Synthesis and Biological Evaluation of Novel Substituted 4-Anilinoquinazolines as Antitumor Agents

Article abstract of DOI:10.1111/cbdd.12706

Eleven novel 4-anilinoquinazoline derivatives were synthesized and evaluated for their in?vitro antiproliferative activity. Among them, compound 9a exhibited the best potency, with IC₅₀ values of 25?682?nm against various types of cancer cell l

Full text of DOI:10.1111/cbdd.12706

Received Date : 06-Oct-2015
Revised Date : 15-Nov-2015
Accepted Date : 01-Dec-2015
Article type
: Research Article
Synthesis and Biological Evaluation of Novel Substituted
4
-Anilinoquinazolines as Antitumor Agents
1, §
1, §
1, §
2
1
1
Dong Cao , Xiaoyan Wang , Lei Lei , Liang Ma , Zhuang Yang , Fang Wang , and
1
,
Lijuan Chen *
1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University, and Collaborative Innovation Center of Biotherapy, Chengdu, China
2
Division of Nephrology, Kidney Research Institute, West China Hospital of Sichuan
University, Chengdu, 610041, China
§
These authors contributed equally and should be considered as co-first authors
*
Corresponding author
Prof. Lijuan Chen. Tel: +86-28-85164063; Fax: +86-28-85164060. E-mail address:
chenlijuan125@163.com
2
Key words: 4-anilinoquinazoline derivatives, anti-proliferation, G /M arrest
Abstract
Eleven novel 4-anilinoquinazoline derivatives were synthesized and evaluated for their in
vitro antiproliferative activity. Among them, compound 9a exhibited the best potency, with
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been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12706
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IC50 values of 25−682 nM against various types of cancer cell lines. In addition, 9a was
confirmed that it could arrest the cell cycle at G /M phase and trigger apoptosis. Indirect
2
immunofluorescence staining revealed its anti-tubulin property. Importantly, 9a significantly
inhibited tumor growths in SM-7721 xenograft models (57.0% tumor mass change) without
causing significant loss of body weight, suggesting that 9a is a promising new anticancer
agent to be developed.
Introduction
Chemotherapy agents (1), including paclitaxel and docetaxel, play a predominant role in
the treatment of human cancer, which is one of the most complicated and capricious diseases
(
2-3). They are clinically used to eliminate the remaining tumor population after surgery, or
to cure patients when metastatic diseases were diagnosed. However, their narrow therapeutic
windows and the emergence of drug resistance (4) have encouraged continued efforts to find
safer and more effective agents capable of treating resistant cancer phenotypes (5).
The early discovery of the anticancer agents erlotinib (6) and gefitinib (7) prompted
intensive research on 4-anilinoquinazoline compounds (8), leading to the development of new
attractive compounds such as lapatinib (9), vandetanib (10), and afatinib (11) (Figure 1).
These therapeutic molecules include the tyrosine kinase inhibitors (12), β-catenin/Tcf4
inhibitors (13), G9a inhibitors (14), and the like. Among them, verubulin (MPC-6827) was
reported as a potent apoptosis inducer (Figure 1). Its corresponding hydrochloride salt is
water-soluble and currently showing promising results in phase II human cancer clinical trials
(15).
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The success of previous research base on 4-anilinoquinazoline scaffold encouraged us to
search for more potential drug candidates. In this paper, a series of substituted
4
-anilinoquinazolines were synthesized and evaluated for their cytotoxicity in various types
of tumor cell lines. And their pharmacological mechanism was subsequently investigated.
The most promising compound was further tested for anti-tumor activity and toxicity in vivo
(16–23).
Experimental Methods
Experimental apparatus and reagents
All chemicals were used without purification as commercially available unless otherwise
noted. Column chromatography was performed on silica gel (300-400 mesh) eluting with
ethyl acetate and petroleum ether. TLC was performed on 0.20 mm Silica Gel 60 F254 plates
(
Qingdao Ocean Chemical Factory, Shandong, China). UV light and I were used to visualize
2
1
13
products. NMR datas were obtained for H at 400 MHz, and for C at 100 MHz, on a Bruker
Avance400 spectrometer (Bruker Company, Germany) or Varian spectrometer (Varian, Palo
Alto, CA) , using TMS as an internal standard. Chemical shifts were given in ppm (parts per
million). Mass spectra (MS) were measured by Q-TOF Priemier mass spectrometer
(
Micromass, Manchester, UK). The purity of each compound (>95%) was determined on an
Waters e2695 series LC system (column, Xtimate C18, 4.6 mm×150 mm, 5μm; mobile phase,
methanol (60%)/H O (40%); low rate, 1.0 mL/min; UV wavelength, 254−400 nm;
2
temperature, 25°C; injection volume, 10 μL). The melting point of each compound was
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determined on a SGWX-4A melting point instrument (±1°C, <200°C; ±2°C, 200～300°C.
Shanghai Precision and Scientific Instrument Corporation, Shanghai, China).
General procedure for preparation of 4-anilinoquinazoline derivatives
The synthetic route to compounds 9a−k is shown in Scheme 1. Commercial available 1
(p-nitrophenol) was the starting material for the synthesis. The phenolic hydroxyl group was
first protected by chloromethyl methyl ether (MOMCl) to give 2. Then reduction of 2 led to 3.
