3-Amino-5-phenoxythiophenes: Syntheses and structure-function studies of a novel class of inhibitors of cellular L-triiodothyronine uptake

Article abstract of DOI:10.1021/jm980288r

A series of substituted 3-amino-5-phenoxythiophenes was synthesized starting from malodinitrile and carbon disulfide. The resulting dicyanoketenedithiolate reacts via Thorpe-Dieckmann cyclization with halogen methanes bearing electron-withdrawing groups t

Full text of DOI:10.1021/jm980288r

J . Med. Chem. 1999, 42, 1849-1854
1849
3-Am in o-5-p h en oxyth iop h en es: Syn th eses a n d Str u ctu r e-F u n ction Stu d ies of a
Novel Cla ss of In h ibitor s of Cellu la r L-Tr iiod oth yr on in e Up ta k e
Detlef Briel,*,† Dirk Pohlers,† Mathias Uhlig,‡ Silke Vieweg,‡ Gerhard H. Scholz,*,‡ Michael Thormann,§ and
Hans-J o¨rg Hofmann*,§
Institute of Pharmacy, Faculty of Biosciences, Pharmacy, and Psychology, University of Leipzig, Bru¨derstrasse 34,
D-04103 Leipzig, Germany, Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology,
University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany, and Department of Internal Medicine III,
University of Leipzig, Philipp-Rosenthal-Strasse 29, D-04103 Leipzig, Germany
Received November 12, 1998
A series of substituted 3-amino-5-phenoxythiophenes was synthesized starting from malo-
dinitrile and carbon disulfide. The resulting dicyanoketenedithiolate reacts via Thorpe-
Dieckmann cyclization with halogen methanes bearing electron-withdrawing groups to give
thiophene-2-thiolates, which can be transformed into 3-amino-5-(methylsulfonyl)thiophene-4-
carbonitriles. Replacement of the methylsulfonyl groups by substituted phenolates provides
the substituted 3-amino-5-phenoxythiophenes. Some of the derivatives show a considerable
inhibitory potency for the L-T3 uptake in inhibition studies on human HepG2 hepatoma cells
with maximum values of about 60% at a dose of 10-5 M for the most potent 2-benzoyl derivatives.
The structure of the phenoxythiophenes fits well into a general concept derived for other classes
of L-T3 uptake inhibitors, which postulates an angular and perpendicular orientation of the
ring systems in these compounds as a prerequisite for an inhibitory potency. Docking studies
for the phenoxythiophenes with transthyretin as a receptor model show their preferred attack
at the L-T4/L-T3 binding channel.
In tr od u ction
Hyperthyroidism is characterized by an enhanced
synthesis of L-tetraiodothyronine (L-thyroxine, L-T4) and
L-triiodothyronine (L-T3, 1a in Figure 1), which repre-
sents the biologically active compound.1,2 The relevant
therapeutic agents presently used for the treatment of
hyperthyroidism, e.g., thiamazole, carbimazole, 6-pro-
pylthiouracil, and lithium, which inhibit synthesis or
release of the thyroid hormones, show considerable side
effects.3 In the search for alternatives, substances
inhibiting the cellular uptake of L-T3 might be useful
tools for the suppression of the symptoms caused by
thyroid hormone excess.
Several structurally and functionally unrelated classes
of compounds, e.g., nonsteroidal antiinflammatory drugs
of the N-phenylanthranilic acid type,4 benzodiaz-
epines,5,6 and 4-phenyl-1,4-dihydropyridine calcium chan-
nel blockers,7,8 are able to inhibit the L-T3 uptake in rat
F igu r e 1. Selected conformations of substituted phenoxy-
thiophenes in comparison to L-T3, diphenyl ether, and ben-
zophenone structures.
H4 and human HepG2 hepatoma cells. A series of
oxamic acid and acetic acid derivatives of thyronine
showed in vitro binding to rat liver nuclear and rat
membrane L-T3 receptors.9
to the preferred conformation in the thyroid hormones
General ideas about structure-activity relationships
established in series of L-T3 derivatives10-20 and our
data from L-T3 uptake inhibition studies with substi-
tuted 4-phenyl-1,4-dihydropyridines and N-phenylan-
thranilic acids suggest a structure-activity concept
postulating an angular orientation of two aromatic or
conjugated ring systems, where one ring is approxi-
mately bisecting the plane of the other corresponding
1a but differing from the propeller-like minimum con-
formation of diphenyl ether 1b (Figure 1).8 Following
the lines of this concept, phenoxy-substituted aromatic
heterocycles could possibly provide a promising pool of
compounds for the development of efficient L-T3 uptake
inhibitors in the therapy of hyperthyroidism.
In this paper, a novel class of substances with
3-amino-5-phenoxythiophene as the parent compound
(9 and 10 in Scheme 1) was synthesized and tested for
L-T3 uptake inhibition. The structure of the new com-
pounds was compared with the conformations of the
thyroid hormones, the 4-phenyl-1,4-dihydropyridines
* To whom correspondence should be addressed.
