Non-Palladium-Catalyzed Approach to the Synthesis of (E)-3-(1,3-Diarylallylidene)Oxindoles.

Two novel synthetic approaches for synthesizing (E)-3-(1,3-diarylallylidene)oxindoles from oxindole were developed. All previously reported methods for synthesizing 3-(1,3-diarylallylidene)oxindoles utilized palladium-catalyzed reactions as a key step to form this unique skeleton. Despite high efficiency, palladium-catalyzed reactions have limitations in terms of substrate scope. Especially, an iodoaryl moiety cannot be introduced by the previous methods due to its high reactivity toward the palladium catalyst. Our Knoevenagel/allylic oxidation/Wittig and Knoevenagel/aldol/dehydration strategies complement each other and show broad substrate scope, including substrates with iodoaryl groups. The current methods utilized acetophenones, benzylidene phosphonium ylides, and benzaldehydes that are commercially available or easily accessible. Thus, the current synthetic approaches to (E)-3-(1,3-diarylallyldiene)oxindoles are readily amendable for variety of oxindole derivatives.

Total Synthesis of Cyclopiamide A Using Palladium-Catalyzed Domino Cyclization.

Total synthesis of cyclopiamide A was accomplished using a palladium-catalyzed domino cyclization. Three rings in the tetracyclic skeleton of cyclopiamide A were constructed in a one-step domino reaction incorporating double carbopalladation and C-H activation.

Isolation of six anthraquinone diglucosides from cascara sagrada bark by high-performance countercurrent chromatography.

In this study, high-performance countercurrent chromatography was employed to isolate six anthraquinone diglucosides, namely, cascarosides A-F, from cascara sagrada (Rhamnus purshiana DC [Rhamnaceae]) bark. The n-butanol-soluble extract of cascara sagrada was separated by off-line two-dimensional high-performance countercurrent chromatography. The first-dimensional high-performance countercurrent chromatography resolved the n-butanol-soluble extract (510 mg) of cascara sagrada using the flow-rate gradient method with a chloroform-methanol-isopropanol-water (6:6:1:4, v/v/v/v, normal-phase mode) system to afford four anthraquinone diglucoside fractions (groups I [cascarosides C-D, 71 mg], II [cascarosides E-F, 56 mg], III [cascaroside A, 53 mg], and IV [cascaroside B, 31 mg]). Groups I and II were separated by the second-dimensional high-performance countercurrent chromatography using an ethyl acetate-n-butanol-water (7:3:10, v/v/v, normal-phase mode) system to yield cascarosides C (34 mg), D (26 mg), E (19 mg), and F (15 mg). Additionally, one-step preparative-scale high-performance countercurrent chromatography method was developed to isolate large amounts of cascarosides A (389 mg) and B (187 mg) from the water-soluble extract (2.1 g) of cascara sagrada using an ethyl acetate-n-butanol-water (2:8:10, v/v/v, normal-phase mode) system. The current study demonstrated that high-performance countercurrent chromatography is a powerful technique for the isolation of marker compounds from herbal materials.

Thiazolidine-2,4-dione-based irreversible allosteric IKK-ß kinase inhibitors: Optimization into in vivo active anti-inflammatory agents.

Selective kinase inhibitors development is a cumbersome task because of ATP binding sites similarities across kinases. On contrast, irreversible allosteric covalent inhibition offers opportunity to develop novel selective kinase inhibitors. Previously, we reported thiazolidine-2,4-dione lead compounds eliciting in vitro irreversible allosteric inhibition of IKK-ß. Herein, we address optimization into in vivo active anti-inflammatory agents. We successfully developed potent IKK-ß inhibitors with the most potent compound eliciting IC50 = 0.20 µM. Cellular assay of a set of active compounds using bacterial endotoxin lipopolysaccharide (LPS)-stimulated macrophages elucidated significant in vitro anti-inflammatory activity. In vitro evaluation of microsomal and plasma stabilities showed that the promising compound 7a is more stable than compound 7p. Finally, in vivo evaluation of 7a, which has been conducted in a model of LPS-induced septic shock in mice, showed its ability to protect mice against septic shock induced mortality. Accordingly, this study presents compound 7a as a novel potential irreversible allosteric covalent inhibitor of IKK-ß with verified in vitro and in vivo anti-inflammatory activity.

