Biotransformation of sufentanil in liver microsomes of rats, dogs, and humans

The biotransformation of sufentanil (SUF), an analog of the synthetic opioids fentanyl and alfentanil, was investigated in liver microsomes of rats, dogs, and humans. The drug was extensively metabolized and the metabolism was found to be very similar, both kinetically and metabolically, in the three species. The initial metabolism of SUF occurred monophasically in man and dog and biphasically in the rat over a concentration range of 0.13-20.1 microM. The apparent Vm values were 7.30 and 6.15 nmol metabolized.min-1.mg protein-1, and the apparent Km values were 4.98 microM and 15.2 microM for dog and human microsomes, respectively. In rat microsomes, apparent Km values were 0.10 and 20.8 microM, and the apparent Vm values were 0.10 and 7.32 nmol metabolized.min-1.mg protein-1 for the high and low affinity site, respectively. The major metabolic pathways were similar in the three species and included oxidative N-dealkylation at the piperidine nitrogen, oxidative N-dealkylation of the piperidine ring from the phenylpropanamide nitrogen, oxidative O-demethylation, and aromatic hydroxylation. Desmethyl-SUF was formed at the shorter incubation times but quickly metabolized into secondary metabolites. The major metabolites which could be detected at the end of the incubation were N-[4-(methoxymethyl)-4-piperidinyl]-N-phenylpropanamide, N-[4-(hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide, and N-phenylpropanamide. The relevance of the in vitro results is discussed in relation to previous in vivo studies of the metabolism of SUF in rats, dogs, and humans.     

Excretion and biotransformation of alfentanil and sufentanil in rats and dogs

The excretion and biotransformation of alfentanil (ALF) and sufentanil (SUF), two recent analogues of the synthetic opioid fentanyl, were studied after single iv administration of the tritium-labeled drugs in male rats and dogs. The drugs were almost completely metabolized in the two species, which resulted in a large number of metabolites. The excretion of the metabolites was rapid and exceeded 95% within 4 days, except for that of ALF metabolites in dogs (about 85%). For ALF, excretion of the radioactivity with the urine (73% in rats, about 76% in dogs) exceeded that with the feces. For SUF, excretion of the radioactivity with the urine amounted to 38 and 60% and that with the feces to 62 and 40%, in rats and dogs, respectively. Bile-cannulated rats excreted 68% with the bile within 24 hr after SUF dosing, and about 22% of this biliary radioactivity was subjected to enterohepatic circulation. After an ALF dose, the biliary excretion amounted to 24%, and the enterohepatic circulation was minimal. The main metabolic pathways of the two drugs were the oxidative N-dealkylation at the piperidine nitrogen and at the amide nitrogen, oxidative O-demethylation, aromatic hydroxylation, and the formation of ether glucuronides. N-[4-(Hydroxymethyl)-4-piperidinyl]-N-phenylpropanamide (M6) was the main metabolite of both ALF and SUF in rats. In dogs, the glucuronide of N-(4-hydroxyphenyl)propanamide (M5) was the main metabolite of ALF. After SUF dosing in dogs, N-[4-(methoxymethyl)-4-piperidinyl]-N-phenylpropanamide was more abundant than M5.     

Synthetic analgesics: N-(1-[2-arylethyl]-4-substituted 4-piperidinyl) N-arylalkanamides.

The synthesis of several 4-arylamino-4-piperdinecarboxylic acids is reported. These acids were starting materials for the preparation of alpha-amino esthers, ethers and ketones. Different synthetic approaches are described. Suitable substitution on both nitrogen atoms afforded extremely potent analgesics. Thus, methyl 4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidinecarboxylate (22),N-(4-(methoxymethyl)-2-[2-(2-thienyl)ethyl]-4-piperidinyl)-N-phenylpropranamide (67) and N-[4-acetyl-1-(2-phenylethyl)-4-piperidinyl]-N-phenylpropanamide (82) were found to be respectively 7682, 3987 and 4921 times as potent as morfine. Both cis- and trans-3-methyl homologs of 22 have been prepared. As expected, analgesic activity resides mainly in the cis-isomer.     

N-4-Substituted 1-(2-arylethyl)-4-piperidinyl-N-phenylpropanamides, a novel series of extremely potent analgesics with unusually high safety margin.

