Patai S., Rappoport Z. 1997 The chemistry of functional groups. The chemistry of double-bonded functional groups / 1625 166

Apart from mammalian tissue preparations, microbial systems also appear to be active in the metabolic conversion of arylalkylamines to oximes. Thus, incubation broths of the fungus Cunninghamella bainieri supplemented with amphetamine have been detected to contain phenylacetone oxime (28) as one of the major metabolites36.

B. Formation of Nitrones

Using male hamster hepatic microsomes, Gorrod and Ulgen were able to demonstrate the formation of diarylnitrones (31) from certain diarylimines3. Metabolic studies on the cyclic imine bromazepam have shown that the corresponding nitrone (32) is excreted in trace amounts by dogs, but not by mice, rats or man4. Similarly, the cyclic imino nitrogen in methaqualone affords a nitrone (33), which has been found to be the second most abundant urinary metabolite in healthy addults43,89.

N

2- MeC6 H4 Me

(33)

Moreover, biotransformation of secondary arylalkylamines also affords nitrones. Thus, N-oxygenation of a series of 4-substituted N-benzylanilines in liver microsomal preparations from various animal species has been detected to be a minor pathway of metabolism, usually generating ˛,N-diphenylnitrones (34) in a species-dependent manner26. Nitrone formation was most abundant in liver, kidney and lung53. There was a clear sex difference

in nitrone production using rat tissue, while no difference was evident when using mouse microsomes53. Several N-substituted phenylethylamines, such as N-methyl-, N-ethyl-, N- propyland N-benzylamphetamine as well as benzphetamine have been recognized to give nitrones of the general structure 35 as urinary or in vitro metabolites after incubation with rat, guinea-pig and rabbit liver tissue or fungal cell systems6,24,25,36,39,54. N-(1-Phenyl) cyclobutylphenyl nitrone (36), existing in the trans configuration about the imine bond, has been isolated from reaction mixtures containing rat liver microsomal fraction fortified with N-(1-phenylcyclobutyl)benzylamine55.

Heterocyclic secondary amines, such as phenmetrazine or ( )-anabasine, a minor tobacco alkaloid, undergo metabolic attack at the amino groups to finally yield nitrones 37 and 38, respectively, when incubated with tissue preparations from various mammals90,91.

Incorporation of N-methylbenzamidine in hepatic microsomes from rabbits affords the tautomeric ˛-aminonitrone 39, a completely new type of metabolite50.

C. Formation of N-Oxides

N-Oxygenation of heteroaromatic amines to yield N-oxides is a well-established metabolic route. The pyridyl nitrogen is a likely target for electrophilic enzymatic oxidation because of its relatively high electronegativity and its unshared pair of electrons. Indeed, urinary excretion of pyridine N-oxide (40) has been observed after intraperitoneal administration of pyridine to mice, hamsters, rats, guinea-pigs, rabbits, cats and man7,18,57;

N

O

(40)

the N-oxide accounted for up to 40% of the administered dose in some species18. In studies on the in vitro N-oxidative transformation of pyridine, 40 was isolated from hepatocytes and subcellular fractions from the livers and lungs of various mammals incubated with

the parent amine28,29,41,56,58 60,92,93.

