Synthesis and antitumor activities evaluation of m-(4-morpholinoquinazolin-2-yl)benzamides in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2015.04.037

In the present study, a series of m-(4-morpholinoquinazolin-2-yl)benzamides were designed, synthesized and characterized. The antiproliferative activities of the synthesized compounds were evaluated against two human cell lines (HCT-116 and MCF-7). Compounds with IC₅₀ values below 4 μM were further evaluated against U-87 MG and A549 cell lines. Among these evaluated compounds, compound T10 displayed a remarkable antiproliferative effect in vitro. The hoechst staining assay showed that compound T10 caused morphological changes. The cell cycle and apoptosis assay further indicated that compound T10 can arrest HCT-116 cells in G2/M and G0/G1 phase and induce apoptosis. PI3K enzyme assays indicated that compounds T7 and T10 selectively inhibit PI3K±. A Western bolt assay further suggested that compound T10 can block the PI3K/Akt/mTOR pathway. Moreover, compound T10 inhibited tumor growth on a mice S180 homograft model. These findings directly identify m-(4-morpholinoquinazolin-2-yl)benzamide derivatives as novel anticancer agents.

Full text of DOI:10.1016/j.ejmech.2015.04.037

European Journal of Medicinal Chemistry 96 (2015) 382e395
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activities evaluation of m-(4-
morpholinoquinazolin-2-yl)benzamides in vitro and in vivo
Xiao-Meng Wang a, Min-Hang Xin a, Jing Xu b, Bo-Rui Kang a, Yan Li a, She-Min Lu b,
,
*
San-Qi Zhang a
a Department of Medicinal Chemistry, School of Pharmacy, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, PR China
b Department of Genetics and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an Shaanxi 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a series of m-(4-morpholinoquinazolin-2-yl)benzamides were designed, synthe-
sized and characterized. The antiproliferative activities of the synthesized compounds were evaluated
Received 16 October 2014
Received in revised form
24 March 2015
Accepted 4 April 2015
Available online 17 April 2015
against two human cell lines (HCT-116 and MCF-7). Compounds with IC50 values below 4
mM were
further evaluated against U-87 MG and A549 cell lines. Among these evaluated compounds, compound
T10 displayed a remarkable antiproliferative effect in vitro. The hoechst staining assay showed that
compound T10 caused morphological changes. The cell cycle and apoptosis assay further indicated that
compound T10 can arrest HCT-116 cells in G2/M and G0/G1 phase and induce apoptosis. PI3K enzyme
Keywords:
Benzamide
assays indicated that compounds T7 and T10 selectively inhibit PI3Ka. A Western bolt assay further
suggested that compound T10 can block the PI3K/Akt/mTOR pathway. Moreover, compound T10
inhibited tumor growth on a mice S180 homograft model. These findings directly identify m-(4-
morpholinoquinazolin-2-yl)benzamide derivatives as novel anticancer agents.
© 2015 Elsevier Masson SAS. All rights reserved.
Quinazoline
Synthesis
Antitumor agents
PI3K inhibitor
1. Introduction
target of rapamycin) is an important signaling pathway in that it is
crucial to diverse cell processes, including cell growth, prolifera-
Among heterocyclic systems, the quinazoline ring system has
been regarded as a privileged drug scaffold in drug discovery [1].
Quinazoline derivatives exhibit a broad spectrum of biological ac-
tivities, such as antitumor, anti-inflammatory, antimicrobial and
antimalarial activities [2e6]. Moreover, the kinase inhibitors gifi-
tinib, erlotinib and lapatinib that contain a quinazoline core have
been approved for the treatment of cancer [7,8]. Recently, AZD8931,
a 4,6,7-trisubsituted quinazoline, has been reported as a reversible
inhibitor of the EGFR, HER2 and HER3 receptors and thus is a
promising drug candidate [9]. A series of novel 4-alkoxyquinazoline
derivatives have been reported as potential inhibitors of VEGFR2
tion, survival, metabolism and autophagy [13e15]. The aberrant
activation of PI3K pathway, commonly due to either mutation in
PIK3CA gene or loss regulation of PTEN, is closely linked to the
development and progression of a wide range of cancers [16,17].
Besides, mTOR, a key node of the PI3K pathway is found to be
frequently hyperactivated in human tumors [18]. Therefore, PI3K
(especially PI3Ka) and mTOR have emerged as attractive targets for
cancer therapy. In recent years, several PI3K inhibitors, such as
BKM120 [19], GDC0941 [20], ZSTK474 [21] and NVP-BYL719 [22],
have been developed into clinical trials. Additionally, PI3K and
mTOR dual inhibitors, such as BEZ235 [23], GSK2126458 [24],
GDC0980 [25], PF-04979064 [26] and PKI-587 [27], are also eval-
uated in clinical trials for their therapeutic potentials. Moreover,
the pharmacophore of the PI3K inhibitors has been recently put
forward as well [28].
[10]. Meanwhile, aminoquinazoline derivatives are described as
V600E
potent mutant B-Raf
selective inhibitors [11]. 2-Amino-3-
substituted quinazolinone derivatives have also been identified as
selective inhibitors of PI4KIIIa, which can inhibit HCV replication
in vitro [12].
Anticancer candidates BKM120, VS-5584 [29] and GDC0980
possess the similar pharmacophore. The aminopyridinyl group in
BKM120, aminopyrimidinyl group in VS-5584 and GDC-0980, and
the morpholine in three compounds could form interaction with
PI3K. As a PI3K inhibitor, BKM120 exhibited inhibitory activity
PI3K (phosphatidylinositol-3 kinase)/Akt/mTOR (mammalian
* Corresponding author.
E-mail address: sqzhang@xjtu.edu.cn (S.-Q. Zhang).
against PI3Ka with an IC50 value of 0.03 mM. The co-crystal
http://dx.doi.org/10.1016/j.ejmech.2015.04.037
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
383
structure of BKM120 in PI3K
g
(PDB code 3SD5) indicates that the
The debenzylation of compound T21 produced the key inter-
mediate 5. In the presence of potassium carbonate, the nucleophilic
substitution of intermediate 5 with substituted alkyl halide affor-
ded intermediate 6 or target compounds T22eT26. The ester group
in intermediate 6 was hydrolyzed to yield the carboxylic acid 7,
which was subsequently condensed with the corresponding
amines to produce target compounds T27eT29 (Scheme 2).
Recently, the 2-(2-aminopyrimidin-5-yl)-4-morpholinylpyrid-
opyrimidine has been reported as a novel PI3K/mTOR dual Inhibi-
tor [36]. To compare the antiproliferative effects of our designed
compounds with the analog with an 2-aminopyrimidinyl moiety,
compound T11 was prepared from commercially available 5-
bromopyrimidin-2-amine by Suzuki coupling. The synthetic route
for compound T11 is depicted in Scheme 3.
oxygen atom of a morpholine ring is bound to the hinge at Val882
and the hydrogen atoms from the aminopyridinyl group interact
with Asp836 and Asp841 via hydrogen bonds [19]. PI-103 (Fig. 1) is
described as a multi-targeted PI3K inhibitor. The crystal structure of
the human PI3Ka with PI-103 complex indicates that a substituted
group at the m-position of the phenol hydroxy of PI-103 may
improve its binding affinity by forming a new H-bond [30]. Early, 2-
(3-hydroxyphenyl)-4-morpholinoquinazoline and related de-
rivatives were reported as PI3K
Recently, based upon the docking model of GSK2126458 with
PI3K [24], we proposed that the water molecule bridge could be
a inhibitors [31,32].
g
replaced by the structure of an amide group. Thereupon, we syn-
thesized a series of 2-substituted-3- phenylsulfonylamino-5-(qui-
nazolin-6-yl or quinolin-6-yl)benzamides and discovered that the
designed compounds are novel PI3K inhibitors and anticancer
agents [33]. Thereafter, we combined the benzamide moiety with
2-aminobenzothiazole to discover novel anticancer agents [34].
There are two hydrogen bond donors and one hydrogen bond re-
ceptor in the structure of benzamide, so the enzymatic inhibitor
with a benzamide moiety is east to form interaction with receptor.
In this study, we combined benzamide moiety with quinazoline
scaffold to design, synthesize and evaluate a series of m-(4-
morpholinoquinazolin-2-yl)benzamides as antitumor agents
(Fig. 2).
2.2. Biological evaluations
2.2.1. Antiproliferative activities in vitro
The newly synthesized compounds T1eT29 were evaluated by
applying the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) colorimetric assay for their antiproliferative
activities against colon carcinoma cell line (HCT-116) and breast
adenocarcinoma carcinoma cell line (MCF-7). The PI3K inhibitor
GDC-0941 was used as the positive control. The results expressed as
IC50 values are summarized in Table 1.
As we expected, all the designed compounds displayed potent
antiproliferative activities against the two cancer cell lines.
Generally, most of the title compounds were more potent against
HCT-116 than they were against MCF-7. Compound T1 exhibited
2. Results and discussion
2.1. Synthesis
potent antiproliferative effects against HCT-116 (IC50 ¼ 0.90
mM)
The synthetic route for the title compounds T1eT10 and
T12eT21 is outlined in Scheme 1.
and MCF-7 (IC50 ¼ 2.14 M). The preliminary results indicate that
m
our design idea is reasonable. The fact that compounds T2 and T4
were more potent than compound T1 against HCT-116 and HCF-7
indicates that the title compound with a methoxy at either R2 or
R3 may enhance the antiproliferative activity. Moreover, com-
pounds T5 and T7 with a substituent at R3 displayed more potency
than compounds T6 and T8 with a substituent at R2, which in-
dicates that a substituent at R3 position (m-position of amide
group) may improve antiproliferative activity. Compounds T5 and
T6 with a benzyloxy gave rise to a decreased potency in comparison
with compounds T4 and T2, which suggests that a large substituent
such as benzyloxy at o-position or m-position of the amide group is
disadvantageous for antiproliferative activity. In addition, com-
pound T9 with a trifluoromethyl at R3 showed a decreased activity
compared with compound T4 as well. However, compound T10
2,4-Dichloro-6,7-dialkyloxyquinazoline (1) was used as starting
material. The reaction of 1 with morpholine produced the key in-
termediate 2-chloro-4- (4-morpholinyl)-6,7-dialkyloxyquinazoline
(2). As we previously reported, catalyzed by PdCl2(dppf), the re-
action of bis(pinacolato)diboron with substituted bromobenzene 3
yielded phenylboronic ester 4, which was used for the subsequent
Suzuki coupling to produce target compounds T1eT6, T9eT10, T12
and T14eT21 [35]. The preparation of phenylboronic ester and
Suzuki coupling was performed in one pot. Compounds T7 and T8
with a hydroxy group at either R5 or R4 were obtained by deben-
zylation of compounds T5 and T6 with the aid of hydrogenolysis. In
addition, the ester group in compound T12 was hydrolyzed to
prepare compound T13.
Fig. 1. The structure of several PI3K inhibitors.

384
X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
Fig. 2. The design strategy of m-(4-morpholinoquinazolin-2-yl)benzamides.
