Synthesis and preliminary evaluation of a modified estradiol-core bearing a fused γ-lactone as non-estrogenic inhibitor of 17β-hydroxysteroid dehydrogenase type 1

Article abstract of DOI:10.1016/j.bmcl.2011.06.110

A non-estrogenic inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) was designed based on a modified 3-hydroxy-estra-1,3,5(10)- triene core having an additional five-member lactone ring and a benzamide group. The inhibitor was synthesized, fu

Full text of DOI:10.1016/j.bmcl.2011.06.110

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5510–5513
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and preliminary evaluation of a modified estradiol-core bearing
a fused c-lactone as non-estrogenic inhibitor of 17b-hydroxysteroid
dehydrogenase type 1
⇑
Étienne Ouellet, Diana Ayan, Donald Poirier
Laboratory of Medicinal Chemistry, CHUQ (CHUL) – Research Center and Laval University, 2705 Laurier Blvd., Québec, Canada G1V 4G2
a r t i c l e i n f o
a b s t r a c t
Article history:
A non-estrogenic inhibitor of 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1) was designed based
on a modified 3-hydroxy-estra-1,3,5(10)-triene core having an additional five-member lactone ring and a
benzamide group. The inhibitor was synthesized, fully characterized and tested for its ability to inhibit
the enzyme activity. Estrogenicity was also investigated and tested on estrogen-dependent T-47D cell
line. Interestingly, this steroid derivative showed inhibitory potency towards 17b-HSD1 and did not pres-
ent residual unwanted estrogenic activity.
Received 15 April 2011
Accepted 24 June 2011
Available online 30 June 2011
Key words:
17b-Hydroxysteroid dehydrogenase
Steroid
Ó 2011 Elsevier Ltd. All rights reserved.
Estradiol
Inhibitor
Enzyme
Breast cancer
17b-Hydroxysteroid dehydrogenase type 1 (17b-HSD1) is a ste-
roidogenic enzyme that catalyzes the reduction of estrone (E1) into
estradiol (E2), the most potent estrogen. Therefore, inhibition of
17b-HSD1 is a suitable approach for the treatment of estrogen-
dependent diseases such as breast cancer (Fig. 1).1–6 Moreover, this
enzyme intervenes at the very last step in the production of E2,
meaning that its inhibition does not interfere with the production
of other essential steroids (i.e., corticosteroid and mineralocorti-
coid).7 The development of therapeutic inhibitors of this enzyme
is mainly lacking because the majority of inhibitors have residual
estrogenic activity. Recently, our research group developed a po-
tent inhibitor for 17b-HSD1; namely CC-156 (Fig. 2).8,9 Unfortu-
nately, this compound revealed to be slightly estrogenic when
tested on estrogen-dependent cell lines. In fact, due to its E2 core,
activation of estrogen receptor occurs, thereby causing cell prolif-
eration. Nevertheless, the great potency of this inhibitor has led
researchers to investigate the interactions and bonding, which re-
sulted in crystallization of CC-156 in the active site of 17b-HSD1 by
Mazumdar et al.10 Thereby, they highlighted various significant
interactions responsible for the high affinity between the inhibitor
and the enzyme. More precisely, the crystallized binary and ter-
nary complexes showed strong interactions between the 3-phenol
of the inhibitor and amino acids His221 and Glu282 of the enzyme,
between 17-OH and Ser142 and, more importantly, between the
benzamide moiety and Phe192 and Asn152.
In order to develop a novel inhibitor of 17b-HSD1 based on E2
nucleus deprived of residual estrogenic activity, we considered
using a modified E2 core. The new scaffold employed consists of
a 3-hydroxy-estra-1,3,5(10)-triene nucleus with an additional
substituted E-ring lactone fused at positions 16 and 17 (Fig. 2).
The addition of a substituted five-member lactone moiety is more
likely to eliminate or reduce estrogenicity while keeping the cru-
cial interactions with 17b-HSD1. To validate our strategy we tar-
geted compound 1.
The synthetic methodology for the preparation of compound 1
is outlined in Scheme 1 starting from steroidal lactone 2. The
Estrogenic effects
Estrogen receptor
Cholesterol
Inhibitor
action
O
OH
17β-HSD1
H
H
H
NAD(P)H
H
H
H
HO
HO
Estrone (E1)
Estradiol (E2)
⇑
Corresponding author.
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
Figure 1. Key role of 17b-HSD1 in the synthesis of potent estrogen E2.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.110

