Synthesis and biological evaluation of indane-based fluorescent probes for detection of amyloid-ß aggregates in Alzheimer's disease.

In this article, the development of fluorescent imaging probes for the detection of Alzheimer's disease (AD)-associated protein aggregates is described. Indane derivatives with a donor-π-acceptor (D-π-A) structure were designed and synthesized. The probes were evaluated for their ability to bind to ß-amyloid (Aß) protein aggregates, which are a key pathological hallmark of AD. The results showed that several probes exhibited significant changes in fluorescence intensity at wavelengths greater than 600 nm when they were bound to Aß aggregates compared to the Aß monomeric form. Among the tested probes, four D-π-A type indane derivatives showed promising binding selectivity to Aß aggregates over non-specific proteins such as bovine serum albumin (BSA). The molecular docking study showed that our compounds were appropriately located along the Aß fibril axis through the hydrophobic tunnel structure. Further analysis revealed that the most active compound having dimethylaminopyridyl group as an election donor and dicyano group as an electron acceptor could effectively stain Aß plaques in brain tissue samples from AD transgenic mice. These findings suggest that our indane-based compounds have the potential to serve as fluorescent probes for the detection and monitoring of Aß aggregation in AD.

Synthetic Strategy for Aryl(alkynyl)phosphinates by a Three-Component Coupling Reaction Involving Arynes, Phosphites, and Alkynes.

An efficient and straightforward method for the synthesis of aryl(alkynyl)phosphinates was developed via a three-component coupling reaction involving arynes, phosphites, and alkynes. An array of aryl(alkynyl)phosphinates were produced from both aryl and aliphatic group-substituted acetylenes. This operationally simple reaction is tolerant to many functional groups, affording various aryl(alkynyl)phosphinates in moderate to good yields. The synthetic utility of alkynyl phosphinates afforded by this method was demonstrated by the elaboration of the products into various phosphorus-containing compounds.

"Turn-On" Quinoline-Based Fluorescent Probe for Selective Imaging of Tau Aggregates in Alzheimer's Disease: Rational Design, Synthesis, and Molecular Docking.

Tau aggregation is believed to have a strong association with the level of cognitive deficits in Alzheimer's disease (AD). Thus, optical brain imaging of tau aggregates has recently gained substantial attention as a promising tool for the early diagnosis of AD. However, selective imaging of tau aggregates is a major challenge due to sharing similar ß-sheet structures with homologous Aß fibrils. Herein, four quinoline-based fluorescent probes (Q-tau) were judiciously designed using the donor-acceptor architecture for selective imaging of tau aggregates. In particular, probe Q-tau 4 exhibited a strong intramolecular charge transfer and favorable photophysical profile, such as a large Stokes' shift and fluorescence emission wavelength of 630 nm in the presence of tau aggregates. The probe also displayed a "turn-on" fluorescence behavior toward tau fibrils with a 3.5-fold selectivity versus Aß fibrils. In addition, Q-tau 4 exhibited nanomolar binding affinity to tau aggregates (Kd = 16.6 nM), which was 1.4 times higher than that for Aß fibrils. The mechanism of "turn-on" fluorescence was proposed to be an environment-sensitive molecular rotor-like response. Moreover, ex vivo labeling of human AD brain sections demonstrated favorable colocalization of Q-tau 4 and the phosphorylated tau antibody, while comparable limited staining was observed with Aß fibrils. Molecular docking was conducted to obtain insights into the tau-binding mode of the probe. Collectively, Q-tau 4 has successfully been used as a tau-specific fluorescent imaging agent with lower background interference.

Novel quinoline-based fluorescent bioimaging probe, KSNP117, for sentinel lymph node mapping.

Fluorescent imaging agents with biocompatibility and high sensitivity are urgently required for the accurate detection of sentinel lymph nodes (SLNs). Herein, we report the design of a novel quinoline-based fluorescent probe, designated KSNP117, which can be applied as a biomedical imaging agent in the sensitive and quantitative detection of SLNs. KSNP117 exerted no adverse effects on the proliferation of ovary and immune cells and also showed excellent serum stability with photo-brightening effects. In vivo fluorescent imaging revealed the accumulation of KSNP117 in the SLNs of nude mice within 10 min post injection, without in vivo toxicity, which was consistent with the findings of ex vivo imaging. These results support the potential of KSNP117 as a promising lymphatic tracer for biomedical imaging applications.

Antioxidant Compound, Oxyresveratrol, Inhibits APP Production through the AMPK/ULK1/mTOR-Mediated Autophagy Pathway in Mouse Cortical Astrocytes.

