Design and synthesis of novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives as thyroid hormone receptor β (TR-β) selective ligands

Article abstract of DOI:10.1016/j.bmcl.2008.06.038

Design and synthesis of a novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives are reported and their in vitro thyroid hormone receptor selectivity has been evaluated in the thyroid luciferase receptor assay. The 3-[3,5-dichloro-4-(4-hydroxy-3-isopropylph

Full text of DOI:10.1016/j.bmcl.2008.06.038

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3919–3924
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel 3-hydroxy-cyclobut-3-ene-1,2-dione
derivatives as thyroid hormone receptor b (TR-b) selective ligands
,
*
Saurin Raval , Preeti Raval, Debdutta Bandyopadhyay, Krunal Soni, Digambar Yevale,
Digvijay Jogiya, Honey Modi, Amit Joharapurkar, Neha Gandhi, Mukul R. Jain, Pankaj R. Patel
Zydus Research Centre, Medicinal Chemistry, Sarkhej-Bavla N. H.8A, Moraiya, Ahmedabad, Gujarat 382210, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Design and synthesis of a novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives are reported and their in
vitro thyroid hormone receptor selectivity has been evaluated in the thyroid luciferase receptor assay.
The 3-[3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)-phenylamino]-4-hydroxy-cyclobut-3-ene-1,2-
dione 21 has shown selectivity towards thyroid hormone receptor b.
Received 16 December 2007
Revised 19 May 2008
Accepted 11 June 2008
Available online 14 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
3-Hydroxy-cyclobut-3-ene-1,2-dione
Thyroid hormone receptor
TR-b Selective
Squaric acid
Thyroid receptor assay
Thyroid hormones are important endocrine signalling hor-
mones, which are involved in a number of important physiologi-
cal processes such as lipid metabolism; control of energy
expenditure and in the brain development.1–4 Natural thyroid
hormone, triiodothyronine (T3) exhibits its physiological effect
by acting on a thyroid hormone receptors (THR). It is an impor-
tant endocrine signalling hormone essential for normal develop-
ment, differentiation and maintenance of metabolic balance in
while avoiding the cardiovascular and other toxicities of native
thyroid hormone. Thus, the selectivity for the TR-b1 over TR-a1
remains an important criterion in the development of THR
ligands.
Axitirome ( )-N-[4-[3-(4-fluoro-a-hydroxybenzyl)-4-hydroxy-
phenoxy]-3,5-dimethylphenyl] oxamic acid ethyl ester 1 (Fig. 1)
has been extensively studied oxamic acid thyromimetics, which
was progressed up to phase I.13–16 However, it was discontinued
in clinical trials in 1998 for unknown reasons.17 Similarly 3,5-di-
mammals. The two major subtypes of THR are TR-
a
and TR-b.
Further these two subtypes are classified as a1
,
a2 and b1, b2 iso-
chloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid
2
forms.5 The TR-a1 and TR-b1 isoforms are ubiquitously expressed,
although TR-a1 predominates in heart (70% of TRs), whereas TR-
b1 predominates in the liver (80% of TRs).6 The activation of TR-
a1 isoform mainly affects the heart rate and rhythm whereas,
activation of TR-b1 isoform is known to affect the liver and other
tissues.7,8
(KB-141, Fig. 1) was found to be promising due to its selectivity
for TR-b subtype18,19 and has been reviewed in detail for its biolog-
ical properties.20,21
Replacement of biaryl ether linkage with a methylene link-
age and phenoxy acetic acid led to a potent compound 3 (GC-
1, Fig. 1). It has 10-fold TR-b selectivity in transactivation and
lowers the serum cholesterol levels without affecting the heart
rate.22,23
Hangeland et al. have reported compound 4 (Fig. 2) containing
larger hydrophobic group replacing isopropyl group of KB-141 at
position R, which demonstrated improved selectivity.24 Similarly,
substitution with aromatic hydrophobic group such as phenyl
lead to more potent compound 5 (GC-24, Fig. 2) with higher affin-
ity and stronger selectivity for the TR-b subtype.25 Recently, com-
pounds 6 and 7 containing 6-azauracil and 30-carboxamide and
30-sulfonamide linkage have been reported as potent and TR-b
selective thyromimetics.26 40-Amido derivatives 8 have also been
described as TR-b selective (Fig. 3).27
T3 is nonselective in binding towards both of the THR isoforms
(TR-a1 and TR-b1). Administration of T3 lowers the plasma choles-
terol, low-density lipoprotein (LDL) and triglyceride levels in ani-
mal models,8,9 and in humans.10–12 However, T3 cannot be used
therapeutically to treat hypercholesterolemia and obesity due to
its cardiac side effects.9,10 TR-b1 agonist could lead to specific
therapies for disorders such as obesity and hyperlipidaemia,
* Corresponding author. Tel.: +91 2717 250801; fax: +91 2717 250606.
