Rational design and synthesis of a novel thyroid hormone antagonist that blocks coactivator recruitment

Article abstract of DOI:10.1021/jm0201013

Recent efforts have focused on the design and synthesis of thyroid hormone (T₃) antagonists as potential therapeutic agents and chemical probes to understand hormone-signaling pathways. We previously reported the development of novel first-gene

Full text of DOI:10.1021/jm0201013

3310
J . Med. Chem. 2002, 45, 3310-3320
Ra tion a l Design a n d Syn th esis of a Novel Th yr oid Hor m on e An ta gon ist Th a t
Block s Coa ctiva tor Recr u itm en t
Ngoc-Ha Nguyen,†,‡ J ames W. Apriletti,§ Suzana T. Cunha Lima,§ Paul Webb,§ J ohn D. Baxter,§ and
Thomas S. Scanlan*,‡
Program in Chemistry and Chemical Biology, Departments of Pharmaceutical Chemistry and Cellular and Molecular
Pharmacology, University of California, San Francisco, California 94143-0446, and Metabolic Research Unit,
University of California, San Francisco, California 94143-0540
Received March 5, 2002
Recent efforts have focused on the design and synthesis of thyroid hormone (T3) antagonists
as potential therapeutic agents and chemical probes to understand hormone-signaling pathways.
We previously reported the development of novel first-generation T3 antagonists DIBRT, HY-
4, and GC-14 using the “extension hypothesis” as a general guideline in hormone antagonist
design.1-3 These compounds contain extensions at the 5′-position (DIBRT, GC-14) of the outer
thyronine ring or from the bridging carbon (HY-4). All of these compounds have only a modest
affinity and potency for the thyroid hormone receptor (TR) that limits studies of their
antagonistic actions. Here, we report the design and synthesis of a novel series of 5′-
phenylethynyl derivatives sharing the GC-1 halogen-free thyronine scaffold.4 One compound
(NH-3) is a T3 antagonist with negligible TR agonist activity and improved TR binding affinity
and potency that allow for further characterization of its observed activity. One mechanism
for antagonism appears to be the ability of NH-3 to block TR-coactivator interactions. NH-3
will be a useful pharmacological tool for further study of T3 signaling and TR function.
In tr od u ction
Thyroid hormone (3,5,3′-triido-L-thyronine, T3, Figure
1) is derived from its precursor T4 (3,5,3′,5′-tetraiodo-
thyronine) secreted from the thyroid gland. It regulates
a multitude of physiologic effects ranging from embry-
onic development to maintenance of homeostasis in
adults.5,6 The thyroid hormone excess state, hyperthy-
roidism, results in a number of abnormalities, including
weight loss, muscle wasting, tachycardia, atrial ar-
rhythmias, bone loss, and nervousness. Current thera-
pies for hyperthyroidism include blockade of release of
thyroid hormones by the gland, peripheral conversion
of T4 to T3, and â-adrenergic receptors to ameliorate
some of the symptoms.7 These approaches are not ideal
because treatment usually requires weeks before symp-
toms are relieved or results in incomplete responses.7
Direct blockade of T3 action with thyroid hormone
antagonists would bypass such complications and con-
stitute an important advance in the treatment of hy-
perthyroidism. T3 antagonists should also be useful for
in vivo pharmacological probes for studying thyroid
F igu r e 1. Structures of agonist and antagonist ligands for
the thyroid hormone receptor (TR). Agonists are T3 and GC-1.
Antagonists are HY-4, GC-14, and DIBRT. Also shown is the
general structure of 5′-phenylethynyl GC-1 analogues.
hormone-signaling pathways.
Two different thyroid hormone receptor (TR) sub-
types, TRR and TRâ, mediate effects of T3.8 Like other
nuclear receptors, the TRs have three major structural
domains: a highly variable N-terminal domain, a
central DNA binding domain (DBD), and a C-terminal
ligand binding domain (LBD).9 Genes positively regu-
lated by thyroid hormone contain a cis-acting thyroid
hormone response element (TRE) upstream of the
promoter region. In general, unliganded TRs are local-
ized in the nucleus and bound to the TRE as a monomer,
homodimer, or heterodimer with retinoid X receptor
(RXR).8 Unliganded TRs are associated with a group of
corepressor proteins to repress the basal transcription
machinery; upon binding of ligand, the TRs undergo a
conformational change that allows release of corepres-
* To whom correspondence should be addressed. Phone: (415) 476-
3620. Fax: (415) 502-7220. E-mail: scanlan@cgl.ucsf.edu.
† Program in Chemistry and Chemical Biology.
‡ Departments of Pharmaceutical Chemistry and Cellular and
Molecular Pharmacology.
§ Metabolic Research Unit.
10.1021/jm0201013 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/19/2002

Thyroid Hormone Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3311
sors and subsequent recruitment of coactivator proteins
to regulate transcription of T3-responsive genes.10
Compared to steroid ligands for nuclear receptors, T3
analogue chemistry is relatively unexplored. Previous
generate compounds having 5′-phenylethynyl exten-
sions. We postulated that the weak antagonist activity
of the previous series of compounds might be due to
flexibility of the 5′-substitutions that would still allow
formation of the coactivator-binding surface. The series
of compounds employed in the current studies utilize
an ethynyl group to link extensions to the GC-1 scaffold
based on the hypothesis that the ethynyl group would
act as a “rigid rod” to position the 5′-extension more
effectively to perturb proper packing of H12.
studies have primarily focused on structure-activity
11,12
relationships of T3
and on development of tissue-
and/or receptor-subtype-specific thyroid hormone ago-
nists to obtain the beneficial effects of T3, such as
reduction of weight or cholesterol levels, without induc-
ing the undesired cardiac side effects.13 However, de-
velopment of thyroid hormone antagonists has been
limited. To date, only a handful of T3 antagonists with
moderate to weak potency have been reported. The
cardiac antiarrhythmic agent amiodarone has some
antithyroid effects,14 but there is no direct data that this
compound is a true T3 antagonist. Desethylamiodarone,
the major metabolite of amiodarone, and not amio-
darone itself, inhibits in vitro T3 binding competitively
to TRR15 but noncompetitively to TRâ16 and interferes
with the interaction of TRâ with coactivator GRIP-1 in
vitro.17 However, desethylamiodarone has not been
shown to be an antagonist in cells in culture or in
animals. Both amiodarone and desethylamiodarone
have low affinity for TR and a slow onset of action and
result in serious side effects.18,19 Therefore, these com-
pounds are not ideal drug therapies.
We recently reported the synthesis of three first-
generation T3 antagonists DIBRT,1 HY-4,2 and GC-143
(Figure 1). Like T3, DIBRT contains a biaryl ether
linkage; however, the 1-position of DIBRT is a benzoic
acid rather than the alanine side chain of T3. Conse-
quently, DIBRT is not an attractive compound from a
synthetic point of view for development of analogues.
HY-4 and GC-14 are based on the halogen-free thyro-
mimetic GC-14 that provides an excellent scaffold for
synthesis of additional analogues (Figure 1). All of these
compounds have relatively weak TR binding and low
potency that limit their usefulness.
Ch em ica l Syn th esis. As shown in Scheme 1, the
synthesis began with the GC-1 biarylmethane interme-
diate 125 undergoing ortho metalation with n-BuLi
followed by in situ iodination with N-iodosuccinimide
(NIS) in a 2:1 THF/hexanes solvent mixture, generating
the 5′-iodinated intermediate 2. Trapping of the lithi-
ated intermediate with NIS generally resulted in a 2:1
conversion to the iodinated species, which could not be
isolated from the starting compound 1. Increasing the
amount of NIS did not improve the yield. Furthermore,
use of other iodinating agents such as iodine and iodine
monochloride resulted in lower yields with decomposi-
tion of starting material. Therefore, the mixture of
compounds was carried through to the next step, where
separation was achieved. Palladium-catalyzed Suzuki-
Miyaura coupling26 of 2 with phenylethynyl boronate
derivatives, generated in situ under basic conditions
with MeO-9-BBN, produced 5′-phenylethynyl ana-
logues 3a and 3b in good yields. Desilylation of 3a and
3b with tetrabutylammonium fluoride generated phe-
nols 4a and 4b, which were then monoalkylated using
tert-butylchloroacetate to give esters 5a and 5b . The
desired 5′-phenylethynyl GC-1 derivatives, NH-1 and
NH-2, were obtained by removal of the methoxymethyl
phenolic protecting groups under acidic conditions,
followed by basic saponification of the tert-butyl esters.