Oxyresveratrol (OxyR), a well-known polyphenolic phytoalexin, possesses a wide range of pharmacological and biological properties, comprising antioxidant, anti-inflammatory, free radical scavenging, anti-cancer, and neuroprotective activities. Autophagy is a cellular self-degradation system that removes aggregated or misfolded intracellular components via the autophagosome-lysosomal pathway. Astrocyte accumulation is one of the earliest neuropathological changes in Alzheimer's disease (AD), and amyloid precursor protein (APP) is the hallmark of AD. OxyR could affect APP modulation via the autophagy pathway. Here, we have reported that OxyR promotes autophagy signaling and attenuates APP production in primary cortical astrocytes based on immunofluorescence and immunoblotting assay results. Co-treatment with the late-stage autophagy inhibitor chloroquine (CQ) and OxyR caused significantly higher microtubule-associated protein light chain 3 (LC3)-II protein levels and LC3 puncta counts, demonstrating that OxyR stimulated autophagic flux. We also found that OxyR significantly reduced the levels of the autophagy substrate p62/SQSTM1, and p62 levels were significantly augmented by co-treatment with OxyR and CQ, because of the impaired deficiency of p62 in autolysosome. Likewise, pretreatment with the autophagy inhibitor, 3-methyladenine (3-MA), resulted in significantly fewer OxyR-induced LC3 puncta and lower LC3-II expression, suggesting that OxyR-mediated autophagy was dependent on the class III PI3-kinase pathway. In contrast, OxyR caused significantly lower LC3-II protein expression when pretreated with compound C, an AMP-activated protein kinase (AMPK) inhibitor, indicating that AMPK signaling regulated the OxyR-induced autophagic pathway. Additionally, co-treatment with OxyR with rapamycin intended to inhibit the mammalian target of rapamycin (mTOR) caused significantly lower levels of phospho-S6 ribosomal protein (pS6) and higher LC3-II expression, implying that OxyR-mediated autophagy was dependent on the mTOR pathway. Conversely, OxyR treatment significantly upregulated unc-51-like autophagy activating kinase 1 (ULK1) expression, and ULK1 small interfering RNAs (siRNA) caused significantly lower OxyR-induced LC3 puncta counts and LC3-II expression, indicating that ULK1 was essential for initiating OxyR-induced autophagy. However, we found that OxyR treatment astrocytes significantly increased the expression of lysosome-associated membrane protein 1 (LAMP1). Finally, we established a stress-induced APP production model using corticosterone (CORT) in cortical astrocytes, which produced significantly more APP than the equivalent using dexamethasone (DEX). In our experiment we found that CORT-induced APP production was significantly attenuated by OxyR through the autophagy pathway. Therefore, our study reveals that OxyR regulates AMPK/ULK1/mTOR-dependent autophagy induction and APP reduction in mouse cortical astrocytes.

O-GlcNAcase: Emerging Mechanism, Substrate Recognition and Small-Molecule Inhibitors.

O-GlcNAcylation is the dynamic and ubiquitous post-translational glycosylation of nucleocytoplasmic proteins on serine/threonine residues; it is implicated in regulation of the cell cycle. This protein modification is mainly governed by a pair of enzymes: O-GlcNAc transferase (OGT) adds the N-acetylglucosamine moiety to acceptor proteins, and O-GlcNAcase (OGA) hydrolyses the sugar moiety from protein acceptors. Irregular O-GlcNAcylation is linked to several diseases including cancer, diabetes and neurodegeneration. Recently, the discovery of small-molecule OGA inhibitors has enabled the physiological function of O-GlcNAcylation to be investigated. However, the design of highly potent and selective inhibitors faces several challenges as no full structural data of human OGA has been discovered to date. Moreover, there are a number of mechanistically similar related enzymes such as ß-hexosaminidases (Hex), and the concomitant inhibition of these enzymes leads to undesirable lysosomal-storage disorders. This review highlights recent insights into the structure of human O-GlcNAcase and its isoforms. We focus on the catalytic mechanism and substrate recognition by OGA. In addition, it presents an updated overview of small-molecule OGA inhibitors, with either carbohydrate or noncarbohydrate scaffolds. We discuss inhibitor structures, binding modes, and selectivity towards the enzyme, and potential outcomes in probing O-GlcNAcylation at cellular levels.

T-type calcium channel blockers: a patent review (2012-2018).

