Design, synthesis and biological evaluation of novel thieno[3,2-d] pyrimidine derivatives containing diaryl urea moiety as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.099

Two series of thieno[3,2-d]pyrimidine derivatives containing diaryl urea moiety were designed, synthesized and evaluated for their biological activity. The preliminary investigation showed that most compounds displayed good to excellent potency against four tested cancer cell lines as compared with GDC-0941 and sorafenib. In particular, the most promising compound 29a showed the most potent antitumor activities with IC₅₀ values of 0.081 μM, 0.058 μM, 0.18 μM, and 0.23 μM against H460, HT-29, MKN-45 and MDA-MB-231 cell lines, respectively. The SAR analyses indicated that compounds with mono-halogen groups at 4-position on the terminal phenyl ring were more active than those with double-halogen groups or methyl groups. In addition, the introduction of chlorine atoms into 6,7-position of thieno[3,2-d]pyrimidine moiety led to a slight decline, but more selective activity against H460 and HT-29 cell lines.

Full text of DOI:10.1016/j.ejmech.2014.07.099

European Journal of Medicinal Chemistry 85 (2014) 215e227
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel thieno[3,2-d]
pyrimidine derivatives containing diaryl urea moiety as potent
antitumor agents
Zijian Liu, Yu Wang, Huafang Lin, Dazhuang Zuo, Lihui Wang, Yanfang Zhao, Ping Gong*
Key Laboratory of Structure-Based Drug Design and Discovery, Shenyang Pharmaceutical University, Ministry of Education, 103 Wenhua Road, Shenhe
District, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of thieno[3,2-d]pyrimidine derivatives containing diaryl urea moiety were designed, syn-
thesized and evaluated for their biological activity. The preliminary investigation showed that most
compounds displayed good to excellent potency against four tested cancer cell lines as compared with
GDC-0941 and sorafenib. In particular, the most promising compound 29a showed the most potent
Received 22 April 2014
Received in revised form
26 July 2014
Accepted 26 July 2014
Available online 28 July 2014
antitumor activities with IC50 values of 0.081 mM, 0.058 mM, 0.18 mM, and 0.23 mM against H460, HT-29,
MKN-45 and MDA-MB-231 cell lines, respectively. The SAR analyses indicated that compounds with
mono-halogen groups at 4-position on the terminal phenyl ring were more active than those with
double-halogen groups or methyl groups. In addition, the introduction of chlorine atoms into 6,7-
position of thieno[3,2-d]pyrimidine moiety led to a slight decline, but more selective activity against
H460 and HT-29 cell lines.
Keywords:
Synthesis
Thieno[3,2-d]pyrimidine derivatives
Diaryl urea moiety
Antiproliferative activity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
pyrimidine derivatives and have prompted us to explore this
pharmacophore as novel promising bioactive molecules.
Cancer is one of the most serious health problems all over the
world and one of the leading causes of death [1e3]. Thus, in the
past for several decades, researchers have been struggling to find
effective clinical approaches for the treatment of cancer and search
for novel anticancer agents [4]. Recently, accumulating evidences
have illustrated that heterocyclic scaffolds are considered to be the
most promising molecules as leads for the discovery of novel syn-
thetic drugs [5]. In particular, substituted thieno[3,2-d]pyrimidines,
present in the cores of many physiologically active agents, display
interesting therapeutic properties, especially antitumor activities
with different biotargets and mechanisms by means of inhibiting
several enzymes as well as modulating the activity of many re-
ceptors [6e8]. Recently, thieno[3,2-d]pyrimidine derivatives have
become an important class of chemotherapeutic drugs, especially
for the treatment of cancers, such as PI3K inhibitor 1 (GDC-0941),
PI3K and mTOR dual inhibitor 2, EGFR inhibitor 3, and EGFR and
VEGFR dual inhibitor 4, et al. (Fig. 1) [9e13]. These recent examples
highlight the extent of ongoing interest toward new thieno[3,2-d]
Diaryl urea derivatives have received considerable attention
among anticancer agents, owing to their good inhibitory activities
against receptor tyrosine kinases (RTKs), Raf kinases, protein
tyrosine kinases (PTKs), and NADH oxidase [14]. Among these de-
rivatives [15e17] (Fig. 2), sorafenib 5, the first orally bioavailable
multiple kinase inhibitor against Raf, VEGF and PDGF receptor
tyrosine kinases, was approved by the U.S. Food and Drug Admin-
istration (FDA) for the treatment of advanced renal cell carcinoma
(RCC) in December 2005 [18] and unresectable hepatocellular car-
cinoma (HCC) in November 2007 [19]. The co-crystal structure of
sorafenib in complex with Raf kinase domains revealed that the
diaryl urea framework played key roles in the interaction with Raf
kinase due to their binding modes and the presence of hydrogen
bonds [20]. Thus, the development of such small molecules with
diaryl urea framework, which can readily bind with various en-
zymes and receptors in organisms through forming hydrogen-
bonding interactions, has been shown to be a feasible antitumor
strategy.
In view of these observations, it was thought of interest to study
the influence of thieno[3,2-d]pyrimidine moiety and diaryl urea
framework combination, so that the two combined substructures
could exhibit synergistic antitumor effect. Therefore, two series of
* Corresponding author.
E-mail address: gongpinggp@126.com (P. Gong).
http://dx.doi.org/10.1016/j.ejmech.2014.07.099
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

216
Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
Fig. 1. Structures of representative kinase inhibitors containing thieno[3,2-d]pyrimidine nuclei and their kinase inhibitory activities.
new thieno[3,2-d]pyrimidine derivatives containing diaryl urea
moiety were designed and synthesized (Fig. 3). In addition, various
substituents were introduced to the terminal phenyl ring with the
purpose of exploring the influence of substituents on anticancer
activity by regulating the electronic and steric effects. Further
modifications were performed by introducing various aliphatic
amines into 4-position and chlorine atoms into 6,7-position of
thieno[3,2-d]pyrimidine ring, respectively, in order to regulate the
lipid-water partition coefficient and electron affinity. Accordingly,
With an aim to develop promising antitumor agents, two series of
novel thieno[3,2-d]pyrimidine derivatives containing diaryl urea
moiety were designed and synthesized.
In the current study, all target compounds were evaluated for
their antiproliferative activity in vitro against four cancer cell lines:
H460 (human lung cancer), HT-29 (human colon cancer), MKN-45
(human gastric cancer) and MDA-MB-231 (human breast cancer
cell). Additionally, the enzymatic assays and apoptosis analysis of
the most potent compounds 29a were also performed.
solvent was evaporated in vacuum and the residue was dissolved in
dehydrated CH2Cl2 to add dropwise to a mixture of 3a or 3b, Et3N
and CH2Cl2 in an ice bath. The mixture was then allowed to react at
room temperature for 10 h to obtain 4a,b. Treatment of 4a,b in
ammonium hydroxide at 60 ꢀC for 8 h yielded compounds 5a,b,
which could be cyclized in the presence of sodium hydroxide to
afford 6a,b in 72e74% yields. Next, the chlorination of 6a,b with
phosphoryl chloride afforded intermediates 7a,b [25], which were
subsequently alkylated with an excess of the corresponding
aliphatic amines in THF at 30 ꢀC, using triethylamine as an acid-
binding agent, to provide intermediates 8ae11a, 8be11b. The key
intermediates 12ae15a, 12be15b were obtained via reduction of
compounds 8ae11a, 8be11b with iron powder and catalytic
amounts of ammonium chloride in ethanol at reflux temperature.
Finally, target compounds 16ae35a and 16be35b were successfully
obtained via reaction of the amines 12ae15a, 12be15b with
substituted isocyanatobenzene in CH2Cl2 at room temperature
overnight, respectively.
2. Chemistry
3. Results and discussion
The synthetic route of target compounds 16ae35a and 16be35b
was illustrated in Scheme 1. Acylation of commercially available
methyl 3-amino-2-thiophenecarboxylate with acetyl chloride in
dry pyridine at 0 ꢀC to room temperature provided the interme-
diate 1 [21,22], which was then chlorinated with sulfuryl chloride to
afford 2 as a white solid [23]. Refluxing in dioxane, intermediate 2
was deacetyled in the presence of 20% H2SO4 yielded 3b [24].
Subsequently, the mixture of 4-nitrobenzoic acid and SOCl2 was
heated to 80 ꢀC for 5 h. Upon cooling to room temperature, the
3.1. In vitro cytotoxicity and structure activity relationships
The cytotoxic activities of the target compounds were evaluated
in H460 (human lung cancer), HT-29 (human colon cancer), MKN-
45 (human gastric cancer) and MDA-MB-231 (human breast can-
cer) cell lines by the MTT assay. Using GDC-0941 and sorafenib as
the positive controls, the bioactivity data summarized in Tables 1
and 2 are presented as IC50 values derived from the average of at
Fig. 2. Structures of some representative diaryl urea derivatives.

Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
217
Fig. 3. Design strategy for the thieno[3,2-d]pyrimidine derivatives containing diaryl urea moieties.
least three independent experiments and calculated relative to a
vehicle (DMSO) control.
R2 ¼ dimethylamino), with no substituent (R3 ¼ H) on the terminal
phenyl ring, showed promising cytotoxicity against H460 and HT-
As illustrated in Tables 1 and 2, all target compounds 16ae35a
and 16be35b showed moderate to significant cytotoxic activities
against the different cancer cell lines. Most of these compounds
were more potent than sorafenib and GDC-0941 against one or
more cancer cell lines, which suggested that the combination of
thieno[3,2-d]pyrimidine moiety and diaryl urea framework exhibit
potent synergistic antitumor effect. The IC50 values of the most
29 cell lines with IC50 values of 0.64 mM and 0.62 mM, respec-
tively. The introduction of mono-halogen groups 20ae23a (R3 ¼ 4-
F, 2-F, 4-Cl, 2-Cl, respectively) led to an obvious improvement in
antitumor activity than compound 19a, which could be further
confirmed by compounds 26a and 27ae30a. However, the intro-
duction of double-halogen groups (24a, R3
¼
2,4-(Cl)2,
M against HT-29) or methyl groups (25a, R3 ¼ 4-CH3,
mM against HT-29) caused the potency to be lowered by
IC50 ¼ 1.32
m
promising compound 29a were 0.081
0.23 M against H460, HT-29, MKN-45 and MDA-MB-231 cell lines,
respectively.
mM, 0.058
mM, 0.18
mM, and
IC50 ¼ 2.15
m
2.1- and 3.5-fold, respectively. This could demonstrate that mono-
halogen groups had a positive effect on antitumor activity, but
double-halogen groups and methyl groups showed negative re-
sults. Interestingly, the position of R3 group was closely related to
antitumor activity as well. Compounds with 4-substituted phenyl
Preliminary structureeactivity relationships (SARs) indicated
that the introduction of different amino groups at the C-4 position
of thieno[3,2-d]pyrimidine moiety had a slight influence on activ-
ity, which suggested that the water-soluble R2 group and the length
of side chain contributed little to their potency. For example,
compounds 22a (n ¼ 2, R2 ¼ dimethylamino, R3 ¼ 4-Cl), 29a (n ¼ 3,
R2 ¼ diethylamino, R3 ¼ 4-Cl) and 33a (n ¼ 2, R2 ¼ diethylamino,
R3 ¼ 4-Cl) exhibited comparable cytotoxic activities against the
different cancer cells.
group (20a, R3 ¼ 4-F, IC50 ¼ 0.35
IC50 ¼ 0.095 M against HT-29) displayed higher potency than
those with 2-substituted phenyl group (21a, R3 2-F,
M against HT-29).
mM and 22a, R3 ¼ 4-Cl,
m
¼
IC50 ¼ 0.51
m
M and 23a, R3 ¼ 2-Cl, IC50 ¼ 0.31
m
Among them, 4-chloro group on terminal phenyl ring was the
most favorable substituent for activity.