Similarly, a series of N-oxides derived from simple 3-substituted pyridines as model compounds, bearing substituents such as Me, Et, Cl, Br, F or CN, were identified as urinary18 or in vitro28,41,92 metabolites. 3-Acetylpyridine has been reported to afford 1-(3-pyridyl N-oxide)ethanol as a principal metabolic product in the rat94, while 3- benzoylpyridine undergoes hepatic conversion to 3-hydroxybenzoyl pyridine N-oxide95; there are strong differences in the urinary metabolic profile between rat and dog. The urinary excretion of nicotinamide N-oxide in mice and rats has been confirmed after the animals were given nicotinamide96,97, and the presence of this pyridyl N-oxide has been recognized in mouse liver, kidney, muscle, intestine, lung and heart tissue upon the administration of radioactive nicotinamide98. Similarly, nicotinamide and isonicotinamide N-oxides were detected in liver microsomes from rats and rabbits incubated in the presence of the heteroaromatic parent amines28,56. N,N-Diethylnicotinamide (nikethamide), a strong central nervous stimulant, has been shown to be oxygenated at the pyridyl nitrogen to yield the corresponding N-oxide in hepatic and pulmonary microsomes from various mammalian species28. This oxy product has a much lower toxicity than the parent drug. The pyridyl moiety of cotinine is prone to N-oxygenation both in vivo99 and in vitro28. In the presence of mouse or rat hepatic microsomal enzymes, 2-methyl- 1,2-bis (3-pyridyl)propane-1-one (metyrapone), a drug used as a diagnostic tool for the determination of residual pituitary function, is converted to a mixture of two isomeric metyrapone mono-N-oxides31,42,61. The urinary metabolic profile of the heterocycle has been studied following intraperitoneal administration, and marked species and sex differences in the excretion of the two metyrapone mono-N-oxides have been found100. However, quantitative analysis has revealed that two metyrapol mono-N-oxides, formed after keto reduction, are the major metabolic products, together accounting for about 75% of the administered dose101. Rats and humans treated with radioactive 1-methyl-3-(3- pyridyl)-5-(2-hydroxymethylphenyl)-1H-1,2,4-triazole, a hypnotic, were found to excrete a high percentage of total radioactivity in the form of the pyridyl N-oxide32. Similarly, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen, undergoes oxygenation at the pyridyl nitrogen in human kidney cells62. The NNK N-oxide exhibits significantly less tumorigenic activity as compared with the parent amine, and is, therefore, regarded as a detoxification product102.

Examples of the N-oxygenation of 4-substituted pyridines are given by 2-phenyl-1,3- di(4-pyridyl)-2-propanol and 4,40 -bipyridyl, two potent metabolic inhibitors. The former prochiral compound afforded a levorotatory chiral pyridyl N-oxide excreted in the urine upon the administration to rats, dogs and humans, which was also detected after in vitro incubation of the parent amine in subcellular fractions from rat liver30. Using the TiCl3 reduction technique, N-oxide production has been reported to constitute the major pathway in the metabolic transformation of 4,40 -bipyridyl15. The xanthine oxidase inhibitor 3,5- di(4-pyridyl)-1,2,4-triazole undergoes oxygenation at one pyridyl nitrogen, the resulting N-oxide being excreted in the bile103.

Small amounts of the benzopyridines quinoline and isoquinoline are excreted as their N-oxides (41) by guinea-pigs and are also formed by guinea-pig and rabbit hepatic microsomes15,28. Moreover, the antimalarial agent chloroquine affords an N-oxide as a metabolite of the quinoline ring system104.

The two nitrogens in the pyrimidine structure are also likely candidates for enzymatic oxygenation. Indeed, the antibacterial agent 2,4-diamino-5-(3,4,5-trimethoxybenzyl)

pyrimidine (trimethoprim) affords small amounts of its 1-N-oxide (42) and 3-N-oxide as urinary metabolites in rats, dogs and humans105. A species variation in the relative proportions of the isomeric N-oxides of trimethoprim formed has been reported: whereas man excretes about equal amounts of each isomer, rats and dogs excrete predominantly the 1-N-oxide and 3-N-oxide, respectively105. N-oxygenation in vitro of trimethoprim has also been documented using washed hepatic microsomal preparations from rats, hamsters, mice and guinea-pigs16. The antimalarial agent pyrimethamine, another 5-substituted 2,4-diaminopyrimidine, has been shown to be excreted in rat urine mainly as the 3-N- oxide106. A series of 6-substituted analogues has been demonstrated to yield 3-N-oxides in liver microsomal fractions from various animal species without evidence of 1-N-oxide formation8.

In experiments with various 9-substituted adenines, the 1-N-oxide (43) derived from 9-benzyladenine has been found to be the major metabolite in dog liver microsomes, while 9-benzhydryladenine underwent N-oxygenation at the 1-position to considerably lower extent65. Studies with the two purines conducted with hepatic preparations from other species revealed increasing rates of 1-N-oxide formation in the order guinea-pig < rat < rabbit < mouse < hamster64,65. Similarly, the anticoccidial agent aprinocid, a halogenated congener of 9-benzyladenine, is converted to an active 1-N-oxide by liver microsomes from the chicken and dog107.

