Increasing Thyromimetic Potency through Halogen Substitution

Article abstract of DOI:10.1002/cmdc.201600408

Sobetirome is one of the most studied thyroid hormone receptor β (TRβ)-selective thyromimetics in the field due to its excellent selectivity and potency. A small structural change—replacing the 3,5-dimethyl groups of sobetirome with either chlorine or bro

Full text of DOI:10.1002/cmdc.201600408

DOI: 10.1002/cmdc.201600408
Full Papers
Increasing Thyromimetic Potency through Halogen
Substitution
Jordan Devereaux, Skylar J. Ferrara, Tania Banerji, Andrew T. Placzek, and
[a]
Thomas S. Scanlan*
Sobetirome is one of the most studied thyroid hormone recep-
tor b (TRb)-selective thyromimetics in the field due to its excel-
lent selectivity and potency. A small structural change—replac-
ing the 3,5-dimethyl groups of sobetirome with either chlorine
or bromine—produces significantly more potent compounds,
both in vitro and in vivo. These halogenated compounds
induce transactivation of a TRb-mediated cell-based reporter
with an EC50 value comparable to that of T3, access the central
nervous system (CNS) at levels similar to their parent, and acti-
vate an endogenous TR-regulated gene in the brain with an
EC50 value roughly five-fold lower than that of sobetirome. Pre-
vious studies suggest that this apparent increase in affinity can
be explained by halogen bonding between the ligand and
a backbone carbonyl group in the receptor. This makes the
new analogues potential candidates for treating CNS disorders
that may respond favorably to thyroid-hormone-stimulated
pathways.
Introduction
Thyroid hormone (TH) in the form of 3,3’,5-triiodothyronine
bone demineralization associated with chronic hyperthyroid-
ism. A more promising approach comes from thyroid hormone
analogues that can activate TH-responsive genes while avoid-
ing the associated downsides of TH by exploiting molecular
and physiological properties of thyroid hormone receptors
(T3) derived from deiodination of the pro-hormone tetraiodo-
thyronine (T4) (Figure 1) is a key signal for oligodendrocyte dif-
ferentiation and myelin formation during development and
also stimulates remyelination in adult models of multiple scle-
[
1]
[2]
rosis (MS). However, TH is not an acceptable long-term thera-
py due to the lack of a therapeutic window in which remyeli-
nation can be induced while avoiding the cardiotoxicity and
(TRs). These receptors are expressed in two major forms with
heterogeneous tissue distribution and overlapping but distinct
[3]
sets of target genes. TRa is enriched in the heart, brain, and
[4]
bone, whereas TRb is enriched in the liver. Developing selec-
tive thyromimetics has been challenging due to the high se-
quence similarity of TR subtypes; only one amino acid residue
on the internal surface of the ligand binding domain cavity
varies between the a and b forms. Sobetirome (Figure 1) was
1
1
one of the first potent analogues that demonstrated significant
[5,6]
[7–9]
TRb selectivity in vitro
and in vivo.
While sobetirome was
designed as a cardiac-sparing drug for treating hypercholester-
[10]
olemia by activating TRb in the liver, it is unique within the
thyromimetic field in possessing significant distribution in the
[7,11]
brain.
Recent studies highlight the potential advantage of
this property by demonstrating its activity in in vitro models of
[12]
human CNS demyelination diseases such as MS and X-linked
[13]
adrenoleukodystrophy (X-ALD).
Although sobetirome is a very promising drug candidate for
central nervous system (CNS) indications, its potency is lower
than that of T3 (Figure 1). A more potent analogue would
evoke the same effects at a lower dose or greater stimulation
of the target at the same dose. While many of the structural
features of sobetirome are critical for its binding affinity and
Figure 1. Structures of selected TR agonists.
[6,12]
[a] Dr. J. Devereaux, Dr. S. J. Ferrara, T. Banerji, Dr. A. T. Placzek,
receptor selectivity,
the 3,5-dimethyl constituents have not
Prof. T. S. Scanlan
been investigated. There is a large body of computational and
biological structure–activity relationship (SAR) data demon-
strating that thyromimetics with 3,5-dimethyl substitutions
have significantly lower activity than structurally similar ana-
Department of Physiology & Pharmacology, Program in Chemical Biology,
Oregon Health & Sciences University,
3
181 SW Sam Jackson Park Road, Portland, OR 97239 (USA)
E-mail: scanlant@ohsu.edu
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logues with 3,5-position halogen substitutions. In the TRa-se-
to the halogen substitutions decreasing the nucleophilicity of
the phenol. However, the reaction went to completion and in
good yield after converting the chloroacetate into an iodoace-
tate via an in situ Finklestein reaction. After forming the tert-
butyl oxyacetate intermediate, the carbon–carbon bond forma-
tion proceeded in the same fashion as with sobetirome by
forming an arylmagnesium with 7 that attacked the benzalde-
hyde to form a carbinol intermediate. The arylmagnesium nu-
cleophile was critical to the success of the synthesis, as it does
not exchange with aryl chlorides or bromides at cryogenic
temperatures and is compatible with the tert-butyl ester pro-
lective compounds CO-22 and CO-24 (Figure 1) replacement of
3
,5-dimethyl groups with bromines was found to improve
[
14,15]
binding affinity 15-fold.