Further analysis clearly revealed that different biological prop-
erties were observed when various R3 groups were introduced into
In general, the data listed in Table 2 revealed that the activity of
compounds 16be35b (R1 ¼ 6,7-dichloro) was less effective than
compounds 16ae35a (R1 ¼ H), suggesting that the introduction of
the terminal phenyl ring. Compound 19a (n
¼
2,
Scheme 1. Reagents and conditions: (i) Acetyl Chloride, pyridine, r.t.; (ii) SO2Cl2, CHCl3, reflux; (iii) H2SO4, dioxane, reflux; (iv) a) 4-nitrobenzoic acid, SOCl2, 80 ꢀC; b) Et3N, CH2Cl2,
r.t.; (v) NH4OH, EtOH, 60 ꢀC; (vi) NaOH, i-PrOH, reflux; (vii) POCl3, 80 ꢀC; (viii) NH2(CH2)nR2, THF, Et3N, 30 ꢀC; (ix) Fe, NH4Cl, EtOH, reflux; (x) isocyanatobenzene, CH2Cl2, r.t.

218
Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
Table 1
Cytotoxicity of 16ae35a against H460, HT-29, MKN-45 and MDA-MB-231 cell lines in vitro.
a
Compd.
n
R2
R3
IC50
(
mmol/L) SD
H460
HT-29
MKN-45
MDA-MB-231
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
31a
3
3
3
2
2
2
2
2
2
2
3
3
3
3
3
3
2
2
2
2
e
e
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
e
2-F
0.53 0.038
0.43 0.021
1.02 0.15
0.64 0.045
0.39 0.027
0.41 0.034
0.13 0.012
0.23 0.017
1.65 0.11
1.88 0.13
0.68 0.039
0.29 0.015
0.37 0.024
0.081 0.011
0.20 0.016
1.24 0.12
0.43 0.040
0.19 0.015
0.32 0.022
1.89 0.16
0.87 0.20
2.19 0.11
0.47 0.032
0.39 0.035
1.86 0.13
0.62 0.055
0.35 0.029
0.51 0.043
0.095 0.010
0.31 0.012
1.32 0.11
2.15 0.19
0.73 0.032
0.34 0.018
0.55 0.037
0.058 0.010
0.34 0.025
1.62 0.14
0.61 0.050
0.15 0.013
0.62 0.038
1.94 0.20
0.86 0.081
3.61 0.36
0.73 0.029
0.69 0.028
2.27 0.21
1.12 0.11
0.33 0.012
0.53 0.032
0.25 0.020
0.85 0.040
2.36 0.19
2.38 0.21
0.98 0.10
0.47 0.036
0.58 0.43
0.18 0.014
0.39 0.26
1.76 0.15
1.04 0.11
0.37 0.028
0.75 0.025
1.52 0.13
0.60 0.12
2.32 0.35
0.66 0.030
0.59 0.029
1.86 0.15
1.31 0.11
0.54 0.032
0.60 0.029
0.31 0.018
0.55 0.022
2.45 0.21
2.18 0.14
1.23 0.11
0.53 0.031
0.86 0.033
0.23 0.015
0.50 0.022
1.98 0.13
0.58 0.030
0.55 0.033
0.68 0.041
2.48 0.21
0.28 0.06
0.94 0.13
2-Cl
2,4-(Cl)2
H
4-F
2-F
4-Cl
2-Cl
2,4-(Cl)2
4-CH3
H
4-F
2-F
4-Cl
2-Cl
4-CH3
2-F
4-Cl
2-Cl
2,4-(Cl)2
e
32a
33a
34a
35a
GDC-0941b
Sorafenibb
e
e
a
Data presented is the mean SD value of three independent determinations.
Used as positive control.
b
chlorine atoms into 6,7-position of thieno[3,2-d]pyrimidine ring
led to a slight decline in efficiency. Nevertheless, it is worth
mentioning that compounds 16be35b were more potent against
H460 and HT-29 cell lines than against MKN-45 and MDA-MB-231
cell lines. These results revealed that this series of compounds
possessed selectivity for H460 and HT-29 cancer cell lines, and had
the makings of good drugs for lung and colorectal cancer. Moreover,
examination of the structureeactivity relationships (SARs) indi-
cated that these analogs had similar SARs as summarized before.
In conclusion, the most potent compound 29a showed prom-
ising cytotoxicity against H460 and HT-29 cell lines with IC50
kinases was negligible. Even though it showed more selectivity
against PI3K , the inhibitory activity of compound 29a was still
much less than GDC-0941. These results indicated that the 10
selected kinases might not be the biological target for synthesized
compounds.
a
3.3. Apoptosis analysis
Cell apoptosis is the process of programmed cell death [26,27].
To examine the mechanism of the cell apoptosis induced by the
synthesized compounds, the cleavage of poly (ADP-ribose) poly-
merase (PARP) was investigated. PARP is a polymerase involved in
cell apoptosis. Cleaved PARP (Clv-PARP) is a significant maker in the
apoptosis of different cell lines. Its expression is positively associ-
ated with cell apoptosis [28,29]. Therefore, in the current study, HT-
29 cell line was used to identify the mechanism of drug-induced
cell apoptosis.
values of 0.081
much higher than GDC-0941 by 10.7- and 14.8-fold
(IC50 ¼ 0.87 M against H460, IC50 ¼ 0.86 M against HT-29),
and sorafenib by 27.0- and 62.2-fold (IC50 ¼ 2.19 M against
M against HT-29), separately. These encour-
mM and 0.058 mM, respectively. The potency was
m
m
m
H460, IC50 ¼ 3.61
m
aging results provide a valuable leading compound 29a, and
highlight the potential for further development of novel thieno
[3,2-d]pyrimidine derivatives containing diaryl urea moiety as
potent antitumor agents.
HT-29 cells were treated with 29a at different concentrations (0,
1, 5
mM) for 48 h. Subsequently, the cleavage of PARP was analyzed
with Western blot (
b
-actin as control). As shown in Fig. 4, the
protein expression of Clv-PARP was significantly elevated after the
treatment with 29a in a concentration-dependent manner. The
3.2. In vitro enzymatic assays
ratio between Clv-PARP and
the activity of PARP protein. The ratio was 2.5- and 3.5-fold higher
than that of control group, at concentration of 1 M and 5 M,
respectively. Such elevated ratios suggested that the significant
drug-induced HT-29 cancer cell apoptosis is PARP-dependent.
However, the upstream and proapoptotic signaling pathways still
need further investigation so that a better understanding of the
precise mechanism is achieved.
b-actin expression was used to reflect
To investigate the molecular mechanisms of action preliminar-
ily, the optimal compound 29a was screened against 10 selected
kinases, which are probable biological targets for the designed
compounds [6e17]. The results summarized in Table 3 were
expressed as IC50 value, which are derived from two independent
m
m
experiments. As shown, compound 29a inhibited PI3K
a kinase with
IC50 values of 0.13 M, whereas the impact on the other tested
m

Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
219
Table 2
Cytotoxicity of 16be35b against H460, HT-29, MKN-45 and MDA-MB-231 cell lines in vitro.3
a
Compd.
n
R2
R3
IC50
(
mmol/L) SD
H460
HT-29
MKN-45
MDA-MB-231
16b
17b
18b
19b
20b
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
31b
3
3
3
3
3
2
2
2
2
3
3
3
3
3
3
2
2
2
2
2
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Dimethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
Diethylamino
4-F
4-Cl
2-Cl
2,4-(Cl)2
4-CH3
4-F
4-Cl
2-Cl
2,4-(Cl)2
H
4-F
4-Cl
2-Cl
2,4-(Cl)2
4-CH3
4-F
4-Cl
2-Cl
0.65 0.020
0.23 0.013
0.49 0.031
0.98 0.033
1.11 0.046
0.59 0.026
0.30 0.014
0.44 0.028
1.52 0.053
0.85 0.048
0.45 0.039
0.27 0.022
0.33 0.025
1.17 0.10
1.24 0.11
0.50 0.027
0.32 0.022
0.46 0.029
1.30 0.11
0.84 0.057
0.87 0.20
2.19 0.11
0.43 0.022
0.33 0.017
0.82 0.030
1.32 0.10
1.32 0.093
0.89 0.069
0.46 0.022
0.57 0.030
1.28 0.084
0.82 0.049
0.35 0.026
0.25 0.014
0.41 0.013
1.23 0.11
0.89 0.036
0.65 0.027
0.43 0.023
0.55 0.033
1.14 0.089
0.95 0.055
0.86 0.081
3.61 0.36
1.12 0.11
1.53 0.13
1.33 0.093
2.56 0.17
3.70 0.20
1.35 0.098
0.91 0.063
1.17 0.088
3.46 0.26
1.64 0.12
1.70 0.11
0.72 0.044
1.45 0.10
5.72 0.32
3.32 0.33
1.37 0.14
0.89 0.066
1.13 0.083
1.77 0.13
1.45 0.12
0.60 0.12
2.32 0.35
0.98 0.067
1.16 0.074
1.56 0.099
3.49 0.21
4.98 0.28
1.24 0.11
0.87 0.069
1.35 0.089
3.50 0.25
1.78 0.13
1.63 0.12
0.66 0.036
0.97 0.077
3.94 0.28
3.04 0.15
1.13 0.10
0.75 0.039
1.41 0.11
1.92 0.13
2.01 0.15
0.28 0.06
0.94 0.13
32b
33b
34b
35b
2,4-(Cl)2
4-CH3
GDC-0941b
Sorafenibb
a
Data presented is the mean SD value of three independent determinations.
Used as positive control.
b
4. Conclusion
Western blot analysis of compound 29a demonstrated that it could
inhibit HT-29 proliferation by inducing apoptosis mechanism. Thus,
additional work will be focused on the mechanistic studies to
determine the targets of action at pathway level. In conclusion,
compound 29a is worth further studying as new potential anti-
cancer agent for the treatment of human cancers.
In summary, two series of thieno[3,2-d]pyrimidine derivatives
containing diaryl urea moiety were designed, synthesized and
evaluated for their biological activity. The preliminary investigation
showed that most compounds displayed good to excellent potency
against four tested cancer cell lines as compared with GDC-0941
and sorafenib. In particular, the most promising compound 29a
showed the most potent antitumor activities with IC50 values of
5. Experimental
0.081
mM, 0.058
mM, 0.18
mM, and 0.23
mM against H460, HT-29,
5.1. Chemistry
MKN-45 and MDA-MB-231 cell lines, respectively. The SAR ana-
lyses indicated that compounds with mono-halogen groups at 4-
position on the terminal phenyl ring were more active than those
with double-halogen groups or methyl groups. In addition, the
introduction of chlorine atoms into 6,7-position of thieno[3,2-d]
pyrimidine moiety led to a slight decline, but more selective ac-
tivity against H460 and HT-29 cell lines. Moreover, the enzymatic
assays suggested that the 10 selected kinases might not be the
biological target for synthesized compounds. Nevertheless,
All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were un-
corrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LCeMS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear
magnetic resonance spectroscopy was performed using Bruker
ARX-300, 300 MHz spectrometers (Bruker Bioscience, Billerica, MA,
USA) with TMS as an internal standard. Column chromatography
was run on silica gel (200e300 mesh) from Qingdao Ocean
Table 3
Inhibition of 10 selected kinases by compound 29a.
a
Compd.
IC50 in
m
M
PI3K
0.13
0.003
e
a
mTOR
B-Raf
C-Raf
EGFR
VEGFR
PDGFR
b
FLT3
KIT
KDR
29a
>10
e
>10
e
>10
e
>10
e
>10
e
5.8
e
1.0
e
6.1
e
>10
GDC-0941b
Sorafenibb
e
0.012
0.006
e
e
e
e
e
e
a
Data presented is the mean SD value of two independent determinations.
Used as positive control.
b

220
Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
Fig. 4. Compound 29a inhibits cell growth by inducing apoptosis in the HT-29 cell line. The apoptosis was assessed by Western blot. HT-29 cells were treated with either vehicle
(Con) or different concentration of 29a compound solution (1 or 5 M). The level of different protein expression was quantified with Western blot. The ratio between Clv-PARP and
m
actin protein level was used to reflect the activity of PARP protein. Such ratio in cells treated with 29a solution is significantly higher than the control group, indicating that PARP is
actively involved in the 29a-induced apoptosis.
Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all
materials were obtained from commercially available sources and
were used without further purification.
