Thieno[2,3-d]pyrimidines and -[1,3]oxazines as glutamate antagonists and investigations on the inhibitory potency toward human leukocyte elastase

Article abstract of DOI:10.1002/jhet.375

(Chemical Equation Presented) A series of fused thiophene derivatives, that is, representatives of thieno[2,3-d]pyrimidines, thieno[2,3-d][1,3]oxazines and thieno[2,3-d][1,3]thiazines, with the common 5-methyl-6-phenyl substitution pattern was synthesized. The target compounds, e.g., 7 or 8, were designed as cyclic analogs of ethyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate, an antagonist at the GluR6 kainate receptor. Thieno[2,3-d][1,3]oxazin-4-one 2 (R = C2H5) was identified as new a potent inhibitor (IC50 = 17 lM) of this receptor subtype. The inhibitory potency of 2 (R = C2H5) against human leukocyte elastase was also examined. The compound was characterized as a noncovalent inhibitor with an IC₅₀ value of 8.8 μM.

Full text of DOI:10.1002/jhet.375

634
Thieno[2,3-d]pyrimidines and -[1,3]oxazines as Glutamate
Antagonists and Investigations on the Inhibitory Potency toward
Human Leukocyte Elastase
Vol 47
Detlef Briel,a Anastasiya Rybak,a Christiane Kronbach,b Klaus Unverferth,b
c
Camino M. Gonzalez Tanarro, and Michael Gu¨tschow *
c
´
aPharmaceutical Chemistry, Institute of Pharmacy, University of Leipzig, Bru¨derstraße 34,
D-04103 Leipzig, Germany
bBiotie Therapies GmbH, Meißner Straße 35, D-01445 Radebeul, Germany
cPharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4,
D-53121 Bonn, Germany
*E-mail: guetschow@uni-bonn.de
Received September 14, 2009
DOI 10.1002/jhet.375
Published online 3 May 2010 in Wiley InterScience (www.interscience.wiley.com).
A series of fused thiophene derivatives, that is, representatives of thieno[2,3-d]pyrimidines,
thieno[2,3-d][1,3]oxazines and thieno[2,3-d][1,3]thiazines, with the common 5-methyl-6-phenyl substitu-
tion pattern was synthesized. The target compounds, e.g., 7 or 8, were designed as cyclic analogs
of ethyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate, an antagonist at the GluR6 kainate
receptor. Thieno[2,3-d][1,3]oxazin-4-one 2 (R ¼ C2H5) was identified as new a potent inhibitor (IC50
¼
17 lM) of this receptor subtype. The inhibitory potency of 2 (R ¼ C2H5) against human leukocyte
elastase was also examined. The compound was characterized as a noncovalent inhibitor with an IC50
value of 8.8 lM.
J. Heterocyclic Chem., 47, 634 (2010).
INTRODUCTION
Substituted alkyl thiophene-3-carboxylates have been
identified as a new class of selective GluR6 antagonists
[11]. As a result of a screening of several thiophene
esters, 2-amino-4-methyl-5-phenylthiophene derivatives
proved to be particularly active. The ethyl ester 1a (IC50
¼ 0.75 lM) exceeded other esters, e.g., methyl, propyl,
with respect to selectivity for the GluR6 kainate receptor
[11]. On the basis of these findings, we envisaged cyclic
analogs of 1a and focused on the synthesis of thienopyri-
midines (Scheme 1). Selected compounds were also eval-
uated as inhibitors of human leukocyte elastase (HLE), a
serine protease of the chymotrypsin family. Under normal
conditions, the activity of HLE is regulated by endoge-
nous inhibitors, but uncontrolled activity of HLE may
result in several pathological states, including emphy-
sema, chronic obstructive pulmonary disease, cystic fibro-
sis and rheumatoid arthritis. HLE inhibitors are therefore
of relevance for the therapy of such afflictions [12–15].
