Synthesis of novel dihydropyridothienopyrimidin-4,9-dione derivatives

Article abstract of DOI:10.3390/molecules20035074

A novel molecular scaffold, dihydropyridothienopyrimidin-4,9-dione, was synthesized from benzylamine or p-methoxybenzylamine in six steps involving successive ring closure to form a fused ring system composed of dihydropyridone, thiophene and pyrimidone. The pharmacological versatility of the dihydropyridothenopyrimidin-4,9-dione scaffold was demonstrated by inhibitory activity against metabotropic glutamate receptor subtype 1 (mGluR1), which shows that the title compounds can serve as an interesting scaffold for the discovery of potential bioactive molecules for the treatment of human diseases.

Full text of DOI:10.3390/molecules20035074

Molecules 2015, 20, 5074-5084; doi:10.3390/molecules20035074
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Novel Dihydropyridothienopyrimidin-4,9-dione
Derivatives
Youngjae Kim 1,2, Minjoo Kim 1,3, Mooseong Park 1,4, Jinsung Tae 4, Du-Jong Baek 3,
Ki Duk Park 1,4 and Hyunah Choo 1,4,
*
1
Center for Neuro-Medicine, Korea Institute of Science and Technology, Seongbuk-gu,
Seoul 136-791, Korea; E-Mails: 213501@kist.re.kr (Y.K.); yonlyk@naver.com (M.K.);
h14507@kist.re.kr (M.P.); kdpark@kist.re.kr (K.D.P.)
2
3
Department of Chemistry, Yonsei University, Seodaemun-gu, Seoul 120-749, Korea
Department of Chemistry, College of Natural Sciences, Sangmyung University,
Seoul 110-743, Korea; E-Mail: djbaek@smu.ac.kr
4
Department of Biological Chemistry, University of Science and Technology, Youseong-gu,
Daejeon 305-350, Korea; E-Mail: jstae@yonsei.ac.kr
* Author to whom correspondence should be addressed; E-Mail: hchoo@kist.re.kr;
Tel.: +82-2-958-5157.
Academic Editor: Derek J. McPhee
Received: 27 January 2015 / Accepted: 13 March 2015 / Published: 19 March 2015
Abstract: A novel molecular scaffold, dihydropyridothienopyrimidin-4,9-dione, was
synthesized from benzylamine or p-methoxybenzylamine in six steps involving successive
ring closure to form a fused ring system composed of dihydropyridone, thiophene and
pyrimidone. The pharmacological versatility of the dihydropyridothenopyrimidin-4,9-dione
scaffold was demonstrated by inhibitory activity against metabotropic glutamate
receptor subtype 1 (mGluR1), which shows that the title compounds can serve as an
interesting scaffold for the discovery of potential bioactive molecules for the treatment of
human diseases.
Keywords: dihydropyridothienopyrimidin-4,9-dione; pyridothienopyrimidine; successive
ring formation

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1. Introduction
Much attention has been paid to the thienopyrimidine and thienopyridine scaffolds due to their notable
biological and pharmacological activities (Figure 1) [1–6]. Thienopyrimidines, like (R)-N-(3-chloro-4-
((3-fluorobenzyl)oxy)phenyl)-6-(pyrrolidin-2-ylethynyl)thieno[3,2-d]pyrimidin-4-amine
and
6-(1-benzyl-1H-indol-3-yl)-2-(piperidin-1-ylmethyl)thieno[3,2-d]pyrimidin-4(3H)-one, show potent
anticancer activity by inhibition of epidermal growth factor receptor (EGFR) [1] and vascular endothelial
growth factor receptor-2 (VEGFR-2) [2], respectively. On the other hand, thienopyridine derivatives
such as ticlopidine, clopidogrel, and prasugrel block P2Y12 receptors and thus inhibit platelet activation
and aggregation [3–6].
O
Cl
F
N
N
HN
N
BnN
NH
S
S
N
O
N
H
(R)-N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-
(pyrrolidin-2-ylethynyl)thieno[3,2-d]pyrimidin-4-amine
6-(1-Benzyl-1H-indol-3-yl)-2-(piperidin-1-
ylmethyl)thieno[3,2-d]pyrimidin-4(3H)-one
O
O
OCH3
N
N
N
S
O
Cl
S
CH3
S
F
Cl
O
Ticlopidine
Prasugrel
Clopidogrel
Figure 1. Bioactive thienopyrimidine and thienopyridine derivatives.
