Anti-Cancer Activity of Novel Dihydrotestosterone-Derived Ring A-Condensed Pyrazoles on Androgen Non-Responsive Prostate Cancer Cell Lines.

Regioselective synthesis of novel ring A-fused arylpyrazoles of dihydrotestosterone (DHT) was carried out in two steps under facile reaction conditions. Aldol condensation of DHT with acetaldehyde afforded a 2-ethylidene derivative regio- and stereo-selectively, which was reacted with different arylhydrazines in the presence of iodine via microwave-assisted oxidative cyclization reactions. The 17-keto analogs of steroidal pyrazoles were also synthesized by simple oxidation in order to enlarge the compound library available for pharmacological studies and to obtain structure-activity relationship. The antiproliferative activities of the structurally related heteroaromatic compounds were tested in vitro on human cervical and breast adenocarcinoma cell lines (HeLa, MCF-7 and MDA-MB-231) and on two androgen-independent malignant prostate carcinoma cell lines (PC-3 and DU 145). Based on primary cytotoxicity screens and IC50 assessment, a structure-function relationship was identified, as derivatives carrying a hydroxyl group on C-17 exhibit stronger activity compared to the 17-one counterparts. Cancer cell selectivity of the derivatives was also determined using non-cancerous MRC-5 cells. Furthermore, the proapoptotic effects of some selected derivatives were verified on androgen therapy refractive p53-deficient PC-3 cells. The present study concludes that novel DHT-derived arylpyrazoles exert cancer cell specific antiproliferative activity and activate apoptosis in PC-3 cells.

Fully automated synthesis of [(18) F]fluoro-dihydrotestosterone ([(18) F]FDHT) using the FlexLab module.

Imaging of androgen receptor expression in prostate cancer using F-18 FDHT is becoming increasingly popular. With the radiolabelling precursor now commercially available, developing a fully automated synthesis of [(18) F] FDHT is important. We have fully automated the synthesis of F-18 FDHT using the iPhase FlexLab module using only commercially available components. Total synthesis time was 90 min, radiochemical yields were 25-33% (n = 11). Radiochemical purity of the final formulation was > 99% and specific activity was > 18.5 GBq/µmol for all batches. This method can be up-scaled as desired, thus making it possible to study multiple patients in a day. Furthermore, our procedure uses 4 mg of precursor only and is therefore cost-effective. The synthesis has now been validated at Austin Health and is currently used for [(18) F]FDHT studies in patients. We believe that this method can easily adapted by other modules to further widen the availability of [(18) F]FDHT.

A remote controlled system for the preparation of 7 alpha-[18F]fluoro-17 alpha-methyl 5 alpha-dihydrotestosterone ([18F]FMDHT) using microwave.

For non-invasive imaging of the prostate cancer, we synthesized 7 alpha-fluoro-17 alpha-methyl 5 alpha-dihydrotestosterone ([(18)F]FMDHT) for androgen receptor mediated PET imaging. Preliminary in vitro and in vivo evaluations of this compound show promise. We designed and implemented a remote controlled system for reliable, efficient, and safe handling of radioactivity during the radiochemical synthesis of [(18)F]FMDHT. The key features of this report are the microwave assisted radiochemical synthesis, increased radiochemical yields, improved radiochemical purity, reduced overall synthesis time, and remote controlled automation of the entire synthesis. The overall synthesis using microwave reaction took 60-70 min and provided the desired product in 20-30% radiochemical yields with >99% radiochemical purity.

High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate.

Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17 beta-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17 beta-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.

Synthesis and basic evaluation of 7α-(3-[18F]fluoropropyl)-testosterone and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone.

