Synthesis and evaluation of methylsulfonylnitrobenzamides (MSNBAs) as inhibitors of the thyroid hormone receptor-coactivator interaction

Article abstract of DOI:10.1016/j.bmcl.2012.12.055

We previously identified the methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its obligate transcriptional coactivators and prevent thyroid hormone signaling. As part of our lead optimization work we demonstrated that sulfonylnitrophenylthiazoles (SNPTs), which replace the ester linkage of MSNBs with a thiazole, also inhibited coactivator binding to TR. Here we report that replacement of the ester with an amide (methylsulfonylnitrobenzamides, MSNBA) also provides active TR antagonists.

Full text of DOI:10.1016/j.bmcl.2012.12.055

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1891–1895
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of methylsulfonylnitrobenzamides
(MSNBAs) as inhibitors of the thyroid hormone receptor–coactivator
interaction
⇑
Jong Yeon Hwang, Ramy R. Attia, Angela K. Carrillo, Michele C. Connelly, R. Kiplin Guy
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously identified the methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thy-
roid hormone receptor with its obligate transcriptional coactivators and prevent thyroid hormone signal-
ing. As part of our lead optimization work we demonstrated that sulfonylnitrophenylthiazoles (SNPTs),
which replace the ester linkage of MSNBs with a thiazole, also inhibited coactivator binding to TR. Here
we report that replacement of the ester with an amide (methylsulfonylnitrobenzamides, MSNBA) also
provides active TR antagonists.
Received 28 September 2012
Revised 13 December 2012
Accepted 17 December 2012
Available online 8 January 2013
Keywords:
Thyroid
Ó 2013 Elsevier Ltd. All rights reserved.
Coactivator
Protein interaction
Inhibitor
The thyroid hormone receptors (TRs), which regulate develop-
ment, growth, and metabolism, belong to the nuclear hormone
receptor (NR) superfamily.1,2 The activity of TRs is induced
in vivo by thyroid hormone (T3).3 TRs contain three functional do-
mains: an amino terminal transcription activation domain (AF-1), a
central DNA binding domain (DBD), and a carboxyl terminal ligand
binding domain (LBD) that includes a T3-inducible coactivator
binding domain, AF-2.4 In the absence of T3, TRs associate with
corepressors and cause suppression of basal transcription at thy-
roid response elements (TREs). Upon binding of T3, TRs undergo
a conformational change that releases corepressors and recruits
coactivators, such as the p160 steroid receptor coactivators (SRC),
to activate gene transcription from the TRE.5,6 Members of SRC
family include SRC1 (NcoA1), SRC2 (GRIP1/TIF2), and SRC3 (AIB1/
TRAM1/RAC3/ACTR).7 These coactivators have variable numbers
of a conserved LXXLL motif, called an NR box that mediates binding
to TRs.8,9 The NR boxes interact with the AF-2 region of the TR
LBD.10
ion channel activity of b-aminoketones and intrinsic chemical
instability of both b-aminoketones and MSBNs. Therefore we have
sought to develop new scaffolds. Recently we reported that sul-
fonylnitrophenylthiazoles (SNPTs), which replace the ester-linkage
of MSNB with a bioisosteric thiazole ring (Fig. 1), retain their TR
antagonism.12 In this Letter, we explored the replacement of the
ester with an amide linkage.
The synthesis of methylsulfonylnitrobenzamide (MSNBA) ana-
logs was accomplished using
a parallel chemistry approach
(Scheme 1) with two diversification steps (Fig. 2, building blocks
X and Y). First, five amino-carboxylic esters (building block X), con-
taining one to three methylenes and capped with either a primary
or secondary amine, were introduced to provide a terminal carbox-
ylate. Second, 24 amines (building block Y) were employed to con-
vert the acid to a set of amides. The amines in the building block Y
set were chosen to systematically vary the size, electrostatics, and
hydrophobicity at this position. The methylsulfonyl moiety re-
mained fixed.
We have previously reported two scaffolds, b-aminoketones
and methylsulfonylnitrobenzoates (MSNBs), that act as antagonists
of coactivator binding to TRs by competing with NR boxes for bind-
ing to the receptor. While the two families have different struc-
tures they have a similar mode of action, irreversibly modifying
Cys298 within the AF-2 domain of TR.11 Unfortunately these com-
pounds suffered from multiple liabilities in vivo, including cardiac
Commercially available chloronitrobenzoic ester 4 was con-
verted to compound 5 by treatment with sodium methanethiolate.
Oxidation with m-CPBA gave the sulfonyl compound 6. Finally
hydrolysis gave the common intermediate carboxylic acid 7. Com-
pound 7 was then converted to amides 8 by reaction with one of
the five amino-carboxylic esters (building block X) using PyBOP.
After hydrolysis of compounds 8 with lithium hydroxide, the
resulting carboxylic acids 9 were reacted with the 24 selected
amines (building block Y), PyBOP, and diisopropylethylamine at
room temperature to give the final MSNBAs 3 {X, Y}. Nomenclature
⇑
Corresponding author. Tel.: +1 (901) 595 5714; fax: +1 (901) 595 5715.
E-mail address: kip.guy@stjude.org (R.K. Guy).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.055

