Chem. Res. Chin. Univ. doi: 10.

1007/s40242-015-5202-3

Synthesis, Characterization and Biological Activity of

Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine Derivatives as
Epidermal Growth Factor Receptor Inhibitors

SUN Bing1, YIN Xiu’e1, ZHANG Jin2, HUANG Jian1,2, XU Yue1, ZHANG Furong1,
WANG Jinhui1,2, WANG Guoqing1 and HU Chun1*
1. Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education,
School of Pharmaceutical Engineering, 2. School of Traditional Chinese Materia Medica,
Shenyang Pharmaceutical University, Shenyang 110016, P. R. China

Abstract Based on the molecular docking studies, which were performed to position Erlotinib and the target com-
pounds into the active site of the epidermal growth factor receptor(EGFR) to determine the probable binding model, a
novel series of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives as the novel potential EGFR kinase
inhibitors was designed and synthesized. The antitumor activity of all the target compounds against human pulmonary
carcinoma cell line A549 has been screened. Of all the target compounds, 4-[2-(1-piperidyl)carbonylmethoxyl-
phenthio]-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7j) demonstrated the most potent antitumor activity.
Several of the target compounds exhibited moderate antitumor activity. The preliminary structure-activity relation-
ships of some target compounds were summarized.
Keywords Antitumor activity; Docking; Epidermal growth factor receptor(EGFR)

1 Introduction hydrogen bond can be formed between the N1 or N3 position

of the quinazoline nucleus and the surrounding amino acid
The epidermal growth factor receptor(EGFR) is a trans- residues. In addition, an adjacent hydrophobic pocket can be
membrane glycoprotein and a member of protein kinase super- filled by the aniline ring[9].
family existing on the cell surface, which can be activated by
the binding of its specific ligands, including epidermal growth
factor and transforming growth factor α, and plays a critical
role in regulating cell proliferation, differentiation, survival,
and migration[1]. The overexpression or overactivity of EGFR
associates with a number of cancers, including non-small cell
lung cancer, anal cancers and glioblastoma multiforme[2].
Transfection or activation of high levels of EGFRs in nonma-
lignant cell lines can lead to a transformed phenotype[3].
After the discovery of the function of EGFR, there has
been a surge of interest in developing therapeutic candidates
with novel chemical scaffolds, which manifest better activity Fig.1 Structures of Gefitinib(A) and Erlotinib(B)
profiles than the currently available therapeutics used in the However, the clinical application of the marketed EGFR
treatment and prevention of cancer associated with EGFR[3]. inhibitors, such as Erlotinib has been limited mainly by the
In the last few years, several drugs and compounds have adverse effects of tetter and diarrhea[10]. Moreover, the problem
been developed to inhibit the EGFR activation, block the path- of resistance to EGFR inhibitors which has been listed is also
way and promote the tumor cell apoptosis[4―8]. A well-studied increasingly prominent[11,12]. Therefore, more efficient new
class of these EGFR inhibitors is 4-anilinoquinazoline deriva- drugs as EGFR inhibitors are needed. Based on the retention of
tives, such as Gefitinib and Erlotinib(Fig.1). The studies of the quinazoline scaffold in the marketed EGFR inhibitors, such as
interactions between these EGFR inhibitors with quinazoline Geftinib, Erlotinib, Lapatinib and Vandetanib[13,14], a novel
scaffold and EGFR show that the quinazoline moiety can fit series of small molecules containing 5,6,7,8-tetrahydrobenzo-
into the ATP binding pocket of the kinase domain, where a [4,5]thieno[2,3-d]pyrimidine scaffold was designed according
———————————
*Corresponding author. E-mail: chunhu@syphu.edu.cn
Received May 20, 2015; accepted August 27, 2015.
Supported by the National Natural Science Foundation of China(No.21342006) and the Program for the Innovative Research
Team of the Ministry of Education of China(No.IRT_14R36).
© Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH
2   Chem. Res. Chin. Univ.
to the subsequent arrangements and alternations. Firstly,
the quinazoline ring in the marketed EGFR kinase inhibitors
with 4-anilinoquinazoline scaffold was replaced by the
novel 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine ring.
Secondly, the 4-anilino group was replaced by 4-arylthio group
as the bioisosteres. Thirdly, the side chains in the scaffold
and the 4-arylthio group were chosen conventionally which
have been widely utilized in designing many EGFR inhibitors.
Hence, a novel series of 5,6,7,8-tetrahydrobenzo-
[4,5]thieno[2,3-d]pyrimidine derivatives was synthesized, and
the antitumor activities of them against human pulmonary car-
cinoma cell line A549 were screened for the discovery of novel
potential EGFR inhibitors.