The resulting aniline 3 was further coupled with 4a (2,4-dichloroquinazoline), 4b
(
4-chloro-2-methylquinazoline) or 4c (4-chloroquinazo- line), giving the intermediate
compounds 5a-c. Subsequently, 5a-c were N-methylated by reacting with MeI to obtain the
compounds 7a-c. The intermediates 6a-c and 8a-c were prepared by removing
hydroxyl-protected group. Ethylene carbonate was used to react with 8a-b to obtain 9a-b.
4
-(2-Chloroethyl)morpholine was reacted with 6a, 6b, 8a to prepare 9c-e. Requisite
boc-protected amino acids were coupled with 6c to give 9f-h. And 9i was prepared by
-(methylsulfonyl)propyl4-methyl-benzene- sulfonate coupling with 8a. Then we used the 9a
3
to react with phosphorus oxychloride, directly converted to 9j by treating with sodium
methanolate-methanol solution and converted to 9k by treating with sodium ethoxide-
ethanol solution. (24, 25)
Cell culture and reagents
PI and caspase-3 (C9598) antibody were obtained from Sigma; PARP (AP102) antibody
was purchased from Beyotime Co. (Nantong, Jiangsu, China). Cell lines were purchased from
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American Type Culture Collection (ATCC; Manassas, USA) and cultured in DMEM or RMPI
640 medium following ATCC’s recommendations. The culture medium was supplemented
1
with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1 unit/mL
penicillin-streptomycin (Gibco). The cell lines were maintained at 37 °C under 5% CO2.
Cell proliferation assay
The antiproliferation activities of the compounds were tested in the HepG2 cells, MCF-7
cells, SW620 cells, A549 cells, HCT116 cells, COLO 205 cells, HCT-8 cells, HCC827 cells,
H1993 cells, H460 cells, BEL-7402 cells, SM-7721 cells, ZR-75-1 cells, SKOV-3 cells, PC-3
cells, DU145 cells, A375 cells and A2058 cells. Cells in logarithmic phase were seeded in
9
6-well plates and allowed to adhere (except the K562 cell line, which takes on a suspension
property). Then the cells were incubated with indicated concentrations of the compounds for
4 h. MTT was subsequently added for an extra 2-3 h incubation. The MTT formazan
precipitate was dissolved in DMSO, and the absorbance was measured at a wave length of
70 nm by a Spectramax M5 Microtiter Plate Luminometer (Molecular Devices, Sunnyvale,
2
5
CA, USA).
Flow cytometry
SKOV3 cells were incubated with various concentrations of 9a or DMSO vehicle for 24 or
4
8 h at 37 °C. The cells were collected and washed by PBS, then fixed in cold 70% ethanol
overnight at 4 °C. The cells were washed again by PBS, then the cell DNA was stained with
0 μg/mL PI containing 1 mg/mL of DNase-free RNaseA for a minimum of 10 min. The
5
samples were analyzed by a flowcytometer (BD FACS Calibur, Franklin Lakes, NJ, USA).
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Western blotting
The cells were treated with 9a by the indicated ways. Then the cells were collected and
resuspended in RIPA lysis buffer (Beyotime Co., P0013B, components: 50 mM Tris, pH 7.4,
1
50 mM NaCl,1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium
orthovanadate, sodium fluoride, EDTA, and leupeptin). After 30 min incubation on ice, the
lysates were collected by centrifuging at 12,000 g for 15 min at 4 °C. The protein
concentration was measured. Equivalent samples (30 μg of protein) were subjected to 10% or
1
2% SDS-PAGE, and then the proteins were transferred onto nitrocellulose membranes. After
blocking by 5% non-fat milk for 2 h, the membranes were incubated with the indicated
primary antibodies and subsequently probed by the appropriate secondary antibodies
conjugated to horseradish peroxidase. Immunoreactive bands were visualized using enhanced
chemiluminescence (Millipore, Billerica, USA). The molecular sizes of the proteins detected
were determined by comparison with prestained protein markers (Invitrogen, California,
USA).
Immunofluorescence staining
The immunofluorescence study of microtubule system was conducted generally as describe
(26). SKOV3 cells were seeded into 6-well plates, and then treated as indicated for 16h. The
cells were fixed with 4% paraformaldehyde and then penetrated with PBS containing 0.5%
Triton X-100. After blocking for 30 min in 5% goat serum albumin at room temperature, cells
were incubated with a monoclonal antibody (anti-α-tubulin) at room temperature for 1 h.
Then the cells was washed three times by PBS following staining by fluorescence antibody
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and labeling of Nuclei by 4, 6-diamidino-2- phenylindole (DAPI). Cells were finally washed
thrice and visualized using a fluorescence microscope (OLYMPUS, Tokyo, Japan).
Animal Tumor Models and Treatment
To establish the SM-7721 in vivo model in a method similar as described (27),
7
SM-7721cells (1 × 10 in 100 μl serum-free DMEM) were injected subcutaneously into the
right flanks of 4-6 week old female nude mice. The tumors were allowed to form for 9 days.
