Characterization of thyroid hormone receptor α (TRα)-specific analogs with varying inner- and outer-ring substituents

Article abstract of DOI:10.1016/j.bmc.2007.10.040

Analogs of the TRα-specific thyromimetic CO23 were synthesized and analyzed in vitro using competitive binding and transactivation assays. Like CO23, all analogs bind to both thyroid hormone receptor subtypes with about the same affinity; however, modification of CO23 by derivatization of the 3′ position of the outer-ring or replacement of the inner-ring iodides with bromides attenuates binding. Despite lacking a preference in binding to TRα, all analogs display TRα-specificity in transactivation assays using U2OS and HeLa cells. At best, several agonists exhibit an approximately 6-12-fold preference in transactivation when tested with TRα in HeLa cells. One analog, CO24, showed in vivo TRα-specific action in a tadpole metamorphosis assay.

Full text of DOI:10.1016/j.bmc.2007.10.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 762–770
Characterization of thyroid hormone receptor a
(TRa)-specific analogs with varying inner-
and outer-ring substituents
Cory A. Ocasioa and Thomas S. Scanlanb,*
aChemistry and Chemical Biology Graduate Program, University of California—San Francisco, 600 16th Street,
San Francisco, CA 94143-2280, USA
bDepartment of Physiology and Pharmacology, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road,
Portland, OR 97239-3098, USA
Received 6 August 2007; revised 2 October 2007; accepted 10 October 2007
Available online 18 October 2007
Abstract—Analogs of the TRa-specific thyromimetic CO23 were synthesized and analyzed in vitro using competitive binding and
transactivation assays. Like CO23, all analogs bind to both thyroid hormone receptor subtypes with about the same affinity; how-
ever, modification of CO23 by derivatization of the 30 position of the outer-ring or replacement of the inner-ring iodides with bro-
mides attenuates binding. Despite lacking a preference in binding to TRa, all analogs display TRa-specificity in transactivation
assays using U2OS and HeLa cells. At best, several agonists exhibit an approximately 6–12-fold preference in transactivation when
tested with TRa in HeLa cells. One analog, CO24, showed in vivo TRa-specific action in a tadpole metamorphosis assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
moter usage: TRa1, TRa2, TRb1, and TRb2.4,6 Ligand
binding to TR induces a conformational change in
the LBD allowing it to induce or repress gene expres-
sion by recruitment of coactivator or corepressor pro-
teins.4 Selective thyromimetics are T3 analogs that,
unlike T3, have tissue selective actions.1,2 A current
guiding hypothesis is that TR subtype selectivity
may correlate with tissue selective actions and TRb-
selective compounds such as GC-1 (Fig. 1) are being
developed as potential therapeutic agents for hyperlip-
idemia and obesity. Until recently, little success had
been reported on the development of TRa-selective
thyromimetics.
Thyroid hormone is a classic endocrine signaling
hormone that mediates a wide variety of regulatory
events affecting growth, development, and meta-
bolism.1–3 In circulation, thyroid hormone exists as
a pro-hormone, 3,5,30,50-tetraiodo-L-thyronine (T4,
Fig. 1), but is converted to its principal active form,
3,5,30-triiodo-L-thyronine (T3, Fig. 1), by deiodination
of one outer-ring position by deiodinases.4,5 Com-
pound T3 exerts its actions by translocating into the
nucleus of target cells and binding to the ligand bind-
ing domain (LBD) of thyroid hormone receptors
(TRs) which are members of the nuclear receptor
superfamily of ligand responsive transcriptional regu-
lators.4 There are two genes for TR, TRa and TRb,
that give rise to an ensemble of four different isoforms
by means of alternative splicing or differential pro-
We recently reported on the synthesis and character-
ization of CO23, the first potent thyromimetic with
TRa-specific effects in vitro and in vivo. This com-
pound demonstrated 3- to 5-fold TRa-specificity in
transactivation assays using U2OS and HeLa cells,
respectively.7 Despite not having an overwhelming
preference for TRa activation, CO23 has profound
effects on precocious Xenopus laevis tadpole metamor-
phosis that correlates with the selective activation of
TRa. In this study, we have prepared a focused panel
of CO23 analogs and evaluated them for TRa selec-
tivity in vitro and in vivo.
Keywords: Thyroid hormone receptors; Thyroid hormone receptor a;
Thyroid hormone receptor b; CO23; CO23 analogs; GC-1; Cardiac
specific; Liver specific; Xenopus laevis; Metamorphosis; Induced meta-
morphosis; In vivo.
*
Corresponding author. Tel.: +1 503 494 9292; fax: +1 503 494
9275; e-mail: scanlant@ohsu.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.040

C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
763
Figure 1. Structures of T3, T4, CO23, and GC-1.
2. Chemistry
3,5-dihalo-L-tyrosine methyl esters (7a–b).7 Compounds
7a–b and boronic acids 5a–f were coupled under Evan’s
conditions using cupric acetate as a catalyst leading to
biaryl ethers (8a–f).7 Amidation of 8a–f in methanol sat-
urated with ammonia gas and Boc-deprotection yields
biaryl ethers with amino acid amide side chains (9a–
f).7 This side chain undergoes cyclization to form the
imidazolidinedione after treatment with para-nitrophe-
nylchloroformate, sodium bicarbonate, and water.7
Deprotection of the TIPS groups with tetrabutylammo-
nium fluoride leads to several CO23 analogs (CO24 and
CO26–CO30). The iodination9 or bromination7 of the 30
position of CO30 leads to two further analogs, CO31
and CO32.
