Identification of human cytochrome P450 isoforms involved in the metabolism of S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid

1. To identify the cytochrome P450 (CYP) isoenzymes responsible for the major metabolic pathways of S-2-[4-(3-methyl-2-thienyl)phenyl] propionic acid (S-MTPPA) in man, the metabolism of S-MTPPA was examined using human liver microsomes and microsomes containing cDNA-expressed CYP isozymes (CYP1A2, 2A6, 2B6, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Val, 2E1 and 3A4). 2. S-MTPPA was mainly oxidized to the 5-hydroxylated metabolite of the thiophene ring (MA6) with human liver microsomes in the presence of NADPH. The formation of MA6 was inhibited by SKF 525-A, suggesting that CYP plays role in the formation of MA6. 3. Eadie-Hofstee plots for the 5-hydroxylation of S-MTPPA in the range 5-100 μM were linear for all samples studied, suggesting that the formation of MA6 by human liver microsomes follows simple Michaelis-Menten kinetics. Apparent V_max = 1.42±0.64 nmol/min/mg protein; K_m = 12±5 μM. 4. Among the CYP inhibitors examined (α-naphthoflavone, sulphaphenazole, omeprazole, quinidine and troleandomycin), sulphaphenazole (a CYP2C9 inhibitor) showed the most potent inhibitory effect on the 5-hydroxylation of S-MTPPA by human liver microsomes. 5. When incubated with microsomes containing cDNA-expressed CYP isozymes, S-MTPPA was substantially oxidized to MA6 only by CYP2C9. 6. These results suggest that formation of the major metabolite of S-MTPPA, MA6, in human liver microsomes is catalysed predominantly by a single CYP isoenzyme, namely CYP2C9.     

Untersuchungen zum Mechanismus der Bischler-Napieralski-Reaktion

Investigations Concerning the Mechanism of the Bischler-Napieralski Reaction According to ¹H- and ¹³C-NMR spectroscopic measurements the imidoyl chloride 3 and its hydrochloride 2 are mixtures of the E(syn) and Z(anti) isomers. Equilibration to the more stable isomer can be followed by NMR spectroscopy. The reaction of 1 with POCl₃ leads to the chlorophosphate 9. The relevance of the counter ions of 2 and 9 , for the cyclisation in the Bischler-Napieralski reaction is discussed.     

Cer(IV)oxidationen von β-Aminoketonen, 8. Mitt.: Synthese von im Piperidinteil verschieden substituierten 1,2,3,4-Tetrahydroisochinolinen

Cerium(IV) Oxidations of β-Aminoketones, VIII¹⁾: Synthesis of 1,2,3,4-Tetrahydroisoquinolines with Differently Substituted Piperidine Parts 1- and 3-Alkyl- and/or aryl substituted tetrahydroisoquinolines are obtained by oxidative cyclisation of N-benzyl-β-aminoketones with cerium(IV) sulphate. In nearly all cases formation of mixtures of diastereomeres is observed. If the ketone function is replaced with COOR-, CN-, CHO and NO₂-groups no cyclisation occurs under standard reaction conditions. The products of hydrolysis, N-benzyl-N-methylaminopropionic acid, and of oxidation in benzylic position, benzaldehyde and benzoic acid, are mainly formed during the reaction. The oxidation of the N-benzylaminopropionaldehydacetal 1k yields the isoquinolinedione 4k .     

Investigations on the cytochrome P450 (CYP) isoenzymes involved in the metabolism of the designer drugs N-(1-phenyl cyclohexyl)-2-ethoxyethanamine and N-(1-phenylcyclohexyl)-2-methoxyethanamine