3
When the xenografts reached 100 mm , the mice were randomly divided (6 per group). The
mice in the test group received intravenous (i.v.) injection of 9a (10 mg/kg, dissolved in
physiological saline containing 2.5% ethanol and 2.5% Tween-80) every 2 days. The mice in
the vehicle group received i.v. injection of equal amount of physiological saline containing
2
.5% ethanol and 2.5% Tween-80. Those in the adriamycin group (positive control) received
i.v. injection of adriamycin (5 mg/kg, dissolved in physiological saline) every 2 days. Tumor
burden was measured every 2 days with a caliper. Tumor volume (TV) was calculated using
2
the following formula: TV = length × width × 0.52. The very day that treatment started was
defined as day 1 and the treatment ended at day 33. At the end of the experiment, mice were
sacrificed and tumors were collected and weighed. This animal study was conducted in
conformity with institutional guide for the care and use of laboratory animals, and all mouse
protocols were approved by the Animal Care and Use Committee of Sichuan University
(
Chengdu, Sichuan, China).
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Statistical analysis
The results are presented as mean ± SEM (or mean ± SD in the figures). The software
GraphPad Prism 5 was used for statistical analysis. Data were statistically analyzed using
One-way ANOVA. Differences were considered significant if P < 0.05.
Result and discussion
Chemistry
Preparation of 2-(4-((2-chloroquinazolin-4-yl)(methyl)amino)phenoxy)ethanol (9a)
To a solution of 8a (1 mmol, 1.0 eq) in 5 mL DMF at 0 °C was added cesium carbonate (2
mmol, 2.0 eq) and the mixture stirred at 0 °C for 15 min. The reaction was warmed to 100 °C
and ethylene carbonate (1.5 mmol, 1.5 eq) added. The resulting mixture was stirred at 100 °C
for 4 h. The reaction was monitored by TLC. The solution was cooled to rt and 50 mL H O
2
added. The resulting aqueous phase was extracted with ethyl acetate (3×40 mL).The organic
phase was extracted with brine (3×40 mL), dried with MgSO
4
, filtered and concentrated
1
under reduced pressure. Yield: 63%; light yellow solid, melting point: 133.8~135.5 °C. H
NMR (400 MHz, CDCl3) δ 7.75 (d, J = 8.3 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 8.0
Hz, 2H), 7.05 – 6.99 (m, 1H), 6.97 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.6 Hz, 1H), 4.13 (d, J =
13
3
.7 Hz, 2H), 4.02 (d, J = 3.7 Hz, 2H), 3.61 (s, 3H), 2.27(s, 1H). C NMR (101 MHz, DMSO)
δ 162.47, 158.33, 156.13, 152.95, 139.73, 133.49, 128.10, 128.10, 127.68, 126.54, 125.67,
+
1
16.45, 116.45, 114.85, 70.34, 59.99, 43.42. ESI HRMS: calcd. for C17
30.1004, found 330.1003.
Preparation of 2-(4-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)ethanol (9b)
3 2
H16ClN O +H
3
To a solution of 8b (1 mmol, 1.0 eq) in 5 mL DMF at 0 °C was added cesium carbonate (2
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mmol, 2.0 eq) and the mixture stirred at 0 °C for 15 min. The reaction was warmed to 100 °C
and ethylene carbonate (1.5 mmol, 1.5 eq) added. The resulting mixture was stirred at 100 °C
for 4 h. The reaction was monitored by TLC. The solution was cooled to rt and 50 mL H
2
O
added. The resulting aqueous phase was extracted with ethyl acetate (3×40 mL).The organic
phase was extracted with brine (3×40 mL), dried with MgSO
4
, filtered and concentrated
1
under reduced pressure. Yield: 68%; light yellow solid. melting point: 123.8~125.5 °C. H
NMR (400 MHz, DMSO) δ 7.65 (d, J = 7.9 Hz, 1H), 7.58 (dd, J = 11.0, 4.0 Hz, 1H), 7.18 (d,
J = 8.8 Hz, 2H), 7.06 (t, J = 7.2 Hz, 1H), 6.98 (dd, J = 8.1, 5.8 Hz, 3H), 4.90 (t, J = 5.5 Hz,
13
1
H), 4.00 (t, J = 4.8 Hz, 2H), 3.73 (dd, J = 10.0, 5.1 Hz, 2H), 3.49 (s, 3H), 2.59 (s, 3H). C
NMR (101 MHz, DMSO) δ 162.46, 158.28, 156.11, 152.95, 139.83, 133.49, 128.10, 128.10,
1
27.70, 126.54, 125.69, 116.53, 116.45, 114.87, 70.34, 59.99, 43.42, 24.62. ESI HRMS:
+
calcd. for C18
H N
19 3
O
2
+H 310.1550, found 310.1549.
