C6-(N,N-butyl-methyl-heptanamide) derivatives of estrone and estradiol as inhibitors of type 1 17β-hydroxysteroid dehydrogenase: Chemical synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2006.10.055

A series of estrone and estradiol derivatives having an N-butyl,methyl heptanamide side chain at C6-position were synthesized, tested as inhibitors of type 1 17β-HSD and assessed for their possible estrogenic activity. A better type 1 17β-HSD inhibition w

Full text of DOI:10.1016/j.bmc.2006.10.055

Bioorganic & Medicinal Chemistry 15 (2007) 714–726
C6-(N,N-butyl-methyl-heptanamide) derivatives of estrone and
estradiol as inhibitors of type 1 17b-hydroxysteroid dehydrogenase:
Chemical synthesis and biological evaluation
Christine Cadot,ꢀ Yannick Laplante,ꢀ Fatima Kamal, Van Luu-The and Donald Poirier*
Oncology and Molecular Endocrinology Research Center, CHUQ-Pavillon CHUL and Universite´ Laval, Que´bec, Canada G1V 4G2
Received 5 September 2006; revised 24 October 2006; accepted 25 October 2006
Available online 28 October 2006
Abstract—A series of estrone and estradiol derivatives having an N-butyl,methyl heptanamide side chain at C6-position were syn-
thesized, tested as inhibitors of type 1 17b-HSD and assessed for their possible estrogenic activity. A better type 1 17b-HSD inhi-
bition was obtained for the 6b-side chain orientation over 6a; the C17-alcohols are more potent inhibitors than the corresponding
ketones; introducing a 2-methoxy group decreased the inhibitory potency; and the replacement of a C–S bond by a C–C bond in the
C6b-side chain is not detrimental to inhibition. Interestingly, the new inhibitors were also found less estrogenic than the lead com-
pound in two breast cancer cell lines, T-47D and MCF-7.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
E2 has been observed both in normal human breast
and malignant breast tumours, the reductive activity is
stronger in tumours than in normal breast tissue.11–15
Thus, in the perspective of designing drugs to control
E2 formation, we are interested in synthesizing potent
inhibitors of type 1 17b-HSD.16
Breast cancer is the most frequent cancer in women and
the second cause of cancer death. In fact, one in eight
North American women suffers from breast cancer dur-
ing her lifetime, and 570,280 women were expected to
die of this disease in 2005.1
Our group has recently reported the synthesis and the
inhibitory potency on type 1 17b-HSD of a number of
E2 derivatives functionalised at position 6.17,18 Com-
pound 1, an E2 derivative bearing a 6b-thiaheptanamide
side chain (Fig. 2), was found to be a good inhibitor of
the synthesis of E2 from E1, whereas the corresponding
6a-epimer is inactive. Unfortunately, compound 1 also
exerts a proliferative (estrogenic) activity as illustrated
by its ability to stimulate growth of estrogen-sensitive
cells in culture,17 thus reducing its possible therapeutic
interest. Three strategies to modify the biological profile
(estrogenicity and inhibitory potency) of lead compound
1 were then tested by synthesizing compounds 2–4.
Thus, the replacement of the 3-OH by a hydrogen atom
as well as that of the amide group by a methyl was clear-
ly unfavourable for the inhibition activity. Changing the
thioether for an ether bond decreased 10-fold the estro-
genic profile of lead compound 1 while the inhibitory
potency was only decreased 5-fold.18
The growth of most cancerous breast tissue is stimulated
by estrogens. It is thus of interest that, in adult women, a
large proportion of active estrogens are synthesized in
peripheral target tissues such as breast from inactive
precursor steroids.2 Among enzymes involved in steroi-
dogenesis,3 type 1 17b-hydroxysteroid dehydrogenase
(17b-HSD) is responsible for the conversion of estrone
(E1) into estradiol (E2), the most potent estrogen, and
dehydroepiandrosterone (DHEA) into 5-androstene-
3b,17b-diol (D5-diol) (Fig. 1).4,5 Types 7 and 12 of
17b-HSD are the two other isoforms which convert
the weak E1 into the potent E2.6 The importance of
17b-HSD activity in breast tumour development and
growth is indicated by higher intratumoral levels of
E2.7–10 Moreover, although the conversion of E1 into
Keywords: Breast cancer; 17b-hydroxysteroid dehydrogenase; Inhibi-
tor; Steroid; Chemical synthesis.
*
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.
As another strategy for reducing the estrogenicity of 1,
we tried adding a short alkylether (MeO or EtO) at
ꢀ
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.055

C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
715
Figure 1. Role of type 1 17b-HSD inhibitors.
Figure 2. Chemical structures of type 1 17b-HSD inhibitors 1–6, of potential metabolites 7–10 and of new inhibitors 11–15.
the C2-position, but stability problems prevented the
synthesis and limited the usefulness of 5 and 6.19 The
instability of the thioether bond was hypothesized to
cause the problems in the chemical synthesis of com-
pounds 5 and 6 as well as the estrogenicity of 1. To con-
firm or infirm this last hypothesis, potential estrogenic
metabolites 7–10 resulting from the cleavage of the thi-
oether bond were tested. Furthermore, considering the
chemical and biological results obtained with com-
pounds 1–10, it was concluded that the thioether
(C–S) or ether (C–O) bond must be replaced by a car-
bon-carbon (C–C) bond, which should be much less
likely to be modified by metabolizing enzymes. We also
investigated the role of the 17b-OH versus the 17-ketone
and the presence of a 2-methoxy group to reduce the
estrogenic properties of the best inhibitors. The chemical
synthesis of 11–15, inhibitory potency on types 1, 7 and
12 of 17b-HSD as well as proliferative activity on estro-
gen sensitive (ER+) breast cancer cell lines T-47D and
MCF-7 are reported in this paper.
2. Results and discussion
2.1. Chemistry
As reported in Scheme 1, compounds 11–14 were syn-
thesized from key intermediates 19 and 21, differing only
in their stereochemistry in C6-position. They both were
synthesized from the same precursor 18, which was
obtained from diTHP-E2 (16) by an oxidation at C6
giving 1720,21 and the addition of a Grignard reagent.

716
C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
Figure 3. Attack of in situ generated hydride on the b face of the
intermediate carbocation. The 3D structure was generated with
CSChem 3D std 5.0 (Cambridge Soft Corporation, Cambridge, USA).
Scheme 1. Reagents and conditions: (a) i—BuLi, diisopropylamine,
t-BuOK, steroid 16, ꢀ78 ꢁC, 3 h; ii—B(OMe)3, 0 ꢁC, 2 h; iii—H2O2,
1 h, rt; iv—PCC, NaOAc, CH2Cl2, 0 ꢁC, 15 min, then 1 h, rt (78%); (b)
i—Mg, Br(CH2)7OTHP, Et2O, THF, rt, 1.5 h; ii—CeCl3, THF,
ꢀ78 ꢁC, 1.5 h, then steroid 17, ꢀ78 ꢁC, 5 min; iii—ꢀ40 ꢁC, 4 h
(99%); (c) Et3SiH, BF3–Et2O, CH2Cl2, rt, 1 h (56%); (d) 5% HCl,
EtOH, THF, 90 ꢁC, 1 h (72%); (e) 10% Pd/C (15% w/w), H2, EtOAc, rt,
4.5 h (95%).
The C6-stereochemistry of 19 and 21 was established by
NMR analysis. Using a combination of NMR experi-
ments (COSY, APT, HSQC, HMBC and NOESY),28,29
all proton and carbon signals were fully identified for 19.
Using the signal at 2.88 ppm (6b-CH), the NOESY spec-
tra allowed identifying four signals of different intensity
with 4-CH (medium), 7b-CH (strong), 10-CH2 (medium)
and 8b-CH (strong). Furthermore, no NOE was ob-
served between the C-6 hydrogen and the 9a-CH. Taken
together, these data clearly established the 6b-CH (or
6a-side chain) stereochemistry of 19 (Fig. 4).
An excess of in situ generated tetrahydropyranyloxyhep-
tyl magnesium bromide22 and cerium chloride was used
for the Grignard reaction23,24 that afforded a 5:2 ratio
of diastereomers 18a and 18b in a nearly quantitative
yield.
Compound 19, 6a-(7-hydroxyheptyl)-E2, was obtained
in 56% yield by a treatment of 18 with triethylsilane
and borontrifluoride etherate (Pathway A in Scheme
1).25,26 A secondary minor product of unknown struc-
ture was also isolated. In the conditions of deoxygen-
ation, the tetrahydropyranyl (THP) groups were
cleaved to give the free hydroxyl groups. Only one iso-
mer was obtained because the intermediate carbocation
was attacked by the in situ generated hydride using the
less hindered b-face. Indeed the 7a-H and 9a-H direct
the hydride attack to the b-face (Fig. 3). For the synthe-
sis of 6b-(7-hydroxyheptyl)-E2 (21), the deoxygenation
step was replaced by two steps:27 a dehydration of 18
with 5% HCl in ethanol at reflux to give the olefin 20,
followed by a catalytic hydrogenation with palladium/
carbon in ethylacetate (Pathway B) to produce a 95:5
mixture of the desired 6b-isomer 21 and 6a-isomer 19
in excellent yield. The stereoselectivity of the double
bond reduction is explained by the palladium complex-
ation, which was achieved on the less hindered a-face
of olefin 20.
Figure 4. 2D and 3D representations of 19. The important NOE
results are represented by four arrows. The 3D structure was generated
with CSChem 3D std 5.0 (Cambridge Soft Corporation, Cambridge,
USA).

