Investigation of (E)-3-[4-(2-oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic acids as oral selective estrogen receptor down-regulators

Article abstract of DOI:10.1021/acs.jmedchem.5b00066

A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen rec

Full text of DOI:10.1021/acs.jmedchem.5b00066

Article
pubs.acs.org/jmc
Investigation of (E)‑3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic
Acids as Oral Selective Estrogen Receptor Down-Regulators
Sebastien L. Degorce,*,†,‡ Andrew Bailey,† Rowena Callis,§ Chris De Savi,† Richard Ducray,‡
́
Gillian Lamont,† Philip MacFaul,† Mickael Maudet,‡ Scott Martin,† Remy Morgentin,‡ Richard A. Norman,§
́
Aurelien Peru,‡ Jennifer H. Pink,† Patrick A. Ple,‡ Bryan Roberts,† and James S. Scott†
†Oncology Innovative Medicines Unit, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG,
́
́
United Kingdom
‡Oncology Innovative Medicines Unit, AstraZeneca, Centre de Recherches, Z.I. la Pompelle, BP1050, 51689 Reims Cedex 2,
France
§Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
S
* Supporting Information
ABSTRACT: A novel estrogen receptor down-regulator,
7-hydroxycoumarin (5, SS5020), has been reported with
antitumor effects against chemically induced mammary
tumors. Here, we report on our own investigation of
7-hydroxycoumarins as potential selective estrogen receptor
down-regulators, which led us to the discovery of potent
down-regulating antagonists, such as 33. Subsequent opti-
mization and removal of the 7-hydroxy group led to coumarin
59, which had increased potency and improved rat bio-
availability relative to SS5020.
SS1020 (6), was also reported to be a selective ER down-
regulator (SERD) by the same group.21 3b, 5, and 6 are believed
to function as SERDs due to their phenyl acrylic acid moiety,
which has now also been used in quinolines, such as 7a and 7b,22
and others more recently.23,24 The structural basis for the down-
regulating mechanism of this phenylacrylic acid has been
proposed by Greene et al. and involves a shift in helix 12,
ultimately leading to degradation of the receptor.25
INTRODUCTION
■
The estrogen receptor (ER) has been an oncology target for
the treatment of breast cancer since the 1960s.1 Since then, a
number of selective ER modulators (SERMs) and down-
regulators (SERDs) have been discovered, and this field has
been reviewed extensively.1−4 Among them, tamoxifen (a SERM,
2a)5,6 and fulvestrant (a SERD)7,8 have provided invaluable
treatments for a number of patients. The search for new anti-
estrogen agents is still continuing, however, due to emerging
drug-resistance mechanisms.9 More specifically, there is still an
unmet medical need for an oral SERD. Most SERMs and
SERDs are phenols (mimicking that of estradiol 1) because a
hydroxy group is generally required for potency, but these are
also associated with poor oral bioavailability (mostly due to
glucuronidation and sulfation of the phenols). In short, the
quest for an oral SERD remains a real challenge.
The 7-hydroxycoumarin scaffold has been known to medic-
inal chemists for over 50 years for its antagonistic properties
against the ER.10,11 Together with its reduced versions,
7-hydroxychromenes12 and 7-hydroxychromanes,13−16 they
have been reported as selective ER ligands or modulators (SERMs)
by many academic groups and pharmaceutical companies, including
AstraZeneca.17 Recently, SS5020 (5) had been reported to down-
regulate the estrogen receptor with antitumor effects against chem-
ically induced mammary tumors.18 Structurally (Figure 1), 5 can
be viewed as a hybrid of the known SERMs CHF4227 (4)15 and
GW7604 (3b),19,20 the latter of which is a descendant of
4-hydroxytamoxifen (2b). A closely related analogue of 3b,
RESULTS AND DISCUSSION
■
In our hands, 5 bound strongly to and down-regulated ER
albeit with modest potency (IC50 = 1.18 μM) and had good
overall properties, including notably low lipophilicity (Table 1).
However, although it showed efficacy upon repetitive daily oral
dosing in rats,18 the oral exposure for this molecule in both
mice and rats was suboptimal and variable and would therefore
benefit from further optimization. Although both solubility and
permeability (as measured in Caco2 cells) appeared suitable for
oral absorption, in vitro metabolism data indicated moderate to
high turnover in hepatocytes across all three species of interest.
This translated to very high in vivo clearances in both mice and
rats. No oral levels were subsequently observed from dosing
a 20 mg/kg suspension formulation of 5 in mice or from a
2 mg/kg solution in rats. Poor oral levels were consistent with
the observed high in vivo clearances. However, reasonable
Received: January 14, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b00066
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Journal of Medicinal Chemistry
Article
Figure 1. Chemical structures of certain ER ligands, modulators, and down-regulators.
Table 1. Detailed Profile of 3a, 3b, and 5
plasma levels were detected in rats following oral dosing of a
5 mg/kg suspension. Interestingly, good oral exposure was
observed for the closely related compound 3a lacking a
phenolic group in mice with low in vivo clearance and good
bioavailability observed. In rats, however, very high in vivo
clearance was again observed, consistent with high turnover in
rat hepatocytes. Oral exposure in rats was higher than expected
given the high clearance of the compound potentially due to
the compound undergoing entero-hepatic recirculation.
Although the X-ray structures of 4 and 5 had not been
published at the start of our investigation, that of 3a was
available (PDB ID: 1R5K),25 and we hypothesized that the
common phenyl acrylic acids should overlay whereas the
phenol of the coumarin core might overlay with the phenol
of 2b (PDB ID: 3ERT), as shown in Figure 2. We thus
compound
3a
<0.0032
>1.27
3b
5
a
ER binding IC50 (μM)
0.00015
0.007
0.0081
1.18
ER MCF7 down-regulation IC50
b
(μM)
c
PR MCF7 antagonism IC50 (μM) 4.0
0.012
>30
nd
1.5
d
PR MCF7 agonism IC50 (μM)
MCF7 proliferation EC50 (μM)
>100
>100
0.26
1.5
e
0.30
3.9
f
log D7.4
nd
g
LLE
<2.0
nd
nd
4.4
h
pKa acid/phenol
nd
4.4/7.7
470
i
solubility (μM)
3.3
200
>100
nd
j
hERG (μM)
>33
nd
>100
10
k
Caco2 A−B Papp (10−6 cm/s)
Hu/mu/rat PPB (% free)
l
<0.2/6.4/<0.01 nd/nd/0.09 1.2/1.2/0.98
Hu/mu/rat heps CLint
nd/nd/160
nd/nd/12
18/87/120
m
(μL/min/106 cells)
n
Mu/rat CL (mL/min/kg)
6.2/80
1.1/0.5
nd
nd
nd
210/67
4.7/1.4
n
Mu/rat Vss (L/kg)
o
p
q
r
s
Mu/rat bioavailability (%)
62 /68
0 /0 , 67
a
LanthaScreen TR-FRET ERα competitive binding assay (Life Tech-
b
c
nologies). ERα down-regulation measured in MCF-7 cells. Progesterone
receptore (PR) was measured as a biomarker for ERα antagonism in
d
MCF-7 cells. PR was measured as a biomarker for ERα agonism in
MCF-7 cells. Decrease in proliferation of MCF-7 cells.26 Measured using
e
f
shake-flask methodology with a buffer/octanol volume ratio of 100:1.
g
h
LLE = down-regulation pIC50 − log D7.4. pKa was measured via
potentiometric titrations using a Sirius Analytical GLpKa instrument.
i
Thermodynamic solubility of solid samples in a 0.1 M aqueous phosphate
buffer at pH 7.4 after 24 h at 25 °C.27 Inhibition of hERG channel IC50 in
j
an electrophysiology (IonWorks) assay.28 Compound permeability using
k
Caco2 cells.29 Plasma protein binding (PPB) was assessed by equilibrium
l
dialysis in the appropriate species plasma at 37 °C. Free and bound
m
concentrations were quantified by LC-UV-MS.27 CLint is the intrinsic
n
clearance of hepatocytes (heps).30 In vivo parameters were generated in
fed CD-1 mice and Alderley Park Han Wistar rats and involved two
animals for each study. IV dosing was conducted at 0.5 mg/kg using an
aqueous solution of DMSO and cyclodextrin. PO dosing was conducted as
Figure 2. Overlay of 3a (PDBID: 1R5K, gray carbons) and 2b (PDB
ID: 3ERT, pink carbons). The van der Waals radii of the protein
atoms in the 1R5K structure are shown as a continuous surface,
highlighting the ligand-binding pocket of the ER. Key protein residues
are labeled, and polar interactions are shown as dotted lines.
o
p
follows: A 50 mg/kg 0.5% polysorbate suspension. A 1 mg/kg solution
q
in 5% DMSO, 95% of a 30% aqueous cyclodextrin solution. A 20 mg/kg
r
s
propylene glycol suspension. A 2 mg/kg propylene glycol solution. A
5 mg/kg HPMC/tween suspension.