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J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1891–1895
O
R2
N
N
S
R3
O
previous
S
R1
work
O
R1 R2
N
O
NO2
O
R3
O
SNPTs (2)
O
S
O
NO2
MSNBs (1)
O
O
R1
this work
N
(CH2)n
N
O
S
R2
O
NO2
MSNBAs (3)
Figure 1. Structural modification of MSNBs (1) to SNPTs (2) and MSNBAs (3).
O
O
O
O
O
c
b
OH
O
a
O
O
S
O
O
S
O
Cl
S
NO2
NO2
NO2
NO2
6
7
4
5
O
O
O
O
O
O
R2
f
N
(CH2)n
OEt
e
N
(CH2)n
OH
d
O
S
N
(CH2)n
N
O
S
O
O
S
O
R1
O
NO2
NO2
NO2
3
8
9
Scheme 1. Synthesis of MSNBAs. Reagents and conditions: (a) NaSMe, tetrahydrofuran, 50 °C, 94%; (b) m-chloroperoxybenzoic acid, dichloromethane, rt, 84%; (c) LiOH,
tetrahydrofuran, rt, 94%; (d) amino-carboxylic esters (building block X), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, diisopropylethylamine,
dimethylformamide, rt, 49–98%; (e) LiOH, tetrahydrofuran, rt, 62–84%; (f) amines (building block Y), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate,
diisopropylethylamine, dimethylformamide, rt, 10–99%.
follows the Chemset convention. All reaction mixtures were con-
centrated in vacuo. The phosphine oxide byproduct generated from
the phosphonium coupling reagent was removed by filtration
through silica. The resulting crude products were purified directly
by automated reverse phase preparative high pressure liquid chro-
matography (HPLC). Ninety-five targeted MSNBAs were success-
fully isolated from 120 attempted reactions. All products were of
>95% purity and had the correct identity as established by HPLC/
MS/ELSD. The compounds were dissolved in DMSO to give stan-
dard 10 mM stock solutions for all further (Fig. 2) studies.
The MSNBAs were evaluated for their ability to antagonize the
TR-coactivator interaction using a fluorescence polarization (FP)
assay measuring the interaction of TRb-LBD and Texas Red-labeled
SRC2-2 peptide (Tx-SRC2-2).13,14 The specificity of the MSNBA ser-
ies was examined by comparing the antagonism of TRb–SRC2-2
with that of the vitamin D receptor (VDR) with SRC2-3.15,16 We
also measured the cytotoxicity of the MSNBA series in HepG2, a
hepatocellular carcinoma derived cell line. Compounds were seri-
are reported as average values across all assays as EC50 value with
standard deviations.
The observed EC50 values for each assay are shown as a heatmap
in Figure 3 and the data from active compounds is summarized in
Table 1. Five out 95 MSNBAs were active (<20 lM EC50), while
most of MSNBAs exhibited no inhibition of the TRb–SRC2-2 inter-
action. Among the actives, four compounds contained the methy-
lene linker (3{1,y}) between the two amide moieties (EC50’s:
3{1,1} = 16, 3{1,11} = 5.8, 3{1,12} = 6.6, and 3{1,18} = 3.6 lM).
Interestingly, these four actives have bulky and highly hydrophobic
secondary amines: adamantylamine, N-methylaniline, N-propylan-
iline, and dibenzylamine, respectively. In contrast, compounds
3{4,y}, bearing an N-methyl group on the ‘left’ amide, were inactive
(especially, 3{4,1}, {4,12}, {4,13}, and {4, 18} with EC50’s >60 lM).
Likewise, the ethyl 3{2,y}, propyl 3{3,y}, and piperidinyl 3{5,y}
linkers gave no inhibitory activity. Remarkably, compound 3{3,1}
showed moderate activity (EC50 = 11
propyl linker. The same trend was followed for TR
nism. The only exceptions were compounds 3{1,13} and 3{1,18},
which exhibited slightly more potency against TR . We examined
lM for TRb) although it has
a-COA antago-
ally diluted using a threefold scheme from a 10,000 lM top con-
centration in DMSO. Serially diluted MSNBAs were transferred to
test solutions by 100 H pins (V&P Scientific). All assays were run
in triplicate and the FP assays for TR-COA and VDR-COA antago-
nism were replicated twice, for a total of 6 replicates; the data
a
the NR specificity of the MSNBA series by testing VDR-coactivator
interactions in FP assay. Similar to the TRs, the VDR has a strongly
activated cysteine residue (Cys 284) in the ligand binding