OBJECTIVE: 7α-Substituted androgen derivatives may have the potential to visualize androgen receptors with positron emission tomography. In the present study, we synthesized fluoropropyl derivatives of 7α-(3-[18F]fluoropropyl)-testosterone ([18F]7) and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone ([18F]15), and characterized their in vitro binding, in vivo biodistribution, and performed blocking studies in mature androgen deprived male rats. METHODS: We synthesized [18F]7 and [18F]15. In vitro binding to recombinant rat AR ligand binding domain protein was determined using a competitive radiometric ligand-binding assay with the high-affinity synthetic androgen [17α-methyl-3H]-methyltrienolone ([3H]R1881). In vivo biodistribution was performed in mature male rats treated with diethylstilbestrol (chemical castration). A blocking study was performed by co-administration of dihydrotestosterone (36 µg/animal). RESULTS: 7α-(3-Fluoropropyl)-testosterone (7) and 7α-(3-fluoropropyl)-dihydrotestosterone (15) showed competitive binding to recombinant rat AR ligand binding domain protein. The IC50 value of 15 (13.0 ± 3.3 nM) was higher than 7 (47.8 ± 10.0 nM). In contrast to the AR binding affinity, the ventral prostate uptake of [18F]7 and [18F]15 at 2 h post-injection was similar (0.07 % injected dose/g of tissue). A blocking study indicated that specific binding of [18F]15 is observed in the ventral prostate. [18F]7 and [18F]15 showed moderate levels of bone uptake, which indicates moderate metabolic de-fluorination in rodents. CONCLUSION: [18F]15 is better than [18F]7 in terms of radiochemical yield, in vitro binding affinity, prostate specific binding and stability against in vivo metabolic de-fluorination. However, the net uptake level of [18F]15 in prostate might be insufficient for in vivo visualization. Although [18F]7 and [18F]15 improved in vivo stability against de-fluorination, other basic characterization data in rodents were not superior to the current standard tracer 16ß-[18F]fluoro-5α-dihydrotestosterone. It is also revealed that the shorter side chain length of 7α-[18F]fluoromethyl-dihydrotestosterone is superior to the longer three carbon chain in [18F]15, in terms of net prostate uptake and in vivo metabolic stability.

Recent Advances in Drug Design and Drug Discovery for Androgen- Dependent Diseases.

This article summarizes the importance of different targets such as 5α-reductase, 17ß-HSD, CYP17A, androgen receptor and protein kinase A for the treatment of prostate cancer and benign prostatic hyperplasia. It is a well known fact that dihydrotestosterone (DHT) is associated with the development of androgen-dependent afflictions. At the present time, several research groups are attempting to develop new steroidal and non-steroidal molecules with the purpose of inhibiting the synthesis and biological response of DHT. This review also discusses the most recent studies reported in the literature that describe the therapeutic potential of novel compounds, as well as the new drugs, principally inhibitors of 5α-reductase.

Design and synthesis of new dihydrotestosterone derivative with positive inotropic activity.

There are several reports which indicate that some steroid derivatives have inotropic activity; nevertheless, the cellular site and mechanism of action of steroid derivatives at cardiovascular level is very confusing. In order, to clarify these phenomena in this study, two dihydrotestosterone derivatives (compounds 5 and 10) were synthesized with the objective of to evaluate its biological activity on left ventricular pressure and characterize their molecular mechanism. In the first stage, the Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of the steroid derivatives. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by the compound 5 was evaluated by measuring left ventricular pressure in absence or presence of following compounds; nifedipine, flutamide, indomethacin, prazosin, isoproterenol, propranolol and metoprolol. The results showed that the compound 5 significantly increased the perfusion pressure and coronary resistance in comparison with dihydrotestosterone, compound 10 and the control conditions. Other data indicate that 5 increase left ventricular pressure in a dose-dependent manner (0.001-100 nM); nevertheless, this phenomenon was significantly inhibited only by propranolol or metoprolol at a dose of 1 nM. These data suggest that positive inotropic activity induced by the compound 5 is through ß1-adrenergic receptor however, this effect was independent of cAMP levels. This phenomenon is a particularly interesting because the positive inotropic activity induced by this steroid derivative involves a molecular mechanism different in comparison with other positive inotropic drugs.

Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand.

A microassay for the androgen receptor was developed to investigate the cellular distribution of receptor in freshly isolated testicular cell types. The microassay uses an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is rapid and irreversible, which permits the development of a highly sensitive assay. The androgen receptor microassay is completed within 4 h and detects receptor in as little as 0.5 micrograms cellular protein. There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, including androgen-binding protein. Androgen receptor was found in cellular sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Although the relative distribution of receptor was similar to that obtained using a traditional equilibrium binding assay, the levels of receptor were significantly higher using the microassay. The androgen receptor microassay was subsequently used to investigate the receptor in isolated testicular cell types. Androgen receptor was detected in freshly isolated peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a mixed population of germ cells. Hormones were not found to influence androgen receptor levels in cultured peritubular cells or Sertoli cells. Electrophoretic analysis of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa form of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The size of the androgen receptor species detected in the rat testicular cell types was slightly smaller than the 56-kDa protein detected in a human fibroblast cell line. The current study demonstrates the utility of the microassay and affinity ligand to investigate androgen receptor biology. Data indicate that androgen receptors are present in several testicular cell types and suggest that the control of testicular function by androgens probably involves actions on multiple cell types.

Dihydrotestosterone heptanoate: synthesis, pharmacokinetics, and effects on hypothalamic-pituitary-testicular function.

Dihydrotestosterone heptanoate (DHT-hp), a seven-carbon fatty acid ester of DHT, was synthesized, and its pharmacokinetics and effects on hypothalamic-pituitary-testicular function were determined in men and pubertal boys. Plasma DHT levels markedly increased 24 h after im injection of DHT-hp, reached their peak during the first week, and fell to baseline levels after 4-6 weeks. An estimated 43-55% of DHT-hp was converted to DHT 4-6 weeks after injection. Plasma testosterone, estradiol, LH, and FSH levels decreased by 4 days after DHT-hp injection, were lowest during the second week, and returned to baseline values after 4-6 weeks. The LH and FSH responses to GnRH were diminished by chronic administration of DHT-hp to pubertal boys at 3-week intervals for 15 weeks. The affinity of DHT-hp was 100 times less than the affinity of DHT for the human androgen receptor, and no affinity for the estrogen receptor in breast tissue could be demonstrated. Since DHT is a nonaromatizable androgen, and neither DHT nor DHT-hp binds readily to the estrogen receptor, suppression of LH and FSH secretion by this drug probably occurs via an androgen-dependent mechanism. Receptor binding and pharmacokinetic data indicate that unesterified DHT is the active principle. DHT-hp is a useful derivative of DHT, since prompt, predictable, and sustained rises in DHT occur after its administration.

Synthesis of 11 beta-[18F]fluoro-5 alpha-dihydrotestosterone and 11 beta-[18F]fluoro-19-nor-5 alpha-dihydrotestosterone: preparation via halofluorination-reduction, receptor binding, and tissue distribution.

We have prepared 11 beta-fluoro-5 alpha-dihydrotestosterone (11 beta-F-DHT, 1) and 11 beta-fluoro-19-nor-5 alpha-dihydrotestosterone (11 beta-F-19-nor-DHT, 2) in order to investigate the properties of these new androgens labeled with fluorine-18 as potential androgen receptor (AR)-based imaging agents for prostate cancer. These compounds were synthesized in 6 steps from hydrocortisone and in 13 steps from 1,4-androstadiene-3,11,17-trione, respectively. Relative binding affinities (RBA) of 11 beta-F-DHT and 11 beta-F-19-nor-DHT to AR are 53.1 and 75.3 (R1881 = 100), respectively, the latter being the highest reported among fluorine-substituted androgens. The fluorination step, which involves addition of halogen fluoride across the 9(11)-double bond, followed by reductive dehalogenation at the 9 alpha-position has been adapted to introduce a fluorine-18-label at the 11 beta-position of DHT and 19-nor-DHT. The two high-affinity F-18-labeled ligands [18F]-1 and [18F]-2 were evaluated in vivo, in tissue distribution studies using diethylstilbestrol-pretreated mature male rats. 11 beta-F-DHT shows high prostate uptake and selective prostate to blood and prostate to muscle uptake ratios, the latter two ratios increasing from 5 and 8 at 1 h to 12 and 19 at 4 h postinjection. Moreover, this compound has low uptake in bone, displaying the lowest in vivo defluorination among all androgens labeled with fluorine-18 tested so far. The in vivo properties of 11 beta-F-DHT in rats are thus favorable for imaging of prostate cancer. On the other hand, 11 beta-F-19-nor-DHT shows low prostate uptake with low selectivity and high uptake in liver, kidney, and bladder. Even though this ligand has the highest RBA and undergoes little metabolic defluorination, it appears to suffer from rapid metabolism in vivo. Therefore, it is apparent that the biodistribution properties of androgens are affected by their structure and metabolism as well as by their RBA.

