3-(Naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride attenuates NLRP3 inflammasome-mediated signaling pathway in lipopolysaccharide-stimulated BV2 microglial cells.

The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasome is a multiprotein complex with a role in innate immune responses. NLRP3 inflammasome dysfunction is a common feature of chronic inflammatory diseases. Microglia activation is also associated with neuroinflammatory pathologies. We previously reported that 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) reduced hypoxia-induced toxicity by modulating inflammation. However, no studies have elucidated the precise mechanisms for the anti-inflammatory action of KHG26792, in particular via inflammasome mediation. This study investigated the effects of KHG26792 on the inflammasome-mediated signaling pathway in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. KHG26792 significantly attenuated several inflammatory responses including tumor necrosis factor-α, interleukin-1ß, interleukin-6, reactive oxygen species, and mitochondrial potential in these cells. KHG26792 also suppressed LPS-induced increase NLRP3, activated caspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) levels. Furthermore, KHG26792 successfully blocked LPS-activated adenosine triphosphate (ATP) level, likely through the purinergic receptor P2X ligand-gated ion channel 7 (P2X7) receptor. Our results suggest that the anti-inflammatory functions of KHG26792 may be, at least in part, due to regulation of the P2X7R/NLRP3-mediated signaling pathway during microglial activation.

Triazolyl phenyl disulfides: 8-Amino-7-oxononanoate synthase inhibitors as potential herbicides.

The chemical validation of a potential herbicide target was investigated with 8-amino-7-oxononanoate synthase (AONS, also known as 7-keto-8-aminopelargonate synthase, KAPAS) and triazolyl phenyl disulfide derivatives in vitro and in vivo. AONS activity was completely inhibited by these synthesized compounds, with an IC50 of 48 to 592µM in vitro. Forty five-day old Arabidopsis thaliana plants were completely killed by representative compound KHG23844 {N-(2-fluorophenyl)-3-(phenyldisulphanyl)-1H-1,2,4-triazole-1-carboxamide} at the application rate of 250gha(-1) of foliar treatment in greenhouse conditions. Foliar application of 1000gha(-1) KHG23844 induced 2.3-fold higher l-alanine accumulation in the treated A. thaliana plants. Foliar supplement of 1mM biotin at 1 and 2days before KHG23844 application effectively recovered the growth inhibition of A. thaliana plant treated with KHG23844. The results strongly suggested that representative compound KHG23844 and its derivatives are potential AONS inhibitors.

2-Cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against beta-amyloid-induced activation of the apoptotic cascade in cultured cortical neurons.

Aggregated ß-amyloid, implicated in the pathogenesis of Alzheimer's disease (AD), induces neurotoxicity by evoking a cascade of oxidative damage-dependent apoptosis in neurons. We investigated the molecular mechanisms underlying the protective effect of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride (KHG26377) against the beta-amyloid (Aß25-35)-induced primary cortical neuronal cell neurotoxicity. Treatment with KHG26377 attenuated the Aß25-35-induced apoptosis by decreasing the Bax/Bcl-2 ratio and suppressing the activation of caspase-3. A marked increase in calcium influx and in the level of reactive oxygen species together with a decrease in glutathione levels was found after Aß25-35 exposure; however, KHG26377 treatment reversed these changes in a concentration-dependent manner. In addition, KHG26377 significantly suppressed Aß25-35-induced toxicity concomitant with a reduction in the activation of extracellular signal-regulated kinases 1 and 2 and nuclear factor kappa B. The KHG26377-induced protection of neuronal cells against Aß toxicity was also mediated by suppressing the expression of glycogen synthase kinase-3ß, increasing the levels of ß-catenin, and reducing the levels of phosphorylated tau. Our findings suggest that KHG26377 may modulate the neurotoxic effects of ß-amyloid and provide a rationale for treatment of AD.

Synthesis and biological evaluation of 3-phenethylazetidine derivatives as triple reuptake inhibitors.

We report the synthesis of 3-phenethylazetidine derivatives 2 and their biological activities against 5-HT, NE and DA transporters as well as microsomal stability, CYP inhibition, and hERG inhibition profiles. Compound 2at showed most potent triple reuptake inhibitor with good selectivity as a candidate for depression.

KHG26792 Inhibits Melanin Synthesis in Mel-Ab Cells and a Skin Equivalent Model.

