Ivonne Heuze

Steroid 5α-Reductase Inhibitors

The objective of this study is to synthesize new steroidal compounds based on the progesterone skeleton with a high inhibitory activity for the enzyme 5α-reductase. Presently similar compounds are being used for the treatment of androgen dependent diseases such as: hirsutism, androgenic alopecia, bening prostatic hyperplasia and prostate cancer. Dihydrotestosterone 2 (Fig. (1)), a 55α-reduced metabolite of testosterone 1 has been implicated as a causative factor in the progression of these diseases, largely through the clinical evaluation of males who are genetically deficient of steroid 55α-reductase enzyme. As a result of this study, the inhibition of this enzyme has become a pharmacological strategy for the design and synthesis of new antiandrogenic drugs. The advent of finasteride 8 (Fig. (4)) a 55α-reductase inhibitor has grately alleviated the symptoms associated with benign prostatic hyperplasia. In our laboratory we recently synthesized several new 165β-methyl-pregnadiene-3,20-diones derivatives 27 (Fig.(6)), 38-42 (Fig. (11)), 16β-phenyl-pregnadiene-3,17a-dione derivatives 32-33 (Fig. (7)), 16β-phenylpregnatriene- 3,17a-diones, 30, 31 (Fig. (7)) and 16β-methyl-pregnatriene-3,20-diones 43-46 (Fig. (11)). These compounds were evaluated as 5α-reductase inhibitors in the following biological models: Penicillium crustosum broths, the flank organs of gonadectomized male hamsters, the incorporation of radiolabeled sodium acetate into lipids, the effect of the new steroids on the reduction of the weight of the seminal vesicles and on the in vitro metabolism of [3H]T to [3H]DHT in seminal vesicles homogenates of gonadectomized male hamsters. All trienones 30, 31, and 43-46 in all biological models showed consistently a higher 5α-reductase inhibitory activity than the corresponding dienones 27, 32, 33 and 38-42. We believe that with these compounds the 5α-reductase enzyme is inactivated by an irreversible Michael type addition of the nucleophilic portion of the enzyme to the conjugated double bond of the steroid. The trienones having a more coplanar structure react faster with the enzyme and thus show a higher inhibitory activity.

Steroids with a carbamate function at C-17, a novel class of inhibitors for human and hamster steroid 5α-reductase

In order to study the biological activity of the two novel steroidal carbamates derivatives: 8a and 8b, we determined the concentration of both compounds that inhibit the 50% of the activity of human prostate 5alpha-reductase enzyme, as well as the in vivo effect of these compounds in the weight of hamster prostate and flank organs diameter size. We determined also, the capacity of these steroids to bind to the androgen receptors present in the rat prostate cytosol. Furthermore the activity of these compounds on the mRNA expression of glycerol 3-phosphate acyl transferase (GPAT) in flank organs was analyzed by RT-PCR. This enzyme induces the triglycerides synthesis, which is increased by T in flank organs. The results from this study indicated that steroids 8a and 8b inhibited the human 5alpha-reductase activity. Compound 8b, which contains a bromine atom in the molecule, decreased the inhibitory effect of the human 5alpha-reductase activity, whereas steroid 8a, which lacks a halogen atom did not show any decrease in the activity of this enzyme. The competition studies demonstrated that 8a and 8b did not inhibit mibolerone binding to the androgen receptor present in the rat prostate cytosol. However, the in vivo activity of both steroids was similar; steroids 8a and 8b had a tendency to decrease the weight of the hamster prostate although this parameter was not statistically significant. These compounds also significantly reduced the diameter of the pigmented spot of hamster flank organs, which are androgen dependent skins pilosebaceous structures. Steroids 8a and 8b, decreased the transcription of mRNA encoding for GPAT in intact hamsters flank organs topically treated in a similar way as in gonadectomized non-treated animals. These results suggest that mRNA encoding for GPAT is induced by DHT in this tissue.