The hydantoin moiety attached to position one of the
inner-ring by a methylene linker was deemed necessary
for conferring TRa-specificity,7 and hence to improve
TRa-specificity, additional modifications to inner-
and outer-ring substituents were examined. Substitu-
tion of the outer-ring was achieved by preparation
or purchase of para-brominated phenols with varying
groups in the ortho-position followed by TIPS protec-
tion (Scheme 1). One of the rare para-brominated phe-
nols was generated by protection of 2-bromophenol
(1) using allyl bromide which gives rise to allyl
2-bromophenyl ether (2). Lithiation of 2 causes it to
undergo an intramolecular carbolithiation/1,3-elimina-
tion reaction that gives rise to 2-cyclopropylphenol
(3a).8 After generation of TIPS protected bromophe-
nols (4a–f), they were all converted to boronic acids
(5a–f) by treatment with n-butyllithium followed by
addition of triisopropylborate and 3 N hydrochloric
acid and used at a later stage for the generation of
biaryl ethers (Scheme 2).7
3. Biological evaluation
The biological activity of the aforementioned CO23
analogs was measured in vitro using 125I–T3 competi-
tive binding and transactivation assays. Replacement
of inner-ring iodides with bromides causes a ꢀ10-fold
decrease in binding (CO24 vs CO23). TR ligand acti-
vation in U2OS cells (Table 1) showed that CO24 was
not TRa-specific compared to the T3 control. In
U2OS cells it is important to compare the potencies
At this stage, another level of thyromimetic diversity is
achieved by starting with either diiodo-L-tyrosine or di-
bromo-L-tyrosine (6a–b) and converting them to N-Boc-
Scheme 1. Synthetic route used for the synthesis of TIPS-protected, 4-hydroxyphenyl boronic acid.

764
C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
Scheme 2. Synthesis of CO24 and CO26–CO32.
of test ligands to that of T3 as thyroid hormone shows
a difference in activation of TRa1 and TRb1 using a
synthetic thyroid hormone response element driven
luciferase reporter construct. However, in HeLa cells,
a cell line where thyroid hormone consistently shows
equal activation of both TR subtypes, not only does
CO24 show fourfold TRa-selectivity in potency, it is
also TRa-selective in terms of efficacy in that it causes
transcriptional activity to plateau at a level that is
twice as high as T3 (Table 1 and Fig. 2a). In vivo,
Table 1. Binding affinity and potency of CO23 analogs
Compound
Kd and EC50 values (nM)
Binding affinity (Kd)a
Transactivation in U2OS cells
b
Transactivation in HeLa cells
b
(EC50)
(EC50
)
Tra
TRb
Tra
TRb
TRa
TRb
c
T3
0.058
1.2 0.2
0.081
1.7 0.3
2.4 0.4
4
11
2
2.4 0.5
2.4 0.5
CO23c
CO24
CO26
CO27
CO28
CO29
CO30
CO31
CO32
34
128
390
421
3
3
11
8.4
42
27
10
39
1
58
32
1
3
17
1
18.4
1
5
3
82 21
42 13
119 30
53 17
870 160
146 28
87 14
8000 1800
4000 1000
1400 170
2000 290
>20,000d
6
6
1
4
265 10
180 30
15
25
6
7
18
49
2
5
27
6
145 10
>20,000d
100 14
>10,000d
216 80
530 170
1700 10
2000 360
36
68 10
>10,000d
24
43
3
6
1
105 8
206 41
2000 170
5200 670
21
44
3
1
a Determined by means of an 125I–T3 competitive binding assay and data are reported as mean Kd standard error of the mean, n = 3.
b Determined through use of a TRE-driven dual-luciferase reporter assay in U2OS or HeLa cells and the data are reported as mean EC50
values standard error of the mean, n = 3.
c See Ref. 7.
d Dose–response curves generated using CO30 in transactivation assays did not plateau, and hence the EC50 value is an approximated value based on
extrapolation.

C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
765
Figure 3. In vivo analysis of CO24. Induced metamorphosis of stage-
53/54 tadpoles, n = 3, treated for 4-days with DMSO, 30 nM T3, and
300 nM CO24.
CO23 analogs with outer-ring substitutions all dis-
played a decrease in binding affinity and potency when
tested in U2OS cells compared to CO23 (Table 1). In
U2OS cells, the potency of transactivation of the follow-
ing substituents against TRa decreases from left to
right: Ethyl > iodo > cyclopropyl > methyl > bromo >
methoxy > hydrogen (Table 1). Unlike CO23, display-
ing only modest transcriptional activity in U2OS cells,
they activate in the presence of TRb very poorly. The
best outer-ring analog had an EC50 value of 1.4 lM
compared to 390 nM for CO23 when tested in U2OS
cells in the presence of TRb (Table 1).
