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, 1. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox-
the quinazoline ring in the marketed EGFR kinase inhibitors amide(2) was synthesized from cyclohexanone(1), cyanoace-
with 4-anilinoquinazoline scaffold was replaced by the tamide and sublimed sulfur via Gewald reaction[15]. The prepa-
novel 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine ring. ration of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidin-
Secondly, the 4-anilino group was replaced by 4-arylthio group 4(3H)-one(3) was achieved via the reaction of compound 2
as the bioisosteres. Thirdly, the side chains in the scaffold with formamide [16] . Compound 3 was converted into 4-
and the 4-arylthio group were chosen conventionally which chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidine(4)
have been widely utilized in designing many EGFR inhibitors. by further treatment with phosphorus oxychloride [17] .
Hence, a novel series of 5,6,7,8-tetrahydrobenzo- 4-(Methoxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
[4,5]thieno[2,3-d]pyrimidine derivatives was synthesized, and d]pyrimidines(5) was obtained via the reaction of compound 4,
the antitumor activities of them against human pulmonary car- 4-methoxythiophenol and triethylamine. Finally, compound 5
cinoma cell line A549 were screened for the discovery of novel was treated by anhydrous aluminum trichloride as a demethyla-
potential EGFR inhibitors. tion reagent to prepare 4-(hydroxyphenthio)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidines(6). The target com-
2 Results and Discussion pound derivatives(7) were synthesized from compound 6 via
the ordinary Williamson reaction in satisfactory yields.
2.1 Chemistry The chemical structures of all the target compounds were
The synthetic pathways of the 5,6,7,8-tetrahydrobenzo- fully characterized by ESI-MS, IR spectra, 1H NMR and
13
[4,5]thieno[2,3-d]pyrimidine derivatives are shown in Scheme 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. 3
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
Fig.3 Interaction between compound 7j and
docking algorithm was set at 1500 maximum iterations with a
active site of EGFR
simple evolution population size of 50 and a minimum of 10
runs. Its original inhibitor(Erlotinib) was eliminated and all the
3 Experimental
water molecules were deleted, adding a further degree of
freedom in terms of variability in the docking results. 3.1 Chemistry
Schematic diagrams of the interactions between 1M17 and
small molecules were analyzed by the Discovery Studio 4.0 Starting materials and solvents were purchased from
Client. common commercial suppliers and were used without further
Based on the docking results, the interactions between Er- purification. Melting points were determined in open capillary
lotinib and EGFR are shown in Fig.2. A hydrogen tubes and uncorrected. Reaction progress was monitored by
bond(represented as a green dotted line) has been formed thin layer chromatography on silica gel sheets. The spots were
between N1 position of the parent nucleus and the Met769 visualized with UV light(254 nm). Mass spectra were recorded
by electrospray(ESI) on a Waters spectrometer(Massachusetts,
residue. There are hydrophobic effects(represented as violet
USA). The IR spectra were obtained on a Bruker IFS55 spec-
dotted lines) between Erlotinib and the residues, such as
trometer(Karlsruhe, Germany). NMR spectra were recorded on
Leu694, Ala719, Lys721, Met769 and Leu820.
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%
Fig.2 Interactions between Erlotinib and and used for the next reaction without further purification. m. p.
active site of EGFR 186—188°C(lit.[13]: 189—190 °C) .
According to the predicted docking interactions between Synthesis of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-
compound 7j and EGFR, a hydrogen bond(represented as a pyrimidin-4(3H)-one(3): a stirred mixture of compound 2(11.6
green dotted line, Fig.3) has been formed between N1 position g, 59.2 mmol) and formamide(46.4 g, 1031.0 mmol) was
of the parent nucleus and the Met769 residue. There are hy- heated at 165 °C for 6 h. The reaction mixture was cooled at
drophobic effects(represented as violet dotted lines) between room temperature for crystallization. Isopropanol(110 mL) was
compound 7j and the residues, such as Leu694, Ala719, Lys721, added and the mixture was stirred at room temperature for 1 h.
