Thyroid receptor ligands. 6. A high affinity "direct antagonist" selective for the thyroid hormone receptor

Article abstract of DOI:10.1021/jm060521i

A new high-affinity thyroid hormone antagonist 6 with druglike properties was designed and synthesized. The compound behaved as an antagonist in a cell transactivation assay, and in a first in vivo experiment in rats.

Full text of DOI:10.1021/jm060521i

J. Med. Chem. 2006, 49, 6635-6637
6635
Letters
Thyroid Receptor Ligands. 6. A High Affinity
“Direct Antagonist” Selective for the Thyroid
Hormone Receptor
Konrad Koehler,† Sandra Gordon,† Peter Brandt,†
Bo Carlsson,† Anna Ba¨cksbro-Saeidi,† Theresa Apelqvist,†
Peter Agback,† Gary J. Grover,‡ William Nelson,†
Marlena Grynfarb,† Mathias Fa¨rnega˚rdh,†
Stefan Rehnmark,† and Johan Malm*,†
Karo Bio AB, NoVum, Huddinge SE-141 57, Sweden, and Product
Safety Laboratories, Eurofins Scientific, 2394 Highway 130,
Dayton, New Jersey 08810
ReceiVed May 3, 2006
Abstract: A new high-affinity thyroid hormone antagonist 6 with
druglike properties was designed and synthesized. The compound
behaved as an antagonist in a cell transactivation assay, and in a first
in vivo experiment in rats.
Figure 1. Chemical structures of tetraiodothyronine (1a), triiodothy-
ronine (1b), including ring-numbering, and reported “direct” thyroid
hormone antagonists ligands 2-5.
Nuclear hormone receptors comprise a class of intracellular,
ligand activated transcription factors, which include receptors
for thyroid hormones (THsa). Thyroid receptors (TRs) exert
profound effects on development and homeostasis in mammals.1
Endogenous THs, 3,5,3′,5′-tetraiodo-L-thyronine (T4, 1a), 3,5,3′-
triiodo-L-thyronine (T3, 1b) (Figure 1), and its metabolites,
regulate crucial genes throughout the organism and influence
basal and adaptive metabolism, lipid levels, bone and muscle
metabolism, heart rate, development, mood, and overall sense
of well being.
When the body is exposed to elevated levels of circulating
1a and 1b, this might result in hyperthyroidism. Clinically, this
state often manifests itself by weight loss, hypermetabolism,
lowering of serum lipid levels, cardiac arrhythmias, heart failure,
muscle weakness, bone loss in postmenopausal women, and
anxiety. At present, treatment of hyperthyroidism is directed to
reduce overproduction of THs by inhibiting their synthesis or
release or by ablating thyroid tissue with surgery or radioiodine.
TR antagonists may have significant clinical use such as for
treating hormone excess states, as it might quickly restore the
euthyroid state and consequently improve the clinical manifesta-
tions mentioned above.
believed to distort the folding of the C-terminal helix (helix
12) to the body of the LBD and thus the formation of the
coactivator site. We prefer to describe this class of compounds
as “direct antagonists” in order to separate them from “indirect”
or “passive” antagonists.10 Examples of synthetic TR antagonists
