Impact of structural modifications at positions 13, 16 and 17 of 16β-(m-carbamoylbenzyl)-estradiol on 17β-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity

Article abstract of DOI:10.1016/j.jsbmb.2015.10.020

The chemical synthesis of four stereoisomers (compounds 5a-d) of 16β-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d. These compounds were tested as 17β-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1 μM, respectively), but like the other isomers 5c and d, it was found to be estrogenic. The intermediate 3a, however, was weakly estrogenic at 1 μM, not at all at 0.1 μM, and showed an interesting inhibitory potency on 17β-HSD1 (90, 59 and 22%, at 10, 1 and 0.1 μM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b, 5a-d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17β-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies.

Full text of DOI:10.1016/j.jsbmb.2015.10.020

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SBMB 4555 No. of Pages 12
Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry & Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Impact of structural modifications at positions 13, 16 and 17 of
16 -(m-carbamoylbenzyl)-estradiol on 17 -hydroxysteroid
b
b
dehydrogenase type 1 inhibition and estrogenic activity
René Maltaisa,b, Alexandre Trottiera,b, Xavier Barbeauc,d, Patrick Lagüed,e
,
Martin Perreaulta,b, Jean-François Thériaulta,b, Sheng-Xiang Lina,b, Donald Poiriera,b,
*
a
Endocrinology and Nephrology Unit, CHU de Québec-Research Center (CHUL, T4), Québec City, QC, Canada
Department of Molecular Medicine, Faculty of Medicine, Université Laval, Québec City, QC, Canada
Département de chimie, Institut de biologie intégrative et des systèmes (IBIS), Québec City, QC, Canada
Centre de recherche sur la fonction, la structure et l'ingénierie des protéines (PROTEO),Université Laval, Québec City, QC, Canada
b
c
d
e
Département de biochimie microbiologie et bio-informatique, Institut de biologie intégrative et des systèmes (IBIS), Québec City, QC, Canada
A R T I C L E I N F O
A B S T R A C T
Article history:
The chemical synthesis of four stereoisomers (compounds 5a–d) of 16
b
-(m-carbamoylbenzyl)-estradiol,
Received 27 February 2015
Received in revised form 11 September 2015
Accepted 25 October 2015
Available online xxx
a potent reversible inhibitor of 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1), and two
intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance
signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear
correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c
and d. These compounds were tested as 17b-HSD1 inhibitors and to assess their proliferative activity on
estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4).
Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol
Keywords:
Steroid
Enzyme inhibitor
Estrogen
Chemical synthesis
Estradiol isomer
(95, 82 and 27%, at 10, 1 and 0.1
estrogenic. The intermediate 3a, however, was weakly estrogenic at 1
showed an interesting inhibitory potency on 17 -HSD1 (90, 59 and 22%, at 10, 1 and 0.1
m
M, respectively), but like the other isomers 5c and d, it was found to be
M, not at all at 0.1 M, and
M,
m
m
b
m
respectively). As expected, no compound showed an androgenic activity. The binding modes for
compounds 3a and b, 5a–d and CC-156 were evaluated from molecular modeling. While the non-polar
interactions were conserved for all the inhibitors in their binding to 17
interactions and in binding conformational energies correlated to the inhibitory potencies.
ã 2015 Elsevier Ltd. All rights reserved.
b-HSD1, differences in polar
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1.
Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1.1.
2.1.2.
2.1.3.
2.1.4.
Synthesis of compound 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Synthesis of compounds 3a and b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Synthesis of compounds 5a and b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Synthesis of compounds 5c and d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2.
Biological assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2.1.
2.2.2.
2.2.3.
2.2.4.
17
Estrogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
ER -binding affinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Androgenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
b-HSD1 inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
b
* Corresponding author at: Laboratory of Medicinal Chemistry, CHU de Québec—Research Center (CHUL, T4ꢀ42), 2705 Laurier Boulevard, Québec City, QC G1V 4G2, Canada.
Fax: +1 418 654 2298.
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
http://dx.doi.org/10.1016/j.jsbmb.2015.10.020
0960-0760/ã 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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2.3.
Molecular modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.
4.
Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1.
3.2.
3.3.
3.4.
3.5.
Chemical synthesis of C13-epi CC-156 stereoisomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
NMR characterization of stereoisomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
17b-HSD1 inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Molecular modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Estrogenic and androgenic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction
possesses estrogenic activity when tested on estrogen-sensitive
breast cancer cell lines (T-47D and MCF-7) and tissues (uterine
and vagina) [19]. Different attempts put forth to fix this problem,
such as introducing a 2-methoxy group, removing the 3-OH and
Estrogen-sensitive diseases affect the health of a significant
number of women around the world, from a benign but painful
disease such as endometriosis, to fatal diseases such as breast and
endometrial cancers. The estrogen hormones, especially estradiol
(E2), strongly accelerate tumor growth by stimulating the
proliferation of cancer cells through interaction with the estrogen
receptor (ER) [1]. Among the different proteins involved in
rigidifying the 16b-side chain (Fig. 1B–D), have met with
moderate success [17,20–22]. However, modification of the
phenolic moiety of CC-156 by replacing the OH at C3 by an
ethylbromide side chain has led to the first non-estrogenic
irreversible steroid inhibitor of 17b-HSD1 (Fig. 1E) [19,23,24]. We
estrogen production and modulation, 17
b
-hydroxysteroid dehy-
are now interested in pursuing our modification of the CC-
156 scaffold to obtain a non-estrogenic reversible inhibitor.
Recent work from our group on the estrogenic activity of
E2 epimers has highlighted the potential of the inversion of
the configuration at C13 (18-methyl) and/or C17 (OH) to decrease
drogenase type 1 (17 -HSD1) has been identified as a promising
b
therapeutic target to block E2 biosynthesis [2–8]. This enzyme
transforms the steroid estrone (E1) into E2, the most potent
estrogen in women, but also transforms dehydroepiandrosterone
(DHEA) into 5-androstene-3
estrogen found in large concentration in post-menopausal women
[9,10]. The blockade of 17 -HSD1 activity has solicited great
b
,17
b
-diol (5-diol), a less potent
the binding affinity with ER
activity. Thus, relative binding affinity (RBA) values of 1.6% and
1.2% were obtained for 13-epi-17 -E2 and 13-epi-17 -E2,
a and consequently the estrogenic
b
a
b
interest over the past 20 years and many different strategies have
been reported to obtain active inhibitors [11–16]. However, despite
these intensive efforts, no inhibitor has yet reached the clinical trial
respectively [16]. Based on these results, we then wanted to
validate this strategy in order to decrease the residual estrogenic
activity of 17b-HSD1 reversible inhibitor CC-156. However, the
step. One of the major causes that delayed the emergence of 17
HSD1 inhibitors as new drugs was the lack of potent candidates
with no estrogenic activity.
b
-
consequence of such structural modifications on the inhibitory
activity of CC-156 was difficult to predict, and for this reason, the
chemical synthesis of each stereoisomeric form of CC-156 at
positions 13, 16 and 17 was necessary to assess their potential as a
In the last few years, we have identified the 16
carbamoylbenzyl) estradiol (CC-156) as a strong inhibitor of
17 -HSD1 (Fig. 1A) [17,18]. Unfortunately this E2 derivative
b-(m-
non-estrogenic inhibitor of 17b-HSD1 (Fig. 1F).
b
Fig. 1. Chemical structure of 17b-HSD1 inhibitor CC-156 (A) and structures representing the previously reported strategies (B–E) and novel strategy (F) used to reduce the
residual estrogenic activity of CC-156. The structural modifications target positions 2, 3, 16 and 17 of CC-156.
