Tetrahydroquinoline-based selective estrogen receptor modulators (SERMs)

Article abstract of DOI:10.1016/S0960-894X(03)00306-8

A new series of estrogen receptor ligands based on a 6-hydroxy-tetrahydroquinoline scaffold is described, in addition to their binding affinity and functional activity in MCF-7 cells. Several 1,2-disubstituted tetrahydroquinolines bearing a basic side cha

Full text of DOI:10.1016/S0960-894X(03)00306-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1907–1910
Tetrahydroquinoline-Based Selective Estrogen Receptor
Modulators (SERMs)
Owen B. Wallace,* Kenneth S. Lauwers, Scott A. Jones and Jeffrey A. Dodge
Discovery Chemistry Research and Technologies, Lilly Research Laboratories,
Eli Lilly and Company, Indianapolis, IN 46285, USA
Received 16 December 2002; accepted 17 February 2003
Abstract—A new series of estrogen receptor ligands based on a 6-hydroxy-tetrahydroquinoline scaffold is described, in addition to
their binding affinity and functional activity in MCF-7 cells. Several 1,2-disubstituted tetrahydroquinolines bearing a basic side
chain were shown to be high affinity ligands and antagonists in the MCF-7 proliferation assay. Compounds lacking the basic
side chain were agonists in the MCF-7 assay.
# 2003 Elsevier Science Ltd. All rights reserved.
The estrogen receptor is a ligand activated transcription
factor which plays an important role not only in the
regulation of the female reproductive system, but also in
bone, cardiovascular and central nervous system func-
tion.1 Estrogen receptor ligands, and the selective estro-
gen receptor modulators (SERMs) in particular, are
being extensively studied for the treatment of reproduc-
tive disorders, estrogen responsive cancers and osteo-
porosis. A common structural feature of SERMs is a
bicyclic core containing a phenolic hydroxyl, with a
basic side chain and an aryl ring emanating from the
core. Recently described SERMs from this general class
include the benzothiophenes (e.g., raloxifene),2 indoles
(e.g., pipendoxifene)3 and tetrahydronaphthalenes (e.g.,
lasofoxifene).4 We herein report the design and synth-
esis of a series of substituted tetrahydroquinolines, their
binding affinity to ERa and ERb and antagonism of
estradiol in MCF-7 breast adenocarcinoma cells.5
or methylene linker between the tetrahydroquinoline
core and the aminoalkoxyaryl side chain (Fig. 1). In
addition, several intermediates lacking the basic side
chain were deprotected to liberate the A-ring phenol and
were assessed for ER binding and MCF-7 antagonism.
Figure 1. Generic structure of tetrahydroquinoline SERMS.
We first chose to examine analogues where the side
chain was connected to the tetrahydroquinoline core via
an amide linker. The synthesis began (Scheme 1) with
commercially available 6-methoxyquinoline N-oxide (1)
which was arylated at the 2-position using a modifi-
cation of a literature protocol. Treatment of 1 with
methyl chloroformate followed by addition of an aryl
Grignard reagent afforded the desired 6-methoxy-2-aryl
quinolines 2a–c.7 Alternatively, the 2-aryl quinoline
could be prepared by addition of an aryllithium to
6-methoxyquinoline;8 however, the former approach
was preferred due to the wider availability of aryl
Grignard reagents. Deprotection of the methoxy group
of 2c was readily achieved with BBr3 in CH2Cl2 to
afford quinoline 3. Reduction of the nitrogen-containing
ring of 2a–c was carried out with sodium in ethanol,9
Although N-aryl tetrahydroquinolines which lacked the
A-ring phenol have been previously reported to be anti-
fertility agents in rats,6 their interaction with the estro-
gen receptor has not been characterized. We felt that the
6-hydroxy-tetrahydroquinoline core would be a suitable
structure for the generation of new ER ligands. We not
only wished to investigate N-aryl derivatives, but also
wanted to explore the effect of introducing a carbonyl
*Corresponding author. Fax: +1-317-433-0715; e-mail: wallace_owen@
lilly.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00306-8

1908
O. B. Wallace et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1907–1910
Scheme 1.
thereby yielding tetrahydroquinolines 4a–c. BBr3
deprotection of 4c afforded the free diphenol 5.
