STS inhibitor – N-Dimethylformamide inhibitor

Synthesis of 16β-derivatives of 3-(2-bromoethyl)-estra-1,3,5 (10)-trien-17β-ol as inhibitors of 17β-HSD1 and/or steroid sulfatase for the treatment of estrogen-dependent diseases

Maxime Lesp´erance a, Jenny Roy a, Adrien Djiemeny Ngueta a, Ren´e Maltais a, Donald Poirier a, b,*

A B S T R A C T

17β-HydroXysteroid dehydrogenase type 1 (17β-HSD1) and steroid sulfatase (STS) are involved in the synthesis of the most potent estrogen in the human body, estradiol (E2). These enzymes are known to play a pivotal role in the progression of estrogen-dependent diseases, such as breast cancer and endometriosis. Therefore, the inhi- bition of 17β-HSD1 and/or STS represents a promising avenue to modulate the growth of estrogen-dependent tumors or lesions. We recently established the key role of a bromoethyl side chain added at the C3-position of a 16β-carbamoyl-benzyl-E2 nucleus to covalently inhibit 17β-HSD1. To extend the structure–activity relationship study to the C16β-position of this new selective irreversible inhibitor (PBRM), we synthesized a series of analog compounds by changing the nature of the C16β-side chain but keeping the 2-bromoethyl group at position C3. We determined their 17β-HSD1 inhibitions in T-47D cells (transformation of E1 into E2), but we did not obtain a stronger 17β-HSD1 inhibitor than PBRM. Compounds 16 and 17 were found to be more likely to bind to the catalytic site and showed a promising but moderate inhibitory activity with estimated IC50 values of 0.5 and 0.7 µM, respectively (about 10 times higher than PBRM). Interestingly, adding one or two sulfamate groups in the D- ring’s surroundings did not significantly decrease compounds’ potential to inhibit 17β-HSD1, but clearly improved their potential to inhibit STS. These results open the door to the development of a new family of steroid derivatives with dual (17β-HSD1 and STS) inhibiting actions.

Keywords:
Steroid
Chemical synthesis
17β-hydroXysteroid dehydrogenase type 1 Steroid sulfatase
Inhibitor

1. Introduction

Estradiol (E2) is known as the most potent estrogen in the human body and is responsible for the development and differentiation of estrogen-dependent tissues. Besides its physiological effects [1,2], E2 is also involved in the incidence and progression of estrogen-dependent diseases (EDDs), such as breast cancer [3,4], ovarian tumors [5], endometriosis [6,7], endometrial hyperplasia [8] and uterine leio- myoma [9]. Current hormonal therapies use antiestrogens, selective estrogen receptor modulators or aromatase inhibitors to block the es- trogen receptor or the biosynthesis of estrogens [10–15]. Like the aromatase, 17β-hydroXysteroid dehydrogenase type 1 (17β-HSD1) and steroid sulfatase (STS) are two enzymes involved in the formation of estrogens. 17β-HSD1 drives the reduction of estrone (E1) into E2, the last step in the formation of the most potent natural estrogen, while STS catalyzes the hydrolysis of sulfated 3-hydroXysteroid, such as E1-sulfate and E2-sulfate, into corresponding hydroXylated steroids E1 and E2. For this reason, the inhibition of one of these enzymes, or both, seems to be a promising avenue to treat EDDs, by reducing the levels of E2.
Some work has been done in the past to inhibit these two key en- zymes and many compounds have been synthesized and show promising inhibitory activity [16–25], but only one STS inhibitor reached clinical trials with limited success [26,27]. For 17β-HSD1, four steroidal and non-steroidal inhibitors demonstrated their efficacy in reducing estrogen-dependent breast cancer tumor growth in mouse models (Xe- nografts) but have not yet been reached clinical trials [28].
Compound CC-156 (Fig. 1A) was the most potent 17β-HSD1 inhibitor synthesized by our research group [29], but this compound has demonstrated some estrogenic effects, which were eliminated with the outcome of PBRM (Fig. 1B) [30–32]. This new steroid derivative is the first irreversible non-estrogenic steroidal inhibitor of 17β-HSD1, as confirmed by mechanistic [33] and X-ray analyses [34]. Nevertheless, the enzymatic assay demonstrated a lower affinity for the enzyme compared to the reversible and potent inhibitor CC-156, hence the need MeOH: water from 70:30 to 100:0. The wavelength of the UV detector was 190 nm.