Preparation of 2-chloro-N-(4-(2-morpholinoethoxy)phenyl)quinazolin-4-amine (9c)
A mixture of compound 6a (1 mmol, 1.0 eq) in 8 mL acetonitrile was added cesium
carbonate (3 mmol, 3.0 eq) and the mixture stirred at 80 °C for 15 min and
4
-(2-Chloroethyl)morpholine hydrochloride (1.5 mmol, 1.5 eq) added. The resulting mixture
was stirred at 80 °C for 4 h. The reaction was monitored by TLC. The solution was cooled to
rt and 50 mL H O added. The resulting aqueous phase was extracted with ethyl acetate (3×40
2
mL).The organic phase was extracted with brine (3×40 mL), dried with MgSO , filtered and
4
1
concentrated under reduced pressure. Yield:64 %; light yellow solid, 122.8~124.9 °C. H
NMR (400 MHz, DMSO) δ 9.56 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 7.77 (t, J = 8.6 Hz, 3H),
7
.67 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H), 4.10 (t, J = 5.5 Hz,
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1
3
2
1
H), 3.59 (m, 4H), 2.70 (t, J = 5.5 Hz, 2H), 2.64 (m, 4H). C NMR (101 MHz, CDCl3) δ
58.76, 157.42, 156.28, 151.35, 133.75, 130.50, 128.22, 126.65 123.67, 123.67, 120.60,
115.12, 115.12, 113.37, 66.94, 66.94, 66.11, 57.66, 54.12, 54.12. ESI HRMS: calcd. for
+
C H ClN O +H 385.1426, found 385.1425.
20
21
4
2
Preparation of 2-methyl-N-(4-(2-morpholinoethoxy)phenyl)quinazolin-4-amine (9d)
A mixture of compound 6b (1 mmol, 1.0 eq) in 8 mL acetonitrile was added cesium
carbonate (3 mmol, 3.0 eq) and the mixture stirred at 80 °C for 15 min and
4
-(2-Chloroethyl)morpholine hydrochloride (1.5 mmol, 1.5 eq) added. The resulting mixture
was stirred at 80 °C for 4 h. The reaction was monitored by TLC. The solution was cooled to
rt and 50 mL H O added. The resulting aqueous phase was extracted with ethyl acetate (3×40
mL).The organic phase was extracted with brine (3×40 mL), dried with MgSO , filtered and
2
4
1
concentrated under reduced pressure. Yield:64 %; white solid, 142.8~145.1 °C. H NMR (400
1
MHz, DMSO) H NMR (400 MHz, DMSO) δ 9.56 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 7.77 (t, J
=
8.6 Hz, 3H), 7.67 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H),
4
.10 (t, J = 5.5 Hz, 2H), 3.59 (m, 4H), 2.70 (t, J = 5.5 Hz, 2H), 2.64 (m, 4H), 2.47 (s, 3H).
1
3
C NMR (101 MHz, CDCl
3
) δ 158.78, 157.46, 156.29, 151.36, 133.78, 130.55, 128.28,
1
5
26.66, 123.68, 123.68, 120.58, 115.11, 115.11, 113.37, 66.91, 66.91, 66.08, 57.62, 54.12,
+
4.12, 24.86. ESI HRMS: calcd. for C21
H
24
N
4
O
2
+H 365.1972, found 365.1973.
Preparation of 2-chloro-N-methyl-N-(4-(2-morpholinoethoxy)phenyl)-quinazolin- 4-amine
9e)
A mixture of compound 8a (1 mmol, 1.0 eq) in 8 mL acetonitrile was added cesium
(
carbonate (3 mmol, 3.0 eq) and the mixture stirred at 80 °C for 15 min and
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4
-(2-Chloroethyl)morpholine hydrochloride (1.5 mmol, 1.5 eq) added. The resulting mixture
was stirred at 80 °C for 4 h. The reaction was monitored by TLC. The solution was cooled to
rt and 50 mL H O added. The resulting aqueous phase was extracted with ethyl acetate (3×40
2
mL).The organic phase was extracted with brine (3×40 mL), dried with MgSO , filtered and
4
1
concentrated under reduced pressure. Yield:64 %; light yellow solid, 128.8~130.2 °C. H
NMR (400 MHz, CDCl
3
) δ 7.73 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.14 (d, J = 8.3
Hz, 2H), 7.01 (t, J = 7.7 Hz, 1H), 6.94 (t, J = 9.6 Hz, 3H), 4.16 (t, J = 5.5 Hz, 2H), 3.77 (m,
1
3
4
1
H), 3.60 (s, 3H), 2.87 t, J = 5.5 Hz, 2H), 2.64 (m, 4H). C NMR (101 MHz, DMSO) δ
62.51, 158.08, 156.13, 152.96, 139.85, 133.52, 128.09, 128.09, 127.70, 126.54, 125.69,
116.53, 116.53, 114.87, 66.66, 66.66, 66.12, 57.46, 54.12, 54.12, 43.42. ESI HRMS: calcd.
+
for C21
H
23ClN
4
O
2
+H 399.1582, found 399.1584.