† Institute of Pharmacy.
§ Institute of Biochemistry.
‡ Department of Internal Medicine III.
10.1021/jm980288r CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/30/1999

1850 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Brief Articles
Ta ble 1. Inhibition Data and Partition Coefficients for Various
3-Amino-5-phenoxythiophenes 9 and 10
Sch em e 1
compd
R1
R2
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CONH2
CONH2
CONH2
R3
% Ia
Clog Pb
9a
9b
9c
9d
9e
9f
9g
9h
9i
COOCH3
COOCH3
CONH2
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
41 ( 5
19 ( 3
14 ( 2
23 ( 3
61 ( 7
57 ( 7
18 ( 3
16 ( 2
21 ( 2
26 ( 3
26 ( 3
40 ( 5
60 ( 8
3.66
4.16
2.19
2.69
4.75
5.25
4.53
5.03
2.54
3.04
3.11
3.61
3.60
CONH2
COC6H5
COC6H5
COC6H4-p-NO2
COC6H4-p-NO2
CN
9j
CN
10a
10b
10c
COOCH3
COOCH3
COC6H5
a
Application dose 10-5 M, five independent measurements.
b
Calculations of the partition coefficients are based on the Clog
P software.32
thiophenes showed L-T3 uptake inhibition in a range of
14-61% when applying a dose of 10-5 M. The most
potent compounds were the 2-benzoyl derivatives 9e,f
and 10c.
and the N-phenylanthranilic acids, to examine whether
they fit into the general structure-activity concept.
Ch em istr y
Str u ctu r e-Activity Rela tion sh ip s
Dicyanoketenedithiolate 4 (Scheme 1), which is avail-
able by addition of malodinitrile to carbon disulfide, was
the starting material for the preparation of the thio-
phenes.21 It reacts with equimolar amounts of halogen
methanes substituted with electron-withdrawing groups
followed by a Thorpe-Dieckmann cyclization to give
thiophene-2-thiolates 6.22,23 After methylation and oxi-
dation of the methylthio group employing standard
methods, 3-amino-5-(methylsulfonyl)thiophene-4-carbo-
nitriles24,25 8 are formed. The thiophenes substituted in
the 2-position are suitable for nucleophilic replacement
of the methylsulfonyl group by some C-, O-, S-, and
N-nucleophiles. Thus, the reactions with sodium meth-
oxide provide the 5-methoxythiophenes and with hy-
drazine hydroxide or morpholine the corresponding
substituted thiophenes, respectively.25,26 Therefore, it
should also be possible that substituted phenolates
might be able to replace the leaving group by a nucleo-
philic attack.
The 5-phenoxythiophenes 9 were obtained by heating
of the 5-(methylsulfonyl)thiophenes 8 with phenol or
m-cresol in the presence of a base under reflux or by
stirring the reactants at room temperature. In another
case the sodium salt of phenol was used. To get
4-carbamoylthiophenes 10 in good yields the nitrile
functions of some 4-phenoxythiophenes 9 were hydro-
lyzed by treatment with concentrated sulfuric acid for
24-48 h. Under these conditions the reaction stops at
the amide level.
The conformational basis of the thyroid hormone
structure to realize good binding properties is well-
established and documented.10-20 The two phenyl rings
of the diphenyl ether moiety have to be in an angular
orientation, and one of the rings is approximately
bisecting the plane of the other. In L-T4 and L-T3 the
so-called outer ring bearing the 4’-hydroxy group is
about perpendicular to the inner ring with the ortho
iodine atoms (1a in Figure 1). For comparison, the
propeller-like conformation of diphenyl ether 1b is also
visualized.
It has to be proved whether the 5-phenoxythiophenes
fit into this concept. For this purpose, the conformation
of the model compounds 2 (Figure 1) was examined
employing ab initio MO theory at the MP2/6-31G* and
HF/6-31G* levels.27 Independent of the approximation
level, the conformational analysis for the unsubstituted
compound 2 (R ) H) provides two sets of energetically
rather equivalent conformers with an approximately
perpendicular ring orientation (Table 2). In the one the
phenyl ring is almost bisecting the thiophene ring (2a );
in the other, alternatively, the thiophene ring is bisect-
ing the phenyl ring plane. In the latter case, there are
the two alternatives: 2b with distal and 2c with
proximal orientation of the thiophene sulfur atom to the
phenyl ring. These conformers are kept in the 5-phen-
oxythiophenes with amide or nitrile groups for R in 2.
Thus, it can be concluded that all 5-phenoxythiophenes
are able to realize conformers equivalent to those of the
thyroid hormones.
