Discovery of a sulfamate-based steroid sulfatase inhibitor with intrinsic selective estrogen receptor modulator properties

Article abstract of DOI:10.1016/j.ejmech.2016.04.044

Steroid sulfatase (STS), the enzyme which converts inactive sulfated steroid precursors into active hormones, is a promising therapeutic target for the treatment of estrogen-sensitive breast cancer. We report herein the synthesis and in vitro study of dua

Full text of DOI:10.1016/j.ejmech.2016.04.044

European Journal of Medicinal Chemistry 119 (2016) 169e182
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of a sulfamate-based steroid sulfatase inhibitor with
intrinsic selective estrogen receptor modulator properties
a
a
a
b
c
ꢀ
Charles Ouellet , Ren eꢀ Maltais , Etienne Ouellet , Xavier Barbeau , Patrick Lagüe ,
Donald Poirier
a
a, d, *
Laboratory of Medicinal Chemistry, CHU de Qu ꢀe bec e Research Center (CHUL, T4), 2705 Laurier Boulevard, Qu ꢀe bec, QC, G1V 4G2, Canada
D ꢀe partement de chimie, Institut de biologie int ꢀe grative et des syst ꢁe mes (IBIS), Centre de recherche sur la fonction, la structure et l'ing ꢀe nierie des prot ꢀe ines
b
(
PROTEO), Universit ꢀe Laval, Qu ꢀe bec City, QC, Canada
D ꢀe partement de biochimie microbiologie et bio-informatique, Institut de biologie int ꢀe grative et des syst ꢁe mes (IBIS), Centre de recherche sur la fonction, la
c
structure et l'ing ꢀe nierie des prot ꢀe ines (PROTEO), Universit ꢀe Laval, Qu ꢀe bec City, QC, Canada
d
Department of Molecular Medicine, Faculty of Medicine, Universit ꢀe Laval, Qu ꢀe bec, QC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 March 2016
Received in revised form
Steroid sulfatase (STS), the enzyme which converts inactive sulfated steroid precursors into active hor-
mones, is a promising therapeutic target for the treatment of estrogen-sensitive breast cancer. We report
herein the synthesis and in vitro study of dual-action STS inhibitors with selective estrogen-receptor
modulator (SERM) effects. A library of tetrahydroisoquinoline-N-substituted derivatives (phenolic
compounds) was synthesized by solid-phase chemistry and tested on estrogen-sensitive breast cancer T-
15 April 2016
Accepted 16 April 2016
Available online 20 April 2016
47D cells. Three phenolic compounds devoid of estrogenic activity and toxicity emerged from this
screening. Their sulfamate analogs were then synthesized, tested in STS-transfected HEK-293 cells, and
found to be potent inhibitors of the enzyme (IC50 of 3.9, 8.9, and 16.6 nM). When tested in T-47D cells
they showed no estrogenic activity and produced a moderate antiestrogenic activity. The compounds
were further tested on osteoblast-like Saos-2 cells and found to significantly stimulate their proliferation
as well as their alkaline phosphatase activity, thus suggesting a SERM activity. These results are sup-
ported by molecular docking experiments.
Keywords:
Sulfatase
Enzyme
Inhibitor
Estrogen
Cancer
SERM
©
2016 Elsevier Masson SAS. All rights reserved.
1
. Introduction
breast cancer cells [1]. Selective estrogen receptor modulators
SERMs), such as tamoxifen and raloxifene, are compounds that are
presently used to treat breast cancer [2]. In breast tissues, SERMs
effectively block the activation of estrogen receptor alpha (ER ) by
endogenous ligands and prevent the transcription of genes medi-
ated by estrogen response elements [3]. This class of compounds
(
Hormonal therapy is commonly used for the treatment of
estrogen-sensitive breast cancer. As the majority of breast cancers
are initially estrogen-dependent, with approximately 55% in pre-
menopausal women and 75% in post-menopausal women, this
therapy efficiently blocks the stimulating effect of estrogens in
a
possesses the particularity of having tissue-specific effects on ERa,
resulting in antagonist activity in breast and uterus tissues and
agonist activity in bone. Although tamoxifen and raloxifene possess
the desired SERM activity, they also increase the risk of venous
thromboembolism [4,5]. As a result, the development of new
SERMs is still active and needed in order to obtain SERMs with
fewer side effects [6e9].
Inhibition of steroid sulfatase (STS) is a therapeutic approach for
the treatment of estrogen-dependent breast cancer and different
kinds of inhibitors were developed in this sense over the past years
Abbreviations: ALP, alkaline phosphatase; DEA, diethylamine; DHEA, dehydro-
epiandrosterone; DHEAS, dehydroepiandrosterone sulfate; 5-diol, 5-androstene-
3
b
,17
b-diol; DIPEA, diisopropylamine; E1, estrone; E1S, estrone sulfate; E2, estra-
diol; ER
a
, estrogen receptor alpha; HFIP, hexafluoroisopropanol; HOBt, hydrox-
ybenzotriazole; IC50, concentration inhibiting 50%; LRMS, low-resolution mass
spectrometry; PREGS, pregnenolone sulfate; PyBOP, benzotriazol-1-yl-oxy-
tripyrrolidinophosphonium hexafluorophosphate; SERM, selective estrogen recep-
tor modulator; STS, steroid sulfatase; TEA, triethylamine.
Corresponding author. Laboratory of Medicinal Chemistry, CHU de Qu eꢀ bec e
Research Center (CHUL, T4-42), 2705 Laurier Boulevard, Qu eꢀ bec, QC, G1V 4G2,
*
[10e22]. STS is an enzyme that converts inactive sulfated steroids,
mainly pregnenolone sulfate (PREGS), estrone sulfate (E1S), and
dehydroepiandrosterone sulfate (DHEAS), into corresponding
Canada.
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
http://dx.doi.org/10.1016/j.ejmech.2016.04.044
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

170
C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
unconjugated hormones (Fig. 1) [23,24]. E1S and DHEAS are
particularly abundant in blood circulation and could act as a
reservoir of steroid precursors [25]. It was also shown that STS
activity in breast cancer tumors is much higher than aromatase
activity and that in situ formation of estrone (E1) and estradiol (E2)
is mainly done via the STS pathway rather than by the aromatase
pathway [26e28]. Therefore, blocking STS could prevent estrogen-
sensitive carcinomas from transforming sulfated steroids into
both synthesized by parallel solid-phase chemistry using a multi-
detachable sulfamate linker [35e37]. Three phenolic compounds
showing good results and their corresponding sulfamate com-
pounds were selected for further testing. The six selected com-
pounds were tested in HEK-293 transfected cells as STS inhibitors,
on T-47D cells to evaluate their non-estrogenic and antiestrogenic
properties and on osteoblast-like Saos-2 cells to evaluate their ca-
pacity to stimulate the cell proliferation and alkaline phosphatase
activity. Finally, molecular docking simulations were achieved to
potent estrogens, mainly E1, E2, and 5-androstene-3b,17b-diol (5-
diol). Using a single compound inhibiting both STS and aromatase
activities could be also a promising strategy [29,30].
predict the binding modes of the compounds in the STS and ER
binding sites.
a
The dual blocking of ER
a
and STS is an interesting therapeutic
þ
approach for the treatment of (ER ) breast cancer by achieving
maximum estrogen blockade. However, the maximum estrogen
blockade obtained by such treatment should induce an estrogen
depletion condition that could lead to undesirable side effects such
as osteoporosis [31]. An approach to counter these unwanted ef-
fects resides in developing a sulfamoylated inhibitor of STS with
SERM properties. This kind of compound should reduce the po-
tential problem related to an estrogen depletion induced by STS
inhibitor monotherapy. Indeed, this dual-action strategy was re-
ported by Rasmussen et al. [32], who sulfamoylated the steroidal
SERM SR 16137 to its corresponding sulfamate form (SR 16157) as
STS inhibitor. However, this STS inhibitor was found to be active as
SERM only in its phenolic form, which required the hydrolysis of
2. Results and discussion
2.1. Selection and chemical synthesis of secondary amines as
building blocks
The choice of the secondary amines (compounds 1a-b, 2a-b, 3c,
4c, 5e11) used as building blocks for the preparation of phenolic
derivatives (compounds 19e31) was guided by their potential ca-
pacity to interact either with STS enzyme (hydrophobic sub-
stituents) or with the estrogen receptor (H-bond acceptor groups).
In the case of STS, it is well known that hydrophobic chains are well
tolerated considering the presence of a large hydrophobic pocket in
the active site of the enzyme [38]. We thus selected hydrophobic
secondary amines with a 4-bromophenyl, furanyl, or thiophenyl
group. Interestingly, the 4-bromophenyl and furanyl groups were
previously found to be the most potent substituents from a series of
tetrahydroisoquinoline derivatives synthesized as STS inhibitors in
a first structure-activity relationship (SAR) study [33].
In addition to the use of a hydrophobic side-chain for STS in-
hibition, we were interested by amines that bear hydrogen bond
acceptor group, such as pyridine, imidazole, morpholine, or piper-
idine. These chemical groups would favor interaction with a key
amino acid of the ER. Indeed, it is known that a key amino acid like
Asp351, which is involved in the stabilisation of ER-H12 helix, could
be targeted to induce SERM activity [39]. For that purpose, we
selected amines of different sizes, shapes and hydrogen bond
acceptor capacity. Particularly, we synthesized the phenoxypropyl-
piperidine chains which have been frequently reported as an
important pharmacophore in several SERM compounds [40].
the sulfamate group by STS in order to interact with ERa and to
provide the desired SERM properties. Considering that a very small
quantity of phenol should be released in vivo from the hydrolysis of
the sulfamate group coming from irreversible STS inhibition, this
approach has the disadvantage of not generating a sufficient
physiological concentration of the phenol needed to exert a rele-
vant SERM action.
An alternative approach, investigated in our laboratory [33], is to
obtain a sulfamate-based STS inhibitor directly active as SERM,
whose effect could be additive to the SERM action of the phenol
counterpart released from sulfamate hydrolysis by STS. A first
generation of compounds showed a good inhibition of STS but did
not possess the SERM capacity we were looking for. Indeed, the
compounds were found to be estrogenic on breast cancer cells [34].
Here we report the synthesis of second generation dual-action
compounds designed to inhibit STS and to act as a SERM (Fig. 2).
These non-steroidal compounds were built around a tetrahy-
droisoquinoline scaffold and the rational to use this scaffold was
previously reported [33]. Phenol and sulfamate derivatives were
Fig. 1. Transformation of sulfated steroid E1S and DHEAS into estrogenic hormones (E2 and 5-diol) by steroid sulfatase (STS) and sites of dual-action sulfamate compounds. The
sulfamate compound inhibits the STS and can act as agonist or antagonist of the estrogen receptor (ER) depending on the tissues.

