Selective estrogen receptor modulators with conformationally restricted side chains. Synthesis and structure-activity relationship of ERα-selective tetrahydroisoquinoline ligands

Article abstract of DOI:10.1021/jm040858p

We disclose herein the discovery of estrogen receptor α (ERα) selective estrogen receptor modulators (SERMs) of the tetrahydroisoquinoline series that incorporate novel conformationally restricted side chains as replacement of the aminoethoxy residue typical of SERMs. Molecular modeling studies used in conjunction with the X-ray crystal structure of the ERα ligand binding domain (LBD) with raloxifene (7) suggested a diazadecaline moiety as a viable mimic of the SERM side chain. On the basis of this knowledge, the piperidinylethoxy moiety of our lead compound 60 was replaced by a diazadecaline subunit, providing the novel tetrahydroisoquinoline 29. In addition to exhibiting a binding affinity to ERα and antagonistic properties in the estrogen response element and MCF-7 assays similar to those of the parent compound 60, ligand 29 showed a reduced agonist behavior in the MCF-7 assay in the absence of 17β-estradiol. These data point toward the fact that 29 may have a potential for breast cancer prevention/treatment in vivo, a feature which is particularly attractive in the quest for safe alternatives to hormone replacement therapy. In a pharmacokinetic experiment carried out in rats, 29 displayed an interesting profile, with a bioavailability of 49%. We also disclose the X-ray crystal structure of 29 in complex with ERα-LBD, which reveals the preferred configurations of 29 at the two chiral centers and the details of its interactions with the receptor. Finally, our structure-activity relationship studies show that other analogues bearing constrained side chains retain potency and antagonist activity and that a 3-OH substituted phenyl D-ring increases the selectivity of a set of piperazinyl-containing ligands in favor of ERα over ERβ.

Full text of DOI:10.1021/jm040858p

364
J. Med. Chem. 2005, 48, 364-379
Articles
Selective Estrogen Receptor Modulators with Conformationally Restricted Side
Chains. Synthesis and Structure-Activity Relationship of ERr-Selective
Tetrahydroisoquinoline Ligands
Johanne Renaud,*,† Serge Franc¸ois Bischoff,‡ Thomas Buhl,† Philipp Floersheim,‡ Brigitte Fournier,†
Martin Geiser,§ Christine Halleux,† Joerg Kallen,§ Hansjoerg Keller,† and Paul Ramage§
Novartis Institute for Biomedical Research, WKL-136.6.81, CH-4002 Basel, Switzerland
Received July 1, 2004
We disclose herein the discovery of estrogen receptor R (ERR) selective estrogen receptor
modulators (SERMs) of the tetrahydroisoquinoline series that incorporate novel conformation-
ally restricted side chains as replacement of the aminoethoxy residue typical of SERMs.
Molecular modeling studies used in conjunction with the X-ray crystal structure of the ERR
ligand binding domain (LBD) with raloxifene (7) suggested a diazadecaline moiety as a viable
mimic of the SERM side chain. On the basis of this knowledge, the piperidinylethoxy moiety
of our lead compound 60 was replaced by a diazadecaline subunit, providing the novel
tetrahydroisoquinoline 29. In addition to exhibiting a binding affinity to ERR and antagonistic
properties in the estrogen response element and MCF-7 assays similar to those of the parent
compound 60, ligand 29 showed a reduced agonist behavior in the MCF-7 assay in the absence
of 17â-estradiol. These data point toward the fact that 29 may have a potential for breast
cancer prevention/treatment in vivo, a feature which is particularly attractive in the quest for
safe alternatives to hormone replacement therapy. In a pharmacokinetic experiment carried
out in rats, 29 displayed an interesting profile, with a bioavailability of 49%. We also disclose
the X-ray crystal structure of 29 in complex with ERR-LBD, which reveals the preferred
configurations of 29 at the two chiral centers and the details of its interactions with the receptor.
Finally, our structure-activity relationship studies show that other analogues bearing
constrained side chains retain potency and antagonist activity and that a 3-OH substituted
phenyl D-ring increases the selectivity of a set of piperazinyl-containing ligands in favor of
ERR over ERâ.
Introduction
cardiovascular disease (22%).6,7 The current concerns
regarding treatment with estrogens are having an
impact on women’s willingness to take HRT, with the
risks associated with the treatment overriding the
benefits. In that context, an agent capable of preventing
or treating postmenopausal osteoporosis (PMO) but not
associated with the safety concerns of estrogens and
progestins would be an important addition to the
portfolio of menopausal therapies.
In recent years, substantial efforts have been put into
the discovery of estrogen surrogates with better benefit-
risk profiles. Agents that have the potential to act as
estrogen in some tissues while antagonizing its action
in others are known. The acronym SERMssselective
estrogen receptor modulatorsshas been ascribed to such
compounds.8 In fact, several SERMs are currently in
advanced clinical trials (lasofoxifene (4)8-10 and baze-
doxifene (5)8,11) or on the market (tamoxifen (6) for the
treatment of hormone-dependent breast cancer8 and
raloxifene (7) for prevention and treatment of osteoporo-
sis12,13) (Figure 1).
A number of endogenous estrogens are known, and
three of them, 17â-estradiol (1), estrone (2), and estriol
(3) (Figure 1), are found in greater concentrations in
female plasma.1 The most important endogenous estro-
gen is 17â-estradiol (1), which exerts its action on female
reproductive functions and on numerous tissues,1 in-
cluding bone.2 The efficacy of estrogens in the preven-
tion and treatment of postmenopausal osteoporosis has
been demonstrated by various studies.3-5 Estrogens are
typically administered in combination with progestins
to women with an intact uterus to prevent endometrial
hyperplasia. Recently, the Women’s Health Initiative
study has shown that the incidence of fractures is
reduced in women taking the estrogen-progestin com-
bination.6 However, this study has also demonstrated
that hormone replacement therapy (HRT) leads to
increased risks of breast cancer (26%), heart attacks
(29%), strokes (41%), blood clots (double rate), and total
* To whom correspondence should be addressed. Phone:
+41 61 696 5399. Fax: +41 61 696 6071. E-mail: johanne.renaud@
pharma.novartis.com.
Members of the SERM class are characterized by at
least two common structural features: a phenolic hy-
droxyl group that is required for binding to the estrogen
receptor R (ERR) or â (ERâ) and a side chain containing
† Arthritis and Bone Metabolism Therapeutic Area.
‡ Nervous System Therapeutic Area.
§ Central Technologies.
10.1021/jm040858p CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/22/2004

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 365
studied, there is current evidence pointing toward ERR
as occupying an important role in bone remodeling.
However, ERâ may also be a contributor to this pro-
cess.23 It is not yet clear what effect an ER subtype-
selective ligand would trigger on bone tissue.
Considerable activity in the SERM field has contrib-
uted to the development of compounds of diverse mo-
lecular architectures. Our interest in this area led us
to explore novel side chains because this is a critical
moiety of the ligand. The fact that the side chain has a
strong impact on the tissue-specific agonist/antagonistic
properties of the molecule influences the pharmacologi-
cal profile of the compound. Particularly, the stimulation
of breast and endometrial tissues is a critical safety
issue. Except for a report on the significance of specific
elements of the aminoethoxy fragment of raloxifene
analogues,24 the effects of modification of the side chain
on potency and antagonistic properties of ERR-selective
SERMs have not been explored.25 Herein, we report for
the first time new cyclic residues as replacements of the
aminoethoxy subunit characteristic of SERMs that
retain potency and antagonistic activity. The synthesis
and biological evaluation of these novel piperidinyl and
piperazinyl derivatives of the tetrahydroisoquinoline
series is described below. In addition, the X-ray crystal
structure of one potent ligand, the tetrahydroisoquino-
line 29, is disclosed.
Figure 1. Structures of estrogens and SERMs.
a basic functionality, which confers SERMs their an-
tagonistic properties in some tissues. The spatial ori-
entation of the 2-aminoethoxyphenyl side chain relative
to the central core of the molecule has been postulated
to influence the endometrial properties of SERMs in
women.14 For example, tamoxifen (6), which has a
relatively flat structure, is estrogenic in the uterus and
has been shown to increase the risk of endometrial
cancer. However, raloxifene (7), which is not associated
with uterine side effects in humans, features a side
chain oriented orthogonally to the plane of its ben-
zothiophene framework.14
The mechanisms by which SERMs exert their tissue-
specific actions are only partly elucidated.15-17 SERMs
bind with different affinities and selectivities to the two
currently known estrogen receptors, ERR and ERâ. The
two receptors, which act as ligand-activated transcrip-
tion factors, are found in a wide variety of tissues and
show a distinct but also overlapping distribution
pattern.18-20 They regulate gene transcription either
directly by binding, primarily as homo- or heterodimers,
to the estrogen response element (ERE) or through
protein-protein interaction with other transcription
factors such as AP-1 or NF-κB.19 Gene transcription is
stimulated via interactions that involve the receptor,
chromatin complexes, and coactivators at the ER-
activated promoters.21,22 Conversely, repression of tran-
scription occurs through recruitment of corepressors by
the antagonist-bound ER.21,22 Each SERM may induce
a different conformation of the ER and alter the interac-
tion of the ligand-bound complex with a given set of
coregulators. In addition, the differential expression of
ERR and ERâ or of coregulators in various tissues may
very well contribute to the formation of specific ER-
ligand-coregulator complexes and thus elicit a tissue-
specific effect.15
Chemistry
The tetrahydroisoquinolines were prepared by three
different routes, using either the iodophenyl intermedi-
ate 13 or bromophenyl derivatives 17a-d as starting
material. The syntheses of compounds 13 and 17a-d
are outlined in Schemes 1 and 2.
Synthesis of Intermediate Tetrahydroisoquino-
line 13 via the Bischler-Napieralski Route. 3-Hy-
droxyphenylacetic acid (8) was transformed into tet-
rahydroisoquinoline 13 following a similar strategy as
described in the literature for related compounds (Scheme
1).26 Selective benzylation of 3-hydroxyphenylacetic acid
was accomplished readily by trapping the dianion
formed upon KOH treatment (2.5 equiv) with benzyl
bromide (1.05 equiv).27 Exposure of 3-benzyloxyphenyl-
acetic acid to (COCl)2 provided the corresponding acyl
chloride, which was transformed into amide 10 by
reaction with aniline. Reduction of 2-(3-benzyloxyphen-
yl)-N-phenyl-acetamide (10) with lithium aluminum
hydride (LAH) led to the phenylethylphenylamine de-
rivative 11. Acylation of this substance with 4-iodoben-
zoyl chloride gave amide 12. Ring closure was effected
by refluxing compound 12 in POCl3 to generate an
intermediate iminium ion, which was reduced with
NaBH4 to the iodophenyl intermediate 13 (Scheme 1).
Synthesis of Intermediate Tetrahydroisoquino-
lines 17a-d via the Schmidt Rearrangement. The
bromophenyl intermediates 17a-d were assembled
from 5-methoxyindanone (14) in four steps (Scheme 2),
following a synthetic route that is shorter and is higher
yielding than that described above. In addition, a broad
diversity of structures could be rapidly synthesized by
this flexible approach. Large quantities of bicyclic
lactam 15 were accessed via the Schmidt rearrangement
of indanone 14, according to a published procedure.28,29
The isoquinolin-1-one 15 proved to be a versatile
Although the relative contributions of ERR and ERâ
to the spectrum of SERMs effects have only been partly

366 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Renaud et al.
Scheme 1. Bischler-Napieralski Route to Tetrahydro-
isoquinoline Derivative 13a
Scheme 3. Synthesis of Piperazine Derivatives 29-33
via Amination Reactiona
a Reagents: (a) benzyl bromide (1.05 equiv), KOH (2.5 equiv),
NaI (0.025 equiv), EtOH (0.26 M), reflux, 16 h; HCl, 93%; (b)
(COCl)2, (1.5 equiv), CH2Cl2 (0.5 M), room temperature, 5 h, 99%;
(c) PhNH2 (1.5 equiv), Na2CO3 (3 equiv), PhH (0.1 M), reflux, 6 h,
61%; (d) lithium aluminum hydride (5 equiv), dioxane-Et2O
(2:3), 35 °C, 17 h, 78%; (e) 4-iodobenzoyl chloride (1.2 equiv),
Na2CO3 (3 equiv), benzene (0.1 M), reflux, 1.5 h, 69%; (f) POCl3,
reflux, 18 h; KI; (g) NaBH4 (2.2 equiv), MeOH (0.1 M), room
temperature, 4 h, 73% over 2 steps.
Scheme 2. Schmidt Rearrangement Route to Tetra-
hydroisoquinolines 17a-da
a Reagents: (a) for the preparation of 22 and 24, procedure A:
18 or 2-ethyl-piperazine (20),tBuOK, Pd2(dba)3, (2′-dicyclohexyl-
phosphanyl-biphenyl-2-yl)-dimethylamine, PhMe, 80-85 °C, 12/
19 h, 53/74%. For the preparation of 23, procedure B: 2-methyl-
piperazine (19), tBuOK, Pd2(dba)3, 1,3-bis(2,6-diisopropylphenyl)-
4,5-dihydroimidazolium tetrafluoroborate,dioxane, 27 °C, 40 h; 40
°C, 4 h, 61%. For the preparation of 25, procedure C: 4-methyl-
t
piperazine (21), BuOK, Pd2(dba)3, (()-BINAP, anhydrous THF,
reflux, 38 h; 53%. (b) For the preparation of 26: 37% aqueous
HCHO, AcOH, MeOH, 80 °C, 1 h; NaBH3CN, CH2Cl2; 0 °C, 1 h,
81%. For the preparation of 27: CH3CHO, AcOH, NaBH(OAc)3,
Cl(CH2)2Cl, room temperature, 16 h, 52%. For the preparation of
28: 37% aqueous HCHO, AcOH, NaBH(OAc)3, Cl(CH2)2Cl, room
temperature, 1 h, 86%. (c) 2:1 or 5:3 dioxane/concentrated HCl,
90 °C, 2-2.5 h, 34-73%.
a Reagents: (a) ref 28; (b) ArI or ArBr, CuI, K2CO3, DMF, 150
°C, 72-120 h, 70-95%; (c) [LiArBr] (generated from p-dibro-
mobenzene and n-BuLi), THF, -78 °C, 1.5-3.5 h; HClO4, 10-15
min; (d) NaBH4, MeOH, room temperature, 15-30 min, 70-86%
over two steps.
latter case, the reaction could be carried out at a
temperature reduced to 40 °C. When BINAP was
employed as the ligand, the coupling reactions pro-
ceeded, but the yields were generally lower. tBuOK was
found to be a suitable base for all cases. By use of the
appropriate piperazines 18-21, the analogues 22-25
were synthesized in 53-74% yield from 13. The coupling
of 2-methyl-piperazine (19) and 2-ethyl-piperazine (20)
with 13 proceeded regioselectively via reaction of the
least hindered amine to provide analogues 23 and 24.
The tetrahydroisoquinoline 23 was then converted into
the corresponding N-methyl piperazine derivative 26 by
reaction with HCHO in the presence of AcOH to
generate the corresponding iminium intermediate, which
was subsequently reduced with NaBH3CN. The ethyl
and methyl analogues 27 and 28 were prepared by the
method of Abdel-Magid et al.36 Exposure of 23 to
CH3CHO and 24 to HCHO in the presence of AcOH and
NaBH(OAc)3 in ClCH2CH2Cl provided the N-alkyl pip-
erazine analogues 27 and 28. Debenzylation of 22, 25,
and 26-28 was achieved by treatment with HCl at 90
°C.
intermediate of our synthetic scheme. N-Arylation of 15
was effected under Ullmann-type conditions,30 using the
appropriately substituted phenyl iodide or bromide and
CuI, to provide compounds 16a-d. These compounds
were then converted into the tetrahydroisoquinolines
17a-d by reaction with 4-bromophenyllithium followed
by NaBH4 reduction of the intermediate iminium salts.
Synthesis of Piperazine Derivatives 29-33 via
the Hartwig-Buchwald Reaction. To synthesize
piperazine derivatives 29-33, a palladium-catalyzed
amination of the aryl iodide 13 served as the key step
(Scheme 3). After investigation of a few reaction condi-
tions, Pd2dba3 (dba ) dibenzylidene acetone) was found
to catalyze the transformation efficiently, particularly
in the presence of (2′-dicyclohexylphosphanyl-biphenyl-
2-yl)-dimethylamine31 in toluene or when used in con-
junction with 1,3-bis(2,6-diisopropylphenyl)-4,5-dihy-
droimidazolium tetrafluoroborate32-35 in dioxane. In the

