Isolation and characterization of carbinolamide and phenolic glucuronide conjugates of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide and N-formylmethamphetamine by FAB/MS, LC/MS/MS, and NMR.

The metabolic disposition of (+-)-N-methyl-N-(1-methyl-3,3- diphenyl-propyl)formamide, especially with regard to the formation of water soluble glucuronides, is described. The glucuronide conjugates, (+-)-N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl)formamide glucuronide, (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxyphenyl)-3-phenylpropyl]formamide glucuronide, and (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxy-3'-methoxyphenyl)-3- phenylpropyl]formamide glucuronide were isolated from the bile of rats dosed with the parent compound. These conjugates were characterized spectroscopically by 1H-NMR, FAB/MS, and LC/MS/MS. Because it is becoming more common to isolate the intact glucuronide conjugates of xenobiotics, we investigated some common mass spectral fragmentation patterns of these conjugates, especially by LC/MS/MS. The fragmentation patterns for each of the conjugates were obtained under MS/MS conditions and compared. Specifically, the fragmentation patterns of phenolic glucuronide and an aliphatic O-glucuronide, in particular a carbinolamide glucuronide, were investigated. The data obtained from these studies was used to predict the nature of glucuronide conjugates obtained from rats dosed with the formamide analog, N-formylmethamphetamine. This is the first spectroscopic characterization of an intact carbinolamide glucuronide conjugate isolated from the bile of rats.     

2,N-二甲基-N-(3,3-二苯基丙基)-1-氨基-2-丙醇的合成

目的研究2,N-二甲基-N-(3,3-二苯基丙基)-1-氨基-2-丙醇(1)的合成方法.方法以肉桂酸、氯化亚砜、甲胺等为原料,经烃化、氯化、酰化和还原反应得到N-甲基-3,3-二苯基丙胺(5);以3-氯异丁烯为原料,经加成、水解、环合反应得到环氧异丁烷(7),化合物5与7经烃化反应得到目标产物.结果与结论设计的合成路线以肉桂酸计,5步反应总收率为62.7%,合成路线简便易行,适于大规模制备.所合成的目标产物经ESI-MS和1H-NMR确证.     

Studies on the metabolism of 4-methyl-piperazine-1-carbodithioc acid 3-cyano-3,3-diphenylpropyl ester hydrochloride in rats by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

4-Methyl-piperazine-1-carbodithioc acid 3-cyano-3,3-diphenylpropyl ester hydrochloride(TM208) is a newly synthesized compound, which has shown excellent in vivo and in vitro anticancer activity and low toxicity. In this study, the metabolism of TM208 in rats was studied for the first time by high-performance liquid chromatography coupled with tandem mass spectrometry. Following a single oral administration to rats, TM208 was metabolized to eight metabolites (M1–M8). M1 is the desmethyl metabolite and the acylation of M1 with N-acetyl transferase results in M6 (N-acetyl metabolite), M5 is N-formyl metabolite; M4 is phenyl monohydroxylation metabolite, M2 is the sulfine metabolite of TM208, and M3 is also an odd-oxygen added products which the possible oxidation site has described in this paper; M8 is the metabolite resulting from the replacement of ‘–CS’ with ‘–CO’, M7 is a ring-opened piperazine oxidation products to a kind of acid.     

Transamination in the metabolism of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide in rats.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is nephrotoxic in rats. Due to the involvement of NDPS metabolism in its mechanism of toxicity, the detailed biotransformation of 14C-NDPS in rats was previously evaluated using high-performance liquid chromatography-electrospray ionization-mass spectrometry. In the present report, we describe the identification of two novel amino metabolites of NDPS, which were present in significant amounts in rat kidney tissues. Using liquid chromatography-tandem mass spectrometry and synthetic standards, the two metabolites were identified as N-(3,5-dichlorophenyl)-2-aminosuccinamic acid (2-NDASA) and its N-acetylated derivative (N-acetyl-2-NDASA). The mechanism of formation of 2-NDASA was studied in vitro. Incubations were carried out in rat liver and kidney cytosols using the major oxidative metabolite of NDPS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid, as the substrate. Formation of 2-NDASA in vitro was confirmed using mass spectrometry. Inhibitors of alcohol dehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase (disulfiram) reduced 2-NDASA formation by 40 to 50%. Menadione (an inhibitor of aldehyde oxidase) and quercetin (an inhibitor of carbonyl reductase) did not show any effects. (Aminooxy)acetic acid, an inhibitor of pyridoxal 5'-phosphate-containing enzymes such as aminotransferases, almost completely abolished the formation of 2-NDASA. Using liquid chromatography-mass spectrometry, the transamination mechanism was further supported by the incorporation of a 15N-amino group in 2-NDASA when 15N-glutamic acid was included in the incubation mixture. Results from these studies show that transamination is a metabolic pathway in the clearance of NDPS in rats, and that cytosolic dehydrogenases and aminotransferases may be involved in this process.     

