An Easy Route to Aziridine Ketones and Carbinols.

N,N-Dimethylaziridine-2-carboxamides react with organolithium reagents yielding 2-aziridinylketones. The reaction with one equivalent of organolithium compound is selective to amide carbonyl at a low (-78 °C) temperature. These ketones, in reaction with organolithium reagents, give symmetrical and unsymmetrical aziridinyl carbinols. The usage of excess phenyllithium may serve as a special N-Boc-protecting group cleavage method for acid-sensitive substrates.

3-Arylaziridine-2-carboxylic Acid Derivatives and (3-Arylaziridin-2-yl)ketones: The Aziridination Approaches.

Aziridination reactions represent a powerful tool in aziridine synthesis. Significant progress has been achieved in this field in the last decades, whereas highly functionalized aziridines including 3-arylated aziridine-2-carbonyl compounds play an important role in both medical and synthetic chemistry. For the reasons listed, in the current review we have focused on the ways to obtain 3-arylated aziridines and on the recent advances (mainly since the year 2000) in the methodology of the synthesis of these compounds via aziridination.

Bicyclic Boronates as Potent Inhibitors of AmpC, the Class C ß-Lactamase from Escherichia coli.

Resistance to ß-lactam antibacterials, importantly via production of ß-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) ß-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C ß­lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied ß-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D ß­lactamases, our data support the proposal that bicyclic boronates are broad-spectrum ß­lactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful ß-lactamase inhibition.

Broad Spectrum ß-Lactamase Inhibition by a Thioether Substituted Bicyclic Boronate.

ß-Lactamases comprise the most widely used mode of resistance to ß-lactam antibiotics. Cyclic boronates have shown promise as a new class of ß-lactamase inhibitor, with pioneering potential to potently inhibit both metallo- and serine-ß-lactamases. We report studies concerning a bicyclic boronate ester with a thioether rather than the more typical ß-lactam antibiotic "C-6/C-7" acylamino type side chain, which is present in the penicillin/cephalosporin antibiotics. The thioether bicyclic boronate ester was tested for activity against representative serine- and metallo-ß-lactamases. The results support the broad inhibition potential of bicyclic boronate based inhibitors with different side chains, including against metallo-ß-lactamases from B1, B2, and B3 subclasses. Combined with previous crystallographic studies, analysis of a crystal structure of the thioether inhibitor with the clinically relevant VIM-2 metallo-ß-lactamase implies that further SAR work will expand the already broad scope of ß-lactamase inhibition by bicyclic boronates.

Wedelolactone Targets EZH2-mediated Histone H3K27 Methylation in Mantle Cell Lymphoma.

BACKGROUND/AIM: Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), possesses histone N-methyltransferase (HMT) activity and plays an essential role in cancer initiation and development. The aim of the present study was to investigate the potential of Wedelolactone (WL) to inhibit the methylation activity of EZH2. MATERIALS AND METHODS: The mantle cell lymphoma (MCL) cell line, Mino, was treated with WL, while untreated cells were used as control. HMT activity and EZH2 amount were measured in nuclear extracts from WL-treated and control Mino cells. RESULTS: WL was found to target EZH2-mediated histone H3K27 methylation. Along with the inhibition of H3K27 methylation in vitro (IC50=0.3 µM), WL suppressed HMT activity in Mino cells with an IC50 value of 3.2 µM. We detected a reduced amount of EZH2 in Mino cells treated with WL, compared to untreated control cells. CONCLUSION: This is the first study to show that WL induces inhibition of H3K27 methylation via EZH2 modulation and decreases cell proliferation in MCL, in vitro. WL is proposed as a promising agent and a novel epigenetic approach in MCL investigation and treatment.

Pyrazole-based inhibitors of enhancer of zeste homologue 2 induce apoptosis and autophagy in cancer cells.

Novel pyrazole-based EZH2 inhibitors have been prepared through a molecular pruning approach from known inhibitors bearing a bicyclic moiety as a central scaffold. The hit compound 1o (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxamide) showed low micromolar EZH2/PRC2 inhibition and high selectivity towards a panel of other methyltransferases. Moreover, 1o displayed cell growth arrest in breast MDA-MB231, leukaemia K562, and neuroblastoma SK-N-BE cancer cells joined to reduction of H3K27me3 levels and induction of apoptosis and autophagy.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

The histone methyltransferase EZH2 as a druggable target in SHH medulloblastoma cancer stem cells.

