Synthesis of thieno[2,3-b]pyridinones acting as cytoprotectants and as inhibitors of [3H]glycine binding to the N-methyl-D-aspartate (NMDA) receptor

Article abstract of DOI:10.1021/jm0503493

The standard glycine site antagonist of the N-methyl-D-aspartate (NMDA) receptor, 3-phenyl-4-hydroxyquinolin-2(1H)-one (21), was used as a template for bioisostere benzene/thiophene exchange. Phenylacetylation of aminothiophene carboxylic acid methyl este

Full text of DOI:10.1021/jm0503493

864
J. Med. Chem. 2006, 49, 864-871
Synthesis of Thieno[2,3-b]Pyridinones Acting as Cytoprotectants and as Inhibitors of
[3H]Glycine Binding to the N-Methyl-D-aspartate (NMDA) Receptor
Hans-Peter Buchstaller,† Carsten D. Siebert, Ralf Steinmetz, Ina Frank, Michael L. Berger,*,‡ Rudolf Gottschlich,†
Joachim Leibrock,† Michael Krug,† Dieter Steinhilber, and Christian R. Noe§
Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe UniVersity, D-60439 Frankfurt/Main, Germany
ReceiVed April 14, 2005
The standard glycine site antagonist of the N-methyl-D-aspartate (NMDA) receptor, 3-phenyl-4-hydroxy-
quinolin-2(1H)-one (21), was used as a template for bioisostere benzene/thiophene exchange. Phenylacetylation
of aminothiophene carboxylic acid methyl esters and subsequent cyclization delivered the three possible
thienopyridinone isomers. 4-Hydroxy-5-phenylthieno[2,3-b]pyridin-6(7H)-one (3a), with the shortest distance
between the sulfur and the nitrogen atom, was the most potent isomer (Ki against the binding of [3H]glycine
to rat membranes 16 µM), comparable in potency to the model quinolinone (21, 12 µM). Replacement of
the phenyl substituent of 21 by a 2-thienyl residue resulted in a 2-5-fold loss in potency and was abandoned.
In the thieno part of the thienopyridinone nucleus, the most successful substituents were halogen (Cl or Br)
close to the sulfur atom and short alkyl chains at the other position, resulting in 7h, 8h, 8i, and 8m, with
Ki values between 5.8 and 10.5 nM. Introduction of a 3′-phenoxy moiety yielded several compounds with
still higher potencies (18h, 18i, 18l, and 18m; Ki between 1.1 and 2.0 nM). Quantitative structure-activity
relationship (QSAR) calculations resulted in a consistent interpretation of the potencies of most compounds.
Several of these 3′-phenoxy derivatives protected mouse fibroblast cell lines with transfected NMDA receptors
from glutamate-induced toxicity. In addition, we report in vivo results for four of these compounds.
Introduction
1). Binding affinities were determined in a competition binding
assay with the radioligand [3H]glycine and rat brain membranes.
To demonstrate the in vitro cell protective potency of the com-
pounds, we used the fibroblast cell line L(tk-) stably transfected
with the recombinant human NR subunits NR1a and NR2A in
a cell toxicity assay.11 Finally, we present in vivo data on four
of our most potent compounds.
N-Methyl-D-aspartate (NMDA) receptors (NRs) are ligand-
gated ion channels with a high permeability for Ca2+. They
contribute to excitatory neurotransmission1 and synaptic plasti-
city2 as well as to neurodegenerative diseases3 like stroke,
epilepsy, Parkinson’s disease, Huntington’s chorea, Alzheimer’s
disease, and HIV dementia. The endogenous amino acid glycine
acts as a co-agonist at the NR,4 and the glycine binding site of
the NR has become a target for drug development.5,6
Immediately following the discovery of the glycine site of
the NR,7 it was recognized that kynurenic acid acts as an
antagonist at this site.8 In addition, many derivatives of 2,3-
dihydroxyquinoxaline (QX), originally introduced as antagonists
of non-NMDA receptors, were subsequently shown to block
the glycine site of the NR.9 For the present study, a series of
2-quinolone derivatives was designed with the goal of combining
structural features present in kynurenic acids and QX. Quino-
lones with a phenyl substituent vicinal to the keto function10
belong to the most potent glycine antagonists yet described, both
in vitro and in vivo (e.g., L-701,324; see ref 5 for review). These
compounds lack the carboxylic acid function of kynurenic acid;
it is supposed that the anionic functionality, which is essential
for binding, is contributed by an electron excess in the phenyl
substituent.
Bioisosterism. The pair benzene/thiophene represents one of
the most prominent examples of bioisosterism: the exploratory
replacement in successful drugs of a benzene by a thiophene
moiety has become a routine strategy in modern drug design.
