Benzothiazolyl Ureas are Low Micromolar and Uncompetitive Inhibitors of 17β-HSD10 with Implications to Alzheimer's Disease Treatment

Abstract

Human 17β-hydroxysteroid dehydrogenase type 10 is a multifunctional protein involved in many enzymatic and structural processes within mitochondria. This enzyme was suggested to be involved in several neurological diseases, e.g., mental retardation, Parkinson's disease, or Alzheimer's disease, in which it was shown to interact with the amyloid-beta peptide. We prepared approximately 60 new compounds based on a benzothiazolyl scaffold and evaluated their inhibitory ability and mechanism of action. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution on the phenyl ring moiety, a small substituent at position 6 on the benzothiazole moiety, and the two moieties were connected via a urea linker (4at, 4bb, and 4bg). These compounds exhibited IC₅₀ values of 1-2 μM and showed an uncompetitive mechanism of action with respect to the substrate, acetoacetyl-CoA. These uncompetitive benzothiazolyl inhibitors of 17β-hydroxysteroid dehydrogenase type 10 are promising compounds for potential drugs for neurodegenerative diseases that warrant further research and development.

Keywords: 17β-hydroxysteroid dehydrogenase type 10; ABAD; Alzheimer’s disease; benzothiazole; inhibitor; neurodegeneration.

Figure 1

Structures of known 17β-HSD10 inhibitors and frentizole.

Figure 2

Design of novel 17β-HSD10 inhibitors. The original scaffold was divided into for parts (depicted in violet, blue, green, and red) and each part was modified separately.

Scheme 1

Synthesis of urea and thiourea final products. Reagents and conditions: (i) CDI, DMF/DCM, reflux; (ii) SCDI, DMF/DCM, reflux; (iii) Et₃N, DMF, RT.

Scheme 2

Synthesis of products 4aj and 4bb. Reagents and conditions: (i) CDI, DCM, reflux; (ii) DMF, 60 °C; (iii) AlCl₃, DCM, reflux.

Scheme 3

Synthesis of guanidine analogue. Reagents and conditions: (i) HgO, NH₃/MeOH, RT.

Scheme 4

Synthesis of compound 4bg. Reagents and conditions: (i) Fe, NH₄Cl, THF/MeOH/water, 50 °C.

Scheme 5

Synthesis of intermediates 1a–1e. Reagents and conditions: (i) hydrazine hydrate, 110 °C; (ii for 1a,1e) BrCN, water/MeOH, RT; (iii for 1b) di(1H-imidazol-1-yl)methanimine, dioxane, reflux; (iv for 1c,1d) BrCN, THF, RT.

Scheme 6

Synthesis of 6-hydroxybenzothiazole-2-amine. Reagents and conditions: (i) AlCl₃, toluene, reflux.

Scheme 7

Synthesis of 6-substituted benzothiazole-2-amines. Reagents and conditions: (i = 1g) KSCN, benzyltrimethylammonium dichloroiodate, DMSO/H₂O, RT–70 °C; (ii) KSCN, Br₂, acetic acid, 10 °C–RT.

Scheme 8

Reduction of nitrobenzenes to anilines. Reagents and conditions: (i) H₂, Pd/C, EtOH, RT; (ii = 3c, 3e, 3f) NH₄Cl, Fe, THF/MeOH/H₂O, 50 °C.

Scheme 9

Synthesis of aniline intermediates 3g and 3h. Reagents and conditions: (i) HNO₃, H₂SO₄, 0 °C–RT; (ii) AlCl₃, DCM, reflux; (iii) H₂, Pd/C, EtOH, RT.

Figure 3

SDS-PAGE and western blotting analysis of recombinant human 17β-HSD10. Lanes: 1, purified recombinant human 17β-HSD10 stained by Coomassie Brilliant Blue; 2, purified recombinant 17β-HSD10 transferred onto PVDF membrane and detected by the monoclonal anti-17β-HSD10 antibody, showing monomeric and also dimeric form (54 kDa).

Figure 4

Determination of the mechanism of action for compounds 4at, 4bb, and 4bg. For all compounds, the uninhibited enzymatic reaction was compared with enzymatic reaction at two concentrations of each particular inhibitor. (A) Lineweaver-Burk plot of compound 4at (plots for 4bb, and 4bg in Supplementary Materials); (B) Hanes-Wolf plot of compound 4at (plots for 4bb, and 4bg in supplementary); (C) comparison of kinetic parameters K_m and V_max between uninhibited and inhibited reactions.