Scheme 1. Synthesis of compounds T1eT8, T10eT21. Reagents and conditions: (a) morpholine, Et3N, THF, 0 ꢀC, 0.5 h, rt, 2 h, 55.8e70.5%; (b) bis(pinacolato)diboron, AcOK,
PdCl2(dppf), 1,4-dioxane, reflux, 2 h; (c) 2, Na2CO3, PdCl2(dppf), DME/H2O (4:1), reflux, 2 h, 43.5e83.2%; (c) Pd/C, HCO2NH4, DMF-H2O (2:1), 100 ꢀC, 6 h, 81.8e84.7%; (d) (c) 2 M
NaOH, MeOHeH2O (4:1), 4 h, 85.4%.
with a trifluoromethoxyl at R3 exhibited an increased potency in
comparison with compound T4 against the two cancer cell lines.
Therefore, a hydrogen-bond receptor at R3 may help to improve the
antiproliferative effect of title compounds.
Compound T11 with a 2-aminopyrimidinyl moiety exhibited
potent antiproliferative activity against two cancer cell lines, but its
activity was slightly weaker than compound T10. The latter
exhibited an excellent antiproliferative activity against HCT-116
(IC50 ¼ 0.30
similar to the positive control GDC0941 (IC50 ¼ 0.37
HCT-116; IC50 ¼ 1.09 M against MCF-7). The above results suggest
that a trifluoromethoxy at R3 is the most favorable substituent for
mM) and MCF-7 (IC50 ¼ 1.09
m
M), which is almost
mM against
m
Scheme 2. Synthesis of compounds T22eT27. Reagents and conditions: (a) Pd/C, HCO2NH4, DMFeH2O (2:1), 100 ꢀC, 6 h, 84.5%; (b) RX, K2CO3, KI, DMF, 90 ꢀC, 5e8 h, 63.1e85.2%; (c)
2 M NaOH, MeOHeH2O (4:1), 4 h, 71.2%; (d) R1H, EDCI, HOBt, DMF, 8 h, 49.5e75.4%.

X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
385
Table 2
Antiproliferative activities of compounds T19eT29 (x s, n ¼ 3).
Scheme 3. Synthesis of compound T11. Reagents and conditions: (a) bis(pinacolato)
diboron, AcOK, PdCl2(dppf), 1,4-dioxane, reflux,
PdCl2(dppf), DME/H2O (4: 1), reflux, 2 h, 67.8%.
2 h; (b) intermediate 2, Na2CO3,
Compds
R1
R2
IC50 (mM)
HCT-116
MCF-7
benzamide moiety.
T4
CH3
CH3
CH3
CH2CH2OCH3
Bn
CH2CH2OCH3
0.45 0.15
0.89 0.11
9.04 0.16
6.45 1.67
2.70 0.56
3.14 0.52
1.75 0.32
5.19 1.32
7.52 2.51
10.27 2.75
>20
To probe the structure-activity relationship (SAR) of title com-
pounds, the amide group in T1 was replaced by ester, carboxyl, N-
methylamide, N, N-dimethylamide or acetylamino group to obtain
compounds T12eT16. As we expected, the antiproliferative effects
of compounds T12eT16 were not improved in comparison with
compound T1. Thereafter, Moving the amide or acetamino sub-
stituent from R1 position to R2 position obtained compounds T17
and T18, which displayed a decline in activity. These results sup-
ported our design idea that the formamide moiety at m-position is
important to the maintenance of the antiproliferative activities.
Therefore, we argue that the benzamide moiety is part of phar-
macophore in the structure of title compounds.
To further improve the solubility and antiproliferative potency
of the title compounds, we replaced the methoxy at 6- or 7-
positions of the quinazoline ring with the substituents bearing
more saturated carbon atoms (Table 2). Table 2 shows that only
compound T26 displayed increased activities against MCF-7
T19
T20
T21
T22
T23
CH2CH2OCH3
CH2CH2OCH3
CH3
CH3
CH3
8.52 2.42
T24
T25
CH3
CH3
4.72 0.49
1.28 0.29
5.66 2.41
6.38 0.98
T26
T27
CH3
CH3
0.81 0.33
6.48 0.47
1.01 0.30
24.38 7.13
(IC50 ¼ 1.01
mM), which is similar to T4 and GDC0941 whereas
other compounds exhibited decreased potency in comparison with
T28
CH3
CH3
9.44 0.67
18.74 5.14
T29
21.68 7.35
0.37 0.06
14.90 2.24
1.09 0.15
Table 1
Antiproliferative activities of compounds T1eT18 (x s, n ¼ 3).
GDC0941
compound T4. Therefore, the methoxy at 6- or 7-positions of the
quinazoline ring is
proliferative activity.
a
suitable group for maintaining anti-
The compounds with IC50 values below 4
m
M against both HCT-
116 and MCF-7 were further evaluated against glioma cell line (U-
87 MG) and lung adenocarcinoma epithelial cell line (A549). As
shown in Table 3, all the compounds displayed more potency
against U-87 MG than against A549. Moreover, compound T10
exhibited excellent antiproliferative activity against both U-87 MG
Compds
R1
R2
R3
IC50 (mM)
HCT-116
MCF-7
(IC50 ¼ 0.60
m
M) and A549 (IC50 ¼ 1.68
mM). The results further
T1
T2
T3
T4
T5
T6
T7
T8
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
H
OCH3
Cl
H
H
OBn
H
OH
H
H
H
H
OCH3
OBn
H
OH
H
0.90 0.15
0.47 0.15
2.97 0.58
0.45 0.06
3.83 0.35
5.03 0.49
0.88 0.23
2.77 0.46
0.71 0.09
0.30 0.06
0.42 0.06
1.07 0.16
12.50 2.69
2.52 1.04
4.73 0.68
1.42 0.32
1.25 0.06
1.09 0.09
0.37 0.06
2.14 0.21
1.22 0.21
4.24 0.72
1.75 0.32
6.93 0.75
7.90 0.40
2.64 0.46
7.42 1.51
2.03 0.20
1.09 0.25
1.38 0.39
3.44 0.76
>20
6.20 0.63
5.01 0.12
3.49 0.57
4.60 0.95
3.61 0.84
1.09 0.15
verify that a trifluoromethoxy at the m-position of amide group is
Table 3
Antiproliferative activity of chosen compounds (x s, n ¼ 3).
Compds
IC50 (mM)
T9
CF3
OCF3
U-87 MG
A549
T10
T11
T12
T13
T14
T15
T16
T17
T18
GDC0941
H
T1
T2
T4
T7
1.57 0.05
1.90 0.17
1.01 0.21
1.84 0.48
1.19 0.15
0.60 0.08
5.00 1.35
2.45 0.22
1.24 0.22
26.10 1.80
9.63 0.33
9.22 0.55
17.18 2.45
4.75 1.28
1.68 0.29
3.21 0.17
9.37 0.41
2.28 0.05
CO2CH3
CO2H
CONHCH3
CON(CH3)2
NHAc
H
H
H
H
H
H
H
H
H
H
H
H
T9
H
T10
T11
T26
GDC0941
CONH2
NHAc
H

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X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
advantageous for antiproliferative activity.
From the data in Tables 1e3, we can conclude that the benza-
mide moiety is important to the maintenance of the anti-
Fig. 5, the percentages of apoptotic cells are 22.70% and 27.34% at
M and 3 M for compound T10, respectively, which is higher
than control group. The results indicated that compound T10 can
1
m
m
proliferative activity of title compounds. What's more,
a
induce the apoptosis of HCT-116 cells.
trifluoromethoxy or methoxy or hydroxyl group at m-position of
amide group and methoxy at 6- and 7-positions of the quinazoline
ring are favorable for antiproliferative activity. On account of the
antiproliferative activities of compound T10 are close to those of
GDC0941, compound T10 was selected for further investigation.
2.2.5. PI3K enzymatic activity assay
To elucidate the mechanism of antiproliferative activity of title
compounds, T7 and T10 were chosen to measure inhibitory enzy-
matic activity against PI3Ks in vitro. An ATP depletion assay was
performed preliminarily with GDC0941 as positive control [19]. The
results presented as IC50 are summarized in Table 4. As the data in
2.2.2. The effect of compound T10 on cellular morphology
In the present study, the effect of compound T10 on cellular
morphological changes was also investigated. HCT-116 cells were
selected and treated with compound T10 at the concentrations of
Table 4, compound T7 could selectively inhibit PI3K
a and T10 could
inhibit both PI3K and PI3K , but both T7 and T10 displayed
a
b
weaker activity than that of GDC0941. These results suggested that
compounds T7 and T10 are potential PI3K inhibitors.
0.1 mM, 1 mM and 10 mM for 24 h. The cells were first examined
directly and then photographed under a phase contrast microscope
followed by stained with Hoechst 33258. As shown in Fig. 3A, the
cellular morphological changes such as cell shrinking, vacuolization
It was reported that 2-(3-hydroxyphenyl)-4-morpholin-
oquinazoline is a PI3Ka inhibitor, but its activity against A375 cell
was weak [31]. Generally, the compound with a hydroxyl at phenyl
and fragmentation were observed at 1
mM and 10
m
M. The images in
ring is a weak spot in metabolism. Compound T7 with a phenolic
Fig. 3B indicated that compound T10 induced chromatin conden-
sation and nuclear fragmentation. These results suggested that
compound T10 could significantly inhibit cell growth and induce
apoptosis accompanying cellular morphological changes.
hydroxyl group exhibited higher activity than T10 against PI3Ka,
but displayed weaker activity than T10 in cell-based activity. The
change may relate to the difference of cell penetration ability be-
tween T7 and T10. In this work, the hydroxyl at phenyl ring was
successfully replaced by the carboxamide moiety, which is a sig-
nificant structural optimization.
2.2.3. The effect of compound T10 on cell cycle
To further understand the mechanism of the antiproliferative
activity of compound T10, the effect of compound T10 on cell cycle
2.2.6. The effect on PI3K/Akt/mTOR pathway
distribution was studied. HCT-116 cells were treated with 3
m
M of
To further verify the mechanism of the antiproliferative activity
of compound T10, Western blot assay was employed to evaluate the
blocking activity of compound T10 against PI3K/Akt/mTOR
pathway. HCT-116 cells were treated with compound T10 and
compound T10 for 24 h and then stained with propidium iodide
(PI). DNA contents were measured by flow cytometry and the re-
sults were shown in Fig. 4. The control group treated with DMSO
had approximately 48.31% in the G0/G1 phase, 38.71% in the S
phase and 12.98% in the G2/M phase. After treated with compound
T10, the cells in the G0/G1 and G2/M phase increased to 56.69% and
23.19%, respectively and the cells in the S phase decreased to 20.11%.
These results indicated that compound T10 can induce a cell arrest
at G2/M and G0/G1 phase.
