Synthesis and evaluation of sulfonylnitrophenylthiazoles (SNPTs) as thyroid hormone receptor-coactivator interaction inhibitors

Article abstract of DOI:10.1021/jm201546m

We previously identified a series of methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its coactivators. MSNBs inhibit coactivator binding through irreversible modification of cysteine 298 of the thyroid hormone receptor (TR). Although MSNBs have better pharmacological features than our first generation inhibitors (β-aminoketones), they contain a potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety, yielding sulfonylnitrophenylthiazoles (SNPTs). An array of SNPTs representing optimal side chains from the MSNB series was constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven transcription in reporter constructs and native genes. In addition the selected SNPTs were shown to be selective for TR relative to other nuclear hormone receptors (NRs).

Full text of DOI:10.1021/jm201546m

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Sulfonylnitrophenylthiazoles (SNPTs) as
Thyroid Hormone Receptor−Coactivator Interaction Inhibitors
Jong Yeon Hwang,† Ramy R. Attia,‡ Fangyi Zhu,‡ Lei Yang,‡ Andrew Lemoff,‡ Cynthia Jeffries,‡
Michele C. Connelly,‡ and R. Kiplin Guy*,‡
†Medicinal Chemistry Group, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, Korea
‡Department of Chemical Biology and Therapeutics, St. Jude Children’s Hospital, 262 Danny Thomas Place, Memphis, Tennessee
38105-3678, United States
S
* Supporting Information
ABSTRACT: We previously identified a series of methyl-
sulfonylnitrobenzoates (MSNBs) that block the interaction of
the thyroid hormone receptor with its coactivators. MSNBs
inhibit coactivator binding through irreversible modification of
cysteine 298 of the thyroid hormone receptor (TR). Although
MSNBs have better pharmacological features than our first
generation inhibitors (β-aminoketones), they contain a
potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety,
yielding sulfonylnitrophenylthiazoles (SNPTs). An array of SNPTs representing optimal side chains from the MSNB series was
constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active
compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven
transcription in reporter constructs and native genes. In addition the selected SNPTs were shown to be selective for TR relative
to other nuclear hormone receptors (NRs).
makes the AF-2 domain an ideal target for developing inhibitors
of TR−SRC interactions. Although a number of small molecule
modulators of TR have been developed recently, including
agonists such as GC-1,17−19 TRIAC,20 and KB-14121,22and
antagonists such as NH-3,23−25 most target the ligand binding
pocket in the LBD.
INTRODUCTION
■
The nuclear hormone receptors (NRs) are transcription factors
that are therapeutic targets for metabolic disease, immunology,
reproductive health, and cancer.1−3 The NR superfamily
includes the thyroid hormone receptors (TRs), TRα and
TRβ, that regulate development, growth, and metabolism.4,5
Although the TR isoforms are widely expressed, they follow
tissue specific patterns that vary with developmental stage.6,7
The TR isoforms have distinct regulatory roles.8,9
We have previously reported a series β-aminoketones that
disrupt the TR−coactivator interaction without affecting T3
binding.26−28 Unfortunately these compounds suffered from
multiple liabilities in vivo, thus requiring development of a new
scaffold. The second generation TRβ−SRC2 inhibitors,
methylsulfonylnitrobenzoates (1, MSNBs), were identified in
a quantitative high throughput screen (qHTS).29 Both the β-
aminoketones and MSNBs have a similar inhibition mecha-
nism, irreversibly modifying Cys298 within the AF-2 domain of
TR.30 However, the MSNBs have two major advantages for the
development of TR-coactivator inhibitors for use in vivo. First,
MSNB members are predicted to lack the cardiac activity
exhibited by the β-aminoketones because they lack the basic
tertiary amines that lead to ion channel binding. Second, the
MSNBs are more stable than the β-aminoketones at
physiological pH. The MSNBs have two distinct structural
features: the methylsulfonyl group that acts as a leaving group
and the ester-linked acetamide group that appears to target the
compound to the AF-2 domain.
Thyroid hormone (T3) regulates transcriptional responses
mediated by TR,9 which contains an amino terminal tran-
scription activation domain (AF-1), a central DNA binding
domain (DBD), and a carboxyl terminal ligand binding domain
(LBD) that contains a T3-inducible coactivator binding
domain, AF-2.10 TR usually functions as a heterodimer with
the retinoid X receptor (RXR). At low levels of T3, TR binds
corepressors using the AF-2 domain and suppresses basal
transcription at thyroid-responsive elements (TREs). In
response to increasing concentrations of T3, TR undergoes a
conformational change, releasing corepressor proteins and
binding coactivator proteins, thus activating gene tran-
scription.11,12 The dominant family of coactivators is the
SRCs, which include SRC1 (NcoA1), SRC2 (GRIP1/TIF2),
and SRC3 (AIB1/TRAM1/RAC3/ACTR).13 The SRCs
include both nuclear receptor interaction (NID) and activation
domains. The SRC’s NID includes a variable number of a
conserved NR box motif, containing the LXXLL sequence, that
binds to the TR’s AF-2 domain.14,15 This interaction is
mediated by a small, well-defined binding pocket16 that
Received: November 15, 2011
Published: February 10, 2012
© 2012 American Chemical Society
2301
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
Figure 1. (A) Structural modification of MSNBs leading to SNPTs. (B) The translucent shape is the van der Waals surface of MSNBs and SNPTs.