2 Results and Discussion
2.1 Chemistry
The synthetic pathways of the 5,6,7,8-tetrahydrobenzo-
[4,5]thieno[2,3-d]pyrimidine derivatives are shown in Scheme
1. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox-
amide(2) was synthesized from cyclohexanone(1), cyanoace-
tamide and sublimed sulfur via Gewald reaction[15]. The prepa-
ration of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidin-
4(3H)-one(3) was achieved via the reaction of compound 2
with formamide [16] . Compound 3 was converted into 4-
chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidine(4)
by further treatment with phosphorus oxychloride [17] .
4-(Methoxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidines(5) was obtained via the reaction of compound 4,
4-methoxythiophenol and triethylamine. Finally, compound 5
was treated by anhydrous aluminum trichloride as a demethyla-
tion reagent to prepare 4-(hydroxyphenthio)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidines(6). The target com-
pound derivatives(7) were synthesized from compound 6 via
the ordinary Williamson reaction in satisfactory yields.
The chemical structures of all the target compounds were
fully characterized by ESI-MS, IR spectra, 1H NMR and
13
C NMR.

5a: 4-OCH3; 5b: 2-OCH3; 6a: 4-OH; 6b: 2-OH; R: 7a: 4-(4-morpholinylcarbonylmethoxyl); 7b: 4-(1-piperidylcarbonylmethoxyl); 7c: 4-(diethylaminocarbonyl-
methoxyl); 7d: 4-(1-pyrrolidylcarbonylmethoxyl); 7e: 4-(anilinocarbonylmethoxyl); 7f: 4-(4-fluoroanilinocarbonylmethoxyl); 7g: 4-(4-chloroanilinocarbonyl-
methoxyl); 7h: 4-(4-bromoanilinocarbonylmethoxyl); 7i: 2-(4-morpholinylcarbonylmethoxyl); 7j: 2-(1-piperidylcarbonylmethoxyl); 7k: 2-(diethylaminocar-
bonylmethoxyl); 7l: 2-(1-pyrrolidylcarbonylmethoxyl); 7m: 2-(anilinocarbonylmethoxyl); 7n: 2-(4-fluoroanilinocarbonylmethoxyl).
Scheme 1Synthetic route of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives
In general, except for compound 7j, the target compounds
2.2 Biological Evaluation having substitutions of side chains at the para position in the
phenyl ring(7a—7h) result in a better antitumor activity com-
The antitumor activities of the target compounds were
pared with those at the ortho position(7i, 7k—7n).
evaluated against the human pulmonary carcinoma cell line
Among the target compounds 7a—7h, compound 7f with
A549, which has abundant expression of EGFR using
4-[(4-fluoroanilino)formylmethoxyl] side chain exhibits the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
most potent antitumor activity. The additional data show that
(MTT) colorimetric assay[18―20]. The results are summarized in
the target compounds with p-halo substituent in anilino group
Table 1.
Table 1 Inhibitory ratio and IC50 of the target compounds
Compound Inhibitory ratio(%) IC50/(μmol·L–1) Compound Inhibitory ratio(%) IC50/(μmol·L–1)
a a
7a 27.1 37.0 7i 21.2 89.4
7b 52.9b 47.2 7j 74.0b 33.8
7c 37.0b 67.6 7k 15.6b >100.0
7d 10.9b >100.0 7l 4.0b >100.0
7e 10.2b >100.0 7m 4.7b >100.0
7f 69.5b 36.0 7n 38.3b 65.4
7g 24.3a 41.2 Erlotinib 74.6a 13.4
7h 23.0a 43.5
a. Inhibitory ratio(%) measured at 20 μmol/L; b. inhibitory ratio(%) measured at 50 μmol/L.
 SUN Bing et al.
generally exhibit greater antitumor activity.
2.3 Docking Studies
Docking simulations and molecular dynamics studies on
the EGFR-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine deriva-
tives complexes were carried out with the aim to rationalize the
molecular level interactions between the target molecules and
EGFR and to find the novel EGFR inhibitors.
In 2002, Stamos et al.[21] reported a crystallographic
structure of EGFR-Erlotinib complexes encoded 1M17, which
was selected from the Protein Data Bank(PDB) of Research
Collaboratory for Structural Bioinformatics(RCSB), and
was prepared by Molegro Virtual Docker 2010(v4.1). The
docking algorithm was set at 1500 maximum iterations with a
simple evolution population size of 50 and a minimum of 10
runs. Its original inhibitor(Erlotinib) was eliminated and all the
water molecules were deleted, adding a further degree of
freedom in terms of variability in the docking results.
Schematic diagrams of the interactions between 1M17 and
small molecules were analyzed by the Discovery Studio 4.0
Client.