2. Materials and methods

2.1. General

Chemical reagents were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). The usual solvents were obtained from Fisher Scientific (Montreal, QC, Canada) and were used as received. Anhydrous acetonitrile (ACN), dichloromethane (DCM), dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained from Sigma-Aldrich. Thin- layer chromatography (TLC) and flash-column chromatography (FCC) were performed on 0.20-mm silica gel 60 F254 plates (E. Merck; Darmstadt, Germany) and with 230–400 mesh ASTM silica gel 60 (Silicycle, Quebec, QC, Canada), respectively. Microwave experiments were conducted on a Biotage Initiator microwave instrument (Charlotte, NC, USA). Infrared spectra (IR) were recorded on a Horizon MB 3000 ABB FTIR spectrometer (Quebec, QC, Canada) and the significant band (ν) reported in cm—1. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for 1H and 100.6 MHz for 13C on a Bruker Avance 400 digital spectrometer (Billerica, MA, USA). The chemical shifts (δ) were expressed in ppm and referenced to residual chloroform (7.26 and 77.0 ppm), acetone (2.06 and 29.8 ppm) or dimethylsulfoXide (DMSO) (2.49 and 39.5 ppm) signals for 1H and 13C NMR, respectively. Low- resolution mass spectra (LRMS) were recorded on a Shimadzu Prominence apparatus (Kyoto, Japan) equipped with a LCMS-2020 mass spectrometer (APCI probe) and expressed in m/z. The purity of final compounds to be tested was determined with a Shimadzu HPLC apparatus equipped with SPD-M20A photodiode array detector, a Setima HPC18 reversed-phase column (250 mm × 4.6 mm) (compounds 12–18, 20, 22–27 and 34) or an Alltima HP18 reversed-phase column (250 mm× 4.6 mm, 5 µm) (compounds 35, 36 and 39) and a solvent gradient of hydroXybenzaldehyde, 4-hydroXybenzaldehyde, 3,4-dihydroXybenzal- dehyde and 3,5-dihydroXybenzaldehyde were protected into tetrahy- dropyranyl (THP) ethers in a yield ranging from 85 to 96% according to the procedure reported by Bernady et al [38]. NMR data of intermediate compound 2 as well as aldehydes used as building blocks agree with those published [32,37,38].

2.2. Chemistry

2.2.1. Synthesis of starting compounds for a new generation of PBRM analogs that would have optimal binding

The key steroid intermediate 3-(2-hydroXyethyl-estra-1,3,5(10)-for the enzyme catalytic site. The development of PBRM clearly demonstrated the key role of the 2-bromoethyl group in the C3 position of an estrane nucleus to selec- tively inactivate 17β-HSD1 [32,35]. Therefore, we kept the bromoethyltrien-17-one (2) was prepared from estrone (1) as previously reported by Maltais et al [32]. The sulfonamide group of 3-formylbenzenesulfona- mide was protected into a triphenylmethyl (Trityl; Tr) derivative in a95% yield according to the general procedure reported by Behloul et al moiety in new designed inhibitors and focused on the modification of [37]. The hydroXyl groups of 3-(hydroXymethyl)benzaldehyde, 3- the benzylamide side chain to maximize enzyme-inhibitor interactions and, consequently, inhibitory activity. Based on the crystal structure of CC-156 and PBRM complexed with 17β-HSD1 [36,34], we suspected that amino acids could be targeted in our inhibitor design strategy. As examples, asparagine (Asn)-152 and leucine (Leu)-95 are potential candidates, due to their proXimity to the C16β-benzylamide ring of the estrane nucleus. Thus, Asn would lead to polar interactions because of its amide group, whereas Leu possesses an isobutyl residue that would lead to an apolar (hydrophobic) interaction. Maintaining the 2-bromoethyl side chain at C3, we designed and synthesized different PBRM analogs for potentially targeting an amino acid and promoting a polar or an apolar binding (Fig. 1C). Our design strategy also includes the intro- duction of a sulfamate group to potentially obtain a compound inhib- iting both 17β-HSD1 and STS.

