Discovery of a non-estrogenic irreversible inhibitor of 17β-Hydroxysteroid dehydrogenase type 1 from 3-substituted-16β-(m -carbamoylbenzyl)-estradiol derivatives

Article abstract of DOI:10.1021/jm401639v

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is thought to play a pivotal role in the progression of estrogen-sensitive breast cancer by transforming estrone (E1) into estradiol (E2). We designed three successive series of E2-derivatives at position C3 of the potent inhibitor 16β-(m-carbamoylbenzyl)-E2 to remove its unwanted estrogenic activity. We report the chemical synthesis and characterization of 20 new E2-derivatives, their evaluation as 17β-HSD1 inhibitors, and their proliferative (estrogenic) activity on estrogen-sensitive cells. The structure-activity relationship study provided a new potent and steroidal nonestrogenic inhibitor of 17β-HSD1 named 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestra- 1(10),2,4-trien-16-yl]methyl}benzamide (23b). In fact, this compound inhibited the transformation of E1 into E2 by 17β-HSD1 in T-47D cells (IC ₅₀ = 83 nM), did not inhibit 17β-HSD2, 17β-HSD7, 17β-HSD12, and CYP3A4, and did not stimulate the proliferation of estrogen-sensitive MCF-7 cells. We also discussed the results of kinetic and molecular modeling (docking) experiments, suggesting that compound 23b is a competitive and irreversible inhibitor of 17β-HSD1.

Full text of DOI:10.1021/jm401639v

Article
pubs.acs.org/jmc
Discovery of a Non-Estrogenic Irreversible Inhibitor of 17β-
Hydroxysteroid Dehydrogenase Type 1 from 3‑Substituted-
16β‑(m‑carbamoylbenzyl)-estradiol Derivatives
Rene Maltais,† Diana Ayan,† Alexandre Trottier,† Xavier Barbeau,‡ Patrick Lague,§
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Jean-Emmanuel Bouchard,† and Donald Poirier*,†
†Laboratory of Medicinal Chemistry, Oncology and Nephrology Unit, CHU de Queb
Faculty of Medicine, Laval University, Quebec City, Quebec G1V 4G2, Canada
‡Dep
artement de Chimie, Institut de Biologie Integrative et Des Systemes (IBIS), and Centre de Recherche sur la Fonction, la
Structure et l’Ingenierie des Proteines (PROTEO), Universite Laval, Quebec City, Quebec G1V 4G2, Canada
§Dep
artement de Biochimie Microbiologie et Bio-informatique, Institut de Biologie Integrative et des Systemes (IBIS), and Centre de
Recherche sur la Fonction, la Structure et l’Ingenierie des Proteines (PROTEO), Universite Laval, Quebec City, Quebec G1V 4G2,
Canada
́
ecResearch Center (CHUL, T4-42) and
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ABSTRACT: 17β-Hydroxysteroid dehydrogenase type 1 (17β-
HSD1) is thought to play a pivotal role in the progression of
estrogen-sensitive breast cancer by transforming estrone (E1) into
estradiol (E2). We designed three successive series of E2-derivatives
at position C3 of the potent inhibitor 16β-(m-carbamoylbenzyl)-E2
to remove its unwanted estrogenic activity. We report the chemical
synthesis and characterization of 20 new E2-derivatives, their
evaluation as 17β-HSD1 inhibitors, and their proliferative (estro-
genic) activity on estrogen-sensitive cells. The structure−activity
relationship study provided a new potent and steroidal nonestrogenic
inhibitor of 17β-HSD1 named 3-{[(16β,17β)-3-(2-bromoethyl)-17-
hydroxyestra-1(10),2,4-trien-16-yl]methyl}benzamide (23b). In fact,
this compound inhibited the transformation of E1 into E2 by 17β-
HSD1 in T-47D cells (IC50 = 83 nM), did not inhibit 17β-HSD2,
17β-HSD7, 17β-HSD12, and CYP3A4, and did not stimulate the proliferation of estrogen-sensitive MCF-7 cells. We also
discussed the results of kinetic and molecular modeling (docking) experiments, suggesting that compound 23b is a competitive
and irreversible inhibitor of 17β-HSD1.
HSD1 expression following antiaromatase therapy in breast
cancer patients has also been observed, suggesting a
compensatory response of the enzyme to estrogen depletion.15
These observations suggest that a 17β-HSD1 inhibitor could be
useful in blocking estrogen biosynthesis in a large number of
breast cancers and could be advantageous toward a maximal
estrogen biosynthesis blockade, considering that aromatase
inhibitors are unable to block 5-diol production (Figure 1).
With the emergence of personalized medicine and diagnostic
tests, the arrival of a potent 17β-HSD1 inhibitor in a clinical
setting is highly expected to give a new option for the treatment
of women detected with a high expression of 17β-HSD1 and a
low expression of aromatase in breast cancer tumor biopsies.
Finally, the use of 17β-HSD1 inhibitors is also a promising
approach for the treatment of other estrogen-dependent
diseases, such as endometrial cancer16 and endometriosis,17
where the enzyme has been shown to be overexpressed.18−20
INTRODUCTION
■
Breast cancer is the most frequent cancer among American
women, with an estimate of 288130 new cases diagnosed and
39520 related deaths in 2011.1,2 This disease is more frequent
in postmenopausal women, representing about three-quarters
of total breast cancer cases. Importantly, almost 65% of
postmenopausal breast cancer tumors are known to be
estrogen-dependent in their progression.3 17β-Hydroxysteroid
dehydrogenase type 1 (17β-HSD1) is suspected to play a
pivotal role in the progression of these breast cancers,
transforming estrone (E1) into estradiol (E2), the most potent
endogenous ligand for estrogen receptors (ERα and ERβ).4−6
17β-HSD1 also catalyzes the reduction of dehydroepiandros-
terone (DHEA) into 5-androstene-3β,17β-diol (5-diol), a
weaker estrogen that becomes more important after menopause
and could also stimulate breast cancer cell proliferation.7
Importantly, several immunohistochemical studies have re-
ported the presence of 17β-HSD1 in breast carcinoma tissue
and the enzyme was detected in approximately 50−60% of
cancer cells.8−14 Furthermore, a significant increment of 17β-
Received: October 22, 2013
Published: December 12, 2013
© 2013 American Chemical Society
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Figure 1. Two pathways involved in the formation of strong estrogen E2 (pathway II) and weak estrogen 5-diol (pathway I) from key steroid
DHEA.
Figure 2. Key interactions observed in a ternary complex of 17β-HSD1/inhibitor 1/cofactor NADP and representation of new E2 derivatives
modified at position 3 (series I, II, and III). The scope of this side-chain (R) is dual: (1) reaching a third interaction with an amino acid and (2)
removing the undesirable estrogenic activity of the first generation inhibitor 1.
Over the past 30 years, many efforts were dedicated to
designing potent inhibitors of the key steroidogenic enzyme
17β-HSD1, but it is only recently that lead candidates have
been reported with very high inhibitory activity.21−28 The
presence of residual estrogenic activity associated with steroidal
inhibitors, which are often built around an estrane nucleus, is a
major drawback to their development and their use as
therapeutic agents. Thus, despite the strong potential of 17β-
HSD1 inhibitors for the treatment of estrogen-dependent
diseases, validation of this pharmaceutical target remains to be
confirmed. New potent and specific inhibitors with a
nonestrogenic profile as well as selectivity toward other 17β-
HSD isoforms, especially type 2, are thus strongly needed to
validate the therapeutic in vivo approach and to engage the first
clinical trial with human subjects.
16β-(m-Carbamoylbenzyl)-E2 (1) has already been reported
as a potent inhibitor of 17β-HSD1.29,30 Despite its good
inhibitory potency, this compound was found to stimulate the
MCF-7 and T-47D estrogen-sensitive breast cancer cell lines in
vitro, thus compromising its therapeutic potential.29 To remove
the undesirable residual estrogenic activity of E2 derivative 1,
we explored the impact on both 17β-HSD1 inhibition and
estrogenicity of a series of differently functionalized small
chains 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, to produce an
estrogenic effect.31 Because replacing the 3-OH group by a
hydrogen atom did not allow full blockade of the estrogenic
activity, as assessed by the proliferation of estrogen-sensitive
cells,29,32 additional modifications should be tested. To reach a
new anchoring point with an amino acid in proximity to
position 3 of compound 1 (Figure 2),33 and to potentially
remove the undesirable estrogenic activity by disturbing the
binding on ER, we selected different functional groups and side
chain lengths (0, 1, or 2 carbon spacer) in order to promote
hydrogen bonding (B(OH)2, NH2, F, CONH2, COOH,
CH2OH, CONR1R2, CH2NH2) or hydrophobic interactions
(CH3, CH2Br, CH2Cl, CH2I, CH2Ph).
The successive synthesis of three series of E2 derivatives,
combined with structure−activity relationships (SAR), pro-
vided a new potent and nonestrogenic steroidal inhibitor of
17β-HSD1 named 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydrox-
yestra-1(10),2,4-trien-16-yl]methyl} benzamide (23b). After
publication of preliminary results,34 we now report the full
details of chemical synthesis and characterization of 20 new E2
derivatives, their evaluation as 17β-HSD1 inhibitors, and their
proliferative (estrogenic) activity on estrogen-sensitive cells.
We also determine the selectivity of the best inhibitor 23b for
other enzymes and used molecular modeling to obtain insight
into its binding mechanism.
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a
Scheme 1
a
Reagents and conditions: (a) 3-carboxamide-benzaldehyde, KOH, EtOH, reflux; (b) NaBH4, MeOH, rt; (c) H2, Pd/C, MeOH, rt.
a
Scheme 2
a
Reagents and conditions: (a) R1R2NH, BOP, DIPEA, DMF, rt; (b) (i) BOP, DIPEA, THF, rt, (ii) NaBH4, rt; (c) PPh3, CBr4, DCM, rt; (d)
NHR1R2, Et3N, DCM, rt; (e) (i) NaN3, DMF, 60 °C, (ii) H2, Pd/C (10%), MeOH, rt.
installation of carbamoylbenzyl moiety and was much more
uncertain considering the reactivity of the carboxamide
functionality. Thus, we first prepared the intermediates 2
(boronate-ester),35 3 (amino),36 4 (fluoro),37 5 (carboxa-
mide),38 and 6 (carboxy)39 following previously reported
synthetic procedures (Scheme 1). These intermediates were
then submitted to a sequence of three chemical steps to give
their corresponding 16β-(m-carbamoylbenzyl) derivatives 7, 8,
9, 10, and 11. These three steps, already reported for the
synthesis of 1,29,40,41 consisted in an aldolization reaction with
the 3-carboxamide-benzaldehyde followed by a reduction with
NaBH4 and a catalytic (Pd/C) hydrogenation of the allylic
alcohol. This sequence of reactions gave low to modest yields
(6−49% for 3 steps) depending of the 3-substituted E1
derivative used as starting material, with the lower yields
observed for the boronic acid 7, aniline 8, and carboxamide 10.
The other derivatives of series I, compounds 12a−c, 13, 14,
and 15a−d, were synthesized from 3-carboxy derivative 11
(Scheme 2). An amidation of the mixed anhydride generated
from the 3-carboxy group of 11 with appropriate amines using
BOP as a coupling agent gave the carboxamide derivatives
RESULTS AND DISCUSSION
■
Chemistry. Compounds reported in this SAR study were all
synthesized from E1 as a common synthetic precursor
(Schemes 1−4). The 3-substituted-16β-(m-carbamoylbenzyl)-
E2 derivatives were regrouped in three different series (I−III)
relative to the spacers present between the functional group
and the A-ring of steroid core (Figure 2).
Series I Compounds (Schemes 1 and 2). The functional
groups (B(OH)2, NH2, F, CONH2, COOH, CH2OH,
CONR1R2, CH2Br, and CH2NH2) that constituted the first
series of derivatives were selected with the intention of
exploring the tolerance of the enzyme for substituents of
different natures (H-donor, H-acceptor, hydrophilic, and
hydrophobic) and to provide useful SAR data. These functional
groups were directly attached to position 3 of the A-ring as
illustrated with compounds 7, 8, 9, 10, 11, 12a−c, 13, 14, and
15a−d (Table 1). Our strategy toward obtaining these
compounds consisted in functionalizing E1 with the appro-
priate substituent at C3 and then introducing the m-
carbamoylbenzyl moiety at C16β. The inverse strategy
consisted in introducing the different chains at C3 after the
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peroxide gave the corresponding primary alcohol, which was
protected as the benzyl ether 21. This compound was next
transformed to 22 using the three-step sequence of reactions.
The last step, the catalytic hydrogenation, also allows
regeneration of the free alcohol. The hydroxyl group of 22
was substituted using (chloro-phenylthio-methylene)-
dimethylammonium chloride (CPMA),43 CBr4 and PPh3, or
NaI in acetone to provide the chloride 23a, bromide 23b, or
iodide 23c. The alcohol 22 was also transformed into the
corresponding carboxylic acid 24 in three steps: a) a
Dess−Martin oxidation, b) an in situ oxidation with NaClO2,
and c) a NaBH4 reduction of the C17-ketone. Finally, the N-
methylamide 25 was obtained from 22 by activating the
carboxylic acid group the intermediate keto acid with BOP
reagent to permit the methylamine nucleophilic displacement.
Series III Compounds (Scheme 4). Compounds 27, 28, and
30 contain a long spacer ((CH2)3 or OCH2CH2) at position 3
of the steroid nucleus. This series was designed to see the
impact of spacer length on 17β-HSD1 inhibitory activity of
compound 23b, the best inhibitor identified in the series II. The
3-vinyl-17-dioxolane-estra-1(10),2,4-triene (16) was first sub-
mitted to a metathesis reaction with the allyloxymethyl-benzene
to give 26 in a low yield. The usual sequence of reactions was
next used to install the 16β-side chain to give the diol 27. The
primary alcohol of 27 was then transformed into bromide 28
using CBr4 and PPh3. Finally, compounds 29 and 30, two
analogues of 27 and 28 both bearing an oxygen atom rather
than a CH2 group as point of attachment on the ring A
(OCH2CH2 instead of CH2CH2CH2), were synthesized in
three steps. Starting from 1, an O-allylation with allylbromide
followed by the ruthenium-catalyzed oxidative cleavage of the
terminal olefin gave the corresponding aldehyde,44 which was
then reduced with NaBH4 to give the alcohol 29. This
compound was next transformed to bromide 30. We were
particularly interested by derivative 30 considering the presence
of an oxygen atom directly attached to the steroid A-ring. In
fact, the CH2CH2O side chain of 30 could allow supplemental
interaction with the enzyme compared to the (CH2)3 spacer of
28.
Optimization of the Chemical Synthesis of 23b (Scheme
5). Small quantities of compounds 23b and 23c were initially
obtained for the purpose of in vitro assays, but larger quantities
were thereafter necessary for in vivo assays. We thus designed a
shorter and more efficient chemical route that enabled the
preparation of multigram quantities of 23b or 23c. Briefly, 3-
vinyl-estra-1(10),2,4-triene-17-one (19)45 was oxidized with
oxone46 to give the oxirane 31 in excellent yield. The epoxide
group of 31 was then transformed to primary alcohol 32 by
performing a regiospecific palladium catalyzed transfer hydro-
genation47 using ammonium formate and palladium on
charcoal in refluxing methanol. The usual sequence of three
reactions for the introduction of the 16β-carbamoyl-m-
benzamide side chain34 was used to provide the diol 22 in a
very good yield of 84%. Finally, the bromination of 22 using
triphenylphosphine and carbon tetrabromide in DCM gave the
bromide 23b. With only eight steps compared to 10 steps
(from estrone), this new strategy to generate 23b was found
advantageous over the first one. Importantly, the second
chemical synthesis of 23b was achieved with a global yield of
17% compared to 7% for the first synthesis. Interestingly, only
four chromatographic purifications were required along the
chemical synthesis, thus reducing the time and cost needed to
obtain multigram of 23b in very good HPLC purity of 98.5%.