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16b-(m-carbamoylbenzyl)-
estradiol on 17b-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020

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3
2. Materials and methods
132.9 (C600), 134.9 (C300), 135.1 (C100), 135.5 (C16), 137.3 (C5), 154.9
(C3), 167.5 (CONH2), 208.5 (C17). LRMS for C26H28NO3 [M + H+]
402.4 m/z.
2.1. Chemistry
The reagents for chemical synthesis were purchased from
Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada). IBX polystyrene
was purchased from Novabiochem (US). The usual solvents were
obtained from Fisher Scientific (Montréal, QC, Canada) and were
used as received. Anhydrous tetrahydrofuran (THF) and anhydrous
dichloromethane (DCM) were from Sigma–Aldrich. Thin-layer
chromatography (TLC) and flash-column chromatography were
performed on 0.20 mm silica gel 60 F254 plates and with Silicycle
R10030B 230–400-mesh silica gel (Québec, QC, Canada). Infrared
spectra (IR) were recorded on ABB model MB3000 FT-IR
spectrophotometer (Québec, QC, Canada), and the significant
bands were reported in cmꢀ1. Nuclear magnetic resonance (NMR)
spectra were recorded at 400 MHz for 1H and 100.6 MHz for 13C
with a Bruker Avance 400 digital spectrometer (Billerica, MA, US).
2.1.2. Synthesis of compounds 3a and b
2.1.2.1. Step 1: reduction of the enone 2. To a solution of enone 2
(2.00 g, 4.98 mmol) in MeOH/DCM (75:25, 250 mL) was added
sodium borohydride (380 mg, 10.0 mmol). The solution was stirred
at room temperature for 2 h and then poured into water. The
resulting solution was extracted with EtOAc, washed with brine,
dried over sodium sulfate, filtered and evaporated under reduced
pressure. The crude compound was purified by flash
chromatography (EtOAc/Hexanes: 7:3 to DCM/MeOH: 9:1) to
give a mixture of compounds 3a and b (1.20 g, 60%).
2.1.2.2. Step 2: acetylation of the mixture of compounds 3a
and b. The mixture of 3a and
submitted to acetylation conditions using acetic anhydride
(875 L, 8.51 mmol) in pyridine (70 mL) and stirred overnight at
b (1.14 g, 2.82 mmol) was
The chemical shifts (d) are expressed in ppm and referenced to
residual acetone (2.06 ppm, 1H, and 29.8 ppm, 13C), methanol
(MeOH) (3.31 ppm, 1H, and 49.0 ppm, 13C) or dimethylsulfoxide
(DMSO) (2.50 ppm, 1H, and 39.5 ppm, 13C). In addition to classic 1H
NMR and 13C NMR data reported in experimental procedures, all
compounds were analyzed by COSY, HSQC, HMBC and NOESY
experiments to support the assignment of protons and carbons.
Low-resolution mass spectra (LRMS) were recorded on a Perki-
nElmer Sciex API-150ex apparatus (Foster City, CA, US) equipped
with a turbo ion-spray source and expressed in m/z. The names of
new compounds were obtained using—ACD/Labs (Chemist’s
version) software (Toronto, ON, Canada) and carbon numbering
was assigned as presented in Fig. 2.
m
room temperature. The resulting solution was diluted with EtOAc
and washed with a solution of copper sulfate (10%). The organic
layer was then washed with brine, dried with sodium sulfate,
filtered and evaporated under reduced pressure. The crude mixture
(1.20 g) was purified by flash chromatography using DCM/MeOH
(97:3) to give four different fractions (fraction A (270 mg): 4a as
17-monoacetate; fraction B (120 mg): 4b as 17-monoacetate;
fraction C (350 mg): a mixture of 17-monoacetate and diacetate at
positions C3 and C17); and fraction D (230 mg): diacetate at
positions C3 and C17.
2.1.2.3. Step 3: (A) hydrolysis of the monoacetate 4a. A solution of
potassium carbonate (10%) in MeOH (30 mL) was added to the
purified monoacetate 4a (238 mg, 0.55 mmol) and the mixture was
stirred for 2 h at room temperature. The solution was poured into
water, extracted with EtOAc, washed with brine, dried over sodium
sulfate and evaporated under reduced pressure. The crude
compound was purified by flash chromatography (DCM/MeOH:
97:3) to give 205 mg (92%) of compound 3a.
2.1.1. Synthesis of compound 2
To a solution of epi-estrone [25] (2.00 g, 7.4 mmol) in ethanol
(200 mL) were added 3-formylbenzonitrile (1.94 g, 14.8 mmol) and
aqueous 10% KOH (34 mL). The mixture was heated at 100 ꢁC for
30 min and then poured into cold water. The resulting solution was
neutralized with 10% aqueous HCl, extracted with EtOAc, washed
with brine, dried over sodium sulfate, filtered and evaporated
under reduced pressure. The crude compound was triturated with
3-{(E)-[(13
dene]methyl}benzamide(3a). IR (KBr)
(CONH2), 1605 (C
C). 1H NMR (MeOH-d4)
(qd, J = 11.1, 2.3 Hz, 8-CH), 1.19–1.59 (m, 7
and 14 -CH), 2.08 (m, 7 -CH and 12 -CH2), 2.17–2.36 (m, 9
and 11 -CH), 2.58–2.68 (m, 15
CH2), 2.97 (ddt, J = 18.2, 8.1, 2.7 Hz, 15
a
,16E,17
a
)-3,17-dihydroxyestra-1(10),2,4-trien-16-yli-
: 3418 (OH and NH2), 1659
: 0.86 (s, 18-CH3), 1.01
cold MeOH to give 2.0 g (70%) of compound 2. 3-{(E)-[(13
hydroxy-17-oxoestra-1(10),2,4-trien-16-ylidene]methyl}benzamide
(2). IR (KBr) : 3456 and 3348 (OH and NH2), 1705 (C O), 1659
(CONH2), 1620 (C : 0.49–0.62 (m, 8
C); 1H NMR (DMSO-d6)
CH), 0.74 (qd, J = 13.6, 4.1 Hz, 11 -CH), 1.05 (s, 18-CH3), 1.31 (qd,
J = 11.9, 5.9 Hz, 7 -CH), 1.51 (td, J = 14.3, 13.6, 4.1 Hz, 12 -CH), 1.86
(dd, J = 11.1, 6.7 Hz, 14 -CH), 1.94–2.04 (m, 7 -CH), 2.15–2.31 (m,
-CH, 11 -CH and 12 -CH), 2.58 (m, 6-CH2), 2.78–2.88 (m, 15
CH), 3.18–3.33 (m, 15 -CH), 6.35 (d, J = 2.6 Hz, 4-CH), 6.49 (dd,
a
,16E)-3-
y
¼
d
y
¼
a
-CH2, 11
b
-CH, 12
a
-CH
-CH
¼
d
b-
a
a
b
b
a
b
b-CH), 2.73 (dd, J = 9.0, 3.9 Hz, 6-
a
a
a-CH), 4.63 (q, J = 3.8, 3.1 Hz,
a
b
17b-CH), 6.47 (d, J = 2.6 Hz, 4-CH), 6.57 (dd, J = 8.3, 2.7 Hz, 2-CH and
9
a
a
b
b-
10-CH), 7.14 (d, J = 8.5 Hz, 1-CH), 7.45 (t, J = 7.7 Hz, 500-CH), 7.59 (dt,
J = 7.9,1.4 Hz, 600-CH), 7.70 (dt, J = 7.7, 1.4 Hz, 400-CH), 7.94 (t, J = 1.8 Hz,
a
J = 8.5, 2.6 Hz, 2-CH), 7.04 (d, J = 8.5 Hz, 1-CH), 7.39 (t, J = 2.3 Hz, 10-
CH), 7.48 (s, 1H of CONH2), 7.55 (t, J = 7.7 Hz, 500-CH), 7.81 (d,
J = 7.7 Hz, 600-CH), 7.89 (dt, J = 7.9, 1.4 Hz, 400-CH), 8.05 (s, 1H of
200-CH). 13C NMR (MeOH-d4)
d: 23.4 (C18), 27.9 (C11), 29.9 (C7),
31.4 (C6), 33.0 (C15), 34.0 (C12), 43.7 (C13), 43.8 (C9), 44.0 (C8),
49.1 (C14), 76.9 (C17), 113.9 (C2), 115.9 (C4), 123.4 (C10), 126.1 (C200),
127.8 (C1), 128.4 (C500), 129.5 (C400), 132.1 (C10), 132.6 (C600), 135.0
(C300),139.2 (C5),139.9 (C100),148.2 (C16),155.9 (C3),172.6 (CONH2).