The colors of the translucent portion represent electrostatics of both molecules: red (negative), blue (positive), and white (neutral). Overall there is
good alignment between the MSNBs and the SNPTs, thus indicating their theoretical viability as more stable bioisosteres.
Carboxylic esters are often metabolically unstable in vivo
because of facile hydrolysis by esterases in multiple compart-
ments and intrinsic chemical instability in the stomach. A
common strategy to replace esters is to use heterocyclic
bioisosteres with increased stability to degradation.31,32
A
structural analysis indicated that thiazole-linked MSNBs, called
sulfonylnitrophenylthiazoles (SNPTs), gave good alignments
between the requisite aromatic and side chain groups of the
MSNBs (Figure 1). For this reason, we modified the MSNB
structure to produce SNPTs. Here we report an efficient
method of parallel synthesis of SNPTs and their evaluation as
thyroid hormone receptor−coactivator inhibitors.
RESULTS AND DISCUSSION
■
Chemistry. The bioisostere hypothesis was tested by
constructing a compound array containing all of the variations
of the warhead and side chains that were reasonably potent in
the MSNB background, with replacement of the ester by one of
two thiazole linkages. This allowed any synergistic interactions
affecting potency to adjust to the new core. The SNPT array
was constructed using a parallel chemistry method with three
diversification steps (Figure 2). First, six α-haloketones were
employed to selectively give either the 4- or 5-carboxamides (x
variation). Second, three thiols were used to provide sulfonyl
group diversity (y variation). Finally, 24 amines were employed
as the third building block (z variation). The amine series were
chosen to systematically vary the size, electrostatics, and
hydrophobicity at this position. All three sets of building blocks
selected for this compound array are shown in Figure 3.
The synthetic route to the SNPTs is depicted in Scheme 1.
Commercially available chloronitrobenzonitrile 4 was con-
verted to benzamide 5 through oxidation with hydrogen
peroxide. Benzamide 5 was then converted to thiobenzamide 6
with high yield by treatment with Lawesson’s reagent.
Intermediate 6 was treated with various 2-chlorooxoacetates 7
and 3-bromooxoacetate 12 to give the arrays of 5-carboxyesters
8 and 4-carboxyesters 13{x}, respectively. Next, compound
Figure 2. Plan for the construction of SNPT compound array.
arrays 8 and 13 were reacted with three thiolates (sodium
methanethiolate, n-butanethiol/K2CO3, and benzylthiol/
K2CO3), yielding sulfide compound arrays 9{x,y} and
14{x,y}, respectively. Oxidation with m-CPBA gave the sulfonyl
compound sets 10 and 15, followed by hydrolysis to give 5-
carboxylic acid SNPT array 11{x,y} and 4-carboxylic acid
SNPT array 16{x,y}, respectively. This chemistry was
performed cleanly enough to allow going from starting material
4 to the penultimate step without column purification, with all
intermediates being purified by crystallization. In the last step
carboxylic acids arrays 11 and 16 were treated with 24 amines,
PyBOP, and DIEA at room temperature to give the final SNPT
arrays (2{x,y,z} and 3{x,y,z}). The final products were purified
using flash chromatography followed by automated preparative
HPLC. The identity of all compounds was established using
NMR and MS. The purity was confirmed by LC/MS/UV/
2302
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
Figure 3. Building blocks (R1−R3/R4).
ELSD/CLND. The yield and purity of the SNPTs are available
in Supporting Information.
substituted compounds 2{4,y,z} showed slightly better TR−
SRC2 antagonism (Figure 4A). Two compounds in this
subseries, 2{4,1,5} and 2{4,1,4}, showed the most potent
activity (IC50 values of 0.3 and 0.6 μM, respectively).
The potency was significantly less tolerant of variation in R2
with most n-butyl- and benzyl-SNPTs exhibiting no inhibitory
activity regardless of substitution on R1 and R3/R4. However,
55 out of 136 methyl-SNPTs (R2 = Me) gave detectable
inhibition with potency depending on the substitution pattern
at R1 and R3/R4 (Figure 4B). Only two compounds from the n-
butyl and benzyl-SNPT subseries showed any activity (17 μM
for 2{2,2,4}, 20 μM for 2{2,3,4}, 20 μM for 2{3,2,4}, and 19
μM for 2{3,3,4}in Supporting Information).
Finally, the binding pocket seems to be fairly tolerant of a
wide range of substituents at R3, with both aliphatic amines and
nonaliphatic amines (anilines, benzylamines, and piperazines)
giving reasonable potency (Figure 4C). It is apparent that small
amines are more favorable than large amines. For instance, the
piperidine (z = 4) and 4-methylpiperidine (z = 5) substituted
series showed generally good inhibitory activity while those
series containing larger amines such as anilines, benzylamines,
and phenylpiperazines did not. However, activity is not simply
described by sterics, as hydrophilic aliphatic amines such as
morpholines (z = 3) and 1-methylpiperazine (z = 6) showed
weak or no inhibitory activity. This indicates that both
electrostatic and hydrophobic interactions are important in
this portion of the binding site. These results strongly support
our previous finding that hydrophilic atoms on the amide group
Biology. All of the SNPTs were evaluated to test their TR−
coactivator (COA) antagonism using a previously reported
fluorescence polarization (FP) in replicate dose−response
experiments using TRβ-LBD and Texas Red labeled SRC2-2
peptide (Tx-SRC2-2).26,34 Compounds were serially diluted in
10 3-fold steps from a 10 000 μM DMSO stock. The resulting
concentration series of each SNPT were transferred to the assay
wells using hydrodynamic pins with a final concentration of
0.1% DMSO. All assays were run in triplicate and the entire
experiment was replicated twice, for a total of six replicates; the
data are reported as average values across all assays as IC50 with
95% confidential range (Supporting Information). Fifty-two out
of 291 SNPTs tested showed detectable inhibitory activity
(EC50 < 60 μM). Among the active analogues, 19 had IC50
values below 10 μM (Table 1).
