Crucial role of 3-bromoethyl in removing the estrogenic activity of 17β-HSD1 inhibitor 16β-(m-carbamoylbenzyl)estradiol

Article abstract of DOI:10.1021/ml200093v

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) represents a promising therapeutic target for breast cancer treatment. To reduce the undesirable estrogenic activity of potent 17β-HSD1 inhibitor 16β-(m-carbamoylbenzyl)estradiol (1) (IC₅₀ = 27

Full text of DOI:10.1021/ml200093v

LETTER
pubs.acs.org/acsmedchemlett
Crucial Role of 3-Bromoethyl in Removing the Estrogenic Activity of
17β-HSD1 Inhibitor 16β-(m-Carbamoylbenzyl)estradiol
Renꢀe Maltais, Diana Ayan, and Donald Poirier*
Laboratory of Medicinal Chemistry, Endocrinology and Genomic Unit, CHUQ (CHUL) À Research Center and Laval University,
Quebec (Quebec) G1V 4G2, Canada
S Supporting Information
b
ABSTRACT: 17β-Hydroxysteroid dehydrogenase type 1 (17
β-HSD1) represents a promising therapeutic target for breast
cancer treatment. To reduce the undesirable estrogenic activity
of potent 17β-HSD1 inhibitor 16β-(m-carbamoylbenzyl)-
estradiol (1) (IC50 = 27 nM), a series of analogues with a small
functionalized side chain at position 3 were synthesized and
tested. The 3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-estra-
1,3,5(10)-trien-17β-ol (5) was found to be a potent inhibitor
(IC50 = 68 nM) for the transformation of estrone (E1) into
estradiol (E2) and, most importantly, did not stimulate the
proliferation of estrogen-sensitive MCF-7 cells, suggesting no estrogenic activity. From these results, the crucial role of a bromoalkyl
side chain at carbon 3 was identified for the first time. Thus, this new inhibitor represents a good candidate with an interesting profile
suitable for further studies including pharmacokinetic and in vivo studies.
KEYWORDS: Steroid, estrogen, hormone, enzyme inhibitor, 17β-hydroxysteroid dehydrogenase, cancer
ydroxysteroid dehydrogenase type 1 (17β-HSD1)
17β-Htransforms estrone (E1) into estradiol (E2),1,2
the most potent natural ligand for estrogen receptors (ER).3 This
enzyme also catalyzes the reduction of dehydroepiandrosterone
(DHEA) into 5-androstene-3β,17β-diol (Δ5-diol), a weaker
estrogen that becomes more important after menopause.4 In-
hibitors of 17β-HSD1 are thus potentially interesting therapeutic
agents for the control of estrogen-dependent diseases such as
breast cancer and endometriosis.5À7 During the last 30 years,
many efforts were made in designing potent inhibitors of this key
steroidogenic enzyme, but it is only recently that lead candidates
have been reported with very good inhibitory activity.8À14 The
presence of residual estrogenic activity associated with steroidal
inhibitors, which are often built around an estrane nucleus,
represented a major drawback in their development and their
use as therapeutic agents. From different strategies tested for
reducing the estrogenicity of estrane derivatives in the past,8À14
two approaches gave interesting results. Thus, Sterix Ltd.15À17
used a combination of an ethyl and alkylamide side chain at
positions 2 and 16 of E1, respectively, whereas Solvay Pharma-
ceuticals18À20 used another alkylamide side chain at position 15
of E1, with or without a substituent at position 2. Despite of the
strong inhibitory potential of 17β-HSD1 to treat estrogen-
dependent diseases, the validation of this pharmaceutical target
in a clinical perspective remains to be done. New potent
inhibitors with a nonestrogenic profile are thus strongly needed
to validate the therapeutic approach in vivo and to engage the
first clinical trial in humans.