In h ibition Stu d ies
The inhibitory potency of the 3-amino-5-phenoxy-
thiophenes 9a -j and 10a -c (for detailed structures, see
Table 1) was studied in human HepG2 hepatoma cells.
The results are presented in Table 1. All phenoxy-
In the case of the especially potent tricyclic com-
pounds 9e,f and 10c with the additional 2-benzoyl
moiety, the conformation of the benzophenone-like part
has also to be taken into consideration, since thyroid

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1851
Ta ble 2. Conformation Data for the Phenoxythiophene
Conformers 2a -c and the Benzoylthiophene Conformers 3b,c
from ab Initio MO Theory at the MP2/6-31G* and HF/6-31G*
Levels
b-d
compda
φb-d
φ′
∆Eb,e
2a
2b
2c
3a
3b
3c
83.9; 83.2
179.7; 179.3
-0.1; -0.1
32.0; 32.6
14.4; 15.2
-90.6; -91.3
-91.1; -91.4
32.0; 32.6
37.5; 37.9
39.1; 37.5
0;f 0.8
5.5; 0g
6.8; 6.2
h
-166.2; -165.0
0;i 0j
19.2; 21.0
5.7; 6.3
a
b
See Figure 1. First value, MP2/6-31G*; second value, HF/6-
31G*. c Angles in degrees. Torsion angles in 2a -c, φ S-C2-O-
d
C1′, φ′ C2-O-C1′-C6′; in 3a , φ C6-C1-C(O)-C1′, φ′ C6′-C1′-
C(O)-C1; in 3b,c, φ S-C2-C(O)-C1′, φ′ C2-C(O)-C1′-C6′.
e Relative energies in kJ mol-1 f ET(MP2) ) -857.246747 a.u.
.
h
g
ET(HF) ) -855.679460 a.u. ET(MP2) ) -574.798740 a.u.;
ET(HF)) -572.986525 a.u. i ET(MP2) ) -895.269215 a.u. j ET(HF)
) -893.575291 a.u.
hormone analogues with a benzophenone moiety instead
of the diphenyl ether structure do not lose binding
affinity and show the possibility to arrange the rings
perpendicularly to each other.10,14 Therefore, the torsion
angles in the corresponding 2-benzoylthiophenes 3b,c
(Figure 1) are also given in Table 2. They show close
correspondence to the propeller-like benzophenone con-
formation 3a , but the perpendicular orientation of the
rings can easily be realized also in this part of the
molecules and is supported by additional ortho substit-
uents.
To get deeper insight into the binding behavior of the
various derivatives of the phenoxythiophene series,
docking studies were performed employing the program
AUTODOCK,28,29 which successfully reproduces the
X-ray structures of protein complexes with small ligands.
The thyroxine-binding protein transthyretin (TTR),
which shows two identical binding sites in its tetrameric
form, served as model for a possible receptor. To
increase the reliability of the docking data, two inde-
pendent docking studies were performed for all deriva-
tives. The one is based on the X-ray structure for a
complex of human TTR with L-T4 (1eta);30 the other uses
the rat TTR tetramer (1gke).31 Both structures show a
rather perfect agreement, when fitting their protein
backbones. The rmsd for the 480 CR atoms is 1.842 Å.
The ligands, whose starting positions were chosen far
away from the L-T4 binding site, were able to contact
the protein surface areas around the channel within a
grid of 30 × 30 × 36 Å3 in the docking runs (for further
details, cf. Experimental Section). To test the docking
method for our purposes, it was used to reproduce the
highly interesting X-ray structure of the TTR complex
with 3′,5′-dinitro-N-acetyl-L-thyronine (2roy) coming
from the Cody group.32 In this complex, the ligand is in
an unusual orientation distinctly different from that of
L-T4 in its TTR complex. The ligand arrangement
obtained from this docking study closely corresponds to
that in the X-ray complex (rmsd ) 0.950 Å without the
Ala residue and rmsd ) 2.095 with the Ala residue) and
confirms the reliability of the AUTODOCK technique.
Most important result of both docking series with the
phenoxythiophene derivatives is the strong tendency of
all compounds to occupy the L-T4 binding channel.
Docking with the 1eta TTR, but not with 1gke, provides
another binding site outside the channel, which is
energetically equivalent (for the detailed characteristics
F igu r e 2. Comparison of the arrangements of L-T4 (top) and
the potent L-T4 uptake inhibitor 9e (below) in the TTR binding
channel arising from docking studies.
of both binding sites, see Supporting Information). This
second binding site disappears, however, when allowing
for reorientation of the amino acid side chains in this
region. Obviously, it arises from special side chain
effects in the 1eta crystal structure and is not of
relevance for the discussion of the binding behavior of
our derivatives.