2.2.2. General procedure for aldolization (aldol condensation)

To a stirring solution the steroidal ketone 2 (0.3 mmol) in ethanol (EtOH) (0.04 M) was added the appropriate formyl-benzyl derivative (0.6 mmol) and an aqueous KOH (10%) solution (15% v/v). The solution was heated at refluX for 0.5 to 2 h. The resulting solution was diluted with water, neutralized with aqueous HCl (10%) and extracted with EtOAc. The organic layers were combined, washed with brine, dried with MgSO4, filtered, and evaporated under reduced pressure. The crude compound was purified by FCC to give the corresponding α,β-unsaturated ketone (enone).

2.2.3. General procedure for C17 reduction of enone

To a solution of enone (0.25 mmol) in MeOH (0.03 M) was added NaBH4 (0.38 mmol), and the miXture was stirred at room temperature. After 1 h, the resulting solution was concentrated under vacuo, diluted with EtOAc, washed with water, washed with brine, and dried with MgSO4. The organic phase was filtered and evaporated under reduced pressure and the crude compound was purified by FCC to give the cor- responding allylic alcohol (enol).

2.2.4. General procedure for catalytic hydrogenation of the allylic alcohol

To a solution of enol (0.2 mmol) in EtOH (0.03 M) under argon at- mosphere at room temperature was added palladium on charcoal (10% w/w). The reaction vessel was flushed 3 times with hydrogen and stirred for 24 h at room temperature. The resulting solution was filtered on Celite and then evaporated under reduced pressure. The crude compound was purified by FCC to give the desired 17β-alcohol derivative.

2.2.5. General procedure for bromination

To a solution of 3-(2-hydroXyethyl)-steroid derivative (0.08 mmol) in DCM (0.03 M) at 0 ◦C was added triphenylphosphine (0.32 mmol), imidazole (0.32 mmol) and carbon tetrabromide (0.32 mmol). The re action miXture was left to slowly warm to room temperature over 2 h. The resulting miXture was poured into water and extracted with DCM. Silica gel basified with triethylamine was added, and the solvent was removed under reduced pressure. The resulting dry pack was flushed with a solution of diethyl ether/hexanes (7:3). The collected filtrate was evaporated to give the crude 3-(2-bromoethyl)-steroid derivative, which was purified by FCC.

2.2.6. General procedure for the hydrolysis of protective group

To a solution of trityl (Tr)-protected sulfonamide (0.05 mmol) or tetrahydropyranyl (THP)-protected hydroXyl in acetone (0.002 M) was added a solution of aqueous HCl (10%) (0.002 M). The miXture was stirred at room temperature until completion (~1h). The acetone was removed under reduced pressure and the suspension was extracted with DCM. The organic phase was washed 3 times with water and evaporated. The resulting crude compound was purified by FCC using an isocratic solution of hexanes/EtOAc.

