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.

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.

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 antibacterial activities of new piperidine substituted (5R)-[1,2,3]triazolylmethyl and (5R)-[(4-F-[1,2,3]triazolyl)methyl] oxazolidinones.

A novel series of 5(R)-[1,2,3]triazolylmethyl and (5R)-[(4-F-[1,2,3]triazolyl)methyl]oxazolidinones having various piperidine group were synthesized and evaluated antibacterial activity against clinically isolated resistant strains of Gram-positive and Gram-negative bacteria. The compound 12a having exo-cyanoethylidene group in the 4-position of piperidine ring was found to be two to threefold more potent than the linezolid against penicillin-resistant Staphylococcus pneumonia and Staphylococcus agalactiae, and also exhibited reduced MAO-B inhibitory activity.

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.

Discovery of aryl-biphenyl-2-ylmethylpiperazines as novel scaffolds for 5-HT(7) ligands and role of the aromatic substituents in binding to the target receptor.

It has been reported that 5-HT(7) receptors are promising targets of depression and neuropathic pain. 5-HT(7) receptor antagonists have exhibited antidepressant-like profiles, while agonists have represented potential therapeutics for pain. In the course of our ongoing efforts to discover novel 5-HT(7) modulators, we designed an arylpiperazine scaffold with a substituted biphenyl-2-ylmethyl group. A series of biphenyl-2-yl-arylpiperazinylmethanes were then prepared, which showed a broad spectrum of binding affinities to the 5-HT(7) receptor depending upon the substituents attached to the biphenyl and aryl functionalities. Among those synthesized compounds, the compounds 1-24 and 1-26 showed the best binding affinities to the 5-HT(7) receptor with K(i) values of 43.0 and 46.0 nM, respectively. Structure-activity relationship study in conjunction with molecular docking study proposed that the 5-HT(7) receptor might have two distinctive hydrophobic binding sites, one specific for aromatic 2-OCH(3) substituents within the arylpiperazine and the other for biphenyl methoxy group.

Oxazolopyridines and thiazolopyridines as monoamine oxidase B inhibitors for the treatment of Parkinson's disease.

In Parkinson's disease, the motor impairments are mainly caused by the death of dopaminergic neurons. Among the enzymes which are involved in the biosynthesis and catabolism of dopamine, monoamine oxidase B (MAO-B) has been a therapeutic target of Parkinson's disease. However, due to the undesirable adverse effects, development of alternative MAO-B inhibitors with greater optimal therapeutic potential towards Parkinson's disease is urgently required. In this study, we designed and synthesized the oxazolopyridine and thiazolopyridine derivatives, and biologically evaluated their inhibitory activities against MAO-B. Structure-activity relationship study revealed that the piperidino group was the best choice for the R(1) amino substituent to the oxazolopyridine core structure and the activities of the oxazolopyridines with various phenyl rings were between 267.1 and 889.5nM in IC50 values. Interestingly, by replacement of the core structure from oxazolopyrine to thiazolopyridine, the activities were significantly improved and the compound 1n with the thiazolopyridine core structure showed the most potent activity with the IC50 value of 26.5nM. Molecular docking study showed that van der Waals interaction in the human MAO-B active site could explain the enhanced inhibitory activities of thiazolopyridine derivatives.

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.

Discovery of piperidinyl aminopyrimidine derivatives as IKK-2 inhibitors.

A serine-threonine kinase IKK-2 plays an important role in activation of NF-κB through phosphorylation of the inhibitor of NF-κB (IκB). As NF-κB is a major transcription factor that regulates genes responsible for cell proliferation and inflammation, development of selective IKK-2 inhibitors has been an important area of anti-inflammatory and anti-cancer research. In this study, to obtain active and selective IKK-2 inhibitors, various substituents were introduced to a piperidinyl aminopyrimidine core structure. The structure-activity relationship study indicated that hydrogen, methanesulfonyl, and aminosulfonyl groups substituted at the piperidinylamino functionality provide high inhibitory activity against IKK-2. Also, morpholinosulfonyl and piperazinosulfonyl group substituted at the aromatic ring attached to the aminopyrimidine core significantly increased the inhibitory activity of the resulting derivatives. In particular, compound 17 with the aromatic piperazinosulfonyl substituent showed the most potent (IC(50)=1.30 µM) and selective (over other kinases such as p38α, p38ß, JNK1, JNK2, JNK3, and IKK-1) inhibitory activity against IKK-2.

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.

"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.

Synthesis and antibacterial activity of new 9-O-arylpropenyloxime ketolides.

A novel series of 9-O-arylpropenyloxime ketolide was synthesized and evaluated for their antibacterial activity. This series of ketolide exhibited potent activity against clinically isolated gram-positive strains including Staphylococcus pneumoniae and Straptococcus Pyogenes.

Novel 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives as potential anti-cancer agents.

A novel series of 3,5,6-trisubstituted pyrazolo[4,3-d]pyrimidin-7-one derivatives, especially 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-ones were synthesized and evaluated for their in vitro anticancer activities against various human cancer cell lines. The inhibitory activities for several kinases have also been tested. The prepared compounds library exhibited significant anticancer activity towards HT-29 colon and DU-145 prostate cancer cell lines. The structure-activity relationships of the 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one scaffold at R(1), R(2) and R(3) have been elucidated. Among the synthesized compounds, 12b was the most active compound with GI(50) value of 0.44microM and 1.07microM against HT-29 and DU-145 cell lines, respectively, and 13a was the most selective compound towards colon cancer cell line.

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.

Quinazolindione derivatives as potent 5-HT3A receptor antagonists.

5-HT(3A) receptor antagonists have been used mainly for the treatment of nausea and vomiting. These days, the antagonists are of special interest due to their therapeutic potential to treat other diseases such as depression, psychotic disorder, drug abuse, and irritable bowel syndrome. To discover novel 5-HT(3A) receptor antagonists, we screened our in-house small molecule library, resulting in identifying the quinazolindione derivatives as potent 5-HT(3A) receptor antagonists. For the purpose of structure-activity relationship study, 24 quinazolindione analogues were biologically evaluated against 5-HT(3A) receptor. Among those, KKHT10612 shows the best antagonistic effect against 5-HT(3A) receptor with an IC(50) value of 0.8 microM which is comparable with that of the reference compound, MDL72222, and selectivity over T-type calcium channel as well.

Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins.

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.