Substituted 2-Aminothiopen-derivatives: A potential new class of GluR6-Antagonists

Article abstract of DOI:10.1016/j.ejmech.2009.09.025

In the course of search for new therapeutic agents against epilepsy new inhibitors for the kainate receptor subtypes GluR5 and GluR6 were synthesized. We were able to synthesize new substituted thieno[2,3-d]pyrimidines 3a,b, 4a,b, 5a,b as well as thiophene-3-carboxamides 2a-d and a multitude of substituted 4-methyl-5-phenylthiophene-3-carboxylic acids. All compounds described herein were tested for their antagonistic effect towards the kainate receptor subtypes GluR5 and GluR6. The highest activity was observed for ethyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate 1c with an IC₅₀ = 0.75 μM at the GluR6 receptor.

Full text of DOI:10.1016/j.ejmech.2009.09.025

European Journal of Medicinal Chemistry 45 (2010) 69–77
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Substituted 2-Aminothiopen-derivatives: A potential new class of
GluR6-Antagonists
,
a
b
b
D. Briel a , A. Rybak , C. Kronbach , K. Unverferth
*
a
¨
¨
¨
¨
Pharmazeutische Chemie, Institut fu¨r Pharmazie, Fakultat fur Biowissenschaften, Pharmazie und Psychologie, Universitat Leipzig, Bruderstr. 34, 04103 Leipzig, Germany
b Biotie Therapies GmbH, Radebeul, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the course of search for new therapeutic agents against epilepsy new inhibitors for the kainate
receptor subtypes GluR5 and GluR6 were synthesized.
We were able to synthesize new substituted thieno[2,3-d]pyrimidines 3a,b, 4a,b, 5a,b as well as
Received 24 February 2009
Received in revised form
11 August 2009
Accepted 10 September 2009
Available online 16 September 2009
thiophene-3-carboxamides 2a-d and
a multitude of substituted 4-methyl-5-phenylthiophene-
3-carboxylic acids.
All compounds described herein were tested for their antagonistic effect towards the kainate receptor
subtypes GluR5 and GluR6. The highest activity was observed for ethyl 2-amino-4-methyl-5-phenyl-
Keywords:
thiophene-3-carboxylate 1c with an IC50 ¼ 0.75
m
M at the GluR6 receptor.
2-Aminothiophenes
Thieno[2,3-d]pyrimidines
Kainate receptor
Antiepileptics
Ó 2009 Elsevier Masson SAS. All rights reserved.
Glutamate
1. Introduction
inhibitors of GluR5 and GluR6 have distinct structural similarities to
endogenous agonist glutamate, including pyrrolidine-, isoxazole-,
oxa(thia)diazole-, pyrimidine- and hexahydrofuropyrane deriva-
Epilepsy is a chronic disease and needs continuous or lifelong
therapy [1]. In pathogenesis of epileptic diseases the disbalance
between GABA- and glutamate-based neurotransmission in the
central nervous system plays an essential role. As the most
important excitatory neurotransmitter glutamate is involved in
many different processes in CNS of mammals [2]. Kainate receptors
[3], a subtype of ionotropic glutamate receptors, are of special
interest. Kainate, as analog of glutamate, is able to induce epileptic
seizures in rodents [4]. Thus the inhibition of glutamate receptors is
an approach for the therapy of epileptic diseases. Especially
subtypes GluR5 and GluR6 are discussed in genesis of different
kinds of epilepsy [5–10]. Due to this kainate receptors are inter-
esting potential targets for the development of new antiepileptics
[10–12]. Among these receptors a special significance to the kainate
receptor subtype GluR6 is described. This subtype is primarily
expressed in the excitatoric pyramidal cells of the hippocampus.
There are hints that the GluR6 and the GluR5 subtype play an
opposing role in the hippocampal activation [11,13]. In search for
new antiepileptics some quinalines and thieno[2,3-d]pyrimidines
proved to be successful anticonvulsants [14,15]. All known
tives. EC50 values of glutamate (631
mM GluR5, 270–762 mM GluR6)
and kainate (33.6 M GluR5, 299 M GluR6) on different glutamate
m
m
receptors are summarized in literature [7]. Our researches gave
indications that thieno[2,3-d]pyrimidines are specific kainate
receptor inhibitors [16,17,18]. Another new chemical class of GluR6
kainate receptor antagonists are 2-aminothiophene-3-carboxylic
acid derivatives. In high throughput screenings ethyl 2-amino-4-
methyl-5-phenylthiophene-3-carboxylate 1c was identified as
non-competitive GluR6-subtype specific Inhibitor. This
compound has an IC50 of 0.75 M in vitro and therefore is an
interesting target for further structure optimization and
modification.
a
m
2. Results and discussion
2.1. Chemistry
Based on thiophenes 1a-d different thieno[2,3-d]pyrimidines
3a,b, 4a,b, 5a,b were synthesized (Scheme 1). Conversion of 2-
aminothiophen-3-carboxamides 1a,b using thiophosgene afforded
the 2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-ones 3a,b,
which could be alkylated by iodoethane to give the 2-(Ethylthio)-
thieno[2,3-d]pyrimidin-4(3H)-ones 4a,b. Ethyl 2-aminothiophene-
* Corresponding author.
E-mail address: briel@rz.uni-leipzig.de (D. Briel).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.025

70
D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
R2
R3
R2
R1
R3
NH2
O
O
R2
R1
O
R3
R2
R1
R2
R1
a
X = NH2
a or b
X
NH2
NH
R1
S
N
N
NH2
S
S
X = OEt
e
2a-d
Y
1a-d
8
6
7
1a-d, 9a-l
O
X = NH2
c
O
O
O
O
Ph
Ph
R2
R1
R2
R1
N
Ph
NH
CN
O
S
NC
CN
CN
N
N
H
S
S
H
CN
5a,b
3a,b
10
11
Scheme 2. (a) S8, morpholine or NH(Et)2, EtOH, r.t., 1–3 h.
d
O
R2
R1
Alkaline hydrolysis of 1c with sodium hydroxide solution gave the
carboxylic acid 12. Various experiments to get decarboxylation of 12,
e.g. reflux in different solvents, quinoline, addition of Ba(OH)2 or Cu,
afforded only complex mixtures. Probably the formed 2-amino-
thiophene undergoes consecutive reactions. These instability would
be equivalent to the properties of unsubstituted 2-aminothiophene,
which already polymerizes at room temperature [25–28]. In contrast
the heterocycle of 2-aminothiophenes bearing a acceptor in 3- or 5-
position is stabilized by a ‘‘push-pull-effect’’ [19,29,30].
NH
N
S
S
4a,b
Scheme 1. (a) for Y ¼ O (2a,b): ClCOOEt, K2CO3, toluene, r.t., 3 h; (b) for Y ¼ NH (2c,d):
EtNCO, CH2Cl2, reflux, 5 d; (c) SCCl2, K2CO3, CH2Cl2, r.t., 1.5 h; (d) NaOCH3, EtI, EtOH, r.t.,
30 min; (e) (i) EtNCO, CH2Cl2, reflux, 5 d; (ii) NaOH, EtOH, reflux, 1.5 h.
3-carboxylates 1c,d were used for the synthesis of 3-ethyl-
thieno[2,3-d]pyrimidine-2,4(1H,3H)-diones 5a,b via conversion
with N-ethylisocyanate followed by the cyclization using sodium
hydroxide. 2-N-substituted thiophene-3-carboxamides 2a-d were
synthesized based on 2-aminothiophen-3-carboxamides 1a,b by
conversion with ethyl chloroformate or N-ethylisocyanate. Under
various conditions no cyclization of thiophene-3-carboxamides 2a-
d to the corresponding thienopyrimidines was observed.
Due to instability of 2-aminothiophene decarboxylation of 12
succeeded only with ethyl chloroformate. Simultaneous acylation
of the amino group obviously stabilizes the intermediate. Anyway
the decarboxylation product 13 is only a side product. The main
product is carboxylic acid 14.
Conversion of 1c using carboxylic acid-anhydrides or –chlorides
gave 2-acylaminothiophenes 15a-d. N-ethylisocyanate formed
derivatives 15e,h via acylation. The use of bromoacetyl bromide as
acylating reagent led to derivative 15f.
