Design and syntheses of diarylisoxazoles: Novel inhibitors of cyclooxygenase‐2 (COX‐2) with analgesic‐antiinflammatory activity

A group of 4,5‐diphenylisoxazoles ( 11a–p ), 3,4‐diphenyl‐5‐trifluoromethylisoxazoles ( 15, 21 ), and 4,5‐diphenyl‐3‐methylsulfonamidoisoxazole ( 23 ) possessing a variety of substituents (H, F, MeS, MeSO, MeSO₂) at the para‐position of one of the phenyl rings were synthesized for evaluation as analgesic, and selective COX‐2 inhibitory antiinflammatory (AI), agents. Although the 4,5‐diphenylisoxazole group of compounds (11a–p) exhibited potent analgesic and AI activities, those compounds evaluated ( 11a, 11b, 11m ) were more selective inhibitors of COX‐1 than COX‐2, with the exception of 4‐(4‐methylsulphonylphenyl)‐5‐phenylisoxazole ( 11n ) that showed a modest COX‐2 selectivity index (SI) of 2.1. In contrast, 3‐(4‐methylsulphonylphenyl)‐4‐phenyl‐5‐trifluoromethylisoxazole ( 15 ), which retained good analgesic and AI activities, was a highly potent and selective COX‐2 inhibitor (COX‐1 IC₅₀ > 500 μM; COX‐2 IC₅₀ < 0.001 μM) with a COX‐2 SI of > 500,000, relative to the reference drug celecoxib (COX‐1 IC₅₀ = 22.9 μM; COX‐2 IC₅₀ = 0.0567 μM) with a COX‐2 SI of 404. The 3‐phenyl‐4‐(4‐methylsulphonylphenyl) regioisomer ( 21 ) was a less potent inhibitor (COX‐1 IC₅₀ = 252 μM; COX‐2 IC₅₀ = 0.2236 μM) with a COX‐2 SI of 1122, relative to the regioisomer ( 15 ). The related compound 4,5‐diphenyl‐3‐methylsulfonamidoisoxazole ( 23 ) exhibited similar (to 21 ) potency and COX‐2 selectivity (COX‐1 IC₅₀ > 200 μM; COX‐2 IC₅₀ = 0.226 μM) with an SI of 752. A molecular modeling (docking) study for the most potent, and selective, COX‐2 inhibitor (15) in the active site of the human COX‐2 enzyme showed the C‐5 CF₃ substituent is positioned 3.37 Å from the phenolic OH of Tyr³⁵⁵, and 6.91 Å from the Ser⁵³⁰ OH. The S‐atom of the MeSO₂ substituent is positioned deep (7.40 Å from the entrance) inside the COX‐2 secondary pocket (Val⁵²³). These studies indicate a C‐5 CF₃ ( 15, 21 ), or C‐3 NHSO₂Me ( 23 ), central isoxazole ring substituent is crucial to selective inhibition of COX‐2 for this class of compounds. Drug Dev. Res. 51:273–286, 2000. © 2001 Wiley‐Liss, Inc.     

Kaempferol glycosides with antioxidant activity from <Emphasis Type="Italic">Brassica juncea</Emphasis>

From the leaves of Brassica juncea, three kaempferol glycosides, kaempferol-3-O-(2-O-sinapoyl)-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranoside (1), kaempferol-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (2), and kaempferol-3-O-(2-O-sinapoyl)-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (3) were isolated and the structures elucidated on the basis of spectral and chemical evidences. Antioxidant activities were determined by measuring the scavenging activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and peroxynitrite (ONOO⁻). Compounds 1 and 3 showed good antioxidant activities with respective IC₅₀ values of 28.61 and 36.93 μM toward DPPH; respective IC₅₀ values of 9.79 and 11.40 μM toward ONOO⁻. However, compound 2 showed no DPPH scavenging activity and weak ONOO⁻ scavenging activity with an IC₅₀ value of 32.00 μM.     