The purpose of this study is to characterize the effects of KHG26792 (3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride), a potential skin whitening agent, on melanin synthesis and identify the underlying mechanism of action. Our data showed that KHG26792 significantly reduced melanin synthesis in a dose-dependent manner. Additionally, KHG26792 downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase, the rate-limiting enzyme in melanogenesis, although tyrosinase was not inhibited directly. KHG26792 activated extracellular signal-regulated kinase (ERK), whereas an ERK pathway inhibitor, PD98059, rescued KHG26792-induced hypopigmentation. These results suggest that KHG26792 decreases melanin production via ERK activation. Moreover, the hypopigmentary effects of KHG26792 were confirmed in a pigmented skin equivalent model using Cervi cornus Colla (deer antler glue), in which the color of the pigmented artificial skin became lighter after treatment with KHG26792. In summary, our findings suggest that KHG26792 is a novel skin whitening agent.

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0 +/- 0.02, 1.0 +/- 0.04, 2.1 +/- 0.12, and 2.0 +/- 0.08 microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05 +/- 0.002, 0.153 +/- 0.004, 1.30 +/- 0.10, and 1.29 +/- 0.40 microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52 kcal/mol and formed a single hydrogen bond with a distance of 1.973 A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.

Suppression of glutamate-induced excitotoxicity by 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride in rat glial cultures.

We have screened new drugs with a view to developing effective drugs against glutamate-induced excitotoxicity. In the present work, we show effects of a new drug, 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against glutamate-induced excitotoxicity in primary rat glial cultures. Pretreatment of glial cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride for 2 h significantly protected glial cells against glutamate-induced excitotoxicity in a time- and dose-dependent manner with an optimum concentration of 100 microM. The drug significantly reduced production of proinflammatory cytokines, tumor necrosis factor-alpha, and interlukin-1beta in glutamate-induced excitotoxicity. The drug also prevented glutamate-induced intracellular Ca2+ influx and reduced the subsequent overproduction of nitric oxide and reactive oxygen species. Furthermore, the drug preserved the mitochondrial potential and inhibited the overproduction of cytochrome c. In addition, the drug effectively attenuated the protein level changes of beta-catenin and glycogen synthase kinase-3beta. These results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride effectively protected primary cultures of rat glial cells against glutamate-induced excitotoxicity.

Exploration of novel 2-alkylimino-1,3-thiazolines: T-type calcium channel inhibitory activity.

We have developed combinatorial libraries of new 2-alkylimino-1,3-thiazolines with four diversity points, consisting of more than 500 compounds, in a parallel synthetic fashion. The synthetic strategy was based on the construction of a large library aimed at the discovery of new compounds with T-type calcium channel inhibitory activity through structure modifications of hit compound 2. The syntheses of the compounds of Chemset A with four diversity points were accomplished by the condensation of thioureas 5 with alpha-haloketones 6{1-66} having two diversity points each. A library of phthalimidyl 1,3-thiazolines 24 was synthesized to provide Chemset B, which allowed the introduction of other diversity points through the nucleophilic character of the amino nitrogen. A sublibrary, Chemset C, was constructed from the libraries of Chemset A and Chemset B by functionalization of the C-4 position of the 1,3-thiazoline ring. The products containing ester or acid groups at the C-4 position of the 1,3-thiazoline ring were used in amide synthesis to give a new sublibrary within Chemset C. Deprotection of the phthalimidyl moiety of 24 followed by the reaction with benzoyl chloride gave the corresponding sublibrary in Chemset C. Another sublibrary which includes secondary amino derivatives was obtained by reduction of the amide moiety or reductive amination of 23 with phenyl aldehyde. The selected compounds from the generated libraries were evaluated with respect to inhibition of T-type calcium channels, where some of them have exhibited promising activity.

Ecofriendly Catechol Lipid Bioresin for Low-Temperature Processed Electrode Patterns with Strong Durability.