GDC0941, respectively, at a concentration of 10 mM. Meanwhile, the
control group was treated with vehicle (0.5% DMSO), whose results
are shown in Fig. 6. Compared with the control, both compound
T10 and GDC0941 could reduce the amount of phosphorylated Akt
at Ser-473 without affecting Akt as a whole, but the inhibitory ac-
tivity of compound T10 was weaker than that of GDC0941. All this
suggested that compound T10 can block the PI3K/Akt/mTOR
pathway, and thus might be a potential PI3K inhibitor.
2.2.4. The effect of compound T10 on apoptosis
To characterize whether compound T10 induces cell apoptosis,
the annexin-V/propidium iodide (PI) biparametric cytofluorimetric
assay was performed on HCT-116 cells. The cells were treated with
2.2.7. Anticancer effect on the mice S180 homograft models in vivo
Last, we tested the anticancer effect of compound T10 on the
homograft mice models established for this study. Mice bearing
DMSO and compound T10 at 1 mM and 3 mM for 24 h. As shown in
Fig. 3. The effect of compound T10 on cellular morphology and apoptosis. The HCT-116 cells were treated with compound T10 at 0.1, 1 and 10 mM. (A) The phase-contrast
microscopic view. The yellow arrows indicate the morphological changes of HCT-116. Scale bar ¼ 1.0 mm. (B) The fluorescence microscopic view. The yellow arrows indicate
apoptotic cells. Scale bar ¼ 1.0 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
387
Fig. 4. Effect of compound T10 on the cell cycle distribution of HCT-116 cells. (A) HCT-116 cells were treated with DMSO or 3
mM of compound T10 for 24 h. Result presented is an
average value of three independent experiments. (B) Quantitative analysis of cell cycle distribution of HCT-116 cells by flow cytometry. Data are the means SD of values from three
independent experiments (*p < 0.05).
Fig. 5. The effect of compound T10 on apoptosis. (A) After treated with DMSO, 1 and 3 mM of compound T10 for 24 h, the HCT-116 cells were harvested, stained with Annexin V and
propidium iodide (PI) and analyzed by flow cytometry. The upper left quadrant Q1 represented necrotic cells, the upper right quadrant Q2 represented for late apoptotic cells, the
low left quadrant Q3 represented live cells and the low right quadrant Q4 represented for early apoptotic cells. Result presented is a representative of three independent ex-
periments. (B) The apoptosis rate was the sum of Q2 þ Q4. Data are the means SD of values from three independent experiments (**p < 0.01).

388
X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
Table 4
Enzymatic activity of T7 and T10 (IC50, nM, n ¼ 2).
Kinase
T7
T10
GDC0941
PI3K
PI3K
PI3K
PI3K
a
b
g
d
53
96
128
465
330
18
108
117
163
454
394
260
sarcoma (S180) were treated orally with compound T10 at 15 mg/
kg or 30 mg/kg once a day for 8 days. As it was difficult to measure
the volume of S180 tumor, tumor weights were used as evaluating
indicators illustrated in Fig. 7. The inhibitory ratios of compound
T10 at 15 mg/kg and 30 mg/kg were 49.3% and 64.2%, respectively.
Additionally, it was found that compound T10 had hardly any effect
on the body weights of the tested mice. These results suggested
that T10 can significantly inhibit tumor growth in vivo and be an
effective anticancer agent.
Fig. 7. The antitumor effect of compound T10 on S180 homograft model. Mice bearing
subcutaneous tumors were orally administered vehicle, compound T10 (15 mg/kg or
30 mg/kg doses) once daily for 8 days. **P < 0.01.
3 mM. The PI3K enzymatic activity assay and Western blot assay
suggested that compound T10 can block the PI3K/AKT/mTOR
pathway. Furthermore, compound T10 can suppress tumor growth
on the mice bearing the sarcoma S180 model. These findings
strongly support our assumptions that the title compounds are
novel anticancer agents.
2.3. Molecular docking studies
To explore the binding mode of T7 and T10 with PI3K
DOCKER program within Discovery Studio 2.5 software package
was utilized to dock T7 and T10 with human PI3K (PDB code 3R7R)
[37] and the results were depicted in Fig. 8. By the analysis of the
binding mode of T7 and T10 with PI3K , we observed that the
g, the C-
g
4. Experimental protocols
g
4.1. Chemistry and chemical methods
oxygen in morpholine ring formed a hydrogen bond with Val882,
one hydrogen atom in amide moiety interacted with Ala 885 and
the 7-methoxy of quinazoline formed a hydrogen bond with
Lys833, which further verified that the amide group at phenyl ring
and 7-methoxy are suitable groups to maintain the activity. In
addition, the hydroxyl of T7 can form a hydrogen bond with Thr887,
while one fluorine atom in T10 formed a hydrogen bond with
Lys890.
Unless specified otherwise, all the starting materials, reagents
and solvents were commercially available. All the reactions were
monitored by thin-layer chromatography on silica gel plates (GF-
254) and visualized with UV light. All the melting points were
determined on a micro melting-point apparatus (Beijing) and
thermometer was uncorrected. NMR spectra were recorded on a
400 Bruker NMR spectrometer with tetramethylsilane (TMS) as an
internal reference. All the chemical shifts are reported in parts per
million (ppm). Mass spectra were recorded on a triple quadrupole
LC/MS/MS (API 3200, AB SCIEX). HRMS measurements were per-
formed using electrospray ionization (positive mode) on a quad-
rupole time-of-flight (QTOF) mass spectrometer (Maxis Q-TOF,
Bruker Inc.).
3. Conclusions
In the present study, title compounds were designed, synthe-
sized and characterized and their antiproliferative activities against
HCT-116 and MCF-7 cancer cell lines were evaluated. Most of the
compounds exhibited potent antiproliferative activity. The SAR of
the title compounds reveals that the benzamide moiety is impor-
tant to the maintenance of the antiproliferative activity of the title
compounds. Besides, a trifluoromethoxy at m-position of the amide
group and methoxy at 6- and 7-positions of the quinazoline ring are
also favorable substituents. Compound T10 displayed a remarkable
4.1.1. General procedure for the synthesis of compound 2
To the mixture of 2,4-dichloro-6,7-dialkyloxyquinazoline 1
(10 mmol) and DIPEA (3.49 mL, 20 mmol) in 20 mL of THF was
added morpholine (0.88 mL, 10 mmol) dropwise at 0 ꢀC. The
mixture was stirred at 0 ꢀC for 0.5 h and then stirred at room
temperature for 2 h. The solvent was evaporated to give a residue.
To the residue was added the mixture of ethanol and water (40 mL,
1:1) and stirred at room temperature for 0.5 h. The precipitate was
filtered, washed by ethanol and water and dried to afford com-
pound 2.
antiproliferative activity, and can induce apoptosis at 1
mM and
4.1.1.1. 4-(2-Chloro-6,7-dimethoxyquinazolin-4-yl)morpholine (2a).
Pale yellow solid; Yield 70.5%; mp 213e214 ꢀC; 1H NMR (CDCl3):
d
7.25 (s,1H, AreH), 7.06 (s,1H, AreH), 7.58 (d, J ¼ 8.8 Hz,1H, AreH),
4.02 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 3.91 (m, 4H, CH2 ꢁ 2), 3.79
(m, 4H, CH2 ꢁ 2).
4.1.1.2. 4-(2-Chloro-6,7-Bis(2-methoxyethoxy)quinazolin-4-yl)mor-
pholine (2b). White solid; Yield 55.8%; mp 94e95 ꢀC; 1H NMR
Fig. 6. The effect of compound T10 on the expression of PI3K/AKT/mTOR pathway of
HCT-116 cells at 10 M. (A) Effect of compounds T10 and GDC0941 on the expression of
p-AKT at Ser-473 and total AKT. (B) The quantified effect of compounds T10 and
m
(CDCl3):
d
7.23 (s, 1H, AreH), 7.19 (s, 1H, AreH), 7.58 (d, J ¼ 8.8 Hz,
1H, AreH), 4.27 (m, 4H, CH2 ꢁ 2), 3.91 (m, 8H, CH2 ꢁ 4), 3.76 (m, 4H,
CH2 ꢁ 2), 3.48 (d, J ¼ 1.6 Hz, 6H, CH3 ꢁ 2). MS (ESI, m/z): 400.1
[M þ H]þ.
GDC0941 on the expression of p-AKT at Ser-473 and total AKT. Each experiment was in
triplicate and independently performed. The results were shown as means
SD.
**P < 0.01.

X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
389
Fig. 8. Docking model of compounds T7 and T10 with PI3K
g
. A: The docking mode of T7 with PI3K
g
; B: The docking mode of T10 with PI3K
g
; Selected residues Lys833, Val882, Ala
885, Thr887 and Lys890 are shown. Green dashed lines indicate hydrogen bond. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
4.1.1.3. 4-(2-Chloro-7-methoxy-6-(2-methoxyethoxy)quinazolin-4-
J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz, 1H, AreH), 7.71 (s, 1H, CONH), 7.61 (s, 1H,
CONH), 7.35 (s, 1H, AreH), 7.26 (d, J ¼ 8.8 Hz, 1H, AreH), 7.17 (s, 1H,
AreH), 3.97 (s, 3H, OCH3), 3.94 (s, 3H, OCH3) 3.93 (s, 3H, OCH3), 3.87
yl)morpholine (2c). Off-white solid; Yield 62.9%; mp 112e114 ꢀC; 1H
NMR (CDCl3):
d 7.22 (s, 1H, AreH), 7.20 (s, 1H, AreH), 7.58 (d,
J ¼ 8.8 Hz, 1H, AreH), 4.28 (m, 2H, CH2), 4.00 (s, 3H, OCH3), 3.90 (m,
4H, CH2 ꢁ 2), 3.86 (m, 2H, CH2), 3.77 (m, 4H, CH2 ꢁ 2), 3.48 (s, 3H,
OCH3). MS (ESI, m/z): 354.1 [M þ H]þ.
(m, 4H, CH2 ꢁ 2), 3.70 (s, 4H, CH2 ꢁ 2). 13C NMR (DMSO-d6):
d 166.8
(Cq), 163.8 (Cq), 159.1 (Cq), 156.9 (Cq), 154.9 (Cq), 149.7 (Cq), 148.4
(Cq), 132.1 (CH), 131.1 (Cq), 130.8 (Cq), 123.2 (CH), 112.3 (CH), 109.5
(Cq), 108.1 (CH), 103.8 (CH), 66.5 (CH2 ꢁ 2), 56.5 (CH3), 56.4 (CH3),
56.1 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z): 425.1 [M þ H]þ. ESI-HRMS
m/z: calc'd for C22H24N4O5 [M þ H]þ: 425.1825; found 425.1822.
4.1.1.4. 4-(7-(Benzyloxy)-2-Chloro-6-methoxyquinazolin-4-yl)mor-
pholine (2d). White solid; Yield 57.4%; mp 168e170 ꢀC; 1H NMR
(DMSO-d6):
d
7.50 (d, J ¼ 7.2 Hz, 2H, AreH ꢁ 2), 7.43 (m, 2H,
AreH ꢁ 2), 7.37 (m, 1H, AreH), 7.30 (s, 1H, AreH), 7.17 (s, 1H, AreH),
5.29 (s, 2H, OCH2), 3.92 (s, 3H, OCH3), 3.76 (m, 8H, CH2 ꢁ 4). MS (ESI,
m/z): 386.1 (M þ H)þ.
