Metabolic N1-Oxidation of 9-Benzyladenine and Isomeric 9-(Nitrobenzyl)Adenines by Hamster Hepatic Microsomes

Abstract

The metabolism of 9-benzyladenine (BA) and isomeric 9-(nitrobenzyl) adenines (NBAs), i.e. 9-(2-nitrobenzyl)adenine (2NBA), 9-(3-nitrobenzyl)adenine (3NBA) and 9-(4-nitrobenzyl)adenine (4NBA), was investigated with normal hamster hepatic microsomes. Using HPLC, TLC, UV and MS techniques, it was established that metabolic N¹-oxidation occurred for all BA and NBAs. The N¹-oxidative rate was in the order 2NBA>BA>3NBA and 4NBA. The results suggest that the introduction of a nitro group may interfere with the binding between substrates and related N¹-oxidase(s). This may be the reason for the lower N¹-oxidative rates of 3NB A and 4NB A. It is also suggested that 2NBA may possess the most favourable conformation and binding characteristics for the enzymes among the substrates studied as it was metabolised even faster than BA.

Metabolism of 9-(2-chlorobenzyl)-, 9-(2-methylbenzyl)-and 9-(2-methoxybenzyl)-adenines by hamster hepatic microsomes

“…In addition, like other 9-alkyl and 9-aralkyladenines previously studied (2)(3)(4)8), dealkylation also occurred during incubation for' 2CBA, 2MBA and 2MOBA as there were areas corresponding to authentic adenine on the TLC plates. N-Dealkylation is a commonly observed biotransformation pathway of tertiary and secondary amines as well as related N-alkylated amides, in which the CYP3A subfamily (espe- HPLC chromatographic and UV spectral characterisation of metabolites Figure 2 shows the HPLC chromatograms of the incubates with 2CBA, 2MBA or 2MOBA as substrates, dally CYP3A4) and CYP2D6 are often found to be involved (14).…”

“…The metabolites formed during incubation were extracted using an off-line solid phase extraction (SPE) procedure previously established (8,10). The acidified incubates were centrifuged (3000 rpm for 10 min) using a benchtop centrifuge.…”

“…2 (11)(12)(13): (i) ammoniacal silver nitrate solution (Tollen's reagent); (ii) ferric reagent (3% ferric chloride in water); (iii) picryl chloride-ammonia reagent; and (iv) tripyridyl-striazine-ferric chloride reagent. A previously reported HPLC method was used for analysis of the extracts of microsomal incubates (8,10). A Spherisorb 5 ODS (2) column (25 x 0.46 em i.d.…”

“…The metabolic incubation procedure was as previously described (8). Briefly, each incubate consisted of microsomal suspension (0.5 ml, equivalent to 0.25 g tissue), NADP (I umol), glucose-6-phosphate (5 umol), glucose-6-phosphate dehydrogenase (0.5 unit), magnesium chloride (10 umol) and phosphate buffer (1.0 ml, 0.2 M, pH 7.4) with a final volume of 1.5 ml.…”

“…Among the possible controlling factors of the NI-oxidation of these adenine derivatives, it was believed that BA possesses a conformation favourable to the corresponding N-oxidase(s) (4,7). A recent investigation on the N 1-oxidation of isomeric 9-(nitrobcnzyl)adenines (NBAs) confirmed that further substitution in the benzyl moiety may greatly modulate the formation rate of NI-oxides from the substrates (8). For example, 9-(2-nitrobenzyl)adenine was Table I : 9-Aralkyladenines (AAs) and their potential metabolites studied Substrates 9-(2-chlorobenzyl)adenine (2CBA) 9-(2-methylbenzyl)adenine (2MBA) 9-(2-methoxybenzyl)adenine (2MOBA) 9-(4-nitrobenzyl)adenine (4NBA) NI-oxidised much faster than BA, whereas 9-(3-nitrobenzyl)adenine and 9-(4-nitrobenzyl)adenine were NI-oxidised significantly slower than BA by a hamster hepatic microsomal system (8).…”