Roch P. Boivin

A concerted, rational design of type 1 17beta-hydroxysteroid dehydrogenase inhibitors: estradiol-adenosine hybrids with high affinity

Human estrogenic 17β‐hydroxysteroid dehydrogenase (17β‐HSD type 1) catalyzes the final step in the synthesis of active estrogens that stimulate the proliferation of breast cancer cells. Based on the initial premise to make use of the binding energies of both the substrate and cofactor sites, and molecular modeling starting from the enzyme structure, several estradiol‐adenosine hybrids were designed and synthesized. Among these hybrids, EM‐1745 with a linker of 8‐CH2 groups is proved to be the best competitive inhibitor with a Ki of 3.0 ± 0.8 nM. The crystal structure of the EM‐1745 enzyme complex at 1.6 Å provides evidence at atomic resolution of strong interactions between both the steroid and cofactor moieties and the enzyme molecule, as illustrated by a σAweighted 2Fo‐Fc electron density map contoured at 3.0 σ. The substrate entry loop is further stabilized in this complex compared with previous complexes of the enzyme. These results confirm our initial strategy of combining studies of structural biology and enzyme mechanism in the inhibitor design, which may be applied to other steroidogenic enzymes involved in human diseases.

17α-Alkyl- or 17α-substituted benzyl-17β-estradiols: A new family of estrone-sulfatase inhibitors

A series of 17α-derivatives of 17β-estradiol was synthesized and tested for their ability to inhibit the estrone-sulfatase activity transforming estrone sulfate to estrone. A strong inhibitory activity was obtained when an alkyl side chain or a substituted benzyl was introduced at position 17α of estradiol. The 17α-(3′-bromobenzyl)-estradiol (26) and 17α-(4′-t-butylbenzyl)-estradiol (30) were the most potent estrone-sulfatase inhibitors obtained in our study with IC50 values of 24 and 28 nM, respectively. They also represent a new family of estrone-sulfatase inhibitors. These compounds are about 300-fold more effective in interacting with the enzyme than the substrate estrone sulfate itself.

Inhibitors of type 1 17β-hydroxysteroid dehydrogenase with reduced estrogenic activity: Modifications of the positions 3 and 6 of estradiol

Breast cancer is the second most frequent cancer affecting women. Among all endocrine therapies for the treatment of breast cancer, inhibition of estrogen biosynthesis is becoming an interesting complementary approach to the use of antiestrogens. The enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a critical role in the biosynthesis of estradiol catalyzing preferentially the reduction of estrone into estradiol, the most active estrogen. Consequently, this enzyme is an interesting biological target for designing drugs for the treatment of estrogen-sensitive diseases such as breast cancer. Our group has reported the synthesis and the biological evaluation of N-methyl, N-butyl 6β-(thiaheptamamide)estradiol as a potent reversible inhibitor of type 1 17β-HSD. Unfortunately, this inhibitor has shown an estrogen effect, thus reducing its possible therapeutic interest. Herein three strategies to modify the biological profile (estrogenicity and inhibitory potency) of the initial lead compound were reported. In a first approach, the thioether bond was replaced with a more stable ether bond. Secondly, the hydroxyl group at position 3, which is responsible for a tight binding with the estrogen receptor, was removed. Finally, the amide group of the side-chain was changed to a methyl group. Moreover, the relationship between the inhibitory potency and the configuration of the side-chain at position 6 was investigated. The present study confirmed that the 6β-configuration of the side chain led to a much better inhibition than the 6α-configuration. The replacement of the 3-OH by a hydrogen atom as well as that of the amide group by a methyl was clearly unfavorable for the inhibition of type 1 17β-HSD. Changing the thioether for an ether bond decreased by 10-fold the estrogenic profile of the lead compound while the inhibitory potency on type 1 17β-HSD was only decreased by 5-fold. This study contributes to the knowledge required for the development of compounds with the desired profile, that is, a potent inhibitor of type 1 l7β-HSD without estrogen-like effects. †These authors contributed equally to the work ‡Current address: Infinity Pharmaceuticals Inc., 780 Memorial Drive, Cambridge MA, 02139, USA ¶Current address: Eisai Research Institute of Boston, 4 Corporate Drive, Andover MA, 01810, USA

Synthesis and steroid sulphatase inhibitory activity of C19- and C21-steroidal derivatives bearing a benzyl-inhibiting group