LRMS for C27H36NO4 [M + CH3OH + H+] 436.4 m/z. HPLC purity of
97.4% (retention time = 11.6 min).
CONH2), 8.10 (s, 200-CH), 8.99 (s, 3-OH). 13C NMR (DMSO-d6)
d: 25.0
(C18), 27.6 (C7), 28.0 (C11), 29.6 (C6), 29.8 (C15), 31.7 (C12), 40.6
(C9), 43.0 (C8), 46.3 (C14), 49.3 (C13), 112.9 (C2), 114.7 (C4), 126.6
(C1), 128.3 (C400), 128.9 (C500), 129.2 (C200), 129.6 (C10), 132.5 (C10),
2.1.2.4. Step 3: (B) hydrolysis of the monoacetate 4b. A solution of
potassium carbonate (10%) in MeOH (30 mL) was added to the
purified monoacetate 4b (110 mg, 0.26 mmol) and the mixture was
stirred for 2 h at room temperature. The solution was poured into
water, extracted with EtOAc, washed with brine, dried over sodium
sulfate and evaporated under reduced pressure. The crude
compound was purified by flash chromatography using DCM/
MeOH (97:3) to give 75 mg (73%) of compound 3b.
Fig. 2. Carbon numbering of a steroidal derivative.
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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SBMB 4555 No. of Pages 12
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R. Maltais et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
3-{(E)-[(13
dene]methyl}benzamide (3b). IR (KBr)
(CONH2); 1605 (C
C); 1H NMR (MeOH-d4)
(m, 12 -CH), 1.42 (q, J = 10.7 Hz, 8 -CH), 1.58 (m, 7
CH), 1.72 (m, 11 -CH), 2.03 (d, J = 11.8,12 -CH), 2.15 (d, J = 13.3, 7
CH), 2.24 (m, 9 -CH and 11
J = 18 Hz, 15 -CH), 2.95 (m, 15
a
,16E,17
b
)-3,17-dihydroxyestra-1(10),2,4-trien-16-yli-
: 3356 (OH and NH2), 1659
: 0.95 (s, 18-CH3), 1.22
(C100),155.8 (C3),176.4 (CONH2). LRMS for C27H38NO4 [M + CH3OH +
y
H+] 438.4 m/z. HPLC purity of 94.5% (retention time = 8.8 min).
¼
d
a
b
a
-CH and 14
a
b
-
-
2.1.3.4. Step 3. (B) Hydrolysis of the mixture of 6b and c (monoacetate
and diacetates). A solution of potassium carbonate (10%) in MeOH
(3 mL) was added to the mixture of compounds 6b and c (23 mg)
and the solution was stirred for 2 h at room temperature. The
reaction mixture was poured into water, extracted with EtOAc,
washed with brine, dried over sodium sulfate and evaporated
under reduced pressure. The crude mixture was purified by flash
chromatography (EtOAc) to give 15 mg (75%) of a mixture of
compounds 5a and b. The proportion of both compounds was
measured by HPLC (5a: 58% and 5b: 42%) and by NMR (5a: 54% and
b
b
a
a
a
-CH), 2.68 (m, 6-CH2), 2.76 (d,
-CH), 4.07 (s, 17 -CH), 6.43 (d,
b
a
J = 2.7 Hz, 4-CH), 6.53 (m, 2-CH), 6.67 (s, 10-CH2), 7.08 (d, J = 8.5 Hz,
1-CH), 7.44 (t, J = 7.7 Hz, 500-CH), 7.57 (d, J = 7.8, 600-CH), 7.70 (d, J = 7.7,
400-CH), 7.90 (s, 200-CH). 13C NMR (MeOH-d4)
d: 30.1 (C12), 30.2
(C18), 30.6 (C11), 31.5 (C6), 33.9 (C7), 34.2 (C15), 42.3 (C9), 43.8
(C13), 44.2 (C8), 52.8 (C14), 87.5 (C17), 113.9 (C2), 115.7 (C4), 126.5
(C10), 126.5 (C400), 128.2 (C1), 128.8 (C200), 129.5 (C500), 132.9 (C600),
133.0 (C10), 135.1 (C300), 139.3 (C5), 139.8 (C100), 150.4 (C16), 155.7
(C3), 172.6 (CONH2). LRMS for C27H36NO4 [M + CH3OH + H+] 436.4
m/z. HPLC purity of 97.1% (retention time = 7.8 min).
5b: 46%, calculated from the ratio of 17
b-CH at 4.02 ppm for 5a and
of 17 -CH at 3.83 ppm for 5b). The retention times are 24.8 min for
b
5b and 24.3 min for 5a. LRMS for both compounds C27H38NO4
[M + CH3OH + H+] 438.4 m/z.
2.1.3. Synthesis of compounds 5a and b
2.1.4. Synthesis of compounds 5c and d
2.1.3.1. Step 1: catalytic hydrogenation of compound 3a. To a
solution of compound 3a (65 mg, 0.16 mmol) in MeOH (50 mL) was
added palladium on charcoal (10%) (40 mg) under an argon
atmosphere. The solution was purged three times with hydrogen,
and stirred under a hydrogen atmosphere at room temperature
overnight. The solution was then filtered under celite pad and
evaporated under reduced pressure. The crude compound was
purified by flash chromatography (EtOAc/Hexanes: 7:3) to give
60 mg (95%) of a mixture of two isomers (5a + b).
To a solution of compound 3b (200 mg, 0.5 mmol) in MeOH
(100 mL) was added palladium on charcoal (10%) (50 mg) under an
argon atmosphere. The solution was purged three times with
hydrogen, and stirred under a hydrogen atmosphere at room
temperature overnight. The solution was then filtered under a
celite pad and evaporated under reduced pressure. The crude
compounds were purified by flash chromatography (EtOAc/
Hexanes: 7:3) to give 25 mg (13%) of compound 5c, 15 mg (8%)
of compound 5d and 105 mg (53%) of a mixture of 5c and d.
