Impact of androstane A- and D-ring inversion on 17β-hydroxysteroid dehydrogenase type 3 inhibitory activity, androgenic effect and metabolic stability

Article abstract of DOI:10.1016/j.bmc.2017.02.008

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a major player in human endocrinology, being one of the most important enzymes involved in testosterone production. To capitalize on the discovery of RM-532-105, a steroidal 17β-HSD3 inhibitor, we explored the effect of its backbone configuration on inhibitory activity, androgenic profile, and metabolic stability. Two modifications that greatly alter the natural shape of steroids, i.e. inversion of the methyl on carbon 13 (13α-CH₃ instead of 13β-CH₃) and inversion of the hydrogen on carbon 5 (5β-H instead of 5α-H), were tested after the syntheses in 6 steps of 2 isomeric forms (5α/13α-RM-532-105 (6a) and 5β/13β-RM-532-105 (6b), respectively) of the 17β-HSD3 inhibitor RM-532-105 (5α/13β-configurations). For compound 6b, a cis/trans junction of the A/B rings did not significantly alter the inhibitory activity on 17β-HSD3 (IC₅₀?=?0.15?μM) as well as the liver microsomal stability (16.6% of 6b remaining after 1?h incubation) compared to RM-532-105 (IC₅₀?=?0.11?μM and 14.1% remaining). In contrast, a trans/cis junction of C/D rings reduced the inhibitory activity on 17β-HSD3 (IC₅₀?=?1.09?μM) but increased the metabolic stability with 29.4% of compound 6a remaining after incubation. The structural modifications represented by compounds 6a and 6b did not change the non-androgenicity profile of an androsterone derivative such as RM-532-105, but slightly increased its cytotoxic activity.

Full text of DOI:10.1016/j.bmc.2017.02.008

Accepted Manuscript
Impact of androstane A- and D-ring inversion on 17β-hydroxysteroid dehydro-
genase type 3 inhibitory activity, androgenic effect and metabolic stability
Francisco Cortés-Benítez, Jenny Roy, Rene Maltais, Donald Poirier
PII:
S0968-0896(16)31491-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.008
BMC 13536
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 December 2016
31 January 2017
6 February 2017
Please cite this article as: Cortés-Benítez, F., Roy, J., Maltais, R., Poirier, D., Impact of androstane A- and D-ring
inversion on 17β-hydroxysteroid dehydrogenase type 3 inhibitory activity, androgenic effect and metabolic stability,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.008
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Bioorg. Med. Chem. (revised)
Impact of androstane A- and D-ring inversion on 17β-hydroxysteroid
dehydrogenase type 3 inhibitory activity, androgenic effect and metabolic
stability
Francisco Cortés-Benítez a,c, Jenny Roy a,b, Rene Maltais a,b, Donald Poirier a,b,*
a Laboratory of Medicinal Chemistry, Endocrinology and Nephrology Unit, CHU de Québec – Research
Center, (Québec), Canada
b Department of Molecular Medicine, Faculty of Medicine, Université Laval, (Québec), Canada
c
Department of Pharmacy, Faculty of Chemistry, National Autonomous University of Mexico, Mexico
City, 04510, Mexico
(*) Corresponding Author:
Donald Poirier
Laboratory of Medicinal Chemistry
CHU de Québec – Research Center (CHUL, T4-42)
2705 Laurier Boulevard
Québec (Québec), G1V 4G2, Canada
Tel.: 1-418-654-2296; Fax: 1-418-654-2298; E-mail: donald.poirier@crchul.ulaval.ca
1

Abstract
17β-Hydroxyst roid d hydrog nas typ 3 (17β-HSD3) is a major player in human
endocrinology, being one of the most important enzymes involved in testosterone production. To
capitalize on the discovery of RM-532-105, a steroidal 17β-HSD3 inhibitor, we explored the
effect of its backbone configuration on inhibitory activity, androgenic profile, and metabolic
stability. Two modifications that greatly alter the natural shape of steroids, i.e. inversion of the
m thyl on ar on 13 (13α-CH3 inst ad of 13β-CH3) and inversion of the hydrogen on carbon 5
(5β-H inst ad of 5α-H), were tested after the syntheses in 6 steps of 2 isomeric forms (5α/13α-
RM-532-105 (6a) and 5β/13β-RM-532-105 (6b), respectively) of th 17β-HSD3 inhibitor RM-
532-105 (5α/13β-configurations). For compound 6b, a cis/trans junction of the A/B rings did not
signifi antly alt r th inhi itory a tivity on 17β-HSD3 (IC50 = 0.15 μM) as w ll as th liv r
microsomal stability (16.6% of 6b remaining after 1 h incubation) compared to RM-532-105
(IC50 = 0.11 μM and 14.1% remaining). In contrast, a trans/cis junction of C/D rings reduced the
inhi itory a tivity on 17β-HSD3 (IC50 = 1.09 μM) t in r as d th m ta oli sta ility with
29.4% of compound 6a remaining after incubation. The structural modifications represented by
compounds 6a and 6b did not change the non-androgenicity profile of an androsterone derivative
such as RM-532-105, but slightly increased its cytotoxic activity.
Keywords: Steroid, Androsterone derivatives, Hydroxysteroid dehydrogenase, LNCaP cells,
LAPC-4 cells, Prostate cancer
2

