Estradiol and estrone C-16 derivatives as inhibitors of type 1 17β-hydroxysteroid dehydrogenase: Blocking of ER+ breast cancer cell proliferation induced by estrone

Article abstract of DOI:10.1016/j.bmc.2007.11.007

Estrogens play an important role in the development of breast cancer. Inhibiting 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1)-the enzyme responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E₂)-would thus allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16β-benzyl-E₂ (1) as a lead compound for developing inhibitors of the transformation of estrone (E₁) into E₂, we modified the benzyl group of 1 to improve its inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line T-47D, which exerts a strong endogenous 17β-HSD1 activity. In this intact cell model, 16β-m-carbamoylbenzyl-E₂ (4m) emerged as a potent inhibitor of 17β-HSD1 with an IC₅₀ value of 44 nM for the transformation of [¹⁴C]-E₁ (60 nM) into [¹⁴C]-E₂ (24-h incubation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with 4m at a concentration of 0.5 μM induced some proliferation (38%) of T-47D estrogen-sensitive (ER⁺) breast cancer cells. Interestingly, when 4m (0.5 μM) was given with E₁ (0.1 nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E₁ after its reduction to E₂ by 17β-HSD1. Thus, in addition to generating useful structure-activity relationships for the development of 17β-HSD1 inhibitors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E₂ and consequently its proliferative effect in T-47D ER⁺ breast cancer cells.

Full text of DOI:10.1016/j.bmc.2007.11.007

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1849–1860
Estradiol and estrone C-16 derivatives as inhibitors of type 1
17b-hydroxysteroid dehydrogenase: Blocking of ER+ breast
cancer cell proliferation induced by estrone
Yannick Laplante, Christine Cadot, Michelle-Audrey Fournier and Donald Poirier*
Medicinal Chemistry Division, Oncology and Molecular Endocrinology Research Center, CHUQ-Pavillon CHUL and
Universite´ Laval, Que., Canada G1V 4G2
Received 31 August 2007; revised 30 October 2007; accepted 1 November 2007
Available online 5 November 2007
Abstract—Estrogens play an important role in the development of breast cancer. Inhibiting 17b-hydroxysteroid dehydrogenase type
1 (17b-HSD1)—the enzyme responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2)—would thus
allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16b-benzyl-E2 (1) as a lead com-
pound for developing inhibitors of the transformation of estrone (E1) into E2, we modified the benzyl group of 1 to improve its
inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing
the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to
be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line
T-47D, which exerts a strong endogenous 17b-HSD1 activity. In this intact cell model, 16b-m-carbamoylbenzyl-E2 (4m) emerged
as a potent inhibitor of 17b-HSD1 with an IC50 value of 44 nM for the transformation of [14C]-E1 (60 nM) into [14C]-E2 (24-h incu-
bation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with 4m at a concentration of
0.5 lM induced some proliferation (38%) of T-47D estrogen-sensitive (ER+) breast cancer cells. Interestingly, when 4m (0.5 lM)
was given with E1 (0.1 nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E1 after its reduction
to E2 by 17b-HSD1. Thus, in addition to generating useful structure–activity relationships for the development of 17b-HSD1 inhib-
itors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E2 and consequently its pro-
liferative effect in T-47D ER+ breast cancer cells.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
sible for the last enzymatic step in the synthesis of potent
estrogen E2, which is the reduction of estrone (E1)
(Fig. 1). Activation of the estrogen receptor (ER) by
It is now well recognized that estrogenic hormones are
important stimulators of most types of breast cancers.1,2
Estrogens are synthesized from circulating inactive ste-
roids by enzymes such as human 17b-hydroxysteroid
dehydrogenases (17b-HSDs).3 These regulate the level
of active steroids by catalyzing the interconversion of
oxidized (17-ketone) and reduced (17b-hydroxy)
forms.4–6 There are 14 known isoforms in this family;
each has a specific tissue distribution and displays selec-
tive substrate affinity.7–10 Type 1 (17b-HSD1) is respon-
Keywords: 17b-hydroxysteroid dehydrogenase; Enzyme; Inhibitor; St-
eroid; Cancer; Estrogen; T-47D cells; MCF-7 cells; Medicinal
chemistry.
*
Corresponding author. Tel.: +1 418 654 2296; fax: +1 418 654
2761; e-mail: donald.poirier@crchul.ulaval.ca
These authors contributed equally to the work.
Figure 1. Role of 17b-HSD1 in the synthesis of E2.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.007

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Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
binding of E2 is crucial to the growth and development
of many mammary tumors.11 There are literature re-
ports indicating that 17b-HSD1 is expressed in a large
proportion of breast cancer tissues (61–100%),12–15
whereas others indicate the opposite;16,17 a high 17b-
HSD1 expression is nonetheless associated with a poor
prognosis.16 Inhibiting 17b-HSD1 activity could thus
constitute a valuable way of reducing E2 level,18–20 with
the aim of shrinking breast tumors. Indeed, it was re-
cently reported that, in immunodeficient mice inoculated
with MCF-7 breast cancer cells stably expressing 17b-
HSD1, an inhibitor of 17b-HSD1 was able to reduce tu-
mor size by 86% after 28 days.21,22 Such inhibition of
17b-HSD1 activity could notably complement the use
of an antiestrogen in the treatment of breast cancer.
used previously for the synthesis of 1.23 First, an aldol
condensation using meta-amido or para-amidobenzalde-
hyde29 afforded 2m and 2p, respectively. Second, the car-
bonyl of 2m and 2p was stereoselectively reduced into
the 17b-OH derivatives 3m and 3p. Third, the double
bond at C16 was stereoselectively reduced by a catalytic
hydrogenation to give the saturated E2 derivatives 4m
and 4p, respectively, bearing a 16b-oriented 3-car-
bamoylbenzyl and 4-carbamoylbenzyl group. To investi-
gate the E1 scaffold as well, alcohols 4m and 4p were
oxidized with Jones’ reagent to give the ketones 5m
and 5p. We alternatively tried performing a catalytic
hydrogenation with palladium on carbon to transform
in one step the a,b-unsaturated ketones 2m and 2p di-
rectly into 5m and 5p, but a mixture of 16a- and 16b-ori-
ented side chain as well as C17 alcohol and ketone was
obtained. The 3-OH group of 4m and 4p was also meth-
ylated using cesium carbonate and methyl iodide to ob-
tain the methoxy derivatives 6m and 6p. The latter
compounds were then oxidized at C17 with Jones’ re-
agent to afford 7m and 7p. The C16b/C17b-stereochem-
istry of final alcohols 4m, 4p, 6m, and 6p was easily
determined using the characteristic 1H NMR
(ꢀ3.8 ppm) and 13C NMR (ꢀ83 ppm) signals for posi-
tion 17. These two signals are clearly characteristic of
a C16b/C17b-stereochemistry as previously published
for 16b-allyl-E2.30 The C16b-stereochemistry of final ke-
tones 5m, 5p, 7m, and 7p was confirmed by 2D NMR
experiments showing nuclear Overhauser effects between
16a-CH and three characteristic protons (14a-CH, 15a-
CH, and one proton of CH2Ph).
Our group has previously reported the synthesis of a
number of E2 derivatives modified at position 16
(C16).23–28 In one of these studies, compound 1
(Fig. 2) was identified as a good inhibitor of 17b-
HSD1 with an IC50 value of 0.79–1.0 lM using purified
enzyme.23 This study also suggested that the 16b-phenyl
group interacts with residues Leu96 and Val196 located
near the nicotinamide residue of the NADPH cofactor.
Based on these results, we introduced different modifica-
tions at C3, C16, and C17 of compound 1 (Fig. 2; com-
pounds 4m,p–7m,p) in an attempt to modulate
interaction with important amino acids belonging to
the catalytic site. Another desirable characteristic of an
inhibitor of 17b-HSD1 is that it must be devoid of estro-
genic activity. Thus, with the aim of reducing such activ-
ity, we modified the E2 nucleus of inhibitor 4m using
three strategies (Fig. 2; compounds 17–19): (1) replacing
the hydroxyl group by a hydrogen at C3; (2) adding a
methoxy group at C2; and (3) adding an alkylamide side
chain at C7a. We now report the chemical synthesis,
inhibitory potency on 17b-HSD1, and proliferative
activity on ER+ breast cancer cell lines of a series of
new C16 derivatives of E2 and E1.
As a next step, in order to reduce the estrogenic effects
associated with the E2 nucleus of 4m (See Section 2.3),
we tested three strategies, resulting in the synthesis of
alcohols 17–19 (Scheme 2). In the first strategy, we re-
moved the hydroxyl group at C3. The phenol of E1
was thus converted into a triflate derivative, which was
used for the palladium-mediated reduction giving 3-
deoxy-E1 (8).31 The latter was submitted to the sequence
of three reactions described above (aldolization with
meta-amidobenzaldehyde, reduction of C-17 ketone,
and catalytic hydrogenation), to afford 11, 14, and 17,
respectively, with a 33% yield for three steps. In the sec-
ond strategy, a methoxy group was added at C2. The
starting 3-O-benzyl-2-methoxy-E1 (9) was synthesized
as reported in the literature.32 Therefore the aldolization
2. Results and discussion
2.1. Chemical synthesis
Compounds 4m and 4p were easily obtained from E1
(Scheme 1) following the sequence of three reactions
Figure 2. Inhibitors of 17b-HSD1. The stereogenic centers are illustrated only for steroid 1, but they are the same for all other steroid derivatives
reported in this paper. Partial numbering of carbons is represented on steroid 1.

Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
1851
Scheme 1. Chemical synthesis of the first series of inhibitors, 4m,p–7m,p. Reagents and conditions: (a) meta- or para-amidobenzaldehyde, KOH 10%,
EtOH, 110 ꢁC, 0.5 h; (b) NaBH4, CH2Cl2, MeOH, 0 ꢁC, 2–4 h; (c) H2, Pd/C 10%, EtOH, rt, 12 h; (d) Jones’ reagent (2.7 M), acetone, rt, 5 min; (e)
Cs2CO3, MeI, acetone, 70 ꢁC, 1 h.
Scheme 2. Chemical synthesis of the second series of inhibitors, 17–19. Reagents and conditions: (a) meta-amidobenzaldehyde, KOH 10%, EtOH,
110 ꢁC, 0.5 h; (b) NaBH4, CH2Cl2, MeOH, 0 ꢁC, 2–4 h; (c) H2, Pd/C 10%, EtOH, rt, 12 h.
of 9 with meta-amidobenzaldehyde and its reduction
with sodium borohydride was performed in the presence
of benzylether, which was removed during the catalytic
hydrogenation step. Then the 2-methoxy-E2 C-16 deriv-
ative 18 was obtained with a 57% yield for the three
steps. In the third strategy, the E2 derivative 19 having
two functionalized side chains—a 16b-(meta-car-
bamoylbenzyl) and a 7a-(N-methyl,N-butyl undecana-
mide)—was synthesized from the ketone 10,33 which
was previously obtained from 19-nortestosterone as

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Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
reported in the literature.34,35 The aldolization of 10
with meta-amidobenzaldehyde and a 10% KOH solu-
tion, the reduction of the 17-ketone, and the catalytic
hydrogenation afforded the desired functionalized prod-
uct 19 in good overall yield (58%). The C16b/C17b-ste-
reochemistry of alcohols 17–19 was established by NMR
spectroscopy as discussed above.
indicate that the carbamoylphenyl moiety is involved
in key interactions with amino acids within the enzyme
catalytic site. It is also known that a 17b-OH and a
17-ketone induce two different orientations of the phe-
nyl ring and consequently of the amide group. Prelimin-
ary results not presented herein also indicate that a
longer spacer between the 16b-steroidal carbon and
the phenyl ring—two or three CH2 groups instead of
one for 4m—reduces the inhibitory potency. Thus, the
17b-OH/meta-carbamoylbenzyl constitutes the best
arrangement for inhibition of 17b-HSD1. In our study,
we also tested the effect of a methoxy group at position
3 instead of the hydroxyl group: it considerably de-
creases the inhibitory potency, from 77% for compound
4m to 16% for compound 6m at 0.1 lM. This observa-
tion was also made for compounds with a 17b-OH or
a 17-ketone as well as meta- or para-amide positioning.
In fact, these results are in agreement with the known
role of phenolic OH group at C3 of E2, which is known
to interact with His221.38–40 For the most active inhibi-
tors 4m and 5m, we determined an IC50 value of 44 nM
and 171 nM (Table 1). These results were found to be
quite satisfactory considering the great improvement
of the inhibitory potency when compared to compound
1 (IC50 = 0.79–1.0 lM).23 Furthermore, we also deter-
mined that compound 4m did not inhibit at a concentra-
tion of 10 lM the oxidative transformation of E2
(60 nM) into E1 catalyzed by 17b-HSD2 overexpressed
in intact HEK-293 cells (data not shown). This is an
interesting characteristic of 4m because an inhibitor of
17b-HSD1 should preferably not inhibit the 17b-HSD2
activity that inactivates the potent estrogen E2.
2.2. 17b-HSD1 inhibition studies
The first series of compounds were tested for their abil-
ity to inhibit the transformation of labeled E1 (60 nM)
into E2 catalyzed by 17b-HSD1 in intact breast cancer
T-47D cells, which are known to express the enzyme.36
All inhibitors were tested at three concentrations (0.1,
1, and 10 lM) and results are reported in Table 1.
At a concentration of 10 lM, 4m, 5m, and 6m show
more than 90% of inhibition, 7m, 4p, and 5p show
around 75% of inhibition and 6p and 7p are weaker
inhibitors (38% and 56%, respectively). A better discrim-
ination between compounds was obtained at lower con-
centrations: 4m and 5m clearly constitute the best
inhibitors with 77% and 51% inhibition, respectively,
at 0.1 lM. Furthermore, only 4m and 5m are better
inhibitors than unlabeled E1 at all three concentrations.
These results also indicate that, at position 17, a b-hy-
droxyl group is better than a ketone for the inhibition
of 17b-HSD1 (see compounds 4m and 6m versus 5m
and 7m). This result is in contradiction with the known
affinity data of E1 (ketone) and E2 (17b-OH) for 17b-
HSD1, which clearly favor E1 (Km = 0.03 lM) over E2
(Km = 4.6 lM).37 However, the tendency observed with
4m–7m was reversed with the weaker inhibitors 4p–7p,
which have a CONH2 in para position on the 16b-aryl
group. The positioning of the amido (CONH2) group
has a drastic effect on enzyme inhibition since com-
pounds 4m–7m, with a meta-amido group, are clearly
better inhibitors than compounds 4p–7p bearing a
para-amido group. Taken together, the above results
A 17b-HSD1 inhibitor must be devoid of estrogenic
activity to be considered useful in the treatment of
breast cancer. Given its E2 nucleus, and based on our
previous work on E2 derivatives modified at C6, C16,
and C17,28,31,32 inhibitor 4m was expected to display
some estrogenic activity. Three strategies were then ap-
plied to reduce this unwanted activity. First, we removed
Table 1. Inhibitors of 17b-HSD1 (first series) and their inhibitory potency in T-47D intact cells (transformation of [14C]-E1 into [14C]-E2)
X
R2
R3
R1O
Compound
R1
R2
R3
X 17b-OH or O
Inhibitiona (%) 0.1 lM/1 lM/10 lM
IC50b (nM)
44
171 22
4m
5m
6m
7m
4p
H
CONH2
CONH2
CONH2
CONH2
H
H
OH
O
77/94/96
51/88/95
16/57/90
10/28/76
8/20/74
8/39/76
0/10/38
5/19/56
37/85/94
7
H
H
CH3
CH3
H
H
OH
O
—
—
H
CONH2
CONH2
CONH2
CONH2
—
OH
O
—
—
5p
6p
H
H
CH3
CH3
—
H
OH
O
—
—
7p
E1
H
—
—
218 20
a The experiment was performed in triplicate. SD < 5%. The inhibitors were tested at three concentrations of 0.1, 1, and 10 lM.
b Mean SD of an experiment performed in triplicate.

Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
1853
the hydroxyl group at position 3. Second, we introduced
a methoxy group at position 2. This strategy rises from
the use of 2-MeO-E2, Panzemꢂ, as a potential chemo-
therapeutic agent for treatment of cancer. Previous re-
ports demonstrated that 2-MeO-E2 can reduce growth
of many cancer cell lines.41–43 The last strategy was to
introduce a 7a-side chain known for its antiestrogenic
property.44 The chemical structure and inhibitory po-
tency of compounds 17–19 are reported in Table 2. Re-
sults indicate that none of these compounds is a more
efficient inhibitor of 17b-HSD1 activity than 4m in T-
47D cells. In fact, removing the hydroxyl group at posi-
tion 3 and introducing a pure antiestrogen side chain at
position 7a decreases significantly the inhibitory po-
tency, down to 24% and 4%, respectively, for 17 and
19 at 0.1 lM. Nevertheless, compound 18 bearing a
methoxy group at position 2 shows the best inhibition
in this second series of inhibitors (81%, 69%, and 37%
at 10, 1, and 0.1 lM, respectively). It however remains
less potent than 4m. The presence of a 2-MeO group
has also been reported to reduce the inhibitory activity
of a potent 17b-HSD1 inhibitor, an E2 derivative, by
10-fold.40
5m exert the most important proliferative activities
at 1 and 0.1 lM but none at 0.01 lM in both cell
lines. As expected, the E2-derivative 4m induces cell
proliferation somewhat more strongly than E1-deriva-
tive 5m. This result is in agreement with the stronger
binding affinity of E2 for ER.45,46 Compounds 17 and
18 exert a same pattern of proliferative activity at all
three concentrations and in both cell lines. In T-47D
cells, removing the hydroxyl at C3 (compound 17)
and introducing a methoxy at C2 (compound 18) sig-
nificantly reduce the proliferative activity of our lead
compound 4m by 47% and 41% at 1 lM, respectively,
whereas no proliferative activity was observed at 0.1
and 0.01 lM. In MCF-7 cells, compounds 17 and 18
exert 31% and 21% less estrogenic activity than com-
pound 4m at 0.1 lM. Moreover, compound 19, having
the characteristic side chain of pure antiestrogens EM-
13934 and ICI 164384,35 totally inhibits growth at
0.01 lM and even brings cell density under that of
the control level at 1 and 0.1 lM in T-47D cells. Even
better results were obtained with MCF-7 cells in
which compound 19 brought cell density under that
of the control at all three concentrations tested. This
compound acts more like an antiestrogen possessing
a weak inhibitory activity toward 17b-HSD1. Indeed,
among the compounds resulting from the three strate-
gies for reducing estrogenic activity, compound 19 ex-
erts the weakest inhibition for the transformation of
E1 into E2 (Table 2). Such results are in agreement
with our previous work using an alkylamide chain at
C7a and a short bromobutyl chain at C16a of E2,28
which indicated that 17b-HSD1 does not tolerate well
a long alkylamide side chain at C7a.
2.3. Proliferative activity on ER+ cell lines
To detect any undesirable estrogenic activity in the
new compounds, cell proliferative assays were carried
out on T-47D and MCF-7 cell lines which are known
to express the estrogen receptor (ER+). Proliferative
activities of compounds 4m, 5m, 17, 18, and 19 were
evaluated at 0.01, 0.1, and 1 lM (Fig. 3). From the
data collected, it is clear that compounds 4m and
Table 2. Inhibitors of 17b-HSD1 (second series) and their inhibitory potency in T-47D intact cells (transformation of [14C]-E1 into [14C]-E2)
Compound
Chemical structure
Inhibitiona (%) 0.1 lM/1 lM/10 lM
17
24/44/79
OH
O
NH2
18
37/69/81
OH
O
NH2
CH3O
HO
19
4/45/61
OH
O
NH2
HO
(CH2)10CONBuMe
a The experiment was performed in triplicate. SD < 5%. The inhibitors were tested at three concentrations of 0.1, 1, and 10 lM.

1854
Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
Figure 4. (A) Cell growth of T-47D cells induced by E1 (0.01 to 5 nM)
in the presence of inhibitor 4m (0 to 1 lM) after 8 days of incubation.
The cell proliferation without E1 (control) is fixed as 100%. (B) Growth
of T-47D cells induced by a physiologic concentration of E1 (0.1 nM)
in the presence or absence of inhibitor 4m (0.5 lM). Results are
expressed as means SEM of triplicate. *P 6 0.01 versus control and
versus E1 (0.1 nM).
Figure 3. Effects of selected inhibitors on the growth of estrogen-
starved T-47D and MCF-7 cells after 8 days of treatment. Control is
fixed as 100%. Results are expressed as means SEM of triplicate.
*P 6 0.05 versus control. **P 6 0.01 versus control.
2.4. Inhibition of E1-induced cell proliferation by inhibitor
4m
by 0.1 nM of E1. This concentration is close to the intra-
cellular concentration of E1 in breast cancer cells.47
Although 4m was successful in reducing the cell growth
from 220% to ꢀ150%, it is not possible to reduce the
proliferation at the basal level (100%) because of the
proliferative activity of 4m itself (150%). Given the fact
that inhibitor 4m stimulated by itself cell growth to
ꢀ150% over the basal level—the same level of cell pro-
liferation obtained with a combination of E1 and 4m—
the proliferative activity induced by E1 in T-47D ap-
pears indeed to be the result of the conversion of E1 into
E2 by 17b-HSD1 activity. Thus, although E1 binds the
estrogen receptor, the results shown in Figure 4B sug-
gest that E2 is the only steroid that promotes the ER+
cell growth observed when using E1.
Even though our inhibitors exert some estrogenic effects
when tested in the absence of E1, we investigated the
possibility that such compounds could block the prolif-
erative effect induced by E1 in breast cancer ER+ cell line