3-{[(13
methyl}benzamide (5c). IR (KBr)
(CONH2),1605 (C
C). 1H NMR (MeOH-d4)
1.49 (m, 7 -CH, 14 -CH, 12 -CH, 15 -CH, 8
J = 12.2, 9.1, 6.8 Hz, 11 -CH), 1.94 (ddt, J = 12.4, 4.9, 2.1 Hz, 7
2.11 (ddd, J = 15.5,13.3, 7.8 Hz,15 -CH,12 -CH), 2.30–2.46 (m,11
CH, 9 -CH), 2.40 (m, 16 -CH), 2.61–2.81 (m, 3H, 6-CH2 and 1H of
10-CH2), 2.94 (dd, J = 13.3, 8.0 Hz, 1H of 10-CH2), 3.46 (d, J = 4.3 Hz,
a
,16
a
,17
b
)-3,17-dihydroxyestra-1(10),2,4-trien-16-yl]
: 3195-3480 (OH and NH2), 1659
: 0.97 (s,18-CH3),1.08–
-CH), 1.66 (ddt,
-CH),
2.1.3.2. Step 2: acetylation of the mixture of compounds 5a and
b. The mixture of 5a and b (60 mg, 0.15 mmol) was submitted to
y
¼
d
acetylation conditions using acetic anhydride (50
m
L, 0.05 mmol)
a
a
b
a
b
b
in pyridine (3 mL) and stirred overnight at room temperature. The
solution was diluted with EtOAc and washed with a solution of
copper sulfate (10%). The organic layer was then washed with
brine, dried with sodium sulfate, filtered and evaporated under
reduced pressure. The crude mixture (55 mg) was purified by flash
chromatography using EtOAc/Hexanes (7:3) to give two different
fractions (fraction A (19 mg): 6a as 3-monoacetate and fraction B
(25 mg): a mixture of 6b as 17-monoacetate and 6c as diacetate at
positions C3 and C17).
b
a
b
a-
b
a
17a-CH), 6.46 (d, J = 2.6 Hz, 4-CH), 6.56 (dd, J = 8.4, 2.7 Hz, 2-CH),
7.05 (d, J = 8.7 Hz, 1-CH), 7.38 (t, J = 7.6 Hz, 500-CH), 7.46 (dt, J = 7.6,
1.5 Hz, 400-CH), 7.69 (dt, J = 7.7, 1.5 Hz, 600-CH), 7.78 (s, 200-CH). 13C
NMR (MeOH-d4) d: 29.7 (C11), 30.2 (C7), 31.0 (C18), 31.3 (C12), 31.4
(C6), 37.0 (C10), 37.2 (C15), 40.4 (C9), 44.9 (C8), 45.7 (C16), 45.9
(C13), 53.1 (C14), 84.3 (C17),114.1 (C2),115.6 (C4),125.9 (C600),128.8
(C1), 129.1 (C200), 129.4 (C500), 133.6 (C400), 133.9 (C10), 134.8 (C300),
138.9 (C5), 144.3 (C100), 155.6 (C3), 172.8 (CONH2). LRMS for
2.1.3.3. Step 3: (A) hydrolysis of the monoacetate 6a. A solution of
potassium carbonate (10%) in MeOH (3 mL) was added to the
purified monoacetate 6a (11 mg, 0.55 mmol) and the mixture was
stirred for 2 h at room temperature. The solution was poured into
water, extracted with EtOAc, washed with brine, dried over sodium
sulfate and evaporated under reduced pressure. The crude
compound was purified by flash chromatography (EtOAc) to give
6 mg (54%) of compound 5a.
C
27H38NO4 [M + CH3OH + H+] 438.4 m/z. HPLC purity of 94.8%
(retention time = 13.2 min).
3-{[(13 ,16 ,17 )-3,17-dihydroxyestra-1(10),2,4-trien-16-yl]
methyl}benzamide (5d). IR (KBr) : 3348 (OH and NH2), 1659
(CONH2), 1605 (C : 1.11 (s, 18-CH3 and 7-
C). 1H NMR (MeOH-d4)
a
b b
y
¼
d
CH), 1.14–1.26 (m, 8-CH), 1.26–1.35 (m, 14-CH), 1.40–1.51 (m, 11-
CH), 1.57 (m, 15-CH, 12-CH2), 1.79 (dt, J = 14.0, 7.2 Hz, 11-CH), 1.84–
3-{[(13
methyl}benzamide (5a). IR (KBr)
(CONH2),1605 (C
C). 1H NMR (MeOH-d4)
1.10 (s, 18-CH3), 1.15 (ddd, J = 12.6, 6.8, 2.8 Hz, 7
14 -CH, 12 -CH and 11 -CH), 1.62 (dd, J = 13.3, 7.2 Hz, 15
1.73–1.95 (m, 15 -CH, 12 -CH, 7 -CH), 2.22–2.42 (m, 9 -CH, 11
CH), 2.45–2.61 (m,16
-CH and 1H of 10-CH2), 2.62–2.74 (m, 6-CH2),
3.04–3.16 (m, 1H of 10-CH2), 3.97–4.08 (m, 17
-CH), 6.44 (d,
a
,16
a
,17
a
)-3,17-dihydroxyestra-1(10),2,4-trien-16-yl]
: 3364 (OH and NH2), 1659
: 1.00–1.08 (m, 8 -CH),
-CH), 1.20–1.47 (m,
-CH),
y
1.96 (m, 7-CH), 2.22 (m, 16
(dd, J = 13.2, 9.5 Hz, 1H of 10-CH2), 2.59–2.72 (m, -6-CH2), 3.10 (dd,
J = 13.2, 4.6 Hz, 1H of 10-CH2), 3.38 (d, J = 8.3 Hz, 17
-CH), 6.42 (d,
b-CH), 2.34 (m, 9-CH and 15-CH), 2.54
¼
d
b
a
a
a
a
b
b
J = 2.6 Hz, 4-CH), 6.55 (dd, J = 8.4, 2.7 Hz, 2-CH), 7.03 (d, J = 8.5 Hz, 1-
a
b
b
a
a-
CH), 7.35–7.46 (m, 500-CH and 600-CH), 7.70 (dt, J = 7.3,1.5 Hz, 400-CH),
b
7.74 (d, J = 1.8 Hz, 200-CH). 13C NMR (MeOH-d4)
d: 28.8 (C11), 29.4
b
(C12), 29.6 (C18), 30.2 (C7), 31.2 (C6), 33.1 (C15), 39.4 (C9), 41.2
(C10), 42.5 (C8), 44.4 (C13), 47.3 (C16), 51.3 (C14), 87.6 (C17), 114.1
(C2), 115.5 (C4), 126.2 (C400), 128.5 (C1), 129.2 (C200), 129.4 (C500),
133.6 (C600), 134.0 (C10), 134.9 (C300), 138.8 (C5), 143.2 (C100), 155.6
(C3), 172.7 (CONH2). LRMS for C27H38NO4 [M + CH3OH + H+] 438.4
m/z. HPLC purity of 95.4% (retention time = 10.8 min).
J = 2.7 Hz, 4-CH), 6.55 (dd, J = 8.5, 2.7 Hz, 2-CH), 7.08 (d, J = 8.4 Hz, 1-
CH), 7.38 (t, J = 7.6 Hz,1H, 500-CH), 7.44 (dt, J = 7.7, 1.5 Hz, 600-CH), 7.69
(dt, J = 7.5, 1.5 Hz, 400-CH), 7.76 (s, 200-CH). 13C NMR (MeOH-d4)
d:
24.7 (C18), 28.1 (C11), 30.3 (C7), 31.3 (C6), 34.3 (C12), 34.7 (C15),
38.6 (C10), 41.8 (C9), 42.3 (C8), 42.9 (C16), 46.2 (C13), 51.3 (C14),
77.7 (C17), 114.0 (C2),115.7 (C4), 126.0 (C400), 128.1 (C1),129.1 (C200),
129.4 (C500), 133.1 (C600), 133.6 (C10), 134.8 (C300), 139.0 (C5), 144.3
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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5
2.2. Biological assays
2.2.2. Estrogenic activity
Estrogen-sensitive T-47D cells were suspended in RPMI
medium supplemented with insulin (50 ng/mL), instead of E2,
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
period, the compound to be tested was dissolved in DMSO, diluted
in fresh culture media and added to the well (final DMSO
concentration <0.5%). The culture media was replaced every
2 days until a total of 7 days of treatment. CellTitter 961 Aqueous
One Solution Cell Proliferation Assay was used as an indirect
colorimetric measurement of cell proliferation according to the
manufacturer’s instructions (Promega, Nepean, ON, Canada).