Based on the docking results, the interactions between Er-
lotinib and EGFR are shown in Fig.2. A hydrogen
bond(represented as a green dotted line) has been formed
between N1 position of the parent nucleus and the Met769
residue. There are hydrophobic effects(represented as violet
dotted lines) between Erlotinib and the residues, such as
Leu694, Ala719, Lys721, Met769 and Leu820.
Fig.2 Interactions between Erlotinib and
active site of EGFR
According to the predicted docking interactions between
compound 7j and EGFR, a hydrogen bond(represented as a
green dotted line, Fig.3) has been formed between N1 position
of the parent nucleus and the Met769 residue. There are hy-
drophobic effects(represented as violet dotted lines) between
compound 7j and the residues, such as Leu694, Ala719, Lys721,
Met742, Leu764, Leu768, Met769, Cys773 and Leu820. Com-
pared with Erlotinib, more hydrophobic effects can be found
between compound 7j and amino acid residues. They are
Met742 , Leu764 , Leu768 and Cys773.
Therefore, the target compound 7j has similar active sites
with Erlotinib in the interaction with EGFR and more
interactions have been found between compound 7j and
EGFR.
3
Fig.3 Interaction between compound 7j and
active site of EGFR
3 Experimental
3.1 Chemistry
Starting materials and solvents were purchased from
common commercial suppliers and were used without further
purification. Melting points were determined in open capillary
tubes and uncorrected. Reaction progress was monitored by
thin layer chromatography on silica gel sheets. The spots were
visualized with UV light(254 nm). Mass spectra were recorded
by electrospray(ESI) on a Waters spectrometer(Massachusetts,
USA). The IR spectra were obtained on a Bruker IFS55 spec-
trometer(Karlsruhe, Germany). NMR spectra were recorded on
Bruker 400 MHz and 600 MHz NMR spectrophotome-
ters(Karlsruhe, Germany) using CDCl3 or DMSO-d6 as the
solvent and TMS as the internal standard.
Synthesis of 2-amino-4,5,6,7-tetrahydrobenzo[b]-thio-
phene-3-carboxamide(2): a stirred mixture of compound 1(11.8
g, 120.0 mmol), cyanoacetamide(10.1 g, 120.0 mmol), sub-
limed sulfur(3.8 g, 120.0 mmol) and ethanol(6.0 mL) was
heated at 45 °C. Piperidine(0.8 g, 9.2 mmol) was added drop-
wise and the temperature was controlled at 45—50 °C. Then
the reaction mixture was stirred at 45—50 °C for 5 h. After that
the reaction solution was frozen for 2 h. The precipitated solid
was filtered off and washed twice with ethanol. An orange solid
as the crude product of 11.7 g was obtained in a yield of 49.6%
and used for the next reaction without further purification. m. p.
186—188°C(lit.[13]: 189—190 °C) .
Synthesis of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-
pyrimidin-4(3H)-one(3): a stirred mixture of compound 2(11.6
g, 59.2 mmol) and formamide(46.4 g, 1031.0 mmol) was
heated at 165 °C for 6 h. The reaction mixture was cooled at
room temperature for crystallization. Isopropanol(110 mL) was
added and the mixture was stirred at room temperature for 1 h.
The precipitated solid was filtered off and washed with isopro-
panol. A total of 9.7 g of brown solid was obtained in a yield of
80.0%. m. p. 255—257 °C(lit.[14]: 255—256 °C) .
Synthesis of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno-
[2,3-d]pyrimidine(4): a stirred mixture of compound 3(8.7 g,
42.0 mmol) and phosphorus oxychloride(70.9 g, 467.1 mmol)
was heated under reflux for 30 min. After the solvent was
evaporated under reduced pressure, the black crude product
4   Chem. Res. Chin. Univ.
was purified by column chromatography on silica gel
[V(petroleum ether):V(ethyl acetate)=2:1] and a yellow solid
was obtained(6.7 g, yield 70.5%), m. p. 97—99 °C(lit.[15]:
99—100 °C) .
Synthesis of 4-(methoxyphenthio)-5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidines(5a and 5b): a stirred mix-
ture of compound 4(8.0 g, 35.6 mmol), 4-methoxythiophenol or
2-methoxythiophenol(5.5 g, 39.2 mmol), triethylamine(10.8 g,
106.8 mmol) and n-butanol(102 mL) was heated under reflux
for 1 h. The reaction mixture was cooled at room temperature
for crystallization. The solid was collected by filtration, washed
with water and ethanol to afford compound 5 after drying.