É. Ouellet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5510–5513
5511
O
OH
O
E
C
D
H
NH2
O
A
B
H
H
HO
HO
NH2
O
Compound 1
CC-156
Figure 2. Structures of 17b-HSD1 inhibitors 1 and CC-156 (compound 4m in Ref. 8). The stereogenic centers at C-8, C-9 and C-14 are illustrated only for CC-156, but they are
the same for all other steroid derivatives reported in this paper. See Scheme 1 for the carbon numbering of 1.
O
O
O
O
a
Estrone (E1)
OH
O
TBSO
TBSO
NH2
2
3
b
18
1'
O
O
O
O
17
13
14
12
11
2'
OH
O
1
3'
9
16
10
c
2
4'
7'
15
8
9'
8'
5'
7
5
4
3
HO
HO
6
10'
NH2
NH2
6'
O
1
4
Scheme 1. Chemical synthesis of compound 1. Reagents and conditions: (a) NaHMDS (1.0 M/THF), meta-amidobenzaldehyde, THF, ꢀ78 °C, 2 h; (b) 2% HCl/MeOH, rt, 3.5 h; (c)
1 atm H2, 20% Pd(OH)2/C, THF/AcOH (1:1), rt, 18 h. Carbon numbering used for 1H and 13C NMR signal attribution is showed for compound 1.
synthesis of 2 has already been reported in literature11 and neces-
sitates three steps: (1) the 3-phenol protection of E1 using a t-
butyldimethylsilyl (TBS) ether, (2) the alkylation in position 16
using methyl bromoacetate, (3) the side chain isomerisation from
16a- to 16b-configuration, and (4) the reduction of the 17-ketone
with lactonisation. From the key intermediate 2, three steps are
phenol derivative 4 in a quantitative yield. The third step is the re-
moval of the benzylic alcohol by means of hydrogenolysis using
20% Pd(OH)2/C to afford compound 1 (50% yield). The synthesis
of 1 from the precursor 2 is achieved in an overall yield of 35%
for three steps. What keeps us from having better yield is the reac-
tion of hydrogenolysis of the benzyl alcohol, because it appears
that only one isomer is able to react. One hypothesis to explain
the difference in reactivity is a possible hydrogen bonding between
the benzylic alcohol and the lactone carbonyl, which stabilizes the
structure. Such hydrogen bonding is not favorable with the other
isomer due to steric hindrance between the benzamide substituent
and the lactonic steroidal core. Thereby, the hydrogen bond ex-
tends the C–OH bond, which facilitates its hydrogenolysis. The ste-
reochemistry of 1 at the new generated chiral center (position 20)
was kept from intermediate 3 and no isomerization happened dur-
ing the hydrogenolysis reaction. In fact, the nuclear Overhauser ef-
fect can be observed between protons of methyl 18 and H20
(Fig. 3b). This informs us that the benzamide chain is still in alpha
position (R configuration).
needed to obtain the target compound 1.
In the first step, an aldol addition using meta-amidobenzalde-
hyde afforded 3 in 70% yield. Different bases, temperatures and
reaction times were investigated in order to find the right condi-
tions. Fine-tuning for this reaction was needed because alkylation
in alpha position of a lactone is usually less efficient than alkyl-
ation in alpha position of a ketone. It is important to notice that
alkylation occurs only from one side of the steroidal core, thus
yielding one isomer only, as is represented in Scheme 1. This stere-
oselectivity is due to the orientation of the lactone in beta position
and the presence of the axial methyl in position 18. The stereo-
chemistry of the side chain (position 20) was determined by a series
of NMR experiments. The first step was to determine the signal of
the proton 20, which was done using correlation spectroscopy
(COSY). Clear interaction between proton 20 and the characteristic
benzylic proton (30) made it obvious for the attribution. Then, the
NOESY experiment permitted to see the interactions between the
characteristic C-18 methyl and the proton 20 (Fig. 3a). The two vis-
ible interactions are explained by the fact that two isomers of the
benzylic alcohol are generated during the addition, thus duplicat-
ing the signals. This nuclear Overhauser effect demonstrates that
the side chain is indeed in alpha position (S configuration).
The stereochemistry of 3 being confirmed, we next proceeded
with the second step which is the hydrolysis of the protecting
TBS group. This reaction took place in 2% HCl/MeOH to yield the
Once compound 1 was fully characterized,12 it was tested for its
ability to inhibit the transformation of [14C]-E1 (60 nM) into [14C]-
E2 catalyzed by 17b-HSD1 in intact T-47D cells. This cell line is
well recognized to exert a strong endogenous 17b-HSD1 activ-
ity.13,14 Compound 1 was tested at five concentrations (0.1, 0.3,
1, 3 and 5
lM) and the results are reported in Figure 4. It showed
75% of inhibition at a concentration of 5
lM compared to 90% for
CC-156. The difference of inhibition at this concentration already
shows us that the lead compound is more potent than compound
1. This gap is accentuated at lower concentrations. Indeed, at
0.3
lM, compound 1 displayed 20% of inhibition while the potent
CC-156 inhibited 85% of transformation. The difference of