This synthetic route was also used to make NH-3 by
the Suzuki-Miyaura coupling of 2 with 1-ethynyl-4-
nitrobenzene. However, complications arose in scaling
up this coupling reaction. Since palladium-catalyzed
coupling is typically facilitated by electron-donating
groups in proximity to the alkyl/aryl group of the
boronate species,27,28 we suspected that the electron-
withdrawing nature of the nitro substituent was hin-
dering the transmetalation and/or the reductive elimi-
nation steps of the catalytic cycle. Consequently, the
synthesis was modified to couple 5′-iodinated intermedi-
ate with 4-ethynylaniline as a precursor to the nitro
compound (Scheme 2). Following iodination to give 2,
the silyl-protecting group was removed and monoalky-
lated with methyl bromoacetate to give 6. Suzuki-
Miyaura coupling of 6 with 4-ethynylaniline gave 7,
which was then oxidized with m-chloroperbenzoic acid
to give the nitro compound 8. Removal of the meth-
oxymethylphenolic protecting groups and saponification
of methyl esters 7 and 8 yielded the corresponding final
compounds NH-3 and NH-4.
In the current studies, we describe the synthesis and
preliminary biological activities of a second generation
of GC-1 derivatives having 5′-phenylethynyl extensions.
These analogues all bind TRs, and most retain agonist
activity with TRâ selectivity. Interestingly, one com-
pound, which has a chemical functionality similar to
that of GC-14, was found to be a T3 antagonist with
improved affinity and potency.
Resu lts
Ra tion a le a n d Design . Our design of thyroid hor-
mone antagonists applied the “extension hypothesis”20,21
combined with crystallographic data of human (h) TRâ
LBD bound to agonist GC-1.22 The C-terminal portion
of the LBD corresponding to helix 12 (H12) appears to
act as a molecular switch that packs against the body
of the receptor upon agonist binding to close the ligand
binding pocket and complete formation of a putative
coactivator-binding surface.23,24 The X-ray structure of
hTRâ LBD bound to GC-1 reveals the 5′-position of the
ligand oriented in the direction of the loop between
helices 11 and 12, implying that extensions of the
thyronine ring at this position should perturb the
packing of H12 and result in a disrupted coactivator-
binding surface. Because the synthetic route of GC-1 is
well-suited to generating analogues, it was adopted to
Recep tor Bin d in g a n d Tr a n sa ctiva tion P r op er -
ties. NH-1, NH-2, NH-3, and NH-4 were tested for
binding to hTRR1 and hTRâ1, as summarized in Table
1, which also includes comparative data for GC-1 and
GC-14.3 Binding affinity was measured by an in vitro
radioligand-displacement assay using recombinant hTRR1
and hTRâ1, a fixed concentration of radiolabeled T3, and

3312 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Nguyen et al.
Sch em e 1. Synthetic Route in the Preparation of NH-1 and NH-2
a range of concentrations of each analogue (Experimen-
tal Section). As previously observed with other GC-1
analogues, substitution at the 5′-position decreased
binding affinity for both TRR and TRâ compared to the
parent compound. All of the 5′-analogues bind TR with
at least 1 order of magnitude reduced affinity compared
to GC-1. Although binding is impaired, the TRâ selec-
tivity of these analogues is retained. This result is
consistent with structural and chemical data, which
suggest that the molecular determinant of selectivity
is located on the 1-oxyacetic acid side chain of the GC-1
core structure.22,29,30
The 5′-analogues were tested in human HeLa cells
for transcriptional transactivation properties using
transient transfection of expression plasmids for hTRR1
or hTRâ1 and a luciferase reporter (Experimental Sec-
tion). This reporter contained a TRE with two tandemly
linked copies of direct repeats of the consensus TR DNA
binding site spaced by four base pairs (DR4 elements)
upstream of the thymidine kinase promoter. All of the
analogues tested, except NH-3, were found to be near
full or partial agonists at TRâ relative to T3-induced
transactivation (Table 1). NH-3 induces minimal trans-
activation of reporter expression above the level of
vehicle control (Figure 2A) with either TRR or TRâ.
Furthermore, NH-3 competitively blocked 1 nM T3
induced transactivation in a dose-dependent manner
down to the maximal level of activation observed with
NH-3 alone (Figure 2B). Under these conditions, the
IC50 value of antagonism by NH-3 was 370 nM for TRâ
(Figure 2B) and 950 nM for TRR (data not shown),
making it more potent than GC-14. Thus, NH-3 is TRâ-
selective in both binding and inhibition of T3-induced
transactivation.
Effect of NH-3 on TR In ter a ction w ith NCoR a n d
GRIP -1. Since NH-3 displayed antagonist activity and
its 5′-extension was designed to prevent folding of TR
to form the coactivator-binding surface, the compound
was tested for its effect on TR interaction with coacti-
vator GRIP-1 using a mammalian two-hybrid assay
system. Since the coactivator surface partly overlaps the
corepressor-binding surface, we also asked if NH-3 could
block binding of the corepressor NCoR (Experimental
Section). HeLa cells were transiently transfected with
expression plasmids for the yeast GAL4 DBD linked to
either NCoR (aa1925-2308) or GRIP-1 (aa618-1121),
hTRâ-LBD fused to the VP16 activation domain, and a
GAL4-driven luciferase reporter (Figure 3A). As shown
in Figure 3B, both T3 and NH-3 promoted release of
NCoR. In a dose-response curve (Figure 3C), the EC50

Thyroid Hormone Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3313
Sch em e 2. Synthetic Route in the Preparation of NH-3 and NH-4
values of TR-NCoR interaction with bound T3 versus
bound NH-3 are approximately 1.5 versus 45 nM,
respectively; this difference is likely to reflect the
difference in their binding affinities for TR. NH-3 also
promoted TR release from corepressor SMRT under
similar conditions (data not shown) indicating that the
effect of NH-3 on corepressor release is not specific to
NCoR. However, NH-3 did not promote RAR release
from the corepressors, indicating that its effect is TR-
selective (data not shown). Thus, NH-3 resembles T3 in
its ability to promote TR dissociation from corepressors.
We then examined the effect of NH-3 on coactivator
binding. As expected, T3 stimulated binding of GRIP-1
to TR whereas NH-3 did not (Figure 3B). NH-3 also
blocked T3-induced TR-GRIP-1 interactions in a dose-
dependent manner (Figure 3D). Thus, NH-3 antagonizes
TR-coactivator interactions in a cellular environment.
We also confirmed that NH-3 showed similar effects
on TR interactions with corepressors and coactivators
in in vitro pull-down assays (Experimental Section,
Figure 3E). In accordance with the results obtained in
transfected cells, NH-3 behaved like T3 in promoting
release of liganded TR from bacterially expressed NCoR.
However, NH-3 again failed to promote TR interactions
with bacterially expressed GRIP-1 and also blocked the
T3-induced interactions between TR and this coactiva-

3314 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Nguyen et al.