Both aromatic rings are similar in size (isostere) and elec-
tronic properties. The diameter of the sulfur atom in thiophene
is roughly equivalent to the distance between two neighboring
carbon atoms in benzene. The physiological effects of thiophene
are similar to those of benzene (bioisostere), with frequently
superior pharmacodynamic, pharmacokinetic, or toxicological
properties. Finally, the three different fusion patterns in thio-
phene allow for a fine-tuning not accessible in benzene deriv-
atives (Chart 1). When the three positions of the sulfur atom in
the three thienopyridinone isomers 3a, 3b, and 3c are taken into
account, each isomer allows attachment of two residues with
slightly differing orientations. By this synthetic strategy, six
potential pharmacophore positions can be addressed, whereas
the single benzo[b]pyridinone isomer addresses only four such
positions. Thus, thiophene-containing ligands may become
sublime tools to investigate receptor binding pockets.12
The aim of this work is to present synthetic protocols and
results of binding assays for a series of thieno[2,3-b]pyridin-
6(7H)-one derivatives, derived from quinolones by replacement
of the benzene nucleus by thiophene (for structures see Chart
Chemistry. For the preparation of all compounds described
in this paper, the appropriate substituted aminothiophenes were
required as starting materials. Compounds 1a-c, which were
necessary for the syntheses of the different isomeric thienopy-
ridinones 3a-c, were either obtained according to the method
of Gewald et al.13 (1a) or commercially available (1b, 1c). The
syntheses of compounds 3a-c were accomplished in two steps
as depicted in Scheme 1. Acylation of 1a-c with a small excess
* Correspondence should be addressed to this author at the Center for
Brain Research, A-1090 Spitalgasse 4, Vienna, Austria. Tel (+431) 4277
62892; fax (+431) 4277 62899; e-mail michael.berger@meduniwien.ac.at.
† Present address: Merck KGaA D-64271 Darmstadt, Germany.
‡ Present address: Center for Brain Research, Medical University of
Vienna, A-1090 Wien, Austria.
§ Present address: Institute of Pharmaceutical Chemistry, University of
Vienna, A-1090 Wien, Austria.
10.1021/jm0503493 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/06/2006

Synthesis of Thieno[2,3-b]pyridinones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 865
Chart 1
Scheme 1a
phenoxyphenyl substituent at position 5, were performed by an
analogous two- or three-step strategy, respectively, as outlined
in Scheme 2. The corresponding amides of aminothiophenes
1a, 1q-s, 1u, and 1w were obtained by Mukaiyama coupling
with 3-phenoxyphenylacetic acid in dichloromethane (DCM).
Alternatively, aminothiophenes 1g-p and 1v were treated with
3-phenoxyphenylacetyl chloride in dry dioxane at elevated
temperature. Both methods gave access to the intermediate
amides 13a, 13g-s, and 13u-w in excellent yields. Compounds
13a and 13g-r, which bear no substituent at the 5-position,
were chlorinated in refluxing chloroform with NCS to produce
compounds 14a and 14g-r. Finally, cyclization of compounds
13a, 13g, 13h, 13l, 13m, 13s, 13u-w, and the chlorinated
derivatives 14a and 14g-r with potassium hexamethyldisila-
zide resulted in thienopyridines 16a, 16g, 16h, 16l, 16m, 16s,
16u-w, and 18a, 18g-p, and 18r in moderate to good yields,
while the cyclization of 14q to 18q failed. But alternatively,
18q could be obtained in a low yield when phosphazene-P2-t-
Bu was used for deprotonation. In some cases (13s, 13w, 14v)
lithium diisopropylamide (LDA) was used instead of potassium
hexamethyldisilazide to afford the final compounds 16s, 16w,
and 18v at least in low yield. Compound 18l could also be
obtained from 16l by subsequent chlorination with NCS in
refluxing chloroform. This alternative route provided 18l in 58%
yield and improved the overall yield by 3-fold. Thieno[2,3-b]-
pyridin-6-thione 20 was obtained from 14i, which was selec-
tively converted into the thioamide 15 with 1 equiv of
Lawesson’s reagent in refluxing dioxane, followed by cycliza-
tion to 20 under the typical conditions.
a Reagents: (i) PhCH2COCl, dioxane; (ii) KN(Si(CH3)3)2, THF.
of phenylacetyl chloride in dry dioxane at high temperature
provided the intermediate amides 2a-c in excellent yields.14
Subsequent cyclization in THF at low temperature with potas-
sium hexamethyldisilazide afforded the desired thienopyridine
derivatives. Substituted 2-aminothiophenes (1d-g, 1s-u) with
the appropriate substitution pattern at C-2 and C-3, which gave
access to further thieno[2,3-b]pyridin-6(7H)-one derivatives,
were synthesized according to the methods of Gewald et al.13
or by Gewald-related preparations (1h-r, 1v, 1w), which have
been described elsewhere.15 Following the acylation conditions
described above, aminothiophenes 1d-i, 1k, 1m, and 1q were
converted to the corresponding intermediates 2a-i, 2k, 2m, 2q,
2t, 11d, 11g, and 11t. The amides 2a-i, 2k, 2m, 2t, 11d, 11g,
and 11t were cyclized to thienopyridines 3a-i, 3k, 3m, 3t, 12d,
12g, and 12t, as outlined in Scheme 2. Compounds 2g-i, 2k,
2m, and 2q, which bear no substituent at the 5-position (R1 in
Scheme 2), were halogenated in refluxing chloroform with
N-bromosuccinimide (NBS) or N-chlorosuccinimide (NCS),
respectively. The subsequent cyclization of halogenated deriva-
tives 4g, 4h, 5g-i, 5k, 5m, and 5q was accomplished with
potassium hexamethyldisilazide at low temperature. Compounds
7g, 7h, 8g-i, 8k, and 8m were obtained in moderate yields,
while the cyclization of 5q to 8q failed. Alternatively, 8q could
be obtained at least in a low yield when phosphazene-P2-t-Bu
was used for deprotonation. The synthesis of halogen-substituted
derivatives of 3a via the two-step procedure failed, because
decomposition was observed during cyclization. However, 7a
could be obtained from 3a by subsequent bromination with
bromine in glacial acetic acid. Thieno[2,3-b]pyridin-6-thione 10
was obtained from 5i, which was selectively converted into the
thioamide 6 with 1 equiv of Lawesson’s reagent [2,4-bis(4-
methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide] in
refluxing dioxane, followed by cyclization to 10 under the
typical conditions.
