Efficient and metal-free synthesis of 2-aroyl 7-azaindoles via thermally induced denitrogenative intramolecular annulation of 1,2,3,4-tetrazolopyridines.

A facile and metal-free intramolecular denitrogenative annulation strategy for the preparation of novel 2-aroyl 7-azaindoles has been developed from 3-(tetrazolo[1,5-a]pyridin-8-yl)prop-2-en-1-one in the presence of the deep eutectic solvent Dowtherm A. The valuable features of the protocol include a short reaction time, absence of any metal catalyst, utilization of a eutectic solvent, easy product isolation, and very good yields of novel 2-aroyl 7-azaindoles.

BX795, a TBK1 inhibitor, exhibits antitumor activity in human oral squamous cell carcinoma through apoptosis induction and mitotic phase arrest.

TANK-binding kinase 1 (TBK1), a member of IκB Kinase (IKK)-related kinases, plays a role in regulating innate immunity, inflammation and oncogenic signaling. This study aims to investigate the role of BX795, an inhibitor of TBK1, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antitumor effects and mechanisms of BX795 were assessed by MTT assays, flow cytometry, Western blotting, and confocal microscopy. BX795 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound caused apoptosis as evidenced by PARP cleavage, the presence of pyknotic nuclei in the TUNEL assay, and fragmented DNA tails in the Comet assay. BX795 inhibits Akt and NF-κB signaling, arrests cells in the mitotic phase, and increases generation of autophagy in oral cancer cells. Interestingly, the antiproliferative activity of BX795 does not correlate with TBK1 protein expression level in OSCC cells. We propose that the TBK1-independet effect is related to mitotic phase arrest. Pleiotropic anticancer activity with relative sparing of normal oral keratinocytes underscores the potential value of BX795 and warrants its further study in oral squamous cell carcinoma therapy.

Design and synthesis of novel anti-tuberculosis agents from the celecoxib pharmacophore.

The identification of compounds with anti-mycobacterial activity within classes of molecules that have been developed for other purposes is a fruitful approach for the development of anti-tuberculosis (TB) agents. In this study we used the scaffold of celecoxib which exhibits several activities against different pathogens, for the design and focused synthesis of a library of 64 compounds. For the primary screen, we used a bioluminescence-based method by constructing a luciferase-expressing reporter M.tb strain which contains the entire bacterial Lux operon cloned in a mycobacterial integrative expression vector. Through the screening of this library, we identified 6 hit compounds with high in vitro anti-mycobacterial activity (IC50 ∼0.18-0.48 µM). In particular, compounds 41, 51 and 53 were capable of inhibiting M.tb as effectively as the anti-TB drug isoniazid (INH) at 5 µM over a 72-h period, as analyzed by both bioluminescence- and colony forming unit (CFU)-based assays. All hit compounds also showed anti-M.tb activities against several multi-drug-resistant (MDR) strains. Most of the hit compounds showed no cytotoxicity for human macrophages at concentrations as high as 40 µM, setting the stage for further optimization and development of these anti-TB hit compounds both ex vivo and in vivo.

Exploitation of the ability of γ-tocopherol to facilitate membrane co-localization of Akt and PHLPP1 to develop PHLPP1-targeted Akt inhibitors.

Previously, we reported that Akt inactivation by γ-tocopherol (2) in PTEN-negative prostate cancer cells resulted from its unique ability to facilitate membrane co-localization of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase isoform 1), a Ser473-specific Akt phosphatase, through pleckstrin homology (PH) domain binding. This finding provided a basis for exploiting 2 to develop a novel class of PHLPP1-targeted Akt inhibitors. Here, we used 3 (γ-VE5), a side chain-truncated 2 derivative, as a scaffold for lead optimization. The proof-of-concept of this structural optimization was obtained by 20, which exhibited higher antitumor efficacy than 3 in PTEN-negative cancer cells through PHLPP1-facilitated Akt inactivation. Like 3, 20 preferentially recognized the PH domains of Akt and PHLPP1, as its binding affinities for other PH domains, including those of ILK and PDK1, were an order-of-magnitude lower. Moreover, 20 was orally active in suppressing xenograft tumor growth in nude mice, which underlines the translational potential of this new class of Akt inhibitor in PTEN-deficient cancers.

Antitumor effects of BI-D1870 on human oral squamous cell carcinoma.