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 367
Scheme 4. Synthesis of Piperidine Derivatives
38a-c, e via Suzuki Cross-Coupling Reactiona
Scheme 5. Synthesis of Piperazine Derivatives 45-50
from Lactam 16da
a For the preparation of 35b-d: 17b-d, 4-pyridineboronic acid,
PdCl2(dppf)‚CH2Cl2, K2CO3, toluene/acetone/water (ratios of 3:3:1
or 4:4:1), reflux, 20-78 h, 45-75%. For the synthesis of 34a: 13,
4-pyridineboronic acid, PdCl2(dppf)‚CH2Cl2, CsF, DME, 80 °C, 24
h, 56%. (b) For the syntheses of 36a, 37b, and c: MeI, DMF, 40
°C, 2 h. For the synthesis of 37d: MeI, xylene, 140 °C, 2.5 h. (c)
H2, PtO2, MeOH or MeOH/THF (4:1 or 1:1), room temperature,
10-45 h, 61-100%. For the reduction of 37d: NaBH4, MeOH,
room temperature, 30 min; H2, PtO2, MeOH, r.t., 20 h, 63%. (d)
For the preparation of 38b, c, and e: EtSH, AlCl3, CH2Cl2, room
temperature, 2-17 h, 20-55%. For the synthesis of 38a: 2:1
dioxane/concentrated HCl, 90 °C, 3 h, 37%.
a (a) Phenyllithium reagent (prepared from 1-(4-bromophenyl)-
4-alkylpiperazine, n-BuLi, THF, -78 °C, 0.5-1 h), -78 °C, 1-2.5
h. For the syntheses of 41, 43, and 44: -78 °C, 10-45 min;
-78 °C f room temperature, 40-60 min. (b) NaBH4, MeOH or
8:1 MeOH/CH2Cl2, room temperature, 15-60 min, 59-87% over
two steps. (c) EtSH, AlCl3, CH2Cl2, room temperature, 0.5-4 h,
45-58%. (d) RCHO or RCOR, NaBH(OAc)3, ClCH2CH2Cl, room
temperature, 2-26 h, 74-99%. (e) (ClCH2CH2)2N, THF-acetone-
H2O, reflux, 3.5 h, 12%.
timal. The desired product could be obtained, however,
in quantitative yield by reductive amination of 51,
carried out in the presence of HCHO, AcOH, and
NaBH3CN. The remaining piperazines 54-58 were also
prepared via reductive amination of 51 with the ap-
propriate aldehyde or ketone, but this transformation
was effected according to the protocol of Abdel-Magid
et al.36
Synthesis of Piperidine Derivatives 38a-c,e via
Suzuki Cross-Coupling Reaction. The approach
pursued for the synthesis of piperidine analogues 38a-c
and e is outlined in Scheme 4. Palladium-catalyzed
coupling of phenyl bromides 17b-d with 4-pyridinebo-
ronic acid was effected in the presence of K2CO3 in a
toluene/acetone/water mixture to provide the pyridine
derivatives 35b-d in 45-75% yield. Compound 34a was
prepared in a similar manner using iodide 13 as the
coupling partner. Treatment of 34a and 35b-d with
MeI in dimethylformamide (DMF) at 40 °C or in xylene
at 140 °C provided the pyridinium salts 36a and 37b-
d. Reduction of these salts, followed by removal of the
protecting groups, furnished the desired products 38a-c
and e. Debenzylation was effected via acid treatment
at 90 °C whereas demethylation was promoted by
reaction with AlCl3 in the presence of EtSH.
Synthesis of Piperazine Derivatives 45-50 from
Lactam 16d. The N-alkylpiperazinyl derivatives 45-
50 were synthesized via an alternative sequence to that
followed for the preparation of analogues 29-33. This
route involved condensation of the appropriate 1-(4-
lithiophenyl)-4-alkyl-piperazine with lactam 16d to
generate an iminium derivative that was reduced with
NaBH4. The resultant tetrahydroisoquinolines 39-44
were then deprotected under Lewis-acid conditions
(Scheme 5). The phenyllithium reagents needed for the
transformation of 16d into 39-44 were generated by
metal-halogen exchange of the 1-(4-bromophenyl)-4-
alkyl-piperazines 53-58. 4-Methyl-piperazine reagent
53 was initially synthesized by alkylation of 4-bro-
mophenylamine (52) with bis-(2-chloroethyl)methyl-
amine in a low yield. This protocol was clearly subop-
Biology
As part of our initial screening strategy, our com-
pounds were tested in three in vitro assays. In a first
instance, new derivatives were screened in a [3H]-
estradiol radioligand binding assay using recombinant
human ERR and ERâ to select those substances that
demonstrate affinity for the target receptor and to
assess their selectivity toward one or the other receptor.
The compounds were then profiled in a cellular tran-
scription assay using HeLa cells stably transfected with
human ERR or ERâ and estrogen response elements
(ERE) upstream of a luciferase gene. In this assay, 17â-
estradiol induces ERE reporter gene activity. Thus,
compounds able to inhibit luciferase transcription in the
presence of 17â-estradiol may be classified as antago-
nists in this cell line. Agonists would activate gene
transcription in the absence of estrogen. The ERE
cellular assay also allows one to determine the ability
of target derivatives to regulate transcription through
ERR or ERâ and thus provides information with respect
to the selectivity of the ligand. The third in vitro assay
was used as an indicator of the ligand’s behavior on
tissues/cells affected by estrogen. For this purpose, new
compounds were evaluated for their ability to antago-

368 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Renaud et al.
Figure 2. Lead identification and optimization.
nize the proliferative action of 17â-estradiol (1) on
MCF-7 breast tumor cells. The agonist action of these
analogues was also measured in the same cellular assay
in the absence of estrogen. Finally, to get early insight
into the pharmacokinetic properties of interesting com-
pounds, selected candidates were profiled in rats using
cassette dosing experiments.
3a). Our studies also indicated that the side chain
conformation would be unaltered by replacement of the
oxygen by a carbon. On the basis of this knowledge, it
appeared that we could make use of an analogous
modification in the tetrahydroisoquinoline series, whereby
the piperidine-containing side chain of the lead tetrahy-
droisoquinoline 60 could be substituted by a 1,4-diaza-
decaline system as in 29. A model of 29 was built
accordingly with the molecular modeling program
Witnotp39 and optimized with Mopac/AM1.39 Superposi-
tion of modeled 29 on raloxifene (7) bound to the
carboxymethylated ERR-LBD (Figure 3b) showed that
29 has the potential to bind to ERR via interactions
similar to those established by 7, but with a reduced
loss of torsional freedom. To confirm the validity of the
model, the bicyclic piperazine derivative 29 was syn-
thesized and its affinity for the estrogen receptors, its
selectivity, and its ability to act as an antagonist in
MCF-7 cells were evaluated. Other analogues, in which
the diazadecaline system of 29 was replaced by 1-alkyl-,
1-alkyl-2-alkyl-piperazines, or N-methyl-piperidine resi-
dues, were also prepared to study the influence of the
substitution pattern on potency and selectivity. A few
derivatives with substituted aryl D rings within the
N-methyl-piperidine series were synthesized to assess
the influence of substituents on selectivity. At this stage
of our program, the new ligands were tested either as
racemates or as mixtures of diastereoisomers. The
results of our investigation are reported below.
Results and Discussion
High-throughput screening of our sample collection
provided the fluorophenyl derivative 59 as our starting
hit (Figure 2). Replacement of fluorine by a piperidin-
ylethoxy side chain provided our lead compound 6037
(Figure 2), which displayed a binding affinity compa-
rable to that of raloxifene (7) (IC50 ) 22 and 19 nM, for
compounds 7 and 60, respectively, Table 1, entries 21
and 17). When profiled in the ERE and MCF-7 assays
to assess its antagonist activity, 60 potently inhibited
luciferase expression in the former assay and cell
proliferation in the latter with IC50 of 16.5 and 32 nM,
respectively (Table 1, entry 17).37 In the absence of 17â-
estradiol in the MCF-7 proliferation assay, both 60 and
raloxifene (7) displayed some agonistic effects (37% and
22%, respectively). Having identified a potent lead
containing the typical aminoethoxy residue, we set to
evaluate ligands containing side chains with reduced
conformational flexibility (Figure 2) and to evaluate the
effect of these novel moieties on affinity, selectivity, and
antagonist potential.
Biological Results. A comparison of the in vitro data
obtained for 29 and 60 reveals that the bicyclic side
chain replacement is an effective mimic of the amino-
ethoxy side chain (Table 1, entries 1 and 17). The
tetrahydroisoquinoline 29 displays good binding affinity
for ERR showing an IC50 of 63 nM. When compared to
that of 60, the potency of 29 in this assay is slightly
reduced by ∼3-fold. In this context, it is thus relevant
to point out that 29 was tested as a mixture of four
diastereoisomers whereas 60 was evaluated as the
racemate. Because there is evidence (vide infra) indicat-
ing that one isomer is responsible for the potency of the
mixture, it ensues that the difference of affinity amounts
to only ∼1.5-fold. When profiled in the ERE and MCF-7
assays, 29 and 60 were found to exhibit similar activi-
ties, within a factor of 1.5-fold in favor of 60 in the
former assay and favoring 29 in the latter (Table 1,
entries 1 and 17). The two most notable differences
Design of Cyclic Side Chains: Modeling Studies.
With this aim in mind, we looked for an appropriate
rigidification of the amine bearing side chain while
potentially maintaining the electrostatic interaction
between the alkylammonium nitrogen atom and the
carboxylic acid group of Asp351 (Figure 3a). Inspection
of the X-ray crystal structure of raloxifene (7) bound to
the ERR ligand binding domain (LBD)38 revealed that
the O-C-C-N chain adopts a near gauche conforma-
tion with a torsion angle that matches the N-C-C-N
torsion angles (∼60°) of a piperazine in the chair
conformation. We saw that substitution of the ether
oxygen of raloxifene (7) by a nitrogen atom and addition
of a methylene group to link the 2-piperidine carbon and
the new nitrogen atom leads to a derivative that
incorporates an ideal trans-diazadecaline system with-
out changing the orientation of the basic nitrogen in the
former piperidine ring of raloxifene (7) (Figures 2 and

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 369
Table 1. ER Binding, ER Transcriptional Activation on an ERE, and Inhibition of MCF-7 Cell Proliferation
a Average of two independent experiments run in triplicate. b Average of two to four independent experiments run either in duplicate
or in triplicate except when indicated; the maximum antagonism in the ERE and MCF-7 assays is 100% unless otherwise noted. c Average
of three determinations issued from one experiment. d Maximum antagonism for 38a, 98%; for 38c, 71%; for 47, 91%; for 6, 68%. e n.d.
) not determined. f Tested as the HCl salt.
between the two ligands lie in their selectivity profile
and their agonistic effect in the MCF-7 proliferation
assay. Tetrahydroisoquinoline 60 displays a stronger
preference by ∼18-fold for ERR whereas 29 shows a ∼4-
fold selectivity for ERR over ERâ. In the MCF-7 cellular
assay, analogue 29 clearly shows a reduced agonism
(16%) relative to that displayed by 60 (37%). These data
indicate that 29 may have a potential for breast cancer
prevention/treatment in vivo. This feature is particu-
larly attractive in the quest for a safe SERM as an
alternative to hormone replacement therapy (HRT).
Motivated by these results, we set to expand our
knowledge of the series by evaluating other analogues
in which the piperidinyl ring of the diazadecaline system
was disconnected. The data demonstrate that, among
the newly prepared tetrahydroisoquinolines bearing
1-alkyl-2-alkylpiperazine residues, the 1-methyl- and
1-ethyl-2-methyl-piperazine derivatives 30 and 31 ex-
hibit profiles similar to that of 29 (Table 1, entries 1-3).
The 2-ethyl-piperazine analogue 32 is less active (Table
1, entry 4). Although 31 shows a marginal increase of
binding affinity relative to 29 (IC50 ) 38 and 63 nM,
respectively), this enhancement is not reflected in the
ERE and MCF-7 assays, where the IC50 values are 64
nM and 30 nM for 31 versus 23 and 21 nM for 29. As
observed for 29, the tetrahydroisoquinoline derivatives
30-32 display weaker agonism (10-18%) than 60 in
the MCF-7 assay in the absence of estrogen.
Next, analogues lacking the 2-position substituent
were prepared to evaluate the influence of this group
on binding affinity and potency in vitro. The N-methyl-
piperazine analogue 33 (Table 1, entry 5) shows a 3-
and 10-fold reduction of binding affinity relative to the
parent 1-methyl-2-methyl-piperazine 30 and to the
aminoethoxy derivative 60, respectively. In the ERE and

