Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for optical imaging

Journal Pre-proofs Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for optical imaging René Maltais, Adrien Ngueta Djiemeny, Jenny Roy, Xavier Barbeau, JeanPhilippe Lambert, Donald Poirier PII: DOI: Reference:

S0968-0896(20)30162-0 https://doi.org/10.1016/j.bmc.2020.115368 BMC 115368

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

15 November 2019 28 January 2020 3 February 2020

Please cite this article as: R. Maltais, A. Ngueta Djiemeny, J. Roy, X. Barbeau, J-P. Lambert, D. Poirier, Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for optical imaging, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115368

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© 2020 Published by Elsevier Ltd.

Bioorg. Med. Chem. (revised version)

Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for optical imaging

René Maltais† Adrien Ngueta Djiemeny†, Jenny Roy†, Xavier Barbeau†, Jean-Philippe Lambert‡ and Donald Poirier*†,‡

† Laboratory

of Medicinal Chemistry, Endocrinology and Nephrology Unit, CHU de Québec – Research

Center, Québec, QC, Canada ‡

Department of Molecular Medicine, Faculty of Medicine, Université Laval, Québec, QC, Canada

(*) Corresponding Author: Donald Poirier Laboratory of Medicinal Chemistry CHU de Québec – Research Center (CHUL, T4-42) 2705 Laurier Boulevard Québec (Québec), G1V 4G2, Canada Tel.: 1-418-654-2296; Fax: 1-418-654-2298; E-mail: [email protected] 1

Graphical Abstract

Abstract:

Steroid sulfatase (STS) is an important enzyme regulating the conversion of sulfated steroids into their active hydroxylated forms. Notably, the inhibition of STS has been shown to decrease the levels of active estrogens and was translated into clinical trials for the treatment of breast cancer. Based on quantitative structure-activity relationship (QSAR) and molecular modeling studies, we herein report the design of fluorescent inhibitors of STS by adding a dansyl group on an estrane scaffold. Synthesis of 17α-dansylaminomethyl-estradiol (7) and its sulfamoylated analog 8 were achieved from estrone in 5 and 6 steps, respectively. Inhibition assays on HEK-293 cells expressing exogenous STS revealed a high level of inhibition for compound 7 (IC50 = 69 nM), a value close to the QSAR model prediction (IC50 = 46 nM). As an irreversible inhibitor, sulfamate 8 led to an even more potent inhibition in the low nanomolar value (IC50 = 2.1 nM). In addition, we show that the potent STS inhibitor 8 can be employed as an optical imaging tool to investigate intracellular enzyme sub-localization as well as inhibitory behavior. As a result, confocal microscopy analysis confirmed good penetration of the STS fluorescent inhibitor 8 in cells and its localization in the endoplasmic reticulum where STS is localized.

Keywords: Steroid, Danzyl derivative, Fluorescent agent, Steroid sulfatase, Enzyme inhibitor.

2

1. Introduction

Steroid sulfatase (STS) is the key enzyme that transforms inactive sulfated steroids to their corresponding hydroxyl forms, leading directly or not to active sexual hormones such as estradiol (E2) and dihydrotestosterone (DHT) (Fig. 1A). Therefore, STS has been identified as an interesting pharmaceutical target to control the levels of key estrogens and/or androgens, thus finding potential indications for the treatment of hormones-sensitive diseases like breast and prostate cancer, as well as endometriosis.1-7 Development of potent STS inhibitors has been reported from several research groups, including inhibitors of reversible and irreversible types.8-14 Notably, we previously described STS reversible inhibitors from a series of 17α-substituted-E2 derivatives, with the 17α-tert-butyl-benzyl-E2 derivative (1a) found as the most potent inhibitor of this sub-class with a low IC50 value of 28 nM (Fig. 1B).15,16 This series of phenolic compounds was also used as a dataset to develop a predictive quantitative structure-activity relationship (QSAR) model.17 Otherwise, adding a sulfamoyl group at C3 of 1a or 2methoxy-17α-tert-butylbenzyl-E2 (1b) turned these reversible inhibitors into irreversible ones (compounds 1c and 1d) while improving their potency (IC50 = 0.03 and 0.04 nM for 1c and 1d).18

Although mechanistic studies on the mode of inhibition of STS inhibitors (reversible and irreversible) have already been reported,1,19-21 to our knowledge, no study has directly addressed the intracellular behavior of these inhibitors in relation to STS localization and/or expression. Optical imaging is an interesting tool to track labeled-fluorescent small molecules and assess cellular interactions, tissue or in vivo distribution in regards to biological targets.22-24 With that mindset, we have recently reported the development a fluorescent dansyl-based inhibitor of 17β-hydroxysteroid dehydrogenase type 3 which allowed us to study the molecular behavior of this steroidal inhibitor in prostate cancer cells.25 The dansyl group has the advantage of being one of the smallest among available fluorophores, which favors its integration into a bioactive molecular scaffold and limits detrimental effects on biological activity.26-29 Herein we report the rational design, chemical synthesis and inhibitory potency of fluorescent-labeled STS inhibitor mimics (compounds 7 and 8, Fig. 1B) as well as their cellular molecular behavior by optical imaging.

