Identification of 1,2,4-Oxadiazoles-Based Novel EGFR Inhibitors: Molecular Dynamics Simulation-Guided Identification and in vitro ADME Studies.

Background: In this work, we have identified heterocyclic derivatives with 1,2,4 oxadiazole scaffold mimicking the functions of tyrosine kinase inhibitors. Fourteen molecules that displayed the best fit were picked from the library of compounds and studied under in-silico and in-vitro conditions. Four compounds were selected for further cytotoxicity and ADME (Absorption, Distribution, Metabolism, Elimination) profiling showing IC50 (from 8-13 µM) values against EGFR positive cancer cell line (MCF7). Methods: A molecular dynamics simulation study was performed to understand the correlation of non-covalent binding energies with biological activity. The drug-like properties of the selected four compounds (7a, 7b, 7e, and 7m) were evaluated by in-vitro ADME studies. Compounds 7a, 7b, and 7m were the active compounds in the molecular dynamics simulations study. Further, EGFR binding activity was confirmed with EGFRWT and EGFRT790M kinase assay using a luminescence-based method. Results: These compounds (7a, 7b, and 7m) showed activity against EGFRWT and mutant EGFRT790M, exhibiting IC50 values of <10 and <50 micromolar, respectively. These compounds also possess moderate aqueous solubility in 40-70 µg/mL at pH 7.4 and 30-100 µg/mL at pH 4.0. Further, 7a, 7b, and 7m showed balanced lipophilicity with Log D values ranging from 1-3. They demonstrated a good correlation in Caco-2 permeability with Apparent permeability (Papp) 1 to 5 × 10-6 cm/s in comparison with 7e, which was found to be highly lipophilic (Log D >5) and showed high permeability (Papp 17 × 10-6 cm/s). Lastly, all these compounds were moderately stable in liver microsomes at alkaline pH with a half-life of 30-60 min, while at a highly acidic pH (2.0), the compounds were stable up to 15-20 min. Conclusion: Overall, in-vitro ADME results of these molecules showed good drug-like properties, which are well correlated with the in-silico ADME data, making them ideal for developing an oral drug delivery formulation.

Rational design-aided discovery of novel 1,2,4-oxadiazole derivatives as potential EGFR inhibitors.

A molecular dynamics-based sampling of epidermal growth factor receptor tyrosine kinase (EGFR-TK) was carried out to search for energetically more stable protein, which was then used for molecular docking of a series of 1,2,4-oxadiazole derivatives previously reported from our laboratory. A total of 14 compounds were docked, where compounds 6a and 6b showed better binding to EGFR in silico. Further, physicochemical properties of all the compounds were calculated, which suggested that all the molecules obeyed Lipinski's rule of 5 and had favorable polar surface area and CaCO2 permeability along with the low potential for HERG inhibition. All the compounds were then screened for their ability to produce cytotoxicity in four different cell lines overexpressing EGFR (A549, HCT-116, HEPG2, MCF-7) and one EGFR negative cancer cell line (SW620); at three concentrations: 10, 1, and 0.1 µM. None of the compounds showed activity against SW620, which suggested that the compounds show cytotoxicity through inhibition of EGFR. Compounds that showed promise in this 3-concentration screen were further subjected to multiple dose-response curves to identify the IC50 values for the shortlisted eight compounds. It was encouraging to see 6a and 6b showing the best IC50 values against almost all the cell-lines which further suggests that our design protocol can be applied to optimize this lead (which are currently in the low micromolar range) to design the homologous compounds to achieve the desired potency in the nanomolar range and also to achieve selectivity across a range of kinases.

Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone deacetylase inhibitors.

The discovery and development of isoform-selective histone deacetylase (HDAC) inhibitor is a challenging task because of the sequence homology among HDAC enzymes. In the present work, novel tetrahydro benzo[b]thiophene-3-carbonitrile based benzamides were designed, synthesized, and evaluated as HDAC inhibitors. Pharmacophore modeling was our main design strategy, and two novel series of tetrahydro benzo[b]thiophene-3-carbonitrile derivatives with piperidine linker (series 1) and piperazine linker (series 2) were identified as HDAC inhibitors. Among all the synthesised compounds, 9h with 4-(aminomethyl) piperidine linker and 14n with piperazine linker demonstrated good activity against human HDAC1 and HDAC6, respectively. Both the compounds also exhibited good antiproliferative activity against several human cancer cell lines. Both these compounds (9h and 14n) also induced cell cycle arrest and apoptosis in U937 and MDA-MB-231 cancer cells. Overall, for the first time, this research discovered potent isoform-selective HDAC inhibitors using cyclic linker instead of the aliphatic chain and aromatic ring system, which were reported in known HDAC inhibitors.