Accordingly, combining both the thienopyrimidine and thienopyridine moieties in the same
molecular framework (i.e., pyridothienopyrimidine 1, Figure 2) has served as an attractive strategy for
designing a novel scaffold with more favorable pharmacological properties [7]. In this study, as a part
of our ongoing efforts to synthesize fused tricyclic heterocycles, dihydropyridinothienopyrimidin-4,9-
dione derivatives 2 were prepared starting from benzylamine or p-methoxybenzylamine in six steps
involving successive ring formations. Also, to demonstrate the biological and pharmacological
versatility, the synthesized compounds were biologically evaluated against metabotropic glutamate
receptor subtype 1 (mGluR1) which is one of molecular targets for treatment of neuropathic pain.
1
1
O
3
N
N
R2
9
R1
N
9
N 3
8
N
N
8
4
4
O
S
S
1
2
Figure 2. Pyridothienopyrimidine (1) and dihydropyridothienopyrimidin-4,9-diones 2.
2. Results and Discussion
To synthesize the target compounds 2, the reaction sequence shown in Scheme 1 was used.
Benzylamines 3a and 3b were used as starting materials. Thus, benzylamine 3a or
p-methoxybenzylamine 3b were treated with ethyl acrylate in the presence of catalytic Cu(OAc)2 to give

Molecules 2015, 20
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the corresponding β-aminoester 4a or 4b (47% or 57% yield, respectively). The β-aminoesters 4a and
4b were coupled with cyanoacetic acid using N,Nʹ-dicyclohexylcarbodimide (DCC) and
1-hydroxybenzotriazole (HOBt) in methylene chloride to afford tertiary cyanoacetamides 5a and 5b in
46%~63% yields.
O
NH
CN
N
NH2
a
b
X
X
X
O
OEt
O
OEt
3a X = H
4a X = H
5a X = H
3b X = OMe
4b X = OMe
5b X = OMe
c
O
N
O
N
O
NH2
CN
OMe
CN
OH
d
e
N
CO2Me
S
X
X
X
8a X = H
7a X = H
6a X = H
8b X = OMe
7b X = OMe
6b X = OMe
f
O
N
N
O
N
Y
S
X
9a X = H, Y = H
9b X = H, Y = 4-Cl
9c X = H, Y = 4-OMe
9d X = H, Y = 3-OMe
9e X = OMe, Y = H
9f X = OMe, Y = 4-Cl
9g X = OMe, Y = 4-OMe
9h X = OMe, Y = 3-OMe
Reagents and conditions: (a) ethyl acrylate, Cu(OAc)2, H2O, rt; (b) cyanoacetic acid, DCC, HOBt, DCM, 0 °C
to rt; (c) Amberlyst A-26 resin, MeOH, rt; (d) Me2SO4, NaH, THF, rt to 40 °C; (e) HSCH2CO2Me, NaOMe,
MeOH, 65 °C; (f) anilines, CH(OEt)3, AcOH, 160 °C, sealed tube.
Scheme 1. Synthesis of N3-aryl-N8-benzyldihydropyridothienopyrimidin-4,9-diones 9a–9h.
To form the dihydropyridone ring, the cyanoacetamides 5a and 5b underwent modified Dieckmann
condensation under acidic conditions using Amberlyst A-26 resin as catalyst to give 6a and 6b in
55%~64% yields [8]. The hydroxy groups of the compounds 6a and 6b were methylated by dimethyl
sulfate and NaH (37%~63% yields). The resulting methylated compounds 7a and 7b were then subjected
to a series of heterocyclization reactions [9,10] to give the desired dihydropyridothienopyrimidin-4,9-
diones 9a~9h in 20%~67% combined yields.
The synthesized compounds 9a~9h were biologically evaluated against Chem-3 cells with stably
expressing mGluR1 which has been considered as a potential target for treating neuropathic pain [11],
and the results are summarized in Table 1. Percent inhibitions (%-inhibitions) for all the synthesized
compounds were measured at 1 μM. Overall, the dihydropyridothienopyrimidin-4,9-dione derivatives
showed low to moderate inhibitory activity compared with the reference compound (93.37% inhibition) [9]:

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the compounds 9a, 9d, and 9f showed moderate inhibitory activity against mGluR1 with 23.04%,
16.54%, and 18.47% inhibition, respectively, while others showed negligible inhibitory activities.
Table 1. Inhibitory activities of the synthesized dihydropyridothienopyrimidin-4,9-diones 9
against mGluR1.
O
N
N
N
Y
O
S
X
9
Entry Compound
X
H
Y
%Inhibition at 1 μM a
1
2
3
4
5
6
7
8
9
9a
9b
9c
9d
9e
9f
H
23.04%
−1.59%
3.15%
H
4-Cl
H
4-OMe
3-OMe
H
H
16.54%
8.29%
OMe
OMe
OMe
OMe
4-Cl
18.47%
10.35%
11.86%
93.37%
9g
9h
4-OMe
3-OMe
Reference compound b
a: %-Inhibition of compounds at 1 μM against mGluR1; b: the reference compound is shown below.