Identification of glutathione conjugate formed from loxistatin in rats

1. The metabolic fate of loxistatin, ethyl-(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methyl- butylcarbamoyl)butylcarbamoyl]-2-oxiranecarboxylate, was studied in rats. 2. Following oral administration of 14C-loxistatin the major part of radioactivity was eliminated via the bile into the faeces. 3. The major metabolites in bile were isolated and identified as glutathione and cysteine conjugates of loxistatin acid, by chromatography, proton n.m.r. spectroscopy and mass spectrometry and comparison with those of the authentic reference compounds. 4. Formation of the glutathuone conjugate was also demonstrated in vitro by incubation of 14C-loxistatin acid with rat liver cytosol and glutathione.     

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): isolation and identification of urinary metabolites following intraperitoneal administration to rat.

1. The metabolism of sulphur mustard, 1,1'-thiobis(2-chloroethane), in vivo was investigated following i.p. administration to rat. 2. Approx. 60% of dose was excreted in the 24 h urine. Many metabolites were present; nine have been isolated by h.p.l.c. and characterized by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards. 3. Some metabolites result from initial hydrolysis of the sulphur mustard, but the majority are formed by conjugation with glutathione. These are further metabolized to N-acetylcysteine conjugates, or to methylthio/methylysulphinyl derivatives by a pathway probably involving beta-lyase, accompanied by oxidation of the mustard sulphur atom to sulphoxide or sulphone. 4. Thiodiglycol sulphoxide, 1,1'-sulphonylbis[2-S(N-acetylcysteinyl)ethane] and 1,1'-sulphonylbis[2-methylsulphinyl)ethane] or 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane were the most prevalent metabolites resulting from the three major pathways. Metabolic pathways for the formation of the excretion products are proposed.     

N-(3-甲基-1-吡咯烷基)-1-丁酮基-苯丙酰胺的合成研究

目的:合成心血管疾病新治疗靶点小分子白介素1受体/髓样分化蛋白88-TIR(Toll/IL-1receptor)(IL-1R/MyD88-TIR)拟似物N-(3-甲基-1-吡咯烷基)-1-丁酮基-苯丙酰胺。方法:以N-叔丁氧羰基-L-缬氨酸羟基琥珀酰亚胺酯为原料,先合成3-甲基-2-叔丁氧羰氨基-1-吡咯烷基-1-丁酮,再合成N-(3-甲基-1-吡咯烷基)-1-丁酮基-苯丙酰胺,产物结构经核磁共振(NMR)和质谱(MS)确证。结果:通过2步反应合成了N-(3-甲基-1-吡咯烷基)-1-丁酮基-苯丙酰胺,反应总收率为81.1%,产物结构经NMR和MS证实为目标化合物。结论:该反应条件温和,操作方便,收率较高。     

NMR-Spektroskopische Untersuchungen an diastereomeren 1-Dimethylamino-2-methyl-3,3-diphenyl-alkanolen-(4)

NMR-Study of Diastereoisomeric 1-Dimethylamino-2-methyl-3,3-diphenyl-alkane-4-ols ¹H-NMR-spectra are recorded of 1-dimethylamino-2-methyl-3,3-diphenyl-pentanol-(4) and the two diastereoisomeric 1-dimethylamino-2-methyl-3,3-diphenyl-hexanols-(4) (2S,4R/2R,4S : α-isomethadol and 2S,4S/2R,4R : α-isomethadol) as the free bases. Especially, ¹H-NMR data of the methine and methylene protons of the chain are given.     