A computational SAR study of thy-
roid hormone analogues suggested a potential mechanism for
these findings: halogen atoms at the 3,5-position can form
a dipole–dipole interaction with a backbone carbonyl group in
the TR ligand binding domain, which influences binding affini-
[
16]
ty and selectivity. Halogen bonding interactions are strongly
influenced by the geometry of the donor and halogen, as the
dipoles must be close enough to interact and oriented so that
the lone pair electrons of the donor and the partial positive
[20]
tecting group. Reduction of the carbinol and deprotection
of the tert-butyl ester and methoxymethyl ether protecting
groups proceeded simultaneously with trifluoroacetic acid and
triethylsilane in dichloromethane. The dibromo analogue JD-20
was synthesized in 27% overall yield, and the dichloro ana-
logue JD-21 was synthesized in 17% overall yield, both in five
steps.
[
17]
charge on the halogen face each other. The interaction is
generally stronger with increasing halogen atom size due to
the increasing polarizability of larger halogens producing
a greater partial positive charge on the atom. These data sug-
gest that replacing the 3,5-dimethyl groups of sobetirome with
halogens may result in thyromimetics with higher affinity and
potency at the thyroid hormone receptor.
A electroporation-based transfection system with U2OS or
HeLa cells was previously used for measuring the potency of
[5,15]
TR-mediated transactivation.
The electroporation method
Results and Discussion
was limited by poor transfection efficiencies and decreased dy-
namic range that delivered inconsistent EC50 values for T3 at
each receptor subtype. Previous reports suggested that lipo-
fectamine reagents sequestered lipophilic compounds such as
3
,5-position halogen analogues of sobetirome required a new
[18]
synthetic approach (Scheme 1). Work by Dabrowski et al.
provided a template for producing the necessary 4-hydroxy-
,6-dihalobenzaldehyde intermediates by selectively deproto-
[21]
2
sobetirome. The electroporation-based assay was modified
and optimized using Lipofectamine 2000 and HEK293 cells. In
comparison with the previous method, the new version has
significantly higher transfection efficiencies and a greater dy-
namic range while being operationally simpler and delivering
similar EC50 values for T3 at both subtypes.
nating the 4-position of silyl-protected 3,5-dihalophenols with
lithium amide reagents. The method was improved by replac-
ing the methyl ether and trimethylsilyl ether protecting groups
used by Dabrowski et al. with the sterically bulkier triethylsilyl
ether protecting group, which significantly improved the selec-
tivity of the deprotonation. These intermediates were used in
a slightly altered version of the recently reported improved so-
Transactivation assays showed that JD-20 and JD-21 have
greater potency than their parent compound sobetirome. In-
creases in potency at TRa were significant, with more modest
improvements at TRb, boosting the analogues to match the
EC50 value of T3 (Figure 2, Table 1).
[
19]
betirome synthesis.
The 4-hydroxy-2,6-dihalobenzaldehyde
intermediates could not be alkylated with tert-butyl chloroace-
tate using the standard cesium carbonate/DMF conditions due
Scheme 1. Synthesis of JD-20 9a and JD-21 9b. Reagents and conditions: a) triethylsilyl chloride, imidazole, CH
788C, 2. DMF, 56–67%; c) tert-butylchloroacetate, NaI, Cs CO , acetone, 60–658C, 84–88%; d) NaI, NaOH, NaOCl, MeOH, H
CH Cl , H Cl , 08C!RT, 58–69%.
O, 81%; f) 1. iPrMgCl, THF, 08C!RT, 2. 4a or 4b, ꢀ788C, 54–79%; g) TFA, triethylsilane, CH
2
Cl
2
, 08C, 95%; b) 1. nBuLi, DIA or TMP, THF,
ꢀ
2
3
2
O, 87%; e) MOMCl, TBAI, NaOH,
2
2
2
2
2
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doses of sobetirome, JD-20, or JD-21. Tissue and blood were
collected 1 h post-injection, and the concentration of the
drugs was determined by LC–MS/MS analysis (Figure 3). JD-20
and JD-21 showed brain uptake similar to that of sobetirome,
while the serum levels of JD-20 and JD-21 were both signifi-
cantly (p<0.05) lower than sobetirome. The brain/serum ratio
of JD-21 trended higher than sobetirome (p=0.055), while JD-
20 had a brain/serum ratio similar to sobetirome.
Induction of the TR-regulated gene Hairless (Hr) in the brain
was determined by qPCR and normalized to glyceraldehyde
-phosphate dehydrogenase (GAPDH) mRNA (Figure 4).
Vehicle (1:1 saline/DMSO) was used as a negative control, and
3
ꢀ
1
saturating doses of T3 (0.305 mmolkg ) and sobetirome
ꢀ
1
(
9.14 mmolkg ) were used as positive controls. JD-20 and JD-
ꢀ
1
2
1 at 0.914 mmolkg had similar Hr induction to a 10-fold
higher dose of sobetirome.
The EC50 values of Hr mRNA induction in the brain normal-
ized to GAPDH mRNA expression by sobetirome, JD-20, and
JD-21 (Figure 4) were determined using the same experimental
protocol. The respective EC50 values for sobetirome, JD-20, and
ꢀ
1
JD-21 were 8.20ꢁ12.65, 1.49ꢁ1.08, and 1.21ꢁ1.75 mmolkg ,
making the halogenated analogues roughly six-fold more
potent than sobetirome at inducing Hr mRNA expression in
the brain.
Figure 2. TRE-driven dual luciferase transactivation assays with calculated
sigmoidal dose–response curves against a) hTRa and b) hTRb in transiently
transfected HEK293 cells. Plots show data normalized to T3 response as
mean values of triplicates with standard error.