2.2.1. 17
T-47D cells obtained from the American Type Culture Collection
(ATCC) were used as a source of 17 -HSD1 activity. They were
b-HSD1 inhibition
b
maintained in RPMI medium supplemented with 10% fetal bovine
serum (FBS), 2 nM glutamax, 100 IU/mL penicillin, 100 mg/mL
streptomycin and 1 nM estradiol (E2) in T-75 culture flasks. For
assays, 5000 cells were seeded in each 24 wells of culture plates in
steroid-deprived medium containing 50 ng/mL insulin and 5% FBS
charcoal-stripped instead of 10% complete FBS, and without
E2 supplement contrary to standard culture medium. Cells were
treated 24 h later by adding a DMSO solution of the compound (3a
and b, 5a–d, 5b/a, CC-156 or E2) to obtain a final concentration of
Briefly, after the treatments, 20 mL of 3-(4,5-dimethylthiazol-2-
10
m
M (DMSO concentration <0.5 %) and by adding the radioactive
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-
liumsolution (MTS) were added to each well and the mixture
incubated at 37 ꢁC for 4 h. The absorbance at 490 nm was then
measured with a Thermo max microplate reader (Molecular
Devices, Sunnyvale, CA, US). The control (culture media + DMSO) is
set to 100% of cell proliferation.
substrate (60 nM of [14C]-estrone; American Radiolabeled Chem-
icals, St. Louis, MO, US). Cell media were collected after overnight
incubation for quantification of radiolabeled steroids ([14C]-E1 and
[
14C]-E2). Steroids were extracted with diethyl ether, separated by
thin layer chromatography using toluene/acetone (4:1) as solvent
system and quantified using a Storm 860 system (Molecular
Dynamics, Sunnyvale, CA, US). The percentage of transformation
(E1–E2) and the percentage of inhibition were calculated as follow:
% transformation = 100 ꢂ [14C]-E2/([14C]-E1 + [14C]-E2) and % of
inhibition = 100 ꢂ (% transformation without inhibitor ꢀ % trans-
formation with inhibitor)/% transformation without inhibitor.
2.2.3. ER
A competitive binding assay using purified ER
according to a known methodology [19]. Briefly, each reaction
consisted of 100 nM ER
-LBD and 5 nM [3H]-estradiol in assay
buffer (10 mM Tris,1.5 mM EDTA,1 mM dithiothreitol,10% glycerol,
b-binding affinity
b-LBD was done
b
Scheme 1. Synthesis pathways used to obtain pure compounds 5a–d. Reagents and conditions: (a) 3-formylbenzonitrile, KOH, EtOH,100 ꢁC; (b) NaBH4, MeOH/DCM (75:25), rt;
(c) H2, Pd/C (10%), MeOH, rt; (d) acetic anhydride, pyridine, rt; (e) K2CO3, MeOH, rt.
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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R. Maltais et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
1 mg/mL bovine serum albumin, pH 7.5) with the appropriate
concentration of compound to be tested in 100 L assay buffer.
Stock solutions of compounds were previously prepared in MeOH
and 1 L was added to the reaction mixture to reach the
appropriate concentration. Nonspecific binding was determined
by incubation with an excess of E2 (1 M). After 3 h of incubation at
room temperature, 100 L of 50% hydroxyapatite slurry was added
to bind the receptor/ligand complex and the supernatant was
discarded after centrifugation at 3500 ꢂ g. Three washing steps
were done with 1 mL of 10 mM Tris pH 7.5, 1 mM EDTA, and 1 mM
EDTA and a centrifugation at 3500 ꢂ g during 5 min. Ethanol (1 mL)
was added to the slurry and the solution incubated for 30 min. The
suspension was put into 10 mL of Biodegradable Counting
Scintillant cocktail and radioactivity was measured by Wallac
1411 Liquid Scintillation (Turku, Finland).
total of 100 mL medium in 96-well microtiter plates (Becton–
m
Dickinson Company, Lincoln Park, NJ, US) were pre incubated for
24 h at 37 ꢁC under 5% CO2 humidified atmosphere. Tested
compounds were dissolved in MeOH to prepare the stock solution
of 10ꢀ2 M, the compounds were then diluted at several concen-
trations with culture medium, added to corresponding wells and
incubated for 5 days (50% medium was changed after 3 days). The
final MeOH concentration in each well was adjusted to 0.05%.
Control wells did not contain the compound to be tested but the
same concentration of MeOH. Quantification of cell growth was
determined by MTS method, using CellTitter 961 Aqueous Solution
Cell Proliferation Assay (Promega, Nepean, ON, Canada) and
following the manufacturer’s instructions. The cell proliferative
was expressed in % and the basal cell proliferation (control) fixed at
100%.
m
m
m
2.2.4. Androgenic activity
2.3. Molecular modeling
Androgen-sensitive human prostate cancer LAPC-4 cells were
kindly provided by Robert E. Reiter from University of California
(Los Angeles, CA, US) and maintained at 37 ꢁC under 5% CO2
humidified atmosphere. Cells were grown in RPMI-1640 medium
Docking simulations were performed using MOE 2014.09
(Molecular Operating Environment (MOE), 2013.08; Chemical
Computing Group Inc., Montréal, QC, Canada). The crystal structure
supplemented (v/v) with 5% fetal bovine serum (FBS), 1%
L-
coordinates of 17b-HSD1, including the CC-156 and the co-factor
glutamine and 1% penicillin/streptomycin. They were rinced
3 times and suspended with the medium supplemented with 5%
dextran-coated charcoal treated FBS rather than 10% FBS to remove
the remaining hormones. Triplicate cultures of 10,000 cells in a
NADP, were taken from PDB 3HB5 [26]. Hydrogen atoms were
added using the Protonate 3D tool, and the protein complex was
prepared using the LigX tool, both included in MOE, as previously
described [27]. Water molecules were then removed. Compounds
Fig. 3. 2D- and 3D-representations of compounds 3a and b and 5a–d. The important NOE results are presented by arrows (Figs. 1S–5S, Supplementary Data). The 3D-
structures were generated using CSChem 3D std 5.0. (Cambridge Soft Corporation, Cambridge, MA, US).
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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R. Maltais et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
7
3a and b and 5a–d were built and energy minimized in MOE based
on compound CC-156. Docking simulations were performed in the
presence of NADP using the induced fit protocol, with a radius of
8 Å and a tether force adjusted according to the b-factor. Validation
of the docking protocol was carried out by a self-docking of
compound CC-156, leading to an RMSD of 0.53 Å between the
docked and the crystallographic structures. Because compounds
3a and b and 5a–d have a scaffold similar to CC-156, no further
optimization of the docking protocol was considered. Conforma-
tional sampling for compounds CC-156, 3a and 5a were realized
using the conformational search engine included in MOE,
according to the LowModeMd protocol, with an RMSD cutoff of
0.75 Å and default parameters. These conformations were then
optimized from density functional theory (DFT) calculations at the
B97-D3/Def2-TZVP [28,29] level. Final single point energies were
obtained at the DSD-PBEP86-D3/Def2-TZVP level [30,31]. All
quantum mechanical (QM) calculations were performed using
Orca 3.0.3 software [32] and used the empirical D3 correction
[33,34] (denoted by the suffix-D3), and the Becke-Johnson
damping potential [35], and the RIJCOSX approximation [36], as
recommended [37]. QM optimization was also applied to the best
docking conformation of compounds 3a, 5a and CC-156, with the
bonds and dihedral angles fixed to conserve the binding
conformation. The structural alignment of minimal energy and
docked conformations were realized using the pair_fit function in
PyMOL (The PyMOL Molecular Graphics System, Version
1.5.0.4 Schrödinger, LLC).
18-methyl on the
b
-face, which gave only the 17
b
-OH allylic
alcohol as reduction product [17]. Because 3a and b cannot be
separated by flash chromatography, two synthetic pathways (1 and
2) were then explored to obtain pure compounds 5a–d.
