Design, synthesis and biological evaluation of novel thieno[3,2-d]pyrimidine derivatives possessing diaryl semicarbazone scaffolds as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.10.022

A series of novel thieno[3,2-d]pyrimidine derivatives possessing diaryl semicarbazone scaffolds were designed, synthesized and evaluated for their anticancer activity. Most compounds displayed good to excellent potency against four tested cancer cell lines as compared with GDC-0941 and sorafenib. In this study, a promising compound 36 (PI3Kα IC₅₀ Combining double low line 0.027 μM) was identified, which showed the most potent antitumor activities with IC₅₀ values of 0.057 μM, 0.039 μM, 0.25 μM, and 0.23 μM against H460, HT-29, MKN-45 and MDA-MB-231 cell lines, respectively. In addition, the SAR analyses indicated that compounds with 4-morpholino group at the C-4 position of thieno[3,2-d]pyrimidine moiety exhibited superior activities than compounds bearing chain amino groups. In addition, compounds with mono-methoxy group at the 3-position or dimethyl groups at the 3,5-position on the terminal phenyl ring were more active. The SAR analyses will guide us to further refine the structure of the thieno[3,2-d]pyrimidine derivatives to achieve optimum anticancer activity.

Full text of DOI:10.1016/j.ejmech.2014.10.022

European Journal of Medicinal Chemistry 87 (2014) 782e793
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 possessing diaryl semicarbazone scaffolds as
potent antitumor agents
Zijian Liu, Shasha Wu, Yu Wang, Ruijuan Li, Jian Wang, Lihui Wang, Yanfang Zhao*,
Ping Gong*
Key Lab of New Drug Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 110016
Shenyang, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel thieno[3,2-d]pyrimidine derivatives possessing diaryl semicarbazone scaffolds were
designed, synthesized and evaluated for their anticancer activity. Most compounds displayed good to
excellent potency against four tested cancer cell lines as compared with GDC-0941 and sorafenib. In this
Received 17 July 2014
Received in revised form
7 October 2014
Accepted 9 October 2014
Available online
study, a promising compound 36 (PI3K
a
IC50 ¼ 0.027
m
M) was identified, which showed the most potent
M, 0.25 M, and 0.23 M against H460, HT-29,
antitumor activities with IC50 values of 0.057
m
M, 0.039
m
m
m
MKN-45 and MDA-MB-231 cell lines, respectively. In addition, the SAR analyses indicated that com-
pounds with 4-morpholino group at the C-4 position of thieno[3,2-d]pyrimidine moiety exhibited su-
perior activities than compounds bearing chain amino groups. In addition, compounds with mono-
methoxy group at the 3-position or dimethyl groups at the 3,5-position on the terminal phenyl ring
were more active. The SAR analyses will guide us to further refine the structure of the thieno[3,2-d]
pyrimidine derivatives to achieve optimum anticancer activity.
Keywords:
Synthesis
Thieno[3,2-d]pyrimidine derivatives
Diaryl semicarbazone scaffolds
Antitumor activity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
examples highlight the ongoing interest toward new thieno[3,2-d]
pyrimidine derivatives and have prompted us to investigate this
Cancer is one of the most serious worldwide health problems
and is also one of the leading causes of death [1,2]. Despite the
efforts to discover and develop small molecule anticancer drugs in
the last decade [3e6], the development of new antitumor agents
with improved tumor selectivity, efficiency, and safety remains
desirable. Recently, thienopyrimidine derivatives have attracted
great attention due to their broad bioactivities, including antitumor
[7e10], anti-microbial [11], anti-inflammatory activities [12].
Among these active compounds, thieno[3,2-d] pyrimidines have
been reported to show remarkable antitumor activities by means of
inhibiting multiple enzymes as well as modulating the activity of
many receptors [8e10]. Therefore, substituted thieno[3,2-d]pyr-
imidins, such as PI3K inhibitor 1 (GDC-0941), PI3K and mTOR dual
inhibitor 2, EGFR and VEGFR dual inhibitor 3, and VEGFR inhibitor 4
(Fig. 1), have become an important class of chemotherapeutic drugs
especially for the treatment of cancers [13e18]. These recent
pharmacophore in drug discovery programs aiming at synthesizing
novel bioactive molecules.
In recent years, diaryl urea derivatives have received consider-
able attention in anticancer agents due to their good inhibitory
activity against receptor tyrosine kinases (RTKs), Raf kinases, pro-
tein tyrosine kinases (PTKs), and NADH oxidase, which play critical
roles in the regulation of tumor genesis [19]. Among these de-
rivatives (Fig. 2) [20e22], sorafenib 5 has been reported to
demonstrate significant inhibitory activity against several receptor
tyrosine kinases involved in neovascularization and tumor pro-
gression, including Raf-1, vascular endothelial growth factor (VEGF)
and platelet derived growth factor (PDGF) receptor families [23].
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 [24]. Thus, the development of
such small molecules with diaryl urea framework, which can
readily bind with various enzymes and receptors through hydrogen
bonds, is
a feasible approach to develop new antitumor
* Corresponding authors.
compounds.
E-mail addresses: yanfangzhao@126.com (Y. Zhao), gongpinggp@126.com
(P. Gong).
http://dx.doi.org/10.1016/j.ejmech.2014.10.022
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
783
Fig. 1. Structures of representative kinase inhibitors containing thieno[3,2-d]pyrimidine nuclei and their kinase inhibitory activities.
Similarly, N-Acylarylhydrazone scaffold, and its analogues such
as PAC-1, was also widely used as building blocks in several anti-
tumor agents due to the presence of hydrogen bond donors and
acceptors as well as its flexible skeleton (Fig. 2) [25e27]. Therefore,
in the current study, the resultant semicarbazone moiety was
introduced as a modification of the diaryl urea framework in an
effort to provide more potent antitumor agents.
In the current study, all target compounds were evaluated for
their anti-proliferative 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 in vitro enzymatic assays and apoptosis
analysis of the most potent compounds 36 were also performed.
In view of these observations, the diverse biological potencies of
both pharmacophores prompted us to synthesize the title com-
pounds, presuming that their incorporation in a single structural
entity could potentially produce novel compounds with significant
synergistic antitumor properties. Moreover, on the molecular
design level, various substituents were introduced to the terminal
phenyl ring with the purpose of exploring the influence of sub-
stituents on anticancer activity by regulating the electronic and
steric effects. Further modifications were also performed by intro-
ducing various aliphatic amino groups into 4-position of thieno
[3,2-d]pyrimidine ring, in order to regulate the lipid-water parti-
tion coefficient. Accordingly, With an aim to develop promising
antitumor agents, a series of novel thieno[3,2-d]pyrimidine de-
rivatives containing diaryl semicarbazone scaffolds were designed
and synthesized (Fig. 3).
2. Chemistry
The synthetic route of target compounds 17e44 was illus-
trated in Scheme 1. The commercially available 4-nitrobenzoic
acid was refluxed in SOCl2 for 8 h to provide 4-nitrobenzoyl
chloride, which was condensed with 3-amino-2-thiophene
carboxylate in the presence of triethylamine in dichloro-
methane at room temperature for 10 h to obtain 9. Treatment of 9
in ammonium hydroxide at 60 ꢀC for 8 h yielded intermediate 10,
which could be cyclized to afford 11. Next, the chlorinated reac-
tion of 11 with phosphoryl chloride afforded 12 [28], which was
subsequently alkylated with an excess of the corresponding
aliphatic amines at 30 ꢀC to provide 13ae13c. The key in-
termediates 14ae14c were obtained via the reduction reaction of
intermediates 13ae13c with iron powder and catalytic amounts
Fig. 2. Structures of representative diaryl urea and N-Acylarylhydrazone derivatives and design strategy for diaryl semicarbazone scaffold.

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Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
Fig. 3. Design strategy for the thieno[3,2-d]pyrimidine derivatives containing diaryl semicarbazone scaffolds.
of ammonium chloride in ethanol at reflux temperature. By
further treatment with phenyl chloroformate, 14ae14c were
converted into amides 15ae15c. Subsequently, semicarbazides
16ae16c were obtained via hydrazinolysis of 15ae15c with 80%
hydrazine monohydrate in refluxing 1,4-dioxane. Finally, target
compounds were successfully obtained via the condensation of
16ae16c with appropriate aldehydes in isopropanol at 80 ꢀC with
catalytic amounts of acetic acid.
The chemical structures of the target compounds were
confirmed by IR, 1H NMR, 2D NOESY NMR, 13C NMR, and MS
spectrum. In addition, the stereochemistry of the double bond in
the imine of 17e44 was determined. In 1H NMR spectra, the
eNaCH- signal was seen as a single peak for each compound,
indicating that only one isomer was generated. In order to confirm
the stereochemistry more precisely, the NOESY experiment of
compound 36 was performed. The result indicates that a single NOE
signal was observed between the proton of aNeNH-
3. Results and discussion
3.1. In vitro cytotoxic activities and structure-activity relationships
The cytotoxic activities of the target compounds 17e44 were
evaluated in H460 (human lung cancer), HT-29 (human colon
cancer), MKN-45 (human gastric cancer) and MDA-MB-231 (hu-
man breast cancer) cell lines using the MTT assay. GDC-0941 and
sorafenib were used as the positive control. The results of the mean
values of experiments performed in triplicate, expressed as half-
maximal inhibitory concentration (IC50) values, were presented in
Table 1.
