Piperazine Trityl Resin

Two libraries, each consisting of 48 16beta-aminopropyl estradiol derivatives, phenols and sulfamates, respectively, were synthesized by solid-phase parallel chemistry through a seven-step reaction sequence. Following the attachment of a C18-steroid sulfamate precursor on a trityl chloride resin, diversity elements were first introduced on the 16beta-aminopropyl chain of the steroid by acylation reactions with eight Fmoc-amino acids. After deprotection, the free amine function of the resulting compounds was reacted with six carboxylic acids for the introduction of a second diversity level. The two variants employed for the cleavage of compounds from the solid support, acidic and nucleophilic, allowed the corresponding libraries of sulfamate and phenol derivatives in yields of 8-50 % and 13-58 % to be obtained with an average HPLC purity of 94 % and 91 %, respectively. Potent steroid sulfatase inhibitors and interesting SAR results were generated from the screening of the sulfamate library. Furthermore, moderate inhibitors of type 1 17beta-HSD resulted from the partial screening of phenol library. Thus, these two categories of compounds were synthesized to rapidly identify potential inhibitors of steroid biosynthesis for the hormonal therapy of estrogen-dependent diseases, and also to demonstrate the versatility and efficiency of the recently developed sulfamate linker.

Steroid sulfates are precursors of hormones that stimulate androgen- and estrogen-dependent cancers. Thus, steroid sulfatase, the enzyme that catalyzes conversion of DHEAS and E1S to the corresponding unconjugated steroids DHEA and E1, appears to be one of the key enzymes regulating the level of active androgenic and estrogenic steroids. Since 17alpha-substituted benzylestradiols and 3-O-sulfamate estrone (EMATE) represent two families of steroid sulfatase inhibitors that probably act through different mechanisms, we synthesized compounds 3-O-sulfamate 17alpha-benzylestradiol (4) and 3-O-sulfamate 17alpha-(tert-butylbenzyl)estradiol (5) that contain two kinds of substituents on the same molecule. In our enzymatic assay using a homogenate of human embryonal (293) cells transfected with steroid sulfatase, compounds 4 and 5 were found to be more potent inhibitors than already known steroid sulfatase inhibitors that have only a C17alpha-substituent or only a C3-sulfamate group (EMATE). The IC50 values of 4 and 5 were, respectively, 0.39 and 0.15 nM for the transformation of E1S to E1 and 4.1 and 1.4 nM for the transformation of DHEAS to DHEA. Compound 5 inhibited the steroid sulfatase activity in intact transfected (293) cell culture assays by inactivating the enzyme activity. Compound 5 also inactivates the steroid sulfatase activity at lower concentration than EMATE in microsomes of transfected (293) cells. In this assay, an excess of natural substrate E1S protects enzyme against inactivation by 5 or EMATE. Furthermore, the unsulfamoylated analogue of 5, compound 3, did not inactivate the steroid sulfatase.

The steroid sulfatase or steryl sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3-hydroxy steroids to the corresponding free active 3-hydroxy steroids. Since androgens and estrogens may be synthesized inside the cancerous cells starting from dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E(1)S) available in blood circulation, the use of therapeutic agents that inhibit steroid sulfatase activity may be a rewarding approach to the treatment of androgeno-sensitive and estrogeno-sensitive diseases. In the present study, we report the chemical synthesis and biological evaluation of a new family of steroid sulfatase inhibitors. The inhibitors were designed by adding an alkyl, a phenyl, a benzyl, or a benzyl substituted at position 17alpha of estradiol (E(2)), a C18-steroid, and enzymatic assays were performed using the steroid sulfatase of homogenized JEG-3 cells or transfected in HEK-293 cells. We observed that a hydrophobic substituent induces powerful inhibition of steroid sulfatase while a hydrophilic one was weak. Although a hydrophobic group at the 17alpha-position increased the inhibitory activity, the steric factors contribute to the opposite effect. As exemplified by 17alpha-decyl-E(2) and 17alpha-dodecyl-E(2), a long flexible side chain prevents adequate fitting into the enzyme catalytic site, thus decreasing capacity to inhibit the steroid sulfatase activity. In the alkyl series, the best compromise between hydrophobicity and steric hindrance was obtained with the octyl group (IC(50) = 440 nM), but judicious branching of side chain could improve this further. Benzyl substituted derivatives of estradiol were better inhibitors than alkyl analogues. Among the series of 17alpha-(benzyl substituted)-E(2) derivatives studied, the 3'-bromobenzyl, 4'-tert-butylbenzyl, 4'-butylbenzyl, and 4'-benzyloxybenzyl groups provided the most potent inhibition of steroid sulfatase transformation of E(1)S into E(1) (IC(50) = 24, 28, 25, and 22 nM, respectively). As an example, the tert-butylbenzyl group increases the ability of the E(2) nucleus to inhibit the steroid sulfatase by 3000-fold, and it also inhibits similarly the steroid sulfatase transformations of both natural substrates, E(1)S and DHEAS. Interestingly, the newly reported family of steroid sulfatase inhibitors acts by a reversible mechanism of action that is different from the irreversible mechanism of the known inhibitor estrone sulfamate (EMATE).