Novel C-6 substituted and unsubstituted pregnane derivatives as 5α-reductase inhibitors and their effect on hamster flank organs diameter size

The present study is addressed to ascertain the inhibitory effect of several progesterone derivatives having a chlorine substituent at C-6 (12a-12d), 15 with a bromine substituent at C-6 and 14a-14d, without any halogen atom at C-6 all having an ester side chain at C-17 (benzoate ester bearing a Cl, F and a Br atom at C-4 position of the phenyl ring) on the 5alpha-reductase enzyme activity present in human prostate. In addition, it was also of interest to investigate the pharmacological effect on hamster flank organs diameter size. In order to study the structure-activity relationships of steroids 12a-12d, 14a-14d and 15 we determined the concentration of these steroids that inhibited 50% of the activity of human prostate 5alpha-reductase enzyme (IC(50)), as well as the in vivo effect of these compounds in the hamster flank organs diameter size. We also ascertained, the capacity of these steroids to bind to the androgen receptors present in the rat prostate cytosol using labeled mibolerone (MIB) for monitoring the binding to the androgen receptor. The results from this study indicated that compounds 12a-12d (having a chlorine substituent at C-6), 14a-14d (lacking a halogen atom at C-6), 13 and 15 (having a bromine atom at C-6) showed an increased antiandrogenic effect (lower value for the diameter of the flank organs) as compared to the flank organs from testosterone-treated hamsters. On the other hand, the series of compounds containing a chlorine substituent at C-6 compounds (12a-12d) showed a higher antiandrogenic activity as compared to the compounds lacking a halogen atom at C-6 (14a, 14b and 14d). Although compounds 13 and 15 decreased the flank organs diameter size, however, this increase was not statistically significant as compared to that of the commercially available product finasteride. The steroidal derivatives 13, 14a-14d (lacking the chlorine substituent at C-6) and 15 (having a bromine atom at C-6) exhibited a higher 5alpha-reductase inhibitory activity (lower IC(50) values) as compared to the series of compounds 12a-12d having the halogen substituent at C-6. Finasteride reduced the diameter size of the flank organs. The effect of this steroid and compounds 12a-12d, 13, 14a-14d and 15 on hamster flank organs can be explained by the fact that these steroids did not bind to the androgens receptor, which indicates that its mechanism of action is an inhibiting for the 5alpha-reductase activity. This enzyme is present in the hamster flank organs and was inhibited by the novel steroids in the human prostate homogenates.

Biological activity of novel progesterone derivatives having a bulky ester side chains at C-3

Antiandrogens are widely used agents for the treatment of androgen dependent diseases as inhibitors of androgen receptors (AR) action. Although the precise mechanism of antiandrogen action is not yet elucidated, recent studies indicate the involvement of the structure of the ligand in relation with the nuclear co-repressors. In the present study, we investigated the relationship between logP (the partition coefficient) of four pregnane derivatives 9a-9d and their biological activity. For this purpose, we determined the relative binding affinity (RBA) of steroids 9a-9d to androgen receptor (AR) obtained from rat prostate cytosol, using labeled mibolerone (MIB) as ligand. The IC(50) value of each compound was calculated according to the plots of concentration versus percentage of binding. The in vivo effect of 9a-9d was determined on the weight of the prostate and seminal vesicles from castrated hamsters treated with dihydrotestosterone. The four compounds bind to the androgen receptor with different relative binding affinity (RBA). Compound 9d having a logP of 4.17 showed the highest RBA>100% as compared to compound 9a having a logP of 2.92 which exhibited a RBA of only 2.85%. These data show a very good correlation between the lipophilicity of these compounds represented by logP and the percentage of RBA. The in vivo experiments showed that all new compound 9a-9d reduced the weight of the prostate gland as well as the seminal vesicles. Steroids 9c and 9d having a logP of 3.75 and 4.17, respectively, showed the highest antiandrogenic effect.

Aromatic esters of progesterone as 5α-reductase and prostate growth inhibitors

The aim of this study was to determine the biological activity of 4 steroidal derivatives (9a, 9b and 10a, 10b) prepared from the commercially available 17α acetoxyprogesterone, where 9a, 9b, have the Δ4-3-oxo structure and 10a and 10b an epoxy group at C-4 and C-5. These steroids were tested as inhibitors of 5α-reductase enzyme, which is present in androgen-dependent tissues and converts testosterone to its more active reduced metabolite dihydrotestosterone. The pharmacological effect of these steroids was demonstrated by the significant decrease of the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with steroids 10a and 10b. For the studies in vitro the IC50 values were determined by measuring the steroid concentration that inhibits 50% of the activity of-5α-reductase. In this study we also determined the capacity of these steroids to bind to the androgen receptor present in the rat prostate cytosol. The results from this work indicated that compounds 9a, 9b, 10a, and 10b inhibited the 5α reductase activity with IC50 values of 360, 370, 13 and 4.9 nM respectively. However these steroids did not bind to the androgen receptors since none competed with labeled mibolerone. Steroid 10b, an epoxy steroidal derivative containing bromine atom in the ester moiety, was the most active inhibitor of 5α-reductase enzyme, present in human prostate homogenates with an IC50 value of 4.9 nM and also showed in vivo pharmacological activity since it decreased the weight of the prostate from hamsters treated with testosterone in a similar way as finasteride.