Ta ble 1. Binding and Transcriptional Activation Data of 5′-Phenylethynyl Derivatives at Human TRR1 and TRâ1
a
The KD and standard error (SE) values were calculated by fitting the competition data to the equations of Swillens34 and using the
b
Graph-Pad Prism computer program (Graph-Pad Software Inc.). HeLa cells were cotransfected with either hTRâ or hTRR expression
vector and a TRE-luciferase reporter plasmid. Luciferase activity of 10-5 M analogue is expressed as a percent of the TRâ1 or TRR1
response with 10-7 M T3. Values are the mean ( SD for three separate experiments. See Experimental Section for more details. c The
EC50 value is the concentration of ligand required for half-maximum activation, whereas the IC50 value is the concentration of ligand
required for half-maximum inhibition in competition experiments with 10-9 M T3. EC50 and IC50 values were calculated by nonlinear
regression with the Graph-Pad Prism computer program (Graph-Pad Software Inc.) using a sigmoidal dose-response and single-site
competition models, respectively. Values are the mean for three separate experiments. Refer to Chiellini et al. (ref 3). e These are IC50
d
values for hTRâ1 and hTRR1, respectively. f Compounds exhibiting thyromimetic transcriptional activation through hTRâ1 were not further
g
characterized with hTRR1. For transactivation assays, HeLa cells were cultured in 10% hormone-depleted, heat-treated (80 °C, 20 min)
newborn calf serum during incubation with ligand. NH-4 was not able to antagonize 10-9 M T3-induced activation in a dose-dependent
h
manner.
tor. Similar results were also obtained with bacterially
expressed fragments of the alternate TR coactivator
TRAP22031 (data not shown). The inability of NH-3 to
promote interaction between TR and its target coacti-
vators is likely one of the underlying mechanisms for
its observed antagonist activity.
values greater than 1000 nM while NH-3 has an IC50
value of 370 nM. Compared to GC-14, NH-3 has a
modest 2-fold greater binding affinity and potency for
TRR and TRâ (Table 1), but its main improvement over
GC-14 is loss of partial agonist activity observed with
GC-14. NH-3 is essentially a full antagonist on the basis
of the transactivation assays, whereas GC-14 exhibits
partial agonism with approximately 35% and 20%
transactivation relative to T3 for TRR and TRâ, respec-
tively (Table 1). Thus, NH-3 is a TRâ-selective antago-
nist with decreased partial agonist activity and im-
proved binding affinity and potency over the first-
generation compounds.
The mechanism for antagonist activity of NH-3 can
be further studied as a result of its improved properties.
The weaker binding affinity and potency of HY-4 and
GC-14 have limited such characterization of these
compounds (Chiellini et al. and Yoshihara et al., un-
published results). Since NH-3 was designed to perturb
proper folding of helix 12 (H12) to complete formation
of the putative coactivator-binding surface, we tested
the effect of NH-3 on TR interactions with corepressors
Discu ssion
The 5′-phenylethynyl GC-1 analogue NH-3 is a second-
generation T3 antagonist with improved properties
compared to the previously reported antagonists
DIBRT,1 HY-4,2 and GC-14.3 While DIBRT and HY-4
do not have TRâ selectivity, NH-3 retains TRâ selectiv-
ity with respect to binding. NH-3 has similar binding
affinity for TRR relative to DIBRT and HY-4 but
approximately 5-fold higher affinity for TRâ. In reporter
gene transactivation assays in cultured cells, both HY-4
and NH-3 slightly induce transcriptional activation
(Table 1). DIBRT did not induce detectable activation
with TRâ but did have a very slight activation with
TRR.1 However, in competition assays with TRâ, DIBRT
and HY-4 inhibit T3-induced transactivation with IC50

Thyroid Hormone Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3315
ficient for complete perturbation of H12, thus allowing
partial coactivator binding and the observed partial
agonism.
This series of 5′-phenylethynyl compounds further
contributes to the SAR data of the GC-1 series and
further suggest that the chemical and structural prop-
erties required to confer antagonistic activity are very
subtle and specific. Changing the nitro to amino 5′-
substitution resulted in loss of antagonism (Table 1,
compare NH-3 and NH-4). Large hydrophobic exten-
sions alone are not sufficient to confer antagonist
activity, as with NH-2, indicating that simple steric
blockade of H12 packing is not operating. Thus, the
“extension hypothesis” is applicable to the design of
nuclear receptor antagonists; however, the nature of
chemical groups that convert agonist ligands to antago-
nists will likely depend on specific interactions between
residues of the receptor and the ligand extension to help
stabilize the antagonist conformation.
T3 antagonists such as NH-3 will be useful therapeu-
tic agents in the treatment of hyperthyroidism and other
metabolic disorders.5 NH-3 is the first T3 antagonist to
exhibit potent antagonism in vivo (Lim et al., submitted
for publication) and therefore may prove to be a gener-
ally useful tool for studying the effects of TR inactivation
in a variety of animal models. Until now, such studies
have been done primarily using TR-knockout mice32
because a pharmacological tool for inducing TR inacti-
vation has not been available.
F igu r e 2. (A) Transcriptional activation of NH-3 compared
to that of T3. (B) Dose-response curve with hTRâ1 for NH-3
alone and in competition with 1 nM T3, with an IC50 value of
370 nM. HeLa cells were treated with indicated concentrations
of NH-3 alone or in the presence of 1 nM T3 for 24 h. Values
are the mean ( SD for three separate experiments expressed
as (A) fold activation relative to EtOH vehicle and (B) percent
of the TRâ reponse with 1 nM T3, which is set at 100%. Data
were fitted by nonlinear regression using the Graph-Pad Prism
computer program (Graph-Pad Software Inc.) for a single-site
competition model to generate the IC50 value.
Exp er im en ta l Section
and coactivators. Theoretically, antagonists may func-
tion in two ways to affect TR-protein interactions: (1)
antagonists may enhance TR-corepressor interactions;
(2) antagonists may inhibit TR-coactivator interactions.
DIBRT was found to block coactivator binding; its effects
on corepressor binding were not examined.1 Our results
suggest that blockade of coactivator binding is also
responsible for the antagonism of NH-3; by contrast,
NH-3 resembles T3 in promoting corepressor release
from TR (Figures 3). Thus, the attachment of an
appropriate appendage at the appropriate position of
an agonist core structure may be a general strategy to
block coactivator recruitment and impair nuclear recep-
tor activation of gene expression.
Gen er a l. 1H and 13C NMR were recorded on the Varian
Utility 400 MHz spectrometer in CDCl3 or CD3OD solvent.
Chemical shifts were reported as parts per million downfield
from an internal tetramethylsilane standard (δ 0.0 for 1H
NMR) or from solvent references. HRMS was performed by
the Biomedical Mass Spectrometry Resource at UCSF or the
Mass Spectrometry Facility at UC Berkeley. Anhydrous
solvents and starting reagents were commercially available
and used without further purification. Glassware was oven-
or flame-dried prior to use. Reactions were performed under
argon inert atmosphere. Crude products were purified by
either flash chromatography using 230-400 mesh silica gel
or preparative TLC (Aldrich Chemical Co.). Target compounds
were analyzed for purity by analytical HPLC, which was
performed using a Rainin HP controller and Varian UV
detector with an Alltech Hypersil 100 silica column (250 mm
× 4.6 mm). Condition A is isocratic 60% (v/v) hexanes (spiked
with 0.5% TFA) in ethyl acetate; condition B is isocratic 1%
(v/v) MeOH (spiked with 0.5% TFA) in CH2Cl2.
Ch em istr y. [4-(3-Iod o-5-isop r op yl-4-m eth oxym eth oxy-
ben zyl)-3,5-d im eth ylp h en oxy]tr iisop r op ylsila n e (2). To
a solution of 1 (1.2 g, 2.55 mmol) in 30 mL of a 2:1 mixture of
anhydrous THF/hexanes cooled to -78 °C was added n-BuLi
(2.5M in hexanes, 5.61 mmol). The resulting mixture was
warmed to room temperature, and after 2 h, it was cooled back
to -78 °C before N-iodosuccinimide (0.70 g, 3.06 mmol) was
added as a solution in THF. The reaction mixture was stirred
at room temperature for an additional 5 h, then quenched with
water and extracted with ethyl acetate (2 × 20 mL). The
organic phase was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. 1H NMR of the crude
material showed >60% conversion to the desired product,
which has an Rf identical to that of the starting compound 1.
Thus, the desired product was not isolated. Other impurities
were removed by flash chromatography (100% hexanes) to give
1.35 g of a 2:1 mixture of product/starting material.
GC-14 and NH-3 share a common chemical function-
ality with a p-nitroaryl group attached to the 5′-
extension. In addition, NH-3 has a unique ethynyl
linkage. Previous SAR data suggest that the nitro group
attached to the 5′-extension dictates the antagonistic
property; the size, position, and electronic properties of
this nitro group are essential for the observed activity.3
The presence of the rigid ethynyl linkage in NH-3
results in increased binding affinity and potency com-
pared to GC-14. Thus, the improved properties of NH-3
can be attributed directly to the internal ethynyl moiety.
We postulate that rigidly extending the nitrophenyl
group of NH-3 by two carbon atoms out of the ligand
binding pocket results in additional or amplified ste-
reoelectronic interactions between ligand and receptor
that are not available to GC-14. These NH-3-specific
interactions lead to increased receptor binding and
affinity and greater perturbation of H12 packing. The
5′-nitrophenyl extension of GC-14 is apparently insuf-
Gen er a l P r oced u r e for Su zu k i-Miya u r a Cou p lin g of
Ar yl Iod in es.26 To a well-stirred solution of aryl acetylene

3316 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Nguyen et al.