good inhibitory potency, this compound was found to stimulate
the MCF-7 and T-47D estrogen-sensitive breast cancer cell lines
in vitro, thus greatly reducing its therapeutic potential.22 To
remove the undesirable residual estrogenic activity of E2 deriva-
tive 1, we explored the impact on both 17β-HSD1 inhibition and
estrogenicity of a series of small chains differently functionalized
in the replacement of the hydroxyl (OH) group at position 3. In
fact, this OH is well-known to be very important for ER binding
affinity and, consequently, for producing the estrogenic effect.23
However, replacing the 3-OH group by a hydrogen atom did not
allow a full blockade of the estrogenicity as assessed by the
proliferation of ER+ cells,22,24 but additional modifications must
be tested at this position. Furthermore, from the X-ray analysis of
the crystallized complex of inhibitor 1 with 17β-HSD1, which
showed key interactions for inhibitory activity (Figure 1),25 we
noticed that the 3-OH seems less crucial for 17β-HSD1 inhibi-
tion. In fact, three major interactions were identified in the binary
complex of 17β-HSD1 and inhibitor 1: The 17β-OH forms a
hydrogen bond with the Ser142, the C(O)NH2 group forms a
hydrogen bond with the peptide backbone of Phe192, and the
phenyl ring at C16 forms a πÀπ interaction with Tyr155.
However, contrary to E1, the natural substrate of the enzyme,
the 3-OH of 1 does not form hydrogen bonding with Glu282 or
His221. To reach a third anchoring point with an amino acid in
proximity of position 3 of 1, and thus obtain a better binding with
17β-HSD1, as well as to remove the undesirable estrogenic
Received: April 7, 2011
Accepted: July 17, 2011
Published: July 17, 2011
The 16β-(m-carbamoylbenzyl)estradiol (1; CC-156) has already
been reported as a potent inhibitor of 17β-HSD1.21 Despite its
r
2011 American Chemical Society
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ACS Medicinal Chemistry Letters
LETTER
Table 1. Inhibition of 17β-HSD1 by Compounds 1À5
Figure 1. Key interactions observed in binary complex (17β-HSD1/ 1)
and representation of new E2 derivatives modified at position 3 (2À5).
The scope of this side chain is dual, (1) reaching a third interaction with
an amino acid and (2) removing the undesirable estrogenic activity of
inhibitor 1.
inhibition (%)
compound no.
R
at 0.01 μMa at 0.1 μMa IC50 (nM)b
1
2
3
4
5
ÀOH
35
14
20
7
66
37
36
17
49
27 ( 4
ND
ÀCH2OH
ÀCH2Br
ÀCH2CH2OH
ÀCH2CH2Br
Scheme 1a
430 ( 99
ND
23
68 ( 6
a Inhibition of the transformation of [14C]-E1 (60 nM) into [14C]-E2 by
17β-HSD1 in T-47D intact cells. The experiment was performed in
triplicate (SD < (5%). The inhibitors were tested at two concentrations
of 0.01 and 0.1 μM. b Mean ( SD of an experiment performed in
triplicate. ND, not determined.
Compounds 2 and 3 were synthesized from 3-carboxy-estrone
(6)26 as reported in Scheme 1 (part A). The benzylcarbamide
side chain was introduced at position 16 using an aldol con-
densation reaction with 3-formyl benzamide27 and potassium
hydroxide.22,28 The C17-ketone of 7 was then stereoselectively
reduced with sodium borohydride, and the 16-exo double bound
was reduced by a Pd on charcoal-catalyzed hydrogenation. The
3-carboxylic acid of 8 was activated using Castro's reagent (BOP)
in the presence of N,N-diisopropylethylamine (DIPEA) to
promote the reduction with sodium borohydride, which give
the corresponding methyl alcohol 2. This later was brominated
using triphenylphosphine and carbon tetrabromide to give the
bromomethyl derivative 3.
Compounds 4 and 5 were synthesized from 3-vinyl-17-
dioxolane-estrone (9) (Scheme 1, part B) obtained from carbo-
nylative vinylation of estrone triflate followed by C17 dioxolane
protection.29 The vinyl group was first transformed to alcohol by
an oxidative hydroboration reaction using boraneÀmethylsulfide
complex.30 The alcohol 10 was then protected as benzyl ether
followed by dioxolane deprotection in acid conditions to give 11.