1
1
Conclusions
While sobetirome has become one of the standard TRb-selec-
tive thyromimetics in the field, this study makes clear that
a simple structural change—replacing the 3,5-dimethyl groups
with bromine or chlorine—produces more potent compounds
that largely maintain TR-isoform selectivity and brain uptake
properties of the parent compound. This improvement is po-
tentially due to a halogen bonding interaction between the
ligand and a backbone carbonyl group in the TR ligand bind-
ing domain. These results suggest that JD-20 and JD-21 may
be useful molecules for studying and treating CNS demyelina-
tion diseases such as X-ALD and MS.
Table 1. Subtype selectivity measured by EC50 values from TRE-driven
dual luciferase transactivation assays.
[
a]
Compound
EC50 [nm]
TRa/TRb
TRa
TRb
T3
1.0ꢁ0.4
74.7ꢁ28.9
8.0ꢁ7.0
1.5ꢁ1.6
2.8ꢁ1.8
0.9ꢁ1.1
1.2ꢁ1.3
0.7
26.5
9.0
Sobetirome
JD-20
JD-21
7.8ꢁ3.6
6.3
[a] Data are the meanꢁSEM of n=1 experiment performed in triplicate.
A distribution study was carried out in C57BL/6J mice to de-
The improved potency of JD-20 and JD-21 is consistent with
numerous thyromimetic SAR studies that have found superior
activity for analogues with halogen atoms at the 3,5-position
termine the concentrations in brain and serum after systemic
ꢀ
1
(
i.p.) administration. Mice were given single 9.14 mmolkg
ꢀ1
Figure 3. In vivo concentrations of GC-1, JD-20, and JD-21 in C57BL/6 mouse tissues 1 h after systemic administration (9.14 mmolkg i.p.), measured by LC–
MS/MS in a) brain and b) serum; c) the ratio of brain/serum drug concentration; *p<0.05 relative to sobetirome.
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analogues into the CNS occurs primarily through an active
transport mechanism.
Experimental Section
Biological assays
Transactivation assay: Human epithelial kidney (HEK293) cells
ꢀ1
were grown to 80% confluency in high-glucose (4.5 gL ) Dulbec-
co’s modified Eagle’s medium (DMEM) containing 10% fetal
ꢀ1
ꢀ1
bovine serum (FBS), 50 UmL penicillin, and 50 mgmL strepto-
mycin. Cells were trypsinized with 0.25% trypsin, then diluted to
5
5
ꢁ10 cells per mL with high-glucose DMEM. Cells were added to
4
Costar 3917 96-well plates at 5ꢁ10 cells per well, then incubated
at 378C for 24 h. TR expression vector (1.5 mg; full-length TRa–CMV
or TRb–CMV), reporter plasmid (1.5 mg) containing a DR4 thyroid
hormone response element (TRE) direct repeat spaced by four nu-
cleotides (AGGTCAcaggAGGTCA) cloned upstream of a minimal
thymidine kinase promoter linked to a firefly luciferase coding se-
quence, and a pRL–SV40 constitutive Renilla luciferase reporter
plasmid (0.75 mg) were diluted into OptiMEM (540 mL). Lipofecta-
mine 2000 reagent (27 mL) was diluted into 540 mL OptiMEM. The
plasmid and lipofectamine dilutions were combined, then incubat-
ed at room temperature for 10 min. The mixture was then diluted
into 4.29 mL OptiMEM. Plates were washed with 100 mL phos-
phate-buffered saline (PBS) at pH 7.2 without magnesium or calci-
um chloride per well. Transfection mixtures were added at 50 mL
per well, then incubated at 378C for 4 h. Modified DME/F-12 Ham’s
medium without phenol red containing 15 mm HEPES and bicar-
ꢀ
1
bonate, 5 mm l-glutamine, charcoal-stripped FBS, 50 UmL peni-
ꢀ1
cillin, and 50 mgmL streptomycin was added at 50 mL per well,
then the plates were incubated at 378C for 20 h. Drug stocks were
made at 10 mm in DMSO, then serially diluted to 1ꢁ concentra-
tions in DME/F-12 Ham’s. Plates were washed with 100 mL PBS
(pH 7.2) per well. Each drug stock (100 mL) was added to the wells
in triplicate, then the plates were incubated at 378C for 24 h. Cells
were assayed for luciferase activity using the Promega DualGlo kit.
Luciferase Reagent (50 mL) was added to each well, the plate was
rocked for 15 min at room temperature, and then read for firefly
luciferase activity. Stop & Glo Reagent (50 mL) was added to each
well, then the plate was read for Renilla luciferase activity. Data
normalized to Renilla internal control were analyzed with GraphPad
Prism v.4a using the sigmoidal dose–response model to generate
EC50 values ꢁSEM. Experiments were performed in triplicate.
Animal studies: Experimental protocols were in compliance with
the US National Institutes of Health Guide for the Care and Use of
Laboratory Animals, and were approved by the Oregon Health &
Science University Institutional Animal Care & Use Committee.
Wild-type male C57BL/6J mice, aged 8–10 weeks, were housed in
a climate-controlled room with a 12-h light–dark cycle with ad libi-
tum access to food and water.
Figure 4. a) Expression of TR-regulated gene Hairless (Hr) mRNA normalized
to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA measured
by qPCR in C57BL/6mouse brain (three mice per dose) 2 h after systemic ad-
ꢀ
1
ministration (i.p.) of saturating doses of T3 (0.305 mmolkg ) or sobetirome
ꢀ
1
(
9
9.15 mmolkg ) plus escalating doses of JD-20 and JD-21 (0.9 and
ꢀ
1
.15 mmolkg ). b) Expression of TR-regulated gene Hairless (Hr) mRNA nor-
malized to GAPDH mRNA measured by qPCR in C57BL/6 mouse brain (three
mice per dose) 2 h after systemic administration (i.p.) of GC-1, JD-20, and
JD-21.
compared with similar analogues with methyl groups at those
positions. It is surprising in this instance that by most meas-
ures JD-20 and JD-21 appear to have very similar properties.