Like CO24, in order to get a more direct measure of
TRa-specificity, all analogs were assayed in HeLa cells.
With the exception of CO30-only because its lack of
activity in both U2OS and HeLa cells made it impossible
to calculate and compare EC50 values from their dose–
response curves-all analogs demonstrated TRa-specific-
ity with a few proving superior to CO23. Compounds
CO26, CO27, CO31, and CO32 demonstrated 6-, 6-,
10-, and 12-fold TRa-specificity, respectively, all of
which are an improvement over CO23’s 5-fold TRa-
specificity. Dose–response curves for CO26 and CO32
showing transactivation in the presence of TRa and
TRb clearly demonstrate TRa-specificity despite the
compounds’ inferior potencies compared to CO23
(Fig. 2b and c). The rank order potency from left to
right in HeLa cells is also impressive as some com-
pounds are about equipotent to CO23-mediated TRa
transactivation: Ethyl > iodo > methyl > cyclopropyl >
methoxy > bromo > hydrogen.
Figure 2. TRE-driven dual-luciferase reporter assays showing transac-
tivation curves for T3, (a) CO24, (b) CO26, and (c) CO32 against hTRa1
and hTRb1 in HeLa cells. Plots show mean of triplicates with SD.
X. laevis tadpoles precociously induced to undergo
metamorphosis revealed gross morphological changes
compared to untreated tadpoles, some of which are
consistent with enhanced TRa activity. Tadpoles trea-
ted with 30 nM T3 and 300 nM CO24 both experi-
enced resorption of tissue in the head and tail,
experienced an overall decrease in size, and developed
Meckel’s cartilage (lower jaw); however, CO24 treated
tadpoles exhibited massive hind leg and fore leg devel-
opment, a noticeably larger body size, and less resorp-
tion of tissue in the head compared to T3 treated
tadpoles (Fig. 3). Compared to CO23 induced meta-
morphosis, CO24 had similar effects on tadpole meta-
morphosis with some exceptions. For example, both
CO24 and CO23 treated tadpoles developed more
massive fore and hind limbs than T3 treated tadpoles,
but tadpoles treated with CO24 developed the most
massive limbs overall. Furthermore, CO24 treated tad-
poles experienced slightly greater resorption of larval
tissue compared to CO23 treated tadpoles, particularly
in the head and tail, but this may be due to a slight
decrease in TRa-specificity compared to CO23.7
4. Discussion
CO23, the first potent thyromimetic to demonstrate
TRa-specificity in vitro and in vivo,7 was derivatized
at the 3, 5, and 30 positions in order to generate thyromi-
metics with enhanced TRa-specificity. Small substitu-

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C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
1
ents with varying electronic properties were selected to
be placed on the 30 position as the structure activity data
correlate with a decrease in potency with substituents
larger than an isopropyl group.5 In terms of the 3 and
5 positions, bromides were selected as previous studies
show a dramatic decrease in potency with methyl groups
on the inner-ring.7 In HeLa cells, four of eight ligands
showed greater TRa-specificity with CO32 achieving
greater than a twofold gain in specificity. Although all
analogs bound poorly to both TRa1 and TRb1 and with
about equal binding affinity to both receptor subtypes,
analogs CO24 and CO28 were about equipotent to
CO23 in their ability to cause TRa-mediated transcrip-
tion. This phenomenon brings attention to a mode of
subtype specificity which has gone unnoticed for some
time. Analogs reported herein are not the first to demon-
strate functional specificity. Considering that there is
only one amino acid side chain difference in the TR
LBD, Ser277 (TRa) to Asn331 (TRb), the similarity in
affinity is not surprising.10 However, previous studies
of related estrogen receptors demonstrate that subtle
differences in the induced conformations of amino acid
side chains may result in this selectivity.11 In this case,
the same ligand may cause minor differences in one
receptor LBD that causes amino acid side chains to per-
turb the conformationally mobile helix-12, an important
mediator of coactivator recruitment, gene regulation,
and potentiation of interactions with the transcriptional
machinery.10
1000 lm). H NMR spectra were taken on the Varian
Utility 400 MHz spectrometer in CDCl3 or DMSO-d6
solvents and chemical shifts were reported as d (parts
per million) downfield of the internal control trimethyl-
silane (TMS) for all solvents. High resolution mass spec-
trometry (HRMS) using electrospray ionization was
performed by the National Bio-Organic, Biomedical
Mass Spectrometry Resource at UCSF. All compounds
were at least 95% pure as determined by HPLC analysis
using a Waters 2695 instrument and an Xterra 3.5 lM
reverse-phase C18 2.1 · 50-mm column. HPLC grade
Acetonitrile and H2O were purchased from Fisher.