4.1.2.3. 2-Chloro-5-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)
benzamide (T3). White solid; Yield 56.0%; mp > 250 ꢀC; 1H NMR
(DMSO-d6):
d
8.49 (s, 1H, AreH), 8.47 (d, J ¼ 8.4 Hz, 1H, AreH), 8.03
(s, 1H, CONH), 7.70 (s, 1H, CONH), 7.62 (d, J ¼ 8.4 Hz, 1H, AreH), 7.36
(s, 1H, AreH), 7.18 (s, 1H, AreH), 3.97 (s, 3H, OCH3), 3.94 (s, 3H,
OCH3), 3.86 (d, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2), 3.72 (d, J ¼ 4.0 Hz, 4H,
4.1.2. General procedure for the synthesis of compounds T1eT6,
T9eT10, T12 and T14eT21
CH2 ꢁ 2). 13C NMR (DMSO-d6):
d 168.7 (Cq), 163.8 (Cq), 155.9 (Cq),
A mixture of substituted 3-(or 4-) bromobenzamides or 3-
bromobenzoate or N-(3-(or 4-)bromophenyl)acetamide 3,
(0.5 mmol), bis(pinacolato)diboron (0.14 g, 0.55 mmol), potassium
acetate (0.15 g, 1.5 mmol), PdCl2(dppf) (0.03 g, 0.04 mmol) and 1,4-
dioxane (10 mL) was refluxed for 2 h under nitrogen atmosphere.
The solvent was evaporated under reduced pressure to afford a
dark brown residue. To the residue was added intermediate 2
(0.4 mmol), sodium carbonate (0.16 g, 1.5 mmol), PdCl2(dppf)
(0.03 g, 0.04 mmol), 1,2-dimethoxyethane (8 mL) and water (2 mL)
and the mixture was refluxed for 2 h under nitrogen atmosphere.
The mixture was evaporated under reduced pressure and purified
by silica gel column chromatography using chloroform/
methanol ¼ 30: 1 as the eluent to afford the compounds T1eT6,
T9eT10, T12 and T14eT21 as an off-white or a white solid.
155.0 (Cq), 149.5 (Cq), 148.8 (Cq), 137.7 (Cq), 137.5 (Cq), 131.6 (CH),
130.2 (CH), 129.7 (CH), 127.9 (Cq), 109.8 (Cq), 108.1 (CH), 103.8 (CH),
66.4 (CH2 ꢁ 2), 56.4 (CH3), 56.2 (CH3), 50.2 (CH2 ꢁ 2). MS (ESI, m/z):
411.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H22ClN4O4 [M þ H]þ:
429.1330; found 429.1324.
4.1.2.4. 3-methoxy-5-(6,7-dimethoxy-4-morpholinoquinazolin-2-yl)
benzamide (T4). White solid; Yield 60.3%; mp 238e240 ꢀC; 1H NMR
(DMSO-d6):
d
8.49 (s, 1H, AreH), 8.47 (d, J ¼ 8.4 Hz, 1H, AreH), 8.03
(s, 1H, CONH), 7.70 (s, 1H, CONH), 7.62 (d, J ¼ 8.4 Hz, 1H, AreH), 7.36
(s, 1H, AreH), 7.18 (s, 1H, AreH), 3.97 (s, 3H, OCH3), 3.94 (s, 3H,
OCH3), 3.86 (d, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2), 3.72 (d, J ¼ 4.0 Hz, 4H,
CH2 ꢁ 2). 13C NMR (DMSO-d6):
d 168.3 (Cq), 163.8 (Cq), 159.7 (Cq),
156.8 (Cq), 155.0 (Cq), 149.6 (Cq), 148.8 (Cq), 140.3 (Cq), 136.4 (Cq),
120.0 (CH), 116.2 (CH), 114.5 (CH), 109.8 (Cq), 108.1 (CH), 103.8 (CH),
66.5 (CH2 ꢁ 2), 56.4 (CH3), 56.1 (CH3), 55.9 (CH3), 50.3 (CH2 ꢁ 2). MS
(ESI, m/z): 429.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H24N4O5
[M þ H]þ: 425.1825; found 425.1819.
4.1.2.1. 3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)benzamide
(T1). White solid; Yield 55.3%; mp 244e246 ꢀC; 1H NMR (DMSO-
d6):
d
8.95 (s, 1H, AreH), 8.59 (d, J ¼ 7.6 Hz, 1H, AreH), 8.12 (s, 1H,
CONH), 7.97 (d, J ¼ 7.6 Hz,1H, AreH), 7.58 (m, 1H, AreH), 7.46 (s,1H,
CONH), 7.38 (s, 1H, AreH), 7.20 (s, 1H, AreH), 3.98 (s, 3H, OCH3),
3.95 (s, 3H, OCH3), 3.86 (m, 4H, CH2 ꢁ 2), 3.74 (s, 4H, CH2 ꢁ 2). 13
C
4.1.2.5. 3-(benzyloxy)-5-(6,7-dimethoxy-4-morpholinoquinazolin-2-
yl)benzamide (T5). White solid; Yield 77.4%; mp 165e166 ꢀC; 1H
NMR (DMSO-d6):
d 168.5 (Cq), 163.8 (Cq), 157.0 (Cq), 155.0 (Cq),
149.6 (Cq), 148.7 (Cq) 138.9 (Cq), 135.1 (Cq), 130.7 (CH), 129.1 (CH),
128.7 (CH), 127.5 (CH), 109.8 (Cq), 108.0 (CH), 103.8 (CH), 66.5
(CH2 ꢁ 2), 56.4 (CH3), 56.1 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z):
395.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C21H23N4O4 [M þ H]þ:
395.1719; found 395.1714.
NMR (DMSO-d6): d 8.50 (s, 1H, AreH), 8.18 (s, 1H, CONH), 8.11 (s,1H,
CONH), 7.64 (m, 1H, AreH), 7.53 (d, J ¼ 7.2 Hz, 2H, AreH, CONH),
7.44 (m, 3H, AreH ꢁ 3), 7.37 (m, 2H, AreH ꢁ 2), 7.18 (s, 1H, AreH),
5.29 (s, 2H, OCH2), 3.97 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.86 (d,
J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.71 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2). 13C NMR
(DMSO-d6):
d 168.2 (Cq), 163.7 (Cq), 158.8 (Cq), 156.7 (Cq), 155.0
4.1.2.2. 5-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-2-
(Cq), 149.6 (Cq), 148.8 (Cq), 140.3 (Cq), 137.4 (Cq), 136.4 (Cq), 129.0
(CH ꢁ 2), 128.4 (CH), 128.2 (CH ꢁ 2), 120.2 (CH), 117.0 (CH), 115.5
(CH), 109.8 (Cq), 108.1 (CH), 103.8 (CH2), 70.0(CH2), 65.5 (CH2 ꢁ 2),
methoxybenzamide (T2). White solid; Yield 73.3%; mp 223e225 ꢀC;
1H NMR (DMSO-d6):
d
8.92 (d, J ¼ 2.4 Hz, 1H, AreH), 8.54 (dd,

390
X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
56.4 (CH3), 56.1 (CH3), 49.6 (CH2 ꢁ 2). MS (ESI, m/z): 501.1 [M þ H]þ.
ESI-HRMS m/z: calc'd for C28H29N4O5 [M þ H]þ: 501.2138; found
501.2132.
(CH), 128.7 (CH), 127.0 (CH), 109.8 (Cq), 108.0 (CH), 103.8 (CH), 66.5
(CH2 ꢁ 2), 56.4 (CH3), 56.2 (CH3), 50.3 (2C), 26.8 (CH3). MS (ESI, m/
z): 409.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H25N4O4 [M þ H]þ:
409.1876; found 409.1871.
4.1.2.6. 2-(benzyloxy)-5-(6,7-dimethoxy-4-morpholinoquinazolin-2-
yl)benzamide (T6). White solid; Yield 67.9%; mp 228e230 ꢀC; 1H
4.1.2.11. 3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-N,N-dime-
NMR (DMSO-d6):
d
8.90 (d, J ¼ 2.4 Hz, 1H, AreH), 8.51 (m, 1H,
thylbenzamide (T15). Off-white solid; Yield 64.6%; mp 169e171 ꢀC;
AreH), 7.68 (s, 1H, CONH), 7,63 (s, 1H, CONH), 7.55 (d, J ¼ 7.2 Hz, 2H,
AreH ꢁ 2), 7.37 (m, 3H, AreH ꢁ 3), 7.17 (s, 1H, AreH), 5.34 (s, 2H,
OCH2), 3.97 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.86 (m, 4H, CH2 ꢁ 2),
1H NMR (DMSO-d6):
d
8.52 (d, J ¼ 7.6 Hz, 1H, AreH), 8.46 (s, 1H,
AreH), 7.58 (m, 1H, AreH), 7.51 (d, J ¼ 7.6 Hz, 1H, AreH), 7.35 (s, 1H,
AreH), 7.19 (s, 1H, AreH), 3.97 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.86
(m, 4H, CH2 ꢁ 2), 3.72 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.04 (s, 3H, NH3),
3.70 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2). 13C NMR (DMSO-d6):
d 166.3 (Cq),
163.1 (Cq), 157.3 (Cq), 156.2 (Cq), 154.3 (Cq), 149.1 (Cq), 147.8 (Cq),
136.2 (Cq), 131.2 (Cq), 130.7 (CH), 130.0 (CH), 128.4 (CH ꢁ 2), 127.9
(CH), 127.6 (CH ꢁ 2), 123.3 (Cq), 113.0 (CH), 108.8 (CH), 107.3 (Cq),
103.1 (CH), 70.0 (CH2), 65.8 (CH2 ꢁ 2), 55.7 (CH3), 55.5 (CH3), 49.6
(CH2 ꢁ 2). MS (ESI, m/z): 501.1[M þ H]þ. ESI-HRMS m/z: calc'd for
2.97 (s, 3H, NH3). 13C NMR (DMSO-d6):
d 170.6 (Cq),163.8 (Cq),156.8
(Cq), 155.0 (Cq), 149.6 (Cq), 148.7 (Cq), 138.8 (Cq), 137.1 (Cq), 128.9
(CH), 128.8 (CH), 128.7 (CH), 126.5 (CH), 109.8 (Cq), 108.1 (CH), 103.8
(CH), 66.5 (CH2 ꢁ 2), 56.4 (CH3), 56.1 (CH3), 50.3 (CH2 ꢁ 2), 35.2
(CH3 ꢁ 2). MS (ESI, m/z): 423.1 [M þ H]þ. ESI-HRMS m/z: calc'd for
C
28H29N4O5 [M þ H]þ: 501.2138; found 501.2132.
C
22H27N4O4 [M þ H]þ: 423.2032; found 423.2027.