Comparing potency trends between classes of substituent at
R1, R2, and R3/R4 allowed an initial analysis of structure−
activity relationships (Figure 4). In general, 5-carboxamide-
SNPTs 2{x,y,z} gave better inhibitory potency compared to 4-
carboxamide-SNPTs 3{x,y,z}. Among the 4-carboxamide-
SNPTs 3{x,y,z} the most active compound was compound
3{6,1,13} with 8 μM IC50 value. Among the 5-carboxamide-
SNPTs 2{x,y,z}, a wide range of substituents were tolerated at
R1, but the phenyl substitutions 2{5,y,z} were mostly inactive,
with the exception of 2{5,1,4} (IC50 = 1 μM). The coactivator-
binding site on TR is shallow; thus, it is likely that large phenyl
groups are not favorable for binding. Remarkably CF3
2303
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of SNPT Analogues
a
Reagents and conditions: (a) H2O2, K2CO3, DMSO, 60 °C, 0.5 h; (b) Lawesson’s reagent, 1,4-dioxane, 110 °C, 2 h; (c) 2-chloro-2-ketoacetate 7,
EtOH, reflux, 24−36 h; (d) NaSMe or RSH/K2CO3, THF, 50 °C, 18 h; (e) m-CPBA, DCM, 24−36 h, rt; (f) LiOH, H2O/THF, rt, 3−5 h; (g)
amines, PyBOP, DIEA, DMF, rt, 24 h; (h) bromooxobutanoate 12, EtOH, reflux, 24 h.
of the MSNB series and on side chain of β-aminoketone series
were not favorable in this portion of the binding pocket.28,30
MSNBs inhibit the TR−coactivator interaction through
alkylation of the Cys 298 residue located on the AF-2 cleft
by nucleophilic replacement with methylsulfonyl group.30 Our
results indicate that a bulky sulfonyl group hinders nucleophilic
attack by this cysteine residue. In addition, larger alkyl groups
do not appear to fit within the relatively shallow and small
binding pocket on TRβ surface, thus blocking reaction.
Next, the SNPT compound array was tested for the
compounds’ ability to inhibit T3-mediated transcription of a
luciferase reporter gene assay. This was done using a single
concentration of inhibitor (5 μM), and the data are
summarized in Table 1. Strikingly, biochemically highly potent
compounds 2{4,1,5} and 2{4,1,4} had weak transcriptional
inhibitory activity at 5 μM (8.1% and 2.3% inhibition,
respectively). In addition 2{5,1,4} and 2{1,1,1}, which
exhibited good biochemical inhibitory activity (1.4 and 1.7
μM IC50), completely failed to inhibit T3-response luciferase
expression. Instead biochemically moderately active com-
pounds 2{3,1,2} and 2{2,1,2} significantly inhibited T3-
response luciferase activity..
To further validate transcriptional inhibition of T3-driven
genes by SNPT compounds, we performed RT-PCR experi-
ments using two well-accepted thyroid-responsive genes,
PEPCK and MMP11, which are known to be T3-responsive
in HepG2 cells (Figure 5).30,35 Cells were co-treated with T3
(100 nM) and compound (10 μM). Controls included NH3, a
ligand antagonist of T3, and a representative MSNB (1), a
known T3 antagonist. mRNA was isolated, and real-time PCR
experiments were carried out on the diluted cDNA prepared
from each mRNA sample. Both genes were inhibited by
SNPTs, with efficacy matching that of the ligand antagonist
NH-3. Compound 2{3,1,2} had slightly better inhibitory
potency for both genes in comparison to other inhibitors.
These data provide support that SNPT analogues effectively
block TR-mediated gene transcription at native response
elements in live cells.
Solubility and permeability of the compounds in the SNPT
array were evaluated to elucidate likely relationships between
biochemical assay and cellular assays. Compound solubility was
2304
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
c
Table 1. Summary of Pharmacological Properties of SNPTs
2305
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
Table 1. continued
a
b
c
Values are the mean of two independent experiments in triplicate. Values are the mean of a single triplicate experiment. The IC50 values are for
the inhibition of coregulatory peptide binding (SRC2-2) to the TR-LBD using a fluorescence polarization assay. The EC50 values are for cellular
proliferation inhibition (HepG2) using the measurement of total ATP content with the CellTiter-Glo (Promega) method. T3-response TRE-
luciferase inhibitory activity was evaluated in HEK293 cells transfected with both CMV-TRβ and a DR4-TRE driven luciferase expression vector.
The data were normalized to co-transfected constitutive Renilla luciferase activity. Solubility was measured using the Millipore method at pH 7.4 in
PBS. Permeability was measured using the parallel artificial membrane permeation assay (PAMPA) at pH 7.4. Compounds are ordered by potency of
TRβ and SRC2-2 inhibition.
Figure 4. Analysis of SNPTs based on substitution position and class.
