Steroidal 21-diazo ketones. Photogenerated corticosteroid receptor labels

Abstract

The 21-diazo derivatives of 9 alpha-fluoro- and 9 alpha-bromo-21 deoxycorticosterone, 21-deoxycorticosterone, and progesterone were synthesized for use as photoaffinity labels for corticosteroid receptors. In the isolated toad bladder system, 9 alpha-bromo-21-diazo-21-deoxycorticosterone was as active as d-aldosterone and more active than 9 alpha-fluoro-cortisol in augmenting active Na+ transport. The activities of 21-diazoprogesterone and progesterone were equal; both were much less potent than d-aldosterone, however. These results indicate that the 21-diazo derivatives had significant functional activity in the toad bladder system. The rat kidney slice system was used to estimate the relative affinities of the diazo steroids for aldosterone receptor sites by competition experiments. At 100-fold excess of competitor to [3-H]aldosterone, the order of affinities was 9 alpha-fluoro-21-diazo-21-deoxycorticosterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone. Moreover, 9 alpha-bromo-21-diazo-21-deoxycorticosterone reduced binding of [3-H]aldosterone to cytoplasmic and nuclear forms of the receptor proportionately. On the basis of competition for [3-H]corticosterone binding, presumably to corticosteroid-binding globulin (CBG), the order of affinities was 21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone. These findings indicate that 21-diazo steroids may be suitable as photogenerated affinity labels for mineralocorticoid receptors. The tritiated derivative, [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone (specific activity 25 Ci/mol) was synthesized and used in model experiments on photogenerated covalent binding to rat plasma proteins. Irradiation with uv light resulted in binding of [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone to plasma proteins, that was resistant to extraction with methylene dichloride and did not exchange with unlabeled corticosterone. The diazocorticosteroids, therefore, may have the requisite functional and selectivity properties for photoaffinity labeling of corticosteroid-binding proteins. Further studies are needed, however, to assure that photogenerated labeling with these steroids was site specific.

Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

“…Thin-layer chromatography (TLC) was done as described (5) with the following solvent systems: system A, benzene-ethyl acetate (1:2 vol/vol); system B, benzene-ethanol (6:1 vol/vol); system C, chloroform-ethyl acetate (1:3 vol/vol). Melting points, microanalyses, and.…”

“…Melting points, microanalyses, and. UV spectroscopy were performed as described (5). Catalytic tritiation was performed with the assistance of Dr. Chin-Tzu Peng and Mr. Ray Aune at the Lawrence Radiation Laboratory (Berkeley, Calif.).…”

“…Found: C, 72.54; H, 7.83). Compound IV [mp 251-255°, Xmax 242 nm, (E 1.55 X 104)] was prepared from compound I as described (5). Compound V [mp 1750 (dec), Xmax 247 nm (E 2.3 X 104)], with a shoulder at 280 nm, was prepared from compound IV as described (5).…”

“…Compound IV [mp 251-255°, Xmax 242 nm, (E 1.55 X 104)] was prepared from compound I as described (5). Compound V [mp 1750 (dec), Xmax 247 nm (E 2.3 X 104)], with a shoulder at 280 nm, was prepared from compound IV as described (5). Tritium-labeled compound IV (at C6-C7) was obtained by catalytic tritiation of compound III in benzene-tetrahydrofuran solvent using 10% palladium on charcoal and carrier-free tritium gas in a microhydrogenation apparatus using vacuum line techniques.…”

Biological Magnetic Resonance

“…The syntheses of 4-fluorohistamine (Kirk and Cohen, 1973) and ring-fluorinated tyramines and dopamines (Kirk, 1976) have been achieved. Fluorine-containing compounds that can function as photoaffinity labeling reagents have been reported (Wolff et al, 1975;Chowdry et al, 1976). …”