Lignans from the fruits of the red pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and their antioxidant effects

The fruits of Capsicum annuum were extracted using 80% aqueous MeOH, and fractionated using EtOAc and water. Repeated column chromatography using silica gel, octadecyl silica gel, and Sephadex LH-20 for the EtOAc fraction led to the isolation of a new lignan glycoside and a known one, icariside E₅. From the results of spectroscopic data, including EIMS, FABMS, UV, IR, ¹H and ¹³C-NMR, DEPT, and 2D-NMR (COSY, HSQC, HMBC), the chemical structure of the new lignan glycoside was determined as (8R)-isodehydrodiconiferyl alcohol-4′-O-(6″-vanilloyl)-β-D-glucopyranoside named vanilloylicariside E₅. All isolated compounds were tested for antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Icariside E₅ (2) and (8R)-Isodehydrodiconiferyl alcohol (3) exhibited a strong scavenging effect on DPPH (2: IC₅₀=42.1 μM, 3: IC₅₀=4.5 μM).     

Effects of Calcium Blockers on the Cytosolic Calcium,H2O2 Production and Elastase Release in Human Neutrophils

Abstract: Activated neutrophils are assumed to be one plausible cause of tissue injury in the ischaemic and reperfused myocardium. We studied the inhibitory effects of the calcium antagonists felodipine, nimodipine and verapamil on human neutrophil activation in order to elucidate the mechanisms underlying their myocardioprotective effects and to determine whether calcium antagonists with different chemical structures vary in their effect on neutrophil activation. Neutrophils were stimulated with formyl-Met-Leu-Phe (0.1 μM) or by phorbol myristate acetate (0.16 μM), and the rise in cytosolic calcium and the H₂O₂ production were determined. For felodipine, the inhibitory effect on granulocyte elastase release was also studied. The calcium antagonists reduced formyl-Met-Leu-Phe and phorbol myristate acetate-induced neutrophil activation in a concentration-dependent manner, the order of potency being: felodipine >nimodipine >verapamil. For felodipine, the IC₅₀ (concentration causing 50% reduction) values were 3×10^?6 and 2×10^?6 M for the formyl-Met-Leu-Phe-induced cytosolic calcium increase and H₂O₂ production, respectively. The IC₅₀ -value for the phorbol myristate acetate-induced cytosolic calcium increase was 6×10^?6 and for H₂O₂ production 4×10^?6 M. For formyl-Met-Leu-Phe-induced granulocyte elastase release, the IC₅₀-value was 5×10^?6 M. The inhibitory effect of felodipine on the phorbol myristate acetate-induced granulocyte elastase release did not exceed 50%. Nimodipine was a less potent inhibitor than felodipine for both formyl-Met-Leu-Phe- and phorbol myristate acetate-induced cell activities. Verapamil was even less potent than the other two agents. The present study demonstrates that felodipine potentially suppresses neutrophil activation at micromolar concentrations. However, this observation should not be directly extrapolated to explain the tissue protection by the compounds without evidence of profound local accumulation.     

Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6)

Flavonoids are conjugated by phase II enzymes in humans to form glucuronidated and sulfated metabolites that are excreted in urine via the kidney. In this study, we examined the interaction between metabolites of quercetin and isoflavonoids found in vivo with human organic anion transporters 1 (OAT1) and 3 (OAT3) and their potential in attenuating OAT-induced cytotoxicity of adefovir. Accumulation of flavonoid conjugates was studied in human embryonic kidney 293H cells overexpressing OAT1 or OAT3. OAT1-overexpressing cells exhibited an increased uptake of the sulfated conjugates, genistein-4′-O-sulfate and quercetin-3′-O-sulfate. OAT3-overexpressing cells demonstrated enhanced uptake of glucuronide conjugates, such as daidzein-7-O-glucuronide, genistein-7-O-glucuronide, glycitein-7-O-glucuronide and quercetin-3′-O-glucuronide. Position of conjugation was important since quercetin-3-O-glucuronide and quercetin-7-O-glucuronide were poorly transported. Kinetic analysis revealed high affinity uptake of quercetin-3′-O-sulfate by OAT1 (K_m = 1.73 μM; V_max = 105 pmol/min/mg). OAT3 transported isoflavone glucuronides with lower affinity (K_m = 7.9–19.1 μM) but with higher V_max (171–420 pmol/min/mg). Quercetin-3′-O-sulfate strongly inhibited OAT1-mediated p-aminohippuric acid uptake with an IC₅₀ of 1.22 μM. Transport of 5-carboxyfluorescein by OAT3 was potently inhibited by quercetin-3-O-glucuronide, quercetin-3′-O-glucuronide and quercetin-3′-O-sulfate (IC₅₀ = 0.43–1.31 μM). In addition, quercetin-3′-O-sulfate was shown to effectively reduce OAT1-mediated cytotoxicity of adefovir, an antiviral drug, in a dose-dependent manner. These data suggest that OAT1 and OAT3 are responsible for basolateral uptake of flavonoid conjugates in kidney, and flavonoid conjugates inhibit OAT1 and OAT3 activity at physiologically relevant concentrations. Interaction with OATs limits systemic availability of flavonoids and may be a mechanism of food–drug interaction via inhibition of renal uptake.     