4-(4-Methoxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]-
thieno[2,3-d]pyrimidine(5a): a yellow solid(10.1 g), yield
86.2%, m. p. 151—152 °C. ESI-MS, m/z: calcd. 328.07(M+);
found 328.9([M+H]+), 351.1([M+Na]+). IR(KBr), ߥ෤ /cm–1:
2930(s), 1640(m), 1591(s), 1492(s), 1411(s), 1173(s), 1129(s),
825(s). 1H NMR(600 MHz, CDCl3), δ: 1.92—1.96(m, 4H),
2.87(s, 2H), 3.20(s, 2H), 3.86(s, 3H), 7.00(d, J=8.8 Hz, 2H),
7.49(d, J = 8.8 Hz, 2H), 8.53(s, 1H).
4-(2-Methoxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]-
thieno[2,3-d]pyrimidine(5b): a yellow solid(11.1 g), yield
94.5%, m. p. 161—162 °C. ESI-MS, m/z: calcd. 328.07(M+);
found 329.3([M+H]+). IR(KBr), ߥ෤ /cm–1: 2936(s), 1643(m),
1584(s), 1496(s), 1415(s), 1168(s), 1129(s), 832(s). 1H NMR
(600 MHz, CDCl3), δ: 1.92—1.96(m, 4H), 2.87(s, 2H), 3.24(s,
2H), 3.79(s, 3H), 7.01―7.07(m, 2H), 7.49(t, 1H), 7.55(dd,
J1=7.5 Hz, J2=1.6 Hz, 1H), 8.49(s, 1H).
Synthesis of 4-(hydroxyphenthio)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines(6a and 6b): a
stirred mixture of 5a or 5b(9.0 g, 27.4 mmol), anhydrous alu-
minum trichloride(11.0 g, 82.2 mmol) and toluene(193 mL)
was heated under reflux for 1 h. After cooling down to room
temperature, water(280 mL) was added to the system and the
mixture stood for a night. The precipitate was filtered off and
purified by column chromatography on silica gel[V(methanol):
V(dichloromethane)=1:100].
4-(4-Hydroxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]thie-
no[2,3-d]pyrimidine(6a): a yellowish solid(5.0 g), yield 58.0%,
m. p. 230—231 °C. ESI-MS, m/z: calcd. 314.05(M+); found
314.9([M+H]+), 337.2([M+Na]+). IR(KBr), ߥ෤/cm–1: 3421(m),
2930(s), 1640(m), 1599(s), 1492(s), 1413(s), 1165(s), 1130(s),
831(s). 1H NMR(400 MHz, CDCl3), δ: 1.92—1.98(m, 4H),
2.88(s, 2H), 3.20(s, 2H), 6.87(d, J=8.6 Hz, 2H), 7.42(d, J=8.6
Hz, 2H), 8.56(s, 1H).
4-(2-Hydroxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]-
thieno[2,3-d]pyrimidine(6b): a yellowish solid(5.6 g), yield
64.5%, m. p. 175—176 °C. ESI-MS, m/z: calcd. 314.05(M+);
found 315.3([M+H]+). IR(KBr), ߥ෤/cm–1: 3421(m), 2932(m),
1632(m), 1596(m), 1473(s), 1409(s), 1384(s), 1142(s), 830(m).
1
H NMR(400 MHz, CDCl3), δ: 1.95—1.99(m, 4H), 2.90(s, 2H),
3.23(s, 2H), 6.94(t, 1H), 7.17(dd, J1=8.1 Hz, J2=1.0 Hz, 1H),
7.42(t, 1H), 7.49(dd, J1=7.8 Hz, J2=1.4 Hz, 1H), 8.61(s, 1H).
Synthesis of the target compounds(7a―7n): a stirred mix-
ture of 6a or 6b(0.5 g, 1.6 mmol), 2-chloroacetamide(1.9
mmol), anhydrous potassium carbonate(2.2 g, 16.0 mmol),
potassium iodide(0.1 g, 0.8 mmol) and acetone(50 mL) was
heated under reflux for 12 h. After the reaction mixture was
evaporated, water(30 mL) was added and the solution was ex-
tracted with dichloromethane(3×20 mL), dried with anhy-
drous MgSO4 and evaporated again. Crude products were
purified by column chromatography on silica gel
[V(methanol):V(dichloromethane)=1:80].