The intravenous analgesic activity and toxicity of a novel series of N-[4-substituted 1-(2-arylethyl)-4-piperidinyl]-N-phenylpropanamides was studied in rats. Onset, potency and duration of analgesic action were assessed in the tail withdrawal test and compared with the activity of fentanyl, (+)-cis-3-methylfentanyl (R 26 800), morphine, and pethidine. All compounds studied were found to be extremely potent analgesics characterized by an unusually high safety margin. Methyl 4-[N-(1-oxopropyl)-N-phenyl-amino]-1-(2-phenylethyl)-4-piperidinecarboxylate (R 31 833; lowest ED50 = 0.00032 mg/kg) is the most potent compound (10 031 times morphine). cis-Methyl 3-methyl-4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidine carboxylate (R 32 792) is the longest acting compound (more than 8 h at 4 times the lowest ED50) and N-[4-(1-oxopropyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (R 33 352) is the shortest acting compound (0.74 h at 4 times the lowest ED50) of the 4-substituted fentanyl derivatives. N-[4-(Methoxymethyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (R 30 730) was selected for further investigation. R 30 730 has a rapid onset of action and is 4521 times more potent than morphine at the time of peak effect; it has a relatively short duration of action comparable to that of fentanyl and its safety margin (LD50/lowest ED50 = 25 211) is unusually high.     

Alfentanil (R 39 209) — a particularly short-acting intravenous narcotic analgesic in rats

Alfentanil, N-{1-[2-(4-ethyl-4,5-dihydro-5-oxo-1H-tetrazol-l-yl)ethyl]-4-(methoxymethyl)-4-piperidinyl}-N-phenyl propanamide monohydrochloride monohydrate, is a chemically novel, potent, extremely short-acting and safe intravenous morphine-like agent in laboratory animals. As an analgesic in rats, alfentanil is 137 times as potent as pethidine (tail withdrawal reaction test: i.v. ED50 is 6.04 mg/kg for pethidine and 0.044 mg/kg for alfentanil). The safety margin in rats is 1,080 (LD50: 47.5 mg/kg i.v.). Alfentanil reaches its peak effect within 2 min after injection, and its duration of action is very short: at two times its lowest ED50 the drug has a duration of action of 10 min. This duration is 30–35 min for pethidine and fentanyl, and 120 min for morphine. The narcotic antagonist naloxone produces an immediate and complete reversal of all morphine-like effects of alfentanil. In dogs, alfentanil is a potent antiemetic (ED50: 0.032 mg/kg i.v.) with a safety margin of over 850 (LD50: > 20 mg/kg i.v.).     

强效镇痛剂研究——Ⅱ.3-甲基芬太尼类衍生物的合成及镇痛活性

本文报道了N-[1-(β-苯乙基)-3-甲基-4-哌啶基]-N-丙酰苯胺(7209)和N-[1-(β-羟基-β-苯乙基)-3-甲基-4-哌啶基]-N-丙酰苯胺(7302)等一系列3-甲基芬太尼类衍生物的合成及镇痛活性。绝大部分该类衍生物均具有典型的吗啡样镇痛活性,是一类结构较简单、易于合成、镇痛作用极强的麻醉镇痛剂。化合物7302的ED₅₀为0.0022mg/kg(ip,小鼠,热板法),比芬太尼强28倍,竟达吗啡的6318倍,为我们至今合成该类衍生物中作用最强者。     

4-取代芬太尼类化合物的晶体结构和构效关系

以芬太尼为典型代表物的4-苯胺哌啶类化合物是一类具有独特结构类型、强度高、作用迅速的高效麻醉镇痛剂。该类化合物哌啶环的3位或4位引入适当取代基团能提高镇痛活性,其活性强度已达微克水平。芬太尼类化合物的构效关系研究表明:分子构象与生物活性之间关系密切。虽Peeters和Koch等人分别对芬太尼(R 4263)和4-甲氧甲基芬太尼类代表物(R 30490,R 30730)     

Identification of new pyrrole alkaloids from the fruits of <Emphasis Type="Italic">Lycium chinense</Emphasis>

Three new minor pyrrole alkaloids, 3-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]pentanedioic acid (1), (2R)-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]-1-methoxy-1-oxobutanoic acid (2), and methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-4-methylpentanoate (3) were isolated from the fruits of Lycium chinense Miller (Solanaceae), along with the known compound, methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(phenyl)propanoate (4). The structures of 1–4 were elucidated by analysis of their 1D- and 2D-NMR and HRMS data. The absolute configurations of 2–4, possessing a stereogenic center in each structure, were determined by comparison of their experimental electronic circular dichroism (ECD) with those of calculated ECD values.     