370 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Renaud et al.
N-methyl piperazines 33 and 45 (Table 1, entries 5 and
10) confirms the trend observed for the piperidine
analogues whereby a meta-OH group increases selectiv-
ity in favor of ERR. The other piperazine ligands 46-
50 (Table 1, entries 11-15) display IC50 values in the
radioligand binding assay that vary from 83 to 177 nM
and show selectivities for ERR over ERâ that range
between 7-16-fold. These new piperazines display
similar potency to that of the diazadecaline derivative
29 in the ERE assay and an activity reduced by 2-5-
fold in the MCF-7 assay. Interestingly, the variation in
the basic character of the nitrogen interacting with
Asp351 by up to 1.4 log units does not significantly affect
affinity. The pKavalues of the alkyl nitrogens for the
N-methyl-piperazine 33, the cyclopropyl-methyl-pipera-
zine 48, and the N-methyl-piperidine 38a are 7.8, 8.0,
and 9.2, respectively. In comparison, the lead tetrahy-
droisoquinoline 60 displays a pKa of 8.5 for its piperidine
nitrogen.
As a result of our studies, we established that the
aminoethoxy subunit of 60 can be replaced effectively
by piperazine or piperidine moieties while maintaining
activity and antagonist potential. In addition, we found
that the analogues with constrained side chains present
a superior profile relative to that of the parent in that
they display reduced agonism in the MCF-7 assay. The
diazadecaline residue was identified as the best side
chain replacement. Because 29 is a mixture of four
diastereoisomers, we questioned whether one or more
isomers were contributing to the activity. There are
literature precedents indicating that one enantiomer of
SERMs such as lasofoxifene (4),10 NNC 45-0781,41 and
EM-65242 is more potent than the other. In the case of
lasofoxifene (4),10 for example, the 1-R, 2-S absolute
configuration was attributed to the most potent enan-
tiomer as illustrated in Figure 1. On the basis of our
own results37 and on published literature data, it
appeared likely that the most active isomer of 29 would
possess an R absolute configuration at C-1 of the
tetrahydroisoquinoline nucleus. However, the impor-
tance of the configuration at the diazadecaline fragment
remained to be confirmed. Our molecular modeling
studies indicated that the preferred absolute configu-
ration of the diazadecaline side chain would be S. An
X-ray crystal structure of 29 in complex with ERR-LBD
corroborated our assumption regarding the absolute
configuration at C1 and the results of the modeling
studies, in addition to providing the details of the
ligand-protein interactions.
Figure 3. Side chain rigidification leading to 29 using
raloxifene (7) bound to ERR-LBD (C atoms in green) as a
template. Salt bridges and hydrogen bonding contacts of
raloxifene (7) are indicated by dotted lines in white. (a) The
conformation of the 2-(1-piperidinyl)ethyloxy fragment of
raloxifene (7) forms a substructure of a trans-1,4-diazadecaline
skeleton that is completed upon addition of one ring atom (the
C atom is shown in white). (b) Model of 29 (C atoms in white)
incorporating the trans-1,4-diazadecaline superimposed on
raloxifene (7). The polar contacts of raloxifene (7) with Asp351,
Glu353, and Arg394 are retained, and the phenolic A rings
and piperidine rings common to both molecules overlap.
MCF-7 assays, its activity is only slightly reduced by
1.6- and 1.3-fold, respectively, relative to that of 30.
In an attempt to further validate our modeling studies
which had suggested piperidines as potential cyclic
versions of the amine bearing side chain, we prepared
piperidine derivative 38a (Table 1, entry 6). The results
indicate that both the piperazine and the piperidine
residues (entries 5 and 6) are acceptable aminoethoxy
side chain replacements, albeit the resulting ligands are
slightly less potent than the parent tetrahydroisoquino-
lines 29 and 60 (entries 1 and 17).
X-ray Crystal Structure
Looking at the impact of the substitution pattern
around the phenyl D ring for a small subset of N-
methyl-piperidine analogues (Table 1, entries 6-9), we
discovered that the presence of a 3-hydroxy group
increased the selectivity toward ERR in both the binding
and the ERE assays (Table 1, entry 9). Aiming to exploit
this finding and to further probe the potential effect of
N-alkyl groups on selectivity, we prepared a number of
tetrahydroisoquinolines, embedding a 3-hydroxyphenyl
D ring. In view of the fact that both the piperazine and
the piperidine side chains have a similar impact on
potency and that the piperazine analogues were more
readily prepared, we carried out our studies with
derivatives of the piperazine series. Comparison of the
Cocrystallization of the Cys f Ser triple mutant
hERR-LBD 301-553 with a mixture containing the four
diastereomers of 29 provided crystals diffracting to at
least 2.05 Å that were suitable for X-ray analysis. This
new crystal form for ERR-LBD contains four monomers
(denoted A, B, C, and D) per asymmetric unit which
form two dimers (AB and CD). The final model contains
residues 307-330, 341-461, 464-528, and 535-548 for
monomers A and C and residues 307-330, 341-461,
464-525, and 535-548 for monomers B and D (the
missing regions correspond to flexible loops), as well as
374 water molecules and 4 ligand molecules 29 (denoted
L, M, N, and O).

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 371
Figure 4. Superposition of the X-ray crystal structures of
(1-R)(S)-29 (blue) in complex with ERR-LBD301-553/CfS
triple mutant (yellow) and (R)-60 (magenta) in complex with
ERR-LBD301-553/CfS triple mutant (gray). For details on
the structure determination, see Supporting Information and
our previous work.37
Figure 5. Superposition of the X-ray crystal structures of
(1-R)(S)-29 (blue) in complex with ERR-LBD301-553/CfS
triple mutant (yellow) and raloxifene (7) (magenta) in complex
with ERR-LBD301-553/carboxymethylated C (grey). For de-
tails on the structure determination, see Supporting Informa-
tion.
The X-ray crystal structure ascertains that only one
diastereoisomer of 29 (that with the R-configuration at
the C1 position of the tetrahydroisoquinoline core and
the S-configuration at the bicyclic piperazine residue)
binds within the cavity of the ERR-LBD (Figure 4). Its
positioning within the binding cleft is reminiscent of
that observed for 6037 and other SERMs.38 The backbone
of tetrahydroisoquinoline 29 establishes a number of
van der Waals contacts with the residues forming the
binding pocket. The tetrahydroisoquinoline ligand 29 is
further anchored within the binding cavity via a net-
work of hydrogen bonds established between its phenolic
hydroxyl and the side chains of Glu353 and Arg394. Its
piperazinyl phenyl side chain protrudes through a
narrow channel and adopts a position which allows a
salt-bridge interaction with Asp351. The electron den-
sity indicates that the two anilinic nitrogens of 29 are
slightly nonplanar as predicted by our model of 29.
An overlay of the X-ray structures of ERR-LBDs
bound to 29, to the parent tetrahydroisoquinoline 60
(Figure 4) or to raloxifene (7) (Figure 5) shows that their
basic side chains adopt similar positions (stabilized by
the interaction with Asp351), despite differences in their
crystal forms. As predicted by molecular modeling, the
diazadecaline moiety of 29 nicely superimposes over the
piperidinylethoxy side chain of 60 (Figure 4). The first
ring of the bicyclic residue of 29, apart from rigidifying
the side chain, also participates in hydrophobic contacts
with Trp383. However, this increased stabilization does
not translate into an improvement in binding affinity.
decaline analogue 29, the N-iPr-piperazine derivative
46 which incorporates a 3-position hydroxyphenyl
residue, and the N-methyl-piperidine 38b bearing a
4-position fluorophenyl moiety were chosen. These tet-
rahydroisoquinolines display good absolute oral bio-
availabilities (BAV) (44-64%, Table 2, entries 3-5) and
maximal blood concentrations which range between 27
and 57 nM (Cmax, po, dose-normalized). The values found
for 29, 38b, and 46 compare very favorably with those
obtained for the parent tetrahydroisoquinoline 60 (BAV,
42%; Cmax, 31 nM; Table 2, entry 6) and are superior to
those exhibited by lasofoxifene (4, Table 2, entry 1, BAV,
38%; Cmax, 15.5 nM) and raloxifene (7, Table 2, entry 2;
BAV, 22%; Cmax, 7.5 nM). The volumes of distribution
at steady state (Vss) for the various tetrahydroisoquino-
lines range from 9 to 16 L/kg, with the exception of the
meta hydroxy derivative 46 that is associated with a
lower Vss (5 L/kg), a value indicating that this com-
pound may have a higher protein binding.
Conclusion
In this paper, we reported the results of our investi-
gations aimed at the discovery of selective estrogen
receptor modulators with conformationally restricted
side chains. The information resulting from the X-ray
crystal structure of hERR in complex with raloxifene (7)
used in conjunction with modeling studies directed our
synthetic work toward the preparation of analogues of
the tetrahydroisoquinoline series that incorporate cyclic
side chains as surrogates for the aminoethoxy residue.
Both piperazine and piperidine derivatives were found
to retain affinity for ERR and, importantly, antagonistic
properties when compared to the parent compound 60.
In addition, all of the new derivatives displayed weaker
agonism in the MCF-7 assay relative to that of lasofox-
ifene (4), raloxifene (7), and tetrahydroisoquinoline 60.
Pharmacokinetic Properties
To examine the pharmacokinetic properties of our
ligands, we profiled a few selected candidates in a
pharmacokinetic (PK) cassette dosing experiment car-
ried out in rats (Table 2). For that purpose, the diaza-

372 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Table 2. Pharmacokinetic Data for Selected Compoundsa
Renaud et al.
maxi blood concn
po dose-normalized
(Cmax) (nM)
vol of distribn
total clearance
(CL) (mL/min kg)
at steady state
entry
no.
4c
7c
29
38b
46
60
oral BAV (%)b
(Vss) (L kg-1
)
1
2
3
4
5
6
38 ( 5
22 ( 4
49 ( 6
64 ( 7.5
44 ( 6
42 ( 10
15.5 ( 5.3
7.5 ( 2.3
29.7 ( 5.4
26.5 ( 7.7
57.2 ( 5.9
31 ( 7
55.7 ( 4.3
157.7 ( 11.7
28.4 ( 4.2
52.4 ( .8
17.9 ( 4.0
51.9 ( 3.2
14.7 ( 1.3
17.6 ( 1.8
9.2 ( 1.2
15.8 ( 2.5
5.0 ( 1.0
13.8 ( 0.7
a Pharmacokinetic parameters calculated from blood levels after iv (1 mg/kg) and po (5 mg/kg) administration to conscious rats. b BAV
) bioavailability. c Tested as the HCl salt.
1599, 1503, 1473, 1378, 1325, 1240, 1228, 1216, 1151, 1008,
751, 693 cm-1. 1H NMR (400 MHz, DMSO) δ: 7.58 (d, J ) 8.5
Hz, 2H), 7.43-7.26 (m, 6H), 7.13 (dd, J ) 7.5, 8.5 Hz, 2H),
7.04 (d, J ) 8.5 Hz, 2H), 6.88-6.81 (m, 2H), 6.78 (d, J ) 8.5
Hz, 2H), 6.64 (t, J ) 7.5 Hz, 1H), 5.85 (s, 1H), 5.04 (s, 2H),
3.69-3.61 (m, 1H), 3.41-3.32 (m, 1H), 2.96-2.86 (m, 1H),
2.86-2.75 (m, 1H). 13C NMR (100.7 MHz, DMSO) δ: 157.0,
148.4, 143.6, 136.8, 136.6, 136.2, 129.6, 129.0, 128.7, 128.4,
128.2, 127.55, 127.4, 116.7, 113.6, 113.1, 112.6, 92.4, 69.1, 60.3,
43.1, 27.7. HRMS (ESI, M + H+) calcd for C28H24NOI: 518.0981.
Found: 518.0985. HPLC purity: 100%; tR 7.95 min.
The most potent analogue, tetrahydroisoquinoline 29,
embeds the diazadecaline side chain, which acts as an
excellent mimic of the traditional SERM residue. In
addition to being potent in functional assays, 29 displays
a very good oral bioavailability (49%) in a cassette
dosing experiment carried out in rats. On the basis of
both its in vitro and PK profile, 29 was identified as
the optimal compound within that series and warrants
further in vivo evaluation.
General Procedure for the Amination of 13. Proce-
dure A. Synthesis of 22 and 24. When two different numbers
appear in parentheses, these refer to the quantities used in
the preparation of bicyclic piperazine 22 and ethyl piperazine
derivative 24, respectively. A mixture containing the phenylio-
dide 13 (1 equiv), octahydropyrido[1,2-a]pyrazine (18, 1.54
Experimental Section
Chemistry. General. All of the commercial chemicals and
solvents are reagent grade and were used without further
purification unless otherwise stated. All of the reactions except
those in aqueous media were carried out under an atmosphere
of N2, in flame-dried glassware. Reactions were monitored by
analytical reverse-phase high-performance liquid chromatog-
raphy (RP-HPLC) using a Hewlett-Packard series 1100,
equipped with a diode array spectrometer (λ ) 210-250 nm)
and a Waters Symmetry C-8 column (3.5 µm, 2.1 mm × 50
mm) as the stationary phase. The mobile phases used were
A, 95:5 H2O/CH3CN containing 0.1% TFA, and B, CH3CN
containing 0.1% TFA. The program employed ran as follows:
0-7 min, 5% B f 100% B; 7-8.5 min, 100% B; at 8.5 min, 5%
B for 2.5 min, prior to the next run. The flow rate was
maintained at 0.5 mL/min. Additionally, thin-layer chroma-
tography on 0.25 mm silica gel plates (E. Merck, silica gel 60
t
equiv) or 2-ethyl-piperazine (20, 1.3 equiv), BuOK (1.54/2
equiv), Pd2(dba)3 (2/5% mol), and (2′-dicyclohexylphosphanyl-
biphenyl-2-yl)-dimethylamine (8/10% mmol) in anhydrous
toluene (0.19/0.24 M) was stirred at 80-85 °C for 12/19 h. After
workup and purification by flash chromatography, the pi-
perazine derivatives 22 and 24 were obtained in 53 and 74%
yield, respectively. One detailed procedure is reported below.
General Procedure for Debenzylation. A solution of the
piperazine derivative 22, 25, 26, 27, or 28 in a 2:1 or 5:3
dioxane/concentrated HCl mixture was stirred at 90 °C for
2-2.5 h. After workup and purification, tetrahydroisoquino-
lines 29, 30, 31, 32, or 33 were isolated in yields ranging from
34 to 73%. A representative example is given below.
F254) was used to follow the reactions. Visualization was
accomplished with UV light, KMnO4, or 5% phosphomolybdic
acid in 95% ethanol.
Procedure A. Synthesis of 6-Benzyloxy-1-[4-(3-ethyl-
piperazin-1-yl)phenyl]-2-phenyl-1,2,3,4-tetrahydroiso-
quinoline (24) by Amination. A mixture containing the
phenyliodide 13 (1.0 g, 1.93 mmol), 2-ethyl-piperazine (20, 286
mg, 2.5 mmol), tBuOK (432 mg, 3.86 mmol), Pd2(dba)3 (88 mg,
0.096 mmol), and (2′-dicyclohexylphosphanyl-biphenyl-2-yl)-
dimethylamine (76 mg, 0.193 mmol) in anhydrous toluene (8
mL) was stirred at 85 °C for 19 h. The mixture was cooled to
room temperature and poured into H2O (10 mL)/Et2O (20 mL).
The layers were separated, the organic phase was washed with
brine (10 mL) and dried (MgSO4), and the solvent was
concentrated in vacuo. The residual brown oil was purified by
flash chromatography (silica gel, 19:1 f 9:1 CH2Cl2/MeOH)
to provide the title compound (721 mg, 74%) as a solid. IR
(KBr) νmax: 3427, 3058, 3031, 2957, 2930, 2818, 1597, 1502,
Intermediates and final compounds were purified by flash
chromatography (E. Merck silica gel 60, 230-400 mesh),
crystallization, or semipreparative reverse-phase HPLC
(RP-HPLC) using a Gilson model 306 equipped with a UV-
vis detector (λ usually set to 214 nm) and a Symmetry Prep
RP18 (7 µm, 19 mm × 150 mm column) as the stationary
phase. The eluents employed were A, H2O containing 0.1%TFA,
and B, CH3CN containing 0.1% TFA. The program ran as
follows: 0-0.5 min, 100% B; 0.5-3 min, 10% B; the compound
was automatically injected at 3 min; from 3 to 4 min, 10% B;
thereafter, a linear gradient was then run from 10% B to 100%
B over the next 16 min followed by a 2.0 min hold at 100% B.
The flow rate was held constant at 20 mL/min.
NMR spectra were recorded either on a Varian spectrom-
eter, model Mercury-300 or -400, or on a Bruker DPX-400,
DMX-500, or DRX-500. Signal positions (δ values) were
calibrated using the residual undeuterated solvent resonance
as the internal standard. The multiplicity is indicated by one
of the following: d, doublet; t, triplet; m, multiplet; br, broad.
Infrared spectra were recorded using a Perkin-Elmer i-Series
FT-IR microscope coupled with a SPECTRUM 2000 FT-IR
spectrometer. High-resolution mass spectra (HRMS) were
recorded on a Finnigan MAT900.
1453, 1381, 1231, 1156, 1025, 747, 694 cm-1 1H NMR (400
.
MHz, DMSO) δ: 7.44-7.22 (m, 6H), 7.12 (t, J ) 8.0 Hz, 2H),
7.02 (d, J ) 8.5 Hz, 2H), 6.86-6.74 (m, 6H), 6.61 (t, J ) 7.5
Hz, 1H), 5.77 (s, 1H), 5.04 (s, 2H), 3.67-3.58 (m, 1H), 3.47-
3.25 (m, 3H), 2.97-2.66 (m, 4H), 2.57-2.43 (m, 2H), 2.15 (t, J
) 10.0 Hz, 1H), 1.40-1.27 (m, 2H), 0.89 (t, J ) 7.5 Hz, 3H).
13C NMR (100.7 MHz, DMSO) δ: 156.7, 149.7, 148.7, 136.9,
136.1, 133.5, 130.6, 128.65, 128.3, 128.2, 127.5, 127.4, 127.3,
116.4, 114.9, 113.5, 113.1, 112.4, 69.1, 60.3, 56.0, 54.13/54.06,
48.76/48.70, 45.1, 42.7, 27.6, 26.4, 10.3. HRMS (ESI, M + H+)
calcd for C34H37N3O: 504.3015. Found: 504.3017. HPLC
purity: 96%; tR 5.19 min.
Syntheses of Piperazinyl Derivatives 29-33 via Ami-
nation. Synthesis of 6-Benzyloxy-1-(4-iodophenyl]-2-
phenyl-1,2,3,4-tetrahydroisoquinoline (13). The iodo de-
rivative 13 was synthesized as described in Scheme 1. A
similar route using intermediates with different protecting
groups has been described.26 IR (KBr) νmax: 2915, 2891, 2821,
Synthesis of 6-Benzyloxy-1-[4-(3-ethyl,4-methyl-pip-
erazin-1-yl)phenyl]-2-phenyl-1,2,3,4-tetrahydroisoquino-
line (28) by Reductive Amination. A mixture of piperazin-