Electrophilic attack at one nitrogen in pyridazine and the herbicide 3-(20 - methylphenoxy)pyridazine (credazine) has been recognized to produce pyridazine N-oxide (44) and the analogous credazine N-oxide when the aromatic diazines were incubated with liver microsomes from various sources9,108. Marked species differences in the ability to convert pyridazine to its mono-N-oxide have been observed9. The same holds true for the microsomal formation of pyrazine N-oxide (45) from pyrazine9. The only reported N- oxygenation of a triazine ring appears to be for the vasodilatator N, N-diallylmelamine109, which has been demonstrated to undergo a unique bioactivation in rats and dogs to the highly hypotensive nuclear 5-N-oxide (46).

V. ENZYMOLOGY OF THE FORMATION OF N-OXYGENATED

C=N FUNCTIONALITIES

In experiments with subcellular tissue fractions, microsomal preparations have been detected to be abundant in N-oxygenating activity giving rise to the formation of oximes1,5,29,51, nitrones3,6,26,53 and heteroaromatic N-oxides15,16,28,65; these reactions invariably required the presence of oxygen and NADPH as the electron donor. Using 18O, Parli and coworkers were able to demonstrate that the oxygen atom inserted into 2,4,6-trimethylacetophenone imine to yield the corresponding oxime derived from molecular oxygen and not from water1. Similarly, complete incorporation of 18O into acetophenone oximes generated from amphetamines in microsomal incubates was observed by Beckett and Belanger´110. These findings permitted the conclusion that N- oxygenation of CDN functionalities bears characteristics typical of a monooxygenation reaction111.

Basically, two major microsomal systems can be envisaged to mediate oxygenation of nitrogen functionalities in organic molecules. One of them, the cytochrome P-450- dependent monooxygenase (P-450; EC 1.14.14.1), has been classified into 22 mammalian subfamilies based on deduced amino-acid sequence identities, each representing a cluster of tightly linked genes112. While NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and phospholipid have long been recognized to be essential in electron transfer to the hemoprotein113, the role of microsomal cytochrome b5 as a redox component is more complex114. The catalytic function of the diverse P-450 isozymes is the two-electron reduction of molecular oxygen to form water and a reactive oxygen species, which serves for insertion into substrate115. The multisubstrate flavin-containing monooxygenase (FMO; EC 1.14.13.8), comprising a single gene family composed of five genes116, provides another significant route for the NADPHand oxygen-dependent attack at nucleophilic centers in nitrogenous structures117. However, both types of monooxygenases operate at distinct catalytic mechanisms: while nitrogen oxygenation mediated by P-450 proceeds via the initial formation of a radical species118, the flavin 4a-hydroperoxide intermediate of the FMO waits in a ready position to oxygenate vulnerable nitrogens in a concerted ionic reaction119.

The relative contribution, in intact microsomal preparations, of the two monooxygenases to the formation of N-oxygenated CDN functionalities has been frequently assessed by measurements in the presence and absence of selective enzyme inhibitors or positive effectors. These observations were supplemented by studies using highly purified native or recombinant proteins in reconstituted systems.

A. Enzymology of Oxime Formation

Oxime formation can occur by various mechanisms. One possibility is the direct oxygenation of imino groups. The stoichiometry of this process is given in equation 4, where R2 D H (or alkyl) to yield either aldoximes or ketoximes.