B
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Article
hypothesized that the structure−activity relationship (SAR)
around the 3-phenyl, assumed to overlay with the D-ring of 1,
might be optimizable based on our findings as well as other
groups that have reported beneficial substitutions in this
lipophilic pocket.14,22
(Figure 3). This o-methyl seems to be filling the same small
lipophilic hole as the terminal methyl of 3a and 2a, and both
phenyl acrylic acid down-regulating chains overlay reasonably
well. The coumarin core places the phenol in a similar position
to that of 2b, where it interacts with Glu351 and Arg394.
As lipophilicity increased with the introduction of an
o-methyl group and either a p-halogen or a p-trifluoromethoxy
substituent on the C4-phenyl ring of molecules 24−26, to
reduce log D and improve LLE, we investigated different linkers
between the two aromatic rings (Table 3). O- and N-linked
analogues of 4-benzylcoumarin 24 were prepared as shown
in Scheme 2. Acylation of 3-methoxyphenol with substituted
phenyl acetic acids (28) gave the desired 2-hydroxy-4-
methoxyketone intermediates (29). These could then be
condensed with either diethyl carbonate or ethyl chloroformate
to effect conversion to 4-hydroxycoumarins (30), which in turn
were converted to the key 4-bromocoumarins (31) with SEM
protection of the 7-phenol group. O-Linked compounds were
prepared under displacement conditions with (E)-tert-butyl
3-(4-hydroxyphenyl)acrylate and subsequent deprotection of
the tert-butyl ester. The corresponding N-linked compounds
were prepared by Buchwald coupling of 31a with (E)-ethyl-3-
(4-aminophenyl)acrylate; intermediate 34 could then be either
deprotected to yield 35 or converted to the N-methyl derivative
with methyl iodide and then deprotected to yield 36.
The 4-O-linked coumarins 32 and 33 proved to be better
binders and down-regulators with an additional reduction in
lipophilicity, which gave superior LLEs (6.1 and 6.5,
respectively) compared to 4-C-linked coumarin 24 (LLE =
4.8), 4-N-linked coumarins 35 (LLE = 4.5) and 36 (LLE = 4.9),
or directly linked coumarin 27 (LLE = 4.5). Table 4
summarizes the profiles of 32 and 33, which appeared to be
much improved versions of the initial hit SS5020 in terms of
binding, down-regulation, and antagonistic potencies (33: >15-,
>420-, and >430-fold, respectively). These translated to excellent
inhibition of in vitro proliferation of MCF7 cells (e.g., 33 has an
EC50 < 0.1 nM). However, pharmacokinetic studies performed
on these cells showed no improvement over 5 in terms of oral
levels, although clearances were slightly reduced.
As an initial part of our optimization program, a set of 3-aryl-
7-hydroxycoumarins were prepared as shown in Scheme 1.
Variation of the 3-aryl unit was achieved via Suzuki coupling of
3-chlorocoumarin intermediate 12 with a range of aryl boronic
acids. The core ring system was prepared by condensation of
keto ester 9 with resorcinol under acid catalysis. The phenol
moiety was then pivaloyl protected to prevent over-reaction
of the coumarin core during subsequent chlorination with
N-chlorosuccinimide (NCS) in acetic acid. Carrying out the
chlorination in acetic acid was also found to be advantageous, as
it disfavored chlorination of the 4-benzyl CH2 group. Suzuki
coupling proceeded efficiently either on the tert-butyl ester
intermediate with subsequent deprotection to the acid or by
first deprotecting and carrying out the coupling on the
deprotected acid intermediate. Other analogues in this set
were prepared via an alternative synthesis, as shown later in
Scheme 4.
Meta substitution (Table 2) does not appear to particu-
larly favor better potency: the direct meta analogues 13−15
offered no advantage over parent SS5020 in contrast to
previously reported SARs in analogues of quinoline 7a, where
m-trifluoromethyl greatly enhanced cell potency.22 Lipophilic
groups at the para position analogues 16−20 offered for the
first time single digit nanomolar binders with submicromolar
down-regulation. The p-trifluoromethoxy analogue 20 proved
to be even more potent with a 10-fold improvement in down-
regulation (IC50 = 52 nM), potentially the result of inserting
the trifluoromethyl group deeper into the lipophilic pocket.
We observed a similar potency boost with o-methyl analogues
21−24, which we attributed to filling a lipophilic hole occupied
by the ethyl group of 2a as well as locking the conformation of
this phenyl ring. These two findings could combine to give 26,
a strong binder (1 nM) and potent down-regulator (7 nM). We
obtained an X-ray structure of compound 24 in ERα, which
validated our initial hypothesis and provided a rationale for
the role of o-methyl in the observed potency improvement
To understand the metabolic fate of our 7-hydroxycoumarins,
we obtained experimental evidence through metabolite
a
Scheme 1. Synthesis of 3-Aryl Coumarins via Suzuki Couplings
a
Reagents and conditions: (a) (i) potassium 3-ethoxy-3-oxopropanoate (2.5 equiv), MgCl2 (3 equiv), Et3N (5.3 equiv), MeCN, (ii) 8 (1 equiv),
CDI (1.25 equiv), MeCN, (iii) 80 °C, 3 h, (iv) 2N aq HCl, rt, 5 min (55%); (b) resorcinol (1 equiv), TsOH (0.1 equiv), toluene, 100 °C, 4 h
(61%); (c) PivCl (1 equiv), Et3N (1.1 equiv), DCM, rt, 10 min (99%); (d) NCS (3.1 equiv), AcOH, 75 °C, 18 h (72%); (e) K2CO3 (1.2 equiv),
MeOH, rt, 1 h (88%); (f) t-butyl acrylate (1.6 equiv), DIPEA (1.2 equiv), (dtbpf)PdCl2 (0.07 equiv), dioxane, 90 °C, 15 h (80%); (g) HCO2H,
rt (94%); (h) ArB(OH)2 (1.2 equiv), S-Phos (0.14 equiv), Pd(OAc)2 (0.07 equiv), K2CO3 (3 equiv), dioxane, water, 90 °C, 1 h or ArB(OH)2
(1.3 equiv), DtBPPS Suzuki mix31 (0.05 equiv), K2CO3 (3 equiv), DMF, water, 65 °C, 18 h (13−96%).
C
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a
Scheme 2. Synthesis of Various Linkers
a
Reagents and conditions: (a) 3-methoxyphenol (1 equiv), BF3·Et2O (4 equiv), chlorobenzene, 100 °C, 2−24 h (44−67%); (b) 29a,
diethylcarbonate (15 equiv), NaH (4 equiv), 0−50 °C, 5 h (84%) or 29b, ethyl chloroformate (3 equiv), K2CO3 (4 equiv), acetone, reflux, 4 h
(50%); (c) POBr3 (1.1 equiv), 1,2-dichlorobenzene, 170 °C, 3−24 h (73−81%); (d) 48% aq HBr, AcOH, 110 °C, 1−3 days (80−95%); (e) SEMCl
(1.2 equiv), DIPEA (1.2 equiv), DCM, rt, 1−4 h (60−91%); (f) (E)-tert-butyl 3-(4-hydroxyphenyl)acrylate (1 equiv), Cs2CO3 (1.2 equiv), MeCN,
70 °C, 3−4 h (63−66%); (g) 31a (1) TBAF·H2O (5 equiv), THF, rt-50 °C, 24 h; (2) TFA, DCM, rt, 3 h (100%) or 31b TFA, DCM, rt, 2 h (89%);
(h) (E)-ethyl-3-(4-aminophenyl)acrylate (1.5 equiv), Pd(PtBu)3 (0.08 equiv), K3PO4 (1.5 equiv), dioxane, 125 °C, 2 h (43%); (i) (1) 6N HCl,
iPrOH, MeOH, rt, 2 h (2) 6N NaOH, MeOH, rt, 3 h (35 55%; 36 30% over 2 steps); (j) (1) NaH (1.5 equiv), DMF, 5 min, (2) MeI (6 equiv), rt, 30 min.