C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
171
Fig. 2. Second generation of dual-action STS inhibitors and SERMs. A) Conversion of sulfamate compounds into phenolic compounds by STS or by chemical hydrolysis. B) The
sulfamate compound (I) binds to the active site of STS, thus inactivating the enzyme. The sulfamate compound (I), by itself or via the released phenolic compound (II), binds to ER
and could act as a SERM.
a
2.2. Chemical synthesis of secondary amines as building blocks
reducing the cell number. Amongst the library of phenolic com-
(
compounds 1a-b, 2a-b, 3c, 4c, 5e11)
pounds, only compounds 25, 29, and 31 showed interesting results.
In fact, they showed neither estrogenicity nor cytotoxicity on T-47D
Non commercially available secondary amines 1a-b, 2a-b, 3c,
cells at the concentrations tested (0.01, 0.1 and 1
suggest that these three compounds do not possess the ability to
activate ER in breast cancer cells and consequently to induce un-
mM). Such results
and 4c were synthesized under classic conditions of reductive
amination, which consisted in reacting the aldehydes 1e4 with
appropriate amines a-c followed by the reduction of the interme-
diate imine with sodium borohydride (Scheme 1) [41]. The alde-
hydes 1 and 2 were synthesized beforehand by reacting 2- or 4-
hydroxybenzaldehyde, 1-(3-chloropropyl)piperidine, sodium car-
bonate and sodium iodide in refluxing acetone.
a
wanted cell proliferation.
2.5. Steroid sulfatase inhibition
Following the results obtained in the initial screening with
phenolic compounds 25, 29, and 31, we synthesized the sulfamate
analogs (compounds 32, 33, and 34) and evaluated their capacity to
inhibit the STS activity. For the enzymatic assay, we used homo-
genated HEK-293 cells overexpressing STS and evaluated the ca-
2
.3. Chemical synthesis of compounds 19e34
The general synthetic methodology for the preparation of all
3
3
library members is outlined in Scheme 2. The starting 1,2,3,4-
tetrahydroisoquinolin-7-ol (12) was selectively protected as the
N-Fmoc derivative 13, which after a sulfamoylation of the phenol
group yielded 14. This sulfamate derivative was then reacted with
the trityl chloride resin to give the solid-phase bound compound
pacity of compounds to inhibit the conversion of [ H]-E1S into [ H]-
E1 (Fig. 3). As reported previously in literature [42e46], phenolic
compounds are less potent STS inhibitors than their sulfamoylated
analogs. Our results confirm this affirmation, since only the sulfa-
mate compounds inhibited STS. Effectively, the phenolic com-
pounds showed no significant inhibition while the sulfamate
compounds showed very good inhibition of STS with IC50 values of
16.6 ± 2.7 nM for 32, 8.9 ± 1.2 nM for 33, and 3.9 ± 1.1 nM for 34.
Interestingly, these three sulfamate compounds demonstrated a
higher STS inhibition potency than our previous generation of dual-
action compounds, where one of the best STS inhibitors had an IC50
of 300 nM [34]. Non-steroidal sulfamate compounds 32e34 were
however less potent inhibitors than steroidal sulfamate derivative
EM-1913 [42e44] used as reference compound (IC50 ~ 0.05 nM).
Therefore, only the sulfamate compounds 32e34 and correspond-
ing phenolic compounds 25, 29, and 31 were tested for additional
in vitro properties.
1
5. Removal of the Fmoc protecting group by a treatment with 20%
piperidine in DMF provided the resin 16 with a free NH, which was
acylated with an activated ester of carboxybenzaldehyde to give the
resin 17. Diversification of 17 was obtained by performing a
reductive amination with various secondary amines that yielded
resins 18. Finally, the phenolic derivatives 19e31 were obtained by
a nucleophilic cleavage from resin 18. The sulfamate compounds
3
2e34 were synthesized following the same solid-phase approach
used for the synthesis of 19e31. In that case, however, an acid
cleavage using hexafluoroacetic acid (HFIP) was used at the end of
the sequence of reactions to release 32e34.
þ
2
.4. Screening of phenolic compounds 19e31 in T-47D (ER ) cells
þ
2
.6. Effect on breast cancer T-47D (ER ) cell proliferation of selected
The library of phenolic compounds described above was tested
sulfamate and phenolic compounds (25, 29, 31e34)
on estrogen-sensitive breast cancer T-47D cells (Table 1). Com-
pounds were discarded if they stimulated T-47D cell proliferation
As the three sulfamate compounds 32e34 are potent inhibitors
(
estrogenic activity) or if they showed some cytotoxicity by
of STS, we wanted to know if they were devoid of estrogenic

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C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
Scheme 1. Preparation of building blocks (secondary amines 1a-b, 2a-b, 3c, and 4c) and structures of all commercially available secondary amines 5e11 used for the synthesis of
targeted tetrahydroisoquinoline phenolic derivatives.
activity. Along with their three phenolic analogs 25, 29, and 31, and
to reconfirm the results of the initial screening, we tested the six
compounds on breast cancer estrogen-sensitive T-47D cells
stimulating their proliferation.
We next evaluated the capacity of compounds 25, 29, 31e34 to
block the stimulation of T-47D cells proliferation induced by E2
(Fig. 5A and B). The cells were incubated with our six compounds
and raloxifene at different concentrations in the presence of E2
(0.1 nM). Raloxifene was able to block the E2 (0.1 nM) stimulation at
(Figs. 4A and B). We used the estrogenic natural hormone E2 as a
positive control of cell proliferation stimulation, and raloxifene, a
SERM with no estrogenicity in breast tissue. E2, at a concentration
of 0.1 nM, induced approximately 160% cell proliferation while
raloxifene showed no estrogenic activity. Our six compounds
showed interesting results because none of them stimulated the
proliferation of estrogen-sensitive T-47D cells. On the other hand,
concentrations of 0.01e1
M. For our six compounds, the results show that they all possess
some antiestrogenic activity in T-47D cells. Because compounds
31e34 showed cytotoxicity at 5 M in the previous test, their
mM and still showed some cytotoxicity at
5
m
m
we observed a reduction of cell proliferation at 5
cytotoxic activity for raloxifene and compounds 31e34. This should
not be a problem as 5 M is a high dose hardly achievable in vivo
m
M, suggesting
apparent antiestrogenicity at this concentration was not consid-
ered. The phenol derivative 29, which did not reduce the cell pro-
liferation at 5 mM, possesses the best antiestrogenic activity at this
m
and that even raloxifene, a SERM used in clinic, induced some
cytotoxicity at this concentration. As a result, both sulfamate and
phenolic compounds can be used on breast cancer cells without
concentration since it blocked approximately 84% of the stimula-
tion induced by E2. Even if compound 29 is not as good anti-
estrogenic as raloxifene, it still got the properties we were looking