L-701,324 (Chart 1), which was used as reference compound
to validate the binding assay, was synthesized in three steps
with an overall yield of 10.5%. In short, 2-amino-4-chloroben-
zoic acid was esterified in refluxing methanol containing
hydrogen chloride. Subsequent acylation with 3-phenoxyphen-
ylacetic acid followed the Mukaiyama protocol, followed by
cyclization with potassium hexamethyldisilazide at low tem-
perature in THF. 4-Hydroxy-3-phenylquinolin-2(1H)-one10 (21)
and 7-Cl-4-hydroxy-3-phenylquinolin-2(1H)-one16 (22) were
prepared according to the literature.
Results and Discussion
The comparison of the three isomeric thienopyridinones 3a,
3b, and 3c showed that thieno[2,3-b]pyridinone 3a and thieno-
[3,4-b] 3b yielded the best Ki values for blocking the binding
of [3H]glycine at rat brain membranes [Table 1, not significantly
different in potency from the unsubstituted benzo[b]pyridinone
21, by analysis of variance (ANOVA)]. Thereafter, numerous
derivatives of the isomer 3a were synthesized. Right from the
The syntheses of thienopyridine derivatives 16a, 16g, 16h,
16l, 16m, 16s, 16u-w, 18a, 18g-r, and 20, which bear a

866 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Buchstaller et al.
Scheme 2a
a Reagents: (i) PhCH2COCl, dioxane; or 2-ThCH2COCl, dioxane; or PhCH2COOH, (chloromethyl)pyridinium iodide, DCM; or 3-PhOPhCH2COCl, dioxane;
or 3-PhOPhCH2COOH, SOCl2, dioxane; or 3-PhOPhCH2COOH, (chloromethyl)pyridinium iodide, DCM; (ii) KN(Si(CH3)3)2, THF; or LDA, THF; or
phosphazene-P2-t-Bu, THF; (iii) NBS or NCS, chloroform; (iv) Br2, AcOH; (v) Lawesson’s reagent, dioxane; (vi) HCl, EtOAc.
Table 1. Inhibition of [3H]Glycine Binding by the Compounds
Table 2. Inhibition of [3H]Glycine Binding by 5-Phenyl- and
Illustrated in Chart 1
5-(2-Thienyl)thienopyridinones: Influence of Alkyl Substitution
[3H]glycine binding Ki (µM) ( SD (n)a
3a
3b
3c
21
16.4 ( 2.7 (6)
26.0 ( 13.0 (3)
97.5 ( 31.5 (3)
12.2 ( 5.2 (4)
[3H]glycine binding
22
0.151 ( 0.045 (5)
0.0014 ( 0.0002 (4)
Ki (µM) ( SD (n)a
L-701,324
R1
R2
R3
Ph
2-Th
Ph
Ph
Ph
2-Th
Ph
Ph
Ph
Ph
a Number of experiments.
3d
12d
3e
3f
3g
12g
3h
3i
3k
3m
3t
CH3
CH3
C2H5
CH3
H
H
H
H
H
H
H
H
9.5 ( 3.2 (3)
22.7 ( 2.4 (3)
3.1 ( 3.0 (6)
1.27 ( 0.48 (3)
8.3 ( 1.4 (3)
beginning of this first series, we wanted to investigate the option
of another benzene/thiophene exchange, namely, at position 5.
Three different 5-thienyl analogues 12d, 12g, and 12t were
synthesized in parallel to the respective phenyl analogues 3d,
3g, and 3t, and all of them were considerably weaker than their
5-phenyl bioisosteres (see Table 2). As can be seen from the
Free-Wilson analysis, the activity of the 5-thienyl analogues
drops by a factor of 5.4 (0.73 log unit). Consequently, no further
5-thienyl derivatives were synthesized. From the quinolinone
parent compound, it is known that lipophilic substituents in the
benzene part of the bicyclic aromatic system enhance binding
affinity. Similarly, alkyl substitution at both positions of the
thiophene ring of 3a resulted in compounds with enhanced
potency. A single methyl group increased potency about 2-fold
(p < 0.01, ANOVA), independent of whether it was attached
to position 2 or to position 3 (see Table 2, 3d and 3g). A single
ethyl group, however, increased the potency of 3a by a factor
of 4 in position 2 (3e) but by a factor 25 in position 3, returning
with 3h the first inhibitor with submicromolar potency in our
series. Stepwise increase of the size of the R3 substituent beyond
that of an ethyl group did not improve potency any further (see
Table 2, 3h-3m). Flexible isopropyl (3i) and secondary butyl
CH3
CH3
CH3
C2H5
i-C3H7
c-C3H5
sec-C4H9
-(CH2)4-
-(CH2)4-
46.0, 34.0 (2)
0.53 ( 0.29 (8)
1.53 ( 0.30 (3)
4.75 ( 0.55 (3)
0.94 ( 0.05 (3)
4.3 ( 2.7 (3)
H
Ph
2-Th
12t
23.1 ( 8.3 (3)
a Number of experiments.