The previous thyromimetic SAR studies found that changing
the halogen substitution frequently produced dramatic shifts
[
16]
in both potency and selectivity. However, the in vitro and
in vivo potencies of JD-20 and JD-21 are roughly the same
within error. This is different from most known thyromimetics
and suggests something unique about the chemical context of
the sobetirome scaffold. Additionally, except for the improve-
ments in potency, the only major difference between these
novel compounds and sobetirome is the decreased serum con-
centrations. This hints that these compounds may be distribut-
ed by different mechanisms, such as being actively transported
across the blood–brain barrier rather than by passive diffusion,
where halogen bonding or steric effects might alter the rela-
tive uptake rates through a putative transporter. It will be im-
portant to determine whether uptake of sobetirome and these
Distribution studies: Mice were injected once intraperitoneally
ꢀ
1
(
i.p.) with drugs at 9.14 mmolkg , and analogues at 0.914, 9.14,
ꢀ1
and 30.5 mmolkg . Euthanasia was performed on three mice per
dose at 1 h, and the tissues and blood were harvested. Tissues
were immediately frozen, and blood was kept on ice for a minimum
of 30 min and then centrifuged at 7500 g for 15 min. Serum
(100 mL) was collected and was stored with tissues at ꢀ808C until
samples were processed.
Serum processing: The serum samples were warmed to room
temperature, and 10 mL of internal standard (2.99 mm
[D ]sobetirome) was added to them. Acetonitrile (500 mL) was
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added, and the sample was vortexed for 20 s. The sample was
then centrifuged at 10000 g for 15 min at 48C. Next, 90% of the
upper supernatant was transferred to a glass test tube and concen-
trated by centrifugal evaporation (SpeedVac) for 1.5 h at 458C. The
from a Seca Solvent System. All other solvents used were pur-
chased from Sigma–Aldrich or Fisher. Purity analysis of final com-
pounds was determined to be >95% by HPLC. HPLC analysis was
performed on a Varian ProStar instrument with an Agilent Eclipse
Plus C18 5 mm column (4.6ꢁ250 mm) with a gradient of 10–95%
MeCN (0.1% TFA) over 15 min.
dried sample was then dissolved in 400 mL MeCN/H O (50:50) and
2
vortexed for 20 s. The resulting mixture was transferred to an Ep-
pendorf tube and centrifuged at 10000 g for 15 min. The superna-
tant was filtered with 0.22 mm centrifugal filters and submitted for
LC–MS/MS analysis. The standard curve was made with 100 mL
serum from an 8- to 10-week-old mouse not injected with T3, so-
betirome, or analogues. The processing was performed exactly the
same, except after filtering the sample was split amongst six vials.
To five out of the six vials was added sobetirome, JD-20, and JD-21
to make final concentrations of each compound in a matrix of: 0.1,
(
2
3,5-dibromophenoxy)triethylsilane (2a). Compound 1a (5.04 g,
0 mmol) and imidazole (4.09 g, 60 mmol) were dissolved in 80 mL
of CH Cl . The solution was cooled to 08C, then triethylsilyl chloride
2
2
(5.03 mL, 30 mmol) was added, then the reaction was stirred at
0
8C for 30 min. The reaction was diluted with 160 mL Et O,
2
washed with H O (2ꢁ50 mL) and brine (2ꢁ50 mL), then dried with
2
MgSO , filtered, and concentrated to give 2a in quantitative yield,
4
1
ꢀ
1
which was used without purification. H NMR (400 MHz, CDCl ): d=
3
1
, 10, 100, and 1000 pgmL .
7
.37 (t, 1H, J=1.6 Hz), 7.10 (d, 2H, J=1.6 Hz), 1.02 (t, 9H, J=
Brain processing: The brain samples were warmed to room tem-
perature and transferred to a homogenizer tube with five Gold-
Spec 1/8-inch (diameter) chrome steel balls (Applied Industrial
7.9 Hz), 0.82 ppm (q, 6H, J=7.9 Hz).
4
-hydroxy-2,6-dibromobenzaldehyde (3a). A flask was loaded
with molecular sieves, then flame-dried under vacuum. After cool-
ing under argon, 2a (5.49 g, 15 mmol) was loaded, then the flask
was sealed, evacuated, and flushed with argon. Dry THF (30 mL)
was added and degassed, then the solution was cooled to ꢀ788C.
A second flask was loaded with molecular sieves, then flame-dried
under vacuum. After cooling under argon, diisopropylamine
Technologies). The resulting tube was weighed, and then H O
2
(
1 mL) was added, followed by 10 mL of 2.99 mm internal standard
(
[D ]sobetirome). The tube was homogenized with a Bead Bug ho-
6
mogenizer (Benchmark Scientific, Edison, NJ (USA)) for 30 s and
then transferred to a falcon tube containing 3 mL MeCN. MeCN
(
1 mL) was used to wash the homogenizer tube, and the solution
(4.6 mL, 33 mmol) was added, followed by 60 mL dry THF, then the
was transferred back to the falcon tube. The sample was then pro-
cessed using the same method for the serum processing, except
the sample was concentrated in a glass tube using a SpeedVac for
solution was degassed and cooled to ꢀ788C. n-Butyllithium (2.5m
solution in hexanes; 12 mL, 30 mmol) was added, then the solution
was stirred for 1 h at ꢀ788C. The lithium diisopropylamide solution
was transferred dropwise via cannula to the 2a solution, then the
deprotonation was stirred for 1 h at ꢀ788C. Dry DMF (5.8 mL,
75 mmol) was added, then the reaction was stirred for 1 h at
ꢀ788C. The reaction was decanted into 50 mL 1n aqueous HCl.