After the addition, the reaction was kept at room temperature for
10 h and was monitored by TLC. The mixture was washed with 10%
K2CO3 (100 mL ꢁ 3), brine (100 mL ꢁ 3) in sequence, and the
organic phase was obtained, dried and evaporated to afford the
corresponding compound 4a,4b.
5.2. Methyl 3-acetamidothiophene-2-carboxylate (1)
Methyl 3-amino-2-thiophenecarboxylate (31.4 g, 0.2 mol) was
dissolved in dehydrated pyridine (150 mL) and cooled in an ice
bath. Acetyl chloride (17.2 g, 0.22 mol) was added dropwise and the
cold bath was removed and the resulting suspension was stirred at
room temperature for 3 h. The solvent was evaporated to yield a red
semisolid. The suspension was filtered, washed with water and
suction dried to give the title compound 1 (36.5 g) as a whitþe solid,
92% yield. M.p.: 98e100 ꢀC; MS (ESI) m/z (%): 200.1 [MþH] .
5.5.1. Methyl 3-(4-nitrobenzamido)thiophene-2-carboxylate (4a)
Yellow solid; yield: 84%; M.p.: 156e158 ꢀC; MS (ESI) m/z (%):
307.1 [MþH]þ.
5.5.2. Methyl 4,5-dichloro-3-(4-nitrobenzamido)thiophene-2-
carboxylate (4b)
Yellow solid; yield: 81%; M.p.: 191e194 ꢀC; MS (ESI) m/z (%):
375.2 [MþH]þ.
5.3. Methyl 3-acetamido-4,5-dichlorothiophene-2-carboxylate (2)
5.6. General procedure for preparation of 3-(4-nitrobenzamido)
thiophene-2-carboxamides (5)
To the solution of 1 (30.0 g, 0.15 mol) in chloroform (100 mL),
sulfuryl chloride (30.5 mL, 0.37 mol) was added dropwise, and the
solution heated to reflux for 2 h. The solvent was evaporated and
the dark red semisolid residue was diluted with diethyl ether
(150 mL). Subsequently, the suspension was filtered, washed with
ether and dried to afford the title compound 2 (24.5 g) as a white
solid, 61% yield. M.p.: 120e123 ꢀC; MS (ESI) m/z (%): 268.2 [MþH]þ.
To a solution of an appropriate methyl thiophene-2-carboxylate
(0.05 mol) in ethanol (200 mL) was added ammonia solution
(50 mL, 50%). The reaction mixture was heated with stirring for 8 h
at 60 ꢀC, whereby a yellow precipitate was formed. The precipitant
was filtered, washed with water and dried to afford the corre-
sponding compound 5a,5b.
5.4. Methyl 3-amino-4,5-dichlorothiophene-2-carboxylate (3b)
5.6.1. 3-(4-Nitrobenzamido)thiophene-2-carboxamide (5a)
Yellow solid; yield: 76%; M.p.: 167e169 ꢀC; MS (ESI) m/z (%):
292.1 [MþH]þ.
To the solution of 2 (24.5 g, 0.092 mol) in dioxane (200 mL) was
added 20% H2SO4 (18.0 g, 0.184 mol) and the resulting reaction
mixture was heated to 100 ꢀC for 6 h. The solvent was removed by
distillation under reduced pressure. After addition of about 150 mL
of water, the mixture was stirred for further 15 min at room tem-
perature, filtered and dried to afford the title compound 3b (18.8 g)
as a light yellow solid, 91% yield. M.p.: 151e153 ꢀC; MS (ESI) m/z (%):
226.0 [MþH]þ.
5.6.2. 4,5-Dichloro-3-(4-nitrobenzamido)thiophene-2-
carboxamide (5b)
Yellow solid; yield: 74%; M.p.: 182e184 ꢀC; MS (ESI) m/z (%):
359.9 [MþH]þ.
5.5. General procedure for preparation of methyl 3-(4-
nitrobenzamido)thiophene-2-carboxylates (4a,4b)
5.7. General procedure for preparation of 2-(4-nitrophenyl)thieno
[3,2-d]pyrimidin-4-ols (6a,6b)
A mixture of 4-nitrobenzoic acid (16.7 g, 0.1 mmol), toluene
(150 mL), and SOCl2 (50 mL) was heated to 80 ꢀC for 5 h. Upon
cooling to room temperature, the solvent was evaporated under
vacuum and the residue was dissolved in dehydrated CH2Cl2
(50 mL). Subsequently, a mixture of an appropriate methyl 3-
amino-2-thiophenecarboxylate (0.1 mol), Et3N (0.12 mol) and
CH2Cl2 (150 mL) was added into the CH2Cl2 solution in an ice bath.
To a solution of an appropriate thiophene-2-carboxamide
(0.034 mol) in isopropanol (150 mL) was added 5% NaOH
(0.1 mol) and heated at reflux for 2 h. After cooling, the reaction
mixture was poured into ice water under agitated stirring, acidified
with HCl to pH 2, 3, filtered and dried to afford the corresponding
compound 6a,6b.

Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
221
5.7.1. 2-(4-Nitrophenyl)thieno[3,2-d]pyrimidin-4-ol (6a)
5.9.4. N1,N1-diethyl-N3-(2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-
4-yl)propane-1,3-diamine (11a)
Light yellow solid; yield: 74%; M.p.:153e156 ꢀC; MS (ESI) m/z
(%): 274.1 [MþH]þ.
Gray solid; yield: 85%; M.p.: 181e184 ꢀC; MS (ESI) m/z (%): 386.2
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.74 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.65 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.77 (t,
J ¼ 5.4 Hz, 1H, ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 3.70 (dd, J ¼ 12.2,
6.3 Hz, 2H, CH2), 2.94 (s, 6H, 3CH2), 2.02 (s, 2H, CH2), 1.14 (t,
J ¼ 7.1 Hz, 6H, 2CH3).
5.7.2. 6,7-Dichloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-ol
(6b)
Light yellow solid; yield: 72%; M.p.: 171e174 ꢀC; MS (ESI) m/z
(%): 341.9 [MþH]þ.
5.9.5. N1-(6,7-dichloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-
yl)-N2,N2-dimethylethane-1,2-diamine (8b)
5.8. General procedure for preparation of 4-chloro-2-(4-
nitrophenyl)thieno[3,2-d]pyrimidines (7a,7b)
Gray solid; yield: 77%; M.p.: 179e183 ꢀC; MS (ESI) m/z (%): 412.3
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.74 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
A
mixture of an appropriate thieno[3,2-d]pyrimidin-4-ol
8.63 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.22 (d, J ¼ 5.2 Hz, 1H, NH), 3.72 (dd,
J ¼ 12.2, 6.3 Hz, 2H, CH2), 2.55 (dd, J ¼ 13.5, 6.8 Hz, 2H, CH2), 2.23 (s,
6H, 2CH3).
(0.025 mol) in phosphorus oxychloride (100 mL) were heated to
80 ꢀC for 6 h. The solvent was concentrated under vacuum and the
residue was poured into stirring ice-water (200 mL), basified with
potassium hydroxide to pH 8, 9, filtered and dried to afford the
corresponding compound 7a,7b.
5.9.6. N1-(6,7-dichloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-
yl)-N3,N3-dimethylpropane-1,3-diamine (9b)
Gray solid; yield: 85%; M.p.: 182e186 ꢀC; MS (ESI) m/z (%): 426.1
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.75 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
5.8.1. 4-Chloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidine (7a)
Gray solid; yield: 69%; M.p.: 156e159 ꢀC; MS (ESI) m/z (%): 292.0
[MþH]þ.
8.65 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.24 (d, J ¼ 5.2 Hz, 1H, NH), 3.63 (dd,
J ¼ 12.3, 6.4 Hz, 2H, CH2), 2.39 (t, J ¼ 6.9 Hz, 2H, CH2), 2.20 (s, 6H,
2CH3), 1.88e1.75 (m, 2H, CH2).
5.8.2. 4,6,7-Trichloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidine
(7b)
5.9.7. N1-(6,7-dichloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-
yl)-N2,N2-diethylethane-1,2-diamine (10b)
Gray solid; yield: 72%; M.p.: 178e180 ꢀC; MS (ESI) m/z (%): 359.9
[MþH]þ.
Gray solid; yield: 73%; M.p.: 174e178 ꢀC; MS (ESI) m/z (%): 440.1
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.74 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.63 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.22 (d, J ¼ 5.2 Hz, 1H, NH), 3.67 (dd,
J ¼ 13.5, 6.2 Hz, 2H, CH2), 2.72e2.63 (m, 2H, CH2), 2.56 (q, J ¼ 7.1 Hz,
4H, 2CH2), 0.98 (t, J ¼ 7.1 Hz, 6H, 2CH3).
5.9. General procedure for preparation of 2-(4-nitrophenyl)-4-
aminothieno[3,2-d]pyrimidins (8ae11a, 8be11b)
Triethylamine (0.026 mol) and corresponding aliphatic amine
(0.019 mol) were successively added to a solution of an appropriate
chloro compound (0.017 mol) in THF (80 mL). After stirring at 30 ꢀC
for 2 h, excess THF was removed under reduced pressure. The
residue was poured into water (200 mL), separated by filtration to
afford the corresponding compound 8ae11a, 8be11b.
5.9.8. N1-(6,7-dichloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-
yl)-N3,N3-diethylpropane-1,3-diamine (11b)
Gray solid; yield: 71%; M.p.: 186e188 ꢀC; MS (ESI) m/z (%): 454.2
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.76 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.65 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.23 (d, J ¼ 5.2 Hz, 1H, NH), 3.70 (d,
J ¼ 4.8 Hz, 2H, CH2), 3.18 (s, 2H, CH2), 3.11 (d, J ¼ 5.1 Hz, 4H, 2CH2),
2.07 (s, 2H, CH2), 1.20 (t, J ¼ 6.7 Hz, 6H, 2CH3).
5.9.1. N1,N1-dimethyl-N2-(2-(4-nitrophenyl)thieno[3,2-d]
pyrimidin-4-yl)ethane-1,2-diamine (8a)
5.10. General procedure for preparation of 2-(4-aminophenyl)-4-
aminothieno[3,2-d]pyrimidins (12ae15a, 12be15b)
Gray solid; yield: 83%; M.p.:187e190 ꢀC; MS (ESI) m/z (%): 344.1
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.72 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.65 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.75 (t,
J ¼ 5.4 Hz, 1H, ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 3.81 (d, J ¼ 5.7 Hz,
2H, CH2), 2.86 (s, 2H, CH2), 2.46 (s, 6H, 2CH3).
To a refluxing solution of an appropriate nitro compound
(0.002 mol) in EtOH (25 mL, 10v/w) was added saturation NH4Cl
solution (25 mL) and Fe Powder (0.008 mol) in batches. The
mixture was kept at this temperature for more 5 h. After comple-
tion of the reaction as indicated by TLC, the mixture was filtered
immediately, and the filtrate was cooled, filtered to obtain the
corresponding anilines 12ae15a, 12be15b.
5.9.2. N1,N1-dimethyl-N3-(2-(4-nitrophenyl)thieno[3,2-d]
pyrimidin-4-yl)propane-1,3-diamine (9a)
Gray solid; yield: 82%; M.p.: 182e185 ꢀC; MS (ESI) m/z (%): 358.2
[MþH]þ. 1H NMR (400 MHz, DMSO)
8.74 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
5.10.1. N1,N1-dimethyl-N2-(2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)ethane-1,2-diamine (12a)
8.63 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.09 (d, J ¼ 5.3 Hz, 1H, ArH), 7.77 (t,
J ¼ 5.4 Hz, 1H, ArH), 7.45 (d, J ¼ 5.3 Hz, NH), 3.64 (dd, J ¼ 12.5,
6.6 Hz, 2H, CH2), 2.47 (d, J ¼ 7.1 Hz, 2H, CH2), 1.81 (p, J ¼ 6.9 Hz, 2H,
CH2), 0.98 (t, J ¼ 7.1 Hz, 6H, 2CH3).
Gray solid; yield: 81%; M.p.: 143e146 ꢀC; MS (ESI) m/z (%): 314.1
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.34 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.77 (t, J ¼ 5.4 Hz, 1H, ArH), 7.58 (d,
J ¼ 8.7 Hz, 2H, 2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 6.27 (s, 2H, NH2),
3.82 (d, J ¼ 5.7 Hz, 2H, CH2), 2.87 (s, 2H, CH2), 2.46 (s, 6H, 2CH3).