E-mail addresses: saurinraval@zyduscadila.com, saurin_raval@rediffmail.com
(S. Raval).
ZRC communication # 241.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.038

3920
S. Raval et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3919–3924
Cl
Cl
OH
Me
O
Cl
O
O
COOH
O
HO
Cl
O
COOH
HO
F
HO
Me
N
H
OEt
1
3
GC-1
2
Axitirome
KB-141
Figure 1.
Cl
Cl
R
O
Ph
HO
COOH
Cl
O
COOH
HO
Cl
4
5
GC-24
Figure 2.
Cl
Cl
O
O
O
O
NH
HO
Cl
N
NH
N
N
HO
Cl
N
O
S
O
O
RN
O
O
NR
7
6
H
N
OH
H
Cl
HO
R
HO
N
R
Cl
O
O
O
O
O
Br
O
Cl
Cl
9-24
8
Figure 3.
The X-ray crystallographic structures of the ligand binding do-
compounds 9–24 for TR-a and TR-b were evaluated with respect
mains (LBD) of TR-
a
1 and TR-b1 determined in complex with thyr-
to T3 in a thyroid luciferase receptor assay.35
omimetics are accessible from Protein Data Bank (PDB).18,24–26,28,29
The hormone binding pocket is very similar in both the receptors.
These receptors differ only by one single amino acid residue,
The synthesis of compounds 9–24 has been outlined in
Scheme 1. Phenol derivatives 25 were reacted with 1,2,3-tri-
chloro-5-nitro benzene using sodium hydride to afford nitro
substituted diaryl ether derivatives 26. The nitro-diaryl ether
derivatives 26 were subsequently reduced using tin chloride in
acidic medium to get amino derivatives 27. The demethylation
of methoxy derivatives 27 was achieved using solution of boron
tribromide to afford amine derivatives 28. The resulting amine
derivatives 28 were further reacted with 3,4-dihydroxy-3-cyclob-
utene-1,2-dione(squaric acid) to afford cyclobut-3-ene-1,2-dione
derivatives 9–24.
Asn331 in TR-b, which is substituted by Ser277 in TR-a. This re-
sults in a significantly different hydrogen bonding patterns be-
tween the acidic group of the ligands and the LBD of the two
receptors and hence accounts for selectivity. In continuation of
our thyroid hormone receptor research program,32 we focused on
modification of acidic moieties in the small molecules to find
TRH agonist selective for b-type. Again, the flexibility in the hydro-
phobic aromatic pocket in TR-b is known to accommodate bulky
groups such as benzyl and retain full agonistic activity.24,25 The hy-
droxy-cyclobut-3-ene-1,2-dione derivatives 9–24 were designed
and validated using molecular modeling.33
In vitro % TR-
ene-1,2-dione derivatives 9–24 at different concentrations such
as 0.001, 0.01 and 1 M were evaluated with respect to T3 (Table
1) keeping KB-141 as a positive control. The primary carboxamide
derivatives 9–12 failed to show TR- or TR-b activity as compared
to potent TR-b selective compound 2 even at the concentration of
M (Table 1). Following the unfavourable activity of compounds
a and TR-b activities of 3-hydroxy-cyclobut-3-
l
Molecular docking studies indicated that compounds 21 and
22 make similar interactions in the LBD of TR-
a
and TR-b as that
a
of exhibited by the ligand 3,5-dichloro-4-[(4-hydroxy-3-isopro-
pylphenoxy)phenyl]acetic acid KB-141 in the crystal structures
(Fig. 4).18
On basis of favourable orientation and similar binding of de-
signed compound to TR-b as that of KB-141 in molecular docking
studies, the derivatives 9–24 comprising 3-hydroxy-cyclobut-3-
ene-1,2-dione were synthesized.34 The in vitro activities of the
1
l
9–12, the primary carboxamide groups were replaced by second-
ary carboxamides, which resulted to the derivatives 13–16. The
N,N-dimethyl carboxamide derivative 13 exhibited mild activity
at 1
lM concentration for both the TR-a and TR-b. The cyclopen-
tyl carboxamide derivative 14 remained inactive at lower concen-

S. Raval et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3919–3924
3921
ARG 320
B
A
ASN 331
ASN 331
ARG 320
ARG 282
ARG 282
MET 313
MET 313
HIS 435
HIS 435
Figure 4. Docking of 21 and 22 in ligand binding pocket of TR-b including water molecules. Residues and ligands are shown as sticks. The docked conformers 21 and 22 are
superimposed on compound 2 KB-141(shown in cyan as sticks). (A) The oxygen atoms in compound 21 (shown in orange) makes hydrogen bonding with the side chains of
residues His435, Arg282, Asn331 and water molecules. The amide linkage makes hydrogen bond with the backbone of Met313 (B) Compound 22 shown in magenta makes
similar interactions as that of shown by compound 21.