赛庚啶对氧自由基的清除作用

赛庚啶(Cyp)对Fenton反应生成的·OH有较强的直接清除作用(EC₅₀为54μmol/L),并明显抑制·OH的生成速率(IC₅₀为22 μmol/L),且作用明显比·OH特异性清除剂甘露醇强(其EC₅₀和IC₅₀分别为22.7 mmol/L和10.7mmol/L)。Cyp对大鼠腹腔多形核白细胞(PMNs)产生的O₂也有一定的清除作用,其IC₅₀为179 μmol/L。提示Cyp的抗心肌损伤作用可能至少部分与其清除氧自由基作用相关。     

Syntheses,calcium channel modulation effects,and nitric oxide release studies of O2‐alkyl‐1‐(pyrrolidin‐1‐yl) diazen‐1‐ium‐1,2‐diolate 4‐aryl(heteroaryl)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates

A group of racemic 4‐aryl(heteroary)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxy‐lates possessing a potential nitric oxide donor C‐5 O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester [alkyl=(CH₂)_n, n=1–4] substituent were synthesized using a modified Hantzsch reaction. Compounds having a C‐4 2‐trifluoromethylphenyl ( 16 ), 2‐pyridyl ( 17 ), or benzofurazan‐4‐yl ( 20 ) substituent generally exhibited more potent smooth‐muscle calcium channel antagonist activity (IC₅₀ values in the 0.55 to 38.6 μM range) than related analogs having a C‐4 3‐pyridyl ( 18 ), or 4‐pyridyl ( 19 ) substituent with IC₅₀ values > 29.91 μM, relative to the reference drug nifedipine (IC₅₀=0.0143 μM). The point of attachment of C‐4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2‐pyridyl > 3‐pyridyl and 4‐pyridyl. Subgroups of compounds 16a–d , 17a–d , and 20a–d having alkyl spacer groups of variable chain length [–CO₂(CH₂)_nO–, n=1–4] exhibited small differences in calcium channel antagonist potency. Replacement of the ester “methyl” moiety of Bay K 8644 by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate group provided the Bay K 8644 group of analogs 16a‐d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O²‐ethyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)pyridine‐5‐carboxylate ( 16b , EC₅₀=0.096 μM) is about eightfold more potent positive inotrope (cardiac calcium channel agonist) than the reference compound Bay K 8644 (EC₅₀=0.77 μM). A similar replacement of the ester “isopropyl” group in the C‐4 benzofurazan‐4‐yl group of compounds by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester substituent provided compounds 20 (n=1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202‐791 ( 3 , EC₅₀=9.40 μM). The O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester moiety present in 1,4‐dihydropyridine calcium channel modulating compounds 16–20 is not a suitable ?NO donor moiety because the percent nitric oxide released upon in vitro incubation with either l ‐cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204–216, 2003. © 2003 Wiley‐Liss, Inc.     

Synthesis and Calcium Antagonistic Activity of 8-[N-[2-(3,4-Dimethoxyphenyl)ethyl]-β-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4]thiazepine Fumarate

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl)ethyl]-β-analyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine ( 1 ). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC₅₀ = 23 μM, n = 7), and a negative inotropic effect in papillary muscles (IC₅₀ = 2.7 μM, n = 7) and left atria (IC₅₀ = 4 μM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC₅₀: 70 ± 5 μM) and low (IC₅₀: 129 ± 34 μM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC₅₀: 80 ± 13 μM) in chick dorsal root ganglion neurons. In addition, nicotine-induced currents were potently inhibited (IC₅₀: 6 ± 0.7 μM) in bovine chromaffin cells.     