5.9.3. N1,N1-diethyl-N2-(2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-
4-yl)ethane-1,2-diamine (10a)
Gray solid; yield: 79%; M.p.: 174e178 ꢀC; MS (ESI) m/z (%): 372.1
[MþH]þ. 1H NMR (400 MHz, DMSO)
d
8.75 (d, J ¼ 8.5 Hz, 2H, 2ArH),
5.10.2. N1,N1-dimethyl-N3-(2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)propane-1,3-diamine (13a)
8.64 (d, J ¼ 8.5 Hz, 2H, 2ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.76 (t,
J ¼ 5.4 Hz, 1H, ArH), 7.43 (d, J ¼ 5.3 Hz, 1H, NH), 3.72 (dd, J ¼ 13.4,
6.2 Hz, 2H, CH2), 2.81 (t, J ¼ 7.1 Hz, 2H, CH2), 2.74e2.60 (m, 4H,
2CH2), 1.02 (t, J ¼ 7.1 Hz, 6H, 2CH3).
Gray solid; yield: 74%; M.p.:153e157 ꢀC; MS (ESI) m/z (%): 328.2
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.36 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.09 (d, J ¼ 5.3 Hz, 1H, ArH), 7.77 (t, J ¼ 5.4 Hz, 1H, ArH), 7.60 (d,

222
Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
J ¼ 8.7 Hz, 2H, 2ArH), 7.45 (d, J ¼ 5.3 Hz, 1H, NH), 6.27 (s, 2H, NH2),
3.64 (dd, J ¼ 12.5, 6.6 Hz, 2H, CH2), 2.47 (d, J ¼ 7.1 Hz, 2H, CH2), 1.81
(p, J ¼ 6.9 Hz, 2H, CH2), 0.98 (t, J ¼ 7.1 Hz, 6H, 2CH3).
5.11.1. 1-(4-(4-((3-(Dimethylamino)propyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(2-fluorophenyl)urea (16a)
Yellow solid; yield: 81%; M.p.: 191e194 ꢀC; MS (ESI) m/z (%):
465.2 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.24 (s, 1H, NHCO),
9.17 (s, 1H, NHCO), 8.35 (d, J ¼ 8.8 Hz, 2H, 2ArH), 8.18e8.10 (m, 1H,
ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.95 (t, J ¼ 5.2 Hz, 1H, ArH), 7.60
(d, J ¼ 8.8 Hz, 2H, 2ArH), 7.41 (d, J ¼ 5.3 Hz, 1H, NH), 7.27e7.19 (m,
1H, ArH), 7.14 (t, J ¼ 7.7 Hz, 1H, ArH), 7.05e6.97 (m, 1H, ArH),
3.70e3.58 (m, 2H, CH2), 2.35 (t, J ¼ 6.9 Hz, 2H, CH2), 2.17 (s, 6H,
2CH3), 1.82 (p, J ¼ 7.0 Hz, 2H, CH2). Anal. calcd. for C24H25FN6OS
(%):C, 62.05; H, 5.42; N, 18.09. Found (%): C, 62.08; H, 5.40; N, 18.07.
5.10.3. N1,N1-diethyl-N2-(2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)ethane-1,2-diamine (14a)
Gray solid; yield: 72%; M.p.: 147e150 ꢀC; MS (ESI) m/z (%): 342.1
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.35 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.76 (t, J ¼ 5.4 Hz, 1H, ArH), 7.61 (d,
J ¼ 8.7 Hz, 2H, 2ArH), 7.43 (d, J ¼ 5.3 Hz, 1H, NH), 6.24 (s, 2H, NH2),
3.72 (dd, J ¼ 13.4, 6.2 Hz, 2H, CH2), 2.81 (t, J ¼ 7.1 Hz, 2H, CH2),
2.74e2.60 (m, 4H, 2CH2), 1.02 (t, J ¼ 7.1 Hz, 6H, 2CH3).
5.11.2. 1-(2-Chlorophenyl)-3-(4-(4-((3-(dimethylamino)propyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (17a)
5.10.4. N1,N1-diethyl-N3-(2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)propane-1,3-diamine (15a)
Yellow solid; yield: 83%; M.p.: 184e187 ꢀC; MS (ESI) m/z (%):
481.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.27 (s, 1H, NHCO),
Gray solid; yield: 77%; M.p.: 137e140 ꢀC; MS (ESI) m/z (%): 356.2
8.75 (s, 1H, NHCO), 8.36 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.14 (d, J ¼ 7.4 Hz,
1H, ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.94 (s, 1H, ArH), 7.61 (d,
J ¼ 8.7 Hz, 2H, 2ArH), 7.46 (d, J ¼ 8.0 Hz, 1H, ArH), 7.41 (d, J ¼ 5.3 Hz,
1H, NH), 7.31 (t, J ¼ 7.8 Hz, 1H, ArH), 7.04 (dd, J ¼ 11.2, 4.2 Hz, 1H,
ArH), 3.64 (d, J ¼ 4.7 Hz, 2H, CH2), 2.35 (t, J ¼ 6.9 Hz, 2H, CH2), 2.17
(s, 6H, 2CH3), 1.83 (p, J ¼ 6.9 Hz, 2H, CH2). Anal. calcd. for
C24H25ClN6OS (%):C, 59.93; H, 5.24; N, 17.47. Found (%): C, 59.96; H,
5.23; N, 17.45.
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.34 (d, J ¼ 8.7 Hz, 2H, 2ArH),
d
8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.77 (t, J ¼ 5.4 Hz, 1H, ArH), 7.58 (d,
J ¼ 8.7 Hz, 2H, 2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 6.27 (s, 2H, NH2),
3.70 (dd, J ¼ 12.2, 6.3 Hz, 2H, CH2), 2.94 (s, 6H, 3CH2), 2.02 (s, 2H,
CH2), 1.14 (t, J ¼ 7.1 Hz, 6H, 2CH3).
5.10.5. N1-(6,7-dichloro-2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)-N2,N2-dimethylethane-1,2-diamine (12b)
Gray solid; yield: 70%; M.p.: 161e165 ꢀC; MS (ESI) m/z (%): 382.3
5.11.3. 1-(2,4-Dichlorophenyl)-3-(4-(4-((3-(dimethylamino)propyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl) urea (18a)
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.39 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
Yellow solid; yield: 76%; M.p.: 181e184 ꢀC; MS (ESI) m/z (%):
8.22 (d, J ¼ 5.2 Hz,1H, NH), 7.63 (d, J ¼ 8.7 Hz, 2H, 2ArH), 6.25 (s, 2H,
NH2), 3.72 (dd, J ¼ 12.2, 6.3 Hz, 2H, CH2), 2.55 (dd, J ¼ 13.5, 6.8 Hz,
2H, CH2), 2.23 (s, 6H, 2CH3).
515.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.51 (s, 1H, NHCO),
8.94 (s, 1H, NHCO), 8.36 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.18 (d, J ¼ 9.0 Hz,
1H, ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 8.01 (t, J ¼ 5.0 Hz, 1H, ArH),
7.63 (s, 1H, ArH), 7.61 (d, J ¼ 2.2 Hz, 2H, 2ArH), 7.41 (d, J ¼ 5.3 Hz,1H,
NH), 7.38 (dd, J ¼ 9.0, 2.5 Hz, 1H, ArH), 3.64 (dd, J ¼ 12.5, 6.6 Hz, 2H,
CH2), 2.47 (d, J ¼ 7.1 Hz, 2H, CH2), 1.81 (p, J ¼ 6.9 Hz, 2H, CH2), 0.98
5.10.6. N1-(6,7-dichloro-2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)-N3,N3-dimethylpropane-1,3-diamine (13b)
Gray solid; yield: 69%; M.p.: 157e159 ꢀC; MS (ESI) m/z (%): 396.1
(t, J ¼ 7.1 Hz, 6H, 2CH3); 13C NMR (400 MHz, DMSO)
d 160.68,
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.40 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
160.09, 157.26, 152.70, 141.73, 135.82, 133.12, 132.70, 129.03, 128.95
(2C), 127.96, 126.64, 125.13, 123.72, 123.15, 117.89 (2C), 113.18, 51.03,
49.04, 46.79 (2C), 26.64. Anal. calcd. for C24H24Cl2N6OS (%):C, 55.92;
H, 4.69; N, 16.30. Found (%): C, 55.95; H, 4.67; N, 16.32.
8.24 (d, J ¼ 5.2 Hz,1H, NH), 7.65 (d, J ¼ 8.7 Hz, 2H, 2ArH), 6.25 (s, 2H,
NH2), 3.63 (dd, J ¼ 12.3, 6.4 Hz, 2H, CH2), 2.39 (t, J ¼ 6.9 Hz, 2H, CH2),
2.20 (s, 6H, 2CH3), 1.88e1.75 (m, 2H, CH2).
5.10.7. N1-(6,7-dichloro-2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)-N2,N2-diethylethane-1,2-diamine (14b)
Gray solid; yield: 73%; M.p.: 154e158 ꢀC; MS (ESI) m/z (%): 410.1
5.11.4. 1-(4-(4-((2-(Dimethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-phenylurea (19a)
Yellow solid; yield: 73%; M.p.: 168e170 ꢀC; MS (ESI) m/z (%):
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.38 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
432.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.60 (s, 1H, NHCO),
8.22 (d, J ¼ 5.2 Hz,1H, NH), 7.63 (d, J ¼ 8.7 Hz, 2H, 2ArH), 6.24 (s, 2H,
NH2), 3.67 (dd, J ¼ 13.5, 6.2 Hz, 2H, CH2), 2.72e2.63 (m, 2H, CH2),
2.56 (q, J ¼ 7.1 Hz, 4H, 2CH2), 0.98 (t, J ¼ 7.1 Hz, 6H, 2CH3).
9.40 (s, 1H, NHCO), 8.38 (s, 2H, 2ArH), 8.36 (s, 1H, ArH), 7.60 (d,
J ¼ 7.7 Hz, 2H, 2ArH), 7.49 (d, J ¼ 6.7 Hz, 2H, 2ArH), 7.41 (d,
J ¼ 5.3 Hz, 1H, NH), 7.38 (dd, J ¼ 9.0, 2.5 Hz, 1H, ArH), 7.29 (s, 2H,
2ArH), 6.97 (s, 1H, ArH), 3.73 (d, J ¼ 5.7 Hz, 2H, CH2), 2.55 (t,
J ¼ 6.4 Hz, 2H, CH2), 2.24 (s, 6H, 2CH3). Anal. calcd. for C23H24N6OS
(%):C, 63.87; H, 5.59; N, 19.43. Found (%): C, 63.84; H, 5.61; N, 19.45.
5.10.8. N1-(6,7-dichloro-2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)-N3,N3-diethylpropane-1,3-diamine (15b)
Gray solid; yield: 71%; M.p.: 164e167 ꢀC; MS (ESI) m/z (%): 424.2
[MþH]þ; 1H NMR (400 MHz, DMSO)
8.36 (t, J ¼ 8.7 Hz, 2H, 2ArH),
d
5.11.5. 1-(4-(4-((2-(Dimethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea (20a)
8.23 (d, J ¼ 5.2 Hz,1H, NH), 7.65 (d, J ¼ 8.7 Hz, 2H, 2ArH), 6.24 (s, 2H,
NH2), 3.70 (d, J ¼ 4.8 Hz, 2H, CH2), 3.18 (s, 2H, CH2), 3.11 (d,
J ¼ 5.1 Hz, 4H, 2CH2), 2.07 (s, 2H, CH2), 1.20 (t, J ¼ 6.7 Hz, 6H, 2CH3).