Table 2. Effect of the C3-Aryl on ER Binding,
Down-Regulation, and Lipophilicity
in rat hepatocytes. Acyl glucuronide was also observed but to
a lesser extent. To reduce the formation of these conjugates,
we examined how substitutions proximal to the phenol could
influence metabolism through lowering lipophilicity by altering
the pKa (fluoro substitutions) or increasing steric hindrance
(methyl substitutions).
a
5-, 6-, and 8-Fluoro analogues 39, 42, and 43 were prepared
in a similar manner to the syntheses shown in Schemes 1 and 2
starting from the appropriately substituted fluorophenols
(Scheme 3). Modification of the procedure was found to be
necessary for step d during the synthesis of 43, as the reaction
proved to be very slow under the conditions we had used
previously for Friedel−Crafts acylation (four equiv of BF3·Et2O
in chlorobenezene). We found instead that the reaction pro-
ceeded more efficiently in neat BF3·Et2O; however, cyclization
of the resultant dihydroxyarylketone to coumarin with either
diethyl carbonate or ethyl chloroformate was unsuccessful.
Monomethylation was thus carried out on the dihydroxyketone
to afford intermediate 41b, which then reacted smoothly to
afford the required intermediate coumarin.
The corresponding 6- and 8-methyl substituted analogues
with a C4-carbon link to the aryl acrylic acid unit were prepared
by a route alternative to that of Scheme 1, introducing the C-3
aryl unit at an earlier stage of the synthesis (Scheme 4). This
approach provided efficient, shorter synthesis of the target
compounds but without the flexibility of late stage C-3 aryl
variation. As this method was successful for the synthesis of
compounds 46 and 47 (Table 5), it was also attempted for the
preparation of 5-fluoro compound 39 starting from 3-fluoro-5-
methoxyphenol. In this case, however, the fluorine substituent
was hydrolyzed under the reaction conditions during step c, and
the isolated product was the analogous 5,7-dihydroxycoumarin;
ER binding
ER MCF7 down-
log
c
b
entry R1
R2
R3 IC50 (μM) regulation IC50 (μM) D7.4 LLE
5
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
0.0081
0.017
0.041
1.18
3.09
5.64
1.71
1.5 4.4
2.2 3.3
1.9 3.3
2.9 2.9
nd nd
2.4 4.0
2.6 3.7
1.9 4.6
2.8 4.5
1.7 4.5
nd nd
1.8 3.8
2.1 4.8
nd nd
13
14
15
16
17
18
19
20
21
Me
OMe
OCF3
H
H
H
H
H
H
H
0.0059
0.0090
0.0037
0.0046
0.0038
0.0016
0.0030
0.0022
0.011
F
1.28
Cl
0.471
0.531
0.350
0.052
0.660
0.178
2.26
0.117
0.030
0.007
CF3
OMe
OCF3
F
22 Cl
F
F
F
23 OMe
24 Me
25 Me
26 Me
H
H
H
H
0.0039
0.0015
0.0010
Cl
OCF3
3.1 5.1
a
b
Experimental assays as per Table 1 unless otherwise specified. All
IC50 data are the mean of at least n = 2 independent measurements.
c
Each has an SEM 0.2 log units. All IC50 data are the mean of at least
n = 3 independent measurements. Each has an SEM 0.3 log units.
identification to show that phenol 16 was extensively con-
jugated with glucuronides and that sulfates were being formed
D
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Journal of Medicinal Chemistry
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Figure 3. Overlay of 24 (PDB ID: 5AK2, green carbons) and 3a (PDB ID: 1R5K, gray carbons). The van der Waals radii of the protein atoms in the
1R5K structure are shown as a continuous surface, highlighting the ligand-binding pocket of the ER. Key protein residues are labeled, and polar
interactions are shown as dotted lines.
Table 3. Effect of the C4-Linker on ER Binding,
Down-Regulation, and Lipophilicity
Table 4. Selected Data for O-Linked 7-Hydroxycoumarins
32 and 33
a
a
compound
32
33
0.0039
PR MCF7 antagonism IC50 (μM)
PR MCF7 agonism IC50 (μM)
MCF7 proliferation EC50 (μM)
pKa acid/phenol
0.027
>100
>3.1
0.0045
3.9/7.1
>2000
<0.0001
nd/8.2
97
solubility (μM)
hERG (μM)
>100
>100
ER binding
ER MCF7 down-
log
Caco2 A−B Papp (10−6 cm/s)
Hu/Mu/rat PPB (% free)
19
15
b
b
entry
X
R
IC50 (μM) regulation IC50 (μM)
D7.4 LLE
0.25/2.8/0.51
32/68/50
0.12/0.84/0.37
11/9/41
c
27
24
32
33
35
36
F
F
F
0.0058
0.0039
0.0004
0.0005
0.025
0.256
0.117
0.021
0.0027
1.45
2.0
2.1
1.6
2.1
1.3
2.3
4.6
4.8
6.1
6.5
4.5
4.9
Hu/Mu/rat heps CLint
CH2
O
(μL/min/106 cells)
b
rat CL (mL/min/kg)
35
0.9
5
38
O
OCF3
b
rat Vss (L/kg)
0.7
NH
NMe
F
F
b
rat bioavailability (%)
a
0
0.0099
0.060
b
Experimental assays as per Table 1 unless otherwise specified. IV
dosing was conducted at 0.5 mg/kg using an aqueous solution of DMSO
and cyclodextrin. PO dosing was conducted using HPMC/tween suspen-
sions dosed at 5 and 50 mg/kg for 32 and 33, respectively.
a
b
Experimental assays as per Table 1 unless otherwise specified. All
IC50 data are the mean of at least n = 3 independent measurements.
Each has an SEM 0.1 log units. n = 1.
c
therefore, the route outlined in Scheme 1 was employed instead
to provide 39, as described above (Scheme 3).
despite an expected loss of potency improvements in pharma-
cokinetics in the absence of the 7-hydroxy group may result in
compounds with suitable profiles to function as in vivo tools.
Exploration of the C-7 functionality was achieved for several
compounds in the C4-carbon linked series by carrying out a
variety of palladium-catalyzed reactions on the versatile 7-triflate
intermediate bearing the appropriate C-3 aryl substitution, as
shown in Scheme 5. In the case of 53, the triflate was first
reacted in a Stille coupling to give alkene 51. Ozonolysis of this
product afforded the bis-aldehyde 52, which could be converted
to the 7-difluoromethyl analogue by reaction with Deoxo-Fluor.
The fluorination was found to be selective for the 7-formyl
group, presumably due to electronic differences between the
two aldehydes, although a smaller amount of the bis-
difluoromethyl product was also formed and was separated
Unfortunately, lowering the pKa of the phenol in an attempt
to reduce metabolism seemed to be detrimental to down-
regulation with no significant effect on glucuronidation or sulfa-
tion as observed by metabolism identification in rat hepatocytes.
Notably, 5-fluoro-7-hydroxycoumarin 39 seemed to be the
most tolerated in terms of potency and seemed to stop
sulfation but not glucuronidation of the phenol, which was
identified as the major metabolite. The introduction of fluoro
substituents reduced lipophilicity through the expected effect
on the phenol’s pKa even in the more distant 5-fluoro analogue.