4.1.2.7. 3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-5-(tri-
4.1.2.12. N-(3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)phenyl)
fluoromethyl)benzamide(T9). White solid; Yield 43.8%; mp
acetamide (T16). Off-white solid; Yield 47.8%; mp 228e230 ꢀC; 1H
243e245 ꢀC; 1H NMR (DMSO-d6):
d
9.19 (s, 1H, AreH), 8.84 (s, 1H,
NMR (DMSO-d6): d 10.10 (s, 1H, CONH), 8.60 (s, 1H, AreH), 8.14 (d,
AreH), 8.40 (s, 1H, CONH), 8.32 (s, 1H, AreH), 7.73 (s, 1H, CONH),
7.42 (s, 1H, AreH), 7.20 (s, 1H, AreH), 3.98 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3), 3.87 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.76 (d, J ¼ 4.0 Hz, 4H,
J ¼ 8.0 Hz, 1H, AreH), 7.80 (m, 1H, AreH), 7.41 (d, 1H, AreH), 7.28 (s,
1H, AreH), 7.18 (s, 1H, AreH), 3.98 (s, 3H, OCH3), 3.94 (s, 3H, OCH3),
3.86 (s, 4H, CH2 ꢁ 2), 3.71 (s, 4H, CH2 ꢁ 2), 2.09 (s, 3H, COCH3). 13
C
CH2 ꢁ 2). 13C NMR (DMSO-d6):
d
166.9 (Cq), 163.8 (Cq), 155.4 (Cq),
NMR (DMSO-d6): d 168.9 (Cq), 163.7 (Cq), 157.4 (Cq), 154.9 (Cq),
149.4 (Cq), 149.0 (Cq), 140.2 (Cq), 136.3 (Cq), 131.0 (CH), 130.0 (Cq),
129.7 (CH), 126.6 (Cq), 125.5 (CH), 124.5 (Cq, JC-F ¼ 271 Hz), 110.0
(Cq), 108.14 (CH), 103.8 (CH), 66.4 (CH2 ꢁ 2), 56.5 (CH3), 56.2 (CH3),
50.2(CH2 ꢁ 2). MS (ESI, m/z): 463.1 [M þ H]þ. ESI-HRMS m/z: calc'd
for C22H22F3N4O4 [M þ H]þ: 463.1593; found 463.1588.
149.6 (Cq), 148.6 (Cq), 139.9 (Cq), 139.3 (Cq), 129.1 (CH), 122.9 (CH),
121.0 (CH), 118.9 (CH), 109.7 (Cq), 107.9 (CH), 103.8 (CH), 66.5
(CH2 ꢁ 2), 56.3 (CH3), 56.1 (CH3), 50.3 (CH2 ꢁ 2), 24.5 (CH3). MS (ESI,
m/z): 409.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H25N4O4
[M þ H]þ: 409.1876; found 409.1870.
4.1.2.8. 3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-5-(tri-
4.1.2.13. 4-(6, 7-dimethoxy-4-morpholinoquinazolin-2-yl)benzamide
fluoromethoxy) benzamide (T10). White solid; Yield 57.3%; mp
(T17). Off-white solid; Yield 78.3%; mp 251e253 ꢀC; 1H NMR
222e224 ꢀC; 1H NMR (DMSO-d6):
d
8.96 (s, 1H, AreH), 8.47 (s, 1H,
(DMSO-d6):
d
8.51 (d, J ¼ 8.4 Hz, 2H, AreH ꢁ 2), 8.08 (s, 1H, CONH),
AreH), 8.31 (s, 1H, AreH), 7.94 (s, 1H, CONH), 7.69 (s, 1H, CONH),
7.39 (s, 1H, AreH), 7.15 (s, 1H, AreH), 3.98 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.76 (d, J ¼ 4.0 Hz, 4H,
CH2 ꢁ 2). 13C NMR (DMSO-d6): 166.8 (Cq), 163.8 (Cq), 155.4 (Cq),
155.1 (Cq), 149.4 (Cq), 149.0 (Cq), 141.4 (Cq), 137.3 (Cq), 126.1 (Cq),
122.4 (Cq), 121.9 (CH), 120.6 (CH), 119.4 (CH), 108.1 (CH), 103.8 (CH),
66.4 (CH2 ꢁ 2), 56.4 (CH3), 56.1 (CH3), 50.2 (CH2 ꢁ 2). MS (ESI, m/z):
479.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H22F3N4O5 [M þ H]þ:
479.1542; found 479.1537.
8.00 (d, J ¼ 8.4 Hz, 2H, AreH ꢁ 2), 7.46 (s, 1H, CONH), 7.35 (s, 1H,
AreH), 7.19 (s,1H, AreH), 3.98 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.86
(d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.74 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2). 13C NMR
(DMSO-d6):
d 168.1 (Cq), 163.7 (Cq), 156.7 (Cq), 155.0 (Cq), 149.6
(Cq),148.8 (Cq),141.3 (Cq),135.8 (Cq),128.1 (CH ꢁ 2),127.8 (CH ꢁ 2),
109.0 (Cq), 108.1 (CH), 103.8 (CH), 66.5 (CH2 ꢁ 2), 56.3 (CH3), 56.1
(CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z): 395.1 [M þ H]þ. ESI-HRMS m/z:
calc'd for C21H23N4O4 [M þ H]þ: 395.1719; found 395.1714.
4.1.2.14. N-(4-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)phenyl)
4.1.2.9. Methyl 3-(6, 7-dimethoxy-4-morpholinoquinazolin-2-yl)
acetamide (T18). Off-white solid; Yield 57.4%; mp 244e246 ꢀC; 1H
benzoate (T12). White solid; Yield 77.8%; mp 207e209 ꢀC; 1H NMR
NMR (DMSO-d6):
d
10.14 (s, 1H, NH), 8.39 (d, J ¼ 8.8 Hz, 2H,
(DMSO-d6):
d
9.08 (s, 1H, AreH), 8.71 (d, J ¼ 8.0 Hz, 1H, AreH), 7.66
AreH ꢁ 2), 7.72 (d, J ¼ 8.4 Hz, 2H, AreH ꢁ 2), 7.26 (s, 1H, AreH), 7.16
(s, 1H, AreH), 3.97 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.85 (d,
J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.70 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 2.09 (s, 3H,
(d, J ¼ 8.0 Hz, 1H, AreH), 7.67 (m, 1H, AreH), 7.38 (s, 1H, AreH), 7.18
(s, 1H, AreH), 3.99 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.93 (s, 3H,
OCH3), 3.87 (m, 4H, CH2 ꢁ 2), 3.74 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2). 13
C
COCH3). 13C NMR (DMSO-d6):
d 168.1 (Cq), 163.7 (Cq), 157.2 (Cq),
NMR (DMSO-d6):
d
168.7 (Cq), 163.8 (Cq), 156.4 (Cq), 155.0 (Cq),
154.9 (Cq), 149.7 (Cq), 148.4 (Cq), 141.4 (Cq), 133.3 (Cq), 128.7
(CH ꢁ 2), 118.9 (CH ꢁ 2), 109.5 (Cq), 107.9 (CH), 103.8 (CH), 66.5
(CH2 ꢁ 2), 56.3 (CH3), 56.1 (CH3), 50.3 (CH2 ꢁ 2), 24.6 (CH3). MS (ESI,
m/z): 409.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H25N4O4
[M þ H]þ: 409.1876; found 409.1871.
149.6 (Cq), 148.8 (Cq), 139.3 (Cq), 132.6 (CH), 130.9 (Cq), 130.4 (CH),
129.5 (CH), 128.6 (CH), 109.9 (Cq), 108.1 (CH), 103.8 (CH), 66.5 (2C),
56.5 (CH3), 56.2 (CH3), 52.8 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z):
410.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C22H24N3O5 [M þ H]þ:
410.1716; found 410.1717.
4.1.2.15. 3-Methoxy-5-(7-methoxy-6-(2-methoxyethoxy)-4-
4.1.2.10. 3-(6, 7-dimethoxy-4-morpholinoquinazolin-2-yl)-N-meth-
morpholinoquinazolin-2-yl) benzamide (T19). White solid; Yield
ylbenzamide (T14). Off-white solid; Yield 43.5%; mp 220e221 ꢀC;
62.1%; mp 209e211 ꢀC; 1H NMR (DMSO-d6):
d 8.55 (s, 1H, AreH),
1H NMR (DMSO-d6):
d
8.91 (s, 1H, AreH), 8.59 (m, 2H, AreH ꢁ 2),
8.13 (m, 2H, AreH, CONH), 7.54 (m, 1H, AreH), 7.46 (s, 1H, CONH),
7.37 (s, 1H, AreH), 7.23 (s, 1H, AreH), 4.28 (m, 2H, CH2), 3.98 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.74 (m,
7.91 (d, J ¼ 8.0 Hz,1H, AreH), 7.59 (m, 1H, CONH), 7.37 (s, 1H, AreH),
7.19 (s, 1H, AreH), 3.98 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.87 (m,
4H, CH2 ꢁ 2), 3.74 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 2.84 (d, J ¼ 4.8 Hz, 3H,
6H, CH2 ꢁ 3), 3.35 (s, 3H, OCH3). 13C NMR (DMSO-d6):
d 168.3 (Cq),
NH3). 13C NMR (DMSO-d6):
d
167.2 (Cq), 163.8 (Cq), 157.0 (Cq), 155.0
163.8 (Cq), 159.7 (Cq), 156.8 (Cq), 155.0 (Cq), 149.6 (Cq), 148.0 (Cq),
140.3 (Cq), 136.4 (Cq), 120.0 (CH), 116.2 (CH), 114.5 (CH), 109.8 (Cq),
(Cq), 149.6 (Cq), 148.7 (Cq), 139.0 (Cq), 135.5 (Cq), 130.5 (CH), 128.8

X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
391
108.1 (CH), 104.9 (CH), 70.8 (CH2), 68.3 (CH2), 66.5 (CH2 ꢁ 2), 58.7
(CH3), 56.4 (CH3), 55.9 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z): 469.0
[M þ H]þ. ESI-HRMS m/z: calc'd for C24H29N4O6 [M þ H]þ:
469.2087; found 469.2082.
(Cq), 149.6 (Cq), 148.7 (Cq), 140.2 (Cq), 135.6 (Cq), 118.4 (CH), 117.6
(CH), 116.4 (CH), 109.8 (Cq), 108.0 (CH), 103.8 (CH), 66.5 (CH2 ꢁ 2),
56.4 (CH3), 56.1 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z): 425.1 [M þ H]þ.
ESI-HRMS m/z: calc'd for C21H23N4O5 [M þ H]þ: 411.1668; found
411.1663.