3

A) O HO S O O

B)

Dansyl mimics

STS inhibitors

STS OH

HO

CHOLS

CHOL

X

INH

OH

H H

H

H

R O

STS O HO S O O

H

O

R

1a (R = OH; X = H) 1b (R = OH; X = OCH3) 1c (R = OSO2NH2; X = H)

O

NH O S

H

N

6 (R = OH) 7 (R = OSO2NH2)

1d (R = OSO2NH2; X = OCH3)

HO

PREG

PREGS INH

O

O HO S O O

O

STS

17-HSD

5-diol

ER

HO

Activation of estrogen-specific effects

DHEA

DHEAS

17-HSD 4

 -dione

INH O

O HO S O O

T

DHT

AR

Activation of androgen-specific effects

O

STS

17-HSD

E2

HO

E1S

E1

Figure 1. A) Involvement of steroid sulfatase (STS) in the transformation of inactive sulfated steroids into their active hydroxylated forms, and the sites of action of STS inhibitors (INH). B) Structure of STS inhibitors 1a-1d and proposed dansyl-labeled mimics 7 and 8. AR: androgen receptor, CHOLS: cholesterol sulfate, DHEAS: dehydroepiandrosterone sulfate, DHT: dihydrotestosterone, Δ5-diol: 5androstene-3β,17β-diol, Δ4-dione: 4-androstene-3,17-dione, E1: estrone, E1S: estrone sulfate, E2: estradiol, ER: estrogen receptor, 17β-HSD: 17β-hydroxysteroid dehydrogenase, PREGS: pregnenolone sulfate, T: testosterone.

2. Material and Methods

2.1. QSAR predictions

The QSAR model used for the prediction of the IC50 values of phenolic compounds 7 and 8 has been previously reported.17 It was developed with MDL Elsevier QSAR software version 2.3 from a dataset of 65 compounds, which was constituted of 17α-substituted-estradiol derivatives.16

4

2.2. Molecular Docking

Molecular docking calculations for STS were performed using QuickVina-W.30 The crystal structure of STS (PDB 1P49) was taken from RCSB PDB.31 Compounds 1a, 1c, 7, 8 and estrone sulfate (E1S) were built in the Pymol Open Source version (The PyMOL Molecular Graphics System, opensource version Schrödinger, LLC.) and were minimized with Open Babel 2.4.1

32

using the steepest

descent algorithm and MMFF94s force field for 10,000 steps. STS inhibitors bearing an aryl sulfamate moiety are expected to react with the catalytic residue hydroxyformylglycine 75 (HFG75)7,33,34 leading to a covalent bond with the enzyme and releasing the corresponding phenolic steroid. For our study, two enzyme structures were generated. For the first structure, all water molecules as well as the sulfate group linked to HFG75 (a glycine gem-diol) were removed. For the second structure, only the water molecules were removed prior to docking, conserving the HFG75 residue and bonded sulfate.

2.3 Chemical synthesis

2.3.1 General Methods Chemical reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA), Matrix Scientific (Columbia, SC, USA) and Zhejiang Xianju Pharmaceutical Co. (Xianju, Zhejiang, China). Flash chromatography was performed on SiliaFlash F60 (230-400-mesh) silica gel (Silicycle, Québec, QC, Canada). Thin-layer chromatography (TLC) was performed on Whatman 0.25-mm silica gel 60 F254 plates (Fisher Scientific, Nepean, ON, Canada) and compounds were visualized by exposure to UV light (254 nm) or/and using a solution of ammonium molybdate/sulphuric acid/ethanol (plus heating). Infrared (IR) spectra were recorded on a MB 3000 ABB FTIR spectrometer (Québec, QC, Canada). Only significant bands are reported (in cm-1). 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz using a Bruker AVANCE 400 spectrometer (Billerica, MA, USA). The chemical shifts (δ) are expressed in ppm and referenced to chloroform (7.26 and 77.0 ppm) or acetone (2.05 and 28.9 ppm) for 1H and 13C, respectively. High-resolution mass spectra (HRMS) were provided by Pierre Audet at the Chemistry Department of Université Laval (Québec, QC, Canada). 5

