Functionalized azirine based scaffolds as endothelin inhibitors for the selective anti-angiogenic activity.

Anti-angiogenic therapy has long been used as an adjunct therapy for the resolution of tumor burden. The current findings describe the synthesis of novel marine-based azirine-containing compounds that exhibit anti-angiogenic mediated anti-tumor activity. Azirine-2-carboxylate inhibited HUVEC-mediated tubulogenesis without causing cell death in a dose-dependent manner. Ex-vivo CAM, in-vivo Matrigel implantation, and ear angiogenesis experiments have all shown that azirine-2-carboxylate effectively inhibits angiogenesis. Furthermore, azirine-2-carboxylate inhibits the migration of ECs without disrupting the preformed tubule network. Azirine-2-carboxylate had adequate intramuscular systemic exposure and inhibited tumor growth in a xenograft mouse model. DARTS analysis, competitive binding assay, and gene expression investigations revealed that azirine-2-carboxylate inhibits endothelin-1-mediated angiogenesis. Overall, the discovery of azirine-2-carboxylate demonstrated a potent inhibition of angiogenesis targeting ET1 and a possible application in anti-angiogenic therapy.

Novel diaryl-2H-azirines: Antitumor hybrids for dual-targeting tubulin and DNA.

Multiple-target drugs may achieve better therapeutic effect via different pathways than single-target ones, especially for complex diseases. Tubulin and DNA are well-characterized molecular targets for anti-cancer drug development. A novel class of diaryl substituted 2H-azirines were designed based on combination of pharmacophores from Combretastatin A-4 (CA-4) and aziridine-type alkylating agents, which are known tubulin polymerization inhibitor and DNA damaging agents, respectively. The antitumor activities of these compounds were evaluated in vitro and 6h showed the most potent activities against four cancer cell lines with IC50 values ranging from 0.16 to 1.40 µM. Further mechanistic studies revealed that 6h worked as a bifunctional agent targeting both tubulin and DNA. In the nude mice xenograft model, 6h significantly inhibited the tumor growth with low toxicity, demonstrating the promising potential for further developing novel cancer therapy with a unique mechanism.

Synthesis of Novel Diaziridinyl Quinone Isoxazole Hybrids and Evaluation of Their Anti-Cancer Activity as Potential Tubulin-Targeting Agents.

Two series of diaziridinyl quinone isoxazole derivatives were prepared and evaluated for their cytotoxic activity against MCF7, HeLa, BT549, A549 and HEK293 cell lines and interaction with tubulin. Compounds (6A-M: ) showed promising activity against all the 5 human cancer cell lines. Compounds 6A: , 6E: and 6 M: were potent [IC50 ranging between 2.21 µg to 2.87 µg] on ER-positive MCF7 cell line similar to the commercially available drug molecule Doxorubicin. The results from docking models are in consistent with the experimental values which demonstrated the favourable binding modes of compounds 6A-M: to the interface of α- and ß-tubulin dimer.

Cationic Amphiphilic Tris-Cyclometalated Iridium(III) Complexes Induce Cancer Cell Death via Interaction with Ca2+-Calmodulin Complex.

