Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities.

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16ß-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone. The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65 µM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4 µM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

Rational design of novel antiandrogens for neutralizing androgen receptor function in hormone refractory prostate cancer.

BACKGROUND: The standard hormonal therapy with currently available antiandrogens and the leutinizing hormone releasing hormone (LHRH) analogs is not effective in the hormone-refractory stage of prostate cancer due to changes in androgen receptor (AR) signaling axis. In this refractory stage, AR continues to play a significant role in the growth of cancer cells even though the cancer cells are no longer dependent on the level of circulating androgens. METHODS: A series of 11beta-Delta(9)-19 nortestosterone compounds were designed through structure-based rationale and tested for their binding affinity against AR and glucocorticoid receptor (GR) using fluorescence polarization assays, their agonistic ability to induce AR dependent transcription using PSA-driven report gene assays, and their growth inhibitory affects against a series of AR positive (LAPC4, LNCap, and CWR22R) and negative human prostate cancer cell lines (PC3) using MTT cell proliferation assays. RESULTS: This study proposes the design of novel bifunctional antiandrogens based on the conjugation of 11beta and/or 7alpha-Delta(9)-19 nortestosterone class of steroidal compounds to the synthetic ligand for FK506-binding proteins. As a critical step towards the development of bifunctional antiandrogens, highly potent and AR-specific lead compounds were identified using in vitro data. The lead compounds identified in this study possessed low binding affinity for GR, indicating the absence of undesirable antiglucocorticoid activity. CONCLUSIONS: The results of this study validate our drug discovery rationale based on the structural biology of AR and pave the pay for future development of bifunctional compounds in order to block AR function in hormone refractory stage of prostate cancer.

Evaluating beta-amino acids as enantioselective organocatalysts of the Hajos-Parrish-Eder-Sauer-Wiechert reaction.

A systematic study of the effect of substitution within the beta-amino acid framework indicates that both beta(2)- and beta(3)-amino acids catalyse the Hajos-Parrish-Eder-Sauer-Wiechert reaction with poor to reasonable levels of enantioselectivity. These results led to the evaluation of the conformationally constrained beta-amino acid (1R,2S)-cispentacin, which catalyses the Hajos-Parrish-Eder-Sauer-Wiechert reaction with comparable or higher levels of enantioselectivity to L-proline.

7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties.

7alpha,11beta-Dimethyl-19-nortestosterone, made by 1,6-methyl addition to 17beta-acetoxy-11beta-methylestra-4,6-dien-3-one, was a highly potent and selective androgen response modulator, with enhanced androgen receptor binding, androgenic activity and anabolic:androgenic ratio over its two monomethyl homologs.

11beta-alkyl-Delta9-19-nortestosterone derivatives: high-affinity ligands and potent partial agonists of the androgen receptor.

We report the synthesis of novel steroidal androgen receptor ligands comprising 11beta-alkyl-Delta(9)-derivatives of 19-nortestosterone. These compounds are structurally related to the antiprogestin, antiglucocorticoid, and antiandrogen drug mifepristone (RU486). Nortestosterone analogues bearing 11beta-octyl and 11beta-decyl side-chains bind tightly to recombinant AR protein (IC(50) = 6.6 nM and IC(50) = 0.8 nM), block AR dimerization, exhibit activity against LNCaP prostate cancer cells, and comprise partial AR agonists with low antiglucocorticoid activity.

Synthesis of the 7alpha-cyano-(17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones: potential radioligands for androgen and progesterone receptors.