Synthesis and evaluation of 7 alpha-iodo-5 alpha-dihydrotestosterone as a potential radioligand for androgen receptor.

7 alpha-Iodo-17 beta-hydroxy-5 alpha-androstan-3-one (7 alpha-iodo-5 alpha-dihydrotestosterone, 7 alpha-IDHT) has been synthesized as a potential radioligand for the detection and measurement of androgen receptor and for imaging of androgen-receptor-containing tissues when labeled with the gamma-emitting radionuclides 125I and 123I, respectively. In vitro binding studies show that 7 alpha-IDHT binds with high affinity to the rat and human androgen receptor (RBA = 74) compared to R1881 (RBA = 100). Further, this compound showed high specificity for the androgen receptor. 7 alpha-IDHT showed only a marginal affinity for the progestin receptor and even less affinity for the estrogen receptor. No binding was detected to the glucocorticoid receptor. These characteristics make 7 alpha-IDHT a potentially ideal agent for imaging and evaluation of androgen-receptor-containing tissues.

A potential radioiodinated ligand for androgen receptor: 7 alpha-methyl-17 alpha-(2'-(E)-iodovinyl)-19-nortestosterone.

The presence of androgen receptor (AR) in prostate cancer has been linked to the androgen-dependent nature of the tumor and has also been shown to have prognostic significance; it also appears to be a positive prognostic indicator in breast cancer. However, due to the relatively low AR concentrations in most tumors and the inherently low specific activity of tritium, the assay of AR based on available 3H-ligands is not sensitive enough to measure accurately the amount of receptor in small specimens. A 125I-ligand like those available for the estrogen and progesterone receptors would be helpful, but development of such a ligand for AR has not been very successful. Although several androgen analogues containing iodine, bromine, or selenium have been synthesized specifically as potential probes for AR, none have shown any significant affinity or specificity for the receptor. We therefore undertook the synthesis of new potential AR ligands which could be radioiodinated, and determined their affinities for AR (from rat uterus and MCF-7 human breast cancer cells) by using a competition assay. We have examined both 5 alpha-dihydrotestosterone (5 alpha-DHT) and 19-nortestosterone analogues and have identified two such compounds which showed high AR affinity: (17 alpha,20E)-17 beta-hydroxy-21-iodo-5 alpha-pregn-20-en-3-one (17 alpha-[E)-iodovinyl)-5 alpha-DHT, 9) and 17 beta-hydroxy-7 alpha-methyl-(17 alpha,20E)-21-iodo-19-norpregna-4,20-dien-3- one (7 alpha-methyl-17 alpha-[E)-iodovinyl)-19-nortestosterone, 11). In fact, the affinity of the latter for human AR was found to be superior to that of 5 alpha-DHT itself. These iodovinyl analogues could be easily prepared in the radioiodinated form, and should prove to be extremely useful in assaying low levels of AR in small specimens.

7alpha-Iodo and 7alpha-fluoro steroids as androgen receptor-mediated imaging agents.