C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
173
Scheme 2. Reagents and conditions for the chemical synthesis of phenolic derivatives (compounds 19e31) and sulfamate derivatives (compounds 32e34). Representation of the
four steps (#1e4) behind the identification of dual-action compounds. (a) Fmoc-O-succinimide, NaHCO , H O; (b) NH SO Cl, 2,6-di-tert-butyl-4-methylpyridine, DCM; (c) trityl
3
2
2
2
chloride resin, DIPEA, DMA, DCM; (d) 20% piperidine in DMF; (e) carboxybenzaldehyde, DIPEA, HOBt, PyBOP, DMF; (f) secondary amine (R
R
1 2
3
NH), NaBH(OAc) , 10% AcOH in NMP;
ꢀ
(
g) 30% DEA in THF, 60 C; (h) 30% HFIP in DCM.
for in breast tissue.
.6. Effect on osteoblast-like Saos-2 cells (proliferation)
and its sulfamate analog 33, which both induced cell proliferation
approximately by 160%. The slight cell proliferation observed is
probably due to the osteoblast cells themselves. One of the main
roles of osteoblasts in bone tissue is to construct a calcified extra-
cellular matrix [31]. As such, bone formation is not principally due
to osteoblast proliferation, but rather osteoblast maturation into
mature osteocytes. To further investigate the high cell proliferation
induced by compounds 29 and 33 and to confirm the first results,
Saos-2 cells were incubated 7 days with 29 or 33 at a wider range of
concentrations (Fig. 6B). We observed an apparent dose-dependent
2
As a SERM possesses the ability to block ER
to activate ER in other tissues, such as bone, we investigated the
action of compounds 25, 29, 31e34 on the osteoblast-like Saos-
cell proliferation. To do so, we incubated Saos-2 cells for 7 days
with E2 or raloxifene, as a SERM reference compound, and our six
compounds of interest at concentrations of 0.1 and 1 M (Fig. 6A).
All compounds, including E2 and raloxifene, significantly induced
Saos-2 cell proliferation at 1 M. However, the induced prolifera-
tion is quite low with the exception of the phenolic compound 29
a in breast tissue and
a
2
m
response from 1 nM to 1
raloxifene, 29, and 33. At higher concentrations, however, all three
compounds reduced Saos-2 cell proliferation, suggesting
mM for the cell proliferation induced by
m
a

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C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
Table 1
þ
Structure of phenolic compounds 19e31 and their capacity to modulate breast cancer T-47D (ER ) cell prolifefation at three concentrations.
ID
R
Proliferation of T-47D cells (%)a
.01
0
m
M
0.1
m
M
1 mM
1
9
0
102.8 ± 6.9
99.8 ± 4.2
37.1 ± 4.2
2
105.9 ± 1.0
104.5 ± 4.8
60.7 ± 6.4
21
96.8 ± 1.2
105.9 ± 5.6
0 ± 9.1
2
2
2
3
95.6 ± 1.8
119.7 ± 4.8
112.9 ± 3.7
133.7 ± 3.0
39.2 ± 2.9
102.8 ± 2.2
2
4
5
94.9 ± 1.3
98.5 ± 4.5
114.6 ± 5.9
103.5 ± 6.5
2
104.1 ± 2.3
103.2 ± 2.5
2
2
2
2
6
7
8
9
106.3 ± 0.6
108.2 ± 5.5
100.6 ± 3.8
97.1 ± 2.6
109.4 ± 1.2
105.9 ± 1.5
105.4 ± 4.4
103.2 ± 1.8
59.2 ± 3.1
118.8 ± 9.6
78.9 ± 6.8
96.8 ± 2.2
3
0
1
108.2 ± 5.5
105.6 ± 0.6
138.0 ± 1.2
104.6 ± 7.4
200.3 ± 4.9
100.9 ± 7.9
3
The grey shades represent compounds showing interesting results.
a
Control cell proliferation is set to 100%. Results are expressed as mean ± SD of triplicate measurements.
cytotoxic effect. Even if the mechanism by which 29 and 33 induced
cell proliferation at concentrations lower than 1 M remains un-
2.7. Effect on osteoblast-like Saos-2 cells (alkaline phosphatase
activity)
m
clear, all compounds tested clearly induced a desirable effect in
osteoblasts.
Compounds 25, 29, 31̶ 34 had an apparently beneficial effect on

C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
175
Fig. 3. Effect of sulfamate compounds 32, 33, and 34 and their phenolic analogs 25, 29,
and 31, respectively, on STS activity in homogenates of transfected HEK-293 cells. The
three sulfamate derivatives inhibited STS (IC50 ¼ 16.6 ± 2.7 nM, 8.9 ± 1.2 nM, and
3
.9 ± 1.1 nM for 32, 33, and 34, respectively) while the three phenolic analogs showed
3
no significant inhibition of STS. Results are expressed as % inhibition of [ H]-E1S
conversion into [ H]-E1 by STS. Each point represents the mean ± SD of triplicate
3
measurements.
Fig. 5. Effect of phenolic (A) and sulfamate (B) compounds on estrogen-sensitive T-
þ
4
7D (ER ) cell proliferation after 7 days of treatment. The antiestrogenicity of phenol
derivatives 25, 29, and 31 as well as sulfamate derivatives 32, 33, and 34 was evaluated
by incubating the cells with different concentrations (0.01, 0.1, 1, and 5 M) of each
m
compound and E2 (0.1 nM). The proliferation of control cells is set to 100%. Results are
expressed as means ± SD of triplicate measurements. * p ꢁ 0.01 vs. E2, ** p ꢁ 0.05 vs.
E2.
activity of a differentiation marker, the alkaline phosphatase (ALP).
ALP is an enzyme that releases inorganic phosphate from different
intracellular substrates such as pyrophosphate and pyridoxal 5’-
phosphate [47]. The inorganic phosphate is used in the formation of
hydroxyapatite crystals which are then inserted in the extracellular
protein scaffold to form the solid part of bone tissue. In osteoblast
cells, ALP is regulated by estrogens and is a good indicator of
osteoblast differentiation [48]. We tested our compounds, raloxi-
fene and E2, in Saos-2 cells and investigated their effect on ALP
activity (Fig. 7). All compounds significantly increased ALP activity
in Saos-2 cells, but it is interesting to see that 29 induced the
highest ALP activity (138%).
2.8. Structural investigation
A molecular docking study was achieved to highlight the
structural determinants that are essential for STS inhibition and
SERM activity. As the same conclusions were drawn for all the
compounds of interest, the docking results are presented only for
phenolic compound 29 and sulfamate compound 33 (the results for
compounds 25, 31, 32 and 34 are included in the Supporting
Information).
Fig. 4. Effect of phenolic (A) and sulfamate (B) compounds on estrogen-sensitive T-
þ
47D (ER ) cell proliferation after 7 days of treatment. The estrogenicity of phenol
derivatives 25, 29, and 31 as well as sulfamate derivatives 32, 33, and 34 was evaluated
by incubating the cells with different concentrations (0.01, 0.1, 1, and 5 M) of each
m
compound. The proliferation of control cells is set to 100%. Results are expressed as
means ± SD of triplicate measurements.
Currently, only one experimental structure of human STS is
available (PDB 1P49) [38]. Although there is no inhibitor present in
this structure, a sulfate group is covalently bound to the catalytic
residue hydroxyformylglycine, which is usually involved in a
osteoblasts. Since they induced only a slight stimulation in Saos-
2
cell proliferation, such as raloxifene, we decided to investigate the

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C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
crystallographic hydroxyformylglycine sulfate group. Using this
procedure, the EM-1913 sulfamate moiety of the best docking pose
is located within 0.6 of the crystallographic hydrox-
Å
yformylglycine sulfate group (Fig. 8).
The STS binding site is composed of a long hydrophobic tunnel
that accommodates the steroid scaffold. Fig. 8 presents the best
docking results for sulfamate compound 33 superimposed on the
best docking result of compound EM-1913 in the STS binding site.
As for the sulfamate moiety of EM-1913, H-bond interactions are
observed between the sulfamate moiety of compound 33 with the
calcium ion and Lys134. These docking results clearly show that the
hydrophobic tunnel of STS binding site comfortably accommodates
the elongated structure of compound 33, despite the absence of a
steroidal scaffold. This docking result of compound 33 is in good
agreement with its observed STS inhibition activity.
Many experimental structures of ERa are available for the APO
protein or bound to either an agonist or an antagonist. In this study,
molecular docking was carried out using the raloxifene-bound
structure (PDB 1ERR) [51], and the docking results were
compared to the agonist-bound structure (PDB 1ERE) [51]. These
two structures differ in the conformation of the H12
a-helix: a
conformational change involving this helix occurs in the presence
of raloxifene and, as a result, the helix is no longer located in the
binding pocket (Fig. 9, black cartoon). To induce this conformational
change, the raloxifene core binds to the E2 binding site, interacting
with Glu353, Arg394 on one side and His524 on the other side, and
leaving the piperidine moiety at the entry of the pocket to interact
with Asp351. The best docking conformations for compounds 29
and 33 in the ERa binding site are presented in Fig. 9. From these
results, the furanyl and methylthiofuranyl groups of phenolic
Fig. 6. Effect of phenolic and sulfamate compounds on Saos-2 cell proliferation after 7
days of treatment. A) Saos-2 cells were incubated with E2, raloxifene, 25, 29, 31̶ 34 at
0.1 and 1
mM. B) The cells were incubated with raloxifene, phenolic compound 29, or
sulfamate compound 33 at concentrations ranging from 1 nM to 10
mM. The prolif-
eration of control cells is set to 100%. Results are expressed as means ± SD of triplicate
measurements.
Fig. 7. Effect of phenolic and sulfamate compounds on alkaline phosphatase (ALP)
activity in Saos-2 cells after 3 days of treatment. Saos-2 cells were incubated with E2,
raloxifene, or a compound of interest at a concentration of 0.1 nM. The ALP activity of
control cells is set to 100%. Results are expressed as means ± SD of triplicate mea-
surements. * p ꢁ 0.01 vs. control.
covalent binding to the substrates [49,50]. As detailed in the
Experimental section, we have used EM-1913, a highly potent STS
inhibitor [42], to develop the docking procedure in STS. Shortly,
the hydroxyformylglycine residue was replaced by a glycine and
the corresponding covalently-bound sulfate group was removed.
The coordinates of the EM-1913 molecule were initialized with
its sulfamate moiety located at the position of the former
Fig. 8. The best docking results for the STS inhibitor EM-1913 and the sulfamate
compound 33 in the STS binding site. EM-1913 and the sulfamate compound 33 are
represented by black and cyan sticks, respectively. STS from PDB 1P49 is represented
by gray cartoon, Lys134 by yellow sticks, and the calcium ion by a green sphere. The
hydroxyformylglycine bound sulfate is shown as lines and spheres for reference. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)