Zinc hydroxide induced respiratory burst in rat neutrophils

The effects of zinc hydroxide on the respiratory burst and phagocytosis by rat neutrophils were examined. Zinc hydroxide induced an increase in oxygen consumption and O₂⁻ production. Electronmicroscopy showed that neutrophils engulfed zinc hydroxide particles by phagocytosis. Pertussis toxin (0.25, 0.5, 1.0 μ/ml) and EGTA (1, 2, 5 mM) inhibited zinc hydroxide-induced O₂⁻ production in a dose-dependent manner. The inhibitors of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide inhibited zinc hydroxide-induced O₂⁻ production with IC₅₀ values ranging between 10 μM and 25 μM. The inhibitory study using an inhibitor of myosin light chain kinase, 1-(5-iodonapthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine, showed IC₅₀ values ranging from 5 μM to 10 μM. These findings indicate that zinc hydroxide induces respiratory burst and phagocytosis by rat neutrophils.     

Antioxidant Activity of Anthraquinones and Flavonoids from Flower ofReynoutria sachalinensis

Bioassay-guided fractionation of methanol extract ofReynoutria sachalinensis flower using DPPH assay has led to the isolation of three anthraquinones and three flavonoids. Their structures were identified as emodin (1), emodin-8-O-β-D-glucopyranoside (2), physcion-8-O-β-D-glucopyranoside (3), quercetin-3-O-α-L-arabinofuranoside (4), quercetin-3-O-α-D-galactopyranoside (5), and quercetin-3-O-β-D-glucuronopyranoside (6) by comparing their physicochemical and spectral data with those published in literatures. All isolated compounds were evaluated for antioxidant activities with free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, superoxide radical scavenging and Cu²⁺-mediated low density lipoprotein (LDL) oxidation assay. The results demonstrated that three flavonoids,4, 5, and6 had remarkable antioxidant activities with the IC₅₀ values of 64.3, 54.7, and 46.2 μM (DPPH scavenging), the IC₅₀ values of 6.0, 6.7, and 4.4 μM (superoxide radical scavenging) and the IC₅₀ values of 3.8, 3.2, and 5.4 μM against LDL oxidation, respectively.     

Fluvoxamine is a More Potent Inhibitor of Lidocaine Metabolism than Ketoconazole and Erythromycin in vitro

Abstract CYP3A4 is generally believed to be the major CYP enzyme involved in the biotransformation of lidocaine in man; however, recent in vivo studies suggest that this may not be the case. We have examined the effects of the CYP3A4 inhibitors erythromycin and ketoconazole and the CYP1A2 inhibitor fluvoxamine on the N-deethylation, i.e. formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine by human liver microsomes. The experiments were carried out at lidocaine concentrations of 5 μM (clinically relevant concentration) and 800 μM. The formation of both MEGX and 3-hydroxylidocaine was best described by a two-enzyme model. At 5 μM of lidocaine, fluvoxamine was a potent inhibitor of the formation of MEGX (IC₅₀ 1.2 μM). Ketoconazole and erythromycin also showed an inhibitory effect on MEGX formation, but ketoconazole (IC₅₀ 8.5 μM) was a much more potent inhibitor than erythromycin (IC₅₀ 200 μM). At 800 μM of lidocaine, fluvoxamine (IC₅₀ 20.7 μM) and ketoconazole (IC₅₀ 20.4 μM) displayed a modest inhibitory effect on MEGX formation, whereas erythromycin was a weak inhibitor (IC₅₀>250 μM). The 3-hydroxylation of lidocaine was potently inhibited by fluvoxamine at both lidocaine concentrations (IC₅₀ 0.16 μM at 5 μM and 1.8 μM at 800 μM). Erythromycin and ketoconazole showed a clear inhibitory effect on the 3-hydroxylation of lidocaine at 5 μM of lidocaine (IC₅₀ 9.9 μM and 13.9 μM, respectively), but did not show a consistent effect at 800 μM of lidocaine (IC₅₀>250 μM and 75.0 μM, respectively). Although further studies are needed to elucidate the role of distinct CYP enzymes in the biotransformation of lidocaine in humans, the findings of this study suggest that while both CYP1A2 and CYP3A4 are involved in the metabolism of lidocaine by human liver microsomes, CYP1A2 is the more important isoform at clinically relevant lidocaine concentrations.     