2.3.2 (17β)-17-(aminomethyl)-3-(benzyloxy)estra-1,3,5(10)-trien-17-ol (5) To a solution of oxirane 435 (8.0 g, 21 mmol) in anhydrous dimethylformamide (DMF) (100 mL) under an atmosphere of argon was added sodium azide (15.0 g, 230 mmol) and boric acid (6.0 g, 97 mmol). The reaction mixture was stirred at reflux temperature for 6 h, then poured into cold water and extracted with EtOAc. The combined organic layer was washed with water, brine, and dried over magnesium sulfate. After filtration and evaporation of the solvent under reduced pressure, the resulting crude product was purified by flash chromatography using hexanes/EtOAc (8:2) as eluent to give after crystallization in hexanes/CHCl3 the azido derivative (4.6 g, 52%). This compound (3.0 g, 7.2 mmol) was dissolved under an argon atmosphere in anhydrous tetrahydrofuran (THF) (100 mL), the solution cooled at 0 °C and LiAlH4 (438 mg, 11.5 mmol) added. After 5 h, the reaction was quenched with the addition of water and a solution of 1M NaOH. The mixture was extracted with EtOAc and the combined organic layer was washed with water, brine and dried over magnesium sulfate. After filtration and evaporation of the solvent under reduced pressure, the resulting crude product was purified by flash chromatography using CHCl3/trimethylamine (TEA)/MeOH (90:5:5) as eluent to give the amino compound 5 (2.66 g, 94%) as a white amorphous solid. IR (film)  3371, 3294 and 3186 (OH and NH2). 1H

NMR (CDCl3) δ: 0.93 (s, 3H, 18-CH3), 1.20-2.35 (unassigned CH and CH2, 16H, 2.60 (d, J = 12.0

Hz, 1H of 17α-CH2N), 2.84 (m, 2H, 6-CH2), 3.01 (d, J = 12.0 Hz, 1H of 17α-CH2N), 5.03 (s, 2H, OCH2Ph), 6.71 (d, J = 2.8 Hz, 1H, 4-CH), 6.77 (dd, J1 = 8.6 Hz, J2 = 2.7 Hz, 1H, 2-CH), 7.20 (d, J = 8.4 Hz, 1H, 1-CH), 7.38 (m, 5H, Ph). 13C NMR (CDCl3) δ: 14.5, 22.6, 26.1, 27.2, 29.8, 31.7, 36.1, 39.4, 43.8, 44.6, 49.6, 54.6, 69.9, 91.9, 112.3, 114.8, 126.4, 127.4 (2C), 127.8, 128.5 (2C), 132.7, 137.3, 138.0, 157.0. HRMS for C26H34NO2 [M + H]+: 392.2584 (calculated), 392.2598 (found). 2.3.3 (8β, 9α, 14α, 17β)-17-({[5-(dimethylaminonaphthalen-1-yl)sulfonyl]amino} methyl)-3-(benzyloxy) estra-1, 3, 5(10)-2,4-trien-17-ol (6) To a solution of amino compound 5 (1.0 g, 2.5 mmol) in anhydrous dichloromethane (DCM) (100 mL) at room temperature was added TEA (2 mL, 15 mmol) and dansyl chloride (1.3 g, 5 mmol). The solution was stirred for 2 h at room temperature, poured into water and extracted with DCM. The combined organic layer was washed with brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure. The crude compound was purified by flash chromatography using hexanes/EtOAc (7:3) as eluent to give compound 6 (707 mg, 44%) as a light green fluorescent amorphous solid. IR (film)  3300 (OH and NH). 1H NMR (CDCl3) δ: 0.84 (s, 3H, 18-CH3), 1.20-2.25 (unassigned 6

CH and CH2, 14H), 2.82 (m, 2H, 6-CH2), 2.89 (s, 6H, (CH3)2N), 3.01 (m, 2H, 17α-CH2N) 5.02 (s, 2H, OCH2Ph), 5.10 (m, 1H, NH), 6.70 (d, J = 2.4 Hz, 1H, 4-CH), 6.75 (dd, J1 = 2.5 Hz, J2 = 8.1 Hz, 1H, 2CH), 7.12 (d, J = 8.6 Hz, 1H, 1-CH), 7.20 (d, J = 7.5 Hz, 1H, 7’-CH), 7.40 (m, 5H, Ph), 7.56 (m, 2H, 3’-CH and 8’-CH), 8.25 (d, J = 7.2 Hz, 1H, 2’-CH), 8.31 (d, J = 8.6 Hz, 1H, 9’-CH), 8.55 (d, J = 8.5 Hz, 1H, 4’-CH). 13C NMR (Acetone-d6) δ: 13.7, 22.9, 26.1, 27.2, 31.5, 33.3, 39.6, 43.5, 44.7 (2C), 46.0, 49.5, 49.6, 49.8, 69.3, 81.9, 112.2, 114.6, 115.2, 119.5, 123.3, 126.1, 127.4 (2C), 127.6, 127.9, 128.3 (2C), 128.8, 129.7, 129.8, 129.9, 132.6, 136.2, 137.7, 137.9, 151.9, 156.7. HRMS: for C38H45N2O4S [M + H]+: 625.3095 (calculated), 625.3090 (found).