(3m) substituents were almost as favorable as ethyl (3h), but
the more rigid cyclopropyl residue (3k) missed the ethyl result
by a factor of 9 (p < 0.001). Almost as powerful as these latter
compounds was 3f, with methyl groups at both positions 2 and
3. Thus, although a substituent as large as an ethyl group was
not as welcome in position 2 as in position 3, a smaller size
occupant also close to the sulfur atom appeared very promising.
Joining both positions with a chain of four methylene groups,
annulating a six-membered ring to the thiophene ring (3t), was
less successful than two single methyl groups (3f, p < 0.01).
Apparently, position 2 is confronted with some size limitation,

Synthesis of Thieno[2,3-b]pyridinones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 867
Table 3. Inhibition of [3H]Glycine Binding by
Table 4. Inhibition of [3H]Glycine Binding and Cytotoxicity by
5-Phenylthienopyridinones: Influence of Halogen and Alkyl Substitution
5-(3-Phenoxyphenyl)thienopyridinones: Influence of Alkyl Substitution
[3H]glycine binding
[3H]glycine binding
cytotoxicity
IC50 (µM) (n)
Hal
R
X
Ki (µM) ( SD (n)a
R1
R2
Ki (µM) ( SD (n)a
7a
7g
7h
8g
8h
8i
10
8k
8m
9
Br
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
O
O
O
O
O
O
S
O
O
O
1.77 ( 0.28 (3)
16a
16g
16h
16l
16m
16s
16u
16v
17
H
H
H
H
H
H
CH3
C2H5
CH2OCH3
sec-C4H9
-(CH2)3-
-(CH2)4-
-S(CH2)3-
CH3
0.368 ( 0.274 (5)
0.330 ( 0.172 (8)
0.179 ( 0.120 (5)
0.036 ( 0.019 (6)
0.096 ( 0.048 (4)
0.118 ( 0.067 (6)
0.392 ( 0.144 (4)
0.037 ( 0.009 (4)
1.98 ( 1.12 (4)
89.2 (2)
>250
CH3
C2H5
CH3
C2H5
i-C3H7
i-C3H7
c-C3H5
sec-C4H9
-(CH2)2NH2
0.39 ( 0.17 (5)
0.0105 ( 0.0059 (3)
0.267 ( 0.116 (3)
0.0089 ( 0.0024 (5)
0.0066 ( 0.0038 (4)
0.89 ( 0.16 (4)
0.058 ( 0.017 (3)
0.0058 ( 0.0019 (3)
4.55, 5.35 (2)
>50
55.8 (2)
>50
>30
>250
CH2NH2
a Number of experiments.
a Number of experiments.
Table 5. Inhibition of [3H]Glycine Binding and Cytotoxicity by
5-(3-Phenoxyphenyl)thienopyridinones: Influence of Alkyl Substitution
more so than position 3. However, this impression was not
corroborated by quantitative structure-activity relationship
(QSAR) calculations: Hansch analysis on 45 compounds
returned no significant term for MR (molar refractivity ≈
volume) at position 2 but a strong influence of lipophilicity (π)
and electron-withdrawing potency (σ).
[3H]glycine
cytotoxicity
IC50 (µM) (n)
Our next series of compounds had chlorine or bromine in
position 2, in combination with alkyl substituents of increasing
size in position 3. Comparison with the previous series of
compounds informed us that chloro and bromo substitution in
position 2 were at least as effective as methyl and even as ethyl
substitution; thus, 7a (bromo, Ki 1.77 µM) was of similar
potency as 3e (ethyl, Ki 3.1 µM), and compounds with Br and
methyl (7g) or with Cl and methyl (8g) in positions 2 and 3,
respectively, were 3-5 times more potent than the dimethyl
analogue 3f (p < 0.01). Since chloro and methyl groups occupy
roughly the same space, the higher potencies of the halogen-
substituted analogues were most likely related to the higher
electronegativity of the halogens. Also for quinolinone-type
glycine antagonists, chloro substitution in the benzo part of the
ring system is more effective than methyl substitution.17 If the
substituent in position 3 was an ethyl group, the additional
introduction of Br respective to Cl into position 2 increased
the potency by a factor of 50-60 (compare 7h and 8h, Table
3, to 3h, Table 2). Also here, the 3-ethyl substituent could be
exchanged to isopropyl or secondary butyl without loss in
potency, finally returning from this series of syntheses four high-
affinity inhibitors of [3H]glycine binding (7h, 8h, 8i, and 8m),
with Ki values from 6 to 10 nM. And again, as in the halogen-
free series shown in Table 2, 3-cyclopropyl substitution (8k)
was not as welcome as the other alkyl substitutions (p < 0.001).
