A Microwave-Enhanced Synthesis and Biological Evaluation of N-Aryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amines

Article abstract of DOI:10.21577/0103-5053.20190044

A series of N-aryl-5,6,7,8-tetra-hydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amines were synthesized in moderate to good yield by using a microwave-enhanced conditions. The selected compounds were evaluated for their cytotoxic effects (IC₅₀ values) on human pulmonary carcinoma (A549), murine BALB/c spontaneous colon adenocarcinoma (CT26) and human hepatocellular liver carcinoma (HepG2) cell lines in vitro. Amongst these compounds, one compound was found to have the better cytotoxic activity with reference to the standard Erlotinib hydrochloride (Tarceva^TM) against A549 (IC₅₀ = 16.06 ± 0.09 μM) and HepG2 (IC₅₀ = 15.01 ± 0.31 μM) cell lines. Especially, two compounds showed best cytotoxic effects against CT26 (IC₅₀ = 11.38 ± 0.44 μM) and HepG2 (IC₅₀ = 8.51 ± 0.52 μM) cell lines, respectively. The preliminary structure-activity relationships were disclosed and the thieno[2,3-d]pyrimidine skeleton could be exploited to potential antitumor agents in the future.

Full text of DOI:10.21577/0103-5053.20190044

http://dx.doi.org/10.21577/0103-5053.20190044
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-15, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
A Microwave-Enhanced Synthesis and Biological Evaluation of
N-Aryl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amines
Qing Han, a,b Zijian Yin,c Jingjiao Sui,d Qingming Wanga and Yaquan Sun*,a,b,e
aSchool of Pharmacy, Yancheng Teachers University, 224051 Yancheng, PR China
bInstitute of Chemical Technology, Nanjing Tech University, 210000 Nanjing, PR China
cSchool of Pharmaceutical Engineering & Life Science, Changzhou University,
213164 Changzhou, PR China
dInstitute of Chemical Technology, Shanghai University of Engineering Science,
201620 Shanghai, PR China
eJiangsu Marine Industry Research Institute, 224051 Yancheng, PR China
A series of N-aryl-5,6,7,8-tetra-hydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amines were
synthesized in moderate to good yield by using a microwave-enhanced conditions. The selected
compounds were evaluated for their cytotoxic effects (IC50 values) on human pulmonary carcinoma
(A549), murine BALB/c spontaneous colon adenocarcinoma (CT26) and human hepatocellular liver
carcinoma (HepG2) cell lines in vitro. Amongst these compounds, one compound was found to
have the better cytotoxic activity with reference to the standard Erlotinib hydrochloride (TarcevaTM)
againstA549 (IC50 = 16.06 0.09 μM) and HepG2 (IC50 = 15.01 0.31 μM) cell lines. Especially,
two compounds showed best cytotoxic effects against CT26 (IC50 = 11.38 0.44 μM) and HepG2
(IC50 = 8.51 0.52 μM) cell lines, respectively. The preliminary structure-activity relationships
were disclosed and the thieno[2,3-d]pyrimidine skeleton could be exploited to potential antitumor
agents in the future.
Keywords: thieno[2,3-d]pyrimidine, microwave-enhanced, Gewald reaction, Dimroth
rearrangement, biological evaluation
Introduction
anti-leukemia,13,14 antiviral,15 anti-inflammatory16 and
anti-diabetic properties.17 As such, many antitumor agents
based on thieno[2,3-d]pyrimidine have been reported
by researchers. Yang et al.18 synthesized two series
of thieno[2,3-d]pyrimidine derivatives and evaluated
for cytotoxic activity in human pulmonary carcinoma
cell line (A549). Of these compounds, one showed the
most cytotoxic effect on A549 cells with half maximal
inhibitory concentration (IC50) value of 4.87 μM (Figure 2,
compound VII). Gryshchenko et al.19 reported the synthesis
of N-phenylthieno[2,3-d]pyrimidin-4-amines, and evaluated
for their ability to inhibit enzymatic activity of the fibroblast
growth factor receptor 1 (FGFR1). In these compounds,
compounds VIII and IX exhibited most effects, with IC50
values of 0.18 and 0.16 μM, respectively. Abbas et al.20
synthesized two series of new tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidines (2,3-disubstituted derivatives and
2,4-disubstituted ones) and evaluated for their cytotoxic
In investigation, we found the presence of several
marketed anticancer drugs such as gefitinib I (Iressa™),1
vandetanib II (Caprelsa™),2 lapatinib III (Tykerb™),3
erlotinib IV (Tarceva™),4 tandutinib V (MLN518)
(phase II clinical trials)5 and icotinib VI (Conmana™)6
(Figure 1), which have a quinazoline nucleus, the
thieno[2,3-d]pyrimidine core can be considered a
bioisostere of this quinazoline core.7
In literatures,8-10 thieno[2,3-d]pyrimidin-4-amine
derivatives exhibit a broad range of biological and
pharmacological activities associated with their heterocyclic
scaffold and have been widely used in the medical field.
Thieno[2,3-d]pyrimidin-4-amine displays a wide range of
biological activity including anti-tumor,11 anti-bacterial,12
*e-mail: sunyaquan@hotmail.com

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A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
Figure 1. Representative examples of potent anticancer quinazoline drugs.
activity. According to the results, compound X was found
to have the highest cytotoxic activity against breast MCF-7
with IC50 = 15.01 μM, and XI showed to be the most active
one to inhibit human hepatocellular liver carcinoma cell
line (HepG2) with IC50 = 1.29 μM. Besides, compound
XII21 showed the best FLT3 (FMS-like receptor tyrosine
kinase 3) cytotoxic activity, with IC50 value of 0.065 μM.
Herein, we take into consideration that the compounds
of thieno[2,3-d]pyrimidine skeleton express an important
biological cytotoxic activity, and intend to synthesize a
series of thieno[2,3-d]pyrimidine derivatives.
via different synthetic methods. Four strategies used for
the synthesis of various thieno[2,3-d]pyrimidine scaffolds
have been reported in literatures (Figure 3).11,14,22-29 The
first method involves the synthesis of thieno[2,3-d]
pyrimidin-4(3H)-one (XVII) from compounds XIV,
XV and XVI, then, compound XVII is converted to
4-chlorothieno[2,3-d]pyrimidine (XIX), and the target
compound XX is obtained from XIX. The second
route is that the compound XIII is directly converted
to compound XVII, and the final compound XX can
be obtained from XVII. The third protocol is the direct
synthesis of 4-chlorothieno[2,3-d]pyrimidine (XIX)
from compound XV. The fourth method is the synthesis
In this study, we are interested in the widely reported
cytotoxic activity of thieno[2,3-d]pyrimidine derivatives
Figure 2. Thieno[2,3-d]pyrimidine derivatives in literatures.18-21