5.2. 125I–T3 competitive binding assay
Full-length hTRa1 and hTRb1 were expressed using a
TNT T7 quick-coupled transcription translation system
(Promega). Competition assays for binding of unlabeled
T3 and CO23 analogs were performed using 1 nM 125I–
T3 in a gel filtration binding assay as described.12
5.3. Transactivation assay
Human bone osteosarcoma epithelial (U2OS) cells or
human uterine cervix cancer (HeLa) cells (Cell Culture
Facility, UCSF) were grown to ꢀ80% confluency in
Dulbecco’s modified Eagle’s (DME)/H-21, 4.5 g/L glu-
cose medium containing 10% newborn calf serum
(NCS) or fetal bovine serum (FBS), respectively, (both
heat-inactivated), 2 mM glutamine, 50 U mlꢁ1 penicil-
lin, and 50 lg mlꢁ1 streptomycin. Cells (ꢀ1.5–2 · 106)
were collected and resuspended in 0.5 ml of electropor-
ation buffer (Dulbecco’s phosphate-buffered saline con-
taining 0.1% glucose and 10 mg/ml bioprene) with
1.5 lg of a TR expression vector (full-length hTRa1-
CMV or hTRb1-CMV), 0.5 lg of pRL-TK constitutive
Renilla luciferase reporter plasmid (Promega), 5 lg of a
reporter plasmid containing a synthetic TR response
element (DR-4) containing two copies of a direct re-
peat spaced by four nucleotides (AGGTCAcag-
In vivo, CO24 led to changes that are consistent with en-
hanced TRa-activity compared to T3 treated controls,
particularly as demonstrated by the massive hind and
fore leg emergence in precociously induced X. laevis tad-
poles. The inner-ring bromides probably confer resis-
tance to dehalogenation by deiodinases, and therefore
provide one explanation for CO24’s potency and effi-
cacy in vivo and in vitro. Although it is a leap to suggest
from studies on amphibian metamorphosis that these
analogs would have beneficial effects in mammals and
possibly humans, other studies with TRa and cardiac-
selective ligands suggest that TRa-specific thyromimet-
ics may have therapeutic utility in the area of heart dis-
ease. Finally, TRa-specific thyromimetics, like their
predecessor TRb-selective counterparts, may be useful
probes of TR biology.
gAGGTCA) cloned immediately upstream of
a
minimal thymidine kinase promoter linked to a lucifer-
ase coding sequence.13 Cells were electroporated using
a Bio-Rad gene pulser at 350 V and 960 microfarads
in 0.4-cm cuvettes, pooled in DME/F-12 Ham’s 1:1
without phenol red (U2OS) or DME/H-21 (HeLa) sup-
plemented as above except that NCS and FBS were
hormone stripped using dextrose-coated charcoal, and
plated in 96-well (U2OS) or 12-well (HeLa) plates to
a final density of 20,000 cells/well and 100,000 cells
per well, respectively. After a 2-h incubation period,
compounds in 1% dimethylsulfoxide (DMSO) were
added to the cell culture medium in triplicate. After
an additional 16-h incubation period, cells were har-
vested and assayed for luciferase activity using the Pro-
mega dual-luciferase kit (Promega) and an Analyst AD
(Molecular Devices). Data normalized to the Renilla
internal control were analyzed with GraphPad Prism,
v4, using the sigmoid-dose response model to generate
EC50 values; EC50 values were obtained by fitting data
to the following equation: Y = Bottom + (Top ꢁ Bot-
5. Experimental
5.1. General
All chemicals used for organic synthesis were purchased
from Aldrich, Sigma-Aldrich, Fluka, or Acros and were
used without further purification. Anhydrous conditions
were maintained under argon using standard schlenk
line techniques and oven-dried glassware. Anhydrous
THF, DCM, pyridine, and diisopropyl ethylamine were
available in house and dispensable from a solvent puri-
fication system. Compounds were purified by either flash
chromatography using silica gel (VWR Scientific) or
through preparatory thin layer chromatography (prep
TLC) using Analtech prep-TLC plates (20 · 20 cm,
tom)/(1 + 10((log EC ꢁX) HillSlope)).
50
*

C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
767
5.4. Preparation of chemicals
added N,N,N0,N0-tetramethylethylenediamine (17 ml,
113.5 mmol) and stirring commenced for 45 min before
warming to room temperature. The reaction mixture
was allowed to stir overnight before addition of water.
The aqueous phase was extracted with EtOAc. The com-
bined organic layers were washed with water, brine, and
3 N HCl, dried over MgSO4, concentrated in vacuo, and
purified by flash chromatography (silica gel, hexane/
ethyl acetate, 20:80) to give 3a (5.1 g, 38.0 mmol, 74%)
as a white solid. 1H NMR (CDCl3) d 7.36 (d,
J = 8 Hz, 1H), 7.06 (m, 2H), 6.85 (d, J = 8 Hz), 5.45
(s, 1H), 1.81 (m, 1H), 0.95 (m, 2H), 0.64 (m, 2H).
Stocks of T3 and CO23 analogs were prepared with
DMSO at a concentration of 10 mM and stored at
ꢁ20 ꢁC until use. All other chemicals were purchased
from Sigma unless otherwise indicated. About 0.1%
aminobenzoic acid ethyl ester (Tricaine or MS222) was
made fresh in sterile ddH2O and kept at 4 ꢁC for no
longer than 1 week.