We demonstrated catechol lipid-based bioresin, which is collected from lacquer trees, to produce conductive pastes that can be processed at low temperatures, which are highly adhesive and multidurable. Our conductive paste, which consists of catechol lipid-based urushiol resin and a multimodal mixture of silver fillers, exhibited stable dispersion with shear thinning properties. The urushiol lacquer induced spontaneous reduction of silver salt at the surface of the silver fillers, thereby contributing to lower the contact resistance between conductive fillers in the electrical conduction. Furthermore, the directional volume shrinkage of the urushiol lacquer matrix in a cross-linking reaction resulted in a highly ordered microstructure of the silver fillers with layer-by-layer stacking of the silver flakes. This structure contributed to the improvement of the electrical contact between fillers as well as excellent mechanical hardness, anti-scratch capability, and the long-term environmental stability of the conductive films. Conductive films based on the silver paste with urushiol lacquer exhibited low electrical resistivity below 4.4 × 10-5 Ω cm, 5B-class strong adhesion strength, and high hardness exceeding 200 MPa. Finally, we demonstrated the facile room-temperature processability and screen printability of the UL-Ag paste by fabricating a printed antenna and three-dimensional (3D) electrode assembly based on a plastic 3D block.

Quantitative structural-activity relationship (QSAR) study for fungicidal activities of thiazoline derivatives against rice blast.

For the development of new fungicides against rice blast, the quantitative structural-activity relationship (QSAR) analyses for fungicidal activities of thiazoline derivatives were carried out using multiple linear regression (MLR) and neural network (NN). We have studied the substituent effects at para site of R(1) and at three sites (ortho, meta, or para) of R(2) aromatic rings in compounds. The results of MLR and NN analyses in the training set of Set-3 showed good correlations (r(2) values of 0.829 and 0.966, respectively) between the descriptors and the fungicidal activities. Five descriptors including the non-overlap steric volume SV(R2C2)), Connolly surface area SA(R1), hydrophobicity Sigma pi(R2), and Hammett substituent constants (sigma(pR1) and sigma(mR2)) were selected as important factors of fungicidal activities. Although the descriptors of optimum MLR model were used in NN, the results were improved by NN. This means that the descriptors used in MLR model include non-linear relationships.

Protective effect of benzothiazole derivative KHG21834 on amyloid beta-induced neurotoxicity in PC12 cells and cortical and mesencephalic neurons.

We have investigated the effect of KHG21834, a benzothiazole derivative, on the amyloid beta protein (Abeta)-induced cell death in rat pheochromocytoma (PC12) cells and rat cortical and mesencephalic neuron-glia cultures. KHG21834 attenuated the Abeta(25-35)-induced apoptotic death in PC12 cells determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL). In the cortical neuron-glia cultures, KHG21834 reduced the Abeta(25-35)-induced apoptosis determined by TUNEL staining. Immunocytochemical analysis and Western blot analysis of Abeta(25-35)-induced neurotoxicity in mesencephalic neuron-glia cultures with anti-tyrosine hydroxylase (TH) antibody showed that Abeta(25-35) decreased the expression of TH protein by 60% and KHG21834 significantly attenuated the Abeta(25-35)-induced reduction in the expression of TH. Moreover, KHG21834 attenuates Abeta(25-35)-induced toxicity concomitant with the reduction of activation of extracellular signal-regulated kinase (ERK)1/2 to a lesser extent. ERK1 was more sensitively affected than ERK2 in attenuation of Abeta(25-35)-induced phosphorylation by KHG21834. These results demonstrated that KHG21834 was capable of protecting neuronal cells from Abeta(25-35)-induced degeneration.

Neuroprotective effects of N-adamantyl-4-methylthiazol-2-amine against amyloid ß-induced oxidative stress in mouse hippocampus.

We previously reported that N-adamantyl-4-methylthiazol-2-amine (KHG26693) suppresses amyloid beta (Aß)-induced neuronal oxidative damage in cortical neurons. Here we investigated the mechanism and antioxidative function of KHG26693 in the hippocampus of Aß-treated mice. KHG26693 significantly attenuated Aß-induced TNF-α and IL-1ß enhancements. KHG26693 decreased Aß-mediated malondialdehyde formation, protein oxidation, and reactive oxygen species by decreasing the iNOS level. KHG26693 suppressed Aß-induced oxidative stress through a mechanism involving glutathione peroxidase, catalase, and GSH attenuation. Aß-induced MMP-2, cPLA2, and pcPLA2 expressions were almost completely attenuated by KHG26693 treatment, suggesting that Aß-induced oxidative stress reduction by KHG26693 is, at least partly, caused by the downregulation of MMP-2 and cPLA2 activation. Compared with Aß treatment, KHG26693 treatment upregulated Nrf2 and HO-1 expressions, suggesting that KHG26693 protects the brain from Aß-induced oxidative damage, likely by maintaining redox balance through Nrf2/HO-1 pathway regulation. KHG26693 significantly attenuated Aß-induced oxidative stress in the hippocampus of Aß-treated mice.