C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
717
Table 1. Characteristic 1H NMR signals (ppm) observed for compounds 19 and 21 in CDCl3 and compound 1 and its epimer in acetone-d6
Signal
19 (6a-side chain)
21 (6b-side chain)
Epimer of 1a (6a-side chain)
1a (6b-side chain)
4-CH
18-CH3
6.75
0.74
6.54
0.78
7.22
0.76
6.88
0.81
a Data from Ref. 17.
The combination of NMR experiments reported above
was also performed for 21, but the 6a-CH (or 6b-side
chain) stereochemistry was not clearly established. The
chemical shift of two protons (4-CH and 18-CH3) of
lead compound 1 and its epimer was next compared to
the corresponding signals of 19 and 21 (Table 1). For
these signals, the chemical displacement tendencies were
the same for the two a isomers (19 and epimer of 1) as
well as for the two b isomers (21 and 1).
bond and is typical of such alkylamide compounds.31,32
In the process, the 17b-hydroxyl group was oxidized to a
ketone and the phenolate group was protected as an
isobutylcarbonate group. The deprotection was easily
performed using a solution of aqueous potassium car-
bonate in methanol to give 11 and 12 in 94% and 96%
yield, respectively. These compounds were next treated
with sodium borohydride to stereoselectively reduce
the C17-carbonyl into alcohols 13 and 14 in excellent
yields.
In the next synthesis step, triols 19 and 21 were oxidized
with Jones’ reagent to give the corresponding carboxylic
acid which, upon treatment with tributylamine, isobu-
tylchloroformate and N-methyl-N-butylamine,30 yielded
50% and 59% of amides 22 and 23, respectively (two
The synthesis of the 2-methoxy analogue of 12, com-
pound 15, is reported in Schemes 3 and 4. In the first
strategy (Scheme 3), triol 21 was protected by three
methoxymethylether (MOM) groups to obtain 24 which
was treated with sodium carbonate, iodine and an excess
of silver trifluoroacetate33 to give in quantitative yield a
mixture of 2-iodo and 4-iodo compounds with a 95/5
regioselectivity. The major product 25 was then trans-
formed into C2-methoxy derivative 26 in 74% yield by
a treatment with sodium methylate, 15-crown-5 ether
1
steps, Scheme 2). In the results of H NMR spectrosco-
py, the methyl of the amide group (CONCH3) appeared
as two singlets at 2.91/2.96 and 2.91/2.97 ppm for 22 and
23, respectively, while the methylene of CONCH2 exhib-
ited two triplets at 3.25/3.35 and 3.26/3.36 ppm. More-
over, the CH3 of the N-butyl group as well as protons
and carbons surrounding the amide function were also
duplicated. The duplication of these NMR signals can
be explained by the two conformations of the amide
Scheme 2. Reagents and conditions: (a) i—Jones’ reagent, acetone, rt,
20 min; ii—Bu3N, (CH3)2CHCH2OCOCl, CH2Cl2, ꢀ10 ꢁC, 40 min,
then BuMeNH, ꢀ10 ꢁC, 5 min; iii—3 h, rt (50% for 22 and 59% for
23); (b) 1% K2CO3, H2O, MeOH, rt, 3 h (94% for 11 and 96% for 12);
(c) NaBH4, MeOH, 0 ꢁC, 0.5 h (99% for 13 and 91% for 14). 6a and 6b
correspond to the isomer with a 6a- and a 6b-side chain, respectively.
Scheme 3. Reagents and conditions: (a) MOMCl, diisopropylethyl-
amine, CH2Cl2, THF, rt, 18 h (59%); (b) NaHCO3, CF3COOAg, I2,
CH2Cl2, ꢀ40 ꢁC, 4 h (100%); (c) MeONa, 15-crown-5, CuI, MeOH,
DMF, 120 ꢁC, 12 h (74%); (d) 5% HCl, MeOH, 90 ꢁC, 2.5 h (91%); (e)
Jones’ reagent, rt or TPAP, NMO, rt or Dess–Martin, rt or IBX
polystyrene, 0 ꢁC.

718
C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
however inhibited the type 1 17b-HSD overexpressed
in HEK-293 cells (data not shown). In addition of con-
firming the importance of the C6-side chain for enzyme
inhibition, this result also indicated that the residual
estrogenicity observed for 117 was not the result of
metabolites such as 7–10, which are known to be estro-
genic compounds.35 Although we concluded that a
metabolite of 1 was not responsible for its estrogenicity,
the presence of a thioether at the C6 position of E2 pre-
vented the synthesis of methoxy derivative 5. The thioe-
ther (C–S) bond was then replaced by a more stable C–C
bond allowing the chemical synthesis of 11–15.
2.2.1. Inhibitory potency in homogenated HEK-293 cells.
We used homogenated HEK-293 cells overexpressing
type 1 17b-HSD to evaluate the ability of compounds
11–14 to inhibit the transformation of E1 into E2 (Table
2). We first compared the inhibitory potency of 17-ke-
tones 11 and 12 and that of 17b-alcohols 13 and 14.
For compounds 12 and 14 (b-oriented side chain at po-
sition C6), it is clear that the alcohol 14 was a better
inhibitor (46% of inhibition, as compared to 23%) at
1 lM. Compounds 11 and 13 with an a-oriented C6 side
chain produced equivalent and very low inhibition val-
ues (16% and 17%). We next determined the effect of
the a and b orientation of the C6 side chain (13 vs 14
and 11 vs 12). Results previously obtained clearly dem-
onstrated that the b orientation was crucial to the inhib-
itory activity of type 1 17b-HSD.17 As expected, the new
results indicated that compound 14 with a b-oriented
side chain is a better inhibitor at 1 lM than compound
13 with an a orientation. At 0.1 lM, the difference of
inhibitory activity was however not significant. For less
potent 17-ketone inhibitors 11 and 12, the differences
were not significant at both concentrations, but the
inhibitory values are too low to allow a good compari-
son. Moreover, we compared 1 (C–S bond at position
6) with its C–C analogue 14 and this modification de-
creased the inhibition from 74% to 46% at 1 lM.
Scheme 4. Reagents and conditions: (a) Cs2CO3, benzyl bromide,
CH3CN, 100 ꢁC, 2.5 h (100%); (b) NaHCO3, CF3COOAg, I2, CH2Cl2,
ꢀ40 ꢁC, 4 h (85%); (c) MeONa, CuI, MeOH, DMF, 120 ꢁC, 12 h
(66%); (d) NaBH4, MeOH, 0 ꢁC, 0.5 h (99%); (e) Pd(OH)2 (10% w/w),
H2, EtOAc, rt, 12 h (98%).
and a catalytic amount of copper iodide warmed at
120 ꢁC overnight.34 After removal of MOM groups with
5% HCl in methanol, triol 27 was submitted to oxida-
tion using a variety of reagents. Unfortunately, all the
methods we tried (Jones’ reagent, TPAP/NMO, Dess–
Martin or IBX-polystyrene) failed to provide 28 as the
corresponding carboxylic acid or aldehyde. Contrary
to phenols 19 and 21, which were oxidized with Jones’
reagent, the 2-methoxy phenol nucleus of 27 is too sen-
sitive to oxidants and undergoes degradation. Although
a selective protection of the phenol over the two remain-
ing alcohols would also be a valuable alternative strate-
gy, we decided to use instead the second strategy
reported in Scheme 4.
2.2.2. Inhibitory potency in intact HEK-293 cells. Purified
enzymes, as well as homogenated cells overexpressing a
functional enzyme, are certainly useful models for devel-
opment of inhibitors. However, they differ much from
actual intact breast cancer cells exerting a 17b-HSD
activity, an experimental model that constitutes a better
approximation of physiological conditions. So we also
performed the inhibition assay in intact HEK-293 cells
overexpressing type 1 17b-HSD, and in a tumoral cell
line (next section). All inhibition values obtained with
11–14 are weaker in intact cells than in homogenated
cells. The significant drop in inhibitory potency could
be explained by the fact that compounds must cross
the cell membrane and resist to metabolization. None-
theless, inhibition values obtained with 11–14 showed
the same pattern of inhibition as in homogenated cells
(Table 2). Inhibitor 1 with a C–S bond has again a better
inhibitory potency (63% and 45%) than 14 (49% and
21%) at 10 and 1 lM, respectively. The b orientation
of the side chain is clearly important for the inhibitory
activity as well as the presence of a 17b-hydroxyl group.
Since the steroidal nucleus may confer estrogenic activ-
ities to inhibitors 11–14, we synthesized an additional
In the second approach, we started by a C3-benzylation
of 12, yielding compound 29 (Scheme 4). The iodation at
the C2-position of 29 gave the 2-iodo (85%) and 4-iodo
(3%) compounds after chromatography. Nucleophilic
substitution of the iodide of compound 30 with sodium
methylate and a stoichiometric amount of copper iodide
afforded 31, bearing the C2-methoxy group, in 66%
yield. It was not possible to increase this yield when
we tried milder conditions using a catalytic amount of
copper iodide (0.3 or 0.7 equiv). Under such conditions,
the nucleophilic substitution was obtained in 50%
yield, but the mixture of starting material 30 and meth-
oxylated product 31 was not separable by chromatogra-
phy. Finally, the reduction of 17-ketosteroid 31 gave
32, which after removal of benzyl ether protection
afforded 15.
2.2. Biological activity
The four possible metabolites of 1, compounds 7–10,
were tested as inhibitors in intact cells. None of them