In this series (and in the following), we included one
compound with an oxo/thio exchange at position 6 of the
bicyclic ring system. The substitution pattern of this compound
(10) was analogous to 8i, with Cl in position 2 and isopropyl
in position 3, but not so was the observed potency: 10 was
more than 100 times weaker than 8i. Thio analogues of
kynurenate-type glycine antagonists have been used success-
fully,18 but for thienopyridinone-type antagonists the higher
electronegativity of the carbonyl oxygen seems to be of high
importance. Also the second thio analogue (20) was 100 times
weaker than the respective oxo congener (18i, see Table 5). In
addition, we tested in the series illustrated in Table 3 the
feasibility of charged substituents in position 3 as inhibitors of
[3H]glycine binding; however, the IC50 of a phenylthienopyri-
R
X
Ki (µM) ( SD (n)a
18a
18g
18h
18i
H
CH3
C2H5
i-C3H7
i-C3H7
n-C3H7
c-C3H5
CH2OCH3
sec-C4H9
tert-C4H9
n-C6H13
c-C6H11
CH2OH
(CH2)2NH2
O
O
O
O
S
O
O
O
O
O
O
O
O
O
0.0166 ( 0.0119 (6)
0.023 ( 0.016 (10)
0.00111 ( 0.00024 (5)
0.00146 ( 0.00054 (5)
0.139 ( 0.042 (3)
0.00412 ( 0.00122 (4)
0.0095 ( 0.0027 (3)
0.00160 ( 0.00097 (7)
0.0020 ( 0.0002 (3)
0.0033 ( 0.0004 (3)
0.080 ( 0.043 (5)
15.4 (2)
>200
5.2 (2)
20
18j
18k
18l
18m
18n
18o
18p
18r
19
15.9 (2)
15.7 (2)
7.9 (2)
15.5 (2)
>50
>30
>50
0.069 ( 0.048 (5)
16.5 ( 5.3 (3)
a Number of experiments.
dinone with aminoethyl in position 3 (9) was weaker by 3 orders
of magnitude than that of analogues with uncharged residues
in position 3 of comparable size (such as 8i or 8m). The binding
of [3H]raclopride to rat striatal dopamine receptors was inhibited
by 9 with IC50 > 10 µM (not illustrated).
Also, our last series of syntheses followed the experience
made with quinolinone-type glycine antagonists. Elongation of
the 5-phenyl substituent by addition of a 3-phenoxy residue
increased the potencies of halogen-free compounds by factors
of 3-44 (Table 4) and of compounds with Cl in position 2 by
factors 3-12 (Table 5). In general, compounds with affinities
already in the low nanomolar range, like 8h, 8i, and 8m, did
not profit from this kind of substitution to the same extent as
weaker compounds like 3a, 3g, or 8g. Thus, no subnanomolar
affinities were reached. Nevertheless, the structure/activity
relationships seen without the 3′-phenoxy substitution (in Tables
2 and 3) were nicely reproduced: The highest potencies were
seen with small and flexible alkyl substituents in position 3.
As in the previous series of compounds, we took the occasion
to study the influence of introduction of a positively charged
substituent. In the phenoxy series without Cl (Table 4),
aminomethyl was introduced in position 2, together with methyl
in position 3 (17). In comparison to the analogous compound

868 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Buchstaller et al.
Table 7. Hansch Analysis: Activity Contribution of Substituents
Table 6. Free-Wilson Analysis: Activity Contribution of Substituentsa
Nb coeff.c std errord
factore
coeff.
std error
core structure (3a)
4.86
1.75
1.30
0.64
0.47
0.18
0.13
0.21
0.35
0.23
0.34
core structure (3a)
R1
R1
4.44
1.10
1.82
0.20
0.15
0.32
R1
R1
R1
R1
R1
Cl
Br
C2H5
CH3
CH2NH2
21
3
1
3
1
56.29
19.74
4.41
2.98
-2.61
p
σp
b
R2
MR
2.15
-0.71
-3.66
0.32
0.13
0.33
R2
R2
MR2
HDon
-0.42
R2
R2
R2
R2
R2
R2
R2
R2
R2
R2
R2
R2
CH2OCH3
C2H5
sec-C4H9
i-C3H7
tert-C4H9
n-C3H7
c-C3H7
2
5
4
5
1
1
3
8
1
1
2
1
1.40
1.38
1.26
1.25
0.88
0.79
0.50
0.27
-0.44
-0.50
-2.55
-3.30
0.27
0.20
0.22
0.24
0.35
0.35
0.24
0.18
0.35
0.35
0.28
0.35
25.05
24.26
18.39
17.84
7.67
6.15
3.17
1.88
R3
R3
2-Th
Ph-O-Ph
-0.74
1.19
0.28
0.14
XdS
-1.67
0.32
a For substitution scheme see Table 2. N ) 43, r2 ) 0.92, s ) 0.42, DF
) 34, F ) 48.8. b R2: MRmax ) 1.47.
CH3
tion of a H-bond active group (17; CH2NH2) decreases activity
by a factor of 2.6.
CH2OH
n-C6H13
(CH2)2NH2
c-C6H11
-2.73
-3.16
-356.81
-1974.42
At position 3 a very sound QSAR for the size of the
substituent was found: the quadratic term in the HA revealed
that the optimal size of a substituent was that of ethyl. Bridging
both positions does not enhance activity significantly, as can
be seen from the summation of the best substituents from
positions 2 and 3: [R1-R2 ) -S(CH2)3-] < (R1 ) Cl) + (R2 )
CH2OCH2). Elongation at position 5 from phenyl to phenoxy-
phenyl enhanced activity by about 1 order of magnitude (0.98
log), whereas bioisosteric substitution of phenyl by thiophene
reduced activity by a factor of 5. HA returned two compounds
as outliers. For 18p (cyclohexyl in position 3), the HA most
likely did not consider sufficiently its increased rigidity in
comparison to the linear analogue (18o). For the hydroxyethyl
derivative (18r), the HA predicted a pronounced decrease in
activity, due to the H-bond donating potential of this substituent.
