Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17alpha-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones.

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17alpha-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11beta-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17alpha-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17alpha-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17alpha-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17alpha-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17alpha-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. (ABSTRACT TRUNCATED)

Hormonal properties of norethisterone, 7alpha-methyl-norethisterone and their derivatives.

Norethisterone (NET) is a progestagenic compound with very weak androgenicity and estrogenicity. These low androgenic and estrogenic activities may be attributed to NET itself or induced by metabolites of NET. In order to improve the bioactivity of NET, the effects of a 7alpha-methyl substitution were studied. Thus this study has two objectives: first the comparison between biological activities of NET and 7alpha-methyl-NET (MeNET), and second the biological activity of tentative metabolites of NET and those of MeNET. The metabolites consist of a 3-keto-, 3alpha- or 3beta-hydroxy-group located next to a carbon 4 to 5 double bond (Delta(4)) or a 5alpha-hydrogen atom. The 7alpha-methyl substitution was of special interest as it prevents 5alpha-reduction. The biological activities of NET, MeNET and their potential metabolites were assessed by in vitro binding, transactivation and proliferation assays on progesterone (PR), androgen (AR), estrogen (ER) and glucocorticoid (GR) receptors and by in vivo progestagenic McPhail, androgenic Hershberger, estrogenic Allen-Doisy tests and combined estrogenic and progestagenic ovulation inhibition tests. NET is a compound with five- to eight-fold weaker PR binding and transactivation activities than the reference compound Org 2058 (100%) and two-fold stronger than progesterone. Binding and transactivation activities of NET for AR (DHT=100%) are 3.2 and 1.1%, respectively, for ER none (E2=100%) and for GR below 1% (DEX=100%). MeNET is 1.5- to two-fold less progestagenic and ten- to 20-fold more androgenic than NET, while it does not show activity for ER and GR. The relative binding affinity of 5alpha-NET was seven-fold lower for PR and 1.5-fold higher for AR than for NET, while in transactivation assays 5alpha-NET was only active at levels below 1% for all tested receptors. 3beta-Hydroxy-(5alpha-reduced)-metabolites showed clear ER binding and transactivation activities, while 3alpha-hydroxy-(5alpha-reduced)-metabolites did hardly possess these characteristics. These hydroxy metabolites did not bind or activate other receptors. Substitution of 7alpha-methyl to NET metabolites led to similar characteristics, but with higher activities for AR and ER and weaker activity for PR. The outcome of in vivo tests showed a remarkable effect for MeNET. Progestagenic activity in rabbits appeared for NET equipotent to or eight-fold higher than for MeNET, after subcutaneous or oral treatment, respectively. On the other hand, MeNET showed in rats a ten-fold higher androgenicity and eight-fold higher estrogenicity than NET. Ovulation inhibition was induced at very low oral or subcutaneous dose levels, being 120- or ten-fold lower than for NET, respectively. The estrogenicity can also be induced by 3alpha- or 3beta-hydroxy metabolites of MeNET, which are 15 or even more than 40-fold stronger than those of NET, respectively. In conclusion, after the introduction of a 7alpha-methyl substituent to NET an increased estrogenicity and androgenicity and a reduced progestagenic activity was found. The in vivo estrogenicity is mainly due to 3beta-hydroxy-MeNET and to a lesser extent to 3alpha-hydroxy-MeNET, while the androgenicity and progestagenicity are most likely caused by MeNET itself. Since the 7alpha-methyl substituent inhibits 5alpha-reductase, 5alpha-reduced MeNET metabolites can be excluded from biological activities. As MeNET is a very effective ovulation inhibitor, due to its mixed progestagenic and estrogenic profile, a further reduction of androgenicity of MeNET may yield new contraceptives with an attractive profile for contraception.

Human progesterone receptor A and B isoforms in CHO cells. I. Stable transfection of receptor and receptor-responsive reporter genes: transcription modulation by (anti)progestagens.