5.5. General Xenopus laevis tadpole procedures
Xenopus laevis stage-53/54 tadpoles were purchased
from NASCO, Inc. and staged according to Nieuwkoop
and Faber.14 Upon receipt, tadpoles were allowed to set
overnight at room temperature (18–25 ꢁC) in order to
recover from shipping shock, after which half of the ini-
tial rearing water was replaced with 0.1· Marc’s Modi-
fied Ringer’s (MMR) buffer (10· solution consists of
100 mM NaCl (Fisher), 2 mM KCl (Fisher), 1 mM
MgCl2, 2 mM CaCl2, 0.1 mM EDTA, and 5 mM Hepes,
pH 7.8). Tadpoles were ultimately maintained in fresh
0.1· MMR buffer, changed every 2-days. After comple-
tion of experiments, live tadpoles were euthanized by
treatment with 0.01% Tricaine, exposure to an ice-bath,
and either fixed in phosphate-buffered saline containing
3.5% formalin or decapitated in order to ensure death.
Animals were photographed with a Canon PowerShot
A510 and images were processed with Adobe Photoshop
CS, v8, and Adobe Illustrator CS, v11. All tadpole
experiments were conducted in accordance with Institu-
tional Animal Care and Use Committee approval (ani-
mal protocol #: A7228-23070-01).
5.8. General procedure for preparation of TIPS-pro-
tected, 4-hydroxyphenyl boronic acids substituted at the
3-position (5a–f)
2-Isopropylphenol 4 (10.0 g, 73.4 mmol) was added to a
dry three-neck round-bottom flask fitted with an addi-
tion funnel and an exhaust line that runs into a base trap
(6 M KOH). This solution was allowed to stir at 0 ꢁC
after which Br2 (4.5 ml, 88.1 mmol) was added drop-
wise over a period of 15 min. Stirring commenced for
an additional 3-h before addition of sat. NaHCO3,
water, and EtOAc. The aqueous phase was extracted
with EtOAC and the combined organic layers were
washed with brine and then dried over MgSO4. After
concentration of the organic phase in vacuo, the crude
product was purified by flash chromatography (silica
gel, hexane/ethyl acetate, 10:90) to give a slightly yellow
clear oil (11.8 g, 55.1 mmol, 75%). This material (5 g,
23.3 mmol) was combined with TIPS-Cl (5.9 ml,
27.8 mmol) in a dry round-bottom flask containing
50 ml of anhydrous DCM and stirred at 0 ꢁC. To this
solution was added imidazole (3.9 g, 58 mmol) and stir-
ring commenced for an additional 18-h. The next day,
the reaction was quenched with water and the aqueous
phase extracted with EtOAc. The combined organic lay-
ers were washed with brine, dried over MgSO4, concen-
trated in vacuo, and then purified with a short-path
distillation column under high vacuum (0.5 mTorr).
Pure fractions were collected at 130 ꢁC to give 4b as a
clear white solid (6.9 g, 18.6 mmol, 80%). This material
was carried on to make 5b. A dry round-bottom flask
was charged with 4b (6.9 g, 18.6 mmol) and 100 ml of
anhydrous THF and stirred at ꢁ78 ꢁC. To this solution
was added drop-wise 2.5 M n-BuLi in hexanes (9.7 ml,
24.2 mmol) after which stirring commenced for 30 min.
To this solution was added triisopropyl borate (8.7 ml,
37.2 mmol) and stirring commenced for 45 min before
warming to room temperature. After 1 h, the reaction
was quenched with 3 N HCl and the aqueous phase ex-
tracted with EtOAc. The combined organic layers were
washed with water and brine, dried over MgSO4, con-
centrated in vacuo, and purified by flash chromatogra-
phy (silica gel, hexane/ethylacetate, 10–40%) to give 5b
(5.5 g, 16.4 mmol, 88%) as a white solid.
5.6. Induced metamorphosis experiments
Stage-53/54 tadpoles were added to Extra-Deep Petri
dishes (Fisher) in triplicate containing 50 mL of 0.1·
MMR buffer and vehicle or the appropriate concentra-
tion of ligand (T3 or CO23 analog). The final DMSO
concentration was 0.1%. Induced metamorphosis exper-
iments were repeated at least threefold.
5.7. Chemistry
5.7.1. 2-Cyclopropylphenol (3a). To 2-bromophenol 1
(8 g, 46.2 mmol) in 100 ml of dimethylformamide at
0 ꢁC was added NaH (2.6 g of a 60% suspension,
64.7 mmol). The reaction mixture was stirred for about
10 min after which allyl bromide was added dropwise.
After 30 min, the reaction mixture was treated with
water and extracted with diethyl ether. The organic por-
tion was dried over MgSO4, filtered, and concentrated in
vacuo to give the crude product, which was purified by
flash chromatography (silica gel, hexane/ethyl acetate,
5:95) to give allyl 2-bromophenyl ether 2 (9.7 g,
45.5 mmol, 99%) as a white solid. This material was car-
ried on to the next reaction to make 3a. A dry round-
bottom flask was charged with 2 (11 g, 51.6 mmol) and
260 ml of anhydrous diethyl ether and stirred at
ꢁ78 ꢁC. To this solution was added drop-wise 1.7 M
tert-BuLi in hexanes (60.7 ml, 103.3 mmol) after which
stirring commenced for 30 min. To this solution was
5.9. General procedure for preparation of N-Boc-3,5-
dihalo-L-tyrosine methyl esters (7a–b)
L-Diiodotyrosine (6a) (5 g, 11.5 mmol) was added to a
round-bottom flask and dissolved in MeOH/H2O (2:1).

