Catalytic Decarboxylative Radical Sulfinylation.

Sulfoxides are ubiquitous in both naturally and synthetically bioactive molecules. We report herein a redox-neutral and mild approach for radical sulfinylation of redox-active esters via dual photoredox and copper catalysis, furnishing a series of functionalized sulfoxides. The reaction could accommodate a range of tertiary, secondary, and primary carboxylic acids, as well as exhibit wide functional group compatibility. The chemistry features a high degree of practicality, is scalable, and allows late-stage modification of bioactive pharmaceuticals.

Necrostatin-1 Analog DIMO Exerts Cardioprotective Effect against Ischemia Reperfusion Injury by Suppressing Necroptosis via Autophagic Pathway in Rats.

AIM: It has been reported that necrostatin-1 (Nec-1) is a specific necroptosis inhibitor that could attenuate programmed cell death induced by myocardial ischemia/reperfusion (I/R) injury. This study aimed to observe the effect and mechanism of novel Nec-1 analog (Z)-5-(3,5-dimethoxybenzyl)-2-imine-1-methylimidazolin-4-1 (DIMO) on myocardial I/R injury. METHODS: Male SD rats underwent I/R injury with or without different doses of DIMO (1, 2, or 4 mg/kg) treatment. Isolated neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment with or without DIMO (0.1, 1, 10, or 100 µM). Myocardial infarction was measured by TTC staining. Cardiomyocyte injury was assessed by lactate dehydrogenase assay (LDH) and flow cytometry. Receptor-interacting protein 1 kinase (RIP1K) and autophagic markers were detected by co-immunoprecipitation and Western blotting analysis. Molecular docking of DIMO into the ATP binding site of RIP1K was performed using GLIDE. RESULTS: DIMO at doses of 1 or 2 mg/kg improved myocardial infarct size. However, the DIMO 4 mg/kg dose was ineffective. DIMO at the dose of 0.1 µM decreased LDH leakage and the ratio of PI-positive cells followed by OGD/R treatment. I/R or OGD/R increased RIP1K expression and in its interaction with RIP3K, as well as impaired myocardial autophagic flux evidenced by an increase in LC3-II/I ratio, upregulated P62 and Beclin-1, and activated cathepsin B and L. In contrast, DIMO treatment reduced myocardial cell death and reversed the above mentioned changes in RIP1K and autophagic flux caused by I/R and OGD/R. DIMO binds to RIP1K and inhibits RIP1K expression in a homology modeling and ligand docking. CONCLUSION: DIMO exerts cardioprotection against I/R- or OGD/R-induced injury, and its mechanisms may be associated with the reduction in RIP1K activation and restoration impaired autophagic flux.

Radical alkylation of para-quinone methides with 4-substituted Hantzsch esters/nitriles via organic photoredox catalysis.

A novel photocatalytic protocol is herein described for the preparation of functionalized phenols via radical alkylation of para-quinone methides under transition-metal-free conditions. The reaction is external oxidant free and performed at ambient temperature upon visible light irradiation, allowing the access to various desired products in satisfactory yields. The readily available 4-alkyl-1,4-dihydropyridines serve as the effective alkyl radical precursors.

The cystathionine ß-synthase/hydrogen sulfide pathway contributes to microglia-mediated neuroinflammation following cerebral ischemia.