C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
Table 2. Percentage of inhibition of type 1 17b-HSD using several inhibition assays
719
Compound
C17 group
C6 side chain
HEK-293
homogenateda (%)
HEK-293 intact cellsb (%)
T-47D intact cellsc (%)
1 lM
0.1 lM
10 lM
1 lM
0.1 lM
10 lM
1 lM
0.1 lM
1
11
12
13
14
15
E1
17b-OH
C@O
C@O
17b-OH
17b-OH
17b-OH
C@O
b
a
b
a
b
b
—
74
16
33
3
63
14
31
15
49
28
83
45
5
11
3
95
71
92
74
95
81
93
82
19
57
21
82
32
85
31
7
23
17
12
9
20
11
21
15
38
8
19
9
3
46
15
N/D
19
11
13
8
29
11
36
N/D
59
N/D, not determined.
a SD < 5%. One run in triplicate.
b SD < 8%. Mean of five experiments in quadruplicate.
c SD < 5% . Mean of two experiments in triplicate.
compound, the 2-MeO analogue of 14, compound 15.
The 2-MeO group decreased the inhibitory activity of
14 from 49% to 28% at 10 lM.
Table 3. Percentage of inhibition of other 17b-HSD isoforms using
intact cells HEK-293 overexpressing type 7 or 12 17b-HSD
Compound
Type 7a (%)
Type 12b (%)
10 lM
1 lM
10 lM 1 lM
2.2.3. Inhibitory potency in breast cancer cell line T-47D.
After we performed inhibition assays of compounds 11–
15 with HEK-293 cells overexpressing type 1 17b-HSD
in homogenated and intact cells, we repeated the assays
with the ER+ breast cancer cell line T-47D (Table 2).
This cell line, which is known for its expression of type
1 17b-HSD,36 yet constitutes a more physiological mod-
el than transfected laboratory HEK-293 cells. All inhib-
itors have a better inhibitory activity in T-47D cells than
in homogenated or intact HEK-293 cells. The better
inhibitory effect on T-47D intact cells (specific activi-
ty = 10 pmol/h/106 cells) than intact HEK-293 cells (spe-
cific activity = 12.5 nmol/h/106 cells) could probably be
due to higher amount of enzymes found in transfected
HEK-293 cells (1250-fold). Unlike results obtained
above with 1 and 14 either in homogenates or in intact
HEK-293 cells, in T-47D cells these compounds have
the same inhibitory potency at 10, 1 and 0.1 lM. Chang-
ing from a C–S bond to a C–C bond did not influence
the inhibitory activity. Results also indicated that inhib-
itors 11 and 13, with the a-oriented side chain, showed
weaker inhibition values with about 70% and 20% at
10 and 1 lM, respectively. Among the C-6b series of
inhibitors, the best inhibitory activity at 1 lM (82%)
was obtained for compound 14 with the hydroxyl group
at position 17. The keto analogue 12 inhibited 57% of
the transformation of E1 into E2. As in the HEK-293
model, alcohol 13 and ketone 11, with an a-orientation
of the side chain, gave similar low percentages of inhibi-
tion. Again, compound 15 (2-MeO) has a lower inhibi-
tory activity than 14 (2-H) with 82% and 32% of
inhibition at 1 lM, respectively.
1
11
12
13
14
15
E1
25
29
31
24
27
17
0
9
5
22
44
40
30
30
22
0
0
3
0
1
1
0
0
7
11
0
0
0
a SD < 5%. One run in triplicate.
b SD < 5%. Mean of two experiments in triplicate.
centration of 10 lM. Type 12 17b-HSD was not inhibit-
ed by any compound at 1 lM but inhibition values of
22% to 44% were obtained at 10 lM. Enzymatic assays
performed in our laboratory with other compounds hav-
ing an alkylamide chain have however demonstrated
that this kind of inhibitor was less selective at higher
concentrations.
2.3. Proliferative activity
In order to determine the possible undesirable estrogenic
activity of our inhibitors, we selected compounds 12, 14
and 15 as well as lead compound 1 for performing pro-
liferative assays with two breast cancer cell lines, T-47D
and MCF-7, which are known to express the estrogen
receptor (ER). For comparison purposes, cell growth
in absence of E2 was assigned to be 100% and in pres-
ence of E2 or other compounds was expressed as per-
centage of control (Fig. 5). Our data showed that a
treatment of estrogen-starved T-47D cells with 10 nM
of E2 for 10 days increased the cell growth 3-fold over
basal level. Only compound 1 at 1 lM produced a sim-
ilarly strong proliferative effect. At lower concentra-
tions, 0.1 and 0.01 lM, compound 1 augmented
2.3-fold the cell proliferation. Switching from a C–S
bond (compound 1) to a C–C bond (compound 14) de-
creased significantly the proliferative activity. Although
we were expecting a much weaker proliferative effect
with 12 because of the lower affinity of a ketone (like
E1 derivative) for the ER, the ketone analogue 12 stim-
ulated cell growth similarly as 14. Results showed a
2.2.4. Selectivity towards other reductive isoforms of 17b-
HSD (types 7 and 12). We studied the selectivity of 11–
15 over other reductive isoforms of 17b-HSDs, types 7
and 12. All enzymatic assays were performed with intact
HEK-293 cells overexpressing type 7 or 12, and we test-
ed the ability of compounds to inhibit the transforma-
tion of E1 into E2 (Table 3). Compounds 11–15
weakly inhibited type 7 17b-HSD with inhibition values
of 0% to 11% at 1 lM and 17% to 31% at the higher con-

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C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
level. These results showed the same tendencies as prolif-
erative activities on T-47D.
3. Conclusion
Five new inhibitors of type 1 17b-HSD, compounds 11–
15, were efficiently prepared from E2, purified by chro-
matography and fully characterized by spectroscopic
analysis (IR, NMR and MS). For the inhibitory activity
on type 1 17b-HSD, a better enzyme inhibition was ob-
tained for compounds 12 and 14 having a 6b-orientation
of the alkylamide side chain (compared to 6a-analogues
11 and 13). A slightly better enzyme inhibition was ob-
tained for compounds 13 and 14 having a 17b-hydroxy,
when compared to 17-ketone analogues 11 and 12. The
presence of a 2-methoxy group on compound 15 re-
duced the inhibitory activity compared to that of com-
pound 14. Compounds 14 and 1, with a C–C and a C–
S bond, respectively, gave the same inhibitory potency
for type 1 17b-HSD in T-47D cells, but the C–C bond
is more stable. Selectivity of compounds 11–15 over
types 7 and 12 17b-HSDs was also investigated, and
they did not inhibit these isoforms at 1 lM.
For the proliferative activity on ER+ cell lines, the three
C–C inhibitors tested (compounds 12, 14 and 15) were
clearly less estrogenic than the C–S lead compound 1.
The results were less impressive in MCF-7 cells but fol-
lowed the same pattern observed in T-47D. The use of a
more stable C–C alkylamide side chain is not detrimen-
tal for enzyme inhibition in T-47D cells and allowed the
synthesis of a 2-MeO analogue (which was not possible
for the C–S lead compound 1). Even if the 2-MeO ana-
logue 15 exerted a weak inhibition of type 1 17b-HSD at
1 lM, we succeeded in eliminating all the remaining
estrogenic activity in both ER+ cell models. Further-
more, at 1 lM, compound 14 inhibited over 80% of type
1 17b-HSD activity and it is 2.1-fold less estrogenic than
compound 1 in T-47D breast cancer cells. Compound 14
arises from this study as the most active inhibitor of type
1 17b-HSD.
Figure 5. Effects of selected inhibitors on the growth of estrogen-
starved T-47D and MCF-7 cells after 10 days of treatment. Control is
fixed as 100%. SEM 6 5%.
slight decrease of the proliferative activity at 0.1 and
0.01 lM for 12 when compared with 14. This result is
supported by the stronger binding affinity on ER for
E2 than for E1.37,38 The 2-MeO derivative 15 has no pro-
liferative activity on T-47D cells at 1 lM, but we ob-
served a higher cell growth at lower concentrations
that reached 2.3-fold over basal level at 0.01 lM. This
increasing proliferative activity with decreasing concen-
tration is supported by the fact that 2-MeO-E2 decreases
cell growth of many cancer cell lines at high concentra-
tions but exerts some affinity for the ER at lower con-
centrations.39–41
4. Experimental
4.1. Chemistry
To improve the evaluation of proliferative activity of
our compounds, we used another ER+ breast cancer cell
line, MCF-7 (Fig. 5; lower panel). The results indicated
that a 10-day treatment of estrogen-starved MCF-7 cells
with 10 nM E2 increased the cell growth 1.8-fold over
control level. As observed in T-47D cells, compound 1
was the only one to stimulate cell growth to this level.
At a lower concentration of 0.01 lM, data showed that
the proliferative effect decreased to 1.6-fold over control.
Comparison between 1 and 14 indicated a decrease of
proliferative activity to 1.3-fold at 1 lM only by switch-
ing the C–S bond to a C–C bond. As expected, the ke-
tone (C@O) group at C17-position of 12 decreased the
proliferative activity at all three concentrations when
compared to 14 (17b-OH). Results for the 2-MeO com-
pound 15 indicated an increasing proliferative activity
from 1.1-fold at 1 lM to 1.7-fold at 0.01 lM over basal
Compounds 8–10 and starting material (estradiol) for
the synthesis of 11–15 were purchased from Steraloids
Inc. (Newport, RI), whereas compound 7 was available
in our laboratory. Anhydrous reactions were performed
in oven-dried glassware under positive argon pressure
using commercially available anhydrous solvents (Gib-
bstown, NJ, USA), except THF which was distilled from
sodium/benzophenone ketyl under argon. Chemical re-
agents were purchased from Aldrich Chemical Co (Mil-
waukee, WI, USA). Flash chromatography was
´
performed on Silicycle 60 230–400-mesh silica gel (Que-
bec, Qc, Canada). Thin-layer chromatography (TLC)
was performed on 0.25-mm silica gel 60 F254 plates
(Nepean, Ontario, Canada) and compounds were visual-
ized by exposure to UV light (254 nm), a solution of