As illustrated in Table 1, all target compounds 17e44 showed
moderate to significant cytotoxic activities against the different
cancer cell lines. Most of these compounds were more potent than
GDC-0941 and sorafenib against one or more cancer cell lines,
which confirmed our hypothesis: the incorporation of the diaryl
semicarbazone scaffold into thieno[3,2-d]pyrimidine moiety in a
single structural entity could produce novel compounds with sig-
nificant synergistic antitumor effects. The IC50 values of the most
(
d
¼ 10.59 ppm) and the proton of -CH]N- ( ¼ 7.83 ppm), which
d
existed only in the E isomer due to the appropriate intramolecular
HeH distance (Fig. 4 and see also Supporting information). Thus,
the target compounds were unequivocally confirmed as the E
isomer.
promising compound 36 were 0.057
mM, 0.039 mM, 0.25 mM, and
0.23 M against H460, HT-29, MKN-45 and MDA-MB-231 cell lines,
m
Scheme 1. Reagents and conditions: (i) a) 4-nitrobenzoic acid, SOCl2, 80 ꢀC; b) Et3N, CH2Cl2, r.t.; (ii) NH4OH, EtOH, 60 ꢀC; (iii) NaOH, i-PrOH, reflux; (iv) POCl3, 80 ꢀC; (v)
NH2(CH2)nR1, THF, Et3N, 30 ꢀC; (vi) Fe, NH4Cl, EtOH, reflux; (vii) phenyl chloroformate, anhydrous K2CO3, dry acetone, 0 ꢀC to r.t.; (viii) 80% hydrazine hydrate, 1,4-dioxane, reflux;
(ix) aldehydes, acetic acid, i-PrOH, reflux.

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
785
Fig. 4. NOESY effect of the representative compound 36.
respectively. Interestingly, most of the compounds were more
potent against H460 and HT-29 cell lines than MKN-45 and MDA-
MB-231 cell lines. These results reflected that this series of com-
pounds possessed selectivity for H460 and HT-29 cancer cell lines,
and had the makings of good drugs for lung and colon cancer.
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 notable influence on ac-
tivity, which suggested that the water-soluble R1 group and the
length of side chain make a great contribution to their potency. For
example, compounds 27e37 with 4-morpholino group (n ¼ 0,
R1 ¼ 4-morpholino) exhibited superior activities than compounds
bearing chain amino groups (17e26, n ¼ 3, R1 ¼ 4-morpholino;
38e44, n ¼ 2, R1 ¼ diethylamino). However, compared to 4-
Table 1
Cytotoxicity of 17e44 against H460, HT-29, MKN-45 and MDA-MB-231 cell lines in vitro.
a
Compd.
n
R1
R2
IC50
(
mmol/L) SD
H460
HT-29
MKN-45
MDA-MB-231
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
3
3
3
3
3
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
e
e
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
4-morpholine
diethylamino
diethylamino
diethylamino
diethylamino
diethylamino
diethylamino
diethylamino
e
2,4-(Cl)2
2,3-(Cl)2
4-OH
3-OH
1.43 0.11
1.32 0.15
1.03 0.15
0.99 0.067
1.41 0.24
1.13 0.12
0.83 0.067
1.45 0.11
0.88 0.13
1.68 0.39
0.64 0.045
0.24 0.021
0.68 0.041
0.59 0.056
1.04 0.12
0.43 0.040
0.39 0.015
0.13 0.012
0.59 0.16
0.057 0.011
0.60 0.032
1.20 0.16
1.24 0.12
1.43 0.40
2.39 0.19
0.89 0.12
1.88 0.21
0.72 0.052
0.87 0.20
2.19 0.11
1.39 0.15
1.86 0.13
0.81 0.052
0.95 0.079
1.21 0.11
0.95 0.10
0.91 0.042
1.72 0.11
0.75 0.10
1.33 0.12
0.62 0.055
0.18 0.017
0.58 0.030
0.74 0.065
1.22 0.14
0.41 0.050
0.35 0.013
0.095 0.010
0.66 0.10
0.039 0.008
0.56 0.050
1.34 0.15
1.62 0.14
1.61 0.30
2.85 0.23
0.72 0.10
1.63 0.12
0.68 0.048
0.86 0.081
3.61 0.36
2.29 0.21
2.27 0.21
1.22 0.15
1.33 0.15
2.53 0.22
1.75 0.20
1.45 0.040
4.36 0.19
1.38 0.21
2.48 0.30
1.12 0.11
0.48 0.023
1.18 0.14
1.39 0.26
1.76 0.15
0.74 0.11
0.57 0.028
0.37 0.020
1.09 0.13
0.25 0.019
1.14 0.13
1.39 0.28
1.76 0.15
2.04 0.38
4.37 0.38
1.75 0.35
2.91 0.25
0.85 0.055
0.60 0.12
2.32 0.35
2.59 0.27
1.86 0.15
1.47 0.18
1.54 0.18
3.60 0.29
2.31 0.18
1.72 0.022
4.45 0.21
1.18 0.14
3.23 0.41
1.31 0.11
0.46 0.033
1.23 0.15
1.50 0.22
1.98 0.13
0.88 0.030
0.65 0.033
0.31 0.018
1.48 0.11
0.23 0.020
1.51 0.17
1.50 0.31
1.98 0.13
2.58 0.40
5.55 0.33
2.48 0.41
3.27 0.33
0.78 0.041
0.28 0.06
0.94 0.13
3-F, 4-OH
3-Br, 4-OH
3-OCH3, 4-OH
3,5-(Br)2, 4-OH
3,5-(CH3)2, 4-OH
3,5-(OCH3)2, 4-OH
2,3-(Cl)2
4-OH
3,4-(OH)2
2,4-(OH)2
2,3,4-(OH)3
3-F, 4-OH
3-Br, 4-OH
3-OCH3, 4-OH
3,5-(Br)2, 4-OH
3,5-(CH3)2, 4-OH
3,5-(OCH3)2, 4-OH
2,3-(Cl)2
3,4-(OH)2
2,4-(OH)2
2,3,4-(OH)3
3-F, 4-OH
3,5-(Br)2, 4-OH
3,5-(CH3)2, 4-OH
e
GDC-0941b
Sorafenibb
e
e
a
Data presented is the mean SD value of three independent determinations.
Used as positive control.
b

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Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
morpholino group, the introduction of diethylamino group at the
terminal of chain amino moiety only had a slight impact on activity
(17e26 vs 38e44).
Further analysis clearly revealed that different biological prop-
erties were obtained when various R2 groups were introduced into
the terminal phenyl ring. Compound 28 (n ¼ 0, R1 ¼ 4-morpholino),
with 4-hydroxy group (R2 ¼ 4-OH) on the terminal phenyl ring,
showed promising cytotoxicity against H460 and HT-29 cell lines
experiments. As shown, compound 36 exhibited dual activity on
PI3K and mTOR with IC50 values of 0.027 M and 0.77 M,
respectively, whereas the effect on the other tested kinases was
negligible. Especially, compared with the highly selective PI3K in-
hibitor, GDC-0941, compound 36 showed promising enzymatic
inhibition which was only slightly lower than the IC50 value of GDC-
0941 against PI3Ka kinase. These results indicated that the PI3Ka
kinase might be the potential biological target for synthesized
compounds.
a
m
m
with IC50 values of 0.24 mM and 0.18 mM, respectively. While the
introduction of more hydroxyl groups (29e31, n ¼ 0, R1 ¼ N-mor-
pholino) resulted in a dramatic loss in activity, suggesting that
multi-hydroxyl groups were not tolerated for this region. In addi-
3.3. Apoptosis analysis
tion, compared to compound 28 (R2 ¼ 4-OH, IC50 ¼ 0.18
HT-29), the introduction of mono-methoxy group at the 3-position
M against HT-29) led to a
mM against
Apoptosis is the process of programmed cell death that may
occur in multicellular organisms. Apoptosis plays a pivotal role in
the pathogenesis of many diseases such as cancer, when insuffi-
cient apoptosis occur. The mechanism of apoptosis is complex and
involves multiple pathways [29]. To examine the mechanism of the
apoptosis induced by the synthesized compounds, the cleavage of
poly (ADP-ribose) polymerase (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 associated with cell apoptosis [30,31].
Therefore, in the current study, HT-29 cell line was used to identify
the mechanism of drug-induced cell apoptosis.
(34, R2 ¼ 3-OCH3, 4-OH, IC50 ¼ 0.095
m
significant improvement in antitumor activity, which could be
further confirmed by compounds 19 and 23. However, the intro-
duction of mono-halogen group at the 3-position (32, R2 ¼ 3-F, 4-
OH, IC50
IC50 ¼ 0.35
¼
0.42
m
M
against HT-29; 33, R2
¼
3-Br, 4-OH,
m
M against HT-29) caused the potency to be lowered by
2.3- and 1.9-fold, respectively. This could demonstrate that mono-
methoxy group at 3-position had a positive effect on antitumor
activity, but mono-halogen group showed negative results.
Another interesting phenomenon is that, when introducing
disubstituted groups into the 3,5-position, whether the substituted
groups were strong electron-withdrawing groups (35, R2 ¼ 3,5-
HT-29 cells were treated with compound 36 at different con-
centrations (0, 1, 5
mM) for 48 h. Subsequently, the cleavage of PARP
(Br)2, 4-OH, IC50 ¼ 0.66
mM against HT-29) or strong electron-
was analyzed with Western blot (
b
-actin as control) [32]. As shown
donating groups (37, R2 ¼ 3,5-(OCH3)2, 4-OH, IC50 ¼ 0.56
mM
in Fig. 5, after the treatment with 36, the significant increased
protein expression of Clv-PARP indicated that 36 could induce
apoptosis in a concentration-dependent manner. The ratio between
against HT-29), their potency was much lower comparing with
compound 28. Meanwhile, the introduction of weak electron-
donating groups (36, R2 ¼ 3,5-(CH3)2, 4-OH, IC50 ¼ 0.039
mM
Clv-PARP and
PARP protein. The ratio was 1.2- and 3.2- 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 was PARP-dependent. However, the
upstream and proapoptotic signaling pathways still need further
investigation so that a better understanding of the precise mech-
anism is achieved.
b-actin expression was used to reflect the activity of
against HT-29) at the 3,5-position led to an unexpected improve-
ment in antitumor activity. According to the pharmacological data,
we concluded that a proper degree of electron density on the ter-
minal phenyl ring was beneficial to achieve better antitumor
activity.
m
m
In general, the most potent compound 36 showed promising
cytotoxicity against H460 and HT-29 cell lines with IC50 values of
0.057
than GDC-0941 by 15.3- and 22.1-fold (IC50 ¼ 0.87
H460, IC50 ¼ 0.86 M against HT-29), and sorafenib by 38.4- and
92.6-fold (IC50 ¼ 2.19 M against HT-
m
M and 0.039
m
M, respectively. The potency was much higher
mM against
3.4. Binding model analysis
m
mM against H460, IC50 ¼ 3.61
m
To further elucidate the binding mode of compounds,
modeling study of 36 docked in PI3K homology model (PDB ID:
3IBE) based on PI3K crystal structures was performed. The dock-
a
29), separately. These encouraging results provide a valuable
leading compound 36, and highlight the potential for further
development of novel thieno[3,2-d]pyrimidine derivatives con-
taining diaryl semicarbazone scaffolds as potent antitumor agents.
a
g
€
ing simulation was conducted with Glide XP (Schrodinger 2014),
since Glide uses a hierarchical series of filters to search for possible
locations of the ligand in the active-site region of the receptor. The
shape and properties of the receptor were represented on a grid by
several different sets of fields that provide progressive accurate
scoring of the ligand poses. The image files were generated by
Accelrys DS visualizer 4.0 system. The binding model was exem-
plified by the interaction of compound 36 with PI3Ka.