Optimization study towards more potent thiazolidine-2,4-dione IKK-ß modulator: Synthesis, biological evaluation and in silico docking simulation.

Inhibition of IKK-ß (inhibitor of nuclear factor kappa-B kinase subunit beta) has been broadly documentedas a promising approach for treatment of acute and chronic inflammatory diseases, cancer, and autoimmune diseases. Recently, we have identified a novel class of thiazolidine-2,4-diones as structurally novel modulators for IKK-ß. Herein, we report a hit optimization study via analog synthesis strategy aiming to acquire more potent derivative(s), probe the structure activity relationship (SAR), and get reasonable explanations for the elicited IKK-ß inhibitory activities though an in silico docking simulation study. Accordingly, a new series of eighteen thiazolidine-2,4-dione derivatives was rationally designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as noteworthy IKK-ß potential modulators. Successfully, new IKK-ß potent modulators were obtained, including the most potent analog up-to-date 7m with IC50 value of 260 nM. A detailed structure activity relationship (SAR) was discussed and a mechanistic study for 7m was carried out indicating its irreversible inhibition mode with IKK-ß (Kinact value = 0.01 (min-1). Furthermore, the conducted in silico simulation study provided new insights for the binding modes of this novel class of modulators with IKK-ß.

Duodenal Niemann-Pick C1-like 1 expression was negatively correlated with liver X receptor expression in nonalcoholic fatty liver disease.

BACKGROUND/AIMS: Intestinal cholesterol absorption includes intestinal Niemann-Pick C1-like 1 (NPC1L1) and is an important target pathway in nonalcoholic fatty liver disease (NAFLD). We investigated the expression of NPC1L1 and its correlation with liver X receptor (LXR) expression in peripheral mononuclear (PMN) cells in patients with NAFLD. METHODS: We evaluated intestinal expression of NPC1L1 in 25 NAFLD patients and 28 healthy controls. We calculated the mRNA expression levels of LXR and farnesoid X receptor (FXR), which are master players of cholesterol metabolism in PMN cells. The protein expression of ABCA1, ABCG5/8, NPC1L1, SREBP, LXR, FXR, and CD36 was measured on tissue samples from the duodenum and ileum. RESULTS: The expression of LXR (p = 0.01) and FXR (p = 0.03) in PMN cells was increased in the NAFLD group compared to the control group. Duodenal NPC1L1 decreased in the NAFLD group compared to the healthy controls (3.38 ± 1.4 vs. 2.42 ± 1.2, p = 0.05). NPC1L1 expression in the duodenum was negatively correlated with LXR expression in PMN cells. Expression of LXR and FXR in the ileum was also negatively correlated with the expression of LXR in PMN cells. CONCLUSION: Duodenal NPC1L1 expression was decreased in NAFLD and was negatively correlated with LXR expression in PMN cells.

Novel Rhodanine Derivative, 5-[4-(4-Fluorophenoxy) phenyl]methylene-3-{4-[3-(4-methylpiperazin-1-yl) propoxy]phenyl}-2-thioxo-4-thiazolidinone dihydrochloride, Induces Apoptosis via Mitochondria Dysfunction and Endoplasmic Reticulum Stress in Human Colon Cancer Cells.

We previously reported that 5-[4-(4-fluorophenoxy) phenyl] methylene-3-{4-[3-(4-methylpiperazin-1-yl)propoxy]phenyl}-2-thioxo-4-thiazolidinone dihydrochloride (KSK05104) has potent, selective and metabolically stable IKKß inhibitory activities. However, the apoptosis-inducing of KSK05104 and its underlying mechanism have not yet been elucidated in human colon cancer cells. We show that KSK05104 triggered apoptosis, as indicated by externalization of Annexin V-targeted phosphatidylserine residues in HT-29 and HCT-116 cells. KSK05104 induced the activation of caspase-8, -9, and -3, and the cleavage of poly (ADP ribose) polymerase-1 (PARP-1). KSK05104-induced apoptosis was significantly suppressed by pretreatment with z-VAD-fmk (a broad caspase inhibitor). KSK05104 also induced release of cytochrome c (Cyt c), apoptosis inducing factor (AIF), and endonuclease G (Endo G) by damaging mitochondria, resulting in caspase-dependent and -independent apoptotic cell death. KSK05104 triggered endoplasmic reticulum (ER) stress and changed the intracellular calcium level ([Ca2+]i). Interestingly, treatment with KSK05104 activated not only ER stress marker proteins including inositol-requiring enzyme 1-alpha (IRE-1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), but also µ-calpain, and caspase-12 in a time-dependent manner. KSK05104-induced apoptosis substantially decreased in the presence of BAPTA/AM (an intracellular calcium chelator). Taken together, these results suggest that mitochondrial dysfunction and ER stress contribute to KSK05104-induced apoptosis in human colon cancer cells.