Inhibitory Effects of Silibinin on Cytochrome P‐450 Enzymes in Human Liver Microsomes

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P‐450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7–300 μM) at 37° in 0.1 M KH₂PO₄ buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC₅₀ values were determined for each substrate at respective K_M concentrations. Silibinin had little effect (IC₅₀>200 μM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)‐mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC₅₀=173 μM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC₅₀=29 μM and 46 μM) and S(?)‐warfarin 7‐hydroxylation (CYP2C9; IC₅₀=43 μM and 45 μM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K_i,c=2.3 μM and 2.4 μM). Inhibition was competitive for S(?)‐warfarin 7‐hydroxylation (K_i,c=18 μM and 19 μM) and mainly non‐competitive for denitronifedipine oxidation (K_i,n=9 μM and 12 μM). With therapeutic silibinin peak plasma concentrations of 0.6 μM and biliary concentrations up to 200 μM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.     

Design,syntheses, and evaluation of novel 1,1‐dihalo‐2,3‐diphenylcyclopropanes as potential cyclooxygenase‐2 (COX‐2) inhibitors with analgesic‐antiinflammatory activity

A group of (Z)‐ and (E)‐1,1‐dihalo‐2‐(4‐substituted‐phenyl)‐3‐phenylcyclopropane [ (Z)‐10 , (E)‐11 ] stereoisomers having a variety of substituents (H, Br, Cl, F, NO₂, SO₂Me) at the para‐position of the C‐2 phenyl ring in conjunction with either two chloro or bromo substituents at C‐1 were synthesized for in vivo evaluation as analgesic and antiinflammatory (AI) agents, and as potential selective cyclooxygenase‐2 (COX‐2) inhibitors. This group of compounds ( 10‐11 ) exhibited significant analgesic activity since 4% NaCl‐induced abdominal constriction was reduced by 44–73% at 30 min, and 48–77% at 60 min, post‐drug administration relative to the reference drugs aspirin and celecoxib (58 and 32% inhibition at 30 min post‐drug administration) for a 50 mg/kg intraperitoneal dose. In the 1,1‐dichloro group of compounds, a Cl or MeSO₂ substituent at the para‐position of the C‐2 phenyl ring generally provided superior analgesic activity. The most active analgesic compound, (E)‐1,1‐dichloro‐2‐(4‐methanesufonylphenyl)‐3‐phenylcyclopropane ( 11h ) inhibited abdominal constriction by 72 and 77% at 30 and 60 min post‐drug administration, respectively. AI activities, determined using the carrageenan‐induced rat paw edema assay, showed that this class of ( Z)‐10 and ( E)‐11 compounds exhibited AI activities in the inactive‐to‐moderate activity range (1.5–45% inhibition) for a 50 mg/kg oral dose. The AI potency order, with respect to the para‐substitutent on the C‐2 phenyl ring, for the ( Z)‐10 compounds was NO₂ > MeSO₂ ≈ H ≥ Cl, and for the ( E)‐11 compounds was H ≥ MeSO₂ > Cl ≈ Br. (E)‐1,1‐dibromo‐2‐(4‐methanesufonylphenyl)‐3‐phenylcyclopropane ( 11l ), which was the most active AI compound, reduced inflammation by 45 and 37% at 3 and 5 h post‐drug administration, respectively. The ( E)‐11 stereoisomer was generally a more potent AI agent than the corresponding ( Z)‐10 stereoisomer. In vitro COX‐1 and COX‐2 inhibition studies showed that (E)‐1,1‐dichloro‐2‐(4‐nitrophenyl)‐3‐phenylcyclopropane ( 11c ) inhibited COX‐1 (IC₅₀ = 278.8 μM) and COX‐2 (IC₅₀ = 80.5 μM) for a COX‐2 selectivity index of 3.5, whereas (E)‐1,1‐dichloro‐2‐(4‐methanesulfonylphenyl)‐3‐phenylcyclopropane ( 11h ) was a more potent inhibitor of COX‐1 and COX‐2, but it was more selective for COX‐1 (COX‐1 IC₅₀ = 0.59 μM, COX‐2 IC₅₀ = 3.04 μM). A molecular modeling (docking) study for (E)‐1,1‐dichloro‐2‐(4‐methanesulfonylphenyl)‐3‐phenylcyclopropane ( 11h ) on the active site of the human COX‐2 isozyme shows it binds in the center of the active site with the 1,1‐dichloro substituents oriented in the direction of the mouth of the channel towards Arg¹²⁰, and the C‐2 MeSO₂ moiety oriented towards the apex of the active site with the S‐atom of the MeSO₂ substituent positioned about 6.56 Å inside the entrance to the secondary pocket (Val⁵²³) of COX‐2. In contrast, the corresponding (Z)‐10h stereoisomer assumes a different position in the COX‐2 binding site where the S‐atom of the MeSO₂ moiety is near (4.02 Å) the Ser⁵³⁰ OH, but a much greater distance from the COX‐2 secondary pocket (Val⁵²³). The results from these docking studies are consistent with the observation that (E)‐11h is an inhibitor of both COX isozymes, whereas the (Z)‐10h stereoisomer is an inactive COX inhibitor (COX‐1 IC₅₀ > 100 μM, COX‐2 IC₅₀ > 200 μM). Drug Dev. Res. 55:79–90, 2002. © 2002 Wiley‐Liss, Inc.     