NH
N
N
N
OMe
S
O
3. Experimental Section
3.1. General Methods
All reactions were carried out under dry nitrogen or argon unless otherwise indicated. Commercially
available reagents were used without further purification. Solvents and gases were dried according to
standard procedures. Organic solvents were evaporated with reduced pressure using a rotary evaporator.
Analytical thin layer chromatography (TLC) was performed using glass plates precoated with silica gel
(0.25 mm). TLC plates were visualized by exposure to UV light (UV), and then were visualized with a
KMnO4 stain followed by brief heating on hot plate. Flash column chromatography was performed using
silica gel 60 (230–400 mesh, Merck, Kenilworth, NJ, USA) with the indicated solvents. The melting
points of solid products was measured by using capillary tubes for the determination of melting points
(Marienfeld-Superior, Lauda-Königshofen, Germany) and an automated melting point system
(OptiMelt, Stanford Research Systems, Inc. Sunnyvale, CA, USA). The tubes containing solid
compounds were inserted in OptiMelt, and the temperature was slowly raised from 100 °C to 400 °C.
1H- and 13C-NMR spectra were recorded on Bruker 300 or 400 spectrometers (Bruker Co. Billerica, MA,
1
USA). H-NMR spectra are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, td = triplet of doublet, brs = broad singlet,
1
brt = broad triplet), integration, and coupling constant (J) in Hertz (Hz). H-NMR chemical shifts are
13
reported relative to CDCl3 (7.26 ppm). C-NMR was recorded relative to the central line of CDCl3

Molecules 2015, 20
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(77.0 ppm). HRMS analyses were performed on Bruker compact ESI+ positive mode. HPLC
purifications were performed on an Alliance Waters 2489 UV/Visible detector, e2695 Separations
module using a Waters Xterra prep RP-18 10 μM, 10 × 250 mm column (Waters Inc. Milford, MA,
USA) and a 30%–100% acetonitrile in water solvent gradient.
Ethyl 3-(benzylamino)propanoate (4a). To a solution of benzylamine 3a (2.6 g, 24.0 mmol) in H2O
(100 mL) were added ethyl acrylate (2 g, 20.0 mmol), and Cu(OAc)2 (182 mg, 1.0 mmol). The resulting
mixture was stirred at room temperature for 6 h. After termination of the reaction, the solution was
extracted with EtOAc, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (n-hexane-EtOAc = 1:1) to afford the desired product 4a (2.34 g,
1
11.3 mmol, 47% yield) as a yellow oil: H-NMR (300 MHz, CDCl3) δ 7.36–7.23 (m, 5H), 4.14 (q,
J = 7.2 Hz, 2H), 3.81 (s, 2H), 2.90 (t, J = 6.3 Hz, 2H), 2.53 (t, J = 6.3 Hz, 2H), 1.66 (s, 1H), 1.25 (t,
J = 7.2 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 172.79, 140.17, 128.42, 128.09, 126.96, 60.41, 53.77,
44.51, 34.79, 14.23; LC/MS (ESI+): m/z: calcd for C12H17NO2: 207.27, [M+H]+; found: 208.00.
Ethyl 3-((4-methoxybenzyl)amino)propanoate (4b). Following the same procedure used for the synthesis
of 4a, the reaction of (4-methoxyphenyl)methanamine 3b (7.8 mL, 60.0 mmol), ethyl acrylate (5 g,
50.0 mmol), and Cu(OAc)2 (454 mg, 2.5 mmol) gave the title compound 4b (6.72 g, 28.3 mmol, 57%
1
yield) as a brown oil: H-NMR (400 MHz, CDCl3) δ 7.24–7.22 (m, 2H), 6.87–6.85 (m, 2H), 4.13 (q,
J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.74 (s, 2H), 2.88 (t, J = 6.4 Hz, 2H), 2.52 (t, J = 6.4 Hz, 2H), 1.63 (s, 1H),
1.25 (t, J = 7.2 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 172.80, 158.66, 132.30, 129.26, 113.81, 60.40,
55.26, 53.16, 44.41, 34.78, 14.22; LC/MS (ESI+): m/z: calcd for C13H19NO3: 237.30, [M+H]+;
found: 238.30.