1. Introduction
Prostate cancer (PCa) is the second most common cancer in men, the fourth most
common cancer in both sexes combined, and its incidence is highest in developed areas such as
North America, Australia, as well as Western and Northern Europe.1 For instance, in the United
States, there will be an estimated 180,890 new cases and 26,120 deaths in 2016.2 Thus, PCa is a
major disease, and there are currently several therapies to improve health or help to prolong life
expectancy. Hormonal therapy is one of the first choices to treat PCa.3 Unfortunately, this cancer
often evolves toward a castration-resistant form.4 Therefore, hormonal therapy must be improved
by discovering new selective and potent drug candidates to take the relay to these first-line
therapies.
The initial growth of prostate carcinomas depends on testosterone (T) and its most
androg ni m ta olit 5α-dihydrot stost ron (5α-DHT), which both stimulate the growth of
hormone-dependent prostate cancer tumors through interaction with the androgen receptor
(AR).5,6 Th r for , s ppr ssing T and 5α-DHT action in prostate cancer cells through the
inhibition of the key enzymes involved in their synthesis is an essential approach to decrease the
survival and progression of PCa tumors. In this ont xt, 17β-hydroxysteroid dehydrogenase type
3 (17β-HSD3) is a key steroidogenic macromolecule involved in the synthesis of androgens (Fig.
1).7-10 This enzyme is well-known to catalyze the reduction of 4-androstene-3,17-dione (4-dione)
to potent androgen T using NADPH as a cofactor,11 but it could be also involved in the reduction
of oth r st roids (5α-androstane-3,17-dione, androsterone (ADT) and dehydroepiandrosterone),
thus contributing to alt rnativ pathways for th synth sis of 5α-DHT.12-15 Altho gh 17β-HSD3
is expressed almost exclusively in testis,16 there have been some reports of its up-regulation in
prostate tumors. For example, Pfeiffer et al have reported that in clinical Castrate-Resistant
Prostate Cancer (CRPC) samples, the xpr ssion of 17β-HSD3 was significantly increased, even
in long-term hormone depletion.17 Th s, 17β-HSD3 is an interesting target for hormonal therapy,
and its inhibition should be an effective strategy for the treatment of androgen-dependent PCa.
In previous studies,18-20 our research group reported the synthesis and pharmacological
activity of RM-532-105, which is a 3β-substituted-androsterone derivative (Fig. 1). This steroid
was identified as a potent 17β-HSD3 inhibitor on transfected HEK-293[17β-HSD3] as well as in
3

LNCaP[17β-HSD3] cells.18, 21 Moreover, RM-532-105 has been shown to decrease the level of T
and 5α-DHT in plasma at 2 h in rats, after subcutaneous injection.20
Figure 1. Contri tion of 17β-hydroxyst roid d hydrog nas typ 3 (17β-HSD3) to the biosynthesis of
the androg ns t stost ron (T) and 5α-dihydrot stost ron (5α-DHT) from 4-androstene-3,17-dione (4-
dione). RM-532-105 is a 3β-androsterone derivativ that inhi its th st roidog ni nzym 17β-HSD3.
However, it is also known that spatial modifications of the steroidal backbone, such as
inversion of the A-ring or D-ring (Fig. 2), exert a substantial effect on its biological properties.22-
26
For example, changing the 18-methyl (CH3) gro p of stradiol (E2) from th β-fa to α-face
modifies the D-ring shape22 and this 13α-E2 (or 18-epi-E2) was reported to be significantly less
binding to th strog n r ptor alpha (ERα) than E2 with a r lativ inding affinity (RBA) of
1.2 and 100%, respectively.23 Moreover, this unnatural steroid did not exert a significant
proliferative effect on estrogen-sensitive MCF-7 cells (ER+) as well as on estrogenic activity in
mice.23,24 Similarly, the cis-trans A/B fused rings of 5β-DHT (or etiocholan-17β-ol-3-one)
exhibit 173-fold less RBA to AR than the trans-trans onformation of 5α-DHT.26
4

Figure 2. Conformational representations of 5α-DHT (potent androgen), 5β-DHT (weak androgen), 13β-
E2 (pot nt strog n) and 13α-E2 (weak estrogen).
Based on the facts reported above, we were interested to test 2 major modifications of the
androstane backbone of RM-532-105 (13α-CH3 vs 13β-CH3 and 5β-H vs 5α-H). Because 17β-
HSD3 is a membrane enzyme, its crystal structure has not yet been resolved and molecular
modelling studies have been limited to homology model constructions.27-29 Therefore, a classical
structure-activity-relationship (SAR) study remains an efficient strategy to explore the tolerance
of an enzyme for structural modification of an inhibitor backbone. Herein, we describe the
chemical synthesis and characterization of compounds 6a (13α-CH3) and 6b (5β-H). We also
address th impa t of th C13 and C5 st r o h mistry on 17β-HSD3 inhibitory activity, the
androgenic effect and metabolic stability.
5

2. Material and methods
2.1. Chemical Synthesis
2.1.1. General information
Compounds 1a and 1c were prepared by the procedure described in reference,30 whereas
compound 1b was purchased from Steraloids (Wilton, NH, USA). The reagents for chemical
synthesis were purchased from Sigma–Aldrich Canada Ltd (Oakville, ON, Canada). The usual
solvents were obtained from Fisher Scientific (Montreal, QC, Canada) and were used as
received. Anhydrous tetrahydrofuran (THF), dichloromethane (DCM), dimethylsulfoxyde
(DMSO) and toluene 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 with a Horizon MB 3000 ABB FTIR spectrometer (Quebec, QC, Canada), and only the
significant bands reported in cm-1. Samples were prepared as KBr pellets. Nuclear magnetic
1
resonance (NMR) spectra were recorded at 400 MHz for H and 100.6 MHz for 13C with a
Bruker Avance 400 digital spectrometer (Billerica, MA, USA). The chemical shifts (δ) are
1
13
1
expressed in ppm and referenced to chloroform (7.26 ppm, H and 77.0 ppm, C). H NMR
signals were reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and broad
(br). Low-resolution mass spectra (LRMS) were recorded on a Perkin-Elmer Sciex API-150ex
apparatus (Foster City, CA, USA) equipped with a turbo ion-spray source and expressed in m/z.
High-resolution mass spectra (HRMS) were provided by Pierre Audet at the Laval University
Chemistry Department (Quebec, QC, Canada). High-performance liquid chromatography
(HPLC) analyses were carried out using a Waters system (Milford, MA, USA) equipped with a
UV detector (207 nm), a reverse-phase column (Luna C18(2) 100A, 100 X 4.6 mm, 3 μm) from
Phenomenex (Torrance, CA, USA) and using an appropriate system of solvents (methanol and
water). HPLC purity was determined for the final compounds. The names of new compounds
w r o tain d sing ACD/La s (Ch mist’s v rsion) softwar (Toronto, ON, Canada). The
numbering reported in Figure 3 was used for the assignment of 1H and 13C NMR signals.
6