INTRODUCTION: T-type calcium channels are attractive targets for potential treatment of epilepsy inflammatory or neuropathic pain, insomnia, Parkinson's disease, and cancer. Three isoforms having different biophysical functions are expressed in peripheral and central nerve. Since the withdrawal of mibefradil, the first compound marketed for selective T-type calcium channel blockade, extensive efforts have been made to identify more selective T-type calcium channel blockers. AREAS COVERED: This review covers the 43 patents describing 'organic small molecules as T-type calcium channel blockers'-published since 2012. The most recent similar patent review was published in 2011. Information from a recent review article and relevant research papers has been included, as well as biological data and clinical trial results where available. EXPERT OPINION: Triazinone derivatives, carbazole compounds, and aryl triazole/imidazole amide derivatives display potent blockade activity α1H, α1G, and pan T-type calcium channel subtypes, respectively, though the specificity of the letter is still unsatisfactory. Nonetheless, improvements seen in the efficacy of compounds targeting α1H T-type calcium channels indicate significant progress. Ongoing clinical trials are for the candidates Z944 (Phase II) and ACT-709478 (Phase II) appear promising. These studies may lead to a new generation of inhibitors with higher selectivity, improved physicochemical properties, and reduced side effects.

The phosphodiesterase 5 inhibitor, KJH-1002, reverses a mouse model of amnesia by activating a cGMP/cAMP response element binding protein pathway and decreasing oxidative damage.

BACKGROUND AND PURPOSE: Inhibition of PDE5 improves synaptic plasticity and memory via enhancing cGMP expression, thus activating the cGMP/cAMP response element binding protein (CREB) signalling pathway. This study investigated the effects of a PDE5 inhibitor on scopolamine-induced cognitive dysfunction, using memory-related behavioural tests and biochemical assays. EXPERIMENTAL APPROACH: In mice were pretreated with PDE5 inhibitor, amnesia was induced by scopolamine. The learning and memory abilities of mice were tested using the Morris water maze test, the Y-maze test, the passive avoidance test and the novel object recognition test in sequence. Expression of memory-related bio-molecules and oxidative stress parameters in brain tissue were measured using Western blot and spectrophotometry respectively. KEY RESULTS: KJH-1002, a novel and potent inhibitor of PDE5 (IC50 0.059 ± 0.04 nmol·L-1 ), was synthesized. In the behavioural tests, it markedly improved the memory performance impaired by scopolamine, indicating a restoration of cognitive function in the mice. Moreover, KJH-1002 increased cGMP levels in the cortex and the scopolamine-reduced expression of phosphorylated CREB, Levels of ERK 1/2, Akt and brain-derived neurotrophic factor in the cortex and hippocampus were restored by KJH-1002 treatment. In addition, KJH-1002 administration increased the activities of SOD, glutathione peroxidase and glutathione reductase, and decreased the level of malondialdehyde. CONCLUSION AND IMPLICATIONS: KJH-1002 restored cognitive function in scopolamine-induced amnesia mice by activating the cGMP/CREB signalling pathway and attenuating oxidative stress. The beneficial effects of KJH-1002 on cognition indicate its potential as a therapeutic candidate for Alzheimer's disease.

Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides as 5-HT6 receptor antagonists.

A novel series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazoles was evaluated for serotonin receptor subtype 6 (5-HT6R) antagonistic effects in vitro. We also investigated their neuropathic pain-alleviating effects in vivo using a rat spinal nerve ligation (SNL) model. Bicyclic aromatic sulfonamino groups, such as naphthalene and quinolin-substituted derivatives, showed good 5-HT6 inhibitory activity in vitro. Among them, selected compounds, 12 and 13, having 8-quinoylsulfonamino groups, showed potent neuropathic pain-alleviating effects in the rat model.

Synthesis and diabetic neuropathic pain-alleviating effects of 2N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide derivatives.

A novel series of fused-benzensulfonamide 2-N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide derivatives was designed and synthesized as metabolically stable T-type calcium channel inhibitors. Several compounds, 9, 10, and 17, displayed potent T-type channel inhibitory activity. Among them, compounds 10 and 17 showed good metabolic stability in human liver microsomes, and low hERG channel and CYP450 inhibition. Compound 10 exhibited diabetic neuropathic pain-alleviating effects in a streptozotocin-induced peripheral diabetic neuropathy (PDN) model. The maximum efficacy of compound 10, which was 3-fold more potent than gabapentin, was observed at 1h after administration, and co-administration of compound 10 with gabapentin showed a considerable synergic effect.

Synthesis and evaluation of 6-pyrazoylamido-3N-substituted azabicyclo[3,1,0]hexane derivatives as T-type calcium channel inhibitors for treatment of neuropathic pain.

