Metabolites Of Carvedilol Research Articles

In silico drug repurposing carvedilol and its metabolites against SARS-CoV-2 infection using molecular docking and molecular dynamic simulation approaches

The pandemic of coronavirus disease 2019 (COVID-19) caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a significant impact on the economy and public health worldwide. Therapeutic options such as drugs and vaccines for this newly emerged disease are eagerly desired due to the high mortality. Using the U.S. Food and Drug Administration (FDA) approved drugs to treat a new disease or entirely different diseases, in terms of drug repurposing, minimizes the time and cost of drug development compared to the de novo design of a new drug. Drug repurposing also has some other advantages such as reducing safety evaluation to accelerate drug application on time. Carvedilol, a non-selective beta-adrenergic blocker originally designed to treat high blood pressure and manage heart disease, has been shown to impact SARS-CoV-2 infection in clinical observation and basic studies. Here, we applied computer-aided approaches to investigate the possibility of repurposing carvedilol to combat SARS-CoV-2 infection. The molecular mechanisms and potential molecular targets of carvedilol were identified by evaluating the interactions of carvedilol with viral proteins. Additionally, the binding affinities of in vivo metabolites of carvedilol with selected targets were evaluated. The docking scores for carvedilol and its metabolites with RdRp were − 10.0 kcal/mol, − 9.8 kcal/mol (1-hydroxyl carvedilol), − 9.7 kcal/mol (3-hydroxyl carvedilol), − 9.8 kcal/mol (4-hydroxyl carvedilol), − 9.7 kcal/mol (5-hydroxyl carvedilol), − 10.0 kcal/mol (8-hydroxyl carvedilol), and − 10.1 kcal/mol (O-desmethyl carvedilol), respectively. Using the molecular dynamics simulation (100 ns) method, we further confirmed the stability of formed complexes of RNA-dependent RNA polymerase (RdRp) and carvedilol or its metabolites. Finally, the drug-target interaction mechanisms that contribute to the complex were investigated. Overall, this study provides the molecular targets and mechanisms of carvedilol and its metabolites as repurposed drugs to fight against SARS-CoV-2 infection.

Low-volume LC-MS/MS method for the pharmacokinetic investigation of carvedilol, enalapril and their metabolites in whole blood and plasma: Application to a paediatric clinical trial.

Evidence-based pharmacotherapy with carvedilol and enalapril in children suffering from heart failure is insufficient owing to limited pharmacokinetic data. Although a few data sets regarding enalapril, its metabolite enalaprilat and carvedilol in children have been published, pharmacokinetic data on carvedilol metabolites are missing. However, for both drug substances, their active metabolites contribute substantially to drug efficacy. As data can hardly be derived from adults owing to the unknown impacts of enzymatic maturation and ontogeny during childhood, customised assays are important to facilitate paediatric evidence-based pharmacotherapy. Considering ethical paediatric constraints, a low-volume liquid chromatography coupled to mass spectrometry (LC-MS/MS) assay was developed using whole blood or plasma for the quantification of enalapril, enalaprilat, carvedilol, O-desmethyl carvedilol, 4- and 5-hydroxyphenyl carvedilol as well as 3- and 8-hydroxy carvedilol. To facilitate broader applications in adults, the elderly and children, a wide calibration range-between 0.024/0.049 and 50.000 ng/ml-was achieved with good linearity (r ≥ 0.995 for all analytes). In compliance with international bioanalytical guidelines, accuracy, precision, sensitivity and internal standard normalised matrix effects were further successfully validated with the exception of those for 3-hydroxy carvedilol, which was therefore assessed semi-quantitatively. Distinct haematocrits did not impact matrix effects or recoveries when analysing whole blood. Blood-to-plasma ratios were determined for all analytes to form the basis for pharmacokinetic modelling. Finally, incurred sample reanalysis of paediatric samples confirmed the reproducibility of the developed low-volume LC-MS/MS method during study sample analysis. The assay facilitates the reliable generation of important data and contributes towards a safe drug therapy in children.