Vol. 00, No. 00, 2019
Han et al.
3
Figure 3. A summary of the synthetic approaches for thieno[2,3-d]pyrimidines in literatures.11,14,22-29
of compound XVIII from XV, subsequently, compound
XV is converted to compound XIX, therefore, the target
compound XX can be synthesized by XV.
(Figure 4, compound XXIV)34 was evaluated to inhibit
EGFR (epidermal growth factor receptor) kinase activity
with the enzyme inhibition IC50 = 2.6 μM. Herein,
thieno[2,3-d]pyrimidine skeleton of compounds XXIII
and XXIV were used as a template, and the structure
was modified: (Figure 4a) hexatomic ring was linked to
thiophene ring instead of five membered ring (compound
XXIII); (Figure 4b) substituted aromatic group was linked to
secondary amine and –CH2 group was subtracted (compound
XXIV). Subsequently, compounds 3a-x (Scheme 1) were
obtained.
In this study, two questions would be solved: (i) a
series of N-aryl-5,6,7,8-tetra hydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amines (3a-x) were obtained with
the aid of microwave irradiation technology (Figure 5);
(ii) the cytotoxic activity of synthesized compounds were
evaluated againstA549, murine BALB/c spontaneous colon
adenocarcinoma cell line (CT26), and HepG2.
Microwave irradiation technology in organic synthesis
is a powerful tool for drug synthesis as it can speed up
the reactions under mild conditions allowing the rapid
preparationofcompounds.30Asmentionedabove,microwave
irradiation technology has been successfully applied to the
synthesis of N-arylthieno[2,3-d]pyrimidine derivatives.
Our recent endeavors involving green chemical techniques
have focused on the synthesis of small organic molecular
compounds. Then, in earlier work from our lab, we have
reported the synthesis of 6-bromopyrido[2,3-d]pyrimidine
derivatives (XXI),31 pyrido[2,3-d]pyrimidine derivatives
(XXII),32 cyclopenta[4,5]thieno[2,3-d]pyrimidine
derivatives (XXIII)33 by microwave irradiation technology
(Figure 4).
Interestingly, a compound of similar structure
Figure 4. Structures of compounds XXI, XXII, XXIII, XXIV. (a) Hexatomic ring was linked to thiophene ring instead of five membered ring (compound
XXIII); (b) substituted aromatic group was linked to secondary amine and –CH2 group was subtracted (compound XXIV).

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A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
Scheme 1. Synthetic route of target compounds 3a-y. Reagents and conditions: (a) S8, K2CO3, EtOH, microwave irradiation (MWI), 85 °C, 15 min, 95%;
(b) DMF-DMA, MWI, 78 °C, 5 min, 98%; (c) ArNH2, AcOH, MWI, 90 °C, 10 min, 28-83%.a3y: this compound could not be obtained under the reaction
conditions.
product in 95% yield in 15 min of reaction. Compared to
the results in the literature,35-40 our method has a shorter
reaction time and high yield. So, under the assistance of
the microwave irradiation, K2CO3 can act as a cheap, highly
efficient, and green catalyst used to prepare compound 1
in high yield over a short reaction time.
As for the synthesis of the intermediate compound 2
(N’-(3-cyano-4,5,6,7-tetrahydrobenzo[b]-thio phen-2-yl)-
N,N-dimethyl-formimidamide), Loidreau et al.41 have
reported that 2-amino-5-bromothiophene-3-carbonitrile
reacts with N,N-dimethylformamide dimethyl acetal
(DMF-DMA) to give N’-(3-cyanothiophen-2-yl)-
N,N-dimethylformimidamide under conventional heating.
Meanwhile, Loidreau et al.41 described the synthesis
of N’-(2-cyano-benzo[b]thiophen-3-yl)-N,N-dimethyl-
Figure 5. Main structure of synthesized compounds (example: new
compound 3n).
formimidamide in high yield upon the reaction of 3-amino-
benzo[b]thiophene-2-carbonitrile and DMF-DMA with the
aid of microwave irradiation. Inspired by these methods,
we attempted to use 2-amino-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carbonitrile to react with DMF-DMA under
the microwave irradiation and obtained compound 2 in
excellent yields (98%). This method not only reduces the
reaction time (only 5 min) with good yield, but also has an
easy work-up procedure.
The synthesis of compounds 3a-x (Table 1) involved
the acid-catalyzed Dimroth rearrangement between
compound 2 and an appropriate aromatic amine under
microwave irradiation. It was found that the reaction
between m, p-substituted aromatic amines and compound 2
can easily occur in high yield with a short reaction time.
However, the reaction between o-substituted aromatic
Results and Discussion
Synthesis
The synthetic strategy used in this study is outlined in
Scheme 1. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-
3-carbonitrile was prepared using a modified Gewald
procedure via the reaction of cyclohexanone, malononitrile
and sulfur in the presence of potassium carbonate (K2CO3),
which serves as a heterogeneous solid basic catalyst,
in ethanol. To show the merits of the present work, we
compared the as-obtained results using K2CO3 with those
previously reported in the literature.35-40
It was found that our method can achieve the target

Vol. 00, No. 00, 2019
Han et al.
5
amines and compound 2 only formed trace amounts of the
desired compounds under the same reaction conditions.
This new synthetic route involves only three steps making
this procedure simple and efficient. Hence, this method
is an efficient method for the synthesis of N-arylthieno-
5,6,7,8-tetrahydro-benzothieno[2,3-d]pyrimidin-4-amines.
The proposed mechanism for this rearrangement41 is
described in Scheme 2.
The structures of all compounds 1, 2 and 3a-x were
confirmed by infrared (IR), 1H nuclear magnetic resonance
(NMR), 13C NMR spectroscopy, and high-resolution mass
spectrometry (HRMS). The IR spectrum of compound 1
shows bands at 3444 and 2193 cm-1 corresponding to the
–NH and –C≡N groups, respectively. For compound 2,
the IR spectrum shows bands at 2199, 1627 and 1106 cm-1
corresponding to the –C≡N, –C=N and –C–N groups,
Table 1. Preparation of the compounds 3a-x
mp/mp literature /
Compound
R1
Ar
Yielda / %
°C
3a
3b
3c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Ph
177.7-178.7/176-17822
83
69
82
71
67
66
75
69
79
66
51
48
56
65
72
65
80
75
62
73
61
53
79
28
1
m-MePh
p-MePh
m-ClPh
p-ClPh
m-BrPh
p-BrPh
m-FPh
138.1-139.5/–
respectively. For the sake of the assignment of H NMR
151.4-153.1/141-14323
153.4-154.8/16842
142.2-144.1/136-13822
168.6-170.3/16942
156.1-157.2/154-15622
123.3-125.0/210.642
169.4-170.6/167-16922
105.7-106.6/19342
spectra, we chose compound 3h as the representative
1
3d
3e
example. The H NMR spectrum of the compound 3h
recorded in DMSO-d6 shows the multiplet peaks at
d 7.74-7.60, 7.54-7.43, 7.42-7.30, and 6.96-6.80 assignable
to the aryl protons (–CH) and the other multiplets at
d 3.24-3.04, 2.96-2.74, and 1.96-1.75 correspond to
alicyclic portions protons (–CH2). However, there are two
peaks at d 8.38 and 8.13, probably assigned to 2-position
hydrogen on the pyrimidine ring (–CH) and the 4-position
at pyrimidine ring (–NH), respectively. In order to solve
this assignment problem, D-H exchange technology was
applied. One drop of D2O was added into the NMR sample
of the compound 3h and it was found that the peak at d
8.13 disappeared. Thus, the peak at d 8.13 was assigned to
the hydrogen of –NH and the other peak at d 8.38 assigned
to the –CH signal.
To further confirm the structures of these compounds
and provide a basis for studies on the structure activity
relationships of these compounds, the crystal structures
of compounds 3a and 3c were determined using single
crystal X-ray diffraction. The molecular structures of the
two compounds are illustrated in Figure 6. Colorless single
crystals of the compound 3a (0.35 × 0.20 × 0.10 mm)
and 3c (0.36 × 0.30 × 0.17 mm) were selected for
X-ray diffraction analysis. The analysis reveals that the
3f
3g
3h
3i
p-FPh
3j
m-MeOPh
3k
3l
p-MeOPh 122.9-124.3/145-14723
m-AcPh
p-NO2Ph
142.7-144.1/–
203.1-204.6/–
183.5-185.4/–
120.6-121.8/–
196.5-197.9/–
144.7-145.9/–
170.6-171.8/–
216.4-217.8/–
199.6-200.7/–
195.2-196.3/–
121.9-123.0/224-22543
108.9-110.1/–
181.9-182.5/–
3m
3nb
3o
3p
3q
3r
m-acetylenePh
Ph
3,5-DiMePh
p-t-BuPh
3,4-DiClPh
3,5-DiClPh
3,4-DiFPh
3,5-DiFPh
m-CF3Ph
3s
3tb
3ub
3v
3wb
3xb
m-t-BuPh
p-CF3Ph
aIsolated yield; bnew compounds.
Scheme 2. Proposed mechanism for the Dimroth rearrangement.