In pathway 1, we submitted the mixture of 3a and b to a
catalytic hydrogenation to yield a new mixture of compounds
5a–d. However, after many attempts with different elution
conditions, we were unable to isolate all isomers as pure
compounds from the mixture by flash chromatography and HPLC,
and only isomers 5a and
d were obtained in pure form.
Consequently, we changed our initial strategy by performing a
selective acetylation of the phenolic OH (pathway 2) in order to
change the polarity of compounds and increase our chances to
obtain a better separation. Fortunately, we were able to separate
the acetylated compounds 4a and b by simple flash column
chromatography. The acetate protecting group of 4a and b were
then easily hydrolyzed with potassium carbonate in MeOH to give
the corresponding phenolic compounds 3a and b, respectively.
Both compounds were then submitted to a catalytic hydrogena-
tion, but whereas 3b provided a mixture of 5c and d, which was
separable by flash column chromatography, the reduction of 3a
gave an inseparable mixture of compounds 5a and b, even by HPLC.
Consequently, we proceeded once again with the acetylation
strategy hoping to change the polarity properties of the mixture of
compounds 5a and b. Thus, the mixture of 5a and b was acetylated
giving a mixture of compounds 6a–c, but only 6a was separable by
flash column chromatography. Despite different attempts with
preparative HPLC, it was however impossible to separate the
mixture of 6b and c. Thus in summary, the synthetic pathway 2
provided pure compounds 5a, c and d,–and a mixture of 5a and b.
3. Results and discussion
3.1. Chemical synthesis of C13-epi CC-156 stereoisomers
3.2. NMR characterization of stereoisomers
The strategy behind the synthesis of stereoisomers 5a–d, the
four C13-epimers of CC-156, is described in Scheme 1. Epi-estrone
(1) [25] was submitted to an aldol condensation with 3-
formylbenzonitrile and potassium hydroxide in refluxing ethanol
to give the enone 2 in good yield. Under these conditions, the
benzonitrile functionality was found to be completely hydrolyzed
in situ to the corresponding carboxamide. Reduction of enone 2
using sodium borohydride in MeOH gave a mixture (60:40) of the
two allylic alcohols 3a and b in quantitative yield. This reduction
was in fact less stereoselective for the enone with the 18-methyl on
The first step to elucidate the stereochemistry of compounds 5a,
c and d was to correctly assign the stereochemistry of inter-
mediates 3a and b. Firstly, all proton and carbon signals were
assigned by 2D NMR spectroscopy experiments (HSQC, HMBC,
COSY and NOESY) for each compound (Table 1S and Figs. 1S–5S,
Supplementary Data). Using NOESY experiments, we observed a
strong NOE correlation between the C18 and the 17a-H for 3b, but
none at all for 3a, thus confirming the reported stereochemistry for
both compounds. The configuration of the steroid C-ring was also
investigated by NOE experiment (Fig. 3). The correlations found in
the
a-steroid face than for the corresponding enone with
Fig. 4. Inhibition of 17b-HSD1 transformation of estrone (E1) to estradiol (E2) by compounds 5a–d at 10, 1.0 and 0.1 mM, in intact T-47D cells. See Experimental section for the
details of the assay.
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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compound 3a between 8
ring was in the chair conformation. In the case of compound 3b, the
strong correlation found between 8 -H and 12 -H was only
possible if the C-ring was in the boat conformation. We were then
interested in confirming the stereochemistry at positions 16 and 17
of pure compounds 5a, c and d. In the case of compound 5c (Fig. 3),
b
-H and 17
b
-H was only possible if the C-
compounds (5b and a in proportions 40:60), we can therefore
conclude that pure compound 5b should be less active than pure
compound 5a. Indeed a significant lower inhibition value was
obtained for the mixture of 5a and b.
b
b
Interestingly, the unsaturated compound 3a (17
inhibition values of 91, 59 and 22%, which represent a much higher
inhibition value than 17 -OH isomer 3b (49, 1 and 0%). This result
seems to point out toward a better tolerance of the enzyme for the
17 -OH configuration. This result is also supported by 17
HSD1 inhibitory results found in the saturated series (compounds
5a and d), which showed better inhibition for derivative 5a (17a-
a-OH) gave
the 18-CH3 showed clear NOE correlations with 17
but not with 10-CH2 as well as with any aromatic H of the
carbamoylbenzyl moiety. Also, the 17 -H produced NOE correla-
tions with 16
-H and 1H of 10-CH2. The absence of a NOE
correlation between 14 -H and 12 -H, confirmed to us that the
a-H and 16
a
-H,
b
a
a
b-
a
a
a
steroid C-ring was in a boat conformation instead of a chair
conformation. This conclusion was also supported by the fact that a
boat conformation was reported for the corresponding
C16-unsubstituted E2 compound [16].
OH). On the other side, the configuration of the carbamoybenzyl
side chain at position 16 seems to also play a role, giving different
inhibitory tendencies according to the 17-OH configuration
(5a > 5b + 5a for 17 -OH and 5c > 5d for 17
a
b-OH). Thus, the
The isomer 5d showed important NOE correlations that
supported the structure assignment. The 18-CH3 showed strong
configuration 16 /17a-OH (compound 5a) was found as the best
a
combination over the four isomeric possibilities (5a–d). Table 1
summarizes the inhibition activities related to structural charac-
teristics of the different isomers. However, the inhibition values of
5a at 10, 1.0 and 0.1 mM (97, 81 and 27%, respectively) are lower
than that of the reference compound CC-156 (100, 96, and 86%).
NOE correlations with 17
with 14 -H. However, the 18-CH3 did not show interaction with
16
-H and 10-CH2, which are much too distant to allow a NOE
effect (Fig. 3). Also, the 17 -H was found to interact with both 16
H and 1H of 10-CH2. As observed in the case of 5c, the absence of
correlation between 14 -H and 12 -H supports the boat
a-H, with both 11a and 12a-H, as well as
a
b
a
b
-
a
a
3.4. Molecular modeling
conformation of a C-ring for 5d. Finally, the stereochemistry of
compound 5a, which was obtained from intermediate compound
Molecular docking simulations were performed to compare the
structural differences between the binding modes of compounds
3a and b, 5a–d and CC-156. Fig. 5 presents a global view of CC-
3a (17
H, which was obviously impossible if the C16 chain was in
-position (Fig. 3; 5a vs b). However, the NOESY spectrum of
b-H), was confirmed by the interaction of 17b-H with 16b-
b
156 bound to the E2 pocket of 17b-HSD1. The predicted binding
compound 5a provided less opportunity than the NOESY spectra of
compounds 5c and d to identify relevant NOE interactions related
to the steroid C-ring configuration. In fact, 14a-H and 12a-H have
modes of compounds 3a and b and 5a–d are presented in Fig. 6
while the inhibition activities are reported in Table 1 for the
different compounds.
very close chemical shifts and no other correlations could support
this conformation assignment. However, based on the reported
epi-estradiol (17a-OH/18a-CH3) derivative without a chain at C16,
the chair conformation was the most favored in this case [16].