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 373
yl derivative 24 (115 mg, 0.23 mmol), 37% HCHO (76 µL, 1.01
mmol), AcOH (14 µL, 0.25 mmol), and NaBH(OAc)3 (73 mg,
0.34 mmol) in Cl(CH2)2Cl (6 mL) was stirred at room temper-
ature for 1 h. The reaction mixture was diluted with CH2Cl2
(10 mL) and washed with 1 N aqueous NaOH (6 mL). The
aqueous layer was extracted with CH2Cl2 (1 × 10 mL), the
combined organic phases were dried (MgSO4), and the solvent
was removed in vacuo. The residue was purified by flash
chromatography (silica gel, 50:1:0.1 CH2Cl2/MeOH/NH4OH) to
(18.4 mg, 0.0386 mmol) was added. The mixture was stirred
at 40 °C for 4 h. After being cooled to room temperature, the
mixture was poured into H2O (10 mL)/Et2O (20 mL). The layers
were separated, and the aqueous layer was extracted once
again with Et2O (20 mL). The combined organic phases were
washed with brine (10 mL) and dried (MgSO4), and the solvent
was removed in vacuo. The residual orange oil was purified
by flash chromatography (silica gel, 9:1 CH2Cl2/MeOH) to
provide the title compound (290 mg, 61%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ: 7.51-7.35 (m, 5H), 7.31-7.10
(m, 5H), 6.94-6.75 (m, 7H), 5.78 (s, 1H), 5.09 (s, 2H), 3.74-
3.65 (m, 1H), 3.57-3.46 (m, 3H), 3.14 (dt, J ) 12.0, 2.5 Hz,
1H), 3.08-2.85 (m, 4H), 2.71 (td, J ) 2.5, 12.0 Hz, 1H), 2.40
(m, 1H), 1.85 (br s, 1H), 1.15 (d, J ) 6.5 Hz, 3H). 13C NMR
(125.8 MHz, DMSO) δ: 157.5, 150.5, 149.5, 137.6, 136.8,
134.15, 131.3, 129.35, 129.1, 128.9, 128.2, 128.1, 128.0, 117.05,
115.4, 114.2, 113.8, 113.1, 69.6, 60.8, 56.4, 50.6, 48.91/48.88,
45.7, 43.2, 28.0, 19.9. MS (APCI) m/z ) 490 [M + H+]. HPLC
purity: 97.8%; tR 5.08 min.
Synthesis of 6-Benzyloxy-1-[4-(3,4-dimethyl-piperazin-
1-yl)phenyl]-2-phenyl-1,2,3,4-tetrahydroisoquinoline (26)
by Reductive Amination. A mixture of piperazin-yl deriva-
tive 23 (450 mg, 0.919 mmol), 37% aq HCHO (829 µL, 11.0
mmol), and AcOH (63 µL, 1.1 mmol) in MeOH (8 mL) was
stirred at 80 °C for 1 h. The reaction mixture was cooled to 0
°C and diluted with CH2Cl2 (1 mL), and NaBH3CN (95%, 881
mg, 13.3 mmol) was added. The resultant mixture was stirred
at this temperature for 1 h and poured into saturated aqueous
NH4Cl (20 mL)/CH2Cl2 (40 mL), and the layers were separated.
The aqueous layer was extracted with CH2Cl2 (3 × 15 mL),
the combined organic phases were dried (Na2SO4), and the
solvent was removed in vacuo. The residual light brown solid
was purified by crystallization (AcOEt/Et2O) and flash chro-
matography (silica gel, 9:1 CH2Cl2/MeOH) to furnish the title
compound (372 mg, 81%) as a beige solid. Acid-catalyzed
removal of the benzyl group, followed by purification by flash
chromatography (silica gel; 19:1 CH2Cl2/MeOH), provided the
debenzylated product 30 as a pale yellow solid in 70% yield.
1
furnish the title compound (101 mg, 86%) as an oil. H NMR
(500 MHz, DMSO) δ: 7.43 (d, J ) 7.0 Hz, 2H), 7.38 (t, J ) 7.0
Hz, 2H), 7.34-7.29 (m, 1H), 7.27 (d, J ) 8.5 Hz, 1H), 7.14 (t,
J ) 7.0 Hz, 2H), 7.04 (d, J ) 8.5 Hz, 2H), 6.77-6.88 (m, 6H),
6.83 (t, J ) 7.0 Hz, 1H), 5.79 (s, 1H), 5.06 (s, 2H), 3.64 (ddd,
J ) 5.5, 5.5, 11.5 Hz, 1H), 3.43-3.30 (m, 3H), 2.915 (ddd, J )
5.5, 5.5, 15.5 Hz, 1H), 2.87-2.74 (m, 2H), 2.68-2.61 (m, 1H),
2.44-2.37 (m, 1H), 2.20 (dm, J ) 11.0 Hz for d, 1H), 2.17 (s,
3H), 1.99-1.92 (m, 1H), 1.63-1.53 (m, 1H), 1.44-1.34 (m, 1H),
0.85 (t, J ) 7.5 Hz, 3H). 13C NMR (125.8 MHz, DMSO) δ:
157.5, 150.1, 149.5, 137.6, 136.8, 134.4, 131.3, 129.3, 129.0,
128.9, 128.2, 128.1, 128.0, 117.1, 115.5, 114.2, 113.8, 113.1,
69.6, 62.8, 60.8, 55.3, 52.6/52.5, 48.45, 43.2, 42.4, 28.1, 22.9,
9.7. HPLC purity: 98.8%; tR 5.27 min.
Synthesis of 1-[4-(3-Ethyl-4-methyl-piperazin-1-yl)phen-
yl]-2-phenyl-1,2,3,4-tetrahydroisoquinolin-6-ol (32) by
Debenzylation. A solution of the piperazine derivative 28
(100 mg, 0.193 mmol) in a 2:1 dioxane/concentrated HCl
mixture (15 mL) was stirred at 90 °C for 2 h. The mixture
was cooled to room temperature and extracted with Et2O. The
aqueous layer was adjusted to pH 10 with 4 N aqueous NaOH
(14 mL) and extracted with CH2Cl2 (3 × 10 mL). The combined
organic phases were dried (MgSO4) and concentrated. The
residue was purified by flash chromatography (silica gel, 19:1
f 9:1 CH2Cl2/MeOH) to provide the tetrahydroisoquinolinol
32 (38 mg, 46%) as a solid. IR (CH2Cl2 solution) νmax: 3582,
3041, 2969, 2838, 2801, 1609, 1598, 1504, 1452, 1384, 1244,
1
1148, 928, 800 cm-1. H NMR (500 MHz, DMSO) δ: 9.27 (s,
1H), 7.17-7.10 (m, 3H), 7.04 (d, J ) 8.5 Hz, 2H), 6.82-6.77
(m, 4H), 6.65-6.56 (m, 3H), 5.72 (s, 1H), 3.65-3.58 (m, 1H),
3.42-3.30 (m, 3H), 2.84 (ddd, J ) 5.5, 5.5, 15.5 Hz, 1H), 2.80-
2.72 (m, 2H), 2.69-2.61 (m, 1H), 2.45-2.37 (m, 1H), 2.25-
2.15 (m, 1H), 2.17 (s, 3H), 2.00-1.93 (m, 1H), 1.64-1.54 (m,
1H), 1.45-1.34 (m, 1H), 0.85 (t, J ) 7.5 Hz, 3H). 13C NMR
(125.8 MHz, DMSO) δ: 156.4, 150.05, 149.5, 136.6, 134.7,
129.3, 128.9, 127.9, 116.9, 115.5, 114.65, 113.7, 113.5, 62.8,
60.9, 55.3, 52.6/52.5, 48.5/48.4, 43.3, 42.3, 28.0, 22.9, 9.7.
HRMS (ESI, M + H+) calcd for C28H33N3O: 428.2702. Found:
428.2702. HPLC purity: 100%; tR 3.81 min.
1-[4-(3,4-Dimethyl-piperazin-1-yl)phenyl]-2-phenyl-
1,2,3,4-tetrahydroisoquinolin-6-ol (30). IR (KBr) νmax
:
3600-2200, 2951, 2822, 1597, 1503, 1381, 1244, 1149, 748
1
cm-1. H NMR (400 MHz, DMSO) δ: 9.24 (s, 1H), 7.16-7.08
(m, 3H), 7.02 (d, J ) 8.5 Hz, 2H), 6.82-6.74 (m, 4H), 6.64-
6.54 (m, 3H), 5.71 (s, 1H), 3.65-3.57 (m, 1H), 3.45-3.30 (m,
3H), 2.88-2.71 (m, 3H), 2.69-2.60 (m, 1H), 2.35-2.24 (m, 1H),
2.22-2.13 (m, 1H), 2.18 (s, 3H), 2.12-2.02 (m, 1H), 1.00 (d, J
) 6.0 Hz, 3H). 13C NMR (100.7 MHz, DMSO) δ: 155.4, 148.9,
148.6, 135.7, 133.7, 128.45, 128.1, 127.1, 116.1, 114.55, 113.8,
112.9, 112.7, 60.2, 56.9, 54.97/54.96, 54.6, 48.0/47.97, 42.6,
41.9, 27.35, 16.6. HRMS (ESI, M + H+) calcd for C27H31N3O:
414.2545. Found: 414.2546. HPLC purity: 100%; tR 3.56 min.
Synthesis of 6-Benzyloxy-1-[4-(4-ethyl-3-methylpi-
perazin-1-yl)phenyl]-2-phenyl-1,2,3,4-tetrahydroisoquinoline
(27) by Reductive Amination. A mixture of piperazin-yl
derivative 23 (290 mg, 0.59 mmol), CH3CHO (401 µL, 7.1
mmol), AcOH (37 µL, 0.65 mmol), and NaBH(OAc)3 (188 mg,
0.887 mmol) in Cl(CH2)2Cl (6 mL) was stirred at room
temperature for 16 h. The reaction mixture was diluted with
CH2Cl2 (40 mL) and poured into 1 N aqueous NaOH (20 mL).
The layers were separated. The aqueous layer was extracted
with CH2Cl2 (1 × 15 mL), the combined organic phases were
dried (MgSO4), and the solvent was removed in vacuo. The
residual orange oil was purified by flash chromatography
(silica gel, 19:1 CH2Cl2/MeOH) to furnish the title compound
(160 mg, 52%) as a yellow solid. Acid-catalyzed removal of the
benzyl group, followed by purification by flash chromatography
(silica gel, 19:1 CH2Cl2/MeOH), provided the debenzylated
product 31 in 55% yield.
1-[4-(Octahydropyrido[1,2-a]pyrazin-2-yl)phenyl]-2-
phenyl-1,2,3,4-tetrahydroisoquinolin-6-ol (29). Derivative
22 was prepared in 53% yield from 13 using procedure A.
Debenzylation provided 29 in 73% yield. IR (KBr) νmax: 3600-
2200, 3022, 2932, 2819, 1596, 1503, 1382, 1254, 1231, 927, 748,
1
691 cm-1. H NMR (400 MHz, DMSO) δ: 9.24 (s, 1H), 7.19-
7.06 (m, 3H), 7.01 (d, J ) 8.0 Hz, 2H), 6.84-6.70 (m, 4H),
6.64-6.51 (m, 3H), 5.70 (s, 1H), 3.67-3.27 (m, 4H), 2.88-2.57
(m, 5H), 2.32-2.10 (m, 2H), 2.02-1.85 (m, 2H), 1.73-1.37 (m,
4H), 1.30-1.05 (m, 2H). 13C NMR (100.7 MHz, DMSO) δ:
155.6, 149.1, 148.8, 135.8, 133.9, 128.6, 128.4, 127.2, 116.2,
114.8, 114.0, 113.0, 112.9, 60.4/60.3, 54.8, 54.3, 54.1, 48.1, 42.8,
29.1, 27.5, 25.2, 23.5. HRMS (ESI, M + H+) calcd for
C29H33N3O: 440.2702. Found: 440.2702. HPLC purity: 97%;
tR 3.67 min.
Procedure B. Synthesis of 6-Benzyloxy-1-[4-(3-methyl-
piperazin-1-yl)phenyl]-2-phenyl-1,2,3,4 -tetrahydroiso-
quinoline (23) by Amination. A mixture containing the
phenyliodide 13 (500 mg, 0.966 mmol), 2-methyl-piperazine
(19, 116 mg, 1.16 mmol), tBuOK (162 mg, 1.44 mmol), Pd2(dba)3
(8.8 mg, 0.0096 mmol), and 1,3-bis(2,6-diisopropylphen-
yl)-4,5-dihydroimidazolium tetrafluoroborate32-35 (18.4 mg,
0.0386 mmol) in anhydrous dioxane (3 mL) was stirred at 27
1-[4-(4-Ethyl-3-methyl-piperazin-1-yl)phenyl]-2-phen-
yl-1,2,3,4-tetrahydroisoquinolin-6-ol (31). IR (KBr) νmax
:
3650-2200, 2967, 2820, 1597, 1503, 1456, 1382, 1326, 1242,
t
1
°C for 40 h. An additional quantity of BuOK (54 mg, 0.48
1173, 1154, 923, 748, 692 cm-1. H NMR (400 MHz, DMSO)
mmol), Pd2(dba)3 (8.8 mg, 0.0096 mmol), and 1,3-bis(2,6-
diisopropylphenyl)-4,5-dihydroimidazolium tetrafluoroborate
δ: 9.22 (s, 1H), 7.16-7.08 (m, 3H), 7.01 (d, J ) 8.5 Hz, 2H),
6.81-6.73 (m, 4H), 6.64-6.53 (m, 3H), 5.70 (s, 1H), 3.65-3.55

