Martin R. Tremblay

Overview of a rational approach to design type I 17β-hydroxysteroid dehydrogenase inhibitors without estrogenic activity : Chemical synthesis and biological evaluation

Hormone-sensitive diseases such as breast cancer are health problems of major importance in North America and Europe. Endocrine therapies using antiestrogens for the treatment and the prevention of breast cancer are presently under clinical trials. Antiestrogens are drugs that compete with estrogens for the estrogen receptor without activating the transcription of estrogen-sensitive genes. However, an optimal blockade of estrogen action could ideally be achieved by a dual-action compound that would antagonize the estrogen receptor and inhibit the biosynthesis of estradiol. Type I 17beta-hydroxysteroid dehydrogenase (17beta-HSD) was chosen as a key steroidogenic target enzyme to inhibit the formation of estradiol, which is the most potent estrogen. This article describes a rational approach that could lead to the development of compounds that exhibit both actions. The chemical syntheses of estradiol derivatives bearing a bromoalkyl and a bromoalkylamide side chain at the 16alpha-position are summarized. Two parameters were studied for biological evaluation of our synthetic inhibitors: (1) the inhibition of estrone reduction into estradiol by type I 17beta-HSD, and (2) the proliferative/antiproliferative cell assays performed on the estrogen-sensitive ZR-75-1 breast tumor cell line. First, the substitution of the 16alpha-position of estradiol by bromoalkyl side chain led to potent inhibitors of type I 17beta-HSD, but the estrogenic activity remained. Secondly, an alkylamide functionality at the 16alpha- or 7alpha-position of estradiol cannot abolish the estrogenic activity without affecting considerably the inhibitory potency on type I 17beta-HSD. In conclusion, the best dual-action inhibitor synthesized showed an IC50 of 13 +/- 1 microM for type I 17beta-HSD, while displaying antiestrogenic activity at 1.0 microM. Despite the fact that we did not obtain an ideal dual-action blocker, we have optimized several structural parameters providing important structure-activity relationship.

Spironolactone-related inhibitors of type II 17β-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.

Inhibitors of type II 17β-hydroxysteroid dehydrogenase

Abstract The 17β-hydroxysteroid dehydrogenases (17β-HSDs) are involved in the last step of the biosynthesis of sex steroids from cholesterol. This family of steroidogenic enzymes constitutes an interesting target in the control of the concentration of estrogens and androgens. Among the isoforms of 17β-HSD, type II preferentially catalyzes the oxidation of estradiol (E2), testosterone (T), dihydrotestosterone (DHT), and 20α-dihydroprogesterone (20α-DHP). Based on structure–activity relationship studies, we have developed steroidal spirolactones as inhibitors of type II 17β-HSD using different steroid nuclei: a C18-steroid (lactones 1 and 10), an antiestrogenic nucleus (lactone 2), and a C19-steroid (lactone 28). We know these inhibitors are selective for type II 17β-HSD as no or only weak inhibition was observed for types I and III. They also have no proliferative (androgenic) activity on androgen sensitive (AR+) Shionogi cells whereas their proliferative (estrogenic) activity on estrogen sensitive (ER+) ZR-75-1 cells depends on the nature of the steroid nucleus. Lactones 1 and 10 are weak estrogens, while lactones 2 and 28 do not exert estrogenic activity, in fact lactone 2 is an antiestrogen. Lactones 1, 2, 10 and 28 were also tested in an identical assay with a series of enzyme substrates, C19-steroid diols, and known inhibitors, for the oxidation of testosterone and estradiol into androstenedione and estrone, respectively. From this comparative study, the best inhibitors of type II 17β-HSD (oxidase activity) were identified, but none of them were clearly more potent than the hydroxylated (reduced) forms of enzyme substrates, E2, T, and DHT. Such inhibitors remain, however, useful tools to, (1) further elucidate the role of type II 17β-HSD, and (2) regulate the level of active estrogens, androgens and progesterone.

Synthesis of 16-(bromoalkyl)-estradiols having inhibitory effect on human placental estradiol 17β-hydroxysteroid dehydrogenase (17-β-HSD type 1)

The activity of 17β-HSD type 1, the enzyme that converts estrone into its more potent metabolite estradiol, has been demonstrated in all classical steroidogenic tissues and almost all peripheral tissues from both rat and human. Since 17β-HSD is one of the most important enzymes involved in active steroid hormone formation, its inactivation could be a clinical approach to the treatment of hormono-dependent diseases like breast cancer. Herein we report the synthesis of 16-(bromoalkyl)-estradiols and their potency to inhibit the human placenta cytosolic estradiol 17β-HSD (type 1). Synthetic analogues possess various side chain lengths and orientation (α or β) at position 16 of the steroidal D ring. The most potent inhibitory effect was observed when the length of the side chain was 3 or 4 carbons. However, the 16β-(bromopropyl)-estradiol easily undergoes cyclization and its effect on 17β-HSD is lost. Consequently, 16α-(bromopropyl)-E2, 16α-(bromobutyl)-E2, and 16β-(bromobutyl)-E2 were the best inhibitors discussed in this paper.

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

Chemical synthesis of 16β-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17β-hydroxysteroid dehydrogenase and binding affinity on steroid receptors

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.

Rapid access to N-Boc phenylglycine derivatives via benzylic lithiation reactions

Abstract We report a novel and efficient method for the enantioselective synthesis of N-Boc protected phenylglycines. Yields and enantiomeric ratios vary widely depending on the nature of the solvent, the substrate and on the method of forming the chiral complex. Results show that the major reaction pathway is an enantioselective deprotonation/substitution process. The enantioselectivity appears to be limited by the chiral discrimination ability of the s-BuLi–(−)-sparteine complex. The synthetic method described is one of the shortest route to useful enantioenriched N-Boc phenylglycine derivatives.

A Convenient Synthetic Method for Alpha-Alkylation of Steroidal 17-Ketone: Preparation of 16β(THPO-Heptyl)-Estradiol

Abstract A sequence of three key-steps (activation of steroidal 17-ketone, alkylation and decarboxyalkoxylation) provided an interesting synthetic device to obtain, after reduction, 16β-(THPO-heptyl)-estradiol.

Parallel solid-phase synthesis of a model library of 7α-alkylamide estradiol derivatives as potential estrogen receptor antagonists

The C17-THP derivative of 7alpha-(11-azidoundecanyl)-estradiol (4) was synthesized and coupled to an aminomethyl resin via a photolabile o-nitrobenzyl linker. Reduction of the azide by the Staudinger reaction to its corresponding amine followed by acylation using four activated NFmoc protected amino acids gave a first level of diversity. Subsequent deprotection of the Fmoc followed by a second acylation with five activated carboxylic acids produced, after photocleavage, a model library of twenty antiestrogen-related 7alpha-alkylamide estradiol derivatives in acceptable overall yields and very good purities.

Solid-phase synthesis of phenolic steroids: Towards combinatorial libraries of estradiol derivatives

Abstract The 16β-(azidopropyl) derivative of estradiol (7) was synthesized and coupled to aminomethyl resin via a photolabile o-nitrobenzyl linker. Condensation of the corresponding iminophosphorane with activated acids successfully gave amides. Photocleavage resulted in good yield recovery of estradiol derivatives 15 and 16 in acceptable purities for biological screening.