C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
177
Fig. 9. Docking results for phenolic compound 29 (left, cyan sticks) and sulfamate compound 33 (right, green sticks) in the ER
a
binding site. ER
a/raloxifene complex structure (PDB
1ERR) is represented by gray cartoon, H12 by black cartoon, and raloxifene, used as reference, by black sticks. H-bonds between ER
a
and raloxifene are identified by dashed yellow
lines. ER
a/E2 complex structure (PDB 1ERE) is represented by pink cartoon and H12 by purple cartoon. The black arrows emphasize the H12 conformational change between both
PDB structures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
compound 29 and sulfamate compound 33 are positioned in the
estrogen pocket, orienting the phenol or sulfamate moiety toward
the solvent. While compounds 29 and 33 realize no H-bond with
and sulfamate compound 33 being the best stimulator of each
family. These molecules also increased the ALP activity. Results
from molecular docking simulations suggest that the hydrophobic
tunnel of the STS binding site accommodates the elongated struc-
ture of compound 33 despite the absence of a steroidal scaffold.
This result is in agreement with its observed inhibition activity.
Moreover, docking of phenolic compound 29 and sulfamate com-
pound 33 led to a binding similar to that of raloxifene, i.e. the fur-
anyl group of compound 29 and methylthiofuranyl group of
compound 33 positioned in the estrogen pocket and the phenol or
sulfamate moiety oriented toward the solvent. These results sug-
gest that compounds 29 and 33 bind in a competitive manner with
E2. The results obtained in this study are promising considering
that compound 33 represents the first example of a sulfamate-
based inhibitor with intrinsic SERM activity. This suggests that
this compound could be used in future in vivo testing as a dual-
action compound for the treatment of estrogen-dependent breast
cancer.
ERa, this conformation is still in agreement with the hydrophobic
character of the pocket and correlates with the geometry of ral-
oxifene, suggesting a competitive binding mode with E2. In addi-
tion, the best binding conformation of both compounds occupy a
similar location as raloxifene in reference with H12, suggesting that
compounds 29 and 33 would also induce a H12 conformational
change as does raloxifene, supporting the SERM activity observed
in Figs. 4e6.
3
. Conclusion
In summary, we described the chemical synthesis and in vitro
biological evaluation of tetrahydroisoquinoline derivatives
designed to inhibit STS and to act as SERMs. The phenolic com-
pounds and three sulfamate analogs were all synthesized by par-
allel solid-phase chemistry using a multidetachable sulfamate
linker. The library of phenols was tested on estrogen-sensitive
breast cancer T-47D cells to discard compounds bearing estro-
genicity or cytotoxicity at concentrations tested. In the library,
phenolic derivatives 25, 29, and 31 showed no such undesirable
activities and were selected for further testing. Their sulfamate
analogs 32, 33, and 34 were tested on homogenated HEK-293 cells
overexpressing STS and demonstrated to be strong STS inhibitors
with IC50 values of 16.6, 8.9, and 3.9 nM, respectively. All six com-
pounds were tested on T-47D cells and showed no estrogenicity and
even some antiestrogenic activity. Following this, we tested our
compounds on osteoblast-like Saos-2 cells as SERMs must be active
in tissues where estrogenic activity is beneficial. All compounds
stimulated Saos-2 cell proliferation with phenolic compound 29
4. Experimental section
4.1. Chemistry (general information)
Chemical reagents were purchased from Aldrich Chemical Co.
(Milwaukee, WI, USA) and solvents were obtained from Fisher
Scientific (Montr eꢀ al, QC, Canada) and VWR (Ville Mont-Royal, QC,
Canada). Trityl chloride resin was supplied by EMD Biosciences
(Novabiochem, La Jolla, CA, USA). Flash chromatography was per-
formed on Silicycle 60 230e400-mesh silica gel (Qu eꢀ bec, QC,
Canada). Thin-layer chromatography 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