Investigations using insect cell microsomes with cDNA-expressed human cytochrome P450 (CYP)s and human liver microsomes (HLM) are reported on the CYP isoenzymes involved in the metabolism of the designer drugs N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA) to O-deethyl PCEEA and N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) to O-demethyl PCMEA. Gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry was used for the analysis of the incubation samples. PCEEA O-deethylation was catalyzed by CYP2B6, CYP2C9, CYP2C19, and CYP3A4, while PCMEA O-demethylation was catalyzed only by CYP2B6 and CYP2C19. Considering the relative activity factor approach, these enzymes accounted for 53%, 25%, 4%, and 18% of net clearance for PCEEA and 91% and 9% of net clearance for PCMEA, respectively. The chemical CYP2B6 inhibitor 4-(4-chlorobenzyl)pyridine (CBP) reduced the metabolite formation in pooled HLM by 63% at 1 μM PCEEA. At 10 μM PCEEA, CBP reduced metabolite formation by 61%, while inhibition of CYP3A4 by ketoconazole and inhibition of CYP2C9 by sulfaphenazole showed no inhibitory effect. At 1 μM PCMEA, CBP reduced metabolite formation in pooled HLM by 70% and at 10 μM PCMEA by 78%, respectively. In conclusion, the main metabolic step of both studied drugs was catalyzed by different CYPs.     

Darstellung,Kristallstruktur und Wirkung von 2-Methyl-3-(4-oxo-3-phenyl-thiazolidin-2-ylidenamino)-4-(3H)-chinazolinon

Quinazolinones, I: Preparation, Crystal Structure, and Activity of 2-Methyl-3-(4-oxo-3-phenylthiazolidine-2-ylidenamino)-4(3H)-quinazolinone The cyclisation of 1-(3,4-dihydro-2-methyl-4-oxo-3H-quinazoline-3-yl)-3-phenylthiourea (2) with chloroacetic acid leads to 2-methyl-3-(4-oxo-3-phenyl-thiazolidine-2-ylidenamino)-4(3H)-quinazolinone (3) and not to the isomer 4 . The crystal structure of 3 has been determined and refined until R = 0.0715. Compound 3 possesses good hypnotic and anticonvulsant activities.     

Schwefelhaltige Heterocyclen aus Benzothienyläthylamin

Sulphur Containing Heterocycles from Benzothienylethylamine An improved synthesis of benzo[b]thien-3-ylethylamine ( 1 ) is described as well as a series of cyclisation reactions following the methods of Bischler-Napieralski and Pictet-Spengler.     

Dihydroisochinolinumlagerung, 33. Mitt.1) Ein Heteroanalogon des N-Methylpavins aus 4-(3,4-Dimethoxybenzyl)-5-methyl-4,5-dihydrothieno [3,2-c] pyridin

Dihydroisoquinoline Rearrangement, XXXIII: A Heteroanalogue of N-Methylpavine from 4-(3,4-Dimethoxybenzyl)-5-methyl-4,5-dihydrothieno[3,2-c]pyridine By reaction of the title compound 1 with a mixture of formic and phosphoric acids the heteroanalogue 3 of N-methylpavine is obtained with 33% yield by intramolecular cyclisation.     

In vitro Metabolism of Two Heterocyclic Amines, 2‐Amino‐9H‐pyrido[2,3‐b]indole (AαC) and 2‐Amino‐3‐methyl‐9H‐pyrido[2,3‐b]indole (MeAαC) in Human and Rat Hepatic Microsomes

Abstract: 2‐Amino‐9H‐pyrido[2,3‐b]indole (AαC) and 2‐amino‐3‐methyl‐9H‐pyrido[2,3‐b]indole (MeAαC) are two mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. In this study, we have investigated the in vitro metabolism of tritium‐labelled AαC and MeAαC in hepatic microsomes from human pools, rats induced with polychlorinated biphenyl (PCB) (Aroclor 1254) and control rats. The microsomes were incubated with AαC and MeAαC and the detoxified and activated metabolites of AαC and MeAαC were separated and characterised by HPLC‐MS. AαC is metabolised to two major and three minor detoxified metabolites, while MeAαC is metabolised to three major and one minor detoxified metabolites. Some AαC and MeAαC are activated by oxidation to the reactive metabolites N²‐OH‐AαC and N²‐OH‐MeAαC, respectively. These reactive N²‐OH‐metabolites react partially in the incubation system with formation of protein adducts, dimers and the parent compound by reduction of the N²‐OH‐metabolites. The distribution between the detoxified and activated metabolites in the different types of hepatic microsomes showed same pattern for both AαC and MeAαC. In PCB‐induced rat microsomes, the major part of the metabolites are detoxified, only a little amount is activated. In control rat microsomes there is a fifty‐fifty distribution between detoxification and activation, while the major part of the metabolites from the human microsomes are activated and reacts to form dimers and protein adducts. These data show that, in human hepatic microsomes compared to rat hepatic microsomes, a major part of AαC and MeAαC are metabolically activated to the reactive N²‐OH‐AαC and N²‐OH‐MeAαC.     