Yellow solid; yield: 76%; M.p.: 181e184 ꢀC; MS (ESI) m/z (%):
451.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.41 (s, 1H, NHCO), 9.34
(s, 1H, NHCO), 8.36 (d, J ¼ 8.5 Hz, 2H, 2ArH), 8.12 (t, J ¼ 10.8 Hz, 1H,
ArH), 7.88 (s, 1H, ArH), 7.58 (d, J ¼ 8.5 Hz, 2H, 2ArH), 7.50 (dd,
J ¼ 8.5, 4.9 Hz, 2H, 2ArH), 7.43 (d, J ¼ 5.3 Hz, 1H, NH), 7.13 (t,
J ¼ 8.7 Hz, 2H, 2ArH), 3.82 (d, J ¼ 5.7 Hz, 2H, CH2), 2.87 (s, 2H, CH2),
5.11. General procedure for preparation of the target compounds
(16ae35a, 16be36b)
An appropriate amine (5.3 mmol) in CH2Cl2 (50 mL) was added
substituted isocyanatobenzene (6.4 mmol), and the resulting
mixture was stirred at room temperature overnight. The resulting
solid was collected by filtration and washed with CH2Cl2 to afford
the target compounds 16ae35a, 16be35b.
2.46 (s, 6H, 2CH3); 13C NMR (101 MHz, DMSO)
d 160.87, 160.10,
157.25, 156.58, 153.13, 142.05, 136.61, 133.47, 132.24, 128.95 (2C),
125.07, 120.23, 120.16, 117.81 (2C), 115.86, 115.64, 113.29, 57.67,
44.82 (2C), 37.73. Anal. calcd. for C23H23FN6OS (%):C, 61.32; H, 5.15;
N, 18.65. Found (%): C, 61.34; H, 5.13; N, 18.67.

Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
223
5.11.6. 1-(4-(4-((2-(Dimethylamino)ethyl)amino)thieno[3,2-d]
5.11.11. 1-(4-(4-((3-(Diethylamino)propyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(2-fluorophenyl)urea (21a)
pyrimidin-2-yl)phenyl)-3-phenylurea (26a)
Yellow solid; yield: 73%; M.p.: 173e175 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 82%; M.p.: 186e189 ꢀC; MS (ESI) m/z (%):
451.5 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
10.02 (s, 1H, NHCO),
475.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.60 (s,1H, NHCO), 9.40
9.14 (s, 1H, NHCO), 8.36 (d, J ¼ 8.6 Hz, 2H, 2ArH), 8.12 (t, J ¼ 7.4 Hz,
1H, ArH), 8.09 (d, J ¼ 5.4 Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.61 (d,
J ¼ 8.6 Hz, 2H, 2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 7.28e7.20 (m, 1H,
ArH), 7.14 (d, J ¼ 7.5 Hz, 1H, ArH), 7.02 (dd, J ¼ 12.9, 6.6 Hz, 1H, ArH),
3.73 (d, J ¼ 5.9 Hz, 2H, CH2), 2.57 (t, J ¼ 6.8 Hz, 2H, CH2), 2.24 (s, 6H,
2CH3). Anal. calcd. for C23H23FN6OS (%):C, 61.32; H, 5.15; N, 18.65.
Found (%):C, 61.35; H, 5.13; N, 18.63.
(s,1H, NHCO), 8.38 (s, 2H, 2ArH), 8.36 (s,1H, ArH), 7.60 (d, J ¼ 7.7 Hz,
2H, 2ArH), 7.49 (d, J ¼ 6.7 Hz, 2H, 2ArH), 7.41 (d, J ¼ 5.3 Hz, 1H, NH),
7.38 (dd, J ¼ 9.0, 2.5 Hz, 1H, ArH), 7.29 (s, 2H, 2ArH), 6.97 (s, 1H,
ArH), 3.70 (dd, J ¼ 12.2, 6.3 Hz, 2H, CH2), 2.94 (s, 6H, 3CH2), 2.02 (s,
2H, CH2), 1.14 (t, J ¼ 7.1 Hz, 6H, 2CH3). Anal. calcd. for C26H30N6OS
(%):C, 65.80; H, 6.37; N, 17.71. Found (%): C, 65.78; H, 6.38; N, 17.74.
5.11.12. 1-(4-(4-((3-(Diethylamino)propyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea (27a)
5.11.7. 1-(4-Chlorophenyl)-3-(4-(4-((2-(dimethylamino)ethyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (22a)
Yellow solid; yield: 76%; M.p.: 190e193 ꢀC; MS (ESI) m/z (%):
493.7 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.20 (s,1H, NHCO), 9.10
Yellow solid; yield: 79%; M.p.: 186e188 ꢀC; MS (ESI) m/z (%):
(s, 1H, NHCO), 8.36 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.09 (d, J ¼ 5.3 Hz, 1H,
ArH), 7.96 (t, J ¼ 5.3 Hz, 1H, ArH), 7.56 (d, J ¼ 8.7 Hz, 2H, 2ArH),
7.52e7.46 (m, 2H, 2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 7.13 (t,
J ¼ 8.9 Hz, 2H, 2ArH), 3.67 (dd, J ¼ 12.4, 6.5 Hz, 2H, CH2), 2.75 (s, 6H,
3CH2), 1.91 (d, J ¼ 3.9 Hz, 2H, CH2), 1.06 (d, J ¼ 7.0 Hz, 6H, 2CH3).
Anal. calcd. for C26H29FN6OS (%):C, 63.39; H, 5.93; N, 17.06. Found
(%): C, 63.36; H, 5.92; N, 17.04.
467.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.42 (s, 2H, 2NHCO),
8.35 (d, J ¼ 8.6 Hz, 2H, 2ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.77 (t,
J ¼ 5.1 Hz, 1H, ArH), 7.58 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.53 (d, J ¼ 8.8 Hz,
2H, 2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 7.33 (d, J ¼ 8.8 Hz, 2H, 2ArH),
3.72 (dd, J ¼ 12.5, 6.3 Hz, 2H, CH2), 2.56 (t, J ¼ 6.8 Hz, 2H, CH2), 2.24
(s, 6H, 2CH3). Anal. calcd. for C23H23ClN6OS (%):C, 59.16; H, 4.96; N,
18.00. Found (%): C, 59.14; H, 4.97; N, 18.03.
5.11.13. 1-(4-(4-((3-(Diethylamino)propyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(2-fluorophenyl)urea (28a)
5.11.8. 1-(2-Chlorophenyl)-3-(4-(4-((2-(dimethylamino)ethyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (23a)
Yellow solid; yield: 78%; M.p.: 183e185 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 73%; M.p.: 162e165 ꢀC; MS (ESI) m/z (%):
493.5 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.44 (s, 1H, NHCO),
467.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
10.29 (s, 1H, NHCO),
8.69 (s, 1H, NHCO), 8.38 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.17 (t, J ¼ 7.7 Hz,
1H, ArH), 8.09 (d, J ¼ 5.3 Hz, 1H, ArH), 7.97 (d, J ¼ 5.3 Hz, 1H, ArH),
7.57 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 7.35e7.23
(m, 1H, ArH), 7.17 (dd, J ¼ 21.5, 13.8 Hz, 1H, ArH), 7.02 (dd, J ¼ 12.9,
6.3 Hz, 1H, ArH), 3.66 (dd, J ¼ 12.1, 6.3 Hz, 2H, CH2), 2.58 (t,
J ¼ 18.3 Hz, 6H, 3CH2), 1.87 (dd, J ¼ 16.1, 9.5 Hz, 2H, CH2), 1.02 (t,
J ¼ 7.1 Hz, 6H, 2CH3). Anal. calcd. for C26H29FN6OS (%):C, 63.39; H,
5.93; N, 17.06. Found (%):C, 63.42; H, 5.91; N, 17.05.
8.79 (s, 1H, NHCO), 8.36 (d, J ¼ 8.6 Hz, 2H, 2ArH), 8.14 (d, J ¼ 8.2 Hz,
1H, ArH), 8.09 (d, J ¼ 5.3 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.62 (d,
J ¼ 8.6 Hz, 2H, 2ArH), 7.46 (d, J ¼ 8.0 Hz, 1H, ArH), 7.42 (d, J ¼ 5.3 Hz,
1H, NH), 7.31 (t, J ¼ 7.8 Hz,1H, ArH), 7.04 (t, J ¼ 7.6 Hz,1H, ArH), 3.73
(d, J ¼ 4.3 Hz, 2H, CH2), 2.57 (t, J ¼ 6.7 Hz, 2H, CH2), 2.24 (s, 6H,
2CH3). Anal. calcd. for C23H23ClN6OS (%):C, 59.16; H, 4.96; N, 18.00.
Found (%): C, 59.15; H, 4.98; N, 18.02.
5.11.14. 1-(4-Chlorophenyl)-3-(4-(4-((3-(diethylamino)propyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (29a)
5.11.9. 1-(2,4-Dichlorophenyl)-3-(4-(4-((2-(dimethylamino)ethyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl) urea (24a)
Yellow solid; yield: 75%; M.p.: 179e182 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 76%; M.p.: 166e168 ꢀC; MS (ESI) m/z (%):
509.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.70 (s, 2H, 2NHCO),
501.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.08 (s, 1H, NHCO),
8.37 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.09 (d, J ¼ 5.3 Hz, 1H, ArH), 7.99 (s,
1H, ArH), 7.59 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.54 (d, J ¼ 8.8 Hz, 2H, 2ArH),
7.42 (d, J ¼ 5.3 Hz, 1H, NH), 7.33 (d, J ¼ 8.8 Hz, 2H, 2ArH), 3.68 (d,
J ¼ 5.5 Hz, 2H, CH2), 2.88 (dd, J ¼ 18.6, 11.5 Hz, 2H, CH2), 2.81 (dd,
J ¼ 14.2, 7.1 Hz, 4H, 2CH2), 1.94 (dd, J ¼ 15.2, 8.0 Hz, 2H, CH2),
8.78 (s, 1H, NHCO), 8.37 (d, J ¼ 8.4 Hz, 2H, 2ArH), 8.19 (d, J ¼ 8.9 Hz,
1H, ArH), 8.09 (d, J ¼ 5.2 Hz, 1H, ArH), 7.78 (s, 1H, ArH), 7.61 (s, 2H,
2ArH), 7.59 (s, 1H, ArH), 7.42 (d, J ¼ 5.5 Hz,1H, NH), 7.38 (s, 1H, ArH),
3.73 (d, J ¼ 4.2 Hz, 2H, CH2), 2.56 (t, J ¼ 6.6 Hz, 2H, CH2), 2.24 (s, 6H,
2CH3); 13C NMR (101 MHz, DMSO)
d 160.79, 160.02, 157.30, 152.55,
1.14e1.03 (m, 6H, 2CH3); 13C NMR (101 MHz, DMSO)
d 160.79,
141.57, 135.75, 133.28, 132.80, 129.04, 128.92 (2C), 128.03, 126.70,
125.10, 123.57, 123.03, 118.04 (2C), 113.20, 58.53, 45.87 (2C), 38.76.
Anal. calcd. for C23H22Cl2N6OS (%):C, 55.09; H, 4.42; N, 16.76. Found
(%): C, 55.07; H, 4.40; N, 16.75.
160.14, 157.27, 153.01, 141.95, 139.36, 133.28, 132.38, 129.08 (2C),
128.94 (2C), 125.66, 125.12, 119.98 (2C), 117.82 (2C), 113.21, 50.00,
46.70 (2C), 38.92, 25.09, 10.50 (2C). Anal. calcd. for C26H29ClN6OS
(%):C, 61.34; H, 5.74; N, 16.51. Found (%):C, 61.32; H, 5.76; N, 16.53.
