Ligands for the glucocorticoid receptor labeled with high-energy isotopes are highly desired for their potential applications in nuclear medical studies of the brain where the dysregulation of this receptor system is thought to be involved in various neurodegenerative disorders. Analogues of the glucocorticoid cortivazol have previously been prepared as target compounds for labeling with high-energy isotopes. However, the phenyl rings of arylpyrazoles of this type are not sufficiently activated for nucleophilic substitution reactions that are generally required for the synthesis of radiohalogenated analogues. Since suitably substituted aromatic nitrogen heterocyclic groups are amenable to nucleophilic substitution, the goal of this study was the synthesis of pyridylpyrazolo and pyrimidylpyrazolo analogues similar to cortivazol that could be labeled with radiohalogens in the pyridine or pyrimidine rings. We describe the synthesis of several [3,2-c]pyrazolo steroids containing pyridyl, halopyridyl, and pyrimidyl substituents at the 2' position of the pyrazole ring. These compounds were tested for binding to the glucocorticoid receptor and for biological activity in glucocorticoid responsive HeLa cells grown in tissue culture. Of the pyridyl and pyrimidyl derivatives, 2'-(3-pyridyl)-11 beta,17,21-trihydroxy-16 alpha-methyl-20-oxopregn-4-eno[3,2-c]pyrazole showed superior activity in both assays and it was used as the basis for the synthesis of several analogues that were halogenated in the pyridine ring. These halogenated compounds were all tested for their binding to the glucocorticoid receptor and for their biological activity. One, a fluorinated compound 2'-(2-fluoro-5-pyridyl)-11 beta,17,21-trihydroxy-16 alpha-methyl-20-oxopregn-4-eno[3,2-c]pyrazole had excellent activity, considerably better than the potent glucocorticoid dexamethasone. Most importantly, fluorination was achieved using a nucleophilic exchange reaction, a method that is adaptable to radiolabeling with the positron-emitting isotope fluorine-18. Thus, considering its superior biological activity and adaptability for facile radiosynthesis, this target compound has the potential for imaging of glucocorticoid receptor containing tissues using positron emission tomography.
We have synthesized several 7alpha-fluoro (F) and 7alpha-iodo (I) analogues of 5alpha-dihydrotestosterone (5alpha-DHT) and 19-nor-5alpha-dihydrotestosterone (5alpha-NDHT) and tested them for binding to the androgen receptor and for their biological activity in an in vitro assay with cells that have been engineered to respond to androgens. The relative binding affinity to the androgen receptor determined in competition assays showed that in the androstane series the fluoro steroids have the highest affinity and that F-17alpha-CH3-DHT (4) has a higher affinity than 5alpha-DHT. All other steroids were somewhat less potent than 5alpha-DHT with F-DHT (2) = I-17alpha-CH3-DHT (3) >/= F-NDHT (6) > F-17alpha-CH3-NDHT (8) = I-DHT (1) >/= I-NDHT (5) > I-17alpha-CH3-NDHT (7). The relative biological activity in cells transfected with the androgen receptor and an androgen responsive reporter gene is 4 >> 5alpha-DHT > 2 > 6 > 3 >/= 1 >/= 8 >/= 5 > 7. The iodinated compound, I-17alpha-CH3-DHT (3), with the highest binding activity was synthesized labeled with 125I and was shown to bind with high affinity, Ka = 1.9 x 10(10) L/mol, and low nonspecific binding to the androgen receptor in rat prostatic cytosol. However, when radiolabeled [125I]-17alpha-CH3-DHT ([125I]3) was injected into castrated male rats, it showed very poor androgen receptor-mediated uptake into the rat prostate. This was unexpected in light of its superior receptor binding properties and its protection by the 17alpha-methyl group from metabolic oxidation at C-17. However, the biological potency of I-17alpha-CH3-DHT (3) was not as high as would have been expected. When I-DHT (1) and I-17alpha-CH3-DHT (3) were incubated in aqueous media at 37 degrees C they rapidly decomposed, but they were stable at 0 degrees C. The fluorinated analogue 4 treated similarly at 37 degrees C was completely stable. The products of the decomposition reaction of I-DHT (1) at 37 degrees C were identified as iodide and principally 17beta-hydroxy-5alpha-androst-7-en-3-one. The temperature dependence of this elimination reaction explains the inconsistency between the high binding to the androgen receptor (measured at 0 degrees C) and the low biological activity, as well as the poor androgen receptor mediated concentration in vivo. The fluorinated analogue F-17alpha-CH3-DHT (4) has both high affinity for the androgen receptor and high stability in aqueous media. Of the compounds tested, 4 has the highest affinity for the androgen receptor as well as the highest androgenic activity. Thus it is likely that F-17alpha-CH3-DHT 4 labeled with 18F will be an excellent receptor-mediated diagnostic imaging agent.
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