Met742, Leu764, Leu768, Met769, Cys773 and Leu820. Com- The precipitated solid was filtered off and washed with isopro-
pared with Erlotinib, more hydrophobic effects can be found panol. A total of 9.7 g of brown solid was obtained in a yield of
between compound 7j and amino acid residues. They are 80.0%. m. p. 255—257 °C(lit.[14]: 255—256 °C) .
Met742 , Leu764 , Leu768 and Cys773. Synthesis of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno-
Therefore, the target compound 7j has similar active sites [2,3-d]pyrimidine(4): a stirred mixture of compound 3(8.7 g,
with Erlotinib in the interaction with EGFR and more 42.0 mmol) and phosphorus oxychloride(70.9 g, 467.1 mmol)
interactions have been found between compound 7j and was heated under reflux for 30 min. After the solvent was
EGFR. evaporated under reduced pressure, the black crude product
4   Chem. Res. Chin. Univ.
was purified by column chromatography on silica gel heated under reflux for 12 h. After the reaction mixture was
[V(petroleum ether):V(ethyl acetate)=2:1] and a yellow solid evaporated, water(30 mL) was added and the solution was ex-
was obtained(6.7 g, yield 70.5%), m. p. 97—99 °C(lit.[15]: tracted with dichloromethane(3×20 mL), dried with anhy-
99—100 °C) . drous MgSO4 and evaporated again. Crude products were
Synthesis of 4-(methoxyphenthio)-5,6,7,8-tetrahydro- purified by column chromatography on silica gel
benzo[4,5]thieno[2,3-d]pyrimidines(5a and 5b): a stirred mix- [V(methanol):V(dichloromethane)=1:80].
ture of compound 4(8.0 g, 35.6 mmol), 4-methoxythiophenol or 4-[4-(4-Morpholinyl)carbonylmethoxylphenthio]-5,6,7,8-
2-methoxythiophenol(5.5 g, 39.2 mmol), triethylamine(10.8 g, tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7a): a yellowish
106.8 mmol) and n-butanol(102 mL) was heated under reflux solid(0.6 g), yield 78.6%, m. p. 141—144 °C. ESI-MS, m/z:
for 1 h. The reaction mixture was cooled at room temperature calcd. 441.12(M+); found 442.0([M+H]+), 463.8([M+Na]+).
for crystallization. The solid was collected by filtration, washed IR(KBr), ߥ෤/cm–1: 3442(m), 2932(s), 2857(s), 1650(s), 1591(s),
with water and ethanol to afford compound 5 after drying. 1492(s), 1440(s), 1409(s), 1380(s), 1236(s), 1110(s), 1031(s),
4-(4-Methoxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]- 829(s), 731(s). 1H NMR(600 MHz, CDCl3), δ: 1.92—1.96(m,
thieno[2,3-d]pyrimidine(5a): a yellow solid(10.1 g), yield 4H), 2.87―2.88(m, 2H), 3.18―3.19(m, 2H), 3.61―3.63(m,
86.2%, m. p. 151—152 °C. ESI-MS, m/z: calcd. 328.07(M+); 2H), 3.65―3.66(m, 2H), 3.68―3.70(m, 4H), 4.75(s, 2H),
found 328.9([M+H]+), 351.1([M+Na]+). IR(KBr), ߥ෤ /cm–1: 7.04(d, 2H, J=8.4 Hz), 7.50(d, 2H, J=8.4 Hz), 8.52(s, 1H);
13
2930(s), 1640(m), 1591(s), 1492(s), 1411(s), 1173(s), 1129(s), C NMR(150 MHz, CDCl3), δ: 22.52, 22.63, 25.90, 26.88,
825(s). 1H NMR(600 MHz, CDCl3), δ: 1.92—1.96(m, 4H), 42.49, 45.95, 66.75(d, J=13.7 Hz), 67.60, 115.63, 119.66,
2.87(s, 2H), 3.20(s, 2H), 3.86(s, 3H), 7.00(d, J=8.8 Hz, 2H), 127.52, 127.97, 136.98, 137.52, 151.65, 158.87, 163.55, 165.71,
7.49(d, J = 8.8 Hz, 2H), 8.53(s, 1H). 166.20.
