2-Methylacrylamide as a bioisoster of thiourea group for 1,3-dibenzylthioureido TRPV1 receptor antagonists.

In order to replace thiourea group with the more drug-like moiety for 1,3-dibenzylthioureas having TRPV1 antagonist activity, we introduced a set of functional groups between the two aromatic rings based on bioisosteric replacement. The synthesized bioisosteres of 1,3-dibenzylthioureas were tested for their antagonist activities on TRPV1 by 45Ca2+-influx assay using neonatal rat cultured spinal sensory neurons. Among the tested 14 kinds of bioisosters, 2-methylacrylamide group was the best candidate to replace thiourea group. Compound 7c, 2-methylacrylamide analog of ATC-120, showed as potent as ATC-120 in its antagonist activity. In addition, 2-methylacrylamide analog 7e having vinyl moiety showed the most potent activity with 0.022 µM of IC50 value, indicating that thiourea group of 1,3-dibenzylthioureas could be replaced to 2-methylacrylamide without loss of their potencies.

Inhibitory and Inductive Effects of Opuntia ficus indica Extract and Its Flavonoid Constituents on Cytochrome P450s and UDP-Glucuronosyltransferases.

Opuntia ficus indica (OFI) is grown abundantly in arid areas and its fruits are regarded as an important food and nutrient source owing to the presence of flavonoids, minerals, and proteins. The previous report that OFI exerts phytoestrogenic activity makes it plausible for OFI-containing supplements to be used as alternative estrogen replacement therapy. In the case of polypharmacy with the consumption of OFI-containing botanicals in post- or peri-menopausal women, it is critical to determine the potential drug-OFI interaction due to the modulation of drug metabolism. In the present study, the modulating effects on the hepatic drug metabolizing enzymes (DMEs) by OFI and its flavonoid constituents (kaempferol, quercetin, isorhamnetin, and their glycosidic forms) were investigated using the liver microsomal fractions prepared from ovariectomized (OVX) rats, human liver microsomes, and human hepatocarcinoma cell line (HepG2). As a result, the oral administration of extracts of OFI (OFIE) in OVX rats induced hepatic CYP2B1, CYP3A1, and UGT2B1. OFIE, hydrolyzed (hdl) OFIE, and several flavonols induced the transcriptional activities of both CYP2B6 and CYP3A4 genes in HepG2 cells. Finally, OFIE did not inhibit activities of cytochrome P450 (CYPs) or uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), whereas hdl OFIE or flavonol treatment inhibited CYP1A2 and CYP3A1/3A4 in rat and human liver microsomes. Our data demonstrate that OFIE may induce or inhibit certain types of DMEs and indicate that drug-OFI interaction may occur when the substrate or inhibitor drugs of specific CYPs or UGTs are taken concomitantly with OFI-containing products.

Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases.

Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug⁻herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.

Psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia.

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERß agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERß. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10 µM was able to induce the maximum reporter gene expression corresponding to that of E2-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin-ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E2, and thus, it could act as an ER agonist in the cellular environment.

Silicon switch approach in TRPV1 antagonist MK-056 and its analogues.

In searching for opportunities to exploit the benefits of silicon in TRPV1 research, we tried to investigate the pharmacological effects of sila-substitution (C/Si exchange) of tert-butyl group in the MK-056 series. Compound 13a, with a 4-positioned trimethylsilanyl group on the B ring in place of tert-butyl group, exhibited the most potent antagonist activity with IC(50) values of 0.15 microM, which is almost equipotent with that of MK-056. This is the first example that tert-butyl group on MK-056 series can be replaced to the other substituent without loss of activity.

In vitro anti-inflammatory activity of lignans isolated from Magnolia fargesii.

The overproduction of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) causes neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Four lignans, (+)-eudesmin (1), (+)-magnolin (2), (+)-yangambin (3) and a new structure named as epimagnolin B (4) were isolated from Magnolia fargesii (Magnoliaceae) as the inhibitors of NO production in LPS-activated microglia. The most potent compound 4 inhibited the production of NO and PGE(2) and the expression of respective enzyme iNOS and COX-2 through the suppression of I-kappaB-alpha degradation and nuclear translocation of p65 subunit of NF-kappaB.

Synthesis of phenylisothiourea derivatives as inhibitors of NO production in LPS activated macrophages.

A series of phenylisothioureas were synthesized as inhibitors of NO production in lipopolysaccharide-activated macrophages. We investigated the effect of lipophilic moiety and N- or S-substituents of the phenylisothioureas on the activity. Inhibitory activities of carbazole-linked phenylisothioureas were superior to the corresponding simple phenylisothiourea derivatives. Among these compounds, 12b having N-ethyl and S-isopropyl groups on phenylisothiourea moiety was the most potent in the inhibition of NO production. They inhibited NO production through the suppression of the LPS-induced translocation of p65 subunit of NF-kappaB and the followed suppression of the iNOS protein and mRNA expression.