Methyl blockers on each side of the phenol also proved
detrimental in terms of potency, and as a result, we decided to
probe phenol replacements instead. Our hypothesis was that
E
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a
Scheme 3. Synthesis of 5-, 6-, and 8-Fluoro Substituted 7-Hydroxycoumarins
a
Reagents and conditions: (a) 9, MeSO3H, 0 °C, rt, 3h (79%); (b) PivCl (1.1 equiv), Et3N (1.1 equiv), DCM, rt, 1 h (65%); (c) (4-fluoro-2-
methylphenyl)acetic acid (1.1 equiv), BF3·Et2O (4 equiv), chlorobenzene, 100 °C, 20 h (52%); (d) (4-fluoro-2-methylphenyl)acetic acid (1.1 equiv),
BF3·Et2O (40 equiv), 80 °C, 24 h (32%); (e) MeOH (1.05 equiv), DIAD (1.3 equiv), PPh3 (1.3 equiv), THF, 0 °C, 30 min (38%).
a
Scheme 4. Synthesis of 6- and 8-Methyl Substituted 7-Hydroxycoumarins
a
Reagents and conditions: (a) 2-(4-bromophenyl)acetic acid (1.1 equiv), BF3·Et2O (3.5 equiv), chlorobenzene, 90 °C, 18 h (36−45%); (b) SEMCl
(0.95 equiv), DIPEA (2 equiv), DCM, rt, 1 h (44−79%); (c) (i) 2-(4-(trifluoromethoxy)phenyl)acetic acid (1.95 equiv), CDI (2 equiv), DMF, rt,
i
45 min, (ii) 45a or 45b, K2CO3 (3 equiv), DMAP (0.2 equiv), 80 °C, 2.5 h, (iii) 5N HCl in PrOH, rt, 2 h (46−55%); (d) (i) tert-butyl acrylate
(1.5 equiv), (dtbpf)PdCl2 (0.1 equiv), DIPEA (1.2 equiv), dioxane, 80 °C, 3 h, (ii) TFA, DCM, rt, 16 h (41−61%).
a
Table 5. Effect of Core Substitutions on ER Binding, Down-Regulation, and in Vitro Properties
a
b,c
Experimental assays as per Table 1 unless otherwise specified. All IC50 data are the mean of at least n = 3 independent measurements. Each has
an SEM 0.1 log units.
from the product during chromatographic purification. Reintro-
duction of the acrylic acid unit was achieved by condensation with
malonic acid and subsequent decarboxylation to afford 53.
Other compounds in this set of C-7 variants included the
7-methoxy compound 54 and its fluoro analogue 55, which
were prepared from 3-methoxyphenol or 3-fluorophenol, respec-
tively, in a manner similar to the route shown in Scheme 4.
Variation at C-7 was also carried out in the C-4 O-linked series.
The 7-ethoxy analogue 64 was prepared via a similar route to that
shown in Scheme 2; conversion of 4-hydroxy-7-methoxycoumarin
F
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a
Scheme 5. Synthesis of Various 7-Substituted, 4-C-Linked Coumarins
a
Reagents and conditions: (a) Tf2NPh (1.2 equiv), Et3N (1.3 equiv), DCM, rt, 18 h or Tf2O (1.1 equiv), Et3N (3 equiv), DCM, 0 °C, rt, 30 min; (b)
t
Pd(OAc)2 (0.2 equiv), PPh3 (0.4 equiv), Et3N (3 equiv), HCO2H, DMF, 60 °C, 1 h (42%); (c) BuOCH2BF3K (1.3 equiv), dichloro(1,5-
cyclooctadiene) Pd(II) (0.05 equiv), dppf (0.1 equiv), K3PO4 (7.2 equiv), tBuOH, H2O, 110 °C, 20 h; (d) TFA, DCM, rt, 2 h (28% over 3 steps); (e)
MeCONH2 (1.2 equiv), Pd(OAc)2 (0.05 equiv), X-Phos (0.1 equiv), K3PO4 (1.6 equiv), toluene, 90 °C, 5 h (95%); (f) 36% aq HCl, THF, MeOH,
65 °C, 3 h (36%); (g) LiOH (1.5 equiv), MeOH, THF, H2O, 75 °C, 1 h (90%); (h) Bu3(CH2CH)Sn (1.2 equiv), Pd2(dba)3 (0.05 equiv), LiCl
(6.5 equiv), PPh3 (1.01 equiv), NMP, 60 °C, 18 h (88%); (i) (1) K2OsO4.2H2O (0.1 equiv), NMO (3 equiv), THF, rt, 5 h, (2) NaIO4 (5 equiv), H2O,
rt, 20 h (quant); (j) Deoxo-Fluor (2 equiv), DCM, rt, 5 h (40%); (k) malonic acid (2.1 equiv), piperidine (0.3 equiv), pyridine, 90 °C, 2 h (85%).
a
Scheme 6. Synthesis of Various 7-Substituted 4-O-Linked Coumarins
a
Reagents and conditions: (a) (E)-tert-butyl 3-(4-hydroxyphenyl)acrylate (1 equiv), K2CO3 (5 equiv), dioxane, 50 °C, 4 h (46%); (b) TFA, 0 °C, 2 h
(97%); (c) 58 (4-fluoro-2-methylphenyl)boronic acid or 59 [2-methyl-4-(trifluoromethoxy)-phenyl]boronic acid (1.5 equiv), Pd(OAc)2 (0.05 equiv),
−
S-Phos-SO3 Na+ (0.1 equiv), Na2CO3 (4 equiv), dioxane, H2O, 100 °C, 2 h (11%); (d) methyl 2-(E)-3-(4-hydroxyphenyl)prop-2-enoate (1.1 equiv),
K2CO3 (2 equiv), MeCN, rt, 4 h (43%); (e) BBr3 (5 equiv), DCM, −78 °C, rt, 18 h (quant.); (f) H2SO4, MeOH, 90 °C, 18 h (77%); (g) Tf2O (1.3
equiv), Et3N (3 equiv), DCM, −10 °C, 2 h (80%); (h) (CH2CH)BF3K (1.5 equiv), Et3N (2 equiv), Pd(dppf)Cl2 (0.1 equiv), 100 °C, 4 h (78%).
G
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a
Table 6. Phenol Replacements
a
b,c
Experimental assays as per Table 1 unless otherwise specified. All IC50 data are the mean of at least n = 2 independent measurements. Each has
an SEM 0.2 log units.
a
30b to 4-bromocoumarin and subsequent deprotection with
HBr in acetic acid gave the phenol intermediate, which could be
alkylated with ethyl iodide. Displacement of the 4-bromo group
with (E)-tert-butyl 3-(4-hydroxyphenyl)acrylate followed by
ester hydrolysis then led to final product 64.
The synthesis of other C-4 O-linked C-7 variants is shown in
Scheme 6. The 7-H analogue was synthesized from 3-bromo-4-
triflate intermediate 56, prepared according to the method
reported by Wu et al.32 Selective displacement of the triflate
with (E)-tert-butyl 3-(4-hydroxyphenyl)acrylate and deprotec-
tion gave acid 57, which could then be converted to 58 or 59
by Suzuki coupling. A similar approach starting from
7-methoxy-4-triflate 60 gave intermediate 61a. Conversion of
the 7-methoxy group to the triflate was then carried out as
shown to provide 61b, which could then be taken through to
7-difluoromethyl compound 63 via 7-vinyl compound 62 in an
analogous route to that for compound 53 (Scheme 5).
Table 7. Detailed Profile of O-Linked Coumarins 58 and 59
compound
58
0.33
59
0.18
PR MCF7 antagonism IC50 (μM)
PR MCF7 agonism IC50 (μM)
MCF7 proliferation EC50 (μM)
pKa
>3.1
nd
>3.1
0.020
3.7
5.3
solubility (μM)
>1500
>100
47
>470
nd
hERG (μM)
Caco2 A−B Papp (10−6 cm/s)
Hu/rat PPB (% free)
39
0.50/1.4
9/27
23
0.16/0.32
7/19
15
Hu/rat heps CLint (μL/min/106 cells)
b
rat CL (mL/min/kg)
b
rat Vss (L/kg)
0.47
47
1.7
b
rat bioavailability (%)
35
a
b
Experimental assays as per Table 1 unless otherwise specified. IV
dosing was conducted at 0.5 mg/kg using an aqueous solution of
DMSO and cyclodextrin. PO dosing was conducted using HPMC/
tween suspensions dosed at 1 mg/kg.
Removal of the 7-hydroxy (compounds 48, 58, and 59,
Table 6) resulted in significant loss of both ER binding and
down-regulation potency. Notably, 59, carrying an optimized
aryl substituent, was the most potent des-phenol synthesized in
this scaffold with a down-regulation IC50 of 100 nM. Replace-
ment with a fluorine (55) was significantly worse than the
corresponding hydrogen (48) and thus offered no way forward.
Similarly, 7-aminocoumarin 50 showed a large loss of down-
regulation potency (>100-fold) compared to phenol 24. The
isolipophilic 7-difluoromethyl analogues 53 and 63 also showed
reduced potency, whereas the extended hydroxymethyl 49,
although less potent, proved to be the most promising
replacement in terms of LLE. However, experimental evidence
showed that metabolism then switched to oxidation of the
benzylic alcohol to the corresponding carboxylic acid with no
observed parent glucuronidation. Capped phenols 54 and 64 as
7-methoxy and 7-ethoxy coumarins, respectively, lost ∼100-fold
potency in terms of down-regulation. We found that the best
compromise was the des-phenol analogues 58 and 59, which
were profiled by pharmacokinetic studies (Table 7).
a down-regulation potency of 100 nM represented an attractive
in vivo tool compound to evaluate the potential of an oral SERD.
