Metabolic Activation of Anthanthrene: Significance of Stable Radicals Derived from its Key Metabolite 3-Hydroxyanthanthrene

Anthanthrene (dibenzo[def,mno]chrysene), a hexacyclic aromatic hydrocarbon lacking the structural element of a bay-region is found to be metabolized to 3-hydroxyanthanthrene (3-HA) and anthanthrene 3,6-quinone by rat liver microsomes. Two further metabolites of anthanthrene are are also derived as metabolites of 3-HA. 3-HA forms stable radicals in the presence of horseradish peroxidase and hydrogen peroxide as revealed by electron spin resonance spectroscopy. These radical species which are also formed spontaneously from 3-HA could be responsible for the genotoxic properties of anthanthrene.     

The Metabolic Fate of ortho-Quinones Derived from Catecholamine Metabolites

ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase or by transition metal ions. Neuromelanin, a dark pigment present in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid (DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible spectrophotometry. HPLC analysis after reduction with NaBH₄ or ascorbic acid enabled measurement of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric examination showed that the ortho-quinones initially formed underwent extensive degradation at pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from DOPE-quinone was DOPEG which was produced through the addition of a water molecule to the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc) and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min. This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are converted to quinone methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The unstable quinone methides afford stable alcoholic or carbonyl products.     

Reactivity of quinones for the dehydrogenation of poly(1,3‐cyclohexadiene)

The reactivity of three types of quinones (3,4,5,6‐tetrachloro‐1,2‐(o‐)‐benzoquinone (TOQ), 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ), and 2,3,5,6,‐tetrachloro‐1,4‐(p‐)‐benzoquinone (TCQ)) was examined for the dehydrogenation of poly(1,3‐cycohexadiene) (PCHD) composed of 1,2‐cyclohexadiene and 1,4‐cyclohexadiene units. The order of reactivity was TOQ > DDQ > TCQ. The steric hindrance of C?O groups in the quinones appeared to be an important factor in determining their reactivity. DDQ was found to be an appropriate dehydrogenation reagent for PCHD. The reactivity of TCQ was considerably low for PCHD containing 1,2‐cyclohexadiene units. The reactivity of TOQ was too high and side reactions occurred, causing the formation of structural defects. Copyright © 2007 Society of Chemical Industry     

New method for controlled synthesis of polylactide block copolymers: organoborane/p-quinone system and reversible-deactivation radical polymerization

We developed a new approach to obtain polylactide hybrid block copolymers with vinyl monomers (styrene, methyl methacrylate, methyl acrylate) through the realization of a reaction sequence using triethylborane and various p-quinones. The method offered includes two stages. In the first stage, a chain-transfer agent was obtained by borylation of the terminal hydroxyl groups of polylactide. The second stage was vinyl monomer radical polymerization in the presence of p-quinone accompanied by S_H2-substitution at the boron atom.1,4-Naphthoquinone, 2,3-dimethyl-1,4-benzoquinone, duroquinone and 2,5-di-tert-butyl-1,4-benzoquinone were used as synthetic polymer chain growth mediators. It is shown that 1,4-naphthoquinone and 2,3-dimethyl-1,4-benzoquinone, similar in their characteristics, are effective agents providing the realization of reversible-deactivation radical polymerization. Realization of reversible-deactivation radical polymerization was proved with the analysis of the kinetics of block copolymerization, molecular weight characteristics and compositional homogeneity of block copolymers as well as its further capability to elongate the polymer chain. Synthesized block copolymers have a high thermal stability compared to the initial borylated polylactide. © 2021 Society of Industrial Chemistry.     

Photoactive mussels inspired polymer coatings: Preparation and antibacterial activity

In this article we report on the facile preparation of photoactive antibacterial mussel inspired polymer coatings deposited on a stainless steel (SS) substrate from water based precursors. The coating is prepared by the sequential deposition of aqueous based solutions of an anchoring layer based on bio-inspired glue, a cationic polymer bearing pendent catechols, a nanogel decorated by ortho-quinones and a photosensitizer of the aminoacridine type. This latter is grafted to the coating by reaction of its amino group with the o-quinone of the deposited nanogel. The deposition of all layers is followed in-line by Quartz crystal microbalance coupled with dissipation (QCM-D) and AFM shows that the thin polymer film repeated the roughness of the SS substrate. The prepared coatings show good mechanical properties applying nanoindentation techniques. The established antibacterial activity of the prepared photoactive polymer coatings on SS against Gram-negative E. coli strain demonstrate their potential as a power tool for medical applications.     