4
h at 458C.
Gene activation: Mice were injected once intraperitoneally (i.p.)
ꢀ
1
with vehicle (1:1 saline/DMSO), T3 at 0.305 mmolkg , sobetirome
ꢀ
1
ꢀ1
at 9.14 mmolkg , and analogues at 0.914 and 9.14 mmolkg . Eu-
thanasia was performed on three mice per dose at 2 h, and the tis-
sues were harvested. The brain tissues collected for qPCR analysis
were processed according to a protocol for RNA extraction using
Trizol reagent and the PureLink RNA mini kit, using a Qiagen
RNase-free DNase kit during the optional DNase treatment step.
Extracted RNA (1 mg) was used to synthesize cDNA via a reverse
transcription (RT) reaction using the Qiagen QuantiTect Reverse
Transcription kit. DNA contamination was controlled for by dupli-
cating one sample without the addition of RT. Expression of the
Hairless (Hr) gene was measured by qPCR using the QuantiTect
SYBR green PCR kit from Qiagen. The primer sequences for hairless
The aqueous layer was extracted with Et O (3ꢁ90 mL). The organic
2
fractions were combined, washed with brine (2ꢁ50 mL), then
dried with MgSO , filtered, and concentrated to give the crude
4
product, which was precipitated from hexanes at ꢀ788C to give
1
2.8 g of 3a (67% yield). H NMR (400 MHz, [D ]acetone): d=10.16
6
(s, 1H), 7.27 ppm (s, 2H).
tert-butyl 2-(3,5-dibromo-4-formylphenoxy)acetate (4a). Com-
pound 3a (2.8 g, 10 mmol), sodium iodide (3 g, 20 mmol), and
cesium carbonate (3.24 g, 10 mmol) were dissolved in 40 mL ace-
tone. tert-Butyl chloroacetate (2.86 mL, 20 mmol) was added, then
the reaction was held at reflux at 658C for 2 h. The reaction was di-
(
Fwd: CCA AGT CTG GGC CAA GTT TG; Rev: TGT CCT TGG TCC GAT
[22]
luted with 80 mL of Et O, washed with water (2ꢁ30 mL) and brine
2
TGG AA) were previously described by Barca-Mayo et al.
The
(
2ꢁ30 mL), then dried with MgSO , filtered, and concentrated. The
4
template cDNA was diluted two-fold to minimize the interference
of RT reagents in the qPCR reaction. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was the housekeeping gene used for nor-
malizing between samples. Data analysis for single-dose experi-
product was precipitated from hexanes and collected by filtration,
then dried under vacuum to give 3.49 g of 4a (88% yield). H NMR
1
(
400 MHz, CDCl ): d=10.23 (s, 1H), 7.19 (s, 2H), 4.59 (s, 2H),
3
1
.52 ppm (s, 9H).
ments was done using the comparative C method to monitor the
T
relative differences in Hr gene expression. Data analysis for dose–
response experiments was done using GraphPad Prism v.4a with
the sigmoidal dose–response model to generate EC50 values ꢁ
SEM. Experiments were performed in triplicate.
(
4
3,5-dichlorophenoxy)triethylsilane (2b). Compound 1b (6.54 g,
0 mmol) and imidazole (8.18 g, 120 mmol) were dissolved in
160 mL of CH Cl . The solution was cooled to 08C, then triethylsilyl
chloride (10 mL, 60 mmol) was added, then the reaction was stirred
2
2
at 08C for 30 min. The reaction was diluted with 320 mL of Et O,
2
washed with H O (2ꢁ100 mL) and brine (2ꢁ75 mL), then dried
2
with MgSO , filtered, and concentrated to give 2b, which was used
Chemistry
4
without purification and weighed 10.58 g after drying (95% yield).
1
General: H NMR spectra were taken on a Bruker 400 instrument.
1
H NMR (400 MHz, CDCl ): d=6.98 (t, 1H, J=1.8 Hz), 6.76 (d, 2H,
3
1
13
All H and C NMR data were calibrated to the NMR solvent refer-
ence peak ([D ]acetone, CDCl ). High-resolution mass spectrometry
J=1.8 Hz), 1.02 (t, 9H, J=7.9 Hz), 0.77 ppm (q, 6H, J=7.9 Hz).
6
3
4
-hydroxy-2,6-dichlorobenzaldehyde (3b). A flask was loaded
(
HRMS) with electrospray ionization was performed by the Bioana-
with molecular sieves, then flame-dried under vacuum. After cool-
ing under argon, 2b (3.6 g, 13 mmol) was loaded, then the flask
lytical MS Facility at Portland State University. Anhydrous tetrahy-
drofuran (THF) and N,N-dimethylformamide (DMF) were obtained
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was sealed, evacuated, and flushed with argon. Dry THF (13 mL)
was added and degassed, then the solution was cooled to ꢀ788C.