The comparison of the preferred orientations of the
most potent inhibitor of the 5-phenoxythiophene series
9e with the arrangement of L-T4 in its binding channel,
which was well-characterized in several papers,14,18,19
shows considerable similarity (Figure 2). Obviously, the
phenoxythiophene part and not the benzophenone-like
moiety occupies the binding site with the thiophene ring
more outside, corresponding to the inner ring of L-T4,
and the phenoxy group representing the outer ring. As
could be expected from the small energy differences

1852 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Brief Articles
Ta ble 3. Synthesis Data for the 3-Amino-5-phenoxythiophenes
9 and 10
between the conformers 2a -c reflected by the quantum
chemical calculations (Table 2), several energetically
rather equivalent complex alternatives are indicated
with the different ligand conformers. Thus, the most
stable complex of 9e visualized in Figure 2 shows the
thiophene ring approximately bisecting the phenoxy
ring, while in the case of the L-T4 complex the ligand
conformer with the outer ring bisecting the inner one
predominates due to steric restrictions. Most phenox-
ythiophene derivatives show a strong tendency to realize
an arrangement similar to that of 9e in Figure 2. For
the high inhibition potency of the 2-benzoyl derivatives
9e and 10c, additional electrostatic interactions between
the benzoyl carbonyl group and the side chain am-
monium group of the Lys 15 (Lys 15′) residue and
hydrophobic contacts of the benzoyl moiety with the
alkyl side chain of Lys 15′ (Lys 15) might be responsible.
The introduction of the nitro group at the 4-position of
the benzoyl group leads to the derivatives 9g,h with
distinctly lower inhibition potency. Docking of these
derivatives provides two alternative ligand orientations
of comparable energy. The one is different from the
typical L-T4 orientation in Figure 2 and shows the nitro-
substituted benzoyl part in the channel, which re-
sembles the above-mentioned ligand arrangement in the
2roy structure. The other is more similar to the L-T4
complex, but the ligand cannot effectively fill the bind-
ing site because of specific interactions between the nitro
group and Arg 104 outside the channel. (The corre-
sponding complex structures can be obtained as Sup-
porting Information.) To generalize the binding concept
of L-T4 uptake inhibitors from different structure classes,
prototypes of substituted 4-phenyl-1,4-dihydropyridines
and N-phenylanthranilic acids, examined in our former
inhibition studies,4,8 were also subject to docking runs.
It is clearly demonstrated in these simulations that
these derivatives also show a remarkable preference for
the interaction with the L-T4 binding site (see Support-
ing Information).
The studies on substituted 4-phenyl-1,4-dihydro-
pyridines and N-phenylanthranilic acids indicated spe-
cial conformational aspects as a presumption for inhibi-
tory potency. However, the differences between the
inhibition data for various derivatives, which are all able
to realize the demanded angular and perpendicular ring
orientations,8 turn the attention to further properties
of the compounds, which might influence the binding
behavior. In the series of substituted N-phenylanthra-
nilic acids,4 but not for the dihydropyridine calcium
antagonists,8 a correlation between inhibition strength
and hydrophobicity was found with an optimum Clog P
value of 5.7 calculated for the octanol-water partition
coefficient. The comparison of the inhibition data in the
phenoxythiophene series with the calculated Clog P
values (Table 1) shows relatively high Clog P values for
the most potent inhibitors 9e,f. Nevertheless, a strict
correlation between inhibitory potency and hydropho-
bicity cannot be seen.
yield
(%)
molecular
formula
compda
mp (°C)
anal.b
method
9a
9b
9c
9d
9e
9f
9g
9h
9i
183-184c
126-128c
208-212c
196-198d
158-160e
191-192c
175-177e
196-198c
151-153c
159-162c
210-215f
190-195f
220-224f
65
62
71
44
85
62
55
57
82
72
85
87
85
C13H10N2O3S C,H,N,O,S
C14H12N2O3S C,H,N,O,S
C12H9N3O2S C,H,N,O,S
C13H11N3O2S C,H,N,O,S
C18H12N2O2S C,H,N,O,S
C19H14N2O2S C,H,N,O,S
C18H11N3O4S C,H,N,O,S
C19H13N3O4S C,H,N,O,S
A1
A1
A1
A2
A1
A1
B
B
D
C
E
C12H7N3OS
C13H9N3OS
C,H,N,O,S
C,H,N,O,S
9j
10a
10b
10c
C13H12N2O4S C,H,N,O,S
C14H14N2O4S C,H,N,O,S
C17H14N2O3S C,H,N,O,S
E
E
a
b
For structures, see Table 1. Analytical results are within
(0.4% of theoretical values. c Recrystallized from ethanol. Recrys-
d
tallized from methanol. e Recrystallized from n-propanol. f Recrys-
tallized from ethanol/water.
orientation with their planes nearly perpendicular to
each other. Thus, the phenoxythiophenes fit well into a
general structure-activity concept derived for N-phen-
ylanthranilic acids and 4-phenyl-1,4-dihydropyridine
calcium antagonists which also exhibit a considerable
inhibition potency for L-T3 uptake. Potent inhibitors in
the phenoxythiophene series are characterized by con-
siderable hydrophobicity. Docking studies with trans-
thyretin confirm the L-T4 binding site to be distinctly
preferred for an attack of the phenoxythiophene deriva-
tives.