Even well-established anticonvulsants, such as carba-
mazepine, valproic acid, phenytoin, or benzodiazepines
can cause undesired side effects. Moreover, certain
forms of epilepsy, that is, focal seizures, cannot be
treated sufficiently with such drugs. This provided the
impetus behind the development of new drugs to
improve the prospects for mono- and combined therapy.
In the course of the continuing search for new anticon-
vulsants, substituted quinazolines and the bioisosteric
thieno[2,3-d]pyrimidines have been reported to be active
[1,2]. Kainate glutamate receptors may represent an
interesting new target for the development of innovative
anticonvulsants [3,4]. The kainate receptor subtype
GluR6, expressed in the excitatory pyramid cells of the
hippocampus, appears to be particularly significant. The
GluR6 and GlyR5 subtypes might play opposing roles
during the hippocampal excitation [3,5].
The know antagonists of the GluR6 kainate receptor
comprise two groups, compounds which contain glutamate
or its isosterically modified fragments on the one hand, and
compounds with a structure not related to glutamate on the
other hand. Diarylureas and quinoxalinediones are predom-
inant examples of the latter group [6–10].
RESULTS AND DISCUSSION
The synthetic routes to bicyclic thiophenes with the com-
mon 5-methyl-6-phenyl substitution pattern are outlined in
Scheme 1. Using known methods, the fused pyrimidine ring
was formed in the reaction of the thiophene-3-carboxamide
C
V
2010 HeteroCorporation

May 2010
Thieno[2,3-d]pyrimidines and -[1,3]oxazines as Glutamate Antagonists and
Investigations on the Inhibitory Potency toward Human Leukocyte Elastase
635
Scheme 1. Conditions: i) HC(OC2H5)3, 12 h, reflux, or (RCO)2O, 1.5 h, reflux; ii) 1. HC(OC2H5)3, 12 h, reflux, 2. POCl3, 5 h, reflux; iii)
NH2CH2CH3 (70% aqueous solution); iv) P4S10 / toluene, 3 h, reflux; v) NaOCH2CH3 / CH2CH3OH, 12 h, or NH2CH2CH3 (70% aqueous solution)
/ CH2Cl2; vi) SOCl2, CHCl3, 30 min, reflux; vii) NH2CH2CH3 (70% aqueous solution).
1b [16] with ethyl orthoformate. Subsequent treatment with
phosphorous oxychloride afforded the 4-chlorothienopyri-
midine 3. The conversion of 3 with sodium ethoxide gave
6a, in which the carboxylic ester moiety of 1a is replaced
by a semicyclic ethyl imidate substructure. The correspond-
ing replacement by an ethyl amidine substructure in 6b was
accomplished when 3 was treated with ethylamine. The
reactions of the thiophene-3-carboxylic acid 1c [17] with
carboxylic anhydrides or carboxylic acid ortho esters pro-
vided an access to thieno[2,3-d][1,3]oxazin-4-ones 2. Oxazi-
nones 2 bear two electrophilic sites, C-2 and C-4. Ethyl-
amine exclusively attacked 2 at the C-2 carbon, leading to
the formation of amidino carboxylic acids 4. Treatment of 4
with thionyl chloride furnished compounds 7a, b with N-
ethyl lactam structure. The thieno[2,3-d][1,3]thiazine-4-thi-
one 5, accessible by thionation of the corresponding oxazi-
none 2b with diphosphorous pentasulfide [17], was reacted
with ethylamine to give the thienopyrimidine 8 with N-ethyl
thiolactam structure. It should be noted that 8 was directly
produced and a corresponding ring-open thiophene deriva-
tive could not be isolated. Such a compound with amidine
and dithiocarboxylate moieties, formed through an attack of
ethylamine at C-2 of 5, might remain in solution or undergo
different transformations, thus accounting for the low yield
of 8. In the mass spectra of the thienopyrimidines 7 and 8,
the molecular peaks appear with highest intensities. Elimi-
nation of C2H4 was the main fragmentation reaction leading
to the detection of fragment ions with the anticipated struc-
ture 9. Similarly, the loss the ethyl chain was the predomi-
nant fragmentation of 6.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

´
D. Briel, A. Rybak, C. Kronbach, K. Unverferth, C. M. Gonzalez Tanarro, and M. Gu¨tschow
636
Vol 47
Table 1
(IC50 ¼ 8.8 lM, Fig. 2), whereas 2a was not sufficiently
soluble in the assay medium, and 2b failed to inhibit
HLE (IC50 > 40 lM). It can be suspected, that the ethyl
group in 2c interacts with the S1 pocket of HLE, thus
reflecting the primary substrate specificity for small ali-
phatic amino acids at P1 position of a substrate.