That such a decrease did not occur to the predicted extent may
have been due to internal H-bond formation with the adjacent
-OH group in position 4. As already mentioned, the optimal
size of a substituent in position 3 expressed as molar refractivity
was MR ) 1.47, very close to the value for isopropyl or n-propyl
(MR ) 1.49).
A series of these antagonists was tested in a cell toxicity assay
with mouse fibroblast cell expressing human NRs. Cell death
was dose-dependently inhibited by nine of the tested thienopy-
ridinones, including the benzene analogue L-701,324 (IC50 for
L-701,324 was 3.79 µM, with n ) 2; value not shown in the
table). The test compounds were about a thousand times more
potent in the binding assay than in the cell toxicity assay,
presumably due to the high (micromolar) concentration of
glycine in the cell culture medium. With some compounds, no
results could be obtained in the cell toxicity assay, due to direct
toxicity of the compounds or to poor solubility in the incubation
medium. IC50 values in the cell toxicity assay were well
correlated to the Ki values obtained from the binding assays.
For the nine compounds thath were active in both settings, a
correlation coefficient r ) 0.89 (P ) 0.001) was observed.
A first series of compounds, 18a, 18h, 18i, and 18l, were
subjected to in vivo testing in the maximal e-shock mouse
model, in direct comparison with the standard compound
L-701,324. L-701,324 protected against tonic extension in 50%
of the mice at 0.70 mg/kg, in agreement with the literature (see
ref 5). Much higher doses of 18h (15.5 mg/kg with e-shock
after 30 min, 13.5 mg/kg after 60 min) and of 18i (43 mg/kg
with e-shock after 30 min) were required to achieve the same
degree of protection (Figure 1). 18a and 18l protected only 10%
and 30%, respectively, at the highest dose tested (30 mg/kg).
18i and L-701,324 were subsequently quantified in plasma and
in brain tissue after systemic application to rats. Both of
them reached much lower levels in brain tissue than in plasma
R3
R3
Ph-O-Ph
2-Th
24
3
0.98
-0.73
0.13
0.21
9.57
-5.35
R1-R2
R1-R2
R1-R2
S(CH2)3-
(CH2)3-
(CH2)4-
1
1
3
1.59
1.08
0.52
0.35
0.35
0.24
38.55
12.08
3.32
XdS
2
-1.90
0.28
-80.04
a For substitution scheme see Table 2. N ) 46, r2 ) 0.97, s ) 0.31, DF
) 22, F ) 36.8. b Frequency of occurrence. c Activity coefficient in log
scale. d Standard error of coefficient. e Activity contribution () exp(coeff);
-exp(1/coeff) for negative values of coeff).
without that aminomethyl group (i.e., with 16g), we observed
a serious drop in potency (Table 4). Since the similarly sized
ethyl has been fine in this position (see 3e, Table 2), the positive
charge (as would occur at pH 7.0) seems to be unfavorable. In
the phenoxy series with Cl (Table 5), the bigger aminoethyl
was introduced in position 3 (the position allowing somewhat
bulkier substituents than position 2), resulting in the very weak
inhibitor 19. Again, similarly sized substituents at this position
had produced inhibitors at least 3 orders of magnitude more
potent, pointing once more to the handicap of a positive charge
at this part of the structure. The binding of [3H]raclopride to
rat striatal dopamine receptors was inhibited by 19 with IC50
30 µM (not illustrated).
>
In the phenoxy compounds only, hydroxymethyl (18r) and
methoxymethyl (16l, 18l) were tested as substituents at position
3 of the thienopyridine. In the phenoxy series without Cl (Table
4), 16l with methoxymethyl in position 3 was among the most
potent compounds, interestingly only equaled by 16v with a
sulfur at position 2. In the phenoxy series with Cl (Table 5),
18l with methoxymethyl in position 3 had the same potency as
other compounds with medium-sized substituents in position 3
(18h, 18i, and 18m). It may be speculated that, in the series
without Cl, the absence of the (very favorable) electronegative
substituent is to some extent compensated by the presence of
O or S in the substituents, whereas in the series with Cl,
methoxymethyl has no additional effect. A hydrophilic sub-
stituent containing O is unfavorable as exemplified by 18r with
hydroxymethyl in position 3 (Table 5).
The results in Tables 2-5 were finally subjected to Free-
Wilson (FWA) and Hansch (HA) analyses, exploring the
individual contributions of functional groups at distinct positions
(FWA) and the influence of molecular properties at specific sites
(HA), with 3a as the reference. The results of the FWA are
shown in Table 6, and those of the HA are shown in Table 7.
As already discussed, the best substituent at position 2 was
chlorine, with a 56-fold better activity than hydrogen. Introduc-

Synthesis of Thieno[2,3-b]pyridinones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 869
solution, dried (Na2SO4), and concentrated in vacuo. The resulting
crude products were crystallized by digestion in diethyl ether and
recrystallized.
Halogenation Reaction: General Procedure. Compounds
2g-i, 2k, 2m, 2q, 13a, 13q-r (1 equiv) and NBS or NCS (1-1.9
equiv), respectively, were dissolved in dry chloroform and refluxed
until no starting material was observed (TLC). The reaction mixture
was diluted with DCM and extracted with water. The combined
organic layers were dried (Na2SO4), and concentrated in vacuo,
and the residue was recrystallized or purified by VFC.
Cyclization Reaction: General Procedure. (A) Method A.