Table 1. Inhibition of 17β-HSD1 (Compounds of Series I)
b
estrogenicity
a
compd
no.
inhibition % at
at
at
R
0.1 μM
0.1 μM
1.0 μM
1
−OH
−B(OH)2
−NH2
−F
−CONH2
−COOH
−CON(CH3)2
−CON(Et)Pr
−CON(CH2)4
−CH2OH
72
36
31
10
2
6
4
6
5
2
3
4
+++
+++
−
−
−
+++
+++
+
7
8
9
+
10
11
12a
12b
12c
13
14
15a
15b
15c
15d
++
+++
++
−
−
+
3
+
1
+
10 12
−
−
−
−
−
−
−
+++
3
3
37 14
30 12
−CH2Br
−
−
−
+
−CH2N(CH3)2
−CH2N(Et)Pr
−CH2N(CH2)4
−CH2NH2
19
9
14 12
10 12
44
8
+++
a
Inhibition of the transformation of [14C]-E1 (60 nM) into [14C]-E2
by 17β-HSD1 in T-47D intact cells. Inhibition values are represented
as means ( SD) of at least two independent experiments performed in
b
triplicate. Effect of inhibitors on the growth of estrogen-starved
estrogen-sensitive MCF-7 cells after 7 days of treatment. Legend for
estrogenicity: “−” = no (0−5%), “+” = weak (5−15%), “++” =
medium (15−30%), “+++” = strong (>30%) vs control (basal cell
proliferation fixed at 0%).
12a−c in low yields (13−30%). The 3-methylalcohol derivative
13 was obtained by activating the carboxy group with BOP and
then reducing the anhydride with NaBH4. The bromide
derivative 14 was obtained in good yield (60%) by bromination
of 13 using triphenylphosphine and carbon tetrabromide in
DCM. From 14, we generated the corresponding amines 15a−
c by a nucleophilic displacement reaction using the appropriate
amines in DCM and TEA as base. The amine 15d was however
synthesized in two steps by using first the sodium azide in DMF
for a substitution of the bromide and next by reducing the
intermediate azide.
Series II Compounds (Scheme 3). In this second series of
compounds represented by 18, 20, 22, 23a−c, 24, and 25, one
additional carbon spacer (CH2) was introduced between the
functional group (CH2Ph, CH3, CH2OH, CH2Cl, CH2Br,
CH2I, COOH, and CONHCH3) and the A-ring of the steroid
core (Scheme 3). The vinyl group was identified as the
common functionality for the synthesis of each compound of
this series. Thus, the key intermediate 16 was obtained from E1
by a dioxolane protection of the C17-ketone,42 an activation of
the phenol by a triflate formation, and a carbonylative
vinylation. The 2-phenylethyl derivative 18 was obtained
from a metathesis reaction between the vinyl intermediate 16
and styrene using the Grubb (II) catalyst, followed by
deprotection of dioxolane to give 17, and the addition of the
16β-(m-carbamoylbenzyl) moiety using the previously de-
scribed three-step sequence of reactions. The synthesis of the
ethyl derivative 20 was generated by deprotection of 16 and
installation of the 16β-side chain. The oxidative hydroboration
of 16 using a dimethylsulfide borane complex and hydrogen
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a
Scheme 3
a
Reagents and conditions: (a) styrene, Grubb (II) catalyst, dichloroethane, reflux; (b) HCl 10% in MeOH, rt; (c) 3-carboxamide-benzaldehyde,
KOH, EtOH, reflux; (d) NaBH4, MeOH, rt; (e) H2, Pd/C (10%), MeOH, rt; (f) (i) BH3-DMS, THF, −78 °C, (ii) H2O2, NaHCO3; (g) NaH,
benzylbromide; (h) CPMA, DCM, rt; (i) PPh3, CBr4, DCM, rt; (j) NaI, acetone, rt; (k) (i) Dess−Martin reagent, DCM, rt, (ii) NaClO2, t-BuOH, 2-
methyl-butene, KH2PO4, rt; (l) CH3NH2 in THF, BOP, DIPEA, DMF, rt.
Structure−Activity Relationship Study. Series I Com-
pounds (Inhibition of 17β-HSD1 and Estrogenicity). The first
series of 3-substituted-16β-(m-carbamoylbenzyl)-E2 derivatives
contains 15 compounds with substituents of different natures
that were selected to study the impact on enzyme inhibition of
various factors like hydrophobicity, H-bond donor/acceptor
capacity, electrostatic charge, and steric hindrance. Compounds
were tested for their ability to inhibit the transformation of E1
into E2 by 17β-HSD1 in intact T-47D cells, a cell line that
expresses endogenous 17β-HSD1.30,48 As an initial observation
(Table 1), five compounds of this series showed a significant
inhibition of 17β-HSD1 (>30% at 0.1 μM), including boronic
acid 7, amines 8 and 15d, methylalcohol 13, and bromomethyl
14, whereas nine compounds gave weak inhibition values
(<30% at 0.1 μM), including fluoride 9, amides 10, 12a, 12b,
and 12c, carboxylic acid 11, and tertiary amines 15a, 15b, and
15c. When we consider these results more closely, we observe a
good inhibition for derivatives with a potential H-bond donor
group like primary amines, boronic acid, and alcohol. This
could be due to the capacity of these substituents to form H-
bond with Glu-282 or His-221 as previously observed for the
phenolic derivative 1 (Figure 2). For example, the aromatic
amine 8 (31% of inhibition at 0.1 μM) showed a lower
inhibition value than the aminomethyl 15d (44% at 0.1 μM).
This could be explained by the fact that the NH2 of aniline 8 is
a poor H-bond acceptor group contrary to the CH2NH2 group
of 15d, which has an acceptor/donor capacity close to that of
the OH of 1, a phenol that can form two H-bond interactions
with Glu-282 and His-221. A protonation of the CH2NH2
group of 15d could also lead to a stronger interaction with Glu-
282 and His-221. This limited H-bond acceptor capacity of 8
could thus explain its lower activity compared to 1 and 15d.
Having a H-bond acceptor/donor capacity, the primary alcohol
13 gave an inhibition close to that of the corresponding primary
amine 15d (37 and 44% at 0.1 μM) and lower than the
inhibition of phenol 1 (70%). Also, the interaction between the
substituent at position C3 and the enzyme seems to be
influenced by the distance between the functionality and the
amino acid to favor the formation of H-bond as seen by the
difference of activity between the alcohol 13 and phenol 1.
The very low inhibition at 0.1 μM of the carboxamide 10
(2%) compared to aminomethyl 15d (44%), H-donor and H-
acceptor, and of dimethylcarboxamide 12a (1%) compared to
corresponding amine 15a (19%), only H-acceptor, reveals that
the flexibility and orientation of the substituent to deliver the
H-donor group could be an important factor for enzyme
inhibition. The presence of a CO, responsible for the
difference of basicity between the amide and amine
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a
Scheme 4
a
Reagents and conditions: (a) Grubb II catalyst, allyloxymethyl-benzene; (b) HCl 10% in MeOH, rt; (c) 3-carboxamide-benzaldehyde, KOH, EtOH,
reflux; (d) NaBH4, MeOH, rt; (e) H2, Pd/C (10%), MeOH, rt; (f) PPh3, CBr4, DCM, rt; (g) allylbromide, NaOH, acetone, reflux; (h) (i) NaIO4,
RuCl3−H2O, EtOAc/ACN, 0 °C, (ii) NaBH4, THF:H2O (1:1), rt.
a
Scheme 5
a
Reagents and conditions: (a) oxone, NaHCO3, acetone/ACN (1:2), rt; (b) Pd/C (10%), ammonium acetate, MeOH, 70°C; (c) 3-carboxamide-
benzaldehyde, KOH, EtOH, reflux; (d) NaBH4, MeOH, rt; (e) H2, Pd/C (10%), MeOH, rt; (f) PPh3, CBr4, DCM, rt.
functionalities or producing a repulsive interaction with Glu-
282, could also explain the lower inhibitory activity of amides
10 and 12a. Furthermore, in the case of a tertiary amine like
dimethylamine 15a, where the amine acted only as H-bond
acceptor group via the lone electron pair on nitrogen, the
interaction with His-221 seems to be less favored than in the
case of the corresponding primary amine 15d (H-donor and H-
acceptor groups). However, the lower inhibition of tertiary
amines could also be explained by steric hindrance of the
dimethyl group of 15a (182.5 cm3/mol), and a loss of
inhibitory activity becomes obvious with more bulky chains like
diethylamine 15b (294.5 cm3/mol) and pyrolidine derivative
15c (238.5 cm3/mol). Despite these first interesting SAR
observations, the most surprising result of the series was the
good inhibition (30% at 0.1 μM) obtained with the
hydrophobic bromomethyl substituent (compound 14),
which we had not anticipated.
After investigating the inhibitory potency on 17β-HSD1, we
evaluated the proliferative (estrogenic) activity of synthesized
compounds on MCF-7 estrogen-sensitive cell line. In fact, for a
potential use in breast cancer, an enzyme inhibitor should be
devoid of estrogenic activity. Despite the promising 17β-HSD1
inhibitory results obtained with boronic acid 7, aniline 8,
methylalcohol 13, and methylamine 15d, these compounds
were all estrogenic at 1 μM and, consequently, they were
disqualified for their use in the context of breast cancer
treatment. However, we were pleased to see that bromomethyl
derivative 14 did not stimulate the cell proliferation at 0.1 and 1
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μM. Other compounds, such as 12b, 12c, 15a, and 15b, were
nonestrogenic on MCF-7 cells, but they inhibit weakly 17β-
HSD1. Finally, compounds 9, 10, 11, and 12a were found to be
estrogenic and weak inhibitors. Thus, on the basis of the 17β-
HSD1 inhibition and estrogenicity results of series I
compounds, we planned the synthesis of new compounds
(series II) in order to investigate the effect of an additional
carbon spacer to move away the functionality from A-ring on
both enzyme inhibition and estrogenic activity.
Series II Compounds (Inhibition of 17β-HSD1 and
Estrogenicity). To extend our SAR studies, we added a
methylene (CH2) spacer between some of the most active
functionalities of the first series of compounds and the steroid
A-ring. The tolerance of this enzyme to different types of
hydrophobic substituents (Ph, H, Cl, Br, and I) was also
investigated. The results obtained in this second series were
very interesting (Table 2) because the bromoethyl derivative
be the most important as observed with the poor inhibition
value (12% at 0.1 μM) obtained with chloroethyl derivative
23a, the more electronegative substituent of the series. The
leaving group capability could represent a more coherent
explanation for the stronger inhibition observed with bromide
and iodide atoms compared to lower leaving capability of the
chloride atom. This hypothesis suggests an irreversible
inhibition of the enzyme by a nucleophilic attack of the
bromide or iodide atom by an amino acid residue. In fact, it was
previously reported that a bromopropyl chain added at position
16 of E2 provided an irreversible inhibition of 17β-HSD1.49,50
The estrogenicity of series II compounds was assessed by
evaluating the proliferation of estrogen-sensitive cells. The
presence of OH group (compound 22) is sufficient to induce a
cell proliferation that is higher than the level observed with
alcohol 13. A weak estrogenicity was also obtained for the
carboxylic acid derivative 24. None of the other compounds
stimulated the proliferation of cells at 0.1 and 1 μM, including
the two most active inhibitors of the series II (compounds 23b
and 23c). These two compounds have a hydrophobic group at
position 3 of steroid core that, contrary to 22 and 24, cannot
generate a hydrogen bonding with ER. In fact, the C3-OH of
E2 is well-known to be involved in binding with ER.51,52
Series III Compounds (Inhibition of 17β-HSD1 and
Estrogenicity). A short series of compounds was designed to
study the tolerance of the enzyme for a longer side chain at
position 3 (Table 3). We first added a CH2 group to the
Table 2. Inhibition of 17β-HSD1 (Compounds of Series II)
b
estrogenicity
a
inhibition % at
at
at
compd no.
R
0.1 μM
0.1 μM 1.0 μM
11 (series I) −COOH
13 (series I) −CH2OH
14 (series I) −CH2Br
3
3
+
−
−
+++
+
Table 3. Inhibition of 17β-HSD1 (Compounds of Series III)
37 14
30 12
−
c
c
18
−CH2CH2Ph
−CH2CH3
17
6
5
−
−
+
−
20
8
−
22
−CH2CH2OH
−CH2CH2Cl
−CH2CH2Br
−CH2CH2I
−CH2COOH
−CH2CONHCH3
18
12
51
54
7
1
7
2
2
+
c
c
23a
23b
23c
24
−
−
−
b
−
estrogenicity
c
c
−
−
−
−
a
inhibition % at
at
at
7
+
compd no.
R
X
0.1 μM
0.1 μM 1.0 μM
25
11
8
−
23b (series II) −Br
CH2
51
20
41
37
2
5
5
3
−
−
−
−
−
+
a
Inhibition of the transformation of [14C]-E1 (60 nM) into [14C]-E2
27
28
30
−CH2OH CH2
−CH2Br
−CH2Br
by 17β-HSD1 in T-47D intact cells. Inhibition values are represented
CH2
−
−
as means ( SD) of at least two independent experiments performed in
O
b
triplicate. Effect of inhibitors on the growth of estrogen-starved
a
Inhibition of the transformation of [14C]-E1 (60 nM) into [14C]-E2
c
estrogen-sensitive MCF-7 cells after 7 days of treatment. Effect of
by 17β-HSD1 in T-47D intact cells. Inhibition values are represented
inhibitors on the growth of estrogen-starved estrogen-sensitive T-47D
cells after 7 days of treatment. Legend for estrogenicity: “−” = no (0−
5%), “+” = weak (5−15%), “++” = medium (15−30%), “+++” =
strong (>30%) vs control (basal cell proliferation fixed at 0%).
as means ( SD) of at least two independent experiments performed in
b
triplicate. Effect of inhibitors on the growth of estrogen-starved
estrogen-sensitive T-47D cells after 7 days of treatment at different
concentrations. Legend for estrogenicity: “−” = no (0−5%), “+” =
weak (5−15%), “++” = medium (15−30%), “+++” = strong (>30%)
vs control (basal cell proliferation fixed at 0%).
23b and the iodoethyl derivative 23c gave good 17β-HSD1
inhibition values (51 and 54% at 0.1 μM, respectively). An
improvement in inhibitory activity was thus observed with 23b
(2 × CH2) over 14 (1 × CH2) when tested at 0.1 μM (51 and
30%, respectively). However, replacement of the bromide of
23b by a lipophilic and nonelectronegative substituent like
phenyl or methyl produced very weak inhibitions at 0.1 μM (17
and 16% for 18 and 20, respectively). As seen with tertiary
amide 25, the size of the phenyl ring substituent is also
detrimental to inhibition. In the same manner, the chain
extension was ineffective for carboxylic acid 11 and alcohol 13
because the homologue compounds 24 and 22 had a weak
inhibitory effect on 17β-HSD1.
bromoethyl side chain of compound 23b, the most potent
inhibitor and nonestrogenic compound of the first two series,
to obtain the bromopropyl derivative 28. We also tested the
intermediate alcohol 27 to validate the tendency observed in
series I and II with alcohol derivatives 13 and 22. Finally, we
tested compound 30, a derivative that integrated an oxygen
atom in the bromopropyl side chain to reach additional
interactions with the enzyme. The inhibition assay revealed that
the longer (CH2)3Br side chain did not increase the inhibition
compared to the shorter (CH2)2Br side chain (41 and 51% at
0.1 μM for 28 and 23b, respectively). For the different alcohol
derivatives synthesized in the three series, the proximity of the
Despite the results pointing toward the importance of
electronegativity of the substituent, this factor does not seem to
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hydroxyl group to the steroid A-ring was an important factor
and inhibition values decreased with the spacer length (72, 37,
18, and 20% of inhibition at 0.1 μM for n = 0−3 or compounds
1, 13, 22, and 27, respectively). As a second relevant result of
series III, the presence of an oxygen atom in the side chain of
compound 28 (compound 30) did not increase the inhibition
(41 and 37% for 28 and 30, respectively). Regarding the ability
of series III compounds to stimulate estrogen-sensitive cell
proliferation, the alcohol derivative 27 was found to be
estrogenic but interestingly not the bromide derivatives 28
and 30. This result once again illustrates the importance of a
hydrogen bonding between the C-3 OH and an amino acid
known to be involved in the binding of E2 to ER.51,52
Thus, the bromoethyl derivative 23b was the most potent 17β-
HSD1 inhibitor of the bromide series with IC50 value of 68 nM
in this assay (Figure 3) and 97 nM in another assay both
performed in T-47D cells (mean value = 83 nM). On the basis
of these screening results, 23b was selected for subsequent
biological in vivo assays.53
Selectivity of 23b over Other Enzymes. Inhibition of
17β-HSD2, 17β-HSD7, and 17β-HSD12. The selectivity of
the17β-HSD1 inhibitor 23b over 17β-HSD2 was evaluated to
ensure that it does not deactivate the oxidation of E2 into E1 by
inhibiting 17β-HSD2 (Table 4). The assay was performed with
stably transfected 17β-HSD2 in intact HEK-293 cells using
[14C]-E2 as substrate.54 As a result, compound 23b did show a
very low inhibition of 17β-HSD2 (only 7% at the higher
concentration of 10 μM) for the conversion of E2 to E1. We
were also interested in the selectivity of compound 23b for
other 17β-HSDs, such as type 7 and type 12,55,56 which have
been reported to convert E1 to E2 in breast cancer cells.30
Similarly to 17β-HSD2, the assays were done with stably
transfected 17β-HSD7 or 17β-HSD12 in HEK-293 cells but
using [14C]-E1 as substrate instead of [14C]-E2. The compound
23b was found to be highly selective over 17β-HSD7 (9% of
inhibition at 10 μM) and less selective over 17β-HSD12 (34%
of inhibition at 10 μM).