F igu r e 3. Effect of NH-3 on TR interactions with corepressors and coactivators. (A) Schematic of the transfection components
in mammalian two-hybrid assays. (B) T3 and NH-3 promote TR dissociation from NCoR, but only T3 is able to induce GRIP-1
association. (C) In a dose-response curve, T3 and NH-3 inhibit TR-NCoR interaction by 50% (EC50) at 1.5 and 45 nM, respectively.
TR-NCoR interaction in the presence of EtOH vehicle is defined as 100% maximal luciferase activity. (D) In a competition assay,
1.5 µM NH-3 inhibits 50% TR-GRIP-1 association induced by 10 nM T3. TR-GRIP-1 interaction in the presence of 10 nM T3 is
defined as 100% maximal luciferase activity. Values are expressed as the mean ( SD for three separate experiments and analyzed
by nonlinear regression using a sigmoidal dose-response model with the Graph-Pad computer program (Graph-Pad Software
Inc.). (E) Autoradiogram of 10% SDS-polyacrylamide gel showing products of in vitro binding reactions between 35S-labeled
hTRâ and bacterially expressed GST control and GST fusions to the nuclear receptor binding regions of NCoR (aa1944-2453)
and GRIP-1 (aa563-1121). A 10% input labeled TR is shown as a control. T3 concentration was 100 nM; NH-3 concentration was
10 µM.
(1.2 equiv) in THF at -78 °C was added KHMDS (0.5 M in
toluene, 1.2 equiv). After 30 min, methoxy-9-BBN (1.0 M in
hexanes, 1.2 equiv) was added, and after 2 h, this solution was
transferred via cannula to a second solution consisting of
PdCl2(PPh3)2 (0.03 equiv) and aryl iodide (1.0 equiv) in THF.
The reaction mixture was heated at reflux for ca. 18 h
(overnight), allowed to cool to room temperature, and diluted
with ethyl acetate (20 mL). The organic phase was washed
with water (3 × 50 mL) and brine (3 × 50 mL), dried over
MgSO4, filtered through Celite, and concentrated in vacuo. The
crude material was purified by flash chromatography on silica
gel.
116.4, 119.6, 123.5, 126.6, 128.3, 129.1, 129.8, 131.4, 136.0,
138.2, 141.9, 153.8, 154.1. HR-MS calcd for C37H50O3Si:
570.3529. Found: 570.3528.
Triisopropyl-{4-[3-isopropyl-4-methoxymethoxy-5-(4-pen-
t ylp h en ylet h yn yl)b en zyl]-3,5-d im et h ylp h en oxy}sila n e
(3b). The coupling of 2 with 1-ethynyl-4-pentylbenzene (0.15
mL, 0.80 mmol) was effected using the general procedure to
afford 312 mg (49%, two steps from 1) of the title compound
1
as a colorless oil. H NMR (CDCl3, 400 MHz): δ 0.89 (t 3H, J
) 6.8 Hz), 1.1 (d 18H, J ) 7.2 Hz), 1.4 (d 6H, J ) 6.8 Hz), 1.3
(m 9H), 1.6 (m 2H), 2.60 (t 2H, J ) 7.6 Hz), 3.39 (heptet 1H,
J ) 6.8 Hz), 3.60 (s 3H), 3.90 (s 2H), 5.25 (s 2H), 6.61 (s 2H),
6.87 (s 2H), 7.13 (d 2H, J ) 8.0 Hz), 7.39 (d 2H, J ) 8.0 Hz).
HR-MS calcd for C42H60O3Si: 640.4312. Found: 640.4300.
4-(3-Isop r op yl-4-m et h oxym et h oxy-5-p h en ylet h yn yl-
ben zyl)-3,5-d im eth ylp h en ol (4a ). Compound 3a (30 mg,
0.05 mmol) and Bu4NF (0.08 mL, 1.0 M in THF) were
combined in 2 mL of THF. Deprotection was nearly instanta-
neous, as determined by TLC. The reaction mixture was
diluted with ethyl acetate (10 mL), washed with water (2 ×
15 mL) and brine (2 × 15 mL), dried over MgSO4, and
concentrated. The crude product was purified by flash column
Tr iisopr opyl-[4-(3-isopr opyl-4-m eth oxym eth oxy-5-ph e-
n yleth yn ylben zyl)-3,5-d im eth ylp h en oxy]sila n e (3a ). The
coupling of 2 with phenylacetylene (0.06 mL, 0.56 mmol) was
effected using the general procedure to afford 202 mg (50%,
1
two steps from 1) of the title compound as a colorless oil. H
NMR (CDCl3, 400 MHz): δ 1.11 (d 18H, J ) 7.2 Hz), 1.15 (d
6H, J ) 6.8 Hz), 1.25 (m 3H), 2.17 (s 6H), 3.40 (heptet 1H, J
) 6.8 Hz), 3.61 (s 3H), 3.91 (s 2H), 5.25 (s 2H), 6.62 (s 2H),
6.90 (s 2H), 7.33 (m 3H), 7.48 (m 2H). 13C NMR (CDCl3, 400
MHz): δ 12.7,18.0, 20.4, 23.4, 26.4, 33.7, 57.6, 86.9, 92.7, 99.8,

Thyroid Hormone Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3317
chromatography (5% ethyl acetate in hexanes) to yield 4a (19
mg, 87%). 1H NMR (CDCl3, 400 MHz) δ 1.18 (d 6H, J ) 6.8
Hz), 2.19 (s 6H), 3.41 (heptet 1H, J ) 6.8 Hz), 3.61 (s 3H),
3.91 (s, 2H), 5.26 (s 2H), 6.57 (s 2H), 6.87 (s 1H), 6.96 (s 1H),
7.3 (m 3H), 7.5 (m 2H). 13C NMR (CDCl3, 400 MHz) δ 20.3,
23.4, 26.4, 33.7, 57.6, 86.9, 92.8, 99.8, 114.8, 116.5, 123.3, 126.8,
128.2, 128.3, 128.9, 129.6, 131.4, 135.9, 138.7, 141.9, 153.6,
153.8. HR-MS calcd for C28H30O3: 414.2195. Found: 414.2181.
organic portions were dried with MgSO4 and concentrated to
yield 14 mg of the corresponding O-methoxymethyl-depro-
tected phenol, which was used directly in the following step.
To the resulting phenol in 1 mL of methanol was added 150
µL of 1 N aqueous NaOH. The reaction mixture was stirred
at room temperature for 4 h, acidified with 1 N aqueous HCl
to pH 6, and diluted with ethyl acetate (15 mL). The organic
portion was extracted with water (2 × 15 mL) and brine (2 ×
15 mL), dried over MgSO4, and concentrated in vacuo to yield
10 mg of desired product (80%, two steps) as a yellow solid.
1H NMR (CDCl3, 400 MHz): δ 1.22 (d 6H, J ) 6.8 Hz), 2.23 (s
6H), 3.26 (heptet 1H, J ) 6.8 Hz), 3.89 (s 2H), 4.67 (s 2H),
6.66 (s 2H), 6.72 (s 1H), 6.95 (s 1H), 7.34 (m 3H), 7.48 (m 2H).