Reduced glutathione effects on α-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent lipid peroxidation

Factors involved in reduced glutathione (GSH) and vitamin E-mediated inhibition of NADPH-dependent rat liver microsomal lipid peroxidation were examined. Lipid peroxidation was monitored over a time-course of 180 min by thiobarbituric acid reactive product formation. The addition of 5 mM GSH to the reaction system containing microsomes from rats fed a diet supplemented with 150 IU/kg of α-tocopherol acetate for eight weeks produced a lag in peroxidation of >30 min. This effect was not observed for microsomes prepared from rats fed a diet deficient in vitamin E. Indeed, a prooxidant effect of 5 mM GSH was observed in assays containing microsomes from rats fed a diet deficient in vitamin E. The inhibition by GSH of lipid peroxidation in microsomes prepared from livers of vitamin E supplemented rats was not restricted by its availability, for it was found that approximately 92% of the GSH remained in the reduced form after 60 min. Additional experiments revealed that the α-tocopherol content of peroxidizing microsomes decreased rapidly in the absence of GSH. The addition of 5 mM GSH to the assay system markedly depressed the loss of microsomal α-tocopherol. The results ofin vivo labeling of liver microsomes with [¹⁴C] α-tocopherol demonstrated that i) GSH addition to thein vitro peroxidizing medium reduced the disappearance of α-tocopherol, and ii) a compound that interfered with the determination of α-tocopherol was separated by HPLC and was not an oxidation product of α-tocopherol. A portion of the microsomal¹⁴C-labeled α-tocopherol was converted to an unidentified product with HPLC retention characteristics that was similar, but not identical, to α-tocopherol quinone.     

Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione

Iron-ascorbate stimulated lipid peroxidation in rat liver microsomes can be inhibited by glutathione (GSH). The role of protein thiols and vitamin E in this process was studied in liver microsomes isolated from rats fed diets either sufficient or deficient in vitamin E and incubated at 37°C unde 100% O₂. Lipid peroxidation was induced by adding 400 μM adenosine 5′-triphosphate, 2.5 to 20 μM FeCl₃, and 450 μM ascorbic acid. One mL of the incubation mixture was removed at defined intervals for the measurement of thiobarbituric acid reactive substances (TBARS), protein thiols and vitamin E. In vitamin E sufficient microsomes, the addition of GSH enhanced the lag time prior to the onset of maximal TBARS accumulation and inhibited the loss of vitamin E. Treatment of these microsomes with the protein thiol oxidant diamide resulted in a 56% loss of protein thiols, but did not significantly change vitamin E levels. However, diamide treatment abolished the GSH-mediated protection against TBARS formation and loss of vitamin E during ascorbate-induced peroxidation. Liver microsomes isolated from rats fed a vitamin E deficient diet contained 40-fold less vitamin E and generated levels of TBARS similar to vitamin E sufficient microsomes at a 4-fold lower concentration of iron. GSH did not affect the lag time prior to the onset of maximal TBARS formation in vitamin E deficient microsomes although total TBARS accumulation was inhibited. Similar to what was previously found in vitamin E sufficient microsomes [Palamanda and Kehrer, (1992)Arch. Biochem. Biophys. 293, 103–109], GSH prevented the loss of protein thiols in vitamin E deficient microsomes. However, GSH did not protect efficiently against the loss of residual vitamin E in deficient microsomes. These data provide support for the concept that GSH protects against microsomal lipid peroxidation by maintaining protein thiols, and consequently vitamin E, in the reduced state. The lack of protection in vitamin E deficient microsomes may be related to the inability of such low levels of vitamin E to inhibit peroxidation.     

Oxokohlenstoffe und verwandte Verbindungen. 27. Synthese von Dihydrocyclobuta[a]naphthalen-1,2-dionen und Cyclobuta[a]naphthalen-1,2-dionen durch Anellierung von Alkoxy-(1-alkenyl)benzolen mit 3-Chlor-3-cyclobuten-1,2-dion. Anwendungsbereich und Grenzen