Figure 5. Regulation of native T3-controlled genes in HepG2 cells by treatment with SNPTs. The cells were exposed to compounds at a fixed
concentration in the presence of T3 for 24 h. RT-PCR was carried out to determine transcription levels of the PEPCK (A) and MMP11 (B) genes.
The ΔΔCt method was used to calculate fold induction of expression. Error bars represent the standard errors of two independent experiments
performed in triplicate: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.005.
determined in PBS buffer containing 1% DMSO, reflecting the
conditions of the biochemical assays. SNPTs generally
possessed relatively poor solubility (0.4−39 μM), but most of
the potent compounds showed reasonable solubility, being
freely soluble at concentrations well above their biochemical
potency. Permeability of compounds was measured using a
PAMPA at pH 7.4. All of the compounds showed acceptable to
good permeability (>40 × 10−6 cm/s) except 2{4,1,2} (8 ×
10−6 cm/s). No correlation was observed between cellular
activity and solubility or permeability.
We also measured the cytotoxicity of selected inhibitors in
HepG2, a hepatocellular carcinoma derived line. Most of
2306
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
SNPTs showed no cytotoxic effects in the concentration range
being evaluated (EC50 > 27 μM). Only three compounds,
2{3,1,4}, 2{3,1,2}, and 2{2,1,1}, showed weak cytotoxicity
potency (11.2, 10.1, and 6.4 μM, respectively)
The association of TRβ with SRC2-2 is ligand dependent.36
Previously, we reported that MSNBs could not block the
binding of T3 to TRβ.30 In order to confirm the suspected
mechanism of action, we tested the antagonism of T3 binding
by SNPTs using a ([125I]T3) competition assay.37 None of
SNPTs blocked T3 binding at concentrations up to 60 μM
(data not shown). Thus, SNPTs do not appear to act as T3
competitive antagonists.
We examined the NR specificity of the SNPT series by
testing the following NR−coactivator interactions in FP assays:
TRα with SRC2-2 and peroxisome proliferator-activated
receptor γ (PPARγ) with vitamin D receptor interacting
protein 2 (DRIP-2). The coactivators were selected on the basis
of previously published work mapping the preferred interaction
partners.38,39 Additionally, PPARγ was chosen because it
contains a reactive cysteine in the ligand binding pocket that
can be alkylated by electrophilic reagents and might potentially
give poor selectivity. A set of 27 compounds was surveyed
(Figure 6). In general, most the compounds were selective for
the TRs but exhibited similar antagonistic potency toward both
TRα and TRβ. However, 2{4,1,4}, 2{4,1,2}, 2{4,1,3}, and
2{4,1,15}, which possess a 4-CF3 at the 4-position, are more
potent against TRα by at least 5-fold. Compounds 2{3,1,1},
2{2,1,1}, and 2{1,1,2} were more potent against TRβ by 4- to
5-fold, although they are only modestly potent (IC50 ranging
from 11 to 15 μM). This set of compounds had almost no
effect on PPARγ.
When the aggregate of all the data is considered, the best-
balanced two compounds are 2{2,1,2} and 2{3,1,2}. These two
compounds showed both good thyroid hormone receptor
coactivator interaction inhibitory potency and reasonable
cellular toxicity. In addition these molecules significantly
inhibited T3- mediated target gene expression in both luciferase
based transcription assay and real-time PCR experiments.
While their physiochemical properties are not ideal, they are
both class II compounds40 with high permeability and low
solubility. Reasonable formulation strategies exist for handling
class II compounds.41 Further optimization will likely focus on
careful studies of the amine substitution pattern where the SAR
is apparently subtle.
Figure 6. Biochemical selectivity of SNPT analogues in inhibiting
coregulator binding to other NR family members. Compounds are
ordered by potency against TRβ. The coactivator and NR interactions
tested were SRC2-2 with TRα, and DRIP-2 with PPARγ. All values are
the mean of two independent fluorescence polarization experiments,
each carried out in triplicate.
signaling was confirmed on native response elements using RT-
PCR. Interestingly, moderately active compounds in the FP
assay significantly inhibited in the cellular level, whereas
biochemically highly potent compounds showed weak inhib-
itory activity. While a number of potent cellular antagonists
were identified, they tended not to be among the most potent
from the biochemical assay, which is attributed to overly high
reactivity of some of the compound array members leading to
poor selectivity. The best two compounds 2{2,1,2} and
2{3,1,2} showed moderate inhibitory activity in biochemical
assay but exhibited stronger inhibitory activity in cellular level.
We successfully changed the metabolically unfavorable ester
group of MSNBs to a thiazole group without any loss of activity
in both biochemical assay and cellular assays. This result
indicated that the SNPTs can be used as new tools for use in
further TR biology studies. Currently we are studying their
activity in in vivo models.
CONCLUSION
■
This paper describes the structural modification of the
previously reported MSNB series of thyroid receptor
antagonists using a bioisosteric approach to remove a
potentially labile ester group linking the two critical
pharmacophore elements for the inhibitors. In order to identify
the best combination of linker and optimal versions of each
portion of the molecule, the candidate array of inhibitors
(SNPTs) was constructed using a parallel chemistry method
with three diversification steps. Antagonism of SNPTs toward
TR−coactivator binding was evaluated, revealing the most
potent biochemical inhibitors of this interaction reported to
date. Among 291 SNPT analogues tested, 60 compounds
inhibited the interaction between TRβ and SRC2-2 peptide.