Design, synthesis, and biological evaluation of novel diarylalkyl amides as TRPV1 antagonists.

We have developed a new class of diarylalkyl amides as novel TRPV1 antagonists. They exhibited potent (45)Ca(2+) uptake inhibitions in rat DRG neuron. In particular, the amide 59 was identified as a potent antagonist with IC(50) of 57 nM. The synthesis and structure-activity relationship of the diarylalkyl amides are also described.

Synthesis and structural optimization of multiple H-bonding region of diarylalkyl (thio)amides as novel TRPV1 antagonists.

Structural optimization of multiple H-bonding region and structure-activity relationship of diarylalkyl amides/thioamides as novel TRPV1 antagonists are described. In particular, we identified amide 34o and thioamides 35o and 35r, of which antagonistic activities were highly enhanced by an incorporation of cyano or vinyl-substituent to the multiple H-bonding region. They exhibited potent (45)Ca(2+) uptake inhibitions in rat DRG neuron with IC(50)s of 25, 32 and 28 nM, respectively.

Synthesis and cytotoxic activities of C-benzylated flavonoids.

Some C-benzylated flavonoids based on gericudranin A were synthesized and evaluated their cytotoxic activities for the elucidation of structure-activity relationship. 2,4,6-Trihydroxyacetophenone was converted to target molecules in 6 approximately 7 steps via sequential protection, aldol condensation, cyclization, regioselective C-benzylation, and deprotection. The cellular growth inhibition of the synthetic C-benzylated flavonoids was investigated against sixteen human cancer cell lines. Among these compounds, 5b showed the most potent cytotoxicities against several cell lines, especially as potent as adriamycin against SNB19 cell lines with an IC(50) value of 0.7 microM.

Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase and cyclooxygenase-2 expression by xanthanolides isolated from Xanthium strumarium.

Three sesquiterpenoids, xanthatin (1), xanthinosin (2), and 4-oxo-bedfordia acid (3) were isolated from Xanthium strumarium as inhibitors of nitric oxide synthesis in activated microglia (IC(50) values: 0.47, 11.2, 136.5 microM, respectively). Compounds 1 and 2 suppressed the expression of iNOS and COX-2 and the activity of NF-kappaB through the inhibition of LPS-induced I-kappaB-alpha degradation in microglia.

Structure-activity relationships of 2-chloro-N6-substituted-4'-thioadenosine-5'-N,N-dialkyluronamides as human A3 adenosine receptor antagonists.

On the basis of potent and selective A(3) adenosine receptor (AR) antagonist, 2-chloro-N(6)-(3-iodobenzyl)-4'-thioadenosine-5'-N,N-dimethyluronamide, structure-activity relationships were studied for a series of 5'-N,N-dialkyluronamide derivatives, synthesized from D-gulonic gamma-lactone. From this study, it was revealed that removal of the hydrogen bond-donating ability of the 5'-uronamide was essential for the pure A(3)AR antagonism. 5'-N,N-Dimethyluronamide derivatives exhibited higher binding affinity than larger 5'-N,N-dialkyl or 5'-N,N-cycloalkylamide derivatives, indicating that steric factors are crucial in binding to the human A(3)AR. A N(6)-(3-bromobenzyl) derivative 6c (K(i)=9.32 nM) exhibited the highest binding affinity at the human A(3)AR with very low binding affinities to other AR subtypes.

In vitro and in vivo study of poly(ethylene glycol) conjugated ketoprofen to extend the duration of action.

Ketoprofen-polyethylene glycol (PEG) conjugates (KPEG) were prepared and their potential as a prolonged release system was investigated. Three KPEG conjugates were synthesized from ketoprofen and methoxy PEG with three different molecular weights by esterification in the presence of DCC. The KPEG conjugates were characterized by FT-IR and (1)H NMR spectroscopy. The rate of hydrolysis profile showed a specific acid-base catalysis pattern with a minimum at pH 4-5. The pharmacokinetic study after the intravenous and intramuscular administration of KPEG750 showed that the plasma levels of KP increased slowly and reached a maximum concentration at later time. The AUC of KPEG750 was higher than that after administering an equivalent dose of ketoprofen except 40mg/kg dose of intramuscular administration. The tail-flick experiment and paw edema test after intramuscular administration showed that KPEG750 had extended analgesic and anti-inflammatory effects compared with ketoprofen. These results suggest that KPEG could be a promising NSAID prodrug with an extended pharmacological effect owing to delayed-release of parent drug.

Stereoselective synthesis of (-)-hydroxyclemastine as a versatile intermediate for the H1 receptor antagonist clemastine.

Hydroxyclemastine was targeted as a versatile analogue of clemastine with H1 receptor antagonist activity. Stereoselective synthesis of (-)-hydroxyclemastine was performed in which the key step was chelation-controlled diastereoselective 1,2-addition of Grignard reagent to alpha-alkoxyketone.