Figure 3. Carbon numbering used for the assignment of 1H NMR and 13C NMR signals.
2.1.2. Synthesis of 2a and 2b
To a solution of (3β,5α,13α)-3-hydroxyandrostan-17-one (1a, 100 mg, 0.34 mmol) or
(3β,5β)-3-hydroxyandrostan-17-one (1b, 150 mg, 0.52 mmol) in anhydrous toluene (5 mL) was
added ethylene glycol (10.0 eq) and p-toluenesulfonic acid (0.1 eq). The reaction was refluxed
under a Dean-Stark trap for 12 h. The solution was poured into cold water (100 mL) and
extracted with EtOAc (3 X 20 mL). The organic phase was washed with a 20% solution of
sodium acetate (3 X 20 mL) and brine (2 X 20 mL), dried over Na2SO4 and filtered. The
resulting solution was evaporated and purified by flash chromatography column using
hexanes/EtOAc (95:5 to 80:20) to give 2a (90 mg, 79%) or 2b (130 mg, 74%), respectively.
(3β,5α,13α)-17-(1,3-dioxolan-2-yl)-3-hydroxyandrostane (2a). IR (KBr) νmax: 3348 (OH), 2924,
1
2854 (C-H, aliphatic). H NMR (400 MHz, CDCl3) δ: 0.62 (m. 1H), 0.74 (s, 19-CH3), 0.94 (s,
18-CH3), 0.60-1.95 (m, unassigned CH and CH2), 3.59 (m, 3α-H), 3.88 (m, OCH2CH2O). LRMS
for C21H35O3 [M+H]+: 335.25.
(3β,5β)-17-(1,3-dioxolan-2-yl)-3-hydroxyandrostane (2b). IR (KBr) νmax: 3286 (OH), 2932 (C-
1
H, aliphatic). H NMR (400 MHz, CDCl3) δ: 0.83 (s, 19-CH3), 0.96 (s, 18-CH3), 1.00-1.99 (m,
unassigned CH and CH2), 3.88 (m, OCH2CH2O), 4.09 (m, 3α-H). LRMS for C21H35O3 [M+H]+:
335.15.
7

2.1.3. Synthesis of 3-keto derivatives 3a and 3b
To a solution of compound 2a (90 mg, 0.27 mmol) or 2b (130 mg, 0.39 mmol) in DCM
(5 mL) was added Dess-Martin periodinane (1.3 eq). The reaction was stirred at room
temperature for 1 h, the resulting white suspension was evaporated. The residue was diluted with
EtOAc (20 mL) and washed with a saturated solution of sodium bicarbonate (3 X 20 mL) and
brine (2 X 20 mL). The organic phase was dried with Na2SO4 and evaporated. The crude product
was purified by flash chromatography column, using hexanes/EtOAc (95:5 to 80:20) to give 3a
(40 mg, 44%) or 3b (110 mg, 84%), respectively.
(5α,13α)-17-(1,3-dioxolan-2-yl)-androstan-3-one (3a). IR (KBr) νmax: 2924, 2870 (C-H,
aliphatic), 1705 (C=O). 1H NMR (400 MHz, CDCl3) δ: 0.94 (s, 19-CH3), 0.95 (s, 18-CH3), 0.65-
2.42 (m, unassigned CH and CH2), 3.89 (m, OCH2CH2O). LRMS for C21H33O3 [M+H]+: 333.15.
(5β)-17-(1,3-dioxolan-2-yl)-androstan-3-one (3b). IR (KBr) νmax: 2932, 2862 (C-H, aliphatic),
1
1713 (C=O). H NMR (400 MHz, CDCl3) δ: 0.87 (s, 19-CH3), 1.02 (s, 18-CH3), 1.05-2.19 (m,
unassigned CH and CH2), 2.33 (td, J1 = 14.6 Hz, J2 = 5.3 Hz, 2-CH), 2.68 (t, J = 14.6 Hz, 4-CH),
3.91 (m, OCH2CH2O). LRMS for C21H33O3 [M+H]+: 333.25.
2.1.4. Synthesis of the oxirane derivatives 4a and 4b
To a solution of trimethylsulfoxonium iodide (2.0 eq), in dry DMSO (3 mL) was
carefully added sodium hydride 60% in oil (2.0 eq). The solution was stirred at room temperature
under argon atmosphere for 1 h before adding 3a (40 mg, 0.12 mmol) or 3b (100 mg, 0.30
mmol) dissolved in THF (1 mL). The reaction mixture was stirred at room temperature for 5 h
and poured in ice/water (100 mL) and extracted with EtOAc. The combined organic layer was
washed with brine and dried with Na2SO4. The resulting solution was filtered, evaporated and the
crude product purified by flash chromatography column using hexanes/EtOAc (85:15) to give 4a
(30 mg, 72%) or 4b (82 mg, 79%), respectively.
8

(5α,13α)-17-(1,3-dioxolan-2-yl)-(3R)-oxiran-2-yl-androstane (4a). IR (KBr) νmax: 2939, 2862
1
(C-H, aliphatic). H NMR (400 MHz, CDCl3) δ: 0.77 (s, 19-CH3), 0.95 (s, 18-CH3), 0.75-2.08
(m, unassigned CH and CH2), 2.61 (s, 20-CH2), 3.88 (m, OCH2CH2O). LRMS for Chemical
Formula: C22H35O3 [M+H]+ = 347.20.
(5β)-17-(1,3-dioxolan-2-yl)-(3S)-oxiran-2-yl-androstane ( (4b). IR (KBr) νmax: 2932, 2862 (C-H,
1
aliphatic). H NMR (400 MHz, CDCl3) δ: 0.85 (s, 19-CH3), 0.99 (s, 18-CH3), 1.03-2.01 (m,
unassigned CH and CH2), 2.32 (t, J = 13.8 Hz, 4-CH), 2.62 (s, 20-CH2), 3.88 (m, OCH2CH2O).
LRMS for C22H35O6 [M+H]+: 347.25.
2.1.5. Synthesis of intermediates 5a and 5b
To a solution of 4a (28 mg, 0.08 mmol) or 4b (80 mg, 0.23 mmol) in anhydrous ethanol
(4 mL) was added trans-2,5-dimethylpiperazine (5.0 eq). The solution was stirred 12 h at 70 °C.
The resulting solution was poured in ice/water (50 mL). The white precipitate was separated by
vacuum filtration, and the solid was purified by flash chromatography column using
DCM/MeOH/TEA (96:2:2) to give 5a (20 mg, 54%) or 5b (52 mg, 50%), respectively.
(3α,5α,13α)-17-(1,3-dioxolan-2-yl)-3-[(trans-2,5-dimethylpiperazin-1-yl)methyl]-3-
1
hydroxyandrostane (5a). IR (KBr) νmax: 3425 (NH and OH), 2939 (C-H, aliphatic). H NMR
(400 MHz, CDCl3) δ: 0.67 (s, 19-CH3), 0.94 (s, 18-CH3), 0.98-1.01 (m, 2 X CH3CH) 1.10-2.08
(m, unassigned CH and CH2), 2.30 (m, 1H), 2.51-2.62 (m, 1H), 2.82-2.87 (m, 1H), 2.88-2.93 (m,
1H), 3.88 (m, OCH2CH2O). LRMS for C28H49N2O3 [M+H]+: 461.30.
(3β,5β)-17-(1,3-dioxolan-2-yl)-3-[(trans-2,5-dimethylpiperazin-1-yl)methyl]-3-
hydroxyandrostane (5b). IR (KBr) νmax: 3433, 3317 (NH and OH), 2932, 2862 (C-H, aliphatic).
9