Based on the good results of compound 1c has an IC50 of 0.75 mM
the substituents R1 and R2 were modified stepwise. For R1 phenyl-
groups and for R2 H or phenyl-groups should be established.
Furthermore R1eR2 was replaced by phenyl tetra methylen-residue.
The thiophene derivatives 1a-d and 9a-l were obtained via
Gewald reaction (Scheme 2). [19,20] The first step was a Knoeve-
nagel Cope [21] condensation between ketone 6 and a acceptor-
substituted acetonitrile 7 to produce the crotonic acid nitrile 8.
Subsequent reaction of 8 with sulfur in the presence of diethyl-
amine or morpholine gave the cyclization to the thiophenes 1a-
d and 9a-l. Sometimes it was possible to perform the reaction as an
one-pot-reaction without isolation of 8. But in most cases it was
convenient to isolate 8 first.
By the method of Gewald 9a-f were synthesized and tested. For
these compounds a decreased GluR6-antagonism was observed in
comparison to 1c (see 2.2). For that reason phenylacetone 6 was
used for further synthesis to give 9g-l with R1 ¼ phenyl and
R2 ¼ methyl.
Substitution of bromine with morpholine, benzylamine and
butylamine delivered 16a-c (Scheme 4).
To prepare a thiourea derivative 1c was converted with thio-
phosgene to the isothiocyanate 17 followed by reaction with eth-
ylamine to give 18.
Methylation of the weak basic amine group was carried out with
methyl iodide under pressure to afford 15g.
Reaction of 1c with 2,5-dimethoxy-tetrahydrofurane formed
pyrrol derivative 19, where the amino-group of thiopene is
involved in a pyrrol-system and lost the ability to build hydrogen
bonds. Additional variations of the basic substituent in 2-position
were obtained by conversion with sodium nitrite/HCl to the inter-
mediate diazoniumsalt followed by the reaction with dimethyl-
amine, morpholine, cupper chloride or cyanide. Thereby the
diazo-derivatives 20a-c and chloride 21 were synthesized.
Reaction of phenylacetone 6 with benzoylacetonitrile 7 formed
the crotonic acid nitrile 8l in 25% yield. The main product 11 which
was formed via condensation of three molecules of benzoylaceto-
nitrile 7. Thereby benzoylacetonitrile 7 acts not only as the
carbonyl-compound but also as methyl-compound (Scheme 2).
If acetylacetonitrile was used instead of benzoylacetonitrile only
10 could be isolated in 33% yield, because the carbonyl reactivity is
much stronger (Scheme 2). These unwanted reactions are well
2.2. Biology
The synthesized thiophene derivatives were studied for their
antagonistic activity towards the kainate-receptor subtypes GluR5
and GluR6. Therefore we established a test system. Human
embryonic kidney cells (HEK-cells) characterized by a stable
recombinant expression of aequorin together with the GluR5 or
GluR6, respectively, kainate receptor were used as test system. The
antagonistic effect of substances on kainate receptors was deter-
mined using a luminescence reporter assay. The application of
kainate receptor agonist glutamate (at its respective EC50 concen-
known in literature [22,23,24] for
group in position 2.
b
-cyanketones with CH-acidic
Using 1c as starting material
a
multitude of thiophene
compounds could be synthesized bearing different modifications
especially in 2-position (Scheme 3).
tration, 275
ion channel which lead to a luminescent signal. The signal was
m
M) caused an influx of Ca2þ ions through the opened

D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
71
O
O
OH
NH
OH
b
NH
NH2
S
S
S
O
O
14
13
12
O
O
a
O
O
O
O
O
O
c
h
R4
Cl
NH2
S
S
S
21
15a-f
1c
d
g
f
O
O
O
O
O
O
e
S
O
N
C
N
R
N
O
N
NH
S
S
S
NH
20a-c
17
18
S
S
19
Scheme 3. (a) NaOH, EtOH, reflux, 2.5 h; (b) ClCOOEt, K2CO3, CH3CN, r.t., 8 h; (c) acid chloride, K2CO3, r.t., 3 h (15c,d,f), acid anhydride, reflux, 2.5 h (15a,b,g) or EtNCO, CH2Cl2,
reflux, 5 d (15e,h); (d) CSCl2, K2CO3, CH2Cl2, r.t., 1 h; (e) NH2Et, CH2Cl2, r.t., 1 h; (f) 2,5-dimethoxytetrahydrofuran, AcOH, reflux, 20 min; (g) (i) NaNO2/HCl, 0 ꢀC, 20 min; (ii) NH(CH3)2
(20a), morpholine (20b) or NaCN (20c), 0 ꢀC, 30 min; (h) (i) NaNO2/HCl, 0 ꢀC, 20 min; (ii) CuCl/HCl, 0 ꢀC, 30 min.
caused by the reaction of Ca2þ with coelenterazine, the cofactor of
the Ca2þ affinic photoprotein aequorine. Antagonistic activity of
compounds was determined by inhibition of the glutamate specific
luminescence signal. The overall luminescence of the cells was
detected after lysis by triton-X100.
It appeared that the thienopyrimidine derivatives 3a,b, 4a,b and
5a,b and the thiophene derivatives 2a-d have only less activity
(Table 1). In contrast compound 1c showed a surprising result. The
IC50 value in GluR6 kainate-receptor-assay of derivative 1c was
The most active members of 4-methyl-5-phenylthiophenes
proved to be the 2-aminothiophenes having a alkylester-group in
3-position (9g-i). For these compounds IC50 of 2.4–4.1 mM were
obtained in in vitro kainate-receptor-assay. The inhibition-values of
methylester 9g and propylester 9h show reduced activity in GluR-
assay in comparison to ethylester 1c.
Exchange of ethylester-group in 3-position against amide (9j),
nitrile (9k), benzoyl (9l) or carboxylic acid (12) leads to decreased
antagonistic activity.
0.75
(6-cyano-7-nitroquinoxaline-2,3-dione) has a IC50 of 23
the same test conditions. NBQX, SYM 2081 and miconazole were
m
M. The known glutamate receptor antagonist CNQX
Also by acylation of 2-aminofunction it was not possible to
enhance the antagonistic activity. The acylated compounds 13, 14,
15a-e and 16 a-c showed only weak inhibition of fractional lumi-
nescence. In some cases the inhibition was lower than 25% and thus
within the margin of error of the bioluminescence methode.
Equally weak inhibition was observed for the 2-thioureido-
thiophene 18 and 2-methylaminothiophene 15g.
m
M using
also tested in this assay. The IC50-values are 74
respectively.
mM, 4 mM and 6.5 mM
To gain more information about the structure-activitiy-rela-
tionship more thiophene derivatives were synthesized.
The results of GluR5- and GluR6-aequorin-assay of all thiophene
compounds synthesized are summarized in Table 2.
Change of substituents in 4- or 5 position leads to a decreased
antagonistic activity against GluR6-receptor. This applies for the
introduction of substituents at phenyl residues in 5-position (9a-c)
as well as the replacement of methyl-group in 4-position against H
or phenyl (9d,e) or anellation of a phenyltetramethylene-residue
(9f). For that reason these positions remained unchanged in
a majority of tested thiophenes ain analogy to lead structure 1c.
Table 1
Substituted thiophenes and thienopyrimidines.
R1
R2
X
Y
c (
m
M)
GluR5
GluR6
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
4a
4b
5a
5b
C6H5
(CH2)4
C6H5
(CH2)4
C6H5
(CH2)4
C6H5
(CH2)4
C6H5
(CH2)4
C6H5
(CH2)4
C6H5
(CH2)4
CH3
NH2
NH2
COOC2H5
COOC2H5
100
10
10
10
10
10
10
10
10
10
10
10
10
10
2a
4a
3a
13a
0.75b
11a
12a
17a
17a
12a
ꢁ2a
0a
CH3
CH3
CH3
CH3
CH3
CH3
12a
7a
O
O
NH
NH
19a
14a
5.8a
0.1a
10a
17a
1a
O
O
O
O
a
NH
NH
S
S
0a
8a
4a
11.8a
6a
9.5a
7.1a
O
O
Br
R
16a-c
15f
a
Scheme 4. (a) morpholine (16a), benzylamine (16b) or butylamine (16c) was used,
CH3CN, r.t., 1 h.