A new series of aryls, including benzo[d]imidazole/isoxazole/pyrazole, conjugated to 3N-substituted-azabicyclo[3.1.0]hexane derivatives were designed and synthesized as inhibitors of T-type calcium channels. Among the synthesized compounds, 3N-R-substituted azabicyclo[3.1.0]hexane carboxamide derivatives containing 5-isobutyl-1-phenyl-pyrazole ring exhibited potent and selective T-channel inhibition and good metabolic stability without CYP450 inhibition. Compounds 10d and 10e contained hydrophobic substituents at the 3N-position and exhibited potent in vitro efficacy, as well as neuropathic pain alleviation in rats.

Synthesis and T-type calcium channel-blocking effects of aryl(1,5-disubstituted-pyrazol-3-yl)methyl sulfonamides for neuropathic pain treatment.

A novel series of aryl(1,5-disubstituted pyrazol-3-yl)methyl sulfonamide derivatives was designed, synthesized, and evaluated for T-type calcium channel (α1G and α1H) inhibitory activity. We identified several compounds, 9a, 9b, 9g, and 9h that displayed good T-type channel inhibitory potency with low hERG channel and CYP450 inhibition. Among them, 9a and 9b exhibited neuropathic pain alleviation effects in mechanical and cold allodynia induced in the SNL rat model. Compounds 9a and 9b displayed better efficacy than mibefradil and gabapentin against cold allodynia. In particular, compound 9a seemed to be valuable as shown fast acting performance in mechanical neuropathic pain model.

Discovery of a Nanomolar Multikinase Inhibitor (KST016366): A New Benzothiazole Derivative with Remarkable Broad-Spectrum Antiproliferative Activity.

Herein we report the discovery of compound 6 [KST016366; 4-((2-(3-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-6-yl)oxy)picolinamide] as a new potent multikinase inhibitor through minor structural modification of our previously reported RAF kinase inhibitor A. In vitro anticancer evaluation of 6 showed substantial broad-spectrum antiproliferative activity against 60 human cancer cell lines. In particular, it showed GI50 values of 51.4 and 19 nm against leukemia K-562 and colon carcinoma KM12 cell lines, respectively. Kinase screening of compound 6 revealed its nanomolar-level inhibitory activity of certain oncogenic kinases implicated in both tumorigenesis and angiogenesis. Interestingly, 6 displays IC50 values of 0.82, 3.81, and 53 nm toward Tie2, TrkA, and ABL-1 (wild-type and T315I mutant) kinases, respectively. Moreover, 6 is orally bioavailable with a favorable in vivo pharmacokinetic profile. Compound 6 may serve as a promising candidate for further development of potent anticancer chemotherapeutics.

Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors.

Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked ß-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.

Synthesis and in vitro evaluation of the antitubercular and antibacterial activity of novel oxazolidinones bearing octahydrocyclopenta[c]pyrrol-2-yl moieties.

A novel series of oxazolidinone-class antimicrobial agents with 5-substituted octahydrocyclopenta[c]pyrrole moieties at the C-ring of linezolid and an acetamide or 1,2,3-triazole ring as the C-5 side chain of the oxazolidinone ring were prepared. The resulting series of compounds were evaluated for in vitro antimicrobial activity against Mycobacterium tuberculosis and a panel of clinically important resistant Gram-positive and -negative bacteria. Among them, endo-alcohol 2a and exo-alcohol 2b showed potent inhibitory activity against M. tuberculosis H37Rv, which was superior to that of linezolid. Several analogues in this series showed potent in vitro antibacterial activity against the clinically important vancomycin-resistant bacteria and showed similar or better potency against linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) strains. The hydroxyl group in the azabicyclic C-ring interacted with the same hydrophobic pocket as linezolid based on a docking study. Selected compounds with high antimicrobial activity showed good human microsomal stability and low CYP isozyme and monoamine oxidase (MAO) inhibition.

Synthesis and biological evaluation of 1-(isoxazol-5-ylmethylaminoethyl)-4-phenyl tetrahydropyridine and piperidine derivatives as potent T-type calcium channel blockers with antinociceptive effect in a neuropathic pain model.

New tetrahydropyridinyl and piperidinyl ethylamine derivatives were designed with hypothetical mapping on pharmacophore model generated from ligand-based virtual screening. The designed compounds were synthesized, and their inhibitory activities on T-type calcium channel were assayed using FDSS and patch-clamp assay. Among them, compounds 7b and 10b showed potent T-type calcium current blocking activity against Ca(v)3.1 (α(1G)) and Ca(v)3.2 (α(1H)) channel simultaneously. With hERG and pharmacokinetics studies, compounds 7b and 10b were evaluated for the antinociceptive effect on rat model of neuropathic pain. They were significantly effective in decreasing the pain responses to mechanical and cold allodynia induced by spinal nerve ligation. These results suggest that modulation of α(1G) and α(1H) subtype T-type calcium channels may provide a promising approach for the treatment of neuropathic pain.