Design, synthesis and biological evaluation of novel thiazolidinedione derivatives as irreversible allosteric IKK-ß modulators.

The kinase known as IKK-ß activates NF-κB signaling pathway leading to expression of several genes contributing to inflammation, immune response, and cell proliferation. Modulation of IKK-ß kinase activity could be useful for treatment and management of such diseases. Starting from a discovered weakly active hit compound, twenty four thiazolidinedione-scaffold based chemical entities belonging to five series have been designed, synthesized and evaluated as potential IKK-ß modulators. Among them, compounds 6q, 6r and 6u showed low micromolar IC50 values while compounds 6v, 6w, and 6x elicited submicromolar IC50 values equal to 0.4, 0.7 and 0.9 µM respectively. These submicromolar IC50 values are 243, 139 and 105 folds the value of the reported IC50 of the starting hit compound. Kinetic study of compounds 6v and 6w confirmed this class of modulators as irreversible inhibitors. LPS-treated RAW 264.7 macrophages proved the anti-inflammatory activity of compounds 6q and 6v. Assay of hERG inhibition demonstrated a safe profile of compound 6q suggesting it as a lead for further development of IKK-ß modulators.

Isolation and structural characterization of a novel sibutramine analogue, chlorosipentramine, in a slimming dietary supplement, by using HPLC-PDA, LC-Q-TOF/MS, FT-IR, and NMR.

A novel sibutramine analogue was detected in a slimming formula by high performance liquid chromatography with a photo diode detector array (HPLC-PDA). The unknown compound exhibited an ultraviolet (UV) spectrum that was similar to that of chlorosibutramine, despite having a different HPLC retention time. Further analysis of the slimming formula by LC-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) showed that the unknown compound had the formula C18H27Cl2N. To elucidate the structure of this new sibutramine analogue, the target compound in the slimming formula was isolated on a preparative-LC system equipped with a PDA. After analysis by fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the unknown compound was identified as a sibutramine analogue in which the iso-butyl group on the side chain is replaced with an iso-pentyl group. This new sibutramine analogue was identified to be 1-(1-(3,4-dichlorophenyl)cyclobutyl)-N,N,4-trimethylpentan-1-amine and has been named as chlorosipentramine.

Identification and structural elucidation of a new sildenafil analogue, dithiopropylcarbodenafil, from a premixed powder intended as a dietary supplement.

A new sildenafil analogue was detected during the monitoring of a premixed powder intended as a dietary supplement. The ultraviolet (UV) spectrum of the unknown compound was similar to that of dithiodesmethylcarbodenafil and dithiodesethylcarbodenafil, although their corresponding HPLC peaks were observed at different retention times. The chemical structure of the unknown compound was characterized by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF/MS), followed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. The comparison of its structure with that of dithiodesmethylcarbodenafil, revealed that the N-methyl group on the piperazine ring is replaced by a propyl group. This new sildenafil analogue was identified as 5-(2-ethoxy-5-(4-propylpiperazine-1-carbonothioyl)phenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidine-7-thione and designated as a dithiopropylcarbodenafil. To the best of our knowledge, this is the first study reporting the identification and characterization of dithiopropylcarbodenafil.

Consecutive One-Pot versus Domino Multicomponent Approaches to 3-(Diarylmethylene)oxindoles.

Based on consecutive one-pot conditions combining three palladium-catalyzed reactions (Sonogashira, Heck and Suzuki-Miyaura reactions), a more efficient domino multicomponent method has been successfully developed to access a wide variety of 3-(diarylmethylene)oxindoles. Microwave irradiation and use of a silver salt were the most important factors to achieve high yields and stereoselectivity.