C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
721
ammonium molybdate/sulfuric acid/water and/or a solu-
tion of para-anisaldehyde/sulfuric acid/acetic acid/etha-
nol (plus heating). Infrared (IR) spectra (Perkin-Elmer
1600, Norwalk, CT, USA) were obtained from a thin
film of the solubilized compound on NaCl pellets (usual-
ly in CH2Cl2) or in KBr pellets containing the solid com-
pound. Only significant bands are reported (in cmꢀ1). 1H
and 13C spectra were recorded at 300 (1H) and 75.5 (13C)
MHz or at 400 (1H) and 100 (13C) MHz using a Bruker
AC/F 300 or a Brucker AVANCE 400 spectrometer
(Billerica, MA, USA). The chemical shifts (d) are ex-
pressed in ppm and referenced to chloroform (7.26 and
diastereomer 18b: Rf = 0.44 (hexanes/EtOAc, 70:30);
Colorless oil; IR (film) m 3358 (OH); 1H NMR
(400 MHz, CDCl3) d 0.81 and 0.82 (2s, 18-CH3), 1.15–
2.35 (m, 43H, CH and CH2 of steroid skeleton, aliphatic
chain and THP groups), 3.38 (m, 1H), 3.50 (m, 2H), 3.64
(m, 1H), 3.72 (m, 2H), 3.90 (m, 3H) (9H of 17a-CH and
4· OCH2 of aliphatic chain and THP groups), 4.57 (m,
O–CH–O), 4.66 (m, O–CH–O), 5.42 (m, ArO–CH–O),
6.92 (dd, J2 = 8.5 Hz, J1 = 2.5 Hz, 2-CH), 7.17 (2d,
J = 8.6 Hz, 1-CH), 7.23 (tapp, J = 2.7 Hz, 4-CH); LRMS
calcd for C40H62O7Na [M+Na]+ 677.4.
1
77.0 ppm) or methanol (3.31 and 49.0 ppm) for H and
4.1.2. Synthesis of 7-(30,17b0-dihydroxy-estra-10,30,50(100)-
trien-60a-yl)-heptanol (19). To a stirred solution of 18b
(334 mg, 0.518 mmol) in CH2Cl2 (11 mL) were added
triethylsilane (2.5 mL) and BF3ÆEt2O (5.3 mL) at 0 ꢁC.
The reaction mixture was stirred under nitrogen for 1 h
at rt, poured into ice-cold aq 10% K2CO3 (5 mL) and
extracted with CH2Cl2. The combined organic layer
was dried over Na2SO4 and evaporated. The crude prod-
uct was purified by chromatography (hexanes/EtOAc,
40:60 then 30:70) to afford 19 (113 mg, 56% yield) as
colourless oil. When this reaction was performed with
18a, compound 19 was also obtained. IR (film) m 3315
13C, respectively. Carbon assignments were reported
only for key intermediates 19 and 21 and for final com-
pounds 11–15. Low-resolution mass spectra (LRMS)
were recorded with an LCQ Finnigan apparatus (San
Jose, CA, USA) equipped with an atmospheric pressure
chemical ionisation (APCI) source on positive mode.
High-resolution mass spectra (HRMS) were provided
by the Regional Laboratory for Instrumental Analysis
´
´
´
(Universite de Montreal, Montreal, Canada). High-per-
formance liquid chromatography (HPLC) analyses were
carried out using a Waters Associates System (Milford,
MA, USA), a Nova-Pack C18 column (150 · 3.9 mm,
1
(OH); H NMR (400 MHz, CDCl3) d 0.75 (s, 180-CH3),
˚
4 lm, 60 A) and a solution of MeOH containing
0.90–2.35 (m, 25H, CH and CH2 of steroid skeleton
and aliphatic chain), 2.88 (m, 60b-CH), 3.66 (m,
CH2OH), 3.72 (t, J = 8.5 Hz, 170a-CH), 6.62 (dd,
J2 = 8.4 Hz, J1 = 2.5 Hz, 20-CH), 6.75 (d, J = 2.2 Hz,
40-CH), 7.14 (d, J = 8.4 Hz, 10-CH);13C NMR
(75 MHz, CD3OD) d 11.6 (C180), 24.1 (C150), 27.0
(C3), 27.1, 30.6 and 31.1 (C4–C6), 27.7 (C110), 30.7
(C160), 33.6 (C2), 35.5 (C70), 37.9 (C120), 39.0 (C7),
39.2 (C60), 40.1 (C80), 44.1 (C130), 45.0 (C90), 51.4
(C140), 63.0 (C1), 82.5 (C170), 113.5 (C20), 115.0 (C40),
126.9 (C10), 133.4 (C100), 142.8 (C50), 156.0 (C30); LRMS
calcd for C25H39O3 [M+H]+ 387.2.
20 mM AcONH4 as eluent (1 mL/min flow rate).
4.1.1. Synthesis of 3,17b-(ditetrahydro-200-H-pyran-200-
yloxy)-6-[70-(tetrahydro-200-H-pyran-200-yloxy)-heptyl]-
estra-1,3,5(10)-trien-6-ol (18). To a stirred solution of
magnesium (528 mg, 22.3 mmol) in dry Et2O (7.5 mL)
was added 7-(tetrahydro-20-H-pyran-20-yloxy)-heptyl
bromide22 (3.07 g, 11.0 mmol) in Et2O (7.5 mL) and
THF (2.5 mL) over a period of 0.5 h. The reaction mix-
ture was stirred under nitrogen for 1 h at rt then THF
(1 mL) was added. A mixture of anhydrous CeCl3
(2.71 g, 11.0 mmol) in dry THF (150 mL) was stirred un-
der nitrogen for 18 h at rt, then cooled at ꢀ78 ꢁC, and
the Grignard reagent solution was added slowly to this
reaction mixture. After 1.5 h at ꢀ78 ꢁC, ketone 1718–21
(1.07 g, 2.35 mmol) in THF (80 mL) was added at
ꢀ78 ꢁC. After being stirred at ꢀ40 ꢁC for 4 h, the reac-
tion mixture was quenched with saturated aq NH4Cl
(100 mL) and extracted with EtOAc. The combined
organic layer was washed with brine, dried over MgSO4
and evaporated. The crude alcohols 18 were a 5:2 mix-
ture of two diastereomers. Purification of this mixture
by flash chromatography (hexanes/EtOAc, 90:10 then
85:15) afforded three fractions containing 0.41 g of
18a, 0.08 g of a mixture of 18a and 18 b and 1.03 g of
18 b (1.52 g, 99% yield). Minor diastereomer 18a:
Rf = 0.56 (hexanes/EtOAc, 70:30); Colorless oil; IR
(film) m 3363 (OH); 1H NMR (400 MHz, CDCl3) d
0.79 and 0.81 (2s, 18-CH3), 1.00–2.35 (m, 43H, CH
and CH2 of steroid skeleton, aliphatic chain and THP
groups), 3.36 (m, 1H), 3.49 (m, 2H), 3.60 (m, 1H),
3.71 (m, 2H), 3.89 (m, 3H) (9H of 17a-CH and 4·
OCH2 of aliphatic chain and THP groups), 4.56 (m,
O–CH–O), 4.66 (m, O–CH–O), 5.36 (m, ArO–CH–O),
6.96 (dd, J2 = 8.5 Hz, J1 = 2.5 Hz, 2-CH), 7.18 (d,
J = 2.5 Hz, 4-CH), 7.22 (2d, J = 8.4 Hz, 1-CH); LRMS
calcd for C40H63O7–H2O [M+H–H2O]+ 637.4. Major
4.1.3. Synthesis of 7-(30,17b0-dihydroxy-estra-10,30,50(100),
60(70)-tetraen-6-yl)-heptanol (20). A solution of 18a and
18b (150 mg, 0.233 mmol) in ethanol (2.6 mL) was treat-
ed with concentrated HCl (0.13 mL). The resulting mix-
ture was stirred at reflux for 1 h. The reaction mixture
was neutralized with saturated aq NaHCO3, extracted
with CH2Cl2, dried over Na2SO4 and evaporated. The
crude product was purified by chromatography (hex-
anes/EtOAc, 70:30 then 55:45) to afford 20 (65 mg,
1
72% yield) as a white solid. IR (film) m 3352 (OH); H
NMR (400 MHz, CD3OD) d 0.75 (s, 180-CH3), 0.80–
2.65 (m, 23H, CH and CH2 of steroid skeleton and ali-
phatic chain), 3.53 (m, CH2OH), 3.67 (t, J = 8.5 Hz,
170a-CH), 5.74 (s, 70-CH), 6.60 (dd, J2 = 8.2 Hz,
J1 = 2.5 Hz, 20-CH), 6.73 (d, J = 2.4 Hz, 40-CH), 7.07
(d, J = 8.3 Hz, 10-CH); 13C NMR (75 MHz, CD3OD)
d 11.5, 24.0, 25.6, 26.9, 29.7, 30.4, 30.5, 30.6, 33.6,
33.9, 37.5, 40.0, 43.7, 44.1, 44.8, 63.0, 82.3, 111.3,
113.9, 125.2, 129.6, 132.7, 137.3, 137.9, 156.6; LRMS
calcd for C25H37O3 [M+H]+ 385.3.
4.1.4. Synthesis of 7-(30,17b0-dihydroxy-estra-10,30,50(100)-
trien-60b-yl)-heptanol (21). A mixture of 20 (135 mg,
0.35 mmol) and 10% palladium on charcoal (20 mg,
15% in weight) in EtOAc (3.5 mL) under hydrogen