Inhibition of luminescence (% of control).
IC50 (mM).
b

72
D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
Table 2
Substituted thiophenes.
R1
R2
R3
R4
c (
m
M)
GluR5
GluR6
0.75b
8.6b
12b
5.7b
1c
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
C6H5
CH3
CH3
CH3
CH3
H
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOCH3
COOC3H7
COOC(CH3)3
CONHC2H5
CN
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHCOOC2H5
NHCOOC2H5
NHCOCH3
NHCOCF3
NHCOC6H5
NHCOCOOC2H5
NHCONHC2H5
NHCH3
10
10
10
10
100
10
10
10
10
10
10
10
10
100
10
10
10
10
10
10
10
10
12a
1a
4-CH3eOeC6H4
4-FeC6H4
2-CleC6H4
C6H5
0a
10a
25a
13a
1a
-1a
C6H5
C6H5
4.5b
4a
CH2CH(C6H5)CH2CH2
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
30b
5a
4.1b
2.4b
3.6b
4a
4a
9a
9k
9l
17a
17a
20a
1a
71b
COC6H5
COOH
H
10.6b
44a
18a
7a
12
13
14
15a
15b
15c
15d
15e
15g
COOH
12a
3a
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
4a
18a
ꢁ3a
4a
20a
5a
10a
9a
0.5a
4.5a
19a
NHCOCH2N
O
16a
C6H5
CH3
COOC2H5
10
14a
9a
16b
16c
17
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
COOC2H5
COOC2H5
COOC2H5
COOC2H5
NHCOCH2NHCH2C6H5
NHCOCH2NHCH2C4H9
NCS
10
10
10
10
25a
ꢁ3a
0a
4a
10a
3.5b
ꢁ6.5a
18
NHCSNHC2H5
7a
N
19
C6H5
C6H5
C6H5
CH3
CH3
CH3
COOC2H5
COOC2H5
COOC2H5
10
10
10
ꢁ1a
10a
4a
2a
38b
54b
20a
20b
N]NeN(CH3)2
N=N-N
O
20c
21
C6H5
C6H5
CH3
CH3
COOC2H5
COOC2H5
N]NeCN
Cl
10
10
7a
0a
21b
10b
a
Inhibition of luminescence (% of control).
IC50 (mM).
b
We found potent compounds in the group of thiophene deriv-
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
atives having a 2-amino-group, which can act as H-donor of
a hydrogen bond. This applies for the 2-diazenylthiophenes 20a-c,
2-chlorothiophene 21 and isothiocyanate 17. The most effective
compound of this group proved to be 17 having an IC50 of 3.5 mM in
GluR6-assay.
The effect of thiophenes 1c, 9e,g,k and 17 on metabolic activity
of cells was determined using a MTT-assay [31]. Toxic effects of
thiophene derivatives against N2A- and COS7-cells were detectable
N2A-cells
COS7-cells
at a concentration of 100
24 h (Scheme 5).
mM only and after an incubation time of
Compounds 1c, 9h and 17 inhibited the activity of mitochondria
in both cell lines in this concentration in comparison to control.
However 9e showed only slightly effect towards N2A-cells. In
the presence of 9g no significant differences of formazan formation
in COS7-cell line was observed.
3. Conclusion
We were able to identify 2-aminothiophenes with hydrophobic
substituents in 4- and 5-position as a new class of selective GluR6-
Antagonists. The aim of this work was to increase the selectivity of
GluR6-specific inhibition. The lead compound of previous studies of
control
1c
9e
9g
9h
17
Scheme 5. Influence of thiophenes 1c, 9e, 9g, 9h and 17 (100
mitochondrial dehydrogenases in N2A- and COS7-cells.
mM) on activity of

D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
73
5-phenylthiopene-3-carboxylic acid methylesters was modified. By
this way the influence of decreasing the log P-value was analysed.
Without changing the chemical structure a variety of substitution
patterns were realized keeping a hydrophobic region (4- and 5-
position) and a hydrophilic region (2- and 3- position). To gather
information about the influence of the amino group for receptor
binding the electronic properties as well as the hydrogen donor
properties in this position were changed.
4.1.1.5. Ethyl 2-amino-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-
3-carboxylate 9f. Yield 86%; mp 101–103 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.35 (t, J ¼ 7.2 Hz, 3 H), 1.91 (m, 1 H), 2.10 (m, 1 H), 2.75 (m, 3
H), 2.99 (m, 2 H), 4.29 (q, J ¼ 7.2 Hz, 2 H), 5.96 (s, 2 H), 7.28 (m, 5 H);
13C-NMR (CDCl3):
(ppm) 14.6, 24.4, 30.2, 32.5, 40.9, 59.6, 107.0,
d
118.4,126.4,127.0,128.6,132.5,146.1,160.2,166.1; LRMS-EI: m/z calcd
301.1, found 301.0; IR (KBr): 1244,1258,1480,1572,1663, 2834, 2988,
3355, 3471 cmꢁ1
.
In MTT-assay toxic effects were observed only in high concen-
trations (100
m
M) and long incubation times (24 h). Having these
4.1.1.6. General method B. Crotonic acid nitrile (0.1 mol), sulfur
(0.1 mol) and diethylamin or morpholin (5–10 ml) in ethanol
(30 ml) were converted and isolated like described in general
method A.
preliminary results the tested compounds can be considered as less
toxic.
4. Experimental
4.1.1.7. 2-Amino-4-methyl-5-phenylthiophene-3-carboxamide
4.1. Chemistry
1a. Yield 60%; mp 185 ꢀC; 1H-NMR (DMSO-d6):
d (ppm) 2.26 (s, 3
H), 6.86 (s, 2 H), 6.94 (s, 2 H), 7.34 (m, 5 H); LRMS-EI: m/z calcd
Unless otherwise noted all reagents and solvents were used as
supplied commercially (Merck, Sigma–Aldrich, Fluka, Lancaster).
All reactions were performed in oven-dried glassware. Analytical
thin layer chromatography (TLC) was performed on Merck Silica gel
60 F254 plates. Flash column chromatography was performed on
232.1, found 232.1; IR (KBr): 1279, 1413, 1468, 1552, 1563, 1592,
1651, 3170, 3312, 3440, 3501 cmꢁ1
.
4.1.1.8. Ethyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate
1c. Yield 77%; mp 95–97 ꢀC; 1H-NMR (CDCl3):
(ppm) 1.37 (t,
d
Merck Silica gel 60 (particle size 63
m
M–200
m
M) and Lancaster
J ¼ 7.2 Hz, 3 H), 2.33 (s, 3 H); 4.32 (q, J ¼ 7.2 Hz, 2 H), 6.10 (s, 2 H),
7.34 (m, 5 H); LRMS-EI: m/z calcd 261.1, found 261.1; IR (KBr):
1070, 1103, 1232, 1256, 1326, 1405, 1475, 1557, 1568, 1596, 1655,
Silica gel 60 (particle size 40 M). Nuclear magnetic resonance
m
spectra were recorded on a Varian Gemini-300, Bruker DRX-400 or
Bruker DRX-600. The chemical shifts are reported in ppm and the
residual solvent signal is used as an internal standard. Infrared
spectra were collected on a Perkin-Elmer FT-IR PC 16 spectrometer.
For mass spectra a VG Analytics VG ZAB-HSQ (EI, 70 eV), Bruker
Daltonics 7 T ApexÔ II FT-ICR-MS (ESI-HRMS, EI, 70 eV), Perkin-
Elmer Micro-HPLC 200/PE SCIEX API 3000 (LC/MS) or Hewlett
Packard HP 1050/MS Engine HP 5989 A (LC/MS) (negative and
positive chemical ionisation, EI, 70 eV) were used. Melting points
2926, 2988, 3356, 3477 cmꢁ1
.