Stereoselective Synthesis of 3-(1,3-Diarylallylidene)oxindoles via a Palladium-Catalyzed Tandem Reaction.

We have developed an efficient three-component tandem reaction for the synthesis of 3-(1,3-diarylallylidene)oxindoles combining three palladium-catalyzed reactions: the Sonogashira, Heck, and Suzuki-Miyaura reactions. This method allows a stereoselective approach to each (E)- and (Z)-isomer by ligand change and controlling the reaction temperature.

Aspirination of α-Aminoalcohol (Sarpogrelate M1).

Aspirination of α-aminoalcohol (sarpogrelate M1) has been performed under various general esterification conditions. In most cases, the desired aspirinate ester was obtained at a low yield with unexpected byproducts, the formation of which was mostly derived from the chemical properties of the tertiary α-amino group. After systematic analysis of those methods, the aspirinated sarpogrelate M1 was prepared using a two-step approach combining salicylate ester formation and acetylation.

Protective effects of a dimeric derivative of ferulic acid in animal models of Alzheimer's disease.

Ferulic acid is a compound with potent anti-oxidant and anti-inflammatory activities. We previously reported the protective effects of ferulic acid administration against two animal models of Alzheimer's disease (AD): intracerebroventricular (i.c.v.) injection of Aß1-42 in mice and APP/PS1 mutant transgenic mice. In this study using the same AD animal models, we examined the effect of KMS4001, one of dimeric derivatives of ferulic acid. Intragastric pretreatment of mice with KMS4001 (30mg/kg/day) for 5 days significantly attenuated the Aß1-42 (i.c.v.)-induced memory impairment both in passive avoidance test and in Y-maze test. APP/PS1 mutant transgenic mice at KMS4001 doses of 3 and 30mg/kg/day via drinking water showed the significantly enhanced novel-object recognition memory at both 1.5 and 3 months after the start of KMS4001 treatment. Treatment of APP/PS1 mutant transgenic mice with KMS4001 for 3 months at the doses of 3 and 30mg/kg/day markedly decreased Aß1-40 and Aß1-42 levels in the frontal cortex. The KMS4001 dose-response relationships for Aß decrease and for improvement in novel-object recognition test corresponded to each other. Taken together, these results suggest that KMS4001 could be an effective drug candidate against AD.

Identification and characterization of potent, selective and metabolically stable IKKß inhibitor.

We have previously reported the identification of a rhodanine compound (1) with well-balanced inhibitory activity against IKKß and collagen-induced TNFα activated cells. However, we need more optimized compounds because of its instability over plasma and microsome. As part of a program directed toward the optimization of IKKß inhibitor, we modified a substituent of parent compound to a series of functional groups. Among substituted compounds, fluorine substituent (12) on the para position of phenyl ring restored the stability toward plasma and microsome while retaining inhibitory potency and selectivity against IKKß over other kinases. Also, we have demonstrated that compound 12 is an ATP non-competitive inhibitor and safe enough to apply to animal experiment from an acute toxicity test.

Discovery of a potent enoyl-acyl carrier protein reductase (FabI) inhibitor suitable for antistaphylococcal agent.

We report the discovery, synthesis, and biological activities of phenoxy-4-pyrone and phenoxy-4-pyridone derivatives as novel inhibitors of enoyl-acyl carrier protein reductase (FabI). Pyridone derivatives showed better activities than pyrone derivatives against FabI and Staphylococcus aureus strains, including methicillin-resistant Staphylococcus aureus (MRSA). Among the pyridone derivatives, compound 16l especially exhibited promising activities against the MRSA strain and good pharmacokinetic profiles.

Cinnamamides, Novel Liver X Receptor Antagonists that Inhibit Ligand-Induced Lipogenesis and Fatty Liver.