1H NMR (400 MHz, CDCl3) δ: 0.84 (s, 19-CH3), 0.96 (s, 18-CH3), 0.98-1.01 (m, 2 X CH3CH)
1.02-2.10 (m, unassigned CH and CH2), 2.30 (m, 1H), 2.51-2.56 (m, 2 X CH3CH), 2.72 (m, 1H)
2.82-2.87 (m, 2H), 3.84-3.96 (m, OCH2CH2O). LRMS for C28H49N2O3 [M+H]+: 461.45.
2.1.6. Synthesis of sulfonamides 6a and 6b
To a solution of 5a (20 mg, 0.04 mmol) or 5b (44 mg, 0.09 mmol) in anhydrous DCM (3
mL) was added triethylamine (4.0 eq) and 2-(trifluoromethyl)benzenesulfonyl chloride (2.0 eq).
The reaction mixture was stirred at room temperature for 4 h, then evaporated, and the remaining
oily residue was diluted with acetone (5 mL) and concentrated hydrochloric acid (0.1 mL). The
reaction mixture was stirred into an ice bath for 30 min. The resulting solution was evaporated
and diluted with EtOAc (50 mL), washed with water, dried with Na2SO4 and evaporated. After
filtration, the residue was purified by flash chromatography column using hexanes/EtOAc (90:10
to 60:40) to give 6a (11 mg, 44% for 2 steps) or 6b (29 mg, 52% for 2 steps), respectively.
(3α,5α,13α)-3-[(trans-2,5-dimethyl-4-{[2-(trifluoromethyl)phenyl]sulfonyl}piperazin-1-
yl)methyl]-3-hydroxyandrostan-17-one (6a). IR (KBr) νmax: 3448 (OH), 2924, 2854 (C-H,
1
aliphatic), 1736 (C=O). H NMR (400 MHz, CDCl3) δ: 0.56 (s, 19β-CH3), 0.60-0.75 (m, 7-CH
and 9α-H), 0.85-0.90 (m, 8β-H), 0.86 and 0.88 (2d, J = 6.5 Hz, 25-CH3), 0.96 (s, 18α-CH3), 1.16
and 1.19 (2d, J = 5.4 Hz, 26-CH3), 1.15-1.45 (m, 1-CH, 2-CH, 15-CH, 6-CH and 12-CH), 1.45-
1.70 (m, 1-CH, 7-CH and 14α-H) 1.80 (m, 1H), 1.95-2.05 (m, 2H). 2.10-2.23 (m, 12-CH, 16-CH
and 20-CH), 2.32 (m, 16-CH, 20-CH and 24-CH), 2.80 (br, OH), 2.89 (m, 21-H), 3.08 ( br d, J =
10.2 Hz, 24-CH), 3.35 (m, 22-CH), 3.49 (dd, J1 = 12.9 Hz, J2 = 2.9 Hz, 22-CH), 4.05 (m, 23-
13
CH), 7.68 (m, 30-CH and 31-CH), 7.87 (m, 29-CH), 8.17 (m, 32-CH). C NMR (100.6 MHz,
CDCl3) δ: 8.6 (C25), 10.9 (C19), 15.7 (C26), 21.2 (C11), 22.4 (C15), 25.2 (C18), 28.6 (C6), 29.7
(C7), 32.2 (C12), 32.3 (C2), 33.6 (C1), 33.8 (C16), 35.9 (C10), 37.8 (C8), 39.4 (C4), 40.1 (C5),
46.0 (C22), 49.5 (C23), 50.1 (C13), 50.8 (C14), 51.4 (C9), 52.4 (C24), 54.7 (C21), 65.7 (C20),
70.9 (C3), 122.6 (q, JC-F = 274.4 Hz, C33), 127.5 (q, JC-C-F = 43.8 Hz, C28), 128.5 (q, JC-C-C-F
=
6.4 Hz, C29), 131.9 (C32), 132.1 (C31), 132.5 (C30), 139.3 (C27), 221.4 (C17). HRMS for
10