Protective effect of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride on hypoxia-induced toxicity by suppressing microglial activation in BV-2 cells.

We recently reported the anti-inflammatory effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on the ATP-induced activation of the NFAT and MAPK pathways through the P2X7 receptor in microglia. To further investigate the underlying mechanism of KHG26792, we studied its protective effects on hypoxia-induced toxicity in microglia. The administration of KHG26792 significantly reduced the hypoxia-induced expression and activity of caspase-3 in BV-2 microglial cells. KHG26792 also reduced hypoxia-induced inducible nitric oxide synthase protein expression, which correlated with reduced nitric oxide accumulation. In addition, KHG26792 attenuated hypoxiainduced protein nitration, reactive oxygen species production, and NADPH oxidase activity. These effects were accompanied by the suppression of hypoxia-induced protein expression of hypoxia-inducible factor 1-alpha and NADPH oxidase-2. Although the clinical relevance of our findings remains to be determined, these data results suggest that KHG26792 prevents hypoxia-induced toxicity by suppressing microglial activation. [BMB Reports 2016; 49(12): 687-692].

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics.

Design and synthesis of novel antidiabetic agents.

The synthesis and structure-activity relationships of a novel series of substituted quercetins that activates peroxisome proliferator-activated receptor gamma (PPARgamma) are reported. The PPARgamma agonistic activity of the most potent compound in this series is comparable to that of the thiazolidinedione-based antidiabetic drugs currently in clinical use.

N-adamantyl-4-methylthiazol-2-amine suppresses lipopolysaccharide-induced brain inflammation by regulating NF-κB signaling in mice.

We report that N-adamantyl-4-methylthiazol-2-amine (KHG26693), a novel thiazole derivative, can prevent lipopolysaccharide (LPS)-induced brain inflammation in mice. In this LPS-induced model of brain inflammation, administration of KHG26693 effectively prevented increases in the levels of IL-1ß, TNF-α, prostaglandin E2, malondialdehyde, and nitric oxide, and mitigated reductions in the levels of superoxide dismutase in the hippocampus. KHG26693 also prevented reductions in the levels of hippocampal brain-derived neurotrophic factors. Furthermore, pretreatment with KHG26693 prior to LPS treatment dramatically attenuated the elevation of inducible nitric oxide synthase and cyclooxygenase-2 protein levels. Moreover, pretreatment with KHG26693 significantly suppressed LPS-induced phosphorylation of NF-κB and IκBα through the inactivation of IKKß. Additionally, KHG26693 caused the downregulation of LPS-induced cystathionine-b-synthase gene expression in the brain. Although the clinical relevance of our findings remains to be determined, our data suggest that KHG26693 might prevent neuronal cell injury via the reduction of inflammation and oxidative stress in the brain.

The azetidine derivative, KHG26792 protects against ATP-induced activation of NFAT and MAPK pathways through P2X7 receptor in microglia.

Azetidine derivatives are of interest for drug development because they may be useful therapeutic agents. However, their mechanisms of action remain to be completely elucidated. Here, we have investigated the effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on ATP-induced activation of NFAT and MAPK through P2X7 receptor in the BV-2 mouse microglial cell line. KHG26792 decreased ATP-induced TNF-α release from BV-2 microglia by suppressing, at least partly, P2X7 receptor stimulation. KHG26792 also inhibited the ATP-induced increase in IL-6, PGE2, NO, ROS, CXCL2, and CCL3. ATP induced NFAT activation through P2X7 receptor, with KHG26792 reducing the ATP-induced NFAT activation. KHG26792 inhibited an ATP-induced increase in iNOS protein and ERK phosphorylation. KHG26792 prevented an ATP-induced increase in MMP-9 activity through the P2X7 receptor as a result of degradation of TIMP-1 by cathepsin B. Our data provide mechanistic insights into the role of KHG26792 in the inhibition of TNF-α produced via P2X7 receptor-mediated activation of NFAT and MAPK pathways in ATP-treated BV-2 cells. This study highlights the potential use of KHG26792 as a therapeutic agent for the many diseases of the CNS related to activated microglia.