Screening of non‐steroidal anti‐inflammatory drugs for inhibitory effects on the activities of six UDP‐glucuronosyltransferases (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) using LC‐MS/MS

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are used widely to relieve pain and to decrease inflammation. Several clinical studies have reported that NSAIDs inhibit uridine 5′‐diphospho‐glucuronosyltransferase (UGT) enzymes. Therefore, the study evaluated the inhibitory potential of 15 NSAIDs on the activities of six UGT isoforms (i.e. UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes (HLMs). Among the 15 NSAIDs tested here, mefenamic acid and diclofenac inhibited all UGTs tested in this study. Piroxicam and niflumic acid inhibited UGT1A9 activity (IC₅₀ = 73.8 μm and 0.38 μm , respectively) and naproxen selectively inhibited UGT2B7 activity (IC₅₀ = 53.1 μm ), whereas it did not inhibit the other UGTs tested (IC₅₀ > 200 μm ). Diflunisal inhibited the UGT1A1 (IC₅₀ = 33.0 μm ) and UGT1A9 (IC₅₀ = 19.4 μm ). Acetaminophen, fenoprofen, ibuprofen, ketoprofen, meloxicam, phenylbutazone, salicylic acid and sulindac showed negligible inhibitory effects on the six UGTs (IC₅₀ > 100 μm ). These results suggest that some NSAIDs have the potential to inhibit UGTs in vitro. Copyright © 2014 John Wiley & Sons, Ltd.     

Influence of temperature on the subcritical water extraction of Actinidia arguta leaves: A screening of pro-healthy compounds

Actinidia arguta is a species disseminated in Europe and classified by the Chinese Herbal Medicine as a medicinal plant. The fruit (kiwiberry) has been extensively exploited for multiple purposes, while leaves where discarded. The objective of this study was to evaluate the optimal Subcritical Water Extraction (SWE) temperature (110 °C - 160 °C) of antioxidants and polyphenols from A. arguta leaves. The optimal temperature of extraction was 123 °C, revealing the highest phenolic and flavonoid contents and good scavenging efficiencies against HOCl (IC₅₀ = 17.06 μg/mL) and O₂^●- (IC₅₀ = 335.2 μg/mL), without toxicity on intestinal cells. The phenolic profile was characterized by high amounts of phenolic acids (e.g., gallic acids), flavanols (catechin) and flavonols (e.g., quercetin-3-O-galactoside). This work allows to conclude that SWE can be a useful extraction technique for the recovery of polyphenolics from A. arguta leaves.     

