Synthesis of 1β‐hydroxydeoxycholic acid in H‐2 and unlabeled forms

1β–hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for use as a biomarker for Cytochrome P450 3A4/5 activity. A lengthy synthesis was undertaken to deliver the unlabeled compound and in the process, to develop a route to the deuterium labeled compound. The synthesis of the unlabeled compound was completed but in a very low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and this approach proved to be much more rapid and afforded the compound in both unlabeled and deuterium labeled forms in a 1‐step oxidation from deoxycholic acid and [D₄]deoxycholic acid, respectively.     

In vitro metabolism of an estrogen‐related receptor γ modulator,GSK5182, by human liver microsomes and recombinant cytochrome P450s

GSK5182 (4‐[(Z)‐1‐[4‐(2‐dimethylaminoethyloxy)phenyl]‐hydroxy‐2‐phenylpent‐1‐enyl]phenol) is a specific inverse agonist for estrogen‐related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N‐desmethyl‐GSK5182 and GSK5182 N‐oxide, respectively, on the basis of liquid chromatography‐tandem mass spectrometric (LC‐MS/MS) analysis. M2 was suggested to be hydroxy‐GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin‐containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N‐demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N‐oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Crucial role of cytochrome P450 in hepatotoxicity induced by 2,3‐dimethoxy‐1,4‐naphthoquinone in rats

Quinone toxicity is induced by two principal mechanisms: arylation/alkylation and a redox cycle. We have previously shown that increases in intracellular levels of superoxide anion and cell death induced by 2,3‐dimethoxy‐1,4‐naphthoquinone (DMNQ), a redox cycling quinone, are enhanced by pretreatment of rat primary hepatocytes with cytochrome P450 inhibitors. This indicates a novel interaction of quinones with cytochrome P450, and is thus worthy of further investigation using an in vivo model. The aim of this study was to examine the effects of cytochrome P450 inhibitors on DMNQ‐induced hepatotoxicity in rats. When DMNQ was administered intraperitoneally, the activities of serum alanine aminotransferase and aspartate aminotransferase were found to increase in a dose‐dependent manner, indicating that hepatotoxicity was induced by treatment with DMNQ. Pretreatment with the cytochrome P450 inhibitors SKF‐525A (SKF), cimetidine and ketoconazole potentiated the DMNQ‐induced hepatotoxicity. The blood concentration of DMNQ was not affected by administration of SKF. Pretreatment with the antioxidant α‐tocopherol almost completely attenuated the hepatotoxicity induced by DMNQ and by the combination of DMNQ with SKF. Levels of reduced glutathione in the liver were decreased and levels of oxidized glutathione were increased by treatment with DMNQ. These effects were potentiated by pretreatment with SKF. DMNQ‐induced lipid peroxidation in the liver was also enhanced by pretreatment with SKF. Taken together, these results indicate that DMNQ‐induced hepatotoxicity is augmented by inhibition of cytochrome P450 and that this augmentation is due to the enhancement of oxidative stress. Copyright © 2010 John Wiley & Sons, Ltd.     

The xenobiotic inhibitor profile of cytochrome P4502C8

AIMS: To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A). METHODS: CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure. RESULTS: At concentrations of the CYP inhibitor 'probes' employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 microM, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 microM. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82-91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 microM. CONCLUSIONS: Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.     