Excavation of lead compounds that inhibit mast cell degranulation by combinatorial chemistry and activity-guided.

An allergic reaction ensues after antigen binds to mast cell or basophil high affinity IgE receptor, Fc epsilonRI, resulting in degranulation of various inflammatory mediators that produce various allergic symptoms. In this study, i) we isolated the active component for the inhibition of mast cell degranulation from the extract of leaves of Castanea crenata and identified it as quercetin; ii) we established the total synthesis procedure of quercetin; iii) using quercetin as positive control, we excavated some lead compounds that possess inhibitory activities for mast cell degranulation by screening the chemical libraries of 1,3-oxazolidine derivatives prepared by solid phase combinatorial chemistry. Some of 1,3-oxazolidine compounds possessing acetyl and 3',4'-dichlorophenyl group displayed strong inhibitory activities on Fc epsilonRI-mediated mast cell degranulation, suggesting that they can be used as lead compounds for the development of anti-allergic agents.

Exploration of 3-Aminoazetidines as Triple Reuptake Inhibitors by Bioisosteric Modification of 3-α-Oxyazetidine.

For a development of broad spectrum antidepressant 3-aminoazetidine derivatives, two series of compounds were explored by bioisosteric modification of 3-α-oxyazetidine. We synthesized 166 novel 3-aminoazetidine derivatives in series A and B, starting from Boc-protected 3-azetidinone (3) and Boc-protected 3-azetidinal (9) respectively, through parallel syntheses. The inhibitory reuptake activities against serotonin (5-HT), norepinephrine (NE), and dopamine (DA) neurotransmitters were measured by the Neurotransmitter Transporter Uptake Assay Kit using the human embryonic kidney 293 (HEK293) cells stably transfected with the respective three kinds of human transporters (hSERT, hNET, and hDAT). Our study aimed to identify compounds having relative inhibitory activities against hSERT > hNET > hDAT. Lead optimization including microsomal stability, CYP, hERG assay, Ames test, BBB, and PK study resulted in the identification of compound 10dl as a candidate for further studies.

Effects of N-adamantyl-4-methylthiazol-2-amine on hyperglycemia, hyperlipidemia and oxidative stress in streptozotocin-induced diabetic rats.

Thiazole derivatives are attractive candidates for drug development because they can be efficiently synthesized and are active against a number of diseases and conditions, including diabetes. In our present study, we investigated the anti-inflammatory and antioxidant properties of N-adamantyl-4-methylthiazol-2-amine (KHG26693), a new thiazole derivative, in a streptozotocin (STZ)-induced model of diabetes mellitus. STZ-induced diabetic rats were intraperitoneally administered KHG26693 (3mg/kg-body weight/day) for 4 weeks. KHG26693 administration significantly decreased blood glucose, triglycerides, and cholesterol and increased insulin. KHG26693 also suppressed several inflammatory responses in STZ-induced diabetic rats, as evidenced by decreased levels of serum tumor necrosis factor-α, interleukin-1ß, and nitric oxide. Additionally, KHG26693 significantly modulated hepatic lipid peroxidation, catalase and superoxide dismutase activity, and the nonenzymatic antioxidant status (e.g., vitamins C and E), and reduced the glutathione content. These anti-inflammatory/antioxidative actions occurred as a result of the downregulation of inducible nitric oxide synthase and nuclear factor-kappa B. Taken together, our results suggest that KHG26693 successfully reduces the production of oxidative stress in STZ-induced diabetic rats by regulating the oxidation-reduction system, specifically increasing antioxidant capacity. Furthermore, KHG26693 treatment significantly reverted the key enzymes of glucose metabolism, such as glucokinase, glucose-6-phosphatase, glycogen synthase, glycogen phosphorylase, and fructose-1,6-bisphosphatase, to near-normal levels in liver tissues. These results indicate that KHG26693 normalizes disturbed glucose metabolism by enhancing glucose utilization and decreasing liver glucose production via insulin release, suggesting the possibility of future diabetes treatments.