4-(2-Methoxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]- 4-[4-(1-Piperidyl)carbonylmethoxylphenthio]-5,6,7,8-
thieno[2,3-d]pyrimidine(5b): a yellow solid(11.1 g), yield tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7b): a yellowish
94.5%, m. p. 161—162 °C. ESI-MS, m/z: calcd. 328.07(M+); solid(0.6 g), yield 91.4%, m. p. 84—86 °C. ESI-MS, m/z: calcd.
found 329.3([M+H]+). IR(KBr), ߥ෤ /cm–1: 2936(s), 1643(m), 439.14(M+); found 440.5([M+H]+), 461.8([M+Na]+). IR(KBr),
1584(s), 1496(s), 1415(s), 1168(s), 1129(s), 832(s). 1H NMR ߥ෤/cm–1: 3443(m), 2935(s), 2855(s), 1639(s), 1592(s), 1493(s),
(600 MHz, CDCl3), δ: 1.92—1.96(m, 4H), 2.87(s, 2H), 3.24(s, 1383(s), 1241(s), 1178(s), 1128(s), 1036(m), 826(s), 727(s).
1
2H), 3.79(s, 3H), 7.01―7.07(m, 2H), 7.49(t, 1H), 7.55(dd, H NMR(400 MHz, CDCl3), δ: 1.58—1.66(m, 6H),
J1=7.5 Hz, J2=1.6 Hz, 1H), 8.49(s, 1H). 1.91—1.97(m, 4H), 2.86—2.88(m, 2H), 3.19(brs, 2H), 3.50(t,
Synthesis of 4-(hydroxyphenthio)-5,6,7,8- 2H), 3.58(t, 2H), 4.73(s, 2H), 7.04(d, 2H, J=8.4 Hz), 7.49(d,
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines(6a and 6b): a 2H, J=8.4 Hz), 8.51(s, 1H); 13C NMR(150 MHz, CDCl3), δ:
stirred mixture of 5a or 5b(9.0 g, 27.4 mmol), anhydrous alu- 22.51, 22.62, 24.44, 25.52, 25.89, 26.49, 26.89, 43.25, 46.46,
minum trichloride(11.0 g, 82.2 mmol) and toluene(193 mL) 67.70, 115.69, 119.20, 127.53, 127.94, 136.88, 137.39, 151.65,
was heated under reflux for 1 h. After cooling down to room 159.23, 163.68, 165.67.
temperature, water(280 mL) was added to the system and the 4-(4-Diethylincarbonylmethoxylphenthio)-5,6,7,8-
mixture stood for a night. The precipitate was filtered off and tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7c): a yellowish
purified by column chromatography on silica gel[V(methanol): solid(0.6 g), yield 90.8%, m. p. 88—91 °C. ESI-MS, m/z: calcd.