722
C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
atmosphere was stirred 4.5 h at rt. Then the palladium
catalysis was removed by filtration on Celite, washed
with EtOAc and the filtrate was concentrated under re-
duced pressure. The crude product was purified by flash
chromatography (hexanes/EtOAc, 50:50 then 40:60) to
afford a 5:95 mixture of diastereomers 19 and 21
(128 mg, 95%) as colourless oil. IR (film) m 3333 (OH);
1H NMR (400 MHz, CD3OD) d 0.78 (s, 180-CH3),
1.10–2.35 (m, 25 H, CH and CH2 of steroid skeleton
and aliphatic chain), 2.65 (m, 60a-CH), 3.55 (t,
J = 6.6 Hz, CH2OH), 3.65 (t, J = 8.6 Hz, 170a-CH),
6.54 (m sharp, 20-CH and 40-CH), 7.06 (d, J = 9.1 Hz,
(400 MHz, CDCl3) d 0.92 (s, 180-CH3), 0.94 (m,
CH2CH3), 1.00 (d, J = 6.7 Hz, CH(CH3)2), 1.20–2.45
(m, 29 H, CH and CH2 of steroid skeleton, chain and
isopropylcarbonate),
2.51
(dd,
J2 = 18.8 Hz,
J1 = 8.3 Hz, 160b-CH), 2.79 (m, 60a-CH), 2.91 and 2.97
(2s, NCH3), 3.26 and 3.36 (2m, NCH2), 4.03 (d,
J = 6.6 Hz, OCH2CH), 6.95 (m sharp, 20-CH and 40-
CH), 7.27 (d, J = 7.7 Hz, 10-CH); LRMS calcd for
C35H54NO5 [M+H]+ 568.2.
4.1.6. Procedure for deprotection of 22 and 23 (synthesis
of 11 and 12). Carbonate 22 or 23 (58 mg, 0.102 mmol)
was dissolved in MeOH (2.75 mL) and a solution of
K2CO3 (1%, w:v) in MeOH/H2O [(25:75, v:v),
2.75 mL] was added. The resulting mixture was stirred
at rt for 3 h. Then, the mixture was acidified with aq
1 M HCl; MeOH was evaporated under reduced pres-
sure and the aq phase was extracted with EtOAc. The
combined organic layer was dried over MgSO4 and
evaporated. The crude residue was purified by chroma-
tography (hexanes/EtOAc, 60:40) to afford phenol 11
or 12 (96% and 94% yield, respectively).
13
10-CH); C NMR (75 MHz, CD3OD) d 11.8 (C180),
24.1 (C150), 26.9 (C3), 27.4 (C110), 29.0, 30.5 and 30.8
(C4–C6), 30.7 (C160), 31.6 (C70), 33.7 (C2), 35.2 (C80),
38.0 (C120), 39.1 (C60), 39.4 (C7), 44.6 (C130), 45.9
(C90), 51.0 (C140), 63.0 (C1), 82.4 (C170), 113.8 (C20),
116.3 (C40), 127.0 (C10), 132.2 (C100), 144.0 (C50),
155.9 (C30); LRMS calcd for C25H39O3–H2O [M+H-
H2O]+ 369.2.
4.1.5. Procedure for amidation of 19 and 21 (synthesis of
22 and 23). A solution of 19 or 21 (110 mg, 0.284 mmol)
and Jones’ reagent (0.22 mL, 0.594 mmol) in acetone
(36 mL) was stirred for 20 min at rt. The reaction was
quenched by addition of isopropanol (2 mL) and ace-
tone was evaporated under reduced pressure at rt. Water
(15 mL) was added to the slurry and the aq phase was
extracted with EtOAc. The combined organic layer
was dried over MgSO4 and evaporated. To a solution
of the crude acid in dry CH2Cl2 (28 mL) and tributyl-
amine (0.13 mL, 0.54 mmol) was added isobutylchloro-
formate (0.065 mL, 0.675 mmol) at ꢀ10 ꢁC and the
mixture was stirred for 40 min at ꢀ10 ꢁC. N-Methylbu-
tylamine (0.615 mL, 5.175 mmol) was then added slowly
at ꢀ10 ꢁC and the mixture was stirred for 3 h at rt. The
mixture was poured into an ice-cold aq 1 M HCl, the aq
phase was extracted with CH2Cl2, the organic phase was
washed with saturated aq NaHCO3, dried over MgSO4
and evaporated. The crude compound was next purified
by flash chromatography (hexanes/EtOAc, 70:30) as elu-
ent to give the desired compound 22 or 23 (59% and 50%
yield, respectively).
4.1.6.1.
N-Butyl-N-methyl-7-(30-hydroxy-170-oxo-
estra-10,30,50(100)-trien-60a-yl)-heptanamide (11). White
amorphous solid (96%). IR (film) m 3276 (OH), 1738
(C@O, ketone), 1621 (C@O, amide); 1H NMR
(400 MHz, CDCl3) d 0.88 (s, 180-CH3), 0.92 and 0.95
(2t, J = 7.2 Hz, CH2CH3), 1.20–2.40 (m, 28H, CH
and CH2 of steroid skeleton and chain), 2.50 (dd,
J2 = 18.7 Hz, J1 = 8.6 Hz, 160b-CH), 2.91 (m, 60b-
CH), 2.95 and 2.99 (2s, NCH3), 3.27 and 3.40 (2m,
NCH2), 6.67 (dd, J2 = 8.4 Hz, J1 = 2.4 Hz, 20-CH),
6.93 (dd, J2 = 7.8 Hz, J1 = 2.2 Hz, 40-CH), 7.12 (d,
J = 8.5 Hz, 10-CH); 13C NMR (75 MHz, CDCl3) d
13.8 (C180), 13.9 (C400), 19.9 and 20.0 (C300), 21.6
(C150), 24.4 and 24.7 (C3), 26.0 (C110), 27.9, 28.0
and 28.5 (C4–C6), 29.4 and 30.6 (C200), 31.5 (C120),
32.7 and 33.3 (C2, C80), 33.7 and 35.9 (NCH3), 35.3
(C70), 35.5 (C160), 37.8 (C7), 38.2 (C60), 44.0 (C90),
47.7 (C100), 47.8 (C130), 47.8 and 50.0 (C100), 50.5
(C140), 112.9 (C20), 114.3 (C40), 126.1 (C10), 131.7
(C100), 141.3 (C50), 154.8 (C30), 173.4 (C1), 221.2
(C170); LRMS calcd for C30H46NO3 [M+H]+ 468.3;
HRMS calcd for C30H46NO3 [M+H]+ 468.34722,
found 468.34686; HPLC purity of 89% (tR =
31.45 min).
4.1.5.1. N-Butyl-N-methyl-7-(30-isobutyloxycarbonyl-
oxy-170-oxo-estra-10,30,50(100)-trien-60a-yl)-heptanamide
(22). Yellow oil (59%). IR (film) m 1760 and 1739 (C@O,
1
carbonate and ketone), 1620 (C@O, amide); H NMR
(400 MHz, CDCl3) d 0.88 (s, 180-CH3), 0.93 (qapp
,
4.1.6.2.
N-Butyl-N-methyl-7-(30-hydroxy-170-oxo-
J = 7.4 Hz, CH2CH3), 1.00 (d, J = 6.7 Hz, CH(CH3)2),
1.00–2.40 (m, 29H, CH and CH2 of steroid skeleton,
chain and isopropylcarbonate), 2.51 (dd, J2 = 18.3 Hz,
J1 = 8.6 Hz, 160b-CH), 2.94 (m, 60b-CH), 2.91 and 2.96
(2s, NCH3), 3.25 and 3.35 (2m, NCH2), 4.03 (d,
J = 6.7 Hz, OCH2CH), 6.95 (dd, J2 = 8.5 Hz,
J1 = 2.3 Hz, 20-CH), 7.06 (d, J = 2.1 Hz, 40-CH), 7.27
(d, J = 6.7 Hz, 10-CH); LRMS calcd for C35H54NO5
[M+H]+ 568.3.
estra-10,30,50(100)-trien-60b-yl)-heptanamide (12). White
amorphous solid (96%). IR (film) m 3265 (OH), 1734
(C@O, ketone), 1621 (C@O, amide); 1H NMR
(400 MHz, CDCl3) d 0.91 (s, 180-CH3), 0.93 and 0.95
(2t, J = 7.2 Hz, CH2CH3), 1.20–2.40 (m, 28 H, CH and
CH2 of steroid skeleton and chain), 2.50 (dd,
J2 = 18.8 Hz, J1 = 8.2 Hz, 160b-CH), 2.76 (m, 60a-CH),
2.94 and 2.99 (2s, NCH3), 3.28 and 3.37 (2m, NCH2),
6.67 (dd, J2 = 8.4 Hz, J1 = 2.5 Hz, 20-CH), 6.81 (s, 40-
CH), 7.12 (d, J = 8.4 Hz, 10-CH); 13C NMR (75 MHz,
CDCl3) d 13.9 (C180 and C400), 19.9 and 20.0 (C300),
21.6 (C150), 24.8 and 25.0 (C3), 25.6 (C110), 26.9 and
28.4 (2C) (C4–C6), 29.4 and 30.6 (C200), 30.8 (C70),
31.5 (C120), 32.5 and 33.2 (C2), 33.8 and 35.8 (NCH3),
4.1.5.2. N-Butyl-N-methyl-7-(30-isobutyloxycarbonyl-
oxy-170-oxo-estra-10,30,50(100)-trien-60b-yl)-heptanamide
(23). Yellow oil (50%). IR (film) m 1761 and 1740 (C@O,
1
carbonate and ketone), 1624 (C@O, amide); H NMR