6
A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
Figure 6. X-ray structures of compounds 3a (left) and 3c (right).
thieno[2,3-d]pyrimidine ring, which exhibits good coplanar
nature, forms a dihedral angle with the benzene ring.
The crystal data for 3a: monoclinic, a = 10.7726(5) Å,
b = 8.6739(4)Å, c = 15.3075(7)Å, α = 90°, β = 106.897(2)°,
γ = 90°,V = 1368.59(11)Å3, wavelength = 0.71073Å, space
group P2(1)/c, Z = 4, absorption coefficient = 0.229 mm-1,
calculated density = 1.366 mg cm-3, θ range for data
collection (2.73 to 27.68°), F(000) = 592, 3072 unique
(Rint = 0.0190) were used in all calculations. The final R1
was 0.0810 (I > 2sigma(I)) and wR2 was 0.2202 (all data).
The crystal data for 3c: monoclinic, a = 11.8384(3) Å,
b = 8.6429(2)Å, c= 14.8165(4)Å, α= 90°, β= 98.8890(10)°,
γ = 90°,V = 1497.79(7)Å3, wavelength = 0.71073Å, space
group P2(1)/c, Z = 4, absorption coefficient = 0.213 mm-1,
calculated density = 1.310 mg cm-3, θ range for data
collection (1.74 to 27.41°), F(000) = 624, 3381 unique
(Rint = 0.0201) were used in all calculations. The final R1
was 0.0707 (I > 2sigma(I)) and wR2 was 0.1982 (all data).
Crystallographic data were deposited with the Cambridge
Crystallographic Data Centre (see Supplementary
Information section below).
and 17.84 0.74%, respectively. Compounds 3l and 3x
displayed better cytotoxic activity (37.18 0.26 and
55.84 0.05%) compared with the positive drug Erlotinib.
Surprisingly, the most outstanding compound is 3q, which
displayed best cytotoxic activity against A549 with the
inhibition of 85.83 0.31%.
Besides, most of target compounds (3b, 3d, 3e,
3f, 3g, 3h, 3j, 3m, 3n, 3o, 3p, 3q, 3r, 3s, 3t, 3u, 3v,
3w and 3x) were shown to have cytotoxic activities
(at 25 μM) against CT26. In these compounds, the
selected six compounds (3e, 3g, 3o, 3p, 3q and 3u)
demonstrated inapparent cytotoxic activities compared
with the positive control Erlotinib (24.52 0.13%),
with inhibition of 2.25 0.19, 1.65 0.04, 2.45 0.39,
1.90 0.20, 5.96 0.51 and 3.04 0.59%, respectively. In
addition, compounds 3b, 3f, 3h, 3m, 3n and 3s inhibited
14.21 0.44, 13.57 0.55, 14.79 0.63, 19.52 0.18,
10.65 0.23, 12.95 0.08% of the cytotoxic activities
of tested CT26 cell line at compound concentration of
25 μM, respectively. Besides, compounds 3d, 3j, 3r, 3t,
3v, 3w and 3x outperformed the positive control Erlotinib
against CT26 cell line, with inhibition of 40.81 0.05,
49.48 0.06, 55.51 0.62, 64.72 0.37, 55.40 0.17,
62.16 0.54 and 34.08 0.01%, respectively.
Biological activity
Taking Erlotinib as reference compound, the synthesized
compounds 3a-x were evaluated for cytotoxic activities of
A549, CT26 and HepG2 cell lines using 3-(4,5-dimethyl-
thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay.
The inhibition (at 25 μM) of all target compounds and
cytotoxic activities (IC50, in μM) for selected compounds
were summarized in Table 2.
As showed in Table 2, compounds 3b, 3d, 3k, 3l, 3q,
3w and 3x displayed cytotoxic activities against A549
with drug concentration of 25 μM. Among the seven
compounds, compounds 3b, 3d, 3k and 3w showed lower
cytotoxicity (at 25 μM) than Erlotinib (25.18 0.28%),
with inhibition of 9.21 0.12, 15.88 0.45, 1.87 0.21
Interestingly, all of synthesized compounds were
shown to have cytotoxic activities (at 25 μM) against
HepG2. In these compounds, 3m (8.45
0.59%)
and 3o (2.72 0.35%) demonstrated the lowest
cytotoxic activities. Whereas, ten compounds (3a, 3c,
3f, 3g, 3h, 3k, 3l, 3p, 3s and 3u) depicted moderate
cytotoxic effects, with the inhibition of 18.24 0.38,
18.72 0.08, 23.38 0.44, 20.64 0.15, 16.28 0.04,
19.17 0.63, 17.48 0.32, 21.29 0.07, 14.58 0.39 and
19.88 0.69%, respectively. There are four compounds
(3e, 3i, 3n and 3t) which exhibited almost similar cytotoxic
activities (27.37 0.55, 28.69 0.29, 27.49 0.11
and 26.81 0.09%) compared with the positive drug