Exp er im en ta l Section
Ch em istr y. All melting points were determined on
a
Boe¨tius melting point apparatus. They are uncorrected. The
IR spectra were registered as KBr pellets on a Perkin-Elmer
16PC FT-IR spectrometer. All absorption values are expressed
in wavenumbers (cm-1). Proton (1H NMR) and carbon (13C
NMR) nuclear magnetic resonance spectra were recorded on
Varian Gemini 200 and 300 NMR spectrometers in the
solvents indicated. Chemical shifts (δ) are in parts per million
(ppm) relative to Si(CH3)4. The results of the elemental
analysis were within (0.4% of the calculated values.
Key In ter m ediates: 3-Am in o-5-(m eth ylth io)th ioph en e-
4-ca r bon itr iles 7. These compounds were synthesized as
previously described in ref 22.
Exa m p le P r oced u r e for th e P r ep a r a tion of 3-Am in o-
5-(m eth ylsu lfon yl)th ioph en e-4-car bon itr iles 8.23 3-Am in o-
2-(4′-n it r ob en zoyl)-5-(m et h ylsu lfon yl)t h iop h en e-4-ca r -
bon itr ile. 7h (10 g, 31.3 mmol) was suspended in a mixture
of 100 mL of acetic acid and 10 mL of hydrogen peroxide (30%)
and stirred for 14 days. The product was filtered off, washed
with ethanol and water, and recrystallized from dioxane:
1
yellow crystals; yield 8.8 g (79%); mp 192-194 °C; H NMR
(DMSO-d6) δ 8.37 (s, NH2), 8.06-7.96 (m, aromatic H), 3.53
(s, SO2CH3); 13C NMR (DMSO-d6) δ 186.35 (CdO), 156.18 (C-
Benzoyl), 153.73 (C-SO2CH3), 149.26 (Benzoyl-C1), 143.91
(Benzoyl-C4), 129.01 (Benzoyl-C2,6), 124.05 (Benzoyl-C3,5),
111.52 (CN), 110.51, 102.83 (C-NH2, C-CN), 44.22 (SO2CH3);
IR 3404, 3268, 2228, 1610, 1522, 1308, 1150. Anal. (C13H9-
N3O5S2) C, H, N, O, S.
Gen er a l P r oced u r es for th e P r ep a r a tion of 3-Am in o-
5-(a r yloxy)th iop h en e-4-ca r bon itr iles 9. For the selected
methods and general synthesis data, see Table 3.
Meth od A1. 5-(Methylsulfonyl)thiophene 8 (8.15 mmol) was
added to a solution of 32.6 mmol of sodium methoxide and 40.8
mmol of the phenol in 10 mL of methanol. The suspension was
heated under reflux for 10 min. The precipitate was filtered
off after cooling and washed with ethanol and water.
Meth od A2. Same as with method A1, but acetonitrile used
as solvent.
Con clu sion s
Some of the newly synthesized derivatives of 3-amino-
5-phenoxythiophenes proved to be potent inhibitors of
L-T3 uptake The conformation of these representatives
corresponds well to that of the thyroid hormones and
shows the thiophene and phenoxy parts in an angular

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1853
Meth od B. 5-(Methylsulfonyl)thiophene 8 (5.7 mmol) was
stirred for 2 h in a solution of 28.5 mmol of sodium methoxide
and 34.2 mmol of the phenol in 10 mL of methanol at room
temperature. The product was filtered off and recrystallized.
Meth od C. 5-(Methylsulfonyl)thiophene 8 (4.4 mmol) was
stirred for 24 h together with 5 mmol of the phenol and 7.2
mmol of potassium carbonate in 5 mL of acetonitrile. Water
was given to the solution or suspension, and the precipitate
was filtered off.
Meth od D. 5-(Methylsulfonyl)thiophene 8 (4.4 mmol) was
added to a solution of 8.8 mmol of the sodium phenolate in 10
mL of acetonitrile. The suspension was heated under reflux
for 10 min. The precipitate was filtered off after cooling and
washed with ethanol and water.
(s, CH3); 13C NMR (DMSO-d6) δ 184.19 (CdO), 178.35 (C-O-
Phe), 155.36 (Phe-C1), 154.81 (C-Benzoyl), 148.58 (Benzoyl-
C1), 145.16 (Benzoyl-C4), 140.84 (Phe-C3), 130.32 (Phe-C2),
128.62 (Phe-C4), 128.22 (Benzoyl-C2,6), 123.89 (Benzoyl-C3,5),
120.52 (Phe-C5), 117.09 (Phe-C6), 111.15 (CN), 96.96, 86.24
(C-NH2, C-CN), 20.72 (CH3); IR 3432, 3324, 2218, 1604, 1518,
1264.