C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
723
33.9 (C80), 35.5 (C160), 36.6 (C7), 37.2 (C60), 44.1 (C90),
47.8 and 49.9 (C100), 48.2 (C130), 50.3 (C140), 112.9 (C20),
115.5 (C40), 125.8 (C10), 131.0 (C100), 142.7 (C50), 154.6
(C30), 173.2 (C1), 221.2 (C170); LRMS calcd for
C30H46NO3 [M+H]+ 468.4; HRMS calcd for
C30H46NO3 [M+H]+ 468.34722, found 468.34685;
HPLC purity of 77% for 12 (tR = 31.24 min).
4.1.8. Synthesis of N-butyl-N-methyl-7-(30-benzyloxy-
170-oxo-estra-10,30,50(100)-trien-60b-yl)-heptanamide (29).
A mixture of 12 (41 mg, 0.087 mmol), cesium carbonate
(88 mg, 0.27 mmol) and benzyl bromide (0.06 mL,
0.50 mmol) in acetonitrile (1.2 mL) was stirred for
2.5 h at reflux. The reaction mixture was cooled and
diluted with EtOAc. Then the organic layer was washed
with water, dried over MgSO4, and evaporated. The
crude residue was purified by chromatography (hex-
anes/EtOAc, 90:10 then 30:70) to afford 29 (50 mg,
quantitative yield) as yellow oil. IR (film) m 1738
(C@O, ketone), 1644 (C@O, amide); 1H NMR
(300 MHz, CDCl3) d 0.92 (s, 180-CH3), 0.91 (m,
CH2CH3), 1.20–2.40 (m, 28H, CH and CH2 of steroid
skeleton and chain), 2.51 (dd, J2 = 18.2 Hz,
J1 = 8.1 Hz, 160b-CH), 2.77 (m, 60a-CH), 2.91 and 2.96
(2s, NCH3), 3.26 and 3.36 (2m, NCH2), 5.04 (s,
OCH2), 6.79 (m sharp, 20-CH and 40-CH), 7.19 (d,
J = 9.2 Hz, 10-CH), 7.38 (m, 5H-phenyl); 13C NMR
(75 MHz, CDCl3) d 13.8, 13.9, 20.0, 21.6, 25.4, 25.6,
28.0, 29.5, 29.6, 29.8, 30.5, 31.6, 32.9, 33.4 (2C), 33.5,
35.4, 35.8 (2C), 37.8, 38.1, 44.4, 47.8, 48.2, 49.7, 50.2,
70.0, 112.0, 115.4, 125.9, 127.5 (2C), 127.9, 128.6 (2C),
132.1, 137.2, 143.0, 156.9, 173.0, 220.9; LRMS calcd
for C37H52NO3 [M+H]+ 558.3.
4.1.7. Procedure for reduction of 11 and 12 (synthesis of
13 and 14). NaBH4 (4 mg, 0.105 mmol) was added to a
cooled (0 ꢁC) solution of 11 or 12 (34 mg, 0.073 mmol)
in MeOH (2 mL). After the mixture was stirred for
0.5 h at 0 ꢁC, the reaction was quenched by addition
of water and extraction was performed with CH2Cl2.
The organic phase was dried over Na2SO4, and evapo-
rated to dryness. The crude residue was purified by chro-
matography (hexanes/EtOAc, 50:50) to afford 13 or 14
(91% and 99% yield, respectively).
4.1.7.1. N-Butyl-N-methyl-7-(30,17b0-dihydroxy-estra-
10,30,50(100)-trien-60a-yl)-heptanamide (13). White amor-
phous solid (91%). IR (film) m 3298 (OH), 1622 (C@O,
1
amide); H NMR (400 MHz, CD3OD) d 0.74 (s, 180-
CH3), 0.93 and 0.97 (2t, J = 7.3 Hz, CH2CH3), 1.00–
2.40 (m, 29H, CH and CH2 of steroid skeleton and
chain), 2.86 (m, 60b-CH), 2.89 and 3.02 (2s, NCH3),
3.32 (m, NCH2), 3.65 (t, J = 8.6 Hz, 170a-CH), 6.53
(dd, J2 = 8.4 Hz, J1 = 2.5 Hz, 20-CH), 6.67 (d,
J = 2.3 Hz, 40-CH), 7.07 (d, J = 8.5 Hz, 10-CH); 13C
NMR (75 MHz, CD3OD) d 11.6 (C180), 14.2 (C400),
20.9 and 21.0 (C300), 24.1 (C150), 26.3 and 26.7 (C3),
26.9 and 30.4 (2C) (C4–C6), 27.7 (C110), 30.7 (C160),
30.8 and 31.7 (C200), 33.7 (C2), 33.9 and 36.0 (NCH3),
34.4 (C2), 35.4 (C70), 37.9 (C120), 38.9 (C7), 39.1
(C60), 40.1 (C80), 44.1 (C130), 45.0 (C90), 48.6 and 51.0
(C100), 51.5 (C140), 82.5 (C170), 113.5 (C20), 115.0
(C40), 126.9 (C10), 133.4 (C100), 142.8 (C50), 156.1
(C30), 175.5 (C1); LRMS calcd for C30H48NO3
[M+H]+ 470.3; HRMS calcd for C30H48NO3 [M+H]+
470.36287, found 470.36383; HPLC purity of 89%
(tR = 31.06 min).
4.1.9. Synthesis of N-butyl-N-methyl-7-(30-benzyloxy-20-
iodo-170-oxo-estra-10,30,50(100)-trien-60b-yl)-heptanamide
(30). To a solution of 29 (58 mg, 0.104 mmol), NaHCO3
(49 mg, 0.585 mmol) and silver trifluoroacetate (28 mg,
0.129 mmol) in dry CH2Cl2 (0.42 mL) was added iodine
(33 mg, 0.129 mmol) dropwise at ꢀ40 ꢁC, and the mix-
ture was stirred for 4 h at ꢀ40 ꢁC. The reaction was
quenched by addition of Et3N, then the mixture was
poured into a column of silica gel and flash chromatog-
raphy (hexanes/EtOAc, 90:10 then 30:70) gave the 4-
iodo product (2 mg, 3% yield) and the desired 2-iodo
product 30 (56 mg, 85 % yield) as yellow oil. IR (film)
1
m 1738 (C@O, ketone), 1644 (C@O, amide); H NMR
(300 MHz, CDCl3) d 0.92 (s, 180-CH3), 0.91 (m,
CH2CH3), 1.20–2.40 (m, 28 H, CH and CH2 of steroid
skeleton and chain), 2.52 (dd, J2 = 19.0 Hz,
J1 = 8.0 Hz, 160b-CH), 2.75 (m, 60a-CH), 2.91 and
2.96 (2s, NCH3), 3.25 and 3.35 (2m, NCH2), 5.13 (s,
OCH2), 6.62 (s, 40-CH), 7.32 (d, J = 7.1 Hz, 400-CH of
phenyl), 7.38 (tapp, J = 7.3 Hz, 200- and 600-CH of phen-
yl), 7.51 (dapp, J = 7.3 Hz, 300- and 500-CH of phenyl),
7.65 (s, 10-CH); 13C NMR (75 MHz, CDCl3) d 13.9
(2C), 20.1, 21.5, 25.0, 25.4, 25.6, 27.9, 29.5, 29.7, 30.6,
31.5, 32.9, 33.2, 33.4, 33.5 (2C), 35.3, 35.8 (2C), 37.9,
38.2, 44.2, 47.4, 48.1, 49.7, 50.1, 71.0, 83.9, 113.6,
127.0 (2C), 127.8, 128.5 (2C), 134.4, 136.1, 136.8,
143.2, 155.2, 172.7, 220.6; LRMS calcd for C37H51INO3
[M+H]+ 684.2.
4.1.7.2. N-Butyl-N-methyl-7-(30,17b0-dihydroxy-estra-
10,30,50(100)-trien-60b-yl)-heptanamide (14). White amor-
phous solid (99%). IR (film) m 3272 (OH), 1618
1
(C@O, amide); H NMR (400 MHz, CD3OD) d 0.78
(s, 180-CH3), 0.93 and 0.97 (2t, J = 7.1 Hz, CH2CH3),
1.10-2.45 (m, 29H, CH and CH2 of steroid skeleton
and chain), 2.69 (m, 60a-CH), 2.90 and 3.03 (2s,
NCH3), 3.36 (m, NCH2), 3.65 (t, J = 8.6 Hz, 170a-
CH), 6.54 (m, 20-CH and 40-CH), 7.06 (d, J = 9.0 Hz,
13
10-CH); C NMR (75 MHz, CD3OD) d 11.8 (C180),
14.2 (C400), 21.0 (C300), 24.1 (C150), 26.3 and 26.7
(C3), 27.4 (C110), 28.9 and 30.4 (2C) (C4–C6), 30.6
(C200), 30.7 (C160), 31.7 (C70), 33.7 (C2), 33.9 and
36.0 (NCH3), 34.4 (C2), 35.3 (C80), 38.0 (C120), 39.1
(C60), 39.4 (C7), 44.6 (C130), 45.9 (C90), 48.6 and 51.0
(C100), 51.1 (C140), 82.4 (C170), 113.9 (C20), 116.3
(C40), 127.0 (C10), 132.2 (C100), 144.0 (C90), 155.9
(C30), 175.5 (C1); LRMS calcd for C30H48NO3
[M+H]+ 470.3; HRMS calcd for C30H48NO3 [M+H]+
470.36287, found 470.36402; HPLC purity of 95%
(tR = 30.91 min).
4.1.10. Synthesis of N-butyl-N-methyl-7-(30-benzyloxy-
20-methoxy-170-oxo-estra-10,30,50(100)-trien-60b-yl)-hep-
tanamide (31). Na (88 mg, 3.8 mmol) was dissolved in
MeOH (4 mL) under an inert atmosphere at rt. Then
DMF (4 mL), 30 (121 mg, 0.190 mmol) in MeOH/
DMF (1:1, 6 mL) and CuI (28 mg, 0.195 mmol) were
added. The reaction mixture was warmed at 120 ꢁC
overnight, diluted with EtOAc, and quenched by addi-