Synthesis and docking studies of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide analogues as potential alkaline phosphatase inhibitors

A series of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i were synthesized as alkaline phosphatase inhibitors. The intermediate 5-substituted 1,3,4-oxadiazole-2-thione 4 was synthesized starting with hippuric acid. Hippuric acid in the first step was converted into corresponding methyl ester 2 which upon reaction with hydrazine hydrate furnished the formation of hydrazide 3 . The hippuric acid hydrazide was then cyclized into 5-substituted 1,3,4-oxadiazole-2-thione 4 . The intermediate 4 was then reacted with alkyl or aryl halides 5a–5i to afford the title compounds N-(5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i . The bioassay results showed that compounds 6a–i exhibited good to excellent alkaline phosphatase inhibitory activity. The most potent activity was exhibited by the compound 6i having IC₅₀ value 0.420 μM, whereas IC₅₀ value of standard (KH₂PO₄) was 2.80 μM. Molecular docking studies was performed against alkaline phosphatase enzyme (PDBID 1EW2) to check binding affinity of the synthesized compounds 6a–i against target protein. The docking results showed that three compounds 6c , 6e , and 6i have maximum binding interactions with binding energy values of −8 kcal/mol. The compound 6i displayed the interactions of oxadiazole ring nitrogen with amino acid His265 having a binding distance 2.13 Ǻ. It was concluded from our results that synthesized compounds, especially compound 6i may serve as lead structure to design more potent inhibitors of human alkaline phosphatase.     

Zur Bildung von 2-(2-Hydroxy-benzoyl)-xanthon

Formation of 2-(2-Hydroxybenzoyl)xanthone (3) The formation of 2-(2-hydroxybenzoyl)xanthone (3) , starting from Ω-acetyl-2-hydroxy-acetophenone (1) and N,N-dimethylformamide dimethylacetal is described and the mechanism is discussed. The structure of 3 is elucidated by rearrangement of 5 .     

Contribution to the synthesis of aureobasidin A. Synthesis of cyclopeptolides containing the sequence leucyl-N-methyl-β-hydroxyvalyl- (2R)-oxy-(3R)-methyl-pentanoic acid *

The efficient antimycotic agent aureobasidin A, isolated from the culture broth of Aureobasidium pullulans R 106, and the [(R)-Pro⁹]-aureobasidin A were prepared starting from benzyl N-Boc-N-methyl-(S)-β-triethylsiloxyvalinate, the synthesis of which is described here. The easy accessibility of the tripeptolide benzyl Boc-leucyl-N-methyl-β-hydroxyvalyl-(2R)-oxy-(3R)-methylpentanoate [Boc-Leu-HOMeVal-(R)-HMP-OBn] facilitates the construction of the cyclopeptolides 28, 34, 45 and 47. The peptide bonds of the N-methylamino acids were formed with the help of O-(7-azabenzotria-zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophophate. The rings of [(R)-Pro⁹]-aureobasidin A and of cyclopeptolides 28, 34, 45 and 47 were closed by way of pentafluorophenyl esters. The ring of aureobasidin A could only be formed with bromo-tris-pyrrolidino-phosphonium hexafluorophosphate.     

Syntheses of some norcholanylisoquinoline derivatives.

N-Phenethyl- and N-[2-(3′,4′-dimethoxyphenyl)ethyl]amides 5-8 of cholanic acid and 3α-acetoxy-cholanic acid were subjected to the Bischler-Napieralski reaction to yield the respective 3′,4′-di-hydroisoquinolines, 9-12. Reaction of 5 also yielded N,N-bis(phenethyl)cholanamidine 13 . The 3′,4′-dihydroisoquinoline 11 was converted to the tetrahydro derivative 14 and to the aromatic compounds 15 and 16 .     

Mechanism of formation of mercapturic acids from (1-bromoethyl)benzene and (2-bromoethyl)benzene in the rat

1. Three hypotheses have been proposed for the mechanism of metabolism of alkylhalides to hydroxy-alkylmercapturic acids, two of which involve the intermediate step of dehydrohalogenation and formation of an epoxide.