N‐nitrosamines induced infertility and hepatotoxicity in male rabbits

N‐nitrosamines are widely spread environmental pollutants of well‐known toxicity and carcinogenicity in various animal species. These compounds are metabolically activated by cytochrome P450 system predominantly in the liver and in other tissues into more active metabolites leading to generation of both alkylating agents that alkylate DNA and reactive oxygen species. In the current study, we investigated the influence of four types of N‐nitrosamines that are commonly present in the environment [methyethylnitrosamine, (MEN), diethylnitrosamine (DEN), diphenylnitroasamine (DPN) and dimethylnitrosamine (DMN)] on both livers and testes of male rabbits through assessment of 17 β‐hydroxysteroid dehydrogenase (17 β‐HSD) activity. The protein expression of the three cytochrome P450s (CYP11A1, CYP19A1, and CYP21A2) is involved in the steroidogenesis. The levels of testosterone (T) and estradiol (E2) were also determined in the plasma of N‐nitrosamines‐treated rabbits after one, four‐, eight‐ and twelve weeks of treatment of male New Zealand rabbits with an oral dose of 0.5 mg/kg B.W/day of each compound. In addition, activities of glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT) and levels of free radicals measured as thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH) level were quantified in both livers and testes. The present study showed that levels of free radicals (TBARS) were markedly increased, whereas GSH levels were depleted in the tissues of both livers and testes after treatment of rabbits with any of N‐nitrosamines. In addition, all tested N‐nitrosamines inhibited the activities of antioxidant enzyme activities (GR, GST, SOD, and CAT) in hepatic and testicular tissues of rabbits after 12 weeks of treatment. Histopathological examination showed that N‐nitrosamines caused lymphocytic infiltration with vascular degeneration and necrosis, congestion of central vein with RBCs hemolysis, dilated sinusoids, as well as fibrosis around portal areas were seen in hepatic tissues. In the testes, histopathological examination displayed disorganized seminiferous tubules with degeneration of germinal epithelium and Sertoli cells. Also, spermatogenic cells had pyknotic nuclei and others were detached from basement membranes of seminiferous tubules, edema was seen between seminiferous tubules. Moreover, the present data showed that MEN and DEN down‐regulated the protein expression of both CYP19A1 and 21A2 in both livers and testes of male rabbits. In addition, both MEN and DEN decreased levels of testosterone and estradiol in plasma of treated rabbits. On the one hand, DMN and DPN markedly up‐regulated the protein expression of CYP19A1 in both hepatic and testicular tissues of treated rabbits. These compounds potentially increased estradiol and decreased testosterone levels. On the other hand, no correlation was found between the expression of CYP11A1 and levels of both testosterone and estradiol. It is concluded that most of tested N‐nitrosamines induce different changes, which could be a new mechanism of infertility due to exposure to N‐nitrosamines from different environmental sources.     

2-(2,4-二氟苯基)-3-(N-甲基-N-取代基酰胺基)-1-(1H 1,2,4-三唑-1-基)-2-丙醇类化合物的合成及抗真菌活性的研究

目的:寻找新的高效、低毒、广谱的抗真菌药物。方法:设计合成了21 个三唑类化合物作为真菌细胞色素P450 14α-去甲基化酶的抑制剂,并通过体外抗真菌实验测定其抗真菌活性。结果:21 个化合物均为新化合物。体外抗真菌试验表明所有目标化合物对试验真菌均有不同程度的抑制作用,特别是对白色念珠菌和近平滑念珠菌具有很好的活性。结论:所有化合物都不同程度地对真菌细胞色素P450 14α-去甲基化酶有抑制作用,化合物15 对8 种不同真菌均显示了较高的活性,有进一步研究价值。     

Inhibitory effects of uraemic toxins 3‐indoxyl sulfate and p‐cresol on losartan metabolism in vitro

Objectives The purpose of this study was to clarify the cause of decreased metabolic clearance of losartan in patients with end‐stage renal failure. The influence of serum from haemodialysis patients (uraemic serum) and uraemic toxins on the metabolism of losartan to EXP‐3174 was investigated in vitro. Methods The formation of EXP‐3174 was estimated using pooled human liver microsomes. 3‐Carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid, hippuric acid, indole‐3‐acetic acid, 3‐indoxyl sulfate and p‐cresol were used as uraemic toxins. Key findings Uraemic serum potently decreased the formation of EXP‐3174 in pooled human liver microsomes. In addition, 3‐indoxyl sulfate and p‐cresol significantly decreased the formation of EXP‐3174 in a concentration‐dependent manner. Furthermore, normal serum (10% v/v) with both 3‐indoxyl sulfate and p‐cresol (both 20 μmol/l) significantly decreased the formation of EXP‐3174 by 46%, which was similar to the level of inhibition with uraemic serum (10% v/v). Conclusions These results suggest that decreased the metabolic clearance of losartan in patients with end‐stage renal failure is partly due to high concentrations of 3‐indoxyl sulfate and p‐cresol.     