768
C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
To this mixture was added NaHCO3 (2.9 g, 34.5 mmol)
followed by Boc2O (3.97 ml, 17.3 mmol). The reaction
mixture was allowed to stir until completion as deter-
mined by TLC analysis (product should turn blue when
tested with p-anisaldehyde). Upon completion, the mix-
ture was acidified to pH 4.5 and extracted with EtOAc.
The combined organic layers were washed with water
and brine and then dried over MgSO4. The crude mate-
rial (5.9 g, 11.1 mmol, 96%) was then utilized in the next
reaction. To this material (5.9 g, 11.1 mmol) in toluene/
MeOH (9:1) was added TMSCHN2 (0.5 M, 22 ml,
11.6 mmol) drop-wise over 30 min at room temperature
using a syringe pump. The reaction mixture was allowed
to stir until completion as determined by TLC analysis
and then washed with 0.5 M HCl and water. The aque-
ous phase was extracted with EtOAc and the organic
layers washed with brine, dried over MgSO4, and con-
centrated in vacuo to give N-Boc-3,5-diiodo-L-tyrosine
methyl ester (7a) (5.4 g, 9.9 mmol, 89%).
16–18 h, the mixture was purged with argon, concen-
trated in vacuo, and dissolved in anhydrous 3 N HCl
in EtOAc/Ether. After 3 h, the mixture was quenched
with water, the pH was adjusted to 4.5, and the aqueous
phase extracted with EtOAc. The organic layers were
washed with water and brine, dried over MgSO4, and
the crude material 9b (410 mg, 0.67 mmol, 99%) was
carried on to make CO24. Compounds 9b (410 mg,
0.67 mmol), 4-nitrophenyl chloroformate (160 mg,
0.78 mmol), and NaHCO3 (218 mg, 2.6 mmol) were
added to a dry round-bottom flask containing 10 ml
anhydrous MeCN. The reaction mixture was allowed
to stir overnight followed by addition of 6.5 ml of
H2O. The solution should quickly turn yellow due to
generation of nitrophenol. After 6 h, the reaction mix-
ture was roto-vapped in order to remove MeCN, acidi-
fied to pH 5, and then extracted with EtOAc. The
combined organic layers were washed with brine, dried
over MgSO4, and reconstituted in 10 ml THF. Depro-
tection of the TIPS group afforded CO24 (204 mg,
0.41 mmol, 63%, 2-steps from 9b) after purification by
prep TLC (silica gel, hexane/ethyl acetate, 40:60).
5.9.1. N-Boc-3,5-diiodo-L-tyrosine methyl ester. The
preparation of 7a was effected using the general proce-
dure for the preparation of Boc-protected dihalo-L-tyro-
sine methyl esters to give 5.4 g (89%) of the titled
5.11.1. Preparation of CO24. The preparation of CO24
was effected using the general procedure for the prepara-
tion of 5-(4-(4-hydroxyphenoxy)-3,5-dihalobenzyl)imi-
dazolidine-2,4-diones to give 204 mg (63%, 2-steps
1
compound as a white solid. H NMR (CDCl3) d 7.44
(s, 1H), 5.01 (s, 1H), 4.49 (dd, J = 4.0 Hz, J = 8.0 Hz,
1H), 3.74 (s, 3H), 3.00 (dd, J = 4.0 Hz, J = 14.0 Hz,
1H), 2.91 (dd, J = 8.0 Hz, J = 14.0 Hz, 1H), 1.45 (s, 9H).
1
from 9b) of the titled compound as a white solid. H
NMR (DMSO-d6) d 10.65 (s, 1H), 9.06 (s, 1H), 7.99
(s, 1H), 7.58 (s, 2H), 6.64 (d, J = 8.0 Hz, 1H), 6.63 (d,
J = 4.0 Hz, 1H), 6.20 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
4.39 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 3.15 (heptet,
J = 8.0 Hz, 1H), 2.99 (dd, J = 4.0 Hz, J = 14.0 Hz,
1H), 2.87 (dd, J = 8.0 Hz, J = 14.0 Hz, 1H), 1.10 (d,
J = 8.0 Hz, 6H). HPLC (MeCN/water, 50–100%,
15 min): retention time 3.1 min HR-MS calcd for
C19H18Br2N2O4: 497.9613. Found: 497.9607.
5.9.2. N-Boc-3,5-dibromo-L-tyrosine methyl ester. The
preparation of 7b was effected using the general proce-
dure for the preparation of Boc-protected dihalo-L-tyro-
sine methyl esters to give 1.1 g (87%) of the titled
1
compound as a white solid. H NMR (CDCl3) d 7.22
(s, 1H), 5.02 (s, 1H), 4.50 (dd, J = 4.0 Hz, J = 8.0 Hz,
1H), 3.74 (s, 3H), 3.05 (dd, J = 4.0 Hz, J = 14.0 Hz,
1H), 2.93 (dd, J = 8.0 Hz, J = 14.0 Hz, 1H), 1.44 (s, 9H).