C33H48F3N2O4S [M+H]+: 625.3281 (calculated), 625.3271 (found). HPLC purity of 95.5%
(retention time = 17.6 min, 70:30 MeOH/H2O-isocratic, Luna C18 column).
(3β,5β)-3-[(trans-2,5-dimethyl-4-{[2-(trifluoromethyl)phenyl]sulfonyl}piperazin-1-
yl)methyl]-3-hydroxyandrostan-17-one (6b). IR (KBr) νmax: 3502 (OH), 2932, 2862 (C-H,
aliphatic), 1736 (C=O). 1H NMR (400 MHz, CDCl3) δ: 0.85 (s, 18β-CH3), 0.88 and 0.89 (2d, J =
6.5 Hz, 25-CH3), 0.98 (s, 19β-CH3), 1.05-1.15 (m, 4β-CH and 7-CH), 1.17 and 1.18 (2d, J = 6.7
Hz, 26-CH3), 1.20-1.37 (m, 2-CH2, 6α-CH, 9α-H, 11α-CH and 14α-H), 1.40-1.64 (m, 1-CH, 7-
CH, 8β-CH, 11β-CH, 12-CH and 15β-CH), 1.72-1.80 (5β-H and 12β-CH), 1.88-1.95 (m, 6β-CH
and 15α-CH), 2.04 (m, 16α-CH), 2.12 (d, J = 13.9 Hz, 20-CH), 2.30 (d, J = 14.1 Hz, 20-CH),
2.38 (m, 24-CH), 2.44 (dd, J1 = 19.1 Hz, J2 = 8.7 Hz, 16β-CH), 2.80 (br, OH), 2.89 (m, 21-CH),
3.08 (dd, J1 = 12.0 Hz, J2 = 3.7 Hz, 24-CH), 3.35 (m, 22-CH), 3.49 (dd, J1 = 12.9, J2 = 2.8 Hz,
13
22-CH), 4.05 (m, 23-CH), 7.68 (m, 30-CH and 31-CH), 7.87 (m, 29-CH), 8.17 (m, 32-CH). C
NMR (100.6 MHz, CDCl3) δ: 8.6 (C25), 13.8 (C18), 15.7 (C26), 20.3 (C11), 21.8 (C15), 23.5
(C19), 25.2 (C7), 26.4 (C6), 34.4 (C2), 31.6 (C1), 31.7 (C12), 34.9 (C10), 35.2 (C8), 35.9 (C16),
36.9 (C4), 38.0 (C5), 40.0 (C9), 46.0 (C22), 47.9 (C13), 49.5 (C23), 51.5 (C14), 52.3 (C24), 54.6
(C21), 65.7 (C20), 71.4 (C3), 122.6 (q, JC-F = 274.2 Hz, C33), 127.5 (q, JC-C-F = 33.1 Hz, C28),
128.5 (q, JC-C-C-F = 6.4 Hz, C29), 131.9 (C32), 132.1 (C31), 132.5 (C30), 221.4 (C17). HRMS for
C33H48F3N2O4S [M+H]+: 625.3281 (calculated), 625.3250 (found). HPLC purity of 99.6%
(retention time = 16.8 min, 70:30 MeOH/H2O-isocratic, Luna C18 column).
2.1.7. Synthesis oftrans-2,5-dimethyl-1-{[2-(trifluoromethyl)phenyl]sulfonyl}piperazine (7)
A solution of trans-2,5-dimethylpiperazine (3.0 g, 26.3 mmol) in DCM (20 mL) was
cooled to -5 °C in an ice-salt bath. The stirrer was started, and 2-
(trifluoromethyl)benzenesulfonyl chloride (2.1 mL, 13.6 mmol) was slowly added to the
solution. After the addition, the reaction was stirred for 1 h at room temperature. The resulting
solution was evaporated and purified by flash chromatography column using DCM/MeOH/TEA
11

(from 98:1:1 to 90:8:2) to give 2.9 g (66%) of compound 7 as a y llow solid. IR (KBr) νmax
:
1
3348 (NH), 3086 (CH aromatic), 2970, 2932, 2878 (CH aliphatic). H NMR (400 MHz, CDCl3)
δ: 1.06 (d, J = 6.6 Hz, 25-CH3), 1.07 (d, J = 6.7 Hz, 26-CH3), 1.51 (br, 1H), 2.58 (dd, J1 = 12.7
Hz, J2 = 5.6 Hz, 1H of 24-CH2), 2.96 (dd, J1 = 12.7 Hz, J2 = 5.5 Hz, 1H of 24-CH2), 3.04 (m, 21-
CH), 3.11 (dd, J1 = 12.7 Hz, J2 = 3.6 Hz, 1H of 22-CH2), 3.63-3.68 (m, 1H of 22-CH2 and 23-
CH), 7.68 (m, 30-CH and 31-CH), 7.87 (m, 29-CH), 8.17 (m, 32-CH). LRMS for
C13H18F3N2O2S [M+H]+: 323.10.
2.1.8. Synthesis of (trans-2,5)-1,2,5-trimethyl-1-{[2-(trifluoromethyl)phenyl]sulfonyl}
piperazine (8)
To a solution of compound 7 (150 mg, 0.46 mmol) in DCM and cooled into an ice bath
was add d diisopropylamin (0.1 mL). To this sol tion was add d m thyl iodid (84 μL, 1.3
mmol) and the reaction mixture was stirred at 0 °C for 3 h and then evaporated. The crude
product was purified by chromatography column using DCM/MeOH/TEA (from 99:0.5:0.5 to
92:7:1) to give 75 mg (48%) of 8 as a whit solid. IR (KBr) νmax: 3086 (CH aromatic), 2978,
1
2932, 2854 (CH aliphatic), 1165 (CF3). H NMR (400 MHz, CDCl3) δ: 0.87 (d, J = 2.6 Hz, 25-
CH3), 1.11 (d, J = 2.6 Hz, 26-CH3), 2.17 (dd, J1 = 12.7 Hz, J2 = 5.6 Hz, 1H of 24-CH2), 2.25 (s,
3H, N-CH3), 2.66 (m, 21-CH), 2.72 (dd, J1 = 12.7 Hz, J2 = 5.6 Hz, 1H of 24-CH2), 3.15 (dd, J1 =
12.7 Hz, J2 = 3.6 Hz, 1H of 22-CH2), 3.59 (dd, J1 = 12.8 Hz, J2 = 3.1 Hz, 1H of 22-CH2), 3.88
(m, 23-CH), 7.68 (m, 30-CH and 31-CH), 7.87 (m, 29-CH), 8.17 (m, 32-CH). 13C NMR (100.6
MHz, CDCl3) δ: 9.3 (C25), 16.3 (C26), 42.5 (C20), 48.4 (C22), 50.9 (C23), 54.6 (C21), 55.1
(C24), 122.6 (q, JC-F = 274.4 Hz, C33), 126.7 (q, JC-C-F = 43.8 Hz, C28), 128.5 (q, JC-C-C-F = 6.4
Hz, C29), 131.9 (C32), 132.1 (C29), 132.5 (C30), 140.1 (C27). HRMS for C14H20F3N2O2S
[M+H]+: 337.1192 (calculated), 337.1189 (found). HPLC purity of 99.9% (retention time = 18.6
min, 40:60 MeOH/H2O-isocratic with 0.1% of formic acid, Luna C18 column).
2.2. Inhibition of 17β-HSD3 (intact LNCaP cells overexpressing 17β-HSD3)
12