The histone methyltransferase EZH2 plays a role in maintenance of the stem component of cancer, and its overexpression and/or mutation typically drives tumor aggressiveness, drug resistance and patients' poor prognosis. In this study, we use mouse and human medulloblastoma stem-like cells belonging to the Sonic Hedgehog subgroup (SHH MB-SLCs) and demonstrate that genetic suppression of EZH2 reduces the level of its histone mark H3K27me3 and lowers proliferation and self-renewal. We designed an EZH2 inhibitor (EZH2i) as a simplified analog of EPZ005687 and GSK2816126, MC3629, and we tested its biological activity in SHH MB-SLCs. Pharmacological inhibition of EZH2 impairs SHH MB cells proliferation and self-renewal, and induces apoptosis in vitro. Finally, we generated xenograft MB-SLCs orthotopic tumors in nude mice to test MC3629 in vivo. In treated mice, we observed impairment of tumor growth, together with induction of apoptosis and reduction of proliferation and stemness. Overall, these findings describe EZH2 as a druggable target in MB and provide insight into the biological activity of MC3629 as an EZH2i.

Depletion of carbonic anhydrase IX abrogates hypoxia-induced overexpression of stanniocalcin-1 in triple negative breast cancer cells.

Carbonic anhydrase IX (CAIX) is a pH-regulating enzyme that plays a key role in maintaining an alkaline intracellular pH under hypoxic conditions. It is overexpressed in a variety of solid cancers, including breast cancer (BC), and has been implicated in the migration, invasion and stemness of breast cancer cells. Therefore, CAIX recently emerged as a novel therapeutic target for the treatment of BC. To gain an insight into the mechanism of action of CAIX inhibitors, we investigated the impact of CAIX knock-down on the transcriptional response to hypoxia in 2 BC cell lines - MCF7 and MDA-MB-231, by performing a global gene expression analysis. This showed that CAIX knock-down had a relatively minor effect on the global transcriptional response to hypoxia, however it blocked hypoxia-induced upregulation of stanniocalcin-1 (STC1), a secreted glycoprotein that has been shown to promote tumor progression and metastasis in BC. Kaplan-Meier survival analysis showed that high STC1 expression is significantly associated with poor survival in patients with basal-type breast cancer but not luminal A and HER2+ subtypes. Moreover, the association was particularly high in a subgroup of basal-type BC patients with TP53 mutations thus revealing a putative cooperation of STC1 with mutated TP53 in generating highly aggressive BC subgroup. Taken together, these findings show that CAIX inhibitors at least partially act through blocking STC1 induction in BC cells and reveal a subgroup of BC patients, who potentially would benefit most from the treatment with CAIX inhibitors.

A Novel Aziridine-based Bruton's Tyrosine Kinase Inhibitor Induces Apoptosis Through Down-regulation of p65/RelA Phosphorylation on Serine 536 and ERK1/2 in Mantle Cell Lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma characterized by hyperactive neoplastic B-cells and extended tumor cell survival. Bruton's tyrosine kinase (BTK), a crucial kinase in the B-cell antigen receptor signaling pathway, has emerged as a novel target of MCL therapy. A novel BTK-targeting inhibitor, JuSt-23F was prepared. MATERIALS AND METHODS: The WST-8 assay was used to determine cytotoxicity and half-maximal inhibitory concentration (IC50) values for JuSt-23F against the MCL cell lines Mino and Maver-1. JuSt-23F-mediated apoptosis was assessed using the annexin V assay. We detected phosphorylation of p65/RelA on serine 536 in whole Jurkat, Mino and Maver-1 cells treated with JuSt-23F and stimulated with tumor necrosis factor (TNFα). We assessed JuSt-23F-mediated phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in T-cell lymphoma and MCL cells stimulated by phorbol-12-myristate-13-acetate (PMA). RESULTS AND CONCLUSION: Our study suggests that JuSt-23F inhibits apoptosis selectively in B-cell lymphoma cells. JuSt-23F exerts its antiproliferative effects on MCL cells through targeting the downstream BTK signaling cascade via down-regulation of nuclear factor kappa-light-chain-enhancer of activated B-cells and ERK1/2 pathways. Thus, our findings propose the novel BTK inhibitor JuSt-23F as an attractive potential agent for investigation and treatment of MCL.

Structures of plasmepsin II from Plasmodium falciparum in complex with two hydroxyethylamine-based inhibitors.

