Synthesis of 17ß-hydroxysteroid dehydrogenase type 10 steroidal inhibitors: Selectivity, metabolic stability and enhanced potency.

17beta-Hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is the only mitochondrial member of 17ß-HSD family. This enzyme can oxidize estradiol (E2) into estrone (E1), thus reducing concentration of this neuroprotective steroid. Since 17ß-HSD10 possesses properties that suggest a possible role in Alzheimer's disease, its inhibition appears to be a therapeutic strategy. After we identified the androsterone (ADT) derivative 1 as a first steroidal inhibitor of 17ß-HSD10, new analogs were synthesized to increase the metabolic stability, to improve the selectivity of inhibition over 17ß-HSD3 and to optimize the inhibitory potency. From six D-ring derivatives of 1 (17-CO), two compounds (17ß-H/17α-OH and 17ß-OH/17α-CCH) were more metabolically stable and did not inhibit the 17ß-HSD3. Moreover, solid phase synthesis was used to extend the molecular diversity on the 3ß-piperazinylmethyl group of the steroid base core. Eight over 120 new derivatives were more potent inhibitors than 1 for the transformation of E2 to E1, with the 4-(4-trifluoromethyl-3-methoxybenzyl)piperazin-1-ylmethyl-ADT (D-3,7) being 16 times more potent (IC50 = 0.14 µM). Finally, D-ring modification of D-3,7 provided 17ß-OH/17α-CCH derivative 25 and 17ß-H/17α-OH derivative 26, which were more potent inhibitor than 1 (1.8 and 2.4 times, respectively).

Identification of fused 16ß,17ß-oxazinone-estradiol derivatives as a new family of non-estrogenic 17ß-hydroxysteroid dehydrogenase type 1 inhibitors.

A new family of cyclic carbamate-estradiol derivatives has been designed to remove the intrinsic estrogenic activity of a parent acyclic compound reported as a potent inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). The synthesis of two series of fused 16ß,17ß-oxazinone-estradiol derivatives, saturated compounds 7a-d and unsaturated compounds 10a-d, led to the identification of 10b, a 17ß-HSD1 inhibitor (IC50 = 1.4 µM) without estrogenic activity in estrogen-sensitive T-47D cells. Interestingly, this compound was found selective over 17ß-HSD2 and 17ß-HSD12. A computational analysis of inhibitors into 17ß-HSD1 by molecular docking also revealed interesting structure-activity relationships that could be helpful in the design of new generation of 16ß,17ß-oxazinone-estradiol analogs.

In vitro and in vivo evaluation of a 3ß-androsterone derivative as inhibitor of 17ß-hydroxysteroid dehydrogenase type 3.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3 or HSD17B3) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T), by stereoselectively reducing the C17 ketone of 4-androstene-3,17-dione (4-dione), with NADPH as cofactor. Since T plays an important role in androgen-sensitive diseases, this enzyme is thus an interesting therapeutic target. In an attempt to design compounds to lower the level of T, we synthesized androsterone derivatives substituted at position 3 as inhibitors of 17ß-HSD3, and selected one of the most potent compounds for additional studies. In an enzymatic assay in homogenized and whole HEK-293 cells overexpressing 17ß-HSD3, the inhibitor RM-532-105 efficiently inhibited the conversion of natural substrate 4-dione (50nM) into T with an IC50 of 26nM and 5nM, respectively. Moreover, the inhibitor RM-532-105 (10mg/kg) reached a plasma concentration of 250ng/mL at 7h (AUC 24h: 3485ngh/mL) after subcutaneous (s.c.) injection in the rat. In order to mimic the human situation in which 4-dione is converted to T in the testis, we used intact rats. Treatment for 7 days with 17ß-HSD3 inhibitor RM-532-105 by s.c. injection or oral gavage exerted no effect on the testis, prostate and seminal vesicle weight and no modification in the levels of plasma steroids. However, after this treatment, the concentration of inhibitor in plasma increased depending on the dose. We thereafter determined the concentration of inhibitor in the testis and we discovered that the compound was slightly present. In fact, at 10mg/kg, the inhibitor RM-532-105 seems to have difficulty penetrating inside the testis and was found to be concentrated in the testicular capsule, and therefore unable to inhibit the 17ß-HSD3 located inside the testis. However, with a higher dose of 50mg/kg injected s.c. in rats, RM-532-105 significantly decreased the level of T and dihydrotestosterone measured in plasma at 2h.

Synthesis and optimization of a new family of type 3 17 beta-hydroxysteroid dehydrogenase inhibitors by parallel liquid-phase chemistry.

Type 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) transforms 4-androstene-3,17-dione (Delta(4)-dione) into the androgen testosterone. To produce potent inhibitors of this key steroidogenic enzyme, we performed parallel liquid-phase synthesis of 3beta-substituted androsterone (ADT) libraries (A-D) in good yields and average high-performance liquid chromatography (HPLC) purities of 92-94%. The first library (A) of 3 beta-amidomethyl-ADT derivatives (168 members), including two levels of molecular diversity on the amide (R(1) and R(2)), was synthesized with a parallel liquid-phase method (method I) in less time than with the classic chemistry method. The screening of library A revealed that relatively small hydrophobic chains at R(1) (5-8 carbons) and small hydrophobic substituents at R(2) (1-4 carbons) provided the most potent inhibitors. In accordance with these inhibition results, a second library (B) of 3 beta-amidomethyl-ADT derivatives (56 members) was generated in a very short time using an improved method based on scavenger resins and liquid-phase parallel chemistry. Library B produced more potent inhibitors than library A and provided useful structure-activity relationships that directed the design of a third library (C) of 49 members. Once again, very potent inhibitors were identified from library C and 3 beta-[(N-adamantylmethyl-N-butanoyl)aminomethyl]-3 alpha-hydroxy-5 alpha-androstan-17-one (C-7-3) was identified as the most potent inhibitor of the three libraries with an inhibitory activity (IC(50) = 35 nM) 18-fold higher than that of the natural substrate of the enzyme, Delta(4)-dione, (IC(50) = 650 nM) used itself as inhibitor. Finally, we designed a library (D) of 3-carbamate-ADT derivatives (25 members) using the efficient parallel liquid-phase method III, which allowed the synthesis of more rigid molecules with two levels of molecular diversity (R(1)/R(2) and R(3)) in the local area occupied by the adamantane group of C-7-3. Interestingly, one of the most potent inhibitors of library D, the 3R-spiro-[3'-[3' '-N-morpholino-2' '-(3' "-cyclopentyl-propionyloxy)propyl]-2'-oxo-oxazolidin-5'-yl]-5 alpha-androstan-17-one (D-5-4), showed an inhibitory activity on type 3 17 beta-HSD similar to that of compound C-7-3, while exhibiting a nonandrogenic profile.