A second flask was loaded with molecular sieves, then flame-dried
under vacuum. After cooling under argon, 2,2,6,6-tetramethylpiper-
didine (1.84 g, 13 mmol) was added, followed by 13 mL of dry THF,
then the solution was degassed and cooled to ꢀ788C. n-Butyllithi-
um (2.5m solution in hexanes; 5.2 mL, 13 mmol) was added, then
the solution was stirred for 20 min at 08C. The lithium TMP solu-
tion was transferred dropwise via cannula to the 2b solution, then
the deprotonation was stirred for 30 min at ꢀ788C. Dry DMF
evacuated, and flushed with argon. Dry THF (24 mL) was added
and degassed, then the solution was cooled to 08C. Isopropylmag-
nesium chloride (2m THF, 5.5 mL, 7.2 mmol) was added, then the
reaction was stirred for 2 h at room temperature. A second flask
was loaded with 4 ꢂ molecular sieves and flame-dried under
vacuum. Compound 4a (946 mg, 2.4 mmol) was loaded and the
flask was sealed, evacuated, and flushed with argon. Dry THF
(12 mL) was added and degassed. The arylmagnesium solution was
cooled to ꢀ788C, then the 4a solution was added dropwise via
cannula, and the reaction was stirred for 1 h at ꢀ788C. The reac-
tion was quenched with 10 mL of 1n aqueous HCl. The aqueous
layer was extracted with EtOAc (3ꢁ10 mL). The organic fractions
were combined and washed with brine (2ꢁ10 mL). The organic
(
5 mL, 65 mmol) was added, then the reaction was stirred for
3
0 min at ꢀ788C. The reaction was decanted into 15 mL 1n aque-
ous HCl. The aqueous layer was extracted with EtOAc (3ꢁ15 mL).
The organic fractions were combined, washed with brine (2ꢁ
layer was dried with MgSO , filtered, and concentrated to give the
4
1
5 mL), then dried with MgSO , filtered, and concentrated to give
crude product, which was purified by flash chromatography (silica
4
the crude product, which was recrystallized from hexanes at
gel, hexanes/EtOAc 4–40%) to give 1.089 g of 8a (79% yield).
1
1
ꢀ
208C to give 1.39 g of 3b (56% yield). H NMR (400 MHz,
D ]acetone): d=10.37 (s, 1H), 7.01 ppm (s, 2H).
6
H NMR (400 MHz, CDCl ): d=7.24 (d, 1H, J=1.9 Hz), 7.17 (s, 2H),
3
[
7.00 (d, 1H, J=8.5 Hz), 6.90 (dd, 1H, J=8.7 Hz, 1.9 Hz), 6.51 (d, 1H,
J=10.8 Hz), 5.21 (s, 2H), 4.53 (s, 2H), 3.49 (s, 3H), 3.36 (d, 1H, J=
tert-butyl 2-(3,5-dichloro-4-formylphenoxy)acetate (4b). Com-
pound 3b (1.15 g, 6 mmol), sodium iodide (1.8 g, 12 mmol), and
cesium carbonate (1.94 g, 6 mmol) were dissolved in 24 mL of ace-
tone. tert-Butyl chloroacetate (1.72 mL, 12 mmol) was added, then
the reaction was held at reflux at 608C for 24 h. The reaction was
1
6
0.8 Hz) 3.34 (m, 1H, J=6.9 Hz), 1.52 (s, 9H), 1.21 ppm (t, 6H, J=
.5 Hz).
2-(3,5-dibromo-4-((3-isopropyl-4-hydroxyphenyl)methyl)phe-
noxy)acetic acid (9a). Compound 8a (1.089 g, 1.9 mmol) was dis-
diluted with Et O (30 mL), washed with water (2ꢁ10 mL) and brine
solved in 19 mL of CH Cl2 with 1.21 mL of triethylsilane
2
2
(
2ꢁ10 mL), then dried with MgSO , filtered, and concentrated. The
(7.58 mmol). The solution was cooled to 08C, then TFA (4.35 mL,
56.9 mmol) was added, and the reaction was stirred for 30 min at
08C, then 2 h at room temperature. The solvent was removed
under vacuum, then the product was precipitated by the addition
of hexanes and collected by filtration. The solid was dried under
4
crude oil was re-dissolved in a minimal amount of Et O then added
2
dropwise to 100 mL of vigorously stirring hexanes at ꢀ788C. The
precipitate was collected by filtration and dried under vacuum to
1
give 1.545 g of 4b (84% yield). H NMR (400 MHz, CDCl ): d=10.43
(
3
1
s, 1H), 6.92 (s, 2H), 4.59 (s, 2H), 1.52 ppm (s, 9H).
vacuum to give 505 mg of JD-20 (9a) (58% yield). H NMR
(
1
2
400 MHz, CDCl ): d=7.19 (s, 2H), 7.10 (d, 1H, J=1.9 Hz), 6.82 (dd,
3
4
-iodo-2-isopropylphenol (6). Compound 5 (6.8 g, 50 mmol) and
H, J=8.7 Hz, 1.9 Hz), 6.64 (d, 1H, J=10.8 Hz), 4.68 (s, 2H), 4.28 (s,
NaI (7.5 g, 50 mmol) were dissolved in 70 mL of MeOH. Aqueous
NaOH (10m; 5 mL, 50 mmol) was added, then the solution was
cooled to 08C. Aqueous NaOCl (6.25% w/v; 62.5 mL, 50 mmol) was
added dropwise over 24 h at 08C. The reaction was acidified to
pH 7 with 12n aqueous HCl, then quenched with 10 mL saturated
aqueous Na S O . The aqueous layer was extracted with Et O (3ꢁ
13
H), 3.18 (m, 1H, J=6.9 Hz), 1.24 ppm (d, 6H, J=6.9 Hz); C NMR
(
400 MHz, CDCl ): d=173.12, 156.17, 151.03, 134.18, 133.67, 130.33,
3
1
2
4
26.90, 126.08, 126.02, 118.98, 115.13, 64.87, 28.04, 27.04,
+
2.57 ppm; HRMS exact mass calculated for C H Br O [M+H] :
18
17
2
4
59.94687, found: 459.94647.