2.3.4 (8β,9α,14α,17β)-17-({[5-(dimethylaminonaphthalen-1-yl)sulfonyl]amino}methyl)estra-1,3,5(10)triene-3,17-diol (7) A mixture of dansyl derivative 6 (450 mg, 0.72 mmol) and 10% palladium on charcoal (225 mg) in MeOH/AcOH (75:25) (30 mL) was stirred for 2 h under hydrogen atmosphere. The palladium reagent was then removed by filtration on celite, washed with MeOH, and the filtrate was evaporated under reduced pressure. Purification by flash chromatography using hexanes/EtOAc (7:3) as eluent afforded compound 7 (335 mg, 55%) as a light green fluorescent amorphous solid. IR (film) : 3448 and 3333 (OH and NH2). 1H NMR (Acetone-d6) δ: 0.87 (s, 3H, 18-CH3), 1.18-2.20 (unassigned CH and CH2, 13H), 2.73 (m, 2H, 6-CH2), 2.88 (s, 6H, (CH3)2N), 3.02 (m, 2H, 17α-CH2N), 3.58 (s, 1H, OH), 6.16 (m, 1H, NH), 6.51 (s, 1H, 4-CH), 6.57 (dd, J1 = 8.4 Hz, J2 = 2.6 Hz, 1H, 2-CH), 7.02 (d, J = 8.4 Hz, 1H, 1-CH), 7.28 (d, J = 7.5 Hz, 1H, 7’-CH), 7.63 (m, 2H, 3’-CH and 8’-CH), 7.89 (s, 1H, OH), 8.24 (dd, J1 = 7.3 Hz, J2 = 0.8 Hz, 1H, 2’-CH), 8.45 (d, J = 8.7 Hz, 1H, 9’-CH), 8.57 (d, J = 8.5 Hz, 1H, 4’-CH). 13C NMR (Acetone-d6) δ: 13.8, 22.9, 26.1, 27.3, 29.4, 31.5, 33.3, 39.7, 43.5, 44.7 (2C), 46.0, 49.6, 49.8, 82.0, 112.6, 114.9, 115.2, 119.5, 123.3, 126.1, 127.9, 128.8, 129.7, 129.8, 129.9, 131.0, 136.1, 137.5, 151.9, 155.0. HRMS for C31H39N2O4S [M+H]+: 535.2625 (calculated), 535.2633 (found). HPLC purity: 95.2% (RT = 12.9 min; Alltima HP18 reversed-phase column (250 mm x 4.6 mm, 5 µm); solvent gradient of MeOH/H2O from 70:30 to 100:0).

2.3.5

(8β,9α,14α,17β)-17-({[5-(dimethylaminonaphthalen-1-yl)sulfonyl]amino}methyl)-17-hydroxy

estra-1,3,5(10)-trien-3-yl sulfamate (8) 7

Under an argon atmosphere and at room temperature, compound 7 (200 mg, 0.4 mmol) was dissolved in anhydrous DCM (20 mL). To this solution was added 2,6-di-tert-butyl-4-methylpyridine (DTMP) (460 mg, 2.2 mmol) and sulfamoylchloride (130 mg, 1.12 mmol). The mixture was stirred at room temperature for 2 h and the resulting solution was poured into water and extracted with EtOAc. The combined organic layer was washed with brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure. The crude compound was purified by flash chromatography using hexanes/EtOAc (7:3) as eluent to give 8 (20.4 mg, 10%) as light green fluorescent amorphous solid. IR (film) :   and    and . 1H NMR (Acetone-d6) δ: 0.88 (s, 3H, 18-CH3), 1.252.25 (unassigned CH and CH2, 13H), 2.83 (m, 2H, 6-CH2), 2.88 (s, 6H, (CH3)2N), 3.05 (m, 2H, 17αCH2N), 3.60 (s, 1H, OH), 6.16 (m, 1H, NH), 6.99 (s, 3H, 4-CH and NH2), 7.03 (d, J = 8.6 Hz, 1H, 2CH), 7.28 (t, J = 8.3 Hz, 2H, 1-CH and 7’-CH), 7.65 (m, 2H, 3’-CH and 8’-CH), 8.24 (d, J = 7.2 Hz, 1H, 2’-CH), 8.45 (d, J = 8.7 Hz, 1H, 9’-CH), 8.57 (d, J = 8.5 Hz, 1H, 4’-CH). 13C NMR (Acetone-d6) δ: 13.7, 22.9, 25.9, 26.9, 29.2, 31.4, 33.3, 39.2, 43.6, 44.7 (2C), 46.0, 49.6, 49.7, 81.9, 115.2, 119.2, 119.5, 122.0, 123.3, 126.4, 127.9, 128.8, 129.7, 129.8, 129.9, 136.2, 138.3, 138.6, 148.4, 151.9. HRMS for C31H40N3O6S2 [M+H]+: 614.2353 (calculated), 614.2347 (found). HPLC purity: 93.4% (RT = 13.7 min; Alltima HP18 reversed-phase column (250 mm x 4.6 mm, 5 µm); solvent gradient of MeOH/H2O from 70:30 to 100:0).