Determination of IC50 Values (17β-HSD1 Inhibition).
The IC50 values of bromide derivatives 14, 23b, 28, and 30, the
most promising compounds for inhibitory as well as
nonestrogenic activity, were determined to compare their
potency to inhibit 17β-HSD1 in T-47D cells (Figure 3). The
Inhibition of CYP3A4. We were concerned about the
selectivity of action of compound 23b toward CYP3A4
(Table 4), one of the most important liver enzymes involved
in drug metabolism. We used the P450 Inhibition Kit CYP3A4/
DBF as suggested by the manufacturer with the exception that
23b was dissolved in a mixture of DMSO/ACN (5:95) instead
of only acetonitrile. Ketoconazole was used as a positive
reference inhibitor giving an IC50 of 0.024 μM, which is in
agreement with reported values.57 When tested for CYP3A4
inhibition, the bromoethyl derivative 23b and the phenolic
derivative 1, we obtained IC50 values of 3.9 0.5 μM for 23b
and 1.5 0.4 μM for 1, thus indicating a very low risk of drug−
drug interactions for both compounds but especially for 23b.57
Competitive Nature and Irreversibility of Inhibitor
23b. The leaving group ability of halogen atoms (Cl, Br, and I)
and the potency of corresponding compounds to inhibit the
transformation of [14C]-E1 to [14C]-E2 by 17β-HSD1 suggests
the formation of a covalent bond. To verify this hypothesis and
to provide some information about the mechanism of action,
we performed an inactivation assay with a representative
inhibitor and purified 17β-HSD1. Figure 4 shows the inhibition
curves for the bromide derivative 23b (0, 0.1, and 0.5 μM)
according to preincubation time. As expected, a slight decrease
of the enzyme activity was observed in the absence of inhibitor,
but a progressive inhibition of 17β-HSD1 was observed with
Figure 3. 17β-HSD1 inhibitory potency of compounds 1, 14, 23b, 28,
and 30 in T-47D intact cells. Breast cancer cells expressing 17β-HSD1
were incubated with various concentrations of inhibitor for 24 h in
presence of labeled [14C]-E1 (60 nM). IC50 represents the
concentration that inhibited the transformation of E1 into E2 by
50%. Results are representative of two experiments performed in
triplicate except for compound 14 (tested one time).
bromomethyl derivative 14 with a shorter spacer (1 × CH2)
was found to be six times less potent than the bromoethyl
derivative 23b (2 × CH2) (IC50 = 430 nM and 68 nM,
respectively), and the bromopropyl derivative 28 with a longer
spacer (3 × CH2) was found to be half as potent (IC50 = 153
nM) than 23b. The presence of an oxygen atom instead of a
CH2 group in the side chain of 30 did not increase the
inhibition value of 28 (IC50 = 172 and 153 nM, respectively).
a
Table 4. Selectivity of Compounds 1 and 23b on Four Enzymes (17β-HSD2, 17β-HSD7, 17β-HSD12, and CYP3A4)
b
inhibition of 17β-HSD1
IC50 (μM)
inhibition of 17β-HSD2%
at 10 μM
inhibition of 17β-HSD7%
at 10 μM
inhibition of 17β-HSD12%
at 10 μM
inhibition of CYP3A4
compd no.
IC50 (μM)
1
0.036 0.012
0.083 0.021
0
7
12
16
0
9
1
5
6
7
1.5 0.4
3.9 0.5
23b
34 10
c
INH-2
INH-7
48 13
d
81
3
e
INH-12
ketoconazole
39
3
0.024 0.004
a
b
c
Inhibition values are represented as means ( SD) of at least two experiments performed in triplicate. 17β-HSD1 in T-47D cells. Potent inhibitor
d
e
of 17β-HSD2 (see compound 1 in ref 54). Potent inhibitor of 17β-HSD7 (see compound 81 in ref 55). Weak inhibitor of 17β-HSD12 (see
compound 55 in ref 56).
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enzymatic activity would be recovered after washing out the
inhibitor independently of the preincubation time. In the
inactivation assay discussed above, we also addressed the
competitiveness of inhibitor 23b. Moreover, the rate of 17β-
HSD1 inactivation ([14C]-E1 to [14C]-E2) is slowed by the
presence of unlabeled E1 (0.5 μM), which thus appear to
compete with 23b (0.5 μM) for the substrate binding site of the
enzyme.
Molecular Modeling. Since we demonstrated the com-
petitive nature of the inhibitor 23b for the substrate binding site
(Figure 4), we were interested in studying the potential 17β-
HSD1/23b interactions by performing some docking experi-
ments. For discussion purposes, we also studied the competitive
reversible inhibitor 1, an E2 derivative that was crystallized with
17β-HSD1.33 The hydroxyl group at position C3 of compound
1 produces H-bonds with side chains of residues Glu-282 and
His-221 (Figure 5). Compound 23b differs from compound 1
as it does not have an H-bond donor group at position C3. Its
inhibition potency was however shown to be very good,
suggesting a different mode of action.
The first docking attempt for compound 23b led to a root-
mean-square deviation (RMSD) value of 1.49 Å when
compared to the compound 1 position obtained from the
crystallographic structure. As shown in Figure 6A, the Glu-282
side chain is oriented toward the binding site to make an H-
bond with compound 1, leaving no space for the bromoethyl
side chain of compound 23b. However, Glu-282 is a solvent
exposed flexible residue as indicated by its high B-factor value of
45. To account for this flexibility in the docking calculations, we
generated a binding site conformation for which the Glu-282
side chain is exposed to the solvent (see Experimental Section).
As shown in Figure 6B, this latter conformation accommodates
Figure 4. Time-dependent inactivation of 17β-HSD1 by compound
23b. The transformation of [14C]-E1 to [14C]-E2 by purified enzyme
was assessed after preincubation with compound 23b (0, 100, or 500
nM), with or without the natural substrate E1 (500 nM), and
expressed as the percentage of initial enzyme activity. See the
Experimental Section for the conditions of the enzymatic assay.
the time at both concentrations for 23b. The rate of this time-
dependent inhibition was higher at 0.5 μM than at 0.1 μM of
inhibitor. The very fact that the preincubation time affects the
enzyme activity indicates an irreversible effect and strongly
suggests the inactivation of the enzyme caused by a covalent
binding of the inhibitor considering its nature. As previously
reported,58 a reversible inhibitor would generate an inhibition
curve closely similar to that of the control because the same
Figure 5. Representation of compound 1 in the active site of 17β-HSD1. Coordinates are from PDB 3HB5.33 Compound 1 is represented by the
thick purple sticks, NADP+ by the small cyan sticks, and the amino acids are represented by the small green sticks. H-bonds between compound 1
hydroxyl at position C3 and Glu-282/His-221 are highlighted.
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Figure 6. Results from the docking of compound 23b at the binding site of 17β-HSD1: (A) using the crystallographic binding site conformation, (B)
with Glu-282 side chain solvent-oriented, (C) with Glu-282 side chain solvent-oriented and His-221 mutated into Ala, and (D) superposition of
receptor from (B) with the docked compound 23b from (C). The structure of compound 1 is shown for purposes of comparison with the docked
compound 23b and is not included in the docking calculations. Atoms from PDB 3HB5 are represented in green (protein in cartoon and compound
1 in sticks), the modified residues are represented by the purple sticks, and docked compound 23b by the orange (A), white (B), and cyan (C,D)
sticks.
Figure 7. Proposed irreversible mechanism of compound 23b (X = Br) and 23c (X = I) on 17β-HSD1.
the docking of compound 23b with a much better RMSD of
0.76 Å. Nonetheless, the distance between His-221 side chain
and the bromide of compound 23b is 3.8 Å, too long to
account for an irreversible binding as hypothesized earlier. To
get around the force field limitations that do not allow for
covalent reactions between the bromoethyl moiety and His-221
side chain, this latter residue was mutated into an Ala, which
has a smaller side chain. The docking results using this binding
site conformation are presented in Figure 6C,D. The RMSD of
the resulting compound 23b structure is 0.59 Å, and the
distance between the CH2 of the bromoethyl side chain and the
NH of reconstituted His-221 side chain is now 1.7 Å, indicating
the possibility of a covalent reaction (Figure 6D). This doubly
mutated docking was made to demonstrate that without the
conformational limitations of His-221 and Glu-282, the core
with the bromoethyl moiety is very well placed in the enzyme
pocket to generate a covalent bond.
On the basis of the results of kinetics and molecular
modeling with bromide derivative 23b, and the similar
reactivity of both bromide and iodide, we are confident that
the iodide of 23c will be released in the enzyme active site after
the formation of a covalent bond with an amino acid.
Therefore, it could be caught by this amino acid to form the
corresponding iodoimidium salt59 and to be retained within the
active site of 17β-HSD1 (Figure 7). In the event that the iodide
would be expelled from the enzyme active site and then to the
cell, it could be caught by the NIS symporter,60 a specialized
protein known to fix iodide and to be selectively expressed in
thyroid and breast cancer cells.61 Interestingly, the use of an
irreversible inhibitor of 17β-HSD1 could open the door to
molecular imaging and radiotherapeutics by adding the
appropriate iodo-radioisotope (123I and 131I, respectively) on
parent inhibitor 23c. Advantageously, the iodo-radioisotope
derivatives could be readily accessible from a simple
substitution reaction between compound 23b and Na*I.
CONCLUSION
■
Three successive series (I−III) of 3-substituted-16β-(m-
carbamoylbenzyl)-E2 derivatives has provided important SAR
data regarding enzyme tolerance for substituents of different
natures (hydrophilic, hydrophobic, H-bond donor, H-bond
acceptor, basic, acidic, etc.) and different chain lengths (0−3
atom spacer). In the first series of synthesized compounds,
different types of substituents like alcohol and amine gave
acceptable inhibition levels but their significant estrogenic
activity disqualified them for treatment of estrogen-related
diseases. The most promising substituent of the first series was
undoubtedly the bromomethyl derivative 14, which showed an
acceptable level of inhibition and a nonestrogenic profile. The
second series of compounds thus focused on the carbon chain
extension of bromomethyl derivative 14 as well as other
promising compounds of the first series. These new series of
compounds converged toward the identification of 3-(2-
bromoethyl)-16β-(m-carbamoylbenzyl)-estra-1(10),2,4-trien-
17β-ol (23b) as potent inhibitor of 17β-HSD1 in T-47D cells
(IC50 = 68 and 97 nM) without any estrogenic activity detected
on estrogen-sensitive cells. This bromo derivative was found to
be a competitive and irreversible inhibitor of 17β-HSD1.
Molecular modeling with 23b docked in 17β-HSD1 showed the
potential key interactions with His-221, highlighting the
possibility of a nucleophilic attack of the His-221 on the
bromoethyl group. Otherwise, this novel inhibitor represents an
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and extracted with EtOAc. The organic layers were combined, washed
with brine, dried with MgSO4, and evaporated under reduced pressure.
The crude compound was purified by flash chromatography to give the
corresponding enone. (b) Reduction of enone: To a solution of enone
(0.25 mmol) in MeOH (0.03 M) was added NaBH4 (0.38 mmol), and
the mixture was stirred at room temperature. After 1 h, the resulting
solution was concentrated under vacuo, diluted with EtOAc, washed
with water and brine, and dried with MgSO4. After the organic phase
was evaporated under reduced pressure, the crude compound was
purified by flash chromatography to give the corresponding allylic
alcohol. (c) Catalytic hydrogenation of allylic alcohol: To a solution of
allylic alcohol (0.2 mmol) in EtOH (0.03 M) under argon atmosphere
at room temperature was added palladium on charcoal (10% w/w).
The reaction vessel was flushed three times with hydrogen and stirred
for 24 h at room temperature. The resulting solution was filtered on
Celite and then evaporated under reduced pressure. The crude
compound was purified by flash chromatography using DCM/MeOH
(95:5) as eluent to give the desired 16β-carbamoyl-benzamide
derivative 7, 8, 9, 10, or 11.
[(16β,17β)-16-(3-Carbamoylbenzyl)-17-hydroxyestra-1(10),2,4-
trien-3-yl]boronic Acid (7). Yield: 13 mg, 8% (for 3 steps). IR (KBr):
1659 (CO, amide), 3380 (OH and NH2). 1H NMR (CD3OD): 0.91
(s, 18-CH3), 1.13 (m, 14α-CH and 15β-CH), 1.20−1.55 (m, 7α-CH,
12α-CH, 8β-CH, and 11β-CH), 1.69 (t, J = 6.0 Hz, 15α-CH), 1.84
(broad d, J = 11.8 Hz, 7β-CH), 2.05 (d, J = 12.2 Hz, 12β-CH), 2.26
(m, 9α-CH), 2.35 (m, 11α-CH), 2.47 (q, J = 12.5 Hz, 1H of 1′-CH2),
2.52 (m, 16α-CH), 2.83 (m, 6-CH2), 3.17 (d, J = 12.1 Hz, 1H of 1′-
CH2), 3.84 (d, J = 9.4 Hz, 17α-CH), 7.22−7.50 (m, 4-CH, 2-CH, 1-
CH, 5″-CH, and 6″-CH), 7.70 (dd, J1 = 1.2 Hz, J2 = 7.4 Hz, 4″-CH),
7.76 (s, 2″-CH). 13C NMR (CD3OD): 13.3 (C18), 27.2 (C11), 28.6
(C7), 30.5 (C6), 33.0 (C15), 38.8 (C1′), 39.0 (C12), 39.6 (C8), 43.4
(C16), 45.4 (C13), 46.0 (C9), 50.1 (C14), 83.0 (C17), 125.4 (C1),
126.0 (C4″), 129.1 (C2″), 129.4 (C5″), 131.9 (C2), 133.5 (C6″),
134.8 (C3 and C3″), 135.4 (C4), 136.5 (C5), 136.7 (C10), 144.4
(C1″), 172.7 (CONH2). LRMS for C27H37BNO5 [M + CH3OH +
H]+ = 466.3. HPLC purity of 97.9% (retention time = 10.6 min).
3-[(16β,17β)-3-Amino-17-hydroxyestra-1(10),2,4-trien-16-yl]-
methyl}benzamide (8). Yield: 5 mg, 8% yield (for 3 steps). IR (KBr):
1663 (CO, amide), 3364 (OH and NH2). 1H NMR (CD3OD): 0.91
(s, 18-CH3), 1.14 (m, 14α-CH and 15β-CH), 1.20−1.50 (m, 7α-CH,
12α-CH, 8β-CH, and 11β-CH), 1.66 (t, J = 7.0 Hz, 15α-CH), 1.84 (m,
J = 11.8 Hz, 7β-CH), 2.02 (d, J = 12.4 Hz, 12β-CH), 2.15 (m, 9α-
CH), 2.30 (m, 11α-CH), 2.47 (q, J = 12.3, 1H of 1′-CH2), 2.50 (m,
16α-CH), 2.72 (m, 6-CH2), 3.17 (dd, J1 = 2.2 Hz, J2 = 12.0 Hz, 1H of
1′-CH2), 3.83 (d, J = 9.4 Hz, 17α-CH), 6.46 (d, J = 2.1 Hz, 4-CH),
6.54 (dd, J1 = 2.3 Hz, J2 = 8.2 Hz, 2-CH), 7.04 (d, J = 8.3 Hz, 1-CH),
7.40 (m, 5″-CH and 6″-CH), 7.70 (d, J = 7.4 Hz, 4″-CH), 7.75 (s, 2″-
CH). 13C NMR (CD3OD): 13.3 (C18), 27.5 (C11), 28.7 (C7), 30.7
(C6), 33.0 (C15), 38.9 (C12), 39.0 (C1′), 40.0 (C8), 43.4 (C16), 45.1
(C13), 45.5 (C9), 49.9 (C14), 83.1 (C17), 114.9 (C2), 117.2 (C4),
126.0 (C4″), 126.8 (C1), 129.2 (C2″), 129.5 (C5″), 132.0 (C10),
133.5 (C6″), 134.8 (C3”), 138.2 (C5), 144.4 (C1″), 145.5 (C3), 172.7
(CONH2). LRMS for C27H37N2O3 [M + CH3OH + H]+ = 437.3.