(4)

Closer inspection of oxime formation from the stable 2,4,6-trimethylacetophenone imine revealed the N-oxygenating activity to be sensitive to the presence of prototypic inhibitors of the P-450 system, such as carbon monoxide, SKF 525A and DPEA1.

to mediate benzamidoxime formation to an appreciable exent2,49; neither superoxide nor H2O2 are directly involved in N-oxygenation of the amidine nitrogen49. Moreover, N-oxidative transformation of benzamidine is blocked by antibody against NADPHcytochrome P-450 reductase86. Studies with a series of para-substituted benzamidines have disclosed a correlation between maximum rates of N-oxygenation and the Hammettp constants47,86. Generally, the presence of electron-donating substituents on the aromatic ring increased reaction rates, whereas electron-withdrawing substituents decreased them. From the slope of the line depicted in Figure 1, a reaction constant of D 0.88 could be taken, the negative sign of the value lending support to the notion of a radical mechanism operative in P-450-catalyzed N-oxygenation47,86, as outlined in Scheme 1. Here, the putative oxene species takes up an electron to produce a cation radical, which is stabilized by proton abstraction and exists in a mesomeric state. The latter species undergoes hydroxylation to yield hydroxyamidine, which tautomerizes to furnish the amidoxime. Unequivocal proof of the participation of P-450 in benzamidoxime formation has been provided by experiments with reconstituted systems consisting of hemoprotein and NADPH-cytochrome P-450 reductase embedded in a phospholipid matrix. Using this technique, the constitutive rabbit liver P-450 2C3 and its variants 6ˇH and 6ˇL were shown to N-oxygenate benzamidine48 with a turnover number of 0.63 min 1. This isozyme also accounts for the metabolic conversion of N-methylbenzamidine50, pentamidine51, debrisoquine52 and some aminoguanidines14 to the N-oxy products 24b, 25, 26 and 27, respectively.

SCHEME 1

It has to be pointed out that imines do not only serve as substrates for the N- oxygenase(s) when added exogenously to assay mixtures, but are also acted upon by these enzymes when metabolically formed from primary amines. Thus, it is interesting to note that benzylmethyl ketimine has been proposed as an intermediate in microsomal

amphetamine metabolism assumed to undergo, in turn, oxygenation to yield phenylacetone oxime34,110,120 (equation 5).

A second possibility for metabolic oxime formation comes from biotransformation of aliphatic primary hydroxylamines bearing at least one ˛-hydrogen87. Indeed, 28 was identified in rat and rabbit liver microsomal incubates of N-hydroxyamphetamine33,35,121. Oxime formation was NADPH/O2-dependent, but did not appear to involve H2O2 or superoxide35,121. The R N-hydroxy enantiomer was converted at a faster rate than the S C form33. It has to be mentioned that 28 has not been unequivocally accepted as a product generated by enzymatic catalysis: based on chemical stability studies, 28 has been contended to arise from chemical oxidation of N-hydroxyamphetamine during the analytical workup procedure122. However, the quantitative difference in conversion between the N-hydroxyamphetamine enantiomers as well as the inability of boiled enzyme to produce substantial amounts of oxime have been advocated to prove the enzymatic nature of the process33. The latter has been shown to be a P-450-independent reaction, since oxime formation from the hydroxylamine precursor was insensitive to the presence of CO, SKF 525A or DPEA and was unaffected by pretreatment of the animals with phenobarbital35,121. This finding is in accord with the observation that highly purified FMO can catalyze oxidation of primary alkylhydroxylamines to oximes123, as illustrated in equation 6. Thus, FMO1 from hog liver brings about oxidation of N- hydroxydidesmethylpromethazine124 to the corresponding oxime (47) with Km D 160 M and a kcat value of 34 (equation 7). Similarly, cDNA-expressed human FMO3 forms oximes from a series of aliphatic primary hydroxylamines possessing chromophores125.

(47)

Oximes have been isolated along with primary hydroxylamines from incubation mixtures containing arylalkylamines22,33,87,126 and related compounds23. This fostered the notion that, for example, oxime formation from amphetamine might proceed via a hydroxylamine as an alternative intermediate to imine34 (equation 5). Another possibility was offered by assuming dehydration of an ˛,N-dihydroxyamphetamine metabolite to give the oxime34. In a study on the biotransformation of a number of ˛-substituted amphetamines, oxime levels were found to be highest with the ˛-Me compound and substantially lower with amines bearing ˛-Et, ˛-Pri or ˛-But substituents126.