As shown in Fig. 6, the oxygen atom of morpholine in compound
36 formed one H-bond interactions with residue VAL882, which
3.2. In vitro enzymatic assays
To investigate the molecular mechanisms of action preliminar-
ily, the optimal compound 36 was screened against 10 selected
kinases, which are the potential biological targets for the designed
compounds [6e17]. The results, summarized in Table 2, were
expressed as IC50 value, which are derived from two independent
Table 2
Inhibition of 10 selected kinases by compound 36.
a
Compd.
IC50 in
m
M
PI3K
a
mTOR
B-Raf
C-Raf
EGFR
VEGFR
PDGFR
b
FLT3
KIT
KDR
36
0.027
0.003
e
0.77
e
>10
e
6.5
e
>10
e
>10
e
7.0
e
2.3
e
5.6
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

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
787
Fig. 5. Compound 36 inhibited 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 36 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 36 solution was significantly higher than the control group, indicating that PARP
was actively involved in the 36-induced apoptosis.
could be the explanation of that compounds 27e37 with 4-
morpholino groups at the C-4 position of thieno[3,2-d]pyrimidine
moiety exhibited superior activities than compounds bearing chain
amino groups. In addition, the oxygen and nitrogen atoms of
semicarbazone in compound 36 formed two H-bond interactions
with residue LYS833 and residue ASP841, respectively. Its favorable
biologic activity might be attributed to the presence of hydrogen
bond donors and acceptors of semicarbazones. In the meantime,
synthesized and evaluated for their biological activity. The pre-
liminary investigation showed that, compared with GDC-0941 and
sorafenib, the most promising compound 36 showed potent anti-
tumor activities with IC50 values of 0.057
mM, 0.039 mM, 0.25 mM,
and 0.23 M against H460, HT-29, MKN-45 and MDA-MB-231 cell
m
lines, respectively. The SAR analyses indicated that compounds
with 4-morpholino group at the C-4 position of thieno[3,2-d]py-
rimidine moiety exhibited superior activities than compounds
bearing chain amino groups. In addition, compounds with mono-
methoxy group at the 3-position or weak electron-donating
groups at the 3,5-position on the terminal phenyl ring were more
the following interactions were formed: 1, four
between the morpholine ring and residue ILE881, VAL882, MET953
and ILEE963, respectively; 2, two -Alkyl interactions and three
Sigma interactions between the thieno[3,2-d]pyrimidine and res-
idue MET953, ILE963, ILE 831 and MET804, respectively; 3, one
Alkyl interaction and one -Sigma interaction between the phenyl
p-Alkyl interactions
p
p-
active. Moreover, the enzymatic assays suggested that the PI3K
a
p
-
kinase might be the potential biological target for synthesized
compounds. In conclusion, it is worth studying compound 36
further as new potential anticancer agent for the treatment of hu-
man cancers.
p
ring and residue ILE963 and ILE879, respectively. Multiple hydro-
phobic interactions between 36 and receptor contribute to the
stabilization of the lowest energy conformation of ligandereceptor
complex.
5. Experimental
5.1. Chemistry
4. Conclusions
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-400, 400 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
Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all
materials were obtained from commercially available sources and
were used without further purification.
In summary, a series of novel thieno[3,2-d]pyrimidine de-
rivatives possessing diaryl semicarbazone scaffolds was designed,
5.2. Methyl 3-(4-nitrobenzamido)thiophene-2-carboxylate (9)
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-thiophene carboxylate (0.1 mol), Et3N (0.12 mol) and
CH2Cl2 (150 mL) was added into the CH2Cl2 solution in an ice bath.
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
Fig. 6. The PI3Ka active site in complex with compound 36 and its hydrophobic sur-
face (brown: highly hydrophobic, blue: highly hydrophilic). The protein was displayed
by secondary structure. Compound 36 was shown in colored sticks (cyan: carbon atom,
blue: nitrogen atom, red: oxygen atom, white: hydrogen atom). The H-bond interac-
tion was shown in green dotted lines,
p-Alkyl interaction was shown in pink dotted
lines, and -Sigma interaction was shown in purple dotted lines. (For interpretation of
p
the references to color in this figure legend, the reader is referred to the web version of
this article.)

788
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
organic phase was obtained, dried and evaporated to afford the
compound 9 as a yellow solid, 84% yield. m.p.: 156e158 ꢀC; MS (ESI)
m/z(%): 307.1 [M þ H]þ.
5.7. General procedure for preparation of 2-(4-aminophenyl)-4-
aminothieno[3,2-d]pyrimidins (14ae14c)
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 14ae14c.
5.3. 3-(4-nitrobenzamido)thiophene-2-carboxamide (10)
To a solution of 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 compound 10 as a yellow
solid, 76% yield. m.p.: 167e169 ꢀC; MS (ESI) m/z(%): 292.1 [M þ H]þ.
5.7.1. 4-(4-morpholinothieno[3,2-d]pyrimidin-2-yl)aniline (14a)
Gray solid; yield: 69%; m.p.: 186e190 ꢀC; MS (ESI) m/z(%): 313.2
5.4. 2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-ol (11)
[M þ H]þ; 1H NMR (400 MHz, DMSO)
8.35 (d, J ¼ 8.7 Hz, 2H), 8.08
d
(d, J ¼ 5.3 Hz,1H), 7.76 (t, J ¼ 5.4 Hz,1H), 7.61 (d, J ¼ 8.7 Hz, 2H), 6.23
(s, 2H), 4.01 (d, J ¼ 4.3 Hz, 4H), 3.82 (d, J ¼ 4.3 Hz, 4H).
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 2e3, filtered and dried to afford the corresponding
compound 11 as a light yellow solid, 74% yield. m.p.:153e156 ꢀC;
MS (ESI) m/z(%): 274.1 [M þ H]þ.
5.7.2. 2-(4-aminophenyl)-N-(3-morpholinopropyl)thieno[3,2-d]
pyrimidin-4-amine (14b)
Gray solid; yield: 67%; m.p.: 201e204 ꢀC; MS (ESI) m/z(%): 370.1
[M þ H]þ; 1H NMR (400 MHz, DMSO)
8.35 (d, J ¼ 8.7 Hz, 2H), 8.09
d
(d, J ¼ 5.3 Hz,1H), 7.76 (t, J ¼ 5.4 Hz,1H), 7.62 (d, J ¼ 8.7 Hz, 2H), 7.43
(d, J ¼ 5.3 Hz, 1H), 6.22 (s, 2H), 3.68 (dd, J ¼ 12.9, 6.5 Hz, 2H), 3.61 (t,
J ¼ 4.5 Hz, 4H), 2.42 (dd, J ¼ 14.8, 7.9 Hz, 6H), 1.85 (s, 2H).
5.5. 4-chloro-2-(4-nitrophenyl)thieno[3,2-d]pyrimidine (12)
A
mixture of an appropriate thieno[3,2-d]pyrimidin-4-ol
5.7.3. N1,N1-diethyl-N2-(2-(4-aminophenyl)thieno[3,2-d]
pyrimidin-4-yl)ethane-1,2-diamine (14c)
(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 8e9, filtered and dried to afford the
corresponding compound 12 as a gray solid, 69% yield. m.p.:
156e159 ꢀC; MS (ESI) m/z(%): 292.0 [M þ H]þ.
Gray solid; yield: 72%; m.p.: 196e199 ꢀC; MS (ESI) m/z(%): 342.1
[M þ H]þ; 1H NMR (400 MHz, DMSO)
8.35 (d, J ¼ 8.7 Hz, 2H), 8.08
d
(d, J ¼ 5.3 Hz,1H), 7.76 (t, J ¼ 5.4 Hz,1H), 7.61 (d, J ¼ 8.7 Hz, 2H), 7.43
(d, J ¼ 5.3 Hz, 1H), 6.24 (s, 2H), 3.72 (dd, J ¼ 13.4, 6.2 Hz, 2H), 2.81 (t,
J ¼ 7.1 Hz, 2H), 2.74e2.60 (m, 4H),1.91 (s, 2H), 1.02 (t, J ¼ 7.1 Hz, 6H).
5.6. General procedure for preparation of 2-(4-nitrophenyl)-4-
aminothieno[3,2-d]pyrimidins (13ae13c)
5.8. General procedure for preparation of phenylcarbamates
(15ae15c)
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 13ae13c.