HPLC purity of 96.9% (retention time = 5.3 min).
important evolution relative to lead compound 1, which was a
potent inhibitor of 17β-HSD1 but with an unwanted estrogenic
activity. Also, compound 23b had a selectivity of action over
17β-HSD2, 17β-HSD7, 17β-HSD12, and liver enzyme
CYP3A4, thus showing a promising profile toward in vivo
assays. Importantly, the evaluation of bromo derivative 23b in
vivo reveals the efficiency of this inhibitor to completely block
the tumor growth of estrone stimulated cancer cells (T-47D)
expressing the 17β-HSD1 enzyme.53 Finally, as interesting
perspective, the iodide derivative 23c could provide an
opportunity for molecular imaging of tissues expressing 17β-
HSD1 as well as selective radiotherapeutic treatment. These
new 17β-HSD1 inhibitors 23b and 23c, developed through a
SAR study, thus represent promising candidates toward clinical
studies for the treatment and diagnosis of estrogen-dependent
diseases like breast cancer and endometriosis.
EXPERIMENTAL SECTION
General. Chemical reagents were purchased from Sigma-Aldrich
Canada Ltd. (Oakville, ON, Canada). The usual solvents were
■
́
obtained from Fisher Scientific (Montreal, QC, Canada) and were
used as received. Anhydrous dichloromethane (DCM), dimethylfor-
mamide (DMF), and tetrahydrofuran (THF) were obtained from
Sigma-Aldrich. Thin-layer chromatography (TLC) and flash-column
chromatography were performed on 0.20- mm silica gel 60 F254 plates
(E. Merck; Darmstadt, Germany) and with 230−400 mesh ASTM
silica gel 60 (Silicyle, Queb
spectra (IR) were recorded on a Horizon MB 3000 ABB FTIR
́
ec, QC, Canada), respectively. Infrared
́
spectrometer (Quebec, QC, Canada), and only the significant bands
reported in cm−1. Samples were prepared as KBr pellet. Nuclear
magnetic resonance (NMR) spectra were recorded at 400 MHz for 1H
and 100.6 MHz for 13C on a Bruker Avance 400 digital spectrometer
(Billerica, MA, USA). The chemical shifts (δ) were expressed in ppm
and referenced to chloroform (7.26 and 77.0 ppm), acetone (2.06 and
1
29.1 ppm), or methanol (3.33 and 49.0 ppm) for H and 13C NMR,
respectively. The numbering reported in Figure 8 was used for the
Figure 8. Carbon numbering used for the assignment of representative
1H NMR signals.
1
assignment of H and 13C NMR signals. Low-resolution mass spectra
(LRMS) were recorded on a PE Sciex API-150ex apparatus (Foster
City, CA, USA) equipped with a turbo ion-spray source. The HPLC
purity of the final compounds to be tested was determined with a
Shimadzu apparatus using a Shimadzu SPD-M20A photodiode array
detector, an Altima HPC18 reversed-phase column (250 mm × 4.6
mm, 5 μM), and a solvent gradient of MeOH:H2O. The wavelength of
the UV detector was selected between 190 and 205 nm. All final
compounds shown a purity ≥95% (95.0−99.9%) except for
compounds 13, 15b, 15c, 18, 20, 24, 25, and 27 (90.2−93.8%).
The IUPAC nomenclature was used in the experimental part and the
names of steroid derivatives were generated using ACD/Laboratories
(Chemist’version) software (Toronto, ON, Canada).
Synthesis of 7, 8, 9, 10, and 11. General Procedure for the
Introduction of 16β-Carbamoyl-m-benzamide Side Chain. (a)
Aldolization reaction: To a solution of the appropriate steroidal
ketone 2, 3, 4, 5, or 6 (0.3 mmol) in EtOH (0.04 M) was added 3-
formyl-benzamide (0.6 mmol) and an aqueous KOH (10%) solution
(15% v/v). The solution was heated at reflux for 30 min. The resulting
solution was diluted with water, neutralized with aqueous HCl (10%),
3-{[(16β,17β)-3-Fluoro-17-hydroxyestra-1(10),2,4-trien-16-yl]-
methyl}benzamide (9). Yield: 59 mg, 26% (3 steps). IR (KBr): 1639
1
(CO, amide), 3356 (OH and NH2). H NMR (CD3OD): 0.91 (s,
18-CH3), 1.15 (m, 14α-CH and 15β-CH), 1.22−1.56 (m, 7α-CH,
12α-CH, 8β-CH, and 11β-CH), 1.67 (t, J = 7.0 Hz, 15α-CH), 1.82 (m,
7β-CH), 2.04 (d, J = 12.2 Hz, 12β-CH), 2.21 (m, 9α-CH), 2.35 (m,
11α-CH), 2.47 (q, J = 12.5, 1H of 1′-CH2), 2.52 (m, broad, 16α-CH),
2.81 (m, 6-CH2), 3.17 (dd, J1 = 3.2 Hz, J2 = 12.6 Hz, 1H of 1′-CH2),
3.84 (d, J = 9.4 Hz, 17α-CH), 6.75 (dd, J1 = 2.6. Hz, J2 = 9.8 Hz, 4-
CH), 6.81 (dt, J1 = 2.8 Hz, J2 = 8.6 Hz, 2-CH), 7.27 (dd, J1 = 6.0 Hz, J2
= 8.4 Hz, 1-CH), 7.40 (5″-CH and 6″-CH), 7.71 (td, J1 = 1.5 Hz, J2 =
7.5 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD): 13.3 (C18),
27.4 (C11), 28.3 (C7), 30.5 (C6), 33.0 (C15), 38.8 (C1′ and C12),
39.4 (C8), 43.3 (C16), 45.2 (C9), 45.3 (C13), 49.8 (C14), 82.9
(C17), 113.1 (d, JCC‑F = 21.0 Hz, C2), 115.7 (d, JCC‑F = 20.2 Hz, C4),
125.9 (C4″), 127.9 (d, JCC‑F = 8.0 Hz, C1), 129.1 (C2″), 129.4 (C5″),
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1
133.5 (C6″), 134.7 (C3″), 137.3 (C10), 140.1 (d, JC−F = 6.8 Hz, C5),
144.3 (C1″), 162.2 (d, JC−F = 242.0 Hz, C3), 172.6. LRMS for
C27H35FNO3 [M + CH3OH + H]+ = 440.3. HPLC purity of 98.1%
(retention time = 17.5 min).
(KBr): 1666 (CO, amide), 3209 and 3406 (OH and NH2). H
NMR (CD3OD): 0.78 and 1.00 (2t, J = 7.1 Hz, CH3CH2CH2N), 0.92
(s, 18-CH3), 1.12 and 1.26 (2t, J = 6.4 Hz, CH3CH2N), 1.17 (m, 14α-
CH and 15β-CH), 1.20−1.75 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-
CH, 15α-CH and CH3CH2CH2N), 1.85 (m, 7β-CH), 2.05 (d, J = 12.2
Hz, 12β-CH), 2.30 (m, 9α-CH), 2.48 (m, 11α-CH, 1H of 1′-CH2 and
16α-CH), 2.85 (m, 6-CH2), 3.10−3.60 (m, 1H of 1′-CH2,
CH3CH2CH2N and CH3CH2N), 3.85 (d, J = 9.4 Hz, 17α-CH),
7.11 (s, 4-CH), 7.04 (d, J = 8.0 Hz, 2-CH), 7.35−7.44 (m, 1-CH, 5″-
CH, and 6″-CH), 7.70 (d, J = 7.5 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C
NMR (CD3OD): 11.7 (CH3CH2N), 13.3 (C18), 14.3
(CH3CH2CH2N), 23.0 (CH3CH2CH2N), 27.2 (C11), 28.3 (C7),
30.4 (C6), 33.0 (C15), 38.8 (C12), 38.9 (C1′), 39.4 (C8), 43.3 (C16),
45.4 (C13), 45.8 (C9), 47.5 (CH3CH2N), 50.0 (C14), 51.9
(CH3CH2CH2N), 83.0 (C17), 124.6 (C2), 126.1 (C1), 126.5
(C4”), 127.8 (C4), 129.2 (C2″), 129.4 (C5″), 133.5 (C6″), 134.3
(C3), 134.8 (C3″), 138.3 (C5), 143.5 (C10), 144.3 (C1″), 172.6
(CONH2), 174.2 (CON). LRMS for C32H43N2O3 [M + H]+ = 503.3.
HPLC purity of 98.8% (retention time = 15.4 min).
(16β,17β)-16-(3-Carbamoylbenzyl)-17-hydroxyestra-1(10),2,4-tri-
ene-3-carboxamide (10). Yield: 21 mg, 6% (for 3 steps). IR (KBr):
1647 (CO, amide), 3213, 3329, 3483, and 3533 (OH and NH2). 1H
NMR (CD3OD): 0.93 (s, 18-CH3), 1.18 (m, 14α-CH and 15β-CH),
1.27−1.60 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.70 (m, 15α-
CH), 1.88 (m, 7β-CH), 2.07 (d, J = 12.6 Hz, 12β-CH), 2.32 (m, 9α-
CH), 2.40 (m, 11α-CH), 2.48 (q, J = 12.4, 1H of 1′-CH2), 2.52 (m,
broad, 16α-CH), 2.89 (m, 6-CH2), 3.17 (m, 1H of 1′-CH2), 3.86 (d, J
= 9.4 Hz, 17α-CH), 7.35−7.45 (m, 1-CH, 5″-CH and 6″-CH), 7.58 (s,
4-CH), 7.62 (dd, J1 = 1.8 Hz, J2 = 8.2 Hz, 2-CH), 7.70 (dd, J1 = 1.4
Hz, J2 = 7.0 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD): 13.3
(C18), 27.2 (C11), 28.4 (C7), 30.5 (C6), 33.1 (C15), 38.9 (C12 and
C1″), 39.3 (C8), 43.4 (C16), 45.4 (C13), 45.9 (C9), 50.0 (C14), 82.9
(C17), 126.0 (C1 and C2), 126.5 (C4″), 129.2 (C2″), 129.3 (C5″),
129.5 (C4), 132.0 (C3), 133.6 (C6″), 134.8 (C3″), 138.2 (C5), 144.4
(C1″), 146.0 (C10), 172.6 (2 × CONH2). LRMS for C28H37N2O4 [M
+ CH3OH + H]+ = 465.3. HPLC purity of 96.4% (retention time = 6.4
min).
(16β,17β)-16-(3-Carbamoylbenzyl)-17-hydroxyestra-1(10),2,4-tri-
ene-3-carboxylic Acid (11). Yield: 320 mg, 49% (for 3 steps). IR
(KBr): 1666 (CO, amide, and acid), 3198, 3283, 3383, and 3553
(OH and NH2). 1H NMR (CD3OD): 0.91 (s, 18-CH3), 1.17 (m, 14α-
CH and 15β-CH), 1.26−1.58 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-
CH), 1.70 (t, J = 7.0 Hz, 15α-CH), 1.87 (m, 7β-CH), 2.06 (d, J = 12.4
Hz, 12β-CH), 2.31 (m, 9α-CH), 2.42 (m, 11α-CH), 2.48 (q, J = 12.6,
1H of 1′-CH2), 2.53 (m, 16α-CH), 2.88 (m, 6-CH2), 3.18 (dd, J1 = 3.2
Hz, J2 = 12.6 Hz, 1H of 1′-CH2), 3.86 (d, J = 9.4 Hz, 17α-CH), 7.36−
7.45 (m, 1-CH, 5″-CH and 6″-CH), 7.69 (d, J = 7.3 Hz, 2-CH),
7.71(s, 4-CH), 7.75 (d, J = 7.4 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C
NMR (CD3OD): 13.3 (C18), 27.2 (C11), 28.3 (C7), 30.4 (C6), 33.0
(C15), 38.8 (C12), 38.9 (C1′), 39.3 (C8), 43.3 (C16), 45.4 (C13),
46.1 (C9), 50.0 (C14), 82.9 (C17), 126.0 (C1), 126.4 (C4”), 128.0
(C2), 129.1 (C2″), 129.4 (C5″), 131.3 (C4), 133.5 (C6″), 134.8
(C3″), 138.0 (C3 and C5), 144.3 (C1″), 146.8 (C10), 171.0
(COOH), 172.7 (CONH2). LRMS for C28H36NO5 [M + CH3OH +
H]+ = 466.3. HPLC purity of 95.1% (retention time = 13.8 min).
Synthesis of 12a−c. (General Procedure for N-Acylation of 11).
To a solution of acid 11 (0.12 mmol) in DMF (3 mL) was added BOP
(0.14 mmol), the appropriate amine (0.36 mmol), and DIPEA (28 μL,
0.17 mmol). The solution was stirred at room temperature for 2 h.
The mixture was poured into water, extracted with EtOAc, washed
with water and brine, dried over MgSO4, and evaporated under
reduced pressure. The crude N-acylated derivative 12a, 12b, or 12c
was purified by flash chromatography (typically DCM/MeOH, 95:5 to
9:1).
3-{[(16β,17β)-17-Hydroxy-3-(pyrrolidin-1-ylcarbonyl)estra-1-
(10),2,4-trien-16-yl]methyl}benzamide (12c). Yield: 17 mg, 30%. IR
(KBr): 1666 (CO, amide), 3402 (OH and NH2). 1H NMR
(CD3OD): 0.91 (s, 18-CH3), 1.15 (m, 14α-CH and 15β-CH), 1.25−
1.60 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.69 (t, J = 7.0 Hz,
15α-CH), 1.89−1.99 (2t, J = 6.5 Hz, CH2CH2 of pyrolidine and 7β-
CH), 2.03 (m, 12β-CH), 2.29 (m, 9α-CH), 2.39 (m, 11α-CH), 2.47
(q, J = 12.5, 1H of 1′-CH2), 2.52 (m, 16α-CH), 2.85 (m, 6-CH2), 3.17
(dd, J1 = 3.0 Hz, J2 = 12.6 Hz, 1H of 1′-CH2), 3.47 and 3.58 (2t, J =
7.0 Hz, 2 × CH2N of pyrolidine), 3.85 (d, J = 9.4 Hz, 17α-CH), 7.21
(s, 4-CH), 7.26 (d, J = 8.2 Hz, 2-CH), 7.34−7.44 (m, 1-CH, 5″-CH
and 6″-CH), 7.70 (d, J = 7.5 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR
(CD3OD): 13.3 (C18), 25.3 (CH2 of pyrolidine), 27.2 (C11 and CH2
of pyrolidine), 28.3 (C7), 30.4 (C6), 33.0 (C15), 38.8 (C12), 38.9
(C1′), 39.3 (C8), 43.3 (C16), 45.4 (C13), 45.8 (C9), 47.4 and 50.0 (2
× CH2N of pyrolidine), 50.0 (C14), 82.9 (C17), 125.3 (C2), 126.0
(C1), 126.4 (C4”), 128.6 (C4), 129.1 (C2″), 129.2 (C5″), 133.5
(C6″), 135.1 (C3″), 134.9 (C3), 138.2 (C5), 144.1 (C10), 144.3
(C1″), 172.1 (CON), 172.6 (CONH2). LRMS for C31H39N2O3 [M +
H]+ = 487.3. HPLC purity of 95.1% (retention time = 13.3 min).