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.

7 alpha-iodine-125-iodo-5 alpha-dihydrotestosterone: a radiolabeled ligand for the androgen receptor.

UNLABELLED: We describe the preparation of 7 alpha-[125I]iodo-5 alpha-dihydrotestosterone (7 alpha-[125I]IDHT) and its characterization as a ligand for the androgen receptor. METHODS: We designed a route to prepare the radioiodine-labeled androgen on microscale through treatment of the 7 beta-tosylate of 7 beta-hydroxy-5 alpha-dihydrotestosterone-17 beta-p-nitro-benzoate with Na125I, followed by alkaline hydrolysis. The radiolabeled steroid was tested as a ligand for the androgen receptor in cytosol from MCF-7 cells, and for its in vivo tissue distribution in the rat. In addition, we tested 7 alpha-[125I]IDHT as a ligand in a novel assay for the detection and quantification of the ligand activated androgen receptor by in vitro autoradiography. RESULTS: The above synthetic route produced the 17 beta-p-nitrobenzoate of 7 alpha-[125I]IDHT in carrier-free form and in good yield. The 17 beta-ester was removed with alkali and the resulting 7 alpha-[125I]IDHT was purified by HPLC. 7 alpha-[125I]IDHT bound with high affinity, Kd = 0.26 nM, to the androgen receptor and showed low nonspecific binding. Since the ligand was carrier free and thus of very high specific activity, approximately 2,200 Ci/mmole, the sensitivity of the assay was much greater than with [3H]R1881, the classical androgen receptor ligand with which it was compared. When tested as a ligand for in vitro autoradiography, 7 alpha-[125I]IDHT produced excellent autoradiograms of the activated receptor with very low nonspecific binding and with only overnight exposure of the film. CONCLUSION: These studies demonstrate that 7 alpha-[125I]IDHT is an excellent ligand for the androgen receptor.

[7alpha-18F]fluoro-17alpha-methyl-5alpha-dihydrotestosterone: a ligand for androgen receptor-mediated imaging of prostate cancer.

We have synthesized a 18F-labeled androgen, [7alpha-18F]fluoro-17alpha-methyl-5alpha-dihydrotestosterone, in a no-carrier-added radiosynthesis by exchange of 18F- (tetrabutylammonium fluoride) with the 7beta-tosyloxy of 17alpha-methyl-5alpha-dihydrotestosterone. The nonradioactive steroid binds with high affinity and specificity to the androgen receptor and binds poorly, if at all, to other steroid receptors and plasma sex hormone binding globulin. The 7alpha-18F-androgen concentrates markedly in the prostate of rats by an androgen receptor-dependent mechanism. It is likely that [7alpha-18F]fluoro-17alpha-methyl-5alpha-dihydrotestosterone will be an excellent positron emission tomography imaging agent for prostate cancer.

Dihydrotestosterone derivatives: relative binding affinity versus affinity purification.

Mono esters of a homologous series of diacids of dihydrotestosterone were synthesized and converted to the corresponding n-butyl amides. The relative binding affinities of these amides to androgen receptor were compared with the degree of purification of rat prostate androgen receptor by affinity columns prepared by linking the steroidal acid to amino Sepharose. There was good correlation between binding of the amide model to androgen receptor and the extent of purification by the affinity resin.

Syntheses and ligand-binding studies of 1 alpha- and 17 alpha-aminoalkyl dihydrotestosterone derivatives to human sex hormone-binding globulin.