The cellular activity of SNPT analogues was explored using a
TRE responsive luciferase reporter gene assay, demonstrating
that a number of compounds were potent inhibitors of T3
induction of this element. This antagonism of TR-mediated T3
EXPERIMENTAL SECTION
■
Chemistry. All materials were obtained from commercial suppliers
and used without further purification. All solvents used were dried
using an aluminum oxide column. Thin layer chromatography was
2307
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
performed on precoated silica gel 60 F254 plates. Purification of
intermediates was carried out by normal phase column chromatog-
raphy (SP1 [Biotage], silica gel 230−400 mesh). Chromatographic
separation was performed using a UPLC−MS (BEH C18 1.7 μm, 2.1
mm × 50 mm column, Waters Corp.). Data were acquired using
Masslynx, version 4.1, and analyzed using the Openlynx software suite.
The flow was then split to an evaporative light scattering detector
(ELSD) and an SQ mass spectrometer. The total flow rate was 1.0
mL/min. The gradient program started at 90% A (0.1% formic acid in
H2O), changed to 95% B (0.1% formic acid in ACN), then to 90% A.
The mass spectrometer was operated in positive-ion mode with
electrospray ionization. NMR spectra were recorded on a Bruker 400
MHz instrument and NMR peaks were assigned by MestReNova
(version 5.2.2). The identity of all final compounds was confirmed by
proton NMR and by mass spectrometry. The purity of all final
compounds was assessed using LC/MS/UV/ELSD, with the purity
(>95%) being assigned as the average determined by UV/ELSD (see
Supporting Information for details).
4-Chloro-3-nitrobenzamide (5). To a solution of 4-chloro-3-
nitrobenznitrile 4 (30 g, 164 mmol) and potassium carbonate (27.3 g,
197 mmol) in DMSO (400 mL) was cautiously added hydrogen
peroxide (27.9 mL, 30% aqueous solution). The reaction mixture was
heated for 15 min at 60 °C, then cooled to room temperature. The
reaction mixture was poured to ice−water to afford a precipitate. Then
the precipitate was washed with water to give the desired product 5
(25 g, 76%). 1H NMR (400 MHz, DMSO) δ 8.52 (d, J = 2.1 Hz, 1H),
8.27 (d, J = 24.5 Hz, 1H), 8.16 (dd, J = 8.4, 2.1 Hz, 1H), 7.92−7.87
(m, 1H), 7.78 (s, 1H); 13C NMR (101 MHz, DMSO) δ 164.81,
147.31, 134.37, 132.52, 131.90, 127.85, 124.65.
(0.27 g, 91%). 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 8.24−8.18
(m, 2H), 4.32 (q, J = 7.1 Hz, 2H), 3.40 (s, 3H), 2.74 (s, 3H), 1.34 (t, J
= 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 163.91, 161.77,
161.52, 149.71, 139.21, 134.99, 132.37, 129.87, 124.90, 122.70, 61.87,
45.18, 17.50, 14.32.
4-Methyl-2-(4-(methylsulfonyl)-3-nitrophenyl)oxazole-5-
carboxylic Acid (11{1,1}). To a solution of compound 10{1,1} (40
mg, 0.11 mmol) in H2O/THF (4 mL, 1:3 v/v) was added lithium
hydroxide (3.2 mg, 0.14 mmol). The reaction mixture was stirred for 3
h at room temperature and neutralized by 1 N HCl to pH 5−6,
resulting in formation of a solid precipitate. The solid product was
washed with water to give a pure, white solid product (20 mg, 54%).
1H NMR (400 MHz, DMSO) δ 8.67 (d, J = 1.8 Hz, 1H), 8.58 (s, 1H),
8.55 (dd, J = 8.3, 1.8 Hz, 1H), 8.28−8.25 (m, 1H), 3.54 (s, 3H); 13C
NMR (101 MHz, DMSO) δ 167.26, 161.67, 149.08, 148.86, 138.18,
133.88, 133.17, 132.34, 130.46, 122.44, 44.43.
Amides 2{x,y,z} and 3{x,y,z}. To a solution of carboxylic acid (50
mmol) in DMF (0.25 mL) in a 48-position Mettler Toledo XT
reaction block were added PyBOP (50 mmol, 0.2 mL of 0.3 M
solution in DMF) and TEA (75 mmol, 0.05 mL of 1.5 M solution in
DMF) followed by the appropriate amine (55 mmol, 0.55 mL of 1 M
solution in DMF). The mixtures were stirred at room temperature for
24 h and concentrated using a GeneVac HT-4. The crude product
mixtures were dissolved in EtOAc (1 mL), filtered through a silica-
packed column, and washed with EtOAc (2 × 3 mL). The organic
solutions were concentrated using a GeneVac HT-4 and dissolved in
DMSO (1 mL). The crude products were purified by automated
HPLC/MS. NMR and MS results of all final compounds are available
in Supporting Information.
Compound Evaluation. All biological and pharmacological
methods have been previously published and followed the established
procedures. For details, see the Supporting Information.