2.4 Biological evaluation 2.4.1 Cell Culture Human embryonic kidney (HEK)-293 cells overexpressing STS (HEK-293[STS]) were obtained from Dr. Van Luu-The (CHU de Quebec - Research Center)36 and maintained in culture flasks (175 cm2 growth area, BD Falcon) at 37 °C in a 5% CO2 humidified atmosphere. These cells were maintained in Minimum Essential Medium with phenol red supplemented with 10% FBS, penicillin (100 IU/mL), streptomycin (100 mg/mL), L-glutamine (2 mM), non-essential amino acids (0.1 mM), sodium pyruvate (1 mM) and geneticin (G418 sulfate) (700 mg/mL). The culture media was changed every two to three days, and the cells were split once a week to maintain cell propagation.

2.4.2 Steroid sulfatase inhibition assay

8

This assay was performed according to a previously described procedure.37 Briefly, HEK-293 cells overexpressing the steroid sulfatase (HEK-293[STS]) 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 an ethanolic solution of test compound. Only ethanol 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 3,000 rpm for 20 min to separate organic and aqueous phases. An aliquot (200 µL) of each phase was used for radioactivity measurement using a Wallac 1400 scintillation counter (Ramsey, MN, USA). The results were expressed as the percentage of estrone (E1) produced (100% for control without inhibitor) and the percentage of inhibition then calculated at each concentration (0.03, 0.3, 3 and 30 nM).

2.5 Fluorescent properties of compounds 7 and 8

Compounds 7 and 8 were first dissolved in dimethylsulfoxide (DMSO) at a concentration of 10-2 M and then diluted in phosphate buffer saline (PBS) to obtain a 50 µM solution containing 0.5% DMSO. The excitation and the emission spectra were recorded on a spectrophotometer (Fluorolog-3, model FL321, Hariba Scientific, Irvine, CA, USA).

2.6 Optical imaging

2.6.1 Wide field fluorescent microscopy HEK-293[STS] cells were plated in 12 wells at 16 x 103 cells per well in the same medium described above but without phenol red. After 48 h of pre-incubation at 37 C to allow the cells to attach properly, they were incubated 2 and 4 h with compounds 7 and 8 at final concentrations of 1, 10 and 30 µM. The cells were analyzed under the EVOS M5000 Imaging System, a digital inverted microscope (Thermo Fisher Scientific, Waltham MA, USA). A fluorite 40X long working distance phase contrast 9

objective was used and the localization of the compound was seen using the DAPI LED light cube (excitation 357 nm and emission 447 nm).

2.6.2. Confocal microscopy The cellular localization of compound 8 was determined by evaluating its co-localization with an endoplasmic reticulum (ER) specific dye for live-cell imaging (ER-Tracker Red (BODIPY, TR Glibenclamide), Invitrogen, Carlsbad, CA, USA) and a cell membrane stain (CellMask Deep Red, Invitrogen, Carlsbad, CA, USA). For the tests, 1 x 104 HEK-293[STS] cells were plated into sterile Nune Lab-Tek II Chambered coverglass-8 wells 200 µL using the same medium described above but without phenol red. After 48 h of pre-incubation at 37 C to allow the cells to attach properly, they were incubated for 4 h with compound 8 at a final concentration of 30 µM. ER-Tracker (1 µM final) and CellMask (4.5 µg/mL) were added in the medium for the last 45 and 10 minutes, respectively. At the end of staining procedure, the cells were placed in a Chamlide live cell system (LCI, Seoul, Republic of Korea) on an Olympus IX81 inverted microscope. The objective used was a 60X oil PlanApo N (NA 1.42). Image acquisition was made with the Quorum WaveFX spinning disk confocal system (Quorum Technologies, Guelph, ON, Canada) with MetaMorph version 7.8.12.0 (Molecular Devices, San Jose, CA, USA). The localization of compound 8 was performed using the 403 nm laser and 460/50 nm emission filter. The same area was excited with 561 nm laser and 593/40 nm emission filter for ER specific localization, whilst for CellMask; the wavelengths for excitation and emission were 642 nm and 700/75 nm, respectively. The image analysis and deconvolution were made with the software Volocity 4.0 (Quorum Technologies, Guelph, ON, Canada).

3. Results and Discussion

3.1. QSAR model and prediction of steroid sulfatase inhibition

A QSAR model was previously developed by our research group and successfully used for the prediction of STS inhibition of 17α-E2 derivatives.17 This model was built with a data set of 65 molecules consisting in 17α-substitued-E2 derivatives (Fig. 1S, Supplementary data). Since the addition of a hydrophobic substituent at position C-17 of E2 was generally beneficial to inhibit STS,16 we explored 10

the possibility of adding a fluorescent dansyl substituent at this position. To test this proposal, we used our previous QSAR model to predict the IC50 value of the 17α-dansylaminomethyl-E2 (7). As a result, this proposed dansyl derivative was predicted to be a highly potent STS inhibitor with an IC50 value (46 nM) in the low nanomolar range. This predicted IC50 value was encouraging since the predicted IC50 value (69 nM) of the reference inhibitor 1a was in the same range (Table 1), and the validity of the prediction was acceptable, being close from the experimental IC50 value of 1a (28 nM).16 These results motivated us to start the synthesis of two dansyl E2-derivatives, phenol 7 and its sulfamate analog 8.