Synthesis of 13. To a solution of acid 11 (300 mg, 0.70 mmol) in
anhydrous THF (20 mL) was successively added BOP (338 mg, 0.76
mmol) and DIPEA (145 μL, 0.84 mmol) under an argon atmosphere
at room temperature. The solution was stirred for 10 min and NaBH4
(30 mg, 0.79 mmol) was added in one portion and stirred again for 1
h. The resulting solution was poured into water, extracted with EtOAc,
washed with brine, dried with MgSO4, and evaporated under reduced
pressure. The crude compound was purified by two successive flash
chromatography procedures, first using DCM/MeOH (95:5) and
second acetone/hexanes (1:1) to give 88 mg (30%) of 13 (3-
{[(16β,17β)-17-Hydroxy-3-(hydroxymethyl)estra-1(10),2,4-trien-16-
yl]methyl}benzamide). IR (KBr): 1663 (CO, amide), 3356 (OH
(16β,17β)-16-(3-Carbamoylbenzyl)-17-hydroxy-N,N-dimethyles-
tra-1(10),2,4-triene-3-carboxamide (12a). Yield: 7 mg, 13%. IR
1
1
and NH2). H NMR (CD3OD): 0.92 (s, 18-CH3), 1.16 (m, 14α-CH
(KBr): 1666 (CO, amide), 3379 and 3456 (OH and NH2). H
and 15β-CH), 1.24−1.54 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH),
1.69 (t, J = 7.0 Hz, 15α-CH), 1.85 (m, 7β-CH), 2.05 (m, J = 12.2 Hz,
12β-CH), 2.26 (m, 9α-CH), 2.37 (m, 11α-CH), 2.47 (q, J = 12.4, 1H
of 1′-CH2), 2.53 (m, broad, 16α-CH), 2.83 (m, 6-CH2), 3.17 (dd, J1 =
3.6 Hz, J2 = 13.0 Hz, 1H of 1′-CH2), 3.85 (d, J = 9.5 Hz, 17α-CH),
7.03 (s, 4-CH), 7.09 (d, J = 8.0 Hz, 2-CH), 7.27 (d, J = 8.0 Hz, 1-CH),
7.38−7.45 (m, 5″-CH and 6″-CH), 7.70 (d, J = 6.0 Hz, 4″-CH), 7.76
(s, 2″-CH). 13C NMR (CD3OD): 13.3 (C18), 27.4 (C11), 28.6 (C7),
30.5 (C6), 33.0 (C15), 38.8 (C1′), 39.0 (C12), 39.7 (C8), 43.4 (C16),
45.4 (C13), 45.8 (C9), 50.0 (C14), 65.1 (CH2OH), 83.0 (C17), 125.5
(C1), 126.0 (C2), 126.3 (C4”), 128.6 (C4), 129.1 (C2″), 129.4 (C5″),
133.5 (C6″), 134.8 (C3″), 137.6 (C3), 139.6 (C5), 140.6 (C10),
144.4 (C1″), 172.7 (CONH2). LRMS for C28H38NO4 [M + CH3OH
+ H]+ = 452.3. HPLC purity of 91.8% (retention time = 9.7 min).
Synthesis of 14. To a solution of alcohol 13 (65 mg, 0.15 mmol)
in DCM (7 mL) was added at 0 °C triphenylphosphine (61 mg, 0.23
mmol), DIPEA (58 mg, 80 μL, 0.45 mmol), and carbon tetrabromide
(77 mg, 0.23 mmol). The solution was stirred at room temperature for
NMR (CD3OD): 0.92 (s, 18-CH3), 1.17 (m, 14α-CH and 15β-CH),
1.22−1.60 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.70 (t, J = 7.0
Hz, 15α-CH), 1.87 (m, 7β-CH), 2.08 (dt, J1 = 2.9 Hz, J2 = 12.3 Hz,
12β-CH), 2.30 (m, 9α-CH), 2.35−2.70 (m, 11α-CH, 1H of 1′-CH2
and 16α-CH), 2.86 (m, 6-CH2), 3.02 (s, NCH3), 3.10 (s, NCH3), 3.17
(dd, J1 = 3.2 Hz, J2 = 12.5 Hz, 1H of 1′-CH2), 3.87 (d, J = 9.4 Hz, 17α-
CH), 7.11 (s, 4-CH), 7.17 (d, J = 8.0 Hz), 7.38−7.44 (m, 1-CH, 5″-
CH and 6″-CH), 7.70 (d, J = 7.5 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C
NMR (CD3OD): 13.3 (C18), 27.2 (C11), 28.3 (C7), 30.4 (C6), 33.0
(C15), 38.8 (C12), 38.9 (C1′), 39.4 (C8), 39.7 and 40.1 (N(CH3)2),
43.3 (C16), 45.4 (C13), 45.8 (C9), 50.0 (C14), 83.0 (C17), 125.3
(C1), 126.0 (C2), 126.5 (C4″), 128.5 (C4), 129.2 (C2″), 129.4
(C5″), 133.5 (C6″), 134.3 (C3), 134.8 (C3″), 138.3 (C5), 143.6
(C10), 144.3 (C1″), 172.6 (CONH2), 174.2 (CON(CH3)2). LRMS
for C29H37N2O3 [M + H]+ = 461.3. HPLC purity of 97.6% (retention
time = 10.2 min).
(16β,17β)-16-(3-Carbamoylbenzyl)-N-ethyl-17-hydroxy-N-propy-
lestra-1(10),2,4-triene-3-carboxamide (12b). Yield: 7 mg, 30%. IR
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15 h. The resulting solution was poured into water, extracted with
DCM, dried with MgSO4, and evaporated under reduced pressure.
The crude compound was purified by flash chromatography (DCM/
MeOH, 97:3) to give 45 mg (60%) of 14 (3-{[(16β,17β)-3-
(bromomethyl)-17-hydroxyestra-1(10),2,4-trien-16-yl]methyl}-
benzamide). 1H NMR (CD3COCD3): 0.92 (s, 18-CH3), 1.16 (m, 14α-
CH and 15β-CH), 1.20−1.56 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-
CH), 1.69 (t, J = 6.2 Hz, 15α-CH), 1.84 (m, 7β-CH), 2.02 (m, under
solvent peak, 12β-CH), 2.24 (m, 9α-CH), 2.35 (m, 11α-CH), 2.47 (q,
J = 12.2 Hz, 1H of 1′-CH2), 2.52 (m, broad, 16α-CH), 2.81 (m, 6-
CH2), 3.22 (dd, J1 = 2.9 Hz, J2 = 12.4 Hz, 1H of 1′-CH2), 3.87 (m,
17α-CH and 17β−OH), 4.58 (s, CH2Br), 6.5 (broad s, 1H of
CONH2), 7.12 (s, 4-CH), 7.19 (d, J = 8.1 Hz, 2-CH), 7.29 (d, J = 8.0
Hz, 1-CH), 7.35 (t, J = 7.6 Hz, 5″-CH). 7.41 (d, J = 7.6 Hz, 6″-CH),
7.42 (broad s, 1H of CONH2), 7.75(d, J = 7.5 Hz, 4″-CH), 7.83 (s, 2″-
CH). 13C NMR (CD3COCD3): 12.4 (C18), 26.1 (C11), 27.3 (C7),
29.2 (C6), 32.1 (C15), 34.0 (CH2Br), 37.8 (C1′ and C12), 38.2 (C8),
42.3 (C16), 44.4 (C13), 44.6 (C9), 48.9 (C14), 81.3 (C17), 124.8
(C4″), 125.8 (C1), 126.6 (C2), 128.0 (C2″), 128.2 (C5″), 129.7 (C4)
131.9 (C6″), 134.5 (C3″), 135.4 (C3), 137.1 (C5), 141.0 (C10),
143.3 (C1″), 168.4 (CONH2). LRMS for C28H37BrNO3 [M +
CH3OH + H]+ = 514.3 and 516.3. HPLC purity of 97.8% (retention
time = 18.3 min).
(CONH2). LRMS for C32H45N2O2 [M + H] = 489.4. HPLC purity
of 91.5% (retention time = 2.1 min).
3-{[(16β,17β)-17-Hydroxy-3-(pyrrolidin-1-ylmethyl)estra-1-
(10),2,4-trien-16-yl]methyl}benzamide (15c). Yield: 7 mg, 28%. IR
1
(KBr): 1663 (CO, amide), 3205 and 3383 (OH and NH2). H
NMR (CD3OD): 0.91 (s, 18-CH3), 1.16 (m, 14α-CH and 15β-CH),
1.25−1.57 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.68 (t, J = 7.0
Hz, 15α-CH), 1.82 (broad s, 7β-CH and CH2CH2 of pyrolidine), 2.04
(d, J = 12.3 Hz, 12β-CH), 2.25 (m, 9α-CH), 2.37 (m, 11α-CH), 2.47
(q, J = 12.3 Hz, 1H of 1′-CH2), 2.55 (broad s, 2 × CH2N of pyrolidine
and 16α-CH), 2.82 (m, 6-CH2), 3.17 (dd, J1 = 3.0 Hz, J2 = 12.6 Hz,
1H of 1′-CH2), 3.57 (s, CH2NAr), 3.85 (d, J = 9.4 Hz, 17α-CH), 7.02
(s, 4-CH), 7.08 (d, J = 8.1 Hz, 2-CH), 7.26 (d, J = 8.0 Hz, 1-CH),
7.36−7.44 (m, 5″-CH and 6″-CH), 7.70 (d, J = 7.5 Hz, 4″-CH), 7.76
(s, 2″-CH). 13C NMR (CD3OD): 13.3 (C18), 24.0 (CH2CH2 of
pyrolidine), 27.3 (C11), 28.6 (C7), 30.5 (C6), 33.0 (C15), 38.8
(C12), 39.0 (C1′), 39.6 (C8), 43.4 (C16), 45.4 (C13), 45.8 (C9), 50.0
(C14), 54.8 (2(CH2N), 61.1 (ArCH2N), 83.0 (C17), 126.0 (C4″),
126.3 (C1), 128.2 (C2), 129.1 (C2″), 129.4 (C5″), 131.0 (C4), 133.5
(C6″), 134.9 (C3″), 136.0 (C3), 137.7 (C5), 140.8 (C10), 144.4
(C1″), 172.7 (CONH2). LRMS for C31H41N2O2 [M + H]+ = 473.3.
HPLC purity of 93.8% (retention time = 2.0 min).
Synthesis of 15d. To a solution of bromide 14 (30 mg, 0.06
mmol) in anhydrous DMF (3 mL) was added sodium azide (12 mg,
0.18 mmol). The solution was stirred at 60 °C for 3 h under an argon
atmosphere. The resulting solution was poured into water, extracted
with EtOAc, washed with brine, dried with MgSO4, and evaporated
under reduced pressure. The crude compound (25 mg) was dissolved
in ethanol (3 mL), and palladium on charcoal (10%) was added (10
mg). The reaction vessel was then flushed three times with hydrogen
and the solution stirred for 24 h under an argon atmosphere at room
temperature. The resulting solution was filtered on Celite and
evaporated under reduced pressure. The crude compound was purified
by flash chromatography using DCM/MeOH (95:5) as eluent to give
15d (3-{[(16β,17β)-3-(aminomethyl)-17-hydroxyestra-1(10),2,4-
trien-16-yl]methyl}benzamide). Yield: 15 mg, 58%. IR (KBr): 1663
Synthesis of 15a−c. General Procedure for N-Akylation of
Bromide 14. To a solution of 14 (25 mg, 0.06 mmol) in DCM (3 mL)
was added triethylamine (43 μL, 3.0 mmol) and the appropriate amine
(3.0 mmol). The solution was stirred at room temperature for 3 h. The
resulting solution was poured into water, extracted with DCM, dried
over a phase separator device (Biotage), and evaporated under reduced
pressure to give the desired N-alkylated derivative after purification by
flash chromatography (typically DCM/MeOH, 95:5 to 9:1).
3-{[(16β,17β)-3-[(Dimethylamino)methyl]-17-hydroxyestra-1-
(10),2,4-trien-16-yl]methyl}benzamide (15a). Yield: 10 mg, 43%. IR
1
(KBr): 1663 (CO, amide), 3364 and 3429 (OH and NH2). H
NMR (CDCl3): 0.91 (s, 18-CH3), 1.16 (m, 14α-CH and 15β-CH),
1.25−1.57 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.69 (t, J = 7.0
Hz, 15α-CH), 1.84 (m, 7β-CH), 2.05 (d, J = 12.2 Hz, 12β-CH), 2.24
(s, 2 × NCH3), 2.26 (m, 9α-CH), 2.37 (m, 11α-CH), 2.47 (q, J = 12.3
Hz, 1H of 1′-CH2), 2.52 (m, 16α-CH), 2.83 (m, 6-CH2), 3.17 (dd, J1
= 2.7 Hz, J2 = 12.5 Hz, 1H of 1′-CH2), 3.41 (s, CH2N(CH3)2), 3.85
(d, J = 9.4 Hz, 17α-CH), 7.00 (s, 4-CH), 7.05 (d, J = 8.0 Hz, 2-CH),
7.27 (d, J = 8.0 Hz, 1-CH), 7.35−7.45 (m, 5″-CH and 6″-CH), 7.70
(d, J = 6.0 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD): 13.3
(C18), 27.3 (C11), 28.6 (C7), 30.5 (C6), 33.0 (C15), 38.8 (C12),
39.0 (C1′), 39.6 (C8), 43.4 (C16), 45.1 (N(CH3)2), 45.4 (C13), 45.8
(C9), 50.0 (C14), 64.6 (CH2N), 83.0 (C17), 126.0 (C4″), 126.3
(C1), 128.0 (C2), 129.2 (C2″), 129.4 (C5″), 131.3 (C4), 133.5
(C6″), 134.8 (C3″), 135.4 (C3), 137.7 (C5), 140.9 (C10), 144.4
(C1″), 172.7 (CONH2). LRMS for C29H39N2O2 [M + H]+ = 447.2.
HPLC purity of 97.4% (retention time = 4.1 min).
1
(CO, amide), 3418 (OH and NH2). H NMR (CD3OD): 0.91 (s,
18-CH3), 1.16 (m, 14α-CH and 15β-CH), 1.22−1.56 (m, 7α-CH,
12α-CH, 8β-CH, and 11β-CH), 1.69 (t, J = 7.0 Hz, 15α-CH), 1.84 (m,
7β-CH), 2.04 (m, 12β-CH), 2.24 (m, 9α-CH), 2.37 (m, 11α-CH),
2.47 (q, J = 12.4 Hz, 1H of 1′-CH2), 2.52 (m, broad, 16α-CH), 2.82
(m, 6-CH2), 3.17 (dd, J1 = 2.9 Hz, J2 = 12.4 Hz, 1H of 1′-CH2), 3.84
(d, J = 9.4 Hz, 17α-CH), 6.94 (s, 4-CH), 7.00 (d, J = 9.2 Hz, 2-CH),
7.25 (d, J = 8.0 Hz, 1-CH), 7.35−7.46 (m, 5″-CH and 6″-CH), 7.70
(d, J = 6.0 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD): 13.3
(C18), 27.4 (C11), 28.5 (C7), 30.5 (C6), 33.0 (C15), 38.8 (C12),
39.0 (C1′), 39.7 (C8), 43.4 (C16), 45.4 (CH2NH2), 45.7 (C9), 45.9
(C13), 50.0 (C14), 83.0 (C17), 126.0 (C4″), 126.2 (C1), 126.7 (C2),
129.1 (C4), 129.2 (C2″), 129.4 (C5″), 133.5 (C6″), 134.8 (C3″),
138.0 (C5), 138.8 (C3), 140.7 (C10), 144.4 (C1″), 172.7 (CONH2).
LRMS for C27H35N2O2 [M + H]+ = 419.3. HPLC purity of 95.5%
(retention time = 2.0 min).
3-{[(16β,17β)-3-[Ethyl(propyl)amino)methyl]-17-hydroxyestra-1-
(10),2,4-trien-16-yl]methyl}benzamide (15b). Yield: 5 mg, 21%. IR
(KBr): 1663 (CO, amide), 3398 (OH and NH2). 1H NMR
(CD3OD): 0.90 (t, J = 7.2 Hz, CH3CH2CH2N), 0.92 (s, 18-CH3),
1.10 (t, J = 7.2 Hz, CH3CH2N), 1.16 (m, 14α-CH and 15β-CH),
1.25−1.60 (m, 7α-CH, 12α-CH, 8β-CH, 11β-CH, and CH2CH2CH3),
1.69 (t, J = 7.1 Hz, 15α-CH), 1.85 (m, 7β-CH), 2.05 (d, J = 12.8 Hz,
12β-CH), 2.26 (m, 9α-CH), 2.33−2.60 (11α-CH, 1H of 1′-CH2, 16α-
CH, NCH2CH3, and NCH2CH2CH3), 2.83 (m, 6-CH2), 3.17 (dd, J1 =
3.1 Hz, J2 = 12.6 Hz, 1H of 1′-CH2), 3.58 (s, ArCH2N), 3.85 (d, J =
9.4 Hz, 17α-CH), 7.01 (s, 4-CH), 7.07 (d, J = 8.1 Hz, 2-CH), 7.26 (d,
J = 8.0 Hz, 1-CH), 7.36−7.45 (m, 5″-CH and 6″-CH), 7.70 (d, J = 7.5
Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD): 11.1 (CH3), 12.1
(CH3), 13.3 (C18), 20.1 (CH3CH2CH2N), 27.3 (C11), 28.6 (C7),
30.5 (C6), 33.0 (C15), 38.8 (C12), 39.0 (C1′), 39.6 (C8), 43.4 (C16),
45.4 (C13), 45.8 (C9), 48.1 (CH3CH2CH2N), 50.0 (C14), 55.8
(CH3CH2N), 58.4 (ArCH2N), 83.0 (C17), 126.0 (C4″), 126.3 (C1),
128.1 (C2), 129.1 (C2″), 129.4 (C5″), 131.3 (C4), 133.5 (C6″), 134.8
(C3 and C3″), 137.7 (C5), 140.8 (C10), 144.4 (C1″), 172.7
Synthesis of 17. To a solution of compound 1634 (350 mg, 1.07
mmol) in DCM (75 mL) under argon atmosphere was added styrene
(257 μL, 233 mg, 2.24 mmol). The solution was purged by argon
bubbling for 5 min, and Grubb (II) catalyst (1,3-bis(2,4,6-
trimethylphenyl)-2-imidazolidinylidene)dichloro(phenylmethylene)-
(tricyclohexylphosphine)ruthenium) (48 mg, 0.056 mmol) was added.