2.3. Biology

2.3.1. 17β-HSD1 inhibition assay

T-47D breast cancer cells were grown in RPMI medium supple- mented with 10% (v/v) fetal bovine serum (FBS), L-glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 µg/mL) and 17β-estradiol (E2) (1 nM). The cells were seeded in a 24-well plate (3000 cells/well). T-47D cells were suspended in the RPMI medium supplemented with insulin (50 ng/mL). A 5% (v/v) FBS treated with dextran-coated char- coal was used to remove the endogenous steroids. Stock solution of each compound to be tested was previously prepared in dimethylsulfoXide (DMSO) and diluted with culture medium to achieve appropriate con- centrations prior to use. After 24 h of incubation, a diluted solution was added to the cells to obtain the appropriate final concentration (0.1 or 1μM for screening and ranging from 1 nM to 5 μM for IC50 value determination). The final concentration of DMSO in the well was adjusted to 0.1%. Additionally, a solution of [14C]-estrone (E1) (American Radio- labeled Chemicals, Inc., St. Louis, MO, USA) was added to obtain a final concentration of 60 nM. Cells were incubated for 24 h, and each inhibitor was assessed in triplicate. After incubation, the culture medium was removed and labeled steroids ([14C]-E1 and [14C]-E2) were extracted with diethyl ether. The organic phase was evaporated to dryness with nitrogen stream. Residues were dissolved in DCM, dropped on silica gel thin layer chromatography plates (EMD Chemicals Inc., Gibbstown, NJ, USA), and eluted with toluene/acetone (4:1) as solvent system. Substrate [14C]-E1 and metabolite [14C]-E2 were identified by comparison with reference steroids (E1 and E2) and quantified using the Storm 860 system (Molecular Dynamics, Sunnyvale, CA, USA). The percentage of transformation and the percentage of inhibition were calculated as follow: % transformation = 100[14C]-E2/([14C]-E1 + [14C]-E2) and % of inhibition = 100 (% transformation without inhibitor— % transformation with inhibitor) / % transformation without inhibi- tor. IC50 values were calculated using GraphPad Prism 6 solfware.

2.3.2. Steroid sulfatase inhibition assay

HEK-293 cells overexpressing the steroid sulfatase (HEK-293[STS]) [39] were homogenized by repeated freezing at 80 ◦C (five times) and defrosting at 4 ◦C. The enzymatic reaction was carried out using tritiated estrone sulfate ([3H]-E1S (10 nM) (American Radiolabeled Chemicals Inc., St. Louis, MO, USA), adjusted to a final concentration of 1 µM with E1S (Sigma-Aldrich, Oakville, ON, Canada) in tris–acetate buffer at pH 7.4 (1 mL), containing 5 mM EDTA and 10% glycerol and a DMSO so- lution of test compound. DMSO alone was used for control. After 2 h of incubation at 37 ◦C, the reaction was stopped by adding xylene (1 mL). Tubes were vortexed and centrifuged at 3000 rpm for 20 min to separate organic and aqueous phases. An aliquot (500 µL) of each phase was used for radioactivity measurement using a Wallac 1400 scintillation counter (Ramsey, MN, USA). The percentage of tritiated estrone (([3H]-E1) produced from [3H]-E1S (100% for control without inhibitor) was calculated and next the percentage of inhibition determined at each concentration (0.1, 1, 5 and 10 µM) of compounds tested. IC50 values were calculated using GraphPad Prism 6 software.