178
C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
(
254 nm) or/and to a solution of ammonium molybdate/sulphuric
(d, J ¼ 8.0 Hz, 1H), 6.90 (t, J ¼ 7.4 Hz, 1H), 7.14 (m, 1H), 7.21 (m, 1H),
1
acid/ethanol (plus heating). H NMR spectra were recorded at
00 MHz, using a Bruker AVANCE 400 spectrometer (Billerica, MA,
USA). The chemical shifts ( ) are expressed in ppm and referenced
to chloroform (7.26 ppm), acetone (2.05 ppm), or methanol
3.31 ppm). Low-resolution mass spectra (LRMS) were recorded on
7.27 (m, 1H), 7.37 (d, J ¼ 6.8 Hz,1H), 7.63 (td, J ¼ 1.8, 7.7 Hz, 1H), 8.54
þ
4
(ddd, J ¼ 0.9, 1.8, 4.9 Hz, 1H). APCI-MS for C21
340.3 m/z.
H30ON
3
[MþH] :
d
(
4.3.2. 1-{4-[3-(Piperidin-1-yl)propoxy]phenyl}-N-(pyridin-2-
a Shimadzu Prominence apparatus (Kyoto, Japan) equipped with an
atmospheric pressure chemical ionization (APCI) source on positive
mode.
ylmethyl)methanamine (2a)
1
H NMR (400 MHz, CDCl
.98 (m, 2H), 2.42 (broad s, 4H), 2.49 (t, J ¼ 7.5 Hz, 2H), 3.78 (s, 2H),
.91 (s, 2H), 4.00 (t, J ¼ 6.4 Hz, 2H), 6.85 (d, J ¼ 8.6 Hz, 2H), 7.16 (ddd,
J ¼ 1.2, 4.9, 7.5 Hz,1H), 7.25 (d, J ¼ 8.5 Hz, 2H), 7.31 (d, J ¼ 7.8 Hz,1H),
3
)
d
: 1.45 (m, 2H),1.60 (p, J ¼ 5.6 Hz, 4H),
1
3
4
.2. Synthesis of aldehydes 1 and 2
7
.64 (td, J ¼ 1.8, 7.7 Hz, 1H), 8.56 (ddd, J ¼ 1.0, 1.9, 4.9 Hz, 1H). APCI-
To a solution of 2- or 4-hydroxy-benzaldehyde (1.64 mmol) in
þ
MS for C21
H30ON
3
[MþH] : 340.3 m/z.
anhydrous acetone (25 mL) was added cesium carbonate
4.92 mmol) and the solution was stirred at room temperature for
0 min under an argon atmosphere. Chloropropylpiperidine hy-
(
1
4.3.3. 1-(Furan-2-yl)-N-{2-[3-(piperidin-1-yl)propoxy]benzyl}
drochloride (2.46 mmol) and sodium iodide (0.82 mmol) was
added to the solution and the mixture was heated at reflux over-
night. The resulting solution was filtered and evaporated to dry-
ness. The crude compound was diluted with EtOAc, the solution
was washed successively with a saturated sodium carbonate solu-
tion and water, dried over MgSO , filtered, and evaporated under
4
reduce pressure. Purification by flash chromatography (hexanes/
acetone/TEA, 80:19:1 to 70:29:1) yielded the desired compound 1
methanamine (1b)
1
H NMR (400 MHz, CDCl
3
)
d
: 1.45 (m, 2H),1.59 (p, J ¼ 5.6 Hz, 4H),
1
.99 (m, 2H), 2.40 (broad s, 4H), 2.48 (t, J ¼ 7.5 Hz, 2H), 3.76 (s, 2H),
3
.81 (s, 2H), 4.03 (t, J ¼ 6.2 Hz, 2H), 6.18 (dd, J ¼ 1.0, 3.2 Hz, 1H), 6.31
(dd, J ¼ 1.8, 3.1 Hz, 1H), 6.86 (d, J ¼ 8.0 Hz, 1H), 6.90 (t, J ¼ 7.4 Hz,
1
H), 7.22 (m, 2H), 7.36 (dd, J ¼ 0.6, 1.6 Hz, 1H). APCI-MS for
þ
C
20
29
H O
2
N
2
[MþH] : 329.3 m/z.
(
360 mg, 89%) or 2 (400 mg, 99%).
4.3.4. 1-(Furan-2-yl)-N-{4-[3-(piperidin-1-yl)propoxy]benzyl}
4
.2.1. 2-[3-(piperidin-1-yl)propoxy]benzaldehyde (1)
methanamine (2b)
1
1
H NMR (400 MHz, CDCl
3
)
d
: 1.45 (q, J ¼ 6.0 Hz, 2H), 1.59 (p,
H NMR (400 MHz, CDCl
3
)
d
: 1.44 (m, 2H),1.59 (p, J ¼ 5.6 Hz, 4H),
J ¼ 5.6 Hz, 4H), 2.05 (m, 2H), 2.40 (broad s, 4H), 2.50 (t, J ¼ 7.4 Hz,
1.97 (m, 2H), 2.40 (broad s, 4H), 2.47 (t, J ¼ 7.5 Hz, 2H), 3.72 (s, 2H),
3.77 (s, 2H), 4.00 (t, J ¼ 6.4 Hz, 2H), 6.18 (dd, J ¼ 0.91, 3.2 Hz, 1H),
6.32 (dd, J ¼ 1.9, 3.2 Hz, 1H), 6.85 (d, J ¼ 8.6 Hz, 2H), 7.22 (d,
2
H), 4.14 (t, J ¼ 6.3 Hz, 2H), 7.01 (t, J ¼ 8.1 Hz, 2H), 7.53 (m, 1H), 7.83
(
[
dd, J ¼ 1.8, 7.8 Hz, 1H), 10.51 (s, 1H). APCI-MS for C15
22 2
H O N
þ
MþH] : 248.3 m/z.
J ¼ 8.6 Hz, 2H), 7.37 (dd, J ¼ 0.8,1.9 Hz,1H). APCI-MS for C20
29 2 2
H O N
þ
[
MþH] : 329.3 m/z.
4.2.2. 4-[3-(piperidin-1-yl)propoxy]benzaldehyde (2)
1
H NMR (400 MHz, CDCl
3
)
d
: 1.45 (m, 2H),1.59 (q, J ¼ 5.6 Hz, 4H),
4.3.5. 3-(1H-imidazol-1-yl)-N-(pyridin-2-ylmethyl)propan-1-
2
.00 (m, 2H), 2.40 (broad s, 4H), 2.47 (t, J ¼ 7.4 Hz, 2H), 4.10 (t,
amine (3c)
J ¼ 6.4 Hz, 2H), 7.00 (d, J ¼ 8.7 Hz, 2H), 7.82 (d, J ¼ 8.7 Hz, 2H), 9.88
þ
1H NMR (400 MHz, CDCl
3
)
d: 1.97 (p, J ¼ 6.8 Hz, 2H), 2.64 (t,
(
s, 1H). APCI-MS for C15
H
22
O
2
N [MþH] : 248.3 m/z.
J ¼ 6.7 Hz, 2H), 3.88 (s, 2H), 4.06 (t, J ¼ 6.9 Hz, 2H), 6.91 (s, 1H), 7.04
(
(
C
s, 1H), 7.18 (ddd, J ¼ 1.2, 4.9, 7.6 Hz, 1H), 7.26 (d, J ¼ 7.8 Hz, 1H), 7.47
4
.3. Synthesis of secondary amines 1a, 2a, 1b, 2b, 3c and 4c
s, 1H), 7.65 (td, J ¼ 1.8, 7.7 Hz, 1H), 8.56 (m, 1H). APCI-MS for
(
general procedure)
þ
12
H
17
N
4
[MþH] : 217.3 m/z.
To a solution of aldehyde 1, 2, 3, or 4 (1.21 mmol) in absolute
ethanol (12 mL) was added the appropriate amine a (1-(pyridin-2-
yl)methanamine), b (1-(furan-2-yl)methanamine), or c (3-(1H
imidazol-1-yl)propan-1-amine) (1.45 mmol) and 4 Å molecular
sieves. The solution was stirred at room temperature for 2.5 h and
filtered to remove molecular sieves. Sodium borohydride
4
.3.6. N-(furan-2-ylmethyl)-3-(1H-imidazol-1-yl)propan-1-amine
(4c)
1H NMR (400 MHz, CDCl
3
)
d
: 1.92 (p, J ¼ 6.9 Hz, 2H), 2.59 (t,
J ¼ 6.7 Hz, 2H), 3.75 (s, 2H), 4.04 (t, J ¼ 6.9 Hz, 2H), 6.15 (dd, J ¼ 0.6,
3
.2 Hz, 1H), 6.32 (dd, J ¼ 1.9, 3.2 Hz, 1H), 6.89 (t, J ¼ 1.3 Hz, 1H), 7.05
(
2.90 mmol) was then added in small portion to the resulting
(
C
s, 1H), 7.37 (dd, J ¼ 0.8, 1.8 Hz, 1H), 7.45 (s, 1H). APCI-MS for
ꢀ
ethanol solution at 0 C and the mixture allowed to return at room
temperature and stirred overnight. Water (15 mL) was added and
the solution stirred for 15 min before to be concentrated under
reduced pressure. The aqueous layer was extracted with DCM
þ
11
H
16ON
3
[MþH] : 206.3 m/z.
4.4. Synthesis of phenol derivatives 19e31
(
5 ꢂ 5 mL), and the combined organic phase was washed with
brine, dried with MgSO , filtered, and evaporated to dryness. Pu-
rification by flash chromatography (DCM/MeOH/TEA, 98:1:1 to
4
The compounds 19e31 were synthesized in good quantity
(28e39 mg) following the solid-phase strategy we previously
developed and published for similar phenolic derivatives [33]. All
these compounds were purified by reverse phase chromatography
on LC-MS preparative system (Model Prominence, Shimadzu,
Kyoto, Japan) equipped with a photodiode detector (SPD M 20A)
and mass analyser (MS 2020) with atmospheric-pressure chemical
positive ionisation (APCI) systems with a synergi Hydro-RP column
9
7
0:9:1) yielded 1a (370 mg, 90%), 2a (318 mg, 78%), 1b (320 mg,
0%), 2b (347 mg, 76%), 3c (400 mg, 99%), or 4c (400 mg, 97%) ac-
cording to the aldehyde and primary amine used.
4.3.1. 1-{2-[3-(Piperidin-1-yl)propoxy]phenyl}-N-(pyridin-2-
ylmethyl)methanamine (1a)
1
H NMR (400 MHz, CDCl
.89 (broad s, NH), 2.01 (m, 2H), 2.38 (broad s, 4H), 2.48 (t,
J ¼ 7.5 Hz, 2H), 3.87 (s, 2H), 3.92 (s, 2H), 4.03 (t, J ¼ 6.2 Hz, 2H), 6.85
3
)
d
: 1.44 (m, 2H),1.59 (p, J ¼ 5.6 Hz, 4H),
(250 mm ꢂ 21.2 mm x 4
mm). Each compound was purified in a 50
1
min-run using a solvent gradient (70e100% MeOH, 30e0% water)
at flow rate of 10 mL/min.