Table 1. Predicted IC50 values from QSAR model and experimental IC50 values for a series of estrane derivatives.

Compound

Structure

Predicted IC50 (nM)a

Experimental IC50 (nM)b

69

45 (28)c

OH

1a

H H

H

HO OH

1c

H H

O H 2N S O O

H

N/A

0.8

46

69

N/A

2.1

N/A

7600c

OH HN

H

7

H

O

H

S

O

HO N OH HN

H

8

O H 2N S O O

H

O

H

S

O

N

O

Estrone sulfate

H O HO S O O

H

H

a) Data generated by the QSAR model reported in reference 17. b) For the transformation of a mixture of [3H]-E1S and E1S (total 1 µM) into E1 by STS presents in HEK-293[STS] cells. c) Data from reference 16. N/A: not applicable. In fact, IC50 values cannot be predicted for sulfamate derivatives, as the QSAR model was built from phenolic derivatives only. 11

3.2. Molecular docking

As a complementary evaluation of compounds 1a, 1c, 7 and 8, molecular docking was performed to model their interactions in the STS catalytic site. We used the experimental structure of human STS (PDB 1P49),38 a structure having a sulfate group bound to the catalytic residue hydroxyformylglycine 75 (HFG75). The STS catalytic site presents a long hydrophobic cavity to accommodate the steroid scaffold and a polar region at the end of the cavity, which bear a calcium ion and the HFG75. The gem-diol of this key catalytic residue, reacts with the arylsulfamate moiety of STS inhibitors.1,7,33,34 The first docking experiment (# 1) was performed for compound 8 using the model of free STS protein (with the HFG75 residue without the sulfate group) to assess whether the dansyl group would impact binding with the enzyme as compared to compound 1c, bearing rather a tert-butyl-benzyl group at position 17α. The second docking experiment (# 2) was performed with the model of STS bound to a sulfate to assess whether the released phenolic compounds 1a and 7, obtained from the reaction of sulfamate compounds 1c and 8 with the HFG75 residue, can still be accommodated in the catalytic site. The best docking results for sulfamate compounds 1c and 8 as well as phenolic compounds 1a and 7 are represented in Fig. 2.

Figure 2. Key interactions observed in the STS binding site for the best docking results of STS inhibitors. A) Docking of sulfamate 1c (docking experiment 1), B) docking of sulfamate 8 (docking experiment 1), C) docking of phenol 1a (docking experiment 2) and D) docking of phenol 7 (docking experiment 2).

12

As seen in Fig. 2, the hydrophobic cavity of the catalytic site easily accommodates the steroid scaffold of all inhibitors. The tert-butyl-benzyl group of compounds 1a and 1c, as well as the dansyl group of compounds 7 and 8 bind in a hydrophobic groove at the entrance of the catalytic site, being composed of residues Leu103, Phe104, Phe178, Phe182, Phe223 and Phe553. On the other end of the catalytic site, the sulfamate moiety of both compounds 1c and 8 is positioned within H-bond distance of the HFG75, Lys368 and His290 (Fig. 2A and 2B). In the second docking experiment, for both compounds 1a and 7, the phenol is positioned within H-bond distance of Lys368 and the sulfate of the HFG75 residue (Fig. 2C and 2D).

3.3. Chemical synthesis

Compounds 7 and 8 were both synthesized from commercially available estrone (2) using a sequence of 5 or 6 steps represented in Scheme 1. Compound 2 was first protected as the benzyl-ether derivative 3 using benzylbromide and cesium carbonate in THF and, next, epoxidation of the 17-ketone of 3 under Corey-Chaykovsky conditions (trimethyl-sulfonium iodide and sodium hydride) stereoselectively afforded the oxirane 4.35 Subsequent opening of the epoxide with sodium azide in anhydrous DMF followed by reduction with LiAlH4 gave primary amine 5, which was then reacted with dansyl chloride in the presence of TEA to obtain sulfonamide 6. The next step was to cleave the benzylether group of 6 to obtain the phenol 7 using a palladium-catalyzed hydrogenation. However, the poor solubility of compound 6 in MeOH led to a long reaction time and an incomplete reaction. Fortunately, the addition of AcOH to MeOH (25:75) was found to be highly beneficial to increase both solubility and reactivity, resulting in a complete hydrogenation in 2 h. Finally, sulfamoylation of phenol 7 using sulfamoyl chloride and DTMP in DCM provided sulfamate 8 in low yield (10%). This result contrasts with sulfamoylation of estrone, which gave a high yield (80%) of estrone sulfamate when performed in parallel to sulfamoylation of 7 by using the same conditions and reagents. This low yield could be explained by the formation of a complex mixture of side products without the side chain at position C17. Alternatively, we also tried another sulfamoylation methodology using sulfamoyl chloride in dimethylacetamide,39 unsuccessfully. In fact, the sulfamoylation of a phenol derivative in presence of a tertiary alcohol at position C17 is difficult to achieve, as it has been observed for the sulfamoylation of 17α-piperazinylmethyl-E2, a phenol derivative closely related to 7, which mainly produced side products having lost the C17-side chain. In this case, we found that prior protection (trifluoromethylacetylation) 13