Synthesis and biological effect of halogen substituted phenyl acetic acid derivatives of progesterone as potent progesterone receptor antagonists

In this paper, we report the relative binding affinities to the progesterone receptor (PR) of several progesterone derivatives containing an acetoxyphenyl substituent at C-17 and their structure-bioactivity relationship. The inhibitory effect to ovulation as well as their function as interrupters of endometrial maturation is also described. The biological activity of the novel steroids was determined in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. The cytosol used for the determination of the PR, was obtained from the uteri of adult estrogen-primed rabbits and the androgen (AR), mineralocorticoid (MR) and glucocorticoid (GR) receptors were determined in the cytosolic fractions from the prostate of castrated rats and from the kidneys and livers of adenalectomized male rats. We evaluated six related steroidal compounds 8a-8f differing in the nature of the 17alpha ester side chain for the inhibition of [3H] R5020 binding to the PR. The IC50 values for the displacement of [3H] R5020 binding to the PR and its relative binding affinities (RBAs) were determined. Progesterone and R5020 had similar IC(50) values; steroids 8a, 8f and 8c bind to the progesterone receptor with RBAs of 100%, whereas 8e, 8b and 8d have RBA values <100%. These data indicate that there is a relationship between the structure of these steroids and their binding activity to the progesterone receptors. Having demonstrated in this study that steroids 8a-8f bind to the PR, we also evaluated the receptors selectivity, since some progesterone derivatives bind to AR, MR, GR receptors. We demonstrated that the tested steroids did not bind to the AR, MR, GR, since none of the steroids inhibited the labeled mibolerone, aldosterone or dexamethasone binding to the AR, MR or GR, respectively. These results show that the novel compounds have certain selectivity for the PR. After LHRH treatment, the mice of the control group showed the presence of ova in the oviduct, whereas the animals treated with steroids 8a, 8f, 8e and 8c with RBAs of 92-100%, did not exhibit any ovum in the oviducts. As a result of this study, it is evident that the novel steroids 8a, 8f, 8e and 8c inhibited the ovulation in these animals at dose of 0.22 mg/kg. After the treatment with LHRH, the uterus of the control group showed the typical progestational activity with an enlarged endometrial thickness with secretory activity. However, the endometrium of the mice treated with steroids 8a, 8f, 8e and 8c (with RBAs of 92-100%) neither did show any enlargement of the endometrium, nor a secretory activity could be detected. The diameter of the uterus was also significantly reduced compared to those of the control group, thus indicating that compounds 8a, 8f, 8e and 8c had antagonistic activity in this tissue. The overall data showed that steroids 8a, 8f, 8e and 8c have a high and selective binding activity to the PR. Furthermore there is a relationship between the structure of these steroids and their binding activity, since the presence of fluorine atom in meta position in the acetoxyphenyl substituent at C-17, improved the binding activity as compared to that for the ortho and para positions. These data also demonstrated that 8a-8f have an anti-progestational activity in vivo, and therefore they have better characteristics than the compounds previously reported.

Antiandrogenic and apoptotic effects of RU-486 on animal prostate.