4.1.1.9. Ethyl 2-amino-5-(4-methoxyphenyl)-4-methylthiophene-3-
carboxylate 9a. Yield 53%; mp 102–105 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.37 (t, J ¼ 7.2 Hz, 3 H), 2.28 (s, 3 H), 3.83 (s, 3 H), 4.31 (q,
J ¼ 7.2 Hz, 2 H), 6.06 (s, 2 H), 6.92 (m, 2 H), 7.26 (m, 2 H); LRMS-EI:
m/z calcd 291.3, found 291.3; IR (KBr): 1180, 1270, 1467, 1570, 1654,
2925, 2984, 3322, 3447 cmꢁ1
.
¨
were determined on a BUCHI Melting Point 535 or on a Boetius. The
declared values are not corrected.
4.1.1.10. Ethyl
carboxylate 9b. Yield 49%; mp 79–82 ꢀC; 1H-NMR (CDCl3):
1.37 (t, J ¼ 7.2 Hz, 3 H), 2.28 (s, 3 H), 4.31 (q, J ¼ 7.2 Hz, 2 H), 6.09 (s, 2
H), 7.07 (m, 2 H), 7.30 (m, 2 H); 19F-NMR (CDCl3):
(ppm) ꢁ37.1;
LRMS-EI: m/z calcd 279.2, found 279.2; IR (KBr): 1134, 1225, 1269,
1466, 1560, 1654, 2929, 2985, 3320, 3447 cmꢁ1
2-amino-5-(4-fluorophenyl)-4-methylthiophene-3-
d
(ppm)
4.1.1. Synthesis of 2-aminothiophenederivatives 1a-d and 9a-l by
Gewald
d
4.1.1.1. General method A. To a stirring mixture of ketone (0.1 mol),
nitrile (0.1 mol) and powdered sulfur (0.1–0.11 mol) in ethanol
(30 ml) diethylamine or morpholine (10 ml) was added dropwise,
keeping the temperature lower than 50 ꢀC. After 1–3 hours the
reactions were complete and the reaction mixtures were cooled in
a fridge for crystallization. If no crystallization takes place the
mixtures were poured in to 2–3 fold volume of water. The precip-
itates were filtered and recrystalized from ethanol.
.
4.1.1.11. Ethyl
2-amino-5-(2-chlorophenyl)-4-methylthiophene-3-
carboxylate 9c. The crude product was purified by column chro-
matography (toluene); yield 28%; mp 96-98 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.11 (t, J ¼ 7.2 Hz, 3 H), 2.11 (s, 3 H), 4.14 (q, J ¼ 7.2 Hz, 2 H),
6.12 (s, 2 H), 7.24 (m, 4 H); 13C-NMR (CDCl3):
d
(ppm) 14.7,16.6, 36.0,
59.9,105.2,127.0,127.6,129.4,130.0,130.4,133.8,138.9,164.9,166.6;
4.1.1.2. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox-
HRMS-ESI: m/z calcd 318.03260 [M þ Na]þ, found 318.03265; IR
amide 1b. Yield 61%; mp 190 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.68
(KBr): 1064, 1134, 1277, 1490, 1593, 1649, 2976, 3309, 3424 cmꢁ1
.
(m, 4 H), 2.51 (m, 4 H), 6.52 (bs, 2 H), 6.89 (s, 2 H); LRMS-EI: m/z
calcd 196.1, found 196.0; IR (KBr): 1420, 1481, 1589, 1645, 2854,
4.1.1.12. Ethyl 2-amino-4,5-diphenylthiophene-3-carboxylate 9e. Yield
59%; mp 146 ꢀC; 1H-NMR (DMSO-d6):
3157, 3388, 3405, 3482, 3937 cmꢁ1
.
d
(ppm) 0.73 (t, J ¼ 7.2 Hz, 3 H),
3.82 (q, J ¼ 7.2 Hz, 2 H), 7.11 (m, 10 H), 7.55 (s, 2 H); HRMS-ESI: m/z
4.1.1.3. Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox-
ylate 1d. Yield 73%; mp 111–115 ꢀC; 1H-NMR (CDCl3):
(ppm) 1.33 (t,
calcd 346.08722 [M þ Na]þ, found 346.08738; IR (KBr): 1235, 1275,
d
1465, 1484, 1547, 1576, 1645, 2978, 3059, 3312, 3425 cmꢁ1
.
J ¼ 7.2 Hz, 3 H), 1.76 (m, 4 H), 2.50 (t, J ¼ 5.7 Hz, 2 H), 2.69 (t, J ¼ 5.7 Hz,
2 H), 4.25 (q, J ¼ 7.2 Hz, 2 H), 5.93 (s, 2 H); LRMS-EI: m/z calcd 225.1,
found 225.1; IR (KBr): 1028, 1154, 1179, 1276, 1295, 1412, 1491, 1499,
4.1.1.13. Methyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate
9g. Yield 30%; mp 118 ꢀC; 1H-NMR (DMSO-d6):
(ppm) 2.24 (s, 3
H), 3.73 (s, 3 H), 7.37 (m, 12 H); 13C-NMR (DMSO-d6):
(ppm) 16.6,
d
1576, 1596, 1647, 2841-2986, 3299, 3404 cmꢁ1
.
d
51.2, 105.5, 117.9, 127.4, 129.4, 129.8, 131.3, 134.7, 164.5, 166.2; LRMS-
4.1.1.4. Ethyl 2-amino-5-phenylthiophene-3-carboxylate 9d. Yield
75%; mp 123-124 ꢀC; 1H-NMR (CDCl3):
EI: m/z calcd 247.1, found 247.1; IR (KBr): 1239, 1271, 1437, 1480,
d
(ppm) 1.37 (t, J ¼ 7.2 Hz, 3
1550, 1581, 1670, 2948, 3304, 3469 cmꢁ1
.
H), 4.30 (q, J ¼ 7.2 Hz, 2 H), 6.00 (s, 2 H), 7.33 (m, 6 H); LRMS-EI: m/z
calcd 247.2, found 247.2; IR (KBr): 1094, 1204, 1264, 1413, 1480,
4.1.1.14. Propyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate
9h. The crude product was purified by column chromatography
1496, 1548, 1585, 1667, 2932, 2978, 3322, 3455 cmꢁ1
.

74
D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
(toluene); yield 20%; mp 66 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.03 (t,
was removed and the residue was solved in DMF. Water was added to
J ¼ 7.5 Hz, 3 H), 1.78 (m, 2 H), 2.34 (s, 3 H), 4.23 (t, J ¼ 6.6 Hz, 2 H),
6.12 (s, 2 H), 7.33 (m, 5 H); HRMS-ESI: m/z calcd 298.08722
[M þ Na]þ, found 298.08736; IR (KBr): 1235, 1272, 1479, 1551, 1576,
precipitate the decarboxylated compound 13 (8%), which was
recrystallized from ethanol; mp 132–134 ꢀC; 1H-NMR (CDCl3):
d (ppm)
1.32 (t, J ¼ 7.2 Hz, 3 H), 2.25 (s, 3 H), 4.26 (q, J ¼ 7.2 Hz, 2 H), 6.45 (s, 1
1666, 2964, 3317, 3427 cmꢁ1
.
H), 6.96 (s,1 H), 7.35 (m, 5 H); LRMS-EI: m/z calcd 261.0, found 261.0; IR
(KBr): 1264, 1311, 1586, 1690, 3432 cmꢁ1
.
4.1.1.15. tert-Butyl 2-amino-4-methyl-5-phenylthiophene-3-carbox-
ylate 9i. The crude product was purified by column chromatog-
raphy (CH2Cl2); yield 20%; mp 101-103 ꢀC; 1H-NMR (CDCl3):
4.1.2.5. 2-(Ethoxycarbonylamino)-4-methyl-5-phenylthiophene-3-
carboxylic acid 14. Originated from 12 and ethyl chloroformate,
acetonitrile was used instead of toluene, reaction time was 8 h;
yield 40% containing 5% decarboxylated compound 13; mp 174–
d
(ppm) 1.59 (s, 9 H), 2.31 (s, 3 H), 6.06 (bs, 2 H), 7.37 (m, 5 H);
HRMS-ESI: m/z calcd 312.10287 [M þ Na]þ, found 312.10308; IR
(KBr): 1169, 1251, 1453, 1577, 1664, 2928, 2975, 3328, 3441 cmꢁ1
.