CONCLUSION
■
7-Hydroxycoumarins proved to be very potent SERD antag-
onists with good overall properties but were prone to metab-
olism via sulfation and glucuronidation, leading to generally
poor oral bioavailability. Through optimization of the scaffold
with regard to the linker and aryl substituents, highly potent
phenols, such as 33, were identified, although bioavailability
remained poor. Removal of the 7-hydroxy groups in des-
phenols 58 and 59 resulted in improvements in pharmacoki-
netic profiles with reasonable levels of potency being retained.
These compounds have the potential to be used as in vivo tools
to explore the potential of oral SERDs.
EXPERIMENTAL SECTION
■
General Procedures. All solvents and reagents were purchased
from commercial sources and used without further purification. Flash
silica chromatography was typically performed on an Isco Companion,
using Silicycle silica gel, 230−400 mesh 40−63 μm cartridges, Grace
Pleasingly, in rats, the compounds were characterized by low
clearances and much improved bioavailabilities (47% and 35%
for 58 and 59, respectively). Des-phenol 59, in particular, with
H
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Resolv silica cartridges, or Isolute Flash Si or Si II cartridges. Reverse
phase chromatography was performed using a Waters XBridge Prep
C18 OBD column (5 μm silica, 19 mm diameter, 100 mm length)
using decreasingly polar mixtures of either water containing 1% NH3
and MeCN or water containing 0.1% formic acid and MeCN as
eluents. Analytical LC-MS was performed on a Waters 2790 LC with a
996 PDA and 2000 amu ZQ Single Quadrupole Mass Spectrometer
using a Phenomenex Gemini 50 × 2.1 mm, 5 μm C18 column, or
UPLC was performed on an Waters Acquity Binary Solvent Manager
with Acquity PDA and an SQD mass spectrometer using a 50 ×
2.1 mm, 1.7 μm BEH column from Waters. Purities were measured by
UV absorption at 254 nm or TIC and are ≥95% unless otherwise
stated. NMR spectra were recorded on a Bruker Av400, Av500, or
DRX400 spectrometer at 400 or 500 MHz in d6-DMSO at 303 or
4-Bromo-3-(4-fluoro-2-methylphenyl)-7-((2-(trimethylsilyl)-
ethoxy)methoxy)-2H-chromen-2-one (31a).
30a (16.48 g, 54.9 mmol) and POBr3 (6.14 mL, 60.4 mmol) in 1,2-
dichlorobenzene (288 mL, 2.6 mol) were stirred at 170 °C for 3 h.
After cooling, the solvent was removed under vacuum. The residue
was diluted with DCM, quenched with saturated aqueous NaHCO3
and extracted with DCM. The combined organic phases were dried
over MgSO4, filtered, and concentrated. The residual solid was
triturated with petroleum ether and dried under vacuum at 50 °C to
afford 4-bromo-3-(4-fluoro-2-methylphenyl)-7-methoxy-2H-chromen-
1
297 K unless otherwise indicated. H NMR spectra are reported as
chemical shifts in parts per million (ppm) relative to an internal solvent
reference.
1
2-one (16.12 g, 81%) as a gray solid. H NMR (400 MHz, DMSO,
303 K) 2.13 (3H, s), 3.92 (3H, s), 7.05−7.16 (3H, m), 7.19 (1H, dd),
All IC50 data are quoted as geometric mean values, and statistical
analyses are available in the Supporting Information. All experimental
activities involving animals were carried out in accordance with
AstraZeneca animal welfare protocols, which are consistent with The
American Chemical Society Publications rules and ethical guidelines.
Synthesis of Representative Key Example Compounds 32,
33, 58, and 59. 2-(4-Fluoro-2-methylphenyl)-1-(2-hydroxy-4-
methoxyphenyl)ethanone (29a).
7.27 (1H, dd), 7.82 (1H, d); m/z 363 [M + H]+.
A suspension of 4-bromo-3-(4-fluoro-2-methylphenyl)-7-methoxy-
2H-chromen-2-one (14.98 g, 41.25 mmol) and HBr, 48% in H2O
(327 mL, 2.9 mol) in acetic acid (100 mL), was stirred at 110 °C for
3 days. The reaction mixture was allowed to cool to room temperature
and concentrated to dryness. Water was added to the residue; the solid
was filtered, washed twice with water, and then taken up in EtOAc.
The organic layer was washed with water, brine, dried over MgSO4,
filtered, and concentrated to afford a gray solid. The crude product was
adsorbed onto silica and purified by flash silica chromatography eluting
with 0 to 10% EtOAc in DCM. The solvent was evaporated to dryness
to afford 4-bromo-3-(4-fluoro-2-methylphenyl)-7-hydroxy-2H-chromen-
1
2-one (9.59 g, 80%) as a colorless solid. H NMR (400 MHz, DMSO,
303 K) 2.12 (3H, s), 6.82 (1H, d), 6.92 (1H, dd), 7.10 (1H, td), 7.18
(1H, dd), 7.25 (1H, dd), 7.74 (1H, d), 10.86 (1H, s); m/z 349 [M + H]+.
(2-(Chloromethoxy)ethyl)trimethylsilane (3.40 mL, 19.2 mmol)
was added dropwise to a stirred solution of 4-bromo-3-(4-fluoro-2-
methylphenyl)-7-hydroxy-2H-chromen-2-one (5.59 g, 16.0 mmol) and
DIPEA (3.35 mL, 19.2 mmol) in DCM (57.3 mL) at 0 °C. The
resulting mixture was stirred at room temperature for 1 h. The reaction
mixture was quenched with water; the phases were separated, and the
organic layer was washed with 0.5 M HCl, brine, dried over MgSO4,
filtered, and concentrated. The crude product was purified by flash
silica chromatography eluting with 100% DCM and then further
purified by flash silica chromatography with an elution gradient of 0 to
100% DCM in heptane. Product-containing fractions were evaporated
to dryness to afford 31a (6.17 g, 60%) as a pale yellow oil, which
solidified upon standing, containing 0.25 equiv of 2-trimethylsilyle-
thanol. 1H NMR (400 MHz, DMSO, 303 K) −0.01 (9H, s), 0.91 (2H,
t), 2.13 (3H, s), 3.68−3.82 (2H, m), 5.40 (2H, s), 7.06−7.17 (3H, m),
7.18 (1H, dd), 7.26 (1H, dd), 7.77−7.89 (1H, m); m/z 479 [M + H]+.
(E)-3-(4-((3-(4-fluoro-2-methylphenyl)-7-hydroxy-2-oxo-2H-chromen-
4-yl)oxy)phenyl)acrylic acid (32).
BF3·Et2O (53.4 mL, 432.5 mmol) was added dropwise to a stirred
mixture of 3-methoxyphenol (11.87 mL, 108.12 mmol) and (4-fluoro-
2-methylphenyl)acetic acid (20 g, 118.9 mmol) in chlorobenzene
(232 mL) at 100 °C under argon. The resulting mixture was stirred
at 100 °C for 20 h. The reaction mixture was quenched with water
(340 mL), diluted with EtOAc (200 mL), and washed with brine
(2 × 200 mL). The organic layer was dried over MgSO4, filtered, and
evaporated. The crude product was adsorbed on silica gel and purified
by flash chromatography on silica gel eluting with 0 to 10% EtOAc in
heptane. The solvent was evaporated to dryness to afford 29a (19.89 g,
1
67%) as a yellow solid. H NMR (400 MHz, CDCl3, 303 K) 2.27
(3H, s), 3.86 (3H, s), 4.23 (2H, s), 6.46 (1H, d), 6.49 (1H, dd), 6.88
(1H, td), 6.95 (1H, dd), 7.09 (1H, dd), 7.77 (1H, d), 12.62 (1H, s);
m/z 275 [M + H]+.
3-(4-Fluoro-2-methylphenyl)-4-hydroxy-7-methoxy-2H-chromen-
2-one (30a).