Antibacterial activity of high concentrations of carvedilol against Gram-positive and Gram-negative bacteria

Many drugs used to treat non-infectious diseases have also shown excellent antibacterial activity or the ability to enhance the action of antibiotics. The aim of this study was to investigate the antibacterial activity of a popular β-blocker, carvedilol, and its mechanism of antibacterial action. The antibacterial activity of carvedilol was evaluated using the microdilution method and its influence on the viability of bacterial cells was investigated by the alamarBlue® test. Changes in phospholipid and fatty acid composition were analysed using LC-MS/MS and GC-MS techniques. The permeability of bacterial cell membranes following exposure to carvedilol was studied using propidium iodide staining and confocal microscopy. The ability of the tested bacteria to degrade carvedilol was examined by LC-MS/MS. In this study, the antibacterial activity of carvedilol is described for the first time, with a decrease in the viability of all assayed bacteria observed following treatment with the β-blocker. Staphylococcus aureus and Staphylococcus epidermidis were found to be the most sensitive among the tested strains. Significant modifications to fatty acid composition were observed in S. aureus incubated with carvedilol. Moreover, the cell membrane permeability of bacteria incubated with carvedilol was higher for Gram-positive bacteria than for Gram-negative bacteria. Furthermore, Gram-negative Escherichia coli and Pseudomonas aeruginosa strains, which were highly resistant to carvedilol, exhibited an ability to eliminate carvedilol from the growth medium. In addition, three carvedilol metabolites were identified in E. coli and P. aeruginosa cultures. The antibacterial activity of carvedilol may suggest its potential usefulness in the synthesis of new antibacterial drugs.

Microbial detoxification of carvedilol, a β-adrenergic antagonist, by the filamentous fungus Cunninghamella echinulata

Beta adrenergic antagonists like carvedilol are typical environmental pollutants detected in wastewater and surface water. Human metabolism of carvedilol is well investigated, while its environmental fates are still unknown. In recent years, there have been appearing reports on high toxicity of β-blockers toward aquatic organisms. In this paper the ability of the filamentous fungus C. echinulata to eliminate the β-blocker has been described for the first time. An 83% loss of carvedilol was observed after 120 h incubation of the tested fungus with the compound, where hydroxylated carvedilol metabolites were identified as the major biotransformation products. Carvedilol degradation by C. echinulata was proceeded by hydroxylation and conjugation reactions similar to its mammalian metabolism. Glucose conjugate was found in the fungi cultures, whereas glucuronide conjugates were detected in mammals. The impact of carvedilol on the functionality of fungal cells was also evaluated. A 2-fold decrease in the PC/PE ratio was noticed in the C. echinulata cell membrane after the exposition to carvedilol compared to control mycelium incubated without the β-blocker. The change can denote perturbation of fungal cell membrane integration by carvedilol. Moreover, 2.8-fold lower toxicity of postcultures supernatants toward D. magna were shown in contrast to abiotic control.

Role of cytochrome P450 2D6 genetic polymorphism in carvedilol hydroxylation in vitro.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that catalyzes the metabolism of a great number of therapeutic drugs. Up to now, >100 allelic variants of CYP2D6 have been reported. Recently, we identified 22 novel variants in the Chinese population in these variants. The purpose of this study was to examine the enzymatic activity of the variants toward the CYP2D6 substrate carvedilol in vitro. The CYP2D6 proteins, including CYP2D6.1 (wild type), CYP2D6.2, CYP2D6.10, and 22 other novel CYP2D6 variants, were expressed from insect microsomes and incubated with carvedilol ranging from 1.0 μM to 50 μM at 37°C for 30 minutes. After termination, the carvedilol metabolites were extracted and detected using ultra-performance liquid chromatography tandem mass-spectrometry. Among the 24 CYP2D6 variants, CYP2D6.92 and CYP2D6.96 were catalytically inactive and the remaining 22 variants exhibited significantly decreased intrinsic clearance values (ranging from ~25% to 95%) compared with CYP2D6.1. The present data in vitro suggest that the newly found variants significantly reduced catalytic activities compared with CYP2D6.1. Given that CYP2D6 protein activities could affect carvedilol plasma levels, these findings are greatly relevant to personalized medicine.

The Effect of Apatinib on the Metabolism of Carvedilol Both in vitro and in vivo

Background: In light of the growing number of cancer survivors, the incidence of cardiovascular complications in these patients had also increased, while the effect of apatinib on the pharmacokinetic of cardioprotective drug (carvedilol) in rats or human is still unknown. The present work was to study the impact of apatinib on the metabolism of carvedilol both in vitro and vivo. Methods: A specific and sensitive ultra-performance liquid-chromatography tandem mass spectrometry method was applied to determine the concentration of carvedilol and its metabolites (4′-hydroxyphenyl carvedilol [4′-HPC], 5′-hydroxyphenyl carvedilol [5′-HPC] and o-desmethyl carvedilol [o-DMC]). Results: The inhibition ratios in human liver microsomes were 10.28, 10.89 and 5.94% for 4′-HPC, 5′-HPC and o-DMC, respectively, while in rat liver microsomes, they were 3.22, 1.58 and 1.81%, respectively. The data in vitro of rat microsomes were consistent with the data in vivo that the inhibition of 4′-HPC and 5′-HPC formation was higher than the control group. Conclusion: Our study showed that apatinib could significantly inhibit the formation of carvedilol metabolites both in human and rat liver microsomes. It is recommended that the effect of apatinib on the metabolism of carvedilol should be noted and carvedilol plasma concentration should be monitored.

