Protective effect of secretory APE1/Ref-1 on doxorubicin-induced cardiotoxicity via suppression of ROS and p53 pathway.

AIMS: The clinical application of doxorubicin (DOX), a potent anthracycline anticancer drug that effectively treats various malignancies, is limited by its side effects, such as cardiomyopathy. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that can be secreted and is a promising target for the reduction of DOX-induced inflammation and oxidative stress. We aimed to investigate the protective role of secretory APE1/Ref-1 against DOX-induced cardiac injury. METHODS AND RESULTS: Designated adenoviral preprotrypsin-leading sequence APE1/Ref-1 (Ad-PPTLS-APE1/Ref-1) was used to overexpress secretory APE1/Ref-1 and assess its role in preventing DOX-induced cardiomyopathy in vitro. Our findings revealed that exposure to secretory APE1/Ref-1 significantly decreased N-terminal pro-B-type natriuretic peptide levels in DOX-treated H9C2 cells. In addition, secretory APE1/Ref-1 reduced the severity of cardiomyocyte injury and apoptosis in both in vitro and in vivo DOX-induced cardiotoxicity models. The observed cardioprotective effects of secretory APE1/Ref-1 were mediated via inhibition of the p53 signalling pathway and enhancement of cell viability through attenuation of oxidative stress in DOX-treated cardiomyocytes. CONCLUSIONS: Our study provides evidence that secretory APE1/Ref-1 has the potential to inhibit DOX-induced cardiac toxicity by inhibiting oxidative stress and p53 related apoptosis both in vitro and in vivo. These findings suggest that secretory APE1/Ref-1 supplementation is a promising strategy to attenuate DOX-induced cardiomyocyte damage in a preclinical model. Further clinical investigations are essential to validate the therapeutic efficacy and safety of the intervention in human subjects.

Design and synthesis of novel arylpiperazine derivatives containing the imidazole core targeting 5-HT(2A) receptor and 5-HT transporter.

Serotonin antagonist reuptake inhibitor (SARI) drugs that block both 5-HT(2) receptors and the serotonin transporters have been developed. The human 5-HT(2A/2C) receptor has been implicated in several neurological conditions, and potent selective 5-HT(2A/2C) ligands may have therapeutic potential for treatment of CNS diseases such as depression. An imidazole moiety usually provides good pharmacokinetic properties as a drug substance, and thus considerable efforts have been devoted to develop imidazole derivatives into drug candidates. The imidazole series of compounds was evaluated against 5-HT(2A/2C) and serotonin reuptake inhibition. A few of the compounds in the series showed promising IC(50) values and antidepressant-like effect in in vivo forced swimming test (FST). On the basis of these results, further lead optimization studies resulted in identifying promising compounds potentially for therapeutic use.

Synthesis and SAR of Thiazolylmethylphenyl Glucoside as Novel C-Aryl Glucoside SGLT2 Inhibitors.

Novel C-aryl glucoside SGLT2 inhibitors containing the thiazole motif were designed and synthesized for biological evaluation. Among the compounds assayed, thiazole containing furanyl moiety 14v and thiophenyl moiety 14y demonstrated the best in vitro inhibitory activity against SGLT2 in this series to date (IC50 = 0.720 nM for 14v and IC50 = 0.772 nM for 14y). Both of these compounds have been further evaluated on a urinary glucose excretion test and the urine volumes excreted.

Characterization of Deinococcus radiophilus thioredoxin reductase active with both NADH and NADPH.