Ethyl 3-(N-benzyl-2-cyanoacetamido)propanoate (5a). DCC (1.39 g, 6.75 mmol) were added to the a
solution of ethyl 3-(benzylamino)propanoate 4a (1 g, 4.82 mmol), cyanoacetic acid (492 mg,
5.79 mmol), and HOBt (782 mg, 5.79 mmol) in MC (30 mL) at 0 °C. The solution was stirred at room
temperature for 12 h. After termination of the reaction, the solution was filtered. The filtrate was washed
with NaHCO3, and brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (n-hexane- EtOAc = 1:2) to afford the desired product 5a (858 mg,
1
3.13 mmol, 65% yield) as a yellow oil: H NMR (300 MHz, CDCl3) δ 7.42–7.15 (m, 5H), 4.62 (d,
J = 2.7 Hz, 2H), 4.12 (q, J = 7.2 Hz, 2H), 3.87 (s, 1H), 3.70 (t, J = 6.6 Hz, 1H), 3.54 (t, J = 6.3 Hz, 1H),
3.47 (s, 1H), 2.66 (t, J = 6.6 Hz, 1H), 2.56 (t, J = 6.6 Hz, 1H), 1.28–1.22 (m, 3H); 13C-NMR (100MHz,
CDCl3) (isomers) δ 171.71, 171.07, 162.63, 162.57, 136.24, 135.39, 129.34, 128.86, 128.23, 127.95,
127.85, 126.08, 114.42, 113.92, 61.27, 60.81, 52.94, 48.35, 44.11, 43.00, 32.55, 32.51, 25.35, 25.23,
14.16, 14.10; LC/MS (ESI+): m/z: calcd for C15H18N2O3: 274.32, [M+H]+; found: 275.15.
Ethyl 3-(2-cyano-N-(4-methoxybenzyl)acetamido)propanoate (5b). Following the same procedure used
for the synthesis of 5a, the reaction of ethyl 3-((4-methoxybenzyl)amino)propanoate 4b (6.72 g,
28.3 mmol), cyanoacetic acid (2.89 g, 34.0 mmol), DCC (8.17 g, 39.6 mmol) and HOBt (4.59 g,
34.0 mmol) in MC (125 mL) gave the title compound 5b (3.99 g, 13.1 mmol, 46% yield) as a yellow oil:
1H-NMR (300 MHz, CDCl3) δ 7.13 (dd, J = 8.6, 26.1 Hz, 2H), 6.88 (dd, J = 8.6, 16.6 Hz, 2H), 4.55 (s,
2H), 4.12 (q, J = 7.1 Hz, 2H), 3.83–3.80 (m, 4H), 3.67 (t, J = 6.6 Hz, 1H), 3.51 (t, J = 6.4 Hz, 1H), 3.47

Molecules 2015, 20
5079
13
(s, 1H), 2.64 (t, J = 6.7 Hz, 1H), 2.56 (t, J = 6.4 Hz, 1H), 1.28–1.22 (m, 3H); C-NMR (100 MHz,
CDCl3) (isomers) δ 171.70, 171.07, 162.48, 162.41, 159.53, 159.29, 129.46, 128.26, 127.52, 127.13,
114.70, 114.43, 114.22, 113.99, 61.25, 60.79, 55.37, 55.29, 52.44, 47.70, 43.81, 42.69, 32.51, 25.36,
25.24, 14.15, 14.09; LC/MS (ESI+): m/z: calcd for C16H20N2O4: 304.35, [M+H]+; found: 305.15.
Benzyl-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile (6a). To a solution of ethyl
3-(N-benzyl-2-cyanoacetamido)propanoate 5a (850 mg, 3.10 mmol) in MeOH (20 mL) were added
Amberlyst A-26 resin (1.5 g). The resulting mixture was stirred at room temperature for 16 h. After
termination of the reaction, the resin was filtered and washed with MeOH. Then, the resin was added to
the solution of TFA (2 mL) in MeOH (20 mL), and stirred at room temperature for 30 min. The mixture
solution was filtered, and concentrated in vacuo to afford the desired product 6a (390 mg, 1.71 mmol,
55% yield) as a white solid: mp 225–227 °C; 1H-NMR (300 MHz, DMSO-d6) δ 7.37–7.24 (m, 5H), 4.51
13
(s, 2H), 3.30 (t, J = 7.2 Hz, 2H), 2.68 (t, J = 6.9 Hz, 2H); C-NMR (100 MHz, DMSO-d6) δ 181.05,
163.16, 137.98, 128.97, 128.05 127.60, 115.57, 83.49, 49.35, 42.82, 29.01; LC/MS (ESI+): m/z: calcd
for C13H12N2O2: 228.25, [M+H]+; found: 229.15.