Finally, consequently to the above assignment of 5a, c and d, and
considering that compound 5b was not available in a pure form
(mixture of 5a and b), the stereochemistry of 17
was assigned by default to compound 5b.
a-OH and 16a-H
3.3. 17b-HSD1 inhibition
Compounds 5a, c and d and the mixture of 5a + b (40:60) were
tested at concentrations of 10, 1.0 and 0.1 M for their capacity to
inhibit the transformation of E1 to E2 by 17 -HSD1 in intact T-47D
cells (Fig. 4), a breast cancer cell line that expresses 17 -HSD1 [18].
m
b
b
Among the isomers of CC-156, the derivative 5a appears as the best
inhibitor of the series with inhibition values of 95, 82 and 27% at
concentrations of 10, 1.0 and 0.1 mM, respectively, followed by less
active derivatives 5c (81, 51 and 2%) and 5d (79, 26 and 20%). Also,
considering that the entry 5a + b is in fact a mixture of two
Fig. 5. A global view of CC-156 in the E2 pocket of 17b-HSD1, from molecular
docking. The protein is represented as grey cartoon, the amino acid side chains
involved in the polar interactions with CC-156, identified in the figure, are in yellow
sticks, CC-156 in green sticks and NADP in purple sticks. The hydrogen bonds are
represented by yellow dashed lines. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Table 1
Inhibition activities and structural characteristics. Columns 2, 3 and 4 present the inhibition values for compounds 3a and b, 5a–d and CC-156 at concentrations of 10, 1.0 and
0.1 M, respectively. Columns 5 and 6 present the presence or absence of the two main structural characteristics modulating inhibition activity of compounds 3a and b and
5a–d.
m
Inhibition (%) for compound concentration of
10
91
Binding conformation compared to CC-156 for
Compound
m
M
1
m
M
0.1
m
M
10-C
17-OH
3a
3b
5a
5b
5c
5d
CC-156
59
22
0
27
n/a
2
Similar
Similar
49
95
n/a
81
1
Similar
Similar
Different
Similar
82
n/a
51
Different
Similar
Different
Reference
Similar
Different
Similar
79
100
26
96
20
86
Reference
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17
org/10.1016/j.jsbmb.2015.10.020
b
-(m-carbamoylbenzyl)-
b
-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.

G Model
SBMB 4555 No. of Pages 12
R. Maltais et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
9
Fig. 6. Predicted binding modes for compounds 3a and b and 5a–d (left to right, respectively).17
b
-HSD1 is represented as grey cartoon. CC-156, used as reference, is in green
sticks while compounds 3a and b and 5a–d are in purple sticks. Ser-142 is in yellow sticks. The 17-OH and 10-C are highlighted with black and red arrows for good and bad
conformations, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
According to the docking calculations, the bindings are driven
by both hydrophobic and polar interactions. The hydrophobic
interactions occur between the E2-like scaffold and residues Leu-
149, Pro-187, Met-193, Val-225, Phe-226 and Phe-259. As these
interactions are similar for all the compounds, the differences
observed experimentally in inhibition activities are not likely to
arise from the E2-like scaffold. Next, the polar interactions arise
from three different chemical groups from the compounds. First,
the Hꢀꢀbond between the 3ꢀꢀOH and Glu-282 and His-221 is
conserved for all the compounds (Figs. 5 and 6, top). The second
interaction involves the benzamide moiety linked to the
E2 scaffold through carbon 16 (compounds 3a and b, 5a–d).
The interaction of the benzamide function with Asn-152 and
Leu-95 backbone amides is conserved for all the compounds
(Figs. 5 and 6, top), but a different torsion angle for the benzamide
linker, at the 10-C position, is predicted (Fig. 6, bottom). This
difference in torsion angle is more pronounced for compounds 5b
and d, for which lower or no inhibition activities were observed
(Table 1). The third polar interaction involves Ser-142 and 17-OH
from the compounds, and lead to structural differences between
Although compounds 3a and 5a present a conformation similar
to CC-156 in the E2 pocket, their respective inhibition activities are
lower than that of CC-156. A comparison of the binding and
minimal energy conformations for 3a, 5a and CC-156 was realized
from QM calculations (Table 2).
Fig. 7 compares the binding and minimal energy conformations.
The main conformational difference for these three compounds
resides in the orientation of the benzamide moiety. Thus, for this
functional group to interact with Asn-152 and Leu-95 backbone
amides, torsion of the 16-C/10-C and 10-C/1*ꢀꢀC bonds is required.
Because the benzamide of compound 3a is conjugated with the
double bond from 16-C to 10-C, the required energy is the highest
and 3a is the less active of the three compounds (Tables 1 and 2).
On the other side, CC-156 has the lower energy conformation
penalty and has the best inhibition activity.
3.5. Estrogenic and androgenic activities
To see the impact of the structural modifications of CC-156 on
estrogenic activity, compounds 3a and b, 5a, c and d were tested on
estrogen-sensitive T-47D cells (Fig. 8A ), which naturally express
the estrogen receptors (ER). As a result, we observed a slight
decrease in estrogenic activity (cell proliferation) for compounds
the compound binding modes. The 17
and b, makes a H-bond with the backbone carbonyl of Ser-142
(Fig. 6, top, black arrows). On the other side, the 17 -OH of
a-OH of compounds 3a, 5a
b
compounds 3b and c is unable to interact with Ser-142 (Fig. 6, top,
5a, c and d at both concentrations tested (0.1 and 1.0 mM) relatively
red arrows). However, because of the torsion induced on the
to reference compound CC-156. However, their residual estrogenic
scaffold to accommodate for the benzamide interaction, the 17
b-
activity remains significant versus the control group. Interestingly,
OH of 5d is positioned close enough to Ser-142 to form a H-bond.
Most interestingly, the loss of the H-bond with Ser-142 for
compounds 3b and c concurs with lower inhibition activities
(Table 1). From this table, compounds 3a and 5a are the only ones
to satisfy the required conformation for both the 17-OH and 10-C,
and consequently have the best inhibition activities.
the allylic alcohols 3a and b, showed
estrogenicity. In fact, compound 3a very slightly stimulated the
T-47D cell proliferation at 1.0 M and not at all at 0.1 M, whereas
a better profile for
m
m
compound 3b was not estrogenic at both concentrations tested.
It is known that the proliferation resulting from a treatment
with an estrogen, such as E2, involves ER
predominant ER-mRNA expressed in T-47D cells, but ER
found although moderately (in proportion 1:9, ER :ER ) [38–40].
In fact, ER is responsible for the proliferative effect of
E2 whereas ER has been proposed to reduce ER -induced
proliferation. Thus, the activation of ER inhibits the T-47D cell
proliferation and inhibits also the growth of T-47D breast cancer
xenografts [38,41]. To determine the influence of ER on the
T-47D cell proliferation results, we then measured the binding
affinity of compounds 3a and b, 5a, c and d on ER (Fig. 6S,
Supplementary Data) using known methodology [19]. At
concentration of 1.0 M, unlabeled E2 used as a positive control
a
, which is the
b
is also
b
a
a
Table 2
b
a
Differences between the binding and the minimum energy
b
conformations, calculated as
E(global
D
Econf = E (docking conformation)
ꢀ
,
from QM calculations, for
minimal conformation)
compounds 3a, 5a and CC-156.
b
Compounds
DEconf (kcal/mol)
b
3a
5a
CC-156
7.21
6.74
5.90
a
m
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

G Model
SBMB 4555 No. of Pages 12
10
R. Maltais et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
Fig. 7. Structural alignment of minimum energy conformation (green) and docked conformation (purple) for compounds 3a,5a and CC-156. The orientation of the benzamide
moiety represents the main structural difference between the conformations. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Fig. 8. Proliferation of estrogen-sensitive T-47D cells (A) and androgen-sensitive LAPC-4 cells (B) induced by compounds 3a and b, 5a, c and d at 0.1 and 1.0
mM. See
Experimental section for the details of the assays. CTL: control; E2: estradiol; DHT: dihydrotestosterone;
D
4: 4-androstene-3,17-dione. *p < 0.01 vs CTL; ~ p < 0.01 vs E2.