2-(2,4-二氟苯基)-3-(N 甲基-N-取代基酰胺基)-1-(1H-1,2,4-三唑-1-基)-2-丙醇类化合物的合成及抗真菌活性的研究

目的：寻找新的高效、低毒、广谱的抗真菌药物。方法：设计合成了２１ 个三唑类化合物作为真菌细胞色素Ｐ４５０ １４α去甲基化酶的抑制剂,并通过体外抗真菌实验测定其抗真菌活性。结果：２１ 个化合物均为新化合物。体外抗真菌试验表明所有目标化合物对试验真菌均有不同程度的抑制作用,特别是对白色念珠菌和近平滑念珠菌具有很好的活性。结论：所有化合物都不同程度地对真菌细胞色素Ｐ４５０ １４α去甲基化酶有抑制作用,化合物１５ 对８ 种不同真菌均显示了较高的活性,有进一步研究价值     

From β‐N‐tert‐butyloxycarbonyl N‐carboxyanhydrides to β‐aminoacid derivatives

Abstract: β‐N‐tert‐butyloxycarbonyl‐N‐carboxyanhydrides are very reactive β‐amino acid derivatives. They react cleanly and smoothly with different nucleophiles like aminoesters, enolates, N‐methyl‐d ‐glucamine, amidoximes to afford in good to excellent yields peptides, β‐amino ketocompounds, β‐aminosugars and functionalized disubstituted 1,2,4‐oxadiazoles.     

Impact of six fruits—banana,guava, mangosteen,pineapple, ripe mango and ripe papaya—on murine hepatic cytochrome P450 activities

The effects of six Thai fruits, namely banana, guava, mangosteen, pineapple, ripe mango and ripe papaya, on cytochrome P450 (P450) activities were investigated. The median inhibitory concentrations (IC₅₀) of each of the fruit juices on CYP1A1, CYP1A2, CYP2E1 and CYP3A11 activities were determined. Pineapple juice showed the strongest inhibitory effect against all the evaluated P450 isozyme activities in mouse hepatic microsomes, followed by mangosteen, guava, ripe mango, ripe papaya and banana. The study was further performed in male ICR mice given pineapple juice intragastrically at doses of 10, 20 and 40 mg kg^?1 per day for 7 or 28 days. In a concentration‐dependent fashion, the pineapple juice raised ethoxyresorufin O‐deethylase, aniline hydroxylase and erythromycin N‐demethylase activities, which are marker enzymatic reactions responsible for CYP1A1, CYP2E1 and CYP3A11, respectively. The effect of pineapple juice on the expression of CYP1A1, CYP2E1 and CYP3A11 mRNAs corresponded to their enzymatic activities. However, the pineapple juice significantly decreased methoxyresorufin O‐demethylase activity. These observations supported that the six Thai fruits were a feasible cause of food–drug interaction or adverse drug effects owing to their potential to modify several essential P450 activities. Individuals consuming large quantities of pineapple for long periods of time should be cautioned of these potential adverse effects. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis,characterization, and antimicrobial activity of some new N‐aryl‐N’‐(2‐oxoindolin‐3‐ylidene)‐benzohydrazonamides

A green approach was developed for synthesizing a series of (isatin‐3‐ylidene)‐hydrazonamides 3a–j  from the reaction between isatin, (isatin‐3‐ylidene)malononitrile, or 2‐cyano‐2‐(2‐isatin‐3‐ylidene)acetate and benzohydrazonamide in ethyl acetate solutions at ambient temperature. The structures of the new compounds were confirmed on the basis of spectral data. In this eco‐friendly medium, a variety of (isatin‐3‐ylidene)hydrazonamides were obtained free of catalyst in good to excellent yields. All the synthesized products were evaluated for their antimicrobial activity. Among the compounds tested, 3b and 3d exhibited good antibacterial activity against Staphylococcus aureus, whereas others responded moderately with reference to the standard drug ciprofloxacin.     