We report the preparation of the 7alpha-cyano derivative of the isomeric (17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones (IVNT) together with their binding affinity for the androgen receptor (AR) and their biodistribution in two different animal models. The cyano group was introduced at the 7alpha-position by hydrocyanation of 4,6-estradien-17beta-ol-3-one with diethylaluminum cyanide. Selective protection of the A-ring enone system as the dienol ether followed by ethynylation and deprotection under base and acid hydrolysis condition gave 7alpha-cyano-17alpha-ethynyl-19-nortestosterone. The stannyl derivatives were prepared by addition of tri-n-butylstannyl hydride and converted stereospecifically to the corresponding [125I]iodovinyl analog using [125I]NaI and H2O2. The [125I]iodovinylsteroids were intravenously administered to male rats and estrogen-primed immature female rats and tissue uptake was measured up to 6h post-injection. Co-administration of NLP-004 or ORG-2058, highly selective ligands for the progesterone receptor, to the female rats did not affect uterus uptake of the 125I-ligands. However co-injection of testosterone to DES-primed male rats induced a marked increase in prostate uptake of the 20Z-isomer of 7alpha-cyano-[125I]-IVNT. The relative binding affinity (RBA) of either 7alpha-cyano-(17alpha,20E/Z)-IVNT isomer for the AR is low (RBA=4 and 3, respectively, versus 100 for 5alpha-dihydrotestosterone (DHT)), suggesting the absence of a possible role of the AR in the localization process. These findings contrast previously reported data for the analogous 7alpha-methyl-[125I]-IVNT where co-administration of testosterone was shown to result in a 50% drop in prostate uptake. These data indicate that the addition of an electron withdrawing 7alpha-cyano group to 123I-labeled nortestosterone derivatives does not improve their potential to serve as SPECT agents for the imaging of AR densities in the prostate.

Synthesis of 17 beta-hydroxy esters of 4-estren-17 beta-ol-3-one and carbenicillin, ticarcillin, or functionalized oxacillin: potentially useful conjugates for beta-lactamase-based homogeneous immunoassays.

On the basis of the large range of kinetic constants of their substrates, beta-lactamases seem to be interesting enzymes for the development of homogeneous immunoassays. For this purpose, hapten-penicillin or -cephalosporin conjugates have to be prepared. The aim of this work is to couple the anabolizing steroid nandrolone to several penicillins characterized by extremely low K(m) and kcat values: ticarcillin, carbenicillin, and oxacillin. The easy decarboxylation of derivatives of phenylmalonic acid (carbenicillin) and thienylmalonic acid (ticarcillin) imposes the choice of very mild procedures which have been specifically adapted to each substance investigated. 4-Estren-17 beta-ol-3-one hemiphenylmalonate is conjugated to 6-aminopenicillanic acid after 1,1'-carbonyldiimidazole activation, while 4-estren-17 beta-ol-3-one hemi(3-thiophene)malonate is coupled to 6-aminopenicillanic acid after activation using methanesulfonyl chloride. Before conjugation of oxacillin, a carboxylated analogue of its side chain has been prepared. A procedure resorting to tert-butyl ester protection of the carboxyl group present on the isoxazole ring allows the binding of nandrolone to the remaining carboxyl followed, after specific deprotection, by the conjugation to 6-aminopenicillanic acid giving the oxacillin derivative. In this way, conjugates retaining immunological properties of nandrolone and high inhibiting power of beta-lactamases should be obtained.

Synthesis of (17 alpha,20E/Z)iodovinyl testosterone and 19-nortestosterone derivatives as potential radioligands for androgen and progesterone receptors.

To develop androgen and progesterone receptor-based radioligands for SPECT imaging we synthesized several radioiodinated 17 alpha-iodovinyl testosterone and 19-nortestosterone analogs and evaluated their biological properties. The synthesis of these compounds proceeds via the (17 alpha,20E/Z)stannyl intermediates and involves addition of tri-n-butyltin hydride to the 17 alpha-ethynyl group of the steroid using either azobisiso butyronitrile or triethylborane as a catalyst. The stannyl derivatives are stereospecifically converted to the corresponding (17 alpha,20E/Z)iodovinyl derivatives using molecular iodine, or to the [125I]iodovinyl analogs using [125I]NaI and H2O2. Androgen and progesterone receptor (AR and PgR) binding affinities were measured via a competitive in vitro binding assay. In general 19-nortestosterone derivatives showed higher receptor affinities as compared to the testosterone derivatives. In the latter series the highest PgR binding affinities were observed with the (17 alpha,20Z)iodovinyl-19-nortestosterone (IVNT) (92 vs 100 for R5020) followed by the 7 alpha-methyl analog, whereas the highest AR binding affinity was observed with the 7 alpha-Me-(17 alpha,20Z)IVNT (54 vs 100 for 5 alpha-dihydrotestosterone). These derivatives were also labeled with 125I and evaluated for their in vivo target organ uptake (prostate and estrogen-primed uterus). The highest PgR-mediated target tissue uptake was observed with the (17 alpha,20Z)-[125I]IVNT and its 7 alpha-methyl derivatives whereas only one derivative, the 7 alpha-Me-(17 alpha,20Z)-[125I]IVNT, showed AR-mediated dorsal prostate retention. Although some of the IVNT derivatives have interesting binding properties, the lack of in vivo selectivity does suggest that the 123I-labeled analogs are unlikely to be suitable for imaging of AR and PgR-rich tissues.