3-Am in o-5-p h en oxyth iop h en e-2,4-d ica r bon itr ile (9i):
white crystals; 1H NMR (DMSO-d6) δ 7.56-7.39 (m, aromatic
H), 7.09 (s, NH2); 13C NMR (DMSO-d6) δ 174.75 (C-O-Phe),
155.05 (Phe-C1), 154.22 (C2-CN), 130.69 (Phe-C2,6), 127.79
(Phe-C4), 120.05 (Phe-C3,5), 114.41(C2-CN), 111.17 (C4-CN),
86.14 (C4-CN), 65.12 (C-NH2); IR 3402, 3346, 3246, 2228,
2202, 1210.
3-Am in o-5-(3′-m eth ylp h en oxy)th iop h en e-2,4-d ica r bo-
3-Am in o-4-cyan o-5-ph en oxyth ioph en e-2-car boxylic acid
m eth yl ester (9a ): white crystals; 1H NMR (DMSO-d6) δ
7.43-7.54 (m, 5H, aromatic H), 7.01 (s, 2H, NH2), 3.65 (s, 3H,
OCH3); 13C NMR (DMSO-d6) δ 175.65 (C-O-Phe), 163.05
(COOCH3), 155.05 (C-COOCH3), 151.91 (Phe-C1), 130.63 (Phe-
C3,5), 127.69 (Phe-C4), 120.22 (Phe-C2,6), 111.5 (CN), 86.4,
86.13 (C-NH2, C-CN), 51.2 (OCH3); IR 3334, 3426, 2230, 1682,
1254.
1
n itr ile (9j): white crystals; H NMR (DMSO-d6) δ 7.47-7.12
(m, NH2, aromatic H), 2.37 (s, CH3); 13C NMR (DMSO-d6) δ
175.09 (C-O-Phe), 155.37 (Phe-C1), 154.50 (C2-CN), 141.15
(Phe-C3), 130.64 (Phe-C2), 128.71 (Phe-C4), 120.61 (Phe-C5),
117.29 (Phe-C6), 114.64 (C2-CN), 111.49 (C4-CN), 86.37 (C4-
CN), 65.37 (C-NH2), 21.05 (CH3); IR 3396, 3344, 3244, 2228,
2198, 1238.
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Am in o-
5-(a r yloxy)t h iop h en e-4-ca r b oxylic Acid Am id es 10:
Meth od E. The thiophene-4-carbonitrile (4 mmol) was given
to 5 mL of concentrated sulfuric acid. This solution was kept
for 24-48 h at room temperature. After addition of ice-water
the precipitate was filtered off and recrystallized.
3-Am in o-4-ca r ba m oyl-5-p h en oxyth iop h en e-2-ca r boxy-
lic a cid m eth yl ester (10a ): white crystals; 1H NMR (DMSO-
d6) δ 7.69-7.30 (m, aromatic H, CONH2, NH2), 3.62 (s, OCH3);
13C NMR (DMSO-d6) δ 170.37 (C-O-Phe), 163.99, 163.49
(CONH2, COOCH3), 155.56 (Phe-C1), 154.18 (C-COOCH3),
130.29 (Phe-C3.5), 127.29 (Phe-C4), 120.79 (Phe-C2,6), 106.22
(C-CONH2), 86.52 (C-NH2), 50.75 (OCH3); IR 3470, 3454,
3178, 1684, 1610, 1224.
3-Am in o-4-ca r ba m oyl-5-(3′-m eth ylp h en oxy)th iop h en e-
2-ca r boxylic a cid m eth yl ester (10b): light-yellow crystals;
1H NMR (DMSO-d6) δ 7.66-7.19 (m, NH2, CONH2, aromatic
H), 3.62 (s, OCH3), 2.35 (s, CH3); 13C NMR (DMSO-d6) δ 170.42
(C-O-Phe), 163.99, 163.50 (CONH2, COOCH3), 155.25 (Phe-
C1), 154.18 (C-COOCH3), 140.30 (C-CH3), 129.93 (Phe-C2),
127.88 (Phe-C4), 121.04 (Phe-C5), 117.60 (Phe-C6), 106.15 (C-
CONH2, C-NH2); IR 3472, 3370, 3170m, 1682, 1604, 1242.
3-Am in o-2-b en zoyl-5-p h en oxyt h iop h en e-4-ca r b oxyl-
ic a cid a m id e (10c): light-yellow crystals; 1H NMR (DMSO-
d6) δ 8.44 (s, CONH2), 7.77 (s, NH2), 7.52-7.35 (m, aromatic
H); 13C NMR (DMSO-d6) δ 186.07 (CdO), 172.82 (C-O-Phe),
163.96 (CONH2), 156.96 (C-Benzoyl), 155.05 (Phe-C1), 140.83
(Benzoyl-C1), 130.25 (Benzoyl-C4), 130.33 (Benzoyl-C2,6),
128.42 (Phe C3, 5), 127.47 (Phe-C4), 126.69 (Benzoyl-C3, 5),
120.86 (Phe-C2, 6), 105.84 (C-CONH2), 96.73 (C-NH2); IR 3476,
3422, 3310, 3182, 1658, 1600, 1224.