4-[4-(4-Morpholinyl)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7a): a yellowish
solid(0.6 g), yield 78.6%, m. p. 141—144 °C. ESI-MS, m/z:
calcd. 441.12(M+); found 442.0([M+H]+), 463.8([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3442(m), 2932(s), 2857(s), 1650(s), 1591(s),
1492(s), 1440(s), 1409(s), 1380(s), 1236(s), 1110(s), 1031(s),
829(s), 731(s). 1H NMR(600 MHz, CDCl3), δ: 1.92—1.96(m,
4H), 2.87―2.88(m, 2H), 3.18―3.19(m, 2H), 3.61―3.63(m,
2H), 3.65―3.66(m, 2H), 3.68―3.70(m, 4H), 4.75(s, 2H),
7.04(d, 2H, J=8.4 Hz), 7.50(d, 2H, J=8.4 Hz), 8.52(s, 1H);
13
C NMR(150 MHz, CDCl3), δ: 22.52, 22.63, 25.90, 26.88,
42.49, 45.95, 66.75(d, J=13.7 Hz), 67.60, 115.63, 119.66,
127.52, 127.97, 136.98, 137.52, 151.65, 158.87, 163.55, 165.71,
166.20.
4-[4-(1-Piperidyl)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7b): a yellowish
solid(0.6 g), yield 91.4%, m. p. 84—86 °C. ESI-MS, m/z: calcd.
439.14(M+); found 440.5([M+H]+), 461.8([M+Na]+). IR(KBr),
ߥ෤/cm–1: 3443(m), 2935(s), 2855(s), 1639(s), 1592(s), 1493(s),
1383(s), 1241(s), 1178(s), 1128(s), 1036(m), 826(s), 727(s).
1
H NMR(400 MHz, CDCl3), δ: 1.58—1.66(m, 6H),
1.91—1.97(m, 4H), 2.86—2.88(m, 2H), 3.19(brs, 2H), 3.50(t,
2H), 3.58(t, 2H), 4.73(s, 2H), 7.04(d, 2H, J=8.4 Hz), 7.49(d,
2H, J=8.4 Hz), 8.51(s, 1H); 13C NMR(150 MHz, CDCl3), δ:
22.51, 22.62, 24.44, 25.52, 25.89, 26.49, 26.89, 43.25, 46.46,
67.70, 115.69, 119.20, 127.53, 127.94, 136.88, 137.39, 151.65,
159.23, 163.68, 165.67.
4-(4-Diethylincarbonylmethoxylphenthio)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7c): a yellowish
solid(0.6 g), yield 90.8%, m. p. 88—91 °C. ESI-MS, m/z: calcd.
427.14(M+); found 428.2([M+H]+). IR(KBr), ߥ෤/cm–1: 3577(s),
3489(s), 2971(s), 2934(s), 1647(s), 1614(s), 1594(s), 1494(s),
1410(s), 1250(s), 1078(s), 835(s), 728(s), 616(s). 1H NMR(400
MHz, CDCl3), δ: 1.16(t, 3H), 1.24(t, 3H), 1.94(t, 4H), 2.87(t,
2H), 3.18(t, 2H), 3.41(m, 4H), 4.72(s, 2H), 7.04(d, 2H, J=8.8
Hz), 7.49(d, 2H, J=8.8 Hz), 8.52(s, 1H); 13C NMR(150 MHz,
CDCl3), δ: 12.84, 14.40, 22.61(d, J=17.0 Hz), 25.93, 26.93,
40.42, 41.63, 67.51, 115.77, 119.20, 127.59, 128.00, 136.94,
137.44, 151.69, 159.36, 163.78, 165.67, 166.63.
4-[4-(1-Pyrrolidyl)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7d): a yellowish
solid(0.6 g), yield 83.8%, m. p. 125—127 °C. ESI-MS, m/z:
calcd. 425.12(M+); found 426.1([M+H]+), 448.2([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3556(s), 3476(s), 2936(s), 2882(s), 1649(s),
1595(s), 1494(s), 1410(s), 1251(s), 1175(s), 833(s), 720(s).
1
H NMR(400 MHz, CDCl3), δ: 1.84―2.02(m, 8H), 2.87(t, 2H),
3.19(t, 2H), 3.55(t, 4H), 4.67(s, 2H), 7.04(d, 2H, J=8.8 Hz),
7.49(d, 2H, J=8.8 Hz), 8.52(s, 1H); 13C NMR(150 MHz,
CDCl3), δ: 22.52(d, J=16.7 Hz), 23.79, 25.85, 26.21, 26.85,
45.98, 46.22, 67.80, 115.67, 119.15, 127.50, 127.90, 136.87,
137.36, 151.60, 159.22, 163.65, 165.60, 166.08.
 SUN Bing et al.