Phenolic constituents of Pulicaria undulata (L.) C.A. Mey. sub sp. undulata (Asteraceae): Antioxidant protective effects and chemosystematic significances

One new naturally isoflavone compound, 5,7,2′,3′,4′ penta hydroxyl isoflavone-4′-O-β-glucopyranoside (1) was isolated from the aqueous methanol extract (AME) of Pulicaria undulata subsp. undulata, together with seven known compounds: kaempferol (2), kaempferol 3-O-β-glucoside (3), quercetin (4), quercetin 3-O-β-glucoside (5), quercetin 3-O-β-galactoside (6), quercetin 3,7-di OCH₃ (7), and caffeic acid (8). Their structures were established through chemical (acid hydrolysis) and spectral analysis (UV, NMR, and ESIM). The AME and some isolated compounds were evaluated as protective agents. Free radical scavenging using a microscaled 2,2-diphenyl-1-picrylhydrazyl assay was used to assess the direct antioxidant properties that were evaluated by the ability to protect murine Hepa1c1c7 liver cells against damage induced by the organic peroxide tert-butyl hydroperoxide. The neutral red uptake assay (NRU) was used to record the activity. Results of the 2,2-diphenyl-1-picrylhydrazyl assay recorded differential scavenging properties in ascending order: 5,7,2′,3′,4′ penta hydroxyl isoflavone-4′-O-β-glucopyranoside > quercetin > quercetin 3-O-galactoside > caffeic acid > quercetin 3,7-di-OCH₃ > kaempferol with 50% inhibitory concentrations of 3.9 μM, 7.5 μM, 11.4 μM, 12.2 μM, 78.1 μM, and 252.3 μM, respectively. The antioxidative potential reveals the potency of AME, quercetin, and quercetin 3,7-di-OCH₃. The latter compound showed full protection at 100 μM (33 μg/mL) against the induced toxicant effect where the 50% effective concentration was calculated as 33.6 ± 1.7 μM (11.1 μg/mL). In addition to quercetin, which was extensively shown previously as a cytoprotective agent, AME was less potent; it was capable of protecting 75% at 100 μg/mL with 50% effective concentration of 92.3 ± 4 μg/mL. Moreover, the isolated flavonoids were found to be significantly chemosystematic markers.     

Selective induction and inhibition of the components of 7-ethoxycoumarin O-deethylase activity in the rat

1. Treatment of rats with 3-methylcholanthrene increased V_max of the high-affinity component of 7-ethoxycoumarin O-deethylase activity 60-fold. There was also an increase in V_max of the low-affinity component.

2. Treatment with phenobarbitone increased V_max of the high-affinity component sixfold whilst not affecting the K_m of this component. Modest changes were also observed in the kinetics of the low-affinity component.

3. Following treatment with 3-methylcholanthrene, the sensitivity of both the high- and low-affinity components of activity to inhibition by α-naphthoflavone was considerably increased, with the IC₅₀ decreasing from >250μM to < 10 μM in both instances.

4. Following treatment with phenobarbitone, the sensitivity of the low-affinity component to inhibition by metyrapone was considerably increased, with the IC₅₀ decreasing from > 1000 μM to 96 μM. There was also a modest, but significant, increase in sensitivity of the high-affinity component to metyrapone.

5. These results indicate that both components of 7-ethoxycoumarin O-deethylase activity are catalysed by more than one form of cytochrome P-450.     

Design and syntheses of methyl 2‐methyl‐2‐[2‐(4‐benzoyl‐5‐phenyl‐7‐halo‐2‐azabicyclo[4.1.0]hept‐3‐ene)]acetates: novel inhibitors of cyclooxygenase‐2 (COX‐2) with analgesic‐antiinflammatory activity

