Chemical synthesis of 16beta-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17beta-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.

Inhibitors of type II 17beta-hydroxysteroid dehydrogenase.

The 17beta-hydroxysteroid dehydrogenases (17beta-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 17beta-HSD, type II preferentially catalyzes the oxidation of estradiol (E(2)), testosterone (T), dihydrotestosterone (DHT), and 20alpha-dihydroprogesterone (20alpha-DHP). Based on structure-activity relationship studies, we have developed steroidal spirolactones as inhibitors of type II 17beta-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 17beta-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 17beta-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 17beta-HSD, and (2) regulate the level of active estrogens, androgens and progesterone.

Solid-phase synthesis of hydroxysteroid derivatives using the diethylsilyloxy linker.

Four different types of hydroxysteroids (primary alcohol, secondary alcohols, and phenol), bearing either an oxirane or an azide as a precursor of molecular diversity, were linked in good yields to solid support using the butyldiethylsilane polystyrene (PS-DES) resin. These molecules were then used as scaffolds to generate hydroxysteroid derivatives containing two levels of diversity. The proposed libraries were tested by running steroidal alcohols through a model sequence of reactions (solid-phase coupling, aminolysis of oxirane or reduction of azide, amidation, and final cleavage). As a result, two linked secondary alcohols (17beta-hydroxy-spiro-3(R)-oxirane-5alpha-androstane and 3beta-hydroxy-spiro- 17(S)-oxirane-5alpha-androstane) and a primary alcohol (spiro-17(S)-oxirane-3-(hydroxymethyl)-1,3,5(10)-estratriene) afforded good overall yields (>45%) and high HPLC purities (>90%) of hydroxysteroids derivatized as alkylamides without purification. One limitation was noted for the fourth library: the phenolic steroid linked by the diethylsilyloxy linker gave a poor overall yield of 8% of the desired model compound. Finally, the diethylsilyloxy linker was used successfully for a rapid solid-phase synthesis of a model library of twenty C19-steroid derivatives (3beta-amido-3alpha-hydroxy-5alpha-androstane-17-ones), with an average yield of 53% and average HPLC purity of 97% without purification steps.

Parallel solid-phase synthesis and structural characterization of a library of highly substituted chiral 1,3-oxazolidines.

The rapid parallel synthesis and characterization of diverse chirally defined 1,3-oxazolidines is reported. Three diversity elements were incorporated in a 6 x 4 x 4 block approach to generate a 96-member 1,3-oxazolidine library. The synthetic route involved initial attachment of six nonracemic phenylglycidols, (2S,3S)1A-C and (2R,3R)-2A-C, to 2% cross-linked polystyrene resin via a chlorodiethylsilane linker (PS-DES), followed by regio- and stereoselective oxirane ring opening with four primary amines (3a-d). The key condensation reaction between the resulting polymer-bound beta-amino alcohols and four aldehydes (4a-d) was found to occur optimally in warm benzene (60 degrees C) in the presence of anhydrous magnesium sulfate. Cleavage of the oxazolidines from the resin support was achieved with TBAF to give the individual members (2R,4R,5R)-5Aaa-Cdd and (2S,4S,5S)-6Aaa-Cdd in good to excellent yields (51-99%) based on mass recovery. Purities of all these crude products was generally >85% (as measured by LCMS). 1H, 13C NMR, and 1D difference nOe of the library members confirmed the structural and stereochemical integrity of the substituents around the 1,3-oxazolidine core. The asymmetric induction at C-2 (cis or trans to the C-4 substituent) ratio ranged from 4 to I to 49 to 1 across the library. This report highlights the versatility of the 1,3-oxazolidine heterocycle as a scaffold for concise parallel library construction and opens the way for high-throughput screening of such compounds in the biological sphere.

Solid-phase synthesis of phenolic steroids: from optimization studies to a convenient procedure for combinatorial synthesis of biologically relevant estradiol derivatives.