Compounds 2a-i, 2k, 2m, 2t, 4g, 4h, 5g-i, 5k, 5m, 5q, 6, 11d,
11g, and 11t (1 equiv) were dissolved in dry THF and the solution
was cooled to -65 °C. KN(Si(CH3)3)2 (0.5 M in toluene or 1 M in
THF, 2.2 equiv) was added dropwise. The reaction mixture was
warmed to room temperature overnight and evaporated to dryness.
The residue was dissolved in water and extracted several times with
diethyl ether. The aqueous layer was acidified with 2 M HCl, the
precipitate was filtered by suction, washed with water, and dried.
The resulting products were triturated with diethyl ether, filtered,
and dried.
Figure 1. Protection of mice against maximal e-shock, by 18h (0),
18i (3), and the reference compound L-701,324 (O).
Table 8. Plasma and Brain Tissue Levels after Intravenous Application
of 18i and of L-701,324 to Rats
(B) Method B. Compounds 13a, 13g, 13h, 13l, 13m, 13s, 13u-
w, 14a, 14g-r, and 15 (1 equiv) were dissolved in dry THF and
the solution was cooled to -65 °C. KN(Si(CH3)3)2 (0.5 M in toluene
or 1 M in THF; 2.2 equiv) or LDA [2 M in THF, 2.2 equiv (3.3
equiv for 13w)] was added dropwise. The reaction mixture was
warmed to 0-10 °C overnight and worked up depending on the
formation of side products and the solubility of the products (for
details, see Supporting Information).
after 6 min
after 1 h
L-701,324 (5 mg/kg)
125 ( 5.6 (3)
plasma (µM)
64 ( 5 (3)
1.4 ( 0.3 (3)
brain (µmol/kg)
5.5 ( 0.5 (3)
18i (10 mg/kg)
97 ( 21 (3)
3.5 ( 0.9 (3)
plasma (µM)
brain (µmol/kg)
9.7 ( 4.5 (3)
0.28 ( 0.13 (3)
3-Phenoxyphenylacetylation of Thiophenes 1a, 1g-s, and 1u-
w: (A) Method A (Compounds 13a, 13q-s, 13u, and 13w). Et3N
(2.2 equiv) was added to a solution of the appropriate ami-
nothiophene (1a, 1q-s, 1u, or 1v; 1 equiv), 3-phenoxyphenyl-
acetic acid (1.2 equiv), and 2-chloro-1-methylpyridiniumiodide (1.2
equiv) in dry DCM. After complete addition, the temperature was
raised to reflux and the mixture was stirred at this temperature for
3 h. The mixture was diluted with DCM and washed with 6 M
HCl (except 13q and 13w, which were extracted with water) and
NaHCO3 solution, dried (Na2SO4), and concentrated in vacuo. The
resulting crude products were purified by VFC and dried.
(B) Method B (Compounds 13g-i and 13p). 3-Phenoxy-
phenylacetic acid (1.3 equiv) was dissolved in dry dioxane, SOCl2
(1.95 equiv) was added, and the mixture was heated to 78 °C for
3 h. The excess SOCl2 was removed in vacuo, and the residue was
diluted with dry dioxane and added dropwise to a dry dioxane
solution of the appropriate aminothiophene (1g-i, 1p) (1 equiv) at
70 °C. After complete addition, the temperature was raised to 98
°C and the mixture was stirred at this temperature until the formation
of HCl gas ceased. The solution was concentrated in vacuo and
the residue was dissolved in DCM, washed with NaHCO3 solution,
dried, and concentrated in vacuo. The resulting crude products were
purified by VFC and dried.
(Table 8). Although 18i was applied at a dose twice as high as
L-701,324, its plasma and its brain tissue levels were lower than
that of the reference compound, especially after the longer time
delay. The weaker in vivo efficiency of 18i as compared to
L-701,324 may, thus, be a consequence of both poorer bio-
availability and faster elimination.
Conclusion
In summary, a set of novel ligands with high affinity at the
glycine binding site of the NR complex is presented. Optimiza-
tion of the substitution pattern in the thieno part of the thieno-
[2,3-b]pyridinone finally yielded glycine antagonists with in
vitro potencies comparable to the most potent quinolinone-type
antagonists known, hence confirming the chosen benzene/
thiophene bioisostere approach. Our detailed survey of optimized
substitution patterns may be of particular relevance since the
binding pocket has been crystallized and resolved with 3.2 Å
resolution.19 Further studies will have to show whether the weak
in vivo efficiency demonstrated for four of them was representa-
tive for this new class of compounds.
(C) Method C (Compounds 13j-o and 13v). To a solution of
the appropriate aminothiophene (1j-o or 1v) (1 equiv) in dry
dioxane was added 3-phenoxyphenylacetylchlorid20 (1.1 equiv),
dissolved in dry dioxane, dropwise at 70 °C. The further procedure
was as described for the preceding Method B.
Experimental Section
Phenylacetylation of Thiophenes 1a-i, 1k, 1m, and 1t:
General Procedure. To a solution of the appropriate ami-
nothiophene (1a-i, 1k, 1m, or 1t) (1 equiv) in dry dioxane was
added phenylacetyl chloride (1.3 equiv), dissolved in dry dioxane,
dropwise at 70 °C. After complete addition the mixture was stirred
at 98 °C until the formation of HCl gas ceased. The solution was
concentrated in vacuo and the residue was dissolved in DCM,
washed with NaHCO3 solution, dried (Na2SO4), and concentrated
in vacuo. The resulting crude products (except 2a, 2b, and 2n) were
recrystallized and dried.