178 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.27 (t, J ¼ 7.2 Hz, 3 H), 2.31 (s,
3 H), 4.23 (q, J ¼ 7.2 Hz, 2 H), 7.43 (m, 5 H), 10.73 (s, 1 H); HRMS-ESI:
m/z calcd 328.06140 [M þ Na]þ, found 328.06143; IR (KBr): 1206,
4.1.1.16. 2-Amino-N-ethyl-4-methyl-5-phenylthiophene-3-carbox-
amide 9j. The product was purified by column chromatography
(1,4-dioxane/toluene 1:2); yield 20%; mp 65 ꢀC; 1H-NMR (CDCl3):
1253, 1410, 1460, 1530, 1690, 1734, 3200, 3400, 3298 cmꢁ1
.
d
(ppm) 1.24 (t, J ¼ 7.2 Hz, 3 H), 2.34 (s, 3 H), 3.47 (m, 2 H), 5.77 (bs, 1
4.1.2.6. Ethyl 2-benzamido-4-methyl-5-phenylthiophene-3-carboxylate
H), 5.97 (s, 2 H), 7.35 (m, 5 H); 13C-NMR (CDCl3):
d
(ppm) 15.2, 16.3,
15c. Originated from 1c and benzoyl chloride (0.5 ml, 4.3 mmol); yield
34.4, 111.8, 127.1, 127.5, 128.6, 129.8, 130.4, 134.3, 159.4, 166.7;
70%; mp 143 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.45 (t, J ¼ 7.2 Hz, 3 H), 2.42
HRMS-ESI: m/z calcd 283.08755 [M þ Na]þ, found 283.08795; IR
(s, 3 H), 4.44 (q, J ¼ 7.2 Hz, 2 H), 7.47 (m, 8 H), 8.04 (m 2 H), 12.46 (s 1
H); LRMS-EI: m/z calcd 365.1, found 365.0; IR (KBr): 1222, 1268, 1298,
(KBr): 1452, 1675, 2972, 3289 cmꢁ1
.
1530, 1559, 1599, 1654, 1670, 2928, 2977, 3440 cmꢁ1
.
4.1.1.17. 2-Amino-4-methyl-5-phenylthiophene-3-carbonitrile
9k. The crude product was purified by column chromatography
(EtOAc/n-hexane, 1:9); yield 10%; mp 137 ꢀC; 1H-NMR (CDCl3):
4.1.2.7. Ethyl
2-(2-ethoxy-2-oxoacetamido)-4-methyl-5-phenyl-
thiophene-3-carboxylate 15d. Originated from 1c and ethyl 2-
d
(ppm) 2.27 (s, 3 H), 4.75 (s, 2 H), 7.35 (m, 5 H); HRMS-ESI: m/z
chloro-2-oxoacetate, benzene was used instead of toluene; yield
calcd 237.04569 [M þ Na]þ, found 237.04591; IR (KBr): 1517, 1654,
70%; mp 123–124 ꢀC; 1H-NMR (CDCl3):
d (ppm) 1.45 (m, 6 H), 2.41
2198, 2922, 3336, 3426 cmꢁ1
.
(s, 3 H), 4.45 (m, 4 H), 7.41 (m, 5 H), 12.55 (s, 1 H); LRMS-EI: m/z
calcd 361.1, found 361.1; IR (KBr): 1237,1282,1381,1444,1526,1556,
4.1.1.18. (2-Amino-4-methyl-5-phenylthiophen-3-yl)(phenyl)metha-
1673, 1702, 1734, 2936, 2978, 3440 cmꢁ1
.
none 9l. The crude product was purified by column chromatog-
raphy (CH2Cl2); yield 22%; mp 80 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.72
(s, 3 H), 6.53 (s, 2 H), 7.45 (m, 10 H); LRMS-EI: m/z calcd 293.1, found
292.9; IR (KBr): 1178,1268,1447,1567,1654, 2923, 3282, 3421 cmꢁ1
4.1.2.8. ethyl 2-(2-bromoacetamido)-4-methyl-5-phenylthiophene-3-
carboxylate 15f. originated from 1c and 2-bromoacetyl bromide
(2.00 ml, 23 mmol), benzene was used instead of toluene; yield 71%;
.
mp 53–54 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.34 (t, J ¼ 6.9 Hz, 3 H),
4.1.2. Synthesis of 2-acylamino-thiophene-3-carboxylic adic
derivatives
2.32 (s, 3 H), 4.35 (q, J ¼ 6.9 Hz, 2 H), 4.42 (s, 2 H), 7.42 (m, 5 H), 11.57
(s, 1 H); LRMS-EI: m/z calcd 381.0, found 383.0; IR (KBr): 1161, 1198,
4.1.2.1. Method A Acylation via carboxylic acid chlorides. To a stirred
mixture of the corresponding 2-aminothiophene-3-carboxylic acid
derivative (0.50 g) and K2CO3 (10 eq.) in toluene (10 ml) the cor-
responding carboxylic acid chloride (1.2–10 eq) was added at 0 ꢀC.
After 3 h at r.t. the inorganic solid were filtered off, solvent was
removed by filtration and the residue was recrystallized from 1,4-
dioxane.
1252,1375, 1441, 1531,1552,1626,1670,1747, 2986, 3212, 3444 cmꢁ1
.
4.1.2.9. Method B Acylation via carboxylic acid anhydrides. The corre-
sponding carboxylic acid anhydride (50 mmol) and the corresponding
2-amino-3-carboxylic acid derivative (2 mmol) were heated under
reflux for 2.5 h. The solvent was removed under reduced pressure and
the residues were dried and recrystallized from ethanol.
4.1.2.2. Ethyl 3-carbamoyl-4-methyl-5-phenylthiophen-2-ylcarbamate
4.1.2.10. Ethyl 2-acetamido-4-methyl-5-phenylthiophene-3-carbox-
2a. Originated from 1a 24 and ethyl chloroformate (0.25 ml,
ylate 15a. Originated from 1c and acetic anhydride; yield 71%; mp
2.63 mmol); yield 23%; mp 194–195 ꢀC; 1H-NMR (CDCl3):
d (ppm)
121–126 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.42 (t, J ¼ 7.2 Hz, 3 H), 2.29
(s, 3 H), 2.38 (s, 3 H), 4.38 (q, J ¼ 7.2 Hz, 2 H), 7.41 (m, 5 H), 11.41 (s, 1
H); 13C-NMR (CDCl3):
(ppm) 14.6, 15.9, 24.0, 61.0, 113.2, 127.7,
1.33 (t, J ¼ 7.2 Hz, 3 H), 2.42 (s, 3 H), 4.28 (q, J ¼ 7.2 Hz, 2 H), 5.82 (bs, 2
H), 7.37 (m, 5 H),11.20 (s,1 H); LRMS-EI: m/z calcd 304.1, found 304.1;
IR (KBr): 1045, 1078, 1117, 1205, 1331, 1546, 1584, 1520, 1635, 1727,
d
128.8, 129.3, 129.7, 130.3, 134.1, 149.3, 167.1, 167.5; LRMS-EI: m/z
2990, 3329 cmꢁ1
.
calcd 303.1, found 303.1; IR (KBr): 1206, 1248, 1306, 1404, 1439,
1523, 1554, 1598, 1657, 1688, 2939, 2988, 3240 cmꢁ1
.
4.1.2.3. Ethyl 3-carbamoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylcar-
bamate 2b. Originated from 1b 25 and ethyl chloroformate (0.25 ml,
4.1.2.11. Ethyl 4-methyl-5-phenyl-2-(2,2,2-trifluoroacetamido)thiophene-
2.63 mmol); yield 31%; mp 209–210 ꢀC; 1H-NMR (CDCl3):
d (ppm) 1.31
3-carboxylate 15b. Originated from 1c and trifluoroacetic anhydride;
yield 97%; mp 88–92 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.44 (t, J ¼ 7.2 Hz, 3
(t, J ¼ 7.2 Hz, 3 H), 1.84 (m, 2 H), 2.68 (m, 2 H), 4.25 (q, J ¼ 7.2 Hz, 2 H),
H), 2.40 (s, 3 H), 4.44 (q, J ¼ 7.2 Hz, 2 H), 7.41 (m, 5 H), 12.44 (s, 1 H); 19F-
5.64 (bs, 2 H), 11.19 (s, 1 H); LRMS-EI: m/z calcd 268.1, found 268.1; IR
(KBr): 1209, 1534, 1587, 1629, 1725, 2990, 3180, 3380 cmꢁ1
.