To the mixture of an appropriate amine 14ae14c (10.0 mmol)
and anhydrous K2CO3 (2.76 g, 20.0 mmol) in dry acetone (60 mL),
phenyl chloroformate (1.88 mL, 15.0 mmol) was added dropwise
while maintaining the temperature between 0 ꢀC and 5 ꢀC. After the
addition was completed, the mixture was warmed to room tem-
perature for another 3 h, and the solvent was evaporated under
reduced pressure. The residue was dissolved in CH2Cl2 (20 mL), and
washed with water (3e10 mL), dried over anhydrous Na2SO4,
concentrated under reduced pressure to afford corresponding
phenylcarbamates 15ae15c, which were immediately used in the
next step without further purification.
5.6.1. 4-(2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-4-yl)morpholine
(13a)
Yellow solid; yield: 65%; m.p.: 148e151 ꢀC; MS (ESI) m/z(%):
343.3 [M þ H]þ. 1H NMR (400 MHz, DMSO)
8.75 (d, J ¼ 8.5 Hz, 2H),
d
8.64 (d, J ¼ 8.5 Hz, 2H), 8.07 (d, J ¼ 5.3 Hz, 1H), 7.76 (d, J ¼ 5.4 Hz,
1H), 4.01 (d, J ¼ 4.3 Hz, 4H), 3.82 (d, J ¼ 4.3 Hz, 4H).
5.6.2. N-(3-morpholinopropyl)-2-(4-nitrophenyl)thieno[3,2-d]
pyrimidin-4-amine (13b)
5.8.1. Phenyl (4-(4-morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)
carbamate (15a)
Yellow solid; yield: 66%; m.p.: 164e168 ꢀC; MS (ESI) m/z(%):
Yellow oil; yield: 87%; m.p.: 177e181 ꢀC; MS (ESI) m/z(%): 433.1
[M þ H]þ.
400.2 [M þ H]þ. 1H NMR (400 MHz, DMSO)
8.76 (d, J ¼ 8.5 Hz, 2H),
d
8.63 (d, J ¼ 8.5 Hz, 2H), 8.08 (d, J ¼ 5.3 Hz, 1H), 7.75 (d, J ¼ 5.4 Hz,
1H), 7.43 (d, J ¼ 5.3 Hz, 1H), 3.68 (dd, J ¼ 12.9, 6.5 Hz, 2H), 3.61 (t,
J ¼ 4.5 Hz, 4H), 2.42 (dd, J ¼ 14.8, 7.9 Hz, 6H), 1.85 (s, 2H).
5.8.2. Phenyl (4-(4-((3-morpholinopropyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)carbamate (15b)
Yellow oil; yield: 89%; m.p.: 190e193 ꢀC; MS (ESI) m/z(%): 490.3
[M þ H]þ.
5.6.3. N1,N1-diethyl-N2-(2-(4-nitrophenyl)thieno[3,2-d]pyrimidin-
4-yl)ethane-1,2-diamine (13c)
Gray solid; yield: 79%; m.p.: 174e178 ꢀC; MS (ESI) m/z(%): 372.1
[M þ H]þ. 1H NMR (400 MHz, DMSO)
d
8.75 (d, J ¼ 8.5 Hz, 2H), 8.64
5.8.3. Phenyl (4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)carbamate (15c)
(d, J ¼ 8.5 Hz, 2H), 8.08 (d, J ¼ 5.3 Hz, 1H), 7.76 (d, J ¼ 5.4 Hz, 1H),
7.43 (d, J ¼ 5.3 Hz, 1H), 3.72 (dd, J ¼ 13.4, 6.2 Hz, 2H), 2.81 (t,
J ¼ 7.1 Hz, 2H), 2.74e2.60 (m, 4H), 1.02 (t, J ¼ 7.1 Hz, 6H).
Yellow oil; yield: 87%; m.p.: 197e201 ꢀC; MS (ESI) m/z(%): 462.2
[M þ H]þ.

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
789
5.9. General procedure for preparation of N-(4-(4-aminothieno[3,2-
(101 MHz, DMSO) d 160.73, 160.09, 157.34, 153.15, 141.01, 137.08,
d]pyrimidin-2-yl)phenyl)hydrazine carboxamides (16ae16c)
134.50, 133.36, 133.19, 132.59, 131.38, 130.87, 128.62, 128.51 (2C),
126.60, 125.14, 119.78 (2C), 113.27, 66.72 (2C), 56.74, 53.88 (2C),
39.24, 26.14. Anal. calcd. for C27H27Cl2N7O2S (%):C, 55.48; H, 4.66; N,
16.77. Found (%):C, 55.46; H, 4.68; N, 16.78.
A
mixture of an appropriate phenylcarbamates 15ae15c
(8.01 mmol) and 80% hydrazine monohydrate (10 mL) in 1,4-
dioxane (20 mL) was refluxed overnight with vigorous agitation.
Most of the solvent was evaporated under reduced pressure when
white solid appeared. After cooling to 10 ꢀC, the resulting precipi-
tate was filtered off, washed with water, and dried under vacuum to
afford the title compound 16ae16c.
5.10.3. (E)-2-(4-hydroxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (19)
White solid; yield: 79%; m.p.: 185e188 ꢀC; MS (ESI) m/z(%):
532.3 [M þ H]þ; IR (KBr) cmꢂ1: 3453.5, 2920.7, 2851.5, 1642.9,
1533.8, 1468.3, 1384.1, 1273.9, 1113.1, 846.3, 778.2, 620.2; 1H NMR
5.9.1. N-(4-(4-morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (16a)
(400 MHz, DMSO)
d 10.62 (s, 1H), 9.84 (s, 1H), 8.98 (s, 1H), 8.35 (d,
White solid; yield: 57%; m.p.: 184e188 ꢀC; MS (ESI) m/z(%):
371.4 [M þ H]þ.
J ¼ 8.8 Hz, 2H), 8.08 (t, J ¼ 5.8 Hz, 1H), 7.93e7.88 (m, 1H), 7.88 (s,
1H), 7.80 (d, J ¼ 8.8 Hz, 2H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.41 (dd, J ¼ 9.3,
5.3 Hz, 1H), 6.82 (d, J ¼ 8.6 Hz, 2H), 3.68 (dd, J ¼ 12.9, 6.5 Hz, 2H),
3.60 (dd, J ¼ 13.3, 8.8 Hz, 4H), 2.43 (t, J ¼ 6.2 Hz, 2H), 2.40 (d,
J ¼ 8.7 Hz, 4H), 1.93e1.79 (m, 2H); 13C NMR (101 MHz, DMSO)
5.9.2. N-(4-(4-((3-morpholinopropyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)hydrazinecarboxamide (16b)
White solid; yield: 54%; m.p.: 193e197 ꢀC; MS (ESI) m/z(%):
428.2 [M þ H]þ.
d
160.74, 160.15, 159.37, 157.32, 153.45, 141.85, 141.35, 133.19, 132.94,
129.25 (2C), 128.52 (2C), 125.87, 125.14, 119.31 (2C), 115.95 (2C),
113.57, 66.73 (2C), 56.75, 53.88 (2C), 39.23, 26.15. Anal. calcd. for
5.9.3. N-(4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)hydrazinecarboxamide (16c)
White solid; yield: 62%; m.p.: 204e207 ꢀC; MS (ESI) m/z(%):
400.1 [M þ H]þ.
C27H29N7O3S (%):C, 61.00; H, 5.50; N, 18.44. Found (%):C, 61.03; H,
5.48; N, 18.45.
5.10.4. (E)-2-(3-hydroxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (20)
5.10. General procedure for preparation of the target compounds
17e44
White solid; yield: 83%; m.p.: 191e194 ꢀC; MS (ESI) m/z(%):
532.5 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.77 (s, 1H), 9.57 (s,
To
a
solution of an appropriate acylhydrazine (16ae16c)
1H), 9.05 (s, 1H), 8.36 (d, J ¼ 8.7 Hz, 2H), 8.09 (d, J ¼ 5.3 Hz, 1H), 7.90
(s, 1H), 7.88 (s, 1H), 7.79 (d, J ¼ 8.8 Hz, 2H), 7.43 (d, J ¼ 5.3 Hz, 1H),
7.26 (d, J ¼ 7.1 Hz, 2H), 7.22 (d, J ¼ 7.5 Hz, 1H), 6.85e6.79 (m, 1H),
3.68 (dd, J ¼ 12.6, 6.5 Hz, 2H), 3.61 (t, J ¼ 4.4 Hz, 4H), 2.43 (t,
J ¼ 5.9 Hz, 2H), 2.40 (d, J ¼ 8.0 Hz, 4H), 1.90e1.79 (m, 2H). 13C NMR
(0.414 mmol) in isopropanol (2 mL), 1.1 equiv of aldehydes or ke-
tones and acetic acid (1 drop) were added, and the mixture was
refluxed for 5e6 h until TLC showed the completion of the reaction.
After cooling to room temperature, the resultant precipitate was
filtered and dried under vacuum to afford the target compounds
17e44.