Vol. 00, No. 00, 2019
Han et al.
7
Table 2. Cytotoxic activities of the compounds 3a-x
Inhibition (at 25 μM) / %
IC50 / μM
HepG2
Compound
R1
Ar
A549
NAb
CT26
NA
HepG2
A549
CT26
NT
AML-12a
NT
3a
3b
3c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
–
Ph
m-MePh
p-MePh
m-ClPh
18.24 0.38
NTc
NT
NT
NT
9.21 0.12
NA
14.21 0.44 33.57 0.23
NA 18.72 0.08
NT
NT
NT
NT
NT
NT
3d
3e
15.88 0.45 40.81 0.05 33.94 0.11
NT
45.71 0.23 29.81 0.11
NT
p-ClPh
NA
NA
2.25 0.19
13.57 0.55 23.38 0.44
1.65 0.04 20.64 0.15
14.79 0.63 16.28 0.04
NA 28.69 0.29
49.48 0.06 47.98 0.06
27.37 0.55
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
3f
m-BrPh
NT
NT
3g
p-BrPh
NA
NT
NT
3h
3i
m-FPh
NA
NT
NT
p-FPh
NA
NT
NT
3j
m-MeOPh
p-MeOPh
m-AcPh
p-NO2Ph
m-acetylenePh
Ph
NA
NT
26.92 0.18 22.35 0.36
NT
3k
3l
1.87 0.21
37.18 0.26
NA
NA
NA
19.17 0.63
17.48 0.32
8.45 0.59
NT
70.90 0.22
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
3m
3n
3o
19.52 0.18
NT
NT
NA
10.65 0.23 27.49 0.11
NT
NT
NT
NA
2.45 0.39
1.90 0.20
5.96 0.51
2.72 0.35
21.29 0.07
61.53 0.26
NT
NT
NT
3p
3q
3r
3,5-DiMePh
p-t-BuPh
3,4-DiClPh
3,5-DiClPh
3,4-DiFPh
3,5-DiFPh
m-CF3Ph
m-t-BuPh
p-CF3Ph
–
NA
NT
NT
NT
85.83 0.31
NA
16.06 0.09
NT
15.01 0.31
> 100
NT
55.51 0.62 35.76 0.43
12.95 0.08 14.58 0.39
64.72 0.37 26.81 0.09
22.91 0.16 36.21 0.22
3s
NA
NT
NT
19.05 0.05
NT
NT
NT
NT
NT
3t
NA
NT
95.50 0.83
NT
3u
3v
NA
3.04 0.59
19.88 0.69
NT
NA
55.40 0.17 63.64 0.17
NT
22.39 0.32 14.02 0.15 61.66 0.59
3w
3x
17.84 0.74 62.16 0.54 32.94 0.54
55.84 0.05 34.08 0.01 66.66 0.03
> 100
21.38 0.69
11.38 0.44
> 100
> 100
56.23 0.71
> 100
8.51 0.52
Erlotinibd
25.18 0.28 24.52 0.13 29.83 0.21
> 100
51.99 0.19 43.05 0.09
> 100
b
c
d
aAML-12: murine non-transformed hepatocyte cell line; NA: no cytotoxic activity (at 25 μM); NT: IC50 value was not measured; used as a positive
control (standard Erlotinib hydrochloride). A549: human pulmonary carcinoma cell line; CT26: murine BALB/c spontaneous colon adenocarcinoma cell
line; HepG2: human hepatocellular liver carcinoma cell line.
Erlotinib (29.83 0.21%). Besides, compounds 3b, 3d,
3j and 3w (33.57 0.23, 33.94 0.11, 47.98 0.06 and
32.94 0.54%) showed better cytotoxic activities than
the positive drug. The most promising compounds 3q, 3v
and 3x exhibited the best cytotoxic effects against HepG2
cell line with inhibition of 61.53 0.26, 63.64 0.17 and
66.66 0.03%, respectively.
According to the analysis of above activity data, five
compounds (3b, 3d, 3q, 3w and 3x) exhibited cytotoxic
activities against all selected tumor cell lines (A549, CT26
and HepG2). Especially, compound 3q depicted better
cytotoxic effects against A549 and HepG2 cell lines. In
addition, 3t and 3x displayed the best cytotoxic effects
against CT26 and HepG2 cell lines, respectively.
3x), which have better cytotoxic activities compared to
Erlotinib (at 25 μM), were subsequently severally selected
to investigate the IC50 values (Table 2) of cytotoxic activity
against A549, CT26 and HepG2 cell lines, respectively.
The present study investigated the effect of several
different structures and from the results of cytotoxic
activities of the compounds, the following preliminary
structure-activity relationships can be derived:
(i) The compound 3q showed the most outstanding
cytotoxic activity (IC50 = 16.06 0.09 μM) and the next
most promising compound was 3x, which displayed good
cytotoxic activity (IC50 = 21.38 0.69 μM) against A549
cell line. Compound 3l showed moderate cytotoxic effects
(IC50 = 70.90 0.22 μM). Based on this (Figure 7), it is
possible to surmise: when introducing a group with strong
electron-donating (3q, Ar = p-t-BuPh) or strong electron-
Three (3l, 3q and 3x), seven (3d, 3j, 3r, 3t, 3v, 3w
and 3x) and seven compounds (3d, 3j, 3q, 3r, 3v, 3w and

8
A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
withdrawing (3x, Ar = p-CF3Ph) into the 4-position, the
cytotoxic activity (A549) of compounds could be boosted.
It was also observed that the presence of π-π conjugate
effect (3l, Ar = m-AcPh) played an important role in the
cytotoxic effect.
or strong electron-donating groups (3q, Ar = p-t-BuPh)
exhibited far superior cytotoxic activity (IC50 = 8.51 0.52,
14.02 0.15 and 15.01 0.31 μM, respectively) than other
compounds (Figure 9). Moreover, the cytotoxic activity
against HepG2 cell line was enhanced because of p-π
conjugate effect (3d, Ar = m-ClPh; 3j, Ar = m-MeOPh),
with IC50 values of 29.81 0.11 and 22.35 0.36 μM,
respectively.
(iv) To sum up, all of the synthesized compounds
exhibited cytotoxic activities against HepG2 cell line with
drug concentration of 25 μM, most of target compounds
were shown to have cytotoxic activities against CT26 cell
line, while only seven compounds demonstrated cytotoxic
activities against A549 cell line. In general, it was found
that all synthesized compounds showed significant selective
cytotoxic effect against the HepG2.
The effects of selected compounds (IC50 values of
selected compounds > IC50 values of Erlotinib against
A549, CT26 and HepG2) on cell viability were assessed.
The detailed results are provided in Figures 10, 11 and 12,
respectively.
In relation to selectivity index (SI), Erlotinib showed
SI > 1.92 and 2.32 for CT26 and HepG2 cells (Table 3,
entry 1), respectively. Compound 3v showed moderate
SI = 2.75 for CT26 cell line (Table 3, entry 4) and
(ii) Subsequently, the introduction of strong electron-
withdrawing group (3r, 3t and 3v, Ar = 3,4-DiClPh,
3,4-DiFPh and m-CF3Ph, respectively) led to a better
cytotoxic activity (IC50 = 22.91 0.16, 19.05 0.05,
22.39 0.32 μM, respectively) against CT26 cell line
(Figure 8). Furthermore, the introduction of strong
electron-donating (3w, Ar = m-t-BuPh) also improves the
cytotoxic effect (IC50 = 11.38 0.44 μM) of the compound.
Another interesting phenomenon was found that when
introducing a double chlorine or a double fluorine (3r,
Ar = 3,4-DiClPh; 3t,Ar = 3,4-DiFPh) into the 3,4-position,
the resulting activity was higher than that of compounds 3s
and 3u (Ar = 3,5-DiClPh; Ar = 3,5-DiFPh) with drug
concentration of 25 μM. Besides, compounds 3d and 3j
also displayed good cytotoxic activity (IC50 = 45.71 0.23
and 26.92 0.18 μM, respectively) against CT26 cell line,
which might be due to the presence of p-π conjugate effect
(3j, Ar = m-MeOPh).
(iii) Meanwhile, compounds substituted with bulky
electron-withdrawing (3x,Ar = p-CF3Ph; 3v,Ar = m-CF3Ph)
Figure 7. Comparison of cytotoxic activities (IC50 values) against A549 cell line.
Figure 8. Comparison of cytotoxic activities (IC50 values) against CT26 cell line.
Figure 9. Comparison of cytotoxic activities (IC50 values) against HepG2 cell line.