Apart from mammalian systems, hydroxylamine/oxime interconversion also occurs in higher plants. Microsomal fractions prepared from etiolated seedlings of Sorghum bicolor catalyze the transformation of the amino acid L-tyrosine to the cyanogenic glucoside dhurrin, N-hydroxytyrosine and p-hydroxyphenylacetaldoxime (48) being key intermediates in biosynthesis127. Cytochrome P-450 79 (P-450TYR) has been demonstrated to account for metabolism of L-tyrosine all the way to the aldoxime with Km D 140 M and a turnover number of 200 min 1, the dehydration and decarboxylation steps apparently being nonenzymatic reactions21,45 (equation 8).

(8)

NOH

(48)

A third pathway for oxime formation is given by tautomerization of nitroso compounds possessing an ˛-hydrogen (equation 9). Such a process involves an intramolecular redox reaction, in which the nitrogen undergoes a formal two-electron reduction, while the ˛-carbon is oxidized. Kinetic analysis of this conversion, as performed with a set of ˛- substituted 2-nitroso-1-phenylethane compounds, has revealed sensitivity toward both the bulkiness of the substituents and the initial concentration of the nitroso dimers128. For instance, tautomerization of 2-nitroso-1-phenylpropane to 28 has been proposed to play a role in the metabolism of methamphetamine by fortified rat liver tissue129.

B. Enzymology of Nitrone Formation

Diarylnitrone (31) formation from N-substituted, diaromatic imines has been recognized to require the presence of NADPH/O2, and has been proposed to proceed via the intermediacy of an oxaziridine3 possibly arising from reaction of the parent imines with the putative P-450 [FeO]3C species in analogy to the oxidation of olefins118. Ring cleavage of the oxaziridine then yields nitrone or amide (equation 10).

O

Moreover, secondary hydroxylamines, generated by N-oxygenation of secondary amine compounds, can afford nitrones after oxidative attack. In this way, a series of N-substituted amphetamines has been found to produce the corresponding nitrones (35) in liver preparations fortified with NADPH6,24,25,54,110,129,130 . When N-hydroxy-N-methylamphetamine was used as the substrate, considerably more nitrone was formed in the presence of liver supernatant containing NADPH than with supernatant or cofactor alone129; on the other hand, appreciable quantities of nitrone were obtained, when N-hydroxy-N- propylamphetamine was incubated in mixtures containing supernatant but no cofactor, or boiled supernatant with NADPH130. These findings clearly indicate that both enzymatic and nonenzymatic processes are operative in the oxidative transformation of the hydroxylamines to the nitrones. While there was only a small enantiomeric preference, during nitrone formation, for the S- and R-isomers of N-benzylamphetamine6,131, R - N-propylamphetamine strongly favored nitrone production in liver supernatant130; no correlation between nitrone and P-450 levels could be established130.

Similarly, hamster hepatic preparations convert N-benzyl-4-chloroaniline53 to a mixture of intermediary hydroxylamine and stable ˛,N-diphenylnitrone (34) end product with Km values ranging from 300 to 420 M. Velocity of nitrone formation from N-benzylaniline is enhanced when the pH of the reaction medium is raised from 7.4 to 8.5 and is diminished by the presence of methimazole53,132, as is typical of a FMO-catalyzed process133. Indeed, the flavin-containing monooxygenase has been reported to metabolize secondary hydroxylamines, such as N-methyl-N-benzylhydroxylamine19,134, N-methyl-N- benzhydrylhydroxylamine19 or N-hydroxynorzimeldine135, to the corresponding nitrones, as illustrated by the general equation 11. Reactions probably proceed via unstable hydroxylamine N-oxides, which readily dehydrate to yield nitrones, asymmetric secondary hydroxylamines affording two isomeric N-oxy products134. FMO-dependent hydroxylamine oxidase activity requires the presence of NADPH and oxygen; cofactor cannot be replaced with H2O219.

However, with certain secondary hydroxylamines, P-450, too, can catalyze transformation to nitrones. Thus, incubation of N,N-(1-phenylcyclobutyl)benzyl hydroxylamine with P-450 2B1 in the presence of NADPH-cytochrome P-450 reductase and NADPH has been demonstrated to trigger nitrone 36 formation with Km D 48 M by direct oxidation of the N-hydroxy compound; exogenous catalase did not change the amount of product generated55.