5.10. General procedure for the preparation of biaryl
ethers (8a–f)
5.11.2. Preparation of CO26. The preparation of CO26
was effected using the general procedure for the prepara-
tion of 5-(4-(4-hydroxyphenoxy)-3,5-dihalobenzyl)imi-
dazolidine-2,4-diones to give 80 mg (41%, 2-steps from
4 molecular sieves were flame-dried under high vacuum
in a dry round-bottom flask. To this flask were added
boronic acid (3 mmol) and copper acetate (dried to a
verdigris color). These components were dissolved in
10 ml anhydrous DCM after which anhydrous pyridine
(5 mmol) and diisopropyl ethylamine (5 mmol) were
added. This mixture was then allowed to stir at room
temperature for 5 min before addition of phenol
(1 mmol) in three portions separated by 5 min each. At
this point, the flask was fitted with a drying tube con-
taining drierite and allowed to stir under ambient air
overnight, ꢀ16–24-h. After this time, the reaction mix-
ture was concentrated in vacuo and purified by flash
chromatography to give products 8a–f (yield generally
from 55% to 83%).
1
9e) of the titled compound as a white solid. H NMR
(DMSO-d6) d 10.61 (s, 1H), 8.65 (s, 1H), 7.99 (s, 1H),
7.75 (s, 2H), 6.64 (d, J = 8.0 Hz, 1H), 6.50 (d,
J = 4.0 Hz, 1H), 5.90 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
4.37 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 3.72 (s, 3H),
2.94 (dd, J = 4.0 Hz, J = 14.0 Hz, 1H), 2.81 (dd,
J = 8.0 Hz, J = 14.0 Hz, 1H). HPLC (MeCN/water,
50–100%, 15 min): retention time 1.8 min HR-MS calcd
for C17H14I2N2O5: 579.8992. Found: 579.9004.
5.11.3. Preparation of CO27. The preparation of CO27
was effected using the general procedure for the prepara-
tion of 5-(4-(4-hydroxyphenoxy)-3,5-dihalobenzyl)imi-
dazolidine-2,4-diones to give 50 mg (25%, 2-steps from
1
9c) of the titled compound as a white solid. H NMR
5.11. General procedure for the preparation of 5-(4-(4-
hydroxyphenoxy)-3,5-dihalobenzyl)imidazolidine-2,4-
diones (CO24 and CO26–CO30)
(DMSO-d6) d 10.65 (s, 1H), 9.09 (s, 1H), 7.97 (s, 1H),
7.77 (s, 2H), 6.66 (d, J = 8.0 Hz, 1H), 6.48 (d,
J = 4.0 Hz, 1H), 6.26 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
4.40 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 2.94 (dd,
J = 4.0 Hz, J = 14.0 Hz, 1H), 2.81 (dd, J = 8.0 Hz,
J = 14.0 Hz, 1H), 2.07 (s, 3H). HPLC (MeCN/water,
Biaryl ether 8b (510 mg, 0.69 mmol) was dissolved in
20 ml MeOH and saturated with ammonia gas. After

C. A. Ocasio, T. S. Scanlan / Bioorg. Med. Chem. 16 (2008) 762–770
769
50–100%, 15 min): retention time 1.9 min HR-MS calcd
for C17H14I2N2O4: 563.9043. Found: 563.9045.
J = 8.0 Hz, J = 14.0 Hz, 1H). HPLC (MeCN/water,
50–100%, 15 min): retention time 2.1 min HR-MS calcd
for C16H11I3N2O4: 675.7853. Found: 675.7877.
5.11.4. Preparation of CO28. The preparation of CO28
was effected using the general procedure for the prepara-
tion of 5-(4-(4-hydroxyphenoxy)-3,5-dihalobenzyl)imi-
dazolidine-2,4-diones to give 91 mg (35%, 2-steps from
5.11.8. Preparation of 5-(4-(4-hydroxy-3-iodophenoxy)-
3,5-diiodobenzyl)imidazolidine-2,4-dione (CO32). 5-(4-(4-
Hydroxyphenoxy)-3,5-diiodobenzyl)imidazolidine-2,4-
dione (CO30, 100 mg, 0.2 mmol) was added to a round-
bottom flask and dissolved in 2 ml of DCM and 0.25 ml
of glacial acetic acid at 0 ꢁC. To this mixture was added
drop-wise bromine (12.3 ll, 0.24 mmol) in 1 ml of
DCM. After 1 h, the reaction mixture was extracted
with EtOAc, concentrated in vacuo, and purified by
flash chromatography (silica gel, hexane/ethyl acetate,
40:60) to give CO32 (109 mg, 0.18 mmol, 88%). 1H
NMR (DMSO-d6) d 10.61 (s, 1H), 9.90 (s, 1H), 7.98
(s, 1H), 7.76 (s, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.82 (d,
J = 4.0 Hz, 1H), 6.55 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
4.36 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 2.96 (dd,
J = 4.0 Hz, J = 14.0 Hz, 1H), 2.79 (dd, J = 8.0 Hz,
J = 14.0 Hz, 1H). HPLC (MeCN/water, 50–100%,
15 min): retention time 2.0 min HR-MS calcd for
C16H11BrI2N2O4: 627.7992. Found: 627.7981.