Cl
Cl
OH
O
O
Cl
Cl
NO2
(b)
(a)
+
O
O
Cl
NO2
O
Cl
NH2
R
R
R
Cl
27
25
26
(c)
Cl
Cl
O
O
O
O
(d)
HO
Cl
N
HO
Cl
NH2
H
OH
R
R
28
9-24
Scheme 1. Reagents and conditions: (a) NaH, DMF, 120 °C, 3–4 h, 95%; (b) SnCl2ꢀ2H2O, concd HCl, EtOH, 65–70 °C, 3 h, 95%; (c) 1 M BBr3, CH2Cl2, 25 °C, 3 h, 20–50%;
(d) C4H2O4, H2O, 100 °C, 4–6 h, 30–40%.
trations of 0.001 and 0.01
was seen at 1 M concentration (50% for TR-
b), which is almost similar to compound 2 at the concentration
of 0.001 M. Changing the ring size of 14 from five- to six-mem-
bered, cyclohexyl carboxamide derivative 15 showed diminished
activities for both TR- and TR-b. The carboxamide with one
l
M; however, the improved activity
Contrary to carboxamide derivatives 9–16, sulfonamide deriva-
tives 17–19 and compound without amidic linkage 20, the com-
pounds substituted by isopropyl 21 and tert-butyl 22 gave the
l
a
and 80% for TR-
l
encouraging in vitroactivitypatternfor TR-
aand TR-battheconcen-
trationof 1 M (Table1). Thetert-butylderivative22wasfoundtobe
l
a
equipotent to isopropyl derivative 21 (Table 1). Further, the benzyl
derivative 23 and phenoxy derivative 24 were found to be inferior
to the isopropyl derivative 21 and the tert-butyl derivatives 22. Fol-
lowing the satisfactory in vitro selectivity of compounds 21 and 22,
the EC50 values of compounds 21 and 22 were calculated for both TR-
more heteroatom, the morpholino derivative 16 remained inac-
tive against thyroid receptor luciferase assay.
Due to the unfavourable in vitro TR-a and TR-b activities for pri-
mary and secondary carboxamide derivatives 9–16, we evaluated
some of the sulfonamide derivatives 17–19 in the same assay and
compared with compound 2. The cyclohexyl sulfonamide derivative
17 showed similar trend of inactivity as that of exhibited by its cor-
responding carboxamide derivative 10. Interestingly, the secondary
a
and TR-b (Table 2). Both thecompounds21 and 22exhibitednearly
similar selectivities for TR-a and TR-b as that of shown by the com-
pound 2.
In summary, a rational structure activity relationship for 3-hy-
droxy-cyclobut-3-ene-1,2-dione compounds 9–24 as thyroid hor-
mone receptor b culminated to compounds 21 and 22, which
sulfonamide derivative 18 showed mild activity at 1 lM concentra-
tion; however, it remained inferior to the compound 2. Similarly, the
morpholino sulfonamide derivative 19 showed weak activities at
exhibited similar selectivities for TR-a and TR-b as that of shown
1
l
M concentration for both TR-
Furthermore, we tested unsubstituted analogue 20, which did
not show pronounce activities for TR- and TR-b; however, the
selectivity towards TR-b was noteworthy.
a
and TR-b.
by the compound 2 (KB-141). The more elaborative SAR, lead opti-
mization, in vitro, in vivo studies and pharmacokinetics in rodent
models is in progress at our centre and will be published in future
course of time.