4-Hydroxy-1-(4-methoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile (6b). Following the
same procedure used for the synthesis of 6a, the reaction of ethyl 3-(2-cyano-N-(4-
methoxybenzyl)acetamido)propanoate 5b (200 mg, 0.66 mmol), Amberlyst A-26 resin (354 mg), and
TFA (2 mL) in MeOH (10 × 2 mL) gave the title compound 6b (108.7 mg, 0.42 mmol, 64% yield) as a
white solid: mp 170–173 °C; 1H-NMR (400 MHz, DMSO-d6) δ 7.30–7.26 (m, 2H), 7.01–6.98 (m, 2H),
13
4.53 (s, 2H), 3.83 (s, 3H), 3.36 (t, J = 7.1 Hz, 2H), 2.74 (t, J = 7.0 Hz, 2H); C-NMR (100 MHz,
DMSO-d6) δ 180.87, 163.02, 158.95, 129.82, 129.56, 115.54, 114.37, 83.59, 55.52, 48.70, 42.53, 28.93;
LC/MS (ESI+): m/z: calcd for C14H14N2O3: 258.28, [M+H]+; found: 259.15.
1-Benzyl-4-methoxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile (7a). To a solution of benzyl-4-
hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile 6a (100 mg, 0.44 mmol) and THF (3 mL), NaH
(21 mg, 0.88 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. Then
dimethylsulfate (0.071 mL, 0.75 mmol) was added to the mixture and the resulting solution was stirred
at 40 °C for 12 h. The mixture was quenched with H2O and evaporated. Then it was diluted with EtOAc,
extracted with EtOAc, and dried over MgSO4 and concentrated, the crude product was purified by silica
gel column chromatography (n-hexane-EtOAc = 1:1) to give product 7a (67 mg, 0.28 mmol, 63% yield)
as a white solid: mp 153–157 °C; 1H-NMR (300 MHz, CDCl3) δ 7.37–7.26 (m, 5H), 4.62 (s, 2H), 4.14
(s, 3H), 3.33 (t, J = 6.9 Hz, 2H), 2.61 (t, J = 6.9 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 177.55, 162.08,
136.66, 128.77, 128.19, 127.76, 114.15, 86.77, 58.13, 49.85, 42.07, 26.91; LC/MS (ESI+): m/z: calcd for
C14H14N2O2: 242.28, [M+H]+; found: 243.15.
4-Methoxy-1-(4-methoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile (7b). Following the
same procedure used for the synthesis of 7a, the reaction of 4-hydroxy-1-(4-methoxybenzyl)-2-oxo-
1,2,5,6-tetrahydropyridine-3-carbonitrile 6b (1.74 g, 6.70 mmol), NaH (321 mg, 13.4 mmol), and
dimethylsulfate (1.1 ml, 11.4 mmol) gave the title compound 7b (677 mg, 2.45 mmol, 37% yield) as a
white solid: mp 145–148 °C; 1H-NMR (300 MHz, CDCl3) δ 7.23 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 8.4 Hz,
13
2H), 4.57 (s, 2H), 4.14 (s, 3H), 3.82 (s, 3H), 3.35 (t, J = 6.7 Hz, 2H), 2.65 (t, J = 6.7 Hz, 2H); C-NMR

Molecules 2015, 20
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(75 MHz, CDCl3) δ 177.20, 161.95, 159.28, 129.62, 128.73, 114.16, 86.85, 58.14, 55.31, 49.29, 41.87,
27.10; LC/MS (ESI+): m/z: calcd for C15H16N2O3: 272.30, [M+H]+; found: 273.15.
Methyl 3-amino-5-benzyl-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate (8a). NaOMe
(5M in MeOH, 36.0 mL, 17.9 mmol) in MeOH followed by methyl thioglycolate (1.8 mL, 20.5 mmol)
were added to 1-benzyl-4-methoxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile 7a (3.1 g, 12.8 mmol).
The reaction mixture was stirred and refluxed at 65 °C for 12 h. After the reaction, the mixture was
filtered using Celite and washed with MC. The crude compound was then purified by by silica gel
column chromatography (n-hexane-EtOAc = 4:1) to give white solid product 8a (2.44 g, 7.38 mmol,
1
58% yield) as a yellowish solid: mp 110–112 °C; H-NMR (300 MHz, CDCl3) δ 7.37–7.27 (m, 5H),
6.91 (brs, 2H), 4.70 (s, 2H), 3.82 (s, 3H), 3.53 (t, J = 6.9 Hz, 2H), 2.93 (t, J = 6.9 Hz, 2H); 13C-NMR
(100 MHz, CDCl3) δ 164.55, 162.73, 153.78, 151.00, 137.12, 128.77, 127.99, 127.63, 119.74, 96.83,
51.16, 49.03, 45.82, 25.00; LC/MS (ESI+): m/z: calcd for C16H16N2O3S: 316.38, [M+H]+; found: 317.05.