J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1891–1895
1893
Figure 2. Building blocks for testing potential amide linkages (X and Y).
pocket.17,18 Recently, Arnold and co-workers reported the first irre-
versible inhibitor for VDR–coactivator interaction.19 Most of
MSNBA series were inactive against the VDR–SRC2-3 interaction.
The exception was 3{3,24} (EC50 = 7 lM), which has 4-trifluoro-
methylphenyl piperazine and three-carbon side chain. Compound
3{3,24} was inactive against TR-SRC. Most of MSNBAs showed no
cytotoxicity against HepG2 in the tested range (>27
few compounds exhibited moderate toxicity (5–17
plementary data).
l
M), while a
lM, see Sup-
To validate compounds as truly inhibiting TR–coactivator inter-
actions, we tested the inhibition of the induction of transcription of
PEPCK, a well-known TR-responsive gene in HepG2 cells, using RT-
PCR experiments (Fig. 4).20 Cells were co-treated with T3 (100 nM)
and compound (1 l a
M and 100 nM). Controls included NH3,21,22
ligand antagonist of T3, and a representative MSNB (1), a known
coactivator antagonist. We have previously shown that compounds
with this mechanism of action are not partial agonists of PEPCK.
mRNA was isolated, and real-time PCR experiments were carried
out on the diluted cDNA prepared from each mRNA sample. Tran-
scription of the PEPCK gene was inhibited by the MSNBA 3{1,18} in
dose-dependent manner, with reasonable efficacy compared to
that of the ligand antagonist NH-3 and significantly better efficacy
and potency than control compound 1. Thus, the MSNBA effec-
tively blocked TR-mediated gene transcription at native response
elements in live cells.
In summary, we describe the replacement of the potentially la-
bile ester of MSNBs with an amide linkage. Antagonism of MSNBA
toward TR was evaluated in FP assay with fluorescently labeled
SRC-2-2 peptide. Among 95 MSNBA analogs five compounds inhib-
ited the interaction between TRb and SRC2-2 peptide; all of these
Figure 3. Biochemical potency, biochemical selectivity, and cytotoxicity of MSN-
BAs. Compounds are ordered by potency of antagonism of the TRb and SRC2-2
interaction. The coactivator and NR interactions were: SRC2-2 with TRa and SRC2-3
with VDR. Cytotoxicity was determined by the measurement of total ATP content
using CellTiter-GloÒ (Promega) after incubation with compound for 72 h.

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J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1891–1895
Table 1
Activity profiles of all active MSNBAs
No.
Structure
FP competition assaya IC50
(l
M)
VDR
Cytotoxicityb EC50
HepG2
(lM)
TRb
TRa
O
O
H
N
N
H
3 {1,1}
16
4
9.6 3.8
4.2 5.1
1.9 0.4
1.7 0.8
>60
>60
>60
>60
>60
>60
>27
>27
>27
O
S
O
O
O
O
NO2
NO2
N
N
H
O
S
3 {1, 11}
3 {1, 12}
3 {1, 18}
3 {3, 1}
5.8 2.9
6.6 3.5
3.6 1.5
O
O
O
N
N
N
H
O
O
O
S
S
S
O
O
O
NO2
NO2
N
H
O
12
1
H
N
N
H
O
11
5
>27
O
O
NO2
CF3
O
N
N
3 {3, 24}
>60
>60
7.0 2.7
13
1
N
H
O
S
O
NO2
a
Values are the mean of two independent experiments each carried out in triplicate with standard deviations.
Values are the mean of a single triplicate experiment each carried out in triplicate with standard deviations.
b
were selective for TR relative to VDR. The antagonism of TR-med-
iated T3 signaling on thyroid-regulated genes in cells was con-
firmed by RT-PCR. The MSNBAs can be used as a new tool for
studying TR biology.
Acknowledgments
This work was supported by NIH/NIAID (Grant Al075517,
DK58080), the American Lebanese Syrian Associated Charities (AL-
SAC), and St. Jude Children’s Research Hospital.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.055.
Figure 4. Regulation of T3-controlled gene expression by treatment with MSNBAs
in HepG2 cells. The cells were exposed to compounds in the presence of T3
(100 nM) for 24 h. RT-PCR was carried out to determine transcription levels of the
PEPCK gene. The DDCt method was used to calculate fold induction of expression.
Error bars represent the standard errors of two independent experiments in
performed in triplicate. ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.005.
References and notes
1. Cheng, S. Y.; Leonard, J. L.; Davis, P. J. Endocr. Rev. 2010, 31, 139.
2. Kress, E.; Samarut, J.; Plateroti, M. Mol. Cell. Endocrinol. 2009, 313, 36.
3. Harvey, C. B.; Williams, G. R. Thyroid 2002, 12, 441.