4-(4-Anilinocarbony lmethoxy lphenthio)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7e): a yellowish
solid(0.6 g), yield 77.5%, m. p. 196—198 °C. ESI-MS, m/z:
calcd. 447.11(M+); found 448.1([M+H]+). IR(KBr), ߥ෤ /cm–1:
3374(s), 2929(s), 1684(s), 1599(s), 1534(s), 1492(s), 1438(s),
1408(s), 1244(d), 1180(s), 820(s), 761(s), 695(s). 1H NMR(400
MHz, CDCl3), δ: 1.94―1.95(m, 4H), 2.88(s, 2H), 3.19(s, 2H),
4.68(s, 2H), 7.10(d, 2H, J=8.8 Hz), 7.18(t, 1H), 7.38(t, 2H),
7.56―7.61(m, 4H), 8.24(s, 1H), 8.52(s, 1H); 13C NMR(150
MHz, CDCl3), δ: 22.62(d, J=17.1 Hz), 25.95, 26.92, 67.64,
115.86, 120.27, 120.65, 125.07, 127.52, 129.19, 136.77, 137.19,
137.79, 151.63, 158.09, 163.36, 165.78.
4-[4-(4-Fluoroanilino)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7f): a yellowish
solid(0.6 g), yield 85.1%, m. p. 195—197 °C. ESI-MS, m/z:
calcd. 465.10(M+); found 466.2([M+H]+), 488.2([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3270(s), 2929(s), 1680(s), 1537(s), 1510(d),
1412(s), 1257(s), 1216(s), 827(s). 1H NMR(400 MHz, CDCl3),
δ: 1.95(t, 4H), 2.88(s, 2H), 3.19(s, 2H), 4.67(s, 2H),
7.05―7.10(m, 4H), 7.55―7.58(m, 4H), 8.22(s, 1H), 8.52(s,
1H); 13C NMR(150 MHz, CDCl3), δ: 22.62(d, J=17.3 Hz),
25.96, 26.92, 67.58, 115.83(d, J=6.5 Hz), 115.95, 120.72,
122.16(d, J=8.0 Hz), 127.53, 137.25, 137.80, 151.61, 158.05,
159.03, 160.65, 163.34, 165.79.
4-[4-(4-Chloroanilino)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7g): a yellowish
solid(0.3 g), yield 41.0%, m. p. 208—210 °C. ESI-MS, m/z:
calcd. 481.07(M+); found 482.1([M+H]+), 504.1([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3262(s), 2926(s), 1682(s), 1593(s), 1532(s),
1492(s), 1413(s), 1258(s), 1090(s), 827(s). 1H NMR(600 MHz,
CDCl3), δ: 1.92―1.95(m, 4H), 2.86―2.88(m, 2H),
3.17―3.19(m, 2H), 4.66(s, 2H), 7.08(d, 2H, J=8.4 Hz), 7.33(d,
2H, J=8.4 Hz), 7.56(d, 4H, J=8.4 Hz), 8.24(s, 1H), 8.51(s, 1H);
13
C NMR(150 MHz, CDCl3), δ: 22.62(d, J=17.4 Hz), 25.97,
26.93, 67.60, 115.86, 120.78, 121.49, 126.21, 127.53, 129.22,
129.68, 130.12, 135.37, 137.28, 137.82, 151.58, 158.00, 163.32,
165.83.
4-[4-(4-Bromoanilino)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7h): a yellowish
solid(0.5 g), yield 74.7%, m. p. 198—199 °C. ESI-MS, m/z:
calcd. 525.02(M+); found 526.4([M+H]+). IR(KBr), ߥ෤ /cm–1:
3261(s), 2925(s), 1682(s), 1654(s), 1531(s), 1492(s), 1412(s),
1255(s), 1071(s), 813(d). 1H NMR(400 MHz, CDCl3), δ: 1.95(s,
4H), 2.88(s, 2H), 3.19(s, 2H), 4.66(s, 2H), 7.08(d, 2H, J=8.4
Hz), 7.47―7.59(m, 6H), 8.24(s, 1H), 8.52(s, 1H); 13C NMR
(150 MHz, CDCl3), δ: 22.62(d, J=17.3 Hz), 25.96, 26.92, 67.60,
115.85, 120.80, 121.18, 121.79, 126.46, 127.52, 132.17, 132.64,
135.88, 137.25, 137.80, 138.41, 151.61, 157.98, 163.30, 163.71,
165.83.