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome of Plasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence of P. falciparum strains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII-NU655 complex. Structural analysis of the PMII-PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275-Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII-NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1' position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.

Morphological and immunohistochemical profile of pancreatic neuroendocrine neoplasms.

The study represents a comprehensive retrospective morphological and immunohistochemical profiling of pancreatic neuroendocrine neoplasms (PNENs) in order to reveal the associations between morphological and molecular parameters. The local tumour spread (T), presence of metastases in regional lymph nodes (N) and distant organs (M), tumour grade (G) and resection line status (R) by pathology findings (pTNMGR), mitotic activity, perineural, vascular and lymphatic invasion were assessed in 16 surgically resected PNENs. By immunohistochemistry, expression of Ki-67, p53, p27, p21, cyclin D1, Bcl-2, E-cadherin, CD44, vimentin, cyclooxygenase 2 (COX-2), microvascular density, and cytokeratin (CK) spectrum, along with neuroendocrine, intestinal and squamous markers were detected. Descriptive statistics, Chi-square test, Spearman's rank correlation, Mann-Whitney and Kruskal-Wallis methods were applied; p<0.05 was considered significant. Ki-67, CK19, p63, vimentin and COX-2 were significantly up-regulated in PNENs in comparison to benign pancreatic islets. A complex network of morphological and molecular associations was identified. Ki-67 correlated with PNEN size (p=0.022), the World Health Organization 2004 and 2010 classification grades (p=0.021 and p=0.002), stage (p=0.028) and mitotic count (p=0.007) but among molecular markers--with CK19 (p=0.033) and vimentin (p=0.045). CK19 was significantly up-regulated in PNENs, having higher pT (p=0.018), pR (p=0.025), vascular (p=0.020), perineural (p=0.026) and lymphatic invasion (p=0.043). In conclusion, proliferation activity (by Ki-67), E-cadherin, vimentin and CK19 are important molecular characteristics of PNENs due to significant associations with morphological tumour characteristics, pTNMGR and invasive growth.

Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells.

PURPOSE: Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme with extracellular catalytic domain that is overexpressed in a variety of cancers including breast cancer and plays a crucial role in maintaining favourable intracellular pH and reducing extracellular pH. The purpose of the current study was to elucidate the prognostic significance of CAIX in the intrinsic subtypes of breast cancer and to characterise CAIX as a drug target in breast cancer. METHODS: The prognostic significance of CAIX mRNA expression was interrogated in a cohort of 3,455 breast tumours by using an online tool, Kaplan-Meier plotter. The functional effects of stable CAIX depletion by shRNA in three breast cancer cell lines­MDA-MB-231, MCF7 and SKBR-3, representing basal-like, luminal A and HER2+ subtypes, respectively­were studied by proliferation, invasion, clonal spheroid formation and chemosensitivity assays under normoxia and hypoxia. Finally, the effect of pharmacological CA inhibition alone or in the combination with doxorubicin on self-renewal was assessed by spheroid-forming assay. RESULTS: High CAIX mRNA expression was significantly associated with poor survival in patients with basal-like, luminal B and triple-negative breast cancer, but not luminal A and HER+ subtypes. Silencing of CAIX expression had no significant effect on the cell proliferation or viability upon treatment with doxorubicin in any of the cell lines studied, while it inhibited spheroid formation in hypoxic conditions. Furthermore, pharmacological inhibition of CAs using acetazolamide had a synergistic effect with doxorubicin on decreasing the spheroid-forming efficiency in MDA-MB-231 cells. CONCLUSIONS: Inhibition of CAIX reduces the self-renewal capacity of breast cancer cells, and the combination of doxorubicin and CAIX inhibition is an attractive therapeutic strategy in basal-like and triple-negative breast cancer, which warrants further investigations.

5-membered cyclic hydroxamic acids as HDAC inhibitors.

The new histone deacylases inhibitors (HDACi) were synthesized in the class of 5-membered cyclic hydroxamic acids (5-CHA), showing medium size CHA as a new Zn-binding group. New reaction sequence was proposed for the synthesis of 5-membered alkylidene-cyclic-hydroxamic acids starting from butyrolactone. Compound 10c showed low µM activity on HeLa cell extracts. From these results, cyclic hydroxamic acids will be further investigated to find more potent compounds.

Saccharin sulfonamides as inhibitors of carbonic anhydrases I, II, VII, XII, and XIII.