5.11.10. 1-(4-(4-((2-(Dimethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(p-tolyl)urea (25a)
5.11.15. 1-(2-Chlorophenyl)-3-(4-(4-((3-(diethylamino)propyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (30a)
Yellow solid; yield: 80%; M.p.: 176e178 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 81%; M.p.: 185e187 ꢀC; MS (ESI) m/z (%):
447.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
9.54 (s,1H, NHCO), 9.33
509.8 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.02 (s, 1H, NHCO),
(s, 1H, NHCO), 8.34 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.08 (d, J ¼ 5.3 Hz, 1H,
ArH), 7.77 (t, J ¼ 5.4 Hz, 1H, ArH), 7.58 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.42
(d, J ¼ 5.3 Hz, 1H, NH), 7.39 (d, J ¼ 8.4 Hz, 2H, 2ArH), 7.09 (d,
J ¼ 8.3 Hz, 2H, 2ArH), 3.72 (dd, J ¼ 12.7, 6.5 Hz, 2H, CH2), 2.56 (t,
J ¼ 6.8 Hz, 2H, CH2), 2.24 (s, 9H, 3CH3); 13C NMR (101 MHz, DMSO)
8.56 (s, 1H, NHCO), 8.39 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.17 (dd, J ¼ 8.3,
1.1 Hz, 1H, ArH), 8.10 (d, J ¼ 5.3 Hz, 1H, ArH), 8.01 (t, J ¼ 5.2 Hz, 1H,
ArH), 7.61 (d, J ¼ 8.8 Hz, 2H, 2ArH), 7.47 (dd, J ¼ 8.0, 1.2 Hz, 1H, ArH),
7.43 (d, J ¼ 5.3 Hz, 1H, NH), 7.31 (dd, J ¼ 12.0, 4.8 Hz, 1H, ArH),
7.11e6.99 (m, 1H, ArH), 3.70 (dd, J ¼ 12.2, 6.3 Hz, 2H, CH2), 2.94 (s,
6H, 3CH2), 2.02 (s, 2H, CH2), 1.14 (t, J ¼ 7.1 Hz, 6H, 2CH3); 13C NMR
d
160.81,160.15,157.28,153.13,142.25,137.80,133.22,132.15,130.94,
129.61 (2C), 128.84 (2C), 125.10, 118.62 (2C), 117.71 (2C), 113.09,
58.54, 45.87 (2C), 38.75, 20.82. Anal. calcd. for C24H26N6OS (%):C,
64.55; H, 5.87; N, 18.82. Found (%): C, 64.52; H, 5.89; N, 18.81.
(101 MHz, DMSO) d 160.81, 160.08, 157.28, 152.69, 141.82, 136.44,
133.43, 132.55, 129.70, 129.01 (2C), 127.96, 125.11, 123.88, 122.83,
122.14, 117.89 (2C), 113.27, 49.52, 46.61 (2C), 38.56, 24.82, 9.81 (2C).

224
Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
Anal. calcd. for C26H29ClN6OS (%):C, 61.34; H, 5.74; N, 16.51. Found
(%):C, 61.33; H, 5.75; N, 16.54.
5.11.21. 1-(4-(6,7-Dichloro-4-((3-(dimethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea (16b)
Yellow solid; yield: 69%; M.p.: 185e187 ꢀC; MS (ESI) m/z
(%):533.5 [MþH]þ; 1H NMR (400 MHz, DMSO)
9.58 (d, J ¼ 41.4 Hz,
d
5.11.16. 1-(4-(4-((3-(Diethylamino)propyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(p-tolyl)urea (31a)
2H, 2NHCO), 8.46 (s, 1H, NH), 8.37 (d, J ¼ 8.6 Hz, 2H, 2ArH), 7.60 (d,
J ¼ 8.6 Hz, 2H, 2ArH), 7.49 (dd, J ¼ 8.7, 5.0 Hz, 2H, 2ArH), 7.13 (t,
J ¼ 8.7 Hz, 2H, 2ArH), 3.70 (d, J ¼ 5.6 Hz, 2H, CH2), 3.18 (s, 2H, CH2),
2.75 (d, J ¼ 4.6 Hz, 6H, 2CH3), 2.10 (s, 2H, CH2). Anal. calcd. for
Yellow solid; yield: 78%; M.p.: 182e184 ꢀC; MS (ESI) m/z (%):
489.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.39 (s, 1H, NHCO), 9.15
(s, 1H, NHCO), 8.36 (d, J ¼ 8.5 Hz, 2H, 2ArH), 8.10 (d, J ¼ 5.2 Hz, 1H,
ArH), 7.98 (s, 1H, ArH), 7.57 (d, J ¼ 8.5 Hz, 2H, 2ArH), 7.43 (d,
J ¼ 5.3 Hz, 1H, NH), 7.37 (d, J ¼ 8.2 Hz, 2H, 2ArH), 7.09 (d, J ¼ 8.1 Hz,
2H, 2ArH), 3.70 (d, J ¼ 5.7 Hz, 2H, CH2), 3.03 (s, 6H, 3CH2), 2.25 (s,
3H, CH3), 2.05 (s, 2H, CH2), 1.17 (t, J ¼ 6.5 Hz, 6H, 2CH3). Anal. calcd.
for C27H32N6OS (%):C, 66.36; H, 6.60; N,17.20. Found (%):C, 66.32; H,
6.62; N, 17.23.
C24H23Cl2FN6OS (%):C, 54.04; H, 4.35; N, 15.75. Found (%):C, 54.01;
H, 4.34; N, 15.78.
5.11.22. 1-(4-Chlorophenyl)-3-(4-(6,7-dichloro-4-((3-
(dimethylamino)propyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
urea (17b)
Yellow solid; yield: 70%; M.p.: 190e193 ꢀC; MS (ESI) m/z
(%):549.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
9.85 (d, J ¼ 7.2 Hz,
5.11.17. 1-(4-(4-((2-(Diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-3-(2-fluorophenyl)urea (32a)
2H, 2NHCO), 8.41 (s, 1H, NH), 8.36 (d, J ¼ 8.1 Hz, 2H, 2ArH), 7.61 (d,
J ¼ 8.0 Hz, 2H, 2ArH), 7.53 (d, J ¼ 8.1 Hz, 2H, 2ArH), 7.33 (d,
J ¼ 8.2 Hz, 2H, 2ArH), 3.67 (s, 2H, CH2), 2.89 (s, 2H, CH2), 2.53 (d,
J ¼ 16.4 Hz, 6H, 2CH3), 2.00 (s, 2H, CH2); 13C NMR (400 MHz, DMSO)
Yellow solid; yield: 74%; M.p.: 183e186 ꢀC; MS (ESI) m/z (%):
479.6 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.65 (s, 1H, NHCO),
8.79 (s, 1H, NHCO), 8.39 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.17 (d, J ¼ 7.0 Hz,
1H, ArH), 8.13 (d, J ¼ 5.3 Hz, 1H, ArH), 8.06 (s, 1H, ArH), 7.59 (d,
J ¼ 8.7 Hz, 2H, 2ArH), 7.45 (d, J ¼ 5.3 Hz, 1H, NH), 7.25 (dd, J ¼ 11.1,
8.8 Hz,1H, ArH), 7.15 (t, J ¼ 7.7 Hz,1H, ArH), 7.02 (dd, J ¼ 13.8, 6.7 Hz,
1H, ArH), 3.96 (s, 2H, CH2), 3.18 (s, 6H, 3CH2), 1.19 (s, 6H, 2CH3).
Anal. calcd. for C25H27FN6OS (%):C, 62.74; H, 5.69; N, 17.56. Found
(%):C, 62.70; H, 5.70; N, 17.58.
d
161.18, 156.16, 154.36, 152.99, 142.53, 139.33, 132.01, 131.39, 129.21
(2C), 129.09 (2C), 125.66, 121.22 (2C), 119.88 (2C), 117.77, 110.41,
55.83, 43.56 (2C), 38.50, 25.24. Anal. calcd. for C24H23Cl3N6OS (%):C,
52.42; H, 4.22; N, 15.28. Found (%): C, 52.45; H, 4.20; N, 15.26.
5.11.23. 1-(2-Chlorophenyl)-3-(4-(6,7-dichloro-4-((3-
(dimethylamino)propyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
urea (18b)
5.11.18. 1-(4-Chlorophenyl)-3-(4-(4-((2-(diethylamino)ethyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (33a)
Yellow solid; yield: 76%; M.p.: 191e194 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 78%; M.p.: 173e176 ꢀC; MS (ESI) m/z (%):
549.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.22 (s, 1H, NHCO),
495.2 [MþH]þ; 1H NMR (400 MHz, DMSO)
9.40 (d, J ¼ 7.1 Hz, 2H,
d
8.68 (s, 1H, NHCO), 8.37 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.32 (s, 1H, NH),
8.16 (d, J ¼ 8.2 Hz, 1H, ArH), 7.64 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.46 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.31 (t, J ¼ 7.8 Hz, 1H, ArH), 7.04 (t, J ¼ 7.7 Hz,
1H, ArH), 3.63 (dd, J ¼ 12.3, 6.4 Hz, 2H, CH2), 2.39 (t, J ¼ 6.9 Hz, 2H,
CH2), 2.20 (s, 6H, 2CH3),1.88e1.75 (m, 2H, CH2); 13C NMR (101 MHz,
2NHCO), 8.37 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH),
7.79 (s, 1H, ArH), 7.61 (t, J ¼ 9.1 Hz, 2H, 2ArH), 7.54 (d, J ¼ 8.8 Hz, 2H,
2ArH), 7.42 (d, J ¼ 5.3 Hz, 1H, NH), 7.31 (t, J ¼ 11.0 Hz, 2H, 2ArH),
3.72 (dd, J ¼ 13.4, 6.2 Hz, 2H, CH2), 2.81 (t, J ¼ 7.1 Hz, 2H, CH2),
2.74e2.60 (m, 4H, 2CH2), 1.02 (t, J ¼ 7.1 Hz, 6H, 2CH3); 13C NMR
DMSO) d 161.14, 156.17,154.29,152.67, 142.38, 136.42, 131.89, 131.66,
129.69, 129.17 (2C), 127.94, 123.91, 122.88, 122.21, 121.23, 117.91
(2C), 110.38, 57.22, 45.47 (2C), 39.26, 26.95. Anal. calcd. for
(101 MHz, DMSO)
d 160.79, 160.15, 157.31, 152.95, 141.93, 139.33,
133.28, 132.44, 129.04, 128.88, 125.76, 125.12, 120.22, 117.99, 113.25,
51.44, 47.35 (2C), 38.46, 11.95 (2C). Anal. calcd. for C25H27ClN6OS
(%):C, 60.66; H, 5.50; N, 16.98. Found (%):C, 60.62; H, 5.52; N, 16.99.
C24H23Cl3N6OS (%):C, 52.42; H, 4.22; N, 15.28. Found (%): C, 52.43;
H, 4.21; N, 15.26.
5.11.19. 1-(2-Chlorophenyl)-3-(4-(4-((2-(diethylamino)ethyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (34a)
5.11.24. 1-(4-(6,7-Dichloro-4-((3-(dimethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(2,4- dichlorophenyl)urea
(19b)
Yellow solid; yield: 74%; M.p.: 163e165 ꢀC; MS (ESI) m/z (%):
495.5 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.08 (s, 1H, NHCO),
Yellow solid; yield: 83%; M.p.: 194e196 ꢀC; MS (ESI) m/z (%):
8.59 (s, 1H, NHCO), 8.39 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.17 (dd, J ¼ 8.3,
1.2 Hz, 1H, ArH), 8.12 (d, J ¼ 5.3 Hz, 1H, ArH), 8.04 (s, 1H, ArH), 7.62
(d, J ¼ 8.7 Hz, 2H, 2ArH), 7.46 (dd, J ¼ 8.1, 1.3 Hz, 1H, ArH), 7.44 (d,
J ¼ 5.4 Hz, 1H, NH), 7.31 (t, J ¼ 7.8 Hz, 1H, ArH), 7.10e6.99 (m, 1H,
ArH), 3.96 (d, J ¼ 34.2 Hz, 2H, CH2), 3.25e2.70 (m, 6H, 3CH2), 1.19 (d,
J ¼ 21.7 Hz, 6H, 2CH3). Anal. calcd. for C25H27ClN6OS (%):C, 60.66; H,
5.50; N, 16.98. Found (%):C, 60.63; H, 5.51; N, 16.97.
583.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.13 (s, 1H, NHCO),
8.68 (s, 1H, NHCO), 8.39 (d, J ¼ 8.2 Hz, 2H, 2ArH), 8.35 (s, 1H, NH),
8.21 (d, J ¼ 9.0 Hz, 1H, ArH), 7.64 (s, 1H, ArH), 7.62 (dd, J ¼ 4.0,
2.5 Hz, 2H, 2ArH), 7.40 (dd, J ¼ 9.0, 2.5 Hz, 1H, ArH), 3.68 (d,
J ¼ 5.8 Hz, 2H, CH2), 2.88 (d, J ¼ 7.3 Hz, 2H, CH2), 2.52e2.48 (m, 6H,
2CH3), 2.04e1.95 (m, 2H, CH2). Anal. calcd. for C24H22Cl4N6OS (%):C,
49.33; H, 3.79; N, 14.38. Found (%):C, 49.36; H, 3.80; N, 14.36.