7 alpha-Methyl-17 alpha-(E-2'-[125I]iodovinyl)-19-nortestosterone: a new radioligand for the detection of androgen receptor.

We have synthesized two gamma-emitting, 125I-labeled steroids, E- and Z-7 alpha-methyl-17 alpha-(2'-[125I]iodovinyl)-19-nortestosterone [125I](E- and Z-MIVNT) for specific labeling of androgen receptors. [125I]E- and [125I]Z-MIVNT were synthesized stereospecifically from E- and Z-7 alpha-methyl-17 alpha-(2'-tri-n-butylstannyl-vinyl)-19-nortestosterone. The tin adducts were prepared by addition of tri-n-butyltin hydride to 7 alpha-methyl-17 alpha-ethynyl-19-nortestosterone, and after purification they were converted in high yield to the [125I]MIVNT isomers by reaction with 125I (generated in situ by oxidation of [125I]iodide with chloramine T). The 125I-labeled products were purified by high-performance liquid chromatography, and their mass determined with an ultraviolet detector (specific activity of both, approximately 2,200 Ci/mmol). In rat prostate cytosol, [125I]E-MIVNT bound with high affinity to a single class of binding sites. Nonspecific binding in the presence of 5 alpha-dihydrotestosterone was relatively low, and compared favorably with that obtained in parallel studies with [3H]methyltrienolone (R1881). The E-isomer bound prostate cytosol with at least twice the affinity of the Z-isomer; therefore, the interaction of the E-isomer with the androgen receptor as well as other steroid receptors was studied in greater detail. Complexes of the androgen receptor with [125I]E-MIVNT as well as [3H]R1881 dissociate very slowly at 4C (kdiss for both = 0.04 h-1). Displacement studies showed that the interaction of [125I]E-MIVNT with the androgen receptor is highly specific. Competition studies showed that unlabeled E-MIVNT binds poorly to other steroid receptors in rat tissue cytosols. These binding properties make [125I]E-MIVNT a promising ligand for study of the androgen receptor, and [123I]E-MIVNT a potential imaging agent for the detection of androgen-dependent tumors, such as prostate cancer.

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase.

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

20-[18F]fluoromibolerone, a positron-emitting radiotracer for androgen receptors: synthesis and tissue distribution studies.

To develop an androgen receptor-based, positron-emitting imaging agent for prostate tumors, we have prepared 20-fluoromibolerone (F-Mib) and evaluated its tissue distribution. This compound was synthesized in eight steps from 7 alpha-methyl-19-nortestosterone, with fluorine introduced in the penultimate step by fluoride ion displacement on a spirocyclic sulfate. Fluoromibolerone was obtained in 9%-19% radiochemical yield (decay corrected), at 1.5 hr after bombardment, with an effective specific activity of 217-283 Ci/mmol. The relative binding affinity of F-Mib is 53 (versus R1881 = 100 or mibolerone = 118). In tissue distribution studies in diethylstilbestrol-treated male rats, 18F-Mib demonstrates high target/tissue uptake efficiency and selectivity: the prostate uptake at 0.5 hr and 4 hr is 1.0%-1.3% injected dose/gram tissue (ID/g) and 0.5%-0.6% ID/g, respectively; the prostate-to-blood and the prostate-to-muscle (non-target) ratios are both ca. 4 at 0.5 hr, and increase to ca. 12 by 4 hr after injection. The observed distribution of 18F-Mib suggests that it may be useful for in vivo imaging of prostatic tumors in man by positron emission tomography.

Reaction of thiol nucleophiles with 1,2-epoxy- and 4,5-epoxy-estrene-3-one-17 beta-ols.

Four ring A steroidal epoxyenones as probable intermediate in the formation of catechol estrogens were synthesized. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one (9) and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (8) were synthesized from 17 beta-hydroxy-5 alpha-estra-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one (11) and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one (10) were prepared from 19-nortestosterone. The reaction of 9 and 10 with sodium/ethanethiol resulted in the formation of three types of reactions leading to multiple products: 1,4-addition, opening of epoxide, and epoxide opening followed by dehydration. Reaction of 8 with ethanethiol gave only one compound identified as 2-ethanethio-1,4-estradien-17 beta-ol-3-one, while reaction of 9 with ethanethiol gave an unusual product identified as 4-estren-1 alpha,17 beta-diol-3-one. Unlike reaction of ethanethiol with 9 and 10, reaction with N-acetylecysteine or glutathione results in epoxide opening followed by dehydration leading to the formation of estradiol-4-thioethers.