Methyl 3-amino-5-(4-methoxybenzyl)-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate (8b).
Following the same procedure used for the synthesis of 8a, the reaction of 4-methoxy-1-(4-
methoxybenzyl)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile 7b (354 mg, 1.30 mmol), NaOMe (5M
in MeOH, 0.40 mL, 2.08 mmol), and methyl thioglycolate (0.21 mL, 2.34 mmol) gave the title compound
8b (281 mg, 0.81 mmol, 62% yield) as a yellow oil: 1H-NMR (400 MHz, CDCl3) δ 7.24–7.22 (m, 2H),
6.88–6.86 (m, 2H), 4.62 (s, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.50 (t, J = 6.9 Hz, 2H), 2.91 (t, J = 6.9 Hz,
13
2H); C-NMR (100 MHz, CDCl3) δ 164.52, 162.63, 159.13, 153.74, 150.99, 129.37, 129.13, 119.77,
114.11, 96.80, 55.28, 51.12, 48.38, 45.59, 24.95; LC/MS (ESI+): m/z: calcd for C17H18N2O4S: 346.40,
[M+H]+; found: 347.05.
8-Benzyl-3-phenyl-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-4,9(3H,6H)-dione (9a). To a
pressure bottle containing methyl 3-amino-5-benzyl-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-
carboxylate 8a (1g, 3.03 mmol), CH(OEt)3 (10 mL) was added, followed by aniline (0.524 mL,
5.75 mmol) and AcOH (1 mL). The reaction mixture was stirred and refluxed at 160 °C for 18 h. After
the reaction, the mixture was evaporated then solidified with EtOAc and Et2O. The produced solid was
filtered and dried in vacuo to give the title product 9a (743 mg, 1.92 mmol, 63% yield) as a reddish solid:
mp 172–173 °C; 1H-NMR (300 MHz, DMSO-d6) δ 8.44–7.51 (m, 5H), 7.39–7.26 (m, 5H), 4.71 (s, 2H),
13
3.62 (t, J = 6.6 Hz, 2H), 3.23 (t, J = 6.6 Hz, 2H); C-NMR (100 MHz, DMSO-d6) δ 159.61, 156.61,
156.48, 154.82, 149.69, 138.26, 137.32, 129.71, 129.49, 129.20, 128.08, 128.02, 127.63, 126.01, 122.13,
49.19, 46.26, 25.79; HRMS (ESI TOF-mass) calcd for C22H17N3O2S [M+Na]+ 410.0934, found
410.0936; purity (HPLC) 83.91%, tR 18.00 min.
8-Benzyl-3-(4-chlorophenyl)-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-4,9(3H,6H)-dione (9b).
Following the same procedure used for the synthesis of 9a, the reaction of methyl 3-amino-5-benzyl-4-
oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8a (100 mg, 0.32 mmol), CH(OEt)3 (1 mL),
4-chloroaniline (72.7 mg, 0.57 mmol) and AcOH (0.1 mL) gave the title compound 9b (89 mg,
1
0.21 mmol, 66% yield) as a yellowish solid: mp 220–223 °C; H-NMR (400 MHz, CDCl3) δ 8.27 (s,
13
1H), 7.54–7.27 (m, 9H), 4.80 (s, 2H), 3.63 (t, J = 6.7 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H); C-NMR
(100 MHz, CDCl3) δ 160.06, 156.36, 155.24, 154.74, 148.31, 137.33, 135.55, 135.10, 129.93, 128.72,

Molecules 2015, 20
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128.46, 128.39, 127.65, 126.17, 122.65, 49.53, 45.64, 25.89; HRMS (ESI TOF-mass) calcd for
C22H16ClN3O2S [M+Na]+ 444.0544, found 444.0545; purity (HPLC) 100.00%, tR 19.21 min.
8-Benzyl-3-(4-methoxyphenyl)-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-4,9(3H,6H)-dione (9c).
Following the same procedure used for the synthesis of 9a, the reaction of methyl 3-amino-5-benzyl-4-
oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8a (100 mg, 0.32 mmol), CH(OEt)3 (1 mL),
p-anisidine (72.7 mg, 0.57 mmol) and AcOH (0.1 mL) gave the title compound 9c (44 mg, 0.11 mmol,
33% yield) as a reddish solid: mp 223–225 °C; 1H-NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.40–7.04
13
(m, 9H), 4.81 (s, 2H), 3.87 (s, 3H), 3.63 (t, J = 6.7 Hz, 2H), 3.14 (t, J = 6.7 Hz, 2H); C-NMR
(100 MHz, CDCl3) δ 160.19, 160.17, 156.84, 154.92, 154.81, 149.05, 137.41, 129.35, 128.71, 128.39,
128.23, 127.61, 126.13, 122.79, 114.88, 55.64, 49.48, 45.64, 25.89; HRMS (ESI TOF-mass) calcd for
C23H19N3O3S [M+Na]+ 440.1039, found 440.1039; purity (HPLC) 100.00%, tR 18.09 min.