A solution of 2-(4-fluoro-2-methylphenyl)-1-(2-hydroxy-4-
methoxyphenyl)ethanone (19.89 g, 0.65 mol) in diethyl carbonate
(119 mL, 0.98 mol) at room temperature was added dropwise to a
stirred suspension of sodium hydride (10.44 g, 0.26 mol) in diethyl
carbonate (119 mL, 0.98 mol) at 0 °C. The resulting mixture was
stirred at 50 °C for 5 h. The reaction mixture was allowed to cool to
0 °C and then quenched with water (100 mL). The remaining diethyl
carbonate was extracted into Et2O. The aqueous layer was carefully
acidified to pH 3 with 2N aq HCl and extracted with DCM. The
combined organic phases were washed with brine, dried over MgSO4,
filtered, and concentrated. The yellow solid residue was triturated with
Et2O, and the solid product was collected by filtration and dried under
vacuum to give 30a (16.48 g, 84%) as an off-white solid, which was
used without further purification. 1H NMR (400 MHz, DMSO,
303 K) 2.13 (3H, s), 3.88 (3H, s), 6.98 (1H, dd), 7.00 (1H, d), 7.04
(1H, td), 7.14 (1H, dd), 7.20 (1H, dd), 7.88 (1H, d), 11.12 (1H, s);
m/z 301 [M + H]+.
(E)-tert-Butyl 3-(4-hydroxyphenyl)acrylate (179 mg, 0.8 mmol) was
added to 31a (390 mg, 0.8 mmol) in MeCN (10 mL). Cs2CO3
(318 mg, 1.0 mmol) was added, and the solution was stirred at 70 °C
for 4 h. The mixture was allowed to cool, poured into water, and
extracted with EtOAc twice. The combined extracts were dried over
MgSO4, filtered, and evaporated. The residue was purified by flash
silica chromatography, eluting with 1:4 EtOAc/heptane. Product-
containing fractions were evaporated to give (E)-tert-butyl 3-(4-((3-(4-
fluoro-2-methylphenyl)-2-oxo-7-((2-(trimethylsilyl)ethoxy)methoxy)-
2H-chromen-4-yl)oxy)phenyl)acrylate (320 mg, 64%) as a colorless
solid. 1H NMR (400 MHz, DMSO, 303 K) 0.00 (9H, s), 0.87 (2H, t),
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1.48 (9H, s), 2.13 (2H, s), 3.70−3.81 (2H, m), 5.39 (2H, s), 6.36
(1H, s), 6.40 (1H, s), 6.90 (1H, td), 6.96 (1H, s), 6.99 (1H, d), 7.03
(1H, dd), 7.18 (1H, s), 7.19 (1H, d), 7.21 (1H, s), 7.42 (1H, s), 7.46
(1H, s), 7.50 (1H, s), 7.54 (1H, s).
A solution of tert-butyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate
(650 mg, 3.0 mmol) in MeCN was stirred at 45 °C for 3 h and
then added dropwise to a solution of 3-bromo-2-oxo-2H-chromen-4-yl
trifluoromethanesulfonate (1.0 g, 2.7 mmol) in MeCN (100 mL) and
K2CO3 (1.85 g, 13.4 mmol), and the resulting solution was stirred
for another 1 h at 50 °C. The reaction was then quenched by the
addition of water (200 mL) and extracted with EtOAc (3 × 100 mL).
The organic layers were combined, dried over Na2SO4, filtered, and
concentrated under vacuum. The residue was purified by silica gel
chromatography, eluting with 10% EtOAc in petroleum ether. Product
fractions were evaporated to afford tert-butyl (2E)-3-(4-((3-bromo-2-
oxo-2H-chromen-4-yl)oxy)phenyl)prop-2-enoate (550 mg, 46%) as
(E)-tert-Butyl 3-(4-((3-(4-fluoro-2-methylphenyl)-2-oxo-7-((2-
(trimethylsilyl)ethoxy)methoxy)-2H-chromen-4-yl)oxy)phenyl)-
acrylate (100 mg, 0.16 mmol) was added to a solution of TFA (1 mL,
13.5 mmol) in DCM (2 mL) and cooled in an ice bath to 0 °C. The
yellow solution was stirred for 1 h, and then the solvent was removed
under reduced pressure. Toluene was added to the residue, and the
mixture was evaporated to dryness to afford 32 (70 mg, 100%) as a
colorless solid. 1H NMR (400 MHz, DMSO, 303 K) 2.11 (3H, s), 6.33
(1H, s), 6.37 (1H, s), 6.76 (1H, d), 6.79 (1H, d), 6.85 (1H, d), 6.93 (1H,
d), 6.95 (1H, s), 7.14−7.18 (1H, m), 7.23 (1H, d), 7.37 (1H, d), 7.45
(1H, d), 7.50 (1H, d), 10.72 (1H, s), 12.26 (1H, s); m/z 433 [M + H]+.
(2-Methyl-4-(trifluoromethoxy)phenyl)acetic Acid (28b).
1
a colorless solid. H NMR (300 MHz, CDCl3, 303 K) 1.52 (9H, s),
6.32 (1H, d), 6.96 (2H, d), 7.28−7.30 (2H, m), 7.51−7.66 (5H, m);
m/z 465 [M + Na]+.
A solution of tert-butyl (2E)-3-(4-((3-bromo-2-oxo-2H-chromen-4-
yl)oxy)phenyl)prop-2-enoate (924 mg, 2.08 mmol) in TFA (5 mL)
was stirred at 0 °C for 2 h. The resulting mixture was evaporated to
dryness under vacuum to afford 57 (781 mg, 97%) as a colorless solid.
m/z 423 [M + MeCN + H]+.
(2E)-3-(4-[[3-(4-Fluoro-2-methylphenyl)-2-oxo-2H-chromen-4-yl]-
Pd2(dba)3 (1.145 g, 1.25 mmol), 2′-(dicyclohexylphosphino)-N,N-
dimethyl-2-biphenylamine (0.984 g, 2.5 mmol), and 2-methyl-4-
(trifluoromethoxy)bromobenzene (4.09 mL, 25.0 mmol) were added
to degassed THF (50 mL). (2-tert-Butoxy-2-oxoethyl)zinc(II) chloride
(0.5 M in Et2O, 100 mL, 50.0 mmol) was then added, and the reaction
mixture was degassed with nitrogen for 5 min before being heated at
reflux under nitrogen for 2 h and then allowed to cool to room
temperature. Silica was added to the mixture, and then the volatiles were
removed under vacuum. The residue was purified via silica chromatog-
raphy, eluting with 1:30 EtOAc/pentane to give tert-butyl 2-(2-methyl-4-
(trifluoromethoxy)phenyl)acetate (5.8 g, 80%) as a colorless solid.
1H NMR (400 MHz, DMSO, 303 K) 1.40 (9H, s), 2.26 (3H, s), 3.62
(2H, s), 7.13 (1H, d), 7.18 (1H, d), 7.29 (1H, d); m/z 277 [M + H]+.
tert-Butyl 2-(2-methyl-4-(trifluoromethoxy)phenyl)acetate (5.8 g,
20.0 mmol) was stirred in DCM (30 mL), and 2,2,2-trifluoroacetic
acid (20 mL, 269.24 mmol) was added. The mixture was stirred
at room temperature for 3 h. The volatiles were removed under
vacuum; toluene was added, and the mixture was evaporated to
oxy]phenyl)acrylic Acid (58).
To a 25 mL round-bottom flask purged and maintained with an inert
atmosphere of nitrogen were placed (4-fluoro-2-methylphenyl)boronic
acid (104 mg, 0.7 mmol), 57 (200 mg, 0.5 mmol), sodium
2′-dicyclohexylphosphino-2,6-dimethoxy-1,1′-biphenyl-3-sulfonate hy-
drate (54. Eight mg), a solution of Na2CO3 (218 mg, 2.1 mmol) in
water (5 mL), and a solution of Pd(OAc)2 (11.60 mg, 0.05 mmol)
in 1,4-dioxane (10 mL). The resulting solution was stirred for 2 h at
100 °C in an oil bath and was then diluted with water (20 mL) and
extracted with EtOAc (3 × 20 mL). The combined extracts were dried
over Na2SO4, filtered, and concentrated under vacuum. The crude
product (292 mg) was purified by preparative HPLC to afford 58
(95 mg, 44%) as a colorless solid. 1H NMR (300 MHz, CDCl3, 303 K)
2.10 (3H, s), 6.33 (1H, d), 6.71−6.89 (4H, m), 7.11−7.15 (1H, m),
7.36−7.48 (3H, m), 7.50−7.76 (2H, m), 7.70−7.79 (2H, m); 13C
NMR (176 MHz, DMSO, 303 K) 19.2, 112.1 (d), 116.1 (d), 116.1,
116.6, 116.7, 117.0, 118.6, 123.8, 124.5, 126.5, 129.6, 129.7, 132.3 (d),
132.7, 140.1 (d), 142.5, 152.9, 157.2, 158.4, 160.9, 161.7 (d), 167.5;
anal. calcd for C25H17FO5 [M + H]+ 417.10881, found 417.11292.