4-甲氧羰基-4-N-丙酰苯胺基哌啶1位衍生物的合成及其镇痛作用

本文报道了一系列N-[-1(2-苯乙基-4-甲氧羰基-4-哌啶基]-N-丙酰苯胺(4-甲氧羰基芬太尼)哌啶环1位取代衍生物的合成及其镇痛活性;讨论了结构与镇痛活性之间的关系。药理试验结果表明,大部分化合物具有典型的吗啡样镇痛活性,是一类作用极强的麻醉性镇痛剂。特别是哌啶环1位β-苯环被取代乙烯基替代的化合物具有相当或接近子母体化合物的镇痛活性。其代表物1321的镇痛活性(ED_(50)=0.005mg/kg ip,小鼠,热板法)略强于4-甲氧羰基芬太尼(ED_(50)=0.0063 mg/kg)。     

Metabolism of 3-(Hydroxymethyl)-8-methoxychromone in the Rat: II. Classification and Identification of Urinary Drug Metabolites

1. Five metabolites were isolated from the urine of dogs dosed with 3-(hydroxymethyl)-8-methoxy[4-¹⁴C]chromone. These were identified as 8-methoxychromone, 2-hydroxy-3-methoxyacetophenone, 3-(hydroxymethyl)-8-hydroxychromone, 8-hydroxychromone and 2,3-dihydroxyacetophenone.

2. These compounds were also present in the urine of rats treated with labelled drug, together with unchanged drug and two intermediate metabolites, 3-carboxy-8-methoxychromone and 3-(carboxymethyl)-8-hydroxychromone.

3. In addition to the unconjugated labelled compounds, glucuronides and sulphates were identified.

4. Quantitative data were obtained for all of the 20 labelled compounds in rat urine.

5. A scheme is presented for the biotransformation of 3-(hydroxymethyl)-8-methoxychromone in rats and dogs, and a mechanism for scission of the γ-pyrone ring is suggested.     

强效镇痛剂研究——Ⅴ.N-[1-(β-羟基-β-苯乙基)-3-甲基-4-哌啶基]-N-丙酰苯胺(7302)酯类衍生物的合成及镇痛活性

本文报道了一系列N-[1-(β-酰氧基-β-苯乙基)-3-甲基-4-哌啶基]-N-丙酰苯胺类衍生物及其化学结构与镇痛强度之间的关系,并测定了几个代表化合物的镇痛作用时间及与阿片受体亲和力。实验结果表明,7302的β-羟基酯化后,均能维持一定的镇痛强度,其镇痛作用时间与母体化合物7302相近。从受体结合试验来看,酯化后与受体亲和的能力显著下降。     

Disposition and metabolism of timiperone in the rat, dog, and monkey

The disposition and metabolism of timiperone (TP), a new butyrophenone antipsychotic, were studied in the rat, dog, and monkey given [14C]TP orally at different dose levels. [14C]TP was absorbed by the three species; the elimination of radioactivity from the blood was most rapid in the rat, and slowest in the dog. In the rat, blood and tissue concentrations of radioactivity were dose-dependent, and persisted after low and high doses. Tissue/blood ratios of radioactivity concentration in most of the rat tissues were much larger than 1.0, indicating that TP has a high affinity for the tissues. Approximately 19, 36, and 54% of the administered dose were excreted into dog, rat, and monkey urine, respectively, within 3 days, and the residue into the feces. [14C]TP was metabolized by N-dealkylation and the reduction of the butyrophenone sidechain to form 4-[4-(2,3-dihydro-2-thioxo-1H-benzimidazol-1-yl)-1-piperidinyl]-1-(4-fluorpheny l)-1-butanol (M-II), 2,3-dihydro-1-(4-piperidinyl)-2-thioxo-1H-benzimidazole (M-V), and N-(4-fluorophenylacetyl)glycine (M-b-l). Urinary major metabolites in all the species were M-V and M-b-l. Metabolite patterns in the rat brain indicated that unchanged TP accounted for the major part of total radioactivity. Similar patterns were obtained in rat and dog plasma, but not in monkey plasma.     