13C NMR (CDCl3, 400 MHz): δ 20.6, 22.3, 27.6, 29.7, 33.7, 83.9,
95.9, 108.9, 114.2, 122.5, 127.1, 127.5, 128.5, 128.7, 130.8,
131.4, 131.6, 134.2, 138.9, 152.2, 155.3. HR-MS calcd for
4-[3-Isop r op yl-4-m eth oxym eth oxy-5-(4-p en tylp h en yl-
eth yn yl)ben zyl]-3,5-d im eth ylp h en ol (4b). Compound 3b
(310 mg, 0.5 mmol) and Bu4NF (0.8 mL, 1.0 M in THF) were
combined in 10 mL of THF. Deprotection was nearly instan-
taneous, as determined by TLC. The reaction mixture was
diluted with ethyl acetate (20 mL), washed with water (2 ×
25 mL) and brine (2 × 25 mL), dried over MgSO4, and
concentrated. The crude product was purified by flash column
chromatography (5% ethyl acetate in hexanes) to yield 4b (215
C
28H28O4: 428.1988. Found: 428.1980. HPLC: condition A
1
retention time 4.6 min; condition B retention time 4.5 min;
95.2% pure.
mg, 90%). H NMR (CDCl3, 400 MHz): δ 0.89 (t 3H, J ) 6.8
Hz), 1.3 (m 4H), 1.6 (m 2H), 2.19 (s 6H), 2.60 (t 2H, J ) 7.6
Hz), 3.41 (heptet 1H, J ) 7.0 Hz), 3.60 (s 3H), 3.90 (s 2H),
4.62 (s broad 1H), 5.25 (s 2H), 6.56 (s 2H), 6.86 (s 1H), 6.94 (s
1H), 7.13 (d 2H, J ) 8.0 Hz), 7.39 (d 2H, J ) 8.0 Hz). 13C NMR
(CDCl3, 400 MHz): δ 14.0, 20.3, 20.8, 22.5, 23.4, 26.4, 30.9,
31.4, 33.7, 35.8, 57.5, 86.2, 93.0, 99.7, 114.8, 116.7, 120.4, 126.5,
128.4, 129.5, 131.3, 135.9, 138.7, 141.8, 143.4, 153.6, 153.8.
HR-MS calcd for C33H40O3: 484.2977. Found: 484.2976.
{4-[4-Hyd r oxy-3-isop r op yl-5-(4-p en tylp h en yleth yn yl)-
ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid (NH-2). To ester
5b (50 mg, 0.08 mmol) in 2 mL of 50% (v/v) mixture of i-PrOH
and THF was added 1 N aqueous HCl (0.2 mL). The reaction
mixture was stirred for 6 h at room temperature, diluted with
water, neurtralized with 1 N aqueous NaOH to pH 6, and then
extracted with ethyl acetate (2 × 15 mL). The combined
organic portions were dried with MgSO4 and concentrated to
yield 40 mg of the corresponding O-methoxymethyl-depro-
tected phenol, which was used directly in the following step.
To the resulting phenol in 1 mL of methanol was added 300
µL of 1 N aqueous NaOH. The reaction mixture was stirred
at room temperature for 4 h, acidified with 1 N aqueous HCl
to pH 6, and diluted with ethyl acetate (15 mL). The organic
portion was extracted with water (2 × 15 mL) and brine (2 ×
15 mL), dried over MgSO4, and concentrated in vacuo to yield
27 mg of desired product (64%, two steps) as a yellow solid.
1H NMR (CDCl3, 400 MHz): δ 0.88 (t 3H, J ) 6.8 Hz), 1.21 (d
6H, J ) 6.8 Hz), 1.3 (m 4H), 1.6 (m 2H), 2.23 (s 6H), 2.60 (t
2H, J ) 7.6 Hz), 3.25 (heptet 1H, J ) 6.8 Hz), 3.88 (s 2H),
4.67 (s 2H), 6.65 (s 2H), 6.71 (s 1H), 6.93 (s 1H), 7.14 (d 2H, J
) 8.0 Hz), 7.41 (d 2H, J ) 8.0 Hz). 13C NMR (CDCl3, 400
MHz): δ 14.0, 20.5, 22.3, 22.5, 27.5, 30.9, 31.4, 33.6, 35.9, 64.8,
83.1, 96.2, 109.1, 114.2, 119.5, 127.0, 127.3, 128.6, 130.8, 131.3,
131.4, 134.1, 138.8, 143.9, 152.0, 155.4. HR-MS calcd for
C33H38O4: 498.2770. Found: 498.2766. HPLC: condition A
retention time 4.2 min; condition B retention time 5.5 min;
95.2% pure.
{4-[3-(4-Am in op h e n yle t h yn yl)-5-isop r op yl-4-m e t h -
oxym et h oxyb en zyl]-3,5-d im et h ylp h en oxy}a cet ic Acid
Meth yl Ester (7). Compound 2 (2:1 mixture with 1, 1.35 g,
2.3 mmol) and Bu4NF (3.4 mL, 1.0 M in THF) were combined
in 30 mL of THF. Deprotection was nearly instantaneous, as
determined by TLC. The reaction mixture was diluted with
ethyl acetate (20 mL), washed with water (2 × 50 mL) and
brine (2 × 50 mL), dried over MgSO4, and concentrated.
Filtration through a silica gel plug (5% ethyl acetate in
hexanes) yields the silyl-deprotected phenol (2:1 mixture, 0.72
g). The crude mixture was then dissolved in 20 mL of DMF,
and Cs2CO3 (2.7 g, 8.2 mmol) was added followed by methyl
bromoacetate (0.2 mL, 2 mmol). The reaction mixture was
stirred for 30 min at room temperature and then neutralized
with cold 1 N aqueous HCl to pH 7 and extracted with ethyl
acetate (3 × 25 mL). The combined organic portions were
washed with brine (3 × 75 mL), dried over MgSO4, and
concentrated. The resulting product was filtered through a
silica gel plug (5% ethyl acetate in hexanes) to yield the methyl
ester 6 (2:1 mixture, 0.73 g).
[4-(3-Isop r op yl-4-m et h oxym et h oxy-5-p h en ylet h yn yl-
ben zyl)-3,5-d im eth ylp h en oxy]a cetic Acid ter t-Bu tyl Es-
ter (5a ). To a dry solution of 4a (15 mg, 0.036 mmol) and
Cs2CO3 (59 mg, 0.18 mmol) in 2 mL of DMF was added tert-
butyl chloroacetate (7 µL, 0.045 mmol). The reaction mixture
was stirred for 30 min at room temperature, neutralized with
cold 1 N aqueous HCl to pH 7, and extracted with ethyl acetate
(3 × 10 mL). The combined organic portions were washed with
brine (3 × 15 mL), dried over MgSO4, and concentrated. Crude
product was purified by preparative TLC (5% ethyl acetate in
hexanes) to yield 5a (16 mg, 82%). 1H NMR (CDCl3, 400
MHz): δ 1.17 (d 6H, J ) 6.8 Hz), 1.49 (s 9H), 2.21 (s 6H), 3.41
(heptet 1H, J ) 6.8 Hz), 3.61 (s 3H), 3.91 (s 2H), 4.51 (s 2H),
5.25 (s 2H), 6.63 (s 2H), 6.85 (s 1H), 6.95 (s 1H), 7.32 (m 3H),
7.48 (m 2H). 13C NMR (CDCl3, 400 MHz): δ 20.6, 23.4, 26.4,
28.0, 33.8, 57.6, 65.7, 82.2, 86.8, 92.8, 99.8, 114.2, 116.4, 123.3,
126.7, 128.2, 128.3, 129.6, 131.4, 135.8, 138.4, 141,9, 153.9,
156.0, 168.3. HR-MS calcd for C34H40O5: 528.2876. Found:
528.2876.
{4-[3-Isop r op yl-4-m eth oxym eth oxy-5-(4-p en tylp h en yl-
eth yn yl)ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid ter t-
Bu tyl Ester (5b). To a dry rbf containing a solution of 4b
(100 mg, 0.21 mmol) and Cs2CO3 (336 mg, 1.05 mmol) in 10
mL of DMF was added tert-butyl bromoacetate (35 µL, 0.23
mmol). The reaction mixture was stirred for 30 min at room
temperature, neutralized with cold 1 N aqueous HCl to pH 7,
and extracted with ethyl acetate (3 × 10 mL). The combined
organic portions were washed with brine (3 × 15 mL), dried
over MgSO4, and concentrated. Crude product was purified
by flash column chromatography (5% ethyl acetate in hexanes)
to yield 5b (102 mg, 82%). 1H NMR (CDCl3, 400 MHz): δ 0.88
(t 3H, J ) 6.8 Hz), 1.17 (d 6H, J ) 6.8 Hz), 1.3 (m 6H), 1.48 (s
9H), 1.60 (m, 2H), 2.21 (s 6H), 2.59 (t 2H, J ) 7.6 Hz), 3.41
(heptet 1H, J ) 6.8 Hz), 3.60 (s 3H), 3.91 (s 2H), 4.51 (s 2H),
5.25 (s 2H), 6.63 (s 2H), 6.85 (s 1H), 6.94 (s 1H), 7.13 (d 2H, J
) 8.0 Hz), 7.39 (d 2H, J ) 8.0 Hz). 13C NMR (CDCl3, 400
MHz): δ 14.0, 20.5, 22.4, 23.3, 26.3, 28.0, 30.8, 31.4, 33.7, 35.8,
57.5, 65.6, 82.1, 86.1, 93.0, 99.7, 114.1, 116.6, 120.4, 126.5,
128.4, 129.5, 129.6, 131.2, 135.7, 138.4, 141.8, 143.3, 153.8,
155.9, 168.3. HR-MS calcd for C39H50O5: 598.3658. Found:
598.3658.