724
C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
tion of water. The organic layer was washed with water,
dried over MgSO4 and evaporated. The crude residue
was purified by chromatography (hexanes/EtOAc,
60:40) to afford 31 (75 mg, 66% yield) as yellow oil. IR
and 21.0 (C300), 24.1 (C150), 26.3 and 26.6 (C3), 27.6
(C110), 28.9 and 30.4 (2C) (C4–C6), 30.6 (C200), 30.7
(C160), 31.6 (C70), 33.8 and 34.4 (C2), 33.9 and 36.0
(NCH3), 35.1 (C80), 38.1 (C120), 38.5 (C60), 39.5
(C7), 44.6 (C130), 46.3 (C90), 48.6 and 51.0 (C100),
51.0 (C140), 56.5 (OCH3), 82.4 (C170), 109.9 (C10),
116.8 (C40), 132.3 (C50), 135.4 (C100), 145.3 (C30),
147.0 (C20), 175.5 (C1); LRMS calcd for C31H50NO4
[M+H]+ 500.3; HRMS calcd for C31H50NO3 [M+H]+
500.37344, found 500.37253; HPLC purity of 82%
(tR = 31.17 min).
1
(film) m 1738 (C@O, ketone), 1644 (C@O, amide); H
NMR (300 MHz, CDCl3) d 0.92 (s, 180-CH3), 0.93 (m,
CH2CH3), 1.20–2.45 (m, 28H, CH and CH2 of steroid
skeleton and chain), 2.50 (dd, J2 = 19.0 Hz,
J1 = 8.1 Hz, 160b-CH), 2.67 (m, 60a-CH), 2.91 and 2.96
(2s, NCH3), 3.25 and 3.36 (2t, J = 7.4 Hz, NCH2), 3.86
(s, OCH3), 5.13 (d of AB system, J = 3.3 Hz, OCH2),
6.66 (s, 40-CH), 6.81 (s, 10-CH), 7.29 (d, J = 7.3 Hz, 400-
CH of phenyl), 7.36 (tapp, J = 7.2 Hz, 200- and 600-CH
of phenyl), 7.45 (dapp, J = 7.0 Hz, 300- and 500-CH of
phenyl); 13C NMR (75 MHz, CDCl3) d 13.9, 14.0,
20.0, 21.5, 25.1, 25.5, 25.9, 28.0, 29.5, 29.7, 29.9, 30.6,
31.6, 32.9, 33.2, 33.4, 33.6, 35.3, 35.8 (2C), 37.3, 38.3,
44.8, 47.5, 48.2, 49.8, 50.2, 56.1, 71.3, 109.0, 115.4,
127.3 (2C), 127.7, 128.5 (2C), 132.1, 133.6, 137.5,
146.2, 147.7, 172.9, 220.9; LRMS calcd for C38H54NO4
[M+H]+ 588.3.
4.2. Cell culture
4.2.1. HEK-293 intact cells (overexpressing type 1, 7 or
12 17b-HSD). Stable transfected HEK-293 cells over-
expressing isoform 1, 7 or 12 were provided by Dr.
Van Luu-The. Cells were maintained in 75 cm2 culture
flask at 37 ꢁC under 5% CO2 humidified atmosphere in
minimum essential medium (MEM) containing non-es-
sential amino acids (0.1 mM), L-glutamine (2 mM), sodi-
um pyruvate (1 mM), 10% foetal bovine serum (FBS),
penicillin (100 IU/mL), streptomycin (100 lg/mL) and
geneticin (G418 sulfate) (Gibco, Burlington, On, Cana-
da) (700 lg/mL).
4.1.11. N-butyl-N-methyl-7-(30-benzyloxy-17b0-hydroxy-
20-methoxy-estra-10,30,50(100)-trien-60b-yl)-heptanamide
(32). The procedure reported above for the synthesis of
13 and 14 was used for the reduction of 31 into 32.
Yellow oil (99%). IR (film) m 3419 (OH), 1633 (C@O,
4.2.2. Breast cancer cell lines. Two ER-positive breast
cancer cell lines, T-47D and MCF-7, were obtained from
the American Type Culture Collection (ATCC) and
maintained in 75 cm2 culture flask at 37 ꢁC under 5%
CO2 humidified atmosphere. The T-47D cells were
grown in RPMI medium supplemented with 10%
FBS, L-glutamine (2 mM), penicillin (100 IU/mL),
streptomycin (100 lg/mL) and estradiol (1 nM). The
MCF-7 cells were propagated in DME-F12 medium
supplemented with 5% FBS, glutamine (2 mM), penicil-
lin (100 IU/mL), streptomycin (100 lg/mL) and estradi-
ol (1 nM).
1
amide); H NMR (400 MHz, CDCl3) d 0.80 (s, 180-
CH3), 0.92 and 0.95 (2t, J = 7.3 Hz, CH2CH3), 1.10–
2.35 (m, 29 H, CH and CH2 of steroid skeleton and
chain), 2.62 (m, 60a-CH), 2.91 and 2.97 (2s, NCH3),
3.26 and 3.36 (2t, J = 7.5 Hz, NCH2), 3.74 (t,
J = 8.5 Hz, 170a-CH), 3.86 (s, OCH3), 5.12 (d of AB
system, J = 5.2 Hz, OCH2), 6.65 (s, 40-CH), 6.82 (s,
10-CH), 7.29 (d, J = 7.2 Hz, 400-CH of phenyl), 7.36
(tapp, J = 7.3 Hz, 200- and 600-CH of phenyl), 7.45 (dapp
,
J = 7.2 Hz, 300- and 500-CH of phenyl); 13C NMR
(75 MHz, CDCl3) d 11.2, 13.9, 20.0, 20.1, 23.1, 23.9,
25.1, 25.5, 26.2, 28.0, 29.4, 29.5, 29.6, 30.6 (2C), 33.0,
33.4, 33.5, 33.6, 35.3, 36.7, 37.4, 38.3, 43.4, 44.8,
47.4, 49.8 (2C), 56.1, 71.3, 81.8, 109.1, 115.4, 127.4,
127.7 (2C), 128.5 (2C), 132.7, 133.9, 137.5, 146.1,
147.6, 172.9; LRMS calcd for C38H56NO4 [M+H]+
590.3.
4.3. Cell proliferation assay
Cell growth was measured using CellTitter 96ꢂ AQueous
Solution Cell Proliferation Assay (Promega, Nepean,
ON, Canada) following the manufacturer’s instructions.
T-47D and MCF-7 cells were resuspended in their
respective medium supplemented with insulin (50 ng/
mL) and 5% dextran-coated charcoal treated FBS to re-
move 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) in triplicate.
After 48 h, the medium was changed with an appropri-
ate dilution of the inhibitor in growth medium and
was replaced every 2 days. Cells were grown in absence
or presence of inhibitors for 10 days in triplicate.
4.1.12. Synthesis of N-butyl-N-methyl-7-(30,17b0-dihy-
droxy-20-methoxy-estra-10,30,50(100)-trien-60b-yl)-hep-
tanamide (15). A mixture of 32 (24 mg, 0.041 mmol) and
Pd(OH)2 (10% in weight, 3 mg) in EtOAc (0.6 mL) un-
der hydrogen atmosphere was stirred for 12 h at rt.
The palladium catalysis was removed by filtration on
Celite and washed with EtOAc. The filtrate was evapo-
rated and the crude product was purified by chroma-
tography (hexanes/EtOAc/MeOH, 67:28:5) to afford
15 (19.6 mg, 98% yield) as yellow amorphous solid.
IR (film) m 3350 (OH), 1620 (C@O, amide); 1H
NMR (400 MHz, CD3OD) d 0.79 (s, 180-CH3), 0.93
and 0.97 (2t, J = 7.1 Hz, CH2CH3), 1.10–2.40 (m,
29 H, CH and CH2 of steroid skeleton and chain),
2.64 (m, 60a-CH), 2.90 and 3.03 (2s, NCH3), 3.35
(m, NCH2), 3.66 (t, J = 8.6 Hz, 170a-CH), 3.80 (s,
OCH3), 6.55 (s, 40-CH), 6.79 (s, 10-CH); 13C NMR
(75 MHz, CD3OD) d 11.8 (C180), 14.2 (C400), 20.9
4.4. Assay of inhibition of isoforms 1, 7 or 12 of 17b-HSD
4.4.1. Type 1 17b-HSD (homogenated HEK-293 cells).
Compounds were evaluated for their ability to inhibit
the reductive transformation of E1 (100 nM) into E2
by type 1 17b-HSD (an homogenate of HEK-293 cells)
in the presence of cofactor NADH according to an
established procedure.18