Suppression of store overload-induced calcium release by hydroxylated metabolites of carvedilol.

Carvedilol is a drug widely used in the treatment of heart failure and associated cardiac arrhythmias. A unique action of carvedilol is its suppression of store overload-induced calcium release (SOICR) through the cardiac ryanodine receptor (RyR2), which can trigger ventricular arrhythmias. Since the effects of carvedilol metabolites on SOICR have not yet been investigated, three carvedilol metabolites hydroxylated at the 3-, 4' and 5'-positions were synthesized and assayed for SOICR inhibition in mutant HEK 293 cells expressing the RyR2 mutant R4496C. This cell line is especially prone to SOICR and calcium release through the defective RyR2 channel was measured with a calcium-sensitive fluorescent dye. These results revealed that the 3- and 4'-hydroxy derivatives are slightly more effective than carvedilol in suppressing SOICR, while the 5'-analog proved slightly less active. Metabolic deactivation of carvedilol via these hydroxylation pathways is therefore insignificant.

Synthesis of Active Metabolites of Carvedilol, an Antihypertensive Drug

A simple synthetic route for active metabolites of carvedilol is reported. The metabolites 4′-hydroxycarvedilol and 5′-hydroxycarvedilol have exhibited high activity for β-blockade. We have disclosed syntheses of 4′-hydroxycarvedilol and 5′-hydroxycarvedilol from commercially available vanillin and isovanillin, respectively.

Stereoselective glucuronidation of carvedilol by Chinese liver microsomes.

To study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes. The metabolites of CARV were identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylisothiocyanate. Two CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of K(m) and V(max) for (S)-CARV and (R)-CARV enantiomers were (118+/-44) micromol/L, (2 500+/-833) pmol/(min.mg protein) and (24+/-7) micromol/L, (953+/-399) pmol/(min.mg protein), respectively. These results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.

Metabolite identification by data‐dependent accurate mass spectrometric analysis at resolving power of 60 000 in external calibration mode using an LTQ/Orbitrap

Performance evaluation of accurate mass measurement by the LTQ/Orbitrap, at a resolving power of 60,000 and in external calibration mode, indicated that the Orbitrap is capable of providing high mass accuracy of <2 ppm for over 24 h post-calibration. This, together with limited trade-off between sensitivity and resolving power plus a wide dynamic range for mass accuracy, suggested that the LTQ/Orbitrap is an ideal analytical tool for structural elucidation of metabolites. The application of the LTQ/Orbitrap to identification of human liver microsomal metabolites of carvedilol was evaluated, using parent mass list triggered data-dependent multiple-stage accurate mass analysis, at a resolving power of 60,000 in external calibration mode. A metabolite identification workflow was developed to utilize chemical formulas from high-resolution accurate mass measurements to confirm structures of product ions of a drug proposed by Mass Frontier, illustrated by identification of structures used to establish lineage of product ions of carvedilol, which later served as a template for identification of its metabolites. A total of 58 in vitro metabolites of carvedilol were detected using 5-ppm mass tolerance filters for theoretical m/z of protonated molecules of predicted metabolites in addition to product ions and neutral mass losses diagnostic of carvedilol. The chemical formulas with unsaturation numbers calculated from the accurate m/z of precursor and product ions can be used to assign, with a high degree of confidence, the structures of metabolites and the sites of metabolism. The mass accuracies obtained for all full scan MS and MSn spectra were <2 ppm. The majority of the metabolites identified agreed with those previously reported except for those that have not been reported before. For example, several glutathione conjugates of carvedilol were reported for the first time, which may explain the reported hepatotoxicity during clinical trials and recent clinical use.

Carvedilol: a new candidate for reversal of MDR1/P-glycoprotein-mediated multidrug resistance.