Biologica l Ma ter ia ls. Tissue culture media and supple-
ments were from GIBCO BRL (Eggenstein, Germany). The
source for [125I]-L-T3 was BRAHMS Diagnostica GmbH (Berlin,
Germany). The unlabeled L-T3 was purchased from SIGMA
(Deisenhofen, Germany). The human HepG2 cells (ATCC CRL
8065-HB) came from the American Type Culture Collection
(Rockville, MD).
3-Am in o-4-cya n o-5-(3′-m et h ylp h en oxy)t h iop h en e-2-
ca r boxylic a cid m eth yl ester (9b): white crystals; 1H NMR
(DMSO-d6) δ 7.23-7.43 (m, aromatic H), 7.03 (s, NH2), 3.67
(s, OCH3), 2.37 (s, Phe-CH3); 13C NMR (DMSO-d6) δ 176.01-
(C-OPhe), 163.37 (COOCH3), 155.37 (C-COOCH3), 152.22
(Phe-C1), 141.06 (Phe-CH3), 130.57 (Phe-C2), 128.60 (Phe-C4),
120.78 (Phe-C5), 117.36 (Phe-C6), 111.83 (CN), 86.62 (C-NH2,
C-CN), 51.49 (OCH3), 21.05 (Phe-CH3); IR 3462, 3336, 2228,
1682, 1254.
3-Am in o-4-cya n o-5-p h en oxyt h iop h en e-2-ca r b oxylic
a m id e (9c): white crystals; 1H NMR (DMSO-d6) δ 7.56-7.39
(m, 5H, aromatic H), 6.96 (4H, NH2, CONH2); 13C NMR
(DMSO-d6) δ 172.50 (C-O-Phe), 165.48 (CONH2), 155.35
(C1′), 150.26 (C-CONH2), 130.63 (C2′,6′), 127.36 (C4′), 120.18
(C3′,5′), 111.92 (CN), 89.76, 87.06 (C-NH2, C-CN); IR 3454,
3396, 3352, 3198, 2236, 1670, 1264.
3-Am in o-4-cya n o-5-(3′-m et h ylp h en oxy)t h iop h en e-2-
ca r boxylic a m id e (9d ): white crystals; 1H NMR (DMSO-d6)
δ 7.41-7.2 (m, aromatic H), 6.96 (s, CONH2), 6.94 (s, NH2),
2.35 (s, CH3); 13C NMR (DMSO-d6) δ 172.57 (C-OPhe), 165.48
(CONH2), 155.37 (C-CONH2), 150.25 (Phe-C1), 140.69 (Phe-
C3), 130.27 (Phe-C2), 127.99 (Phe-C4), 120.46 (Phe-C5), 117.05
(Phe-C6), 111.93 (CN), 89.73, 86.97 (C-NH2, C-CN), 20.76
(CH3); IR 3470, 3426, 3354, 3204, 2220, 1682, 1278.
3-Am in o -2-b e n zo y l-5-p h e n o x y t h io p h e n e -4-c a r b o -
n itr ile (9e): white crystals; 1H NMR (DMSO-d6) δ 7.36-7.54
(m, aromatic H), 8.17 (s, NH2); 13C NMR (DMSO-d6) δ 186.43
(CdO), 177.58 (C-O-Phe), 154.87, 154.68 (C-Benzoyl and
Phe-C1), 139.90 (Benzoyl-C1), 131.03 (Benzoyl-C4), 130.66
(Benzoyl-C2,6), 128.57 (Benzoyl-C3,5), 127.84 (Phe-C4), 126.76
(Phe-C3,5), 120.25 (Phe-C2,6), 111.33 (CN), 97.09, 86.37 (C-
NH2, C-CN); IR 3404, 3302, 2228, 1596, 1256.
3-Am in o-2-ben zoyl-5-(3′-m eth ylp h en oxy)th iop h en e-4-
1
ca r bon itr ile (9f): white crystals; H NMR (CDCl3, DMSO-
d6) δ 7.63-7.04 (m, aromatic H, NH2), 2.38 (s, CH3); 13C NMR
(DMSO-d6, CDCl3) d 187.78 (CdO), 177.42 (C-O-Phe), 155.38
(Phe-C1), 154.29 (C-Benzoyl), 141.04 (Phe-C3), 139.97 (Ben-
zoyl-C1), 131.24 (Benzoyl-C4), 130.10 (Phe-C2), 128.67 (Ben-
zoyl-C2,6), 128.39 (Phe-C4), 127.37 (Benzoyl-C3,5), 120.61
(Phe-C5), 116.94 (Phe-C6), 111.39 (CN), 98.69 (C-CN), 87.22
(C-NH2), 21.41 (CH3); IR 3414, 3310, 2226, 1600, 1256.