Oxocarbons and Related Compounds. 27. Synthesis of Dihydrocyclobuta[a]naphthalene-1,2-diones and Cyclobuta[a]naphthalene-1,2-diones via Annulation of Alkoxy-(1-alkenyl)benzenes with 3-Chloro-3-cyclobutene-1,2-dione. Scope and Limitations The reaction of alkoxy-(1-alkenyl)benzenes with semisquaric chloride ( 3 ) has been investigated systematically. 1,2-Dialkoxy- and 1-alkoxy'-2-alkoxy″-4-(1-alkenyl)benzenes ( 6a–j ) and ( 11a–i ) react with 3 to give the 3,4-dihydrocyclobuta[a]naphthalene-1,2-diones ( 8a–j ) and ( 12a–i ). Treatment of the dihydrocyclobuta[a]naphthalene-1,2-diones with 1.2 equiv. bromine effects dehydrogenation and affords cyclobuta[a]naphthalene-1,2-diones ( 9a–e ) and ( 13b–f ). Any efforts to extend this annulation reaction to dimethoxy-(1-alkenyl)benzenes with the methoxy groups in other than the 1,2-positions, e. g. 14a, b, 16a, b have been unsuccessful. The reaction of 1,2,3-trimethoxy-4-(1-propenyl) [and 4-(1-butenyl)]-benzenes ( 18a ) and ( 18b ) with semisquaric chloride ( 3 ) leads to the elimination of HCl and CH₃OH and gives 5,6-dimethoxy-3-methyl [and 3-ethyl]-cyclobuta[a]naphthalene-1,2-diones ( 20a ) and ( 20b ). The reaction pathway of this novel annulation reaction is discussed.     

Atmospheric Distribution of Gas- and Particle-Phase Quinones in Southern California

Quinones are reactive organic compounds known to initiate reactions associated with a host of toxicological events. Their presence in different atmospheres has been demonstrated although their sources remain uncertain. As a result of their reactivity and instability during chemical analysis, only a limited number of studies have reported on atmospheric concentrations of quinones in ambient air. Furthermore, besides the limited information on quinones associated with particulate matter, no previous studies have quantified vapor-phase quinones. We report vapor- and particle-phase concentrations of 1,2- and 1,4-naphthoquinones (1,2-NQ, 1,4-NQ), 9,10-phenanthraquinone (9,10-PQ), and 9,10-anthraquinone (9,10-AQ), measured over a 5-year period in Southern California. The results showed that vapor-phase concentrations of the target quinones were in general higher than those in the particle-phase. Vapor-phase concentrations ranged from 80 pg/m ³ for the AQ to 1747 pg/m ³ for the 1,4-NQ, and the particle-phase concentrations between 13 pg/m ³ for the 1,2-NQ and 250 pg/m ³ for 9,10-AQ. The target quinones were found to be distributed between vapor- and particle-phase, with the exception of 9,10-PQ found only in the particle-phase. The differences observed in the concentrations among sites and seasons suggest different source contributions; source sites were dominated by primary sources, while downwind locations showed a high contribution from photochemical activity.     

Benzene‐di‐N‐substituted carbamates as conformationally constrained analogs of Pseudomonas lipase substrates

Benzene‐1,2‐, ‐1,3‐, and ‐1,4‐di‐N‐substituted carbamates ( 1 – 15 ) are synthesized as the constrained analogs of gauche, eclipsed, and anti conformations of diesters of ethylene glycol, respectively. Carbamates 1 – 15 are characterized as the pseudo‐substrate inhibitors of Pseudomonas species lipase. Long‐chain carbamates are more potent inhibitors than short‐chain ones. Different geometries of benzene‐di‐substituted carbamates, such as benzene‐1,2‐di‐N‐octylcarbamate ( 3 ) (ortho compound), benzene‐1,3‐di‐N‐octylcarbamate ( 8 ) (meta compound), and benzene‐1,4‐di‐N‐octylcarbamate ( 13 ) (para compound), show similar inhibitory potencies for the enzyme. In other words, kinetic data suggest that the enzyme does not discriminate ortho, meta, and para geometries of these constrained analogs.     

Activity of phospholipid-synthesizing enzymes in rat liver plasma membranes and the source of biliary lecithin

The potential for the synthesis of phosphatidylcholine by the bile canalicular membrane of the liver cell was assessed by measuring the activity of a number of phospholipid synthesizing enzymes in isolated bile canalicular membrane fractions from rat liver. The activity of these various enzymes was compared to that present in noncanalicular liver cell plasma membranes and in microsomes. The CDP-choline: 1,2-diacyl-sn-glycerol-cholinephosphotransferase was virtually absent from the bile canalicular membranes but the specific activities of S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase and acyl-CoA:1-acyl-sn-glycero-3-phosphoryl-choline acyltransferase were 11–15% of those found in the microsomes. The bile canalicular membranes also contained detectable acyl-CoA:sn-glycero-3-phosphate acyltransferase activity and the ability to potentiate the Ca⁺⁺-stimulated exchange of bases between different phospholipids. These findings indicate that the bile canalicular membranes have a very limited capacity for the formation of phosphatidylcholine under the assay conditions employed. A preliminary report of this paper was given at the AOCS Spring Meeting, Dallas, April 1975.     