In 1983, carvedilol [1-[carbazolyl-(4)-oxy]-3-[(2-methoxyphenoxyethyl)amino]-2-propanol] was designed and developed as a beta-adrenoceptor antagonist with vasodilating activity for efficacious and safe treatment of hypertension and coronary artery disease. Carvedilol belongs to the 'third generation' of beta-adrenoceptor antagonists and shows selectivity for the beta1- rather than beta2-adrenoceptor. Carvedilol is also an alpha1-blocking agents, with around 2- to 3-fold more selectivity for beta1- than alpha1-adrenoceptors. This degree of alpha1-blockade is responsible for the moderate vasodilator properties of carvedilol, being different from other beta-adrenoceptor antagonists. In addition, carvedilol is a potent antioxidant, with a 10-fold greater activity than vitamin E. Some carvedilol metabolites found in human plasma also exhibit antioxidative activity approximately 50- to 100-fold greater than carvedilol and other antioxidants. These unique properties of carvedilol, i.e. adrenergic (beta1, beta2 and alpha1) blockade and antioxidative activity, may be important in preventing progressive deterioration of left ventricular dysfunction and chronic heart failure. Recently, carvedilol has been demonstrated to reverse multidrug resistance (MDR) to anticancer drugs in tumor cells in vitro and its reversal effects were comparable with verapamil, which has been used in the first clinical trial for the reversal of MDR. This review introduces the reversal activity and usefulness against MDR, as well as an overview of the pharmacological and pharmacokinetic properties, of carvedilol.

Chenodeoxycholate induction of mitochondrial permeability transition pore is associated with increased membrane fluidity and cytochrome c release: protective role of carvedilol

Chenodeoxycholate (CDCA) is a primary bile acid mostly implicated in cholestatic liver injury. In this study, we have investigated the involvement of membrane fluidity and cytochrome c release in CDCA-induced mitochondrial permeability transition (MPT), and the preventive role of carvedilol. Treatment of calcium-loaded hepatic mitochondria with CDCA was found to cause osmotic swelling and release of cytochrome c, associated with an increase in membrane fluidity, in both protein and lipid regions. Carvedilol and cyclosporine A (CyA) reduced both cytochrome c release and alterations in membrane fluidity induced by CDCA. The hydroxylated metabolite of carvedilol, BM-910228, had no effect. Thus, modulation of membrane fluidity, plays an important role in MPT pore opening promoted by CDCA. As a result, we have delineated a pathway for the preventive role of carvedilol in mitochondrial dysfunction induced by CDCA.

Effects of Carvedilol on Isolated Heart Mitochondria: Evidence for a Protonophoretic Mechanism

Carvedilol ({1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-propanol-(2)}) is a novel compound used in clinical practice for the treatment of congestive heart failure, mild to moderate hypertension, and myocardial infarction. Carvedilol was also shown to protect cardiac mitochondria from oxidative stress events. Because mitochondria are the main suppliers of ATP for cardiac muscle work, a study of the effects of carvedilol in mitochondrial bioenergetics is necessary to fully understand the basis of its protective role in myocardial energetics. In this work we show that carvedilol acts as an uncoupler of oxidative phosphorylation, decreasing mitochondrial electric potential (ΔΨ) by a weak protonophoretic mechanism. Theoretical studies were carried out to determine the relevance of conformation and proton affinity of the protonable amino side-chain group in the proton-shuttling activity across the inner mitochondrial membrane. BM910228, a hydroxylated metabolite of carvedilol, was also studied for comparison with the parent compound. Implications for the protective role of carvedilol in heart mitochondrial bioenergetics are discussed.

Steady-state pharmacokinetics of carvedilol and its enantiomers in patients with congestive heart failure.

Carvedilol is a relatively new drug with beta- and alpha 1-receptor blocking activity and antioxidant effects recently approved for the treatment of congestive heart failure (CHF). An ascending, multiple-dose study was completed in 20 male patients with stable New York Heart Association (NYHA) Class III or IV CHF. The pharmacokinetics of carvedilol, S(-)-carvedilol, R(+)-carvedilol, and the active metabolites of carvedilol was assessed at steady state after twice-daily oral administration of carvedilol for 7 days at 6.25, 12.5, 25, and 50 mg doses. Carvedilol exhibited stereoselective pharmacokinetics in CHF patients with dose-proportional increases in steady-state plasma concentrations of carvedilol and its enantiomers. Mean AUC and Cmax values for carvedilol were up to twofold higher in patients with Class IV CHF as compared to those with Class III CHF. Steady-state plasma concentrations of the active metabolites also increased in a dose-proportional manner and were typically 10% or less of that observed for carvedilol. In general, carvedilol was adequately tolerated by adult male CHF patients at the dose levels (6.25-50 mg) evaluated in this study as adverse events were consistent with those frequently observed in patients with CHF.