4.1.2.16. 3-(6,7-Bis(2-methoxyethoxy)-4-morpholinoquinazolin-2-
yl)-5-methoxybenzamide(T20). White solid; Yield 63.1%; mp
4.1.3.2. 5-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-2-
124e126 ꢀC; 1H NMR (DMSO-d6):
d
8.54 (s, 1H, AreH), 8.12 (s, 2H,
hydroxybenzamide (T8). White solid; Yield 81.8%; mp > 250 ꢀC; 1H
AreH, CONH), 7.53 (s, 1H, AreH), 7.46 (s, 1H, CONH), 7.38 (s, 1H,
AreH), 7.26 (s, 1H, AreH), 4.32 (m, 4H, CH2), 3.90 (s, 3H, OCH3), 3.86
(d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.75 (m, 4H, CH2 ꢁ 2), 3.37 (s, 6H,
NMR (DMSO-d6): d 9.77 (s,1H, AreH), 8.39 (s,1H, AreH), 8.05 (s,1H,
CONH), 8.00 (s, 1H, CONH), 7.33 (s, 3H, AreH ꢁ 2, OH), 7.19 (s, 1H,
AreH), 3.98 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.87 (d, J ¼ 4.4 Hz, 4H,
CH2 ꢁ 2), 3.82 (s, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2). 13C NMR (DMSO-d6):
OCH3 ꢁ 2). 13C NMR (DMSO-d6):
d 168.3 (Cq), 163.8 (Cq), 159.7 (Cq),
156.8 (Cq), 154.3 (Cq), 149.5 (Cq), 148.0 (Cq), 140.3 (Cq), 136.4 (Cq),
120.0 (CH), 116.3 (CH), 114.4 (CH), 109.8 (Cq), 108.9 (CH), 105.5 (CH),
70.9 (CH2), 70.5 (CH2), 68.7 (CH2), 68.6 (CH2), 66.5 (CH2 ꢁ 2), 58.9
(CH3), 58.8 (CH3), 55.9 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z): 513.2
[M þ H]þ. ESI-HRMS m/z: calc'd for C26H33N4O7 [M þ H]þ:
513.2349; found 513.2350.
d 172.1 (Cq), 163.7 (Cq), 163.6 (Cq), 162.8 (Cq), 157.0 (Cq), 154.9 (Cq),
148.3 (Cq), 133.8 (CH), 128.6 (CH), 124.6 (CH), 117.8 (Cq), 115.3 (Cq),
109.2 (Cq), 107.6 (CH), 103.9 (CH), 66.5 (CH2 ꢁ 2), 56.3 (CH3), 56.1
(CH3), 50.3 (CH2 ꢁ 2). MS (ESI, m/z): 411.1 [M þ H]þ. ESI-HRMS m/z:
calc'd for C21H23N4O5 [M þ H]þ: 411.1668; found 411.1663.
4.1.4. 3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)benzoic acid
(T13)
4.1.2.17. 3-(7-(benzyloxy)-6-methoxy-4-morpholinoquinazolin-2-
yl)-5-methoxybenzamide (T21). White solid; Yield 83.2%; mp
To a solution of compound T12 (0.4 mmol) in the mixture of
methanol (10 mL) and water (8 mL) was added 2 M NaOH (2 mL,
4 mmol) and the mixture was stirred at room temperature for 4 h.
The solvent was removed and 20 mL water was added to the
mixture. The suspension was adjusted to pH 4e5 with 6 M hy-
drochloric acid whereupon a white precipitate formed. The sus-
pension was filtered and dried to give a Off-white solid; Yield
240e242 ꢀC; 1H NMR (DMSO-d6):
d 8.54 (s, 1H, AreH), 8.12 (m, 2H,
AreH, CONH), 7.53 (d, J ¼ 7.2 Hz, 3H, AreH ꢁ 3), 7.46 (m, 4H, CONH,
AreH ꢁ 3), 7.38 (s, 1H, AreH), 7.19 (s, 1H, AreH), 5.34 (s, 2H, OCH2),
4.32 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.87 (m,
4H, CH2 ꢁ 2), 3.74 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2). 13C NMR (DMSO-d6):
d
168.3 (Cq), 163.8 (Cq), 159.7 (Cq), 156.8 (Cq), 153.9 (Cq),149.5 (Cq),
148.9 (Cq), 140.3 (Cq), 136.8 (Cq), 136.5 (Cq), 129.0 (CH ꢁ 2), 128.6
(CH), 128.4 (CH ꢁ 2), 120.0 (Cq), 116.3 (CH), 114.4 (CH), 109.9 (Cq),
109.3 (CH), 104.1 (CH), 70.5 (CH2), 66.5 (CH2 ꢁ 2), 56.2 (CH3), 55.9
(CH3), 50.2 (CH2 ꢁ 2). MS (ESI, m/z): 501.1 [M þ H]þ. ESI-HRMS m/z:
calc'd for C28H29N4O5 [M þ H]þ: 501.2138; found 501.2132.
85.4%; mp > 250 ꢀC; 1H NMR (DMSO-d6):
d 8.92 (s, 1H, AreH), 8.69
(d, J ¼ 7.6 Hz, 1H, AreH), 8.21 (d, J ¼ 7.6 Hz, 1H, AreH), 7.77 (m, 1H,
AreH), 7.72 (s, 1H, AreH), 7.36 (s, 1H, AreH), 4.15 (s, 4H, CH2 ꢁ 2),
4.00 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 3.85 (d, J ¼ 4.0 Hz, 4H,
CH2 ꢁ 2). 13C NMR (DMSO-d6):
d
167.1 (Cq ꢁ 2), 163.8 (Cq), 156.0
(Cq), 153.3 (Cq), 148.7 (Cq ꢁ 2), 133.5 (Cq), 133.4 (CH), 132.0 (CH),
129.8 (CH), 126.7 (CH), 109.9 (Cq), 105.9 (CH), 103.7 (CH), 66.4
(CH2 ꢁ 2), 56.8 (CH3), 56.5 (CH3), 49.8 (CH2 ꢁ 2). MS (ESI, m/z):
4.1.2.18. 5-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-2-yl)pyr-
imidin-2-amine (T11). Compound T11 was synthesized from 5-
bromopyrimidin-2-amine (0.05 g, 0.13 mmol) using the general
procedure described above. Off-white solid; Yield 67.8%;
396.1 [M
þ
H]þ. ESI-HRMS m/z: calc'd for C21H2N3O5
[M þ H]þ:396.1559; found 396.1554.
mp > 250 ꢀC; 1H NMR (DMSO-d6):
d
9.16 (s, 2H, AreH ꢁ 2), 7.25 (s,
1H, AreH), 7.14 (s, 1H, AreH), 7.10 (s, 2H, NH2), 3.95 (s, 3H, OCH3),
3.92 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.69 (d,
4.1.5. 3-(7-Hydroxy-6-methoxy-4-morpholinoquinazolin-2yl)-5-
methoxybenzamide (5)
J ¼ 4.0 Hz, 4H, CH2 ꢁ 2). 13C NMR (DMSO-d6):
d
164.5 (Cq), 163.6
Compound 5 was prepared from compound T21 as the same
(Cq), 158.2 (CH ꢁ 2), 155.4 (Cq), 154.9 (Cq), 149.6 (Cq), 148.2 (Cq),
120.8 (Cq), 109.4 (Cq), 107.7 (CH), 103.9 (CH), 66.4 (CH2 ꢁ 2), 56.3
(CH3), 56.1 (CH3), 50.2 (CH2 ꢁ 2). MS (ESI, m/z): 369.1 [M þ H]þ. ESI-
HRMS m/z: calc'd for C18H21N6O3 [M þ H]þ: 369.1675; found
369.1670.
procedure with T7. White solid; Yield 84.5%; mp 236e237 ꢀC; 1H
NMR (DMSO-d6):
d 10.53 (bm, 1H, OH), 8.50 (s, 1H, AreH), 8.10 (m,
2H, AreH, CONH),7.52 (m, 1H, AreH), 7.45 (s, 1H, CONH), 7.21 (s, 1H,
AreH), 7.18 (s,1H, AreH), 3.95 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.86
(d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.71 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2). MS (ESI,
m/z): 411.1 [M þ H]þ.
4.1.3. General procedure for the synthesis of compounds T7 and T8
To the solution of compound T5 or T6 (0.4 mmol) in the mixture
of DMF (6 mL) and water (3 mL) was added ammonium formate
(0.25 g, 4 mmol) and 5% Pd on carbon (0.1 mmol) and the above
mixture was stirred at 100 ꢀC for 6 h. The mixture was filtered and
the filtrate was evaporated under vacuum to give a residue. The
residue was dissolved in 10 mL water. The water suspension was
extracted with ethyl acetate (10 mL ꢁ 3). The combined organic
extracts was washed by saturated sodium chloride, dried, filtered
and evaporated under vacuum to afford compounds T7eT8.
4.1.6. General procedure for the synthesis of compounds 6 and
T22eT26
To the solution of compound 5 (1 mmol) in DMF (5 mL) was
added potassium carbonate (0.55 g, 4 mmol) and the mixture
was stirred at room temperature for 10 min. Then alkyl halides
(2 mmol) and potassium iodide (0.17 g, 0.1 mmol) was added to
the above mixture and the mixture was stirred at 90 ꢀC for 5-8 h
under nitrogen atmosphere. The solvent was removed under
vacuum and the residue was suspended in water. The suspension
was extracted by dichloromethane (10 mL ꢁ 3) and the combined
organic layer was evaporated under reduced pressure to afford
the crude product. The crude product was recrystallized from
ethanol to afford compound 6 or T22eT26 as a white or an off-
white solid.
4.1.3.1. 3-(6,7-Dimethoxy-4-morpholinoquinazolin-2-yl)-5-
hydroxybenzamide (T7). White solid; Yield 84.7%; mp 238e240 ꢀC;
1H NMR (DMSO-d6):
d 9.77 (s, 1H, AreH), 8.39 (s, 1H, AreH), 8.05 (s,
1H, CONH), 8.00 (s, 1H, CONH), 7.33 (s, 3H, AreH ꢁ 2, OH), 7.19 (s,
1H, AreH), 3.98 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.87 (d, 4H,
J ¼ 4.4 Hz, CH2 ꢁ 2), 3.82 (d, 4H, J ¼ 4.0 Hz, CH2 ꢁ 2). 13C NMR
4.1.6.1. Ethyl 2-((2-(3-carbamoyl-5-methoxyphenyl)-6-methoxy-4-
morpholinoquinazolin-7-yl)oxy)acetate (6). Off-white solid; Yield
(DMSO-d6):
d
168.7 (Cq), 163.8 (Cq), 157.8 (Cq), 157.1 (Cq), 154.9

392
X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
85.2%; mp 199e201 ꢀC; 1H NMR (DMSO-d6):
d
8.53 (s, 1H, AreH),
z): 522.2 [M þ H]þ. ESI-HRMS m/z: calc'd for C28H36N5O5 [M þ H]þ:
8.11 (d, J ¼ 1.6 Hz, 2H, AreH, CONH), 7.54 (m, 1H, AreH), 7.46 (s, 1H,
CONH), 7.29 (s, 1H, AreH), 7.23 (s, 1H, AreH), 5.06 (s, 2H, OCH2),
4.22 (m, 2H, CH2), 3.97 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.87 (m, 4H,
CH2 ꢁ 2), 3.75 (d, J ¼ 2.4 Hz, 4H, CH2 ꢁ 2), 1.25 (t, J1 ¼ 6.8 Hz,
J2 ¼ 7.2 Hz, 3H, CH3). MS (ESI, m/z): 497.8 [M þ H]þ.