J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1891–1895
1895
4. Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz, G.; Umesono,
K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83,
835.
14. 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.
Sci STKE 2006, 2006, 13.
5. Alonso, M.; Goodwin, C.; Liao, X.; Ortiga-Carvalho, T.; Machado, D. S.;
Wondisford, F. E.; Refetoff, S.; Weiss, R. E. Endocrinology 2009, 150, 3927.
6. Paul, B. D.; Buchholz, D. R.; Fu, L.; Shi, Y. B. J. Biol. Chem. 2007, 282, 7472.
7. Xu, J.; Li, Q. Mol. Endocrinol. 2003, 17, 1681.
15. Moore, J. M.; Galicia, S. J.; McReynolds, A. C.; Nguyen, N. H.; Scanlan, T. S.; Guy,
R. K. J. Biol. Chem. 2004, 279, 27584.
16. Feau, C.; Arnold, L. A.; Kosinski, A.; Zhu, F.; Connelly, M.; Guy, R. K. ACS Chem.
Biol. 2009, 4, 834.
8. Savkur, R. S.; Burris, T. P. J. Pept. Res. 2004, 63, 207.
9. Ding, X. F.; Anderson, C. M.; Ma, H.; Hong, H.; Uht, R. M.; Kushner, P. J.; Stallcup,
M. R. Mol. Endocrinol. 1998, 12, 302.
10. Darimont, B. D.; Wagner, R. L.; Apriletti, J. W.; Stallcup, M. R.; Kushner, P. J.;
Baxter, J. D.; Fletterick, R. J.; Yamamoto, K. R. Genes Dev. 1998, 12, 3343.
11. 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. J. Biol. Chem. 2011, 286, 11895.
17. Gampe, R. T., Jr.; Montana, V. G.; Lambert, M. H.; Miller, A. B.; Bledsoe, R. K.;
Milburn, M. V.; Kliewer, S. A.; Willson, T. M.; Xu, H. E. Mol. Cell 2000, 5, 545.
18. Vanhooke, J. L.; Benning, M. M.; Bauer, C. B.; Pike, J. W.; DeLuca, H. F.
Biochemistry 2004, 43, 4101.
19. Nandhikonda, P.; Lynt, W. Z.; McCallum, M. M.; Ara, T.; Baranowski, A. M.;
Yuan, N. Y.; Pearson, D.; Bikle, D. D.; Guy, R. K.; Arnold, L. A. J. Med. Chem. 2012,
55, 4640.
20. Park, E. A.; Song, S.; Olive, M.; Roesler, W. J. Biochem. J. 1997, 322(Pt 1), 343.
21. Nguyen, N. H.; Apriletti, J. W.; Cunha Lima, S. T.; Webb, P.; Baxter, J. D.; Scanlan,
T. S. J. Med. Chem. 2002, 45, 3310.
12. Hwang, J. Y.; Attia, R. R.; Zhu, F.; Yang, L.; Lemoff, A.; Jeffries, C.; Connelly, M. C.;
Guy, R. K. J. Med. Chem. 2012, 55, 2301.
13. 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. J. Biol. Chem. 2005, 280, 43048.
22. Shah, V.; Nguyen, P.; Nguyen, N. H.; Togashi, M.; Scanlan, T. S.; Baxter, J. D.;
Webb, P. Mol. Cell. Endocrinol. 2008, 296, 69.