of both the secondary amine of piperazine and the 17β-tertiary alcohol was required to succeed sulfamoylation of the phenol at C3 in a very good yield (78%).35 Thus, two additional steps (protection and deprotection) in the sequence of reactions starting with the phenol 7 would be a strategy to improve the low yield of sulfamate 8. Nevertheless, the quantity of sulfamate 8 obtained was judged sufficient to engage inhibition assays and optical imaging experiments. O

a

H H

O

O

H

H

HO

H

b

H

H

H

BnO

4

3

2 (estrone) OH H H

H

BnO

OH

d

HN SO 2

H

H

H

BnO

c

H

BnO

N

6

NH2

5

e OH

OH H H

f H

HN SO2 H

H

HO

H

N SO2 H

H2NO2SO

N

N

8 (sulfamate)

7 (phenol)

Scheme 1. Chemical synthesis of compounds 7 and 8. Reagents and conditions: (a) Benzyl bromide, Cs2CO3, THF, rt, 2-4 h; (b) Tetramethylsulfonium iodide, NaH, DMSO / THF (70:30), rt, 18 h; (c) 1) NaN3, H3BO3, DMF, reflux, 6 h; 2) LiAlH4, THF, 0°C, 5 h; (d) Dansyl chloride, TEA, DCM, rt, 2 h; (e) H2, Pd/C 10%, MeOH/AcOH (75:25), rt, 2 h; (f) DTMP, sulfamoyl chloride, DCM, rt, 2 h.

3.4. Inhibition of steroid sulfatase activity

The inhibitory activity of dansyl-labeled derivatives 7 and 8 was determined following the conditions reported in previous STS inhibition assays.37 A high level of inhibition for the transformation of E1S to E1 was obtained for the phenol 7 (IC50 = 69 nM) with a comparable value to lead compound 1a (IC50 = 45 nM) in the same assay (Table 1). The experimental IC50 value of 7 was close to the predicted value obtained from the QSAR model (IC50

exp

= 69 nM vs IC50

pred

= 46 nM), thus validating our 14

predictive approach. As anticipated, the sulfamate analog 8, an irreversible STS inhibitor, was 31 times more potent (IC50 = 2.1 nM) than phenol 7, a reversible STS inhibitor. Indeed, even if we did not formally address their type of inhibition by enzyme kinetic study, we assumed that phenol 7 and sulfamate 8 are reversible and irreversible STS inhibitors, respectively, based on previous reports.1,7,14,21,33,40 The inhibitory activities of these two compounds acting differently were judged adequate to engage optical imaging studies.

3.5. Fluorescence spectra The fluorescence emission spectra of compounds 7 and 8 were obtained at a concentration of 50 µM (Fig. 3). The maximum excitation (major peak amplitude) was first determined as 350 nm for both compounds. When excited at this wavelength, the maximum emission peak observed for compounds 7 and 8 were at 493 and 494 nm, respectively. These values are in the same range as those found when a dansylaminomethyl group was added at position C3 of androsterone.25

Figure 3. Emission spectra of phenol 7 (left) and sulfamate 8 (right) (50 µM in PBS).

3.6. Optical imaging of cells expressing the steroid sulfatase

15

3.6.1. Wide field fluorescent microscopy Optical imaging was attempted with dansyl-labeled phenol 7 and dansyl-labeled sulfamate 8 in HEK-293[STS] cells with varying times (1.5 and 4 h) of exposure and compound concentrations (1, 10 and 30 µM) using a wide field fluorescent microscope (Fig. S6, supplementary data). This experiment led to the visualization of the cellular penetration of both compounds at the longest exposure time (4 h). Following 4 h of incubation, an increase in the concentration of STS inhibitor treatment correlated with the signal intensity. We obtained a stronger signal for sulfamate 8 than for phenol 7, which is in accord with the better STS binding (lower IC50 values) obtained for irreversible inhibitor 8 (2.1 nM) over reversible inhibitor 7 (69 nM). Despite the interesting results, as illustrated in Fig. 4, sub-cellular localization was difficult to assess with this low-resolution microscope, and confocal imaging was judged necessary to obtain more informative images.

Figure 4. Fluorescence observed in HEK-293STS cells treated for 4 h with compound 7 at 30 µM.