Preparation of 4-(quinazolin-4-ylamino)phenyl 2-((tert-butoxycarbonyl)amino)- acetate
9f)
Boc-glycine (0.45 mmol) was added to a stirred mixture of compound 6c (147.3 mg, 0.4
mmol), EDCI (115.0 mg, 0.6 mmol), DMAP (24.4 mg, 0.2 mmol) in anhydrous CH Cl (5
mL). The mixture was stirred at room temperature for 12 h. On completion, the slurry was
partitioned between water (20 mL) and CH Cl (20 mL), and the water was extracted with
CH Cl (3 × 10mL). The combined extract of CH Cl and the solvent was removed under
(
2
2
2
2
2
2
2
2
reduced pressure to yield a yellow solid. Chromatographic separation (petroleum ether-ethyl
acetate, 10:1) gave the product as a pale yellow solid. Yield: 71%; light yellow solid,
1
1
1
91.2~193.5 °C. H NMR (400 MHz, DMSO) δ 9.87 (s, 1H), 8.60 (s, 1H), 8.55 (d, J = 8.4 Hz,
H), 7.96 – 7.84 (m, 3H), 7.80 (d, J = 8.2 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.41 (t, J = 5.7
This article is protected by copyright. All rights reserved.

13
Hz, 1H), 7.15 (d, J = 8.2 Hz, 2H), 3.98 (d, J = 5.8 Hz, 2H), 1.42 (s, 9H). C NMR (101 MHz,
DMSO) δ 170.06, 158.23, 156.41, 154.92, 150.12, 146.63, 137.29, 133.54, 128.27, 126.76,
1
24.01, 124.01, 123.45, 121.98, 121.98, 115.57, 78.93, 42.68, 28.63, 28.63, 28.63. ESI
+
HRMS: calcd. for C H N O +H 395.1714, found 399.1715.
21
22
4
2
Preparation
of
4-(quinazolin-4-ylamino)phenyl
3-((tert-butoxycarbonyl)amino)-
propanoate (9g)
Boc-beta-alanine (0.45 mmol) was added to a stirred mixture of compound 6c (147.3 mg,
.4 mmol), EDCI (115.0 mg, 0.6 mmol), DMAP (24.4 mg, 0.2 mmol) in anhydrous CH Cl
5 mL). The mixture was stirred at room temperature for 12 h. On completion, the slurry was
partitioned between water (20 mL) and CH Cl (20 mL), and the water was extracted with
CH Cl (3 × 10mL). The combined extract of CH Cl and the solvent was removed under
0
2
2
(
2
2
2
2
2
2
reduced pressure to yield a yellow solid. Chromatographic separation (petroleum ether-ethyl
acetate, 10:1) gave the product as a pale yellow solid. Yield:64 %; light yellow solid,
1
1
1
7
66.5~168.2 °C. H NMR (400 MHz, DMSO) δ 9.87 (s, 1H), 8.60 (s, 1H), 8.55 (d, J = 8.4 Hz,
H), 7.87(d, J = 8.4 Hz, 1H), 7.85 (t, J = 8.5 Hz, 2H), 7.80 (d, J = 8.2 Hz, 1H), 7.65 (t, J =
.6 Hz, 1H), 7.18 (d, J = 8.5 Hz, 2H), 7.03 (s, 1H), 3.31 (t, J = 6.5 Hz, 2H), 2.71 (t, J = 6.5
13
Hz, 2H),1.40 (s, 9H). C NMR (101 MHz, DMSO) δ 170.05, 158.23, 156.41, 154.92, 150.13,
1
46.61, 137.26, 133.52, 128.27, 126.77, 124.05, 124.05, 123.45, 121.93, 121.93, 115.55,
+
7
8.93, 36.68, 35.72, 28.61, 28.61, 28.61. ESI HRMS: calcd. for C H N O +H 409.1870,
22
24
4
2
found 409.1875.
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Preparation
of
4-(quinazolin-4-ylamino)phenyl
2-((tert-butoxycarbonyl)amino)-
propanoate (9h)
N-(tert-butoxycarbonyl)-L-alanine (0.45 mmol) was added to a stirred mixture of
compound 6c (147.3 mg, 0.4 mmol), EDCI (115.0 mg, 0.6 mmol), DMAP (24.4 mg, 0.2
mmol) in anhydrous CH
On completion, the slurry was partitioned between water (20 mL) and CH
the water was extracted with CH Cl (3 × 10mL). The combined extract of CH
2
Cl
2
(5 mL). The mixture was stirred at room temperature for 12 h.
Cl (20 mL), and
Cl and the
2
2
2
2
2
2
solvent was removed under reduced pressure to yield a yellow solid. Chromatographic
separation (petroleum ether-ethyl acetate, 10:1) gave the product as a pale yellow solid. Yield:
1
7
1
0%; light yellow solid, 153.8~155.5 °C. H NMR (400 MHz, DMSO) δ 9.89 (s, 1H), 8.60 (s,
H), 8.55 (d, J = 8.3 Hz, 1H), 7.88 (t, J = 7.4 Hz, 3H), 7.84 – 7.76 (m, 1H), 7.65 (t, J = 7.5
Hz, 1H), 7.53 (d, J = 6.9 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 4.33 – 4.14 (m, 1H), 1.44(m, 3H),
1
3
1
1
.40 (d, J = 16.9 Hz, 9H). C NMR (101 MHz, DMSO) δ 170.08, 158.25, 156.47, 154.98,
50.21, 146.69, 137.31, 133.58, 128.32, 126.82, 124.07, 124.07, 123.45, 121.96, 121.96,
+
24 4 2
115.52, 78.95, 53.58, 28.67, 28.67, 28.67, 17.57. ESI HRMS: calcd. for C22H N O +H
4
09.1870, found 409.1875.
of
quinazolin-4-amine (9i)
mixture of compound 8a (1 mmol, 1.0 eq), 3-(methylsulfonyl)propyl
-methylbenzenesulfonate (1.1 mmol, 1.1 eq) in 5 mL DMF was added cesium carbonate (2
mmol, 2.0 eq) and the mixture stirred at 100 °C for 2 h. The reaction was monitored by TLC.