The mechanisms underlying neuroinflammation following cerebral ischemia remain unclear. Hydrogen sulfide (H2S), a newly identified gasotransmitter, has been reported to regulate inflammation. In the current study, we investigated whether the endogenous H2S production pathway contributed to microglia-mediated neuroinflammation following stroke. We used a mouse middle cerebral artery occlusion (MCAO) model and an in vitro cellular model to mimic ischemia-induced microglial neuroinflammation. Expression of the H2S synthase cystathionine ß-synthase (CBS) and H2S synthetic activity were rapidly decreased in the ischemic brain tissue following MCAO. Consistently, when cultured microglia were polarized toward a pro-inflammatory phenotype with conditioned medium collected from neurons that had been subjected to oxygen-glucose deprivation (OGD neuron CM), they displayed reduced CBS expression and H2S production. Enhancing H2S bioavailability either by overexpressing CBS or by supplementing with exogenous H2S donors promoted a shift in microglial polarization from ischemia-induced pro-inflammatory phenotypes toward anti-inflammatory phenotypes. Mechanistically, microglia that were exposed to OGD neuron CM displayed reduced activation of AMP-activated protein kinase (AMPK), which was rescued by overexpressing CBS or by supplementing with H2S donors. Moreover, the promoting effects of H2S donors on microglial anti-inflammatory polarization were abolished by an AMPK inhibitor or CaMKKß inhibitor. Our results suggested that reduced CBS-H2S-AMPK cascade activity contributed to microglia-mediated neuroinflammation following stroke. Targeting the CBS-H2S pathway is a promising therapeutic approach for ischemic stroke.

Discovery of novel curcumin derivatives targeting xanthine oxidase and urate transporter 1 as anti-hyperuricemic agents.

A series of curcumin derivatives as potent dual inhibitors of xanthine oxidase (XOD) and urate transporter 1 (URAT1) was discovered as anti-hyperuricemic agents. These compounds proved efficient effects on anti-hyperuricemic activity and uricosuric activity in vivo. More importantly, some of them exhibited proved efficient effects on inhibiting XOD activity and suppressing uptake of uric acid via URAT1 in vitro. Especially, the treatment of 4d was demonstrated to improve uric acid over-production and under-excretion in oxonate-induced hyperuricemic mice through regulating XOD activity and URAT1 expression. Docking study was performed to elucidate the potent XOD inhibition of 4d. Compound 4d may serve as a tool compound for further design of anti-hyperuricemic drugs targeting both XOD and URAT1.

Synthesis and biological evaluation of novel hydrogen sulfide releasing nicotinic acid derivatives.

Twelve novel hybrids of slowly releasing hydrogen sulfide donor ADT-OH combined with nicotinic acid were synthesized. All of their structures had been confirmed by 1H NMR, 13C NMR and MS spectra. The target compounds were evaluated for their neuroprotective effects on hippocampal neuron HT22 cells against glutamate-induced injury at the concentrations of 1-100µM with MTT assay, and their toxicity on HT22 cells untreated by glutamine at the concentration of 100µM. The active compound was further investigated for its effect on ischemic infarct volume by intraperitoneal injection at 3h after ischemia in mice models of permanent middle cerebral artery occlusion (pMCAO). The results showed that all the compounds significantly protected HT22 cells from glutamate-induced damage at most of the experimental concentrations, and had no or little neurotoxicity on normal HT22 cells at the high concentration. More importantly, compound A6 significantly reduced infarct volume in the pMCAO model. These results suggested that compound A6 may be promising for further evaluation for the intervention of cerebral ischemic injury.

Synthesis and biological evaluation of novel hydrogen sulfide releasing glycyrrhetic acid derivatives.

A series of hybrids, which are composed of glycyrrhetic acid (GA) and slowly hydrogen sulfide-releasing donor ADT-OH, were designed and synthesized to develop anticancer and anti-inflammatory agents. Most of the compounds, whose inhibitory rates were comparable to or higher than those of GA and aspirin, respectively, significantly inhibited xylene-induced ear edema in mice. Especially, compound V4 exhibited the most potent inhibitory rate of 60.7%. Furthermore, preliminary structure-activity relationship studies demonstrated that 3-substituted GA derivatives had stronger anti-inflammatory activities than the corresponding 3-unsubstituted GA derivatives. In addition, anti-proliferative activities of compounds V1-9 were evaluated in three different human cancer cell lines. Compound V4 showed the most high potency against all three tumor cell lines with IC50 values ranging from 10.01 µM in Hep G2 cells to 17.8 µM in MDA-MB-231 cells, which were superior to positive GA.