3.6.2. Confocal microscopy Giving the relative low resolution of the images obtained from wide field fluorescent microscopy experiments, we were interested in taking advantage of confocal microscopy to obtain more resolute images.,41 We decided to use the sulfamate based inhibitor 8 for confocal microscopy imaging considering its better signal intensity in wide field fluorescent microscopy experiments, and also because of its better affinity for the enzyme (IC50 = 2.1 nM) compared to phenol inhibitor 7 (IC50 = 69 nM). 16

Furthermore, even if phenol 7 will be released from sulfamate 8 following STS inhibition, this hydrolysis product should stay in the active site of STS and be able to have favorable hydrophobic interactions with the hydrophobic part of STS and potentially hydrogen bond with His290 and Lys368 (Fig. 2D). Thus, with a higher affinity than the endogenous competitive substrate E1S (IC50 = 7600 nM), phen 7 will continue to give a fluorescence signal. This fact can explain why a clear fluorescence signal is still obtained even after sulfamate 8 has been hydrolysed by the enzyme. In the imaging experiments, HEK-293STS cells were treated with sulfamate 8 at 30 µM for 4 h, the optimal conditions obtained in the preliminary wide field fluorescent microscopy experiments. As initially observed, the penetration of compound 8 was effective as shown in a four-cell aggregation and illuminated in the light blue color region giving a satisfactory resolute image (Fig. 5A). To assess the sub-localization of STS inhibitor 8, we next proceeded to label cell organelles using fluorescent dyes to specifically stain the cell membrane (CellMask; Fig. 5B) and ER (ER-tracker red; Fig. 5C). In doing so, we observed that inhibitor 8 is distributed around the nucleus and is highly co-localized with the ERtracker red signal, leading us to conclude that the ER is the potential host of STS and of compound 8. Superimposition of images reported in Fig. 5A, 5B and 5C (Fig. 5D) unequivocally confirms this hypothesis with a clear co-localization of inhibitor 8 with ER leading to a pink color. This result is in agreement with previous studies reporting STS mainly into ER.20,42 Otherwise, inhibitor 8 is not present in the plasmatic membrane (light green) or nucleus. The images revealed that sulfamate inhibitor 8, or its corresponding phenol 7 released from sulfamate hydrolysis by STS, are retained into the ER for up to four hours after the initial treatment.The signal of unreacted sulfamate probe (background fluorescence), or corresponding phenol after sulfamate cleavage by STS, is not seen in organelles other than endoplasmic reticulum (ER). It seems that inhibitor 8 is only found in the area of cells bearing STS, in our case ER. Thus, we presume that a concentration effect results from the retention of sulfamate inhibitor 8 and/or released of phenol inhibitor 7 by STS located in ER, as suggested by the successful colocalization results with the ER red tracker. Organelles lacking STS are simply not illuminated by the sulfamate probe since the concentration of accumulated compound in these areas is too weak. For instance, this is the case for plasmatic membrane, where hydrophobic compounds should be retrieved, but as seen in co-localization experiments with membrane stain, no fluorescence signal is seen from sulfamate 8, or its released phenolic counterpart 7, thus suggesting a very specific binding to STS in ER. To better appreciate the weak background fluorescence observed and specific binding of sulfamate 8, images without imaging treatment (deconvolution) have been added to the supplementary data (Fig. S7). 17

Figure 5. A) HEK-293[STS] cells treated with STS inhibitor 8 at 30 µM for 4 h, which illuminate in light blue, B) membrane stain (in light green), C) ER stain (in red) and D) superimposition of compound 8 stain with ER-tracker stain, leading to a pink color, and with a membrane marker stain.

4. Conclusion

The development of a fluorescent inhibitor of STS was successfully achieved assisted by the QSAR model and molecular modeling. Chemical synthesis of 17α-dansylaminomethyl-E2 (7) and 17αdansylaminomethyl-3-sulfamoyl-E2 (8) led to highly potent inhibitors of STS (IC50 = 69 and 2.1 nM, respectively). These two compounds have appropriate fluorescent properties (excitation

max;

350 nm;

emission max: 494 nm) allowing further optical imaging experiments. Wide field fluorescent microscopy demonstrates good penetration of both compounds in HEK-293[STS] cells. Subsequent confocal microscopy revealed sub-localization of compound 8 in the ER of those cells, which supports previous

18

reports on STS subcellular localization. This first example of STS fluorescent inhibitor provides a new tool for the cellular study of STS and related inhibitor behavior.

Acknowledgments

Adrien Ngueta would like to thank the Fondation du CHU de Québec (Endocrinology and Nephrology Unit) for the fellowship awarded. We acknowledge the Bioimaging platform of the Infectious Disease Research Centre, funded by an equipment and infrastructure grant from the Canadian Foundation for Innovation (CFI) and Leader’s Opportunity Funds from the CFI (37454) to J.-P.L. J.-P.L. is supported by a Junior 1 salary award from the Fonds de Recherche du Québec-Santé (FRQ-S). We are also grateful to Micheline Harvey for careful reading of this manuscript.

Supplementary data

QSAR model for STS-inhibition prediction, NMR (1H and 13C) spectra of phenolic compound 7 and sulfamate compound 8, and preliminary optical imaging data with STS inhibitors 7 and 8.

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Highlights Design of dansyl-labeled STS inhibitors assisted by QSAR and molecular docking A short chemical synthesis led to phenolic and sulfamate dansyl-labeled inhibitors Low nanomolar (IC50) inhibitors of steroid sulfatase (STS) were obtained Confocal microscopy shows the STS inhibitor located in the endoplasmic reticulum

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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