NMR (CDCl3):
d (ppm) 2.36; LRMS-EI: m/z calcd 357.2, found 357.2; IR
(KBr): 1240, 1322, 1525, 1565, 1668, 1719, 2934, 2996, 3426 cmꢁ1
.
4.1.2.4. Ethyl4-methyl-5-phenylthiophen-2-ylcarbamate13. Originated
from 12 and ethyl chloroformate, acetonitrile was used instead of
toluene, reaction time was 8 h, the solvent was removed under
reduced pressure, the solid (mixture of 13 and 14) was solved in CHCl3
and washed with NaOH (0.5 M). The phases were separated, solvent
4.1.3. Synthesis of ethyl 4-methyl-2-(methylamino)-5-
phenylthiophene-3-carboxylate 15g
A mixture of 1c (0.15 g, 0.60 mmol), iodomethane (0.36 ml,
6 mmol) and triethylamine (0.84 ml, 6 mmol) in acetonitrile (3 ml)

D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
75
were converted in a high pressure reactor (20 bar) at 105 ꢀC for 30 h.
The solvent was removed under reduced pressure and the crude
product was recrystallized from ethanol to give 15g 139 (0.10 g,
10.4 mmol) was added at 0 ꢀC. After 1 h at r.t. the inorganic solid
was removed by filtration, solvent was removed under reduced
pressure and the residue was washed with n-hexane to give 17
0.36 mmol, 61%). mp 140 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.38 (t,
(0.52 g, 1.71 mmol, 45%); mp 58-60 ꢀC; 1H-NMR (CDCl3):
d
(ppm)
1.44 (t, J ¼ 7.2 Hz, 3 H), 2.37 (s, 3 H), 4.40 (q, J ¼ 7.2 Hz, 2 H), 7.40 (m,
5 H); 13C-NMR (CDCl3):
(ppm) 14.6, 15.2, 61.2, 128.4, 128.5, 128.9,
129.9, 132.7, 133.2, 133.8, 162.5; LRMS-EI: m/z calcd 303.0, found
303.0; IR (KBr): 1196, 1302, 1456, 1541, 1707, 2102, 2979 cmꢁ1
J ¼ 7.2 Hz, 3 H), 2.34 (s, 3 H), 3.01 (d, J ¼ 4.7 Hz, 3 H), 4.29 (q,
J ¼ 7.2 Hz, 2 H), 7.32 (m, 5 H), 7.79 (s, 1 H); LRMS-EI: m/z calcd 275.1,
d
found 275.0; IR (KBr): 1082,1225,1528,1596,1647, 2978, 3307 cmꢁ1
.
.
4.1.4. Synthesis of 2-(3-ethylureido)-thiophene-carboxylic acid
derivatives
4.1.7. Synthesis of ethyl 2-(3-ethylthioureido)-4-methyl-5-
phenylthiophene-3-carboxylate 18
4.1.4.1. General procedure. To a solution of the corresponding 2-
aminothiophene (0.80 mmol) in dry CH2Cl2 (5 ml) N-ethyl-
isocyanate (1.00 ml, 12.8 mmol) was added and the mixture was
refluxed for 5 days. After cooling down to r.t. 50% aqueous ethanol
was added and the precipitate was filtered off and recrystallized
from ethanol.
To a solution of 17 (0.50 g, 1.65 mmol) in CH2Cl2 (4.5 ml) ethyl-
amine (0.2 ml, 3.06 mmol) was added dropwise. After 1 h at r.t. the
reaction mixture was acidified using HCl (1 M in ethanol). The
precipitate was filtered off and recrystallized from ethanol to give
18 (0.40 g, 1.15 mmol, 70%); mp 178–180 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.15 (t, J ¼ 6.6 Hz, 3 H), 1.34 (t, J ¼ 7.5 Hz, 3 H), 2.31 (s, 3 H),
3.44 (m, 2 H), 4.35 (q, J ¼ 7.5 Hz, 2 H), 7.41 (m, 5 H), 9.52 (s, 1 H),
11.58 (s, 1 H); LRMS-EI: m/z calcd 348.1, found 348.0; IR (KBr): 1067,
4.1.4.2. 2-(3-Ethylureido)-4-methyl-5-phenylthiophene-3-carbox-
amide 2c. Originated from 1a; yield 83%; mp 223–225 ꢀC; 1H-NMR
1227, 1442, 1550, 1578, 1654, 2926, 2973, 3228 cmꢁ1
.
(DMSO-d6):
d
(ppm) 1.05 (t, J ¼ 7.2 Hz, 3 H), 2.29 (s, 3 H), 3.11 (m, 2
H), 7.38 (m, 7 H), 7.59 (s, 1 H), 10.17 (s, 1 H); LRMS-EI: m/z calcd
4.1.8. Synthesis of ethyl 4-methyl-5-phenyl-2-(1H-pyrrol-1-
yl)thiophene-3-carboxylate 19
303.1, found 303.0; IR (KBr): 1154, 1247, 1294, 1523, 1557, 1594,
1645, 1664, 2931, 2974, 3100, 3399 cmꢁ1
.
A solution of 1c (0.50 g, 1.92 mmol) and 2,5-dimethoxyte-
trahydrofuran (0.32 ml, 2.5 mmol) in acetic acid (5 ml) was refluxed
for 20 min. The reaction mixture was poured into crushed ice, made
basic and extracted with CH2Cl2. The organic phase was washed
several times with water, died (Na2SO4) and evaporated. Column
chromatography (CH2Cl2/acetone 9.5:0.5) of the residue gave 19
4.1.4.3. 2-(3-Ethylureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxamide 2d. Originated from 1b; yield 92%; mp 194–198 ꢀC;
1H-NMR (DMSO-d6):
2.52 (m, 4 H), 3.08 (m, 2 H), 6.97 (bs, 2 H), 7.53 (s, 1 H), 10.48 (s, 1 H);
d
(ppm) 1.03 (t, J ¼ 7.2 Hz, 3 H), 1.70 (m, 4 H),
LRMS-EI: m/z calcd 267.1, found 267.0; IR (KBr): 1228, 1278, 1298,
(0.51 g, 1.64 mmol, 85%) as an oil; 1H-NMR (CDCl3):
d (ppm) 1.16 (t,
1524, 1559, 1592, 1637, 1677, 2856, 2974, 3088, 3473 cmꢁ1
.
J ¼ 7.2 Hz, 3 H), 2.37 (s, 3 H), 4.20 (q, J ¼ 7.2 Hz, 2 H), 6.30 (m, 2 H),
6.78 (m, 2 H), 7.42 (m, 5 H); LRMS-EI: m/z calcd 311.1, found 311.0; IR
(CHCl3): 1078, 1218, 1328, 1379, 1405, 1464, 1514, 1560, 1716, 2927,
4.1.4.4. Ethyl 2-(3-ethylureido)-4-methyl-5-phenylthiophene-3-carbox-
ylate 15e. Originated from 1c; yield 75%; mp 138–140 ꢀC; 1H-NMR
2980 cmꢁ1
.
(DMSO-d6):
d
(ppm) 1.07 (t, J ¼ 7.2 Hz, 3 H),1.33 (t, J ¼ 7.2 Hz, 3 H), 2.29
(s, 3 H), 3.13 (m, 2 H), 4.31 (q, J ¼ 7.2 Hz, 2 H), 7.39 (m, 5 H), 7.91 (s, 1 H),
10.36 (s, 1 H); LRMS-EI: m/z calcd 332.1, found 332.0; IR (KBr): 1034,
4.1.9. Conversion of 15f with amines
4.1.9.1. General procedure. To a solution of 15f in acetonitrile
(3.5 ml) the corresponding amine (10 eq.) was added dropwise at
0 ꢀC. The precipitate was filtered off after 1 h, dried and recrystal-
lized from ethanol.
1337, 1534, 1557, 1648, 1664, 2931, 2974, 3286 cmꢁ1
.