In our previous paper, we reported on the preparation of some cationic amphiphilic Ir complexes (2c, 2d) containing KKGG peptides that induce and detect cell death of Jurkat cells. Mechanistic studies suggest that 2c interacts with anionic molecules and/or membrane receptors on the cell surface to trigger an intracellular Ca2+ response, resulting in the induction of cell death, accompanied by membrane disruption. We have continued the studies of cell death of Jurkat cells induced by 2c and found that xestospongin C, a selective inhibitor of an inositol 1,4,5-trisphosphate receptor located on the endoplasmic reticulum (ER), reduces the cytotoxicity of 2c, suggesting that 2c triggers the release of Ca2+ from the ER, leading to an increase in the concentration of cytosolic Ca2+, thus inducing cell death. Moreover, we synthesized a series of new amphiphilic cationic Ir complexes 5a-c containing photoreactive 3-trifluoromethyl-3-phenyldiazirine (TFPD) groups, in an attempt to identify the target molecules of 2c. Interestingly, it was discovered that a TFPD group functions as a triplet quencher of Ir complexes. It was also found that 5b is useful as a turn-on phosphorescent probe of acidic proteins such as bovine serum albumin (BSA) (pI = 4.7) and their complexation was confirmed by luminescence titrations and SDS-PAGE of photochemical products between them. These successful results allowed us to carry out photoaffinity labeling of the target biomolecules of 5b (2c and analogues thereof) in Jurkat cells. A proteomic analysis of the products obtained by the photoirradiation of 5b with Jurkat cells suggests that the Ca2+-binding protein "calmodulin (CaM)" is one of target proteins of the Ir complexes. Indeed, 5b was found to interact with the Ca2+-CaM complex, as evidenced by luminescence titrations and the results of photochemical reactions of 5b with CaM in the presence of Ca2+ (SDS-PAGE). A plausible mechanism for cell death induced by a cationic amphiphilic Ir complex is discussed on the basis of our results.

Two new compounds from an Indonesian sponge Dysidea sp.

On our joint bioprospecting research on Indonesian marine invertebrates, we found moderate cytotoxicity on an extract of the sponge Dysidea sp. collected at Biak, West Papua. Separation of the extract provided two new compounds, biaketide (1) and debromoantazirine (2), along with four known molecules 3-6. The new structures were elucidated by spectroscopic analyses and by comparison with those reported. Compounds 1 and 2 showed moderate cytotoxicity against NBT-T2 cells with IC50 values of 8.3 and 4.7 µg ml(- 1), respectively.

Novel chiral ferrocenylpyrazolo[1,5-a][1,4]diazepin-4-one derivatives--synthesis, characterization and inhibition against lung cancer cells.

A series of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one derivatives with optical activity (2) was synthesized in the microwave-assisted condition and characterized by means of IR, (1)H NMR and mass spectroscopy, and furthermore confirmed by X-ray analysis of a representative compound (R)-2a. Preliminary biological evaluation showed that some compounds could suppress the growth of A549, H322 and H1299 lung cancer cells. Among the tested compounds, 2b-d were more effective and might perform their action through cell cycle arrest for A549 cell. Whereas these compounds inhibited growth of H1299 and H322 cells by inducing apoptosis. The anti-tumor activities of these compounds were related to the nature of substituents in benzene moiety. In addition, the results indicated also that compounds 2b-d possessed notable cytotoxicity and selectivity for A549 vs H1299 and H322 lung cancer cells.

Synthesis of a potent photoreactive acidic γ-secretase modulator for target identification in cells.

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aß fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.

Selective inhibition of BET bromodomains.

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Motualevic acids A-F, antimicrobial acids from the sponge Siliquariaspongia sp.

Seven new antibacterials, motualevic acids A-F (1-6) and (4E)-(R)-antazirine (7), have been isolated from the marine sponge Siliquariaspongia sp. and their structures elucidated by spectroscopic methods. Motualevic acids A-D are the first glycyl conjugates of the omega-brominated lipid (E)-14,14-dibromotetradeca-2,13-dienoic acid, and motualevic acid F is the first long-chain 2H-azirine 2-carboxylic acid to be found in nature. Carboxylic acid-containing compounds 1 and 6 inhibit the growth of Staphylococcus aureus and methicillin-resistant S. aureus at 1.2-10.9 microg/mL.

Synthesis and antifungal activity of (-)-(Z)-dysidazirine.

A short, flexible synthesis of the marine natural product (2 R)-(Z)-dysidazirine (-)-1 has been completed. (-)-1 shows significant antifungal activity across a panel of seven human pathogens, whereas the structural analogue (-)-2, featuring a terminal tert-butyl group, is essentially inactive.

Long-chain 2H-azirines with heterogeneous terminal halogenation from the marine sponge Dysidea fragilis.