V(dichloromethane)=1:100]. 427.14(M+); found 428.2([M+H]+). IR(KBr), ߥ෤/cm–1: 3577(s),
4-(4-Hydroxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]thie- 3489(s), 2971(s), 2934(s), 1647(s), 1614(s), 1594(s), 1494(s),
no[2,3-d]pyrimidine(6a): a yellowish solid(5.0 g), yield 58.0%, 1410(s), 1250(s), 1078(s), 835(s), 728(s), 616(s). 1H NMR(400
m. p. 230—231 °C. ESI-MS, m/z: calcd. 314.05(M+); found MHz, CDCl3), δ: 1.16(t, 3H), 1.24(t, 3H), 1.94(t, 4H), 2.87(t,
314.9([M+H]+), 337.2([M+Na]+). IR(KBr), ߥ෤/cm–1: 3421(m), 2H), 3.18(t, 2H), 3.41(m, 4H), 4.72(s, 2H), 7.04(d, 2H, J=8.8
2930(s), 1640(m), 1599(s), 1492(s), 1413(s), 1165(s), 1130(s), Hz), 7.49(d, 2H, J=8.8 Hz), 8.52(s, 1H); 13C NMR(150 MHz,
831(s). 1H NMR(400 MHz, CDCl3), δ: 1.92—1.98(m, 4H), CDCl3), δ: 12.84, 14.40, 22.61(d, J=17.0 Hz), 25.93, 26.93,
2.88(s, 2H), 3.20(s, 2H), 6.87(d, J=8.6 Hz, 2H), 7.42(d, J=8.6 40.42, 41.63, 67.51, 115.77, 119.20, 127.59, 128.00, 136.94,
Hz, 2H), 8.56(s, 1H). 137.44, 151.69, 159.36, 163.78, 165.67, 166.63.
4-(2-Hydroxyphenthio)-5,6,7,8-tetrahydrobenzo[4,5]- 4-[4-(1-Pyrrolidyl)carbonylmethoxylphenthio]-5,6,7,8-
thieno[2,3-d]pyrimidine(6b): a yellowish solid(5.6 g), yield tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7d): a yellowish
64.5%, m. p. 175—176 °C. ESI-MS, m/z: calcd. 314.05(M+); solid(0.6 g), yield 83.8%, m. p. 125—127 °C. ESI-MS, m/z:
found 315.3([M+H]+). IR(KBr), ߥ෤/cm–1: 3421(m), 2932(m), calcd. 425.12(M+); found 426.1([M+H]+), 448.2([M+Na]+).
1632(m), 1596(m), 1473(s), 1409(s), 1384(s), 1142(s), 830(m). IR(KBr), ߥ෤/cm–1: 3556(s), 3476(s), 2936(s), 2882(s), 1649(s),
1
H NMR(400 MHz, CDCl3), δ: 1.95—1.99(m, 4H), 2.90(s, 2H), 1595(s), 1494(s), 1410(s), 1251(s), 1175(s), 833(s), 720(s).
1
3.23(s, 2H), 6.94(t, 1H), 7.17(dd, J1=8.1 Hz, J2=1.0 Hz, 1H), H NMR(400 MHz, CDCl3), δ: 1.84―2.02(m, 8H), 2.87(t, 2H),
7.42(t, 1H), 7.49(dd, J1=7.8 Hz, J2=1.4 Hz, 1H), 8.61(s, 1H). 3.19(t, 2H), 3.55(t, 4H), 4.67(s, 2H), 7.04(d, 2H, J=8.8 Hz),
Synthesis of the target compounds(7a―7n): a stirred mix- 7.49(d, 2H, J=8.8 Hz), 8.52(s, 1H); 13C NMR(150 MHz,
ture of 6a or 6b(0.5 g, 1.6 mmol), 2-chloroacetamide(1.9 CDCl3), δ: 22.52(d, J=16.7 Hz), 23.79, 25.85, 26.21, 26.85,
mmol), anhydrous potassium carbonate(2.2 g, 16.0 mmol), 45.98, 46.22, 67.80, 115.67, 119.15, 127.50, 127.90, 136.87,
potassium iodide(0.1 g, 0.8 mmol) and acetone(50 mL) was 137.36, 151.60, 159.22, 163.65, 165.60, 166.08.
