Yvonne Heuze

Recent advances in the chemistry and pharmacological activity of new steroidal antiandrogens and 5α-reductase inhibitors

The object of this paper is to summarize for the past two years the most recent development in the field of prostate cancer and 5 alpha-reductase inhibitors. In addition we are also including some results on the synthesis and pharmacological evaluation of new steroidal compounds developed in our laboratory. Most of the new steroidal derivatives are based on the progesterone skeleton and showed a high inhibitory activity for the enzyme 5 alpha-reductase. Presently, similar compounds are used for the treatment of androgen dependent diseases such as: hirsutism, androgenic alopecia, benign prostatic hyperplasia and prostate cancer. Dihydrotestosterone 2 (Fig. 1) a 5 alpha-reduced metabolite of testosterone 1 has been implicated as a causative factor for the progression of these diseases, largely through the clinical evaluation of males who are genetically deficient of the enzyme steroid 5 alpha-reductase. 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 7 (Fig. 3) a 5 alpha-reductase inhibitor has greatly alleviated the symptoms associated with benign prostatic hyperplasia. In our laboratory, we recently synthesized several new 16 beta-methylpregnadiene-3,20-diones: 40, 41 (Fig. 8), 16 beta-phenylpregnadiene-3,17a-dione derivatives 46 and 47 (Fig. 9) and 49 (C-4 bromoderivative) (Fig. 11), 52-56 (Fig. 13). The analogue pregnatriene derivatives were also prepared: 44, 45 (Fig. 9) 50, 51 (Fig. 11) and 57-60 (Fig. 13) These compounds were evaluated as 5 alpha-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 of homogenates of gonadectomized male hamsters. All trienones 44, 45, 50, 51 and 57-60 in all biological models showed consistently a higher 5 alpha-reductase inhibitory activity than the corresponding dienones: 40, 41, 46, 47, 49 and 52-56. We believe that with these compounds the 5 alpha-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 thus showing a higher inhibitory activity.

Molecular interactions of progesterone derivatives with 5α-reductase types 1 and 2 and androgen receptors

The aim of this study was to ascertain the inhibitory effect of several progesterone derivatives for 5 alpha-reductase types 1 and 2 isozymes and to determine the binding to the androgen receptor. The 3,20-dioxopregna-4-ene-17 alpha-yl acetate 4 containing an acetoxy group in C-17 and steroid 17 alpha-hydroxypregn-4-ene-3,20-dione 5 having a hydroxyl group in the same position inhibited both isozymes. On the other hand, 17 alpha-hydroxy-4,5-epoxypregnan-3,20-dione 6 with an epoxy function at C-4, inhibited only the type 1 enzyme. Steroid 4-chloro-17 alpha-hydroxypregn-4-ene-3,20-dione 7a and 4-bromo-17 alpha-hydroxypregn-4-ene-3,20-dione 7b having the C-4 conjugated system and a chlorine or a bromine atom at C-4 respectively, inhibited both types of 5 alpha-reductase. These results indicate that an increase in the electronegativity of ring A produces a major inhibitory activity for 5 alpha-reductase type 1; however this increase was not observed for type 2 enzyme. When the free hydroxyl group of 7a or 7b was esterified, compounds 3,20-dioxo-4-chloropregn-4-ene-17 alpha yl-4-ethylbenzoate 8a and 3,20-dioxo-4-bromopregn-4-ene-17 alpha yl-4-ethylbenzoate 8b were obtained; these steroids inhibited only the 5 alpha-reductase type 2 enzyme. Finasteride and steroids 4, 5, 7b, 8a showed a comparable in vivo pharmacological activity, however the IC(50) values of these compounds were higher as compared to that of finasteride. These results indicated also that steroids 4, 5, 7a, and 7b bind to the androgen receptor whereas compounds 6, 8a and 8b failed to do so. The overall data from this study showed that steroids 5 and 7b bind to the AR and decreased of the growth of prostate and seminal vesicles. Moreover, 4 decreased also the growth of seminal vesicles.

Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties.

Biological Evaluation of Androstene Derivatives

The effect of several new dihydroepiandrosterone ester derivatives A2–A6 was demonstrated using female cycling mice, which were synchronized for estrus with luteinizing hormone‐releasing hormone (LHRH) and injected with the steroids. The binding to the progesterone receptor (PR), was obtained from the cytosol of uteri from adult estrogen‐primed rabbits. A1 binds to the PR and inhibited the ovulation in cycling mice stimulated with LHRH. The activity of the endometrium and mammary glands in these mice was markedly reduced as compared to the control. A2, A4, and A5 were not active; nevertheless, A3 binds to the PR with high affinity. However, this steroid did not produce any effect as compared to that observed for the control in the endometrial and mammary glands. A6 binds to the PR with the highest affinity and induces a synergistic activity with progesterone in these tissues. Furthermore, A6 inhibited the ovulation in the same manner as A1. These results suggested that A1 and A6 are blocking the gonadotropin secretion. A1 inhibited the conversion of progesterone to 5α‐progesterone. As a result of this, a blockage of the ductal and alveolar epithelial cell proliferation in the mammary and endometrial glands, which depends on 5α‐progesterone, was also observed.

In vivo and in vitro effect of androstene derivatives as 5α-reductase type 1 enzyme inhibitors

The aim of these studies was to synthesize twelve ester derivatives of dehydroepiandrosterone with therapeutic potential. The effect of 1–12 was demonstrated in the flank organs of gonadectomized hamsters treated with testosterone and the synthesized steroids. In vitro studies were carried out determining the IC50 values for the inhibition of the activity of 5α-reductase type 1 and 2, which are present in rat liver and human prostate respectively. The binding of 1–12 to the androgen receptors (AR) was determined using rat’s prostate cytosol. Steroids 1–12 containing different substituents in the phenyl group of the ester moiety in C-3 reduced the flank organs and inhibited the activity of 5α-R type 1; however only steroids 1 and 2 inhibited 5α-R type 2. 1–12 did not bind to the AR. The modification of one atom of the substituents in the phenyl group of the ester moiety in C-3 changed their biological potency (IC50).

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([3H] 4-dione) to [3H]dihydrotestosterone ([3H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [3H] 4-dione to [3H]DHT, homogenates of human prostate were incubated with [3H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [3H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [3H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [3H] 4-dione to [3H]DHT, showing IC50 values of 2 and 1.6 nM, respectively.