The solution was heated at reflux for 24 h under argon atmosphere.
The solution was poured into water, extracted two times with DCM,
filtered under separator phase device (Biotage), and evaporated under
reduced pressure. The crude compound was purified by flash
chromatography (EtOAc/hexanes: 95:5) to give 50 mg (11%) of
metathesis product. This later compound (42 mg, 0.105 mmol) was
then dissolved in methanol (3 mL), and an aqueous solution of HCl
10% (1 mL) was added. The reaction mixture was stirred at room
temperature for 2 h. The solution was poured into a saturated
NaHCO3 solution (50 mL), extracted with EtOAc, washed with brine,
dried with MgSO4, and evaporated under reduced pressure to give
compound 17 (3-[(E)-2-phenylethenyl]estra-1(10),2,4-trien-17-one).
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Yield: 40 mg, 78%. IR (KBr): 1736 (CO). 1H NMR (CDCl3): 0.92
(s, 18-CH3), 1.40−2.58 (residual CH and CH2), 2.95 (m, 6-CH2),
7.08 (s, CHCH), 7.23−7.38 (m, 1-CH, 2-CH, 4-CH, and 3-CH of
styrene), 7.51 (d, J = 7.2 Hz, 2CH of styrene). 13C NMR (CDCl3):
13.8 (C18), 21.6 (C15), 25.7 (C11), 26.5 (C7), 29.4 (C6), 31.6
(C12), 35.9 (C16), 38.2 (C8), 44.5 (C14), 48.0 (C13), 50.5 (C9),
124.0 (C4), 125.7 (C2), 126.4 (CH of Ph), 127.1 (C1), 127.4 (CH of
Ph), 128.0 (CH of Ph), 128.4 (CHCH), 128.6 (CH of Ph), 134.9
(C10), 136.7 (C5), 137.5 (C of Ph), 139.4 (C3), ∼225 (too weak not
recorded). LRMS for C26H29O [M + H]+ = 357.3.
Synthesis of 21 and 22. These compounds were prepared from
16 as previously published in our preliminary report.34
Synthesis of 23a. To a solution of alcohol 2234 (20 mg, 0.05
mmol) in DCM (1.0 mL) was added chlorodimethyl(phenylthio)-
chloride methanaminium (CPMA) (45 mg, 0.19 mmol) at 0 °C under
an argon atmosphere. The solution was then allowed to return at room
temperature and stirred for 3 h. The crude compound was purified by
flash chromatography (DCM/MeOH, 97:3) to give 12 mg (57%) of
chloride 23a (3-{[(16β,17β)-3-(2-chloroethyl)-17-hydroxyestra-
1(10),2,4-trien-16-yl]methyl}benzamide). IR (KBr): 1639 (CO,
1
amide) and 3364 (OH and NH2). H NMR (CD3OD) 0.91 (s, 18-
Synthesis of 18. Compound 18 (6 mg, 11% yield) was prepared
from 17 using the general three-step procedure for introducing the
16β-carbamoyl-m-benzamide side chain. 3-{[(16β,17β)-17-hydroxy-3-
(2-phenylethyl)estra-1(10),2,4-trien-16-yl]methyl}benzamide (18). IR
(KBr): 1643 (CO, amide), 3379 (OH and NH2). 1H NMR
(CDCl3): 0.88 (s, 18-CH3), 1.15 (m, 14α-CH and 15β-CH), 1.26−
1.60 (m, 7α-CH, 12α-CH, 8β-CH and 11β-CH), 1.75 (t, J = 7.2 Hz,
15α-CH), 1.83 (m, 7β-CH), 2.00 (m, 12β-CH), 2.25 (m, 9α-CH),
2.35 (m, 11α-CH), 2.48 (q, J = 12.8, 1H of 1′-CH2), 2.55 (m, 16α-
CH), 2.80 (m, 6-CH2 and PhCH2CH2Ph), 3.17 (dd, J1 = 3.8 Hz, J2 =
12.9 Hz, 1H of 1′-CH2), 3.88 (m, 17α-CH), 5.65 and 6.10 (2 broad s,
CONH2), 6.94 (s, 4-CH), 7.01 (d, J = 8.0 Hz, 2-CH), 7.18−7.42 (m,
1-CH, 5″-CH, 6″-CH and PhCH2), 7.60 (d, J = 7.3 Hz, 4″-CH), 7.72
(s, 2″-CH). 13C NMR (CD3OD): 13.3 (C18), 27.4 (C11), 28.7 (C7),
30.5 (C6), 33.0 (C15), 38.7 (PhCH2), 38.8 (C12), 39.0 (C1′), 39.2
(PhCH2), 39.7 (C8), 43.4 (C16), 45.4 (C13), 45.7 (C9), 50.0 (C14),
83.0 (C17), 126.0 (C4″), 126.1 (C2), 126.8 (C1), 129.1
(PhCH2CH2), 129.2 (2 × PhCH2CH2), 129.4 (PhCH2CH2), 129.5
(2 × PhCH2CH2), 129.9 (C4), 133.5 (C6″), 134.8 (C3″), 137.4 (C3),
139.0 (C5), 140.1 (C10), 143.3 (PhCH2CH2), 144.4 (C1″), 167.6
(CONH2). LRMS for C34H40NO2 [M + H]+ = 494.3. HPLC purity of
93.5% (retention time = 21.6 min).
CH3), 1.15 (m, 14α-CH and 15β-CH), 1.25−1.56 (m, 7α-CH, 12α-
CH, 8β-CH, and 11β-CH), 1.68 (t, J = 7.0 Hz, 15α-CH), 1.83 (m, 7β-
CH), 2.04 (m, 12β-CH), 2.23 (m, 9α-CH), 2.36 (m, 11α-CH), 2.47
(q, J = 12.3, 1H of 1′-CH), 2.53 (m, 16α-CH), 2.81 (m, 6-CH2), 2.96
(t, J = 7.3 Hz, CH2CH2Cl), 3.17 (dd, J1 = 2.9 Hz, J2 = 12.4 Hz, 1H of
1′-CH2), 3.70 (t, J = 7.4 Hz, CH2CH2Cl), 3.84 (d, J = 9.4 Hz, 17α-
CH), 6.92 (s, 4-CH), 6.98 (d, J = 8.0 Hz, 2-CH), 7.22 (d, J = 8.0 Hz,
1-CH), 7.36−7.44 (m, 5″-CH and 6″-CH), 7.70 (d, J = 7.5 Hz, 4″-
CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD): 13.3 (C18), 27.3 (C11),
28.6 (C7), 30.5 (C6), 33.0 (C15), 38.8 (C1′), 39.0 (C12), 39.6 (C8),
39.8 (CH2CH2Cl), 43.3 (C16), 45.4 (C13), 45.7 (C9), 46.0
(CH2CH2Cl), 50.0 (C14), 83.0 (C17), 126.0 (C4″), 126.4 (C1),
127.1 (C2), 129.1 (C2″), 129.4 (C5″), 130.3 (C4), 133.5 (C6″), 134.8
(C3″), 136.7 (C3), 137.8 (C5), 140.0 (C10), 144.4 (C1″), 172.7
(CONH2). LRMS for C29H39ClNO3 [M + CH3OH + H]+ = 484.2.
HPLC purity of 98.7% (retention time = 19.2 min).
Synthesis of 23b. This compound was prepared from 22 as
previously published in our preliminary report.34
Synthesis of 23c. To a solution of bromide 23b (35 mg, 0.07
mmol) in acetone (5 mL) was added sodium iodide (15 mg, 0.1
mmol), and the solution was stirred at room temperature under argon
atmosphere for 24 h. Another portion of sodium iodide (52 mg,
mmol) was added and the solution stirred for an additional 24 h. The
resulting solution was poured into water (100 mL) and extracted with
EtOAc. The combined organic layer was washed with brine, dried with
MgSO4, and evaporated under reduced pressure. The crude
compound was purified by flash chromatography (DCM/MeOH,
95:5) to give 18 mg (47%) of iodure 23c (3-{[(16β,17β)-17-hydroxy-
3-(2-iodoethyl)estra-1(10),2,4-trien-16-yl]methyl} benzamide). IR
(KBr): 1636 (CO, amide), 3379 (OH and NH2). 1H NMR
(CD3OD): 0.91 (s, 18-CH3), 1.14 (m, 14α-CH and 15β-CH), 1.22−
1.55 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.69 (t, J = 7.1 Hz,
15α-CH), 1.82 (m, 7β-CH), 2.04 (m, 12β-CH), 2.22 (m, 9α-CH),
2.36 (m, 11α-CH), 2.47 (q, J = 12.3 Hz, 1H of 1′-CH), 2.52 (m, 16α-
CH), 2.82 (m, 6-CH2), 3.07 (t, J = 7.6 Hz, CH2CH2I), 3.17 (dd, J1 =
3.0 Hz, J2 = 12.5 Hz, 1H of 1′-CH2), 3.36 (t, J = 7.6 Hz, CH2CH2I),
3.84 (d, J = 9.4 Hz, 17α-CH), 6.89 (s, 4-CH), 6.95 (d, J = 8.0 Hz, 2-
CH), 7.22 (d, J = 8.0 Hz, 1-CH), 7.36−7.44 (m, 5″-CH and 6″-CH),
7.69 (dd, J1 = 1.4 Hz, J2 = 6.0 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR
(CD3OD): 6.3 (CH2I), 13.3 (C18), 27.3 (C11), 28.4 (C7), 30.5 (C6),
33.0 (C15), 38.8 (C1′), 39.0 (C12), 39.6 (C8), 41.1 (CH2CH2I), 43.4
(C16), 45.4 (C13), 45.7 (C9), 50.0 (C14), 83.0 (C17), 126.0 (C4″),
126.5 (C1), 126.6 (C2), 129.1 (C4), 129.4 (C2″), 129.8 (C5″), 133.5
(C6″), 134.8 (C3″), 137.9 (C5), 139.2 (C3), 140.0 (C10), 144.4
(C1″), 172.7 (CONH2). LRMS for C29H39INO3 [M + CH3OH + H]+
= 576.2. HPLC purity of 95.0% (retention time = 16.6 min).
Synthesis of 24. Dess−Martin periodinane (67 mg, 0.16 mmol)
was added in one portion to a solution of alcohol 22 (50 mg, 0.12
mmol) in DCM (4 mL) at room temperature. After 1 h, the reaction
mixture was treated with saturated NaHSO3 (0.25 mL), followed by a
saturated solution of NaHCO3 (5 mL) and the aqueous layer was
extracted with EtOAc. The combined organic phase was dried over
MgSO4, filtered, and evaporated to give a crude compound (49 mg)
that was taken up in t-BuOH (2.2 mL) and water (0.2 mL). 2-Methyl-
2-butene (64 μL, 0.76 mmol), NaClO2 (13 mg, 0.14 mmol), and
KH2PO4 (19 mg, 0.14 mmol) were then sequentially added, and the
mixture was stirred for 12 h at room temperature. The organic solvent
was evaporated under vacuum, and the resulting aqueous solution was
acidified using 1 N HCl (1 mL) and extracted with EtOAc. The
Synthesis of 19. To a solution of compound 1634 (180 mg, 0.64
mmol) in acetone (18 mL) was added an aqueous solution of HCl
10% (2 mL), and the mixture was stirred at room temperature for 2 h.
The solution was then poured into a saturated solution of NaHCO3
(50 mL), extracted with EtOAc, washed with brine, dried with MgSO4,
and evaporated under reduced pressure to give 140 mg of deprotected
compound 19 (3-ethenylestra-1(10),2,4-trien-17-one).42 1H NMR
(CD3COCD3): 0.90 (s, 18-CH3), 1.37−2.48 (residual CH and CH2),
2.88 (m, 6-CH2), 5.16 (d, J = 10.9 Hz, 1H of CHCH2), 5.74 (d, J =
17.6 Hz, 1H of PhCHCH2), 6.69 (dd, J1 = 10.9 Hz, J2 = 17.6 Hz,
CHCH2), 7.16 (s, 4-CH), 7.26 (m, 1-CH and 2-CH). 13C NMR
(CD3COCD3): 13.4 (C18), 21.4 (C15), 25.8 (C11), 26.5 (C7), 29.3
(C6, under solvent peaks), 31.9 (C12), 35.3 (C16), 38.3 (C8), 44.6
(C14), 47.7 (C13), 50.5 (C9), 112.4 (CHCH2), 123.6 (C4), 125.7
(C2), 126.9 (C1), 135.2 (C10), 136.7 (C5), 137.1 (CHCH2), 139.9
(C3), 218.6 (C17). LRMS for C20H25O [M + H]+ = 281.2.
Synthesis of 20. Compound 20 (40 mg, 54% yield) was prepared
from 19 using the general procedure for introduction of the 16β-
carbamoyl-m-benzamide side chain. 3-{[(16β,17β)-3-ethyl-17-hydrox-
yestra-1(10),2,4-trien-16-yl]methyl}benzamide (20). IR (KBr): 1666
1
(CO, amide), 3186 and 3367 (OH and NH2). H NMR (CDCl3):
0.87 (s, 18-CH3), 1.12 (m, 14α-CH and 15β-CH), 1.22 (t, J = 7.6 Hz,
CH3CH2), 1.20.-1.65 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH),
1.74 (t, J = 7.0 Hz, 15α-CH), 1.83 (m, 7β-CH), 2.00 (m, 12β-CH),
2.24 (m, 9α-CH), 2.34 (m, 11α-CH), 2.48 (q, J = 12.3, 1H of 1′-CH2),
2.53 (m, 16α-CH), 2.58 (q, J = 7.6 Hz, CH3CH2Ph), 2.82 (m, 6-CH2),
3.16 (dd, J1 = 4.3 Hz, J2 = 12.8 Hz, 1H of 1′-CH2), 3.87 (m, 17α-CH),
5.75 and 6.12 (broad s, CONH2), 6.92 (s, 4-CH), 6.99 (d, J = 8.1 Hz,
2-CH), 7.22 (d, J = 8.0 Hz, 1-CH), 7.34−7.42 (m, 5″-CH and 6″-CH),
7.60 (d, J = 8.8 Hz, 4″-CH), 7.72 (s, 2″-CH). 13C NMR (CD3OD):
13.3 (C18), 16.3 (CH3CH2), 27.4 (C11), 28.7 (C7), 29.4 (CH3CH2),
30.5 (C6), 33.0 (C15), 38.9 (C1′), 39.0 (C12), 39.7 (C8), 43.3 (C16),
45.4 (C13), 45.6 (C9), 50.0 (C14), 83.0 (C17), 126.0 (C4″), 126.1
(C2), 126.2 (C1), 129.1 (C4), 129.2 (C2″), 129.4 (C5″), 133.5
(C6″), 134.8 (C3″), 137.4 (C3), 138.6 (C5), 142.4 (C10), 144.3
(C1″), 172.7 (CONH2). LRMS for C29H40NO3 [M + CH3OH + H]+
= 450.3. HPLC purity of 93.6% (retention time = 19.8 min).
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organic phase was washed with brine, dried over MgSO4, and
evaporated. The crude compound was purified by trituration from
MeOH to give 30 mg of an aldehyde. To a solution of the later
compound (30 mg, 0.067 mmol) in MeOH (5 mL) was added NaBH4
(7 mg, 0.18 mmol). The solution was stirred at room temperature for
2 h, and two other portions of NaBH4 were sequentially added (7 mg,
0.18 mg) over a period of 2 h. The resulting solution was concentrated
under vacuo, diluted with DCM (25 mL), washed with water, dried
with MgSO4, and evaporated under reduced pressure. The crude
compound was purified by flash chromatography (DCM/MeOH, 9:1)
to give 15 mg (50%) of compound 24 ([(16β,17β)-16-(3-
carbamoylbenzyl)-17-hydroxyestra-1(10),2,4-trien-3-yl]acetic acid).