The solution was cooled to rt and 50 mL H O added. The resulting aqueous phase was
Preparation
2-chloro-N-methyl-N-(4-(3-(methylsulfonyl)propoxy)phenyl)-
A
4
2
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extracted with ethyl acetate (3×40 mL).The organic phase was extracted with brine (3×40
mL), dried with MgSO
4
, filtered and concentrated under reduced pressure. Yield: 64%; light
1
yellow solid. 144.6~146.1 °C. H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.4 Hz, 1H), 7.56 (t,
J = 7.7 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.02 (t, J = 7.7 Hz, 1H), 6.93 (d, J = 8.4 Hz, 3H),
4
2
1
4
.16 (t, J = 5.7 Hz, 2H), 3.61 (s, 3H), 3.28 (dd, J = 17.2, 9.5 Hz, 2H), 2.97 (d, J = 7.6 Hz, 3H),
1
3
.45 – 2.30 (m, 2H). C NMR (101 MHz, DMSO) δ 162.52, 157.85, 156.12, 152.96, 140.05,
33.53, 128.16, 128.16, 127.71, 126.53, 125.72, 116.55, 116.55, 115.06, 66.42, 50.96, 43.41,
+
0.71, 22.40. ESI HRMS: calcd. for C19
H20ClN
3
O
3
S+H 406.0987, found 406.1000.
Preparation of 2-(4-((2-chloroquinazolin-4-yl)(methyl)amino)phenoxy)ethyl dimethyl
phosphate (9j)
Under argon atmosphere, POCl
3
(186 μL,2 mmol) was dissolved in anhydrous CH
2 2
Cl (8
mL); the mixture was then cooled to 0 °C, and anhydrous triethylamine (700 μL, 5 mmol)
was added. After 5 min of stirring, compound 9a (111 mg, 0.39 mmol dissolved in 1 mL of
anhydrous CH Cl ) was added dropwise to the reaction mixture. The resulting mixture was
2
2
stirred for 1 h at 0°C and then allowed to warm to room temperature overnight. The crude
mixture was then evaporated to dryness under reduced pressure to remove the excess NEt
and POCl . The residue was dissolved in CH Cl and evaporated to dryness again. The
3
3
2
2
procedure was repeated twice. Followly, the residue was dissolved in 6 mL 30% sodium
methoxide – methanol solution and the mixture was vigorously stirred for 1 h at 0°C. The
crude mixture was then evaporated to dryness under reduced pressure. The residue was
cooled to rt and 20 mL H
2
O added. The resulting aqueous phase was extracted with ethyl
acetate (3×20 mL).The organic phase was extracted with brine (3×20 mL), dried with MgSO
4
,
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filtered and concentrated under reduced pressure. Yield: 68%; light yellow solid.
1
1
10.5~111.8 °C. H NMR (400 MHz, CDCl
3
) δ 7.77 (d, J = 8.3 Hz, 1H), 7.57 (t, J = 7.6 Hz,
1
H), 7.16 (d, J = 8.3 Hz, 2H), 7.05 – 6.99 (m, 1H), 6.96 (d, J = 8.3 Hz, 2H), 6.92 (d, J = 8.5
13
Hz, 1H), 4.47 – 4.38 (m, 2H), 4.24 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.61 (s, 3H). C NMR
101 MHz, DMSO) δ 162.51, 157.68, 156.12, 152.96, 140.18, 133.52, 128.19, 128.19, 127.70,
(
1
26.53, 125.70, 116.55, 116.55, 114.85, 67.52, 66.21, 54.60, 54.56, 43.38. ESI HRMS: calcd.
+
for C19
H
21ClN
3
O
5
P+H 438.0980, found 438.0998.