4-Chloro-3-nitrobenzothioamide (6). To a solution of com-
pound 5 (25 g, 125 mmol) in 1,4-dioxane (300 mL) was added
Lawesson’s reagent (25.2 g, 62.3 mmol). The reaction mixture was
stirred for 2 h at 110 °C. The reaction mixture was concentrated in
vacuo, then diluted with ethyl acetate, and washed with water. The
organic solution was concentrated and the resulting solid product
crystallized from dichloromethane to afford a yellow solid product (25
g, 93%). 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 2.2 Hz, 1H), 8.04
(dd, J = 8.4, 2.2 Hz, 1H), 7.66 (br, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.19
(br, 1H); 13C NMR (101 MHz, CDCl3) δ 198.17, 138.54, 132.14,
131.31, 130.50, 123.56.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, tabulated activity data, and character-
ization data for intermediates and final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Ethyl 2-(4-Chloro-3-nitrophenyl)-4-methylthiazole-5-car-
boxylate (8{2}). To a solution of compound 6 (3 g, 13.9 mmol) in
EtOH (40 mL) was added ethyl 2-chloro-3-oxobutanoate (2.9 mL,
20.8 mmol). The reaction mixture was stirred overnight at 110 °C and
then cooled to room temperature. The resulting precipitate was
isolated by filtration and washed with MeOH to give a pure, white
solid product (3.5 g, 77%). 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J =
2.1 Hz, 1H), 8.07 (dd, J = 8.4, 2.1 Hz, 1H), 7.66−7.56 (m, 1H), 4.38
(q, J = 7.1 Hz, 2H), 2.79 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 165.26, 161.77, 161.40, 148.40, 132.88, 132.63,
130.53, 129.00, 123.54, 123.44, 61.65, 17.48, 14.33.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (901) 595-5714. Fax: (901) 595-5715. E-mail: kip.
guy@stjude.org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Ethyl 4-Methyl-2-(4-(methylthio)-3-nitrophenyl)thiazole-5-
carboxylate (9{2,1}). To a solution of compound 8{2} (0.5 g, 1.5
mmol) in THF (5 mL) was added sodium methane thiolate (0.13 g,
1.8 mmol). The reaction mixture stirred for 1 h at room temperature.
The resulting precipitate was filtered and washed with THF and ethyl
This work was supported by NIH Grant DK58080, the
American Lebanese Syrian Associated Charities (ALSAC), and
St. Jude Children’s Research Hospital (SJCRH).
1
acetate to afford a yellow solid product (0.5 g, 97%). H NMR (400
ABBREVIATIONS USED
■
MHz, CDCl3) δ 8.83 (d, J = 2.0 Hz, 1H), 8.16 (dd, J = 8.5, 2.0 Hz,
1H), 7.45 (d, J = 8.6 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 2.79 (s, 3H),
2.56 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
166.45, 161.97, 161.33, 145.54, 142.27, 130.91, 129.48, 126.22, 124.11,
122.61, 61.51, 17.53, 16.16, 14.36.
Ethyl 4-Methyl-2-(4-(methylsulfonyl)-3-nitrophenyl)-
thiazole-5-carboxylate (10{2,1}). To a solution of compound
9{2,1} (0.27 g, 0.8 mmol) in DCM was added m-chloroperoxybenzoic
acid (0.49 g, 2.0 mmol, <70% maximum activity). The reaction
mixture was stirred for 6 h at room temperature and washed with
water and saturated sodium bicarbonate. The organic solution was
dried over MgSO4 and concentrated in vacuo. The crude product was
purified by recrystallization with DCM to give a white solid product
MSNB, methylsulfonylnitrobenzoate; SNPT, sulfonylnitrophe-
nylthiazole; NR, nuclear hormone receptor; TR, thyroid
hormone receptor; RXR, retinoid X receptor; PPAR,
peroxisome proliferator-activated receptor; AF, transcription
activation; NID, nuclear receptor interaction domain; LBD,
ligand binding domain; TRE, thyroid-responsive element; T3,
triiodothyronine; SRC, steroid receptor coactivator; COA,
coactivator; PAMPA, parallel artificial membrane permeability
assay; FP, fluorescence polarization; PDK, pyruvate dehydro-
genase kinase; PEPCK, phosphoenolpyruvate carboxykinase;
MMP11, matrix metallopeptidase 11
2308
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
(22) Garg, N.; Li, Y. L.; Garcia Collazo, A. M.; Litten, C.; Ryono, D.
E.; Zhang, M.; Caringal, Y.; Brigance, R. P.; Meng, W.; Washburn, W.
N.; Agback, P.; Mellstrom, K.; Rehnmark, S.; Rahimi-Ghadim, M.;
Norin, T.; Grynfarb, M.; Sandberg, J.; Grover, G.; Malm, J. Thyroid
receptor ligands. Part 8: Thyromimetics derived from N-acylated-
alpha-amino acid derivatives displaying modulated pharmacological
selectivity compared with KB-141. Bioorg. Med. Chem. Lett. 2007, 17,
4131−4134.
(23) Shah, V.; Nguyen, P.; Nguyen, N. H.; Togashi, M.; Scanlan, T.
S.; Baxter, J. D.; Webb, P. Complex actions of thyroid hormone
receptor antagonist NH-3 on gene promoters in different cell lines.
Mol. Cell. Endocrinol. 2008, 296, 69−77.
(24) Webb, P.; Nguyen, N. H.; Chiellini, G.; Yoshihara, H. A.; Cunha
Lima, S. T.; Apriletti, J. W.; Ribeiro, R. C.; Marimuthu, A.; West, B. L.;
Goede, P.; Mellstrom, K.; Nilsson, S.; Kushner, P. J.; Fletterick, R. J.;
Scanlan, T. S.; Baxter, J. D. Design of thyroid hormone receptor
antagonists from first principles. J. Steroid Biochem. Mol. Biol. 2002, 83,
59−73.