The benzylcarbamide side chain was introduced at position 16
using the sequence of reactions reported above for the synthesis
of 8. The cleavage of 3-O-benzylether afforded the alcohol 4,
which was brominated using triphenylphosphine and carbon
tetrabromide to give the bromoethyl derivative 5.
During the synthesis of compounds 2À5, two new stereo-
genic centers were generated at positions 16 and 17. Their C16
β-R and C17β-OH configurations were however easily con-
firmed by NMR spectroscopy. As illustrated with 5, the chemical
shifts for 16R-H and 17R-H (3.17 and 3.84 ppm) in 1H NMR are
identical to those (3.14 and 3.81 ppm) published for 16β-m-
carbamoylbenzyl-estradiol (1).31 Similarly, in 13C NMR, the
signal at 83.0 ppm for 5 is also characteristic of the 16β-R and
17β-OH configuration (83.0 ppm for 1).
a Reagents and conditions: (a) 3-Formyl benzamide, KOH, EtOH,
reflux, 0.5 h. (b) NaBH4, MeOH, CH2Cl2, room temperature, 1 h. (c)
H2, 10% Pd/C, MeOH, room temperature, 36 h. (d) (1) BOP, DIPEA,
DMF, room temperature; (2) NaBH4, MeOH, room temperature. (e)
PPh3, CBr4, CH2Cl2, 0 °C, 1.7 h. (f) (1) BH3-(CH3)2S, THF, À78 °C to
room temperature; (2) NaHCO3, H2O2, room temperature, 3 h. (g)
NaH, benzylbromide, DMF, 0 °C to room temperature, 18 h. (h) 10%
HCl:acetone (1:1), room temperature, 5 h.
activity by disturbing the binding on ER, we chose different
functional groups and different side chain lengths that could
promote additional hydrogen bonding (alkylalcohol chains) or
hydrophobic interactions (alkylbromide chains). From the struc-
tureÀactivity relationship results obtained with 25 derivatives of
inhibitor 1, we have selected compounds 2À5 to demonstrate
the relevant results highlighted by compound 5.
Compounds 1À5 were tested for their ability to inhibit the
transformation of E1 into E2 by 17β-HSD1 in T-47D cells
(Table 1), a cell line well-known to express endogeneous 17
β-HSD1.32,21 In the alcohol series, we made the observation that
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ACS Medicinal Chemistry Letters
LETTER
between a nucleophilic amino acid residue of the enzyme and
compound 5 (irreversible inhibitor) by a nucleophilic substitu-
tion of the bromide. However, the alkylating property of
bromoalkyls is greatly variable according to the environment
and the nature of the alkyl moiety, and our bromoethyl inhibitor
5 is much more stable with a less alkylating (electrophilic)
potency than a bromobenzyl derivative such as 3. So, it is also
possible that the hydrophobic bromoethyl side chain interacts
reversibly with an hydrophobic pocket, such as this identified by
Lawrence et al.15 in the proximity of position 2 of natural enzyme
substrate or another hydrophobic pocket that remains to be
identified. It can also be highlighted that the X-ray analysis of
crystallized binary complex of 1 with 17β-HSD1 showed no
interaction of 3-OH with Glu282 or His221, which can be
explained by the presence of three interactions between the
16β-side chain and the17β-OH with the enzyme that slightly
moves the inhibitor toward the right side. Transposed to the
inhibitor 5, this could free up space to accommodate the
bromoethyl group added at position 3. Whatever the case, it is
clear that additional experiments such as enzyme kinetic and
crystallization studies will be necessary to properly address all of
the questions regarding the mechanism of 17β-HSD1 by new
inhibitor 5.