 SUN Bing et al. 5
4-(4-Anilinocarbony lmethoxy lphenthio)-5,6,7,8- 3.54―3.57(m, 2H), 3.60―3.62(m, 2H), 3.65(s, 2H), 7.20(dd,
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7e): a yellowish 1H, J1=8.0 Hz, J2=1.2 Hz), 7.28―7.32(m, 1H), 7.35―7.40(m,
solid(0.6 g), yield 77.5%, m. p. 196—198 °C. ESI-MS, m/z: 1H), 7.65(dd, 1H, J1=8.0 Hz, J2=1.2 Hz), 8.45(s, 1H);
13
calcd. 447.11(M+); found 448.1([M+H]+). IR(KBr), ߥ෤ /cm–1: C NMR(150 MHz, CDCl3), δ: 22.45, 22.96, 25.71, 26.06,
3374(s), 2929(s), 1684(s), 1599(s), 1534(s), 1492(s), 1438(s), 35.66, 42.35, 46.72, 66.45, 66.74, 119.08, 123.19, 126.85,
1408(s), 1244(d), 1180(s), 820(s), 761(s), 695(s). 1H NMR(400 127.31, 128.33, 128.67, 132.14, 136.44, 151.23, 151.96, 162.90,
MHz, CDCl3), δ: 1.94―1.95(m, 4H), 2.88(s, 2H), 3.19(s, 2H), 166.80, 168.80.
4.68(s, 2H), 7.10(d, 2H, J=8.8 Hz), 7.18(t, 1H), 7.38(t, 2H), 4-[2-(1-Piperidyl)carbonylmethoxylphenthio]-5,6,7,8-
7.56―7.61(m, 4H), 8.24(s, 1H), 8.52(s, 1H); 13C NMR(150 tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7j): a yellowish
MHz, CDCl3), δ: 22.62(d, J=17.1 Hz), 25.95, 26.92, 67.64, solid(0.5 g), yield 85.7%, m. p. 127—129 °C. ESI-MS, m/z:
115.86, 120.27, 120.65, 125.07, 127.52, 129.19, 136.77, 137.19, calcd. 439.14(M+); found 440.2([M+H]+). IR(KBr), ߥ෤ /cm–1:
137.79, 151.63, 158.09, 163.36, 165.78. 3426(m), 2939(s), 2856(s), 1642(s), 1624(s), 1556(s), 1506(s),
4-[4-(4-Fluoroanilino)carbonylmethoxylphenthio]-5,6,7,8- 1471(s), 1390(s), 1309(s), 1193(s), 1138(s), 1070(s), 978(s),
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7f): a yellowish 774(s). 1H NMR(400 MHz, CDCl3), δ: 1.40―1.52(m, 4H),
solid(0.6 g), yield 85.1%, m. p. 195—197 °C. ESI-MS, m/z: 1.55―1.60(m, 2H), 1.92―1.97(m, 4H), 2.89(m, 2H), 3.14(m,
calcd. 465.10(M+); found 466.2([M+H]+), 488.2([M+Na]+). 2H), 3.25(t, 2H), 3.49(t, 2H), 3.68(s, 2H), 7.19(dd, 1H, J1=7.6
IR(KBr), ߥ෤/cm–1: 3270(s), 2929(s), 1680(s), 1537(s), 1510(d), Hz, J2=1.6 Hz), 7.27―7.37(m, 2H), 7.64(dd, 1H, J1=7.6 Hz,
1412(s), 1257(s), 1216(s), 827(s). 1H NMR(400 MHz, CDCl3), J2=1.6 Hz), 8.45(s, 1H); 13C NMR(150 MHz, CDCl3), δ: 22.46,
δ: 1.95(t, 4H), 2.88(s, 2H), 3.19(s, 2H), 4.67(s, 2H), 22.98, 24.37, 25.49, 25.73, 26.06, 26.32, 36.18, 43.17, 47.48,
7.05―7.10(m, 4H), 7.55―7.58(m, 4H), 8.22(s, 1H), 8.52(s, 119.16, 123.09, 126.83, 127.45, 128.16, 129.07, 131.64, 136.26,
1H); 13C NMR(150 MHz, CDCl3), δ: 22.62(d, J=17.3 Hz), 150.88, 152.00, 162.97, 166.28, 168.72.