4-[2-(4-Morpholinyl)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7i): a yellowish
solid(0.5 g), yield 82.1%, m. p. 164—165 °C. ESI-MS, m/z:
calcd. 441.12(M+); found 441.8([M+H]+), 464.1([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3441(m), 2943(s), 1643(s), 1626(s), 1557(s),
1504(s), 1471(s), 1436(s), 1305(s), 1198(s), 1115(s), 1070(s),
977(s), 777(s). 1H NMR(400 MHz, CDCl3), δ: 1.94―1.95(m,
4H), 2.90(m, 2H), 3.13(m, 2H), 3.32(t, 2H), 3.50(t, 2H),
5
3.54―3.57(m, 2H), 3.60―3.62(m, 2H), 3.65(s, 2H), 7.20(dd,
1H, J1=8.0 Hz, J2=1.2 Hz), 7.28―7.32(m, 1H), 7.35―7.40(m,
1H), 7.65(dd, 1H, J1=8.0 Hz, J2=1.2 Hz), 8.45(s, 1H);
13
C NMR(150 MHz, CDCl3), δ: 22.45, 22.96, 25.71, 26.06,
35.66, 42.35, 46.72, 66.45, 66.74, 119.08, 123.19, 126.85,
127.31, 128.33, 128.67, 132.14, 136.44, 151.23, 151.96, 162.90,
166.80, 168.80.
4-[2-(1-Piperidyl)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7j): a yellowish
solid(0.5 g), yield 85.7%, m. p. 127—129 °C. ESI-MS, m/z:
calcd. 439.14(M+); found 440.2([M+H]+). IR(KBr), ߥ෤ /cm–1:
3426(m), 2939(s), 2856(s), 1642(s), 1624(s), 1556(s), 1506(s),
1471(s), 1390(s), 1309(s), 1193(s), 1138(s), 1070(s), 978(s),
774(s). 1H NMR(400 MHz, CDCl3), δ: 1.40―1.52(m, 4H),
1.55―1.60(m, 2H), 1.92―1.97(m, 4H), 2.89(m, 2H), 3.14(m,
2H), 3.25(t, 2H), 3.49(t, 2H), 3.68(s, 2H), 7.19(dd, 1H, J1=7.6
Hz, J2=1.6 Hz), 7.27―7.37(m, 2H), 7.64(dd, 1H, J1=7.6 Hz,
J2=1.6 Hz), 8.45(s, 1H); 13C NMR(150 MHz, CDCl3), δ: 22.46,
22.98, 24.37, 25.49, 25.73, 26.06, 26.32, 36.18, 43.17, 47.48,
119.16, 123.09, 126.83, 127.45, 128.16, 129.07, 131.64, 136.26,
150.88, 152.00, 162.97, 166.28, 168.72.
4-(2-Diethylaminocarbonylmethoxylphenthio)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7k): a yellowish
solid(0.4 g), yield 72.2%, m. p. 117—119 °C. ESI-MS, m/z:
calcd. 427.14(M+); found 428.2([M+H]+). IR(KBr), ߥ෤ /cm–1:
3428(m), 2936(s), 1649(s), 1559(s), 1503(s), 1466(s), 1390(s),
1309(s), 1193(s), 1136(s), 1069(s), 974(s), 781(s). 1H NMR
(400 MHz, CDCl3), δ: 1.06―1.10(m, 6H3), 1.91―1.96(m, 4H),
2.88―2.91(m, 2H), 3.12―3.15(m, 2H), 3.18―3.24(m, 2H),
3.31―3.36(m, 2H), 3.66(s, 2H), 7.19(dd, 1H, J1=8.0 Hz,
J2=1.6 Hz), 7.26―7.30(m, 1H), 7.33―7.37(m, 1H), 7.62(dd,
1H, J1=8.0 Hz, J2=1.6 Hz), 8.45(s, 1H); 13C NMR(150 MHz,
CDCl3), δ: 12.87, 14.29, 22.40, 22.92, 25.66, 25.99, 36.29,
40.46, 42.47, 119.11, 123.02, 126.69, 127.41, 128.19, 128.99,
131.95, 136.18, 151.02, 151.95, 162.94, 167.12, 168.69.
4-[2-(1-Pyrrolidyl)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7l): a yellowish
solid(0.6 g), yield 80.9%, m. p. 146—147 °C. ESI-MS, m/z:
calcd. 425.12(M+); found 426.0([M+H]+). IR(KBr), ߥ෤ /cm–1:
3432(m), 2937(s), 2878(m), 1646(s), 1628(s), 1557(s), 1506(s),
1471(s), 1435(s), 1391(s), 1307(s), 1194(s), 1070(s), 977(s),
776(s). 1H NMR(400 MHz, CDCl3), δ: 1.77―1.88(m, 4H),
1.91―1.99(m, 4H), 2.89(m, 2H), 3.13(m, 2H), 3.32(t, 2H),
3.43(t, 2H), 3.60(s, 2H), 7.19(dd, 1H, J1=7.6 Hz, J2=1.6 Hz),
7.27―7.36(m, 2H), 7.66(dd, 1H, J1=7.6 Hz, J2=1.6 Hz), 8.45(s,
1H); 13C NMR(150 MHz, CDCl3), δ: 22.44, 22.96, 24.32, 25.71,
26.03, 26.18, 37.10, 46.21, 46.87, 119.15, 123.03, 126.79,
127.47, 128.11, 129.13, 131.70, 136.25(d, J=7.8 Hz), 150.84,
151.98, 162.96, 166.45, 168.71.
