Biological evaluation of [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone as potential antineoplastic agent in 2D and 3D breast cancer models.

Targeting tubulin polymerization and depolymerization represents a promising approach to treat solid tumors. In this study, we investigated the molecular mechanisms underlying the anticancer effects of a structurally novel tubulin inhibitor, [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone (ARDAP), in two- and three-dimensional MCF-7 breast cancer models. At sub-cytotoxic concentrations, ARDAP showed a marked decrease in cell proliferation, colony formation, and ATP intracellular content in MCF-7 cells, by acting through a cytostatic mechanism. Additionally, drug exposure caused blockage of the epithelial-to-mesenchymal transition (EMT). In 3D cell culture, ARDAP negatively affected tumor spheroid growth, with inhibition of spheroid formation and reduction of ATP concentration levels. Notably, ARDAP exposure promoted the differentiation of MCF-7 cells by inducing: (i) expression decrease of Oct4 and Sox2 stemness markers, both in 2D and 3D models, and (ii) downregulation of the stem cell surface marker CD133 in 2D cell cultures. Interestingly, treated MCF7 cells displayed a major sensitivity to cytotoxic effects of the conventional chemotherapeutic drug doxorubicin. In addition, although exhibiting growth inhibitory effects against breast cancer cells, ARDAP showed insignificant harm to MCF10A healthy cells. Collectively, our results highlight the potential of ARDAP to emerge as a new chemotherapeutic agent or adjuvant compound in chemotherapeutic treatments.

Novel N-(Heterocyclylphenyl)benzensulfonamide Sharing an Unreported Binding Site with T-Cell Factor 4 at the ß-Catenin Armadillo Repeats Domain as an Anticancer Agent.

Despite intensive efforts, no inhibitors of the Wnt/ß-catenin signaling pathway have been approved so far for the clinical treatment of cancer. We synthesized novel N-(heterocyclylphenyl)benzenesulfonamides as ß-catenin inhibitors. Compounds 5-10 showed strong inhibition of the luciferase activity. Compounds 5 and 6 inhibited the MDA-MB-231, HCC1806, and HCC1937 TNBC cells. Compound 9 induced in vitro cell death in SW480 and HCT116 cells and in vivo tumorigenicity of a human colorectal cancer line HCT116. In a co-immunoprecipitation study in HCT116 cells transfected with Myc-tagged T-cell factor 4 (Tcf-4), compound 9 abrogated the association between ß-catenin and Tcf-4. The crystallographic analysis of the ß-catenin Armadillo repeats domain revealed that compound 9 and Tcf-4 share a common binding site within the hotspot binding region close to Lys508. To our knowledge, compound 9 is the first small molecule ligand of this region to be reported. These results highlight the potential of this novel class of ß-catenin inhibitors as anticancer agents.

Rimonabant-Based Compounds Bearing Hydrophobic Amino Acid Derivatives as Cannabinoid Receptor Subtype 1 Ligands.

In this study, 1H-pyrazole-3-carboxylic acids related to the cannabinoid type 1 (CB1) receptor antagonist rimonabant were amidated with valine or tert-leucine, and the resulting acids were further diversified as methyl esters, amides, and N-methyl amides. In vitro receptor binding and functional assays demonstrated a wide series of activities related to the CB1 receptors (CB1Rs). Compound 34 showed a high CB1R binding affinity (K i = 6.9 nM) and agonist activity (EC50 = 46 nM; E max = 135%). Radioligand binding and [35S]GTPγS binding assays also demonstrated its selectivity and specificity to CB1Rs. Moreover, in vivo experiments revealed that 34 was slightly more effective than the CB1 agonist WIN55,212-2 in the early phase of the formalin test, indicating a short duration of the analgesic effect. Interestingly, in a mouse model of zymosan-induced hindlimb edema, 34 was able to maintain the percentage of paw volume below 75% for 24 h following subcutaneous injection. After intraperitoneal administration, 34 increased the food intake of mice, suggesting potential activity on CB1Rs.

RS6077 induces mitotic arrest and selectively activates cell death in human cancer cell lines and in a lymphoma tumor in vivo.

We synthesized a new inhibitor of tubulin polymerization, the pyrrole (1-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxy-phenyl)methanone 6 (RS6077). Compound 6 inhibited the growth of multiple cancer cell lines, with IC50 values in the nM range, without affecting the growth of non-transformed cells. The novel agent arrested cells in the G2/M phase of the cell cycle in both transformed and non-transformed cell lines, but single cell analysis by time-lapse video recording revealed a remarkable selectivity in cell death induction by compound 6: in RPE-1 non-transformed cells mitotic arrest induced was not necessarily followed by cell death; in contrast, in HeLa transformed and in lymphoid-derived transformed AHH1 cell lines, cell death was effectively induced during mitotic arrest in cells that fail to complete mitosis. Importantly, the agent also inhibited the growth of the lymphoma TMD8 xenograft model. Together these findings suggest that derivative 6 has a selective efficacy in transformed vs non-transformed cells and indicate that the same compound has potential as novel therapeutic agent to treat lymphomas. Compound 6 showed good metabolic stability upon incubation with human liver microsomes.

Discovery of a Novel Class of Norovirus Inhibitors with High Barrier of Resistance.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting in ~219,000 deaths annually and a societal cost of ~USD60 billion. There are no antivirals or vaccines available to treat and/or prevent HuNoV. In this study, we performed a large-scale phenotypical antiviral screening using the mouse norovirus (MNV), which included ~1000 drug-like small molecules from the Drug Design and Synthesis Centre (Sapienza University, Rome). Compound 3-((3,5-dimethylphenyl)sulfonyl)-5-chloroindole-N-(phenylmethanol-4-yl)-2.carboxamide (compound 1) was identified as an inhibitor of MNV replication with an EC50 of 0.5 ± 0.1 µM. A series of 10 analogs were synthesized of which compound 6 showed an improved potency/selectivity (EC50 0.2 ± 0.1 µM) against MNV; good activity was also observed against the HuNoV GI replicon (EC50 1.2 ± 0.6 µM). Time-of-drug-addition studies revealed that analog 6 acts at a time point that coincides with the onset of viral RNA replication. After six months of selective pressure, two compound 6res variants were independently selected, both harboring one mutation in VPg and three mutations in the RdRp. After reverse engineering S131T and Y154F as single mutations into the MNV backbone, we did not find a markedly compound 6res phenotype. In this study, we present a class of novel norovirus inhibitors with a high barrier to resistance and in vitro antiviral activity.

Discovery of pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia.

Long-term survivors of glioblastoma multiforme (GBM) are at high risk of developing second primary neoplasms, including leukemia. For these patients, the use of classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate, is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. We aimed to develop agents useful for the treatment of patients with GBM and chronic myeloid leukemia (CML) using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization. Compounds 7 and 25, as planned, not only inhibited tubulin polymerization, but also inhibited the proliferation of both GMB and CML cells, including those expressing the T315I mutation, at nanomolar concentrations. In in vivo experiments in BALB/cnu/nu mice injected subcutaneously with U87MG cells, in vivo, 7 significantly inhibited GBM cancer cell proliferation, in vivo tumorigenesis, and tumor growth, tumorigenesis and angiogenesis. Compound 7 was found to block human topoisomerase II (hTopoII) selectively and completely, at a concentration of 100 µM.