A hormone-dependent transcription modulation system was established on the basis of a two-step transfection procedure of the human progesterone receptor isoforms (hPR-A and hPR-B, respectively) and a progesterone receptor-responsive reporter (MMTV-Luc). In the first step, stable transfection of the hPR-A and hPR-B isoform-encoding cDNAs was performed in the steroid receptor-negative CHO K1 cell line. Individual clones were characterized for hPR-isoform expression with respect to Western immuno-blotting, transcriptional activation and hormone binding. With respect to the latter characteristic, individual hPR-isoforms demonstrated similar dissociation constants (Kd for hPR-A: 0.5 +/- 0.3 and hPR-B: 0.8 +/- 0.3 nM, respectively) irrespective of the amount of receptor isoform expressed (Bmax varying from 4.1 to 33.2 nM). The Kd values observed for individual hPR-isoforms were comparable to those found for human breast tumor MCF-7 cells (Kd for hPR-A + hPR-B: 0.6 +/- 0.3 nM). In the second step, hPR-isoform expressing CHO clones were supertransfected with a MMTV-Luc reporter construct resulting in permanent cell lines useful for testing the activity of natural and synthetic steroids in their ability to modulate gene transcription. Both isoform-specific reporter cell lines responded in a similar ranking order towards different progesterone reference compounds such as Org 2058, progesterone (Prog), R5020, norethisterone (NE), and medroxy progesterone acetate (MPA). Moreover, a good correlation was observed between the relative binding affinity (RBA) and the transcriptional activation potency of these compounds towards the individual hPR-isoforms. The latter correlation could not only be demonstrated for the progestagenic agonist reference compounds but was also observed for the progestagenic antagonist reference compounds like Org 33628, Org 31710, RU 38486 and ZK 98299. The major difference observed between the individual PR-isoforms was related to the degree of stimulation of the reporter gene (MMTV-based) within the cellular CHO context. Therefore, these cell lines can be used for the determination and quantitation of the activity of (anti)progestagenic compounds in vitro but may also be useful to predict the activity of compounds in vivo (see also II Comparison of binding, transactivation and ED50 values of several synthetic (anti) progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats).

Human progesterone receptor A and B isoforms in CHO cells. II. Comparison of binding, transactivation and ED50 values of several synthetic (anti)progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats.

The human progesterone receptor A and B isoforms (hPR-A and hPR-B) were stably transfected in Chinese Hamster Ovary (CHO) cells in the presence or absence of the mouse mamma tumor virus (MMTV) promoter and luciferase (LUC) reporter gene. In this way four stably transfected CHO cell lines, i.e. hPR-A, hPR-B, hPR-A-MMTV-LUC and hPR-B-MMTV-LUC cells, were prepared. hPR-A and -B isoforms were compared by binding and transactivation analysis for 14 progestagens and 7 antiprogestagens. Thereby Org 2058 was used as standard in both agonistic and binding assays and Org 31710 in antagonistic assays. The obtained data were compared with relative binding affinities (RBA) for both hPR-A and -B, which are present in human breast tumor MCF-7 cells, and with biopotency estimations with McPhail tests in rabbits and ovulation inhibition and pregnancy interruption tests in rats. The relative binding affinities of 14 progestagens and 7 antiprogestagens towards hPR-A, hPR-B or hPR-A/B of either CHO or MCF-7 cells were highly correlated with respect to ranking. This was also shown by the high correlation coefficients between the RBA's of hPR-B and hPR-A in CHO cells (r = 0.983) and between those of hPR-B of CHO and hPR A/B of MCF-7 cells (r = 0.957). The transactivation data of the 14 progestagens and 7 antiprogestagens for the hPR-B-MMTV-LUC cells were compared with those for hPR-A-MMTV-LUC cells and showed no differences between both cell lines with exception of the progestagens Org 32704 and 33766 and the antiprogestagen Org 33245. Therefore only the relative agonistic activities (RAA) and relative antagonistic activities (RANTA) of hPR-B-MMTV-LUC cells were compared with RBA values of hPR-B, showing a high similarity in ranking for the tested compounds, and high correlation coefficients of r = 0.91 and r = 0.97, respectively. Remarkably, RBA's were 1.6 fold higher than RAA's and RANTA's. These in vitro RBA, RAA and RANTA values for hPR-B were checked for their pharmacological relevance by in vivo biopotency measurements with the 14 progestagens and 7 antiprogestagens in rabbits and rats. The in vitro binding and transactivation potencies of progestagens appeared to be very predictive for in vivo analysis on endometrium proliferation in rabbits in the McPhail test with correlation coefficients of r = 0.81 and r = 0.87, while ovulation inhibition in rats correlated less well with r = 0.516 and r = 0.65. On the other hand, the antiprogestagenic potencies found with binding and transactivation assays had a good correlation with the potencies in the pregnancy interruption test in rats for all antiprogestagens tested, being r = 0.849 and r = 0.744, respectively. In conclusion, the binding and transactivation potencies for the tested compounds in hPR-A and -B containing cell lines showed in general a good resemblance. The transactivation studies with hPR-B-MMTV-LUC cells indicated that ranking of compounds was fairly identical to binding analysis and could be used for pre-screening of the (anti)-progestagenic bioactivity in the McPhail test in rabbits, the ovulation inhibition test and the pregnancy interruption test in rats. Therefore this transactivation assay can replace binding assays. Moreover, direct pre-screening of agonists, antagonists and partial antagonists is even possible in this in vitro bioassay, making transactivation assays for a particular class of chemical compounds to a valuable pre-screening tool for in vivo studies.