Synthesis and antiallergy activity of 4-(diarylhydroxymethyl)-1-[3-(aryloxy)propyl]piperidines and structurally related compounds

A series of 4-(diarylhydroxymethyl)-1-[3-(aryloxy)propyl]piperidines was synthesized and evaluated for antiallergy activity. Several analogues had potent activity in the passive foot anaphylaxis (PFA) assay, an IgE-mediated model useful in the detection of compounds possessing antiallergic activity. In particular 1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1-piperidinyl] propoxy]-3-methoxyphenyl]ethanone (1, AHR-5333) was more potent than oxatomide and terfenadine in this assay.     

Metabolism and Cytotoxicity of 7,12-Dimethylbenz[a] anthracene by Hamster,Rat and Rabbit Embryo Cell Cultures

1. The metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by rat, hamster and rabbit fibroblasts has been studied and compared with the metabolism of the hydrocarbon by liver homogenates. The metabolism of DMBA by cell cultures and by liver homogenates was very similar. The ethyl acetate-soluble metabolites identified included dihydrodiols, phenols and hydroxymethyl derivatives. Other more polar metabolites and unidentified water-soluble metabolites were also formed.

2. High yields of phenols and other more polar metabolites, which may be tetrahydrotetrols, were produced by rabbit fibroblasts.

3. Kinetic studies showed that the metabolic activity of rabbit fibroblasts was high but that the conversion of DMBA into water-soluble metabolites was lower than with hamster and rat fibroblasts.

4. 7,12-Dimethylbenz[a]anthracene-induced cytotoxicity was inversely related to the conversion of metabolized DMBA to water-soluble derivatives.     

New 1-(heterocyclylalkyl)-4-(propionanilido)-4-piperidinyl methyl ester and methylene methyl ether analgesics.

A series of new 1-(heterocyclyalkyl)-4-(propionanilido)-4-piperidinyl methyl esters and methylene methyl ethers have been synthesized and pharmacologically evaluated. In the mouse hot-plate test, the majority of compounds exhibited an analgesia (ED50 less than 1 mg/kg) superior to that of morphine. These studies revealed a pharmacological accommodation for many more structurally diverse and far bulkier aromatic ring systems than the corresponding components of the arylethyl groups of the prototypic methyl ester (carfentanil, 2) and methylene methyl ether (sufentanil, 3 and alfentanil, 4) 4-propionanilido analgesics. Compound 9A (methyl 1-[2-(1H-pyrazol-1-yl)-ethyl]-4-[(1-oxopropyl)phenylamino]-4- piperidinecarboxylate), which exhibited appreciable mu-opioid receptor affinity, was a more potent and short-acting analgesic, than alfentanil with less respiratory depression in the rat. On the other hand, the phthalimides 57A and 57B, which exhibited negligible affinity for opioid receptors associated with the mediation of nociceptive transmission (i.e., mu-, kappa-, and delta-subtypes), displayed analgesic efficacy in all antinociception tests. In addition, while 57B, compared to clinical opioids, showed a superior recovery of motor coordination after regaining of righting reflex from full anesthetic doses in the rat rotorod test, 57A showed significantly less depression of cardiovascular function at supraanalgesic doses in the isoflurane-anesthetized rat.     

In vitro and in vivo metabolism of 14C-AZ11, a novel inhibitor of bacterial DNA gyrase/type II topoisomerase

Abstract

1.?(2R,4S,4aS)-11-Fluoro-2,4-dimethyl-8-((S)-4-methyl-2-oxooxazolidin-3-yl)-2,4,4a,6-tetrahydro-1H,1'H-spiro [isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione (AZ11) is a novel mode-of-inhibition bacterial topoisomerase inhibitor that entered preclinical development for the treatment of Gram-positive bacteria infection.

2.?The in vitro biotransformation studies of AZ11 using mouse, rat, dog and human hepatocytes showed low-intrinsic clearance in all species attributed to microsomal metabolism.