We report on the syntheses of 1 alpha- and 17 alpha-aminoalkyl dihydrotestosterone (DHT) derivatives and the particularly high binding affinity of the 1 alpha-aminohexyl ligand for human sex hormone-binding globulin (SHBG). The two 17 alpha-aminopropyl-17 beta-hydroxy-5 alpha-androstan-3-one (1) and 17 alpha-aminocaproylamidoethyl-17 beta-hydroxy-5 alpha-androstan-3-one (2) derivatives were synthesized via a 17beta-spirooxirane intermediate in high yields. The 1 alpha-aminohexyl-17 beta-hydroxy-5 alpha-androstan-3-one compound (3) was obtained in a seven step synthesis using a copper-catalyzed conjugate addition of a omega-silyloxyhexyl Grignard reagent to 17 beta-benzoyloxy-5 alpha-androst-1-en-3-one. All structures were elucidated based on 1H NMR spectroscopy and mass spectral analyses. The three aminosteroid derivatives were tested as ligands for SHBG by competition experiments with tritiated testosterone as tracer under equilibrium conditions. The association constants of the two 17 alpha-DHT derivatives were approximately 1 x 10(7) M(-1), whereas the 1 alpha-DHT derivative showed a remarkably high binding affinity to SHBG with an association constant of 1.40 x 10(9) M(-1). These aminoalkyl derivatives, substituted either at the D-ring or the A-ring of the steroid skeleton, can be easily coupled onto a carboxymethylated solid state surface of a biosensor. Such a device lends itself to kinetic and thermodynamic studies aimed to provide a better understanding of the biospecific interaction of steroids with SHBG.

Analogues of androgen hormones with inverted configuration at carbons 5, 9, and 10.

On catalytic hydrogenation of Delta(9)-steroids (e.g. 3beta-hydroxy-5-methyl-19-nor-5beta-androst-9-en-17-one), four isomers were formed: 9alpha,10alpha-, 9alpha,10beta-, 9beta,10alpha- and 9beta,10beta-adducts. The product distribution was affected by the nature of the C-3 substituent. A chair conformation of A, B, and C rings was found in all of the products with the exception of the 9alpha,10alpha-adduct whose B ring adopts a twist boat conformation. The products were utilized for the synthesis of dihydrotestosterone analogues.

Preparation of 17 alpha-iodoethynylandrosta- and 17 alpha-(2-iodoethenyl)androsta-4,6-dien-17 beta-ol-3-ones as active site-directed photoaffinity ligands for androgen-binding proteins.

Unsaturated analogues of androst-4-en-17 beta-ol-3-one, each with a 17 alpha-iodoethynyl or 17 alpha-(2-iodoethenyl) substituent, were prepared, and their relative binding affinities (RBAs) for androgen-binding protein (ABP) were compared with those of 5 alpha-androstan-17 beta-ol-3-one, androst-4-en-17 beta-ol-3-one, androsta-4,6-dien-17 beta-ol-3-one, and androsta-1,4,6-trien-17 beta-ol-3-one. These binding studies indicate that the iodine[125I] analogues of 17 alpha-iodoethynyl and 17 alpha-[(E)-2-iodoethenyl] derivatives of androsta-4,6-dien-17 beta-ol-3-one and androsta-1,4,6-trien-17 beta-ol-3-one will have RBAs at least twice as great as that of 5 alpha-androstan-17 beta-ol-3-one. They can be prepared from 17 alpha-ethynylandrosta-4-en-17 beta-ol-3-one, the final synthetic step using N-[125I]iodosuccinimide, and are potential radioiodinated, active site-directed photoaffinity ligands for ABP and testosterone-binding globulin.

Synthesis and androgen receptor binding of dihydrotestosterone hemisuccinate homologs.

Dihydrotestosterone-succinyl-agarose is the most common form of androgen affinity column. To investigate the effect of variation in the number of methylene groups between the ester and amide functions, a homologous series, varying the number of methylenes between the functional groups, has been synthesized and evaluated. In addition, since structure studies show 17 alpha-methyldihydrotestosterone binds with high affinity, a 17 alpha-carboxymethyl ligand (3) was studied. Relative binding affinities of the dicarboxylates (assayed as the n-butyl amides) range from 0.003 to 0.044 (dihydrotestosterone = 1.00), while there was no detectable binding for 3. Only the suberate binds better than the much-used succinate, and it would be a likely candidate for an affinity ligand.