1
9d) of the titled compound as a white solid. H NMR
(DMSO-d6) d 10.60 (s, 1H), 8.94 (s, 1H), 7.98 (s, 1H),
7.75 (s, 2H), 6.64 (d, J = 8.0 Hz, 1H), 6.53 (d,
J = 4.0 Hz, 1H), 6.24 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
4.36 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 3.16 (q, 2H),
2.95 (dd, J = 4.0 Hz, J = 14.0 Hz, 1H), 2.85 (dd,
J = 8.0 Hz, J = 14.0 Hz, 1H), 1.11 (t, 3H). HPLC
(MeCN/water, 50–100%, 15 min): retention time
2.1 min HR-MS calcd for C18H16I2N2O4: 577.9199.
Found: 577.9198.
5.11.5. Preparation of CO29. The preparation of CO29
was effected using the general procedure for the prepara-
tion of 5-(4-(4-hydroxyphenoxy)-3,5-dihalobenzyl)imi-
dazolidine-2,4-diones to give 1.0 g (67%, 2-steps from
1
9a) of the titled compound as a white solid. H NMR
(DMSO-d6) d 10.60 (s, 1H), 9.00 (s, 1H), 7.98 (s, 1H),
7.74 (s, 2H), 6.62 (d, J = 8.0 Hz, 1H), 6.24 (d,
J = 4.0 Hz, 1H), 6.13 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
4.37 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 3.17 (m, 1H),
2.94 (dd, J = 4.0 Hz, J = 14.0 Hz, 1H), 2.85 (dd,
J = 8.0 Hz, J = 14.0 Hz, 1H), 0.84 (m, 2H), 0.53 (m,
2H). HPLC (MeCN/water, 50–100%, 15 min): retention
time 2.0 min HR-MS calcd for C19H16I2N2O4: 589.9199.
Found: 589.9209.
Acknowledgments
We would like to thank Professor J. David Furlow,
Eric Neff, and Cindy Chen for their advice, guidance,
and taking time to critically analyze the X. laevis in-
duced metamorphosis experiments. We are also grate-
ful to Suzana T. Cunha Lima, Ph.D., for her technical
expertise with the 125I–T3 competitive binding assay.
Finally, we are grateful to the NIH (DK-52798,
T.S.S.) and the Ford Foundation for financial
support.
5.11.6. Preparation of CO30. The preparation of CO30
was effected using the general procedure for the prepara-
tion of 5-(4-(4-hydroxyphenoxy)-3,5-dihalobenzyl)imi-
dazolidine-2,4-diones to give 1.3 g (65%, 2-steps from
1
9f) of the titled compound as a white solid. H NMR
(DMSO-d6) d 10.61 (s, 1H), 9.09 (s, 1H), 7.98 (s, 1H),
7.76 (s, 2H), 6.67 (d, J = 8.0 Hz, 2H), 6.51 (d,
J = 8.0 Hz, 2H), 4.36 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
2.95 (dd, J = 4.0 Hz, J = 14.0 Hz, 1H), 2.84 (dd,
J = 8.0 Hz, J = 14.0 Hz, 1H). HPLC (MeCN/water,
50–100%, 15 min): retention time 1.7 min HR-MS calcd
for C16H12I2N2O4: 549.8886. Found: 549.8900.
References and notes
1. Scanlan, T. S.; Yoshihara, H.; Nguyen, N.-H.; Chiellini,
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5.11.7. Preparation of 5-(4-(4-hydroxy-3-iodophenoxy)-
3,5-diiodobenzyl)imidazolidine-2,4-dione (CO31). 5-(4-(4-
Hydroxyphenoxy)-3,5-diiodobenzyl)imidazolidine-2,4-
dione (CO30, 100 mg, 0.2 mmol) in 0.2 ml of MeOH
was added to a round-bottom flask at ꢁ5 ꢁC and dis-
solved in 5 ml of a 70% solution of aqueous ethylamine.
To this mixture was added drop-wise Iodine (I2) as a 1 N
aqueous solution saturated with KI (0.24 ml,
0.24 mmol). After 6 h, the reaction mixture was acidified
to pH 4.5, extracted with EtOAc, concentrated in vacuo,
and purified by flash chromatography (silica gel, hexane/
ethyl acetate, 40:60) to give CO31 (85 mg, 0.13 mmol,
7. Ocasio, C. A.; Scanlan, T. S. ACS Chem. Biol. 2006, 1,
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´
8. Barluenga, J.; Fan˜anas, F. J.; Sanz, R.; Marcos, C. Chem.
Eur. J. 2005, 11, 5397.
9. Hart, M.; Suchland, K.; Miyakawa, M.; Bunzow, J.;
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gen, J. A.; Agard, D. A.; Greene, G. L. Nat. Struct. Biol.
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1
62%). H NMR (DMSO-d6) d 10.62 (s, 1H), 9.97 (s,
1H), 8.00 (s, 1H), 7.77 (s, 2H), 7.02 (d, J = 4.0 Hz,
1 H), 6.81 (d, J = 8.0 Hz, 1H), 6.58 (dd, J = 4.0 Hz,
J = 8.0 Hz, 1H), 4.37 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H),
2.95 (dd, J = 4.0 Hz, J = 14.0 Hz, 1H), 2.84 (dd,

770
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