3,3',4,4'-Tetrachlorobiphenyl. Excretion and tissue retention of hydroxylated metabolites in the mouse

The coplanar 3,3',4,4'-tetrachlorobiphenyl (TCB) was given orally to mice and the metabolite patterns in feces, urine, liver, and adipose tissue were examined. In feces, 80% of the dose was excreted within 5 days. 5-Hydroxy-, 6-hydroxy-TCB, 4-hydroxy-3,3',4',5-tetrachlorobiphenyl, and unmetabolized TCB were identified by comparison to synthetic standards (GC/MS). 4-Hydroxy-trichlorobiphenyl and a dihydroxy-trichlorobiphenyl were indicated by the fragmentation pattern of the corresponding methylated derivatives by GC/MS. In urine, 4.9% of the TCB dose was excreted mainly as conjugates. After hydrolysis, TCB and seven hydroxylated metabolites were detected; 2-, 5-, and 6-hydroxy-TCB and 4-hydroxy-3,3',4',5-tetrachlorobiphenyl were identified and two dihydroxy-tetrachlorobiphenyls were indicated. The major compound detected after hydrolysis of urine was a dihydroxy-trichlorobiphenyl. TCB was the major compound present in the liver, while a minor portion was due to 4-hydroxy-3,3',4',5-tetrachlorobiphenyl. TCB, 4-hydroxy-3,3',4',5-tetrachlorobiphenyl, and 5- and 6-hydroxy-TCB were present in adipose tissue. In addition, radiolabeled material was present in a lipid fraction obtained after gel permeation chromatography of all samples except urine, indicating the presence of TCB metabolites with lipid characteristics.     

Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile

High-resolution accurate MS with an LTQ-Orbitrap was used to identify quinone imine metabolites derived from the 5-hydroxy (5-OH) and 4 prime-hydroxy (4'-OH) glutathione conjugates of diclofenac in rat bile. The initial quinone imine metabolites formed by oxidation of diclofenac have been postulated to be reactive intermediates potentially involved in diclofenac-mediated hepatotoxicity; while these metabolites could be formed using in vitro systems, they have never been detected in vivo. This report describes the identification of secondary quinone imine metabolites derived from 5-OH and 4'-OH diclofenac glutathione conjugates in rat bile. To verify the proposed structures, the diclofenac quinone imine GSH conjugate standards were prepared synthetically and enzymatically. The novel metabolite peaks displayed the identical retention times, accurate mass MS/MS spectra, and the fragmentation patterns as the corresponding authentic standards. The formation of these secondary quinone metabolites occurs only under conditions where bile salt homeostasis was experimentally altered. Standard practice in biliary excretion experiments using bile duct-cannulated rats includes infusion of taurocholic acid and/or other bile acids to replace those lost due to continuous collection of bile; for this experiment, the rats received no replacement bile acid infusion. High-resolution accurate mass spectrometry data and comparison with chemically and enzymatically prepared quinone imines of diclofenac glutathione conjugates support the identification of these metabolites. A mechanism for the formation of these reactive quinone imine containing glutathione conjugates of diclofenac is proposed.     

Pharmacological studies of 3-[bis(3,3-diphenylpropyl)-amino]-propan-1-ol hydrochloride, (PF-244), a new cerebral vasodilator.

Pharmacological effects of 3-[bis(3,3-diphenylpropyl)-amino]-propan-1-ol-hydrochloride (PF-244) mainly on cerebral and cardiovascular systems, were studied. PF-244 indicated a potent cerebral vasodilation effect and a marked increase in oxygen supply to cerebral tissue with only weak inotropic action and depressor effect. This cerebral vasodilation was more potent and specific than that of papaverine hydrochloride in regional cerebral flow. PF-244 also indicated an antibarium action, although it did not show the properties related to the cholinergic and/or adrenergic mechanism. PF-244 appears to be one of the potent cerebral vascular dilators, and the nature of its mechanism was discussed.     

