NADPH → (4aR,4bS,6aS,7S,9aS,9bS,11aR)-N-(tert-butyl)-4a,6a-dimethyl-2-oxohexadecahydro-1H-indeno(5,4-f)quinoline-7-carboxamide

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To obtain sufficient transformed inhibitor for mass spectral analysis, a preparation of baculovirus-expressed enzyme was employed that consisted of all protein sedimenting between 1,000 to 100,000×g. The enzyme-[3H]finasteride complex was formed in a solution containing 1.39 μM enzyme (41,000 units, 141 mg protein), 1.14 μM unlabeled finasteride (15.0 nmol), 0.0113 uM tracer [3H]finasteride (1.56 μCi), and 573 μM NADPH, in a buffer composed of 0.1M MOPS, 1M sucrose, and 10 mM EDTA at pH 7.20 in a total volume of 13.1 mL. The enzyme was dispersed in the solution with a dounce and stirred for 45 min as it warmed to room temperature, which produced 73% inhibition. The solution was then dialyzed overnight at 4° C. in 14,000 Da cutoff dialysis tubing (SPECTRAPOR 2) against 2 changes of 4L water containing 1 mM MOPS and 1 mM EDTA at pH 7.20. This resulted in 88% retention of the radioactivity. (Analysis of the radioactivity lost to dialysis indicated 62% chromatographed as [3H]dihydrofinasteride, and the remainder as [3H]finasteride. This corresponds to loss of 8% of the total starting radioactivity as [3H]dihydrofinasteride, implying a partition ratio of 1.08 in this instance.)

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