Inhibition (% of Control) of fractional luminescence or IC50 value.
Compd.
GluR5
GluR6
2c
6a
6b
7a
7b
8
11
9
IC50 ¼ 17 lM
14
3
ꢁ7
ꢁ6
7
To elucidate the mechanism of elastase inhibition by
2c, we examined a possible enzyme-catalyzed degrada-
tion of the inhibitor by means of HPLC. Compound 2c
was incubated at 25ꢀC, pH 7.8, with HLE in a 200-fold
higher concentration compared to that used in the inhibi-
tion assays. Despite the inhibition of HLE by 2c, an
accelerated hydrolysis in the presence of such a high
amount of enzyme could be expected [24]. However,
the decrease in concentration of 2c was weak (<20%
within 4 h) and similarly observed in the control experi-
ment, where 2c was incubated in the absence of HLE. It
can therefore be concluded that 2c does not act as an
alternate substrate inhibitor of HLE, but most probably
as a competitive noncovalent inhibitor. This behavior
differs from that of thieno[1,3]oxazin-4-ones with
alkoxy, alkylthio, or (di)alkylamino substituents at 2-
position. Such compounds have been characterized [22]
to interact with HLE in the way outlined in Figure 1.
9
5
ꢁ11
0
The thienopyrimidines 6a,b, 7a,b, 8, and the oxazinone
2c were evaluated as antagonists of the kainate receptor
subtypes GluR5 and GluR6 (Table 1). The experiments
were performed with human embryonic kidney (HEK)
cells stably expressing the GluR5 and GluR6 receptor.
The antagonistic activity was determined by measuring
the glutamate-mediated luminescence. The photoprotein
aequorin was used as a bioluminescent reporter. The com-
plete luminescence of the cells was determined with the
detergent triton X-100 [11]. While the thienopyrimidines
6a,b, 7a,b, and 8, at a concentration of 10 lM, did barely
influence the luminescence signal in the GluR5 and
GluR6 assays, 2-ethylthieno[2,3-d][1,3]oxazin-4-one (2c)
inhibited the glutamate response in the GluR6 aequorin
assay with an IC50 value of 17 lM. Thus, this compound
will serve as a lead for further chemical modification to
develop new anticonvulsants.
EXPERIMENTAL
Representatives of 3,1-benzoxazin-4-ones have been
reported as alternate substrate inhibitors of HLE [18–
20]. It has been shown that the introduction of small
alkyl groups connected via an O, S, or N atom to the
position 2 of the heterocyclic system resulted in potent
inhibition [18]. Bioisosteric thieno[1,3]oxazin-4-ones
react in an analogous manner as alternate substrate
inhibitors with serine proteases and esterases [21–24].
The interaction involves the nucleophilic attack of the
active site serine (SerOH), formation of an acyl-enzyme
and hydrolytic cleavage to release the modified ring-
opened inhibitor and the free enzyme (Fig. 1).