522.2716; found 522.2711.
4.1.6.6. 3-Methoxy-5-(6-methoxy-4-morpholino-7-(2-(pyrrolidin-1-
yl)ethoxy) quinazolin-2-yl)benzamide (T26). Off-white solid; Yield
72.7%; mp 195e197 ꢀC; 1H NMR (DMSO-d6):
d 8.54 (s, 1H, AreH),
8.12 (m, 2H, AreH, CONH), 7.53 (d, J ¼ 1.6 Hz, 1H, AreH), 7.45 (s, 1H,
CONH), 7.37 (s, 1H, AreH), 7.19 (s, 1H, AreH), 4.28 (m, 2H, CH2), 3.94
(s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2),
3.73 (d, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2), 2.88 (m, 2H, CH2), 2.56 (s, 4H,
CH2 ꢁ 2), 1.52 (m, 4H, CH2 ꢁ 2), 1.70 (m, 4H, CH2 ꢁ 2). 13C NMR
4.1.6.2. 3-Methoxy-5-(6-methoxy-7-(2-methoxyethoxy)-4-
morpholinoquinazolin-2-yl) benzamide (T22). White solid; Yield
74.1%; mp 210e212 ꢀC; 1H NMR (DMSO-d6):
d 8.55 (s, 1H, AreH),
8.12 (d, J ¼ 1.2 Hz, 2H, AreH, CONH), 7.54 (s, 1H, AreH), 7.46 (s, 1H,
CONH), 7.38 (s, 1H, AreH), 7.19 (s, 1H, AreH), 4.32 (m, 2H, CH2), 3.95
(s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2),
3.75 (m, 6H, CH2 ꢁ 3), 3.35 (s, 3H, OCH3). 13C NMR (DMSO-d6):
(DMSO-d6):
d 168.2 (Cq), 163.7 (Cq), 159.7 (Cq), 156.8 (Cq), 153.7
(Cq), 149.5 (Cq), 148.7 (Cq), 140.3 (Cq), 136.4 (Cq), 120.0 (CH), 116.3
(CH), 114.5 (CH), 110.0 (Cq), 109.0 (CH), 104.1 (CH), 66.5 (CH2 ꢁ 2),
56.4 (CH3), 55.9 (CH3), 54.6 (CH2 ꢁ 3), 53.9 (CH2), 50.3 (CH2 ꢁ 2),
23.4 (CH2 ꢁ 2). MS (ESI, m/z): 508.2 [M þ H]þ. ESI-HRMS m/z: calc'd
for C27H34N5O3 [M þ H]þ: 508.2560; found 509.2555.
d
168.3 (Cq), 163.7 (Cq), 159.7 (Cq), 156.8 (Cq), 154.1 (Cq), 149.5 (Cq),
148.7 (Cq), 140.3 (Cq), 136.4 (Cq), 120.0 (CH), 116.2 (CH), 114.4 (CH),
109.8 (Cq), 108.7 (CH), 103.9 (CH), 70.5 (CH2), 68.4 (CH2), 66.5
(CH2 ꢁ 2), 58.7 (CH3), 56.1 (CH3), 55.9 (CH3), 50.3 (CH2 ꢁ 2). MS (ESI,
m/z): 469.2 [M þ H]þ. ESI-HRMS m/z: calc'd for C24H29N4O6
[M þ H]þ: 469.2087; found 469.2082.
4.1.7. 2-((2-(3-Carbamoyl-5-methoxyphenyl)-6-methoxy-4-
morpholinoquinazolin-7yl)oxy)acetic acid (7)
To a solution of compound 6 (1.00 g, 2 mmol) in the mixture of
methanol (12 mL) and water (3 mL) was added 2 M NaOH (10 mL,
20 mmol) and the mixture was stirred at room temperature for 4 h.
The solvent was removed and 20 mL water was added to the
mixture. The suspension was adjusted to pH 4e5 with 6 M hy-
drochloric acid whereupon a white precipitate formed. The sus-
pension was filtered and dried to give a white solid. Yield 71.2%;
4.1.6.3. 3-Methoxy-5-(6-methoxy-4-morpholino-7-((tetrahydro-2H-
pyran-4-yl) methoxy)quinazolin-2yl)benzamide (T23). White solid;
Yield 67.8%; mp 231e233 ꢀC; 1H NMR (DMSO-d6):
d 8.55 (s, 1H,
AreH), 8.12 (m, 2H, AreH, CONH), 7.53 (d, J ¼ 1.6 Hz,1H, AreH), 7.45
(s, 1H, CONH), 7.36 (s, 1H, AreH), 7.19 (s, 1H, AreH), 4.06 (d,
J ¼ 6.4 Hz, 2H, CH2), 3.94 (s, 3H, OCH3), 3.88 (m, 9H, OCH3, CH2 ꢁ 3),
3.73 (d, J ¼ 3.6 Hz, 4H, CH2 ꢁ 2), 2.12 (m,1H, CH),1.73 (d, J ¼ 12.0 Hz,
mp > 250 ꢀC; 1H NMR (DMSO-d6):
d 8.48 (s, 1H, AreH), 8.23 (s, 1H,
2H, CH2), 1.39 (m, 2H, CH2). 13C NMR (DMSO-d6):
d
168.3 (Cq), 163.8
CONH), 8.05 (s, 1H, AreH), 7.70 (s, 1H, AreH), 7.62 (s, 1H, AreH),
7.55 (s, 1H, CONH), 7.38 (s, 1H, AreH), 4.96 (s, 2H, OCH2), 4.14 (s, 4H,
CH2 ꢁ 2), 3.99 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.85 (d, J ¼ 4.0 Hz,
4H, CH2 ꢁ 2). MS (ESI, m/z): 469.1 [M þ H]þ.
(Cq), 159.7 (Cq), 156.8 (Cq), 154.3 (Cq), 149.6 (Cq), 148.9 (Cq), 140.3
(Cq), 136.4 (Cq), 112.0 (CH), 116.2 (CH), 114.5 (CH), 109.7 (Cq), 108.69
(CH), 103.94 (CH), 73.3 (CH2), 67.0 (CH2 ꢁ 2), 66.4 (CH2 ꢁ 2), 56.2
(CH3), 55.9 (CH3), 50.3 (CH2 ꢁ 2), 34.6 (CH), 29.6 (CH2 ꢁ 2). MS (ESI,
m/z): 509.2 [M þ H]þ. ESI-HRMS m/z: calc'd for C27H33N4O6
[M þ H]þ: 509.2400; found 509.2401.
4.1.8. General procedure for the synthesis of compounds T27eT29
A solution of compound 7 (0.24 g, 0.51 mmol) in DMF (8 mL) was
added alkyl amine (4 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (0.48 g, 2.5 mmol), N-hydroxybenzotrizole (0.41 g,
3 mmol) and the mixture was stirred for 8 h at room temperature
under nitrogen atmosphere. The solvent was removed in vacuum
and the residue was suspended in water (10 mL). The suspension
was extracted by ethyl acetate (10 mL ꢁ 3). The combined organic
layer was washed by sodium bicarbonate and saturated sodium
chloride, dried, filtered and evaporated to afford a crude product,
which was recrystallized from ethanol to afford compounds
T27eT29 as a white or an off-white solid.
4.1.6.4. 3-Methoxy-5-(6-methoxy-4-morpholino-7-(2-
morpholinoethoxy)quinazolin-2-yl) benzamide (T24). Off-white
solid; Yield 69.4%; mp 194e196 ꢀC; 1H NMR (DMSO-d6):
d 8.54 (s,
1H, AreH), 8.12 (m, 2H, AreH, CONH), 7.53 (d, J ¼ 1.6 Hz, 1H, AreH),
7.45 (s, 1H, CONH), 7.38 (s, 1H, AreH), 7.19 (s, 1H, AreH), 4.28 (m,
2H, CH2), 3.94 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz,
4H, CH2 ꢁ 2), 3.73 (d, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2), 2.75 (m, 2H, CH2),
2.48 (s, 4H, CH2 ꢁ 2), 2.79 (m, 4H, CH2 ꢁ 2), 1.52 (m, 4H, CH2 ꢁ 2),
1.40 (m, 2H, CH2). 13C NMR (DMSO-d6):
d 168.3 (Cq), 163.7 (Cq),
159.7 (Cq), 156.8 (Cq), 154.1 (Cq), 149.5 (Cq), 148.8 (Cq), 140.3 (Cq),
136.4 (Cq), 120.0 (CH), 116.2 (CH), 114.4 (CH), 109.8 (Cq), 108.8 (CH),
103.9 (CH), 66.9 (CH2), 66.6 (CH2 ꢁ 2), 66.5 (CH2 ꢁ 2), 57.2 (CH2),
56.2 (CH3), 55.9 (CH3), 54.1 (CH2 ꢁ 2), 50.3 (CH2 ꢁ 2). MS (ESI, m/z):
522.2 [M þ H]þ. ESI-HRMS m/z: calc'd for C27H34N5O6 [M þ H]þ:
524.2509; found 524.2505.
4.1.8.1. 3-Methoxy-5-(6-methoxy-4-morpholino-7-(2-morpholino-2-
oxoethoxy)quinazolin-2-yl)benzamide (T27). Off-white solid; Yield
75.4%; mp 141e143 ꢀC; 1H NMR (DMSO-d6):
d 8.54 (s, 1H, AreH),
8.11 (d, J ¼ 1.2 Hz, 2H, AreH, CONH), 7.53 (m, 1H, AreH), 7.46 (s, 1H,
CONH), 7.32 (s, 1H, AreH), 7.21 (s, 1H, AreH), 5.13 (s, 2H, OCH2),
3.96 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H,
4.1.6.5. 3-Methoxy-5-(6-methoxy-4-morpholino-7-(2-(piperidin-1-
CH2 ꢁ 2), 3.74 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.63 (m, 4H, CH2 ꢁ 2). 13
C
yl)ethoxy) quinazolin-2-yl)benzamide (T25). Off-white solid; Yield
NMR (DMSO-d6): d 168.3 (Cq), 165.8 (Cq), 163.7 (Cq), 159.7 (Cq),
63.1%; mp 192e194 ꢀC; 1H NMR (DMSO-d6):
d
8.54 (s, 1H, AreH),
156.8 (Cq), 153.7 (Cq), 149.3 (Cq), 148.8 (Cq), 140.3 (Cq), 136.5 (Cq),
120.0 (CH), 116.3 (CH), 114.4 (CH), 110.0 (Cq), 109.3 (CH), 101.1 (CH),
66.6 (CH2), 66.5 (CH2 ꢁ 2), 56.3 (CH3), 55.9 (CH3), 50.3 (CH2 ꢁ 2),
45.2 (CH2 ꢁ 2), 42.1 (CH2 ꢁ 2). MS (ESI, m/z): 538.2 [M þ H]þ. ESI-
HRMS m/z: calc'd for C27H32N5O7 [M þ H]þ: 538.2302; found
538.2308.