Laparoscopic Roux‐en‐Y gastric bypass surgery influenced pharmacokinetics of several drugs given as a cocktail with the highest impact observed for CYP1A2, CYP2C8 and CYP2E1 substrates

There is a lack of information about the changes in drug pharmacokinetics and cytochrome P450 (CYP) metabolism after bariatric surgery. Here, we investigated the effects of laparoscopic Roux‐en‐Y gastric bypass (LRYGB) surgery on pharmacokinetics of nine drugs given simultaneously which may reveal changes in the activities of the main CYPs. Eight obese subjects undergoing LRYGB received an oral cocktail containing nine drugs, substrates of various CYPs: melatonin (CYP1A2), nicotine (CYP2A6), bupropion (CYP2B6), repaglinide (CYP2C8), losartan (CYP2C9), omeprazole (CYP2C19/CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A). The 6‐hours pharmacokinetic profiles in serum and urine of each drug or corresponding metabolite as well as their metabolic ratios were compared before surgery with those at a median 1 year later. LRYGB exerted variable effects on the pharmacokinetics of these drugs. The geometric mean AUC_0‐6 (90% confidence interval) of melatonin, bupropion, repaglinide, chlorzoxazone and midazolam after LRYGB was 27 (19%‐41%), 54 (43%‐67%), 44 (29%‐66%), 160 (129%‐197%) and 74 (62%‐90%) of the pre‐surgery values, respectively. The pharmacokinetics of losartan, omeprazole and dextromethorphan did not change in response to surgery. Nicotine was not detected in serum, while geometric mean of AUC_0‐6 of its metabolite, cotinine, increased by 1.7 times after surgery. There were 3.6‐ and 1.3‐fold increases in the AUC ratios of 6‐hydroxymelatonin/melatonin and hydroxybupropion/bupropion, respectively. The cocktail revealed multiple pharmacokinetic changes occurring after LRYGB with the greatest effects observed for CYP1A2, CYP2C8 and CYP2E1 substrates. Future studies should be focused on CYP1A2, CYP2A6, CYP2C8 and CYP2B6 to clarify the changes in activities of these enzymes after LRYGB.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

Isoeugenodilol: A vasorelaxant α/β‐adrenoceptor blocker with antioxidant activity

Isoeugenodilol, derived from isoeugenol, was investigated under in vivo and in vitro conditions. Isoeugenodilol (0.1, 0.5, 1.0, and 3.0 mg kg^–1, i.v.) produced dose‐dependent hypotensive and bradycardia responses in pentobarbital‐anesthetized Wistar rats. Isoeugenodilol (0.5 mg kg^–1, i.v.) also markedly inhibited both the tachycardia effects induced by (‐) isoproterenol and arterial pressor responses induced by phenylephrine. A single oral administration of isoeugenodilol at doses of 10, 30, and 100 mg kg^–1 dose‐dependently reduced blood pressure, with a decrease in heart rate in conscious spontaneously hypertensive rats (SHRs). In the isolated Wistar rat right atria, left atria, and guinea pig tracheal strips, isoeugenodilol competitively antagonized the (‐) isoproterenol‐induced positive chronotropic effects, inotropic effects, and tracheal relaxation effects in a concentration‐dependent manner. The parallel shift to the right of the concentration–response curve of (‐) isoproterenol suggested that isoeugenodilol was a β₁/β₂‐adrenoceptor competitive antagonist. The apparent pA₂ values were 7.33 ± 0.12 in the right atria, 7.80 ± 0.09 in the left atria, and 7.26 ± 0.11 in the trachea, indicating that isoeugenodilol was a nonselective β‐adrenoceptor blocker. In thoracic aorta experiments, isoeugenodilol also produced a competitive antagonism of norepinephrine‐induced contraction with a pA₂ value of 7.47 ± 0.45. In isolated atria of reserpinized rats, cumulative additions of isoeugenodilol and propranolol produced significantly cardiodepressant responses at high concentrations (10^–5 M) and were without intrinsic sympathomimetic activity (ISA). In isolated rat thoracic aorta, isoeugenodilol more potently relaxed the contractions induced by norepinephrine (3 × 10^–6 M) than those by high K⁺ (75 mM). The vasorelaxant effects of isoeugenodilol on norepinephrine‐induced contractions were attenuated by pretreatment with tetraethylammonium (TEA) and glibenclamide, implying the involvement of K⁺ channel opening. In addition, isoeugenodilol inhibited norepinephrine‐induced biphasic contraction; it affected the fast phase significantly more than the slow phase. Furthermore, the binding characteristics of isoeugenodilol and various β‐adrenoceptor antagonists were evaluated in [³H]CGP‐12177 binding to rat ventricle and lung tissues and [³H]prazosin binding to brain membranes. The ranking order of inhibition for [³H]CGP‐12177 binding on β‐adrenoceptor was propranolol > labetalol > isoeugenodilol, and that for [³H]prazosin binding to α‐adrenoceptors was isoeugenodilol > labetalol. Furthermore, isoeugenodilol inhibited lipid peroxidation induced by Fe²⁺ and ascorbic acid with IC₅₀ of 0.74 ± 0.03 mM, indicating that it possesses the antioxidant activity inherent in isoeugenol. In conclusion, isoeugenodilol was found to be a new generation α/β‐adrenoceptor antagonist with vasorelaxant activity by inhibiting Ca²⁺ channel, receptor‐mediated Ca²⁺ mobilization and by K⁺ channel opening, and to have additional potentially antioxidant effects. Drug Dev. Res. 51:29–42, 2000. © 2000 Wiley‐Liss, Inc.     