Melting points were obtained on a Bu¨chi melting point ap-
paratus 535 and are uncorrected. Mass spectra (EI, 70 eV)
were measured on a VG Analytics VG ZAB-HSQ spectrome-
ter. ESI-HRMS spectra were recorded on a Bruker Daltonics
We have selected the thieno[2,3-d][1,3]oxazin-4-ones
2a–c and determined their inhibitory activity against
HLE. Compound 2c was identified as an HLE inhibitor
Figure 2. Inhibition of HLE by 2c in the presence of 100 lM of the
chromogenic substrate MeO-Suc-Ala-Ala-Pro-Val-pNA. The data are
mean values of duplicate measurements. The reactions were followed
over 10 min, and the rates, v, were determined by linear regression.
The rates in absence of inhibitor, v0, were set to 100%. Nonlinear
regression according to the equation v ¼ v0/([I]/(IC50 þ 1) gave a
value IC50 ¼ 8.8 6 0.1 lM.
Figure 1. Enzyme-catalyzed conversion of thieno[2,3-d][1,3]oxazin-4-ones.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

May 2010
Thieno[2,3-d]pyrimidines and -[1,3]oxazines as Glutamate Antagonists and
Investigations on the Inhibitory Potency toward Human Leukocyte Elastase
637
1
7T Apex II FT-ICR mass spectrometer. H NMR spectra were
recorded on a Varian Gemini-300 spectrometer at 300.08
MHz. 13C NMR spectra were recorded on a Varian Gemini-
300 spectrometer at 75.45 MHz. IR spectra were recorded on a
Perkin–Elmer FT-IR PC 16 instrument. 5-Methyl-6-phenyl-
thieno[2,3-d][1,3]oxazin-4-one (2a) [17], 2,5-dimethyl-6-phe-
nylthieno[2,3-d][1,3]oxazin-4-one (2b) [17], and 4-chloro-5-
methyl-6-phenylthieno[2,3-d]pyrimidine (3) [16] were prepared
as reported.
224 (12), 215 (41), 203 (13), 184 (9), 171 (31), 127 (10), 121
1
(17), 115 (27), 89 (9), 77 (14); H NMR (deuteriochloroform):
d 2.61 (s, 3H, CH3), 2.78 (s, 3H, CH3), 7.48–7.51 (m 5H, phe-
nyl-H); 13C NMR (deuteriochloroform): d 19.0 (5-CH3), 25.5
(2-CH3), 127.1 (C-4a), 128.7 (C-40), 128.9 (C-20, C-60), 130.1
(C-30, C-50), 130.8 (C-6), 132.1 (C-10), 132.8 (C-5), 136.8 (C-
7a), 170.1 (C-2), 201.6 (C¼¼S); ir (potassium bromide): m
1545, 1262, 1052 cmꢁ1
.
4-Ethoxy-5-methyl-6-phenylthieno[2,3-d]pyrimidine (6a). Com-
pound 3 [16] (0.5 g, 1.9 mmol) was refluxed in 10 mL of a so-
lution of sodium ethoxide in ethanol (1 mol/L) for 12 h. The
solvent was evaporated, the precipitate was isolated, washed
with water and recrystallized from ethanol. Yield 0.4 g (78%).
