growth factor receptor (EGFR) signaling is considered to be a promising treatment strategy for estrogen receptor (ER)-negative breast tumors. We have investigated here the anti-breast cancer properties of a novel anti-proliferative benzopyran compound namely, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) in ER- negative and EGFR- overexpressing breast cancer cells. The benzopyran compound selectively inhibited the EGF-induced growth of MDA-MB 231 cells and ER-negative primary breast cancer cell culture. The compound significantly reduced tumor growth in xenograft of MDA-MB 231 cells in nude mice. The compound displayed better binding affinity for EGFR than inhibitor AG1478 as demonstrated by molecular docking studies. CDRI-85/287 significantly inhibited the activation of EGFR and downstream effectors MEK/Erk and PI-3-K/Akt. Subsequent inhibition of AP-1 promoter activity resulted in decreased transcription activation and expression of c-fos and c-jun. Dephosphorylation of downstream effectors FOXO-3a and NF-κB led to increased expression of p27 and decreased expression of cyclin D1 which was responsible for decreased phosphorylation of Rb and prevented the transcription of E2F- dependent genes involved in cell cycle progression from G1/S phase. The compound induced apoptosis via mitochondrial pathway and it also inhibited EGF-induced invasion of MDA-MB 231 cells as evidenced by decreased activity of MMP-9 and expression of CTGF. These results indicate that benzopyran compound CDRI-85/287 could constitute a powerful new chemotherapeutic agent against ER-negative and EGFR over-expressing breast tumors.

Benzopyran derivative CDRI-85/287 induces G2-M arrest in estrogen receptor-positive breast cancer cells via modulation of estrogen receptors α- and ß-mediated signaling, in parallel to EGFR signaling and suppresses the growth of tumor xenograft.

In an endeavor to develop novel and improved selective estrogen receptor modulators as anti-breast cancer agents, the benzopyran compounds have been synthesized and identified which act as potent anti-estrogen at uterine level. The present study evaluates the anti-tumor activity of 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) and explores the mechanism of action with a view to describe its potential to inhibit proliferation in ER-positive breast cancer cells MCF-7 and T47D. The compound decreased the expression of ERα while increased the expression of ERß thereby altering ERα/ERß ratio in both cell lines. Although the compound showed low binding affinity to ERs, it acted as ERα antagonist and ERß agonist in decreasing ERE- or AP-1-mediated transcriptional activation in these cells. Transactivation studies in ERα/ß-transfected MDA-MB231 cells suggested that at cyclin D1 promoter, compound antagonized the action of ERα-mediated E2 response while acted as estrogen agonist via ERß. Further, the compound led to decreased expression of ERα-dependent proliferation markers and ERß-dependent cell cycle progression markers. The expression of cell cycle inhibitory protein p21 was increased leading to G2/M phase arrest. In parallel, compound also interfered with EGFR activation, caused inhibition of PI-3-K/Akt pathway and subsequent induction of apoptosis via intrinsic pathway. A significant reduction in tumor mass and volume was observed in 85/287-treated mice bearing MCF-7 xenograft. We conclude that compound 85/287 exhibits significant anti-tumor activity via modulation of genomic as well as non-genomic mechanisms involved in cellular growth and arrested the cells in G2 phase in both MCF-7 and T47D breast cancer cells. Study suggests that CDRI-85/287 may have therapeutic potential in ER-positive breast cancer.

Apoptosis induction and inhibition of hyperplasia formation by 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran in rat uterus.

OBJECTIVE: The study was undertaken to explore the antiproliferative mechanism of action of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) in estradiol-induced rat uterine hyperplasia. STUDY DESIGN: Adult ovariectomized rats received vehicle or estradiol alone (20 µg/kg) or estradiol along with K-1 (100 or 200 µg/kg) for 14 days. Uterine histomorphometric analysis and immunoblotting were performed. Caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining were performed to analyze the apoptotic potential of compound. RESULTS: Compound inhibited estradiol-induced uterine weight and histomorphometric changes pertaining to endometrial growth and down-regulated the expression of estrogen response element and activator protein-1 regulated genes and transcription factors. The compound significantly induced apoptosis, interfered with Akt activation, decreased X-linked inhibitor of apoptosis protein expression leading to an increased cleavage of caspase-9, caspase-3, poly(adenosine diphosphate-ribose) polymerase, increased Bax/Bcl2 ratio, and caspase-3 activity. CONCLUSION: K-1 inhibits endometrial proliferation via nonclassical estrogen receptor signaling mechanisms. It interfered with Akt activation and induced apoptosis via the intrinsic pathway and inhibited estradiol-induced hyperplasia formation in rat uterus.