In conclusion, we have successfully synthesized a series of
C3-functionalized derivatives of the potent 17β-HSD1 in-
hibitor 1. The bromoethyl side chain generated the most
active inhibitor, compound 5, thus demonstrating the good
tolerance of 17β-HSD1 for a hydrophobic chain introduced
at position 3 of this steroid nucleus. Most importantly, the
addition of this small chain removed the residual estrogenic
activity of 1. A more complete investigation of the crucial C3
side chain by the study of different spacers and a larger
diversity of chemical groups [NR1R2, NC(O)R, F, Cl, I, and
Ph] is ongoing in our laboratory and will be reported in due
Figure 2. Effect of inhibitors 1À5 on the growth of estrogen-starved
estrogen-sensitive (ER+) MCF-7 cells after 7 days of treatment. The
proliferation of control cells is fixed at 100%. Results are expressed as
means ( SEMs of triplicate.
moving away the hydroxyl (OH) group from the steroid A ring
had a negative effect on 17β-HSD1 inhibition with % of inhibition
ranging from 66% for phenol 1 to 37% for methyl alcohol 2 and
17% for ethylalcohol 4 when tested at 0.1 μM. These results and
those obtained at 0.01 μM highlight the fact that no additional
favorable interaction was obtained with an amino acid of the
enzyme. On the other side, the bromoalkyl derivatives 3 and 5
were much more promising. In fact, the inhibitory activity of 5
(IC50 = 68 nM) was just 2.5 times lower than that of reference
inhibitor 1 (IC50 = 27 nM). However, the most important finding
was that the estrogenic activity was greatly modulated by the
presence of the C3 side chain (Figure 2). Bearing a phenol at
position 3, compound 1 clearly stimulated at each concentration
tested the proliferation of estrogen-sensitive MCF-7 cells, a breast
cancer cell line known to express ER. By adding one or two
methylene (CH2) groups between the OH and the steroid A ring,
the estrogenicity decreased, but compounds 2 and 4 remained
with a significant estrogenic activity. Substituting the OH by a
bromide atom also reduced the estrogenicity as illustrated with
compounds 2 and 3. Interestingly, the addition of two CH2 groups
and the substitution of the OH by a bromide atom to generate 5
did not stimulate cell proliferation, suggesting no estrogenic effect
at the three concentrations tested.
The results presented above identified the importance of a
bromoethyl side chain at position 3 in modulating both the 17
β-HSD1 inhibition activity and the estrogenic activity of our lead
compound 1, but these results also raise the question of the
mechanism of action of compound 5. For the estrogenic activity,
because substituting the E2 nucleus generally reduces ER bind-
ing, especially when replacing the 3-OH, we expected to obtain a
reduction of estrogenic activity for our E2 derivatives. It however
remains to be determined whether the presence in position 3 of a
bromoethyl group alone is sufficient to block the estrogenicity of
E2 nucleus. It is also possible that the combined effect of two
chains, a bromoethyl at position 3 and a carbamoylbenzyl at
position 16, is necessary to complete blockage of estrogenic effect
measured in vitro on ER+ MCF-7 cells.
course. However, with a very good inhibitory activity (IC50
=
68 nM) and no estrogenic effect observed on estrogen-
sensitive breast cancer cells, compound 5 represents a good
candidate with an interesting profile suitable for further
studies including pharmacokinetic and in vivo studies.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures for
b
the synthesis and characterization of new compounds 2À5 (IR,
1H NMR, 13C NMR, and HRMS), HPLC chromatogram of
compound 5, and description of biological assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 1-418-654-2296. Fax: 1-418-654-2761. E-mail: donald.
poirier@crchul.ulaval.ca.
Funding Sources
We thank the Canadian Institutes of Health Research (CIHR)
for an operating grant.
’ ACKNOWLEDGMENT
For inhibition of 17β-HSD1 activity, the alkylating property of
alkyl bromide suggests the possible formation of a covalent bond
We thank Micheline Harvey for careful reading of the
manuscript.
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ACS Medicinal Chemistry Letters
LETTER
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