25.96, 26.92, 67.58, 115.83(d, J=6.5 Hz), 115.95, 120.72, 4-(2-Diethylaminocarbonylmethoxylphenthio)-5,6,7,8-
122.16(d, J=8.0 Hz), 127.53, 137.25, 137.80, 151.61, 158.05, tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7k): a yellowish
159.03, 160.65, 163.34, 165.79. solid(0.4 g), yield 72.2%, m. p. 117—119 °C. ESI-MS, m/z:
4-[4-(4-Chloroanilino)carbonylmethoxylphenthio]-5,6,7,8- calcd. 427.14(M+); found 428.2([M+H]+). IR(KBr), ߥ෤ /cm–1:
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7g): a yellowish 3428(m), 2936(s), 1649(s), 1559(s), 1503(s), 1466(s), 1390(s),
solid(0.3 g), yield 41.0%, m. p. 208—210 °C. ESI-MS, m/z: 1309(s), 1193(s), 1136(s), 1069(s), 974(s), 781(s). 1H NMR
calcd. 481.07(M+); found 482.1([M+H]+), 504.1([M+Na]+). (400 MHz, CDCl3), δ: 1.06―1.10(m, 6H3), 1.91―1.96(m, 4H),
IR(KBr), ߥ෤/cm–1: 3262(s), 2926(s), 1682(s), 1593(s), 1532(s), 2.88―2.91(m, 2H), 3.12―3.15(m, 2H), 3.18―3.24(m, 2H),
1492(s), 1413(s), 1258(s), 1090(s), 827(s). 1H NMR(600 MHz, 3.31―3.36(m, 2H), 3.66(s, 2H), 7.19(dd, 1H, J1=8.0 Hz,
CDCl3), δ: 1.92―1.95(m, 4H), 2.86―2.88(m, 2H), J2=1.6 Hz), 7.26―7.30(m, 1H), 7.33―7.37(m, 1H), 7.62(dd,
3.17―3.19(m, 2H), 4.66(s, 2H), 7.08(d, 2H, J=8.4 Hz), 7.33(d, 1H, J1=8.0 Hz, J2=1.6 Hz), 8.45(s, 1H); 13C NMR(150 MHz,
2H, J=8.4 Hz), 7.56(d, 4H, J=8.4 Hz), 8.24(s, 1H), 8.51(s, 1H); CDCl3), δ: 12.87, 14.29, 22.40, 22.92, 25.66, 25.99, 36.29,
13
C NMR(150 MHz, CDCl3), δ: 22.62(d, J=17.4 Hz), 25.97, 40.46, 42.47, 119.11, 123.02, 126.69, 127.41, 128.19, 128.99,
26.93, 67.60, 115.86, 120.78, 121.49, 126.21, 127.53, 129.22, 131.95, 136.18, 151.02, 151.95, 162.94, 167.12, 168.69.