Hydrogen sulfide protects against myocardial ischemia and reperfusion injury by activating AMP-activated protein kinase to restore autophagic flux.

How hydrogen sulfide (H2S) protects against myocardial ischemia-reperfusion (I/R) injury is poorly understood. By using a slow-releasing H2S donor, we investigated if H2S protected against myocardial I/R injury by activating AMPK and restoring I/R-impaired autophagic flux. Male rats received anterior descending coronary artery occlusion followed by reperfusion. The H2S donor ADT and/or the AMPK inhibitor, compound C (CC), were administered after occlusion. Infarction was analyzed histologically. AMPK activation was assessed in the ischemic heart by analyzing phosphorylation of AMPK and S6 ribosomal protein. Autophagy was assessed by analyzing the following markers: microtubule-associated protein 1 light chain 3 (LC3) I and II, lysosome associated membrane protein-2 (LAMP-2), P62 and beclin-1. We further investigated if blocking autophagic flux with chloroquine abolished ADT cardioprotection in vivo. Myocardial I/R reduced serum H2S levels, which was elevated by ADT. ADT enhanced AMPK activation and reduced infarction following I/R, and both effects were abolished by AMPK inhibition. Myocardial I/R induced autophagosome accumulation, as evidenced by the increased ratios of LC3-II/LC3-I, upregulation of beclin-1 and P62 and reduction in LAMP-2. ADT blunted these autophagic changes induced by I/R, indicating that ADT restored I/R-impaired autophagic flux. The AMPK inhibitor CC blocked ADT effects on restoring I/R-impaired autophagy flux. Moreover, chloroquine pretreatment abolished cardioprotection of ADT and increased autophagosome accumulation in the ADT-treated heart following I/R. In conclusion, AMPK activation and subsequent restoration of I/R-impaired autophagic flux are unrecognized mechanisms underlying cardioprotective effects conferred by H2S donors.

Hydrogen sulfide protects blood-brain barrier integrity following cerebral ischemia.

By using two structurally unrelated hydrogen sulfide (H2 S) donors 5-(4-methoxyphenyl) -3H-1, 2-dithiole-3-thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H2 S protected blood-brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H2 S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post-ischemic inflammation-induced Matrix Metalloproteimase-9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H2 S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H2 S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro-inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL-1ß while enhanced expression of anti-inflammatory markers arginase 1 and IL-10 in the ischemic brain. Accordingly, ADT attenuated ischemia-induced expression and activity of MMP9. Moreover, ADT reduced NOX-4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa-B (NF-κB) in the ischemic brain. In conclusion, H2 S donors protected BBB integrity following experimental stroke possibly by acting through NF-κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4-derived free radicals. To determine H2 S effects on blood-brain barrier (BBB) disruption following stroke, we used two structurally unrelated H2 S donors ADT and NaHS. Both ADT and NaHS remarkably protected BBB integrity following experimental stroke. The slow-releasing donor ADT also reduced post-ischemic inflammation-induced expression and activity of MMP9 and NOX4 in the ischemic brain possibly by inhibiting NF-κB activation.

Growth inhibitory effect of KYKZL-1 on Hep G2 cells via inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest.

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the inhibitory activity test on Hep G2 growth. We found that KYKZL-1 inhibited the growth of Hep G2 cells via inducing apoptosis. Further studies showed that KYKZL-1 activated caspase-3 through cytochrome c release from mitochondria and down regulation of Bcl-2/Bax ratio and reduced the high level of COX-2 and 5-LOX. As shown in its anti-inflammatory effect, KYKZL-1 also exhibited inhibitory effect on the PGE2 and LTB4 production in Hep G2 cells. Accordingly, exogenous addition of PGE2 or LTB4 reversed the decreases in cell viability. In addition, KYKZL-1 caused cell cycle arrest at the S-G2 checkpoint via the activation of p21(CIP1) protein and down-regulation of cyclin A expression. These data indicate that the growth inhibitory effect of KYKZL-1 is associated with inhibition of AA metabolites and caspase-3 pathway and cell cycle arrest. Combined with our previous findings, KYKZL-1 exhibiting COX/5-LOX inhibition may be a promising potential agent not only for inflammation control but also for cancer prevention/therapy with an enhanced gastric safety profile.