(101 MHz, DMSO)
d 160.74, 160.12, 158.05, 157.32, 153.36, 141.81,
141.24, 136.09, 133.19, 133.07, 130.08, 128.54 (2C), 125.14, 119.41
(2C), 118.76, 117.17, 113.81, 113.27, 66.71 (2C), 56.73, 53.87 (2C),
39.34, 26.13. Anal. calcd. for C27H29N7O3S (%):C, 61.00; H, 5.50; N,
18.44. Found (%):C, 61.02; H, 5.48; N, 18.44.
5.10.1. (E)-2-(2,4-dichlorobenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (17)
White solid; yield: 82%; m.p.: 196e199 ꢀC; MS (ESI) m/z(%):
584.2 [M þ H]þ; IR (KBr) cmꢂ1: 3450.2, 2920.3, 2851.4, 1687.0,
1642.0, 1593.6, 1532.6, 1465.3, 1402.8, 1383.9, 1357.4, 1317.7, 1271.9,
1184.4, 1114.3, 1048.3, 849.2, 821.8, 796.1, 721.0, 619.8, 515.8; 1H
5.10.5. (E)-2-(3-fluoro-4-hydroxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
hydrazinecarboxamide (21)
White solid; yield: 87%; m.p.: 182e184 ꢀC; MS (ESI) m/z(%):
NMR (400 MHz, DMSO)
d
11.12 (s, 1H), 9.21 (s, 1H), 8.46 (d,
550.3 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.72 (s, 1H), 10.26 (s,
J ¼ 8.6 Hz,1H), 8.37 (d, J ¼ 8.8 Hz, 2H), 8.33 (s,1H), 8.08 (t, J ¼ 6.5 Hz,
1H), 7.95e7.85 (m, 1H), 7.81 (t, J ¼ 12.6 Hz, 2H), 7.69 (d, J ¼ 2.1 Hz,
1H), 7.52 (dd, J ¼ 8.6, 1.9 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 3.68 (dd,
J ¼ 12.9, 6.5 Hz, 2H), 3.61 (t, J ¼ 4.5 Hz, 4H), 2.42 (dd, J ¼ 14.8, 7.9 Hz,
1H), 9.05 (s, 1H), 8.36 (d, J ¼ 8.5 Hz, 2H), 8.09 (d, J ¼ 5.3 Hz, 1H), 7.92
(s, 1H), 7.88 (s, 1H), 7.87 (s, 1H), 7.81 (d, J ¼ 8.5 Hz, 2H), 7.43 (d,
J ¼ 5.2 Hz, 1H), 7.36 (d, J ¼ 8.2 Hz, 1H), 6.98 (t, J ¼ 8.5 Hz, 1H), 3.69
(dd, J ¼ 12.4, 6.3 Hz, 2H), 3.61 (d, J ¼ 4.0 Hz, 4H), 2.48e2.42 (m, 2H),
2.41 (d, J ¼ 8.7 Hz, 4H), 1.90e1.81 (m, 2H). 13C NMR (101 MHz,
6H), 1.85 (s, 2H); 13C NMR (101 MHz, DMSO)
d 160.72, 160.09,
157.33, 153.17, 141.04, 136.28, 134.88, 133.70, 133.32, 133.20, 131.15,
129.61, 129.26, 128.50 (2C), 128.12, 125.14, 119.79 (2C), 113.09, 66.72
(2C), 56.74, 53.88 (2C), 39.28, 26.14. Anal. calcd. for C27H27Cl2N7O2S
(%):C, 55.48; H, 4.66; N, 16.77. Found (%):C, 55.46; H, 4.67; N, 16.79.
DMSO) d 160.74, 160.14, 157.33, 153.43, 153.03, 150.63, 146.85,
141.29, 140.69, 133.18, 133.06, 128.48 (2C), 126.81, 125.15, 119.59
(2C), 117.96, 114.22, 113.22, 66.71 (2C), 56.74, 53.87 (2C), 39.34,
26.13. Anal. calcd. for C27H28FN7O3S (%):C, 59.00; H, 5.13; N, 17.84.
Found (%):C, 59.03; H, 5.12; N, 17.82.
5.10.2. (E)-2-(2,3-dichlorobenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (18)
5.10.6. (E)-2-(3-bromo-4-hydroxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
hydrazinecarboxamide (22)
White solid; yield: 85%; m.p.: 190e194 ꢀC; MS (ESI) m/z(%):
584.2 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d
11.16 (s, 1H), 9.19 (s,
White solid; yield: 81%; m.p.: 195e198 ꢀC; MS (ESI) m/z(%):
1H), 8.42 (s, 1H), 8.41 (s, 1H), 8.37 (d, J ¼ 8.5 Hz, 2H), 8.09 (d,
J ¼ 5.3 Hz,1H), 7.89 (t, J ¼ 5.2 Hz,1H), 7.79 (d, J ¼ 8.6 Hz, 2H), 7.68 (d,
J ¼ 7.8 Hz, 1H), 7.47 (d, J ¼ 7.9 Hz, 1H), 7.43 (d, J ¼ 5.5 Hz, 1H), 3.69
(dd, J ¼ 12.5, 6.3 Hz, 2H), 3.61 (t, J ¼ 4.2 Hz, 4H), 2.43 (t, J ¼ 5.6 Hz,
2H), 2.40 (d, J ¼ 8.8 Hz, 4H), 1.85 (p, J ¼ 6.6 Hz, 2H). 13C NMR
610.1 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.70 (s, 1H), 10.66 (s,
1H), 9.08 (s, 1H), 8.35 (d, J ¼ 8.5 Hz, 2H), 8.11 (s, 1H), 8.09 (d,
J ¼ 5.3 Hz,1H), 7.88 (d, J ¼ 5.5 Hz,1H), 7.85 (s,1H), 7.80 (d, J ¼ 8.4 Hz,
2H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.43 (d, J ¼ 5.3 Hz, 1H), 7.00 (d,
J ¼ 8.4 Hz, 1H), 3.69 (dd, J ¼ 12.7, 6.5 Hz, 2H), 3.62 (t, J ¼ 4.0 Hz, 4H),

790
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
2.45 (t, J ¼ 5.6 Hz, 4H), 2.42 (d, J ¼ 9.0 Hz, 4H), 1.92e1.80 (m, 2H). 13
C
3.61 (s, 4H), 2.43 (d, J ¼ 4.5 Hz, 2H), 2.39 (s, 4H), 1.91e1.81 (m, 2H).
13C NMR (101 MHz, DMSO)
160.74, 160.15, 157.31, 156.22, 153.47,
NMR (101 MHz, DMSO)
d
160.75, 160.16, 157.33, 155.71, 153.43,
d
141.31, 140.38, 133.17, 133.05, 131.54, 128.62, 128.49 (2C), 127.63,
125.15, 119.60 (2C), 116.68, 113.22, 110.45, 66.68 (2C), 56.72, 53.84
(2C), 39.36, 26.12. Anal. calcd. for C27H28BrN7O3S (%):C, 53.12; H,
4.62; N, 16.06. Found (%):C, 53.10; H, 4.63; N, 16.09.
148.57 (2C), 142.19, 141.32, 137.91, 137.51, 133.09, 128.55 (2C),
125.45, 125.12, 119.45 (2C), 105.36, 104.91, 66.70 (2C), 56.74, 56.64,
56.55, 53.87 (2C), 39.34, 26.12. Anal. calcd. for C29H33N7O3S (%):C,
62.23; H, 5.94; N, 17.52. Found (%):C, 62.27; H, 5.92; N, 17.49.
5.10.7. (E)-2-(4-hydroxy-3-methoxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2- yl)phenyl)
hydrazinecarboxamide (23)
5.10.11. (E)-2-(2,3-dichlorobenzylidene)-N-(4-(4-morpholinothieno
[3,2-d]pyrimidin-2-yl)phenyl)hydrazine carboxamide (27)
White solid; yield: 85%; m.p.: 175e178 ꢀC; MS (ESI) m/z(%):
White solid; yield: 76%; m.p.: 203e206 ꢀC; MS (ESI) m/z(%):
523.1 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 8.42 (s, 1H), 8.34 (s,
562.3 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.65 (s, 1H), 9.42 (s,
2H), 8.25 (d, J ¼ 5.6 Hz, 1H), 7.78 (d, J ¼ 8.7 Hz, 2H), 7.75e7.70 (m,
2H), 7.67 (d, J ¼ 5.8 Hz, 2H), 7.52 (d, J ¼ 5.5 Hz, 1H), 7.48e7.42 (m,
1H), 4.01 (s, 4H), 3.82 (s, 4H). Anal. calcd. for C24H20Cl2N6O2S (%):C,
54.65; H, 3.82; N, 15.93. Found (%):C, 54.68; H, 3.81; N, 15.91.
1H), 9.03 (s, 1H), 8.36 (d, J ¼ 8.6 Hz, 2H), 8.09 (d, J ¼ 5.3 Hz, 1H), 7.88
(s, 2H), 7.80 (d, J ¼ 8.6 Hz, 2H), 7.50 (s, 1H), 7.42 (t, J ¼ 7.2 Hz, 1H),
7.18 (d, J ¼ 8.2 Hz, 1H), 6.82 (d, J ¼ 8.1 Hz, 1H), 3.88 (s, 3H), 3.68 (dd,
J ¼ 12.5, 6.4 Hz, 2H), 3.61 (t, J ¼ 4.0 Hz, 4H), 2.44 (d, J ¼ 6.6 Hz, 2H),
2.39 (s, 4H), 1.91e1.78 (m, 2H); 13C NMR (101 MHz, DMSO)
d 160.74,
5.10.12. (E)-2-(4-hydroxybenzylidene)-N-(4-(4-morpholinothieno
[3,2-d]pyrimidin-2-yl)phenyl)hydrazine carboxamide (28)
White solid; yield: 79%; m.p.: 180e184 ꢀC; MS (ESI) m/z(%):
160.15, 157.32, 153.46, 148.90, 148.42, 142.08, 141.34, 133.17, 132.98,
128.54 (2C),126.29,125.15,121.93,119.38 (2C),115.83,113.21,110.51,
66.71 (2C), 56.75, 56.28, 53.88 (2C), 39.35, 26.13. Anal. calcd. for
475.3 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.76 (s, 1H), 9.92 (s,
C
28H31N7O4S (%):C, 59.88; H, 5.56; N, 17.46. Found (%):C, 59.85; H,
1H), 9.26 (s, 1H), 8.54 (t, J ¼ 7.8 Hz, 1H), 8.44 (t, J ¼ 13.2 Hz, 2H), 7.94
(d, J ¼ 8.6 Hz, 2H), 7.91 (s, 1H), 7.81 (d, J ¼ 5.5 Hz, 1H), 7.68 (s, 2H),
6.84 (d, J ¼ 8.4 Hz, 2H), 4.19 (s, 4H), 3.87 (d, J ¼ 4.1 Hz, 4H). Anal.
calcd. for C24H22N6O3S (%):C, 60.75; H, 4.67; N, 17.71. Found (%):C,
60.71; H, 4.68; N, 17.73.
5.57; N, 17.48.