A group of methyl 2‐methyl‐2‐[2‐(4‐benzoyl‐5‐phenyl‐7‐halo‐2‐azabicyclo[4.1.0]hept‐3‐ene)]acetates ( 10–15 ), and the related acetamide derivative ( 16 ), that possess a variety of C‐7 substituents (Br, Cl, F, H), were designed for evaluation as analgesic‐antiinflammatory agents. The effect of the C‐7 substituent(s) and the nature of the acetic acid ester (R¹ = Ome) or acetamide (R¹ = NH₂) moiety on analgesic activity was determined using a 4% NaCl‐induced abdominal constriction assay. Compounds 10–16 inhibited writhing by 36–82%, relative to the reference drugs aspirin (58% inhibition) and celecoxib (62% inhibition). The nature of the C‐7 substituents was a determinant of analgesic activity in the 7,7‐dihalo group of compounds where the relative activity profile was 7‐Cl₂ > 7‐Br₂ > 7‐F₂ > 7‐Cl,7‐F, and for 7‐monohalo compounds where the potency order was 7‐Br > 7‐Cl. Elaboration of the 7,7‐dibromo methyl acetate ester ( 10 ) to the corresponding acetamide derivative ( 16 ) enhanced analgesic activity. The nature of the 7‐halo substituent(s) in the 7,7‐dihalo group of compounds was a determinant of antiinflammatory activity, determined using the carrageenan‐induced rat paw edema assay, where the relative potency order was 7‐Br₂ > 7‐Cl₂ > 7‐F₂ > 7‐Cl,7‐F. The most potent 7,7‐dibromo compound ( 10 ) inhibited inflammation by 62%, relative to the reference drug ibuprofen (44%), and 10 inhibited COX‐2 (IC₅₀ = 26.4 μM) and COX‐1 (IC₅₀ = 227 μM) for a COX‐2 selectivity index of 8.6. Docking 10 in the active site of human COX‐2 showed it binds in the center of the COX‐2 binding site with the C‐5 phenyl ring oriented toward the acetylation site (Ser⁵³⁰), and the phenyl group of the C‐4 benzoyl moiety oriented in the vicinity of the COX‐2 secondary binding pocket near Val⁵²³. Drug Dev. Res. 49:75–84, 2000. © 2000 Wiley‐Liss, Inc.     

Selective inhibition of the cyclooxygenase pathway of the arachidonic acid cascade by the nonsteroidal antiinflammatory drug isoxicam

The effects of the nonsteroidal antiinflammatory drug isoxicam on prostaglandin formation by HSDM₁C₁ mouse fibrosarcoma cells grown in culture and 5-HETE(5(S)-5-hydroxy-6-trans, 8,11,14-cis eicosatetraenoic acid) formation by human leukocytes were determined. Isoxicam inhibited the formation of the cyclooxygenase product prostaglandin E₂ with an IC₅₀ of 2.0 μM. In comparison, the reference standards piroxicam and indomethacin had IC₅₀ values of 0.55 μM and 0.14 μM, respectively. Nordihydroguaiaretic acid (NDGA) was inactive up to 10 μM. Isoxicam, piroxicam, and indomethacin were relatively devoid of any significant inhibitory property on 5-lipoxygenase activity exhibiting IC₅₀ values > 500 μM. In comparison, the compounds BW755C and NDGA, known lipoxygenase inhibitors, had IC₅₀ values of 11 μM and 0.6 μM, respectively. The present findings indicated that isoxicam exhibited a selective inhibition of the cyclooxygenase pathway of the arachidonic acid cascade and that this effect may at least in part be responsible for its antiinflammatory activity observed in animals and humans.     

Synthesis,in silico and in vitro studies of hydrazide-hydrazone imine derivatives as potential cholinesterase inhibitors

A series of hydrazide-hydrazone imine derivative compounds (3a–k) were synthesized and their structures characterized using FTIR, ¹H, and ¹³C (NMR) spectroscopic methods. In addition, molecular structures of compounds 3a, 3d, and 3g were elucidated by X-ray diffraction technique. In vitro inhibition activities of hydrazide-hydrazone imine derivatives against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were investigated. Compound 3i (IC₅₀ = 2.01 μM) exhibited the best inhibitory activity against AChE, comparable to the control Galantamine (IC₅₀ = 2.60 μM). Against BChE, compound 3h (IC₅₀ = 2.83 μM) showed the best inhibitory property which is higher control Galantamine (IC₅₀ = 3.70 μM). The Ki values of compound 3i (Ki = 0.63 μM) and compound 3h (Ki = 0.94 μM) that have the strongest inhibitory potential were determined against AChE and BChE, respectively. According to the docking result, the most stable conformation of AChE and compound 3i showed that it has a binding affinity of −10.82 kcal/moL. The binding affinity of the most stable conformation formed by BChE and compound 3h is −8.60 kcal/moL. Finally, in silico results and pharmacokinetic parameters of ADME showed that these compounds have good oral bioavailability properties.