374 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Renaud et al.
(m, 1H), 3.40-3.20 (m, 3H), 2.90-2.65 (m, 5H), 2.45-2.35 (m,
2H), 2.33-2.19 (m, 2H), 1.00 (d, J ) 5.0 Hz, 3H), 0.95 (t, J )
7.0 Hz, 3H). 13C NMR (125.8 MHz, DMSO) δ: 155.9, 149.5,
149.0, 136.1, 134.1, 128.9, 128.5, 127.45, 116.4, 115.0, 114.2,
113.2, 113.1, 60.4, 55.5, 53.8, 49.6, 48.4, 46.35, 42.9, 27.5, 15.8,
10.6. HRMS (ESI, M + H+) calcd for C28H33N3O: 428.2702.
Found: 428.2701. HPLC purity: 99%; tR 3.57 min.
141.0, 129.8, 129.6 (d, JC-F ) 9 Hz), 121.5, 120.9 (d, JC-F ) 3
Hz), 112.8, 112.3 (d, JC-F ) 23 Hz), 112.0 (d, JC-F ) 21 Hz),
111.7, 55.4, 48.6, 28.0. HRMS (ESI, M + H+) calcd for C16H14-
NO2F: 272.1087. Found: 272.1088. HPLC purity: 97%; tR 5.38
min.
General Procedure for the Conversion of Lactams
16a-d into Tetrahydroisoquinolines 17a-d. Lactam
16a-d was allowed to undergo reaction with 4-bromophenyl-
lithium in THF (0.08-0.1 M) for 1.5-3.5 h. (The phenyllithium
reagent was generated by treatment of p-dibromobenzene (1.1
equiv) with n-BuLi (1.1 equiv) in THF at -78 °C for 25-150
min.) After aqueous workup and isolation, the iminium salt
was dissolved in MeOH (0.06-0.085 M) and treated with
NaBH4 (2.2 equiv) at room temperature for 15-30 min. After
that period of time, the product was isolated and purified.
Tetrahydroisoquinolines 17a-d were obtained in yields rang-
ing from 70 to 86%. A representative example is given below.
Synthesis of 1-(4-Bromophenyl)-2-(3-fluorophenyl)-6-
methoxy-1,2,3,4-tetrahydroisoquinoline (17c). To a cold
(-78 °C) solution of p-dibromobenzene (2.39 g, 10.14 mmol)
in anhydrous THF (50 mL), a solution of n-BuLi in hexanes
(1.6 M, 6.3 mL, 10.14 mmol) was added over a period of 5 min.
After the resultant suspension was stirred at this temperature
for 25 min, a solution of lactam 16c (2.5 g, 9.22 mmol) in
anhydrous THF (50 mL) was added dropwise over 30 min.
Stirring was continued for an additional 3 h at this temper-
ature. The mixture was poured onto H2O (100 mL)/AcOEt (100
mL). HClO4 (70%, 2.4 mL) was added, the mixture was stirred
for 15 min at room temperature, and the phases were
separated. The aqueous layer was extracted with AcOEt (100
mL), and the combined organic layers were washed with brine
(75 mL) and dried (MgSO4), and the solution was concentrated
in vacuo yielding an iminium salt that was used directly in
the next step.
Procedure C. Synthesis of 6-Benzyloxy-1-[4-(4-methyl-
piperazin-1-yl)phenyl]-2-phenyl-1,2,3,4 -tetrahydroiso-
quinoline (25) by Amination. A mixture containing the
phenyliodide 13 (300 mg, 0.58 mmol), 4-methyl-piperazine (21,
t
80 µL, 0.72 mmol), BuOK (78 mg, 0.81 mmol), Pd2(dba)3 (2
mg, 0.0022 mmol), and (()-BINAP (4 mg, 0.0064 mmol) in
anhydrous tetrahydrofuran (THF) (3 mL) was refluxed for 4
h. Additional quantities of Pd2(dba)3 (4 mg, 0.0044 mmol), (()-
BINAP (8 mg, 0.0128 mmol), and 4-methyl-piperazine (21, 80
µL, 0.72 mmol) were added. The mixture was refluxed for 17
t
h prior to the addition of BuOK (80 mg, 0.83 mmol). After
being stirred for another 17 h at the same temperature, the
mixture was cooled to room temperature and poured into H2O
(10 mL)/AcOEt (30 mL). The layers were separated. The
organic phase was washed with water (10 mL) and dried
(MgSO4), and the solvent was removed in vacuo. The residue
was filtered over silica gel, using MeOH as eluent to provide
the title compound (152 mg, 53%, 87% purity by HPLC), which
was used in the next step (HCl-promoted debenzylation)
without further purification. The debenzylated product 33 was
isolated as a solid in 34% yield after purification by flash
chromatography (silica gel, 19:1 CH2Cl2/MeOH) and recrys-
tallization in AcOEt.
1-[4-(4-Methyl-piperazin-1-yl)phenyl]-2-phenyl-1,2,3,4-
tetrahydroisoquinolin-6-ol (33). IR (KBr) νmax: 3650-2000,
3023, 2977, 2935, 2906, 2815, 2683, 2569, 1596, 1503, 1325,
1287, 1238, 1151, 1006, 989, 925, 859, 785, 749, 694 cm-1. 1H
NMR (400 MHz, DMSO) δ: 9.30 (s, 1H), 7.16-7.08 (m, 3H),
7.04 (d, J ) 8.5 Hz, 2H), 6.82-6.74 (m, 4H), 6.64-6.55 (m,
3H), 5.72 (s, 1H), 3.66-3.57 (m, 1H), 3.42-3.32 (m, 1H), 3.14-
3.03 (m, 4H), 2.88-2.70 (m, 2H), 2.62-2.52 (m, 4H), 2.29 (s,
3H). 13C NMR (100.7 MHz, DMSO) δ: 155.6, 148.9, 148.7,
135.8, 134.2, 128.6, 128.5, 128.2, 127.2, 116.2, 114.9, 114.0,
112.95, 112.85, 60.3, 54.0, 47.45, 44.85, 42.8, 27.5. HRMS (ESI,
M + H+) calcd for C26H29N3O: 400.2389. Found: 400.2388.
HPLC purity: 100%; tR 3.44 min.
Syntheses of Piperidine Derivatives 38a-c, e. General
Procedure for the Synthesis of 16a-d via N-Arylation
of 15.30 6-Methoxy-3,4-dihydro-2H-isoquinolin-1-one (15)28 was
N-arylated by reaction with an aryl iodide or an aryl bromide
(2 equiv) in the presence of CuI (0.1 equiv initially; portions
of 0.1 equiv were added, for a maximal amount of 0.4 equiv,
in cases where the reaction slowed) and K2CO3 (1 equiv) in
DMF (0.5-0.6 M) at 150 °C for 72-120 h. The yields of the
arylated products ranged from 70 to 95%. A representative
procedure is given below.
To a solution of the iminium intermediate in MeOH (150
mL), portions of solid NaBH4 (770 mg, 20.35 mmol) were added
over 5 min. The resultant mixture was stirred at room
temperature for 30 min. The mixture was poured into H2O
(100 mL). The aqueous layer was extracted with CH2Cl2 (2 ×
100 mL). The combined organic phases were washed with brine
(50 mL), dried (MgSO4), and concentrated in vacuo. The
resultant yellow solid was purified by flash chromatography
(75 g silica gel; 19:1 f 9:1 hexanes/AcOEt) to provide the title
compound 17c (3.12 g, 82%) as a white solid. IR (KBr) νmax
:
2958, 2906, 2862, 1615, 1576, 1494, 1475, 1381, 1157, 818, 752
cm-1. 1H NMR (400 MHz, DMSO) δ: 7.46-7.34 (m, 3H), 7.26-
7.10 (m, 3H), 6.81-6.76 (m, 2H), 6.63-6.52 (m, 2H), 6.45-
6.39 (m, 1H), 5.93 (s, 1H), 3.74-3.65 (m, 1H), 3.71 (s, 3H),
3.42-3.32 (m, 1H), 2.96-2.88 (m, 1H), 2.85-2.74 (m, 1H). 13
C
NMR (100.7 MHz, DMSO) δ: 163.1 (d, JC-F ) 239 Hz), 158.0,
150.2 (d, JC-F ) 11 Hz), 142.7, 136.1, 130.9, 130.1 (d, JC-F
)
11 Hz), 129.2, 128.7, 128.3, 119.6, 112.7, 112.0, 108.7, 102.6
(d, JC-F ) 21 Hz), 99.4 (d, JC-F ) 25 Hz), 59.9, 55.0, 43.3, 27.6.
HRMS (ESI, M + H+) calcd for C22H19BrFNO: 412.0707.
Found: 412.0708. HPLC purity: 99%; tR 7.79 min.
Synthesis of 2-(3-Fluorophenyl)-6-methoxy-3,4-dihy-
dro-2H-isoquinolin-1-one (16c). A mixture of 6-methoxy-
3,4-dihydro-2H-isoquinolin-1-one (15, 1.0 g, 5.64 mmol), 1-fluoro-
3-iodobenzene (2.5 g, 11.3 mmol), K2CO3 (780 mg, 5.64 mmol),
and CuI (107 mg, 0.56 mmol) in DMF (10 mL) was heated at
150 °C for 72 h under N2 atmosphere. After being cooled to
room temperature, the mixture was poured into aqueous
NH4OH (50 mL)/AcOEt (50 mL). The layers were separated,
and the aqueous layer was extracted with portions of AcOEt
(3 × 50 mL). The combined organic phases were washed with
brine (75 mL), dried (MgSO4), and concentrated in vacuo. The
resulting solid was recrystallized from CH2Cl2-Et2O to provide
the title compound (1.06 g, 69%) as a beige solid. IR (KBr)
νmax: 2956, 2894, 2839, 1648, 1605, 1492, 1410, 1322, 1270,
General Procedure for the Suzuki Cross-Coupling
Reaction. A solution containing phenylbromide 17b, c, or d
(1 equiv), 4-pyridineboronic acid (1-1.2 equiv), K2CO3 (2
equiv), and PdCl2(dppf)‚CH2Cl2 (0.05-0.1 equiv) in toluene/
acetone/water (ratio 3:3:1 or 4:4:1; ∼0.1 M) was refluxed for
20-78 h. After being cooled to room temperature, the mixture
was poured into saturated aqueous NaHCO3/AcOEt. The
phases were separated. The aqueous layer was extracted with
AcOEt, the combined organic layers were washed with brine
and dried (MgSO4), and the solution was concentrated to
provide pyridines 35b-d in yields ranging from 45 to 75%.
For the synthesis of 34a, the iodo derivative 13 served as the
coupling partner, CsF (3 equiv) was used instead of K2CO3,
and the toluene/acetone/water mixture was replaced by 1,2-
dimethoxyethane (DME) as a solvent.
1
1208, 1025, 897, 779, 687 cm-1. H NMR (400 MHz, DMSO)
δ: 7.86 (d, J ) 9.0 Hz, 1H), 7.41 (ddd, J ) 8.0, 8.0, 8.0 Hz,
1H), 7.28 (ddd, J ) 2.5, 2.5, 11.0 Hz, 1H), 7.23 (dm, J ) 8.0
Hz for d, 1H), 7.04 (ddd, J ) 2.5, 8.0, 8.0 Hz, 1H), 6.92 (d, J )
2.5 Hz, 1H), 6.90 (s, 1H), 3.93 (t, J ) 6.5 Hz, 2H), 3.82 (s, 3H),
3.08 (t, J ) 6.5 Hz, 2H). 13C NMR (100.7 MHz, DMSO) δ:
162.7, 161.9, 161.5 (d, JC-F ) 242 Hz), 144.6 (d, JC-F ) 10 Hz),
Synthesis of 2-(3-Fluorophenyl)-6-methoxy-1-[4-pyri-
din-4-yl-phenyl]-1,2,3,4-tetrahydroisoquinoline (35c). A
solution containing phenylbromide 17c (500 mg, 1.21 mmol),
4-pyridineboronic acid (179 mg, 1.46 mmol), K2CO3 (335 mg,