5.10.8. (E)-2-(3,5-dibromo-4-hydroxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin- 2-yl)phenyl)
hydrazinecarboxamide (24)
White solid; yield: 82%; m.p.: 211e214 ꢀC; MS (ESI) m/z(%):
5.10.13. (E)-2-(3,4-dihydroxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)hydrazine
carboxamide (29)
688.1 [M þ H]þ. 1H NMR (600 MHz, DMSO)
d 10.81 (s, 1H), 10.59 (d,
J ¼ 34.7 Hz, 1H), 9.19 (s, 1H), 8.37 (d, J ¼ 8.7 Hz, 2H), 8.10 (d,
J ¼ 5.3 Hz, 1H), 8.08 (s, 2H), 7.92 (t, J ¼ 5.2 Hz, 1H), 7.82 (d, J ¼ 7.1 Hz,
2H), 7.80 (s, 1H), 7.44 (d, J ¼ 5.3 Hz, 1H), 3.70 (dd, J ¼ 12.3, 6.4 Hz,
2H), 3.64 (s, 4H), 2.56 (d, J ¼ 6.9 Hz, 2H), 2.53 (d, J ¼ 20.5 Hz, 4H),
White solid; yield: 83%; m.p.: 177e180 ꢀC; MS (ESI) m/z(%):
491.1 [M þ H]þ; IR (KBr) cmꢂ1: 3451.1, 2920.2, 2851.3, 1644.4,
1593.9, 1527.7, 1489.1, 1448.2, 1418.9, 1400.6, 1384.1, 1277.8, 1234.0,
1180.0, 1111.8, 1017.7, 936.1, 847.6, 795.8, 778.1, 620.0; 1H NMR
1.93e1.86 (m, 2H); 13C NMR (151 MHz, DMSO)
d 160.73, 160.12,
157.29, 156.13,153.41, 152.85, 152.55e151.80 (m, 0H), 141.28,139.03,
133.24, 133.07, 131.13, 130.90, 128.47 (2C), 125.13, 119.83 (2C),
112.89, 112.79, 66.29 (2C), 56.46, 53.54 (2C), 39.14, 25.82. Anal.
calcd. for C27H27Br2N7O3S (%):C, 47.04; H, 3.95; N, 14.22. Found
(%):C, 47.07; H, 3.96; N, 14.19.
(400 MHz, DMSO)
d
10.72 (s, 1H), 9.28 (s, 1H), 8.53 (d, J ¼ 5.5 Hz,
1H), 8.43 (d, J ¼ 8.6 Hz, 2H), 7.92 (d, J ¼ 8.5 Hz, 2H), 7.84 (d,
J ¼ 3.2 Hz,1H), 7.83 (s, 1H), 7.31 (d, J ¼ 7.3 Hz,1H), 7.06 (d, J ¼ 8.2 Hz,
1H), 6.80 (d, J ¼ 8.1 Hz, 1H), 4.20 (s, 4H), 3.87 (s, 4H). 13C NMR
(101 MHz, DMSO)
d 163.23, 157.05, 155.39, 153.21, 150.07, 147.93,
146.02, 144.41, 142.92, 138.91, 130.02 (2C), 126.18, 120.32, 120.06,
119.22 (2C), 116.01, 114.22, 112.69, 66.27 (2C), 47.51 (2C). Anal. calcd.
for C24H22N6O4S (%):C, 58.76; H, 4.52; N, 17.13. Found (%):C, 58.78;
H, 4.53; N, 17.11.
5.10.9. (E)-2-(4-hydroxy-3,5-dimethylbenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (25)
White solid; yield: 75%; m.p.: 181e184 ꢀC; MS (ESI) m/z(%):
560.2 [M þ H]þ; IR (KBr) cmꢂ1: 3451.1, 2920.6, 2851.9, 1641.2,
1594.2, 1538.5, 1498.0, 1462.0, 1417.4, 1384.2, 1360.0, 1186.2, 1158.7,
5.10.14. (E)-2-(2,4-dihydroxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)hydrazine
carboxamide (30)
1111.5, 847.1, 796.6, 620.5; 1H NMR (400 MHz, DMSO)
d 10.59 (s,
1H), 8.95 (s, 1H), 8.64 (s, 1H), 8.35 (d, J ¼ 8.2 Hz, 2H), 8.09 (d,
J ¼ 5.4 Hz, 1H), 7.88 (d, J ¼ 5.5 Hz, 1H), 7.83 (s, 1H), 7.80 (s, 2H), 7.43
(d, J ¼ 3.8 Hz, 1H), 7.42 (s, 2H), 3.73e3.64 (m, 2H), 3.61 (s, 4H), 2.44
(t, J ¼ 5.6 Hz, 2H), 2.41 (d, J ¼ 9.4 Hz, 4H), 2.22 (s, 6H), 1.85 (dd,
White solid; yield: 77%; m.p.: 169e173 ꢀC; MS (ESI) m/z(%):
491.2 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.72 (s, 1H), 10.28 (s,
0H), 9.39 (s, 0H), 8.48 (dd, J ¼ 9.7, 5.1 Hz,1H), 8.44 (d, J ¼ 8.8 Hz, 0H),
8.19 (s, 0H), 7.86 (d, J ¼ 8.7 Hz, 0H), 7.82 (d, J ¼ 5.0 Hz, 0H), 7.66 (d,
J ¼ 8.8 Hz, 0H), 6.48e6.39 (m, 0H), 6.37 (t, J ¼ 5.2 Hz, 0H), 6.33 (dd,
J ¼ 8.5, 2.2 Hz, 0H), 4.15 (d, J ¼ 4.6 Hz, 4H), 3.88e3.81 (m, 4H). Anal.
calcd. for C24H22N6O4S (%):C, 58.76; H, 4.52; N, 17.13. Found (%):C,
58.79; H, 4.51; N, 17.11.
J ¼ 13.3, 6.4 Hz, 2H). 13C NMR (101 MHz, DMSO)
d 160.91, 160.16,
157.51, 155.37, 153.44, 142.24, 141.35, 133.36, 132.96, 128.51 (2C),
127.85 (2C), 125.73, 125.15, 124.88 (2C), 119.38 (2C), 113.49, 66.72
(2C), 56.75, 53.89 (2C), 39.34, 26.15, 17.01 (2C). Anal. calcd. for
C
29H33N7O3S (%):C, 62.23; H, 5.94; N, 17.52. Found (%):C, 62.26; H,
5.92; N, 17.50.
5.10.15. (E)-N-(4-(4-morpholinothieno[3,2-d]pyrimidin-2-yl)
phenyl)-2-(2,3,4-trihydroxybenzylidene)hydrazine carboxamide
(31)
5.10.10. (E)-2-(4-hydroxy-3,5-dimethoxybenzylidene)-N-(4-(4-((3-
morpholinopropyl)amino)thieno[3,2-d] pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (26)
White solid; yield: 75%; m.p.: 176e179 ꢀC; MS (ESI) m/z(%): 507.1
White solid; yield: 78%; m.p.: 193e197 ꢀC; MS (ESI) m/z(%):
[M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.77 (s, 1H), 10.36 (s, 1H),
592.3 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d
10.72 (s, 1H), 10.49 (s,
9.49 (s, 1H), 9.44 (s, 1H), 8.56 (dd, J ¼ 5.5, 3.6 Hz, 1H), 8.50 (d,
J ¼ 8.9 Hz,1H), 8.44 (d, J ¼ 8.9 Hz,1H), 8.18 (s,1H), 7.70 (d, J ¼ 8.4 Hz,
1H), 6.96 (d, J ¼ 8.6 Hz, 2H), 6.46 (dd, J ¼ 8.6, 3.6 Hz, 2H), 6.40 (d,
J ¼ 8.5 Hz, 1H), 4.20 (s, 4H), 3.90e3.82 (m, 4H). Anal. calcd. for
1H), 9.07 (s, 1H), 8.69 (s, 1H), 8.36 (d, J ¼ 8.4 Hz, 2H), 8.09 (d,
J ¼ 5.3 Hz, 1H), 7.87 (s, 2H), 7.81 (d, J ¼ 8.5 Hz, 2H), 7.75 (s, 1H), 7.43
(d, J ¼ 5.2 Hz, 1H), 3.86 (s, 6H), 3.82 (s, 6H), 3.68 (d, J ¼ 6.0 Hz, 2H),

Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
791
C24H22N6O5S (%):C, 56.91; H, 4.38; N, 16.59. Found (%):C, 56.87; H,
4.40; N, 16.61.
159.54, 158.30, 155.37, 153.42, 142.26, 141.60, 134.17, 132.31, 128.63
(2C), 127.86 (2C), 125.72, 125.26, 124.87 (2C), 119.49 (2C), 112.15,
66.50 (2C), 46.36 (2C), 17.01 (2C). Anal. calcd. for C26H26N6O3S (%):C,
62.13; H, 5.21; N, 16.72. Found (%):C, 62.17; H, 5.20; N, 16.70.