T-47D. We tested the ability of the most potent inhibitor
4m to inhibit the cell growth induced by the transforma-
tion of E1 (0.01–5 nM) into potent estrogen E2
(Fig. 4A). We did not select compounds 5m, 17, 18,
and 19 for this assay, even though they are less estro-
genic, because they are also less potent inhibitors of
the 17b-HSD1 activity.
Results indicated that inhibitor 4m is able to inhibit the
proliferative effect induced by the conversion of E1 into
E2 by 17b-HSD1 activity in a concentration-dependent
manner. The stronger cell growth inhibition, was, how-
ever, obtained at a concentration of 0.5 lM rather than
1 lM. At the latter concentration, the inhibitory effect of
4m is probably counterbalanced by its residual estrogen-
like proliferation effect on ER+ cells. In another experi-
ment (Fig. 4B), the optimal concentration (0.5 lM) of
inhibitor 4m reduced 62% of the cell growth induced
The reduction of E1-induced cell proliferation obtained
when using inhibitor 4m could also be the result of an
antiestrogenic activity of this E2 derivative. Indeed, an
antiestrogenic compound will block the proliferative
(estrogenic) effect of E2 mediated by their action on
the estrogen receptor. As illustrated in Figure 5, the en-
zyme inhibitor 4m does not reverse the proliferative ef-
fect on ER+ cells of E2 (0.1 nM) as the pure

Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
1855
appropriate for X-ray analysis, we are confident to re-
port soon the key interactions established between the
potent inhibitor and the enzyme residues.
4. Experimental
4.1. Chemistry
Chemical reagents were purchased from Aldrich Chem-
ical Co. (Milwaukee, WI, USA). Flash chromatography
was performed on Silicycle 60 230–400-mesh silica gel
´
´
(Quebec, Quebec, Canada). Thin-layer chromatography
(TLC) was performed on Whatman 0.25-mm silica gel
60 F254 plates (Fisher Scientific, Nepean, Ontario, Can-
ada) and compounds were visualized by exposure to UV
light (254 nm), a solution of ammonium molybdate/sul-
phuric acid/water, and/or a solution of para-anisalde-
hyde/sulphuric acid/acetic acid/ethanol (plus heating).
Infrared (IR) spectra were recorded on a Perkin-Elmer
1600 (Norwalk, CT, USA) and obtained from a thin film
of the solubilized compound on NaCl pellets (usually in
CH2Cl2) or in KBr pellets containing the solid com-
pound. Only significant bands are reported (in cmꢁ1).
1H and 13C NMR spectra were recorded, respectively,
at 300 (1H) and 75.5 (13C) MHz or at 400 (1H) and
100 (13C) MHz using a Bruker AC/F 300 or a Bruker
AVANCE 400 spectrometer (Billerica, MA, USA).
The chemical shifts (d) are expressed in ppm and refer-
enced to chloroform (7.26 and 77.0 ppm), methanol
(3.31 and 49.0 ppm), or acetone (2.05 and 29.8 ppm)
for 1H and 13C, respectively (Fig. 6). Low-resolution
mass spectra (LRMS) were recorded with an atmo-
spheric pressure chemical ionization (APCI) source on
positive mode. The purity of tested compounds 4m,p–
7m,p and 17–19 was determined by high-performance li-
quid chromatography (HPLC). The analyses were per-
formed using a Waters 600E pump, a Water 717 plus
Autosampler, and a Waters 996 Photodiode Array
Detector (Milford, MA, USA). A Waters Nova-Pack
Figure 5. Effect of inhibitor 4m and the pure antiestrogen EM-13934 on
the inhibition of E2 (0.1 nM)-induced proliferation (antiestrogenic
activity) of estrogen-sensitive human breast cancer MCF-7 cells. Two
days after plating, the cells were incubated for 8 days with the
indicated concentration of compounds. Media was changed every
second day. Results are expressed as means SEM of triplicate.
antiestrogen EM-139 does. This result suggests that 4m
does not work as an antiestrogenic compound, but acts
instead as an inhibitor of E1-into-E2 transformation
(inhibitor of 17b-HSD1).
3. Conclusion
In this report, we extended results of previous studies of
E1 and E2 derivatives at C16, aimed at developing 17b-
HSD1 inhibitors. Among the eleven inhibitors that were
synthesized, 4m was the most potent inhibitor with an
IC50 value of 44 nM for the transformation of E1 into
E2 in intact T-47D cells. A meta-carbamoylbenzyl
group, which probably mimics the nicotinamide side
chain of cofactor NADPH, appears to be an important
characteristic of this new 17b-HSD1 inhibitor. Interest-
ingly, the proliferative activity induced by a physiologic
concentration of E1 (0.1 nM) in T-47D ER+ cells was re-
duced by 62% by 4m. The cell growth reduction was not
100% because a weak (38%) estrogenic activity was in-
duced by 4m itself, an E2 derivative. Three strategies
were thus tested to decrease the undesirable estrogenic
activity. Although the resulting inhibitors 17–19 were
less estrogenic than our lead compound 4m, they exhib-
ited an important drop in inhibitory activity on 17b-
HSD1.
˚
C18 column (150 · 3.9 mm, 4 lm, 60 A) was used with
the following eluent system (1 mL/min flow rate): (A)
a gradient of 10% MeOH ca 20 mM of AcONH4 and
90% of H2O ca 20 mM of AcONH4 to 100% MeOH
ca 20 mM of AcONH4 (from 0 to 30 min); (B) 100%
MeOH ca 20 mM of AcONH4 (from 30 to 45 min);
and (C) 10% MeOH ca 20 mM of AcONH4 and 90%
of H2O ca 20 mM of AcONH4 (from 45 to 62 min).
4.1.1. General procedure for aldolization at the 16-
position of E1 template. To a stirred solution of E1, 8,31
9,32 or 1033 (0.5 mmol) in EtOH (5.6 mL) were added
meta- or para-amidobenzaldehyde29 (1 mmol) and a
By emerging as a potent inhibitor of 17b-HSD1, 4m
warrants further investigations. Since a crystallization
study has generated an enzyme/inhibitor complex
Figure 6. Carbon numbering used for the assignment of representative 1H NMR signals.

