C-3- and C-4-Substituted Bicyclic Coumarin Sulfamates as Potent Steroid Sulfatase Inhibitors

Abstract

Synthetic routes to potent bicyclic nonsteroidal sulfamate-based active-site-directed inhibitors of the enzyme steroid sulfatase (STS), an emerging target in the treatment of postmenopausal hormone-dependent diseases, including breast cancer, are described. Sulfamate analogs 9-27 and 28-46 of the core in vivo active two-ring coumarin template, modified at the 4- and 3-positions, respectively, were synthesized to expand structure-activity relationships. α-Alkylacetoacetates were used to synthesize coumarin sulfamate derivatives with 3-position modifications, and the bicyclic ring of other parent coumarins was primarily constructed via the Pechmann synthesis of hydroxyl coumarins. Compounds were examined for STS inhibition in intact MCF-7 breast cancer cells and in placental microsomes. Low nanomolar potency STS inhibitors were achieved, and some were found to inhibit the enzyme in MCF-7 cells ca. 100-500 more potently than the parent 4-methylcoumarin-7-O-sulfamate 3, with the best compounds close in potency to the tricyclic clinical drug Irosustat. 3-Hexyl-4-methylcoumarin-7-O-sulfamate 29 and 3-benzyl-4-methylcoumarin-7-O-sulfamate 41 were particularly effective inhibitors with IC₅₀ values of 0.68 and 1 nM in intact MCF-7 cells and 8 and 32 nM for placental microsomal STS, respectively. They were docked into the STS active site for comparison with estrone 3-O-sulfamate and Irosustat, showing their sulfamate group close to the catalytic hydrated formylglycine residue and their pendant group lying between the hydrophobic sidechains of L103, F178, and F488. Such highly potent STS inhibitors expand the structure-activity relationship for these coumarin sulfamate-based agents that possess therapeutic potential and may be worthy of further development.

Figure 1

Structures of STS inhibitors: 1 (EMATE), 1a (E2MATE), and 2 (Irosustat, STX64, 667COUMATE, BN83495).

Figure 2

Structures of bicyclic coumarin sulfamates 3–8, including COUMATE 3. X = OSO₂NH₂.

Figure 3

General structures of bicyclic coumarin sulfamates: A: 4-substituted series, B: 3,4-disubstituted series.

Scheme 1. Synthesis of Various Ethyl Alkanoyl Acetates for the Preparation of 4-Alkylcoumarin Sulfamates

(i) SOCl₂/tetrahydrofuran (THF), reflux; (ii) (a) MeCN, MgCl₂, Et₃N, 10–25 °C, 2.5 h, (b) RCOCl, Et₃N, 0 °C, 0.5 h, room temperature (rt), 12 h; (iii) RCHO, SnCl₂, CH₂Cl₂, rt, 3 h.

Scheme 2. Synthesis of Various α-Alkylacetoacetates for the 3-Alkyl-4-methylcoumarin Sulfamates

(i) RBr, K₂CO₃, H₂O, Bu₄NCl, CH₂Cl₂, reflux, 40 h.

Scheme 3. Synthesis of 4-Substituted-7-O-coumarin Sulfamates

(i) Conc. H₂SO₄/conc. CF₃COOH, 3 h, 0 °C to rt, (ii) anhydrous dimethylformamide (DMF), NaH, N₂, 0 °C and H₂NSO₂Cl (∼5 equiv), 0 °C to rt.

Scheme 4. Synthesis of 4-Methyl-3-substituted-7-O-coumarin Sulfamates

(i) Conc. H₂SO₄/conc. CF₃COOH, 3 h, 0 °C to rt; (ii) (Bu)₄N⁺HSO₄^–, CH₂Cl₂, rt; (iii) anhydrous DMF, NaH, N₂, 0 °C and H₂NSO₂Cl (∼5 equiv), 0 °C to rt.

Figure 4

Diagrammatic comparison of some potent inhibitors evaluated: 4-pentylcoumarin-7-O-sulfamate (10), 4-nonylcoumarin-7-O-sulfamate (14), 4-tridecylcoumarin-7-O-sulfamate (18), 3-hexyl-4-methylcoumarin-7-O-sulfamate (29), 3-nonyl-4-methylcoumarin-7-O-sulfamate (32), 3-benzyl-4-methylcoumarin-7-O-sulfamate (41), 3-phenethyl-4-methylcoumarin-7-O-sulfamate (42), in comparison with EMATE (1), Irosustat (2), and COUMATE (3). Solid lines denote similarity to steroid C and D rings.

Figure 5

Left: docking of EMATE (cyan) and 29 (brown) into the crystal structure of human STS. Right: docking of Irosustat (cyan) and 41 (brown) into the crystal structure of human STS. In both figures, the Ca²⁺ ion is depicted as a yellow sphere and FG75 is the gem-diol form of FGly 75 aldehyde. The dotted yellow lines are potential hydrogen bonds.