(25) Nguyen, N. H.; Apriletti, J. W.; Cunha Lima, S. T.; Webb, P.;
Baxter, J. D.; Scanlan, T. S. Rational design and synthesis of a novel
thyroid hormone antagonist that blocks coactivator recruitment. J.
Med. Chem. 2002, 45, 3310−3320.
(26) Arnold, L. A.; Estebanez-Perpina, E.; Togashi, M.; Jouravel, N.;
Shelat, A.; McReynolds, A. C.; Mar, E.; Nguyen, P.; Baxter, J. D.;
Fletterick, R. J.; Webb, P.; Guy, R. K. Discovery of small molecule
inhibitors of the interaction of the thyroid hormone receptor with
transcriptional coregulators. J. Biol. Chem. 2005, 280, 43048−43055.
(27) Arnold, L. A.; Kosinski, A.; Estebanez-Perpina, E.; Fletterick, R.
J.; Guy, R. K. Inhibitors of the interaction of a thyroid hormone
receptor and coactivators: preliminary structure−activity relationships.
J. Med. Chem. 2007, 50, 5269−5280.
(28) Hwang, J. Y.; Arnold, L. A.; Zhu, F.; Kosinski, A.; Mangano, T.
J.; Setola, V.; Roth, B. L.; Guy, R. K. Improvement of pharmacological
properties of irreversible thyroid receptor coactivator binding
inhibitors. J. Med. Chem. 2009, 52, 3892−3901.
(29) Johnson, R. L.; Hwang, J. Y.; Arnold, L. A.; Huang, R.;
Wichterman, J.; Augustinaite, I.; Austin, C. P.; Inglese, J.; Guy, R. K.;
Huang, W. A quantitative high-throughput screen identifies novel
inhibitors of the interaction of thyroid receptor beta with a peptide of
steroid receptor coactivator 2. J. Biomol. Screening 2011, 16, 618−627.
(30) Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.;
Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.;
Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a
new class of irreversible inhibitors of the interaction of the thyroid
hormone receptor and its obligate coactivators that functionally
antagonizes thyroid hormone. J. Biol. Chem. 2011, 286, 11895−11908.
(31) Warmus, J. S.; Flamme, C.; Zhang, L. Y.; Barrett, S.; Bridges, A.;
Chen, H.; Gowan, R.; Kaufman, M.; Sebolt-Leopold, J.; Leopold, W.;
Merriman, R.; Ohren, J.; Pavlovsky, A.; Przybranowski, S.; Tecle, H.;
Valik, H.; Whitehead, C.; Zhang, E. 2-Alkylamino- and alkoxy-
substituted 2-amino-1,3,4-oxadiazoles-O-alkyl benzohydroxamate es-
ters replacements retain the desired inhibition and selectivity against
MEK (MAP ERK kinase). Bioorg. Med. Chem. Lett. 2008, 18, 6171−
6174.
(32) Diana, G. D.; Volkots, D. L.; Nitz, T. J.; Bailey, T. R.; Long, M.
A.; Vescio, N.; Aldous, S.; Pevear, D. C.; Dutko, F. J. Oxadiazoles as
ester bioisosteric replacements in compounds related to disoxaril.
Antirhinovirus activity. J. Med. Chem. 1994, 37, 2421−2436.
(33) The MOE program (Chemical Computing Group) was used.
(34) Arnold, L. A.; Estebanez-Perpina, E.; Togashi, M.; Shelat, A.;
Ocasio, C. A.; McReynolds, A. C.; Nguyen, P.; Baxter, J. D.; Fletterick,
R. J.; Webb, P.; Guy, R. K. A high-throughput screening method to
identify small molecule inhibitors of thyroid hormone receptor
coactivator binding. Sci. STKE 2006, pl3.
REFERENCES
■
(1) He, J.; Cheng, Q.; Xie, W. Minireview: Nuclear receptor-
controlled steroid hormone synthesis and metabolism. Mol. Endocrinol.
2010, 24, 11−21.
(2) Schulman, I. G. Nuclear receptors as drug targets for metabolic
disease. Adv. Drug Delivery Rev. 2010, 62, 1307−1315.
(3) Sun, G.; Shi, Y. Nuclear receptors in stem cells and their
therapeutic potential. Adv. Drug Delivery Rev. 2010, 62, 1299−1306.
(4) Cheng, S. Y.; Leonard, J. L.; Davis, P. J. Molecular aspects of
thyroid hormone actions. Endocr. Rev. 2010, 31, 139−170.
(5) Kress, E.; Samarut, J.; Plateroti, M. Thyroid hormones and the
control of cell proliferation or cell differentiation: paradox or duality?
Mol. Cell. Endocrinol. 2009, 313, 36−49.
(6) Oppenheimer, J. H.; Schwartz, H. L. Molecular basis of thyroid
hormone-dependent brain development. Endocr. Rev. 1997, 18, 462−
475.
(7) Yaoita, Y.; Brown, D. D. A correlation of thyroid hormone
receptor gene expression with amphibian metamorphosis. Genes Dev.
1990, 4, 1917−1924.
(8) Brent, G. A. Tissue-specific actions of thyroid hormone: insights
from animal models. Rev. Endocr. Metab. Disord. 2000, 1, 27−33.
(9) Harvey, C. B.; Williams, G. R. Mechanism of thyroid hormone
action. Thyroid 2002, 12, 441−446.
(10) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.;
Schutz, G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.;
Chambon, P.; Evans, R. M. The nuclear receptor superfamily: the
second decade. Cell 1995, 83, 835−839.