Vol. 00, No. 00, 2019
Han et al.
9
Table 3. Selectivity index (SI)a for selected compounds
Selectivity index
entry Compound
AML-12 vs.
AML-12 vs.
AML-12 vs.
A549
CT26
HepG2
1
Erlotinibb
NCc
> 6.23
NC
> 1.92
NC
> 2.32
> 6.66
NC
2
3
4
5
6
3q
3t
5.01
2.75
4.94
NC
3v
3w
3x
NC
4.40
NC
NC
> 4.68
> 11.75
aSI, selectivity index: IC50 on normal cells / IC50 on cancer cells;
bpositive control: standard Erlotinib hydrochloride of AML-12 cell;
cNC: not calculated. AML-12: murine non-transformed hepatocyte
cell line; A549: human pulmonary carcinoma cell line; CT26: murine
BALB/c spontaneous colon adenocarcinoma cell line; HepG2: human
hepatocellular liver carcinoma cell line.
Figure 10. Cell viability of selected compounds toward A549 cell line.
Figure 11. Cell viability of selected compounds toward CT26 cell line.
Figure 12. Cell viability of selected compounds toward HepG2 cell line.
Conclusions
In conclusion, we have synthesized and evaluated
a library of thieno[2,3-d]pyrimidine derivatives.
Amongst these compounds (3a-x), 3q was found
to have better cytotoxic activity with reference to
the standard Erlotinib hydrochloride (TarcevaTM
)
against A549 (IC50 = 16.06 0.09 μM) and HepG2
(IC50 = 15.01 0.31 μM) cell lines. Especially, 3w
and 3x showed best cytotoxic effects against CT26
(IC50 = 11.38 0.44 μM) and HepG2 (IC50 = 8.51 0.52 μM)
cell lines, respectively. Compound 3x also showed good
selectivity index (SI > 11.75). Furthermore, additional
preliminary structure-activity relationships could be
responsible for the found potent and selective activity of
new thieno[2,3-d]pyrimidine compounds. Besides, the
synthesized compounds (3a-x) would be evaluated for
their enzyme activities and protein activities in following
work and the more complete structure-activity relationships
would be disclosed.
Experimental
General remarks
All melting points were measured on an MP90
apparatus and are uncorrected. IR spectra were recorded
in a VERTEX 80/Raman II (Bruker, Switzerland,
1
compounds 3t, 3w and 3v showed good selectivity index
for CT26 cell line (Table 3, entry 3, 5; SI = 5.01, 4.94)
and HepG2 cell line (Table 3, entry 4; SI = 4.40). Besides,
compound 3x also displayed good SI > 4.68 for A549
cell line and lower cytotoxicity (Table 3, entry 6), with
SI > 11.75 for HepG2 cell line.
KBr pellets). The H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded in an Avance III HD
(Bruker, Switzerland) spectrometer with tetramethyl-
silane (TMS) as internal reference in CDCl3 or dimethyl
sulfoxide (DMSO-d6) as the solvent. Only discrete or
1
characteristic signals for the H NMR are reported.

10
A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
The MS spectra were obtained on a Triple-TOF™
5600+ spectrometer (AB SCIEX, USA). An X-ray
crystallographic analysis was performed with a Bruker
SMART APEX II (Bruker, Switzerland). The structure
of the single crystals was refined by a Shelx-97.44 All
reactions were monitored by thin layer chromatography
(TLC) with ethyl acetate:hexane (1:4, v/v) as the eluent.
All reagents and solvents had been purchased from
commercial sources and commonly purified before their
usage.
Preparation of N-aryl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amines (3a-x)
Compound 2 (2.33 g, 10 mmol), appropriate aniline
(11 mmol) and acetic acid (8 mL) as the solvent as well as
catalyst were added into a 1-necked 20 mL pressure tube at
room temperature. The mixture was subjected to microwave
irradiation (CEM Explorer Hybrid, USA, 100 W, 200 psi,
90 °C) for 10 min. After reaction, the acetic acid was evapo-
rated in a vacuum and the crude product was recrystallized
from ethyl acetate to give target compounds (3a-x).
General procedure for preparation of 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carbonitrile (1)
N-Phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4-amine (3a)
Cyclohexanone (0.98 g, 10 mmol), malononitrile
(0.66 g, 10 mmol), sulfur powder (0.35 g, 11.0 mmol),
K2CO3 (0.28 g, 2.0 mmol) and 20 mL of dry ethanol were
mixed and placed in a 50 mL pressure tube. The mixture
was subjected to microwave irradiation (CEM Explorer
Hybrid, USA, 100 W, 200 psi, 85 °C) for 15 min. After
the reaction, the insoluble material was filtered out, and the
solvent was removed through evaporation under reduced
pressure. The crude product was washed with water and
recrystallized from ethanol to give yellowish crystals of
1.69 g (9.50 mmol), in 95% yield; mp 146.8-148.0 °C;
1H NMR (400 MHz, CDCl3) d 4.61 (s, 2H), 2.59-2.39
(m, 4H), 1.90-1.69 (m, 4H); 13C NMR (100 MHz, CDCl3)
d 159.87, 132.37, 120.67, 115.43, 88.82, 24.52, 24.13,
23.37, 22.12; IR (KBr) ν / cm-1 3444, 3327, 2907, 2193,
1618, 1520; HRMS (ESI) m/z, calcd. for C9H10N2S
[M + H]+: 179.0637; found: 179.0638.
1H NMR (400 MHz, DMSO-d6) d 8.38 (s, 1H), 8.13 (s,
1H), 7.67-7.65 (m, 2H), 7.39-7.32 (m, 2H), 7.13-7.07 (m,
1H), 3.14 (m, 2H), 2.83 (m, 2H), 1.86 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 166.24, 155.35, 152.56, 139.67,
133.41, 128.90, 127.09, 123.80, 122.58, 117.25, 25.83,
25.55, 22.63, 22.48; IR (KBr) ν / cm-1 3426, 3044, 2921,
1602, 1563, 1500, 1440, 1213, 972, 748, 697, 498; HRMS
(ESI) m/z, calcd. for C16H15N3S [M + H]+: 282.1059; found:
282.1063.
N-(m-Tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3b)
1H NMR (400 MHz, CDCl3) d 8.49 (s, 1H), 7.50-7.40
(m, 2H), 7.30-7.22 (m, 1H), 7.11 (s, 1H), 6.96 (d, J 7.5 Hz,
1H), 3.12-3.02 (m, 2H), 2.90-2.81 (m, 2H), 2.39 (s, 3H),
2.06-1.88 (m, 4H); 13C NMR (100 MHz, CDCl3) d 166.23,
155.10, 152.65, 139.00, 138.35, 134.59, 128.93, 124.94,
124.80, 121.93, 118.51, 116.65, 26.56, 25.56, 22.60,
22.44, 21.59; IR (KBr) ν / cm-1 3450, 2944, 1610, 1565,
1482, 1372, 1165, 777, 517; HRMS (ESI) m/z, calcd. for
C17H17N3S [M + H]+: 296.1216; found: 296.1219.
Preparation of N’-(3-cyano-4,5,6,7-tetrahydrobenzo
[b]thiophen-2-yl)-N,N-dimethylformimidamide (2)
Compound 1 (1.78 g, 10 mmol) and DMF-DMA
(3.57 g, 30 mmol) were added into a 1-necked 20 mL
pressure tube, and then the mixture was subjected to
microwave irradiation (CEM Explorer Hybrid, USA,
100 W, 200 psi, 78 °C) for 5 min. After the reaction, the
DMF-DMA was evaporated in vacuum and the crude
compound was re-crystallized from ethyl acetate to give
the target compound of 2.29 g (9.80 mmol), in 98% yield;
mp 125.8-126.9 °C; 1H NMR (400 MHz, CDCl3) d 7.67
(s, 1H), 3.08 (s, 6H), 2.61-2.52 (m, 4H), 1.87-1.74 (m,
4H); 13C NMR (100 MHz, CDCl3) d 164.07, 153.85,
133.25, 124.39, 116.30, 97.11, 40.59, 34.95, 24.75, 24.44,
23.39, 22.27; IR (KBr) ν / cm-1 2930, 2199, 1627, 1508,
1106; HRMS (ESI) m/z, calcd. for C12H15N3S [M + H]+:
234.1059; found: 234.1099.
N-(p-Tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3c)
1H NMR (400 MHz, CDCl3) d 8.46 (s, 1H), 7.50 (d,
J 5.5 Hz, 2H), 7.19 (d, J 8.2 Hz, 2H), 7.07 (s, 1H), 3.16-2.98
(m, 2H), 2.94-2.74 (m, 2H), 2.35 (s, 3H), 2.11-1.80 (m, 4H);
13C NMR (100 MHz, CDCl3) d 166.15, 155.31, 152.71,
135.70, 134.42, 133.97, 129.62, 124.87, 121.86, 116.53,
26.56, 25.55, 22.60, 22.45, 20.94; IR (KBr) ν / cm-1 3421,
2918, 1599, 1560, 1505, 1222, 977, 815, 496; HRMS (ESI)
m/z, calcd. for C17H17N3S [M + H]+: 296.1216; found:
296.1212.
N-(3-Chlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3d)
1H NMR (400 MHz, DMSO-d6) d 8.45 (s, 1H, –CH),