Microwave‐accelerated synthesis of psychoactive deuterated N,N‐dialkylated‐[α,α,β,β‐d4]‐tryptamines

A large number of N,N‐dialkylated tryptamines are known to induce psychoactive effects in humans. This has resulted in their increased attention within clinical and forensic communities. Deuterated tryptamines are ideal for use as internal standards during MS bioanalysis or of use in biochemical NMR studies. The present study reports on a microwave‐enhanced synthesis of 22 N,N‐dialkylated‐[α,α,β,β‐d4]‐tryptamines via the reduction with lithium aluminium deuteride of glyoxalylamide precursors obtained by the procedure of Speeter and Anthony. Syntheses were carried out using a single‐mode system under elevated pressure conditions where anhydrous tetrahydrofuran was used as the solvent at 150°C. Good yields were obtained within 5 min. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of β and γ-fluorenylmethyl esters of respectively Nα-Boc-L-aspartic acid and Nα-Boc-L-glutamic acid

The orthogonal synthesis of N^x-Boc-L-aspartic acid-γ-fluorenylmethyl ester and N^α-Boc-L-glutamic acid-δ-fluorenylmethyl ester is reported. This is a four-step synthesis that relies on the selective esterification of the side-chain carboxyl groups on N^x-CBZ-l -aspartic acid and N^α-CBZ-l -glutamic acid. Such selectivity is accomplished by initially protecting the a-carboxyl group through the formation of the corresponding 5-oxo-4-oxazolidinone ring. Following side-chain esterification, the α-carboxyl and α-amino groups are deprotected with acidolysis. Finally, the α-amino group is reprotected with the t-butyl-oxycarbonyl (Boc) group. Thus aspartic acid and glutamic acid have their side-chain carboxyl groups protected with the base-labile fluorenylmethyl ester (OFm) and their α-amino groups protected with the acid-labile Boc group. These residues, when used in conjunction with N^x-Boc-N^ε-Fmoc-l -lysine, are important in the formation of side-chain to side-chain cyclizations, via an amide bridge, during solid-phase peptide synthesis.     

Discovery of naphthyl‐N‐acylhydrazone p38α MAPK inhibitors with in vivo anti‐inflammatory and anti‐TNF‐α activity

Protein kinases constitute attractive therapeutic targets for development of new prototypes to treat different chronic diseases. Several available drugs, like tinibs, are tyrosine kinase inhibitors; meanwhile, inhibitors of serine/threonine kinases, such as mitogen‐activated protein kinase (MAPK), are still trying to overcome some problems in one of the steps of clinical development to become drugs. So, here we reported the synthesis, the in vitro kinase inhibitory profile, docking studies, and the evaluation of anti‐inflammatory profile of new naphthyl‐N‐acylhydrazone derivatives using animal models. Although all tested compounds ( 3a–d ) have been characterized as p38α MAPK inhibitors and have showed in vivo anti‐inflammatory action, LASSBio‐1824 ( 3b ) presented the best performance as p38α MAPK inhibitor, with IC₅₀ = 4.45 μm , and also demonstrated to be the most promising anti‐inflammatory prototype, with good in vivo anti‐TNF‐α profile after oral administration.