[4-(4-Hyd r oxy-3-isop r op yl-5-p h en yleth yn ylben zyl)-3,5-
d im eth ylp h en oxy]a cetic Acid (NH-1). To ester 5a (15 mg,
0.03 mmol) in 1 mL of 50% (v/v) mixture of i-PrOH and THF
was added 1 N aqueous HCl (60 µL). The reaction mixture
was stirred for 6 h at room temperature, diluted with water,
neutralized with 1 N aqueous NaOH to pH 6, and then
extracted with ethyl acetate (2 × 15 mL). The combined
The ester 6 was then carried to the next step to undergo
Suzuki-Miyaura coupling with 4-ethynylaniline (200 mg, 1.7
mmol) using the general procedure described above to afford
0.29 g (23%, four steps from 1) of the title compound as a
yellow oil. H NMR (CDCl3, 400 MHz): δ 1.17 (d 6H, J ) 7.2
Hz), 2.21 (s 6H), 3.41 (heptet 1H, J ) 7.2 Hz), 3.60 (s 3H),
3.82 (s 3H), 3.90 (s 2H), 4.63 (s 3H), 5.24 (s 2H), 6.60 (d 2H, J
1

3318 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
) 8.0 Hz), 6.81 (s 1H), 6.91 (s 1H), 7.28 (d 2H, J ) 8.0 Hz). 13
NMR (CDCl3, 400 MHz): δ 20.5, 23.3, 26.4, 33.8, 52.2, 57.5,
65.3, 84.6, 93.6, 99.6, 112.7, 114.2, 114.7, 117.0, 126.1, 129.3,
130.1, 132.7, 135.5, 138.6, 141.7, 146.6, 153.6, 155.9, 169.7.
HR-MS calcd for C31H35NO5: 501.2515. Found: 501.2519.
Nguyen et al.
C
by flash column chromatography (2% MeOH in CHCl3) to yield
30 mg of desired product (78%) as a bright-yellow solid. 1H
NMR (CDCl3, 400 MHz): δ 1.22 (d 6H, J ) 6.8 Hz), 2.23 (s
6H), 3.26 (heptet 1H, J ) 6.8 Hz), 3.90 (s 2H), 4.69 (s 2H), 5.7
(broad 1H), 6.67 (s 2H), 6.71 (s 1H), 7.02 (s 1H), 7.63 (d 2H, J
) 8.8 Hz), 8.20 (d 2H, J ) 8.8 Hz). 13C NMR (CDCl3, 400
MHz): δ 20.5, 22.3, 27.5, 33.6, 64.8, 89.4, 93.9, 107.9, 114.2,
123.7, 127.3, 128.6, 129.4, 130.5, 131.8, 132.1, 134.7, 138.8,
{4-[3-Isop r op yl-4-m et h oxym et h oxy-5-(4-n it r op h en yl-
eth yn yl)ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid Meth -
yl Ester (8). A solution of m-CPBA (60% pure, 260 mg, 0.90
mmol) in 10 mL of CHCl3 was added gradually at 0 °C to an
ice-cooled, stirred solution of 6 (150 mg, 0.30 mmol) in 10 mL
of CHCl3. Stirring was continued for 3 h, during which time
the mixture was allowed to come to room temperature. The
reaction mixture was quenched with water (20 mL) and
extracted with ethyl acetate (3 × 25 mL). The combined
organic phase was washed with saturated NaHCO3 (3 × 50
mL) and brine (3 × 50 mL), dried over MgSO4, and concen-
trated. The crude product was purified by flash column
chromatography (10% ethyl acetate in hexanes) to give the
nitro compound 7 (97 mg, 61%) as a bright-yellow oil. 1H NMR
(CDCl3, 400 MHz): δ 1.20 (d 6H, J ) 6.8 Hz), 2.22 (s 6H),
3.41 (heptet 1H, J ) 6.8 Hz), 3.61 (s 3H), 3.93 (s 2H), 4.64 (s
2H), 5.23 (s 2H), 6.65 (s 2H), 6.85 (s 1H), 7.05 (s 1H), 7.62 (d
2H, J ) 8.4 Hz), 8.19 (d 2H, J ) 8.4 Hz). 13C NMR (CDCl3,
400 MHz): δ 20.5, 22.2, 23.3, 26.5, 33.7, 52.2, 57.6, 65.2, 90.9,
92.4, 99.9, 114.2, 115.5, 123.6, 128.0, 129.6, 130.3, 132.0, 136.0,
138.6, 142.2, 146.9, 154.3, 156.0, 169.6. HR-MS calcd for
147.1, 152.4, 155.5, 173.3. HR-MS calcd for
C28H27NO6:
473.1838. Found: 473.1839. HPLC: condition A retention time
4.5 min; condition B retention time 5.4 min; 98.0% pure.
{4-[3-(4-Am in op h en yleth yn yl)-4-h yd r oxy-5-isop r op yl-
ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid (NH-4). To the
above phenol 9 (34 mg, 0.07 mmol) in 4 mL of methanol was
added LiOH‚H2O (7 mg, 0.16 mmol) and H2O (7 µL, 0.70
mmol). The reaction mixture was stirred at room temperature
for 4 h, acidified with 1 N aqueous HCl to pH 6, and diluted
with ethyl acetate (15 mL). The organic portion was extracted
with water (2 × 15 mL) and brine (2 × 15 mL), dried over
MgSO4, and concentrated in vacuo. The product was purified
by preparative TLC (5% MeOH in CHCl3) to yield 13 mg of
the desired product (41%) as a yellow solid. 1H NMR (1% CD3-
OD, in CDCl3, 400 MHz): δ 1.20 (d 2H, J ) 6.8 Hz), 2.22 (s
6H), 3.25 (heptet 1H, J ) 6.8 Hz), 3.88 (s 2H), 4.60 (s 2H),
6.65 (s 2H), 6.67 (m 3H), 6.91 (s 1H), 7.31 (d 2H, J ) 8.4 Hz).
13C NMR (1% CD3OD, CDCl3, 400 MHz): δ 20.3, 22.1, 27.3,
33.5, 60.5, 64.9, 81.8, 96.1, 109.4, 114.0, 115.1, 126.7, 126.9,
130.2, 131.2, 132.7, 133.9, 138.5, 151.7, 155.7, 171.5, 174.0.
HR-MS calcd for C28H29NO4: 443.2097. Found: 443.2099.
C
31H35NO7: 531.2257. Found: 531.2252.
{4-[4-H yd r oxy-3-isop r op yl-5-(4-n it r op h en ylet h yn yl)-
ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid Meth yl Ester
(9). To ester 7 (95 mg, 0.18 mmol) in 5 mL of 50% (v/v) mixture
of i-PrOH and THF was added 1 N aqueous HCl (0.35 mL).
The reaction mixture was stirred for 6 h at room temperature,
diluted with 10 mL of water, neutralized with 1 N aqueous
NaOH to pH 6, and extracted with ethyl acetate (2 × 15 mL).
The combined organic portions were dried with MgSO4 and
concentrated to yield the corresponding O-methoxymethyl-
deprotected phenol. Purification by flash column chromatog-
raphy (10% ethyl acetate in hexanes) gave the desired product
Th yr oid Hor m on e Recep tor Liga n d Bin d in g Assa ys.
Hormone binding and analogue competition assays were
carried out as described in Apriletti et al.33 The Kd and
standard error (SE) values were calculated by fitting the
competition data to the equations of Swillens34 using the
Graph-Pad Prism computer program (Graph-Pad Software
Inc., San Diego, CA).