2
2
3
2
1
00 mL). The organic fractions were combined, washed with brine
tert-butyl 2-(3,5-dichloro-4-(hydroxy(3-isopropyl-4-(methoxyme-
thoxy)phenyl)methyl)phenoxy)acetate (8b). A flask was loaded
with 4 ꢂ molecular sieves and flame-dried under vacuum. Com-
pound 7 (459 mg, 1.5 mmol) was loaded, and the flask was sealed,
evacuated, and flushed with argon. Dry THF (6 mL) was added and
degassed, then the solution was cooled to 08C. Isopropylmagnesi-
um chloride (2m THF, 1.125 mL, 2.25 mmol) was added, then the
reaction was stirred for 2 h at room temperature. A second flask
was loaded with 4 ꢂ molecular sieves and flame-dried under
vacuum. Compound 4b (305 mg, 1 mmol) was loaded and the
flask was sealed, evacuated, and flushed with argon. Dry THF
(
2ꢁ100 mL), then dried with MgSO , filtered, and concentrated to
4
give the crude product, which was purified by flash chromatogra-
phy (silica gel, hexane/EtOAc, 1–20%) to give 11.35 g of 6 (87%
yield) as a reddish oil. H NMR (400 MHz, CDCl ): d=7.47 (d, 1H,
J=2.1 Hz), 7.36 (dd, 1H, J=8.4 Hz, 2.2 Hz), 6.54 (d, 1H, J=8.4 Hz),
1
3
3
.16 (m, 1H, J=6.9 Hz), 1.25 ppm (d, 6H, J=6.9 Hz).
4
6
1
1
6
-iodo-2-isopropyl-1-(methoxymethoxy)benzene (7): Compound
(2.62 g, 10 mmol) and tetrabutylammonium iodide (369 mg,
mmol) were dissolved in 100 mL of CH Cl . Aqueous NaOH (10m,
2
2
0 mL) was added, followed by chloromethyl methyl ether (5 mL,
m in MeOAc). The reaction was stirred for 30 min at room tem-
(4 mL) was added and degassed. The arylmagnesium solution was
cooled to ꢀ788C, then the 4b solution was added dropwise via
cannula, and the reaction was stirred for 1 h at ꢀ788C. The reac-
tion was quenched with 5 mL of 1n aqueous HCl. The aqueous
layer was extracted with EtOAc (3ꢁ5 mL). The organic fractions
were combined and washed with brine (2ꢁ5 mL). The organic
perature, then diluted with 200 mL of Et O. The organic layer was
washed with H O (2ꢁ100 mL) and brine (2ꢁ100 mL), then dried
with MgSO , filtered, and concentrated to give the crude product,
which was purified by flash chromatography (silica gel, hexane/
EtOAc, 1–20%) to give 2.48 g of 7 (81% yield). H NMR (400 MHz,
CDCl ): d=7.47 (d, 1H, J=2.2 Hz), 7.42 (dd, 1H, J=8.6 Hz, 2.2 Hz),
2
2
4
1
layer was dried with MgSO , filtered, and concentrated to give the
4
3
crude product, which was purified by flash chromatography (silica
6
6
.83 (d, 1H, J=8.6 Hz), 5.18 (s, 2H), 3.47 (s, 3H), 3.27 (m, 1H, J=
.9 Hz), 1.20 ppm (d, 6H, J=7 Hz).
gel, hexanes/EtOAc 2–20%) to give 260 mg of 8b (54% yield).
1
H NMR (400 MHZ, CDCl ): d=7.26 (d, 1H, J=2.1 Hz), 6.99 (d, 1H,
3
tert-butyl 2-(3,5-dibromo-4-(hydroxy(3-isopropyl-4-(methoxyme-
thoxy)phenyl)methyl)phenoxy)acetate (8a). A flask was loaded
with 4 ꢂ molecular sieves and flame-dried under vacuum. Com-
pound 7 (1.47 g, 4.8 mmol) was loaded, and the flask was sealed,
J=8.5 Hz), 6.93 (dd, 1H, J=8.2 Hz, 2.2 Hz), 6.92 (s, 2H), 6.50 (d, 1H,
J=10.8 Hz), 5.21 (s, 2H), 4.53 (s, 2H), 3.50 (s, 3H), 3.33 (m, 1H, J=
6.9 Hz), 3.23 (d, 1H, J=10.8 Hz), 1.52 (s, 9H), 1.21 ppm (t, 6H, J=
6.8 Hz).
&
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www.chemmedchem.org
6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
2
-(3,5-dichloro-4-((3-isopropyl-4-hydroxyphenyl)methyl)phe-
[5] G. Chiellini, J. W. Apriletti, H. A. Yoshihara, J. D. Baxter, R. C. J. Ribeiro,
T. S. Scanlan, Chem. Biol. 1998, 5, 299–306.
noxy)acetic acid (9b). Compound 8b (260 mg, 0.54 mmol) was
[
[
6] H. A. I. Yoshihara, J. W. Apriletti, J. D. Baxter, T. S. Scanlan, J. Med. Chem.
003, 46, 3152–3161.
dissolved in 5.4 mL of CH Cl2 with 0.345 mL of triethylsilane
2
2
(
2.16 mmol). The solution was cooled to 08C, then TFA (1.24 mL,
7] S. U. Trost, E. Swanson, B. Gloss, D. B. Wang-Iverson, H. Zhang, T. Volo-
darsky, G. J. Grover, J. D. Baxter, G. Chiellini, T. S. Scanlan, W. H. Dillmann,
Endocrinology 2000, 141, 3057–3064.
8] G. J. Grover, D. M. Egan, P. G. Sleph, B. C. Beehler, G. Chiellini, N.-H.