Colorless crystals, mp 103ꢀC (C2H5OH); ms: m/z (%) 270
(Mþꢂ, 100), 255 (36), 242 (78); 1H NMR (DMSO-d6): d 1.40
(t, 3H, CH3, J ¼ 7.2 Hz), 2.50 (s, 3H, CH3), 4.53 (q, 2H,
CH2, J ¼ 7.2 Hz), 7.44–7.52 (m, 5H, phenyl-H), 8.60 (s, 1H,
CH); 13C NMR (deuteriochloroform): d 14.9 (CH3), 15.4
(CH3), 63.3 (OCH2), 120.2 (Cq), 126.3 (Cq), 129.3 (CH, Ph),
129.7 (2 ꢃ CH, Ph), 130.2 (2 ꢃ CH, Ph), 133.4 (Cq), 135.4
(Cq), 153.7 (CH), 164.7 (Cq), 167.1 (Cq); ESI-HRMS: m/z
271.09014 ([MþH]þ)ꢂ (C15H15N2OSþ requires 271.08996);
2-Ethyl-5-methyl-6-phenylthieno[2,3-d][1,3]oxazin-4-one
(2c). A mixture of compound 1c [11] (2 g, 8.58 mmol) and
propionic anhydride (10 mL, 78 mmol) was refluxed for 90
min. The precipitate, formed after cooling, was filtered off,
dried, and recrystallized from ethanol. Yield 1.2 g (34þ%ꢂ ). Col-
orless crystals, mp 99–100ꢀC; ms: m/z (%) 271 (M , 100),
1
242 (40); H NMR (deuteriochloroform): d 1.33 (t, 3H, CH3, J
¼ 7.5 Hz), 2.56 (s, 3H, CH3), 2.72 (q, 2H, CH2, J ¼ 7.5 Hz),
7.39–7.46 (m, 5H, phenyl-H); ir (potassium bromide): m 1744
(C¼¼O), 1596, 1158–1075 cmꢁ1. Anal. Calcd. for C15H13NO2S
ꢃ 0.5 H2O: C, 64.26; H, 5.03; N, 5.00; S, 11.44. Found C,
64.03; H, 5.16; N, 5.37; S, 11.48.
2-(Ethylaminomethylenamino)-4-methyl-5-phenylthiophene-
3-carboxylic acid (4a). Ethylamine (10 mL of a 70% aqueous
solution, 0.126 mol) was added dropwise to compound 2a [17]
(1.4 g, 5.7 mmol). The mixture was kept at 0ꢀC until the crys-
tallization was finished. The precipitate was separated and
dried to obtain a crude product which was not further purified.
Yield 1.1 g (70%). Beige solid, mp 180–183ꢀC; ms: m/z (%)
288 (Mþꢂ, 97), 215 (100); ESI-HRMS: m/z 577.19433
([2MþH]þ)ꢂ (C30H33N4O4S2þ requires 577.19377); 289.10042
([MþH]þ)ꢂ (C15H17N2O2Sþ requires 289.10052); 599.17642
563.15462
([2MþNa]þ)ꢂ
(C30H28N4NaO2Sþ2
requires
563.15459); 293.07211 ([MþNa]þ)ꢂ (C15H14N4NaOSþꢂ
requires 293.07190); ir (potassium bromide): m 3446 (br, traces
from water), 3064–2978, 1555–1450, 1332, 1063, 1044 cmꢁ1
.
Anal. Calcd. for C15H14N2OS ꢃ H2O: C, 62.47; H, 5.59; N,
9.71; Found C, 62.69; H, 4.96; N, 9.67.
4-Ethylamino-5-methyl-6-phenylthieno[2,3-d]pyrimidine
(6b). Compound 3 [16] (0.3 g, 1.15 mmol) was dissolved in
dichloromethane (3 mL) and treated with ethylamine (100 mL
of a 70% aqueous solution, 1.26 mol). If no crystallization
occurred, water was added to the mixture. The precipitate was
filtered off, dried, and recrystallized from ethanol. Yield 0.2 g
(65%). Yellow crystals, mp 155–158ꢀC (C2H5OH); ms: m/z
(%) 269 (Mþꢂ, 100), 254 (27), 240 (30); 1H NMR (deuterio-
chloroform): d 1.35 (t, 3H, CH3, J ¼ 7.2 Hz), 2.63 (s, 3H,
CH3), 3.66–3.75 (m, 2H, CH2, J ¼ 7.2 Hz), 5.57 (s, br, NH,
exchangeable with D2O), 7.41–7,50 (m, 5H, phenyl-H), 8.49
(s, 1H, CH); ir (potassium bromide): m 3419 (NH), 2988–2863,
1574–1460, 1128–1012 cmꢁ1. Anal. Calcd. for C15H15N3S ꢃ
0.5 C2H5OH: C, 65.72; H, 6.20; N, 14.37; S, 10.96. Found C,
65.30; H, 5.76; N, 14.75; S, 11.23.