Oxidation of Hydrogen Sulfide by Quinones: How Polyphenols Initiate Their Cytoprotective Effects

We have shown that autoxidized polyphenolic nutraceuticals oxidize H₂S to polysulfides and thiosulfate and this may convey their cytoprotective effects. Polyphenol reactivity is largely attributed to the B ring, which is usually a form of hydroxyquinone (HQ). Here, we examine the effects of HQs on sulfur metabolism using H₂S- and polysulfide-specific fluorophores (AzMC and SSP4, respectively) and thiosulfate sensitive silver nanoparticles (AgNP). In buffer, 1,4-dihydroxybenzene (1,4-DB), 1,4-benzoquinone (1,4-BQ), pyrogallol (PG) and gallic acid (GA) oxidized H₂S to polysulfides and thiosulfate, whereas 1,2-DB, 1,3-DB, 1,2-dihydroxy,3,4-benzoquinone and shikimic acid did not. In addition, 1,4-DB, 1,4-BQ, PG and GA also increased polysulfide production in HEK293 cells. In buffer, H₂S oxidation by 1,4-DB was oxygen-dependent, partially inhibited by tempol and trolox, and absorbance spectra were consistent with redox cycling between HQ autoxidation and H₂S-mediated reduction. Neither 1,2-DB, 1,3-DB, 1,4-DB nor 1,4-BQ reduced polysulfides to H₂S in either 21% or 0% oxygen. Epinephrine and norepinephrine also oxidized H₂S to polysulfides and thiosulfate; dopamine and tyrosine were ineffective. Polyphenones were also examined, but only 2,5-dihydroxy- and 2,3,4-trihydroxybenzophenones oxidized H₂S. These results show that H₂S is readily oxidized by specific hydroxyquinones and quinones, most likely through the formation of a semiquinone radical intermediate derived from either reaction of oxygen with the reduced quinones, or from direct reaction between H₂S and quinones. We propose that polysulfide production by these reactions contributes to the health-promoting benefits of polyphenolic nutraceuticals.     

The Aldo-Keto Reductases and Polycyclic Aromatic Hydrocarbon Activation

The aldo-keto reductases (AKRs), are monomeric 37 kDa oxidoreductases that catalyze the NADP + -dependent oxidation of PAH trans -dihydrodiol proximate carcinogens to their reactive and redox-active o -quinones. Five human isoforms have been cloned and expressed as purified recombinant enzymes (AKR1C1-AKR1C4 and AKR1A1). Of these the general metabolic enzyme aldehyde reductase (AKR1A1) displays the highest utilization ratio for the oxidation of ( m ) R,R -benzo[ a ]pyrene-7,8-diol (BP-7,8-diol) to benzo[ a ]pyrene-7,8-dione (BP-7,8-dione). AKR1A1 is coexpressed with CYP1A1 and epoxide hydrolase suggesting that it is exposed to its trans -dihydrodiol substrates in situ. PAH o -quinones produced by AKRs are highly electrophilic yielding bimolecular rate constants for the addition of GSH of at least 1.3 2 10 3 M m 1 min m 1 . By reacting with DNA, PAH o -quinones form both stable and depurinating adducts in vitro. By entering into futile redox-cycles the PAH o -quinones can amplify the production of reactive oxygen species and increase 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dGuo) formation. The spectrum of DNA-adducts and generation of prooxidants (reactive oxygen) may explain how PAHs can act as complete carcinogens.     

A convenient and efficient method for the synthesis of benzo- and naphthothiazolediones

A series of benzo- and naphthothiazolediones have been synthesized by the reactions of N-substituted thioureas with π-deficient quinones. The products were obtained by the reaction of N-aroylphenylthioureas with 2,3,5,6-tetracholoro-1,4-benzoquinone, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and 2,3-dichloro-1,4-naphthoquinone.     

CDPcholine:1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100,n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.     