IR (KBr): 1659 (CO, amide), 1705 (CO, acid), 2300−3600
metathesis product. To a solution of this compound (120 mg, 0.28
mmol) in acetone (3 mL) was added aqueous 10% HCl (3 mL), and
the solution was stirred at room temperature for 6 h. The resulting
solution was diluted with water (60 mL), neutralized with a saturated
NaHCO3 solution, and extracted with EtOAc. The organic layers were
combined and washed with brine, dried with MgSO4, and evaporated
under reduced pressure. The crude compound was purified by flash
chromatography (EtOAc/hexanes, 5:95) to give 80 mg (69%) of
compound 26 (3-[(1E)-3-(benzyloxy)prop-1-en-1-yl]estra-1(10),2,4-
trien-17-one). 1H NMR (CDCl3): 0.91 (s, 18-CH3), 1.40−2.45
(residual CH and CH2), 2.52 (dd, J1= 8.6 Hz, J2 = 18.7 Hz, 16β-CH),
2.92 (m, 2H, 6-CH2), 4.19 (dd, J1 = 1.1 Hz, J2 = 6.1 Hz, OCH2CH
CH), 4.57 (s, OCH2Ph), 6.30 (m, CHCHCH2O), 6.58 (d, J = 16.0
Hz, CHCHCH2O), 7.14 (s, 4-CH), 7.18−7.39 (m, OCH2Ph, 1-CH,
and 2-CH). 13C NMR (CDCl3): 13.8 (C18), 21.6 (C15), 25.7 (C11),
26.5 (C7), 29.4 (C6), 31.6 (C12), 35.8 (C16), 38.1 C8), 44.4 (C14),
48.0 (C13), 50.5 (C9), 70.8 (OCH2Ph), 72.0 (PhOCH2CH), 123.9
(C4), 125.4 (C2), 125.6 (CH of Ph), 127.1 (C1), 127.6 (CH of Ph),
127.8 (CH of Ph), 128.4 (CH of Ph), 132.4 (Ph-CHCH2), 134.3
(C10), 136.6 (C5), 138.3 (C of Ph), 139.4 (C3), 217.0 (C17). LRMS
for C28H33O2 [M + H]+ = 401.3.
1
(OH, acid), 3388 (OH and NH2). H NMR (CD3OD): 0.91 (s, 18-
CH3), 1.15 (m, 14α-CH and 15β-CH), 1.24 (m, 7α-CH, 12α-CH, 8β-
CH, and 11β-CH), 1.68 (t, J = 7.1 Hz, 15α-CH), 1.82 (m, 7β-CH),
2.04 (m, 12β-CH), 2.23 (m, 9α-CH), 2.36 (m, 11α-CH), 2.47 (q, J =
12.3 Hz, 1H of 1′-CH2), 2.53 (m, 16α-CH), 2.81 (m, 6-CH2), 3.17
(dd, J1 = 3.1 Hz, J2 = 12.7 Hz, 1H of 1′-CH2), 3.51 (s, CH2COOH),
3.84 (d, J = 9.4 Hz, 17α-CH), 6.96 (s, 4-CH), 7.01 (d, J = 8.0 Hz, 2-
CH), 7.23 (d, J = 8.0 Hz, 1-CH), 7.36−7.46 (m, 5″-CH and 6″-CH),
7.70 (td, J1 = 1.5 Hz, J2 = 7.5 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR
(CD3OD): 13.3 (C18), 27.3 (C11), 28.6 (C7), 30.5 (C6), 33.0 (C15),
38.8 (C1′), 39.0 (C12), 39.6 (C8), 43.4 (C16), 45.4 (C13), 45.7 (C9),
49.2 (under solvent peaks (CH2COOH)), 50.0 (C14), 83.0 (C17),
126.0 (C4”), 126.4 (C1), 127.5 (C2), 129.1 (C2″), 129.4 (C5″), 130.8
(C4), 133.3 (C3), 133.5 (C6″), 134.8 (C3″), 137.8 (C5), 140.1
(C10), 144.4 (C1″), 163.0 (COOH), 172.7 (CONH2). LRMS for
C29H38NO5 [M + CH3OH + H]+ = 480.3. HPLC purity of 91.2%
(retention time = 5.7 min).
Synthesis of 27. Compound 27 (38 mg, 55%) was obtained from
26 using the general three-step procedure used for introducing the
16β-carbamoyl-m-benzamide side chain. 3-{[(16β,17β)-17-hydroxy-3-
(3-hydroxypropyl)estra-1(10),2,4-trien-16-yl]methyl}benzamide (27).
1
IR (KBr): 1663 (CO, amide), 3383 (OH and NH2). H NMR
(CD3OD): 0.91 (s, 18-CH3), 1.15 (m, 14α-CH and 15β-CH), 1.20−
1.58 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.68 (t, J = 7.0 Hz,
15α-CH), 1.80 (m, 7β-CH and CH2CH2CH2OH), 2.04 (m, 12β-CH),
2.21 (m, 9α-CH), 2.35 (m, 11α-CH), 2.47 (q, J = 12.2, 1H of 1′-CH),
2.52 (m, 16α-CH), 2.59 (t, J = 7.2 Hz, ArCH2CH2), 2.79 (m, 6-CH2),
3.17 (d, J = 12.5 Hz, 1H of 1′-CH2), 3.56 (t, J = 6.5 Hz, CH2CH2OH),
3.84 (d, J = 9.3 Hz, 17α-CH), 6.87 (s, 4-CH), 6.93 (d, J = 8.0 Hz, 2-
CH), 7.18 (d, J = 8.0 Hz, 1-CH), 7.36−7.44 (m, 5″-CH and 6″-CH),
7.70 (d, J = 6.0 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD):
13.3 (C18), 27.4 (C11), 28.7 (C7), 30.5 (C6), 32.6 (CH2CH2CH2),
33.0 (C15), 35.5 (ArCH2CH2), 38.9 (C1′), 39.0 (C12), 39.7 (C8),
43.4 (C16), 45.4 (C13), 45.7 (C9), 50.0 (C14), 62.3 (CH2OH), 83.0
(C17), 126.0 (C4″), 126.2 (C1), 127.2 (C2), 129.1 (C2″), 129.4
(C5″), 129.9 (C4), 133.5 (C6″), 134.8 (C3″), 137.5 (C5), 138.9
(C3), 140.3 (C10), 144.3 (C1″), 172.7 (CONH2). LRMS for
C30H42NO4 [M + CH3OH + H] = 480.3. HPLC purity of 90.6%
(retention time = 13.5 min).
Synthesis of 25. To the C17-ketonic form of 24 (37 mg, 0.08
mmol), obtained from Dess−Martin oxidation of 22, dissolved in
anhydrous DMF (3 mL) and under argon atmosphere was added BOP
(40 mg, 0.09 mmol), methyl amine (115 μL, 0.03 mmol, 2.0 M in
THF), and DIPEA (18 μL, 0.11 mmol). The solution was stirred at
room temperature. After 3 h, the mixture was poured into water,
extracted with EtOAc, washed with brine, dried with MgSO4, and
evaporated under reduced pressure to give 41 mg of a crude amide.
This compound was taken up into a mixture of MeOH/DCM (9:1),
then treated with NaBH4 (15 mg, 0.40 mmol) and stirred for 30 min
at room temperature. The resulting solution was poured into water,
extracted with EtOAc, washed with brine, dried with MgSO4, and
evaporated under reduced pressure. The crude amide compound was
purified by flash chromatography (DCM/MeOH, 95:5) to give 6 mg
(15%) of 25 (3-{[(16β,17β)-17-hydroxy-3-[2-(methylamino)-2-
oxoethyl]estra-1(10),2,4-trien-16-yl]methyl}benzamide). IR (KBr):
Synthesis of 28. To a solution of compound 27 (28 mg, 0.063
mmol) in DCM (3 mL) was added at 0 °C triphenylphosphine (33
mg, 0.13 mmol) and carbon tetrabromide (42 mg, 0.13 mmol). The
solution was stirred at 0 °C for 40 min, and second portions of
triphenylphosphine (13 mg, 0.05 mmol) and carbon tetrabromide (17
mg, 0.05 mmol) were added. After 1 h at 0 °C, the resulting solution
was poured into water (50 mL), extracted with DCM, dried with
MgSO4, and evaporated under reduced pressure. The crude
compound was purified by flash chromatography (DCM/MeOH:
97:3) to give 8 mg (25%) of 28 (3-{[(16β,17β)-3-(3-bromopropyl)-
17-hydroxyestra-1(10),2,4-trien-16-yl]methyl} benzamide). IR (KBr):
1663 (CO, amide), 3383 (OH and NH2). 1H NMR (CDCl3): 0.88
(s, 18-CH3), 1.12 (m, 14α-CH and 15β-CH), 1.20−1.58 (m, 7α-CH,
12α-CH, 8β-CH, and 11β-CH), 1.75 (t, J = 7.1 Hz, 15α-CH), 1.83 (m,
7β-CH), 2.00 (m, 12β-CH), 2.15 (m, CH2CH2Br), 2.23 (m, 9α-CH),
2.33 (m, 11α-CH), 2.48 (q, J = 12.3, 1H of 1′-CH), 2.53 (m, 16α-
CH), 2.70 (t, J = 7.2 Hz, ArCH2CH2), 2.82 (m, 6-CH2), 3.17 (dd, J1 =
4.3 Hz, J2 = 12.9 Hz, 1H of 1′-CH2), 3.41 (t, J = 6.6 Hz, CH2CH2Br),
3.86 (m, 17α-CH), 5.60 and 6.10 (broad s of CONH2), 6.91 (s, 4-
CH), 6.98 (d, J = 7.9 Hz, 2-CH), 7.22 (d, J = 8.0 Hz, 1-CH), 7.34−
7.42 (m, 5″-CH and 6″-CH), 7.60 (d, J = 7.3 Hz, 4″-CH), 7.72 (s, 2″-
CH). 13C NMR (CD3OD): 13.3 (C18), 27.4 (C11), 28.6 (C7), 30.5
(C6), 33.0 (C15), 33.7 (CH2CH2CH2Br), 34.4 (ArCH2CH2), 35.7
(CH2Br), 38.9 (C1′), 39.0 (C12), 39.7 (C8), 43.3 (C16), 45.4 (C13),
45.7 (C9), 50.0 (C14), 83.0 (C17), 126.0 (C4″), 126.4 (C1), 126.8
(C2), 129.1 (C2″), 129.4 (C5″), 130.0 (C4), 133.5 (C6″), 134.8
(C3″), 137.7 (C5), 139.0 (C3), 139.3 (C10), 144.3 (C1″), 172.7
1
1655 (CO, amide), 3360 (OH and NH2). H NMR (CD3OD):
0.91 (s, 18-CH3), 1.15 (m, 14α-CH and 15β-CH), 1.25−1.50 (m, 7α-
CH, 12α-CH, 8β-CH, and 11β-CH), 1.68 (t, J = 7.0 Hz, 15α-CH),
1.83 (m, 7β-CH), 2.04 (m, 12β-CH), 2.24 (m, 9α-CH), 2.36 (m, 11α-
CH), 2.47 (q, J = 12.3, 1H of 1′-CH2), 2.52 (m, 16α-CH), 2.71 (s,
CH3NHCO), 2.86 (m, 6-CH2), 3.17 (dd, J1 = 2.5 Hz, J2 = 12.3 Hz, 1H
of 1′-CH2), 3.41 (s, ArCH2CO), 3.84 (d, J = 9.4 Hz, 17α-CH), 6.96 (s,
4-CH), 7.01 (d, J = 8.0 Hz, 2-CH), 7.23 (d, J = 8.0 Hz, 1-CH), 7.36−
7.44 (m, 5″-CH and 6″-CH), 7.70 (d, J = 7.4 Hz, 4″-CH), 7.76 (s, 2″-
CH). 13C NMR (CD3OD): 13.3 (C18), 26.5 (CH3NHCO), 27.3
(C11), 28.6 (C7), 30.5 (C6), 33.0 (C15), 38.8 (C1′), 39.0 (C12), 39.7
(C8), 43.3 (C16), 43.4 (CH2CONH), 45.4 (C13), 45.7 (C9), 50.0
(C14), 83.0 (C17), 126.0 (C4″), 126.5 (C1), 127.3 (C2), 129.1
(C2″), 129.4 (C5″), 130.5 (C4), 133.5 (C6″), 133.8 (C3), 134.8
(C3″), 138.0 (C5), 140.2 (C10), 144.4 (C1″), 167.7 (CONHCH3),
175.1 (CONH2). LRMS for C29H37N2O3 [M + H]+ = 461.2. HPLC
purity of 90.2% (retention time = 4.1 min).
Synthesis of 26. To a solution of compound 1634 (1.5 g, 4.82
mmol) in DCM (400 mL) was added allyloxymethyl-benzene (1.3 g,
9.55 mmol), and the mixture was stirred under an argon atmosphere
for 5 min. Grubb’s catalyst (204 mg, 0.24 mmol) was then added, and
the solution was heated at 60 °C under argon atmosphere for 48 h.
The solution was evaporated and the residue purified by flash
chromatography using EtOAc/hexanes (5:95) to give 130 mg (6%) of
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(CONH2). LRMS for C30H41BrNO3 [M + CH3OH + H]+ = 542.2 and
544.3. HPLC purity of 97.5% (retention time = 16.6 min).
(t, J = 5.6 Hz, OCH2CH2Br), 6.62 (s, 4-CH), 6.70 (d, J1 = 8.7 Hz, 2-
CH), 7.19 (d, J = 8.6 Hz, 1-CH), 7.36−7.44 (m, 5″-CH and 6″-CH),
7.70 (d, J = 7.4 Hz, 4″-CH), 7.76 (s, 2″-CH). 13C NMR (CD3OD):
13.3 (C18), 27.5 (C11), 28.6 (C7), 30.7 (C6 and CH2Br), 33.0 (C15),
38.8 (C1′), 39.0 (C12), 39.8 (C8), 43.4 (C16), 45.4 (C9), 45.4 (C13),
49.9 (C14), 69.2 (OCH2CH2Br), 83.0 (C17), 113.3 (C2), 115.6 (C4),
126.0 (C4″), 127.4 (C1), 129.1 (C2″), 129.4 (C5″), 133.5 (C6″),
134.5 (C5), 134.8 (C3″), 139.1 (C10), 144.4 (C1″), 157.5 (C3),
172.7 (CONH2). LRMS for C29H39BrNO4 [M + CH3OH + H]+ =
544.2 and 546.3. HPLC purity of 98.0% (retention time = 18.6 min).
Multigram Synthesis of 23b. Synthesis of 19. 3-Vinyl-estra-
1(10),2,4-trien-17-one (19) was synthesized in two steps (6.4 g, 72%)
from estrone (8.6 g, 31.7 mmol) using a published procedure.45
Synthesis of 31 from 19. To a solution of alkene 19 (6.1 g, 21.7
mmol) in a mixture of acetone and acetonitrile (1:2) 450 mL was
added a saturated aqueous solution of NaHCO3 (300 mL) and oxone
(20.0 g, 65.1 mmol). The solution was stirred at room temperature for
4 h, then poured into water and extracted with EtOAc. The organic
phase was washed with brine, dried over Na2SO4, and evaporated
under reduce pressure. Purification by flash chromatography with
hexanes/EtOAc (8:2) yielded 4.9 g (76%) of 3-(oxiran-2-yl)estra-
1(10),2,4-trien-17-one (31). IR (film): 1736 (CO, ketone). 1H
NMR (CD3COCD3): 0.91 (s, 18-CH3), 1.40−2.50 (residual CH and
CH2), 2.76 and 3.06 (2m, CH2OCH), 2.89 (m, 6-CH2), 3.79 (dd, J1 =
4.0 Hz, J2 = 2.6 Hz, CHOCH2), 7.01 (s, 4-CH), 7.06 (d, J = 8.1 Hz, 2-
CH), 7.29 (d, J = 8.1 Hz, 1-CH). 13C NMR (CD3COCD3): 13.4
(C18), 21.4 (C15), 25.8 (C11), 26.5 (C7), 29.3 (C6) (under solvent
peaks), 31.9 (C12), 35.3 (C16), 38.3 (C8), 44.6 (C14), 47.7 (C13),
50.2 (CH2O of epoxide), 50.5 (C9), 51.6 (CHO of epoxide), 123.0
(C4), 125.6 (C2), 126.2 (C1), 135.6 (C10), 136.8 (C5), 140.0 (C3),
227.0 (C17). LRMS for C20H25O2 [M + H]+ = 297.2. HPLC purity of
99.9% (retention time = 13.2 min).
Synthesis of 29. To a solution of compound 129 (150 mg, 0.37
mmol) in acetone (3 mL) were added NaOH (50 mg, 1.25 mmol) and
allyl bromide (40 μL, 0.46 mmol). After the mixture was stirred at 60
°C for 5 h, the resulting solution was diluted with EtOAc, and the
solution was washed with a saturated aqueous solution of ammonium
chloride, brine, dried with MgSO4, and evaporated under reduced
pressure to give 165 mg of the corresponding 3-O-allyl compound.
