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, (\quad N)-( (p)-azidobenzyl)- (N)-methylphenethylamine (\quad\left(\mathrm{N}{3}-\mathrm{BP}\right), \quad N)-benzyl- (N)-methyl(p)-azidophenethylamine ( (\left.\mathrm{BP}-\mathrm{N}{3}\right)), and disubstituted (N) - (p)-azidobenzyl) (N)-methyl-p- -azidophenethylamine (\left(\mathrm{N}{3}-\mathrm{BP}-\mathrm{N}{3}\right)), in overall yields of 34,38 , and (10 %), respectively. From the comparison of spectral dissociation constants ( (K_{\mathrm{s}}) ) of the azido-compounds (the (K_{\mathrm{s}}) values for which range from 2.3 to (4.2 \times 10^{-5} \mathrm{~mol} /) liter) with the (K_{5}) value for the P450 substrate benzphetamine of (6.0 \times 10^{-5} \mathrm{~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 (K_{m}) (values (1.0-1.2 \times 10^{-4} \mathrm{~mol} /) /iter) for (N)-demethylation of these photolabile compounds and benzphetamine ( (K_{m}=0.9 \times 10^{-5} \mathrm{~mol} /) /iter) . All three azido-compounds are extremely photolabile under irradiation at (254 \mathrm{~nm}) (half-life about (1 \mathrm{~s}) ). Photolysis of (\mathrm{N}_{3}-) 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 P4502BI and (1 \mathrm{Al}) 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. c 1994 Academic Press, Inc.