fully reversed the binding of tritiated-E2 on ER
compounds 3a and b, 5a and c that produced non-significant
inhibitions. In fact, only compound 5d weakly bound ER at
1.0 M. From these results, it seems that the proliferation of T-47D
cells observed at 0.1 and 1.0
(except maybe for 5d). At a higher concentration of 20
however, we observed a significant ER
compounds (Fig. 6S). So, it is possible that the binding to ER
b
,
but not
being used as positive controls. As expected, the synthesized
compounds did not stimulate the LAPC-4 cell proliferation,
b
suggesting
derivatives.
a non-androgenic profile of these epi-estradiol
m
m
M (Fig. 8A) was not mediated by ER
b
m
M,
4. Conclusion
b
-binding for all the
b
The development of a non-estrogenic version of 16
carbamoylbenzyl)-estradiol (CC-156), a potent reversible inhibitor
of 17 -HSD1, was attempted by the synthesis of different C13-
b-(m-
may contribute to reduce the proliferation (estrogenic activity)
observed in T-47D cells, but only at high concentrations.
b
We also tested compounds 3a and b, 5a, c and d on androgen-
sensitive LAPC-4 cells (Fig. 8B). Contrary to LNCaP cells expressing
a mutated androgen receptor (AR), LAPC-4 cells express the wild
type AR. The proliferation of LAPC-4 cells was stimulated by the
potent androgen dihydrotestosterone (DHT) as well as by its
epimeric analogs. The chemical synthesis of four possible stereo-
isomers (compounds 5a–d) by varying the configuration of
17-alcohol, as well as the configuration of the 16-benzylamide
side chain, was tried using two different synthetic pathways. Three
of the four stereoisomers (compounds 5a, c and d) were finally
obtained in high purity (>95%). However, the fourth isomer
precursor 4-androstene-3,17-dione (D
4), these two compounds
Please cite this article in press as: R. Maltais, et al., Impact of structural modifications at positions 13, 16 and 17 of 16
estradiol on 17 -hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity, J. Steroid Biochem. Mol. Biol. (2015), http://dx.doi.
org/10.1016/j.jsbmb.2015.10.020
b-(m-carbamoylbenzyl)-
b

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SBMB 4555 No. of Pages 12
R. Maltais et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx
11
(compound 5b) was obtained as a mixture (40:60) with isomer 5a
and was tested as such. Assignation of the conformation of each
isomer was then realized by 2D NMR analysis. The different
stereoisomers were tested for their inhibitory capacity of 17b-
HSD1 in T-47D intact cells and showed the best inhibition for 5a
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with values of 95, 82 and 27% at concentrations of 10, 1.0, and
0.1 mM, respectively, in the relative inhibitory potency order of
5a > 5c > 5b > 5d. Estrogenic activity was also determined for these
compounds and unfortunately showed significant estrogenic
activity, as was found for the lead compound CC-156. However,
as complements to these four compounds, intermediates 3a and b
were also assessed for their inhibitory potency as well for their
estrogenic activities. As a result, compound 3a was found to be a
good inhibitor (90, 59 and 22% at 10, 1.0 and 0.1
and interestingly, with a very slight estrogenic effect at 1.0
no significant estrogenic activity at 0.1 M. As expected, no
m
M, respectively),
m
M and
m
compound showed an androgenic activity. From molecular
modeling, two interactions were reported as significant to the
binding of the compounds in the E2 pocket of 17b-HSD1: the
interactions of 17-OH with Ser-142 and the benzamide moiety with
Asn-152 and Leu-95. In addition, conformations of lower energies
contributed to the higher inhibition activities of compounds 3a and
5a, and CC-156. Thus, considering that very few examples of C13-
epimer of estradiol have been reported so far, this study provided
instructive information on their chemical synthesis, NMR confor-
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mation, 17b-HSD1 inhibition and estrogenic properties which
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Spadaro, R. Werth, M. Wetzel, K. Xu, et al., 17Beta-hydroxysteroid
dehydrogenases (17beta-HSDs) as therapeutic targets: protein structures,
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modifications (18-methyl and/or 17-hydroxy inversion of configuration) on
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could also be helpful in the synthesis and design of novel steroidal
templates to explore steroid-related targets.
Conflicts of interest
R. Maltais and D. Poirier have ownership interests on a patent
application related to 17
HSD10 inhibitors.
b-HSD1, 17b-HSD3 and 17b-
Acknowledgments
[18] Y. Laplante, C. Rancourt, D. Poirier, Relative involvement of three 17beta-
hydroxysteroid dehydrogenases (types 1, 7 and 12) in the formation of
estradiol in various breast cancer cell lines using selective inhibitors, Mol. Cell.
Endocrinol. 301 (1–2) (2009) 146–153.
[19] D. Ayan, R. Maltais, J. Roy, D. Poirier, A new nonestrogenic steroidal inhibitor of
17beta-hydroxysteroid dehydrogenase type I blocks the estrogen-dependent
breast cancer tumor growth induced by estrone, Mol. Cancer Ther. 11 (10)
(2012) 2096–2104.
[20] D. Ayan, R. Maltais, D. Poirier, Identification of a 17beta-hydroxysteroid
dehydrogenase type 10 steroidal inhibitor: a tool to investigate the role of type
10 in Alzheimer’s disease and prostate cancer, Chem. Med. Chem. 7 (7) (2012)
1181–1184.
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modified estradiol-core bearing a fused gamma-lactone as non-estrogenic
inhibitor of 17beta-hydroxysteroid dehydrogenase type 1, Bioorg. Med. Chem.
Lett. 21 (18) (2011) 5510–5513.
This work was supported by a grant from the Canadian
Institutes of Health Research (grant number MOP43994) to D.P.,
a fellowship from “Fonds de recherche du Québec—Santé” to A.T., a
fellowship from CREMOGH to A.T. and a fellowship to X.B. from the
“Fonds de recherche du Québec—Nature et Technologies”. We
thank Calcul Québec, a division of Compute Canada, for the
provision of computer time on the Colosse supercomputer. Thanks
to Dr. Robert E. Reiter (University of California, CA, US) and Jian
Song (CHU de Québec - Research Center) for providing LAPC-4 cells
and purified ERb-LBD, respectively. Careful reading of the
manuscript by Mrs. Micheline Harvey is also greatly appreciated.
[22] R. Maltais, A. Trottier, A. Delhomme, X. Barbeau, P. Lagüe, D. Poirier,
Identification of fused 16
family of non-estrogenic 17
b
,17
b-oxazinone-estradiol derivatives as a new
Appendix A. Supplementary data
b-hydroxysteroid dehydrogenase type 1 inhibitor,
Eur. J. Med. Chem. 93 (2015) 470–480.
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
jsbmb.2015.10.020.
[23] R. Maltais, D. Ayan, D. Poirier, Crucial role of 3-bromoethyl in removing the
estrogenic activity of 17beta-HSD1 inhibitor 16beta-(m-carbamoylbenzyl)
estradiol, ACS Med. Chem. Lett. 2 (9) (2011) 678–681.
[24] R. Maltais, D. Ayan, A. Trottier, X. Barbeau, P. Lagüe, J.E. Bouchard, D. Poirier,
Discovery of a non-estrogenic irreversible inhibitor of 17beta-hydroxysteroid
dehydrogenase type 1 from 3-substituted-16beta-(m-carbamoylbenzyl)-
estradiol derivatives, J. Med. Chem. 57 (1) (2014) 204–222.
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b-(m-carbamoylbenzyl)-
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