a

3922
S. Raval et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3919–3924
Table 1
In vitro % activity of novel 3-hydroxy-cyclobut-3-ene-1,2-dione compounds 9–24 for TR-
a
and TR-b with respect to T3
OH
H
N
Cl
HO
O
O
R
O
Cl
9-24
M)
Compound
R
Concn (
l
% Activity for TR-aa
% Activity for TR-ba
2
KB-141
0.001
0.01
1
53.25
100.00
84.70
72.56
100.00
111.03
O
9
0.001
0.01
6.16
6.94
7.015
8.34
N
H
1
11.70
19.05
O
10
11
0.001
0.01
1
7.41
7.69
9.40
10.01
12.28
15.59
N
H
H
N
O
0.001
0.01
1
6.23
6.80
6.00
8.50
10.39
11.53
O
12
13
0.001
0.01
1
7.12
7.13
7.19
12.30
12.70
8.80
N
H
O
N
0.001
0.01
1
7.35
7.37
13.45
8.50
9.00
23.20
O
O
14
15
16
0.001
0.01
1
8.56
9.62
50.29
8.60
10.60
80.70
N
N
0.001
0.01
1
5.63
5.96
5.25
5.63
5.30
6.23
O
0.001
0.01
1
6.93
7.91
11.03
13.00
12.70
30.90
N
O
O
O
N
H
17
18
0.001
0.01
1
6.28
6.57
16.72
12.00
13.60
48.10
S
O
O
N
S
0.001
0.01
1
8.56
9.83
32.28
12.80
32.50
63.80
O
O
N
S
19
20
21
22
23
24
a
0.001
0.01
1
8.96
10.55
22.86
15.70
24.70
43.30
O
H
0.001
0.01
1
6.12
6.08
9.36
14.60
15.10
30.90
0.001
0.01
1
22.36
35.04
79.84
30.00
70.00
130.00
0.001
0.01
1
15.30
30.70
61.50
14.20
53.50
107.10
0.001
0.01
1
9.63
13.64
45.48
8.30
29.00
62.30
O
0.001
0.01
1
7.23
7.89
27.22
8.96
9.14
71.49
The TRE-luciferase assay35 has been used for the in vitro TR-
the measurements is on average of 25%.
a and TR-b activities and values are mean of duplicate measurements and expressed in nM. The variability of

S. Raval et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3919–3924
3923
Table 2
M.; Craievich, A. F.; Garratt, R. C.; Baxter, J. D.; Webb, P.; Polikarpov, I. J. Mol.
Biol. 2006, 360, 586.
30. ArgusLab 4.0, Mark A. Thompson, Planaria Software LLC, Seattle. Available
from: http://www.ArgusLab.com.
In vitro EC50 for selected compounds 21 and 22 for TR-a, TR-b
Compound
EC50 TR-aa (nM)
EC50 TR-ba (nM)
EC50 TR-a/b
31. Thompson, M. A. Molecular docking using ArgusLab, an efficient shape-based
search algorithm and the AScore scoring function. ACS meeting, Philadelphia,
2004, 172, CINF 42, PA.
32. Raval, S.; Raval, P.; Lohray, B. B.; Lohray, V. B.; Patel, P. R. PCT WO2007/132475,
2007.
2
21
22
4.5
222.00
1056.00
1.12
70.00
317.00
4.01
3.00
3.33
a
EC50 values are expressed as mean from the duplicate measurements and are
expressed in nM. The variability of the measurements is on average of 25%.
33. The X-ray crystallographic structure of the KB-141 (2) with TR-
a (PDB Code:
1NAV) and TR-b (PDB Code: 1NAX) was selected for the docking study.18
Docking was performed for compounds 9–24 using ArgusLab 4.030 in the
presence of water molecules. Hydrogens and charges were added to the ligand
and receptor using modules from DS Studio 2.0 (Accelrys, Inc.). The binding site
was defined from the coordinates of the ligand in the PDB file. Argusdock
exhaustive search docking engine was used, with grid resolution of 0.20 Å. Five
hundred docking runs were used for each compound. Docking precision was
set to ‘high precision’ and ‘flexible ligand docking’ mode was employed for
each docking run. Docked conformers for each compound were ranked and
scored using Ascore31 implemented in ArgusLab. The interaction of the top
ranked docked poses in the pocket were visualized using DS Visualizer 1.7
(Accelrys, Inc.).
Acknowledgments
Authors are thankful to Dr. Brijesh Kumar Srivastava, Ms. Rina
Soni, and Mr. Sidhartha S. Kar for their help in the preparation of
the manuscript, management of the Zydus Group for encourage-
ment and the analytical department for the support.