Three new omega-halogenated long-chain 2H-azirines were isolated from the sponge Dysidea fragilis. Their structures revealed heterogeneity in both the composition of the terminal 1,1-dihalo-vinyl group and enantiomeric ratios at C2 of the azirine-2-carboxylate ester terminus. Azirine-2-carboxylate esters were shown to racemize spontaneously. A hypothesis is proposed for the biosynthesis of the azirinecarboxylate family of natural products that involves enzyme-catalyzed free radical halogenation followed by elimination of hydrohalic acid.

Array synthesis of progesterone receptor antagonists: 3-aryl-1,2-diazepines.

New non-steroidal chemotypes are required for the development of drugs targeting the steroid hormone receptors. The parallel array synthesis of 3-aryl-1,2-diazepines employing solid-supported reagents is described. The resulting compounds demonstrated high affinity binding to the progesterone receptor.

Synthesis of analogs of (1,4)-3- and 5-imino oxazepane, thiazepane, and diazepane as inhibitors of nitric oxide synthases.

A series of 3- and 5-imino analogs from oxazepane, thiazepane, and diazepane was prepared and evaluated as inhibitors of human nitric oxide synthesis (NOS). The most potent iNOS inhibitor was the thiazepane analog 25 (IC(50) = 0.19 microM).

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

1-Azaadamantanes: pharmacological applications and synthetic approaches.

1-Azaadamantane (1-azatricyclo [3.3.1.1(3,7)]decane) was synthesized in 1953, and the derivatives have been used as rigid models for studies on intramolecular charge transfer phenomena, fluorescence excitation Rydberg states, highly twisted amides, solid electrolyte gas sensors, basicities, and self-organization systems. These structures have also been attracting considerable interest due to their pharmacological activities. The substituted 1-azaadamantanes as conformationally restricted amines have great potential for the therapeutic utilization as anticholinergic agents, serotonergic agents, and squalene synthase inhibitors. However, many steps have been needed for the synthesis of 1-azaadamantanes, and the concise synthetic approaches have been developed. Though double or triple Mannich reactions yield 1-azaadamantanes in moderate yields, the reduction steps are necessary. Our recent research has revealed that trifluoromethanesulfonic anhydride is available for the convenient synthesis of 1-azoniaadamantanes and 1-azaadamantanes without reduction. The new tools for the discovery of novel drugs such as quantitative structure-activity relationship (QSAR) analysis and vibrational circular dichroism (VCD) spectroscopy have also been discussed.

Identification of signal peptide peptidase, a presenilin-type aspartic protease.

Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of some signal peptides after they have been cleaved from a preprotein. In humans, SPP activity is required to generate signal sequence-derived human lymphocyte antigen-E epitopes that are recognized by the immune system, and to process hepatitis C virus core protein. We have identified human SPP as a polytopic membrane protein with sequence motifs characteristic of the presenilin-type aspartic proteases. SPP and potential eukaryotic homologs may represent another family of aspartic proteases that promote intramembrane proteolysis to release biologically important peptides.

Microwave assisted solid support synthesis of novel 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazepines as potent antimicrobial agents.

An environmentally benign and economic synthesis of 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazepines is described from readily accessible substituted 2-mercapto-1-amino triazoles and substituted chalcones on basic alumina that are accelerated by exposure to microwaves. The reaction time has been brought down from hours to seconds with improved yield as compared to conventional heating. The method reported herein is devoid of the hazards of solution phase reactions. All the synthesised compounds were tested for their in vitro antibacterial and antifungal activity. Some compounds showed significant antimicrobial properties. The best activity was observed with compounds 3a, 3c, 4a and 4d.

Site of resting state inhibition of the nicotinic acetylcholine receptor by a hydrophobic inhibitor.