2. After injection of (1-bromoethyl)benzene in rat, the only mercapturic acid appearing in the urine was N-acetyl-S-1 -phenylethylcysteine. After injecting (2-bromoethyl)benzene in the rat only N-acetyl-S-2-phenylethylcysteine and N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine were found in the urine.

3. Since the principal mercapturic acid formed from both styrene and styrene oxide could not be detected in the urine of rats receiving either 1- or 2-bromoethyl benzene, the intermediate formation of styrene or styrene oxide from the arylalkylhalides does not occur.     

Prediction of differences in in vivo oral clearance of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100) between extensive and poor metabolizers from in vitro metabolic data in human liver microsomes lacking CYP2D6 activity and recombinant CYPs

1. It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs).

2. In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152?+?163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4.

3. The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CL_{int-HLM-total}) corresponded to those observed from EM- and PM-HLM (the observed CL_int-HLM).

4. The in vivo oral clearance (CL_oral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50?times higher in extensive metabolizers than poor metabolizers using in vitro–in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.

     

Synthese von 5H-Pyrido[2,3-c]-2-benzazepinen

Synthesis of 5H-Pyrido[2,3-c]-2-benzazepines The title compounds can be obtained by two differend ways: Ring closure of 2-benzazepine enaminonitrile 1 and the C₂-building blocks 2, 7 and 8 gives rise to the title compounds 6, 9 and 10 . - The second way starts with Wolff-Kishner-reduction of the 2-amino-3-benzoylpyridines 16a,b to the 2-amino-3-benzylpyridines 18a,b . Benzoylation of 18a,b leads to the dibenzoylated compounds 20 and 21 , respectively, which can be transformed to the monobenzoylated pyridines 22 resp. 23 . Applying the Bischler-Napieralski-reaction on 22a,b and 23a,b leads to the 5H-pyrido[2,3-c]-2-benzazepines 24a,b and 25a,b . By means of ¹H-, ¹³C- and ¹⁵N-NMR-data it is demonstrated that ethyl benzoylcyanacetimidate ( 12a ) exists as benzoylketene-O,N-acetal 12a AE .     

Reaktionen von N-Alkoxycycliminiumsalzen, 11. Mitt.1) Reaktionen von 2-Chlor-N-methoxy-3-(-4)-nitropyridinium-(perchlorat) mit Aminen

Reactions of N-Alkoxycycliminium Salts, XI: Reactions of 2-Chloro-N-methoxy-3-or-4-nitropyridinium Perchlorate with Amines Reactions of 3-, 4- and 5-nitro-2-chloro-N-methoxypyridinium salts with aromatic and aliphatic amines show a remarkable dependence with regard to reactivity and pathway on the position of the nitro group. While reactions of the 3-nitro derivative 1a are characterized by attack at position 6 of the pyridine ring followed by ring cleavage yielding stable 1-methoxyimino-2,4-pentadiene derivatives, reactions of the 4-nitro compound 1b are more complex and the reactivity is remarkably decreased.     

Comparison of rat and human cytochrome P450 (CYP) sources of N-alkylprotoporphyrin IX. Formation after interaction with porphyrinogenic xenobiotics: studies with cDNA-expressed single CYP enzymes

1. The porphyrinogenicity of certain xenobiotics is due to mechanism-based inactivation of selected cytochrome P450 (CYP) enzymes, with concurrent formation of N -alkylprotoporphyrins (N-alkylPPs), which disrupt control of haem biosynthesis. An ambiguity arises when extrapolating results obtained with such porphyrinogenic xenobiotics in animals to humans owing to species' differences in CYP enzymes. The objective was to use cDNA-expressed individual rat CYP enzyme preparations in microsomes prepared from baculovirus-infected insect cells to determine which rat CYP enzymes were the source of N-alkylPPs after interaction with three porphyrinogenic xenobiotics and to compare the results with formation of N-alkylPPs in individual human CYP enzyme orthologues. 2. A sensitive fluorometric technique was employed to quantitate N-alkylPP formation after interaction of individual CYP enzymes with a porphyrinogenic xenobiotic. 3. N-alkylPP formation was found following the interaction of three porphyrinogenic xenobiotics with CYP1A2, 2B1, 2C6, 2C11 and 3A2, in amounts ranging from 0.45 to 0.07 nmol N-alkylPP nmol^?1 CYP. The results obtained with rat CYP1A2, 2C6, 2C11 and 3A2 were compared with those previously obtained with the human CYP orthologues 1A2, 2C9 and 3A4. 4. Some results corresponded while others did not, reinforcing a previous recommendation that when dealing with xenobiotics whose porphyrinogenicity depends upon interaction with CYP enzymes resulting in N-alkylPP formation, animal experiments should be augmented with studies using human CYP preparations.     