Two series of compounds, benzyl alkylated at position 17alpha and 20 of androstane and pregnane, respectively, were synthesised and tested for steroid sulphatase inhibition. We compared the ability of the compounds to inhibit steroid sulphatase obtained from two different sources (homogenates of transfected HEK-293 cells and Jeg-3 cells) and with two types of substrate (DHEAS or E(1)S). The inhibitory activity of 17alpha-benzyl-5alpha-androstane-3beta,17beta-diol (7), 17alpha-benzyl-5-androstene-3beta,17beta-diol (9), 17alpha-benzyl-4,17beta-dihydroxy-4-androsten-3-one (15) and 20-benzyl-5-pregnene-3beta,20alpha-diol (16) has proven to be superior to that of danazol, the first steroid sulphatase inhibitor to be reported, but still lower than that of the potent inhibitor estrone-3-O-sulphamate. The inhibitory activity of compound 7 was as potent as that of its previously reported estrane analogue, 17alpha-benzyl estradiol. Benzyl alkylated compounds with no OH group on the A-ring (with a 4-OCH(3), 4-Cl, or 4-H and their precursor epoxides), as well as a series of basic steroids without a benzyl group (ADT, epi-ADT, 3alpha-diol, 3beta-diol, DHEA, Delta(5)-diol, DHT, T, Preg and Prog), did not show steroid sulphatase inhibition. We have thus demonstrated that the steroid sulphatase inhibitory effect of a benzyl group, previously observed for an estrane nucleus, can be extended to certain androstane and pregnane nuclei bearing a 3beta-OH or a 4-OH group. Inhibitors 7, 9, 15 and 16 did not induce any proliferative effect on androgen-sensitive Shionogi cells. However, when tested on oestrogen-sensitive ZR-75-1 cells, a proliferative effect was observed for 7 and 9, but not for 15 and 16.

Estrone and estradiol C-16 derivatives as inhibitors of type 1 17β-hydroxysteroid dehydrogenase

Three series of steroid derivatives, enones 1, enols 2 and saturated alcohols 3, were easily synthesized from estrone according to a sequence of three reactions: an aldol condensation with an aromatic aldehyde (R(a-g)CHO) to afford 1, the carbonyl reduction of 1 to obtain the enol 2, and the double bond reduction of 2 to give 3 with the R(a-g) group 16beta-oriented. All compounds were tested as inhibitors of type 1 17beta-HSD. The inhibitory potency increases in the following order 1<2<3, suggesting that the presence of a flexible 16beta-methylene group allows a better positioning of the aryl moiety. With an IC50 of 0.8 microM, the 16beta-benzyl-E2 (3a) is the best inhibitor in this series.

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors.

A series of C19 and C21 steroids bearing one or two inhibiting groups (3 β -sulfamate and 17 α - or 20(S)- t -butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3 β,17 β -diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 μM. With only a t -butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC 50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t -butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3 β -sulfamoyloxy-17 α - t -butylbenzyl-5-androsten-17 β -ol (10) was thus found to be the most active compound with IC 50 values of 46 ± 8 and 14 ± 1 nM, respectively for the transformations of E 1 S to E 1 and DHEAS to DHEA. The IC 50 values of compound 10 are similar to that of 17 α - t -butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3- O -sulfamate (EMATE) and 17 α - t -butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.

16α-propyl derivatives of estradiol as inhibitors of 17β-hydroxysteroid dehydrogenase type 1

Abstract The synthesis of eight 16α-propyl derivatives of estradiol is described, and structure-activity relationships are discussed. Potent inhibitors of cytosolic 17β-hydroxysteroid dehydrogenase of human placenta (type 1) can be obtained when a good leaving group is located at the end of a 16α-propyl side chain; the 16α-(iodopropyl)-estradiol (7) and 16α-(bromopropyl)-estradiol (6) gave the best irreversible inhibitions of 17β-HSD type 1, with IC50 values of 0.42 and 0.46 μM, respectively.

Synthesis of a First Estradiol-Adenosine Hybrid Compound

Abstract An estradiol derivative, bearing an adenosine residue linked at C-16α by a three-carbon side chain, provides access to a new series of substrate-cofactor hybrid compound designed to potentially interact with two binding domains of the enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD). The synthesis of 5′-O-[3-(3′,17′β-dihydroxy-1′,3′,5′(10′)-estratrien-16′α-yl)propanoyl]adenosine (7) is reported focusing on the crucial last steps: the coupling of adenosine residue to estradiol side chain by an ester link and the appropriate final cleavage of three protecting groups.