2-Thienylacetylation of Thiophenes 1d, 1g, and 1t: General
Procedure. To a solution of the appropriate aminothiophene (1d,
1g, or 1t) (1 equiv) in dry dioxane was added 2-thienylacetyl
chloride (1.3 equiv), dissolved in dry dioxane, dropwise at 70 °C.
After complete addition the mixture was stirred at 98 °C until the
formation of HCl gas ceased. The solution was concentrated in
vacuo and the residue was dissolved in DCM, washed with NaHCO3
Radioligand Binding Assays. The affinities of the test com-
pounds for the glycine regulatory site of the NMDA receptor
complex were evaluated by analyzing their abilities to displace [3H]-
glycine in the presence of 10 µM glutamic acid from rat hippo-
campal membranes. We used 4-5 different inhibitor concentrations
inducing 20-90% inhibition of specific binding. In parallel, the
apparent affinity of [3H]glycine was evaluated in each experiment
and used to calculate the concentration of unavoidable background
glycine, assuming a true affinity constant of 40 nM.21 This strategy
allowed the reliable calculation of Ki values from the experimentally
obtained IC50 values. The ethylamines 9 and 19 were, in addition,
tested as inhibitors of specific [3H]raclopride binding22 to rat striatal
membranes. To estimate the significance of differences of various
Ki values, they were comprised into groups of 3-5 values as

870 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Buchstaller et al.
for 20 min at 25 °C. LDH assay mixture contained 1% sodium
L-lactate in Tris, pH 9.0, 1.4 mM NAD+, 20 mM phosphate buffer,
pH 7.4, 0.8 mg of BSA, 18 units of diaphorase, and 4 mg of INT
per milliliter. The coupled enzymatic reaction results in the
conversion of iodonitrotetrazolium salt (INT) into a red formazan
product. After 20 min the reaction was stopped by addition of 50
µL of 1 M acetic acid and absorption was measured at 490 nm in
a Dynatech MR 5000 Elisa-reader. The intensity of the red color
formed is directly proportional to the number of lysed cells.
Cytosolic LDH of surviving cells was released into blank medium
containing 0.1% Triton X-100 and measured as mentioned above.
Data were corrected for cell culture medium background. Cell death
rate (CD) was calculated as follows: CD (%) ) ALDHrel/(ALDHrel
+
A
LDHcyt) × 100. Obtained data were fitted to a four-parameter
logistic function by use of Sigma Plot. To prevent measurement of
nonspecific toxicity, the applicable concentration range of each
compound was determined by trypan blue exclusion with non-
dexamethasone-treated cells. After 24 h incubation, 0.04% trypan
blue in phosphate-buffered saline (PBS) was added and dye
exclusion was checked by light microscopy. Only nontoxic inhibitor
concentrations were used.
Figure 2. Correlation of calculated versus observed activities from
the Hansch analysis. Activities are shown as pKi values [) -log
(1/Ki)]. The labeled compounds (18p and 18r) have been excluded from
model generation.
Mouse Seizure Model. Ten male Swiss mice per dosage received
intraperitoneally six doses of L-701,324 (0.3-100 mg/kg); three
doses of 18a, 18h, and 18l (3-30 mg/kg); six doses of 18i
(0.3-60 mg/kg); or vehicle (8 parts DMSO, 10 parts H2O). Maxi-
mal e-shock was applied 30 or 60 min later, and tonic hind limb
extension was rated as an unprotected response. Dose/response data
were fitted to the function: response (dose ) x) ) response (dose
) 0) × ID50/(ID50 + x), where x is the dose of the compound tested,
and ID50 the dose inhibiting the response in 50% of the animals.
appropriate for the respective argument raised in the discussion and
subjected to statistical analysis (ANOVA with post hoc Newman-
Keuls test).
QSAR. To gain insight into the quantitative structure-activity
relationship (QSAR) of the compounds synthesized, two kind of
analyses were performed. First, the individual contribution of a
functional group at a distinct position was evaluated by means of
a Free-Wilson analysis23 (FWA). For this approach compound 3a
was chosen as reference. In a table for each substituent at the
positions 2, 3, and 5 a column was built as well as for annulated
rings in position 2 and 3 and sulfur at position 6 (10, 20). All
compounds were stored as rows with their activity expressed as
pKi (Figure 2). The resulting Free-Wilson matrix was solved by
conventional multiple linear regression approaches.24 For Hansch
analysis25 (HA), the substituent parameters MR (molar refractivity),
π (lipophilicity), σ (Hammett constant), and L, B1, B5 (Sterimol)
were assigned for each group in positions 2 and 3; one compound,
16v, could not be included because of missing descriptors.
Substitution pattern at positions 5 and 6 were encoded as indicator
variables such as at the FWA. As a quality measure of both
methods, the standard error and the Fischer test variable were
calculated.
Drug Concentrations in Blood and Brain of Rats. In six male
Wistar rats, plasma and brain tissue levels of 18i, one of our most
potent compounds, were measured 6 min (n ) 3) and 1 h (n ) 3)
after intravenous administration of a dose of 10 mg/kg. In another
six rats, similar measurements were performed after application of
5 mg/kg of the reference glycine antagonist L-701,324. Both
compounds were quantified by an analytical HPLC technique.
Supporting Information Available: Experimental details of
all intermediates and target compounds with supporting spectral
data and C, H, N analysis results. This material is available free of
charge via the Internet at http://pubs.acs.org.
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