Vol. 00, No. 00, 2019
Han et al.
11
8.24 (s, 1H), 7.86 (t, J 2.0 Hz, 1H), 7.73-7.53 (m, 1H), 7.36
(t, J 8.1 Hz, 1H), 7.26-6.98 (m, 1H), 3.25-3.03 (m, 2H),
2.98-2.69 (m, 2H), 1.93-1.68 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) d 166.54, 154.82, 152.37, 141.30, 133.90,
133.21, 130.41, 126.96, 123.11, 121.49, 120.55, 117.53,
25.74, 25.56, 22.61, 22.42; IR (KBr) ν / cm-1 3444, 2944,
1599, 1558, 1502, 1437, 1219, 1129, 984, 875, 762, 679,
515; HRMS (ESI) m/z, calcd. for C16H14ClN3S [M + H]+:
316.0670; found: 316.0668.
7.41-7.32 (m, 1H), 6.94-6.84 (m, 1H), 3.20-3.04 (m, 2H),
2.90-2.76 (m, 2H), 1.94-1.77 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) d 166.47, 163.73, 161.34, 154.78, 152.38,
141.44, 134.06, 130.42, 126.98, 117.72 (d, J 2.6 Hz),
117.56, 109.91, 108.63, 25.68, 25.55, 22.58, 22.41; IR
(KBr) ν / cm-1 3452, 2936, 1611, 1577, 1506, 1447, 1377,
1139, 990, 763; HRMS (ESI) m/z, calcd. for C16H14FN3S
[M – H]–: 298.0820; found: 298.0825.
N-(4-Fluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3i)
N-(4-Chlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3e)
1H NMR (400 MHz, DMSO-d6) d 8.35 (s, 1H), 8.13
(s, 1H), 7.70-7.59 (m, 2H), 7.24-7.14 (m, 2H), 3.16-3.07
(m, 2H), 2.87-2.76 (m, 2H), 1.91-1.80 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 166.21, 160.11, 157.73, 155.46,
152.54, 135.90 (d, J 2.6 Hz), 133.32, 127.07, 125.02,
117.03, 115.55, 115.33, 25.85, 25.53, 22.63, 22.46; IR
(KBr) ν / cm-1 3428, 2921, 1614, 1569, 1504, 1438,
1205, 1128, 955; HRMS (ESI) m/z, calcd. for C16H14FN3S
[M – H]–: 298.0820; found: 298.0823.
1H NMR (400 MHz, CDCl3) d 8.48 (s, 1H), 7.73-7.50
(m, 2H), 7.44-7.29 (m, 2H), 7.11 (s, 1H), 3.17-2.95 (m, 2H),
2.96-2.74 (m, 2H), 2.07-1.86 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 166.46, 154.76, 152.43, 137.09, 135.09, 129.06,
128.94, 124.59, 122.48, 116.70, 26.58, 25.57, 22.57,
22.41; IR (KBr) ν / cm-1 3451, 2933, 1606, 1560, 1495,
1434, 1092, 979, 832, 497; HRMS (ESI) m/z, calcd. for
C16H14ClN3S [M + H]+: 316.0670; found: 316.0673.
N-(3-Bromophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3f)
N-(3-Methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3j)
1H NMR (400 MHz, CDCl3) d 8.44 (s, 1H), 7.87 (d,
J 1.8 Hz, 1H), 7.53-7.46 (m, 1H), 7.18-7.10 (m, 2H), 7.07
(s, 1H), 3.03-2.94 (m, 2H), 2.83-2.73 (m, 2H), 1.99-1.81
(m, 4H); 13C NMR (100 MHz, CDCl3) d 166.15, 154.55,
152.17, 139.85, 135.39, 130.27, 126.71, 124.55, 123.70,
122.65, 119.39, 116.79, 26.53, 25.57, 22.53, 22.38; IR
(KBr) ν / cm-1 3446, 2945, 1597, 1500, 1469, 1439, 1378,
1131, 992, 763, 668, 520; HRMS (ESI) m/z, calcd. for
C16H14BrN3S [M – H]–: 358.0019; found: 358.0020.
1H NMR (400 MHz, CDCl3) d 8.50 (s, 1H), 7.47-7.42
(m, 1H), 7.32-7.24 (m, 1H), 7.15 (s, 1H), 7.14-7.10 (m,
1H), 6.72-6.65 (m, 1H), 3.85 (s, 3H), 3.13-3.02 (m, 2H),
2.91-2.79 (m, 2H), 2.07-1.87 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 166.29, 160.25, 154.97, 152.55, 139.71, 134.79,
129.74, 124.73, 116.73, 113.38, 109.27, 107.34, 55.39,
26.55, 25.57, 22.59, 22.42; IR (KBr) ν / cm-1 3443, 2925,
1608, 1565, 1486, 1156, 1043, 779, 679, 521; HRMS (ESI)
m/z, calcd. for C17H17N3OS [M + H]+: 312.1165; found:
312.1161.
N-(4-Bromophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3g)
N-(4-Methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3k)
1H NMR (400 MHz, CDCl3) d 8.41 (s, 1H), 7.59-7.44
(m, 2H), 7.44-7.35 (m, 2H), 7.06 (s, 1H), 3.04-2.95 (m, 2H),
2.84-2.72 (m, 2H), 2.00-1.79 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 166.04, 154.69, 152.17, 137.53, 135.26, 131.99,
124.61, 122.77, 116.76, 116.54, 26.54, 25.57, 22.54,
22.39; IR (KBr) ν / cm-1 3451, 2932, 1605, 1558, 1496,
1432, 1222, 1125, 829, 803; HRMS (ESI) m/z, calcd. for
C16H14BrN3S [M – H]–: 358.0019; found: 358.0017.
1H NMR (400 MHz, CDCl3) d 8.44 (s, 1H), 7.56-7.41
(m, 2H), 7.02 (s, 1H), 6.97-6.87 (m, 2H), 3.83 (s, 3H),
3.14-2.97 (m, 2H), 2.94-2.77 (m, 2H), 2.06-1.83 (m, 4H);
13C NMR (100 MHz, CDCl3) d 176.04, 165.75, 156.79,
155.66, 152.67, 134.42, 131.03, 124.93, 124.21, 116.42,
114.38, 55.57, 26.57, 25.53, 22.59, 22.45, 20.74; IR (KBr)
ν / cm-1 3418, 2935, 1604, 1564, 1503, 1221, 1026, 829,
552; HRMS (ESI) m/z, calcd. for C17H17N3OS [M + H]+:
312.1165; found: 312.1159.
N-(3-Fluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3h)
1H NMR (400 MHz, DMSO-d6) d 8.46 (s, 1H), 8.28 (s,
1H), 7.74-7.60 (m, 1H), 7.54-7.43 (m, 1H), 7.42-7.30 (m,
1H), 6.96-6.80 (m, 1H), 3.24-3.04 (m, 2H), 2.96-2.74 (m,
2H), 1.96-1.75 (m, 4H); 1H NMR (400 MHz, DMSO-d6-
D2O) d 8.44 (s, 1H), 7.72-7.63 (m, 1H), 7.50-7.43 (m, 1H),
1-(3-((5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
4-yl)amino)phenyl)ethan-1-one (3l)
1H NMR (400 MHz, DMSO-d6) d 8.40 (s, 1H), 8.32 (s,
1H), 8.22-8.18 (m, 1H), 8.00-7.95 (m, 1H), 7.71-7.66 (m,
1H), 7.53-7.46 (m, 1H), 3.18-3.12 (m, 2H), 2.86-2.79 (m,