3. Results and discussion

3.1. Chemical synthesis

All compounds were synthesized from the key intermediate 2, which was obtained from commercially available estrone (1) [32]. This inter- mediate ketone was taken forward for the introduction of each appropriate C16β-benzyl side chain using the standard 3-step procedure developed for the synthesis of CC-156 and PBRM [29,30]. Briefly, an aldol condensation of an aldehyde at position C16 of ketone 2 first produced an enone (α,β-unsaturated ketone) intermediate, which was stereoselectively reduced into the corresponding enol (17β-allylic alcohol) derivative, and this latter reduced by a stereoselective catalytic hydrogenation. The running of each of its three stages is monitored using NMR markers established during previous studies on C16 and C17 positions [29,30].
Sulfonamide, methylalcohol, phenolic and trimethoXy benzyl de- rivatives 12–18 were synthesized as reported in Scheme 1. Starting from ketone 2, the standard 3-step procedure provided alcohols 3–5, which after purification and characterization were brominated to afford 6–8. Especially for 4, which will be further used as an intermediate in the synthesis of other compounds (Scheme 2), the bromination conditions were selected to avoid cleavage of the sensitive protecting group during the bromination of the resulting primary alcohol. Thus, the Appel re- action using PPh3 and CBr4 was favored over the reaction using N-bro- mosuccinimide or bromine for the formation of 6–8 as well as all other bromo derivatives. Different bases were added to the standard condi- tions of the Appel reaction to eliminate the acidity of the bromoform generated, and imidazole was selected over pyridine, DIPEA, DMAP and K2CO3, which produced lower yields of bromo derivatives. On the other hand, intermediate compounds 9–11 and final compound 18 were ob- tained from 2 with the above strategy using the 3-step procedure (aldol condensation, NaBH4 reduction and catalytic hydrogenation) followed by the bromination. Final compounds 12–17 were next obtained from 6 to 11 after cleavage of Tr or THP protecting groups with an acid treatment.
Key intermediate compound 4 was also used for the synthesis of 20, 22, 23 and 24, as reported in Scheme 2. The THP protecting group of 4 was first hydrolyzed to generate the trihydroXy derivative 19, and the two primary alcohols of this later were next brominated under the Appel conditions providing the dibromoderivative 20. To generate 22–24, the primary alcohol of 4 was brominated and the OTHP hydrolyzed to provide 13. The two alcohols of 13 were oXidized with Jones’ reagent to afford the keto-carboXylic acid 21, which was treated with NaBH4 to stereoselectively reduce the C17-carbonyl, thus providing 22 as a final compound. The carboXylic acid 22 was easily transformed to the methylester 23 with trimethylsilyldiazomethane, a mild and efficient reagent for the methylation of carboXylic acids. Because bromide is a good leaving group, it was not possible to obtain 24 from 22 using a coupling agent, and we only observed the substitution of the bromoethyl side chain in C3 with the 5-methyl-2-thiazolamine. Compound 24 was however obtained from a Schotten-Baumann acylation using DIPEA, as base, 5-methyl-2-thiazolamine and the acid chloride derivative gener- ated from 22.
Compounds 25–27 and 34 were prepared as reported in Scheme 3. Starting from compound 2 (Scheme 1), the pyridinyl group was added on the steroid D-ring using the standard 3-step C16β-functionalization procedure followed by the bromination of the side chain at C3 to afford lower temperature to avoid this side reaction. The intermediate enone derivative obtained from 2 was next carried forward with the two reduction steps (NaBH4 and H2/cat.) followed by the final bromination providing 27.
Compound 34, the PBRM analog with a methylene spacer between the carboXamide and the 16β-benzyl group, was obtained using the standard 3-step C16β-functionalization of compound 2 followed by a bromination of the C3-side chain (Scheme 3). The building block needed in the first step (aldol condensation), the aldehyde 33, was previously synthesized from isophthalaldehyde (28) in 5 steps. Instead of per- forming a selective reduction of the bis-aldehyde 28, we opted for a selective protection. A mono-ketalization was first performed using the procedure reported by Zhang et al. [40] and the remaining formyl group was reduced into alcohol 30. This alcohol was then brominated to 31 before being substituted by a nitrile to give 32. For the cyanidation, a phase transfer reaction using a miXture of an organic solvent and water [41] was preferred over a procedure using only a solvent, like ethanol. In the last step, the protecting ketal group was hydrolyzed to provide the aldehyde 33.
Sulfamate derivatives 35, 36 and 39 were prepared from phenol derivative 14 (Scheme 1) by a mono- or di-sulfamoylation using sulfa- moyl chloride as a reagent (Scheme 4). Two different methods were generally used for the synthesis of sulfamates: 1) the alcohol in DCM was treated with the reagent in the presence of 2,6-di-tert-butyl-4-methyl- pyridine and 2) the alcohol was treated with the reagent in DMA acting as solvent and base. With the second method, we observed the sulfamate formation starting from 14, but also the substitution of the bromide atom by a chloride. This side reaction was however avoided with the first method using a base in excess to scavenge the source of chloride. Under the first-method conditions, a miXture of 35 (aryl sulfamate) and 36 (disulfamate) was observed and both compounds were easily sepa- rated by chromatography. To obtain the phenol 39 (C17-sulfamate), the phenolic OH of 14 was first protected as a TBDMS ether derivative (compound 37) before being sulfamoylated with the first-method con- ditions to generate 38. The removal of the TBDMS group by a treatment with TBAF afforded the phenol-sulfamate derivative 39.