8.12 (m, 2H, AreH, CONH), 7.53 (d, J ¼ 1.6 Hz, 1H, AreH), 7.45 (s, 1H,
CONH), 7.38 (s, 1H, AreH), 7.19 (s, 1H, AreH), 4.28 (m, 2H, CH2), 3.94
(s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (d, J ¼ 4.4 Hz, 4H, CH2 ꢁ 2),
3.73 (d, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2), 2.75 (m, 2H, CH2), 2.48 (s, 4H,
CH2 ꢁ 2), 2.79 (m, 4H, CH2 ꢁ 2), 1.52 (m, 4H, CH2 ꢁ 2), 1.40 (m, 2H,
CH2). 13C NMR (DMSO-d6):
d 168.3 (Cq), 163.7 (Cq), 159.7 (Cq), 156.8
(Cq), 154.4 (Cq), 149.6 (Cq), 148.8 (Cq), 140.3 (Cq), 136.4 (Cq), 120.0
(CH), 116.2 (CH), 114.5 (CH), 109.8 (Cq), 108.8 (CH), 103.9 (CH), 67.1
(CH2), 66.5 (CH2 ꢁ 2), 57.6 (CH2), 56.2 (CH3), 55.9 (CH3), 54.9
(CH2 ꢁ 2), 50.26 (CH2 ꢁ 2), 26.04 (CH2 ꢁ 2), 24.40 (CH2). MS (ESI, m/
4.1.8.2. 3-Methoxy-5-(6-methoxy-7-(2-(4-methylpiperazin-1-yl)-2-
oxoethoxy)-
Off-white solid; Yield 49.5%; mp 141e143 ꢀC; 1H NMR (DMSO-d6):
8.53 (s, 1H, AreH), 8.11 (s, 2H, AreH, CONH), 7.53 (d, J ¼ 1.2 Hz, 1H,
4-morpholinoquinazolin-2-yl)benzamide
(T28).
d

X.-M. Wang et al. / European Journal of Medicinal Chemistry 96 (2015) 382e395
393
AreH), 7.45 (s,1H, CONH), 7.30 (s,1H, AreH), 7.21 (s,1H, AreH), 5.11
(s, 2H, OCH2), 3.96 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (d,
J ¼ 4.4 Hz, 4H, CH2 ꢁ 2), 3.74 (d, J ¼ 4.0 Hz, 4H, CH2 ꢁ 2), 3.50 (s, 4H,
captured from randomly selected fields under fluorescent micro-
scope to qualitatively determine the morphological changes of
HCT-116 cells based on their relative fluorescence and nuclear
fragmentation.
CH2 ꢁ 2), 2.39 (s, 2H, CH2), 2.29 (s, 2H, CH2), 2.21 (s, 3H, CH3). 13
C
NMR (DMSO-d6):
d 168.3 (Cq), 165.5 (Cq), 163.7 (Cq), 159.7 (Cq),
156.8 (Cq), 153.7 (Cq), 149.3 (Cq), 148.8 (Cq), 140.3 (Cq), 136.5 (Cq),
120.0 (CH), 116.3 (CH), 114.4 (CH), 101.0 (Cq), 109.3 (CH), 101.1 (CH),
66.5 (CH2), 66.5 (CH2 ꢁ 2), 56.3 (CH3), 55.9 (CH3), 55.1 (CH2), 54.8
(CH2), 50.3 (CH2 ꢁ 2), 46.6 (CH2), 44.5 (CH2), 41.7 (CH2). MS (ESI, m/
z): 551.2 [M þ H]þ. ESI-HRMS m/z: calc'd for C28H35N6O6 [M þ H]þ:
551.2618; found 551.2613.
4.2.4. Cell cycle analysis
HCT-116 cells were seeded into 6-well plate at a concentration of
3 ꢁ 105 cells per well. After 24 h, the medium was replaced and the
cells were incubated with compound T10 at a concentration of 3 mM
for 24 h. The controls were treated with vehicle (0.5% DMSO). The
cells were then harvested and fixed with 70% ice-cold ethanol
overnight at ꢂ20 ꢀC. After centrifugation, the cell pellets were
4.1.8.3. 3-Methoxy-5-(6-methoxy-7-(2-(mathylamino)-2-
treated with propidium iodide (5 mg/mL) and RNase A in PBS
oxoethoxy)-4-morpholinoquinazolin-2-yl)benzamide
Off-white solid; Yield 63.7%; mp 193e195 ꢀC; 1H NMR (DMSO-d6):
8.53 (s, 1H, AreH), 8.11 (m, 2H, AreH, CONH), 8.05 (m, 1H, NH),
(T29).
(100 mg/mL) in PBS for 30 min at room temperature. DNA was
observed and analyzed with a flow cytometer. The independent
experiment was repeated three times.
d
7.53 (d, J ¼ 1.2 Hz, 1H, AreH), 7.44 (d, J ¼ 1.6 Hz, 1H, CONH), 7.25 (s,
1H, AreH), 7.22 (s, 1H, AreH), 4.73 (s, 2H, OCH2), 3.97 (s, 3H, OCH3),
3.90 (s, 3H, OCH3), 3.87 (m, 4H, CH2 ꢁ 2), 3.75 (d, J ¼ 4.4 Hz, 4H,
4.2.5. Apoptosis analysis
HCT-116 cells were seeded into 12-well plate at a concentration
of 1.5 ꢁ 105 cells per well and incubated for 24 h. The medium was
replaced and the cells were then incubated with compound T10 at
CH2 ꢁ 2), 2.69 (d, J ¼ 4.8 Hz, 3H, NCH3). 13C NMR (DMSO-d6):
d 168.3
(Cq), 165.5 (Cq), 163.7 (Cq), 159.7 (Cq), 156.8 (Cq), 153.7 (Cq), 149.3
(Cq), 148.8 (Cq), 140.3 (Cq), 136.5 (Cq), 120.0 (CH), 116.3 (CH), 114.4
(CH), 101.0 (Cq), 109.3 (CH), 101.1 (CH), 68.0 (CH), 66.5 (CH2 ꢁ 2),
56.2 (CH3), 55.9 (CH3), 50.2 (CH2 ꢁ 2), 26.0 (CH3). MS (ESI, m/z):
482.1 [M þ H]þ. ESI-HRMS m/z: calc'd for C24H28N5O6 [MþH]þ:
482.2040; found 482.2034.
concentrations of 1 mM and 3 mM for 24 h. The controls were treated
with vehicle (0.5% DMSO). The cells were harvested and performed
with Annexin V-FITC/PI apoptosis detection Kit according to the
manufacturer's instruction. Then the cells were analyzed with a
flow cytometer (Guava easyCyte).
4.2. Biology
4.2.6. PI3Ks enzymatic activities assay
Inhibitory PI3Ks enzymatic activities of compounds T7, T10 and
GDC0941 were evaluated according to process described in
Ref. [19]. Compounds T7, T10 and GDC0941 were dissolved in DMSO
and diluted to a series of concentrations. Different concentrations
of compounds were added to the enzyme reaction buffer contain-
ing 40 mM TriseHCl, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM
4.2.1. Cell culture
The four human tumor cell lines including HCT-116, MCF-7, U-
87 MG and A549 were grown as a monolayer respectively. HCT-116,
MCF-7 and U-87 MG were maintained in DMEM medium supple-
mented with 10% heat inactivated fetal bovine serum (FBS). A549
was maintained in RPMI-1640 medium, with 10% heat inactivated
calf serum. All cancer cells were incubated in a humidified atmo-
sphere containing 5% CO2 at 37 ꢀC.
DTT, 2.5
p110 /p85
ume was 50
m
M ATP, PI3K (p110
) and -phosphatidylinositol. The final reaction vol-
L. After incubation for 40 min at 30 ꢀC, the reaction
a/p85a, p110b/p85a, p110g/PIK3R5 or
d
a
L-a
m
was terminated by addition of stop solution. The amount of ADP
was then detected via luciferase assay. After incubation for 5 min,
the luminescence signal was determined by the multimode reader
(MD-SpectraMax M5). The signal intensity is proportional to the
PI3K activity.
4.2.2. MTT assay
The in vitro antiproliferative activities of compounds were
determined by MTT assay. 1500e4000 cells per well were seeded
into 96-well plates in 200 mL medium and incubated for 24 h. A
series of concentrations of synthesized compounds and GDC0941
were added to the wells with DMSO as vehicle control. The mixture
was incubated at 37 ꢀC, with a final concentration of 1% DMSO.
The percentage of inhibition was calculated based on the
following equation
After 72 h of incubation, 20
m
L of MTT solution (5 mg/mL in PBS)
% inhibition ¼ [1 ꢂ (Lucompound ꢂ Lumin)/(Lumax ꢂ Lumin)] ꢁ 100%,
was added to each well and incubated at 37 ꢀC for 4 h. The super-
natant of each well was removed and the formed blue formazan
where Lucompound is the signal at a given compound concentration,
Lumax is the signal of PI3Ks without compound and Lumin is the
signal of background in the absence of enzyme and compound. The
IC50 values were calculated according to the fit of the dos-
eeresponse curves by using Graph Pad Prism 5.
crystals were dissolved in 200 mL of DMSO. The optical density at
490 nm wavelength was determined by Varioskan Flash Multimode
Reader (Thermo scientific). Three separate experiments with trip-
licate data were performed to obtain mean cell viability. The IC50
value, that is, the concentration (mM) of a compound was able to
cause 50% cell death with respect to the control culture, were
calculated by means of PRISM 5, Graph Pad software.
4.2.7. Western blot assay
HCT-116 cells were seeded into 12-well plates at 5 ꢁ 105 cells
per well and incubated at 37 ꢀC for 16 h. The medium was replaced
and cells were incubated with compounds T10 and GDC0941 at
4.2.3. Hoechst staining
HCT-116 cell line was selected for Hoechst staining assay. 15,000
cells per well were seeded into 96-well plates and incubated for
24 h. The medium was replaced and cells were incubated with
10 m
M at 37 ꢀC for 1 h. The controls were added vehicle (0.5%
DMSO). The cells were lysed into ice-cold cell lysis buffer and the
cell lysates were centrifuged at 12,000 rpm for 20 min at 4 ꢀC. The
supernatant was collected and protein concentrations were deter-
mined by BCA protein Assay Kit. The protein samples were sepa-
rated on SDS-PAGE on 10% gels and then transferred onto
nitrocellulose membrane. The membranes were blocked in block-
ing buffer (10% nonfat dry milk/1% Tween 20 in TBS) for 2 h,
compound T10 at concentrations of 0.1, 1 and 10 mM for 24 h. The
controls were treated with vehicle (0.5% DMSO). Before staining,
the cells were examined directly and photographed under a phase
contrast microscope. Then the cells were treated with Hoechst
33258 for 0.5 h at 37 ꢀC. After washed by PBS, the cells were

394
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