Evaluation of 5-hydroxy-2,3-diaryl (substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anti angiogenic and anticancer agents.

Two potent cis-restricted CA-4 analogues 11 and 42 belonging to 2,3-diaryl-5-hydroxycyclopent-2-en-1-one class were evaluated for anticancer and anti angiogenic activity. The compound 42 displayed potent cytotoxic activity (IC(50) < 1 muM) against a panel of human cancer cell lines viz PTC, MDA.MB.453, PA1, SKOV3, DU145 and Miapaca2, whereas compound 11 displayed cytotoxicity activity (IC(50) < 1 microM) only in Miapaca2. Both the compounds inhibit growth factor stimulated endothelial cell proliferation, migration and capillary tube formation. In all the above parameter compound 42 was superior to 11. Based on the above results compound 42 was assessed for inhibition of vasculature in vivo and showed significant inhibition at 25 mg/kg dose. Further it was evaluated for in vivo anti tumor activity in athymic mice bearing DU145 and SKVO3 tumor xenograft and showed regression in tumor volume (T/C) of 23.8% (CA-4), 50.1% (compound 42) and 23.5% (CA-4), 56% (compound 42) respectively at a dose of 20 mg/kg (i.v.) daily for 14 days.

Anticancer and immunomodulatory activities of novel 1,8-naphthyridine derivatives.

A number of 1,8-naphthyridine derivatives (22-62) have been synthesized and screened for their in vitro cytotoxicity against eight tumors and two non-tumor cell lines. Halogen substituted 1,8-naphthyridine-3-caboxamide derivatives showed potent activity with compound 47 having IC(50) of 0.41 and 0.77 microM on MIAPaCa and K-562 cancer cell lines, respectively while, compound 36 had IC(50) of 1.19 microM on PA-1 cancer cell line. However, one of the unsubstituted 1,8-naphthyridine-C-3'-heteroaryl derivative 29 showed potent cytotoxicity with IC(50) of 0.41 and 1.4 microM on PA-1 and SW620 cancer cell lines, respectively. These compounds were also evaluated for anti-inflammatory activity as suggested by downregulation of proinflammaotory cytokines.

1,8-Naphthyridine-3-carboxamide derivatives with anticancer and anti-inflammatory activity.

A number of 1-propargyl-1,8-naphthyridine-3-carboxamide derivatives (15-35) have been synthesized and screened for their in vitro cytotoxicity and anti-inflammatory activity. Compounds 22, 31 and 34 have shown high cytotoxicity against a number of cancer cell lines, while compound 24 showed significant anti-inflammatory activity.

Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents.

A new series of functionalized amino acid derivatives N-substituted 1-N-(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazolidine carboxamide (1-17) and 1-N-substituted-3-amino-2-hydroxy-3-phenylpropane-1-carboxamide (18-34) were synthesized and evaluated for their in vitro cytotoxicity against human cancer cell lines. Compound 6 has shown interesting cytotoxicity (IC(50) = 5.67 microm) in ovarian cancer, while compound 10 exhibited promising cytotoxicity in ovarian (IC(50) = 6.1 microm) and oral (IC(50) = 4.17 microm) cancers. These compounds could be of use in designing new anti-cancer agents.

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives.

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.

Modulation of key signal transduction molecules by a novel peptide combination effective for the treatment of gastrointestinal carcinomas.