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 375
2.42 mmol), and PdCl2(dppf)‚CH2Cl2 (50 mg, 0.061 mmol) in
3:3:1 toluene/acetone/water (10.5 mL) was refluxed for 42 h.
After being cooled to room temperature, the mixture was
poured into saturated aqueous NaHCO3 (10 mL)/AcOEt (20
mL). The phases were separated. The aqueous layer was
extracted with AcOEt (15 mL), the combined organic layers
were washed with brine (15 mL) and dried (MgSO4), and the
solution was concentrated. The resulting brown oil was puri-
fied by flash chromatography (75 g silica gel; 19:1 CH2Cl2/
MeOH) to furnish the pyridine 35c (368 mg, 74%) as a beige
solid. IR (KBr) νmax: 3027, 2909, 2834, 1615, 1596, 1577, 1496,
1163, 817, 788, 756, 682 cm-1. 1H NMR (400 MHz, DMSO) δ:
8.56 (d, J ) 5.5 Hz, 2H), 7.67 (d, J ) 8.0 Hz, 2H), 7.61 (d, J )
5.5 Hz, 2H), 7.43 (d, J ) 8.0 Hz, 1H), 7.39 (d, J ) 8.0 Hz, 2H),
7.15 (ddd, J ) 8.0, 8.0, 8.0 Hz, 1H), 6.82 (d, J ) 2.5 Hz, 1H),
6.79 (s, 1H), 6.65-6.55 (m, 2H), 6.42 (ddd, J ) 2.5, 8.0, 8.0
Hz, 1H), 6.01 (s, 1H), 3.81-3.68 (m, 4H, includes a 3-H s at δ
) 3.72), 3.45-3.36 (m, 1H), 2.99-2.80 (m, 2H). 13C NMR (100.7
MHz, DMSO) δ: 163.1 (d, JC-F ) 239 Hz), 157.9, 150.2 (d, JC-F
2.75 (m, 4H), 2.40-2.30 (m, 1H), 2.14 (s, 3H), 1.94-1.83 (m,
2H), 1.69-1.50 (m, 4H). 13C NMR (100.7 MHz, DMSO) δ: 163.1
(d, JC-F ) 238 Hz), 157.8, 150.3 (d, JC-F ) 11 Hz), 144.3, 140.8,
136.0, 130.0 (d, JC-F ) 11 Hz), 129.9, 128.3, 126.35, 126.31,
112.6, 111.8, 108.4, 102.2 (d, JC-F ) 21.5 Hz), 99.1 (d, JC-F
)
25 Hz), 60.2, 55.7, 54.9, 46.1, 43.2, 40.7, 32.9, 27.5. HRMS (ESI,
M + H+) calcd for C28H31FN2O: 431.2499. Found: 431.2498.
HPLC purity: 97%; tR 5.39 min.
A mixture of the piperidin-1-yl derivative from above (145
mg, 0.337 mmol), AlCl3 (269 mg, 2.02 mmol), and EtSH (125
µL, 1.69 mmol) in CH2Cl2 (15 mL) was stirred at room
temperature for 17 h. The mixture was diluted with CH2Cl2
(15 mL), and saturated aqueous NaHCO3 (10 mL) was added
to the mixture. The layers were separated, and the aqueous
layer was further extracted with CH2Cl2 (20 mL). The com-
bined organic phases were washed with brine (20 mL), dried
(MgSO4), and concentrated in vacuo. The yellow residue was
purified by reverse phase HPLC, followed by extraction of the
clean fractions with CH2Cl2/aqueous NaHCO3. Compound 38c
) 11 Hz), 149.8, 146.3, 144.5, 136.1, 135.3, 130.1 (d, JC-F
)
was isolated as a yellow solid (77 mg, 55%). IR (KBr) νmax
:
11 Hz), 129.5, 128.4, 127.2, 126.6, 120.8, 112.7, 112.0, 108.6,
102.5 (d, JC-F ) 21.5 Hz), 99.3 (d, JC-F ) 26 Hz), 60.2, 55.0,
43.4, 27.7. HRMS (ESI, M + H+) calcd for C27H23FN2O:
411.1873. Found: 411.1876. HPLC purity: 97%; tR 5.38 min.
General Procedures for Methylation and Reduction
Reactions. Preparation of 38a, b, c, and e. A mixture of
pyridine derivative 34a, 35b, c, or d and MeI (5 equiv) in DMF
(0.2-0.3 M) was stirred at 40 °C for 2 h. Removal of solvent
in vacuo provided the crude pyridinium salt 36a, 37b, c, or d
as a solid, which was used directly in the next step. For the
synthesis of 37d, xylene was used as solvent (M ) 0.5) in
replacement of DMF, and the mixture was heated to 140 °C
for 2.5 h.
A mixture of the pyridinium salt 36a, 37b, or c and PtO2
(20% w/w) in MeOH or MeOH/THF (4:1 or 1:1) was stirred
under an atmosphere pressure of H2 at room temperature for
10-45 h. The reaction mixture was filtered through Celite,
and the solvent was removed in vacuo. The residue was
purified to provide the corresponding piperidine derivative. For
the reduction of 37d, a two-step procedure was used. The
pyridinium salt was reduced with NaBH4 (3.3 equiv) in MeOH
(0.04 M) at room temperature for 30 min. The resultant alkene
(tetrahydropyridine) in MeOH (0.04 M) was hydrogenated at
atmospheric pressure and room temperature in the presence
of PtO2 (20% w/w). Yields of products ranged from 61 to 100%.
Piperidine derivatives 38b, c, and e were obtained after
demethylation, achieved by treatment of the methoxy ana-
logues with AlCl3 (∼6 equiv) in CH2Cl2 (0.020-0.025 M) in
the presence of EtSH (5 equiv) at room temperature for 2-17
h. Yields of purified products ranged from 20 to 55%. Tetrahy-
droisoquinoline 38a was isolated after debenzylation effected
as described above for the preparation of piperazine derivatives
29-33.
3600-2200, 3023, 2937, 2851, 2801, 1618, 1577, 1496, 1380,
1277, 1249, 1162, 991, 820, 764, 682 cm-1. 1H NMR (500 MHz,
DMSO) δ: 9.34 (s, 1H), 7.26 (d, J ) 8.5 Hz, 1H), 7.18-7.10
(m, 5H), 6.63-6.59 (m, 2H), 6.58 (d, J ) 2.5 Hz, 1H), 6.54 (dt,
J ) 13.5, 2.5 Hz, 1H), 6.40 (ddd, J ) 2.5, 8.5, 8.5 Hz, 1H),
5.83 (br s, 1H), 3.74-3.67 (m, 1H), 3.40-3.30 (m, 1H), 2.88-
2.72 (m, 4H), 2.40-2.31 (m, 1H), 2.15 (s, 3H), 1.93-1.85 (m,
2H), 1.69-1.52 (m, 4H). 13C NMR (125.8 MHz, DMSO) δ: 163.4
(d, JC-F ) 239 Hz), 156.2, 150.6 (d, JC-F ) 11 Hz), 144.5, 141.3,
136.1, 130.3 (d, JC-F ) 10 Hz), 128.5, 126.6, 126.5, 114.2, 113.2,
108.5, 102.3 (d, JC-F ) 21 Hz), 99.1 (d, JC-F ) 26 Hz), 60.4,
55.8, 46.2, 43.4, 40.8, 33.0, 27.5. HRMS (ESI, M + H+) calcd
for C27H29FN2O: 417.2342. Found: 417.2339. HPLC purity:
100%; tR 4.42 min.
1-[4-(1-Methyl-piperidin-4-yl)phenyl]-2-phenyl-1,2,3,4-
tetrahydroisoquinolin-6-ol (38a). IR (KBr) νmax
: 3600-
2200, 3021, 2925, 2852, 2800, 2680, 1596, 1503, 1469, 1380,
1325, 1277, 1252, 1223, 1152, 749, 692 cm-1 1H NMR (500
.
MHz, CD3OD) δ: 7.18-7.06 (m, 7H), 6.84 (d, J ) 8.0 Hz, 2H),
6.69 (t, J ) 7.5 Hz, 1H), 6.65 (dd, J ) 2.5, 8.0 Hz, 1H), 6.60 (d,
J ) 2.5 Hz, 1H), 5.74 (s, 1H), 3.66-3.60 (m, 1H), 3.44-3.38
(m, 1H), 3.08 (dm, J ) 12.0 Hz for d, 2H), 2.90-2.78 (m, 2H),
2.53 (tt, J ) 4.0, 12.0 Hz, 1H), 2.43 (s, 3H), 2.34 (td, J ) 12.0,
2.5 Hz, 2H), 1.87-1.71 (m, 4H). 13C NMR (125.8 MHz, CD3OD)
δ: 157.3, 151.1, 145.0, 143.4, 138.1, 130.4, 130.0, 129.9, 128.7,
127.5, 118.6, 115.5, 115.4, 114.2, 63.3, 56.8, 45.8, 44.7, 41.9,
33.6/33.5, 29.1. HRMS (ESI, M + H+) calcd for C27H30N2O:
399.2431. Found: 399.2429. HPLC purity: 100%; tR 4.13 min.
2-(4-Fluorophenyl)-1-[4-(1-methyl-piperidin-4-yl)phen-
yl]-1,2,3,4-tetrahydroisoquinolin-6-ol (38b). IR (KBr)
νmax: 3650-2200, 3049, 2936, 2800, 1610, 1507, 1379, 1276,
1230, 1151, 991, 810 cm-1. 1H NMR (400 MHz, DMSO) δ: 9.27
(s, 1H), 7.17-7.05 (m, 5H), 6.96 (t, J ) 8.5 Hz, 2H), 6.80-6.70
(m, 2H), 6.60-6.53 (m, 2H), 5.71 (s, 1H), 3.64-3.55 (m, 1H),
3.43-3.26 (m, 1H), 2.88-2.69 (m, 4H), 2.42-2.30 (m, 1H), 2.16
(s, 3H), 1.99-1.87 (m, 2H), 1.70-1.49 (m, 4H). 13C NMR (100.7
MHz, DMSO) δ: 155.7, 154.4 (d, JC-F ) 233 Hz), 145.6, 144.1,
141.4, 135.75, 128.3 (d, JC-F ) 16 Hz), 126.7, 126.2, 115.0 (d,
JC-F ) 21.5 Hz), 114.3, 114.2, 114.0, 113.0, 61.1, 55.7, 46.0,
43.4, 40.6, 32.8, 27.5. HRMS (ESI, M + H+) calcd for C27H29-
FN2O: 417.2342. Found: 417.2341. HPLC purity: 99%; tR 4.07
min.
2-(3-Hydroxyphenyl)-1-[4-(1-methyl-piperidin-4-yl)phen-
yl]-1,2,3,4-tetrahydroisoquinolin-6-ol (38e). IR (KBr)
νmax: 3650-2200, 3399, 3023, 2924, 2853, 1678, 1610, 1499,
1468, 1380, 1276, 1248, 1202 cm-1. 1H NMR (400 MHz, DMSO)
δ: 9.26 (s, 1H), 8.98 (s, 1H), 7.19 (d, J ) 8.0 Hz, 1H), 7.14 (d,
J ) 8.5 Hz, 2H), 7.09 (d, J ) 8.5 Hz, 2H), 6.89 (t, J ) 8.0 Hz,
1H), 6.57 (d, J ) 8.0 Hz, 1H), 6.55 (s, 1H), 6.21 (br d, J ) 8.0
Hz, 1H), 6.14 (br s, 1H), 6.05 (br d, J ) 8.0 Hz, 1H), 5.68 (s,
1H), 3.68-3.57 (m, 1H), 3.40-3.20 (m, 1H), 2.95-2.64 (m, 4H),
2.56-2.35 (m, 1H), 2.24 (s, 3H), 2.15-2.00 (m, 2H), 1.75-1.55
(m, 4H). 13C NMR (125.8 MHz, DMSO) δ: 158.1, 156.1, 150.2,
143.9, 142.0, 136.3, 129.5, 128.7, 128.4, 126.7, 126.4, 114.2,
A representative example for this series of transformations
is provided below.
Synthesis of 2-(3-Fluorophenyl)-1-[4-(1-methyl-piperi-
din-4-yl)phenyl]-1,2,3,4-tetrahydroisoquinolin-6-ol (38c).
A mixture of pyridine derivative 35c (300 mg, 0.731 mmol)
and MeI (228 µL, 3.65 mmol) in DMF (4 mL) was stirred at
40 °C for 2 h. Removal of the solvent in vacuo provided the
crude pyridinium salt as a solid, which was used directly in
the next step. A mixture of the pyridinium salt and PtO2 (20%
w/w) in 4:1 MeOH/THF (15 mL) was reduced under an
atmosphere of H2 (1013 mbar) at room temperature for 10 h.
The reaction mixture was filtered through Celite using MeOH
as eluent, and the solvents were removed in vacuo. The residue
was purified by flash chromatography (30 g of silica gel; 13:
1:0.1 CH2Cl2/MeOH/NH4OH) to furnish the corresponding
piperidine (193 mg, 61%) as a white solid. IR (KBr) νmax: 2935,
2842, 2780, 2735, 2678, 1617, 1577, 1497, 1380, 1279, 1163,
1
995, 821, 683 cm-1. H NMR (400 MHz, DMSO) δ: 7.35 (d, J
) 9 Hz, 1H), 7.17-7.07 (m, 5H), 6.79-6.74 (m, 2H), 6.62-
6.50 (m, 2H), 6.39 (ddd, J ) 2.0, 8.0, 8.0 Hz, 1H), 5.88 (br s,
1H), 3.75-3.65 (m, 1H), 3.70 (s, 3H), 3.43-3.31 (m, 1H), 2.95-