Thioredoxin reductase (TrxR, EC 1.6.4.5) of Deinococcus radiophilus was purified by steps of sonication, ammonium sulfate fractionation, 2'5' ADP Sepharose 4B affinity chromatography, and Sephadex G-100 gel filtration. The purified TrxR, which was active with both NADPH and NADH, gave a 368 U/mg protein of specific activity with 478-fold purification and 18% recovery from the cell-free extract. An isoelectric point of the purified enzymes was ca. 4.5. The molecular weights of the purified TrxR estimated by PAGE and gel filtration were about 63.1 and 72.2 kDa, respectively. The molecular mass of a TrxR subunit is 37 kDa. This suggests that TrxR definitely belongs to low molecular weight TrxR (L-TrxR). The Km and Vmax of TrxR for NADPH are 12.5 µM and 25 µM/min, whereas those for NADH are 30.2 µM and 192 µM/min. The Km and Vmax for 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB, a substituted substrate for thioredoxin) are 463 µM and 756 µM/min, respectively. The presence of FAD in TrxR was confirmed with the absorbance peaks at 385 and 460 nm. The purified TrxR was quite stable from pH 3 to 9, and was thermo-stable up to 70°C. TrxR activity was drastically reduced (ca. 70%) by Cu(2+), Zn(2+), Hg(2+), and Cd(2+), but moderately reduced (ca. 50%) by Ag(+). A significant inhibition of TrxR by N-ethylmaleimide suggests an occurrence of cysteine at its active sites. Amino acid sequences at the N-terminus of purified TrxR are H(2)N-Ser-Glu-Gln-Ala-Gln-Met-Tyr-Asp-Val-Ile-Ile-Val-Gly-Gly-Gly-Pro-Ala-Gly-Leu-Thr-Ala-COOH. These sequences show high similarity with TrxRs reported in Archaea, such as Methanosarcina mazei, Archaeoglobus fulgidus etc.

Chronic Administration of Monosodium Glutamate under Chronic Variable Stress Impaired Hypothalamic-Pituitary-Adrenal Axis Function in Rats.

The hypothalamic-pituitary-adrenal (HPA) axis is the primary endocrine system to respond to stress. The HPA axis may be affected by increased level of corticotrophin-releasing factors under chronic stress and by chronic administration of monosodium glutamate (MSG). The purpose of this study was to investigate whether chronic MSG administration aggravates chronic variable stress (CVS)-induced behavioral and hormonal changes. Twenty-four adult male Sprague-Dawley rats, weighing 200~220 g, were divided into 4 groups as follows: water administration (CON), MSG (3 g/kg) administration (MSG), CVS, and CVS with MSG (3 g/kg) administration (CVS+MSG). In addition, for the purpose of comparing the effect on plasma corticosterone levels between chronic stress and daily care or acute stress, 2 groups were added at the end of the experiment; the 2 new groups were as follows: naïve mice (n=7) and mice exposed to restraint stress for 2 h just before decapitation (A-Str, n=7). In an open field test performed after the experiment, the CVS+MSG group significant decrease in activity. The increase in relative adrenal weights in the CVS and CVS+MSG group was significantly greater than those in the CON and/or MSG groups. In spite of the increase in the relative adrenal weight, there was a significant decrease in the plasma corticosterone levels in the CVS+MSG group as compared to all other groups, except the naïve group. These results suggest that impaired HPA axis function as well as the decrease in the behavioral activity in adult rats can be induced by chronic MSG administration under CVS rather than CVS alone.

Discovery of 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) as a potent, selective and orally efficacious cannabinoid-1 receptor antagonist.

Structure-activity relationship studies in a series of diarylpyrazolyl thiadiazoles identified cannabinoid-1 receptor antagonists with excellent potency and selectivity. Based on its exceptional in vivo efficacy in animal models and its favorable pharmacokinetic and toxicological profiles, 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2-chlorophenyl)-1H-pyrazol-3-yl)-5-tert-butyl-1,3,4-thiadiazole (GCC2680) was selected as a preclinical candidate for the treatment of obesity.

Further optimization of novel pyrrole 3-carboxamides for targeting serotonin 5-HT(2A), 5-HT(2C), and the serotonin transporter as a potential antidepressant.

In the continuing search for novel compounds targeting serotonin 5-HT(2A), 5-HT(2C), and serotonin transporter, new arylpiperazine-containing pyrrole 3-carboxamide derivatives were synthesized and evaluated. Based on the lead reported previously, structural modifications regarding N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)-1,2-dimethyl-5-phenyl-1H-pyrrole-3-carboxamide 5, were accomplished for improvements in not only binding affinity against serotonin receptors and transporter, but also in hERG channel inhibition. Along the line, both the forced swimming tests and spontaneous locomotor activity tests were performed to distinguish between antidepressant activity and false positive results. As potential antidepressant agents, both 2,4-dimethyl-5-phenyl-1H-pyrrole-3-carboxamide and 5-tert-butyl-2-methyl-1H-pyrrole-3-carboxamide derivatives exhibited favorable in vitro and in vivo activities, warranting further investigation around these scaffolds.

Novel C-aryl glucoside SGLT2 inhibitors as potential antidiabetic agents: 1,3,4-Thiadiazolylmethylphenyl glucoside congeners.