Preparative separation of crocins and geniposide simultaneously from gardenia fruits using macroporous resin and reversed-phase chromatography.

Gardenia fruits contain valuable natural food colorants including crocins (gardenia yellow) and geniposide. In this study, a process for the enrichment of crocins and geniposide simultaneously from gardenia fruits was developed using macroporous resin and RP chromatography. The performance of eight different types of macroporous resins was evaluated. Static absorption/desorption experiments revealed that LX60 possessed optimal separating capacity. Further dynamic absorption/desorption experiments on LX60 columns were conducted to obtain the optimal parameters. After one run treatment with LX60, the content of crocin-1 in gardenia yellow reached 29.6%, while geniposide in another fraction reached 83.4%. An extract of crocins was obtained from gardenia yellow in a second-stage separation using RP medium-pressure LC, with its color value to be 756 and the content of crocin-1 reaching 60.8%. The separation process was highly efficient, low cost, and compact, which may be informative for purifications of other natural products from complex plant extracts.

Antioxidant properties and PC12 cell protective effects of a novel curcumin analogue (2E,6E)-2,6-bis(3,5- dimethoxybenzylidene)cyclohexanone (MCH).

The antioxidative properties of a novel curcumin analogue (2E,6E)-2,6-bis(3,5-dimethoxybenzylidene)cyclohexanone (MCH) were assessed by several in vitro models, including superoxide anion, hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and PC12 cell protection from H2O2 damage. MCH displayed superior O2•- quenching abilities compared to curcumin and vitamin C. In vitro stability of MCH was also improved compared with curcumin. Exposure of PC12 cells to 150 µM H2O2 caused a decrease of antioxidant enzyme activities, glutathione (GSH) loss, an increase in malondialdehyde (MDA) level, and leakage of lactate dehydrogenase (LDH), cell apoptosis and reduction in cell viability. Pretreatment of the cells with MCH at 0.63-5.00 µM before H2O2 exposure significantly attenuated those changes in a dose-dependent manner. MCH enhanced cellular expression of transcription factor NF-E2-related factor 2 (Nrf2) at the transcriptional level. Moreover, MCH could mitigate intracellular accumulation of reactive oxygen species (ROS), the loss of mitochondrial membrane potential (MMP), and the increase of cleaved caspase-3 activity induced by H2O2. These results show that MCH protects PC12 cells from H2O2 injury by modulating endogenous antioxidant enzymes, scavenging ROS, activating the Nrf2 cytoprotective pathway and prevention of apoptosis.

Inhibition of inflammatory mediators contributes to the anti-inflammatory activity of KYKZL-1 via MAPK and NF-κB pathway.

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the anti-inflammatory activity test focusing on its modulation of inflammatory mediators as well as intracellular MAPK and NF-κB signaling pathways. In acute ear edema model, pretreatment with KYKZL-1 (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice with a higher inhibition than diclofenac. In a three-day TPA-induced inflammation, KYKZL-1 also showed significant anti-inflammatory activity with inhibition ranging between 20% and 64%. In gastric lesion test, KYKZL-1 elicited markedly fewer stomach lesions with a low index of ulcer as compared to diclofenac in rats. In further studies, KYKZL-1 was found to significantly inhibit the production of NO, PGE2, LTB4 in LPS challenged RAW264.7, which is parallel to its attenuation of the expression of iNOS, COX-2, 5-LOX mRNAs or proteins and inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. Taken together, our data indicate that KYKZL-1 comprises dual inhibition of COX and 5-LOX and exerts an obvious anti-inflammatory activity with an enhanced gastric safety profile via simultaneous inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB.

Design, synthesis and molecular docking of α,ß-unsaturated cyclohexanone analogous of curcumin as potent EGFR inhibitors with antiproliferative activity.