12
A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
2H), 2.60 (s, 3H), 1.90-1.83 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) d 198.18, 166.48, 155.17, 152.44, 140.21,
137.67, 133.65, 129.15, 127.21, 127.10, 123.69, 121.76,
117.45, 27.28, 25.81, 25.56, 22.64, 22.45; IR (KBr) ν / cm-1
3443, 2939, 1566, 1508, 1442, 1360, 1262, 964, 775, 680,
534; HRMS (ESI) m/z, calcd. for C18H17N3OS [M + H]+:
324.1165; found: 324.1162.
137.92, 133.30, 127.07, 125.37, 120.10, 117.18, 25.84,
25.54, 22.62, 22.48, 21.55; IR (KBr) ν / cm-1 3451, 2942,
2837, 1614, 1560, 1505, 1450, 1164, 1127, 1042, 820, 519;
HRMS (ESI) m/z, calcd. for C18H19N3S [M + H]+: 310.1372;
found: 310.1377.
N-(4-(tert-Butyl)phenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3q)
N-(4-Nitrophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3m)
1H NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.50-7.43
(m, 2H), 7.37-7.31 (m, 2H), 7.06 (s, 1H), 3.03-2.95 (m,
2H), 2.83-2.74 (m, 2H), 2.00-1.81 (m, 4H), 1.26 (s, 9H);
13C NMR (100 MHz, CDCl3) d 155.25, 152.38, 147.38,
135.49, 134.74, 126.01, 124.87, 121.47, 116.61, 34.45,
31.40, 26.53, 25.55, 22.59, 22.42; IR (KBr) ν / cm-1 3417,
2954, 1598, 1560, 1501, 1435, 1316, 1237, 982, 822;
HRMS (ESI) m/z, calcd. for C20H23N3S [M – H]–: 336.1540;
found: 336.1543.
1H NMR (400 MHz, CDCl3) d 8.60 (s, 1H), 8.36-8.18
(m, 2H), 8.03-7.84 (m, 2H), 7.60 (s, 1H), 3.18-3.07 (m, 2H),
2.96-2.82 (m, 2H), 2.09-1.89 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 167.17, 153.73, 152.02, 144.82, 142.66, 136.49,
125.23, 124.16, 119.33, 117.35, 26.59, 25.64, 22.52, 22.35;
IR (KBr) ν / cm-1 3439, 2932, 2846, 1585, 1557, 1502,
1435, 1326, 1237, 1181, 1116, 851, 778, 534, 490; HRMS
(ESI) m/z, calcd. for C16H14N4O2S [M + H]+: 327.0910;
found: 327.0915.
N-(3,4-Dichlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3r)
N-(3-Ethynylphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3n)
1H NMR (400 MHz, DMSO-d6) d 8.39 (s, 1H), 8.25 (s,
1H), 7.97 (d, J 2.5 Hz, 1H), 7.63 (dd, J 8.8, 2.5 Hz, 1H),
7.52 (d, J 8.8 Hz, 1H), 3.12-2.98 (m, 2H), 2.83-2.69 (m,
2H), 1.85-1.71 (m, 4H); 13C NMR (100 MHz, DMSO-d6)
d 166.69, 154.64, 152.34, 140.03, 134.16, 131.05, 130.62,
126.99, 124.84, 123.29, 122.23, 117.67, 25.72, 25.58,
22.62, 22.40; IR (KBr) ν / cm-1 3447, 2936, 1596, 1554,
1493, 1434, 1374, 1266, 1128, 990, 805; HRMS (ESI)
m/z, calcd. for C16H13Cl2N3S [M – H]–: 348.0134; found:
348.0130.
1H NMR (400 MHz, CDCl3) d 8.50 (s, 1H), 7.80-7.76
(m, 1H), 7.76-7.68 (m, 1H), 7.37-7.29 (m, 1H), 7.26-7.22
(m, 1H), 7.13 (s, 1H), 3.10 (s, 1H), 3.09-3.02 (m, 2H),
2.91-2.81 (m, 2H), 2.05-1.87 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 166.39, 154.66, 152.38, 138.63, 135.04, 129.04,
127.46, 124.62, 124.20, 122.81, 121.50, 116.71, 83.37,
26.52, 25.56, 22.54, 22.38; IR (KBr) ν / cm-1 3443, 3240,
2939, 1559, 1502, 1438, 1133, 991, 889, 774, 650, 519;
HRMS (ESI) m/z, calcd. for C18H15N3S [M + H]+: 306.1059;
found: 306.1055.
N-(3,5-Dichlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3s)
N-Methyl-N-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3o)
1H NMR (400 MHz, DMSO-d6) d 8.50 (s, 1H), 8.38
(s, 1H), 7.84 (d, J 1.9 Hz, 2H), 7.25 (t, J 1.9 Hz, 1H),
3.17-3.07 (m, 2H), 2.91-2.79 (m, 2H), 1.92-1.79 (m, 4H);
13C NMR (100 MHz, DMSO-d6) d 166.83, 154.45, 152.30,
142.46, 134.39, 134.11, 127.00, 122.27, 119.89, 117.90,
25.69, 25.59, 22.62, 22.39; IR (KBr) ν / cm-1 3444, 3103,
2943, 2845, 1598, 1502, 1446, 1226, 1121, 1000, 837,
811; HRMS (ESI) m/z, calcd. for C16H13Cl2N3S [M – H]–:
348.0134; found: 348.0138.
1H NMR (400 MHz, DMSO-d6) d 8.29 (s, 1H), 7.49-
7.37 (m, 4H), 7.29-7.21 (m, 1H), 3.48 (s, 3H), 2.62-2.54
(m, 2H), 2.50-2.47 (m, 2H), 1.84-1.71 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 163.24, 153.16, 143.97, 133.15,
130.00, 126.11, 125.79, 121.11, 116.01, 98.08, 34.80,
24.65, 24.40, 23.27, 22.21; IR (KBr) ν / cm-1 3448, 3074,
2942, 2835, 2208, 1616, 1494, 1352, 1224, 1134, 977, 756,
688; HRMS (ESI) m/z, calcd. for C17H17N3S [M + H]+:
296.1216; found: 296.1222.
N-(3,4-Difluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3t)
N-(3,5-Dimethylphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3p)
1H NMR (400 MHz, DMSO-d6) d 8.39 (s, 1H), 7.97 (s,
1H), 7.31 (s, 2H), 6.74 (s, 1H), 3.16-3.10 (m, 2H), 2.87-
2.77 (m, 2H), 2.28 (s, 6H), 1.90-1.80 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 166.10, 155.38, 152.65, 139.50,
1H NMR (400 MHz, DMSO-d6) d 8.52 (s, 1H), 8.42
(s, 1H), 7.56-7.47 (m, 1H), 6.94-6.84 (m, 1H), 3.17-3.08
(m, 2H), 2.88-2.81 (m, 2H), 1.92-1.83 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 166.86, 164.03, 163.88, 161.62,
161.47, 154.47, 152.29, 142.57, 134.49, 126.93, 117.94,
104.51, 104.22, 98.18, 25.63 (d, J 6.4 Hz), 22.62, 22.39;