Tr a n scr ip tion a l Activa tion Assa ys. The reporter vector
for the luciferase reporter containing a DR-4 TRE promoter
and the expression vectors for the human TRR1 and TRâ1 were
gifts from Dr. M. Privalsky (University of California Davis,
Davis, CA). HeLa cells (UCSF Cell Culture Facility) were
maintained in culture and transfected as described previously3
except for the following changes. Cells were also transfected
with 0.5 µg per transfection of pRL-TK constitutive renilla
luciferase reporter (Promega Corp., Madison, WI) and plated
in 12-well plates in growth medium (DME H-21 with 10%
hormone-depleted newborn calf serum, or 10% hormone-
depleted, heat-treated (80 °C, 20 min) newborn calf serum
where indicated). After 6 h of incubation, ligand or vehicle
(EtOH) was added in triplicate. After an additional 24 h of
incubation, cells were harvested and assayed for luciferase
activity using the Promega Dual Luciferase kit (Promega
Corp., Madison, WI) and Analytical Luminescence Laboratory
Monolight 3010 luminometer. Data were normalized to the
renilla luciferase and analyzed with the Graph-Pad computer
program (Graph-Pad Software Inc., San Diego, CA) using the
sigmoidal dose-response or single-site competition models to
generate EC50 and IC50 values, respectively.
1
9 (55 mg, 63%). H NMR (CDCl3, 400 MHz): δ 1.23 (d 6H, J
) 6.8 Hz), 2.22 (s 6H), 3.26 (heptet 1H, J ) 6.8 Hz), 3.82 (s
3H), 3.90 (s 2H), 4.64 (s 2H), 5.68 (s 1H), 6.65 (s 2H), 6.71 (s
1H), 7.02 (s 1H), 7.64 (d 2H, J ) 8.8 Hz), 8.21 (d 2H, J ) 8.8
Hz). 13C NMR (CDCl3, 400 MHz) δ 20.5, 22.2, 27.5, 33.5, 52.2,
65.2, 89.5, 93.9, 107.9, 114.1, 123.7, 127.3, 128.6, 129.4, 130.0,
131.9, 132.1, 134.6, 138.6, 147.1, 152.4, 155.9, 169.7. HR-MS
calcd for C29H29NO6: 487.1995. Found: 487.1989.
{4-[3-(4-Am in op h en yleth yn yl)-4-h yd r oxy-5-isop r op yl-
ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid Meth yl Ester
(10). To ester 6 (83 mg, 0.16 mmol) in 5 mL of a 50% (v/v)
mixture of i-PrOH and THF was added 1 N aqueous HCl (0.20
mL). The reaction mixture was stirred for 6 h at room
temperature, diluted with 10 mL of water, neutralized with 1
N aqueous NaOH to pH 6, and extracted with ethyl acetate (2
× 15 mL). The combined organic portions were dried with
MgSO4 and concentrated to yield the corresponding O-meth-
oxymethyl-deprotected phenol. Purification by flash column
chromatography (50% ethyl acetate in hexanes) gave the
1
desired product 9 (34 mg, 45%). H NMR (CDCl3, 400 MHz):
δ 1.21 (d 6H, J ) 6.8 Hz), 2.21 (s 6H), 3.25 (heptet 1H, J ) 6.8
Hz), 3.81 (s 3H), 3.88 (s 2H), 4.63 (s 2H), 6.61 (d 2H, J ) 8.8
Hz), 6.63 (s 2H), 6.69 (s 1H), 6.91 (s 1H), 7.29 (d 2H, J ) 8.8
Hz). 13C NMR (CDCl3, 400 MHz): δ 20.6, 23.4, 26.4, 28.0, 33.8,
57.6, 65.7, 82.2, 86.8, 92.8, 99.8, 114.2, 116.4, 123.3, 126.7,
128.2, 128.3, 129.6, 131.4, 135.8, 138.4, 141.9, 153.9, 156.0,
168.3. HR-MS calcd for C29H31NO4: 457.2253. Found: 457.2249.
{4-[4-H yd r oxy-3-isop r op yl-5-(4-n it r op h en ylet h yn yl)-
ben zyl]-3,5-d im eth ylp h en oxy}a cetic Acid (NH-3). To the
above phenol 8 (40 mg, 0.10 mmol) in 3 mL of methanol was
added LiOH‚H2O (10 mg, 0.22 mmol) and H2O (10 µL, 0.10
mmol). The reaction mixture was stirred at room temperature
for 4 h, acidified with 1 N aqueous HCl to pH 6, and diluted
with ethyl acetate (15 mL). The organic portion was extracted
with water (2 × 15 mL) and brine (2 × 15 mL), dried over
MgSO4, and concentrated in vacuo. The product was purified
Ma m m a lia n Tw o-Hybr id Assa ys. The following expres-
sion plasmids were derived as previously described: GAL-
NCoR35 and luciferase reporter containing GAL promoter.36
GAL-SMRT (aa987-1491) and GAL-GRIP (aa618-1121) were
synthesized by standard PCR methods and cloned into the
mammalian GAL4-DBD expression vector pM (Clontech, Palo
Alto, CA) between the EcoRI and SalI sites. The expression
vector for VP16-hTRâ LBD was a gift from Dr. R. Evans
(University of California San Diego, San Diego, CA) and that
for VP16-RAR LBD was a gift from Dr. D. Moore (Baylor
College of Medicine, Houston, TX).
HeLa cells (Cell Culture Facility, UCSF) were maintained
in culture and transfected as described previously.3,35 Briefly,
cells (5 × 10-6) were collected and resuspended in Dulbecco’s
PBS (0.5 mL/transfection) containing 0.1% dextrose and 10
mg/mL bioprene and mixed with 2.5 µg of luciferase reporter,

Thyroid Hormone Antagonist
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3319
(10) Zhang, J .; Lazar, M. A. The mechanism of action of thyroid
hormones. Annu. Rev. Physiol. 2000, 62, 439-466.
(11) J orgensen, E. C. Thyroid hormones and analogs. I. synthesis,
physical properties and theoretical calculations. Hormonal
Proteins and Peptides; Academic Press: New York, 1978; pp 57-
105.
(12) J orgensen, E. C. Thyroid hormones and analogs. II. structure-
activity relationships. Hormonal Proteins and Peptides; Aca-
demic Press: New York, 1978; pp 107-204.
(13) Scanlan, T. S.; Yoshihara, H. A.; Nguyen, N. H.; Chiellini, G.
Selective thryomimetics: Tissue-selective thyroid hormone ana-
logs. Curr. Opin. Drug Discovery Dev. 2001, 4, 614-622.
(14) Martino, E.; Bartalena, L.; Bogazzi, F.; Braverman, L. E. The
effects of amiodarone on the thyroid. Endocr. Rev. 2001, 22, 240-
254.
(15) van Beeren, H. C.; Bakker, O.; Wiersinga, W. M. Desethylamio-
darone is a competitive inhibitor of the binding of thyroid
hormone to the thyroid hormone alpha 1-receptor protein. Mol.
Cell. Endocrinol. 1995, 112, 15-19.
(16) Bakker, O.; van Beeren, H. C.; Wiersinga, W. M. Desethylamio-
darone is a noncompetitive inhibitor of the binding of thyroid
hormone to the thyroid hormone beta 1-receptor protein. Endo-
crinology 1994, 134, 1665-1670.
(17) van Beeren, H. C.; Bakker, O.; Wiersinga, W. M. Desethylamio-
darone interferes with the binding of co-activator GRIP-1 to the
beta 1-thyroid hormone receptor. FEBS Lett. 2000, 481, 213-
216.
(18) Latham, K. R.; Sellitti, D. F.; Goldstein, R. E. Interaction of
amiodarone and desethylamiodarone with solubilized nuclear
thyroid hormone receptors. J . Am. Coll. Cardiol. 1987, 9, 872-
876.
(19) Morse, R. M.; Valenzuela, G. A.; Greenwald, T. P.; Eulie, P. J .;
Wesley, R. C.; et al. Amiodarone-induced liver toxicity. Ann.
Intern. Med. 1988, 109, 838-840.
(20) Ribeiro, R. C.; Apriletti, J . W.; Wagner, R. L.; West, B. L.; Feng,
W.; et al. Mechanisms of thyroid hormone action: insights from
X-ray crystallographic and functional studies. Recent Prog.
Horm. Res. 1998, 53, 351-392, 392-354 (discussion).