The lipophilic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) is a noncompetitive, resting-state inhibitor of the nicotinic acetylcholine receptor (nAChR) that requires tens of milliseconds of preincubation to inhibit agonist-induced cation efflux. At equilibrium, [(125)I]TID photoincorporates into both the ion channel and the lipid-protein interface of the Torpedo nAChR. To determine which of these regions is responsible for resting-state inhibition, we characterized the interactions between [(125)I]TID and nAChR-rich membranes milliseconds after mixing, by use of time-resolved photolabeling. Photolabeling was performed after preincubation times of 2 ms or 600 s (equilibrium), and the efficiencies of incorporation at specific residues were determined by amino-terminal sequence analysis of nAChR-subunit proteolytic fragments isolated by SDS-PAGE and/or reversed-phase HPLC. Equilibration of TID with lipid was complete within a millisecond as determined by both stopped-flow fluorescence quenching of diphenylhexatriene in lipid bilayers and photoincorporation into nAChR-rich membrane phospholipids. Equilibration with the lipid-protein interface (alphaM4) was slightly slower, reaching approximately 50% that at equilibrium after 2 ms preincubation. In contrast, equilibration with the channel region (alpha 2 and deltaM2) was much slower, reaching only 10% that at equilibrium after 2 ms preincubation. Within the ion channel, the ratio of [(125)I]TID incorporation between M2 residues 9', 13', and 16' was independent of preincubation time. We conclude that TID's access to the ion channel is more restricted than to the lipid-protein interface and that TID bound within the ion channel is responsible for flux inhibition upon activation of the nAChR.

Structural differences between foot-and-mouth disease and poliomyelitis viruses influence their inactivation by aziridines.

Inactivation of foot-and-mouth disease virus (FMDV) and poliovirus by ethyleneimine (EI) and N-acetylethyleneimine (AEI) has been studied at 25 degrees and at 37 degrees C and in different ionic conditions. FMDV is inactivated rapidly in 100 mM Tris pH 7.6 by each reagent at both temperatures. Poliovirus is also inactivated rapidly in 100 mM Tris by EI at both temperatures and by AEI at 37 degrees C. However, it is inactivated much more slowly by AEI at 25 degrees C; but if the virus is first incubated overnight at 2 degrees C with AEI before transferring to 25 degrees C inactivation then proceeds rapidly. Moreover, the rate of inactivation at 25 degrees C is markedly increased if the virus is suspended in 1 mM Tris. We had interpreted these differences as being due to the greater penetrability of poliovirus (i) in 100 mM Tris at 37 degrees C compared with 25 degrees C and (ii) at lower ionic strength. This interpretation has been confirmed by electron microscopy of FMDV and poliovirus particles stained with phosphotungstic acid. At the elevated temperature, poliovirus had an average diameter of 34+/-0. 21 nm and the stain outlined the nucleic acid core and the individual subunits, whereas at 25 degrees C it averaged 28+/-0.13 nm and the stain did not penetrate the particle. This study also showed that the particle diameter alters with changes in buffer concentration, being 28+/-0.13 nm in 100 mM Tris, 31+/-0.16 nm in 10 mM Tris and 34+/-0.21 nm in 1 mM Tris. The changes in poliovirus are reversible as addition of 1/10 volume of 1 M Tris to the virus in 1 mM Tris resulted in the return of the diameter to 28+/-0.13 nm. FMDV, on the other hand, was less sensitive to osmotic differences as its particle diameter only varied by 7% over the 100-fold change in buffer concentration compared with the 22% change observed for poliovirus.

A chlorodiazirine analog of pentamidine with anti-trypanosomal activity.

A chlorodiazirine derivative of pentamidine was synthesized and tested for anti-trypanosomal activity using EATRO stock 164 trypanosomes in cell culture. Anti-trypanosomal activity was measured as a decrease in [3H]hypoxanthine incorporation by the organisms. The derivative, 3,3'-[1,5-pentanediylbis(oxy-4,1-phenylene)]bis(3-chloro-3H-diazir ine), at a treatment level of 0.1 microM inhibited isotope incorporation by 40-50% compared to nontreated controls. At this concentration, pentamidine inhibited incorporation only 10-15%. The derivative is a nonionic molecule with much different solubility properties than the parent compound and should readily cross the blood-brain barrier.