LNCaP transf t d lls (LNCaP[17β-HSD3]) kindly provided by IPSEN
INNOVATION (France) were maintained at 37 °C under 5% CO2 humidified atmosphere. Cells
were grown in RPMI-1640 medium supplemented (v/v) with 10% fetal bovine serum (FBS), 1%
penicillin/streptomycin, 2 mM L-glutamine, 4.5 g/L D-glucose, 10 mM Hepes, 1 mM sodium
pyruvate and 250 µg/mL hygromycin. For enzymatic assays, the protocol medium had the same
composition, but hygromycin, used to maintain the clone selection, was not included.
LNCaP[17β-HSD3] cells were plated in a 24-well culture at 10,000 cells per well, in protocol
medium. After 2 days of incubation, 15 nM of [14C]-4-androstene-3,17-dion and 10 μL of a
solution of inhibitor dissolved in DMSO and culture medium were added. The final DMSO
concentration in each well was adjusted to 0.05%. After 1 h of incubation, the culture medium
was removed from wells and steroids (4-androstene-3,17-dione and testosterone) were extracted
with diethyl ether. After evaporating the organic phase to dryness with nitrogen stream, residue
was dissolved in DCM, dropped on silica gel 60 F254 thin layer chromatography plates (EMD
Chemicals Inc, Gibbstown, NJ, USA), and eluted with a mixture of toluene/acetone (4:1). [14C]-
4-androstene-3,17-dione and [14C]-testosterone were identified by comparison with reference
steroids and quantified using the Storm 860 System (Molecular Dynamics, Sunnyvale, CA,
USA). Percentages of transformation and inhibition were then calculated.
2.3. Proliferative activities on LAPC-4 (AR+) cells
Androgen-sensitive human prostate cancer LAPC-4 cells were kindly provided by Robert
E. Reiter from the University of California (Los Angeles, CA, USA). Cells were grown at 37 °C
under 5% CO2 humidified atmosphere in RPMI-1640 medium supplemented (v/v) with 10%
fetal bovine serum (FBS), 1% L-glutamine, 1% insulin and 1% penicillin/streptomycin. To
determine the effect of novel compounds on cell proliferation, LAPC-4 cells were suspended
with the medium supplemented with 5% dextran-coated charcoal treated FBS rather than 10%
FBS, to r mov th r maining hormon s. Tripli at lt r s of 10,000 lls in a total of 100 μL
medium in 96-well microtiter plates (Becton–Dickinson Company, Lincoln Park, NJ, USA) were
pre-incubated for 24 h at 37 °C under 5% CO2 humidified atmosphere. Tested compounds were
dissolved in DMSO to prepare the stock solution of 10−2 M. The compounds were then diluted at
several concentrations with culture medium, added to corresponding wells, and incubated for 3
13

days. Control wells were treated with vehicle DMSO. MTS method was used for the
quantification of cell growth, using CellTitter 96® AQueous Solution Cell Proliferation Assay
(Prom ga, N p an, ON, Canada) and following th man fa t r r’s instr tions. Th prolif rativ
(androgenic) activity was expressed as difference between the cell proliferation (in %) caused by
a given compound and the basal cell proliferation fixed at 100%.
2.4. Metabolic stability assays
Assays were performed for 1 h at 37 °C, with or without 10 mM NADPH, in the presence
of 40 µg of human liver microsomes from Corning (Melrose, MA, USA) and 10 µM of substrate
in a final 100 µL volume of 50 mM Tris buffer supplemented with 10 mM MgCl2. Assays were
ended by adding 100 µL of MeOH, centrifuged at 13,000 g for 10 min to obtain a pellet of
proteins. The supernatant of 2 assays were pooled, filtered and 100 µL submitted to HPLC-MS
analysis (Shimadzu LCMS-2020 APCI, Altima HP C18 (250 mm x 4.6 mm, 5 µm) column,
MeOH:water gradient). The solvent gradient started with a mixture of MeOH:water (70:30,
50:50, 30:70 or 20:80 according to the compound) and finished with MeOH (100%). The
wavelength of the UV detector was selected at 190 nm. Remaining compound (expressed in %)
was calculated by dividing the area under the curve of the substrate for the assays with NADPH
by the one without NADPH, and multiplied by 100. Values represent the average of 4
independent experiments.
3. Results and discussion
3.1. Chemistry
The chemical synthesis of the first RM-532-105 isomer, compound 6a, is disclosed in
Scheme 1. This compound, with an 18-methyl (CH3) gro p in α-steroid face (18- pi or 13α-RM-
532-105, 6a) was prepared from 1a, which was previously obtained from commercially available
14

piandrost ron . Th s, th pim rization of th 13β-CH3 group of epiandrosterone to give the
13α-isomer 1a (13α-CH3) was carried out following the method described by Yaremenko and
Khvat,30 which uses 1,2-phenylenediamine in boiling acetic acid. Treatment of 1a with ethylene
glycol and p-toluenesulfonic (p-TSA) acid in refluxing toluene yielded the protected C17 ketone
as dioxolane 2a. The secondary alcohol at C3 was oxidized in mild conditions by using Dess-
Martin periodinane to give 3a. This steroidal ketone was efficiently reacted under the Corey-
Chaykovsky epoxidation conditions,31 using trimethylsulfoxonium iodide and sodium hydride, to
obtain 4a. Under these conditions, it is known that a 5α-H-androstane-3-ketone give an oxirane
with the R-configuration as the major compound.18, 32-36 The treatment of 4a with trans-2,5-
dimethylpiperazine in refluxing ethanol opened the oxirane group and provided the tertiary
alcohol 5a with the R-configuration at C3, which cannot be modified during the last two steps of
the synthesis. Finally, the free NH of 5a was submitted to a reaction with 2-
(trifluoromethyl)benzenesulfonyl chloride and trimethylamine (TEA) and the dioxolane
protecting group was subsequently removed in acidic conditions to yield the desired product 6a
in an overall yield of 6% (6 steps from 1a).
Scheme 1. Reagents and conditions: a) Ethylene glycol, p-TSA, toluene, reflux, 12 h; b) Dess-Martin
reagent, DCM, rt, 1 h; c) (CH3)3SOI, NaH, DMSO, THF, rt, 5 h; d) trans-2,5-dimethylpiperazine, ethanol,
70 °C, 12 h; e) 1. 2-(trifluoromethyl)benzenesulfonyl chloride, TEA, DCM, rt, 4 h, 2. HCl 36%, acetone,
0 °C, 30 min.
15

