Synthesis of (5-azido-2-nitrobenzoyl)amido, (4-azido-2-nitrophenyl)amino, and (5-azido-2-nitro-3,4,6-trifluorophenyl)amino derivatives of 17 alpha-methylamino-, 17 alpha-ethylamino-, and 17 alpha-propylamino-5 alpha-dihydrotestosterone as reagents of different linker lengths for the photoaffinity labeling of sex hormone binding globulins and androgen receptors

The photoactivable aryl azide reagents, N-(5-azido-2-nitrobenzoyl)oxysuccin imide, 4-azido-1-fluoro-2-nitrobenzene, and 4-azido-1-nitro-2,4,5,6-tetrafl uorobenzene have been condensed at the extremity of three 17 alpha-aminomet hyl, 17 alpha-aminoethyl, and 17 alpha-aminopropyl side-chains introduced o n (17S)-spiro-(3,3-dimethoxy)-5 alpha-androstan-17 beta,2'-oxirane either d irectly, by ammonolysis, in the first case, or by conversion to nitrile int ermediates with cyano or cyanomethyl anions and subsequent reduction to ami nes with lithium aluminum hydride, in the two other cases. The 3,3-dimethox y group of these photoreagents was cleaved by acidolysis to a 3-ketone, whi ch was reduced with sodium borohydride to a 3 beta-alcohol. All of these co mpounds were characterized by H-1- and C-13-NMR as well as by H-1, C-13 het eronuclear 2D NMR, which helped to resolve ambiguous assignments. Significa nt differences of substituent-induced effects on C-13 NMR signals were obse rved according to the 17 alpha-side-chain length, the structure of the term inal aryl azide groups, and the solvent, showing a different behavior of N- 5-azido-2-nitrobenzoyl derivatives as compared with 4-azido-2-nitrophenylam ino and 5-azido-2-nitro-3,4,6-trifluorophenylamino derivatives. The N-5-azi do-2-nitrobenzoyl conjugates of the three 17 alpha-aminomethyl, aminoethyl, and aminopropyl derivatives of 5 alpha-dihydrotestosterone were tested as ligands for purified human sex hormone-binding globulin and for the cytosol ic androgen receptor of rat ventral prostate by competition experiments wit h tritiated 5 alpha-dihydrotestosterone. The increasing lengths of the amin omethyl, aminoethyl, and aminopropyl spacer arms of N-5-azido-2-nitrobenzoy l conjugates were found to correspond to decreasing relative binding affini ties for sex hormone-binding globulin (0.76, 0.47, and 0.10, respectively, versus 1.00 for 5 alpha-dihydrotestosterone) while only the longer aminoeth yl and aminopropyl conjugates interacted significantly with the androgen re ceptors (0.05 and 0.10, respectively). (C) 2000 Elsevier Science Inc. All r ights reserved.