376 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Renaud et al.
113.1, 104.1, 103.9, 99.9, 60.7, 55.4, 45.6/45.5 (maxima of br
signal), 43.25, 32.4 (br signal), 27.5. HRMS (ESI, M + H+) calcd
for C27H30N2O2: 415.2386. Found: 415.2390. HPLC purity:
93%; tR 3.36 min.
General Procedure for the Conversion of Lactam 16d
into Tetrahydroisoquinolines 39-44. The phenyllithium
reagent was generated from the reaction of n-BuLi (0.9-1.4
equiv) with 1-(4-bromophenyl)-4-alkylpiperazine 53, 54, 55, 56,
57, or 58 (1-1.3 equiv) in THF (0.1-0.3 M) for 0.5-1 h.
Lactam 16d was allowed to undergo reaction with the ap-
propriate phenyllithium reagent in THF (0.04-0.05 M) for
1-2.5 h at -78 °C. For the syntheses of 41 and 43, the reaction
mixtures were stirred for 10 min at -78 °C and then allowed
to warm to room temperature, for total reaction times of 60
and 40 min, respectively. In the case of 44, the mixture was
stirred at -78 °C for 45 min following addition of the amide
16d and allowed to warm to room temperature for 45 min.
After aqueous workup and isolation, the iminium salt was
dissolved in MeOH or 8:1 MeOH/CH2Cl2 (0.06-0.09 M) and
treated with NaBH4 (2-2.2 equiv) at room temperature for
15-60 min. After that period of time, the product was isolated
and purified. Tetrahydroisoquinolines 39-44 were obtained
in yields ranging from 59 to 87%. A representative example is
given below.
Synthesis of 6-Methoxy-2-(3-methoxyphenyl)-1-[4-(4-
methyl-piperazin-1-yl)phenyl]-1,2,3,4-tetrahydroisoquin-
oline (39). To a cold (-78 °C) solution of 1-(4-bromophenyl)-
4-methyl-piperazine (53) (100 mg, 0.39 mmol) in anhydrous
THF (1.5 mL), was added a solution of n-BuLi in hexanes (1.6
M, 0.244 mL, 0.39 mmol). After the resultant suspension was
stirred at this temperature for 30 min, a solution of lactam
16d (122 mg, 0.43 mmol) in anhydrous THF (3 mL) was added
dropwise. Stirring was continued for an additional 60 min. The
mixture was poured onto H2O (40 mL). HClO4 (70%, 75 µL)
was added, the mixture was stirred for 10 min, and the organic
solvent was removed in vacuo. The aqueous layer was ex-
tracted with AcOEt (4 × 30 mL) and CH2Cl2 (2 × 20 mL). The
combined organic layers were washed with brine (30 mL) and
dried (MgSO4), and the solution was concentrated in vacuo
yielding an iminium salt that was used directly in the next
step.
To a solution of the iminium intermediate in 8:1 MeOH/
CH2Cl2 (4.5 mL), was added solid NaBH4 (97%, 30.5 mg, 0.78
mmol) in two portions. The resultant mixture was stirred at
room temperature for 15 min. The suspension was poured into
saturated aqueous NaHCO3 (30 mL). The organic solvents
were removed in vacuo. The aqueous layer was extracted with
AcOEt (3 × 20 mL), and the combined organic phases were
washed with brine (20 mL), dried (MgSO4), and concentrated
in vacuo. The resultant brown wax was purified by flash
chromatography (25 g of silica gel, 12:1 CH2Cl2/MeOH) to
provide the title compound 39 (129 mg, 68%) as a yellow wax.
IR (KBr) νmax: 2994, 2935, 2832, 2794, 2746, 1676, 1609, 1496,
1380, 1239, 1167, 1038, 817 cm-1. 1H NMR (500 MHz, DMSO)
δ: 7.26 (d, J ) 8.5 Hz, 1H), 7.06-7.00 (m, 3H), 6.81-6.72 (m,
4H), 6.42 (dd, J ) 2.0, 8.5 Hz, 1H), 6.32 (t, J ) 2.0 Hz, 1H),
6.23 (dd, J ) 2.0, 8.5 Hz, 1H), 5.77 (s, 1H), 3.71 (s, 3H), 3.66
(s, 3H), 3.68-3.60 (m, 1H), 3.42-3.28 (m, 1H), 3.03 (t, J ) 5.0
Hz, 4H), 2.94-2.78 (m, 2H), 2.39 (t, J ) 5.0 Hz, 4H), 2.18 (s,
3H). 13C NMR (125.8 MHz, DMSO) δ: 160.2, 157.8, 150.3,
149.6, 136.2, 134.0, 130.5, 129.5, 128.5, 127.4, 115.0, 112.7,
111.9, 106.2, 101.6, 99.6, 60.3, 55.0, 54.7, 54.6, 48.0, 45.7, 42.9,
27.6. HRMS (ESI, M + H+) calcd for C28H33N3O2: 444.2646.
Found: 444.2644. HPLC purity: 91%; tR 4.30 min.
Syntheses of Piperazines 45-50. Synthesis of 6-Meth-
oxy-2-(3-methoxyphenyl)-3,4-dihydro-2H-isoquinolin-1-
one (16d). A mixture of 6-methoxy-3,4-dihydro-2H-isoquinolin-
1-one (15, 7.2 g, 40.6 mmol), 3-bromoanisole (98%, 10.5 mL,
81.3 mmol), K2CO3 (99%, 5.67 g, 40.6 mmol), and CuI (0.774
g, 4.06 mmol) in DMF (70 mL) was heated at 150 °C for 30 h
under N2 atmosphere. After that period of time, a second
portion of CuI (0.774 g, 4.06 mmol) was added. Heating was
continued for another 40 h prior to the addition of a last portion
of CuI (0.774 g, 4.06 mmol). After being heated for a further
48 h, for a total reaction time of 118 h, the mixture was cooled
to room temperature and poured into aqueous NH4OH (300
mL)/AcOEt (300 mL). The layers were separated, and the
aqueous layer was extracted with portions of AcOEt (3 × 300
mL). The combined organic phases were washed with brine
(500 mL), dried over MgSO4, and concentrated in vacuo until
crystallization started. Hexane was added slowly to the
solution. The resulting solid was filtered, washed with hexane,
and dried in vacuo to provide 16d (10.96 g, 95%). IR (KBr)
νmax: 1653, 1597, 1472, 1403, 1327, 1312, 1295, 1262, 1214,
1
1194, 1172, 1157, 1034, 1022, 851 cm-1. H NMR (500 MHz,
CDCl3) δ: 8.10 (d, J ) 8.5 Hz, 1H), 7.30 (t, J ) 8.0 Hz, 1H),
6.98-6.94 (m, 2H), 6.88 (dd, J ) 2.5, 8.5 Hz, 1H), 6.80 (dm, J
) 8.0 Hz for d, 1H), 6.72 (d, J ) 2.5 Hz, 1H), 3.96 (t, J ) 6.5
Hz, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.10 (t, J ) 6.5 Hz, 2H). 13
C
NMR (125.8 MHz, CDCl3) δ: 163.8, 162.1, 159.5, 144.0, 140.0,
130.6, 129.1, 122.1, 117.0, 112.3, 111.6, 111.5, 110.9, 55.05,
55.0, 49.0, 28.6. HRMS (ESI, M + H+) calcd for C17H17NO3:
284.1287. Found: 284.1285. HPLC purity: 98%; tR 5.43 min.
General Synthesis of 1-(4-Bromophenyl)-4-alkylpi-
perazine 54-58 by Reductive Amination. To a solution of
1-(4-bromophenyl)-piperazine‚HCl (51)(1 equiv) and the ap-
propriate ketone or aldehyde (1-2 equiv) in ClCH2CH2Cl
(0.22-0.26 M), was added NaBH(OAc)3 (1.4-1.5 equiv), and
the resultant mixture was stirred at room temperature for
2-26 h.36 Following workup, piperazines 54-58 were isolated.
They were used without further purification in cases where
the purity was acceptable. Otherwise, the product was either
triturated with solvent, crystallized, or purified by flash
chromatography. The bromophenylpiperazines 54-58 were
obtained in yields ranging from 74 to 99%. A representative
example is given below.
Synthesis of 1-(4-Bromophenyl)-4-isopropylpiperazine
(54). To a solution of 1-(4-bromophenyl)-piperazine‚HCl (51)
(98%, 3.0 g, 10.6 mmol) and anhydrous acetone (98%, 628 mg,
10.6 mmol) in ClCH2CH2Cl (40 mL), was added NaBH(OAc)3
(3.37 g, 15.9 mmol), and the resultant mixture was stirred at
room temperature for 1 h. Another portion of anhydrous
acetone (98%, 628 mg, 10.6 mmol) was added. Stirring was
continued for 25 h. The mixture was then treated with 1 N
aqueous NaOH (20 mL) and diluted with Et2O (100 mL). The
layers were separated, and the aqueous phase was extracted
once again with Et2O (50 mL). The combined organic layers
were washed with brine (75 mL), dried (MgSO4), and concen-
trated in vacuo. Crystallization of the resultant beige solid
from hexane at -4 °C provided the title compound (2.53 g,
84%) as a white solid. 1H NMR (300 MHz, DMSO) δ: 7.30 (d,
J ) 9.0 Hz, 2H), 6.86 (d, J ) 9 Hz, 2H), 3.08 (t, J ) 5 Hz, 2H),
2.65 (septet, J ) 6.5 Hz, 1H), 2.54 (t, J ) 5 Hz, 2H), 0.98 (d,
J ) 6.5 Hz, 6H). 13C NMR (100.7 MHz, DMSO) δ: 150.2, 131.4,
117.15, 109.8, 53.65, 48.2, 47.9, 18.2. MS (ESI) m/z 283 [M +
H+]. HPLC purity: 96%; tR 4.04 min.
General Procedure for Demethylation. Synthesis of
45-50. Demethylation was achieved by treatment of 39-44
with AlCl3 (5.9-6 equiv) in CH2Cl2 (0.02-0.03 M) in the
presence of EtSH (5 equiv) at room temperature for 0.5-4 h.
Yields of purified products varied between 45 and 58%. A
representative example is described below.
Synthesis of 1-(4-Bromophenyl)-4-methyl-piperazine
(53). This compound was synthesized by reductive amination
of 51 according to the conditions described for the preparation
of 26. 1H NMR (300 MHz, DMSO) δ: 7.30 (d, J ) 9.0 Hz, 2H),
6.86 (d, J ) 9.0 Hz, 2H), 3.10 (t, J ) 5.0 Hz, 2H), 2.42 (t, J )
5.0 Hz, 2H), 2.20 (s, 3H). 13C NMR (100.7 MHz, DMSO) δ:
150.1, 131.4, 117.2, 109.8, 54.4, 47.7, 45.7. MS (ESI) m/z 255
[M + H+]. HPLC purity: 91%; tR 3.81 min.
Synthesis of 2-(3-Hydroxyphenyl)-1-[4-(4-methylpi-
perazin-1-yl)phenyl]-1,2,3,4-tetrahydroisoquinolin-6-ol
(45). A mixture of the piperazin-1-yl derivative 39 (120 mg,
0.27 mmol), AlCl3 (99%, 224 mg, 1.66 mmol), and EtSH (97%,
107.5 µL, 1.41 mmol) in CH2Cl2 (12 mL) was stirred at room
temperature for 4 h. The mixture was diluted with CH2Cl2
(50 mL) and poured onto saturated aqueous NaHCO3 (30 mL).
The layers were separated, and the aqueous layer was