5.11.20. 1-(2,4-Dichlorophenyl)-3-(4-(4-((2-(diethylamino)ethyl)
amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)urea (35a)
5.11.25. 1-(4-(6,7-Dichloro-4-((3-(dimethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(p-tolyl) urea (20b)
Yellow solid; yield: 79%; M.p.: 189e192 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 76%; M.p.: 168e170 ꢀC; MS (ESI) m/z
(%):529.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.54 (s, 1H,
NHCO), 8.95 (s, 1H, NHCO), 8.36 (d, J ¼ 8.6 Hz, 2H, 2ArH), 8.18 (d,
J ¼ 8.9 Hz, 1H, ArH), 8.08 (d, J ¼ 5.3 Hz, 1H, ArH), 7.80 (t, J ¼ 5.3 Hz,
1H, ArH), 7.62 (s, 1H, ArH), 7.60 (s, 2H, 2ArH), 7.41 (d, J ¼ 5.3 Hz, 1H,
NH), 7.38 (dd, J ¼ 9.0, 2.3 Hz, 1H, ArH), 3.67 (dd, J ¼ 13.5, 6.1 Hz, 2H,
CH2), 2.78e2.65 (m, 2H, CH2), 2.57 (q, J ¼ 7.1 Hz, 4H, 2CH2), 1.00 (t,
J ¼ 7.1 Hz, 6H, 2CH3). Anal. calcd. for C25H26Cl2N6OS (%):C, 60.66; H,
5.50; N, 16.98. Found (%):C, 60.64; H, 5.53; N, 16.96.
529.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.57 (s,1H, NHCO), 9.31
(s, 1H, NHCO), 8.37 (d, J ¼ 8.3 Hz, 2H, 2ArH), 7.61 (d, J ¼ 8.6 Hz, 2H,
2ArH), 7.38 (d, J ¼ 8.2 Hz, 2H, 2ArH), 7.09 (d, J ¼ 8.5 Hz, 2H, 2ArH),
3.67 (d, J ¼ 5.8 Hz, 2H, CH2), 2.83 (s, 2H, CH2), 2.25 (s, 2H, CH2), 2.23
(s, 9H, 3CH3), 2.04e1.92 (m, 2H, CH2). Anal. calcd. for
C25H26Cl2N6OS (%):C, 56.71; H, 4.95; N, 15.87. Found (%):C, 56.68; H,
4.97; N, 15.85.

Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
225
5.11.26. 1-(4-(6,7-Dichloro-4-((2-(dimethylamino)ethyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea (21b)
Yellow solid; yield: 81%; M.p.: 190e193 ꢀC; MS (ESI) m/z (%):
7.51 (s, 2H, 2ArH), 7.15 (s, 2H, 2ArH), 3.70 (s, 2H, CH2), 3.13 (s, 6H,
3CH2), 2.08 (s, 2H, CH2), 1.21 (s, 6H, 2CH3). Anal. calcd. for
C26H27Cl2FN6OS (%):C, 55.61; H, 4.85; N, 14.97. Found (%): C, 55.62;
519.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
9.81 (s,1H, NHCO), 9.73
H, 4.86; N, 14.95.
(s, 1H, NHCO), 8.72 (s, 1H, NH), 8.38 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.60 (d,
J ¼ 8.7 Hz, 2H, 2ArH), 7.49 (dd, J ¼ 8.9, 4.9 Hz, 2H, 2ArH), 7.13 (t,
J ¼ 8.8 Hz, 2H, 2ArH), 4.01 (d, J ¼ 5.5 Hz, 2H, CH2), 3.39 (dd, J ¼ 12.7,
7.0 Hz, 2H, CH2), 2.85 (s, 6H, 2CH3). Anal. calcd. for C23H21Cl2FN6OS
(%):C, 53.18; H, 4.08; N, 16.18. Found (%):C, 53.16; H, 4.07; N, 16.19.
5.11.32. 1-(4-Chlorophenyl)-3-(4-(6,7-dichloro-4-((3-
(diethylamino)propyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
urea (27b)
Yellow solid; yield: 74%; M.p.: 189e192 ꢀC; MS (ESI) m/z (%):
577.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
9.51 (d, J ¼ 17.1 Hz, 2H,
d
5.11.27. 1-(4-Chlorophenyl)-3-(4-(6,7-dichloro-4-((2-
(dimethylamino)ethyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
urea (22b)
2NHCO), 8.39 (s, 2H, 2ArH), 8.37 (s, 1H, NH), 7.60 (d, J ¼ 8.3 Hz, 2H,
2ArH), 7.52 (d, J ¼ 8.3 Hz, 2H, 2ArH), 7.34 (d, J ¼ 8.3 Hz, 2H, 2ArH),
3.70 (d, J ¼ 4.8 Hz, 2H, CH2), 3.18 (s, 2H, CH2), 3.11 (d, J ¼ 5.1 Hz, 4H,
2CH2), 2.07 (s, 2H, CH2), 1.20 (t, J ¼ 6.7 Hz, 6H, 2CH3); 13C NMR
Yellow solid; yield: 76%; M.p.: 185e188 ꢀC; MS (ESI) m/z (%):
535.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
9.66 (t, J ¼ 17.1 Hz, 2H,
(101 MHz, DMSO) d 161.14, 156.20, 154.43, 152.88, 142.47, 139.18,
2NHCO), 8.38 (s, 2H, 2ArH), 8.36 (s, 1H, NH), 7.60 (d, J ¼ 8.4 Hz, 2H,
2ArH), 7.52 (d, J ¼ 8.5 Hz, 2H, 2ArH), 7.33 (d, J ¼ 8.5 Hz, 2H, 2ArH),
3.85 (d, J ¼ 5.3 Hz, 2H, CH2), 2.94 (s, 2H, CH2), 2.52 (s, 6H, 2CH3).
Anal. calcd. for C23H21Cl3N6OS (%):C, 51.55; H, 3.95; N, 15.68. Found
(%): C, 51.52; H, 3.96; N, 15.67.
132.14, 131.39, 129.25 (2C), 129.12 (2C), 125.80, 121.27, 120.02 (2C),
117.87 (2C), 110.45, 49.07, 46.69 (2C), 38.18, 23.60, 8.98 (2C). Anal.
calcd. for C26H27Cl3N6OS (%):C, 54.03; H, 4.71; N, 14.54. Found (%):C,
54.00; H, 4.72; N, 14.53.
5.11.33. 1-(2-Chlorophenyl)-3-(4-(6,7-dichloro-4-((3-
(diethylamino)propyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
urea (28b)
5.11.28. 1-(2-Chlorophenyl)-3-(4-(6,7-dichloro-4-((2-
(dimethylamino)ethyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
urea (23b)
Yellow solid; yield: 74%; M.p.: 189e192 ꢀC; MS (ESI) m/z (%):
Yellow solid; yield: 78%; M.p.: 198e200 ꢀC; MS (ESI) m/z (%):
577.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.24 (s, 1H, NHCO),
535.1 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
10.11 (s, 1H, NHCO),
10.11 (s, 1H, NHCO), 8.59 (s, 1H, NH), 8.41 (s, 2H, 2ArH), 8.18 (s, 1H,
ArH), 7.65 (s, 2H, 2ArH), 7.46 (s, 1H, ArH), 7.31 (s, 1H, ArH), 7.05 (s,
1H, ArH), 3.71 (s, 2H, CH2), 3.14 (s, 2H, CH2), 3.10 (s, 4H, 2CH2), 2.09
8.65 (s, 1H, NHCO), 8.39 (t, J ¼ 9.7 Hz, 2H, 2ArH), 8.22 (d, J ¼ 5.2 Hz,
1H, NH), 8.16 (d, J ¼ 8.3 Hz, 1H, ArH), 7.63 (d, J ¼ 8.7 Hz, 2H, 2ArH),
7.46 (d, J ¼ 7.8 Hz, 1H, ArH), 7.31 (t, J ¼ 7.7 Hz, 1H, ArH), 7.05 (t,
J ¼ 7.3 Hz, 1H, ArH), 3.72 (dd, J ¼ 12.2, 6.3 Hz, 2H, CH2), 2.55 (dd,
J ¼ 13.5, 6.8 Hz, 2H, CH2), 2.23 (s, 6H, 2CH3). Anal. calcd. for
(s, 2H, CH2), 1.20 (s, 6H, 2CH3); 13C NMR (101 MHz, DMSO)
d 161.11,
156.21, 154.72, 152.60, 142.38, 136.37, 132.16, 131.59, 129.70, 129.29
(2C), 127.98, 123.93, 122.78, 122.09, 121.24, 117.95 (2C), 110.50,
49.06, 46.63 (2C), 38.24, 23.57, 8.98 (2C). Anal. calcd. for
C
23H21Cl3N6OS (%):C, 51.55; H, 3.95; N, 15.68. Found (%): C, 51.53; H,
3.94; N, 15.69.
C26H27Cl3N6OS (%):C, 54.03; H, 4.71; N, 14.54. Found (%):C, 54.01; H,
4.70; N, 14.56.
5.11.29. 1-(4-(6,7-Dichloro-4-((2-(dimethylamino)ethyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(2,4- dichlorophenyl)urea
(24b)
5.11.34. 1-(4-(6,7-Dichloro-4-((3-(diethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(2,4- dichlorophenyl)urea
(29b)
Yellow solid; yield: 74%; M.p.: 194e196 ꢀC; MS (ESI) m/z (%):
569.0 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
10.80 (s, 1H, NHCO),
Yellow solid; yield: 77%; M.p.: 193e196 ꢀC; MS (ESI) m/z (%):
9.00 (s, 1H, NHCO), 8.86 (s, 1H, NH), 8.40 (d, J ¼ 8.6 Hz, 2H, 2ArH),
8.18 (d, J ¼ 9.0 Hz, 1H, ArH), 7.66 (d, J ¼ 8.6 Hz, 2H, 2ArH), 7.60 (d,
J ¼ 2.2 Hz, 1H, ArH), 7.38 (dd, J ¼ 8.9, 2.2 Hz, 1H, ArH), 3.99 (d,
J ¼ 5.5 Hz, 2H, CH2), 2.76 (d, J ¼ 24.4 Hz, 6H, 2CH3), 2.50 (s, 2H, CH2);
611.2 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.15 (s, 2H, 2NHCO),
8.67 (s, 1H, NH), 8.42 (s, 2H, 2ArH), 8.22 (s, 1H, ArH), 7.64 (s, 3H
3ArH), 7.40 (s, 1H, ArH), 3.71 (s, 2H, CH2), 3.12 (s, 6H, 3CH2), 2.09 (s,
2H, CH2), 1.21 (s, 6H, 2CH3); 13C NMR (101 MHz, DMSO)
d 161.07,
13C NMR (101 MHz, DMSO)
d
161.05, 156.07, 154.45, 152.71, 142.48,
156.21, 154.41, 152.44, 142.17, 135.62, 132.18, 131.72, 129.30 (2C),
129.04, 128.05, 126.71, 123.39, 122.81, 121.25, 118.02 (2C), 110.52,
49.05, 46.64 (2C), 38.22, 23.73, 8.97 (2C). Anal. calcd. for
135.76, 132.51, 131.41, 129.31 (2C), 129.02, 127.94, 126.65, 123.76,
123.17, 121.02, 117.94 (2C), 110.89, 55.90, 42.99 (2C), 36.56. Anal.
calcd. for C23H20Cl4N6OS (%):C, 48.44; H, 3.53; N, 14.74. Found (%):C,
48.41; H, 3.54; N, 14.75.
C26H26Cl4N6OS (%):C, 50.99; H, 4.28; N,13.72. Found (%): C, 50.97; H,
4.29; N, 13.75.