([2MþNa]þ)ꢂ
(C30H32N4NaO4Sþ2
([MþNa]þ)ꢂ
requires
(C15H16N2NaO2Sþ
599.17572);
requires
311.08269
1
311.08247); H NMR (DMSO-d6): d 1.16 (t, 3H, CH3, J ¼ 7.2
Hz), 2.36 (s, 3H, CH3), 3.29 (q, 2H, CH2, J ¼ 7.0 Hz), 7.38–
7.50 (m, 5H, phenyl-H), 8.14 (s, 1H, CH), 8.34 (s, br, 0.5H,
NH, exchangeable with D2O), 14.30 (s, br, 0.5H, OH,
exchangeable with D2O); ir (potassium bromide): m 3221
(NAH), 3069–2873, 1694 (C¼¼O), 1632, 1577 cmꢁ1
.
2-(1-Ethylaminopropane-1-ylidenamino)-4-methyl-5-phe-
nylthiophene-3-carboxylic acid (4b). Compound 4b was pre-
pared from 2c (1.2 g, 4.4 mmol) following the aforementioned
procedure. Yield 1.2 g (89%). Beige solid, mp 152–155ꢀC
(crude product); ms: m/z (%) 316 (Mþꢂ, 18), 242 (100); 1H
NMR (DMSO-d6): d 1.20 (t, 6H, 2 CH3, J ¼ 7.5 Hz), 2.41 (s,
3H, CH3), 2.7 (q, 2H, CH2, J ¼ 7.8 Hz), 3.27 (q, 2H, CH2, J
¼ 7.5 Hz), 7.40–7.54 (m, 5H, phenyl-H), 8.42 (s, br, 1H, NH,
exchangeable with D2O); ir (potassium bromide): m 3237
(NAH), 2978–2937, 1684 (C¼¼O), 1592, 1490, 1238, 1073
cmꢁ1. Anal. Calcd. for C17H20N2O2S: C, 64.53; H, 6.37; N,
8.85; S, 10.13. Found C, 64.22; H, 6.34; N, 8.79; S, 10.07.
2,5-Dimethyl-6-phenylthieno[2,3-d][1,3]thiazin-4-thione
(5) [17]. Compound 2b [17] (2.57 g, 10 mmol) was dissolved
in 80 mL of dry toluene. After addition of diphosphorous pen-
tasulfide (22.2 g, 100 mmol), the mixture was refluxed for 2 h.
The formed inorganic precipitate was filtered off and washed
several times with hot toluene. The combined toluene solutions
were evaporated and the precipitated crude product was sepa-
rated and recrystallized from ethanol. Yield 1.16 g (40þ%ꢂ ). Red
crystals, mp 135ꢀC (C2H5OH); ms: m/z (%) 291 ([M þ 2],
26), 289 (Mþꢂ, 100), 274 (10), 256 (12), 247 (15), 230 (11),
3-Ethyl-5-methyl-6-phenylthieno[2,3-d]pyrimidin-4-one
(7a). Compound 4a (0.3 g, 1 mmol) was dissolved in anhy-
drous chloroform (10 mL). After addition of thionyl chloride
(1.19 g, 10 mmol), the mixture was refluxed for 30 min. The
solvent was evaporated at room temperature, the precipitate
formed was filtered off, and dried. The crude product was
purified by column chromatography on silica gel (63–200 lm,
Merck) with ethanol. Yield 0.087 g þ(ꢂ30%). Red solid, mp
90ꢀC (C2H5OH); ms: m/z (%) 270 (M , 100), 242 (42); ESI-
HRMS: m/z 541.17313 ([2MþH]þ)ꢂ (C30H29N4O2S2þ requires
541.17264); 271.08999 ([MþH]þ)ꢂ (C15H15N2OSþ requires
271.08996); 563.15500 ([2MþNa]þ)ꢂ (C30H28N4NaO2Sþ2
requires 563.15459); 293.07208 ([MþNa]þ)ꢂ (C15H14N4NaOSþ
1
requires 293.07190); H NMR (deuteriochloroform): d 1.43 (t,
3H, CH3, J ¼ 7.2 Hz), 2.64 (s, 3H, CH3), 4.07 (q, 2H, CH2, J
¼ 7.3 Hz), 7.39–7.47 (m, 5H, phenyl-H), 7.97 (s, 1H, CH);
13C NMR (deuteriochloroform): d 14.9 (CH3), 15.4 (CH3),
42.1 (NCH2), 124.4 (Cq), 128.3 (CH, Ph), 128.9 (2 ꢃ CH, Ph),
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Vol 47
129.9 (2 ꢃ CH, Ph), 130.3 (Cq), 133.6 (Cq), 135.5 (Cq), 146.0
lL triton X-100 in assay buffer (without Ca2þ) was carried out
after 20 s into the same cavity.