Novel C-aryl glucoside SGLT2 inhibitors containing 1,3,4-thiadiazole moieties were designed and synthesized. Among the compounds tested, biaryl-type compounds containing pyrazine 59, 2-furan 61, and 3-thiophene 71 showed the best in vitro inhibitory activities to date (IC(50) = 3.51-7.03 nM) against SGLT2. A selected compound 61, demonstrated reasonable blood glucose-lowering effects, indicating that the information obtained from the SAR studies in this 1,3,4-thiadiazolylmethylphenyl glucoside series might help to design more active SGLT2 inhibitors that are structurally related.

Arylpiperazine-containing pyrrole 3-carboxamide derivatives targeting serotonin 5-HT(2A), 5-HT(2C), and the serotonin transporter as a potential antidepressant.

Arylpiperzine-containing pyrrole 3-carboxamide derivatives were synthesized and evaluated as novel antidepressant compounds. The various analogues were efficiently prepared and bio-assayed for binding to 5-HT(2A), 5-HT(2C) receptor, and 5-HT transporter. Based on their in vitro and in vivo activities as well as selectivity over other neurotransmitter receptors and PK profiles, 33 and 34 were identified as lead compounds. Consequently, this pyrrole series of compounds appears to be promising enough to warrant further investigation.

Synthesis and structure-activity relationship of 1,2,4-triazole-containing diarylpyrazolyl carboxamide as CB1 cannabinoid receptor-ligand.

Numerous research groups have been engaged in searching for novel CB1 receptor antagonists, since SR141716A (rimonabant), a CB1 receptor antagonist, proved to be efficacious in human for the treatment of obesity. In the present study, a series of 1,2,4-triazole-containing diarylpyrazolyl carboxamides based on the 1,5-diarylpyrazole template of rimonabant, was synthesized and tested for CB1 receptor binding affinity. The structure-activity relationship studies demonstrated that incorporation of 1,2,4-triazole ring onto the pyrazole scaffold via a methylene linker led to a significant improvement for CB1 receptor binding affinity. Importantly, these analogues also exhibited excellent selectivity for CB1 receptor over CB2 receptor.

Pentacycle derivatives as cannabinoid CB1 receptor ligands.

Cannabinoid CB-1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of pentacycle derivatives. Five of the new compounds which displayed high in vitro rCB1 binding affinities were assayed for binding to hCB2 receptor. Noticeably, 2-(5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-(5-methyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)cyclopropyl)-1,3,4-oxadiazole (16l) demonstrated good binding affinity and decent selectivity for rCB1 receptor (IC(50)=1.72 nM, hCB2/rCB1=142).

Substituted pyrimidines as cannabinoid CB1 receptor ligands.

Cannabinoid CB1 receptors have been the avenue of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of substituted pyrimidines based on chemical structure of Merck's taranabant, a cannabinoid CB1 receptor inverse agonist. Noticeably, N4-((2S,3S)-3-(3-bromophenyl)-4-(4-chlorophenyl)butan-2-yl)-N6-butylpyrimidine-4,6-diamine (13b) demonstrated good binding affinity and decent selectivity for CB1 receptor (IC(50)=16.3nM, CB2/CB1=181.6).

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.

Oxadiazole-diarylpyrazole 4-carboxamides as cannabinoid CB1 receptor ligands.

Cannabinoid CB-1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of oxadiazole-diarylpyrazole 4-carboxamides. Six of the new compounds which displayed high in vitro CB1 binding affinities were assayed for binding to CB2 receptor. Noticeably, 5-(4-bromophenyl)-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(2,4-dichlorophenyl)-N-phenyl-1H-pyrazole-4-carboxamide (12q) and 5-(4-bromophenyl)-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(2,4-dichlorophenyl)-N-(pyridin-2-yl)-1H-pyrazole-4-carboxamide (12r) demonstrated good binding affinity and decent selectivity for CB1 receptor (IC(50) = 1.35 nM, CB2/CB1 = 286 for 12q; IC(50) = 1.46 nM, CB2/CB1 = 256 for 12r).

Synthesis and structure-activity relationship of novel diarylpyrazole imide analogues as CB1 cannabinoid receptor ligands.