3-(R)-(碱基)-4-(S)-羟基-5-(R)-羟亚甲基四氢呋喃的合成及抗肿瘤活性

目的：研究一系列3-(R)-单脱氧异核苷的合成和抗肿瘤活性。方法和结果：由L-木糖出发,合成了环氧化物5-(R)-二甲氧甲基-3(S),4(S)-环氧四氢呋喃4;在碱性条件下,利用嘌呤的N⁹位或嘧啶的N¹位对环氧化物进行亲核进攻,得到一系列3-(R)-单脱氧异核苷5a-d和6a-d;并进行了体外抗肿瘤活性筛选。结论：其中3-(R)-单脱氧异核苷5a-d为首次报道;同已报道的3-(S)-单脱氧异核苷合成方法相比,路线缩短,收率提高。在体外抗肿瘤和端粒酶抑制活性筛选中,只有化合物6a显示了对BIU细胞较弱的抑制活性,其余均未显示有意义的抗肿瘤活性和端粒酶抑制活性。     

Metabolic Studies with Trans-5-Amino-3-[2-(5-Nitro-2-Furyl)Vinyl]-1,2,4-[5-14C]Oxadiazole (SQ 18,506): 1. Reductive Cleavage of the 1, 2, 4-oxadiazole ring

Abstract

1. Reductive cleavage of the oxadiazole ring of trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4-[5-¹⁴C]oxadiazole (SQ 18,506-¹⁴C) has been demonstrated both enzymically and chemically.

2. Oral administration of SQ 18,506-¹⁴C to humans and rat resulted in the formation of respiratory ¹⁴CO₂ and the excretion of ¹⁴C-urea in the urine.

3. Formation of ¹⁴CO₂ was demonstrated in vitro by incubation of SQ 18,506-¹⁴C with homogenate of human or mouse liver or with milk xanthine oxidase.

4. ¹⁴C-urea was isolated after reduction of SQ 18,506-¹⁴C by ferrous hydroxide.     

Chemical reactivity of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) in vitro

Ethyl (6R)‐6‐[N‐(2‐chloro‐4‐fluorophenyl)sulfamoyl]cyclohex‐1‐ene‐1‐carboxylate (TAK‐242) was metabolized to cyclohexene and phenyl ring moieties in non‐clinical pharmacokinetic studies and it was suggested that the cyclohexene ring moiety of TAK‐242 is tightly bound to endogenous macromolecules. After incubation of TAK‐242 and glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C, TAK‐242 reacted with GSH to produce a glutathione conjugate of the cyclohexene ring moiety of TAK‐242, which had been observed as a metabolite (M‐SG) in non‐clinical pharmacokinetic studies. Formation of M‐SG was time dependent with a first order reaction and M‐I, a metabolite from the phenyl ring moiety of TAK‐242, was also produced in parallel. The formation of M‐SG was accelerated with increasing pH, therefore it was indicated that TAK‐242 reacted with GSH by a nucleophilic substitution reaction. Because glutathione transferase (GST) enhanced M‐SG formation in vitro, it is expected that the conjugation of TAK‐242 with GSH is also facilitated by GST in vivo in addition to a spontaneous chemical reaction. When radio‐labeled TAK‐242 ([cyclohexene ring‐U‐¹⁴ C]TAK‐242) was incubated with rat serum albumin (RSA) or human serum albumin (HSA) in vitro, the radioactive material was covalently bound to RSA and HSA, and M‐I was generated simultaneously in the reaction mixture. The chemical structure of the TAK‐242 adduct covalently bound to HSA was characterized by the accurate mass spectra that cyclohexene ring moiety of TAK‐242 was covalently bound to the lysine residue in HSA. The adduct was also detected in the plasma of rats and humans after single i.v. dosing of TAK‐242 (in vivo). Copyright © 2011 John Wiley & Sons, Ltd.