Synthesis and biological evaluation of aryloxazole derivatives as antimitotic and vascular-disrupting agents for cancer therapy.

A series of aryloxazole, thiazole, and isoxazole derivatives was synthesized as vascular-targeting anticancer agents. Antiproliferative activity and tumor vascular-disrupting activity of all of the synthesized compounds were tested in vitro using various human cancer cell lines and HUVECs (human umbilical vein endothelial cells). Several compounds with an arylpiperazinyl oxazole core showed excellent cytotoxicity and metabolic stability in vitro. Among this series, two representative compounds (6-48 and 6-51) were selected and tested for the evaluation of anticancer effects in vivo using tumor-bearing mice. Compound 6-48 effectively reduced tumor growth (42.3% reduction in size) at the dose of 100 mg/kg. We believe that compound 6-48 will serve as a good lead compound for antimitotic and vascular-disrupting agents; further investigation to improve the in vivo efficacy of this series is underway.

Aryl biphenyl-3-ylmethylpiperazines as 5-HT7 receptor antagonists.

The 5-HT7 receptor (5-HT7 R) is a promising therapeutic target for the treatment of depression and neuropathic pain. The 5-HT7 R antagonist SB-269970 exhibited antidepressant-like activity, whereas systemic administration of the 5-HT7 R agonist AS-19 significantly inhibited mechanical hypersensitivity and thermal hyperalgesia. In our efforts to discover selective 5-HT7 R antagonists or agonists, aryl biphenyl-3-ylmethylpiperazines were designed, synthesized, and biologically evaluated against the 5-HT7 R. Among the synthesized compounds, 1-([2'-methoxy-(1,1'-biphenyl)-3-yl]methyl)-4-(2-methoxyphenyl)piperazine (28) was the best binder to the 5-HT7 R (pKi =7.83), and its antagonistic property was confirmed by functional assays. The selectivity profile of compound 28 was also recorded for the 5-HT7 R over other serotonin receptor subtypes, such as 5-HT1 R, 5-HT2 R, 5-HT3 R, and 5-HT6 R. In a molecular modeling study, the 2-methoxyphenyl moiety attached to the piperazine ring of compound 28 was proposed to be essential for the antagonistic function.

A novel glucosamine derivative exerts anti-inflammatory actions via inhibition of nuclear factor-kappaB.

Glucosamine suppresses lipopolysaccharide (LPS)-induced upregulation of pro-inflammatory mediators both in vivo and in culture systems of mouse microglia or macrophage. In the present study, we show that the novel glucosamine derivative, 2-deoxy-2-[(o-methylbenzylidene)]-ß-glucopyranoside (NK-4), significantly reduced LPS-induced production of nitric oxide (NO) in BV2 microglia, RAW264.7 macrophage, and primary cultured peritoneal macrophages cells. NK-4 inhibited LPS-induced upregulation of inducible NO synthase (iNOS), cyclooxygenase-2, interleukin-6, tumor necrosis factor-α, and interleukin-1ß in RAW264.7 cells in a time- and concentration-dependent manner. Furthermore, administering NK-4 significantly inhibited the induction of inflammatory cytokine mRNAs in the brains of LPS-injected mice. Although NK-4 inhibited LPS-induced nuclear factor-kappaB (NF-κB) activation, IκB-α degradation was not changed. Instead, NK-4 inhibited LPS-induced DNA-binding activity of NF-κB by suppressing p50 and c-Rel binding to NF-κB binding site of the iNOS promoter.

Synthesis and the 5-HT6 receptor antagonistic effect of 3-arylsulfonylamino-5,6-dihydro-6-substituted pyrazolo[3,4]pyridinones for neuropathic pain treatment.

A novel series of 3-arylsulfonylamino-5,6-dihydro-6-substituted-1H-pyrazolo[3,4-c]pyridine-7-ones was designed and synthesized as 5-HT6 ligands. Among the derivatives synthesized, the lead compound, 12b, having piperidine functionality at the 6-position and (1-naphthyl)sulfonamino at the 3-position of the core structure showed the most potent 5-HT6 inhibitory activity in vitro, good stability without CYP liability, and good neuropathic pain alleviation activity in a rat animal model.