(21) Weatherman, R. V.; Fletterick, R. J .; Scanlan, T. S. Nuclear-
receptor ligands and ligand-binding domains. Annu. Rev. of
Biochem. 1999, 68, 559-581.
(22) Wagner, R. L.; Huber, B. R.; Shiau, A. K.; Kelly, A.; Cunha Lima,
S. T.; et al. Hormone selectivity in thyroid hormone receptors.
Mol. Endocrinol. 2001, 15, 398-410.
(23) Wagner, R. L.; Apriletti, J . W.; McGrath, M. E.; West, B. L.;
Baxter, J . D.; et al. A structural role for hormone in the thyroid
hormone receptor. Nature 1995, 378, 690-697.
(24) Feng, W.; Ribeiro, R. C.; Wagner, R. L.; Nguyen, H.; Apriletti,
J . W.; et al. Hormone-dependent coactivator binding to a
hydrophobic cleft on nuclear receptors. Science 1998, 280, 1747-
1749.
2.5 µg of a constitutive â-galactosidase expression vector, 1
µg of hTRâ LBD fused to the VP16 activation domain, and 1
µg of the appropriate corepressor or coactivator proteins fused
to the GAL4 DBD, per transfection. After electroporation (see
above), cells were resuspended in medium containing 10%
hormone-depleted newborn calf serum and incubated with
ligand for 24 h. Luciferase activity was measured using the
Promega luciferase kit (Promega Corp., Madison, WI), and
data were analyzed with the Graph-Pad computer program
(Graph-Pad Software Inc., San Diego, CA) using the sigmoidal
dose-response model to generate EC50 values.
In Vit r o Bin d in g Assa ys. GST-NCoR and GST-GRIP-1
plasmids were previously described.35 Binding assays were also
previously described in detail.1,35 Briefly, fusion proteins were
expressed from pGEX vectors (Pharmacia, Piuscataway, NJ )
in E. Coli strain BL21 (Novagen Inc., Madison, WI). Bacterial
cultures were grown to OD600 1.5 at room temperature, and
protein production was initiated by addition of IPTG to a final
concentration of 1 mM. After 4 h, bacterial pellets were
obtained, resuspended in 20 mM HEPES, pH 7.9, 80 mM KCl,
6 mM MgCl2, 1 mM dithiothreitol, 1 mM ATP, 0.2 mM
phenylmethylsulfonyl fluoride, and protease inhibitors and
then sonicated mildly. Debris was pelleted by centrifugation
and an SS34 rotor for 1 h at 12 000 rpm. The supernatant
was incubated with glutathione sepharose 4B beads (Amer-
sham Pharmacia Biotech AB, Uppsala, Sweden) and washed
as previously described.35 [35S]-Methionine labeled TR was
produced using coupled in vitro transcription-translation
(TNT kit, Promega Corp., Madison, WI). Binding assays used
3 µg of bacterially expressed proteins or controls and were
performed as previously described.35 Bound, labeled TR was
subjected to sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and then to autoradiography.
Ack n ow led gm en t. This work was supported by
grants from the National Institutes of Health (Grant
DK-52798 to T.S.S; Grant DK-41842 to J .D.B). N.H.N.
is grateful for financial support from the UCSF Depart-
ment of Pharmaceutical Chemistry Training Grant T32
07175-25. We thank Dr. M. Privalsky, Dr. R. Evans, and
Dr. D. Moore for the gifts of plasmids used in the
transfection and mammalian two-hybrid assays and
Phuong Nguyen for technical assistance. J .D.B. has
proprietary interests in and serves as a consultant and
Deputy Director to Karo Bio AB, which has commercial
interests in this area of research.
(25) Chiellini, G.; Nguyen, N. H.; Yoshihara, H. A.; Scanlan, T. S.
Improved synthesis of the iodine-free thyromimetic GC-1. Bioorg.
Med. Chem. Lett. 2000, 10, 2607-2611.
(26) Soderquist, J . A.; Matos, K.; Rane, A.; Ramos, J . Alkynylboranes
in the Suzuki-Miyaura Coupling. Tetrahedron Lett. 1995, 36,
2401-2402.
(27) Rossi, R.; Carpita, A.; Bellina, F. Palladium- and/or copper-
mediated cross-coupling reactions between 1-alkynes and vinyl,
aryl, 1-alkynyl, 1,2-propadienyl, propargyl, and allylic halides
or related compounds. Review. Org. Prep. Proced. Int. 1995, 27,
127-160.
Refer en ces
(1) Baxter, J . D.; Goede, P.; Apriletti, J . W.; West, B. L.; Feng, W.;
et al. Structure-Based Design and Synthesis of a Thyroid
Hormone Receptor (TR) Antagonist. Endocrinology 2002, 143,
517-524.
(2) Yoshihara, H. A.; Apriletti, J . W.; Baxter, J . D.; Scanlan, T. S.
A designed antagonist of the thyroid hormone receptor. Bioorg.
Med. Chem. Lett. 2001, 11, 2821-2825.
(3) Chiellini, G.; Nguyen, N. H.; Apriletti, J . W.; Baxter, J . D.;
Scanlan, T. S. Synthesis and biological activity of novel thyroid
hormone analogues: 5′-aryl substituted GC-1 derivatives. Bioorg.
Med. Chem. 2002, 10, 333-346.
(28) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling
Reactions of Organoboron Compounds. Chem. Rev. 1995, 95,
2457-2483.
(29) Yoshihara, H. A. Unpublished results.
(30) Baxter, J . D.; Dillmann, W. H.; West, B. L.; Huber, R.; Furlow,
J . D.; et al. Selective modulation of thyroid hormone receptor
action. J . Steroid Biochem. Mol. Biol. 2001, 76, 31-42.
(31) Fondell, J . D.; Ge, H.; Roeder, R. G. Ligand induction of a
transcriptionally active thyroid hormone receptor coactivator
complex. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8329-8333.
(32) Hsu, J . H.; Brent, G. A. Thyroid hormone receptor gene
knockouts. Trends Endocrinol. Metab. 1998, 9, 103-112.
(33) Apriletti, J . W.; Baxter, J . D.; Lau, K. H.; West, B. L. Expression
of the rat alpha 1 thyroid hormone receptor ligand binding
domain in Escherichia coli and the use of ligand-induced
conformation change as a method for its purification to homo-
geneity. Protein Expression Purif. 1995, 6, 363-370.
(34) Swillens, S. Interpretation of binding curves obtained with high
receptor concentrations: practical aid for computer analysis.
Mol. Pharmacol. 1995, 47, 1197-1203.
(4) Chiellini, G.; Apriletti, J . W.; al Yoshihara, H.; Baxter, J . D.;
Ribeiro, R. C.; et al. A high-affinity subtype-selective agonist
ligand for the thyroid hormone receptor. Chem. Biol. 1998, 5,
299-306.
(5) Utiger, L. E. B.; Utiger, R. D. The Thyroid: A Fundamental and
Clinical Text; Lippencott: Philadelphia, PA, 1991.
(6) Greenspan, F. S. The Thyroid Gland. Basic & Clinical Endo-
crinology, 5th ed.; Appleton & Lange: Stamford, CT, 1997; pp
192-262.
(7) Utiger, R. D. The thyroid: physiology, thyrotoxicosis, hypothy-
roidism, and the painful thyroid. Endocrinology and Metabolism;
McGraw-Hill: New York, 1995; pp 435-519.
(8) Lazar, M. A. Thyroid hormone receptors: multiple forms,
multiple possibilities. Endocr. Rev. 1993, 14, 184-193.
(9) Moras, D.; Gronemeyer, H. The nuclear receptor ligand-binding
domain: structure and function. Curr. Opin. Cell Biol. 1998, 10,
384-391.

3320 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Nguyen et al.
(35) Webb, P.; Anderson, C. M.; Valentine, C.; Nguyen, P.; Ma-
rimuthu, A.; et al. The nuclear receptor corepressor (N-CoR)
contains three isoleucine motifs (I/LXXII) that serve as receptor
interaction domains (IDs). Mol. Endocrinol. 2000, 14, 1976-
1985.
(36) Webb, P.; Nguyen, P.; Shinsako, J .; Anderson, C.; Feng, W.; et
al. Estrogen receptor activation function 1 works by binding p160
coactivator proteins. Mol. Endocrinol. 1998, 12, 1605-1618.
J M0201013