Mifepristone (RU-486) is a potent antagonist of steroid hormone receptors such as glucocoticoid and progesterone receptors. This compound also is a very strong inducer of the interaction between androgen receptors and corepressors NCoR and SMRT and therefore could be used as selective receptor modulator. In this study we determined the relative binding affinity of RU-486 to androgen receptors (AR) obtained from rat prostate cytosol as well as the in vivo effect of different doses of RU-486 on the prostate weight of hamsters treated with dihydrotestosterone and/or RU-486. We determined also the prostate cell death (apoptosis) in hamster treated with, dihydrotestosterone (DHT) and/or RU-486. The results of this study indicated that the relative binding affinity of RU-486 for AR is 4.3%. The data from the in vivo experiments also showed that RU-486 inhibited the prostate weight significantly in the highest doses thus indicating the antagonistic action of this compound on hamster prostate. The immunohistochemistry analysis showed that after 1 month of castration, the hamster prostate was atrophic. Treatment with DHT produced epithelial cell activity (measured by the increase in the prostate weight) and very low rate of apoptosis. When DHT was administered together with RU-486 (10 mg/kg) no change was observed. On the other hand, DHT plus higher doses of RU-486 (40, 80 mg/kg) resulted in an increase of apoptosis in stromal and secretory epithelial cells. In addition to the increase of the prostate cell apoptosis produced by the treatment with high dose of RU-486, other factors could contribute to the decrease of the prostate weight observed. Another possibility could be a reduction in the ductal fluid due to poor epithelial cell secretory activity more than apoptosis itself. Furthermore, in this experiment, RU-486 could have inhibited the growth of the prostate gland produced by DHT in a greater extent than the induction of atrophy and cell death. This fact could depend on the doses used, due to the low affinity of this compound for the androgen receptors.

New Aromatic Esters of Progesterone as Antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17α-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17α-(p-chlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17α-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17α-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1–4 have a weak inhibitory activity on 5α-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114 μM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds. The binding efficiency of the synthesized steroids 1–4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1–4.

Relative binding affinity of novel steroids to androgen receptors in hamster prostate.

The in vivo and in vitro antiandrogenic activity of four aromatic esters 10a–10d, one aliphatic ester 10e based on the pregna-4,16-diene-6, 20-dione structure and two aromatic 17c, 17d and two aliphatic valeroyloxy esters 17a, 17b based on the more saturated 4-pregnene-6,20-dione skeleton was examined. The biological activity of steroids 9, 10a–10e and 17a–17d, was determined using prostate glands from gonadectomized adult male golden hamsters. In the in vitro studies, the relative binding affinity of these steroids to cytoplasmic androgen receptor (AR) of hamster prostate was determined from, the corresponding IC50 values obtained from the competitive binding plots. The standards dihydrotestosterone (DHT) and cyproterone (CA) acetate used have displaced [3H]DHT from the AR with an IC50 value of 3.2 and 4.4 nM respectively. All steroidal compounds synthesized in this study showed a binding affinity for the androgen receptor, present in the cytosol from prostate hamster; compounds 10a–10c showed the highest affinities for this receptor. The in vivo experiments showed that all steroidal derivatives were subcutaneously active, since they decreased the weight of the prostate gland in gonadectomized hamsters treated with DHT, and are antagonists for the androgen receptor since they block the DHT-induced prostate weight gain. The derivatives having the more conjugated 4,16-pregnadiene-6, 20-dione system (10a–10c) exhibited a higher antiandrogenic activity than the corresponding steroids (17a–17d) based on the more saturated 4-pregnene-6,20-dione system.

New-D-homoandrost-4,6-diene derivatives as potent progesterone receptor antagonist

The aim of this study was to synthesize three different D-homoandrostadiene derivatives (2-4) and study their biological activity. We carried out in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. It was also determined the binding of these compounds to the progesterone receptors (PR). Since these steroids have a new D-homoandrostandienone skeleton in their molecular structure, it was of interest also to study their binding to the androgen receptors (AR). After LHRH treatment, the mice of the control group showed the presence of 14+/-4 corpus lutea in the ovary whereas the animals treated with steroids 2-4, with RBAs of 100%, exhibited 11+/-7, 12+/-2, and 10+/-4 respectively. As a result of this study, it is evident that these steroids did not inhibit the ovulation in these animals. The uterus of the control group, showed the typical progestational activity with an enlarged endometrial thickness with a secretory activity. However, the endometrium of the mice treated with steroids 2-4 did not show an enlargement of the endometrium and no secretory activity could be detected. This fact indicates that compounds 2-4 had antagonistic activity in this tissue. The overall data show that steroids 2-4 are antagonists of the PR. However, they do not bind to the AR. These results also demonstrate that 2-4 have an antiprogestational activity in vivo, but do not decrease the number of corpus lutea in the ovary of mice treated with LHRH.