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Han et al.
13
IR (KBr) ν / cm-1 3449, 3097, 2936, 2853, 1615, 1561,
1509, 1476, 1441, 1148, 1109, 1030, 989, 827; HRMS
(ESI) m/z, calcd. for C16H13F2N3S [M – H]–: 316.0725;
found: 316.0720.
ν / cm-1 3448, 2933, 2846, 1610, 1563, 1501, 1438,
1324, 1241, 1162, 1107, 1066, 983, 838; HRMS (ESI)
m/z, calcd. for C17H14F3N3S [M + H]+: 350.0933; found:
350.0912.
N-(3,5-Difluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (3u)
Biological evaluation
1H NMR (400 MHz, DMSO-d6) d 8.50 (s, 1H), 8.38
(s, 1H), 7.56-7.46 (m, 2H), 6.87 (tt, J 9.3, 2.3 Hz, 1H),
3.12 (m, 2H), 2.84 (m, 2H), 1.85 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 166.83, 154.45, 152.30, 142.46,
134.39, 134.11, 127.00, 122.27, 119.89, 117.90, 25.64
(d, J 10.1 Hz), 22.62, 22.39; IR (KBr) ν / cm-1 3449,
3097, 2937, 2853, 1615, 1561, 1509, 1476, 1441, 1148,
1108, 988, 826; HRMS (ESI) m/z, calcd. for C16H13F2N3S
[M – H]–: 316.0725; found: 316.0728.
Cytotoxicity assay in vitro
At first, the cytotoxic activities of target compounds
3a-x were determined against A549, CT26 and HepG2
cell lines in vitro according to MTT assay with Erlotinib
as the positive control at 25 μM. Then, the selected
compounds, which have better cytotoxic effects
compared to Erlotinib (at 25 μM), were subsequently
severally selected to investigate the IC50 values of
cytotoxic activity against three tumor cell lines tested
at six concentrations (3.125, 6.25, 12.5, 25, 50 and
100 μM). MTT was dissolved at 4 mg mL-1 in phosphate-
buffered saline (PBS).
N-(3-(Trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (3v)
1H NMR (400 MHz, DMSO-d6) d 8.37 (s, 1H), 8.33
The three cancer cell lines were cultured in a Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 100 g mL-1 penicillin and
100 g mL-1 streptomycin. Cancer cell lines in logarithmic
phase were plated at a density of 3 × 103 cells well-1 into each
well of 96-well microtiter plates with 100 μL of DMEM
and incubated at 37 °C for 24 h in humidified atmosphere
containing 5% CO2. Then, compounds 3a-x solution
(100 μL) was added into the culture medium and the cancer
cells were incubated at same condition (37 °C, 5% CO2) for
72 h. The MTT solution (20 μL of 4 mg mL-1 MTT in PBS)
was added into each well and the cells containing target
compounds were further incubated at 37 °C for 4 h in 5%
CO2.After removing the culture medium, DMSO (200 μL)
was then added into each well to dissolve the crystals,
shaken mechanically for 10 min, and then optical densities
(OD) values at a wavelength of 540 nm were measured on
a microplate reader (SpectraMax 190, Molecular Devices,
USA). Survival ratios are expressed in percentages with
respect to untreated cells.
(s, 1H), 8.01 (s, 1H), 7.96-7.88 (m, 1H), 7.56-7.46 (m,
1H), 7.38-7.30 (m, 1H), 3.13-3.03 (m, 2H), 2.81-2.72 (m,
2H), 1.83-1.75 (m, 4H); 13C NMR (100 MHz, DMSO-d6)
d 166.66, 154.90, 152.38, 140.64, 134.01, 129.93, 129.55,
127.06, 126.03, 123.35, 119.79 (d, J 3.6 Hz), 118.36 (d,
J 4.0 Hz), 117.61, 25.75, 25.58, 22.63, 22.42; IR (KBr)
ν / cm-1 3445, 3095, 2947, 2870, 1567, 1510, 1442,
1327, 1164, 1110, 988, 781; HRMS (ESI) m/z, calcd. for
C17H14F3N3S [M – H]–: 348.0788; found: 348.0782.
N-(3-(tert-Butyl)phenyl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (3w)
1H NMR (400 MHz, DMSO-d6) d 8.35 (s, 1H), 8.08 (s,
1H), 7.63-7.57 (m, 1H), 7.57-7.49 (m, 1H), 7.33-7.22 (m,
1H), 7.16-7.05 (m, 1H), 3.19-3.09 (m, 2H), 2.88-2.72 (m,
2H), 1.90-1.80 (m, 4H), 1.30 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) d 166.19, 155.49, 152.59, 151.55, 139.29,
133.22, 128.40, 127.12, 120.83, 120.25, 119.69, 117.17,
34.92, 31.63, 25.84, 25.55, 22.65, 22.49; IR (KBr) ν / cm-1
3453, 3355, 2937, 1603, 1559, 1501, 1438, 1374, 1198,
1131, 992, 784; HRMS (ESI) m/z, calcd. for C20H23N3S
[M + H]+: 338.1685; found: 338.1696.
Supplementary Information
Crystallographic data (excluding structure factors) for
the structures in this work were deposited in the Cambridge
Crystallographic Data Centre as supplementary publication
number CCDC 1542488 (3a) and CCDC 1542147 (3c).
Copies of the data can be obtained, free of charge, via
www.ccdc.cam.ac.uk/conts/rtrieving.html or from the
Cambridge Crystallographic Data Centre, CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
E-mail: deposit@ccdc.cam.ac.uk.
N-(4-(Trifluoromethyl)phenyl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (3x)
1H NMR (400 MHz, CDCl3) d 8.47 (s, 1H), 7.75 (d,
J 8.5 Hz, 2H), 7.55 (d, J 8.6 Hz, 2H), 7.22 (s, 1H), 3.02
(t, J 6.0 Hz, 2H), 2.80 (dd, J 8.1, 3.9 Hz, 2H), 2.00-1.76
(m, 4H); 13C NMR (100 MHz, CDCl3) d 166.74, 154.37,
152.25, 141.76, 135.64, 126.31 (d, J 3.8 Hz), 124.4,
120.21, 116.99, 26.58, 25.60, 22.55, 22.38; IR (KBr)

14
A Microwave-Enhanced Synthesis and Biological Evaluation
J. Braz. Chem. Soc.
Supplementary data associated with this paper can be
found, in the online version, at http://jbcs.sbq.org.br as
PDF file.
Yang, L.; Zhang, X.-Q.; Hu, W.-H.; Eur. J. Med. Chem. 2011,
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Acknowledgments
The authors greatly acknowledge financial support of
this research by the Jiangsu Prospective Joint Research
Project (No. BY2016066-02) and College Students
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Submitted: October 17, 2018
Published online: March 19, 2019
This is an open-access article distributed under the terms of the Creative Commons Attribution License.