3.2. NMR characterization

The final products required for the structure–activity relationship (SAR) study were characterized by IR, 1H NMR, 13C NMR and MS. NMR analysis made it possible to confirm the integrity of the 2-bromoethyl chain, the estrane nucleus as well as the expected stereochemistry of the two new chiral centers formed in positions 16 and 17 and linked to the introduction of the various 16β-side chains. The compilation of all 13C NMR signals (Table S1, Supp. Info.) is particularly important, since it shows the similarity of the signals for each portion of the new inhibitors synthesized. For the 2-bromoethyl side chain at C3, the two methylene groups of the products appear at 39.0–39.6 (CH2CH2Br) and 32.8–34.2 (CH2CH2Br) ppm depending on the solvent used (Acetone‑d6 or CDCl3). For the steroid skeleton, no variations in chemical shifts of carbons 1–15 were observed, suggesting an identical skeleton to the lead compound PBRM. For the stereochemistry in C16 and C17 (Table 1), the chemical shifts observed at 11.3–13.2 ppm (C18), 81.8–82.2 ppm (C17) and 41.7–43.3 ppm (C16) confirmed that the desired 17β-OH and 16β-R products are obtained by carrying out the reaction sequence (aldol condensation, NaBH4 reduction and catalytic reduction) necessary to introduce the benzyl derivative side chain at C16. Indeed, we were able to compare the chemical shifts of the recently obtained final compounds (Table S1) with those coming from a 13C NMR analysis having already identified markers for each of the 4 possible stereoisomers in the case of 16-allyl-estradiols (Table 1). However, for compounds 36 and 39 the characteristic 17-CH signal is deshielded to 90.7 and 91.0 ppm, respectively, due to the sulfamoylation at C17.
Although the structure of the reference inhibitor PBRM was recently confirmed by its 3D structure X-ray analysis [34] and previously by a 2D NMR analysis [30] providing key NMR data, we performed a more exhaustive 2D NMR analysis for one representative of these new in- hibitors. As for PBRM, 2D NMR experiments (COSY, NOESY, HSQC and HMBC) carried out with compound 23 made it possible to confirm the desired structure and to carry out the complete assignment of all protons and carbons (Figs. S1-S6 and Table S2; Supp. Info.).