C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
179
4
(
.4.1. (7-Hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)(4-{[{2-[3-
piperidin-1-yl)propoxy]benzyl}(pyridin-2-ylmethyl)amino]
methyl}phenyl)methanone (19)
4.4.6. [4-({(Furan-2-ylmethyl)[3-(1H-imidazol-1-yl)propyl]amino}
methyl)phenyl](7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)
methanone (24)
1
1
H NMR (400 MHz, CD
3
OD)
d
: 1.48 (broad s, 2H), 1.60 (q, J ¼ 4.7,
H NMR (400 MHz, CD
3
OD)
d
: 1.99 (p, J ¼ 6.8 Hz, 2H), 2.47 (t,
5
2
3
4
.3 Hz, 4H), 1.97 (m, 2H), 2.47 (broad s, 4H), 2.54 (t, J ¼ 7.6 Hz, 2H),
.76 (broad s, 1H), 2.85 (broad s, 1H), 3.58 (broad s, 1H), 3.70 (s, 2H),
.71 (s, 2H), 3.76 (s, 2H), 3.91 (broad s, 1H), 4.02 (t, J ¼ 6.1 Hz, 2H),
.50 (s,1H), 4.74 (s,1H), 6.63 (d, J ¼ 7.5 Hz, 2H), 6.94 (m, 2H), 6.97 (d,
J ¼ 6.7 Hz, 2H), 2.77 (broad s, 1H), 2.86 (broad s, 1H), 3.65 (m, 5H),
3.93 (broad s, 1H), 4.07 (t, J ¼ 6.8 Hz, 2H), 4.52 (s, 1H), 4.77 (s, 1H),
6.23 (d, J ¼ 3.2 Hz, 1H), 6.35 (dd, J ¼ 1.9, 3.2 Hz, 1H), 6.63 (d,
J ¼ 5.2 Hz, 2H), 6.93 (s, 1H), 7.00 (m, 1H), 7.03 (s, 1H), 7.46 (m, 5H),
þ
J ¼ 7.8 Hz, 1H), 7.18 (td, J ¼ 1.5, 7.7 Hz, 1H), 7.26 (m, 1H), 7.38 (d,
J ¼ 6.4 Hz, 2H), 7.46 (d, J ¼ 6.3 Hz, 1H), 7.51 (d, J ¼ 7.9 Hz, 2H), 7.70
7.59 (s, 1H). APCI-MS for C28
purity: 90.0%.
H
31
O
3
N
4
[MþH] : 471.3 m/z. HPLC
(
dt, J ¼ 1.2, 8.0 Hz, 1H), 7.79 (td, J ¼ 1.80, 7.7 Hz, 1H), 8.40 (d,
þ
J ¼ 4.4 Hz, 1H). APCI-MS for C38
H
45
O
3
N
4
[MþH] : 605.4 m/z. HPLC
4
.4.7. (4-{[(Furan-2-ylmethyl)(pyridin-2-ylmethyl)amino]methyl}
phenyl)(7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone
25)
1H NMR (400 MHz, CD
H), 3.60 (s, 1H), 3.70 (s, 2H), 3.71 (s, 2H), 3.80 (s, 2H), 3.91 (s, 1H),
.51 (s, 1H), 4.75 (s, 1H), 6.26 (d, J ¼ 3.1 Hz, 1H), 6.35 (dd, J ¼ 1.8,
.2 Hz, 1H), 6.63 (d, J ¼ 4.6 Hz, 2H), 6.98 (d, J ¼ 7.1 Hz, 1H), 7.28 (m,
H), 7.40 (d, J ¼ 6.1 Hz, 2H), 7.47 (s, 1H), 7.51 (d, J ¼ 7.9 Hz, 2H), 7.69
purity: 91.0%.
(
3
OD) d: 2.77 (broad s, 1H), 2.86 (broad s,
4
(
.4.2. (7-Hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)(4-{[{4-[3-
piperidin-1-yl)propoxy]benzyl}(pyridin-2-ylmethyl)amino]
methyl}phenyl)methanone (20)
1
4
3
1
1
H NMR (400 MHz, CD
J ¼ 5.8 Hz, 4H), 2.02 (p, J ¼ 6.1 Hz, 2H), 2.68 (m, 6H), 2.76 (broad s,
H), 2.85 (broad s, 1H), 3.56 (s, 2H), 3.59 (m, 1H), 3.63 (s, 2H), 3.71
3
OD)
d
: 1.53 (d, J ¼ 6.6 Hz, 2H), 1.67 (p,
(
d, J ¼ 7.9 Hz,1H), 7.81 (td, J ¼ 1.8, 7.7 Hz,1H), 8.42 (d, J ¼ 4.6 Hz,1H).
2
þ
APCI-MS for C28
28
H O
3
N
3
[MþH] : 454.2 m/z. HPLC purity: 94.7%.
(
6
s, 2H), 3.91 (s, 1H), 4.05 (t, J ¼ 6.0 Hz, 2H), 4.50 (s, 1H), 4.75 (s, 1H),
.63 (d, J ¼ 6.3 Hz, 2H), 6.87 (d, J ¼ 8.6 Hz, 2H), 6.97 (d, J ¼ 7.2 Hz,
H), 7.29 (m, 3H), 7.40 (d, J ¼ 7.3 Hz, 2H), 7.50 (d, J ¼ 7.8 Hz, 2H), 7.68
1
4
.4.8. (4-{[(4-Bromobenzyl)(furan-2-ylmethyl)amino]methyl}
phenyl)(7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone
26)
1H NMR (400 MHz, CD
.55e3.95 (m, 8H), 4.51 (s, 1H), 4.76 (s, 1H), 6.33 (s, 1H), 6.40 (s, 1H),
(
d, J ¼ 7.8 Hz,1H), 7.82 (td, J ¼ 1.8, 7.7 Hz,1H), 8.41 (d, J ¼ 4.4 Hz,1H).
þ
APCI-MS for C38
H
45
O
3
N
4
[MþH] : 605.4 m/z. HPLC purity: 90.0%.
(
3
OD)
d
: 2.77 (t, J ¼ 5.6 Hz,1H), 2.86 (s, 1H),
3
6
2
4.4.3. 4-{[(Furan-2-ylmethyl){2-[3-(piperidin-1-yl)propoxy]
.63 (d, J ¼ 4.4 Hz, 2H), 6.98 (d, J ¼ 7.9 Hz, 1H), 7.33 (d, J ¼ 8.2 Hz,
benzyl}amino]methyl}phenyl)(7-hydroxy-3,4-dihydroisoquinolin-
þ
H), 7.40e7.55 (m, 7H). APCI-MS for C29
H
28BrO
3
N
2
[MþH] : 531.5
2(1H)-yl)methanone (21)
1
and 533.5 m/z. HPLC purity: 97.1%.
3
H NMR (400 MHz, CD OD) d: 1.67 (broad s, 2H), 1.85 (broad s,
4
1
2
4
2
1
H), 2.11 (dq, J ¼ 5.88, 11.4 Hz, 2H), 2.77 (broad s, 1H), 2.86 (broad s,
H), 3.14 (m, 2H), 3.61 (broad s,1H), 3.67 (s, 2H), 3.68 (s, 2H), 3.70 (s,
H), 3.93 (broad s,1H), 3.93 (t, J ¼ 6.2 Hz,1H), 4.05 (t, J ¼ 5.8 Hz, 2H),
.51 (s, 1H), 4.77 (s, 1H), 6.28 (d, J ¼ 3.0 Hz, 2H), 6.38 (d, J ¼ 3.1 Hz,
H), 6.63 (d, J ¼ 6.6 Hz, 2H), 6.95 (m, 4H), 7.23 (td, J ¼ 1.6, 7.7 Hz,
4
.4.9. [4-({(Furan-2-ylmethyl)[2-(morpholin-4-yl)ethyl]amino}
methyl)phenyl](7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)
methanone (27)
1
H NMR (400 MHz, CD
3
OD)
d
: 2.44 (s, 4H), 2.53 (dd, J ¼ 5.7,
H), 7.40 (d, J ¼ 7.5 Hz, 2H), 7.49 (m, 4H); APCI-MS for C37
44 4 3
H O N
8
1
4
.2 Hz, 2H), 2.66 (dd, J ¼ 5.8, 8.4 Hz, 2H), 2.77 (broad s, 1H), 2.86 (s,
H), 3.65 (m, 4H), 3.70 (s, 4H), 3.92 (broad s, 1H), 4.53 (s, 1H), 4.65
.77 (s, 1H), 6.27 (d, J ¼ 3.2 Hz, 1H), 6.36 (d, J ¼ 2.9 Hz, 1H), 6.63 (d,
þ
[MþH] : 594.4 m/z. HPLC purity: 89.9%.
J ¼ 6.1 Hz, 2H), 6.98 (d, J ¼ 7.1 Hz, 2H), 7.39e7.54 (m, 6H). APCI-MS
4.4.4. (4-{[(Furan-2-ylmethyl){4-[3-(piperidin-1-yl)propoxy]
þ
for C28
H
34
O
4
N
3
[MþH] : 476.4 m/z. HPLC purity: 91.0%.
benzyl}amino]methyl}phenyl)(7-hydroxy-3,4-dihydroisoquinolin-
2
(1H)-yl)methanone (22)
1
H NMR (400 MHz, CD
3
OD)
d
: 1.51 (d, J ¼ 5.9 Hz, 2H), 1.64 (p,
4
.4.10. (7-Hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)[4-({[(5-
J ¼ 5.6 Hz, 4H), 2.00 (dq, J ¼ 6.05, 11.9 Hz, 2H), 2.61 (m, 6H), 2.77
broad s, 2H), 2.86 (broad s, 1H), 3.54 (s, 2H), 3.60 (s, 2H), 3.62 (m,
H), 3.92 (s,1H), 4.01 (t, J ¼ 6.1 Hz, 2H), 4.52 (s,1H), 4.76 (s,1H), 6.24
dd, J ¼ 0.9, 3.3 Hz, 1H), 6.36 (dd, J ¼ 1.9, 3.2 Hz, 1H), 6.63 (d,
methylthiophen-2-yl)methyl](pyridin-2-ylmethyl)amino}methyl)
phenyl]methanone (28)
(
3
(
1
H NMR (400 MHz, CD
3
OD)
d
: 2.44 (d, J ¼ 1.2 Hz, 3H), 2.76 (t,
J ¼ 5.7 Hz, 1H), 2.85 (broad s, 1H), 3.60 (broad s, 1H), 3.69 (s, 2H),
J ¼ 6.2 Hz, 2H), 6.86 (d, J ¼ 8.6 Hz, 2H), 6.98 (d, J ¼ 8.0 Hz, 1H), 7.28
3
3
5
.75 (2s, 4H), 3.90 (broad s, 1H), 4.50 (s, 1H), 4.75 (s, 1H), 6.62 (m,
H), 6.74 (d, J ¼ 3.3 Hz,1H), 6.97 (d, J ¼ 7.5 Hz,1H), 7.28 (ddd, J ¼ 1.3,
.0, 7.4 Hz, 1H), 7.40 (d, J ¼ 6.5 Hz, 2H), 7.54 (d, J ¼ 7.8 Hz, 2H), 7.73
(
C
d, J ¼ 8.6 Hz, 2H), 7.40 (d, J ¼ 6.7 Hz, 2H), 7.49 (m, 3H). APCI-MS for
þ
37
H
44
O
4
N
3
[MþH] : 594.5 m/z. HPLC purity: 92.0%.
(
dt, J ¼ 1.1, 7.9 Hz, 1H), 7.83 (td, J ¼ 1.8, 7.7 Hz, 1H), 8.42 (ddd,
þ
J ¼ 0.89, 1.7, 5.1 Hz, 1H). APCI-MS for C29
H
30
O
2
N
3
S [MþH] :
4
.4.5. (7-Hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)[4-({[3-(1H-
imidazol-1-yl)propyl](pyridin-2-ylmethyl)amino} methyl)phenyl]
4
84.2 m/z. HPLC purity: 99.4%.
methanone (23)
1
H NMR (400 MHz, CD
3
OD)
d
: 2.01 (p, J ¼ 6.9 Hz, 2H), 2.51 (t,
4.4.11. [4-({(Furan-2-ylmethyl)[(5-methylthiophen-2-yl)methyl]
amino}methyl)phenyl](7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)
J ¼ 6.8 Hz, 2H), 2.77 (broad s, 1H), 2.86 (broad s, 1H), 2.86 (t,
J ¼ 6.1 Hz, 1H), 3.60 (br t, J ¼ 5.0 Hz, 1H), 3.68 (s, 2H), 3.74 (s, 2H),
methanone (29)
1
3
2
.92 (br t, J ¼ 5.0 Hz, 1H), 4.02 (t, J ¼ 6.9 Hz, 2H), 4.50 (s, 1H), 4.76 (s,
H), 6.63 (d, J ¼ 6.4 Hz, 2H), 6.88 (s, 1H), 6.98 (m, 2H), 7.30 (ddd,
3
H NMR (400 MHz, CD OD) d: 2.44 (s, 3H), 2.78 (broad s, 1H),
2.86 (broad s,1H), 3.65 (m, 5H), 3.74 (s, 2H), 3.92 (s,1H), 4.53 (s,1H),
J ¼ 1.3, 5.0, 7.5 Hz, 1H), 7.40 (d, J ¼ 6.0 Hz, 2H), 7.49 (m, 2H), 7.59 (dt,
4.76 (s, 1H), 6.26 (d, J ¼ 3.1 Hz,1H), 6.37 (dd, J ¼ 1.9, 3.2 Hz, 1H), 6.60
J ¼ 1.1, 7.9 Hz, 1H), 7.82 (td, J ¼ 1.8, 7.7 Hz, 1H), 8.44 (ddd, J ¼ 0.9, 1.8,
(m, 3H), 6.73 (d, J ¼ 3.4 Hz, 1H), 6.98 (d, J ¼ 7.4 Hz, 1H), 7.44 (m, 3H),
þ
þ
5
.1 Hz, 1H). APCI-MS for C29
H
32
O
2
N
5
[MþH] : 482.3 m/z. HPLC
7.53 (d, J ¼ 7.7 Hz, 2H). APCI-MS for C28
z. HPLC purity: 98.1%.
H
29
O
3
N
2
S [MþH] : 473.3 m/
purity: 99.0%.