We have reported earlier a novel combination of four structurally designed synthetic neuropeptide analogs of vasoactive intestinal peptide (VIP), bombesin, substance P and somatostatin, code-named DRF 7295 which have anti-tumor efficacy for adenocarcinomas in vitro and in vivo (Jaggi et al., Invest New Drugs, 2008). The discovery, synthesis, in vitro and in vivo efficacy was reported (Jaggi et al., Invest New Drugs, 2008). Gastrointestinal tumor cells of the colon, pancreas and duodenum were found to most sensitive to DRF7295 in vitro and in vivo (Jaggi et al., Invest New Drugs, 2008). We have further investigated and report here the modulation of cellular signaling in gastrointestinal carcinomas by DRF 7295, which may be mediating its observed anticancer activity in these cancer types. DRF 7295 inhibits the binding of specific neuropeptides initiating a cascade of cellular signaling events leading to programmed cell death. It down regulates the second messenger cAMP, epidermal growth factor (EGF) dependent proliferation and the phosphorylated MAP Kinase pERK1/2 in gastrointestinal carcinomas, thus depriving the tumour cells of critical pro-proliferative cellular signals. It triggers bcl2 and Caspase 3 dependent apoptotic cell death and induces p53 tumor suppressor protein in the treated carcinoma cells in vitro. It has significant anti-angiogenic potential as reflected in the inhibition of tube like formation in the endothelial cells and down regulation of VEGF levels. Tumour xenograft studies confirmed the in vivo efficacy of DRF 7295 for gastrointestinal carcinomas (Jaggi et al., Invest New Drugs, 2008). The Phase I clinical trials have shown DRF 7295 to be well tolerated and devoid of systemic toxicities of the conventional cytotoxics (Mukherjee et al., Phase I dose escalating study of DRF7295: a new class of peptide based drugs. "Abstract" ASCO ID:948, 2003). The drug may have a promising role in disease stabilization in colorectal and other cancers. Thus DRF 7295 is a novel targeted drug in the class of signal transduction modulators, with potential for treatment of gastrointestinal carcinomas.

Synthesis and evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anticancer agents.

A new series of 2,3-diaryl-4/5-hydroxy-cyclopent-2-en-1-one analogues replacing the cis double bond of combretastatin A-4 (CA-4) by 4/5-hydroxy cyclopentenone moieties was designed and synthesized. The analogues displayed potent cytotoxic activity (IC50<1 microg/mL) against a panel of human cancer cell lines and endothelial cells. The most potent analogues 11 and 42 belonging to the 5-hydroxy cyclopentenone class were further evaluated for their mechanism of action. Both of the analogues led to cell cycle arrest at G2/M phase and induced apoptosis in endothelial cells. Antitubulin property of 42 was superior to 11 and comparable to CA-4. The compound 42 had better aqueous solubility, metabolic stability, and pharmacokinetic profile than CA-4 and also demonstrated significant tumor regression in the human colon xenograft model. Our data suggests that cis-restricted analogues of CA-4 are a new class of molecules that have the potential to be developed as novel agents for the treatment of cancer.

Bombesin analogs containing alpha-amino-isobutyric acid with potent anticancer activity.

Six octapeptide bombesin (BN) analogs were synthesized by substituting alpha-aminoisobutyric acid (Aib), in place of Ala9 or Gly11, or both, in the [D-Phe6, desMet14]-BN (6-14) sequence: D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-NH2 (P0). Additionally, Leu13 was replaced with isoleucine in two analogs and one of the analogs was butanoylated at the N-terminus. The antiproliferative activity of the analogs was tested in vitro on human pancreatic (MiaPaCa-2) and colon cancer (SW620, HT29 and PTC) cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The analogs demonstrated anticancer activity in the above cell lines at concentrations ranging from 0.01 nM to 1 microM. One of the analogs, P6, was evaluated for in vivo tumor regression in a xenograft model of human primary colon cancer in athymic nude mice and was found to cause significant reduction in tumor volume. NMR and molecular dynamics (MD) simulation studies for this analog revealed the presence of a mixed 3(10)/alpha-helical structure. This study demonstrates that the designed BN analogs retain their anticancer activity after the incorporation of the constrained amino acid, Aib, and are potential molecules for future use in cancer therapy and drug targeting.

Synthesis of 13-amino costunolide derivatives as anticancer agents.

A number of costunolide derivatives (4a-p) have been synthesized and evaluated for their in vitro cytotoxicity against eight tumor and a non-tumor cell lines. Compound 4d showed around 2-fold better cytotoxicity against SW-620 (colon) cell line with improved safety index than costunolide (1). While compounds 4e, 4g, and 4p have shown around 2- to 3-fold better cytotoxicity against MIAPaCa2 (pancreas), K-562 (leukemia) and PA-1 (ovary) cell lines as well as better safety index in comparison to costunolide (1). Compound 4p also exhibited cytotoxicity against HBL100 (breast) cell line with 2-fold better safety index. Structure-activity relationship has been described.