Acylation of the tetrahydroquinoline nitrogen was
readily accomplished by treating 4a–c with 4-(2-piper-
idinyl-ethoxy)-benzoyl chloride and Et3N in CH2Cl2 to
yield amides 6a–c. Removal of the methoxy protecting
group was once again achieved with BBr3 to afford tetra-
hydroquinolines 7a–c.
Scheme 2.
Introduction of flexibility between the tetrahydroquino-
line core and the basic side chain was then explored
(Scheme 2). Reduction of amides 6a–c (R=Me) with
LAH in THF followed by BBr3-promoted deprotection
afforded the benzyl substituted tetrahydroquinolines
8a–c.10 Pyrrolidine-derived analogue 10 was prepared
from intermediate 4c (Scheme 3). Alkylation of the
nitrogen of 4c was effected with 4-benzyloxybenzyl
chloride and phosphazene P4-t-butyl base11 in THF to
yield 9. Removal of the benzyl protecting group (H2,
Pd/C) followed by alkylation of the phenol with 1-(2-
chloro-ethyl)-pyrrolidine and subsequent deprotection
(BBr3) afforded 10.
Finally, we wished to investigate compounds where the
linker between the tetrahydroquinoline core and the side
chain was removed, that is, the effect of aminoalk-
oxyaryl side chain attachment directly to the N(1) posi-
tion. The N-aryl tetrahydroquinolines were thus
prepared using transition metal chemistry12 by treating
4c with arylbromide 1113 in the presence of Pd(OAc)2,
P(t-Bu)3 and t-BuONa in toluene (Scheme 4). De-
methylation with BBr3 afforded N-aryl tetra-
hydroquinolines 12 and 13.
Scheme 3.
The compounds were tested as racemates for their abil-
3
ity to compete with H-estradiol for binding to both
ERa and ERb.14 In addition, the compounds were
examined for their ability to inhibit estradiol-stimulated
proliferation of MCF-7 breast adenocarcinoma cells.15
Raloxifene and 4-hydroxytamoxifen (4-OHT) have been
included for comparison purposes (Table 1).16
Scheme 4.
nity to ERb (Ki: 380 nM). Interestingly, addition of a
hydroxyl on the C2-aryl ring failed to increase binding
or MCF-7 antagonism. The 40-OH analogue (7c) had
weaker binding than the parent, while the 30-OH con-
gener (7b) displayed similar ERa binding and functional
activity to the parent (7a), but weaker ERb binding.
Amide derivative 7a displayed reasonable binding affi-
nity to ERa (Ki: 29 nM), but considerably weaker affi-

O. B. Wallace et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1907–1910
1909
Table 1. Binding and MCF-7 inhibition data
backbone of 4-OHT. Alternatively, the three structural
classes may differentially recruit cofactors, which may
also play a role in functional activity.19
Compd
ERa binding
Ki, nM
ERb binding
Ki, nM
MCF-7
IC50, nM
Raloxifene
4-OHT
3
0.4
0.5
310
170
29
4.3
0.5
35
65
380
>1 mM
>1 mM
4.0
5.5
4.4
0.2
2.6
Addition of the N-benzyl basic side chain is an impor-
tant feature for regulating receptor affinity. For exam-
ple, tetrahydroquinoline 10 has >10-fold higher affinity
to both ERa and ERb than compound 5 which lacks
the entire side chain moiety. Interestingly, both quino-
line 3 and tetrahydroquinoline 5 display modest selec-
tivity for ERb (2- to 8-fold), which is in contrast to the
derivatives bearing a basic side chain which are all a
selective (3- to 10-fold). The small, polar nature of 3 and
5 is consistent with previously reported ERb selective
ligands. Both 3 and 5 showed no antagonism in the
MCF-7 assay and, in fact, both compounds were shown
to be agonists (EC50 15 and 320 nM, respectively) when
tested in the absence of 17b-estradiol.