Heme oxygenase 1 mediates anti-inflammatory effects of 2',4',6'-tris(methoxymethoxy) chalcone.

We report that the synthetic chalcone 2',4',6'-tris(methoxymethoxy) chalcone (TMMC) is an anti-inflammatory compound that reduces nitric oxide (NO) production by inhibiting of inducible NO synthase (iNOS) expression, and that TMMC decreases the degradation of the inhibitory factor kappaB, leading to inhibition of nuclear factor-kappaB translocation into the nucleus in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. We also demonstrate that TMMC by itself is a potent inducer of heme oxygenase 1 (HO-1). Inhibition of HO-1 activity or scavenging of carbon monoxide, a byproduct of heme degradation, significantly attenuated this anti-inflammatory action. Treating cells with the specific p42/44 MAPK inhibitor, PD98059, blocked the TMMC-mediated induction of HO-1 and the inhibition of LPS-stimulated expression of iNOS. TMMC also depleted intracellular GSH. Our data suggest that TMMC exerts an anti-inflammatory effect in macrophages through a mechanism that involves the induction of HO-1, which is mediated by activation of p42/44 MAPK and GSH depletion.

Novel potent antagonists of transient receptor potential channel, vanilloid subfamily member 1: structure-activity relationship of 1,3-diarylalkyl thioureas possessing new vanilloid equivalents.

Recently, 1,3-diarylalkyl thioureas have merged as one of the promising nonvanilloid TRPV1 antagonists possessing excellent therapeutic potential in pain regulation. In this paper, the full structure-activity relationship for TRPV1 antagonism of a novel series of 1,3-diarylalky thioureas is reported. Exploration of the structure-activity relationship, by systemically modulating three essential pharmacophoric regions, led to six examples of 1,3-dibenzyl thioureas, which exhibit Ca(2+) uptake inhibition in rat DRG neuron with IC(50) between 10 and 100 nM.

Synthesis and conformational study of 2-trityloxymethyltetrahydrofurans as key intermediates for antiviral nucleosides.

We wanted to elucidate the reason why the trityloxymethyl substituent in gamma-trityloxymethyl-gamma-butyrolactone takes a sterically unfavorable specific conformation, and so we synthesized 5-trityloxymethyldihydrofuran-3-one, 3-(trityloxymethyl)-4-butanolide and 2-trityloxymethyl-tetrahydrofuran and we then analyzed their conformation by 1H-NMR analysis.

Asymmetric synthesis of H1 receptor antagonist (R,R)-clemastine.

The first asymmetric synthesis of (R,R)-clemastine (1) has been accomplished by the coupling of (R)-tertiary alcohol 2 and (R)-chloroethylpyrrolidine 3 via O-alkylation. (R)-Tertiary alcohol 2 was synthesized by stereoselective alkylation of chiral α-benzyloxy ketone with Grignard reagent via chelation-controlled 1,4-asymmetric induction. In the reaction, chiral benzyl group acts as a chiral auxiliary as well as a protecting group. (R)-Chloroethylpyrrolidine 3 was prepared by asymmetric transformation starting with L-homoserine lactone, in which racemization-minimized N-allylation and ring-closing metathesis were involved as key steps.

N-(3-acyloxy-2-benzylpropyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues: novel potent and high affinity antagonists and partial antagonists of the vanilloid receptor.

Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists with the alkylsulfonamido group provides a series of compounds which are effective antagonists to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin, displayed a K(i) value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX), and showed excellent analgesic activity in mice. Structure-activity analysis of the influence of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy-2-benzylpropyl moieties in the C-region favored agonism.

Pluronic-grafted poly-(L)-lysine as a new synthetic gene carrier.

Genes are attractive candidates as therapeutic agents, and the development of safe and effective gene carriers is essential for the success of human gene therapy. To develop a gene delivery vector that shows low cytotoxicity and high efficiency, we synthesized poly-L-lysine-g-pluronic by conjugating poly-L-lysine (PLL) to pluronic, which is partially functionalized with para-nitrophenyl carbonate groups, and evaluated for its efficiency as a possible nonviral gene carrier candidate. Structural analysis of synthesized polymer was performed by using 1H-NMR. Gel retardation assay, zeta potential and size measurement confirmed that the new gene carrier made a compact complex with plasmid DNA. pCMV-beta-gal was used as a reporter gene, and the in vitro transfection efficiency was measured in HeLa cells by using the o-nitrophenyl-beta-D-galactopyranoside assay. The highest transfection efficiency among those tested was achieved at the 1:1 weight ratio of polymer:DNA, and a 3-fold increase in transfection efficiency was achieved by treatment of a lysosomotropic agent, chloroquine. Compared with unmodified PLL, PLL-g-pluronic showed about 2-fold increase in transfection efficiency with similar cytotoxicity specifically at the 1:1 weight ratio of polymer:DNA.