1856
Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
KOH aq solution (10% w/w, 1.13 mL). The reaction
mixture was stirred for 0.5 h at 130 ꢁC, then H2O
(2 mL) was added and EtOH evaporated under reduced
pressure. The aqueous phase was extracted with EtOAc
and the organic phase was dried over MgSO4 and evap-
orated under vacuum. Purification by flash chromatog-
raphy (hexanes/EtOAc) afforded products 2m, 2p, 11,
12, or 13.
(C@O, ketone), 1673 (C@O, amide); 1H NMR
(400 MHz, CDCl3) d 1.01 (s, 18-CH3), 1.20–2.45 (m,
unassigned CH and CH2), 2.60 and 2.96 (2m, 15-CH2),
2.80 (m, 6-CH2), 3.87 (s, OCH3), 5.11 (s, OCH2), 5.88
and 6.25 (2bs, NH2), 6.65 (s, 1-CH), 6.85 (s, 4-CH),
7.40 (m, CH of BnO, 10-CH and 500-CH), 7.70 (d,
J = 7.7 Hz, 600-CH), 7.77 (d, J = 7.7 Hz, 400-CH), 8.03
(s, 200-CH); 13C NMR (75 MHz, CDCl3) d 14.5, 26.1,
26.9, 28.9, 29.0, 31.6, 37.8, 44.3, 47.9, 48.4, 56.2, 71.0,
109.5, 114.5, 127.2 (2C), 127.6, 127.7, 128.5 (3C),
129.0, 129.1, 131.9, 132.1, 133.5, 133.8, 136.2, 137.3,
137.4, 146.4, 147.6, 168.8, 209.3; LRMS calcd for
C34H36NO4 [M+H]+ 522.1.
4.1.1.1. 3-(3-Hydroxy-17-oxo-estra-1,3,5(10)-trien-16-
ylidenemethyl)-benzamide (2m). Yellow amorphous solid
(80% yield); hexanes/EtOAc: 50/50 for chromatography.
IR (film) m 3304 (OH and NH2), 1706 (C@O, ketone),
1
1652 (C@O, amide); H NMR (400 MHz, MeOH-d4) d
1.00 (s, 18-CH3), 1.30–2.50 (m, unassigned CH and
CH2), 2.66 and 3.00 (2m, 15-CH2), 2.85 (m, 6-CH2),
6.50 (d, J = 2.4 Hz, 4-CH), 6.55 (dd, J2 = 8.4 Hz,
J1 = 2.5 Hz, 2-CH), 7.09 (d, J = 8.4 Hz, 1-CH), 7.44 (s,
10-CH), 7.55 (t, J = 7.7 Hz, 500-CH), 7.78 (d,
J = 7.8 Hz, 600-CH), 7.88 (d, J = 7.8 Hz, 400-CH), 8.11
(s, 200-CH); 13C NMR (100 MHz, MeOH-d4) d 15.0,
27.1, 27.9, 30.0, 30.5, 32.8, 39.5, 45.3, 49.2, 49.8, 113.8,
116.1, 127.1, 129.3, 130.0, 130.4, 132.0, 133.3, 134.5,
135.6, 137.2, 138.7, 138.8, 156.1, 171.7, 211.7; LRMS
calcd for C26H28NO3 [M+H]+ 402.2.
4.1.1.5. N-butyl-N-methyl-11-[16-(3-carbamoylbenzy-
lidene)-3-hydroxy-17-oxo-estra-1,3,5(10)-trien-7a-yl]-unde-
canamide (13). Yellow oil (80% yield); hexanes/EtOAc:
20/80–0/100 for chromatography. IR (film) m 3350 and
3194 (OH and NH2), 1713 (C@O, ketone), 1670 and
1624 (C@O, amides); 1H NMR (400 MHz, CDCl3) d
0.98 (s, 18-CH3), 0.92 and 0.95 (2t, J = 7.4 Hz, CH2CH3),
0.90–3.00 (m, unassigned CH and CH2), 2.94 and 2.98
(2s, NCH3), 3.26 and 3.38 (2t, J = 7.6 Hz, NCH2), 5.95
and 6.80 (2bs, NH2), 6.65 (d, J = 2.3 Hz, 4-CH), 6.68
(dd, J2 = 8.4 Hz, J1 = 2.4 Hz, 2-CH), 7.13 (d,
J = 8.5 Hz, 1-CH), 7.47 (s, 10-CH), 7.50 (d, J = 7.7 Hz,
500-CH), 7.69 (d, J = 7.7 Hz, 600-CH), 7.84 (d, J = 7.6 Hz,
400-CH), 8.07 (s, 200-CH); 13C NMR (75 MHz, CDCl3) d
13.8, 14.5, 19.9 (20.0), 25.0 (25.5), 25.1, 26.8, 27.1, 28.6,
28.8, 28.9, 29.0, 29.1, 29.3 (2C), 29.3 (30.5), 31.8, 32.9
(33.5), 33.6 (35.5), 34.5 (2C), 38.2, 41.2, 45.1, 47.7
(50.0), 48.0, 113.2, 116.2, 126.8, 127.8, 128.9, 129.4,
130.3, 132.0, 133.3 (2C), 136.1, 136.5, 137.2, 154.5,
169.0, 173.7, 209.5; LRMS calcd for C42H57N2O4
[MꢁH]+ 653.3.
4.1.1.2. 4-(3-Hydroxy-17-oxo-estra-1,3,5(10)-trien-16-
ylidenemethyl)-benzamide (2p). Yellow amorphous solid;
hexanes/EtOAc: 40/60–30/70 for chromatography. IR
(film) m 3353 (OH and NH2), 1715 (C@O, ketone), 1660
1
(C@O, amide); H NMR (400 MHz, MeOH-d4) d 1.02
(s, 18-CH3), 1.30–2.50 (m, unassigned CH and CH2),
2.67 and 3.00 (2m, 15-CH2), 2.86 (m, 6-CH2), 6.51 (d,
J = 2.4 Hz, 4-CH), 6.57 (dd, J2 = 8.4 Hz, J1 = 2.5 Hz, 2-
CH), 7.11 (d, J = 8.4 Hz, 1-CH), 7.46 (s, 10-CH), 7.71
(d, J = 8.3 Hz, 200-CH and 600-CH), 7.95 (d, J = 8.3 Hz,
300-CH and 500-CH); 13C NMR (75 MHz, MeOH-d4) d
15.0, 27.1, 27.9, 30.1, 30.5, 32.8, 39.5, 45.3, 49.0, 49.8,
113.8, 116.1, 127.2, 129.1 (2C), 131.4 (2C), 132.0, 133.0,
135.3, 138.7, 139.5, 140.1, 156.1, 171.6, 211.7; LRMS
calcd for C26H28NO3 [M+H]+ 402.2.
4.1.2. General procedure for the reduction of the 17-
ketone group on the E1 template. NaBH4 (0.85 mmol)
was added to a cooled (0 ꢁC) solution of 2m, 2p, 11,
12, or 13 (0.5 mmol) in MeOH (12.7 mL) and CH2Cl2
(2.5 mL). After the mixture was stirred for 3–4 h at
0 ꢁC, the reaction was quenched by addition of H2O
and the extraction was performed with CH2Cl2. The or-
ganic phase was dried over Na2SO4, and evaporated to
dryness. The crude product was purified by flash chro-
matography (hexanes/EtOAc) to afford 3m, 3p, 14, 15,
or 16.
4.1.1.3. 3-(17-Oxo-estra-1,3,5(10)-trien-16-ylidenem-
ethyl)-benzamide (11). Yellow amorphous solid (47%
yield); hexanes/EtOAc: 40/60–30/70 for chromatogra-
phy. IR (film) m 3348 and 3192 (NH2), 1710 (C@O, ke-
tone), 1670 (C@O, amide); 1H NMR (400 MHz,
CDCl3) d 1.02 (s, 18-CH3), 1.40–2.55 (m, unassigned
CH and CH2), 2.62 and 3.00 (2m, 15-CH2), 2.97 (m, 6-
CH2), 5.84 and 6.18 (2bs, NH2), 7.14 (m, 3H aryl),
7.32 (d, J = 6.5 Hz, 1H aryl), 7.49 (d, J = 3.5 Hz, 10-
CH), 7.52 (d, J = 7.7 Hz, 500-CH), 7.71 (d, J = 7.8 Hz,
600-CH), 7.77 (d, J = 7.7 Hz, 400-CH), 8.04 (s, 200-CH);
13C NMR (75 MHz, CDCl3) d 14.5, 25.7, 26.8, 29.1,
29.3, 31.7, 37.6, 44.5, 47.9, 48.6, 125.2, 125.8, 125.9,
127.5, 129.0, 129.1, 131.9, 133.5, 133.8, 136.3, 136.4,
137.4, 139.6, 168.8, 209.3; LRMS calcd for C26H28NO2
[M+H]+ 386.2.
4.1.2.1. 3-(3,17b-Dihydroxy-estra-1,3,5(10)-trien-16-
ylidenemethyl)-benzamide (3m). Yellow amorphous solid
(86% yield); hexanes/EtOAc: 30/70 for chromatography.
IR (film) m 3344 (OH and NH2), 1654 (C@O, amide); 1H
NMR (400 MHz, MeOH-d4) d 0.72 (s, 18-CH3), 1.20–
2.40 (m, unassigned CH and CH2), 2.78 (m, 6-CH2
and 1 H of 15-CH2), 4.06 (s, 17a-CH), 6.48 (d,
J = 2.4 Hz, 4-CH), 6.54 (m, 2-CH and 10-CH), 7.06 (d,
J = 8.5 Hz, 1-CH), 7.40 (t, J = 7.7 Hz, 500-CH), 7.55 (d,
J = 7.8 Hz, 600-CH), 7.66 (d, J = 7.7 Hz, 400-CH), 7.92
(s, 200-CH); 13C NMR (100 MHz, MeOH-d4) d 11.9,
27.6, 28.6, 30.7, 31.6, 37.6, 39.9, 44.4, 45.4, 48.9, 85.7,
113.8, 116.2, 123.3, 126.2, 127.2, 128.5, 129.6, 132.5,
132.6, 135.1, 138.8, 140.0, 148.5, 156.0, 172.6; LRMS
calcd for C26H30NO3 [M+H]+ 404.3.
4.1.1.4.
3-(3-Benzyloxy-2-methoxy-17-oxo-estra-
1,3,5(10)-trien-16-ylidenemethyl)-benzamide (12). Yellow
oil (93% yield); hexanes/EtOAc: 50/50–30/70 for chro-
matography. IR (film) m 3366 and 3163 (NH2), 1704

Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
1857
4.1.2.2. 4-(3,17b-Dihydroxy-estra-1,3,5(10)-trien-16-
ylidenemethyl)-benzamide (3p). Yellow amorphous solid;
hexanes/EtOAc: 35/65 for chromatography. IR (film) m
3352 and 3196 (OH and NH2), 1652 (C@O, amide);
1H NMR (400 MHz, acetone-d6) d 0.74 (s, 18-CH3),
1.30–2.40 (m, unassigned CH and CH2), 2.85 (m, 6-
CH2 and 1 H of 15-CH2), 4.14 (s, 17a-CH), 6.55 (d,
J = 2.5 Hz, 4-CH), 6.61 (m, 2-CH and 10-CH), 7.12 (d,
J = 8.4 Hz, 1-CH), 7.51 (d, J = 8.3 Hz, 200-CH and 600-
CH), 7.93 (d, J = 8.3 Hz, 300-CH and 500-CH); 13C
NMR (75 MHz, acetone-d6) d 11.7, 27.2, 28.2, 30.3,
31.4, 37.2, 39.4, 44.0, 45.0, 48.4, 85.2, 113.6, 115.9,
122.6, 127.0, 128.5 (2C), 128.7 (2C), 131.9, 132.5,
138.4, 142.2, 149.8, 155.9, 168.7; LRMS calcd for
C26H30NO3 [M+H]+ 404.1.
500-CH), 7.55 (d, J = 7.8 Hz, 600-CH), 7.64 (d,
J = 7.7 Hz, 400-CH), 7.87 (s, 200-CH); 13C NMR
(75 MHz, CDCl3) d 11.2, 13.8, 20.0, 25.0, 25.0 (25.5),
27.1, 27.3, 28.8, 29.0, 29.2 (2C), 29.3, 29.4, 29.4 (30.6),
30.2, 32.9 (33.3), 33.6 (35.5), 34.6 (2C), 36.5, 38.2,
41.4, 43.5, 44.0, 47.6 (49.9), 85.0, 113.1, 116.3, 122.4,
125.1, 126.8, 127.5, 128.6, 130.7, 131.4, 133.4, 136.7,
138.4, 147.7, 154.3, 169.7, 173.6; LRMS calcd for
C42H61N2O4 [M+H]+ 657.3.
4.1.3. General procedure for the hydrogenation of the
benzylidene double bond at the 16-position of E2 template.
A mixture of 3m, 3p, 14, 15, or 16 (0.2 mmol) and palla-
dium on charcoal (15% in weight) in anhydrous EtOH
(3.8 mL) under hydrogen atmosphere was stirred 12 h
at rt. Then the palladium reagent was removed by filtra-
tion on celite, washed with MeOH, and the filtrate was
concentrated under reduced pressure. Purification by
flash chromatography (hexanes/EtOAc) afforded 4m,
4p, 17, 18, or 19.
4.1.2.3. 3-(17b-Hydroxy-estra-1,3,5(10)-trien-16-ylide-
nemethyl)-benzamide (14). Yellow amorphous solid (95%
yield); hexanes/EtOAc: 40/60–30/70 for chromatogra-
phy. IR (film) m 3385 and 3180 (OH and NH2), 1670
1
(C@O, amide); H NMR (400 MHz, CDCl3) d 0.75 (s,
18-CH3), 1.30–2.50 (m, unassigned CH and CH2), 2.79
(dd, J2 = 16.8 Hz, J1 = 6.7 Hz, 1 H of 15-CH2), 2.92
(m, 6-CH2), 4.17 (s, 17a-CH), 5.98 and 6.19 (2bs,
NH2), 6.59 (d, J = 1.8 Hz, 10-CH), 7.14 (m, 3H aryl),
7.32 (d, J = 6.9 Hz, 1H aryl), 7.42 (t, J = 7.6 Hz, 500-
CH), 7.57 (d, J = 7.7 Hz, 600-CH), 7.61 (d, J = 7.5 Hz,
400-CH), 7.87 (s, 200-CH); 13C NMR (75 MHz, CDCl3)
d 11.1, 26.0, 27.4, 29.4, 30.6, 36.3, 37.9, 43.3, 44.5,
47.7, 84.9, 122.2, 124.9, 125.2, 125.6 (2C), 125.7, 127.3,
128.6, 129.1, 131.6, 136.5, 138.4, 140.0, 147.7, 169.7;
LRMS calcd for C26H30NO2 [M+H]+ 388.2.
4.1.3.1. 3-(3,17b-Dihydroxy-estra-1,3,5(10)-trien-16b-
ylmethyl)-benzamide (4m). White amorphous solid (98%
yield); hexanes/EtOAc: 20/80–0/100 for chromatogra-
phy. IR (film) m 3330 (OH and NH2), 1652 (C@O,
1
amide); H NMR (400 MHz, MeOH-d4) d 0.89 (s, 18-
CH3), 1.10–2.60 (m, unassigned CH and CH2), 2.72
(m, 6-CH2), 3.14 (d, J = 9.5 Hz, 16a-CH), 3.81 (d,
J = 9.3 Hz, 17a-CH), 6.45 (d, J = 2.5 Hz, 4-CH), 6.52
(dd, J2 = 8.4 Hz, J1 = 2.6 Hz, 2-CH), 7.07 (d,
J = 8.4 Hz, 1-CH), 7.37 (m, 500-CH and 600-CH), 7.67
(dt, J2 = 7.4 Hz, J1 = 1.5 Hz, 400-CH), 7.73 (s, 200-CH);
13C NMR (100 MHz, MeOH-d4) d 13.3, 27.5, 28.6,
30.6, 33.0, 38.8, 38.9, 39.8, 43.3, 45.3, 45.4, 49.8, 83.0,
113.7, 116.0, 125.9, 127.1, 129.1, 129.4, 132.6, 133.5,
134.7, 138.7, 144.3, 155.8, 172.7; LRMS calcd for
C26H32NO3 [M+H]+ 406.1. HPLC purity = 98.6%,
rt = 26.6 min, k = 218 nm.
4.1.2.4. 3-(3-Benzyloxy-17b-hydroxy-2-methoxy-estra-
1,3,5(10)-trien-16-ylidenemethyl)-benzamide (15). Yellow
oil (90% yield); hexanes/EtOAc: 40/60–30/70 for chro-
matography. IR (film) m 3348 and 3198 (OH and
NH2), 1667 (C@O, amide); 1H NMR (400 MHz,
CDCl3) d 0.75 (s, 18-CH3), 1.30–2.40 (m, unassigned
CH and CH2), 2.70 (m, 6-CH2 and 1 H of 15-CH2),
3.87 (s, OCH3), 4.16 (s, 17a-CH), 5.11 (s, OCH2Ph),
5.88 and 6.22 (2bs, NH2), 6.59 (d, J = 2.0 Hz, 10-CH),
6.64 (s, 1-CH), 6.86 (s, 4-CH), 7.36 (m, CH of OCH2Ph
and 500-CH), 7.56 (d, J = 7.8 Hz, 600-CH), 7.60 (d,
J = 7.7 Hz, 400-CH), 7.87 (s, 200-CH); 13C NMR
(75 MHz, CDCl3) d 11.1, 26.4, 27.5, 29.0, 30.5, 36.3,
38.1, 43.3, 44.3, 47.5, 56.3, 71.0, 84.9, 109.6, 114.5,
122.3, 124.9, 127.2 (3C), 127.7 (2C), 128.5 (2C), 128.6,
131.6, 132.5, 133.3, 137.3, 138.4, 146.4, 147.6, 147.7,
169.4; LRMS calcd for C34H38NO4 [M+H]+ 524.1.
4.1.3.2. 4-(3,17b-Dihydroxy-estra-1,3,5(10)-trien-16b-
ylmethyl)-benzamide (4p). White amorphous solid (75%
yield, 3 steps from E1); hexanes/EtOAc: 30/70 for chroma-
tography. IR (film) m 3353 (OH and NH2), 1663 (C@O,
1
amide); H NMR (400 MHz, MeOH-d4) d 0.88 (s, 18-
CH3), 1.10–2.55 (m, unassigned CH and CH2), 2.72 (m,
6-CH2), 3.14 (d, J = 9.5 Hz, 16a-CH), 3.80 (d,
J = 9.3 Hz, 17a-CH), 6.45 (d, J = 2.5 Hz, 4-CH), 6.53
(dd, J2 = 8.4 Hz, J1 = 2.6 Hz, 2-CH), 7.07 (d,
J = 8.4 Hz, 1-CH), 7.30 (d, J = 8.2 Hz, 200-CH and 600-
CH), 7.78 (d, J = 8.2 Hz, 300-CH and 500-CH); 13C NMR
(75 MHz, MeOH-d4) d 13.3, 27.5, 28.7, 30.7, 33.1, 38.9
(2C), 39.9, 43.3, 45.4 (2C), 49.9, 83.0, 113.7, 116.0,
127.2, 128.7 (2C), 130.0 (2C), 132.2, 132.6, 138.8, 148.5,
155.9, 172.5; LRMS calcd for C26H32NO3 [M+H]+
406.2. HPLC purity = 93.5%, rt = 27.2 min, k 209 nm.
4.1.2.5. N-butyl-N-methyl-11-[16-(3-carbamoylbenzy-
lidene)-3,17-dihydroxy-estra-1,3,5(10)-trien-7a-yl]-undecana-
mide (16). Yellow oil (75% yield); hexanes/EtOAc: 20/
80–0/100 for chromatography. IR (film) m 3360 (OH
1
and NH2), 1667 and 1621 (C@O, amides); H NMR
(400 MHz, CDCl3) d 0.74 (s, 18-CH3), 0.92 and 0.94
(2t, J = 7.5 Hz, CH2CH3), 0.90–3.00 (m, unassigned
CH and CH2), 2.93 and 2.97 (2s, NCH3), 3.26 and
3.37 (2m, NCH2), 4.16 (s, 17a-CH), 5.94 and 6.52
(2bs, NH2), 6.59 (d, J = 1.7 Hz, 10-CH), 6.61 (d,
J = 2.4 Hz, 4-CH), 6.68 (dd, J2 = 8.4 Hz, J1 = 2.5 Hz,
2-CH), 7.14 (d, J = 8.5 Hz, 1-CH), 7.41 (t, J = 7.7 Hz,
4.1.3.3.
3-(17b-Hydroxy-estra-1,3,5(10)-trien-16b-
ylmethyl)-benzamide (17). White amorphous solid (75%
yield); hexanes/EtOAc: 40/60–30/70 for chromatogra-
phy. IR (film) m 3346 and 3180 (OH and NH2), 1666
1
(C@O, amide); H NMR (400 MHz, CDCl3) d 0.88 (s,
18-CH3), 1.10–2.65 (m, unassigned CH and CH2), 2.84
(m, 6-CH2), 3.16 (dd, J2 = 12.9 Hz, J1 = 4.3 Hz, 16a-