8-Benzyl-3-(3-methoxyphenyl)-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-4,9(3H,6H)-dione
(9d). Following the same procedure used for the synthesis of 9a, the reaction of methyl 3-amino-5-
benzyl-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8a (200 mg, 0.63 mmol), CH(OEt)3
(2 mL), m-anisidine (135 μL, 1.21 mmol) and AcOH (0.2 mL) gave the title compound 9d (53 mg, 0.13
1
mmol, 20% yield) as a white solid: mp 223–226 °C; H-NMR (400 MHz, CDCl3) δ 8.27 (s, 1H),
7.47–6.95 (m, 9H), 4.81 (s, 2H), 3.85 (s, 3H), 3.63 (t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.7 Hz, 2H); 13C-NMR
(100 MHz, CDCl3) δ 160.43, 160.17, 156.50, 154.98, 154.78, 148.72, 137.72, 137.38, 130.45, 128.72,
128.39, 127.63, 126.18, 122.86, 119.15, 115.39, 112.93, 55.60, 49.49, 45.63, 25.91; HRMS (ESI
TOF-mass) calcd for C23H19N3O3S [M+Na]+ 440.1039, found 440.1038; purity (HPLC) 99.64%, tR
18.31 min.
8-(4-Methoxybenzyl)-3-phenyl-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-4,9(3H,6H)-dione (9e).
Following the same procedure used for the synthesis of 9a, the reaction of methyl 3-amino-5-(4-
methoxybenzyl)-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8b (118 mg, 0.34 mmol),
CH(OEt)3 (1.2 mL), aniline (62 μL, 0.68 mmol) and AcOH (0.12 mL) gave the title compound 9e
(95 mg, 0.23 mmol, 67% yield) as a yellowish solid: mp 227–230 °C; 1H-NMR (400 MHz, DMSO-d6)
δ 8.27 (s, 1H), 7.58–6.85 (m, 9H), 4.73 (s, 2H), 3.79 (s, 3H), 3.61 (t, J = 6.7 Hz, 2H), 3.12 (t, J = 6.8 Hz,
13
2H); C-NMR (100 MHz, CDCl3) δ 160.16, 159.18, 156.56, 154.96, 154.78, 148.80, 136.70, 134.10,
129.76, 129.70, 129.43, 127.06, 126.22, 122.87, 114.09, 55.31, 48.87, 45.43, 25.91; HRMS (ESI
TOF-mass) calcd for C23H19N3O3S [M+Na]+ 440.1039, found 440.1039; purity (HPLC) 98.75%,
tR 17.88 min.
3-(4-Chlorophenyl)-8-(4-methoxybenzyl)-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-4,9(3H,6H)-
dione (9f). Following the same procedure used for the synthesis of 9a, the reaction of methyl 3-amino-
5-(4-methoxybenzyl)-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8b (100 mg, 0.29 mmol),
CH(OEt)3 (1 mL), 4-chloroaniline (74 mg, 0.58 mmol) and AcOH (0.1 mL) gave the title compound 9f
1
(68 mg, 0.15 mmol, 52% yield) as a yellowish solid: mp 269–272 °C; H-NMR (400 MHz, CDCl3) δ
8.24 (s, 1H), 7.55–6.86 (m, 8H), 4.74 (s, 2H), 3.80 (s, 3H), 3.62 (t, J = 6.7 Hz, 2H), 3.13 (t, J = 6.7 Hz,
13
2H); C-NMR (100 MHz, CDCl3) δ 159.98, 159.17, 156.36, 155.19, 154.72, 148.27, 135.53, 135.11,

Molecules 2015, 20
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129.91, 129.77, 129.42, 128.47, 126.24, 122.61, 114.08, 55.31, 48.92, 45.46, 25.88; HRMS (ESI TOF-mass)
calcd for C23H18ClN3O3S [M+Na]+ 474.0650, found 474.0652; purity (HPLC) 100.00%, tR 19.03 min.