(2E)-3-(4-((3-(2-Methyl-4-(trifluoromethoxy)phenyl)-2-oxo-2H-
chromen-4-yl)oxy)phenyl)acrylic Acid (59).
1
dryness to give 28b (4.0 g, 85%) as a light brown solid. H NMR
(400 MHz, DMSO, 303 K) 2.32 (3H, s), 3.68 (2H, s), 7.18 (1H, d),
7.24 (1H, s), 7.36 (1H, d), 12.43 (1H, s); m/z 233 [M − H]−.
(E)-3-(4-((7-Hydroxy-3-(2-methyl-4-(trifluoromethoxy)phenyl)-2-
oxo-2H-chromen-4-yl)oxy)phenyl)acrylic Acid (33).
The title compound was prepared from 28b in a similar manner to that
1
described for 32. H NMR (700 MHz, DMSO, 303 K) 2.14 (3H, s),
6.35 (1H, d), 6.81 (1H, dd), 6.87 (1H, d), 6.93−6.96 (2H, m), 7.03
(1H, d), 7.08 (1H, s), 7.25 (1H, d), 7.43−7.5 (4H, m), 10.76 (1H, s);
13C NMR (176 MHz, DMSO, 303 K) 19.2, 102.3, 107.9, 111.8, 113.4,
116.9, 117.6, 118.2, 119.9 (q), 121.7, 125.3, 129.5, 129.6, 130.0, 132.5,
140.1, 142.8, 147.9, 154.9, 157.2, 159.6, 161.3, 162.1, 167.4; HRMS
(ESI+) anal. calcd for C26H17F3O7 [M + H]+ 499.09991, found 499.09906.
(2E)-3-(4-((3-Bromo-2-oxo-2H-chromen-4-yl)oxy)phenyl)acrylic
Acid (57).
The title compound was prepared from 57 in a similar manner to that
described for 58. H NMR (400 MHz, CDCl3, 303 K) 2.19 (3H, s),
6.32 (1H, d), 6.76 (2H, d), 6.92 (2H, d), 7.10 (1H, d), 7.24−7.38
(3H, m), 7.51 (1H, d), 7.64−7.70 (2H, m), 7.78 (1H, d); m/z 483
[M + H]+.
1
Biology. Details for the suite of assays used to support this project
have previously been published.26
ERα Binding. Test compounds were dispensed into 384 well
nonbinding plates to create a 12 point half-log concentration response
curve. ERα binding was measured using a LanthaScreen ERα binding
assay (Life Technologies), and the TR-FRET emission ratios were
acquired on a microplate reader.
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ERα Down-Regulation. MCF-7 cells were plated in reduced serum
media in a 384 well assay plate the day prior to the assay. Cells were
treated with a 12 point concentration range of each test compound for
24 h before being fixed and immunostained for ERα. The levels of
ERα were measured using a high content imaging reader.
PR Antagonism/Agonism. The ERα regulated gene expression of
PR was measured as a biomarker for ERα antagonism and agonism.
MCF-7 cells were plated in reduced serum media in a 384 well assay
plate the day prior to the assay. Antagonism: cells were pretreated for
30 min with 0.1 nM estradiol followed by a 12 point concentration
range of each test compound for 24 h before being fixed and
immunostained for PR. The levels of PR were detected using a laser
scanning imaging cytometer. Agonism: cells were treated with a
12 point concentration range of each test compound for 24 h before
being fixed and immunostained for PR. The PR levels were detected
using a laser scanning imaging cytometer.
REFERENCES
■
(1) Ariazi, E. A.; Jordan, V. C. Estrogen receptors as therapeutic
targets in breast cancer. Methods Princ. Med. Chem. 2008, 39, 127−199.
(2) Levenson, A. S.; Jordan, V. C. Selective oestrogen receptor
modulation: molecular pharmacology for the millennium. Eur. J.
Cancer 1999, 35, 1628−1639.
(3) Johnston, S. R. D. Endocrinology and hormone therapy in breast
cancer: Selective estrogen receptor modulators and downregulators for
breast cancer - have they lost their way? Breast Cancer Res. 2005, 7,
119−130.
(4) Blizzard, T. A. Selective Estrogen Receptor Modulator Medicinal
Chemistry At Merck. A Review. Curr. Top. Med. Chem. 2008, 8, 792−
812.
(5) Harper, M. J. K.; Walpole, A. L. Contrasting Endocrine Activities
of cis and trans Isomers in a Series of Substituted Triphenylethylenes.
Nature 1966, 212, 87−87.
(6) Jordan, V. C. Tamoxifen: a most unlikely pioneering medicine.
Nat. Rev. Drug Discovery 2003, 2, 205−213.
(7) Wakeling, A. E.; Dukes, M.; Bowler, J. A potent specific pure
antiestrogen with clinical potential. Cancer Res. 1991, 51, 3867−3873.
(8) Johnston, S. J.; Cheung, K. L. Fulvestrant - a novel endocrine
therapy for breast cancer. Curr. Med. Chem. 2010, 17, 902−914.
(9) Toy, W.; Shen, Y.; Won, H.; Green, B.; Sakr, R. A.; Will, M.; Li,
Z.; Gala, K.; Fanning, S.; King, T. A.; Hudis, C.; Chen, D.; Taran, T.;
Hortobagyi, G.; Greene, G.; Berger, M.; Baselga, J.; Chandarlapaty, S.
ESR1 ligand-binding domain mutations in hormone-resistant breast
cancer. Nat. Genet. 2013, 45, 1439−1445.
Proliferation. MCF-7 cells were plated in reduced serum media in a
384 well assay plate the day prior to the assay. Cells were treated with
a concentration response of each test compound for 7 days before
determination of cell number using a nucleic acid stain. The final cell
number was measured using a laser scanning imaging cytometer.
ASSOCIATED CONTENT
■
S
* Supporting Information
Complete experimental details for the syntheses of inter-
mediates and all final compounds are described together
with assay statistical analyses and crystallographic information.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(10) Lednicer, D.; Lester, S. C.; Duncan, G. W. Mammalian
antifertility agents. II. Basic ethers of 3,4-diphenylcoumarins. J. Med.
Chem. 1965, 8, 725−726.
(11) Musa, M. A.; Cooperwood, J. S.; Khan, M. O. F. A Review of
Coumarin Derivatives in Pharmacotherapy of Breast Cancer. Curr.
Med. Chem. 2008, 15, 2664−2679.
AUTHOR INFORMATION
■
Corresponding Author
(12) Jain, N.; Xu, J.; Kanojia, R. M.; Du, F.; Jian-Zhong, G.; Pacia, E.;
Lai, M.; Musto, A.; Allan, G.; Reuman, M.; Li, X.; Hahn, D.;
Cousineau, M.; Peng, S.; Ritchie, D.; Russell, R.; Lundeen, S.; Sui, Z.
Identification and Structure Activity Relationships of Chromene-
Derived Selective Estrogen Receptor Modulators for Treatment of
Postmenopausal Symptoms. J. Med. Chem. 2009, 52, 7544−7569.
(13) Tan, Q.; Blizzard, T. A.; Morgan, J. D.; Birzin, E. T.; Chan, W.;
Yang, Y. T.; Pai, L.; Hayes, E. C.; DaSilva, C. A.; Warrier, S.;
Yudkovitz, J.; Wilkinson, H. A.; Sharma, N.; Fitzgerald, P. M. D.; Li, S.;
Colwell, L.; Fisher, J. E.; Adamski, S.; Reszka, A. A.; Kimmel, D.;
DiNinno, F.; Rohrer, S. P.; Freedman, L. P.; Schaeffer, J. M.;
Hammond, M. L. Estrogen receptor ligands. Part 10: Chromanes: old
scaffolds for new SERAMs. Bioorg. Med. Chem. Lett. 2005, 15, 1675−
1681.
(14) Bury, P. S.; Christiansen, L. B.; Jacobsen, P.; Jørgensen, A. S.;
Kanstrup, A.; Nærum, L.; Bain, S.; Fledelius, C.; Gissel, B.; Hansen, B.
