Structural characterization of the human androgen receptor ligand-binding domain complexed with EM5744, a rationally designed steroidal ligand bearing a bulky chain directed toward helix 12.

Antiandrogens are commonly used to treat androgen-dependent disorders. The currently used drugs unfortunately possess very weak affinity for the human AR (hAR), thus indicating the need to develop new high-affinity steroidal antiandrogens. Our compounds are specially designed to impede repositioning of the mobile carboxyl-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using crystal structures of the hARLBD, we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of 5alpha-dihydrotestosterone-derived molecules bearing various C18 chains were thus synthesized and tested for their capacity to bind hAR and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM5744, was determined at a 1.65-A resolution. We have identified new interactions involving Gln(738), Met(742), and His(874) that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. This structural information will be helpful to refine the structure of the chains placed on the C18 atom to obtain efficient H12-directed steroidal antiandrogens.

Synthesis of libraries of 16beta-aminopropyl estradiol derivatives for targeting two key steroidogenic enzymes.

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.

Inhibition of estrone sulfate-induced uterine growth by potent nonestrogenic steroidal inhibitors of steroid sulfatase.

The present study describes the biological in vitro and in vivo evaluation of 2-methoxy derivatives of estrogenic inhibitors of steroid sulfatase, namely 3-sulfamoyloxy-17alpha-p-tert-butylbenzyl(or benzyl)-1,3,5 (10)-estratrien-17beta-ols. The addition of the 2-methoxy group conserves the potent inhibitory effect on steroid sulfatase activity (IC(50)s of 0.024 and 0.040 nM) while removing the estrogenic action. Using an ovariectomized mouse model, we show that the first generation of steroid sulfatase inhibitors tested, 3-sulfamoyloxy-17alpha-p-tert-butylbenzyl(or benzyl)estra-1,3,5 (10)-trien-17beta-ols and estrone-3-O-sulfamate, are estrogenic compounds stimulating estrogen-sensitive uterine growth. Interestingly, the 2-methoxy-3-sulfamoyloxy-17alpha-benzylestra-1,3,5 (10)-trien-17beta-ol (7) has no estrogenic activity but efficiently blocks (s.c. and p.o.) uterine growth induced by estrone sulfate, which is converted into estrone and then estradiol by steroid sulfatase and type 1 17beta-hydroxysteroid dehydrogenase, respectively. This report clearly shows that a steroid sulfatase inhibitor can efficiently block estrogen action from the inactive precursor estrone sulfate, in vitro and in vivo.

Solid-phase parallel synthesis of 17alpha-substituted estradiol sulfamate and phenol libraries using the multidetachable sulfamate linker.

We report an application of the multidetachable sulfamate linker in the synthesis of two model libraries of N-derivatized 17alpha-piperazinomethyl estradiols (phenols and sulfamates) by solid-phase parallel chemistry. The solid-phase precursor, a 3-sulfamoyl-17alpha-(N-trifluoroacetyl-piperazinomethyl) estradiol, was synthesized in solution from estrone and loaded efficiently onto trityl chloride resin as polymeric support. After cleavage of the trifluoroacetyl protecting group, sequential acylation reactions with five Fmoc-protected amino acids and five carboxylic acids were performed to introduce two levels of molecular diversity. Finally, the resins were split into two parts, and acidic (5% trifluoroacetic acid in dichloromethane) and nucleophilic (piperazine in tetrahydrofuran) cleavages were used to generate libraries A (5 x 5 sulfamates) and B (5 x 5 phenols) members in overall yields of 18-66% and high HPLC purities (87-96%) without purification steps. A preliminary screening test for inhibition of steroid sulfatase showed that the phenols were clearly weaker inhibitors, as compared to their sulfamate analogues. The most potent inhibitors were those with suitable hydrophobic amino acid and carboxylic acid substituents. Thus, compounds with a phenylalanine residue as the first element of diversity inhibited over 90% of steroid sulfatase activity at a concentration of 1 nM in homogenates of HEK-293 transfected cells, being as potent as the leading inhibitor 17alpha-tert-butylbenzyl estradiol 3-O-sulfamate previously reported. These results suggest that the steroid sulfatase inhibitory potency of estradiol derivatives, sulfamoylated or not, can be increased by the hydrophobic effect of a suitable substituent introduced in the proximity of the D ring of the steroid. The present work also demonstrated the efficiency and the cleavage versatility of the sulfamate linker to generate libraries of compounds with relevant biological importance, phenols and sulfamates.

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 (3beta-sulfamate and 17alpha- 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-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. 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 IC50 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 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-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 17alpha-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.

A multidetachable sulfamate linker successfully used in a solid-phase strategy to generate libraries of sulfamate and phenol derivatives.

The sulfamates and phenols constitute two families of compounds with numerous interesting biological properties. Using the ability of a new multidetachable sulfamate linker to generate these two families of compounds from the same resin, we designed and synthesized libraries of estradiol derivatives, sulfamoylated or not. A C-16beta side chain was then judiciously diversified to target two key steroidogenic enzymes, the steroid sulfates and the type 1 17beta-HSD. Four libraries of sulfamate and phenol derivatives were easily obtained by solid-phase parallel synthesis in good crude overall yields (13-62%) and HPLC purities (85-96%). Such strategy using the new two-in-line sulfamate linker could be also extended to other therapeutic targets than steroidogenic enzymes, thus adding to its potential.