34. Spectroscopic data for compounds 9–24:
References and notes
Compound 9: 70% Yield; 96.1% purity by HPLC; mp 203–205 °C; 1H NMR
(300 MHz, DMSO-d6): d 9.61 (s, 1H), 7.94 (s, 2H), 7.53 (d, J = 2.76 Hz, 1H), 6.81
(d, J = 9.0 Hz, 1H), 6.72 (d, J = 9.03 Hz, 1H), 4.10–4.12 (m, 1H), 1.17 (d,
J = 6.57 Hz, 6H); ESI-MS: 449.0 [MꢁH]+.
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6.88 (s, 2H), 2.01–2.03 (br s, 9H), 1.62–1.64 (br s, 6H); ESI-MS: 541.0 [MꢁH]+.
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27. Li, Y. L.; Litten, C.; Koehler, K. F.; Mellstrom, K.; Garg, N.; Collazo, A. M. G.;
Farnegard, M.; Grynfarb, M.; Husman, B.; Sandberg, J.; Malm, J. Bioorg. Med.
Chem. Lett. 2006, 16, 884.
28. Sandler, B.; Webb, P.; Apriletti, J. W.; Huber, B. R.; Togashi, M.; Cunha Lima, S.
T.; Juric, S.; Nilsson, S.; Wagner, R.; Fletterick, R. J.; Baxter, J. D. J. Biol. Chem.
2004, 279, 55801.
(300 MHz, DMSO-d6):
d 9.61 (s, 1H), 9.53 (s, 1H), 7.91 (s, 2H), 6.80 (d,
J = 8.91 Hz, 1H), 6.68–6.72 (dd, J = 8.8 and 3.06 Hz, 1H), 6.45 (d, J = 3.03 Hz, 1H),
2.71–2.79 (br s, 6H); ESI-MS: 435.0 [MꢁH]+.
Compound 14: 61% Yield; 96.9% purity by HPLC; mp >220 °C; 1H NMR
(300 MHz, DMSO-d6):
d 9.69 (s, 1H), 9.63 (s, 1H), 7.92 (s, 2H), 6.81 (d,
J = 8.91 Hz, 1H), 6.70–6.74 (dd, J = 8.79 and 3 Hz, 1H), 6.51 (d, J = 3 Hz,1H),
3.18–3.20 (br s, 4H), 1.77–1.79 (br s, 4H); ESI-MS: 461.1 [MꢁH]+.
Compound 15: 66% Yield; 97.1% purity by HPLC; mp 186–190 °C; 1H NMR
(300 MHz, DMSO-d6): d 11.90 (s, 1H), 9.54 (s, 1H), 8.07 (s, 2H), 6.78 (d,
J = 8.49 Hz, 2H), 6.47 (s, 1H), 3.14–3.16 (br s, 4H), 1.43–1.53 (m, 6H); ESI-MS:
475.1 [MꢁH]+.
Compound 16: 64% Yield; 96.3% purity by HPLC; mp 210–214 °C; 1H
NMR(300 MHz, DMSO-d6): d 9.65 (s, 1H), 9.63 (s, 1H), 7.92 (s, 2H), 6.80 (d,
J = 8.79 Hz, 1H), 6.70 (d, J = 6.21 Hz, 1H), 6.52 (d, J = 2.19 Hz, 1H), 3.53–3.55 (br
s, 4H), 3.14–3.16 (br s, 4H); ESI-MS: 477.0 [MꢁH]+.
Compound 17: 50% Yield; 98.1% purity by HPLC; mp 182–184 °C; 1H NMR
(300 MHz, DMSO-d6): d 11.92 (s, 1H), 8.09 (s, 2H), 7.21 (d, J = 7.65 Hz, 1H),
7.06–7.10 (dd, J = 8.7 and 3 Hz, 1H), 6.93 (d, J = 3 Hz, 1H), 2.78–2.80 (br s, 1H),
1.53–1.56 (m, 4H), 1.02–1.18 (m, 6H); ESI-MS: 524.9 [MꢁH]+.
Compound 18: 87% Yield; 97.5% purity by HPLC; mp 189–193 °C; 1H NMR
(300 MHz, DMSO-d6): d 11.94 (s, 1H), 10.43 (s, 1H), 8.10 (s, 2H), 6.94–7.07 (m,
3H), 3.03–3.05 (br s, 4H), 1.45-1.47 (br s, 6H); ESI-MS: 510.9 [MꢁH]+.