Preparation of 2-(4-((2-chloroquinazolin-4-yl)(methyl)amino)phenoxy)ethyl diethyl
phosphate (9k)
Under argon atmosphere, POCl
3
(186 μL, 2 mmol) was dissolved in anhydrous CH
2 2
Cl (8
mL); the mixture was then cooled to 0 °C, and anhydrous triethylamine (700 μL, 5 mmol)
was added. After 5 min of stirring, compound 9a (111 mg, 0.39 mmol dissolved in 1 mL of
anhydrous CH
stirred for 1 h at 0°C and then allowed to warm to room temperature overnight. The crude
mixture was then evaporated to dryness under reduced pressure to remove the excess Et
and POCl . The residue was dissolved in CH Cl and evaporated to dryness again. The
2 2
Cl ) was added dropwise to the reaction mixture. The resulting mixture was
3
N
3
2
2
procedure was repeated twice. Followly, the residue was dissolved in 6 mL 30% sodium
ethoxide-ethanol solution and the mixture was vigorously stirred for 1 h at 0°C. The crude
mixture was then evaporated to dryness under reduced pressure. The residue was cooled to rt
and 20 mL H
2
O added. The resulting aqueous phase was extracted with ethyl acetate (3×20
mL).The organic phase was extracted with brine (3×20 mL), dried with MgSO
4
, filtered and
1
concentrated under reduced pressure. Yield: 72%; light yellow solid. 69.5~70.5 °C. H NMR
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(
400 MHz, CDCl3) δ 7.76 (d, J = 8.2 Hz, 1H), 7.62 – 7.53 (m, 1H), 7.15 (t, J = 6.1 Hz, 2H),
7
4
1
.07 – 6.99 (m, 1H), 6.99 – 6.89 (m, 3H), 4.46 – 4.36 (m, 2H), 4.27 – 4.21 (m, 2H), 4.21 –
13
.10 (m, 4H), 3.61 (s, 3H), 1.36 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl
57.54, 156.35, 152.66, 140.40, 132.78, 128.19, 128.19, 127.54, 126.35, 125.10, 116.15,
16.15, 114.65, 67.18, 65.47, 64.07, 64.01, 43.34, 16.19, 16.00. ESI HRMS: calcd. for
3
) δ162.40,
1
+
21 3 5
C H25ClN O P+H 466.1293, found 466.1319.
Biological activities
Evaluation of the antiproliferative activity
Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells may
undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell
lysis. The cells can also stop actively growing and dividing, or the cells can activate a genetic
program of controlled cell death (apoptosis) (28). The synthesized derivatives 9a-9k were
evaluated for antiproliferative activities in vitro by MTT assay against HepG2 cells (human
hepatocellular carcinoma), A549 cells (human lung cancer), MCF-7 cells (human breast
cancer) and SW620 cells (human colon cancer) (Table 1). Compounds 9a-9b, 9i-9k displayed
potent antiproliferative activities against above-mentioned tumor cell lines (IC values from
50
0
.025 μM to 3.65 μM), particularly 9a, which showed the strongest activity against HepG2
cells and MCF-7 cells (IC50 0.025 μM and 0.032 μM). Howerver, Componds 9c-9h showed
weak activity. Compound 9a, with a Cl group at the 2-positionin and a NMe linker, was
found to be much more active than the other molecules, indicating that the Cl group at the
2
-position of quinazoline and the NMe linker were important for in vitro activity.
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Then we focused on the evaluation of the antiproliferative activity of 9a across various
tumor cells. As summarized in Table 2, the tumor cells were evaluated including cancer cell
lines coming from colon, lung, liver, breast, ovarian, prostate and melanoma cancers. 9a
possessed potent inhibitory activity, with IC values ranged from 0.025 μM to 0.682 μM
50
against all the tested tumor cells lines. In general, the wide range of cell killing across
multiple tumor types may suggest that this kind of compounds induce tumor cell killing by
inhibiting an intrinsically important process of tumor cell division. It is worth noting that 9a
showed potent activity in three human liver tumor cells, with IC50 0.025 μM, 0.059 μM and
0
.021 μM against HepG2, BEL-7402 and SM-7721 cells. 9a was therefore selected for
further analysis of its effects in SM-7721 cells.
a caused a G /M arrest and induced apoptosis
Due to the best antiproliferation activities against SM-7721 cells (IC50 0.025 μM), the
effect of 9a on cell cycle distribution was examined in SM-7721 cells by Propidium Iodide
PI) staining (Figure 2). The data indicated that 9a treatment for 24h could cause a
remarkable G /M arrest, which appeared in a concentration- dependent manner. In the vehicle
control group, the percent of cells in G /M phase was just 5.4%, while the percent increased
9
2
(
2
2
to 12.3%, 17.87%, 22.8%, 35.6% when cells were exposed to 1.25 nM, 2.5 nM, 5 nM, 10 nM
of 9a. When the dose changed to 20 nM, impressively, 51.8% of the cell population was
blocked in G /M phase. As a result of the anti-mitotic drugs induced cell cycle arrest at G2/M
2
phase, it seems that tubulin might be an effective target for 9a.
The apoptosis-inducing effect of 9a was further demonstrated by western blotting. After
treatment for 48 h, it could be revealed that 9a downregulated the protein level of
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pro-caspase-3, which are regarded as important biomarkers of apoptosis (29), in a
concentration-dependent way (Figure 3). Moreover, the protein level of α-tubulin was
obviously downregulated after expose to at the corresponding concentration of 9a. These
trends were in accordance with the anti-mitotic drugs, further indicating that tubulin probably
be an effective target for 9a.
Immunofluorescence staining of microtubule
Blockage of the cell cycle in G2/M phase hinted that 9a may carry on the
tubulin-disrupting property, as the event of microtubule network turbulence often precedes
G2/M phase arrest. To investigate whether the antiproliferative activities of 9a were derived
from an interaction with tubulin, we examined the effect on the cellular microtubule network.
SM-7721 cells were exposed to vehicle control or 1 μM 9a for 24 h, following visualizing of
microtubule structures by staining for DNA (blue) and α-tubulin (green). In contrast with the
vehicle control group, cells treated by 9a exhibited disrupted microtubule organization.