129.68, 130.12, 135.37, 137.28, 137.82, 151.58, 158.00, 163.32, 4-[2-(1-Pyrrolidyl)carbonylmethoxylphenthio]-5,6,7,8-
165.83. tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7l): a yellowish
4-[4-(4-Bromoanilino)carbonylmethoxylphenthio]-5,6,7,8- solid(0.6 g), yield 80.9%, m. p. 146—147 °C. ESI-MS, m/z:
tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine(7h): a yellowish calcd. 425.12(M+); found 426.0([M+H]+). IR(KBr), ߥ෤ /cm–1:
solid(0.5 g), yield 74.7%, m. p. 198—199 °C. ESI-MS, m/z: 3432(m), 2937(s), 2878(m), 1646(s), 1628(s), 1557(s), 1506(s),
calcd. 525.02(M+); found 526.4([M+H]+). IR(KBr), ߥ෤ /cm–1: 1471(s), 1435(s), 1391(s), 1307(s), 1194(s), 1070(s), 977(s),
3261(s), 2925(s), 1682(s), 1654(s), 1531(s), 1492(s), 1412(s), 776(s). 1H NMR(400 MHz, CDCl3), δ: 1.77―1.88(m, 4H),
1255(s), 1071(s), 813(d). 1H NMR(400 MHz, CDCl3), δ: 1.95(s, 1.91―1.99(m, 4H), 2.89(m, 2H), 3.13(m, 2H), 3.32(t, 2H),
4H), 2.88(s, 2H), 3.19(s, 2H), 4.66(s, 2H), 7.08(d, 2H, J=8.4 3.43(t, 2H), 3.60(s, 2H), 7.19(dd, 1H, J1=7.6 Hz, J2=1.6 Hz),
Hz), 7.47―7.59(m, 6H), 8.24(s, 1H), 8.52(s, 1H); 13C NMR 7.27―7.36(m, 2H), 7.66(dd, 1H, J1=7.6 Hz, J2=1.6 Hz), 8.45(s,
(150 MHz, CDCl3), δ: 22.62(d, J=17.3 Hz), 25.96, 26.92, 67.60, 1H); 13C NMR(150 MHz, CDCl3), δ: 22.44, 22.96, 24.32, 25.71,
115.85, 120.80, 121.18, 121.79, 126.46, 127.52, 132.17, 132.64, 26.03, 26.18, 37.10, 46.21, 46.87, 119.15, 123.03, 126.79,
135.88, 137.25, 137.80, 138.41, 151.61, 157.98, 163.30, 163.71, 127.47, 128.11, 129.13, 131.70, 136.25(d, J=7.8 Hz), 150.84,
165.83. 151.98, 162.96, 166.45, 168.71.
4-[2-(4-Morpholinyl)carbonylmethoxylphenthio]-5,6,7,8- 4-(2-Anilinocarbony lmethoxy lphenthio)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7i): a yellowish tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(7m): a yello-
solid(0.5 g), yield 82.1%, m. p. 164—165 °C. ESI-MS, m/z: wish solid(0.3 g), yield 57.9%, m. p. 187—189 °C. ESI-MS,
calcd. 441.12(M+); found 441.8([M+H]+), 464.1([M+Na]+). m/z: calcd. 447.11(M+); found 448.1([M+H]+), 470.1([M+Na]+).
IR(KBr), ߥ෤/cm–1: 3441(m), 2943(s), 1643(s), 1626(s), 1557(s), IR(KBr), ߥ෤/cm–1: 3440(m), 3256(d), 2936(d), 1659(s), 1604(s),
1504(s), 1471(s), 1436(s), 1305(s), 1198(s), 1115(s), 1070(s), 1552(s), 1502(s), 1470(s), 1390(s), 1334(s), 1306(s), 1194(s),
977(s), 777(s). 1H NMR(400 MHz, CDCl3), δ: 1.94―1.95(m, 1067(s), 974(s), 767(m). 1H NMR(400 MHz, CDCl3), δ:
4H), 2.90(m, 2H), 3.13(m, 2H), 3.32(t, 2H), 3.50(t, 2H), 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, has demonstrated the most potent antitumor activity. In general,
J2=1.2 Hz), 7.25―7.38(m, 6H), 7.46(dd, 1H, J1=8.0 Hz, J2=1.2 a preliminary structure-activity relationship has been clearly
Hz), 8.37(s, 1H), 8.53(s, 1H); 13C NMR(150 MHz, CDCl3), δ: discerned. It is hoped that this series of novel potential EGFR
22.36, 22.89, 25.70, 26.03, 37.05, 119.17, 120.31, 123.29, inhibitors can be used as lead compounds for the further re-
124.83, 127.22(d, J=5.9 Hz), 127.72, 128.63, 128.94, 130.16, search.
136.84, 137.23, 150.95, 151.75, 162.80, 165.61, 169.11.
4-[2-(4-Fluoroanilino)carbonylmethoxylphenthio]-5,6,7,8- References
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4 Conclusions
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