8-(4-Methoxybenzyl)-3-(4-methoxyphenyl)-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-
4,9(3H,6H)-dione (9g). Following the same procedure used for the synthesis of 9a, the reaction of methyl
3-amino-5-(4-methoxybenzyl)-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8b (100 mg,
0.29 mmol), CH(OEt)3 (1 mL), p-anisidine (73 mg, 0.58 mmol) and AcOH (0.1 mL) gave the title
compound 9g (67 mg, 0.15 mmol, 51% yield) as a reddish solid: mp 225–228 °C; 1H-NMR (400 MHz,
CDCl3) δ 8.25 (s, 1H), 7.33–7.30 (m, 4H), 7.06–6.84 (m, 4H), 4.73 (s, 2H), 3.86 (s, 3H), 3.78 (s, 3H), 3.60
13
(t, J = 6.7 Hz, 2H), 3.12 (t, J = 6.7 Hz, 2H); C-NMR (100 MHz, CDCl3) δ 160.18, 160.16, 159.17,
156.85, 154.83, 154.82, 149.04, 129.76, 129.47, 129.35, 128.20, 126.23, 122.82, 114.89, 114.08, 55.63,
55.31, 48.86, 45.44, 25.91; HRMS (ESI TOF-mass) calcd for C24H21N3O4S [M+Na]+ 470.1145, found
470.1146; purity (HPLC) 100.00%, tR 17.95 min.
8-(4-Methoxybenzyl)-3-(3-methoxyphenyl)-7,8-dihydropyrido[3',4':4,5]thieno[3,2-d]pyrimidine-
4,9(3H,6H)-dione (9h). Following the same procedure used for the synthesis of 9a, the reaction of methyl
3-amino-5-(4-methoxybenzyl)-4-oxo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylate 8b (100 mg,
0.29 mmol), CH(OEt)3 (1 mL), m-anisidine (65 μL, 0.58 mmol) and AcOH (0.1 mL) gave the title
compound 9h (66 mg, 0.15 mmol, 51% yield) as a yellowish solid: mp 171–175 °C; 1H-NMR (400 MHz,
CDCl3) δ 8.28 (s, 1H), 7.46–6.84 (m, 8H), 4.74 (s, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 3.61 (t, J = 6.7 Hz,
2H), 3.12 (t, J = 6.8 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 160.42, 160.12, 159.16, 156.51, 154.98,
154.72, 148.77, 137.71, 130.44, 129.76, 129.44, 126.20, 122.81, 119.15, 115.41, 114.08, 112.92, 55.60,
55.31, 48.87, 45.44, 25.89; HRMS (ESI TOF-mass) calcd for C24H21N3O4S [M+Na]+ 470.1145, found
470.1146; purity (HPLC) 98.23%, tR 18.13 min.
3.2. mGluR1 Assay using FDSS6000
Chem-3 cells which stably express mGluR1 were purchased from Milipore Co. (Billerica, MA, USA).
Cells were grown in DMEM medium supplemented with 10% (v/v) fetal bovine serum, penicillin
(100 U/mL), streptomycin (100 μg/mL), and puromycin (10 μg/mL) at 37 °C in a humid atmosphere of
5% CO2 and 95% air. For calcium assay, cells were harvested and dispensed into 96-well black wall
clear bottom plates at a density of 40,000 cells per a well. After 18 h of incubation, cells were treated
with Calcium-5 assay reagent, which is prepared by manufacture’s instruction (Molecular Devices Co.
Sunnyvale, CA, USA). During fluorescence-based FDSS6000 assay, mGluR1 was activated using a high
concentration of L-glutamate (10 μM) in HBSS, and proper concentrations of synthesized compounds
were treated to cells 75 s before mGluR activation. All data were collected and analyzed using
FDSS6000 and related software (Hamamatsu Photonics, Hamamatsu, Japan).
4. Conclusions
In this proof-of-concept study, a synthetic protocol was devised to allow access to a fused tricyclic
heterocycle composed of dihydropyridone, thiophene and pyrimidinone. Also, even though only a small
set of the dihydropyridothienopyrimidin-4,9-dione derivatives were synthesized, promising biological

Molecules 2015, 20
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activity was identified in the title compounds. Thus, by using variously substituted benzylamines and
anilines, an extensive structure-activity relationship study is warranted to investigate the
pharmacological versatility of the dihydropyridothienopyrimidin-4,9-dione derivatives.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/03/5074/s1.
Acknowledgments
This research was supported by the Basic Science Research Program (NRF-2013-
R1A1A2A10009907) funded by the National Research Foundation of Korea (NRF). Additional funding
was provided by the Korea Institute of Science and Technology (KIST) Institutional Program (2E24510,
2E25473, and 2E25240).
Author Contributions
YK, JT, D-JB, and HC designed research; YK and MK performed research; YK, MK, MP, and KDP
analyzed the data; YK and HC wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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