During the course of our studies on therapeutic agents for the treatment of breast cancer, we became interested in the solid-phase combinatorial synthesis of estradiol derivatives that contain a functionalized side chain at either position 16 beta or 7 alpha. Both types of compounds have already demonstrated inhibitory activity toward both biosynthesis and action of estradiol. As a first step, two versatile precursors bearing an azidoalkyl side chain at either position 16 beta or 7 alpha of estradiol were synthesized using standard solution-phase methods. Afterward, the effectiveness of five linkers to attach the phenolic function of these estradiol derivatives to a polystyrene resin was investigated; they were benzylic ether (Merrifield), 4-alkoxy-benzylic ethers (Wang, Sheppard), tetrahydropyranyl ether (Ellman), benzoic ester, and o-nitrobenzyl ether. To test the linker in a synthetic context, a short sequence of reactions, including reduction of the azide and acylation of the corresponding amine, was performed on the polymer-bound estradiol derivative. While all of the tested linkers proved effective in attaching the phenol functionality of the precursor, only the o-nitrobenzyl ether photolabile linker enabled the release of the final products in acceptable purities. Consequently, this linker was used to perform successfully the solid-phase synthesis of four different classes of estradiol derivatives in acceptable yields and excellent purities. This study was preliminary to the combinatorial synthesis of larger libraries of biologically relevant estradiol derivatives.

Parallel solid-phase synthesis of a model library of 7alpha-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.

Spironolactone-related inhibitors of type II 17beta-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.

Overview of a rational approach to design type I 17beta-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.

C16 and C17 derivatives of estradiol as inhibitors of 17 beta-hydroxysteroid dehydrogenase type 1: chemical synthesis and structure-activity relationships.

As a first part of our research focused on the synthesis of 17 beta-HSD type 1 inhibitors without estrogenic activity, we needed to identify a small, easy-to-handle pharmacophore able to block the enzymatic activity. Previous studies on the active site of the enzyme by affinity labeling gave us a basis for the design of steroidal inhibitors derivatives. Several estradiol derivatives bearing a short (three carbons) side chain in position 17 alpha or 16 alpha were synthesized and tested for their ability to inhibit the transformation of estrone into estradiol by 17 beta-HSD type 1 (cytosolic fraction of human placenta). We found that 16 alpha-derivatives of estradiol gave better 17 beta-HSD inhibition than their corresponding 17 alpha analogs. Among several chemical groups used in this study, we conclude that better 17 beta-HSD inhibition was obtained for compounds with a good leaving group at the end of side chain. Thus, an iodopropyl or a bromopropyl side chain at C16 alpha of estradiol (E2) inhibit efficiently the 17 beta-HSD type 1 with IC50 values of 0.42 and 0.46 microM, respectively. Their 17-keto analogs inhibit also the enzyme activity similarly. Since this kind of compounds inhibit the 17 beta-HSD type 1 in time-dependent manner and that enzymatic activity cannot be restored later, we conclude to inhibitor of inactivator type. This conclusion is in accordance with the correlation observed between the ability of leaving group to dissociate and their potency to inhibit 17 beta-HSD type 1. We have also observed that additional addition of untritiated estrone protect the enzyme against the inactivation caused by 16 alpha-bromopropyl-E2 suggesting a competitive inhibitor of 17 beta-HSD. The bromopropyl pharmacophore was then selected to be further added onto an antiestrogenic steroid nucleus.

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

The activity of 17 beta-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 17 beta-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 17 beta-HSD (type 1). Synthetic analogues possess various side chain lengths and orientation (alpha or beta) 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 16 beta-(bromopropyl)-estradiol easily undergoes cyclization and its effect on 17 beta-HSD is lost. Consequently, 16 alpha-(bromopropyl)-E2, 16 alpha-(bromobutyl)-E2, and 16 beta-(bromobutyl)-E2 were the best inhibitors discussed in this paper.