3-Am in o-2-(4′-n itr oben zoyl)-5-ph en oxyth ioph en e-4-car -
bon itr ile (9g): yellow crystals; 1H NMR (DMSO-d6) δ 8.30
(s, NH2), 8.25-7.39 (m, aromatic H); 13C NMR (DMSO-d6) δ
184.20 (CdO), 178.31 (C-O-Phe), 155.37 (Phe-C1), 154.80
(C-Benzoyl), 148.58 (Benzoyl-C1), 145.13 (Benzoyl-C4), 130.71
(Benzoyl-C2,6), 128.20 (Benzoyl-C3,5), 128.00 (Phe-C4), 123.88
(Phe-C3,5), 120.25 (Phe-C2,6), 111.14 (CN), 96.98 (C-CN),
86.30 (C-NH2); IR 3372, 3264, 2226, 1604, 1518, 1260.
3-Am in o-2-(4′-n it r ob e n zoyl)-5-(3′-m e t h ylp h e n oxy)-
th iop h en e-4-ca r bon itr ile (9h ): yellow crystals; 1H NMR
(DMSO-d6) δ 8.29 (s, NH2), 8.26-7.19 (m, aromatic H), 2.31
L-T3 Up ta k e. The screening procedure for the inhibitors of
[
125I]-L-T3 uptake corresponds to that of Chalmers et al.4 The
cells were maintained in DMEM (Dulbecco’s modified of
Eagle’s medium) with 10% fetal calf serum at 37 °C, subcul-
tured for the uptake experiments in 2-mL wells, and grown
to confluence (2×106 cells/well). Cultivation was continued in
serum-free DMEM overnight. This medium was then replaced
by DMEM containing 10-11 M [125I]-L-T3 (specific activity >
3200 µCi/µg). The uptake of [125I]-L-T3 was measured after 2
min at 37 °C. The nondisplaceable uptake was determined
from duplicate incubations containing 10-5 M unlabeled L-T3.
For screening the test substances were applied in a dose of
10-5 M together with [125I]-L-T3. They were dissolved in 100%
DMSO for stock solutions of 10-2 M and added to DMEM to

1854 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
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give the desired final concentration. The controls contained
equivalent amounts of solvent. The uptake was terminated
after incubation by decanting the medium and washing the
cells five times with PBS (2 mL; pH 7.4) at room temperature.
The cells were harvested with 2 mL of 0.1 M NaOH, and the
uptake was measured as the cell-associated radioactivity.
Th eor etica l Ca lcu la tion s. The quantum chemical calcula-
tions on the model compounds 2 and 3 were performed
employing the SPARTAN 4.1 program package.33 The geom-
etries of all structures were completely optimized at the MP2/
6-31G* and HF/6-31G* levels of ab initio MO theory.27 The
octanol-water partition coefficients were calculated on the
basis of the Clog P software, version 1.0.0, from BioByte Corp.34
For the docking studies the transthyretin and ligand
structures were prepared with the QUANTA96 modeling
software35 by addition of polar hydrogens and template
charges. A grid of 30 × 30 × 36 Å3 size was centered at the
L-T4 binding channel to realize the docking employing the
AUTODOCK program, version 2.4.28,29 The grid spacing was
0.3 Å. The starting arrangements of the ligands were randomly
chosen with all torsion angles given free. Each docking
consisted of 25 runs per ligand, 120 cycles per run, and 100 000
accepted or rejected steps per cycle resulting in about 300
million conformations. The annealing temperature, RT, was
600.0 cal mol-1 during the first cycle. The initial translation
step was 0.8 Å per step; quaternion and torsional variations
were 30° per step. The reduction factors applied after each
cycle for each parameter were 0.93 for RT, 0.96 for translation,
0.97 for quaternion, and 0.95 for torsion. Every new cycle start-
ed from the lowest-energy arrangement of the previous one.
Ack n ow led gm en t. Technical assistance by Mrs. S.
Kistner, Mrs. J . Ortwein, and Mrs. E. Trobisch is grate-
fully acknowledged. The authors are obliged to Deutsche
Forschungsgemeinschaft (Innovationskolleg Chemis-
ches Signal und Biologische Antwort and Scho 543/2)
and the German Fonds der Chemischen Industrie.
Su p p or tin g In for m a tion Ava ila ble: PDB files of all rat
and human TTR-ligand complexes and characteristics of the
two most stable binding sites in the 1eta complexes obtained
from the docking studies and discussed in the text. This
information is available free of charge via the Internet at
http://pubs.acs.org.
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