SERMS with Restricted Side Chains
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 377
extracted with CH2Cl2 (3 × 50 mL). The combined organic
phases were washed with brine (50 mL), dried (MgSO4), and
concentrated in vacuo. The yellow residue was purified by flash
chromatography (25 g silica gel, 9:1 CH2Cl2/MeOH) to provide
the title compound as a yellow solid (67.8 mg, 58%). IR (KBr)
νmax: 3600-2200, 3318, 3027, 2947, 2841, 1609, 1512, 1453,
1389, 1366, 1292, 1248, 1220, 1208, 1164, 1000, 922, 829, 812,
6.15 (br s, 1H), 6.05 (br d, J ) 8.0 Hz, 1H), 5.61 (s, 1H), 3.62-
3.53 (m, 1H), 3.42-3.20 (m, 1H), 3.03 (br s, 4H), 2.85-2.65
(m, 2H), 2.58-2.40 (m, 5H), 1.85-1.73 (m, 2H), 1.65-1.42 (m,
4H), 1.41-1.28 (m, 2H). 13C NMR (100.7 MHz, DMSO) δ:
157.8, 155.6, 150.2, 149.2, 136.0, 134.1, 129.2, 128.8, 128.2,
127.1, 114.7, 114.0, 112.8, 104.2, 103.8, 100.0, 66.6, 60.5, 51.6,
48.2, 43.0, 29.8, 27.6, 23.7. HRMS (ESI, M + H+) calcd for
C30H35N3O2: 470.2808. Found: 470.2815. HPLC purity: 98%;
tR 3.64 min.
1-[4-(4-Cyclohexylpiperazin-1-yl)phenyl]-2-(3-hydroxy-
phenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol (50). IR (KBr)
νmax: 3390, 3024, 2931, 2826, 2598, 1612, 1576, 1497, 1380,
1286, 1233, 977, 918 cm-1. 1H NMR (400 MHz, DMSO) δ: 9.24
(s, 1H), 8.98 (s, 1H), 7.13 (d, J ) 8.0 Hz, 1H), 7.01 (d, J ) 8.5
Hz, 2H), 6.89 (t, J ) 8.0 Hz, 1H), 6.76 (d, J ) 8.5 Hz, 2H),
6.60-6.52 (m, 2H), 6.23 (br d, J ) 8.0 Hz, 1H), 6.16 (br s, 1H),
6.06 (br d, J ) 8.0 Hz, 1H), 5.62 (s, 1H), 3.62-3.53 (m, 1H),
3.45-3.25 (m, 1H), 3.01 (br s, 4H), 2.85-2.67 (m, 2H), 2.59
(br s, 4H), 2.30-2.19 (m, 1H), 1.82-1.66 (m, 4H), 1.59-1.51
(m, 1H), 1.26-1.00 (m, 5H). 13C NMR (100.7 MHz, DMSO) δ:
157.8, 155.6, 150.2, 149.3, 136.0, 134.0, 129.2, 128.8, 128.2,
127.1, 114.8, 114.0, 112.8, 104.2, 103.8, 100.0, 62.5, 60.5, 48.6,
48.4, 43.0, 28.3, 27.6, 25.9, 25.3. HRMS (ESI, M + H+) calcd
for C31H37N3O2: 484.2964. Found: 484.2965. HPLC purity:
97%; tR 3.71 min.
792, 750, 686 cm-1 1H NMR (400 MHz, DMSO) δ: 9.25 (s,
.
1H), 8.98 (s, 1H), 7.14 (d, J ) 8.0 Hz, 1H), 7.02 (d, J ) 8.5 Hz,
2H), 6.90 (t, J ) 8.0 Hz, 1H), 6.79 (d, J ) 8.5 Hz, 2H), 6.60-
6.54 (m, 2H), 6.24 (dd, J ) 2.0, 8.0 Hz, 1H), 6.17 (t, J ) 2.0
Hz, 1H), 6.06 (dd, J ) 2.0, 8.0 Hz, 1H), 5.63 (s, 1H), 3.62-
3.55 (m, 1H), 3.40-3.26 (m, 1H), 3.04 (t, J ) 5.0 Hz, 4H), 2.86-
2.69 (m, 2H), 2.40 (t, J ) 5.0 Hz, 4H), 2.19 (s, 3H). 13C NMR
(100.7 MHz, DMSO) δ: 157.8, 155.6, 150.2, 149.2, 136.0, 134.0,
129.2, 128.8, 128.2, 127.1, 114.8, 114.0, 112.8, 104.2, 103.8,
100.0, 60.5, 54.6, 48.0, 45.7, 43.0, 27.6. HRMS (ESI, M + H+)
calcd for C26H29N3O2: 416.2338. Found: 416.2337. HPLC
purity: 96%; tR 2.88 min
2-(3-Hydroxyphenyl)-1-[4-(4-isopropylpiperazin-1-yl)-
phenyl]- 1,2,3,4-tetrahydroisoquinolin-6-ol (46). IR (KBr)
νmax: 3700-2100, 3382, 2968, 2829, 1609, 1511, 1455, 1383,
1237, 1173, 1008, 929, 973, 919, 818, 755, 689 cm-1. 1H NMR
(400 MHz, DMSO) δ: 9.26 (s, 1H), 8.99 (s, 1H), 7.13 (d, J )
8.0 Hz, 1H), 7.02 (d, J ) 8.5 Hz, 2H), 6.90 (t, J ) 8.0 Hz, 1H),
6.77 (d, J ) 8.5 Hz, 2H), 6.61-6.54 (m, 2H), 6.24 (d, J ) 8.0
Hz, 1H), 6.18 (s, 1H), 6.07 (d, J ) 8.0 Hz, 1H), 5.63 (s, 1H),
3.63-3.54 (m, 1H), 3.34-3.25 (m, 1H), 3.04 (br s, 4H), 2.86-
2.63 (m, 3H), 2.56 (br s, 4H), 0.99 (d, J ) 6.5 Hz, 6H). 13C NMR
(100.7 MHz, DMSO) δ: 157.8, 155.7, 150.2, 149.3, 136.0, 134.1,
129.3, 128.8, 128.2, 127.1, 114.8, 114.0, 112.8, 104.2, 103.8,
100.0, 60.5, 53.9, 48.4, 48.0, 43.1, 27.6, 18.2. HRMS (ESI, M
+ H+) calcd for C28H33N3O2: 444.2646. Found: 444.2646.
HPLC purity: 99%; tR 3.14 min.
2-(3-Hydroxyphenyl)-1-[4-(4-isobutylpiperazin-1-yl)-
phenyl]- 1,2,3,4-tetrahydroisoquinolin-6-ol (47). IR (KBr)
νmax: 3700-2200, 3390, 2954, 2826, 1610, 1511, 1456, 1382,
1234, 1172, 1008, 929, 817 cm-1. 1H NMR (400 MHz, DMSO)
δ: 9.25 (s, 1H), 8.99 (s, 1H), 7.14 (d, J ) 8.0 Hz, 1H), 7.02 (d,
J ) 8.5 Hz, 2H), 6.90 (t, J ) 8.0 Hz, 1H), 6.78 (d, J ) 8.5 Hz,
2H), 6.61-6.52 (m, 2H), 6.24 (br d, J ) 8.0 Hz for d, 1H), 6.18
(d, J ) 2.0 Hz, 1H), 6.07 (br d, J ) 8.0 Hz, 1H), 5.63 (s, 1H),
3.63-3.52 (m, 1H), 3.34-3.23 (m, 1H), 3.04 (br s, 4H), 2.86-
2.67 (m, 2H), 2.45 (br s, 4H), 2.08 (br s, 2H), 1.84-1.71 (m,
1H), 0.86 (d, J ) 6.5 Hz, 6H). 13C NMR (100.7 MHz, DMSO)
δ: 157.8, 155.7, 150.2, 149.2, 136.0, 134.1, 129.2, 128.8, 128.2,
127.2, 114.8, 114.0, 112.9, 104.2, 103.8, 100.0, 60.5, 53.0, 48.1,
43.1, 27.6, 24.7, 20.8. HRMS (ESI, M + H+) calcd for
C29H35N3O2: 458.2802. Found: 458.2800. HPLC purity: 100%;
tR 3.41 min.
1-[4-(4-Cyclopropylmethyl-piperazin-1-yl)phenyl]-2-(3-
hydroxyphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol (48).
IR (KBr) νmax: 3600-2100, 3384, 3002, 2916, 2827, 1609, 1511,
1455, 1380, 1336, 1235, 1168, 1002, 920, 817, 792, 753, 687
cm-1. 1H NMR (400 MHz, DMSO) δ: 9.24 (s, 1H), 8.98 (s, 1H),
7.13 (d, J ) 8.0 Hz, 1H), 7.01 (d, J ) 8.5 Hz, 2H), 6.89 (t, J )
8.0 Hz, 1H), 6.78 (d, J ) 8.5 Hz, 2H), 6.60-6.53 (m, 2H), 6.24
(dd, J ) 2.0, 8.5 Hz, 1H), 6.16 (t, J ) 2.0 Hz, 1H), 6.06 (dd, J
) 2.0, 8.0 Hz, 1H), 5.62 (s, 1H), 3.62-3.54 (m, 1H), 3.42-3.24
(m, 1H), 3.05 (t, J ) 5.0 Hz, 4H), 2.85-2.68 (m, 2H), 2.58-
2.50 (m, 4H), 2.20 (d, J ) 6.5 Hz, 2H), 0.88-0.77 (m, 1H),
0.49-0.42 (m, 2H), 0.10-0.04 (m, 2H). 13C NMR (100.7 MHz,
DMSO) δ: 157.8, 155.6, 150.2, 149.3, 136.0, 134.1, 129.2, 128.8,
128.2, 127.1, 114.8, 114.0, 112.8, 104.2, 103.8, 100.0, 62.7, 60.5,
52.6, 48.2, 43.0, 27.6, 8.2, 3.8. HRMS (ESI, M + H+) calcd for
C29H33N3O2: 456.2651. Found: 456.2654. HPLC purity: 100%;
tR 3.42 min.
Crystallography. See Supporting Information. The struc-
ture factors and the coordinates of the refined structure were
deposited in the protein structure database (PDB ID code:
1XQC).
Biological Assays. Radioligand Binding Studies. The
radioligand binding assay was performed by using 96-well
microtiterplates (Picoplates, Packard) in volumes of 0.2 mL
of incubation buffer (50 mM Tris, pH 7.4). The incubation
mixture contained 5 nM ERR (PanVera, USA) or 6 nM ERâ
long form human recombinant receptors (PanVera,), 8 nM [3H]
17â-estradiol (∼180 000 total counts, Perkin-Elmer NET 517,
USA), and the compound to be tested and 0.25 mg/well SPA-
beads (Amersham RPNQ 0001). After incubation at room
temperature for 2-4 h, the reaction was terminated by
centrifugation at room temperature (10 min at 1000g). The
radioactivity was counted at least 3 h after completion of the
experiment in a Packard Topcount scintillation counter.
Nonspecific binding was defined as the remaining radioactivity
in the presence of 10 µM nonradioactive 17â-estradiol (Sigma).
Assays were performed in triplicate.
MCF-7 Proliferation Assay. The human breast adeno-
carcinoma cell line, MCF-7 (ATCC HTB-22), was routinely
cultivated in DMEM (Dulbecco’s modified eagle medium, high
glucose, Gibco Invitrogen Corporation, Paisley, U.K.) supple-
mented with 10% fetal bovine serum (FBS), 2 mM l-glutamine,
10 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid), 50 IU/mL penicillin, and 50 µg/mL streptomycine (pen/
strep) at 37 °C in a 5% CO2 humidified incubator. Three days
prior to an assay, MCF-7 cells were switched to DMEM low
glucose phenol red free,43 supplemented with 10% dextran-
coated charcoal-stripped FBS (sFBS) to deplete internal stores
of steroids, 2 mM L-glutamine, 10 mM Hepes, and penicillin/
streptomycin. Cells were trypsinized from the maintenance
flask with phenol red free trypsin (0.05%)-EDTA (0.02%)
(HyClone, Logan, UT) and seeded in a 96-well plate (Nunc) at
a density of 103 cells per final volume of 100 µL DMEM low
glucose phenol red free, supplemented with 5% sFBS, 2 mM
l-glutamine, 10 mM Hepes, and penicillin/streptomycin. After
24 h, fresh medium, supplemented with serial dilutions of
compounds or DMSO as diluent control, was prepared and
added in the presence or absence of 10-10 M 17â-estradiol to
triplicate microcultures. Cells were incubated for 6 days, and
medium with compounds was changed once after 3 days. At
the end of the incubation time, proliferation was assessed
using the CellTiter 96 aqueous one solution cell proliferation
assay kit from Promega (Madison, WI) according to the
manufacturer’s instructions. The absorbance at 490 nm was
measured. This parameter relates to the amount of formazan
1-[4-(4-Cyclopentylpiperazin-1-yl)phenyl]-2-(3-hydroxy-
phenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol (49). IR (KBr)
νmax: 3650-2200, 3400, 2954, 2827, 1610, 1511, 1453, 1381,
1237, 1170, 1000, 927, 817, 753, 688 cm-1. 1H NMR (400 MHz,
DMSO) δ: 9.23 (s, 1H), 8.97 (s, 1H), 7.12 (d, J ) 8.0 Hz, 1H),
7.01 (d, J ) 8.5 Hz, 2H), 6.88 (t, J ) 8.0 Hz, 1H), 6.77 (d, J )
8.5 Hz, 2H), 6.59-6.51 (m, 2H), 6.22 (br d, J ) 8.0 Hz, 1H),

378 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Renaud et al.
produced, the quantity of which is directly proportional to the
number of living cells in the culture.
potential pharmacokinetic interactions. Deviations exceeding
the typical range of biological variability (approximately (50%
maximum for individual parameters) were considered strongly
indicative for pharmacokinetic interactions between com-
pounds in the cassette, and the respective data were discarded.
ERE-Luciferase Reporter Assay. HELNR and HELNâ,
two human cervix adenocarcinoma cell lines derived from
HeLa cells stably transfected with the reporter gene ERE-
âGlob-Luc-SVNeo and the expression plasmids ERR or ERâ,
respectively, were used to quantify the antiestrogenic and
estrogenic effects of compounds on ERE.44,45 These cells were
routinely cultivated in DMEM phenol red free, supplemented
with 5% sFBS, 2 mM glutamine, 1% penicillin/streptomycin,
1 mg/mL Geneticin, and 0.5 µg/mL puromycin to ensure
appropriate antibiotic selection. For the assay, cells were
trypsinized from the maintenance flask with phenol red free
trypsin (0.05%)-EDTA (0.02%) (HyClone, Logan, UT) and
seeded in an opaque 96-well plate (Nunc) at a density of 7.5 ×
104 cells/well in a final volume of 100 µL of assay medium
(DMEM, phenol red free, supplemented with 3% sFBS, 2 mM
glutamine, and penicillin/streptomycin). Five hours later, cells
were adherent. Serial dilutions of compounds or DMSO as
diluent control were then added in the presence of a fixed
concentration of 17â-estradiol (10-10 M in HELNR and 10-9
M in HELNâ) to triplicate microcultures. Faslodex (Tocris, 10-8
M) was used as a baseline indicator. Cells were incubated for
20 h at 37 °C in a 5% CO2 humidified incubator before being
processed for luciferase determination. Medium was aspirated
and 100 µL of a 1:1 mixture of LucLite (Perkin-Elmer, Life
Science, Boston, MA)/assay medium was added to each well.
Plates were then sealed with a Topseal and left in the dark
for 10 min before luminescence activity was determined by
counting the plates for 6 s in a â-TopCount (Packard Instru-
ment Company, Meriden, CT). Protein concentrations were
determined using the Bradford Micromethod (Bio-Rad). Lu-
ciferase was expressed in relative luminescence units/µg
protein.
Cassette Dosing Experiments. Rat Pharmacokinetic
Cassette Standard Assay. A set of seven novel compounds
plus the internal standard raloxifene (7) was administered
intravenously via the femoral vein at a dose level of 1 mg/kg
of body weight (for each individual compound) to four female
rats. Cannulas were implanted into the femoral vein for
intravenous compound administration and into the femoral
artery for rapid blood sampling. Compounds were adminis-
tered orally by gavage at a dose level of 5 mg/kg of body weight.
Blood was sampled over a 24 h time period postdose (from 5
min for iv and 15 min for po). Blood samples were then
processed through steps involving protein precipitation by
CH3CN addition, centrifugation of the supernatant, and
evaporation of the supernatant in a vacuum centrifuge. Dried
residues were dissolved in MeOH/H2O (60:40 v/v) containing
1% HCOOH and analyzed by HPLC on an Uptisphere C18
reversed-phase HPLC column (particle size, 3 µm; column
dimensions, 2 mm × 50 mm). Eluents used consisted of A, 10%
CH3CN in H2O with 0.1% HCOOH (pH 2.1), and B, 90%
CH3CN with 10% H2O and 0.1% HCOOH (pH 2.1). A linear
gradient was run from 5 to 100% B over 7 min followed by a
3 min hold at 100% B at a constant temperature of 50 °C in
the column compartment. The flow rate was held constant at
0.4 mL/min. Sample injection volume was 10 µL. The flow from
the HPLC system was directly introduced into the ion source
of an Agilent 1100 series MS detector (single quadrupole mass
analyzer) and subjected to atmospheric pressure electrospray
ionization (positive mode). All compounds were detected as
protonated quasi-molecular ions [M + H]+. A structurally
closely related SERM was used as an analytical internal
standard. Quantification of blood levels of the parent com-
pounds was based on a seven-level calibration curve (in
triplicate) using blank rat blood samples spiked with stock
solutions of external and internal standards.
Acknowledgment. We would like to thank our
collaborators for their important contribution to the
work disclosed herein: Nadine Braendlin, Patrick Lerch,
Pierre Martin, Nicole Reymann, Hans Rudolf Zihlmann,
Diana Latscha, Roland Feifel, Jean-Louis Runser, Yves
Seltenmeyer, Tanja Grabenstaetter, and Erika Kuhn.
In addition, the support of M. Mahnke and P. Graff for
the expression and mass spectrometry analysis of ERR-
LBD, the experimental assistance of Lukas Oberer for
NMR experiments, and the staff of the Swiss-Norwe-
gian Beam Lines at the European Synchrotron Radia-
tion Facilities in Grenoble are gratefully acknowledged.
We also wish to thank Drs. J.-C. Nicolas and P.
Balaguer (INSERM U439, Montpellier, France) for their
precious advice on the use of the HELNR and HELNâ
cell lines.
Supporting Information Available: Protein preparation,
crystallization, data collection, and structure resolution. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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