Liviu C. Ciobanu

Steroid sulfatase inhibitors

Steroid sulfatase catalyses the hydrolysis of sulfated steroids to their corresponding hydroxylated forms. Since oestrogens and androgens can be synthesised intracellularly from the abundant circulating sulfated steroids, steroid sulfatase inhibitors should prove to be valuable therapeutic agents for the treatment of hormono-dependent diseases. Furthermore, steroid sulfatase inhibitors have been reported to control brain concentrations of sulfated neurosteroids and also to improve memory. Such therapeutic potential stimulated research and development of steroid sulfatase inhibitors and important progress has been achieved during the last ten years. This review focuses on patented steroid sulfatase inhibitors, but discussion of unpatented inhibitors published in the literature has been included to complement the information presented.

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 5α-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-Å resolution. We have identified new interactions involving Gln738, Met742, and His874 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 16β-Aminopropyl Estradiol Derivatives for Targeting Two Key Steroidogenic Enzymes

Two libraries, each consisting of 48 16β‐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 16β‐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 17β‐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.

Nonsteroidal compounds designed to mimic potent steroid sulfatase inhibitors

Chemical synthesis and enzyme inhibition results are reported for a series of nonsteroidal sulfatase inhibitors, 1-(p-sulfamoyloxyphenyl)-5-(p-t-butylbenzyl)-5-alkanols and the lower active phenolic analogues. These compounds conserve some structural elements from the previously reported potent steroidal inhibitor 3-O-sulfamate-17alpha-(p-t-butylbenzyl)-17beta-hydroxy-estra-1,3,5(10)-triene, while the C18-methyl group and the hydrocarbon backbone represented by the steroid rings B, C, and D were replaced with a free conformational chain. Using estrone sulfate (100 microM) as substrate and homogenate of transfected HEK-293 cells as source of steroid sulfatase activity, the IC(50) values of the best inhibitors, the undecanol derivatives, were 0.4+/-0.1 and >300 nM, respectively, in the sulfamate and phenolic series. Although these sulfamoylated nonsteroidal inhibitors appear a bit less active than their steroidal analogues, they are however more potent than known inhibitors estrone-3-O-sulfamate and p-(O-sulfamoyl)-N-tetradecanoyl tyramine. The optimal side-chain length for the inhibition of steroid sulfatase activity was found to be six carbons, which corresponds to the number of carbons that mimic the B, C and D steroid rings, between C6 and C17. Furthermore, compounds with only the t-butylbenzyl group or the alkyl chain of six carbons are less potent inhibitors compared to the one that include both of these hydrophobic substituents. Such results suggest that compound from this later category better mimic the steroidal inhibitor.

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.

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.

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.