N-(3,5-dichlorophenyl)succinimide nephrotoxicity: evidence against the formation of nephrotoxic glutathione or cysteine conjugates.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity via one or more metabolites. Previous studies suggested that glutathione is important for mediating NDPS-induced nephropathy. The purpose of this study was to examine the possibility that a glutathione or cysteine conjugate of NDPS or an NDPS metabolite might be the penultimate or ultimate nephrotoxic species. In one set of experiments, male Fischer 344 rats were administered intraperitoneally (i.p.) NDPS (0.4 or 1.0 mmol/kg) 1 h after pretreatment with the gamma glutamyltranspeptidase inhibitor AT-125 (acivicin) (10 mg/kg, i.p.) and renal function was monitored at 24 and 48 h. In general, AT-125 pretreatment had few effects on NDPS-induced nephropathy. In a second set of experiments, rats were treated i.p. or orally (p.o.) with a putative glutathione (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)glutathione (NDPSG), a cysteine (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)cysteine (NDPSC) (as the methyl ester) or N-acetylcysteine (S-(2-(N-(3,5-dichlorophenyl)succinimidyl)-N-acetylcysteine (NDPSN) conjugate of NDPS (0.2, 0.4 or 1.0 mmol/kg) or vehicle and renal function was monitored at 24 and 48 h. An intramolecular cyclization product of NDPSC, 5-carbomethoxy-2-(N-(3,5-dichlorophenyl)carbamoylmethyl)-1,4-th iazane-3-one (NDCTO) was also examined for nephrotoxic potential. None of the compounds produced toxicologically important changes in renal function or morphology. The in vitro ability of the conjugates to alter organic ion accumulation by cortical slices was also examined. All of the conjugates tested caused a reduction in p-aminohippurate (PAH) accumulation at a conjugate bath concentration of 10(-4) M, but none of the conjugates reduced tetraethylammonium (TEA) uptake. In a third experiment, the ability of the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid (AOAA) (0.5 mmol/kg, i.p.) to alter the nephrotoxicity induced by two NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA) (0.2 mmol/kg, i.p.), was examined. AOAA pretreatment had no effect on NDHS- or NDHSA-induced nephrotoxicity. These results do not support a role for a glutathione or cysteine conjugate of NDPS or and NDPS metabolite as being the penultimate or ultimate nephrotoxic species.     

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): isolation and identification of urinary metabolites following intraperitoneal administration to rat

1. The metabolism of sulphur mustard, 1,1'-thiobis(2-chloroethane), in vivo was investigated following i.p. administration to rat.

2. Approx. 60% of dose was excreted in the 24 h urine. Many metabolites were present; nine have been isolated by h.p.l.c. and characterized by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards.

3. Some metabolites result from initial hydrolysis of the sulphur mustard, but the majority are formed by conjugation with glutathione. These are further metabolized to N-acetylcysteine conjugates, or to methylthio/methylysulphinyl derivatives by a pathway probably involving β-lyase, accompanied by oxidation of the mustard sulphur atom to sulphoxide or sulphone.

4. Thiodiglycol sulphoxide, 1,1'-sulphonylbis[2-S (N-acetylcysteinyl)ethane] and 1,1'-sulphonylbis[2-methylsulphinyl)ethane] or 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane were the most prevalent metabolites resulting from the three major pathways. Metabolic pathways for the formation of the excretion products are proposed.     

S-(2-hydroxy-3-buten-1-yl)glutathione and S-(1-hydroxy-3-buten-2-yl)glutathione are in vivo metabolites of butadiene monoxide: detection and quantitation in bile.

Administration (ip) of butadiene monoxide, a toxic metabolite of 1,3-butadiene, to rats caused the appearance of two new biliary peaks when analyzed by HPLC chromatography. These peaks were isolated and identified as the regioisomeric glutathione conjugates, S-(2-hydroxy-3-buten-1-yl)glutathione (I) and S-(1-hydroxy-3-buten-2-yl)glutathione (II), by comparison of their HPLC retention times and fast atom bombardment mass spectra to those of synthetic standards. S-(4-Hydroxy-2-buten-1-yl)glutathione, a rearrangement product formed during chemical synthesis or storage of I, was not detected. Whether butadiene monoxide was given at a dose of 14.3 or 143 mumol/kg, the amount of conjugates excreted in 30 min was at least 85% of that excreted in 120 min. Conjugate excretion in 60 min did not exhibit saturation when the butadiene monoxide dose was varied between 14.3 and 286 mumol/kg; the total amount of the butadiene monoxide dose excreted as combined I and II averaged only 7.6 +/- 4.2% (mean +/- SD, n = 12), with approximately a 3:1 ratio of isomers I:II being excreted at all butadiene monoxide doses. Whereas these results indicate a role for glutathione S-transferase-catalyzed reactions in butadiene monoxide metabolism in vivo, biliary excretion of I and II can only account for a small fraction of the butadiene monoxide dose given.     