The chemical synthesis of the RM-532-105 isomer with the cis-trans conformation to the
A/B f s d rings (5β-RM-532-105, 6b) is dis los d in S h m 2. Starting from 5β-epi-
androsterone (1b), the synthetic pathway uses the same sequence of reactions reported for 6a: 1)
protection of C17 ketone as dioxolane 2b, 2) oxidation of the secondary alcohol at C3 to the
ketone 3b, 3) epoxidation to obtain the oxirane 4b, 4) opening of the oxirane to provide 5b, 5)
formation of the sulfonamide and 6) hydrolysis of the dioxolane protecting group to yield the
desired compound 6b in an overall yield of 12% (6 steps from 1b). The only difference between
the sequences of reactions providing 6a and 6b (Schemes 1 and 2) is the configuration of the
oxirane generated at C3. In fact, contrary to the 3R-oxiran o tain d from th 5α-androstan-3-
one (compound 3a), a 3S-oxiran was o tain d from th 5β-androstan-3-one (compound 3b).
Aher et al37 unambiguously confirmed by X-ray analysis the S-oxirane configuration generated
from a 5β-steroidal 3-ketone. Consequently, the configuration at C3 is expected to be S for the
tertiary alcohol 4b. Since the last two steps toward 6b can not modified the stereochemistry at
C3, 5b and 6b will be of 3S-configuration. The C-3 stereochemistry of 6b will be however
addressed in an upcoming section.
16

Scheme 2. Reagents and conditions: a) Ethylene glycol, p-TSA, toluene, reflux, 12 h; b) Dess-Martin
reagent, DCM, rt, 1 h; c) (CH3)3SOI, NaH, DMSO, THF, rt, 5 h; d) trans-2,5-dimethylpiperazine, ethanol,
70 °C, 12 h; e) 1. 2-(trifluoromethyl)benzenesulfonyl chloride, TEA, DCM, rt, 4 h, 2. HCl 36%, acetone,
0 °C, 30 min.
Compound 8, whi h r pr s nts th sid hain introd d at position 3β of th st roid
scaffold of 6a and 6b, was prepared for the purposes of comparison (Scheme 3). Briefly, an
excess of trans-2,5-dimethylpiperazine was treated with 2-(trifluoromethyl) benzenesulfonyl
chloride to give 7, and the free NH was next reacted with methyl iodide to yield the desired
compound 8 in a moderate 34% yield for 2 steps.
Scheme 3. Reagents and conditions: a) DCM, 0 °C, 1 h; b) CH3I, DIPEA, 0 °C, 3 h.
The structures of final compounds 6a, 6b and 8 were confirmed based on their spectral
1
13
data (IR, H NMR, C NMR and MS) and their purity, determined by HPLC and found to be
˃95%. Additionally, 2D-NMR experiments (COSY, NOESY, HSQC and HMBC) were carried
out to obtain a full assignation for H and C atoms, and to find evidence for the conformational
g om try (13α-CH3 and 5β-H) of the2 steroid C3-derivatives (see supporting information). For
compound 6a, th h mi al shift data of 13α-CH3 group is in agreement with previous studies
for 13α-ADT.30 Compared to RM-532-105, this methyl group has shifted downfield from 0.85
ppm (13β-CH3) to 0.96 ppm (13α-CH3) in the 1H NMR spectra and from 13.8 (13β-CH3) to 25.2
ppm (13α-CH3) in the 13C NMR spectra. The chemical shift for C3, the new steroidogenic center,
is also the same (70.9 ppm) suggesting the same R-configuration for 6a and RM-532-105, as
expected.
For compound 6b, the configuration of the asymmetric C3 carbon was expected to be S
per the work of Aher et al,37 but it was addressed and confirmed by NMR analyses. Using the
17

HMBC experiment, we first identified the methyl, producing a correlation with the carbonyl-17
(18-CH3), and that producing no correlation (19-CH3). The 4 correlations observed with 19-CH3,
in combination with 13C NMR data fo nd in lit rat r for 5β-steroids,38 allowed the
identification of the following carbons: 1-CH2, 10-C, 9-CH and 5-CH. Using the HSQC
experiment, the 5-C signal observed at 38.0 ppm was linked to th 5β-H at 1.75 ppm. Th 5β-H
displays also a NOE correlation with 19-CH3 (13β-CH3) but not with the 20-CH2 located at C3
(Fig. 4A). How v r, oth 4α-H and 4β-H produced NOE correlations with 20-CH2, which is only
possible with the S onfig ration at C3 (3β-OH/3α-CH2) instead of the R configuration (Fig. 4B).
The C3-chemical shift (71.5 ppm) for 6b (3S and 5β-H) is also slightly different than the values
(both 70.9 ppm) obtained for 6a and RM-532-105 (both 3R and 5α-H).
Figure 4. Partial NOESY spectra (A) and 3D-representation (B) of compound 6b.
18

3.2. Biological activity
3.2.1. 17β-HSD3 inhibitory activity
Th inhi ition of 17β-HSD3 activity was determined using intact LNCaP cells
ov r xpr ssing 17β-HSD3, by measuring the amount of labeled T formed from natural labeled 4-
dione, and then calculating the percentage of inhibition (Fig. 5). The first general observation
from this screening study is that the steroid backbone alone (compounds 1c and 1b), as well as
the side chain alone (compound 8), did not contribute to the inhibitory activity. However, the
combination of these 2 structural elements on the same molecule (compounds 6a and 6b)
significantly reduced the catalytic a tivity of th nzym . Both th 13α-CH3-derivative 6a (Fig.
5A) and 5β-H-derivative 6b (Fig. 5B) inhibited the enzyme at the 3 concentrations tested (0.1,
0.5 and 1 µM). Thus, the presence of the side chain attached to the C3 position of the steroid
backbone is crucial to th inhi ition of 17β-HSD3 activity.
19

Figure 5. Impact of steroid scaffold (steroids 1c and 1b), 3β-side chain (compound 8) and both structural
elements (steroid derivatives 6a and 6b) on 17β-HSD3 inhibitory activity (transformation of 4-dione to T
in LNCaP lls ov r xpr ssing 17β-HSD3). A) Modifi ation at th C/D ring j n tion (13α-CH3) and B)
modifi ation at th A/B ring j n tion (5β-H).
The IC50 values were obtained for the newly designed compounds 6a and 6b, as well as
for the reference RM-532-105 (Fig. 6). All compounds showed a dose-dependent inhibition of
17β-HSD3 activity that afforded IC50 values of 1.09, 0.15 and 0.11 μM for 6a, 6b and RM-532-
105, respectively. Therefore, 6a (5α/13α-RM-532-105) is 10-fold less potent than the inhibitor
used as reference (RM-532-105, with a 5α-H and 13β-CH3), but interestingly 6b (5β/13β-RM-
532-105) is as almost as potent as the lead inhibitor. Based on these findings, changing the shape
of D ring greatly decreas s 17β-HSD3 inhibitory activity, whereas the A-ring inversion (cis-
trans instead trans-trans conformation) combined to the inversion of the OH at C3 didn’t exhibit
a substantial impact on the inhibitory effect. A possible explanation for this might be that the
ketone at C17 is a key function interacting with a hydrogen bond donor from an amino acid
r sid in th inding po k t of 17β-HSD3. Thus, changing the shape of D-ring disrupts this
interaction resulting in a lower inhibitory potency. On the other hand, the 2 modifications in A-
ring may be tolerated because this change does not affect the interactions performed by the side
chain at C3. In fact, this moiety has a high degree of conformational flexibility that can favor
interactions in the binding cavity of 17β-HSD3, especially in the hydrophobic pocket, which
20