The calculation of specific channel activity induced by an
agonist was determined as fractional luminescence, which was
calculated from respective sum of signals of agonist- and tri-
ton-induced luminescence. For determination of the IC50 value,
a Hill plot (4-parameter model) was used.
(CH), 158.5 (Cq), 163.0 (Cq); ir (potassium bromide): m 1666,
1658 (C¼¼O), 1600, 1576, 1234–1083 cmꢁ1. Anal. Calcd. for
C15H14N2OS: C, 66.64; H, 5.22; N, 10.36. Found: C, 66.32; H,
5.40; N, 10.13.
2,3-Diethyl-5-methyl-6-phenylthieno[2,3-d]pyrimidin-4-one
(7b). Thionyl chloride (2.38 g, 20 mmol) was added to a solu-
tion of 4b (1.2 g, 3.8 mmol) in anhydrous chloroform (30
mL). The mixture was refluxed for 30 min. The solvent was
evaporated at room temperature. The precipitate was filtered
off and dried. The crude product was thoroughly washed with
acetone and recrystallized from acetonitrile. Yield 0.5 g
(40%). Beige solid, mp 175–179ꢀC (CH3CN); ms: m/z (%)
298 (Mþꢂ, 100), 270 (69), 215 (66); 1H NMR (DMSO-d6): d
1.24–1.32 (m, 6H, 2 CH3, J ¼ 7.2 Hz, J ¼ 6.9 Hz), 2.53 (s,
3H, CH3), 2.93 (q, 2H, CH2, J ¼ 7.2 Hz), 4.10 (q, 2H, CH2, J
¼ 6.9 Hz), 7.44–7.53 (m, 5H, phenyl-H); ir (potassium bro-
mide): m 3434 (br, traces of water), 1670 (C¼¼O), 1596, 1543
cmꢁ1. Anal. Calcd. for C17H18N2OS: C, 68.43; H, 6.08; N,
9.39, S 10.74. Found C, 68.16; H, 5.83; N, 8.92; S, 10.27.
3-Ethyl-2,5-dimethyl-6-phenylthieno[2,3-d]pyrimidine-4-
thione (8). Ethylamine (3 mL of a 70% aqueous solution,
0.04 mol) was added dropwise to 5 (0.4 g, 1.38 mmol). The
mixture was kept at 0ꢀC until the crystallization was finished.
The precipitate was separated and dried. Yield 0.1 g (24%).
