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

Article abstract of DOI:10.1016/j.bmc.2020.115368

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 t

Full text of DOI:10.1016/j.bmc.2020.115368

Journal Pre-proofs
Design and synthesis of dansyl-labeled inhibitors of steroid sulfatase for opti-
cal imaging
René Maltais, Adrien Ngueta Djiemeny, Jenny Roy, Xavier Barbeau, Jean-
Philippe Lambert, Donald Poirier
PII:
S0968-0896(20)30162-0
https://doi.org/10.1016/j.bmc.2020.115368
BMC 115368
DOI:
Reference:
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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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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: donald.poirier@crchul.ulaval.ca
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 2-
methoxy-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)
B)
Dansyl mimics
STS inhibitors
STS
O
HO
S
O
OH
OH
NH
HO
O
H
CHOLS
PREGS
O
CHOL
PREG
H
X
R
S
O
H
H
INH
H
H
R
O
O
N
1a
(R = OH; X = H)
STS
6
7
(R = OH)
1b
1c
1d
(R = OH; X = OCH3)
(R = OSO2NH2; X = H)
(R = OSO2NH2; X = OCH3)
(R = OSO2NH2)
O
S
HO
O
HO
O
INH
O
O
STS
17 -HSD

Activation of
estrogen-specific
effects
5
-diol
ER
O
HO
S
O
HO
O
DHEA
-dione
DHEAS
17 -HSD

Activation of
androgen-specific
effects
4
DHT
AR
T
INH
O
O
STS
17 -HSD

O
S
E2
HO
O
HO
O
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: 5-
androstene-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, open-
32
source version Schrödinger, LLC.) and were minimized with Open Babel 2.4.1 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
1
13
significant bands are reported (in cm-1). H- and C-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
13
Hz, 1H, 1-CH), 7.38 (m, 5H, Ph). C 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, 2-
CH), 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), 1.25-
2.25 (unassigned CH and CH2, 13H), 2.83 (m, 2H, 6-CH2), 2.88 (s, 6H, (CH3)2N), 3.05 (m, 2H, 17α-
CH2N), 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, 2-
CH), 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 FL3-
21, 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
Experimental
IC50 (nM)b
IC50 (nM)a
OH
H
1a
1c
69
45 (28)c
H
H
H
HO
OH
H
H
O
S
N/A
46
0.8
69
H2N
O
O
OH
HN
H
O
S
O
H
H
7
8
HO
N
OH
HN
H
O
S
O
H
H
O
N/A
N/A
2.1
H2N
S
O
O
N
O
H
Estrone
sulfate
7600c
H
H
O
S
HO
O
O
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 benzyl-
ether 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
O
O
H
a
b
H
H
H
H
H
H
H
H
BnO
HO
BnO
4
(estrone)
3
2
OH
OH
c
H
HN
d
H
NH2
SO2
H
H
H
H
BnO
BnO
N
6
5
e
OH
OH
f
H
SO2
H
SO2
N
HN
H
H
H
H
H
H2NO2SO
HO
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 ER-
tracker 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 co-
localization 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 17α-
dansylaminomethyl-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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23

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
24

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:
25