using this “extension theory” is depicted in Figure 1 by the
extensively studied O-[4-hydroxy-3,5-diisopropylphenyl]-L-
tyrosine11-17 (2), 3,5-dibromo-4-(3,5-diisopropylphenoxy)ben-
zoic acid18 (3), {4-[4-hydroxy-3-isopropyl-5-(4-nitrophenyl)-
benzyl]-3,5-dimethylphenoxy}acetic acid (4),19 and {4-[4-hy-
droxy-3-isopropyl-5-(4-nitrophenylethynyl)benzyl]-3,5-dimeth-
ylphenoxy}acetic acid (5).20-21
In order to optimize drug feasibility by increasing water
solubility, we decided to substitute the R5′-position with a
pyridyl vinyl group. Furthermore, the alkynyl part in 5 can be
problematic because it potentially can ring-close with the R4′-
hydroxy to form a benzofuran ring at elevated pH,22 perceptibly
not possible with an alkene group. We have also previously
showed that affinity for TR increases in the order formic, acetic,
and propionic acids and that accompanying optimal substitution
patterns for affinity and stability involve bromine groups at the
R3 and R5 positions and an isopropyl group at the R3′-position.6
Structure-based design work intended for the displacement
of helix-12 (H12) revealed that a viable substitution of the R5′-
position indeed could be accomplished through the use of a
pyridylvinyl group. The alkene part extends precisely through
a well-defined hole between helix-3 and helix-11, and the termi-
nal pyridyl group is directed into H12, thus potentially distorting
the folding of H12 to the body of the LBD (Figure 2).23
The designed ligand, 3-{3,5-dibromo-4-[4-hydroxy-3-isopro-
pyl-5-((E)-2-pyridin-4-ylvinyl)phenoxy]phenyl}propionic acid
(6), was prepared as outlined in Scheme 1. The starting material,
methyl 3-[3,5-dibromo-4-(3-isopropyl-4-methoxyphenoxy)phen-
yl]propionate (7),24 was regioselectively nitrated at the R5′-
position, the nitro group reduced, diazotizated, and substituted
with potassium iodide to give the intermediate methyl 3-[3,5-
Several crystallographic structures of the ligand binding
domains (LBDs) have been determined for TR agonists, but
none include a TR antagonist.2-9 Therefore, certain assumptions
have to be made in terms of the design of new TR antagonists.
In the literature, ligand design of TR antagonists has generally
been based on the attachment of a large extension group at the
5′-position of 1b or other close analogues. This extension is
* To whom correspondence should be addressed. Phone: +46-8-608
6046. Fax: +46-8-774 8261. E-mail: johan.malm@karobio.se.
† Karo Bio AB.
‡ Eurofins Scientific.
a Abbreviations: THs, thyroid hormones; TRs, thyroid receptors; LBDs,
ligand binding domains; H12, helix-12; hTRa1, human TRR1; CHO K1
cells, Chinese hamster ovary K1 cells; alkaline phosphate, ALP; TRAFR1,
CHO K1 cells stably transfected with hTRR1 and an ALP reporter gene
downstream the thyroid response element; LDL-C, low-density lipoprotein
cholesterol; SARs, structure-activity relationships; 3D, three-dimensional.
10.1021/jm060521i CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/13/2006