180
C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
4
.4.12. (4-{[(Furan-2-ylmethyl)(4-hydroxybenzyl)amino]methyl}
salt (MTS) was purchased from Promega (Madison, WI, USA).
Radiolabeled [6,7- H] estrone sulfate (54.3 Ci/mmol) was pur-
3
phenyl)(7-hydroxy-3,4-dihydroiso quinolin-2(1H)-yl)methanone
(
30)
1H NMR (400 MHz, CD
s, 1H), 3.49 (s, 2H), 3.62 (3s, 6H), 3.92 (broad s, 1H), 4.52 (s, 1H), 4.76
chased from Perkin Elmer (Woodbridge, ON, Canada). Raloxifene
hydrochloride was bought from Cayman Chemical (Ann Harbor, MI,
USA). Biodegradable Counting Scintillant was purchased from
Amersham Biosciences (Waltham, MA, USA). Penicillin/strepto-
3
OD)
d
: 2.77 (t, J ¼ 5.7 Hz, 2H), 2.86 (broad
(
s, 1H), 6.23 (d, J ¼ 3.0 Hz, 1H), 6.36 (dd, J ¼ 1.9, 3.1 Hz, 1H), 6.63 (m,
2
2
C
H), 6.74 (d, J ¼ 8.0 Hz, 2H), 6.97 (d, J ¼ 7.1 Hz,1H), 7.19 (d, J ¼ 8.3 Hz,
mycin mix, L-glutamine, non-essential amino acids, sodium pyru-
H), 7.40 (d, J ¼ 6.9 Hz, 2H), 7.49 (t, J ¼ 8.1 Hz, 3H). APCI-MS for
vate, normal and charcoal-stripped Fetal Bovine Serum (FBS), and
geneticin (G418 sulfate) were purchased from Wisent, Inc. (St-
Bruno, QC, Canada). All cell culture media were purchased from Life
Technologies (Grand Island, NY, USA) except for phenol-red free
McCoy's 5A medium that was purchased from PromoCell (Heidel-
berg, Germany). STS inhibitor EM-1913 was synthesized in our
Laboratory of Medicinal Chemistry using published procedures
[42]. For the purpose of in vitro assays, all chemicals tested (in-
hibitors and reference compounds) were first dissolved in DMSO
and subsequent dilutions were done in the proper buffer or cell
culture media. The final concentration of DMSO in the culture
medium was 0.1% or less.
þ
H
29 29
O
4
N
2
[MþH] : 469.2 m/z. HPLC purity: 89.3%.
4
.4.13. (7-Hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)(4-{[(4-
methoxybenzyl)(pyridin-2-ylmethyl)amino] methyl}phenyl)
methanone (31)
1
3
H NMR (400 MHz, CD OD) d: 2.76 (broad s, 1H), 2.85 (broad s,
1
3
6
2
H), 3.56 (s, 2H), 3.59 (broad s, 1H), 3.64 (s, 2H), 3.71 (s, 2H), 3.76 (s,
H), 3.91 (broad s, 1H), 4.50 (s, 1H), 4.75 (s, 1H), 6.62 (broad s, 2H),
.87 (d, J ¼ 8.6 Hz, 2H), 6.98 (d, J ¼ 8.1 Hz, 1H), 7.29 (m, 3H), 7.40 (m,
H), 7.51 (d, J ¼ 7.8 Hz, 2H), 7.69 (d, J ¼ 7.9 Hz, 1H), 7.82 (td, J ¼ 1.78,
7
.7 Hz, 1H), 8.40 (d, J ¼ 4.4 Hz, 1H). APCI-MS for C31
32 3 3
H O N
þ
[MþH] : 494.3 m/z. HPLC purity: 98.3%.
4.6.1. Steroid sulfatase inhibition assay
4.5. Synthesis of sulfamate derivatives 32e34
An enzymatic assay previously described was used for the in-
hibition of the transformation of estrone sulfate (E1S) to estrone
The compounds 32e34 were synthesized following a solid-
(E1) by STS [42]. Briefly, the transfected HEK-293 cells were ho-
ꢀ
phase strategy we previously developed and published for similar
sulfamate derivatives [33]. These compounds were purified by flash
chromatography (hexanes/EtOAc, 3:7).
mogenized by repeated (5 times) cycles of freezing (ꢃ80 C) and
ꢀ
thawing on ice (4 C). The homogenates were then incubated for
ꢀ
2 h at 37 C (shaking water bath) with or without inhibitors
3
(0.01
m
Me1
m
M) in presence of [ H]-E1S (9 nM), adjusted to 1
m
M
4
.5.1. 2-[(4-{[(Furan-2-ylmethyl)(pyridin-2-ylmethyl)amino]
with E1S, in a Tris-acetate buffer (pH 7.4) containing 5 mM EDTA
and 10% glycerol. After the incubation, 1 mL of xylene was added to
each tube and the solutions were then centrifuged at 3000 rpm for
methyl}phenyl)carbonyl]-1,2,3,4-tetrahydroisoquinolin-7-yl
sulfamate (32)
1H NMR (400 MHz, CDCl
d
: 2.88 (m, 2H), 3.70 (m, 6H), 3.82 (s,
H), 3.98 (broad s, 1H), 4.60 (broad s, 1H), 4.88 (broad s, 1H), 5.12
broad s, 2H), 6.22 (dd, J ¼ 0.9, 3.2 Hz, 1H), 6.33 (dd, J ¼ 1.8, 3.2 Hz,
20 min to separate the organic ([ H]-E1) and aqueous ([ H]-E1S)
phases. Once 500 L of each phase was added to 10 mL of Biode-
3
3
3
)
2
(
1
m
gradable Counting Scintillant, the radioactivity of samples was
recorded using a Wallac 1411 Liquid Scintillation Counter. The
percentage of inhibition was determined by comparison with the
H), 7.18 (m, 4H), 7.44 (m, 5H), 7.58 (d, J ¼ 7.8 Hz, 1H), 7.69 (td,
J ¼ 1.8, 7.7 Hz, 1H), 8.53 (dd, J ¼ 1.5, 4.7 Hz, 1H). APCI-MS for
þ
3
C
H
28 29
O
5
N
4
S [MþH] : 533.3 m/z. HPLC purity: 79.9%.
control (buffer þ homogenate þ [ H]-E1S) which was set to 0% of
inhibition. IC50 value was obtained using GraphPad Prism 5 soft-
ware (La Jolla, CA, USA).
4.5.2. 2-{[4-({(Furan-2-ylmethyl)[(5-methylthiophen-2-yl)methyl]
amino}methyl)phenyl]carbonyl}-1,2,3,4-tetrahydro-isoquinolin-7-yl
sulfamate (33)
4.6.2. Cell culture
1H NMR (400 MHz, CDCl
)
d
: 2.47 (s, 3H), 2.84 (m, 2H), 3.66 (s,
The ER breast cancer T-47D cells and the osteoblast-like Saos-
þ
3
2
4
H), 3.69 (s, 2H), 3.77 (s, 2H), 3.98 (broad s, 1H), 4.64 (broad s, 1H),
.89 (broad s, 2H), 5.00 (m, 2H), 6.22 (dd, J ¼ 0.83, 3.1 Hz, 1H), 6.35
2 cells were purchased from the American Type Culture Collection
(ATCC) (Manassas, VA, USA). The HEK-293 cell line overexpressing
STS was obtained from Dr. Van Luu-The (CHU de Qu eꢀ bec - Research
(
dd, J ¼ 1.8, 3.2 Hz, 1H), 6.59 (dd, J ¼ 1.4, 3.3 Hz, 1H), 6.72 (d,
J ¼ 3.4 Hz, 1H), 7.15 (m, 3H), 7.42 (d, J ¼ 8.0 Hz, 3H), 7.50 (d,
Center) [52]. All cell lines were maintained in culture flasks
þ
2
ꢀ
J ¼ 8.0 Hz, 2H). APCI-MS for C28
30
H O
5
N
3
S
2
[MþH] : 552.3 m/z.
(175 cm growth area, BD Falcon) at 37 C in a 5% CO
atmosphere. The T-47D cells were grown in phenol red free RPMI
640 medium supplemented with 10% FBS, penicillin (100 IU/mL),
streptomycin (100 g/mL), -glutamine (2 mM), and 17 -estradiol
2
humidified
HPLC purity: 90.9%.
1
4.5.3. 2-[(4-{[(4-Methoxybenzyl)(pyridin-2-ylmethyl)amino]
m
L
b
methyl}phenyl)carbonyl]-1,2,3,4-tetrahydroisoquinolin-7-yl
sulfamate (34)
(1 nM). The Saos-2 cells were grown in phenol red free McCoy's 5A
medium supplemented with 10% FBS, penicillin (100 IU/mL), and
1H NMR (400 MHz, CDCl
)
d
: 2.89 (m, 2H), 3.57 (s, 2H), 3.64 (m,
H), 3.74 (s, 2H), 3.80 (s, 3H), 3.91 (broad s, 1H), 4.59 (s, 1H), 4.8e5.2
m, 3H), 6.87 (d, J ¼ 8.6 Hz, 2H), 7.17 (m, 4H), 7.31 (d, J ¼ 8.6 Hz, 2H),
.39 (m, 2H), 7.46 (m, 2H), 7.58 (d, J ¼ 7.9 Hz, 1H), 7.68 (td, J ¼ 1.8,
streptomycin (100
were maintained in Minimum Essential Medium supplemented
with 10% FBS, penicillin (100 IU/mL), streptomycin (100 g/mL),
glutamine (2 mM), non-essential amino acids (0.1 mM), sodium
pyruvate (1 mM), and geneticin (G418 sulfate) (700 g/mL).
mg/mL). The HEK-293 cells transfected with STS
3
3
(
7
7
m
L-
.7 Hz, 1H), 8.51 (dt, J ¼ 1.3, 5.0 Hz, 1H). APCI-MS for C31
H
O
33 5
N
4
S
m
þ
[
MþH] : 573.3 m/z. HPLC purity: 83.0%.
4
.6.3. Cell proliferation
4.6. Biological assays (general information)
CellTitter 96® Aqueous One Solution Cell Proliferation Assay was
used as an indirect colorimetric measurement of cell proliferation
according to the manufacturer's instructions. After the treatments,
1
7
b
-estradiol, Tris, EDTA, glycerol, insulin, and bovine serum
albumin (BSA) were purchased from SigmaeAldrich Ltd (Oakville,
ON, Canada). 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner
20
m
L of MTS solution was briefly added to each well (100
mL) of the
ꢀ
plates and incubated at 37 C for 2 h (Saos-2) or 4 h (T-47D). The
absorbance at 490 nm was then measured with a Thermo max