NC
NC
620
590
990
490
390
270
68
5
7a
7b
7c
8a
8b
8c
10
12
13
37
88
1.7
0.6
0.6
1.1
0.9
2.5
4.9
3.2
8.0
490
590
NC, not calculated. These compounds were shown to be agonists in
this assay.
Reduction of the amide carbonyl to provide the corre-
sponding benzyl-substituted tetrahydroquinolines affor-
ded compounds with significantly higher binding
affinity. For example, 8a is approximately 17-fold more
potent than the analogous amide 7a for ERa and
94-fold more potent for ERb. The weaker binding affi-
nity of 7a relative to 8a may be due to the amide bond
forcing the side chain into a less favorable conforma-
tion, thereby diminishing binding. An alternative expla-
nation may be that the electronegative amide is not
tolerated in this hydrophobic region of the receptor.17 A
similar observation was made for a series of phenan-
thridines which possessed a lactam moiety in this
region.18 Introduction of a phenol on the C2-aryl ring of
8a somewhat increased ERa binding affinity but had
little effect on ERb binding. Compounds 8b and 8c both
had higher affinity to ERa compared to the unsub-
stituted derivative 8a. As was observed previously with
SERMs from the benzothiophene or indole series,2,3
there is a weak correlation between ERa binding affinity
and MCF-7 inhibition for the tetrahydroquinolines.
In summary, we have investigated the use of 1,2-
disubstituted tetrahydroquinolines as estrogen receptor
ligands. Compounds containing an amide linker
between the core and basic side chain had weaker bind-
ing and somewhat weaker functional activity compared
to the other series investigated. Both the N-benzyl and
N-aryl derivatives displayed high binding affinity with
modest ERa selectivity. Compound 10 is the most
potent inhibitor of MCF-7 proliferation identified in
this series (IC50 68 nM).
Acknowledgements
We wish to thank the Lead Optimization Biology group
for conducting binding and MCF-7 assays.
References and Notes
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Since the pyrrolidine base is a common feature of many
SERMs, we wished to investigate the effect of replacing
the piperidine with a pyrrolidine. As is commonly seen
with SERMs, the base change did not dramatically
affect binding affinity, but had a more significant impact
on functional activity: Tetrahydroquinoline 10 is a high
affinity ER ligand (Ki ERa: 1.1 nM; Ki ERb: 4.9 nM),
and is 4-fold more potent than the piperidine analogue
8c in the MCF-7 proliferation assay.
2. Grese, T. A.; Pennington, L. D.; Sluka, J. P.; Adrian,
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N-Aryl tetrahydroquinoline 12 is likewise a high affinity
ligand; however, the MCF-7 inhibition is approximately
7-fold weaker than 10, which contains a methylene
spacer between the tetrahydroquinoline ring and the
side chain aryl moiety. Piperidine derivative 13 has
weaker binding affinity than 12 and is likewise a weak
antagonist in the MCF-7 proliferation assay. In general,
all of the tetrahydroquinolines displayed weaker MCF-7
antagonism than either raloxifene or 4-hydroxy-
tamoxifen, in spite of a similar binding profile. This may
be due to the increased polarity of the tetra-
hydroquinolines which may limit their cellular penetra-
tion relative to the more lipophilic triaryl ethylene
4. Rosati, R. L.; Jardine, P. D.; Cameron, K. O.; Thompson,
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5. A structurally distinct series of diphenyl quinolines and
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red-free, Gibco BRL) supplemented with 10% fetal bovine
serum (FBS) (V/V), l-glutamine (2 mM), sodium pyruvate (1
mM), HEPES ((N-[2-hydroxyethyl]piperazine-N0-[2-ethane-