3.3. Inhibition of 17β-HSD1

3.3.1. Structure-activity relationships with PBRM analogs (Series 1)

All final compounds had their 17β-HSD1 inhibitory activity evalu- ated in vitro (Table 2). Those enzymatic assays were carried out in T-47D intact cells, a human breast cancer cell line expressing 17β-HSD1 and efficiently transforming E1 into E2 [43]. As previously observed, PBRM efficiently inactivated the enzyme activity by 67 and 99% at 0.1 and 1 μM, respectively. The replacement of the carboXamide group (CONH2) of the PBRM by a sulfonamide group (SO2NH2) caused a significant decrease in inhibitory activity, since compound 12 only inhibited 50% of activity at the concentration of 1 μM. A similar result was obtained with the methyl alcohol 13 since the latter produced a 30% inhibition at 1 μM. The presence of a phenolic OH, instead of CONH2 group, modu- lated the inhibitory activity depending on the positioning (meta > para) and the number (n = 2 > 1) of OH. Thus, inhibition values of 43, 14, 86 and 88% were obtained at 1 μM for the phenols 14 (m-OH), 15 (p-OH), 16 (m, p-di-OH) and 17 (m,m-di-OH), respectively. However, the pres- ence of three methoXy groups (m,p,m-tri-CH3O), bromomethyl (BrCH2) or methylester (COOCH3) is damaging for the inhibitory activity (be- tween 1 and 4% inhibition at 1 μM for compounds 18, 20 and 23). The results also suggest that an acid group able of generating a hydrogen bond is favorable for inhibition. Indeed, the oXidation of methyl alcohol 13 to carboXylic acid 22 increased the inhibitory activity from 5 and 30% to 24 and 50%, at 1 and 10 μM, respectively.
On the other hand, the oXidation of the carboXamide group to nitrile (compound 27) or its N-substitution by a methylthioimidazole (com- pound 24) are two transformations which practically eliminated the inhibitory activity of these PBRM analogs. However, the addition of a methylene spacer between the carboXamide group and the aromatic nucleus (CH2CONH2) only slightly decreased the inhibitory activity, which goes from 57 and 97%, at 1 and 10 μM, to 19 and 62% for the PBRM and its counterpart 34, respectively. Finally, the use of a pyr- idinylmethyl nucleus (compound 25) or the corresponding N-oXime (compound 26) in replacement of the carbamoylbenzyl nucleus of PBRM does not promote better inhibitory activity.
The inhibition percentages of this first series of compounds (Table 2) clearly showed that none of these PBRM analogs is to be a better in- hibitor than PBRM, but the presence of a small group with an H-donor character, the NH of Ar-CONH2, the OH of Ar-COOH or the OH of ArOH, seems important for 17β-HSD1 inhibitory activity. In fact, compounds promoting binding via a hydrogen bond, such as 14, 16, 17, 22 and 34 seem more likely to inhibit 17β-HSD1. According to those results, our best inhibitors are the dihydroXybenzyl derivatives 16 and 17 (IC50 = 0.5 and 0.7 µM), which possess a better inhibition profile than the mono hydroXybenzyl derivatives 14 and 15 (IC50 1.1 and 4.5 µM, respectively) and hydroXymethylbenzyl derivative 13 (30% inhibition at 1 μM). Compound 34, with a methyl carboXamide group showed also good 17β-HSD1 inhibition (IC50 = 0.8 µM). However, with an IC50 value of 0.08 µM, PBRM remains our leading 17β-HSD1 inhibitor.

3.3.2. 17β-HSD1 inhibition with sulfamate derivatives (Series 2)