5.10.16. (E)-2-(3-fluoro-4-hydroxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)hydrazine
carboxamide (32)
5.10.21. (E)-2-(4-hydroxy-3,5-dimethoxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)
White solid; yield: 80%; m.p.: 180e184 ꢀC; MS (ESI) m/z(%):
493.3 [M þ H]þ; IR (KBr) cmꢂ1: 3450.0, 2920.5, 2851.3, 1688.3,
1642.3, 1592.2, 1527.0, 1487.0, 1445.6, 1383.2, 1305.3, 1276.4, 1234.9,
1187.6,1113.1,1018.4, 848.0, 777.3, 619.5; 1H NMR (400 MHz, DMSO)
hydrazinecarboxamide (37)
White solid; yield: 81%; m.p.: 180e184 ꢀC; MS (ESI) m/z(%):
535.1 [M þ H]þ; IR (KBr) cmꢂ1: 3448.9, 2920.0, 2851.2, 1645.4,
1594.0, 1530.1, 1468.3, 1400.2, 1384.2, 1322.3, 1274.7, 1114.8, 847.9,
d
10.88 (s, 1H),10.40 (s, 1H), 9.32 (s, 1H), 8.53 (d, J ¼ 5.5 Hz,1H), 8.43
(s, 2H), 7.95 (d, J ¼ 8.7 Hz, 2H), 7.89 (s, 1H), 7.86 (s, 1H), 7.84 (d,
778.9, 620.1, 515.9, 443.6; 1H NMR (400 MHz, DMSO)
d 10.88 (s,1H),
J ¼ 5.6 Hz, 1H), 7.36 (d, J ¼ 8.2 Hz, 1H), 7.02 (t, J ¼ 8.6 Hz, 1H), 4.19 (s,
9.37 (s, 1H), 8.52 (d, J ¼ 5.5 Hz, 1H), 8.44 (d, J ¼ 8.8 Hz, 2H), 7.95 (d,
J ¼ 8.8 Hz, 2H), 7.89 (s, 1H), 7.81 (d, J ¼ 5.5 Hz, 1H), 7.13 (s, 2H), 4.19
(s, 4H), 3.86 (d, J ¼ 6.9 Hz, 4H), 3.85 (s, 6H). Anal. calcd. for
4H), 3.90e3.80 (m, 4H). 13C NMR (101 MHz, DMSO)
d 157.03,155.23,
153.22, 152.88, 150.59, 147.05, 144.47, 141.35, 130.01 (2C), 129.36,
128.66, 126.57, 126.00, 125.27, 119.54 (2C), 118.01, 114.52, 112.73,
66.26 (2C), 48.29 (2C). Anal. calcd. for C24H21FN6O3S (%):C, 58.53; H,
4.30; N, 17.06. Found (%):C, 58.55; H, 4.31; N, 17.03.
C26H26N6O5S (%):C, 58.41; H, 4.90; N, 15.72. Found (%):C, 58.45; H,
4.88; N, 15.69.
5.10.22. (E)-2-(2,3-dichlorobenzylidene)-N-(4-(4-((2-
(diethylamino)ethyl)amino)thieno[3,2-d]pyrimidin-2-yl) phenyl)
hydrazinecarboxamide (38)
5.10.17. (E)-2-(3-bromo-4-hydroxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)hydrazine
carboxamide (33)
White solid; yield: 85%; m.p.: 190e194 ꢀC; MS (ESI) m/z(%):
White solid; yield: 85%; m.p.: 184e187 ꢀC; MS (ESI) m/z(%):
553.1 [M þ H]þ; IR (KBr) cmꢂ1: 3454.0, 2919.3, 2850.7, 1642.6,
1527.7, 1384.0, 1192.2, 1111.0, 778.4, 619.8; 1H NMR (400 MHz,
556.1 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 11.17 (s, 1H), 9.18 (s,
1H), 8.43 (s, 1H), 8.41 (s, 1H), 8.38 (d, J ¼ 8.7 Hz, 2H), 8.09 (d,
J ¼ 5.2 Hz,1H), 7.80 (d, J ¼ 8.6 Hz, 2H), 7.76 (s, 1H), 7.68 (d, J ¼ 7.8 Hz,
1H), 7.46 (d, J ¼ 7.9 Hz, 1H), 7.43 (d, J ¼ 5.0 Hz, 1H), 3.69 (d,
J ¼ 6.4 Hz, 2H), 3.45 (dt, J ¼ 13.0, 6.7 Hz, 1H), 2.71 (t, J ¼ 7.1 Hz, 2H),
2.59 (dd, J ¼ 13.9, 6.9 Hz, 4H), 1.01 (t, J ¼ 7.0 Hz, 6H). 13C NMR
DMSO)
d
10.85 (s, 1H), 10.76 (s, 1H), 9.35 (s, 1H), 8.52 (d, J ¼ 5.3 Hz,
1H), 8.42 (d, J ¼ 8.6 Hz, 2H), 8.11 (s, 1H), 7.96 (t, J ¼ 9.3 Hz, 2H), 7.88
(s, 1H), 7.79 (s, 1H), 7.61 (d, J ¼ 8.5 Hz, 1H), 7.05 (t, J ¼ 8.7 Hz, 1H),
4.19 (s, 4H), 3.90e3.80 (m, 4H). Anal. calcd. for C24H21BrN6O3S (%):C,
52.09; H, 3.82; N, 15.19. Found (%):C, 52.12; H, 3.80; N, 15.17.
(101 MHz, DMSO)
d 160.74, 160.10, 157.36, 153.14, 141.02, 137.05,
134.49, 133.31, 133.30, 132.58, 131.38, 130.86, 128.60, 128.49 (2C),
126.60, 125.13, 119.72 (2C), 113.30, 51.78, 47.46 (2C), 39.00, 12.57
(2C). Anal. calcd. for C26H27Cl2N7OS (%):C, 56.11; H, 4.89; N, 17.62.
Found (%):C, 56.15; H, 4.86; N, 17.60.
5.10.18. (E)-2-(4-hydroxy-3-methoxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (34)
White solid; yield: 82%; m.p.: 178e182 ꢀC; MS (ESI) m/z(%):
5.10.23. (E)-N-(4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-2-(3,4-dihydroxy benzylidene)
hydrazinecarboxamide (39)
505.1 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.80 (s, 1H), 9.48 (s,
1H), 9.31 (s, 1H), 8.51 (d, J ¼ 5.3 Hz, 1H), 8.42 (d, J ¼ 8.9 Hz, 2H), 7.95
(d, J ¼ 8.8 Hz, 2H), 7.90 (s, 1H), 7.77 (d, J ¼ 5.5 Hz, 1H), 7.51 (d,
J ¼ 1.6 Hz, 1H), 7.17 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.83 (d, J ¼ 8.1 Hz, 1H),
4.19 (s, 4H), 3.88 (s, 3H), 3.84 (dd, J ¼ 12.5, 4.8 Hz, 4H). Anal. calcd.
for C25H24N6O4S (%):C, 59.51; H, 4.79; N, 16.66. Found (%):C, 59.54;
H, 4.77; N, 16.63.
White solid; yield: 87%; m.p.: 193e196 ꢀC; MS (ESI) m/z(%):
520.1 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.61 (s, 1H), 8.98 (s,
1H), 8.36 (d, J ¼ 8.5 Hz, 2H), 8.10 (d, J ¼ 5.4 Hz, 1H), 7.84 (s, 1H), 7.81
(s, 1H), 7.76 (t, J ¼ 9.7 Hz, 2H), 7.43 (d, J ¼ 5.0 Hz, 1H), 7.27 (d,
J ¼ 15.7 Hz, 1H), 7.06 (d, J ¼ 8.0 Hz, 1H), 6.80 (d, J ¼ 8.1 Hz, 1H), 3.74
(s, 2H), 2.82 (s, 2H), 2.69 (d, J ¼ 6.6 Hz, 4H), 1.05 (t, J ¼ 7.0 Hz, 6H).
Anal. calcd. for C26H29N7O3S (%):C, 60.10; H, 5.63; N, 18.87. Found
(%):C, 60.06; H, 5.65; N, 18.90.
5.10.19. (E)-2-(3,5-dibromo-4-hydroxybenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (35)
White solid; yield: 75%; m.p.: 169e173 ꢀC; MS (ESI) m/z(%):
5.10.24. (E)-N-(4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-2-(2,4-dihydroxy benzylidene)
hydrazinecarboxamide (40)
631.0 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.25 (s, 1H), 9.00 (s,
1H), 8.33 (d, J ¼ 8.8 Hz, 2H), 8.24 (d, J ¼ 5.5 Hz, 1H), 7.79 (d,
J ¼ 8.8 Hz, 2H), 7.74e7.66 (m, 2H), 7.63 (s, 1H), 7.62 (s, 1H), 7.52 (d,
J ¼ 5.5 Hz, 1H), 4.07e3.96 (m, 4H), 3.82 (d, J ¼ 4.5 Hz, 4H). Anal.
calcd. for C24H20Br2N6O3S (%):C, 45.59; H, 3.19; N, 13.29. Found
(%):C, 45.62; H, 3.17; N, 13.26.
White solid; yield: 83%; m.p.: 201e204 ꢀC; MS (ESI) m/z(%):
520.2 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.49 (s, 1H), 8.98 (s,
1H), 8.35 (d, J ¼ 8.8 Hz, 2H), 8.32 (s, 1H), 8.17 (s, 1H), 8.09 (d,
J ¼ 5.4 Hz, 2H), 7.75 (d, J ¼ 9.0 Hz, 2H), 7.61 (s,1H), 7.43 (d, J ¼ 5.2 Hz,
2H), 6.35e6.31 (m, 1H), 3.72 (s, 2H), 2.79 (s, 2H), 2.67 (s, 4H), 1.04 (s,
6H). Anal. calcd. for C26H29N7O3S (%):C, 60.10; H, 5.63; N, 18.87.
Found (%):C, 60.07; H, 5.64; N, 18.89.