SB 211475, a metabolite of carvedilol, reduces infarct size after myocardial ischemic and reperfusion injury in rabbits

The aim of this study was to investigate the effect of SB 211475, a metabolite of carvedilol with weak α 1-adrenoceptor antagonism and antioxidant effect, on myocardial reperfusion injury and infarct size in anesthetized rabbits. The rabbits were subjected to 60 min of regional myocardial ischemia and 180 min of reperfusion. SB 211475 was administered either as 0.3, 1.0 or 3.0 mg/kg and compared to vehicle and carvedilol (1 mg/kg) treated animals. The lowest dose of SB 211475 (0.3 mg/kg) did not reduce infarct size compared to vehicle, whereas SB 211475 1.0 or 3.0 mg/kg reduced infarct size significantly compared to vehicle (41.2±2.2% and 40.5±2.8% vs. 59.1±3.9%, p<0.05). Carvedilol reduced infarct size significantly more than SB 211475 1.0 and 3.0 mg/kg (28.8±3.9% vs. 41.2±2.2% and 40.5±2.7%, p<0.05). Carvedilol and SB 211475 1.0 and 3.0 mg/kg reduced myeloperoxidase activity to the same extent, indicative of reduced inflammation. Rate-pressure product did not differ between doses of SB 211475. In conclusion, SB 211475 in the two highest doses reduced infarct size by protecting from reperfusion injury, possibly by reduced neutrophil accumulation. The superior cardiac protective effect of Carvedilol over SB 211475 are most likely due to its adrenergic pharmacology including non-selective β- and α 1-adrenoceptor antagonism.

Effects of a hydroxylated metabolite of the beta-andrenoreceptor antagonist, carvedilol, on post-ischaemic splachnic tissue injury.

1 Reactive oxygen species have been demonstrated to play a critical role in post-ischaemic tissue injury. The present experiment was designed to evaluate the effects of SB 211475, a hydroxylated metabolite of the new beta-adrenoceptor antagonist, carvedilol, on rat splanchnic ischaemia (SI, 60 min) and reperfusion(R)-induced shock and tissue injury. 2 Administration of SB 211475 two min before R attenuated SI/R injury in a dose-dependent manner. At doses of 0.5 mg kg(-1) and 1.0 mg kg(-1), SB 211475 exerted significant anti-shock and endothelial protective effects, characterized by prolonged survival times, increased survival rates, attenuated increases in tissue myeloperoxidase activity and haematocrits, and preserved endothelium-dependent vasorelaxation. 3 Administration of 1 mg kg(-1) carvedilol attenuated shock-induced tissue injury and endothelial dysfunction. However, administration of 0.5 mg kg(-1) carvedilol had no protective effects on post-ischaemic tissue injury. 4 Previous studies have shown that SB 211475 has virtually no beta-blocking activity but possesses more potent antioxidant activity than carvedilol. In the present study, SB 211475 exerted more potent protective effects than the parent compound, suggesting that this metabolite of carvedilol is superior to carvedilol with regard to its protection against post-ischaemia tissue injury.

SB 211475, a metabolite of carvedilol, a novel antihypertensive agent, is a potent antioxidant

The antioxidant effects of SB 211475, a metabolite of carvedilol, a novel antihypertensive agent, were studied and compared with carvedilol and other antioxidants such as U78517F, U74500A and probucol. SB 211475 inhibited Fe 2+-vitamin C-initiated lipid peroxidation, assessed as thiobarbituric acid reactive substance, in brain homogenate with an IC 50 of 0.28 μM. Under the same conditions, the IC 50s of probucol, carvedilol, U74500A and U78517F were 50, 8.1, 0.71 and 0.16 μM, respectively. SB 211475 inhibited oxidation of human low density lipoprotein by mouse macrophages with an IC 50 of 0.043 μM. In the same model, the IC 50s of carvedilol, U78517F and probucol were 3.8, 0.15 and 0.80 μM, respectively. SB 211475 protected cultured bovine pulmonary artery endothelial cells against hydroxyl radical-initiated lipid peroxidation (IC 50= 0.15 μM) and cell damage (lactate dehydrogenase release, (IC 50 = 0.16 μM), and promoted cell survival with an EC 50 of 0.13 μM. SB 211475 also protected endothelial cells against xanthine/xanthine oxidase-initiated cytotoxicity and protected and rat cerebellar neurons from hydroxyl radical-mediated cell death (EC 50 = 0.19 μM). Moreover, SB 211475 inhibited superoxide (O 2 −) release from human neutrophils stimulated by phorbol myristate acetate. These observations indicate that SB 211475 is a potent antioxidant and may potentially contribute to the therapeutic effects of carvedilol in vivo.