Nguyen, J. D. Baxter, T. S. Scanlan, Endocrinology 2004, 145, 1656–1661.
1
0
6.2 mmol) was added, and the reaction was stirred for 30 min at
8C, then 2 h at room temperature. The solvent was removed
under vacuum, then the product was precipitated by the addition
of hexanes and collected by filtration. The solid was dried under
[
1
vacuum to give 137 mg of JD-21 (9b) (69% yield). H NMR
[9] J. D. Baxter, P. Webb, G. Grover, T. S. Scanlan, Trends Endocrinol. Metab.
2004, 15, 154–157.
(
400 MHz, CDCl ): d=7.12 (d, 1H, J=2 Hz), 6.95 (s, 2H), 6.86 (dd,
3
[
10] T. S. Scanlan, Heart Fail Rev. 2010, 15, 177–182.
1
2
H, J=8.2 Hz, 2.2 Hz), 6.64 (d, 1H, J=8.2 Hz), 4.68 (s, 2H), 4.18 (s,
H), 3.17 (m, 1H, J=6.9 Hz), 1.24 ppm (d, 6H, J=6.9 Hz); C NMR
13
[11] N. Takahashi, Y. Asano, K. Maeda, N. Watanabe, Biol. Pharm. Bull. 2014,
7, 1103–1108.
3
(
400 MHz, CDCl ): d=172.18, 155.92, 151.09, 136.36, 134.15, 130.97,
3
[
12] E. G. Baxi, J. T. Schott, A. N. Fairchild, L. A. Kirby, R. Karani, P. Uapinyoy-
1
2
3
30.67, 126.83, 121.46, 115.11, 115.04, 64.84, 27.06, 24.35,
2.53 ppm; HRMS exact mass calculated for C H Cl O [M+H] :
67.04984, found: 367.05053.
ing, C. Pardo-Villamizar, J. R. Rothstein, D. E. Bergles, P. A. Calabresi, GLIA
+
18
17
2
4
2
014, 62, 1513–1529.
[
13] E. C. Genin, C. Gondcaille, D. Trompier, S. Savary, J. Steroid Biochem. Mol.
Biol. 2009, 116, 37–43.
[
[
[
14] C. A. Ocasio, T. S. Scanlan, ACS Chem. Biol. 2006, 1, 585–593.
15] C. A. Ocasio, T. S. Scanlan, Bioorg. Med. Chem. 2008, 16, 762–770.
16] N. F. Valadares, L. B. Salum, I. Polikarpov, A. D. Andricopulo, R. C. Garratt,
J. Chem. Inf. Model. 2009, 49, 2606–2616.
Acknowledgements
This research was supported by a grant from the US National In-
stitutes of Health (T.S.S.). The authors thank D. Koop and L. Bleyle
in the OHSU Pharmacokinetics Core for their technical expertise
and guidance.
[
[
17] P. Auffinger, F. A. Hays, E. Westhof, P. S. Ho, Proc. Natl. Acad. Sci. USA
2
004, 101, 16789–16794.
18] M. Dabrowski, J. Kubicka, S. Luli n´ ski, J. Serwatowski, Tetrahedron Lett.
2005, 46, 4175–4178.
19] A. T. Placzek, T. S. Scanlan, Tetrahedron 2015, 71, 5946–5951.
20] A. Krasovskiy, P. Knochel, Angew. Chem. Int. Ed. 2004, 43, 3333–3336;
Angew. Chem. 2004, 116, 3396–3399.
[
[
Keywords: brain · central nervous system · thyroid hormones ·
[
[
21] H. Yoshihara, J. W. Apriletti, J. D. Baxter, T. S. Scanlan, Bioorg. Med. Chem.
Lett. 2001, 11, 2821–2825.
22] O. Barca-Mayo, X.-H. Liao, M. Alonso, C. Di Cosmo, A. Hernandez, S. Re-
fetoff, R. E. Weiss, Mol. Endocrinol. 2011, 25, 575–583.
thyromimetics
[
1] L. Calzꢃ, M. Fernandez, A. Giuliani, G. D’Intino, S. Pirondi, S. Sivilia, M.
Paradisi, N. Desordi, L. Giardino, Brain Res. Rev. 2005, 48, 339–346.
2] J. Malm, G. J. Grover, M. Fꢄrnegꢅrdh, Mini-Rev. Med. Chem. 2007, 7, 79–
[
86.
[
[
3] P. Yen, Physiol. Rev. 2001, 81, 1097–1142.
4] P. O’Shea, J. H. D. Bassett, S. Cheng, G. R. Williams, Nucl. Recept. Signal.
Received: August 15, 2016
2006, 4, e011.
Published online on && &&, 0000
ChemMedChem 2016, 11, 1 – 8
www.chemmedchem.org
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&
These are not the final page numbers! ÞÞ

FULL PAPERS
J. Devereaux, S. J. Ferrara, T. Banerji,
A. T. Placzek, T. S. Scanlan*
Modifying the structure of the potent
TRb-selective thyroid hormone analogue
sobetirome by replacing the 3,5-dimeth-
yl groups with chlorine or bromine to
exploit halogen bonding interactions
within the TR ligand binding domain
gives compounds with significantly in-
creased potency in vitro and in vivo
while retaining distribution to the CNS.
These improved characteristics make
the new compounds attractive candi-
dates for treating CNS demyelination
diseases influenced by thyroid hormone
stimulation.
&& – &&
Increasing Thyromimetic Potency
through Halogen Substitution
&
ChemMedChem 2016, 11, 1 – 8
www.chemmedchem.org
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!