5.11.30. 1-(4-(6,7-Dichloro-4-((3-(diethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-phenylurea (25b)
Yellow solid; yield: 69%; M.p.: 191e193 ꢀC; MS (ESI) m/z (%):
5.11.35. 1-(4-(6,7-Dichloro-4-((3-(diethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(p-tolyl) urea (30b)
Yellow solid; yield: 74%; M.p.: 193e196 ꢀC; MS (ESI) m/z (%):
543.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
9.60 (s, 1H, NHCO),
557.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.34 (s,1H, NHCO), 9.04
9.40 (s, 1H, NHCO), 8.38 (s, 2H, 2ArH), 8.36 (s, 1H, NH), 7.60 (d,
J ¼ 7.7 Hz, 2H, 2ArH), 7.49 (d, J ¼ 6.7 Hz, 2H, 2ArH), 7.29 (s, 2H,
2ArH), 6.97 (s, 1H, ArH), 3.68 (s, 2H, CH2), 2.81 (d, J ¼ 63.4 Hz, 6H,
3CH2), 1.94 (d, J ¼ 23.6 Hz, 2H, CH2), 1.11 (s, 6H, 2CH3). Anal. calcd.
for C26H28Cl2N6OS (%):C, 48.44; H, 3.53; N, 14.74. Found (%):C,
48.41; H, 3.54; N, 14.75.
(s, 1H, NHCO), 8.40 (s, 1H, NH), 8.38 (s, 2H, 2ArH), 7.60 (s, 2H, 2ArH),
7.36 (s, 2H, 2ArH), 7.10 (s, 2H, 2ArH), 3.71 (s, 2H, CH2), 3.18 (s, 2H,
CH2), 3.12 (s, 4H, 2CH2), 2.25 (s, 3H, CH3), 2.07 (s, 2H, CH2), 1.20 (s,
6H, 2CH3). Anal. calcd. for C27H30Cl2N6OS (%):C, 58.16; H, 5.42; N,
15.07. Found (%): C, 58.13; H, 5.44; N, 15.08.
5.11.36. 1-(4-(6,7-Dichloro-4-((2-(diethylamino)ethyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(4- fluorophenyl)urea (31b)
Yellow solid; yield: 78%; M.p.: 196e198 ꢀC; MS (ESI) m/z (%):
d 9.66 (s, 1H, NHCO), 9.57
(s, 1H, NHCO), 8.34 (s, 2H, 2ArH), 8.27e8.20 (m, 1H, NH), 7.59 (d,
J ¼ 8.8 Hz, 2H, 2ArH), 7.55e7.46 (m, 2H, 2ArH), 7.12 (t, J ¼ 8.9 Hz, 2H,
5.11.31. 1-(4-(6,7-Dichloro-4-((3-(diethylamino)propyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(4-fluorophenyl)urea (26b)
Yellow solid; yield: 71%; M.p.: 193e195 ꢀC; MS (ESI) m/z (%):
547.4 [MþH]þ; 1H NMR (400 MHz, DMSO)
561.2 [MþH]þ; 1H NMR (400 MHz, DMSO)
d
9.90 (s,1H, NHCO), 9.42
(s, 1H, NHCO), 9.31 (s, 1H, NH), 8.40 (s, 2H, 2ArH), 7.60 (s, 2H, 2ArH),

226
Z. Liu et al. / European Journal of Medicinal Chemistry 85 (2014) 215e227
2ArH), 3.68 (dt, J ¼ 13.2, 6.5 Hz, 2H, CH2), 2.74 (dd, J ¼ 14.2, 8.3 Hz,
2H, CH2), 2.62 (dd, J ¼ 13.3, 6.2 Hz, 4H, 2CH2), 1.00 (td, J ¼ 7.1, 2.8 Hz,
suspended in MEM medium, were plated into each well of a 96-
well plate and incubated in 5% CO2 at 37 ꢀC for 24 h. The tested
compounds at the indicated final concentrations were added to the
culture medium and incubated for 72 h. Fresh MTT was added to
6H, 2CH3); 13C NMR (101 MHz, DMSO)
d 161.21, 156.22, 154.35,
153.13, 151.73, 142.70, 136.62, 131.95, 131.27, 129.77, 129.09 (2C),
120.17 (2C), 117.70 (2C), 115.73 (2C), 113.49, 51.45, 47.37 (2C), 38.87,
12.19 (2C). Anal. calcd. for C25H25Cl2FN6OS (%):C, 54.85; H, 4.60; N,
15.35. Found (%):C, 54.86; H, 4.61; N, 15.37.
each well at the terminal concentration of 5
with cells at 37 ꢀC for 4 h. The formazan crystals in each well were
dissolved in 100 L DMSO, and the absorbency at 492 nm (for
mg/mL, and incubated
m
absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with an ELISA reader. All of the com-
pounds were tested three times in each of the cell lines. The results,
expressed as IC50 (inhibitory concentration 50%), were the averages
of three determinations and calculated relative to the vehicle
(DMSO) control by the Bacus Laboratories Incorporated Slide
Scanner (Bliss) software.
5.11.37. 1-(4-Chlorophenyl)-3-(4-(6,7-dichloro-4-((2-
(diethylamino)ethyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
urea (32b)
Yellow solid; yield: 78%; M.p.: 191e193 ꢀC; MS (ESI) m/z (%):
563.6 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.77 (s, 2H, 2NHCO),
8.37 (s, 1H, NH), 8.35 (s, 2H, 2ArH), 7.60 (d, J ¼ 8.8 Hz, 2H, 2ArH),
7.56e7.50 (m, 2H, 2ArH), 7.37e7.30 (m, 2H, 2ArH), 3.82e3.75 (m,
2H, CH2), 2.90 (d, J ¼ 3.5 Hz, 2H, CH2), 2.76 (t, J ¼ 9.0 Hz, 4H, 2CH2),
1.07 (q, J ¼ 6.8 Hz, 6H, 2CH3). Anal. calcd. for C25H25Cl3N6OS (%):C,
53.25; H, 4.47; N, 14.90. Found (%): C, 53.23; H, 4.48; N, 14.91.
5.12.2. In vitro enzymatic assays
The in vitro enzymatic assays of compound 29a were evaluated
by homogeneous time-resolved fluorescence (HTRF) assay. Briefly,
20
m
g/mL poly (Glu, Tyr) 4:1 (Sigma) was preloaded as a substrate in
5.11.38. 1-(2-Chlorophenyl)-3-(4-(6,7-dichloro-4-((2-
(diethylamino)ethyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
urea (33b)
384-well plates. Then 50
diluted in kinase reaction buffer (50 mM HEPES, Ph 7.0, 1 mM DTT,
1 mM MgCl2, 1 mM MnCl2, 0.1% NaN3) was added to each well.
Various concentrations of compounds were diluted in 10 mL of 1%
DMSO (v/v), with blank DMSO solution as the negative control. The
m
L of 10 mM ATP (Invitrogen) solution
Yellow solid; yield: 80%; M.p.: 187e189 ꢀC; MS (ESI) m/z (%):
563.6 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.99 (s, 1H, NHCO),
8.58 (s, 1H, NHCO), 8.36 (s, 2H, 2ArH), 8.18 (s, 1H, NH), 8.17 (d,
J ¼ 8.6 Hz, 1H, ArH), 7.62 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.46 (d, J ¼ 8.0 Hz,
1H, ArH), 7.31 (t, J ¼ 7.8 Hz, 1H, ArH), 7.09e7.00 (m, 1H, ArH), 3.67
(dd, J ¼ 13.5, 6.2 Hz, 2H, CH2), 2.72e2.63 (m, 2H, CH2), 2.56 (q,
J ¼ 7.1 Hz, 4H, 2CH2), 0.98 (t, J ¼ 7.1 Hz, 6H, 2CH3). Anal. calcd. for
kinase reaction was initiated by the addition of purified tyrosine
kinase proteins diluted in 39
Reactions were incubated for 30 min at 25 ꢀC and stopped by the
addition of 5 L Streptavidin-XL665 and 5 L Tk Antibody Cryptate
working solution to all of wells. The plate was read by Envision
(PerkinElmer) at 320 nm and 615 nm. IC50 values were calculated
from the inhibition curves.
mL of kinase reaction buffer solution.
m
m
C
25H25Cl3N6OS (%):C, 53.25; H, 4.47; N,14.90. Found (%): C, 53.22; H,
4.46; N, 14.88.
5.11.39. 1-(4-(6,7-Dichloro-4-((2-(diethylamino)ethyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(2,4-dichlorophenyl)urea
(34b)
5.12.3. Western blot analysis
About 1 ꢁ 107 HT-29 cells were gathered after pre-treatment
with indicated concentrations of compound 29a for 48 h. Protein
lysates were prepared using lysis buffer (10ꢁ) (Cell Signaling
Technology, Danvers, MA, USA) and protease inhibitor cocktail
(SigmaeAldrich). All samples were sonicated at 10 Hz for 10 s. An
equal amount of total protein extracts from cultured cells or tissues
were fractionated by 10% SDS-PAGE and electrically transferred
onto polyvinylidene difluoride (PVDF) membranes. Mouse or rabbit
primary antibodies and horseradish peroxidase (HRP)-conjugated
appropriate secondary antibodies were used to detect the desig-
nated proteins. The bound secondary antibodies on the PVDF
membrane were reacted with ECL detection reagents (Thermo
Scientific) and exposed to X-ray films. The proportion of cleaved
Yellow solid; yield: 83%; M.p.: 194e197 ꢀC; MS (ESI) m/z (%):
597.2 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 10.52 (s, 1H, NHCO),
9.21 (s, 1H, NHCO), 8.36 (d, J ¼ 8.7 Hz, 2H, 2ArH), 8.18 (s, 1H, NH),
8.11 (d, J ¼ 8.9 Hz, 1H, ArH), 7.64 (d, J ¼ 8.7 Hz, 2H, 2ArH), 7.61 (d,
J ¼ 2.3 Hz, 1H, ArH), 7.38 (dd, J ¼ 8.9, 2.3 Hz, 1H, ArH), 3.67 (d,
J ¼ 6.9 Hz, 2H, CH2), 2.69 (t, J ¼ 7.1 Hz, 2H, CH2), 2.56 (q, J ¼ 7.1 Hz,
4H, 2CH2), 0.98 (t, J ¼ 7.1 Hz, 6H, 2CH3). Anal. calcd. for
C
25H24Cl4N6OS (%):C, 50.18; H, 4.04; N, 14.04. Found (%):C, 50.16; H,
4.05; N, 14.06.
5.11.40. 1-(4-(6,7-Dichloro-4-((2-(diethylamino)ethyl)amino)
thieno[3,2-d]pyrimidin-2-yl)phenyl)-3-(p-tolyl)urea (35b)
Yellow solid; yield: 82%; M.p.: 192e195 ꢀC; MS (ESI) m/z (%):
caspase-3 was normalized to the internal control b-actin.
543.3 [MþH]þ; 1H NMR (400 MHz, DMSO)
d 9.60 (s, 1H, NHCO),
Acknowledge
9.33 (s, 1H, NHCO), 8.39 (s, 1H, NH), 8.36 (d, J ¼ 8.0 Hz, 2H, 2ArH),
7.59 (d, J ¼ 7.8 Hz, 2H, 2ArH), 7.37 (d, J ¼ 7.5 Hz, 2H, 2ArH), 7.09 (d,
J ¼ 7.5 Hz, 2H, 2ArH), 3.80 (s, 2H, CH2), 2.95 (s, 2H, CH2), 2.82 (s, 4H,
2CH2), 2.25 (s, 3H, CH3), 1.08 (s, 6H, 2CH3). Anal. calcd. for
The work was supported by Program for Liaoning Excellent
Talents in University (LJQ2013106), National Natural Science
Foundation of China (81102470) and National High Technology
Research and Development Program of China (2012AA020305).
C
26H28Cl2N6OS (%):C, 57.46; H, 5.19; N, 15.46. Found (%): C, 57.44; H,
5.18; N, 15.44.
5.12. Pharmacology
Appendix A. Supplementary data
5.12.1. MTT assay in vitro
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.099.
The antiproliferative activities of compounds 16ae35a and
16be35b were evaluated against HT-29, H460, MKN-45 and MDA-
MB-231 cell lines by the standard MTT assay in vitro, with GDC-
0941 and sorafenib as the positive controls. The cancer cell lines
were cultured in minimum essential medium (MEM) supplement
with 10% fetal bovine serum (FBS). Approximate 4 ꢁ 103 cells,
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