4-(2-Anilinocarbony lmethoxy lphenthio)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7m): a yello-
wish solid(0.3 g), yield 57.9%, m. p. 187—189 °C. ESI-MS,
m/z: calcd. 447.11(M+); found 448.1([M+H]+), 470.1([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3440(m), 3256(d), 2936(d), 1659(s), 1604(s),
1552(s), 1502(s), 1470(s), 1390(s), 1334(s), 1306(s), 1194(s),
1067(s), 974(s), 767(m). 1H NMR(400 MHz, CDCl3), δ:
1.92―1.96(m, 4H), 2.88―2.90(m, 2H), 3.12―3.14(m, 2H),
6   Chem. Res. Chin. Univ.
3.69(s, 2H), 7.08―7.12(m, 1H), 7.20(dd, 1H, J1=8.0 Hz,
J2=1.2 Hz), 7.25―7.38(m, 6H), 7.46(dd, 1H, J1=8.0 Hz, J2=1.2
Hz), 8.37(s, 1H), 8.53(s, 1H); 13C NMR(150 MHz, CDCl3), δ:
22.36, 22.89, 25.70, 26.03, 37.05, 119.17, 120.31, 123.29,
124.83, 127.22(d, J=5.9 Hz), 127.72, 128.63, 128.94, 130.16,
136.84, 137.23, 150.95, 151.75, 162.80, 165.61, 169.11.
4-[2-(4-Fluoroanilino)carbonylmethoxylphenthio]-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7n): a yellowish
solid(0.2 g), yield 39.0%, m. p. 122—124 °C. ESI-MS, m/z:
calcd. 465.10(M+); found 465.9([M+H]+). IR(KBr), ߥ෤ /cm–1:
3275(m), 2934(s), 1670(s), 1556(s), 1508(s), 1469(s), 1436(s),
1390(s), 1305(s), 1197(s), 1067(s), 974(s), 835(s), 777(s).
1
H NMR(400 MHz, CDCl3), δ: 1.92―1.96(m, 4H), 2.89(m,
2H), 3.12(m, 2H), 3.69(s, 2H), 6.96(t, 2H), 7.19―7.29(m, 4H),
7.34―7.39(m, 1H), 7.45(dd, 1H, J1=8.0 Hz, J2=1.6 Hz), 8.32(s,
1H), 8.56(s, 1H); 13C NMR(150 MHz, CDCl3), δ: 22.34, 22.87,
25.68, 26.01, 36.91, 115.58(d, J=22.5 Hz), 119.18, 122.26(d,
J=8.0 Hz), 123.27, 127.21(d, J=6.8 Hz), 127.70, 128.68,
130.27, 133.26, 136.93, 151.03, 151.61, 160.49, 162.79, 165.73,
169.1.
3.2 In vitro Antitumor Activity Assay
A human pulmonary carcinoma cell line A549 was used to
evaluate the antitumor activity of target compounds in vitro by
MTT assay. A549 cells were cultured in a Dulbecco’s modified
Eagle’s medium(DMEM) supplemented with 10% fetal bovine
serum(FBS), 100 U/mL penicillin and 100 g/mL streptomycin.
Cells were grown to 80%—90% confluence and subjected to
no more than 20 cell passages and were left to acclimate for
24 h before any treatments. The cells were seeded into 96-well
plates(8×104 cells/mL) and incubated at 37 °C in humidified
atmosphere containing 5% CO2 overnight. After 24 h, the cells
were treated with 20/50 μmol/L target compounds. After 24-h
incubation, MTT(5 mg/mL) was subsequently added and incu-
bated for 4 h. The culture medium was removed, and the crys-
tals were dissolved in DMSO. The optical densities(OD) at 490
nm were measured on a microplate reader(TECAN SPECTRA,
Wetzlar, Germany). Inhibitory effects were expressed as the
percentage of inhibition and IC50 value.
4 Conclusions
A series of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
derivatives has been synthesized and the antitumor activities of
the derivatives against the human pulmonary carcinoma cell
line A549, which has abundant expression of EGFR were eva-
luated via the MTT colorimetric assay. The results show that
several target compounds exhibit moderate antitumor activity,
and the compound 4-[2-(1-piperidyl)carbonylmethoxylphen-
thio]-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7j)
has demonstrated the most potent antitumor activity. In general,
a preliminary structure-activity relationship has been clearly
discerned. It is hoped that this series of novel potential EGFR
inhibitors can be used as lead compounds for the further re-
search.
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