Biliary excretion and intestinal metabolism in the intermediary metabolism of pentachlorothioanisole

1. Biliary metabolites from rats dosed with pentachlorothioanisole (PCTA) were characterized by fast atom bombardment mass spectrometry and electron impact mass spectrometry. 2. Most of the biliary metabolites from PCTA were mercapturic acid pathway metabolites of methylsulphinyltetrachlorobenzene (51% of the dose); the remaining characterized biliary metabolites (20%) were mainly methylsulphinyltetrachlorothiophenols excreted as unknown conjugates. 3. Pathways are proposed for the intermediary metabolism of PCTA to bis-(methylthio)tetrachlorobenzene (bis-MTTCB) involving glutathione conjugation, biliary excretion, intestinal metabolism, and enterohepatic circulation.     

Arylalkylamines are a novel class of positive allosteric modulators at GABA(B) receptors in rat neocortex

Using grease-gap recording from rat neocortical slices, the gamma-aminobutyric acid(B) (GABA(B)) receptor agonists baclofen (3-100 microM) and SKF 97541 (3-aminopropyl-methylphosphinic acid) (1-30 microM) elicited reversible and concentration-dependent hyperpolarizing responses, with EC(50) values of 10 and 3 microM, respectively. The hyperpolarizations were antagonised by the GABA(B) receptor antagonist Sch 50911 ((+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid) (1, 5 and 10 microM). Fendiline (N-[3,3-diphenylpropyl)-alpha-methylbenzylamine) (5-50 microM) and its congeners, prenylamine (N-[3,3-diphenylpropyl)-alpha-methylphenylethylamine) (10-100 microM) and F551 (N-[3,3-diphenylpropyl)-alpha-methyl-3-methoxybenzylamine) (1-30 microM) reversibly enhanced hyperpolarizing responses to the agonists; such effects were reduced by Sch 50911. These arylalkylamines produced leftward shifts of the concentration-response curves, with a marked increase in the maximal hyperpolarization obtained, compared with the agonists alone, F551 being the most potent. These findings suggest that these arylalkylamines represent a new class of positive modulators of GABA(B) receptor-mediated function.     

A new metabolite of methamphetamine; evidence for formation of N-[(1-methyl-2-phenyl)ethyl]ethanimine N-oxide

1. N-Hydroxyamphetamine and N-hydroxymethamphetamine, metabolic intermediates of amphetamine and methamphetamine, showed no reactivity towards endogeneous protein, amino acids, nucleic acids and fatty acids. In contrast, formaldehyde, acetaldehyde and propionaldehyde reacted well with N-hydroxyamphetamine and slightly with N-hydroxymethamphetamine under mild conditions (pH 7.4, 37 degrees C). 2. Two products were isolated from the reaction mixture of acetaldehyde and N-hydroxyamphetamine. These were characterized as N-[(1-methyl-2-phenyl)ethyl]ethanimine N-oxide and N-[(1-methyl-2-phenyl)ethyl]butenimine N-oxide by mass and n.m.r. spectrometries. 3. N-[(1-Methyl-2-phenyl)ethyl]ethanimine N-oxide was formed by incubating methamphetamine with liver 9000 g supernatants of rats and guinea-pigs. The butenimine N-oxide derivative, however, could not be detected as a metabolite of methamphetamine in vitro. 4. N-[(1-Methyl-2-phenyl)ethyl]ethanimine N-oxide was also detected as a urinary metabolite of methamphetamine in rats and guinea-pigs. Thus, the ethanimine N-oxide was established as a novel metabolite of methamphetamine.     

Urinary metabolites of the antiprotozoal agent cis-3a,4,5,6,7,7a- hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole in the rat

1H-NMR and MS were employed to identify 13 rat urinary metabolites of 14C-labeled cis-3a,4,5,6,7,7a- hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436). The major free (unconjugated) metabolite was cis-3a,4,5,6,7, 7a-hexahydro-3-carboxamido-1,2-benzisoxazole; it was also the second most abundant metabolite released during hydrolysis of the conjugated fraction. All other identified metabolites were hydroxylated analogues substituted at C(4)-C(7a) of the cyclohexane ring. the 4-equatorial,5-axial,7a-triol was the second most abundant metabolite excreted in an unconjugated form. Four monohydroxy (5-axial, 6-axial, 6-equatorial, 7-equatorial) metabolites of the drug were identified; they were found in the conjugated fraction only and were released by hydrolysis. The 5-axial hydroxy compound is the major conjugated metabolite and is overall the most abundant of all the metabolites. Six dihydroxy metabolites were identified: one was found exclusively in the free state, three as conjugates only (including the 7-axial,7a-diol, which is the major dihydroxy species), and two both free and conjugated. A second triol was found both free and conjugated.