A myriad of research groups have been engaged in searching for novel CB1 receptor antagonists, since SR141716A (rimonabant), a CB1 receptor antagonist, was discovered for an obesity treatment. In this research, extended series, based on the 1,5-diarylpyrazole template of rimonabant, was synthesized and tested for CB1 receptor binding affinity. In the present study, N-piperidinylcarboxamide group of rimonabant was replaced with the corresponding sulfonamide, imide, N-methyl imide and methylenediamide, respectively. The SAR studies to optimize the CB1 binding affinity led to the potent imide derivatives. The in vivo efficacy test of a derivative (16f) gave a promising result for this novel scaffold. In order to explore physicochemical properties (hydrophobic, steric and electronic) of the representative imide derivatives responsible for their CB1 receptor binding affinity, quantitative structure activity relationship (QSAR) studies were performed. Hansch QSAR models, which were moderate in the explanation for SAR, were generated with hydrophobic, steric and electronic properties of substituents. Especially, the Taft Es-based parabolic model was obtained with the best correlation result (r(2)=0.846).

Diarylimidazolyl oxadiazole and thiadiazole derivatives as cannabinoid CB1 receptor antagonists.

Since the CB1 receptor antagonist SR141716 (rimonabant) was reported to modulate food intake, CB1 antagonism has been considered as a new therapeutic target in the treatment of obesity. Several series of derivatives based on diarylimidazolyl oxadiazole and thiadiazole scaffolds were synthesized and tested for CB1 receptor binding affinity. SAR studies directed toward the optimization of imidazole scaffolds resulted in the discovery of 10s which showed highest potency for CB1 receptor binding affinity (IC(50)=1.91nM) prepared to date.

Methylsulfonylpyrazolyl oxadiazoles and thiadiazoles as potent, orally bioavailable cannabinoid-1 receptor antagonists for the treatment of obesity.

BACKGROUND: Since the cannabinoid receptor 1 (CB1) antagonist SR141716 (rimonabant) was previously reported to modulate food intake, CB1 antagonism has been considered as a new therapeutic target for the treatment of obesity. DISCUSSION: In the present study, biarylpyrazole analogues based on a sulfur-containing pyrazole core coupled with 1,3,4-oxadiazole and 1,3,4-thiadiazole were synthesized and assayed for rat CB1 receptor binding affinity. RESULTS: The structure-activity relationship studies to optimize pyrazole substituents as well as 1,3,4-oxadiazole or 1,3,4-thiadiazole rings led to four novel CB1 antagonists with IC(50) values of approximately 1 nM for the rat CB1 receptor binding. Among these derivatives, we identified trifluoromethylcyclobutyl analogues 19e and 19l as promising precandidates for the development as anti-obesity agents.

Biarylpyrazolyl oxadiazole as potent, selective, orally bioavailable cannabinoid-1 receptor antagonists for the treatment of obesity.

Since the CB1 cannabinoid receptor antagonist 1 (SR141716, rimonabant) was previously reported to modulate food intake, CB1 antagonism has been considered as a new therapeutic target for the treatment of obesity. In the present study, biarylpyrazole analogues based on a pyrazole core coupled with 1,3,4-oxadiazole were synthesized and tested for CB1 receptor binding affinity. Thorough SAR studies to optimize pyrazole substituents as well as 1,3,4-oxadiazole ring led to several novel CB1 antagonists with IC(50) approximately 1 nM for the CB1 receptor binding. Among these analogues, we identified 2-(4-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)cyclopropyl)-1,3,4-oxadiazole 43c as a promising precandidate for the development as an antiobesity agent.

Tetrazole-biarylpyrazole derivatives as cannabinoid CB1 receptor antagonists.

Cannabinoid CB1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed a new series of tetrazole-biarylpyrazoles. The various analogues were efficiently prepared and bio-assayed for binding to cannabinoid CB1 receptor. Six of the new compounds which displayed high in vitro CB1 binding affinities were assayed for binding to CB2 receptor. Noticeably, cyclopentyl-tetrazole (9a) demonstrated good binding affinity and selectivity for CB1 receptor (IC(50)=11.6nM and CB2/CB1=366).

Novel T-type calcium channel blockers: dioxoquinazoline carboxamide derivatives.

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium channel blockers. A small molecule library of dioxoquinazoline carboxamide derivatives containing 155 compounds was designed, synthesized, and biologically evaluated for T-type calcium channel blocking activity. Among those compounds synthesized, the compound 1n shows the most potent T-type calcium current blocking activity with an IC(50) value of 1.52 microM, which is comparable to that of mibefradil.