6636 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Letters
Table 1. Thyroid Hormone Receptor Binding Affinities of Synthetic
Thyromimetics 3-6 and 9a
hTRR
hTRâ
910
hTRR/hTRâb
3
4
5
6
9
1600
1.0
5.7
4.6
0.96
1.4
200 ( 60
35 ( 12
20 ( 7
22 ( 3
93 ( 29
36 ( 3
460
190
a All values are expressed as nM. The value for 6 is expressed as the
mean IC50 ( SE. Data for 3 and 9 have been published before and are
average mean IC50 values of two runs. Data for 4 and 5 were taken from
ref 20 and are expressed as mean KD ( SE. b Selectivity was “normalized”
for 3, 6, and 9: 1.7 × [IC50(hTRR1)/( IC50(hTRâ1)]. For an explanation,
see ref 6.
Figure 2. Depiction of the designed antagonist (6) docked into the
ligand binding domain of TRâ. The backbone of the protein is displayed
as a green tube (except the loop between H11 and H12, which is colored
cyan, and antagonist conformation of H12 as magenta, and the agonist
conformation of H12 as red). The side chains of residues Arg228,
Arg266, and His381 are displayed as green capped sticks, and the ligand
is displayed as capped sticks (white ) carbon, red ) oxygen, blue )
nitrogen, green ) bromine). Hydrogen bonds between the ligand and
protein are represented by dashed yellow lines, and the surface of the
ligand binding cavity is represented as a yellow mesh. There is a severe
steric clash between the 5′-substituent of 6 and the agonist conformation
of H12, which forces H12 into the antagonist conformation. This figure
was created using the PyMol molecular graphics system.25
Figure 3. Transcriptional effects of 6 + L-T3 (red curve) and 6 only
(blue curve) on CHO K1 cells, stably transfected with hTRR1 and with
an ALP reporter gene downstream to a TRAFR1.26 The y-axis is
expressed as light units emitted from ALP and the x-axis as log of the
concentration of added ligand. The concentration of 6 required for 50%
inhibition of L-T3 is 32 nM. The response value for each concentration
of ligand is the mean of triplicate determinations with (SD for each
value indicated.
Scheme 1a
warrants, however, further investigation and continued structure-
activity relationship work.
The result from a reporter cell assay employing CHO K1
cells (Chinese hamster ovary K1 cells) stably transfected with
hTRR1 and an alkaline phosphate reporter gene downstream the
thyroid response element (TRAFR1), is shown in Figure 3.
Clearly 6 acts as a full antagonist in the TRAFR cell assay and
the IC50 for TRAFâ was similar to that for TRAFR. The IC50
values were 32 nM for both TRAFR1 and TRAFâ1.
In order to confirm the antagonism of 6 in an in vivo
experiment setting, we utilized our “standard screen” for TR
agonists:27 the cholesterol fed rat model. In this model the
animals confirmed the TR antagonism of 6 by a lowering of
the heart rate (-10 ( 2.5% versus vehicle treated animals, p <
0.05) and a possible trend toward an increase of low-density
lipoprotein cholesterol (LDL-C) (+13 ( 13%). This experiment
is, however, highly preliminary, and we need to further confirm
the antagonism in vivo.
We have shown that a potentially druglike high-affinity direct
antagonist can be designed from available crystal structures and
synthesized in reasonable overall yield. Although substantial
additional work on structure-activity relationships (SARs),
synthetic procedures, 3D crystal structural work, and in vivo
experiments is required to ensure its relevance, this approach
represents a promising approach to novel and high-affinity TR
antagonists.
a Reagents and conditions: (a) HNO3, C6H6, room temp; (b) Na2S2O4,
EtOH, 70 °C; (c) NaNO2, HCl, KI; (d) BF3‚Me2S, CH2Cl2; (e) 4-vinylpy-
ridine, Pd(OAc)2, TEA, DMF, 100 °C; (f) LiOH, THF, room temp.
dibromo-4-(3-iodo-5-isopropyl-4-methoxyphenoxy)phenyl]pro-
pionate (8) in moderate yield. After deprotection, the final
product 6 was obtained in good yields via a regioselective cross-
coupling between the R5′-iodo and 4-pyridylvinyl. Total yields
from the starting material 7, using six synthetic steps, were 15%
for 6.
The results of a binding assay for the human TRR1 and TRâ1
for 3-6 and the indirect antagonist 3-(3,5-dibromo-4-cyclo-
hexylmethoxyphenyl)propionic acid (9)10 are summarized in
Table 1. Compound 6 displayed high affinity for both TRR1
and TRâ1 and represents with respect to TRR1-binding an
improvement of affinity compared with the previously known
high-affinity ligands 4 and 5. Furthermore, 6, in contrast to 4
and 5, does not appear to be selective for either TR isoform,
which is likely to be an advantage when treating the hyperthy-
roid state. Also, with the present binding data at hand for the
high-affinity “direct antagonists” 4-6, the value of the design
approach of indirect TR antagonists appears at least in this
context as somewhat less valuable. The viability of this approach
Supporting Information Available: Experimental procedures
and characterization data of 6 and methods of characterization in
vivo and in vitro. This material is available free of charge via the
Internet at http://pubs.acs.org.

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6637
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