Marie Bérubé

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.

Synthesis of a First Estradiol-Adenosine Hybrid Compound

Abstract An estradiol derivative, bearing an adenosine residue linked at C-16α by a three-carbon side chain, provides access to a new series of substrate-cofactor hybrid compound designed to potentially interact with two binding domains of the enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD). The synthesis of 5′-O-[3-(3′,17′β-dihydroxy-1′,3′,5′(10′)-estratrien-16′α-yl)propanoyl]adenosine (7) is reported focusing on the crucial last steps: the coupling of adenosine residue to estradiol side chain by an ester link and the appropriate final cleavage of three protecting groups.

Chemical synthesis and in vitro biological evaluation of a phosphorylated bisubstrate inhibitor of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T) by selectively reducing the C17 ketone of 4-androstene-3,17-dione (Δ4-dione), with NADPH as cofactor. This enzyme is thus an interesting therapeutic target for androgen-sensitive diseases. Using an efficient convergent chemical approach we synthesized a phosphorylated version of the best Δ4-dione/adenosine hybrid inhibitor of type 3 17β-HSD previously reported. An appropriately protected C2′ phosphorylated adenosine was first prepared and linked by esterification to the steroid Δ4-dione bearing an alkyl spacer. After three deprotection steps, the phosphorylated bisubstrate inhibitor was obtained. The inhibitory potency of this compound was evaluated on homogenated HEK-293 cells overexpressing type 3 17β-HSD and compared to the best non-phosphorylated bisubstrate inhibitor. Unexpectedly, the phosphorylated derivative was slightly less potent than the non-phosphorylated bisubstrate inhibitor of type 3 17β-HSD. Two hypotheses are discussed to explain this result: 1) the phosphorylated adenosine moiety does not interact optimally with the cofactor-binding site and 2) the bisubstrate inhibitors, phosphorylated or not, interact only with the substrate-binding site of type 3 17β-HSD.

Design, chemical synthesis, and in vitro biological evaluation of simplified estradiol–adenosine hybrids as inhibitors of 17β-hydroxysteroid dehydrogenase type 1

A series of estradiol (E2) derivatives were designed to interact with both the substrate- and the cofactor-binding sites of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). These analogues of potent E2–adenosine hybrid inhibitor EM-1745, where the adenosine moiety was replaced by a more stable benzene derivative, were synthesized from estrone using alkene cross-metathesis and Sonogashira coupling reactions as key steps. In vitro biological evaluation of these steroid derivatives revealed that a spacer of 13 methylenes, between the 16β-position of E2 and the adenosine mimic bearing a carboxylic acid group, gave the best inhibition of 17β-HSD1.

Design, Synthesis and In Vitro Evaluation of 4-Androstene-3,17-dione/Adenosine Hybrid Compounds as Bisubstrate Inhibitors of Type 3 17β-Hydroxysteroid Dehydrogenase

Steroidogenic enzyme type 3 17β-hydroxysteroid dehydrogenase (17β-HSD) is an important therapeutic target for androgen-sensitive diseases. This enzyme selectively reduces the C17 ketone of 4-androstene-3,17-dione (Δ4-dione), thus producing testosterone (T) using NADPH as cofactor. Our group previously synthesized hybrid (estradiol/adenosine) inhibitors that successfully inhibit the biosynthesis of the potent estrogen estradiol by type 1 17β-HSD. To similarly lower the level of the potent androgen testosterone, inhibitors of type 3 17β-HSD were designed and synthesized applying the same hybrid (substrate/cofactor) strategy. Two chemical approaches were developed to join the three components of the bisubstrate inhibitor (the substrate Δ4-dione, an alkyl spacer and the cofactor moiety adenosine). An alkylation in the α position of steroidal 17-ketone or a cross-metathesis was used as a key step to efficiently join the substrate and the alkyl spacer, whereas an esterification was employed to link the spacer to adenosine. An enzymatic assay in homogenated HEK-293 cells overexpressing type 3 17β-HSD revealed that the best inhibitors of that series are those bearing an alkyl side-chain spacer of 11 or 12 methylenes: inhibition of 69 and 78% at 1 μM were respectively observed. As expected, these bisubstrate inhibitors were less potent in intact cells than in homogenated cells. However, both enzymatic assays revealed that the strategy of substrate/cofactor dual inhibitors seems to work for type 3 17β-HSD, although the inhibitors designed have not been optimized yet.

Preparation of 16β-Estradiol Derivative Libraries as Bisubstrate Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 Using the Multidetachable Sulfamate Linker

Combinatorial chemistry is a powerful tool used to rapidly generate a large number of potentially biologically active compounds. In our goal to develop bisubstrate inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) that interact with both the substrate (estrone or estradiol) and the cofactor (NAD(P)H) binding sites, we used parallel solid-phase synthesis to prepare three libraries of 16β-estradiol derivatives with two or three levels of molecular diversity. From estrone, we first synthesized a sulfamate precursor that we loaded on trityl chloride resin using the efficient multidetachable sulfamate linker strategy recently developed in our laboratory. We then introduced molecular diversity [one or two amino acid(s) followed by a carboxylic acid] on steroid nucleus by Fmoc peptide chemistry. Finally, after a nucleophilic cleavage, libraries of 30, 63 and 25 estradiol derivatives were provided. A library of 30 sulfamoylated estradiol derivatives was also generated by acidic cleavage and its members were screened for inhibition of steroid sulfatase. Biological evaluation on homogenated HEK-293 cells overexpressing 17β-HSD1 of the estradiol derivatives carrying different oligoamide-type chains at C-16 first revealed that three levels of molecular diversity (a spacer of two amino acids) were necessary to interact with the adenosine part of the cofactor binding site. Second, the best inhibition was obtained when hydrophobic residues (phenylalanine) were used as building blocks.

Improved synthesis of EM-1745, preparation of its C17-ketone analogue and comparison of their inhibitory potency on 17β-hydroxysteroid dehydrogenase type 1

Endocrine therapies are widely used for the treatment of estrogen-sensitive diseases. 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the last step of the biosynthesis of potent estrogen estradiol (E2). This enzyme catalyzes the reduction of the C17-ketosteroid estrone (E1) into the C17β-hydroxy steroid E2 using the cofactor NAD(P)H. The X-ray analysis of E2/adenosine bisubstrate inhibitor EM-1745 proven that this compound interacts with both the substrate- and the cofactor-binding sites. However, E1 is a better substrate of 17β-HSD1 than E2. Thus, in order to improve the inhibitory potency of EM-1745, the C17-ketone analogue was prepared. During this work, a new and more efficient method for synthesizing EM-1745 was developed using an esterification and a cross-metathesis as key steps. Contrary to what was expected, the C17-ketone analogue of EM-1745 is a less potent inhibitor (IC50 = 12 nM) than the C17-alcohol (IC50 = 4 nM) in homogenated HEK-293 cells overexpressing 17β-HSD1. Our results contribute to the knowledge of an unexpected observation: the C17-ketone steroidal inhibitors of 17β-HSD1 are less potent than their corresponding C17-alcohol derivatives.

Efficient coupling and solid-phase synthesis of steroidal ketone derivative using polymer-bound glycerol

Abstract High-yield coupling of steroidal hindered ketones onto polymer-bound glycerol by an efficient acetal exchange reaction is reported under mild conditions. A solid-phase model sequence of five steps is thereafter achieved to synthesize a steroidal ketone derivative having two levels of diversity in good yield and purity.