sulfonic acid]10 mM}, non-essential amino acids (0.1 mM) and
Penicillin Streptomycin (1ꢁ). Seven days prior to assay,
MCF-7 cells were switched to assay media which was the same
as maintenance medium except supplemented with 10% dex-
tran-coated charcoal-stripped fetal bovine serum (DCC-FBS)
assay medium in place of 10% FBS. MCF-7 cells were
removed from flasks using 10ꢁ Trypsin EDTA (phenol red
free, Gibco BRL) and diluted to 1ꢁ in (Ca++/Mg++ free
HBSS (phenol red-free)). Cells were adjusted to 80,000 cells/
mL in assay medium. Approximately 8000 cells (100 mL) were
added to each well in 96-well Cytostar T scintillation plates
(Amersham) and incubated at 37 ꢀC in a 5% CO2 humidified
incubator for 24 h to allow cell adherence and equilibration
after transfer. Serial dilutions of drugs were prepared in assay
medium at 4ꢁ the final desired concentration). A 50 mL aliquot
of drug dilutions (at 4ꢁ the final assay concentration) was
transferred to duplicate wells followed by 50 mL assay medium
for the agonist mode or 50 mL of 40 pM of E2 for the antagonist
mode to a final volume of 200 mL. For each of the agonist
plates, a basal level (media) and a maximum stimulated level
(with 1 mM E2) was determined. For each of the antagonist
plates, a basal level (media) and an E2 (10 pM) alone control
was determined. After an additional 48 h at 37 ꢀC in a 5% CO2
humidified incubator, 20 mL of assay medium containing 0.01
mCi of 14C-thymidine (52 mCi/mmol, 50 mCi/uL, Amersham)
was added to each well. The plates were incubated overnight in
the same incubator and then counted on the Wallac Microbeta
counter. The data was averaged to calculate an IC50 and %
inhibition @ 1 mM for the antagonist mode. For the agonist
mode, an EC50 and % of maximum E2 stimulation and con-
centration of maximum stimulation was calculated.
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14. The competition binding assay was run in a buffer con-
taining 50 mM Hepes, pH 7.5, 1.5 mM EDTA, 150 mM NaCl,
10% glycerol, 1 mg/mL ovalbumin and 5 mM DTT, using
0.025 mCi per well 3H-Estradiol (NEN #NET517 at 118 Ci/
mmol, 1mCi/mL), 10 ng/well ER a or ERb receptor (Pan-
Vera). Competing compounds were added at 10 different con-
centrations. Non-specific binding was determined in the
presence of 1 mM of 1 7b Estradiol. The binding reaction (140
mL) was incubated for 4 h at room temperature, then 70 mL of
cold DCC buffer was added to each reaction (DCC buffer
contains per 50 mL of assay buffer, 0.75 g of charcoal (Sigma)
and 0.25 g of dextran (Pharmacia)). Plates were mixed for 8
min on an orbital shaker at 4 ꢀC. Plates were then centrifuged
at 3000 rpm at 4 ꢀC for 10 min. An aliquot of 120 mL of the
mix was transferred to another 96-well, white flat bottom plate
(Costar) and 175 mL of Wallac Optiphase ‘Hisafe 3’ scintilla-
tion fluid was added to each well. Plates were sealed and sha-
ken vigorously on an orbital shaker. After an incubation of 2.5
h, plates were read in a Wallac Microbeta counter. The data
was used to calculate an IC50 and % inhibition at 10 mM. The
16. The compounds were generally tested in duplicate. The
minimum significant ratio (MSR) calculated for the ERa and
ERb binding assays were 2.4 and 2.3, respectively. The MSR
for the MCF-7 assay was 2.9.
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3
Kd for H-Estradiol was determined by saturation binding to
ERa and ERb receptors. The IC50 values for compounds were
converted to Ki using the Cheng–Prusoff equation and the Kd
determined by saturation binding assay.
15. MCF-7 breast adenocarcinoma cells (ATCC HTB 22)
were maintained in MEM (minimal essential medium, phenol
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