S.; Korsgaard, N.; Thorpe, S. M.; Wassermann, K. Synthesis and
pharmacological evaluation of novel cis-3,4-Diaryl-hydroxychromanes
as high affinity partial agonists for the estrogen receptor. Bioorg. Med.
Chem. 2002, 10, 125−145.
(15) Amari, G.; Armani, E.; Ghirardi, S.; Delcanale, M.; Civelli, M.;
Caruso, P. L.; Galbiati, E.; Lipreri, M.; Rivara, S.; Lodola, A.; Mor, M.
Synthesis, pharmacological evaluation, and structure-activity relation-
ships of benzopyran derivatives with potent SERM activity. Bioorg.
Med. Chem. 2004, 12, 3763−3782.
(16) McKie, J. A.; Bhagwat, S. S.; Brady, H.; Doubleday, M.; Gayo,
L.; Hickman, M.; Jalluri, R. K.; Khammungkhune, S.; Kois, A.;
Mortensen, D.; Richard, N.; Sapienza, J.; Shevlin, G.; Stein, B.;
Sutherland, M. Lead identification of a potent benzopyranone selective
estrogen receptor modulator. Bioorg. Med. Chem. Lett. 2004, 14, 3407−
3410.
*E-mail: sebastien.degorce@astrazeneca.com. Tel: +44 (0)1625
514382. Fax: +44 (0)1625 516667.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Scott Boyd, Julie Demeritt, Shaun Fillery,
Jonathan Finlayson, Lorraine Hassall, Barry Hayter, Rosie Isaac,
Patrice Koza, Franck Lach, Trystan Nicholls, Anil Patel, Stuart
Pearson, and chemists at Pharmaron Beijing for the synthesis of
some of the compounds contained herein. We also thank
Christian Delvare and Howard Beeley for NMR assistance,
Caroline McMillan for accurate mass spectral data, and the
Alderley Park purification group for purification of some final
compounds.
ABBREVIATIONS USED
■
ER, estrogen receptor; DR, down-regulation; LLE, ligand
lipophilicity efficiency; PR, progesterone receptor; SAR,
structure−activity relationship; SERM, selective estrogen receptor
modulator; SERD, selective estrogen receptor down-regulator;
AcOH, acetic acid; CDI, carbonyldiimidazole; DCM, dichloro-
methane; DIPEA, N,N-diisopropylethylamine; DMAP, N,N-
dimethylpyridin-2-amine; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; DtBPPS, 3-(di-t-butylphosphonium)-
propanesulfonate; HLPC, high-performance liquid chromatog-
raphy; NCS, N-chlorosuccinimide; SEM, trimethylsilylethoxy-
methyl; SEMCl, 2-(chloromethoxy)ethyl-trimethyl-silane; TBAF,
tetra-N-butylammonium fluoride; Tf, triflate; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TsOH, p-toluensulfonic acid
(17) Barlaam, B. C.; Piser, T. M. Estrogen receptor-β ligands for
therapy. PCT Int. Appl. WO2000062765, 2000.
(18) Suzuki, N.; Liu, X.; Laxmi, Y. R. S.; Okamoto, K.; Kim, H. J.;
Zhang, G.; Chen, J. J.; Okamoto, Y.; Shibutani, S. Anti-breast cancer
K
DOI: 10.1021/acs.jmedchem.5b00066
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
potential of SS5020, a novel benzopyran antiestrogen. Int. J. Cancer
2010, 128, 974−982.
(19) Willson, T. M.; Henke, B. R.; Momtahen, T. M.; Charifson, P.
S.; Batchelor, K. W.; Lubahn, D. B.; Moore, L. B.; Oliver, B. B.; Sauls,
H. R. 3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic Acid: A Non-
Steroidal Estrogen with Functional Selectivity for Bone over Uterus
in Rats. J. Med. Chem. 1994, 37, 1550−1552.
(20) Willson, T. M.; Norris, J. D.; Wagner, B. L.; Asplin, I.; Baer, P.;
Brown, H. R.; Jones, S. A.; Henke, B.; Sauls, H.; Wolfe, S.; Morris, D.
C.; Mcdonnell, D. P. Dissection of the molecular mechanism of action
of GW5638, a novel estrogen receptor ligand, provides insights into
the role of estrogen receptor in bone. Endocrinology 1997, 138, 3901−
3911.
(21) Laxmi, Y. R. S.; Liu, X.; Suzuki, N.; Kim, S. Y.; Okamoto, K.;
Kim, H. J.; Zhang, G.; Chen, J. J.; Okamoto, Y.; Shibutani, S. Anti-
breast cancer potential of SS1020, a novel antiestrogen lacking
estrogenic and genotoxic actions. Int. J. Cancer 2010, 127, 1718−1726.
(22) Hoekstra, W. J.; Patel, H. S.; Liang, X.; Blanc, J. E.; Heyer, D.
O.; Willson, T. M.; Iannone, M. A.; Kadwell, S. H.; Miller, L. A.;
Pearce, K. H.; Simmons, C. A.; Shearin, J. Discovery of Novel
Quinoline-Based Estrogen Receptor Ligands Using Peptide Interaction
Profiling. J. Med. Chem. 2005, 48, 2243−2247.
(23) Shibutani, S.; Yennurajalingam, S. L. Antiestrogens for breast
cancer therapy. PCT Int. Appl. WO2010107474, 2010.
(24) Shibutani, S.; Yennurajalingam, S. L. Pure antiestrogens for
breast cancer therapy. PCT Int. Appl. WO2010107475, 2010.
(25) Wu, Y.; Yang, X.; Ren, Z.; McDonnell, D. P.; Norris, J. D.;
Willson, T. M.; Greene, G. L. Structural Basis for an Unexpected
Mode of SERM-Mediated ER Antagonism. Mol. Cell 2005, 18, 413−
424.
(26) Callis, R.; Walker, G.; Rabow, A.; Tonge, M.; Bradbury, R.;
Chalinor, M.; Roberts, K.; Jones, K. A Screening Assay Cascade to
Identify and Characterize Novel Selective Estrogen Receptor Down-
regulators (SERDs). J. Biomol. Screening 2015, in press.
(27) Buttar, D.; Colclough, N.; Gerhardt, S.; MacFaul, P. A.; Phillips,
S. D.; Plowright, A.; Whittamore, P.; Tam, K.; Maskos, K.;
Steinbacher, S.; Steuber, H. A combined spectroscopic and crystallo-
graphic approach to probing drug−human serum albumin interactions.
Bioorg. Med. Chem. 2010, 18, 7486−7496.
(28) Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme,
A.; Henthorn, D. C.; Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C.
G.; Abi-Gerges, N.; Persson, F.; Jacobson, I.; Sullivan, M.; Albertson,
N.; Hammond, T. G.; Sullivan, E.; Valentin, J. P.; Pollard, C. E.
Optimisation and validation of a medium-throughput electrophysiol-
ogy-based hERG assay using IonWorks HT. J. Pharmacol. Toxicol.
Methods 2006, 54, 189−199.
(29) Camenisch, G.; Alsenz, J.; van de Waterbeemd, H.; Folkers, G.
Estimation of permeability by passive diffusion through Caco-2 cell
monolayers using the drugs’ lipophilicity and molecular weight. Eur. J.
Pharm. Sci. 1998, 6, 313−319.
(30) Soars, M. G.; Grime, K.; Sproston, J. L.; Webborn, P. J. H.;
Riley, R. J. Use of hepatocytes to assess the contribution of hepatic
uptake to clearance in vivo. Drug Metab. Dispos. 2007, 35, 859−865.
(31) DtBPPS Suzuki mix (0.05 M in water) as used was prepared as
follows: sodium tetrachloropalladate (294.2 mg, 1.0 mmol) and 3-(di-
tert-butylphosphino)propane-1-sulfonic acid (537 mg, 2.0 mmol) were
placed in a 20 mL microwave vial containing a stir bar, and the vessel
was sealed. Degassed water (20 mL) was added, and the suspension
was stirred for 5 min under nitrogen to afford a brown/red solution.
(32) Zhang, L.; Meng, T.; Fan, R.; Wu, J. General and Efficient Route
for the Synthesis of 3,4-Disubstituted Coumarins via Pd-Catalyzed
Site-Selective Cross-Coupling Reactions. J. Org. Chem. 2007, 72,
7279−7286.
L
DOI: 10.1021/acs.jmedchem.5b00066
J. Med. Chem. XXXX, XXX, XXX−XXX