A Novel and Convenient Process for the Selective Oxidation of Naphthalenes with Hydrogen Peroxide

A practical ruthenium phase‐transfer catalyst (Ru‐PTC) system for the oxidation of naphthalene derivatives has been developed. Substituted 1,4‐quinones are obtained in good selectivity and yield in water without the addition of any organic solvent and acid. By applying the optimized conditions the feed additive menadione (vitamin K₃) is obtained from 2‐methylnaphthalene with 64 % yield and 73 % selectivity.     

Metabolism Of Benzo[a]Pyrene in Fish Hepatocytes Cultured on Microplates

The metabolism of benzo[a]pyrene (BP) by trout and catfish hepatocytes seeded on 96-well microplates was examined. The 9,10-; 4,5-; and 7,8-dihydrodiols, 1,6-; 3,6- quinones and 3-OH BP were found to be the major metabolites in both the trout and catfish hepatocytes exposed to BP for 4 h. The results were compared with the corresponding long term culture of hepatocytes on porous membranes and in microsomes isolated from the whole liver. It was found that cells cultured on microplates retained their BP metabolizing activities for at least 6 d after seeding. This method has the major advantage that only a small number of cells are required and in parallel, it is possible to perform cytotoxicity and cytogenetic microassays on cell populations characterized for their metabolic capacity.     

Inhibition of diacylglycerol: CDPcholine cholinephosphotransferase activity by dimethylaminoethylp-clorophenoxyacetate

Cholinephosphotransferase [EC 2.7.8.2] activity of rat liver microsomes, with 1,2-di-0-[³H]acyl glycerol or 1-0-hexadecanoyl [U-¹⁴C]ethanediol as substrate, was inhibited byN,N-dimethylaminoethylp-chlorophenoxyacetate (centrophenoxine). Inhibition progressed in a linear fashion with increasing drug levels and was complete at 30 mM concentration. It appears that the microsomal enzyme was largely affected by the drug itself because the hydrolysis products of centrophenoxine, viz.,N,N-dimethylaminoethanol andp-chlorophenoxyacetic acid, were less inhibitory.     

The effect of glutathione on the vitamin E requirement for inhibition of liver microsomal lipid peroxidation

Vitamin E dependent inhibition of rat liver microsomal lipid peroxidation in an NADPH and ADP-Fe⁺³ containing system occurred at lower vitamin E concentrations in the presence of glutathione (GSH). Using microsomes from rats fed a vitamin E deficient diet, vitamin E was shown to be rquired for inhibition. Inhibition also required the presence of a storage labile microsomal component, since no inhibition was observed when using microsomes that had been stored for one moth. This observation provides evidence that direct reduction of reversibly oxidized vitamin E by GSH does not appear to contribute significantly to inhibition of peroxidation. During GSH and vitamin E dependent inhibition of lipid peroxidation, vitamin E (reduced form) concentrations remained constant, indicating that GSH maintained vitamin E concentrations. Without GSH, vitamin E concentrations dropped rapidly. By adding vitamin E to microsomes, it was found that inhibition of lipid peroxidation in the presence of GSH occurred at about five-fold less vitamin E than in the absence of GSH. Inhibition at these lower levels of vitamin E was 85–90% complete. Results indicate that GSH can be used to maintain vitamin E (reduced form) concentrations, thereby lowering the concentration of vitamin E necessary to inhibit microsomal lipid peroxidation.     

N‐(Anilinoethyl)amides: Design and Synthesis of Metabolically Stable,Selective Melatonin Receptor Ligands

The class of N‐(anilinoethyl)amides includes melatonin receptor ligands with varied subtype selectivity and intrinsic activity. One of these ligands, the MT₂‐selective partial agonist UCM765 (N‐{2‐[(3‐methoxyphenyl)phenylamino]ethyl}acetamide), had evidenced hypnotic effects in rodents at doses ≥40 mg kg^?1 (s.c.), in spite of its sub‐nanomolar affinity for human melatonin receptors. Supposing that its low in vivo potency could be due, at least in part, to metabolic liability in rat liver, UCM765 was incubated with rat liver S9 fraction and rat, mouse, or human microsomes, and the major metabolites were identified by LC–MS, synthesized, and in vitro tested for their affinity toward MT₁ and MT₂ receptors. The obtained information was exploited to design novel analogues of UCM765 that are more resistant to in vitro oxidative degradation, while maintaining a similar binding profile. The analogue UCM924 (N‐{2‐[(3‐bromophenyl)‐(4‐fluorophenyl)amino]ethyl}acetamide) displayed a binding profile similar to that of UCM765 on cloned human receptors (MT₂‐selective partial agonist) and a significantly longer half‐life in the presence of rat liver S9 fraction.