Light yellow crystals, mp 89–91ꢀC (CH3CN); ms: m/z (%) 302
HLE inhibition assay. The spectrophotometric assay for
HLE was done on a Varian Cary 50 Bio UV/VIS spectrometer
with a cell holder equipped with a constant temperature water
bath. HLE was available from a previous study [24]. Reactions
were followed at 405 nm at 25ꢀC for 10 min. Stock solutions
of the inhibitors were prepared in DMSO. IC50 values were
calculated from the linear steady-state turnover of the sub-
strate. Assay buffer was 50 mM sodium phosphate buffer, 500
mM NaCl, pH 7.8. An enzyme stock solution of 50 lg/mL
was prepared in 100 mM sodium acetate buffer, pH 5.5 and
diluted with assay buffer. A 50 mM stock solution of the chro-
mogenic substrate MeOSuc-Ala-Ala-Pro-Val-pNA (Bachem,
Bubendorf, Switzerland) was prepared in DMSO and diluted
with assay buffer containing 10% DMSO. The final concentra-
tion of the substrate was 100 lM, of DMSO was 1.5% and of
HLE was 25 ng/mL. Into a cuvette containing 890 lL assay
buffer, 10 lL of an inhibitor solution and 50 lL of a substrate
solution were added and thoroughly mixed. The reaction was
initiated by adding 50 lL of the HLE solution (500 ng/mL).
HLE incubation experiment. Compound 2c, dissolved in
acetonitrile, and a solution of HLE were added to assay buffer
and incubated for 4 h at 25ꢀC in a quartz cuvette. The final
concentration of 2c was 20 lM, of acetonitrile was 2% and of
HLE was 5 lg/mL. In the control experiment, 2c was incu-
bated in assay buffer without HLE. In 60-min intervals, 10 lL
aliquots were injected into the HPLC system (Dionex P580,
Phenomenex Gemini 5 l, C18, mobile phase A: H2O/acetoni-
trile/CF3CO2H (475:25:0.3), mobile phase B: H2O/acetonitrile/
tetrahydrofuran/CF3CO2H (25:465:10:0.3), gradient 0–25 min:
70–10% A, 30–90% B, 27–30 min: 70% A, 30% B, UV detec-
tion, 310 nm). The retention time of 2c was 17.33–17.35 min.
1
([Mþꢂ þ 2], 13), 300 (Mþꢂ, 100), 272 (51); H NMR (DMSO-
d6): d 1.35 (t, 3H, CH3, J ¼ 6.9 Hz), 2.74 (s, 3H, CH3), 2.81
(s, 3H, CH3), 4.79 (s, br, 2H, CH2), 7.52–7.59 (m, 5H, phenyl-
H), ir (potassium bromide): m 3440 (br, traces of water), 2974–
2926, 1557 (strong, C¼¼S), 1493, 1444, 1191, 1093 cmꢁ1
.
Anal. Calcd. for C16H16N2S2 ꢃ 0.5 H2O: C, 62.10; H, 5.54;
N, 9.05; S, 20.72. Found C, 62.40; H, 5.23; N, 9.11; S, 20.88.
Kainate receptor-aequorin assay. Human embryonic kid-
ney 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 re-
ceptor antagonists. GluR5- (or GluR6-) and aequorin-express-
ing HEK 293 cells were cultivated in a MEM growth medium
together with Earle’s salts and Glutamax-I (Life Technologies),
containing 10% FKS, 1% nonessential amino acids, 100 IU/
mL penicillin, 100 lg/mL streptomycin, 600 lg/mL G 418
(Calbiochem) and 500 nM ouabaine. One day before the mea-
surement, 60,000 cells per cavity were seeded into white, non-
transparent 96-well microtiter plates (Costar). On the test day,
cells were incubated with 5 lM coelenterazine for 1 h at
37ꢀC. Then the medium was poured off and replaced by 80
lL assay buffer, and 10 lL test compound was added. The
assay buffer contained 150 mM NaCl, 2.5 mM KCl, 10 mM
HEPES, 1 mM MgCl2, 10 mM glucose, 0.3 mg/mL concanava-
line A (ConA) and (for GluR6) 100 mM CaCl2 (pH ¼ 7.3).
Afterwards the cells were incubated for 10 min at room
temperature.
Acknowledgment. The work was supported by the European
Fund for Regional Development 2000–2006, sector technology
support, and by the Freistaat Sachsen, project number SAB8093.
C.M.G.T. and M.G. are grateful to the German Research Founda-
tion, Graduate College 677 for financial support.
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