Compound 19: 60% Yield; 98.3% purity by HPLC; mp 196–199 °C; 1H NMR
(300 MHz, DMSO-d6): d 11.95 (s, 1H), 10.58 (s, 1H), 8.09 (s, 2H), 6.95–7.09 (m,
3H), 3.55–3.57 (br s, 4H), 3.03–3.05 (br s, 4H); ESI-MS: 513.0 [MꢁH]+.
Compound 20: 65% Yield; 98.1% purity by HPLC; mp 205–208 °C; 1H NMR
(300 MHz, DMSO-d6): d 9.71 (s, 1H), 9.06 (s, 1H), 7.81 (s, 2H), 6.53–6.62 (m,
4H); ESI-MS: 364.0 [MꢁH]+.
Compound 21: 41% Yield; 98.9% purity by HPLC; mp 210–213 °C; 1H NMR
(300 MHz, DMSO-d6): d 9.63 (s, 1H), 8.99 (s, 1H), 7.90 (s, 2H), 6.62–6.65 (m,
2H), 6.25–6.29 (dd, J = 8.67 and 3.03 Hz, 1H), 3.11–3.15 (m, 1H), 1.10 (d,
J = 6.9 Hz, 6H); ESI-MS: 406.0 [MꢁH]+.
Compound 22: 96% Yield; 99.1% purity by HPLC; mp 215–218 °C; 1H NMR
(300 MHz, DMSO-d6): d 9.56 (s, 1H), 9.04 (s, 1H), 7.91 (s, 2H), 6.71 (d, J = 3 Hz,
1H), 6.64 (d, J = 8.7 Hz, 1H), 6.26–6.30 (dd, J = 8.64 and 3.03 Hz, 1H), 1.29 (s,
9H); ESI-MS: 420.1 [MꢁH]+.
Compound 23: 81% Yield; 98.2% purity by HPLC; mp 219–221 °C; 1H NMR
(300 MHz, DMSO-d6): d 9.58 (s, 1H), 9.14 (s, 1H), 7.89 (s, 2H), 7.10–7.26 (m,
5H), 6.69 (d, J = 8.73 Hz, 1H), 6.58 (d, J = 3.03 Hz, 1H), 6.34–6.38 (dd, J = 8.64
and 3.09 Hz, 1H), 3.80 (s, 2H); ESI-MS: 454.1 [MꢁH]+.
Compound 24: 58% Yield;98.6% purity by HPLC; mp >222 °C; 1H NMR
(300 MHz, DMSO-d6): d 9.61 (s, 1H), 9.27 (s, 1H), 7.89 (s, 2H), 7.27–7.32 (m,
2H), 6.98–7.03 (m, 1H), 6.81–6.88 (m, 3H), 6.42–6.48 (m, 2H); ESI-MS: 456.1
[MꢁH]+.
29. Nascimento, A. S.; Dias, S. M. G.; Nunes, F. M.; Aparicio, R.; Ambrosio, A. L. B.;
Bleicher, L.; Figueira, A. C. M.; Santos, M. A. M.; Neto, M. O.; Fischer, H.; Togashi,
35. Thyroid receptor assay: A luciferase receptor assay has been used to find the
TR-a and TR-b selectivities of the compounds, where luciferase gene

3924
S. Raval et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3919–3924
expression is driven by a thyroid receptor binding element (TRE) upstream
media having different concentrations of the agonist. The concentration of
agonist is adjusted in such way that the concentration of the solvent
(DMSO) in each well is maintained at 1%. The plates are incubated at 37 °C
of the luciferase gene. Briefly 6 ꢂ 104 CV-1 cells were plated in each well of
a
a 24-well cell culture plate. The cells were transfected 16 h after seeding
with a plasmid bearing three copies of TRE cloned upstream of luciferase
gene along with a plasmid expressing either the full length human thyroid
for 16 h before lysing and assaying the luciferase activity using
commercially available Glo-lysis kit from Promega and
a standard
receptor-
a
or -b isoform and a third plasmid expressing b-galactosidase. The
luminometer. The b-galactosidase activity was measured by using the b-
galactosidase assay kit from Promega and the absorbance was read at
415 nM.
transfection is carried out using polyfect reagent from Invitrogen, Inc.
(Carlsbad, CA). The medium is replaced 6 h post transfection with fresh