A potent antioxidant, SB209995, inhibits oxygen-radical-mediated lipid peroxidation and cytotoxicity.

The antioxidant activity of SB209995, a metabolite of carvedilol in human, was studied and compared with its parent compound, carvedilol. SB209995 or carvedilol inhibited iron-catalyzed lipid peroxidation assessed as thiobarbituric acid-reactive substance generated in brain homogenate with IC50s of 0.30 and 8.1 mumol/l, respectively. The oxidation of low-density lipoprotein (LDL) by macrophages or that initiated by Cu2+ ions was inhibited by SB209995 with IC50s of 59 nmol/l and 1.7 mumol/l, respectively. Under the same conditions, the IC50s of carvedilol were 3.8 and 17.1 mumol/l, respectively. Furthermore, SB209995 protected cultured endothelial cells against hydroxyl radical (OH.) or superoxide (O2-)-mediated lipid peroxidation and cytotoxicity, assessed as lactate dehydrogenase release and cell death. The results indicate that SB209995 is a more potent antioxidant than carvedilol and may contribute to the therapeutic effects of carvedilol.

Synthesis of the enantiomers and three racemic metabolites of carvedilol labeled to high specific activity with tritium

AbstractCarvedilol (SK&amp;F 105517) possesses unique cardiovascular activity, and is under development for indications such as angina and hypertension. Tritium labeled enantiomers of Carvedilol and racemates of three metabolites were needed for pharmacologic and drug metabolic studies. These compounds were synthesized by catalytic tritium‐halogen exchange using tritium gas and 10% palladium‐on‐carbon catalyst. The precursors were polyhalogenated in the carbazole ring. Direct electrophilic bromination of the enantiomers of Carvedilol gave precursors that were converted to the corresponding tritiated final products by catalytic tritium halogen exchange. Bromination of 4‐(2,3‐epoxypropyloxy)‐9H‐carbazole gave an intermediate that was converted to the halogenated precursors of the racemic metabolites. Elaboration of this intermediate, 1,3,6‐tribromo‐4‐(2,3‐epoxypropyloxy)‐9H‐carbazole, to the desired metabolite precursors was achieved by nucleophilic epoxide opening with suitably functionalized N‐benzyl aryloxyethylamines. Catalytic tritium‐halogen exchange upon the brominated metabolite precursors was accompanied by cleavage of N‐ and Q‐benzyl protecting groups. Radiochemical purities of all tritiated final products were greater than 98% after preparative HPLC. Specific activities of the final products, determined by mass spectrometry, ranged from 35 to 76 Ci/mmol. Optical purity of the Carvedilol enantiomers, determined by chiral HPLC, was greater than 99%

Structural determination of glucuronide conjugates and a carbamoyl glucuronide conjugate of carvedilol: Use of acetylation reactions as an aid to determine positions of glucuronidation

Carvedilol is metabolized via both oxidation and conjugation pathways in dog and rat to more than 12 different products. Several glucuronide conjugates of the parent drug were identified. The drug contains an aliphatic hydroxyl, an aliphatic amine and a carbazole amine, all of which are potential sites for conjugation with glucuronic acid. In order to determine the positions of glucuronidation, a strategy involving acetylation of the metabolites was devised. The metabolites were acetylated using acetic anhydride in either pyridine or aqueous solution, and the products were analyzed by fast atom bombardment mass spectrometry. Carvedilol was acetylated at both the hydroxyl and the aliphatic amine in pyridine and at only the aliphatic amine in aqueous solution. The carbazole nitrogen was unreactive under both conditions. Based on the acetylation patterns observed for the metabolites in pyridine or aqueous solution, the positions of conjugation were determined. Each of the five glucuronide metabolites of carvedilol formed in dog and rat was analyzed and the structures included two diastereomeric carbamoyl glucuronide metabolites formed from addition of CO2 and glcuronic acid to carvedilol, a carbazole-N-linked glucuronide, and two diastereomeric O-linked glucuronides. This approach should be generally applicable in many cases to determine the structures of glucuronide conjugates for compounds which contain more than one potential glucuronidation site.