A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA) inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC). Saccharin bound the CAs weakly, exhibiting the affinities of 1-10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1-10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.

Hydrophobic substituents of the phenylmethylsulfamide moiety can be used for the development of new selective carbonic anhydrase inhibitors.

A new series of compounds containing a sulfamide moiety as zinc-binding group (ZBG) has been synthesized and tested for determining inhibitory properties against four human carbonic anhydrase (hCA) isoforms, namely, CAs I, II, IX, and XII. The X-ray structure of the cytosolic dominant isoform hCA II in complex with the best inhibitor of the series has also been determined providing further insights into sulfamide binding mechanism and confirming that such zinc-binding group, if opportunely derivatized, can be usefully exploited for obtaining new potent and selective CAIs. The analysis of the structure also suggests that for drug design purposes the but-2-yn-1-yloxy moiety tail emerges as a very interesting substituent of the phenylmethylsulfamide moiety due to its capability to establish strong van der Waals interactions with a hydrophobic cleft on the hCA II surface, delimited by residues Phe131, Val135, Pro202, and Leu204. Indeed, the complementarity of this tail with the cleft suggests that different substituents could be used to discriminate between isoforms having clefts with different sizes.

Plasmepsin inhibitory activity and structure-guided optimization of a potent hydroxyethylamine-based antimalarial hit.

Antimalarial hit 1 SR (TCMDC-134674) identified in a GlaxoSmithKline cell based screening campaign was evaluated for inhibitory activity against the digestive vacuole plasmepsins (Plm I, II, and IV). It was found to be a potent Plm IV inhibitor with no selectivity over Cathepsin D. A cocrystal structure of 1 SR bound to Plm II was solved, providing structural insight for the design of more potent and selective analogues. Structure-guided optimization led to the identification of structurally simplified analogues 17 and 18 as low nanomolar inhibitors of both, plasmepsin Plm IV activity and P. falciparum growth in erythrocytes.

A novel matrix metalloproteinase-2 inhibitor triazolylmethyl aziridine reduces melanoma cell invasion, angiogenesis and targets ERK1/2 phosphorylation.

A novel matrix metalloproteinase-2 (MMP-2) inhibitor JaZ-30, which belongs to the class of C(2)-monosubstituted aziridine - aryl-1,2,3-triazole conjugates, was developed. MTT and crystal violet assays were used to determine cytotoxicity- IC(50) values of compound JaZ-30 on melanoma cell line B16 4A5. Our study proves the anti-cancer properties of JaZ-30 with a wide spectrum of activities. JaZ-30 was revealed as selective inhibitor of matrix metalloproteinase-2. JaZ-30-mediated decrease of Vascular Endothelial Growth Factor (VEGF) secretion results in inhibition of angiogenesis, performed with the human umbilical vein endothelial cell line (HUVEC-2) on Matrigel. A novel inhibitor decreases invasive properties of melanoma cells measured in Matrigel chambers assay. JaZ-30 downregulates phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in melanoma cells stimulated by phorbol-12-myristate-13-acetate (PMA). Our findings propose a novel MMP-2 inhibitor JaZ-30 as an attractive potential agent for melanoma treatment.

Rat serum carboxylesterase partly hydrolyses gamma-butyrobetaine esters.

Although described some time ago, gamma-butyrobetaine esters and related compounds have not gained much attention from researchers, and their physiological function remains obscure. Formerly we detected GBB-esterase enzymatic activity in rat blood serum using phenylated gamma-butyrobetaine as an artificial substrate of the enzyme and HPLC. The aim of the present work was to develop an assay that would enable spectrophotometric or colorimetric determination of the reaction products of GBB-esterase activity and to reveal individual proteins performing GBB-esterase activity in rat blood serum. For this purpose gamma-butyrobetaine 1-naphthyl ester was synthesised. Hydrolysis of this ester releases 1-naphthol, which increases the optical absorbance at 322 nm. We have shown that the enzymatic hydrolysis of GBB 1-naphthyl ester to 1-naphthol in rat blood serum is due to GBB-esterase activity. An attempt was done to purify the enzyme from rat blood serum. By combining DEAE Sepharose at pH 4.2 and affinity chromatography with procainamide we achieved a 68-fold enrichment of GBB-esterase activity in our preparations. Separation of fraction proteins in 2D protein electrophoresis with following mass-spectrometry indicated that GBB esterase activity in rat blood serum is performed in part by carboxylesterase.