(11) Alonso, M.; Goodwin, C.; Liao, X.; Ortiga-Carvalho, T.;
Machado, D. S.; Wondisford, F. E.; Refetoff, S.; Weiss, R. E. In vivo
interaction of steroid receptor coactivator (SRC)-1 and the activation
function-2 domain of the thyroid hormone receptor (TR) beta in
TRbeta E457A knock-in and SRC-1 knockout mice. Endocrinology
2009, 150, 3927−3934.
(12) Paul, B. D.; Buchholz, D. R.; Fu, L.; Shi, Y. B. SRC-p300
coactivator complex is required for thyroid hormone-induced
amphibian metamorphosis. J. Biol. Chem. 2007, 282, 7472−7481.
(13) Xu, J.; Li, Q. Review of the in vivo functions of the p160 steroid
receptor coactivator family. Mol. Endocrinol. 2003, 17, 1681−1692.
(14) Savkur, R. S.; Burris, T. P. The coactivator LXXLL nuclear
receptor recognition motif. J. Pept. Res. 2004, 63, 207−212.
(15) Darimont, B. D.; Wagner, R. L.; Apriletti, J. W.; Stallcup, M. R.;
Kushner, P. J.; Baxter, J. D.; Fletterick, R. J.; Yamamoto, K. R.
Structure and specificity of nuclear receptor−coactivator interactions.
Genes Dev. 1998, 12, 3343−3356.
(16) Feng, W.; Ribeiro, R. C.; Wagner, R. L.; Nguyen, H.; Apriletti, J.
W.; Fletterick, R. J.; Baxter, J. D.; Kushner, P. J.; West, B. L. Hormone-
dependent coactivator binding to a hydrophobic cleft on nuclear
receptors. Science 1998, 280, 1747−1749.
(17) Yuan, C.; Lin, J. Z.; Sieglaff, D. H.; Ayers, S. D.; Denoto-
Reynolds, F.; Baxter, J. D.; Webb, P. Identical gene regulation patterns
of T3 and selective thyroid hormone receptor modulator GC-1.
Endocrinology 2012, 153, 501−511.
(18) Grover, G. J.; Egan, D. M.; Sleph, P. G.; Beehler, B. C.; Chiellini,
G.; Nguyen, N. H.; Baxter, J. D.; Scanlan, T. S. Effects of the thyroid
hormone receptor agonist GC-1 on metabolic rate and cholesterol in
rats and primates: selective actions relative to 3,5,3′-triiodo-L-
thyronine. Endocrinology 2004, 145, 1656−1661.
(19) Baxter, J. D.; Webb, P.; Grover, G.; Scanlan, T. S. Selective
activation of thyroid hormone signaling pathways by GC-1: a new
approach to controlling cholesterol and body weight. Trends
Endocrinol. Metab. 2004, 15, 154−157.
(20) Messier, N.; Langlois, M. F. Triac regulation of transcription is
T(3) receptor isoform- and response element-specific. Mol. Cell.
Endocrinol. 2000, 165, 57−66.
(21) Bryzgalova, G.; Effendic, S.; Khan, A.; Rehnmark, S.; Barbounis,
P.; Boulet, J.; Dong, G.; Singh, R.; Shapses, S.; Malm, J.; Webb, P.;
Baxter, J. D.; Grover, G. J. Anti-obesity, anti-diabetic, and lipid
lowering effects of the thyroid receptor beta subtype selective agonist
KB-141. J. Steroid Biochem. Mol. Biol. 2008, 111, 262−267.
(35) Sadana, P.; Hwang, J. Y.; Attia, R. R.; Arnold, L. A.; Neale, G.;
Guy, R. K. Similarities and differences between two modes of
antagonism of the thyroid hormone receptor. ACS Chem. Biol. 2011, 6,
1096−1106.
2309
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310

Journal of Medicinal Chemistry
Article
(36) Jeyakumar, M.; Tanen, M. R.; Bagchi, M. K. Analysis of the
functional role of steroid receptor coactivator-1 in ligand-induced
transactivation by thyroid hormone receptor. Mol. Endocrinol. 1997,
11, 755−767.
(37) Feau, C.; Arnold, L. A.; Kosinski, A.; Guy, R. K. A high-
throughput ligand competition binding assay for the androgen
receptor and other nuclear receptors. J. Biomol. Screening 2009, 14,
43−48.
(38) Moore, J. M.; Galicia, S. J.; McReynolds, A. C.; Nguyen, N. H.;
Scanlan, T. S.; Guy, R. K. Quantitative proteomics of the thyroid
hormone receptor−coregulator interactions. J. Biol. Chem. 2004, 279,
27584−27590.
(39) Feau, C.; Arnold, L. A.; Kosinski, A.; Zhu, F.; Connelly, M.;
Guy, R. K. Novel flufenamic acid analogues as inhibitors of androgen
receptor mediated transcription. ACS Chem. Biol. 2009, 4, 834−843.
(40) Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R. A
theoretical basis for a biopharmaceutic drug classification: the
correlation of in vitro drug product dissolution and in vivo
bioavailability. Pharm. Res. 1995, 12, 413−420.
(41) Pouton, C. W. Formulation of poorly water-soluble drugs for
oral administration: physicochemical and physiological issues and the
lipid formulation classification system. Eur. J. Pharm. Sci. 2006, 29,
278−287.
2310
dx.doi.org/10.1021/jm201546m | J. Med. Chem. 2012, 55, 2301−2310