Liver X receptor (LXR) is a member of the nuclear receptor superfamily, and it regulates various biologic processes, including de novo lipogenesis, cholesterol metabolism, and inflammation. Selective inhibition of LXR may aid the treatment of nonalcoholic fatty liver diseases. In the present study, we evaluated the effects of three cinnamamide derivatives on ligand-induced LXRα activation and explored whether these derivatives could attenuate steatosis in mice. N-(4-trifluoromethylphenyl) 3,4-dimethoxycinnamamide (TFCA) decreased the luciferase activity in LXRE-tk-Luc-transfected cells and also suppressed ligand-induced lipid accumulation and expression of the lipogenic genes in murine hepatocytes. Furthermore, it significantly attenuated hepatic neutral lipid accumulation in a ligand-induced fatty liver mouse system. Modeling study indicated that TFCA inhibited activation of the LXRα ligand-binding domain by hydrogen bonding to Arg305 in the H5 region of that domain. It regulated the transcriptional control exerted by LXRα by influencing coregulator exchange; this process involves dissociation of the thyroid hormone receptor-associated proteins (TRAP)/DRIP coactivator and recruitment of the nuclear receptor corepressor. These results show that TFCA has the potential to attenuate ligand-induced lipogenesis and fatty liver by selectively inhibiting LXRα in the liver.

Palladium-Catalyzed One-Pot Approach to 3-(Diarylmethylene)oxindoles from Propiolamidoaryl Triflate.

3-(Diarylmethylene)oxindoles have been synthesized from propiolamidoaryl triflate utilizing a palladium-catalyzed one-pot reaction consisting of three successive reactions: Sonogashira, Heck, and Suzuki-Miyaura. This method allows for the production of a complex skeleton of 3-(diarylmethylene)oxindole from propiolamidoaryl triflate using a commercially available aryl iodide and arylboronic acid in a simple and efficient way with moderate yield and stereoselectivity.

Induction of NQO1 and Neuroprotection by a Novel Compound KMS04014 in Parkinson's Disease Models.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with a selective loss of the neurons containing dopamine (DA) in the substantia nigra pars compacta. Lines of evidence suggest that oxidative stress is a major factor contributing to the vulnerability of DA cells and that the enzyme NAD(P)H quinone oxidoreductase (NQO1) provides protection in these cells. In the present study, we report the synthesis of a novel compound KMS04014 and show that it induces NQO1 gene expression and protects DAergic neuronal cells in both cell culture and animal models of PD. In vitro, KMS04014 increased both mRNA and protein levels of NQO1 and induced nuclear translocation of Nrf2 in the DAergic neuronal cell line CATH.a. It also protected the cells against oxidative stress generated by tetrahydrobiopterin, 1-methyl-4-phenylpyridinium (MPP(+)), and H2O2. In vivo, KMS04014 attenuated the loss of tyrosine hydroxylase-immunopositive DAergic neurons in the substantia nigra and reduced degeneration of the nigral neurons and striatal fibers in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an animal model of PD. Taken together, KMS04014 may be utilized toward development of neuroprotective therapy for PD.

Expression of liver X receptor correlates with intrahepatic inflammation and fibrosis in patients with nonalcoholic fatty liver disease.

BACKGROUND: Liver X receptor (LXR) is an oxysterol-activated nuclear receptor involved in the control of major metabolic pathways for cholesterol homeostasis and lipogenesis. Although the role of LXR in hepatic steatosis is well known, its correlation with intrahepatic inflammation and fibrosis has not been thoroughly studied. We investigated the association between LXRα, hepatic inflammation, and fibrosis, as well as its correlation with other intrahepatic lipid transporters in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We evaluated clinical characteristics including sex, age, body mass index, and laboratory findings from 40 NAFLD and 16 control patients. Immunohistochemical staining was carried out on liver biopsy samples from all patients. RESULTS: The positive rate of LXRα expression was 30 % in the control group, 50 % in the NAFLD group, and 97 % in NASH groups. LXRα expression was positively correlated with not only the amount of intrahepatic fat, but also with intrahepatic inflammation and hepatic fibrosis. LXRα expression showed positive correlation with intrahepatic expression of ABCG5/8, CD36, and SREBP-1c. The expression of ABCA1, ABCG5/8, SREBP-1c, and CD36 was higher in NAFLD than in controls and there was no further increase in the NASH group. NPC1L1 was abundant in human liver. Expression of NPC1L1 was negatively correlated with intrahepatic inflammation and LXRα intensity. CONCLUSION: LXR expression correlated with the degree of hepatic fat deposition, as well as with hepatic inflammation and fibrosis in NAFLD patients. Our research suggests that LXR is an attractive target for treatment and regulation of hepatic inflammation and fibrosis.