1858
Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
CH), 3.87 (d, J = 9.5 Hz, 17a-CH), 5.90 and 6.24 (2bs,
NH2), 7.10 (m, 3H aryl), 7.30 (d, J = 7.1 Hz, 1H aryl),
7.38 (m, 500-CH and 600-CH), 7.61 (d, J = 6.6 Hz, 400-
CH), 7.72 (s, 200-CH); 13C NMR (100 MHz, CDCl3) d
12.6, 25.9, 27.3, 29.4, 32.1, 37.5, 37.6, 37.9, 41.7, 44.4,
44.5, 48.7, 82.1, 124.5, 125.3, 125.6 (2C), 127.9, 128.5,
129.0, 132.6, 133.2, 136.6, 140.2, 142.9, 169.7; LRMS
calcd for C26H32NO2 [M+H]+ 390.1. HPLC purity =
93.8%, rt = 34.4 min, k = 209 nm.
by chromatography (hexanes/EtOAc) to afford 5m, 5p,
7m, or 7p.
4.1.4.1.
3-(3-Hydroxy-17-oxo-estra-1,3,5(10)-trien-
16b-ylmethyl)-benzamide (5m). White amorphous solid
(49% yield); hexanes/EtOAc: 40/60 for chromatography.
IR (film) m 3454, 3328, 3288 and 3231 (OH and NH2),
1732 (C@O, ketone), 1658 (C@O, amide); 1H NMR
(300 MHz, MeOH-d4 + acetone-d6) d 0.73 (s, 18-CH3),
1.20–2.65 (m, unassigned CH and CH2), 2.78 (m, 6-
CH2 and 1 H of CH2-Aryl), 3.20 (dd, J2 = 13.6 Hz,
J1 = 4.2 Hz, 16a-CH), 6.47 (d, J = 2.4 Hz, 4-CH), 6.54
(dd, J2 = 8.4 Hz, J1 = 2.5 Hz, 2-CH), 7.07 (d,
J = 8.5 Hz, 1-CH), 7.41 (m, 500-CH and 600-CH), 7.75
(m, 400-CH and 200-CH); 13C NMR (75 MHz, MeOH-
d4) d 14.1, 27.1, 27.9, 28.7, 30.5, 33.1, 38.1, 39.4, 45.4,
50.0 (2C), 52.3, 113.8, 116.1, 126.7, 127.1, 129.3, 129.6,
132.0, 133.8, 135.1, 138.7, 141.8, 156.1, 172.4, 223.7;
LRMS calcd for C26H30NO3 [M+H]+ 404.2. HPLC pur-
ity = 93.8%, rt = 28.6 min, k = 208 nm.
4.1.3.4.
3-(3,17b-Dihydroxy-2-methoxy-estra-
1,3,5(10)-trien-16b-ylmethyl)-benzamide (18). Yellow
amorphous solid (68% yield); hexanes/EtOAc: 40/60–
30/70 for chromatography. IR (film) m 3352 and 3204
(OH and NH2), 1664 (C@O, amide); 1H NMR
(400 MHz, CDCl3) d 0.88 (s, 18-CH3), 1.10–2.60 (m,
unassigned CH and CH2), 2.73 (m, 6-CH2), 3.15 (dd,
J2 = 12.8 Hz, J1 = 4.3 Hz, 16a-CH), 3.85 (s, OCH3),
3.87 (d, J = 9.8 Hz, 17a-CH), 5.88 and 6.22 (2bs,
NH2), 6.63 (s, 1-CH), 6.78 (s, 4-CH), 7.38 (m, 500-CH
and 600-CH), 7.60 (d, J = 7.2 Hz, 400-CH), 7.71 (s, 200-
CH); 13C NMR (100 MHz, CDCl3) d 12.6, 26.5, 27.5,
28.9, 32.1, 37.5, 37.7, 38.2, 41.7, 44.2, 44.4, 48.6, 56.0,
82.1, 108.1, 114.6, 124.5, 128.0, 128.5, 129.5, 131.7,
132.6, 133.2, 142.9, 143.5, 144.6, 169.7; LRMS calcd
for C27H34NO4 [M+H]+ 436.1. HPLC purity = 97.5%,
rt = 27.1 min, k = 213 nm.
4.1.4.2.
4-(3-Hydroxy-17-oxo-estra-1,3,5(10)-trien-
16b-ylmethyl)-benzamide (5p). White amorphous solid
(50% yield); hexanes/EtOAc: 45/55 for chromatography.
IR (film) m 3421, 3350, 3184 (OH and NH2), 1729 (C@O,
ketone), 1660 (C@O, amide); 1H NMR (400 MHz,
MeOH-d4) d 0.70 (s, 18-CH3), 1.30–2.65 (m, unassigned
CH and CH2), 2.79 (m, 6-CH2 and 1 H of CH2-Aryl),
3.18 (dd, J2 = 13.5 Hz, J1 = 4.3 Hz, 16a-CH), 6.47 (d,
J = 2.5 Hz, 4-CH), 6.53 (dd, J2 = 8.4 Hz, J1 = 2.6 Hz,
2-CH), 7.06 (d, J = 8.4 Hz, 1-CH), 7.31 (d, J = 8.2 Hz,
200-CH and 600-CH), 7.80 (d, J = 8.2 Hz, 300-CH and 500-
CH); 13C NMR (75 MHz, MeOH-d4) d 14.1, 27.0,
27.9, 28.7, 30.5, 33.1, 37.9, 39.3, 45.4, 50.0 (2C), 52.2,
113.8, 116.1, 127.1, 128.8 (2C), 130.3 (2C), 132.0,
132.9, 138.7, 145.6, 156.1, 172.2, 223.5; LRMS calcd
for C26H30NO3 [M+H]+ 404.1. HPLC purity = 98.9%,
rt = 28.3 min, k = 209 nm.
4.1.3.5. N-butyl-N-methyl-11-[16b-(3-carbamoylben-
zyl)-3,17-dihydroxy-estra-1,3,5(10)-trien-7a-yl]-undecana-
mide (19). White amorphous solid (96% yield);
hexanes/EtOAc: 10/90–0/100 for chromatography. IR
(film)
m 3322 (OH and NH2), 1667 and 1618
(C@O, amides); 1H NMR (400 MHz, CDCl3)
d
0.87 (s, 18-CH3), 0.92 and 0.95 (2t, J = 7.4 Hz,
CH2CH3), 0.90–2.90 (m, unassigned CH and CH2),
2.93 and 2.98 (2s, NCH3), 3.16 (dd, J2 = 13.1 Hz,
J1 = 4.1 Hz, 16a-CH), 3.26 and 3.35 (2m, NCH2),
3.86 (d, J = 9.6 Hz, 17a-CH), 5.90 and 6.40 (2bs,
NH2), 6.59 (d, J = 2.2 Hz, 4-CH), 6.65 (dd,
J2 = 8.3 Hz, J1 = 2.2 Hz, 2-CH), 7.11 (d, J = 8.5 Hz,
1-CH), 7.38 (m, 500-CH and 600-CH), 7.63 (d,
J = 7.4 Hz, 400-CH), 7.73 (s, 200-CH); 13C NMR
(75 MHz, CDCl3) d 12.6, 13.8, 20.0, 24.9 (25.5),
25.1, 27.1, 27.3, 28.8, 28.9, 29.0, 29.1, 29.2, 29.3,
29.4 (30.6), 31.6, 33.0 (33.7), 33.4, 33.6 (35.5), 34.6,
37.6, 37.8, 38.2, 41.5, 41.7, 44.5, 44.9, 47.6 (49.9),
82.2, 113.0, 116.2, 124.6, 126.8, 128.1, 128.5, 131.0,
132.7, 133.0, 136.8, 143.0, 154.2, 169.8, 173.6; LRMS
calcd for C42H63N2O4 [M+H]+ 659.5. HPLC pur-
ity = 95.7%, rt = 35.2 min, k = 220 nm.
4.1.4.3.
3-(3-Methoxy-17-oxo-estra-1,3,5(10)-trien-
16b-ylmethyl)-benzamide (7m). White amorphous solid
(69% yield); hexanes/EtOAc: 40/60 for chromatography.
IR (film) m 3339 and 3191 (NH2), 1734 (C@O, ketone),
1
1660 (C@O, amide); H NMR (400 MHz, MeOH-d4) d
0.70 (s, 18-CH3), 1.25–2.60 (m, unassigned CH and
CH2), 2.80 (m, 6-CH2 and 1 H of CH2-Aryl), 3.20 (dd,
J2 = 13.6 Hz, J1 = 4.2 Hz, 16a-CH), 3.71 (s, OCH3),
6.57 (d, J = 2.6 Hz, 1-CH), 6.64 (dd, J2 = 8.6 Hz,
J1 = 2.7 Hz, 2-CH), 7.14 (d, J = 8.7 Hz, 4-CH), 7.40
(m, 500-CH and 600-CH), 7.71 (m, 400-CH and 200-CH);
13C NMR (100 MHz, MeOH-d4) d 14.1, 27.0, 27.9,
28.8, 30.6, 33.1, 38.1, 39.3, 45.4, 49.8, 50.0, 52.3, 55.5,
112.5, 114.6, 126.7, 127.2, 129.3, 129.6, 133.1, 133.8,
135.1, 138.8, 141.8, 159.0, 172.3, 223.6; LRMS calcd
for C27H32NO3 [M+H]+ 418.1. HPLC purity = 92.4%,
rt = 33.2 min, k = 210 nm.
4.1.4. General procedure for Jones’ oxidation of the 17b-
hydroxy group of the E2 template. To a solution of 4m,
4p, 6m, or 6p (0.120 mmol) in acetone (15 mL) was
added a solution of Jones’ reagent (0.127 mmol) at
0 ꢁC. After the mixture was stirred for 5 min at room
temperature, the reaction was quenched by addition of
isopropanol (0.8 mL) and acetone was evaporated under
reduced pressure. H2O (6 mL) was added to the slurry
mixture and the aqueous phase was extracted with
EtOAc. Then the combined organic layer was dried over
MgSO4 and evaporated. The crude product was purified
4.1.4.4.
4-(3-Methoxy-17-oxo-estra-1,3,5(10)-trien-
16b-ylmethyl)-benzamide (7p). White amorphous solid
(64% yield); hexanes/EtOAc: 50/50 for chromatography.
IR (film) m 3449, 3350 and 3195 (NH2), 1732 (C@O,
ketone), 1662 (C@O, amide); 1H NMR (400 MHz,
MeOH-d4) d 0.68 (s, 18-CH3), 1.30–2.60 (m, unassigned

Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
1859
CH and CH2), 2.81 (6-CH2 and 1 H of CH2-Aryl), 3.17
(dd, J2 = 13.5 Hz, J1 = 4.2 Hz, 16a-CH), 3.71 (s, OCH3),
6.58 (d, J = 2.4 Hz, 4-CH), 6.64 (dd, J2 = 8.6 Hz,
J1 = 2.6 Hz, 2-CH), 7.13 (d, J = 8.6 Hz, 1-CH), 7.31 (d,
J = 8.2 Hz, 200-CH and 600-CH), 7.80 (d, J = 8.2 Hz,
300-CH and 500-CH); 13C NMR (75 MHz, MeOH-d4) d
14.1, 27.0, 27.9, 28.7, 30.6, 33.1, 38.0, 39.2, 45.4, 49.8,
50.0, 52.2, 55.5, 112.5, 114.7, 127.2, 128.8 (2C), 130.3
(2C), 132.9, 133.1, 138.7, 145.6, 159.0, 172.2, 223.4;
LRMS calcd for C27H32NO3 [M+H]+ 418.2. HPLC pur-
ity = 99%, rt = 33.0 min, k = 232 nm.
tained in 75 cm2 culture flasks at 37 ꢁC under 5% CO2
humidified atmosphere. The T-47D cells were grown
in RPMI medium (Sigma–Aldrich, Oakville, Ontario,
Canada) supplemented with 10% fetal bovine serum
(FBS), L-glutamine (2 mM), penicillin (100 IU/mL),
streptomycin (100 lg/mL) and estradiol (1 nM). The
MCF-7 cells were propagated in Dulbecco’s modified
Eagle’s medium containing a nutrient mixture F-12
ham (DME-F12) (Sigma–Aldrich, Ontario, Canada)
and supplemented with 5% FBS, glutamine (2 mM),
penicillin (100 IU/mL), streptomycin (100 lg/mL) and
estradiol (1 nM). Both medium were prepared without
phenol red.
4.1.5. General procedure for the methylation of the
hydroxyl group at the 3-position of E2 template. A solu-
tion of 4m or 4p (0.34 mmol) in acetone (18 mL) was
treated with cesium carbonate (0.51 mmol) and iodo-
methane (4.8 mmol). The resulting mixture was stirred
at reflux for 1 h, then filtered on Celite and washed with
CH2Cl2. The filtrate was evaporated and the crude prod-
uct was purified by chromatography (hexanes/EtOAc)
to afford 6m or 6p.
4.2.2. Type 1 17b-HSD inhibition. T-47D cells were
seeded in 24-well plates at 3 · 104 cells/well in prolifera-
tion assay medium (see Section 4.2.3). After 48 h, 60 nM
of [4-14C]-estrone (American Radiolabeled Chemicals
Inc., St. Louis, MO, USA) and an ethanolic solution
of inhibitor (0.5%, v/v) at concentrations of 0.1, 1, and
10 lM (0.1 nM to 100 lM for IC50) were added to
freshly changed culture medium and the cells were incu-
bated for 24 h. Each inhibitor was assessed in triplicate.
After incubation, labeled steroids (E1 and E2) were ex-
tracted and quantified according to an established
procedure.31,48
4.1.5.1. 3-(17b-Hydroxy-3-methoxy-estra-1,3,5(10)-
trien-16b-ylmethyl)-benzamide (6m). White amorphous
solid (76% yield); hexanes/EtOAc: 45/55 for chromatog-
raphy. IR (film) m 3348 and 3198 (OH and NH2), 1654
1
(C@O, amide); H NMR (400 MHz, MeOH-d4) d 0.88
(s, 18-CH3), 1.10–2.60 (m, unassigned CH and CH2),
2.78 (6-CH2), 3.15 (d, J = 10.8 Hz, 16a-CH), 3.71 (s,
OCH3), 3.81 (d, J = 9.3 Hz, 17a-CH), 6.56 (d,
J = 2.2 Hz, 4-CH), 6.64 (dd, J2 = 8.6 Hz, J1 = 2.5 Hz,
2-CH), 7.15 (d, J = 8.6 Hz, 1-CH), 7.37 (m, 500-CH and
600-CH), 7.67 (d, J = 7.4 Hz, 400-CH), 7.73 (s, 200-CH);
13C NMR (75 MHz, MeOH-d4) d 13.3, 27.5, 28.6,
30.7, 33.0, 38.8, 38.9, 39.8, 43.3, 45.3, 45.4, 49.9, 55.5,
83.0, 112.4, 114.5, 125.9, 127.1, 129.1, 129.4, 133.5,
133.7, 134.7, 138.8, 144.3, 158.8, 172.6; LRMS calcd
for C27H34NO3 [M+H]+ 420.2. HPLC purity = 96.7%,
rt = 33.6 min, k = 212 nm.
4.2.3. Cell proliferation assay. Quantification of cell
growth was determined using CellTitter 96ꢂ Aqueous
Solution Cell Proliferation Assay (Promega, Nepean,
Ontario, Canada) following the manufacturer’s
instructions. T-47D and MCF-7 cells were resus-
pended in their respective medium (RPMI or DME-
F12) supplemented with insulin (50 ng/mL) and 5%
dextran-coated charcoal treated FBS to remove the
remaining estrogen present in the serum and medium.
Aliquots (100 lL) of the cell suspension were seeded
in 96-well plates (3000 cells/well). After 48 h, the med-
ium was changed with appropriate dilution of the dif-
ferent inhibitors and reference compounds in growth
medium and was replaced every 2 days until 8 days
of treatment.
4.1.5.2. 4-(17b-Hydroxy-3-methoxy-estra-1,3,5(10)-
trien-16b-ylmethyl)-benzamide (6p). White amorphous
solid (79% yield); hexanes/EtOAc: 45/55 for chromatog-
raphy. IR (film) m 3336 and 3182 (OH and NH2), 1670
To determine the proliferative (estrogenic) activity, the
estrogen-sensitive T-47D and MCF-7 cells were grown
in absence (control fixed as 100%) or presence of an
inhibitor (4m, 5m, 17, 18, or 19 at 0.01, 0.1, and
1 lM) or estrogen E2 at 10 nM. To determine the
inhibition of E1-induced cell proliferation, the T-47D
(ER+) cells were grown in the presence of E1 (0.01,
0.1, 1, or 5 nM) and inhibitor 4m (0.05, 0.1, 0.5, or
1 lM). The cell proliferation without E1 and inhibitor
4m (control) was fixed as 100%. To determine the po-
tential antiestrogenic activity of inhibitor 4m, the
MCF-7 (ER+) cells were grown in the presence of
estrogen E2 (0.1 nM) and pure antiestrogen EM-
13934 (0.5 lM) or inhibitor 4m (0.5 lM). The cell pro-
liferation without E2 and tested compounds (control)
was fixed as 100%.
1
(C@O, amide); H NMR (400 MHz, MeOH-d4) d 0.87
(s, 18-CH3), 1.10–2.60 (m, unassigned CH and CH2),
2.76 (m, 6-CH2), 3.14 (d, J = 9.5 Hz, 16a-CH), 3.71 (s,
OCH3), 3.81 (d, J = 9.3 Hz, 17a-CH), 6.56 (d,
J = 2.4 Hz, 4-CH), 6.64 (dd, J2 = 8.6 Hz, J1 = 2.6 Hz,
2-CH), 7.15 (d, J = 8.6 Hz, 1-CH), 7.30 (d, J = 8.2 Hz,
200-CH and 600-CH), 7.78 (d, J = 8.2 Hz, 300-CH and 500-
CH); 13C NMR (100 MHz, MeOH-d4) d 13.3, 27.5,
28.6, 30.7, 33.0, 38.8, 38.9, 39.8, 43.3, 45.3, 45.4, 49.9,
55.4, 82.9, 112.4, 114.5, 127.1, 128.6 (2C), 129.9 (2C),
132.2, 133.7, 138.8, 148.4, 158.9, 172.5; LRMS calcd
for C27H34NO3 [M+H]+ 420.1. HPLC purity = 98.9%,
rt = 33.1 min, k = 232 nm.
4.2. Biological assays
4.2.1. Cell culture. Two ER-positive breast cancer cell
lines, T-47D and MCF-7, were obtained from the
American Type Culture Collection (ATCC) and main-
4.2.4. Statistical analysis. Statistical significance was
determined according to the Duncan–Kramer multiple
range test.49

1860
Y. Laplante et al. / Bioorg. Med. Chem. 16 (2008) 1849–1860
22. Husen, B.; Huhtinen, K.; Saloniemi, T.; Messinger, J.;
Acknowledgments
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We thank the Canadian Institutes of Health Research
(CIHR) for an operating grant. We also thank Sylvie
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Methot for careful reading of the manuscript.
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