Anti-implantation effect of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran, a potent antiestrogenic agent in rats.

OBJECTIVE: To investigate the anti-implantation effect and hormonal profile of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) in rats. DESIGN: In vivo assays for anti-implantation activity were performed in pregnant rats. Assays for estrogenicity/antiesrogenicity were performed in immature ovariectomized female rats. In vitro competitive binding of K-1 to human recombinant ERα, transient transfection assay using ERE-luciferase reporter, and alkaline phosphatase (ALP) activity as a measure of estrogenicity and/antiestrogenicity in human endometrial carcinoma cells were performed. SETTING: Research laboratory. ANIMAL(S): Adult female rats for anti-implantation activity, immature ovariectomized female rats, and immature castrated/intact male rats. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Number of implantations, uterine growth, luciferase reporter activity, ER binding affinity, and ALP activity. RESULT(S): Compound K-1 given orally for 1-7 days post coitum at the dose of 100 µg/kg body weight prevented pregnancy in 100% of rats. K-1 was a potent antiestrogenic, and at 50 µg/kg, it could inhibit the effect of 1 µg E(2) in immature rats. Compound was devoid of uterotrophic, androgenic, or antigonadotropic activity. A high affinity binding to ERα was displayed by K-1, with a relative binding affinity of 5% of E(2). In human endometrial carcinoma cells, K-1 did not induce ERα-mediated transcriptional activation that is measured as luciferase reporter activity. K-1 antagonized the E-induced transcriptional activation significantly. K-1 also antagonized E-induced ALP activity in human endometrial cells. CONCLUSION(S): K-1 appeared to exert its antifertility action by virtue of its strong antiestrogenic activity.

NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor.

Mycobacterium tuberculosis codes for an essential NAD+-dependent DNA ligase (MtuLigA) which is a novel, validated, and attractive drug target. We created mutants of the enzyme by systematically deleting domains from the C-terminal end of the enzyme to probe for their functional roles in the DNA nick joining reaction. Deletion of just the BRCT domain from MtuLigA resulted in total loss of activity in in vitro assays. However, the mutant could form an AMP-ligase intermediate that suggests that the defects caused by deletion of the BRCT domain occur primarily at steps after enzyme adenylation. Furthermore, genetic complementation experiments using a LigA deficient E. coli strain demonstrates that the BRCT domain of MtuLigA is necessary for bacterial survival in contrast to E. coli and T. filiformis LigA, respectively. We also report the identification, through virtual screening, of a novel N-substituted tetracyclic indole that competes with NAD+ and inhibits the enzyme with IC50 in the low muM range. It exhibits approximately 15-fold better affinity for MtuLigA compared to human DNA ligase I. In vivo assays using LigA deficient S. typhimurium and E. coli strains suggest that the observed antibacterial activity of the inhibitor arises from specific inhibition of LigA over ATP ligases in the bacteria. In silico ligand-docking studies suggest that the exquisite specificity of the inhibitor arises on account of its mimicking the interactions of NAD+ with MtuLigA. An analysis of conserved water in the binding site of the enzyme suggests strategies for synthesis of improved inhibitors with better specificity and potency.

Synthesis of some new diaryl and triaryl hydrazone derivatives as possible estrogen receptor modulators.

1,2-Bis(4-substituted phenyl)-2-methyl ethanone (2,4-dinitrophenyl)hydrazones and 1-naphthyl-1-(4-substituted phenyl)-methanone (2,4-dinitrophenyl)hydrazones have been synthesized and evaluated for their anti-implantation, uterotrophic, antiuterotrophic, anticancer and antimicrobial activities. Diphenolic hydrazone (compound 6) showed maximum uterotrophic inhibition of 70%, whereas compound 20 exhibited cytotoxicity in the range of 50-70% against MCF-7 and ZR-75-1 human malignant breast cell lines.