A type of novel α,ß-unsaturated cyclohexanone analogous, which designed based on the curcumin core structure, have been discovered as potential EGFR inhibitors. These compounds exhibit potent antiproliferative activity in two human tumor cell lines (Hep G2 and B16-F10). Among them, compounds I(3) and I(12) displayed the most potent EGFR inhibitory activity (IC(50) = 0.43 µM and 1.54 µM, respectively). Molecular docking of I(12) into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.

Autophagy and cathepsin L are involved in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.

AIM: 2-(3',5'-Dimethoxybenzylidene) cyclopentanone (DMBC) is a novel synthetic compound with antinociceptive activities. The aim of this study was to investigate the roles of the autophagic-lysosomal pathway in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model. METHODS: Mouse acetic acid-writhing test and hotplate test were used to assess the antinociceptive effects of DMBC, 3-MA (autophagy inhibitor) and Clik148 (cathepsin L inhibitor). The drugs were administered peripherally (ip) or centrally (icv). RESULTS: Peripheral administration of 3-MA (7.5-30 mg/kg) or Clik148 (10-80 mg/kg) produced potent antinociceptive effect in acetic acid-writhing test. Central administration of 3-MA or Clik148 (12.5-50 nmol/L) produced comparable antinociceptive effect in acetic acid-writhing test. Peripheral administration of DMBC (25-50 mg/kg) produced potent antinociceptive effects in both acetic acid-writhing and hotplate tests. Furthermore, the antinociceptive effect produced by peripheral administration of DMBC (50 mg/kg) in acetic acid-writhing test was antagonized by low doses of 3-MA (3.75 mg/kg) or Clik148 (20 mg/kg) peripherally administered, but was not affected by 3-MA or Clik148 (25 nmol/L) centrally administered. CONCLUSION: Activation of central autophagy and cathepsin L is involved in nociception in mice, whereas peripheral autophagy and cathepsin L contributes, at least in part, to the antinociceptive effect of DMBC in mice.

[Study on stability and degradation kinetics of ketoprofen-paeonol conjugate].

OBJECTIVE: To study the stability and degradation kinetics of Ketoprofen-Paeonol conjugate (Ket-Pae). METHOD: RP-HPLC method was used to determine the solubility and partition coefficient of Ket-Pae. Stability test was carried out to investigate the factors affecting Ket-Pae. The kinetic studies of Ket-Pae degradation were conducted in different pH buffer solutions and 80% rat plasma at 37 degrees C. RESULT: Ket-Pae showed significant degradation phenomenon at high temperature. The solubility of Ket-Pae was decreased about 200 to 300 times compared with parent drugs in water while the lnP increased about 4 times. The degradation curve displayed a V-shape, and kept maximum stability at week acidic (pH 5.0, t(1/2) = 11.4 d). Ket-Pae degraded quickly with very short half life of 1.3 min in plasma, therefore easily released ketoprofen and paeonol. CONCLUSION: The lipophilicity of Ket-Pae is increased, its stability is affected by temperature and pH value.

Copper-Catalyzed Remote C(sp3)-H Amination of Carboxamides.

Here we report a method for the site-selective intermolecular C(sp3)-H amination of carboxamides by merging transition-metal catalysis and the hydrogen atom transfer strategy. The reaction proceeds through a sequence of favorable single-electron transfer, 1,5-hydrogen atom transfer, and C-N cross-coupling steps, thus allowing access to a series of desired products. This reaction could accommodate a wide diversity of nitrogen nucleophiles as well as demonstrate excellent chemoselectivity and functional group compatibility.

SQR mediates therapeutic effects of H2S by targeting mitochondrial electron transport to induce mitochondrial uncoupling.