seems able to accommodate large substituents and greatly contributed to the inhibitory potency
of 3β-substituted-ADT derivatives.39-41
Figure 6. Inhi ition of 17β-HSD3 activity by several concentrations of 6a (5α/13α-RM-532-105, IC50 =
1.09 µM), 6b (5β/13β-RM-532-105, IC50 = 0.15 µM) and 5α/13β-RM-532-105 (IC50 = 0.11 µM).
3.2.2. Proliferative effect on LAPC-4 cells (AR+)
An inhibitor of androgen biosynthesis should be devoid of androgenic activity for use in
prostate cancer therapy. To investigate whether the synthetized compounds display an
androgenic profile, we assessed compounds 6a and 6b on the androgen-sensitive prostate cancer
LAPC-4 (AR+) cells and measured the proliferative (androgenic) activity which was expressed as
the difference between the cell proliferation (in %) caused by a given compound and the basal
cell proliferation fixed at 100% (Fig. 7). The natural androgens DHT and RM-532-105 were also
tested as reference compounds. At 10 nM, the potent androgen DHT increased the cell
proliferation to 190%, thus clearly demonstrating the androgen-dependence of LAPC-4 cells,
which express the wild-type androgen receptor.42 With percentages of cell growth lower than
100% (96, 48 and 33% at 0.1, 0.5 and 1 μM, r sp tiv ly), RM-532-105 did not show an
androgenic effect. As expected, the isomers of RM-532-105 displayed no androgenic activity
with cell growth values of 76.3, 61.6 and 51.0% for 6a and 87.2%, 72.3% and 28.2 % for 6b at
0.1, 0.5 and 1 μM, r sp tiv ly. In fa t, th s 3 compounds reduced cell proliferation when
compared to the control, which suggests a potential cytotoxic activity at least at the 2 higher
21

concentrations. Compound 6b, th 5β-isomer of RM-532-105, is however less cytotoxic than the
other 2 compounds.
Figure 7. Androgenic profile for 6a (5α/13α-RM-532-105), 6b (5β/13β-RM-532-105) and 5α/13β-RM-
532-105 on androgen-sensitive LAPC-4 cells.
3.2.3. Metabolic stability
Another criterion for discriminating different potential drug candidates is to evaluate their
metabolic stability. Compounds 6a and 6b were thus treated for 1 h by a microsomal preparation
of human liver in the presence of NADPH as cofactor to measure the impact of the modifications
at C5 and C13. In this assay, a compound is transformed by phase-I reactions (oxidation,
reduction, hydrolysis) instead of phase-II reactions (glucuronidation, sulfatation, acetylation),
and the remaining compound is measured at the end of the incubation time, and expressed in %.
As observed in Figure 8, the inversion at C5 (compound 6b) did not influence metabolic
stability. However, with only a tertiary alcohol, the A-ring of RM-532-105, 6a and 6b is not
favorable to phase-I reactions. Interestingly, compound 6a is more stable than RM-532-105 and
6b. This result suggests a less important metabolism on D-ring positions 16 and 17 for 6a (13α-
CH3) than for 6b and RM-532-105 ( oth 13β-CH3). In fact, the reduction of the C17-carbonyl
into alcohol and the hydroxylation at position 16 are 2 reactions involved in the formation of
known metabolites of steroidal ketones.43 Thus, the production of D-ring metabolites by the
22

enzymes of the cytochrome P450 family found in the liver is probably reduced by the steric
hindrance involved in the inversion of 18-CH3.
Figure 8. Metabolic stability of 6a (5α/13α-RM-532-105), 6b (5β/13β-RM-532-105) and 5α/13β-RM-
532-105. The results are expressed as the % of the remaining quantity of the tested compound treated with
a microsomal preparation of human liver. The data represent the average of 4 experiments ± SD. *
p<0.05.
4. Conclusion
Two isom rs of 17β-HSD3 inhibitor RM-532-105 (compounds 6a and 6b) were prepared
and characterized to assess the impact of two structural modifications of the steroid scaffold on
biological activities. Th iologi al ass ssm nts on LNCaP lls ov r xpr ssing 17β-HSD3 and
androgen-sensitive prostate cancer LAPC-4 cells allowed the identification of 6b (5β-H; cis-
trans A/B f s d rings) as a 17β-HSD3 inhibitor without androgenic activity and a weak
ytotoxi a tivity. Th 17β-HSD3 inhibitory activity of compound 6a (13α-CH3; trans-cis C/D
fused rings) was however reduced, but its androgenic profile was not affected. Compared to RM-
532-105 (5α-H/13β-CH3), the liver microsomal stability of 6b (5β-H) was not affected, whereas
that of 6a (13α-CH3) was improved. This finding has important implications for developing new
g n rations of mor pot nt 17β-HSD3 inhibitors. Moreover, these results justify the exploration
23

of other structural modifications on the A-ring, since this change seems to be well-tolerated by
th 17β-HSD3 enzyme.
Acknowledgments
This work was supported by Prostate Cancer Canada. Francisco Cortés-Benítez would
like to thank the National Autonomous University of Mexico and the National Council for
Sciences and Technology (CONACyT), Mexico, for the awarded fellowship. The authors would
like to thank Dr. Robert E. Reiter (University of California, CA, USA) for providing LAPC-4
lls and IPSEN INNOVATION (Fran ) for providing LNCAP[17β-HSD3] cells. Our thanks
also to Ms. Marie-Claude Trottier for providing NMR and HPLC data as well as Ms. Micheline
Harvey for the careful reading of this manuscript.
Supplementary data
1
HPLC chromatogram, HRMS data, IR spectra, H NMR, 13C (APT), HSQC, HMBC,
COSY and NOESY spectra for compounds 6a, 6b and 8.
Supplementary data associated with this article can be found, in the online version, at
…………………….
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