5.10.20. (E)-2-(4-hydroxy-3,5-dimethylbenzylidene)-N-(4-(4-
morpholinothieno[3,2-d]pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (36)
White solid; yield: 85%; m.p.: 183e186 ꢀC; MS (ESI) m/z(%):
503.2 [M þ H]þ. IR (KBr) cmꢂ1: 3454.3, 2919.6, 2851.4, 1692.0,
1642.9, 1593.7, 1526.6, 1489.9, 1447.9, 1418.3, 1384.3, 1364.1, 1276.6,
1179.3,1159.7,1115.7,1016.5, 933.9, 795.6, 619.6; 1H NMR (400 MHz,
5.10.25. (E)-N-(4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-2-(2,3,4-trihydroxy benzylidene)
hydrazinecarboxamide (41)
White solid; yield: 79%; m.p.: 211e214 ꢀC; MS (ESI) m/z(%):
DMSO)
d
10.59 (s, 1H), 8.98 (s, 1H), 8.64 (s, 1H), 8.35 (d, J ¼ 8.6 Hz,
536.2 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.61 (s, 1H), 9.12 (s,
2H), 8.24 (d, J ¼ 5.5 Hz, 1H), 7.83 (s, 2H), 7.81 (s, 1H), 7.52 (d,
J ¼ 5.5 Hz, 1H), 7.42 (s, 2H), 4.01 (d, J ¼ 4.3 Hz, 4H), 3.82 (d,
1H), 8.35 (d, J ¼ 8.3 Hz, 2H), 8.16 (s, 1H), 8.09 (d, J ¼ 5.2 Hz, 2H), 7.80
(s, 1H), 7.74 (d, J ¼ 8.4 Hz, 2H), 7.63 (s, 1H), 7.43 (d, J ¼ 5.2 Hz, 2H),
7.13 (d, J ¼ 7.7 Hz, 1H), 6.38 (d, J ¼ 8.5 Hz, 1H), 3.74 (s, 2H), 2.84 (s,
J ¼ 4.3 Hz, 4H), 2.22 (s, 6H); 13C NMR (101 MHz, DMSO)
d 163.01,

792
Z. Liu et al. / European Journal of Medicinal Chemistry 87 (2014) 782e793
2H), 2.71 (d, J ¼ 6.3 Hz, 4H), 1.07 (dt, J ¼ 13.7, 7.0 Hz, 6H). 13C NMR
and incubated for 72 h. Fresh MTT was added to each well at the
terminal concentration of 5
g/mL, and incubated with cells at 37 ꢀC
for 4 h. The formazan crystals in each well were dissolved in 100 mL
DMSO, and the absorbency at 492 nm (for absorbance of MTT for-
mazan) and 630 nm (for the reference wavelength) was measured
with an ELISA reader. All of the compounds 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.
(101 MHz, DMSO)
d
172.53, 160.83, 160.30, 157.34, 153.25, 148.38,
m
147.18, 141.92, 133.50, 133.23, 132.76, 128.63, 125.13 (2C), 119.15
(2C), 118.92, 113.57, 113.20, 108.39, 51.86, 47.40 (2C), 21.59, 11.77
(2C). Anal. calcd. for C26H29N7O4S (%):C, 58.30; H, 5.46; N, 18.31.
Found (%):C, 58.32; H, 5.46; N, 18.30.
5.10.26. (E)-N-(4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-2-(3-fluoro-4-hydroxyl benzylidene)
hydrazinecarboxamide (42)
White solid; yield: 77%; m.p.: 205e208 ꢀC; MS (ESI) m/z(%):
522.2 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 11.11 (s, 1H), 9.40 (s,
5.11.2. In vitro enzymatic assays
The in vitro enzymatic assays of compound 36 were evaluated by
homogeneous time-resolved fluorescence (HTRF) assay. Briefly,
1H), 8.35 (d, J ¼ 8.7 Hz, 2H), 8.08 (d, J ¼ 5.3 Hz, 1H), 7.85 (s, 1H), 7.79
(d, J ¼ 8.8 Hz, 2H), 7.78e7.75 (m, 1H), 7.70 (d, J ¼ 12.3 Hz, 1H), 7.43
(d, J ¼ 5.3 Hz,1H), 7.22 (d, J ¼ 8.2 Hz,1H), 6.91 (t, J ¼ 8.7 Hz,1H), 3.67
(dt, J ¼ 19.4, 9.6 Hz, 2H), 2.72 (d, J ¼ 8.1 Hz, 2H), 2.58 (q, J ¼ 7.1 Hz,
20
mg/mL poly (Glu, Tyr) 4:1 (Sigma) was preloaded as a substrate in
384-well plates. Subsequently, 50
mL of 10 mM ATP (Invitrogen)
4H), 1.01 (t, J ¼ 7.0 Hz, 6H); 13C NMR (101 MHz, DMSO)
d 160.77,
solution 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 kinase reaction was initiated by the addition of purified tyro-
160.23, 157.34, 153.52, 152.14, 151.24, 142.17, 141.63, 133.18, 133.00,
129.48,128.48 (2C),125.14,119.10 (2C),118.79,113.85,113.50,113.18,
51.79, 47.45 (2C), 38.99, 12.58 (2C). Anal. calcd. for C26H28FN7O2S
(%):C, 59.87; H, 5.41; N, 18.80. Found (%):C, 59.89; H, 5.40; N, 18.79.
sine kinase proteins diluted in 39
lution. Reactions were incubated for 30 min at 25 ꢀC and
terminated by the addition of 5 L Streptavidin-XL665 and 5 L Tk
Antibody Cryptate working solution. 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 so-
5.10.27. (E)-2-(3,5-dibromo-4-hydroxybenzylidene)-N-(4-(4-((2-
(diethylamino)ethyl)amino)thieno[3,2-d] pyrimidin-2-yl)phenyl)
hydrazinecarboxamide (43)
m
m
White solid; yield: 82%; m.p.: 197e201 ꢀC; MS (ESI) m/z(%):
660.0 [M þ H]þ. 1H NMR (400 MHz, DMSO)
d 10.60 (s, 1H), 9.10 (s,
1H), 8.37 (d, J ¼ 8.6 Hz, 2H), 8.11 (d, J ¼ 5.3 Hz, 1H), 7.99 (s, 1H), 7.93
(s, 2H), 7.81 (d, J ¼ 8.6 Hz, 2H), 7.77 (s, 1H), 7.45 (d, J ¼ 5.3 Hz, 1H),
3.88 (s, 2H), 3.13 (d, J ¼ 34.2 Hz, 2H), 2.92 (d, J ¼ 21.4 Hz, 4H), 1.13 (t,
5.11.3. Western blot analysis
About 1 ꢁ 107 HT-29 cells were gathered after pre-treatment
with indicated concentrations of compound 36 for 48 h. Protein
lysates were prepared using lysis buffer (10ꢁ) (Cell Signalling
Technology, Danvers, MA, USA) and protease inhibitor cocktail
(SigmaeAldrich). All samples were sonicated at 10 kHz for 10 s. An
equal amount of total protein extracts from cultured cells or tissues
were fractionated in 10% SDSePAGE solution and electrically
transferred onto polyvinylidene difluoride (PVDF) membranes.
Corresponding mouse or rabbit primary antibodies and horseradish
peroxidase (HRP)-conjugated appropriate secondary antibodies
were used to detect the proteins of interest. The bound HRP-
conjugated secondary antibodies on the PVDF membrane were
reacted with ECL detection reagents (Thermo Scientific) and
exposed to X-ray films. The proportion of cleaved poly (ADP-ribose)
J ¼ 6.7 Hz, 6H); 13C NMR (101 MHz, DMSO)
d 160.90, 160.21, 157.28,
153.44, 141.52, 140.22, 133.58, 132.75, 130.99 (2C), 128.54 (2C),
125.12, 119.61 (2C), 114.09 (2C), 113.42, 50.93, 47.40 (2C).), 37.29,
10.78 (2C). Anal. calcd. for C26H27Br2N7O2S (%):C, 47.21; H, 4.11; N,
14.82. Found (%):C, 47.25; H, 4.10; N, 14.80.
5.10.28. (E)-N-(4-(4-((2-(diethylamino)ethyl)amino)thieno[3,2-d]
pyrimidin-2-yl)phenyl)-2-(4-hydroxy-3,5- dimethylbenzylidene)
hydrazinecarboxamide (44)
White solid; yield: 85%; m.p.: 190e194 ꢀC; MS (ESI) m/z(%):
532.3 [M þ H]þ; IR (KBr) cmꢂ1: 3454.6, 2920.4, 2851.3,1691.0,
1645.8, 1594.4, 1535.7, 1499.6, 1467.8, 1419.5, 1384.1, 1352.6, 1271.0,
1186.5, 1112.5, 848.0, 796.0, 718.8, 620.1, 516.7; 1H NMR (400 MHz,
DMSO)
d
10.60 (s, 1H), 8.95 (s, 1H), 8.64 (s, 1H), 8.37 (d, J ¼ 8.4 Hz,
polymerase (PARP) was normalized to the internal control b-actin.
2H), 8.08 (d, J ¼ 5.3 Hz,1H), 7.83 (s,1H), 7.80 (s, 2H), 7.75 (s,1H), 7.43
(d, J ¼ 3.0 Hz, 1H), 7.42 (s, 2H), 3.69 (dd, J ¼ 13.0, 6.1 Hz, 2H), 2.71 (t,
J ¼ 7.1 Hz, 2H), 2.59 (q, J ¼ 7.0 Hz, 4H), 2.22 (s, 6H), 1.01 (t, J ¼ 7.1 Hz,
Acknowledgments
6H). 13C NMR (101 MHz, DMSO)
d 160.77, 160.18, 157.36, 155.37,
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).
153.43, 142.26, 141.35, 133.43e133.13 (m, 0H), 132.94, 128.50 (2C),
127.85 (2C), 125.73, 125.14, 124.88 (2C), 119.34 (2C), 113.47, 51.81,
47.46 (2C), 39.00,17.00 (2C),12.57 (2C). Anal. calcd. for C28H33N7O2S
(%):C, 63.25; H, 6.26; N, 18.44. Found (%):C, 63.22; H, 6.28; N, 18.46.
Appendix A. Supplementary data
5.11. Pharmacology
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.10.022.
5.11.1. MTT assay in vitro
The antiproliferative activities of compounds 17e44 were eval-
uated against H460, HT-29, MKN-45 and MDA-MB-231 cell lines by
the standard MTT assay in vitro, with GDC-0941 and sorafenib as
the positive control [32]. The cancer cell lines were cultured in
minimum essential medium (MEM) supplement with 10% fetal
bovine serum (FBS). Approximate 4 ꢁ 103 cells, suspended in MEM
medium, were plated into each well of a 96-well plate and incu-
bated in 5% CO2 at 37 ꢀC for 24 h. The tested compounds at the
indicated final concentrations were added to the culture medium
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