The sulfamate group (OSO2NH2) is well known to inhibit the steroid sulfatase efficiently and irreversibly when positioned on an aryl deriv- ative [44–46]. This enzyme being involved in the transformation of inactive sulfate steroids into free hydroXylated steroids active on their receptors, or precursor of estrogens and androgens, the development of potent STS inhibitors would be a therapeutic strategy for the treatment of estrogen- and androgen-dependent diseases [20–25]. It would therefore be attractive to obtain a dual-action inhibitor inhibiting both enzymes (17β-HSD1 and STS). Among the PBRM analogs synthesized in Series 1, phenolic derivatives could be sulfamoylated to generate cor- responding sulfamate derivatives potentially inhibiting STS and 17β- HSD1 (in their sulfamate form or as the phenolic derivative released after STS inactivation). Indeed, it is known that arylsulfamates (estrone sulfamate as example) [44] potently inactivate STS and release the corresponding phenolic derivative after inhibiting the enzyme.
To evaluate our hypothesis, we selected the phenol derivative 14 and proceeded to the sulfamoylation of the OH groups to obtain the aryl- monosulfamate 35, the disulfamate 36 and the 17β-monosulfamate 39. The presence of one or two sulfamate groups reduces the ability of these PBRM analogs to inhibit 17β-HSD1 by an alkylating action of the 2-bromoethyl chain positioned at C3 (Table 2). Indeed, at the concen- tration of 1 μM, the sulfamates 35, 36 and 39 inhibited 18, 11 and 42%, respectively, of the transformation of E1 into E2 by 17β-HSD1, while the phenolic derivative 14 inhibited 43% of the enzyme activity. At 5 μM, the inhibition percentages increased to over 70% and their estimated IC50 values are close to that of phenol 14 (2.8, 2.5, 1.5 and 1.1 μM for 35, 36, 39 and 14, respectively). It should be noted that the addition of a sulfamate group at C17 reduces the inhibitory activity less than the addition of a sulfamate to the benzyl nucleus at C16 or of the addition of two sulfamate groups.

3.4. STS inhibition with sulfamate derivatives

The enzymatic assay was performed using a preparation of homogenized transfected HEK-293 cells as source of STS activity [39]. Sulfa- mate derivatives 35, 36 and 39 were tested for their ability to inhibit the transformation of E1S/(3H)-E1S into E1/(3H)-E1 by measuring the formation of labeled E1 in organic phase and remaining labeled E1S in aqueous phase by scintillation counting (Fig. 2). The phenolic com- pound 14 poorly inhibited the STS activity (50% at 5 μM) especially compared to potent STS inhibitor EM-1913 [47–49] used as positive control (100% inhibition at 0.1 μM). However, as expected, adding a sulfamate group on the 16β-phenolic ring of 14 increased the ability of the monoarylsulfamate 35 to inhibit STS by 20-fold (IC50 of 5.0 and 0.23 μM, respectively). Adding a second sulfamate group on the 17β-OH group of 35 by generating the disulfamate 36 produced a slight improvement in STS inhibition by 2-fold (IC50 = 0.23 and 0.10 μM, respectively). The positive effect of adding a sulfamate group at C17β was also observed by transforming the 17β-alcohol of 14 to the mono- sulfamate 39. In this case, the inhibition potency increased by 10-fold with IC50 of 5.0 and 0.55 μM for 14 and 39, respectively. Aryl sulfamates are well known to inhibit STS irreversibly by alkylating the enzyme [44], but this is not the case for alkyl sulfamates, which weakly inhibit STS [50]. Thus, the reason for the improvement of the STS in- hibition that we observed by sulfamoylating the 17β-OH group (2- and 10-fold according to the substrate) remains to be determined, it is however possible that a favorable reversible interaction was generated with STS.

4. Conclusion

We successfully synthesized a series of estrane derivatives that differ from the potent 17β-HSD1 inhibitor PBRM by their C16-side chain. Unfortunately, they seem to show lower inhibitory activity. In fact, we failed to increase the inhibitory activity of our lead compound, PBRM. However, we were able to establish that compounds with hydrophobic (apolar) C16β-side chain are clearly weaker 17β-HSD1 inhibitors than those with a side chain having a polar group, such as hydroXyls, sug- gesting that it is better to target Asn-152 instead of Leu-95. Interestingly, adding one or two sulfamate groups in the steroid D-ring did not significantly decrease the compounds’ potential to inhibit 17β-HSD1, but clearly improved their potential to inhibit STS. Compounds with dual (17β-HSD1 and STS) inhibiting actions were thus obtained, and their potency could be improved, especially by using triol compounds 16 and 17 as starting scaffolds. Molecular docking studies with both 17β- HSD1 and STS would be necessary to explain our inhibitory results and to suggest the synthesis of improved new PBRM analogs as dual inhibitors.

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