Hydrogen sulfide (H2S) is a gasotransmitter and a potential therapeutic agent. However, molecular targets relevant to its therapeutic actions remain enigmatic. Sulfide-quinone oxidoreductase (SQR) irreversibly oxidizes H2S. Therefore, SQR is assumed to inhibit H2S signaling. We now report that SQR-mediated oxidation of H2S drives reverse electron transport (RET) at mitochondrial complex I, which, in turn, repurposes mitochondrial function to superoxide production. Unexpectedly, complex I RET, a process dependent on high mitochondrial membrane potential, induces superoxide-dependent mitochondrial uncoupling and downstream activation of adenosine monophosphate-activated protein kinase (AMPK). SQR-induced mitochondrial uncoupling is separated from the inhibition of mitochondrial complex IV by H2S. Moreover, deletion of SQR, complex I, or AMPK abolishes therapeutic effects of H2S following intracerebral hemorrhage. To conclude, SQR mediates H2S signaling and therapeutic effects by targeting mitochondrial electron transport to induce mitochondrial uncoupling. Moreover, SQR is a previously unrecognized target for developing non-protonophore uncouplers with broad clinical implications.

Neuroprotective Effects of DTIO, A Novel Analog of Nec-1, in Acute and Chronic Stages After Ischemic Stroke.

Receptor-interacting protein 1 kinase (RIP1K) plays a key role in necroptosis. Necrostatin-1 (Nec-1), a specific inhibitor of RIP1K, provides neuroprotection against ischemic brain injury, associating with inhibition of inflammation. Recently, our group synthesized a novel analog of Nec-1, 5-(3',5'-dimethoxybenzal)-2-thio-imidazole-4-ketone (DTIO). The present study investigated the effect of DTIO on ischemic stroke-induced brain injury in both acute and chronic phase and its underlying mechanism. In vivo, DTIO treatment reduced infarct volume and improved neurological deficits in the acute phase after permanent middle cerebral artery occlusion (pMCAO) and it also attenuated brain atrophy and promoted brain functional recovery in the chronic phase post-cerebral ischemia/reperfusion (I/R). In vitro, DTIO treatment decreased lactate dehydrogenase (LDH) leakage and necrotic cell death in the oxygen and glucose deprivation (OGD) or oxygen and glucose deprivation and reoxygenation (OGD/R)-induced neuronal or astrocytic cell injury. Simultaneously, DTIO suppressed the production and release of inflammatory cytokines, and reduced the formation of glial scar. Homology modeling analysis illustrated that DTIO had an ability of binding to RIP1K. Furthermore, immunoprecipitation analysis showed that DTIO inhibited the phosphorylation of RIP1K and decreased the interaction between the RIP1K and RIP3K. In addition, knockdown of RIP1K had neuroprotective effects and inhibited the release of proinflammatory cytokines, but didn't have a significant effect on DTIO-mediated neuroprotection. In conclusion, DTIO has protective effects on acute ischemic stroke and promotes functional recovery during chronic phase, associating with protecting ischemic neurons and astrocytes, inhibiting inflammation, and lessening the glial scar formation via inhibiting of the RIP1K.

Cathepsin B and L inhibitors: a patent review (2010 - present).

INTRODUCTION: Cathepsins play an important role in protein degradation and processing. Aberrant cathepsin B or L is closely associated with many serious diseases such as cancer, osteoporosis and autoimmune disorders. Therefore, development of potent and selective cathepsin B and L inhibitors has aroused much attention in recent years. Although several classes of cathepsin inhibitors are presently available, there are still some problems to solve, such as broad-spectrum inhibition to protease, specially cysteine proteases, which lead to unpredictable side effects in clinical trials. Therefore, it is very necessary to discovery new scaffolds and new application of cathepsin B and L inhibitors for developing therapeutic agents for treating diseases mediated by cathepsin B or L. Areas covered: This updated review summarizes new patents on cathepsin B and L inhibitors from 2010 to present. Expert opinion: The review gives the latest development in the area of inhibitors of cathepsin B and L, which have been considered key therapeutic targets for the development of drugs treating related diseases. This review puts emphasis on the discovery of novel small molecule inhibitors of cathepsin B and L, as well as their new application as new therapeutic agents.