C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
725
6. Luu-The, V.; Tremblay, P.; Labrie, F. Mol. Endocrinol.
2006, 20, 437–443.
7. Theobald, A. J. Int. J. Clin. Pract. 2000, 54, 665.
8. Henderson, B. E.; Ross, R.; Bernstein, L. Cancer Res.
1988, 48, 246.
9. Lippman, M. E.; Dickson, R. B.; Kasid, A.; Gelmann, E.;
Davidson, N.; McManaway, M.; Huff, K.; Bronzert, D.;
Bates, S.; Swain, S. J. Steroid Biochem. 1986, 24, 147.
10. Chetrite, G. S.; Cortes-Prieto, J.; Philippe, J. C.; Wright, F.;
Pasqualini, J. R. J. Steroid Biochem. Mol. Biol. 2000, 72, 23.
11. Speirs, V.; Green, A. R.; Walton, D. S.; Kerin, M. J.; Fox,
J. N.; Carleton, P. J.; Desai, S. B.; Atkin, S. L. Br. J.
Cancer 1998, 78, 1421.
4.4.2. Type 1 17b-HSD (intact HEK-293 cells). Com-
pounds were evaluated for their ability to inhibit the
reductive transformation of E1 (60 nM) into E2 by type
1 17b-HSD (transfected HEK-293 intact cells). Trans-
fected cells were plated at 5000 cells/well in a 24-well
plate. For the inhibitory activity assay, ethanolic solu-
tions of inhibitors were immediately added to each well
and incubated for 24 h. After incubation, [4-14C]-E1
(American Radiolabeled Chemicals Inc., St. Louis,
MO, USA) was added and incubated for 15 min. After
incubation, culture medium was removed and labelled
steroids (E1 and E2) were extracted and quantified
according to an established procedure.42
12. Gunnarsson, C.; Hellqvist, E.; Stal, O. Br. J. Cancer 2005,
92, 547.
13. Gunnarsson, C.; Olsson, B. M.; Stal, O. Cancer Res. 2001,
61, 8448.
4.4.3. Type 1 17b-HSD (intact T-47 D cells). T-47D cells
were seeded in 24-well plate at 3 · 104 cells/well in pro-
liferation assay medium. After 48 h, 60 nM of [4-14C]-
E1 and an ethanolic solution of inhibitor (0.5 %, v/v)
at concentrations of 0.1, 1 and 10 lM were added to
freshly changed culture medium and the cells were incu-
bated for 24 h. After incubation, labelled steroids (E1
and E2) were extracted and quantified according to an
established procedure.42
14. Miyoshi, Y.; Ando, A.; Shiba, E.; Taguchi, T.; Tamaki,
Y.; Noguchi, S. Int. J. Cancer 2001, 94, 685.
15. Suzuki, T.; Moriya, T.; Ariga, N.; Kaneko, C.; Kanazawa,
M.; Sasano, H. Br. J. Cancer 2000, 82, 518.
16. Poirier, D. Curr. Med. Chem. 2003, 10, 453.
17. Poirier, D.; Dionne, P.; Auger, S. J. Steroid Biochem. Mol.
Biol. 1998, 64, 83.
18. Tremblay, M. R.; Boivin, R. P.; Luu-The, V.; Poirier, D.
J. Enzyme Inhib. Med. Chem. 2005, 20, 153.
´
19. Berthiaume, D. In Developpement d’inhibiteurs non oes-
´
4.4.4. Type 7 17b-HSD (intact cells). Compounds were
evaluated for their ability to inhibit the reductive trans-
formation of E1 (60 nM) into E2 by type 7 17b-HSD
(transfected HEK-293 intact cells) according to an
established procedure.43
´
´
´
trogeniques de la 17b-hydroxystero¨ıde deshydrogenase type
1; Memoire de maıtrise. In Faculte des etudes superieures;
Universite Laval, Quebec: Canada, 2000; pp 1–102.
20. Skaddan, M. B.; Wust, F. R.; Katzenellenbogen, J. A.
J. Org. Chem. 1999, 64, 8108.
´
ˆ
´
´
´
´
´
21. Tedesco, R.; Fiaschi, R.; Napolitano, E. Synthesis 1995,
1493.
22. Wang, C.L.J. U.S. Patent 4,456,613, 1984; Chem. Abstr.
1984, 102, 166531.
23. Takeda, N.; Imamoto, T. Org. Synth. 2004, 10, 200.
24. Li, X.; Singh, S. M.; Labrie, F. Tetrahedron Lett. 1994, 35,
1157.
25. Peters, R. H.; Crowe, D. F.; Avery, M. A.; Chong, W. K.;
Tanabe, M. J. Med. Chem. 1989, 32, 2306.
26. Tedesco, R.; Fiaschi, R.; Napolitano, E. J. Org. Chem.
1995, 60, 5316.
27. Droescher, P.; Schwarz, S.; Ring, S.; Menzenbach, B.
Synthesis of Estradiol Derivatives Functionalised at C-6 by
Substituted Aryl and Alkyl Groups Mediated by Transi-
tion Metals. In 11th International Conference on Organic
Synthesis; Amsterdam, Netherlands, June 30–July 4, 1996.
28. Claridge, T. Tetrahedron Org. Chem. Ser. 1999, 19, 382.
29. Dionne, P.; Ngatcha, B. T.; Poirier, D. Steroids 1997, 62,
674.
4.4.5. Type 12 17b-HSD (intact cells). Compounds were
evaluated for their ability to inhibit the reductive trans-
formation of E1 (60 nM) into E2 by type 12 17b-HSD
(transfected HEK-293 intact cells). Transfected cells
were plated at 1.5 · 105 cells/well in a 24-well plate.
For the inhibitory activity assay, ethanolic solutions of
inhibitor and [4-14C]-E1 were added to freshly changed
culture medium and incubated for 24 h. After incuba-
tion, culture medium was removed and labelled steroids
(E1 and E2) were extracted and quantified according to
an established procedure.42
Acknowledgments
The authors thank the Canadian Institutes of Health
Research (CIHR) for financial support. They also thank
Steeves Potvin and Guy Reimnitz for helpful discus-
30. Poirier, D.; Labrie, C.; Merand, Y.; Labrie, F. J. Steroid
Biochem. Mol. Biol. 1991, 38, 759.
31. Dionne, P.; Singh, S. M.; Labrie, F. Steroids 1994, 59, 493.
32. Sam, K.; Labrie, F.; Poirier, D. Eur. J. Med. Chem. 2000,
35, 217.
´
sions. Careful reading of the manuscript by Sylvie Met-
hot is also greatly appreciated.
33. Labrie, F.; Singh, S.; Gauthier, S.; Frechette, Y.; Chenard,
S.; Breton, R. WO 2005/066194 A1, 2005; Chem. Abstr.
2005, 143, 153578.
References and notes
34. Chen, S. H.; Luo, G. R.; Wu, X. S.; Chen, M.; Zhao, H.
M. Steroids 1986, 47, 63.
35. von Angerer, E. In The Estrogen Receptor as a Target for
Rational Drug Design; R.G. Landes Company: George-
town, 1995; pp 1–237.
36. Duncan, L. J.; Reed, M. J. J. Steroid Biochem. Mol. Biol.
1995, 55, 565.
37. Kuiper, G. G.; Carlsson, B.; Grandien, K.; Enmark, E.;
Haggblad, J.; Nilsson, S.; Gustafsson, J. A. Endocrinology
1997, 138, 863.
1. Jemal, A.; Murray, T.; Ward, E.; Samuels, A.; Tiwari, R.
C.; Ghafoor, A.; Feuer, E. J.; Thun, M. J. CA Cancer J.
Clin. 2005, 55, 10.
2. Labrie, F.; Luu-The, V.; Labrie, C.; Belanger, A.; Simard,
J.; Lin, S. X.; Pelletier, G. Endocr. Rev. 2003, 24, 152.
3. Payne, A. H.; Hales, D. B. Endocr. Rev. 2004, 25, 947.
4. Luu-The, V. J. Steroid Biochem. Mol. Biol. 2001, 76, 143.
5. Mindnich, R.; Moller, G.; Adamski, J. Mol. Cell. Endo-
crinol. 2004, 218, 7.

726
C. Cadot et al. / Bioorg. Med. Chem. 15 (2007) 714–726
38. Matthews, J.; Celius, T.; Halgren, R.; Zacharewski, T. J.
Steroid Biochem. Mol. Biol. 2000, 74, 223.
41. Merriam, G. R.; MacLusky, N. J.; Picard, M. K.;
Naftolin, F. Steroids 1980, 36, 1.
39. Cushman, M.; He, H. M.; Katzenellenbogen, J. A.;
Varma, R. K.; Hamel, E.; Lin, C. M.; Ram, S.; Sachdeva,
Y. P. J. Med. Chem. 1997, 40, 2323.
40. Liu, Z. J.; Zhu, B. T. J. Steroid Biochem. Mol. Biol. 2004,
88, 265.
42. Ngatcha, B. T.; Laplante, Y.; Labrie, F.; Luu-The,
V.; Poirier, D. Mol. Cell. Endocrinol. 2006, 248,
225.
43. Bellavance, E.; Luu-The, V.; Poirier, D. Lett. Drug Des.
Discov. 2004, 1, 194.
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