3.?After a single intravenous administration of [¹⁴C]AZ11 in bile duct cannulated rats, the mean percentage of dose recovered in rat urine, bile and feces was approximately 18, 36 and 42%, respectively. Unchanged AZ11 recovered in rat urine and bile was less than 9% of the dose, indicating that AZ11 underwent extensive metabolism in rats.

4.?The most abundant in vivo metabolite detected in urine and bile was M1 formed via ring opening on the piperidine and morpholine rings accounting for 20% of the administered dose. The major fecal metabolite was M5, which accounted for approximately 32% of administered dose. M5 was not formed when AZ11 incubated with rat intestinal microsomes and cytosol but was formed when incubated with fresh rat feces, suggesting that unchanged AZ11 was directly excreted into gut lumen where M5 formed as an intestinal microflora-mediated product. This process could have significant impact on bioavailability or exposure of AZ11 in rat.     

New bronchodilators. Synthesis and bronchodilating activity of some 3-(alkoxymethyl)-alpha-(N-substituted aminomethyl)-4-hydroxybenzyl alcohols.

A series of 3-(alkoxymethyl)-alpha-(N-substituted aminomethyl)-4-hydroxybenzyl alcohols was synthesized as potential bronchodilators. The ability to prevent effects against histamine-induced bronchoconstriction in guinea pigs was studied to determine their bronchodilating activity. Introduction of a methoxymethyl group in place of the m-hydroxyl group of beta-adrenergic catecholamines afforded compounds especially effective in delaying histamine-induced bronchoconstriction in guinea pigs. Appropriate N-substitution also enhanced the potency of these catecholamine analogues. 4-Hydroxy-3-(methoxymethyl)-alpha-[N-[4-(methoxymethyl)-alpha-methylphenyl]aminoethyl]benzyl alcohol hemifumarate (3r) was the most potent compound in this series.     

Synthesis and biological activities of 5-deaza analogues of aminopterin and folic acid

N-[p-[[(2,4-Diaminopyrido[2,3-d]pyrimidin-6-yl)methyl] amino]benzoyl]-L-glutamic acid (1a, 5-deazaaminopterin) and the 5-methyl analogue (1b) were synthesized in 14 steps from 5-cyanouracil (4a) and 5-cyano-6-methyluracil (4b), respectively, by exploitation of the novel pyrimidine to pyrido[2,3-d]pyrimidine ring transformation reaction. The 5-cyanouracils 4 were treated with chloromethyl methyl ether to the 1,3-bis(methoxymethyl)uracils (5, which were treated with malononitrile in NaOEt/EtOH to give the pyrido[2,3-d]pyrimidines 6. Diazotization of 6 in concentrated HCl afforded the 7-chloro derivatives 8 in high yield. After reduction of 8, the 7-unsubstituted products 9 were reduced in the presence of Ac2O and the products, 6-(acetamidomethyl)pyridopyrimidines 10, were converted into the 6-acetoxymethyl derivatives 12 via nitrosation. After removal of the N-methoxymethyl groups from 12, the 6-(acetoxymethyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones 14 were converted into 2,4-diamino-6-(hydroxymethyl)pyrido[2,3-d]pyrimidine (15a) and its 5-methyl analogue 15b by the silylation-amination procedure. Compounds 15 were brominated to the 6-bromomethyl derivatives 16, which were treated with diethyl (p-aminobenzoyl)-L-glutamate, and the products 17 were saponified to afford 5-deazaaminopterin (1a) and its 5-methyl analogue 1b. Compound 1b was also prepared by an alternative procedure in 10 steps from cyanothioacetamide and ethyl beta-(ethoxymethylene)acetoacetate via 2,4-diamino-6-(hydroxymethyl)-5-methylpyrido[2,3-d]pyrimidine (15b). 5-Deaza-5-methylfolic acid (2) was also prepared in four steps from 15b. The aminopterine analogues 1 showed significant anticancer activity in vitro and in vivo, whereas the folic acid analogue 2 did not exhibit any significant toxicity.     

Comparative metabolism of flunarizine in rats, dogs and man: an in vitro study with subcellular liver fractions and isolated hepatocytes.

1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.