The crude compound was found sufficiently pure to pursue to the next
step without further purification. Sodium periodate (108 mg, 0.50
mmol) was added to water (0.5 mL) and the solution stirred at 0 °C
for 5 min followed by the addition of RuCl3−H2O (4 mg, 0.02 mmol),
EtOAc (1 mL), and acetonitrile (1 mL). The 3-O-allyl compound (150
mg, 0.33 mmol) was added to the previous solution and stirred for 90
s. The reaction was quenched by the addition of a saturated aqueous
solution of Na2S2O3 (2 mL). The aqueous layer was extracted with
EtOAc, and the combined organic phase was dried with Na2SO4 and
evaporated under reduced pressure. The residue was dissolved in a
mixture of THF (1 mL) and water (1 mL), and NaBH4 (13 mg, 0.34
mmol) was added. The solution was stirred at room temperature for
20 min, water was then added (10 mL), and the mixture was extracted
with DCM. The organic phase was washed with a saturated NaHCO3
solution, dried with Na2SO4, and evaporated under reduced pressure.
The residue was dissolved in THF (1 mL) and water (1 mL) at 0 °C,
sodium periodate (144 mg, 0.67 mmol) was added in small portions,
and the solution was stirred for 20 min at room temperature. Ethylene
glycol (50 μL) was added, the reaction mixture was diluted with water
(3 mL), and the mixture was extracted with EtOAc. The combined
organic phase was dried with Na2SO4 and evaporated under reduced
pressure. The crude compound was purified by flash chromatography
(EtOAc/hexanes, 9:1) to give 25 mg (15%) of compound 29 (3-
{[(16β,17β)-17-hydroxy-3-(2-hydroxyethoxy)estra-1(10),2,4-trien-16-
Synthesis of 32 from 31. To a solution of oxirane 31 (4.9 g, 16.5
mmol) in MeOH in a Schlenck reactor were added ammonium
formate (10.4 g, 165 mmol) and 10% palladium on charcoal (2.50 g)
under an argon atmosphere at room temperature. The solution was
heated at 70 °C for 2 h. The suspension was filtered over Celite and
the filtrate evaporated under reduced pressure to dryness. Purification
by flash chromatography with hexanes/EtOAc (7:3) yielded 3.6 g
(73%) of 3-(2-hydroxyethyl)estra-1(10),2,4-trien-17-one (32). IR
1
yl]methyl} benzamide). H NMR (CD3OD): 0.91 (s, 18-CH3), 1.14
(m, 14α-CH and 15β-CH), 1.20−1.50 (m, 7α-CH, 12α-CH, 8β-CH,
and 11β-CH), 1.67 (m, 15α-CH), 1.81 (m, 7β-CH), 2.02 (m 12β-
CH), 2.17 (m, 9α-CH), 2.33 (m, 11α-CH), 2.47 (q, J = 12.3, 1H of 1′-
CH2), 2.52 (m, 16α-CH), 2.78 (m, 6-CH2), 3.17 (dd, J1 = 3.0 Hz, J2 =
12.5 Hz, 1H of 1′-CH2), 3.85 (m, 17α-CH and CH2OH), 3.99 (m,
OCH2CH2OH), 6.62 (d, J = 2.7 Hz, 4-CH), 6.70 (dd, J1 = 2.7 Hz, J2 =
8.6 Hz, 2-CH), 7.17 (d, J = 8.6 Hz, 1-CH), 7.36−7.44 (m, 5″-CH and
6″-CH), 7.70 (dd, J1 = 1.5 Hz, J2 = 6.0 Hz, 4″-CH), 7.76 (s, 2″-CH).
13C NMR (CD3OD): 13.3 (C18), 27.5 (C11), 28.6 (C7), 30.8 (C6),
33.0 (C15), 38.8 (C1′), 39.0 (C12), 39.9 (C8), 43.4 (C16), 45.4 (C9),
45.5 (C13), 49.9 (C14), 61.8 (CH2OH), 70.4 (OCH2CH2), 83.0
(C17), 112.2 (C2), 115.4 (C4), 126.0 (C4″), 127.2 (C1), 129.1
(C2″), 129.4 (C5″), 133.5 (C6″), 134.0 (C5), 134.8 (C3″), 138.9
(C10), 144.4 (C1″), 158.2 (C3), 172.7 (CONH2). LRMS for
C29H40NO5 [M + CH3OH + H]+ = 450.3.
Synthesis of 30. To a solution of compound 29 (20 mg, 0.46
mmol) in DCM (2 mL) and anhydrous THF (1 mL) was added at 0
°C triphenylphosphine (23 mg, 0.87 mmol) and carbon tetrabromide
(29 mg, 0.87 mmol). The solution was stirred at 0 °C for 40 min, and
second portions of triphenylphosphine (20 mg, 0.46 mmol) and
carbon tetrabromide (23 mg, 0.87 mmol) were added. The solution
was stirred at 0 °C for 40 min, and then third portions of
triphenylphosphine (20 mg, 0.46 mmol) and carbon tetrabromide
(23 mg, 0.87 mmol) were added. After 1 h at 0 °C, the resulting
solution was poured into water (100 mL), extracted with DCM, dried
with MgSO4, and evaporated under reduced pressure. The crude
compound was purified by flash chromatography (DCM/diethyl
ether/MeOH, 75:20:5) to give 12 mg (52%) of 30 (3-{[(16β,17β)-3-
(2-bromoethoxy)-17-hydroxyestra-1(10),2,4-trien-16-yl]methyl}-
benzamide). IR (KBr): 1663 (CO, amide), 3367 (OH and NH2).
1H NMR (CD3OD): 0.91 (s, 18-CH3), 1.14 (m, 14α-CH and 15β-
CH), 1.24−1.52 (m, 7α-CH, 12α-CH, 8β-CH, and 11β-CH), 1.67 (t, J
= 6.8 Hz, 15α-CH), 1.82 (m, 7β-CH), 2.03 (m, 12β-CH), 2.18 (m, 9α-
CH), 2.33 (m, 11α-CH), 2.47 (q, J = 12.3 Hz, 1H of 1′-CH2), 2.52
(m, 16α-CH), 2.80 (m, 6-CH2), 3.17 (d, J = 10.4 Hz, 1H of 1′-CH2),
3.67 (t, J = 5.6 Hz, OCH2CH2Br), 3.83 (d, J = 9.2 Hz, 17α-CH), 4.25
1
(film): 1728 (CO, ketone), 3464 (OH). H NMR (CDCl3): 0.91
(s, 18-CH3), 1.38−2.44 (residual CH and CH2), 2.51 (dd, J1 = 8.6 Hz,
J2 = 18.8 Hz, 16β-CH), 2.82 (t, J = 6.5 Hz, CH2CH2OH), 2.91 (m, 6-
CH2), 3.86 (t, J = 6.5 Hz, CH2CH2OH), 6.98 (s, 4-CH), 7.03 (d, J =
8.0 Hz, 2-CH), 7.25 (d, J = 9.0 Hz, 1-CH). 13C NMR (CDCl3): 13.8
(C18), 21.6 (C15), 25.7 (C11), 26.5 (C7), 29.4 (C6), 31.6 (C12),
35.9 (C16), 38.2 (C8), 38.7 (CH2CH2OH), 44.3 (C14), 48.0 (C13),
50.5 (C9), 63.6 (CH2OH), 125.6 (C1), 126.4 (C2), 129.7 (C4), 135.8
(C3), 136.7 (C5), 137.9 (C10), 221.5 (C17). LRMS for C20H27O2 [M
+ H]+ = 299.2. HPLC purity of 99.3% (retention time = 9.7 min).
Transformation of 32 to 22 and Then to 23b. The compound
2234 was synthesized in a yield of 84% (4.4 g) from compound 32 (3.6
g, 12.1 mmol) using the three-step general procedure for the
introduction of 16β-carbamoyl-m-benzamide side chain described
above. The bromination of 22 (4.4 g, 10.1 mmol) was performed using
the same procedure described above and provided 23b (2.5 g, 50%) in
excellent HPLC purity (98.5%).
17β-HSD1 Inhibition Assay. Breast cancer T-47D cells were
seeded in a 24-well plate (3000 cells/well) in RPMI medium
supplemented with insulin (50 ng/mL) and 5% dextran-coated
charcoal-treated fetal bovine serum (FBS), which was used rather
than untreated 10% FBS, to remove the remaining steroid hormones.
Stock solution of each compound to be tested was previously prepared
in EtOH and diluted with culture medium to achieve appropriate
concentrations prior to use. After 24 h of incubation, a diluted solution
was added to the cells to obtain the appropriate final concentration
(0.1 or 1 μM for screening and ranging from 1 nM to 10 μM for IC50
value determination). The final concentration of EtOH in the well was
adjusted to 0.1%. Additionally, a solution of [14C]-E1 (American
Radiolabeled Chemicals, Inc., St. Louis, MO, USA) was added to
219
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Journal of Medicinal Chemistry
Article
obtain a final concentration of 60 nM. Cells were incubated for 24 h,
and each inhibitor was assessed in triplicate. After incubation, the
culture medium was removed and labeled steroids (E1 and E2) were
extracted with diethyl ether. The organic phase was evaporated to
dryness with nitrogen. Residues were dissolved in DCM, dropped on
silica gel thin layer chromatography plates (EMD Chemicals Inc.,
Gibbstown, NJ, USA), and eluted with toluene/acetone (4:1) as
solvent system. Substrate [14C]-E1 and metabolite [14C]-E2 were
identified by comparison with reference steroids (E1 and E2) and
quantified using the Storm 860 system (Molecular Dynamics,
Sunnyvale, CA, USA). The percentage of transformation and the
percentage of inhibition were calculated as follow: % transformation =
100[14C]-E2/([14C]-E1 + [14C]-E2) and % of inhibition = 100(%
transformation without inhibitor − % transformation with inhibitor)/
% transformation without inhibitor.
Molecular Modeling. Docking simulations were performed using
MOE 2012.10.62 The crystal structure coordinates of 17β-HSD1,
including inhibitor 1 and cofactor NADP, were taken from PDB ID
3HB5 (PMID: 19929851). Hydrogen atoms were added using the
Protonate 3D tool included in MOE. The protein complex was
prepared using the LigX tool, included in MOE, to adjust H, rotamers,
and to minimize the system’s energy as previously described (PMID:
22566074). Docking simulations were performed using the rigid
receptor protocol and default parameters. Validation of the docking
protocol was carried out by a self-docking of compound 1, leading to
an RMSD of 0.37 Å between the docked and the crystallographic
structures. Because compound 23b shares its core structure with
compound 1, no further optimization of the docking protocol was
considered.
Compound 23b was built in MOE based on compound 1.
Hydrogens were readjusted and molecules were energy-minimized
prior to docking using the same protocol as for compound 1. Three
docking calculations were done for compound 23b, each using a
different binding site conformation: (A) using the crystallographic
conformation, (B) with Glu-282 side chain removed from the binding
site and exposed to the solvent, and (C) in addition to point 2, the
mutation of His-221 to Ala. The Glu-282 conformation was modified
using the Rotamer Explorer tool in MOE, and the lowest energy
conformer not pointing toward the binding site was selected. The
mutation of His-221 to Ala was done using the Sequence tool in MOE,
and no further energy minimization was required.
17β-HSD1 Inactivation Assay. Purified 17β-HSD1 kindly
provided by Dr. Sheng-Xiang Lin (CHU de Queb
́
ecResearch
Center)33 was used for inactivation/competition assays. An enzyme
solution was diluted in physiological buffer (100 mM Tris, 20%
glycerol, 0.2 mg/mL BSA, 2 mM EDTA) containing 1 mM of
NADPH as cofactor and was treated in triplicate with an ethanolic
solution of compound 23b to reach the accurate concentration, with or
without unlabeled E1 (500 nM). The mixture was then preincubated
at 37 °C with shaking before simultaneous dilution 1:20 in
physiological buffer and addition of radiolabeled substrate to a final
concentration of 60 nM [14C]-E1. Transformation of substrate was
stopped after 45 min of incubation at 37 °C with shaking by cooling
down on ice the enzyme medium and adding equal volume of diethyl
ether for further extractions, separation by TLC, and quantification of
radiolabeled steroids as described above.
AUTHOR INFORMATION
■
Corresponding Author
Estrogen-Sensitive Cell Proliferation Assays (Estrogenic
Activity). Quantification of cell growth was determined by using
CellTiter 96 aqueous solution cell proliferation assay (Promega,
Nepean, ON, Canada) following the manufacturer′s instructions.
MCF-7 or T-47D cells were resuspended in their medium (DMEM-
F12 or RPMI, respectively) supplemented with insulin (50 ng/mL)
and 5% dextran-coated charcoal treated FBS to remove remaining
estrogenic hormones present in the serum and medium. Aliquots (100
μL) of the cell suspension were seeded in 96-well plates (3000 cells/
well). After 48 h, the medium was changed for a new one containing
an appropriate concentration of the steroid derivative to be tested. The
medium was replaced every two days. Cells were left to grow for seven
days, either in presence or absence of the compound to be tested.
Inhibition Assays for 17β-HSD2, 17β-HSD7, and 17β-HSD12.
Selectivity of compounds 1 and 23b were assessed as previously
described30 in stably transfected HEK-293 cells kindly provided by Dr.
*Phone: 418-654-2296. E-mail: donald.poirier@crchul.ulaval.
ca.
Notes
The authors declare the following competing financial
interest(s): R. Maltais and D. Poirier have ownership interest
in a patent application. No potential conflicts of interest were
disclosed by the other authors.
ACKNOWLEDGMENTS
■
We thank the Canadian Institutes of Health Research (CIHR)
for an operating grant. We also thank Charles Ouellet for
performing an experiment for the estrogenicity, Dr. Van Luu-
The for providing HEK-293 transfected cells, and Dr. Sheng-
Xiang Lin for providing purified 17β-HSD1. Careful reading of
the manuscript by Micheline Harvey is also greatly appreciated.
Van Luu-The (CHU de Queb
́
ecResearch Center). Cells were
seeded in 24-well plates in protocol medium (MEM medium
supplemented with 5% dextran-coated charcoal stripped, G418 (700
μg/mL), penicillin (100 IU/mL), streptomycin (100 μg/mL), insulin
(50 ng/mL), glutamine (2 mM), nonessential amino acids (0.1 mM),
and pyruvate (1 mM)). After 48 h incubation, treatment with DMSO
solution (<0.5% final) of compound 1 or 23b in protocol medium was
conducted. Radiolabeled substrates ([14C]-E2 for 17β-HSD2 and
[14C]-E1 for 17β-HSD7 and 17β-HSD12), obtained from American
Radiolabeled Chemicals Inc. (St. Louis, MO, USA), were added to
obtain a final concentration of 60 nM. Once a substrate transformation
of 12−25% was reached, radiolabeled steroids (E1 and E2) were
extracted from the supernatant with diethyl ether. Separation and
quantification of substrates and metabolites were conducted as
previously described in the section on 17β-HSD1 inhibition assays.
Inhibition of CYP3A4. We used the commercially available P450
Inhibition Kit CYP3A4/DBF (BD Gentest) of BD Biosciences
(Mississauga, ON, Canada) according to manufacturer’s instructions,
with the exception that 23b was dissolved in a mixture of
dimethylsulfoxide/acetonitrile (5:95) instead of only acetonitrile.
The enzyme activity was measured by the fluorescence caused by
the enzymatic transformation of dibenzylfluorescein (DBF). Inhibitory
potencies were determined as IC50 values, which were calculated with
GraphPad Prism 5 software to express inhibition potency.
ABBREVIATIONS USED
■
ACN, acetonitrile; BOP, (benzotriazol-1-yloxy)tris-
(dimethylamino) phosphonium hexafluorophosphate; DCM,
dichloromethane; DHEA, dehydroepiandrosteone; 5-diol, 5-
androsten-3β,17β-diol; 4-dione, 4-androstene-3,17-dione;
DIPEA, diisopropylethylamine; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; E1, estrone; E2, estradiol; ERα,
estrogen receptor alpha; ERβ, estrogen receptor beta; EtOAc,
ethyl acetate; EtOH, ethanol; FBS, fetal bovine serum; 3β-
HSD, 3β-hydroxysteroid dehydrogenase; 17β-HSD, 17β-
hydroxysteroid dehydrogenase; MeOH, methanol; p-TSA,
para-toluene sulfonic acid; SAR, structure−activity relationship;
TEA, triethylamine; THF, tetrahydrofuran
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