Synthesis and Characterization of Azidobenzphetamine Analogs of the Cytochrome P450 Substrate Benzphetamine

Benzphetamine, an amphetamine with sympathomimetic stimulant activity in the central nervous system, is a substrate for cytochromes P450 with highest metabolic turnover being catalyzed by cytochrome P4502B1. We synthesized three photolabile azido-compound analogs, N-(p-azidobenzyl)-N-methylphenethylamine (N3-BP), N-benzyl-N-methyl-p-azidophenethylamine (BP-N3), and disubstituted N-(p-azidobenzyl)-N-methyl-p-azidophenethylamine (N3-BP-N3), in overall yields of 34, 38, and 10%, respectively. From the comparison of spectral dissociation constants (Ks) of the azido-compounds (the Ks values for which range from 2.3 to 4.2 × 10-5 mol/liter) with the Ks value for the P450 substrate benzphetamine of 6.0 × 10-5 mol/liter, it is clear that the introduction of azido-group(s) into a desmethylbenzphetamine skeleton did not significantly change the cytochrome P450 active site binding affinity in phenobarbital induced microsomes. Similarly, there is almost no difference in Km (values 1.0-1.2 × 10-4 mol/liter) for N-demethylation of these photolabile compounds and benzphetamine (Km = 0.9 × 10-5 mol/liter). All three azido-compounds are extremely photolabile under irradiation at 254 nm (half-life about 1 s). Photolysis of N3-BP in methanol, revealing a N-methoxy compound as a major product (85%) of a nitrene reaction, demonstrates a high reactivity of these compounds after photoactivation. Photoactivated azidodesmethylbenzphetamines produced clear inhibition of cytochrome P4502B1 and 1A1 specific catalytic activities in corresponding microsomal samples, compared to activities in samples irradiated with prephotolyzed probes. Pentoxyresorufin and ethoxyresorufin O-dealkylase activities were inhibited from 13 to 32%, depending on the compound used.