4.1.4.5. Ethyl 2-(3-ethylureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
1
carboxylate 15h. Originated from 1d; yield 84%; mp 165–169 ꢀC; H-
NMR (DMSO-d6):
d
(ppm) 1.06 (t, J ¼ 6.9 Hz, 3 H), 1.29 (t, J ¼ 6.9 Hz, 3
4.1.9.2. Ethyl4-methyl-2-(2-morpholinoacetamido)-5-phenylthiophene-
3-carboxylate 16a. Morpholine was used; yield 31%; mp 142 ꢀC; 1H-
H), 1.70 (m, 4 H), 2.51 (m, 2 H), 2.67 (m, 2 H), 3.11 (m, 2 H), 4.25 (q,
J ¼ 6.9 Hz, 2 H), 7.80 (s, 1 H), 10.24 (s, 1 H); LRMS-EI: m/z calcd 296.1,
found 296.0; IR (KBr): 1040, 1229, 1544, 1565, 1654, 1684, 2931, 2975,
NMR (DMSO-d6):
H), 3.24 (s, 2 H), 3.65 (s, 4 H), 4.37 (q, J ¼ 7.2 Hz, 2 H), 7.43 (m, 5 H),
12.21 (s, 1 H); 13C-NMR (DMSO-d6):
(ppm) 14.9, 16.1, 54.0, 61.3, 61.3,
d
(ppm) 1.35 (t, J ¼ 7.2 Hz, 3 H), 2.33 (s, 3 H), 2.55 (s, 4
3230, 3283 cmꢁ1
.
d
66.9, 114.0, 128.4, 128.6, 129.5, 130.0, 130.2, 134.0, 147.7, 165.7, 169.1;
4.1.5. Synthesis of 2-amino-4-methyl-5-phenylthiophene-3-
carboxylic acid 12
LRMS-EI: m/z calcd 388.1, found 388.2; IR (KBr): 1112, 1244, 1273, 1523,
1552, 1676, 2822, 2964, 3440 cmꢁ1
.
A solution of 1c (67.7 g, 0.26 mol) in ethanol (650 ml) and
aqueous NaOH (10.0 M, 260 ml) was refluxed for 2.5 h. The solvent
was removed under reduced pressure, the precipitate of sodium
salt was filtered off and solved in water. The free acid was precip-
itated by the addition of HCl (3 M), filtered off, washed with cold
water and dried to give 12 (50.4 g, 0.22 mol, 83%); mp 153 ꢀC; 1H-
4.1.9.3. Ethyl 2-(2-(benzylamino)acetamido)-4-methyl-5-phenyl-
thiophene-3-carboxylate 16b. Benzylamine was used; yield 50%;
mp 122–123 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 1.42 (t, J ¼ 7.2 Hz, 3
H), 2.14 (bs, 1 H), 2.41 (s, 3 H), 3.56 (s, 2 H), 3.88 (s, 2 H), 4.42 (q,
J ¼ 7.2 Hz, 2 H), 7.36 (m, 10 H), 12.43 (s, 1 H); LRMS-EI: m/z calcd
408.2, found 408.0; IR (KBr): 1174, 1242, 1305, 1517, 1551, 1668,
NMR (DMSO-d6):
(DMSO-d6):
d
(ppm) 2.24 (s, 3 H), 7.34 (m, 5 H); 13C-NMR
2927, 2985, 3435 cmꢁ1
.
d
(ppm) 16.0, 105.6, 116.8, 126.6, 128.7, 128.0, 131.0,
134.2, 163.9, 167.0; HRMS-ESI: m/z calcd 234.05833 [M þ H]þ, found
234.05866; IR (KBr): 1260, 1375, 1451, 1470, 1579, 1647, 2924, 3380,
4.1.9.4. Ethyl 2-(2-(butylamino)acetamido)-4-methyl-5-phenyl-
thiophene-3-carboxylate 16c. Butylamine was used; yield 38%;
3470 cmꢁ1
.
mp 97-99 ꢀC; 1H-NMR (CDCl3):
d
(ppm) 0.93 (t, J ¼ 7.2 Hz, 3 H),
4.1.6. Synthesis of ethyl 2-isothiocyanato-4-methyl-5-
phenylthiophene-3-carboxylate 17
To a mixture of 1c (1.00 g, 3.83 mmol) and K2CO3 (0.97 g,
7.00 mmol) in CH2Cl2 (5 ml) thiophosgene in CH2Cl2 (0.80 ml,
1.48 (m, 7 H), 2.40 (s, 3 H), 2.69 (t, J ¼ 7.5 Hz, 2 H), 3.53 (s, 2 H),
4.41 (q, J ¼ 7.2 Hz, 2 H), 7.37 (m, 5 H), 12.33 (s, 1 H); LRMS-EI: m/z
calcd 374.0, found 374.0; IR (KBr): 1185, 1239, 1307, 1423, 1441,
1512, 1548, 1670, 2860, 2926, 2956, 3446 cmꢁ1
.

76
D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
4.1.10. Diazotation of 1c
d
(ppm) 21.5, 22.5, 23.9, 24.9, 116.4, 128.2, 130.8, 150.0, 157.0, 173.8;
4.1.10.1. General procedure. To a mixture of 1c (0.5 g, 1.92 mmol),
water (7 ml) and conc. HCl (3 ml) NaNO2-solution (0.20 g in 1 ml
water, 2.68 mmol) was added at 0 ꢀC during 20 min. After 30 more
min in an ice bath the mixture was filtered and the diazonium-salt-
solution was added dropwise to a 0 ꢀC cold solution of the corre-
sponding nucleophile. The reaction mixture was stirred for 30 min,
extracted with Et2O (3 ꢂ10 ml), dried (Na2SO4) and evaporated.
Crude products were purified by column chromatography (Et2O).
LRMS-EI: m/z calcd 238.0, found 238.0.
4.1.12. Synthesis of 2-(Ethylthio)-thieno[2,3-d]pyrimidin-4(3H)-
ones 4a,b
4.1.12.1. General procedure. 2-Thioxo-2,3-dihydrothieno[2,3-d]pyr-
imidin-4(1H)-one 3a,b (1.10 mmol) was solved in a mixture of
ethanol (5 ml) and sodium methanolate solution (1 M, 10 ml).
Iodoethane (3.30 mmol) was added and the mixture was stirred for
30 min. The solvent was evaporated, the precipitate was filtered off,
washed with water and recrystallized from ethanol.
4.1.10.2. Ethyl
2-(3,3-dimethyltriaz-1-enyl)-4-methyl-5-phenyl-
thiophene-3-carboxylate 20a. Dimethylamin (40% aqueous solu-
4.1.12.2. 2-(Ethylthio)-5-methyl-6-phenylthieno[2,3-d]pyrimidin-
tion, 4 ml) was used as nucleophile; yield 52%; mp 88-92 ꢀC; 1H-
4(3H)-one 4a. Originated from 3a; yield 82%; mp > 312 ꢀC; 1H-
NMR (DMSO-d6):
d
(ppm) 1.29 (t, J ¼ 7.2 Hz, 3 H), 2.23 (s, 3 H), 3.19
NMR (DMSO-d6):
d
(ppm) 1.26 (t, J ¼ 7.5 Hz, 3 H), 2.57 (s, 3 H), 2.97
(s, 3 H), 3.49 (s, 3 H), 4.26 (q, J ¼ 7.2 Hz, 2 H), 7.40 (m, 5 H); 13C-NMR
(q, J ¼ 7.5 Hz, 2 H), 7.36 (m, 5 H); 13C-NMR (DMSO-d6):
d (ppm) 15.2,
(DMSO-d6):
d (ppm) 14.9, 15.0, 37.4, 43.8, 60.8, 123.9, 128.3, 129.5,
15.3, 23.8, 120.0, 124.0, 126.6, 128.6, 128.9, 129.4, 134.9, 165.0, 169.0;
129.7, 130.4, 131.9, 134.5, 158.1, 165.0; LRMS-ESI: m/z calcd 340.1
LRMS-EI: m/z calcd 302.1, found 301.9; IR (KBr): 1028, 1055, 1362,
[M þ Na]þ, found 340.1; IR (KBr): 1057, 1100, 1227, 1372, 1443, 1474,
1550, 1600, 1654, 2868, 2968, 3406 cmꢁ1
.