C. Ouellet et al. / European Journal of Medicinal Chemistry 119 (2016) 169e182
181
microplate reader (Molecular Devices, Sunnyvale, CA, USA). The
control (culture media þ DMSO) is set to 100% of cell proliferation.
T-47D cells were suspended in RPMI supplemented with insulin
conformation as the best bound conformation, positioning the
sulfamate moiety at a distance of 0.6 Å from the original sulfate (see
Fig. 8). The best result from EM-1913 docking is used as the refer-
ence for the docking of sulfamate compounds 32, 33, and 34 using
the same procedure.
(
50 ng/mL), instead of 17b-estradiol, and 5% charcoal-stripped FBS
to deprive the media of estrogens. The cells were plated in 96-well
plates at a density of 3000 cells/well and allowed to attach for 48 h.
After this pre-incubation, the inhibitors and the reference com-
pounds diluted in fresh culture media were added to the wells and
replaced every 2 days for 7 days of treatment. Saos-2 cells were
suspended in phenol-red free McCoy's 5A medium supplemented
with 10% charcoal-stripped FBS, penicillin (100 IU/mL) and strep-
4.7.2. Estrogen receptor docking
Experimental structure of human ERa/raloxifene complex was
obtained from PDB 1ERR. Hydrogens were added to the structure
using Protonate 3D in MOE followed by a minimization process
using the LigX procedure with force field MMFF94x, born solvation
and default parameters. Docking was performed using the induced
fit procedure with MMFF94x force field with the distance solvation
model and default parameters. RMSD of 1.5 Å was obtained be-
tween the best docked conformation and the crystal structure of
raloxifene, validating the procedure. The same procedure was used
to perform docking of both phenolic as well as sulfamate com-
pounds 25, 29, 31, 32, 33, and 34.
tomycin (100 mg/mL). The cells were seeded in 96-well plates at a
density of 3000 cells/well and allowed to attach. After 24 h, the
inhibitors and the reference compounds diluted in fresh culture
medium were added to the wells and replaced every 2 days for 7
days of treatment.
4.6.4. Alkaline phosphatase activity
Saos-2 cells were treated similarly as reported in the cell pro-
liferation assay. The cells were seeded at a density of 2000 cells/
well and were treated with the inhibitors and the reference com-
pounds for 3 days. The alkaline phosphatase (ALP) activity was
measured using Sensolyte® pNPP Alkaline Phosphatase Assay Kit
Acknowledgments
We thank the Fondation du Cancer du Sein du Qu eꢀ bec for
granting this study. We would also like to thank Jenny Roy for the
HPLC purification of compounds and Marie-Claude Trottier for
NMR analyses. Careful reading of the manuscript by Micheline
Harvey is also greatly appreciated.
*Colorimetric* (AnaSpec, Fremont, CA, USA) following the manu-
facturer's protocol. Briefly, after the 3 days of treatment, the cells
were washed twice with washing buffer (provided with the kit) and
lysed with 0.2% Triton X-100. The cell lysates were centrifuged and
the supernatants were used to determine the ALP activity. The
supernatants were deposed in a 96-well plate and incubated
Appendix B. Supplementary data
3
0 min with a p-nitrophenyl phosphate solution (provided with the
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.044.
kit). The absorbance at 405 nm was measured with a Thermo max
microplate reader (Molecular Devices, Sunnyvale, CA, USA). The
control (culture medium þ DMSO) is set to 100% of alkaline phos-
phatase activity.
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