PURPOSE: Among the signaling pathways implicated in the tumorigenesis of oral squamous cell carcinoma (OSCC) is the extracellular signal-regulated kinase mitogen-activated protein kinase pathway, a downstream target of which is a family of serine/threonine kinases known as the 90 kDa ribosomal S6 kinases (RSKs). This study aims to investigate the role of BI-D1870, a specific inhibitor of p90 RSKs, in a panel of OSCC cell lines. METHODS: The antitumor effects and mechanisms of BI-D1870 were assessed by MTT assays, flow cytometry, Western blotting, transfection, and confocal microscopy. RESULTS: BI-D1870 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound inhibited the downstream RSK target YB-1 and caused apoptosis as evidenced by PARP cleavage, activation of the caspase cascade, and the presence of pyknotic nuclei in the 4,6-diamidino-2-phenylindole assay. In addition, BI-D1870 also induced G2/M arrest by modulating the expression of p21 and other cell cycle regulators. Other newly discovered anticancer attributes of BI-D1870 included the generation of reactive oxygen species and increases in endoplasmic reticulum stress and autophagy. CONCLUSIONS: Together, these results suggest the translational value of BI-D1870 in oral squamous cell carcinoma therapy.

Development of p21 activated kinase-targeted multikinase inhibitors that inhibit thyroid cancer cell migration.

CONTEXT: The p21 activated kinases (PAKs) are a family of serine/threonine kinases that are downstream effectors of small GTPase Cdc42 and Rac. PAKs regulate cell motility, proliferation, and cytoskeletal rearrangement. PAK isoform expression and activity have been shown to be enhanced in cancer and to function as an oncogene in vivo. PAKs also have been implicated in cancer progression. OBJECTIVE: In thyroid cancer, we have previously determined that PAK overactivation is common in the invasive fronts of aggressive tumors and that it is functionally involved in thyroid cancer cell motility using molecular inhibitors. We report the development of two new PAK-inhibiting compounds that were modified from the structure OSU-03012, a previously identified multikinase inhibitor that competitively blocks ATP binding of both phosphoinositide-dependent kinase 1 (PDK1) and PAK1. RESULTS: Seventeen compounds were created by combinatorial chemistry predicted to inhibit PAK activity with reduced anti-PDK1 effect. Two lead compounds were identified based on the ability to inhibit PAK1 activity in an ATP-competitive manner without discernible in vivo PDK1 inhibitory activity in thyroid cancer cell lines. Both compounds reduced thyroid cancer cell viability. Although they are not PAK-specific on a multikinase screening assay, the antimigration activity effect of the compounds in thyroid cancer cells was rescued by overexpression of a constitutively active PAK1, suggesting this activity is involved in this biological effect. CONCLUSIONS: We have developed 2 new multikinase inhibitors with anti-PAK activity that may serve as scaffolds for further compound development targeting this progression-related thyroid cancer target.

Vitamin E facilitates the inactivation of the kinase Akt by the phosphatase PHLPP1.

Vitamin E is a fat-soluble vitamin with antioxidant properties. Tocopherols are the predominant form of vitamin E found in the diet and in supplements and have garnered interest for their potential cancer therapeutic and preventive effects, such as the dephosphorylation of Akt, a serine/threonine kinase with a pivotal role in cell growth, survival, and metabolism. Dephosphorylation of Akt at Ser473 substantially reduces its catalytic activity and inhibits downstream signaling. We found that the mechanism by which α-tocopherol and γ-tocopherol facilitate this site-specific dephosphorylation of Akt was mediated through the pleckstrin homology (PH) domain-dependent recruitment of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1) to the plasma membrane. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain-containing proteins, which may underlie the preferential recruitment of these proteins to membranes containing tocopherols. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. By describing a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, we provide insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors.

Development of novel antibacterial agents against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health because of its resistance to multiple antibiotics most commonly used to treat infection. In this study, we report the unique ability of the cyclooxygenase-2 (COX-2) inhibitor celecoxib to kill Staphylococcus aureus and MRSA with modest potency. We hypothesize that the anti-Staphylococcus activity of celecoxib could be pharmacologically exploited to develop novel anti-MRSA agents with a distinct mechanism. Examination of an in-house, celecoxib-based focused compound library in conjunction with structural modifications led to the identification of compound 46 as the lead agent with high antibacterial potency against a panel of Staphylococcus pathogens and different strains of MRSA. Moreover, this killing effect is bacteria-specific, as human cancer cells are resistant to 46. In addition, a single intraperitoneal administration of compound 46 at 30 mg/kg improved the survival of MRSA-infected C57BL/6 mice. In light of its high potency in eradicating MRSA in vitro and its in vivo activity, compound 46 and its analogues warrant continued preclinical development as a potential therapeutic intervention against MRSA.

Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells.

Despite recent advances in the clinical evaluation of various poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer (TNBC) patients, data defining potential anti-tumor mechanisms beyond PARP inhibition for these agents are lacking. To address this issue, we investigated the effects of four different PARP inhibitors (AG-014699, AZD-2281, ABT-888, and BSI-201) in three genetically distinct TNBC cell lines (MDA-MB-468, MDA-MB-231, and Cal-51). Assays of cell viability and colony formation and flow cytometric analysis were used to determine effects on cell growth and cell cycle progression. PARP-dependent and -independent signaling mechanisms of each PARP inhibitor were investigated by western blotting and shRNA approaches. Potential synergistic interactions between PARP inhibitors and cisplatin in suppressing TNBC cell viability were assessed. These PARP inhibitors exhibited differential anti-tumor activities, with the relative potencies of AG-014699 > AZD-2281 > ABT-888 > BSI-201. The higher potencies of AG-014699 and AZD-2281 were associated with their effects on G(2)/M arrest and DNA damage as manifested by γ-H2AX formation and, for AG-014699, its unique ability to suppress Stat3 phosphorylation. Abilities of individual PARP inhibitors to sensitize TNBC cells to cisplatin varied to a great extent in a cell context- and cell line-specific manner. Differential activation of signaling pathways suggests that the PARP inhibitors currently in clinical trials have different anti-tumor mechanisms beyond PARP inhibition and these PARP-independent mechanisms warrant further investigation.

Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline derivatives, synthesis and biological activity.

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.

Design, synthesis, and biological evaluation of novel water-soluble N-mustards as potential anticancer agents.

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xenografts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa' at the maximum tolerable dose with relatively low toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa' in rats was also investigated. The current studies suggest that this agent is a promising candidate for preclinical studies.

Water mediated construction of trisubstituted pyrazoles/isoxazoles library using ketene dithioacetals.

A small molecule library of alkyl, sulfone, and carboxamide functionalized pyrazoles and isoxazoles has been developed via a rapid sequential condensation of various alpha-acylketene dithioacetals (1a-o) with hydrazine hydrate or hydroxylamine hydrochloride, followed by oxidation of sulfide to sulfone using water as the reaction medium. An efficient and safe oxidation of sulfides (4/5a-o) to the corresponding sulfones (6/7a-o) using sodium per borate system in aqueous medium is reported. The concise and two step synthesis of trisubstituted pyrazoles and isoxazoles was investigated under variety of reaction condition. The newly developed methodology has the advantage of excellent yield and chemical purity with short reaction time using water as a solvent.

Potent antitumor bifunctional DNA alkylating agents, synthesis and biological activities of 3a-aza-cyclopenta[a]indenes.

A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-8H-3a-azacyclopenta[a]indene-1-yl derivatives were synthesized for antitumor evaluation. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and antitumor therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little or no cross-resistance to either Taxol or Vinblastine. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 13a,b and 14g,h and significant suppression against prostate adenocarcinoma PC3 xenograft by 13b were achieved at the maximum tolerable dose with relatively low toxicity. In addition, these agents induce DNA interstrand cross-linking and substantial G2/M phase arrest in human non-small lung carcinoma H1299 cells. The current studies suggested that these agents are promising candidates for preclinical studies.

Novel DNA-directed alkylating agents: design, synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker.

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC(50) in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.

Synthesis and in vitro cytotoxicity of 9-anilinoacridines bearing N-mustard residue on both anilino and acridine rings.

A series of 9-anilinoacridines having an alkylating N-mustard pharmacophore on both anilino (C-3' or C-4') and acridine (C-4) rings with O-ethyl (O-C(2)) or O-butyl (O-C(4)) spacer were synthesized to evaluate their cytotoxicity against human lymphoblastic leukemia (CCRF-CEM) cell growth in vitro. It was revealed that these conjugates exhibited significant in vitro cytotoxicity. Among these agents, compound 13 was the most cytotoxic with IC(50) value of 1.3 nM and is as potent as taxol (IC(50)=1.1 nM). The structure-activity relationship study showed that the length of the spacer and the position of the substituent do affect their cytotoxicity.

Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage.

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.