125I-ligand for progesterone receptor: 17 alpha-(6'-iodohex-1'-ynyl)-19-nortestosterone.

Progesterone receptor (PgR) is known to be a significant indicator of hormone dependency of a breast tumor and also an important prognostic factor for recurrence and survival. Relatively low specific activity of tritium-labeled ligands makes it difficult to detect the presence of PgR in very small specimens and in specimens with low PgR content. We therefore undertook the synthesis of a radioiodinated progestin. We have synthesized unlabeled 17 alpha-(omega-haloalk-1'-ynyl)-19-nortestosterones with 4, 6, and 8 carbon side chains; these have high affinity for PgR and clear potential for conversion to a 125I form. Since the six-carbon side chain analogue had the highest relative PgR affinity (38% of the affinity of R5020, using PgR from T47D human breast cancer cells), it was selected for preparation of the 125I form by radiohalogen exchange of a omega-bromoalkynyl precursor with carrier-free Na125I. This new ligand was found by Scatchard analysis to have a Kd of 0.47 nM, as compared with [3H]R5020 at 0.22 nM and [3H]progesterone at 2.0 nM. (For affinity studies, all ligands were diluted in dimethylformamide and added to PgR-containing T47D cytosol to give a final DMF concentration of 4%, in order to prevent R5020 and the test compounds from binding nonspecifically to proteins and glass/plasticware.) The new 125I-ligand showed very little instability when stored in ethanol at approximately 10 degrees C even for several months. Thus, 17 alpha-(6'-[125I]iodohex-1'-ynyl)-19-nortestosterone should prove to be a useful ligand for high sensitivity PgR assays.

delta 9-[16 alpha-125I]iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor.

We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one [delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)] labeled with 125I (delta 9-[16 alpha-125I]INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available [3H]R5020. We have characterized the interaction of delta 9-[16 alpha-125I]INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM [3H]progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-[16 alpha-125I]INT and [3H]progesterone estimated the same number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-[16 alpha-125I]INT of about 2.7 nM. The binding of delta 9-[16 alpha-125I]INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-[16 alpha-125I]INT covalently labeled the same mol wt receptor forms as [3H]R5020. Although the efficiency of cross-linking was similar for [3H]R5020 (3%) and delta 9-[16 alpha-125I]INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-[16 alpha-125I]INT and the lack of sample quench. The use of delta 9-[16 alpha-125I]INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.

A progesterone receptor affinity chromatography reagent: 17 alpha-hexynyl nortestosterone sepharose.

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17 alpha-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17 alpha-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17 alpha-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred microliter of these beads adsorbed 71% of the PgR present in 1 ml of cytosol from T47D cells. This adsorption was inhibited by 10 microM progesterone but not cortisol, indicating the specificity of the binding. Comparisons with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.

(Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)-4-estren-3-one: a new specific gamma-emitting ligand for determination of progesterone receptor.

An iodine-125 labeled ligand for progesterone receptor determination was synthesized: (Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)-4-estren-3-one ([125I]SH-D 510). The ligand is stable chemically as well as under the conditions of a receptor assay. The relative binding affinity of the nonradioactive compound towards human uterine progesterone receptor was 7.0 for the Z-isomer (promegestone (R5020) 1.0) and 0.95 for the E-isomer. 4 S and 8 S receptor forms were obtained on sucrose density gradient analysis. Progesterone receptors were assayed in 103 human mammary tumour cytosols, using either [3H]promegestone or [125I]SH-D 510. The coefficient of correlation was r = 0.951.

E-17 alpha-(2-[125I]iodovinyl)-19-nortestosterone: the synthesis of a gamma-emitting ligand for the progesterone receptor.

We have synthesized a gamma-emitting steroid, E-17 alpha-(2-[125I]iodovinyl)-19-nortestosterone (E-IVNT), which is a useful ligand for the sensitive and accurate assay of the progesterone receptor. The synthetic scheme is rapid and is performed with readily available materials. This compound, [125I]E-IVNT, is stable and binds with high affinity to the progesterone receptor.