1596, 1697, 2850, 2919 cmꢁ1
.
4.1.12.3. 2-(Ethylthio)- 5,6,7,8-tetrahydrobenzo[b]-thieno[2,3-d]pyr-
4.1.10.3. Ethyl 4-methyl-2-(morpholinodiazenyl)-5-phenylthiophene-
imidin-4(3H)-one 4b. Originated from 3b; yield 82%; mp 297-
298 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.26 (t, J ¼ 6.9 Hz, 3 H), 1.74
(m, 4 H), 2.65 (m, 2 H), 2.81 (m, 2 H), 3.03 (q, J ¼ 6.9 Hz, 2 H), 12.45
(bs, 1 H); 13C-NMR (DMSO-d6):
(ppm) 15.0, 22.2, 22.9, 23.9, 24.5,
3-carboxylate 20b. Morpholine (10 ml) was used as nucleophile;
yield 83%; 1H-NMR (CDCl3):
d
(ppm) 1.38 (t, J ¼ 7.2 Hz, 3 H), 2.33 (s,
3 H), 3.84 (m, 8 H), 4.38 (q, J ¼ 7.2 Hz, 2 H), 7.42 (m, 5 H); HRMS-ESI:
d
m/z calcd 382.11958 [M þ Na]þ, found 382.11997; IR (KBr): 1109,
25.8, 118.5, 125.3, 130.4, 161.3, 163.9, 164.9; LRMS-EI: m/z calcd
1171, 1221, 1306, 1349, 1377, 1422, 1598, 1706, 2859, 2976 cmꢁ1
.
266.1, found 265.9; IR (KBr): 1028, 1233, 1387, 1489, 1540, 1654,
2856, 2930, 3432 cmꢁ1
.
4.1.10.4. Ethyl 2-(cyanodiazenyl)-4-methyl-5-phenylthiophene-3-
carboxylate 20c. Sodium cyanide (0.94 g, 19.2 mmol) in water
(5 ml) was used as nucleophile; yield 87%, mp 60-62 ꢀC; %; 1H-
4.1.13. Synthesis of 3-Ethylthieno[2,3-d]pyrimidine-2,4(1H,3H)-
diones 5a,b
4.1.13.1. General procedure. Ethyl 2-(3-ethylureido)-thiophene-3-
carboxylates 15e,h (0.30 mmol) and NaOH (4 ml, 10 M in ethanol)
were refluxed for 1.5 h and acidified with HCl (1 M). The precipitate
was filtered off and recrystallized from ethanol.
NMR (CDCl3):
d
(ppm) 1.42 (t, J ¼ 7.2 Hz, 3 H), 2.41 (s, 3 H), 4.48 (q,
J ¼ 7.2 Hz, 2 H), 7.51 (m, 5 H); 13C-NMR (CDCl3):
d (ppm) 14.1, 14.4,
62.8, 116.4, 129.3, 129.4, 130.6, 132.2, 137.1, 145.9, 153.3, 155.0,
163.3; HRMS-ESI: m/z calcd 322.06207 [M þ Na]þ, found
322.06207; IR (KBr): 1069, 1221, 1278, 1328, 1380, 1447, 1654,
1718, 2176, 2988 cmꢁ1
.
4.1.13.2. 3-Ethyl-5-methyl-6-phenylthieno[2,3-d]pyrimidine-
2,4(1H,3H)-dione 5a. Originated from 15e; yield 74%; mp 280-
283 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.23 (t, J ¼ 6.9 Hz, 3 H), 2.44 (s,
4.1.10.5. Ethyl 2-chloro-4-methyl-5-phenylthiophene-3-carboxylate
3 H), 3.89 (q, J ¼ 6.9 Hz, 2 H), 7.43 (m, 5 H), 12.22 (s, 1 H); LRMS-EI:
m/z calcd 286.1, found 286.0; IR (KBr): 1041, 1204, 1525, 1567, 1601,
21. CuCl-solution (0.80 g, 8.08 mmol) in conc. HCl (20 ml) was used
as nucleophile; yield 92%; 1H-NMR (DMSO-d6):
d (ppm) 1.30 (t,
1644, 1706, 2868, 2976 cmꢁ1
.
J ¼ 7.2 Hz, 3 H), 2.39 (s, 3 H), 4.27 (q, J ¼ 7.2 Hz, 2 H), 7.46 (m, 5 H);
LRMS-EI: m/z calcd 280.0, found 282.1; IR (CHCl3): 1065, 1222, 1297,
1306, 1378, 1450, 1600, 1716, 2871, 2958 cmꢁ1
.
4.1.13.3. 3-Ethyl-5,6,7,8-tetrahydrobenzo[b]-thieno[2,3-d]pyrimi-
dine-2,4(1H,3H)-dione 5b. Originated from 15h; yield 72%; mp 260-
364 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.09 (t, J ¼ 7.2 Hz, 3 H), 1.73
4.1.11. Synthesis of 2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-
4(1H)-ones 3a,b
(m, 4 H), 2.61 (m, 2 H), 2.76 (m, 2 H), 3.84 (q, J ¼ 7.2 Hz, 2 H), 12.02
(s, 1 H); LRMS-EI: m/z calcd 250.1, found 250.0; IR (KBr): 1028, 1060,
4.1.11.1. General procedure. To a mixture of 2-aminothiophene-3-
carboxamide 1a,b (3.80 mmol), water (0.50 ml) and K2CO3 (0.97 g,
7 mmol) in CH2Cl2 (5 ml) was added thiophosgene (0.80 ml,
10.4 mmol) in CH2Cl2 (5 ml) at 0 ꢀC. After 1.5 h the precipitate was
filtered off, washed with cold CH2Cl2 and dried.
1207, 1532, 1579, 1634, 1701, 2856, 2981 cmꢁ1
.
4.2. Biology
The kainate-receptor assay was performed by C. Kronbach,
Biotie Therapies GmbH, Radebeul [10].
Human embryonic kidney cells (HEK 293-cells) which stably
express the GluR5 or GluR6 receptor, respectively, together with the
luminescent protein aequorin were used as screening assay for kainate
receptor antagonists. GluR5- (or GluR6-) and aequorin-expressing HEK
293-cells were cultivated in a MEM growing medium together with
Earle’s salts and Glutamax-I (Life Technologies), containing 10% FKS,1%
4.1.11.2. 5-Methyl-6-phenyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyr-
imidin-4(1H)-one 3a. Originated from 1a; yield 93%; mp 296-
300 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 2.44 (s, 3 H), 7.39 (m, 5 H),
12.38 (s, 1 H), 13.47 (s, 1 H); 13C-NMR (DMSO-d6):
d
(ppm) 14.7,
118.6, 128.9, 129.7, 129.8, 130.1, 132.8, 151.7, 158.2, 173.8; LRMS-EI:
m/z calcd 274.0, found 274.0.
non-essential amino acids, 100 IU/ml penicillin, 100
mg/ml strepto-
mycin, 600 g/ml G 418 (Calbiochem) and 500 nM ouabaine.
One day before the measurement 60.000 cells per cavity were
seeded into white, non-transparent 96-well microtiterplates
(Costar). On the test day cells were incubated with 5
lenterazine for 1 h at 37 ꢀC. Then the medium was poured off and
m
4.1.11.3. 5,6,7,8-Tetrahydrobenzo[b]-2-thioxo-2,3-dihydrothieno[2,3-
d]pyrimidin-4(1H)-one 3b. Originated from 1b; yield 82%; mp 287-
290 ꢀC; 1H-NMR (DMSO-d6):
d
(ppm) 1.73 (m, 4 H), 2.64 (m, 2 H),
mM coe-
2.74 (m, 2 H), 12.27 (s, 1 H), 13.30 (s, 1 H); 13C-NMR (DMSO-d6):

D. Briel et al. / European Journal of Medicinal Chemistry 45 (2010) 69–77
77
replaced by 80
ml assay buffer and 10
ml test compound was added.
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This studies were financially supported by ‘‘European Fund for
Regional Development 2000-2006’’, sector technology support and
by Freistaat Sachsen project number SAB8093.