5512
É. Ouellet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5510–5513
Figure 3. Partial 2D NOESY spectrum of compounds 3 (a) and 1 (b).
estrogenicity is explained to be due to the fused lactonic cycle in
combination with the benzamide moiety, which clearly reduces
the ability of compound 1 to bind to the estrogen receptor, thus
depriving it of any estrogenic activity.
In summary, we have designed and synthesized a steroidal 17b-
HSD1 inhibitor (compound 1) deprived of estrogenic activity by
using a new E2-derived scaffold. However, it showed less inhibi-
tory activity towards the enzyme than estrogenic lead compound
CC-156, but its potency could be increased by a judicious lactone
diversification. Further work reporting full details of the diversifi-
cation of the benzyl ring, including the chemistry and SAR study,
will be presented in a full paper in due course.
Figure 4. Inhibition of 17b-HSD1 by CC-156 and compound 1 at five concentrations
(0.1, 0.3, 1, 3 and 5 lM. Results are expressed as mean SEM of triplicate.
Acknowledgments
inhibition strongly suggests that compound 1 did not keep all of
the identified crucial interactions with 17b-HSD1, thus reducing
We wish to thank René Maltais and Jean-Yves Sancéau for help-
ful discussions and suggestions, Marie-Claude Trottier for technical
assistance with the NMR experiments and Micheline Harvey for
careful reading of the manuscript. We gratefully acknowledge the
Canadian Institutes of Health Research (CIHR) for operating grants.
its IC50 value to 1.0 lM compared to 44 nM for CC-156, as previ-
ously determined under the same conditions.8
The next step was to determine the presence or absence of
estrogenic activity. To do so, cell proliferative assays were carried
out on intact T-47D cells. This cell line is known to express the
estrogen receptor (ER).15 This means that molecules possessing
estrogenic activity will stimulate cell growth. Proliferative activity
of compound 1 and CC-156 was evaluated at 0.1 and 1 lM (Fig. 5).
It is clear that compound 1 did not stimulate proliferation at both
concentrations, contrary to CC-156 that did stimulate the cell
growth by 48% at 0.1 lM and 83% at 1 lM. The absence of
Supplementary data
13C NMR spectrum, 1H NMR spectrum and HPLC analysis report
of compound 1. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2011.06.110.
References and notes
1. Poirier, D. Expert Opin. Ther. Pat. 2010, 9, 1123.
2. Marchais-Oberwinkler, S.; Henn, C.; Möller, G.; Klein, T.; Negri, M.; Oster, A.;
Spadaro, A.; Werth, R.; Wetzel, M.; Xu, K.; Frotscher, R. W.; Adamski, J. J. Steroid
Biochem. Mol. Biol. 2011, 125, 66.
3. Day, J. M.; Tutill, H. J.; Purohit, A.; Reed, M. J. Endocr. Relat. Cancer 2008, 15, 665.
4. Moeller, G.; Adamski, J. Mol. Cell. Endocrinol. 2009, 301, 7.
5. Lin, S.-X.; Chen, J.; Mazumdar, M.; Poirier, D.; Wang, C.; Azzi, A.; Zhou, M. Nat.
Rev. Endocrinol. 2010, 6, 485.
6. Penning, T. M. Endocr. Relat. Cancer 1996, 3, 41.
7. Payne, A. H.; Hales, D. B. Endocr. Rev. 2004, 25, 947.
8. Laplante, Y.; Cadot, C.; Fournier, M.-A.; Poirier, D. Bioorg. Med. Chem. 2008, 16,
1849.
9. Poirier, D. J. Steroid Biochem. Mol. Biol. 2011, 125, 83.
10. Mazumdar, M.; Fournier, D.; Zhu, D.-W.; Cadot, C.; Poirier, D.; Lin, S.-X.
Biochem. J. 2009, 424, 357.
Figure 5. Effect of E2, CC-156 and compound 1 on the growth of estrogen-starved
T-47D cells (ER+) after 7 days of treatment. Control is fixed at 100% cell prolifer-
ation. Results are expressed as mean SEM of triplicate. ⁄P 6 0.01 versus control.
11. Bydal, P.; Auger, S.; Poirier, D. Steroids 2004, 69, 325.

É. Ouellet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5510–5513
5513
12. Data for compound 1: White amorphous solid; IR (film)
m
in cmꢀ1: 3352 (OH
43.6 (C-9), 43.7 (C-13), 49.3 (C-20), 51.0 (C-14), 89.6 (C-17), 112.8 (C-2), 115.0
(C-4), 125.7 (C-70), 126.2 (C-1), 128.2 (C-50), 128.5 (C-80), 130.6 (C-10), 131.9
(C-90), 134.7 (C-60), 137.3 (C-5), 139.4 (C-40), 155.1 (C-3), 168.0 (C-100), 178.9
(C-10); HRMS: calcd for C28H32NO4 [M+H]+ 446.2326, found 446.2331. HPLC
purity = 94.3%, rt = 17.2 min.
and NH2), 1753 (C@O, lactone), 1666 (C@O, amide); 1H NMR (400 MHz,
acetone-d6) d in ppm: 0.74 (s, 18-CH3), 1.10–2.80 (unassigned CH and CH2),
2.81 (m, 20-CH), 3.28 (dd, J2 = 13.1 Hz, J1 = 3.7 Hz, 1H of 30-CH2), 4.33 (d,
J = 10.0 Hz, 17a-CH), 6.50 (d, J = 2.5 Hz, 4-CH), 6.58 (dd, J2 = 8.4 Hz, J1 = 2.6 Hz,
2-CH), 6.67 (br s, 1H of NH2), 7.07 (d, J = 8.4 Hz, 1-CH), 7.41–7.51 (m, 80-CH,
90-CH and 1H of NH2), 7.83 (d, J = 7.7 Hz, 70-CH), 7.90 (s, 50-CH), 7.99 (br s, 3-
OH); 13C NMR (400 MHz, acetone-d6) d in ppm: 11.9 (C-18), 26.2 (C-11), 27.4
(C-7), 29.5 (C-6), 32.8 (C-15), 37.2 (C-12), 37.4 (C-30), 38.2 (C-8), 41.5 (C-16),
13. Duncan, L. J.; Reed, M. J. J. Steroid Biochem. Mol. Biol. 1995, 55, 565.
14. Laplante, Y.; Rancourt, C.; Poirier, D. Mol. Cell. Endocrinol. 2009, 301, 146.
15. Keydar, I.; Chen, L.; Karby, S.; Weiss, F. R.; Delarea, J.; Radu, M.; Chaitcik, S.;
Brenner, H. J. Eur. J. Cancer 1979, 15, 659.