Therefor it could be indicated that 9a acted as a microtubule destabilizer (Figure 4). The
ability to disrupt microtubules could be a bonus to the value of 9a, for this class of agents still
stand as the best for cancer chemotherapy (30).
In vivo anticancer activity
To determine whether the anti-tumor potency of 9a could also be reflected in vivo, we
investigated the effect of 9a on the growth of SM-7721 cells in nude mice. Adriamycin
(ADM), the agent for clinical treatment of liver cancer, was selected as a positive control
drug. SM-7721 xenograft model was established. The mice in the test group received
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intravenous (i.v.) injection of 9a (10 mg/kg, dissolved in physiological saline containing 2.5%
ethanol and 2.5% Tween-80) every 2 days. The mice in the vehicle group received i.v.
injection of equal amount of physiological saline containing 2.5% ethanol and 2.5%
Tween-80. Those in the ADM group (positive control) received i.v. injection of adriamycin (5
mg/kg, dissolved in physiological saline) every 2 days. In the vehicle control group, the mean
3
tumor volume increased from 84.19 ± 12.88 to 1421.31 ± 209.38 mm during a period of 33
days, whereas the change of that of 9a-treated group was from 76.81 ± 12.47 to 433.45 ±
3
2
03.80 mm (Figure 5A). The mean tumor weight of 9a-treated mice was 0.45 ± 0.20 g and
the tumor mass change is 57.0%. As a comparison, the mean tumor volume change of ADM
3
3
group was from 73.71 ± 5.97 mm to 414.72 ± 154.88 mm during the course, and the mean
tumor weight of ADM group was 0.31 ± 0.11 g.
During the therapy, 9a was dissolved well, reflecting its good aqueous solubility. In
9
a-treatment group, no serious side-effects, such as weight loss, feeding, abnormal behavior
were observed (Figure 5B), suggesting 9a is safe and convenient to administrate in vivo. On
the contrary, the body weight of the positive control ADM group dropped severely during the
treatment, consistent with reported high toxicity of this drug. All these results showed that 9a
could inhibit tumor growth significantly and seemed more safety than ADM when possessing
a similar tumor suppression. The exact mechanism still needs further research.
Conclusion
In our research, we described the synthesis of a series of substituted 4-anilinoquinazolines,
which induced apoptotic death of a wide variety of tumor cell lines in the double-digit
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nanomolar range. Subsequently, a flow cytometric study showed that compound 9a
significantly induced cell cycle arrest in G2/M phase at 200 nM. Furthermore, in vitro
immunofluorescence staining, 9a was validated to maintain the microtubule disrupting ability.
In the in vivo investigation, compound 9a exhibited potent inhibitory activity in SM-7721
xenograft tumor models.
In the future, we will focus on discovering more potent compounds that can exhibit
upgraded anticancer activity while remaining well solubility. Simultaneity, the precise
molecular mechanisms of the microtubule disrupting ability of 9a and its apoptosis-
triggering efficiency will be studied.
Acknowledgements
The authors greatly appreciate the financial support from: National Key Programs of China
during the 12th Five-Year Plan Period (2012ZX09103101-009); National Natural Science
Foundation of China (81373283 and 81272458); Program for New Century Excellent Talents
in University (NCET)-12-0381; Sichuan Province Science and Technology Support
Program-2014SZ0045.
Conflicts of Interest
The authors declare no conflict of interest.
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SUPPORT INFORMATION
Supplementary data associated with this article can be found in the online version (The
procedures for the synthesis of 2, 3, 5a-5c, 6a-6c, 7a-7c, 8a-8c).
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Figure legends
Scheme Captions
Scheme 1.
2
Reagents and conditions: (a) MOMCl, NaH, DMF, 0 °C, 2 h; (b)10% Pd/C, H , EtOH, r.t.,
1
2
2 h; (c) AcONa, THF/H O, 60 °C, 2 h; (d) MeI, NaH, DMF, 0 °C, 15 min, r.t. 1 h; (e) HCl,
ethyl acetate, 0 °C, 1 h, r.t. 2 h; (f) EDCi, DMAP, DCM, r.t., 12h; (g) Cs CO , DMF, 100 °C,
2
3
2
2 3 3 2 3 3 3
h or Cs CO , CH CN, 80 °C, 4 h; (h) Cs CO , DMF, 100 °C, 4 h; (i) step A: Et N, POCl , 1
h at 0°C, r.t. 12 h; step B: 30% sodium ethoxide-ethanol solution or 30% sodium
ethoxide-ethanol solution, 0 °C, 1 h.
Table Captions
Table 1. The antiproliferative activities of test compounds against four cancer cell lines.
Table 2. Activity of 9a against various human tumor cell lines.
Figure Captions
Figure 1. Some reported anticancer agents.
Figure 2. 9a treatment of cancer cells results in G2/M arrest. SM-7721 cells were treated
with 9a at 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM for 24 h, and control group indicates the
vehicle control.
Figure 3. Protein levels of pro-caspase-3, α-tubulin were determined by Western blotting.
SM-7721 cell lines were treated with 9a at 40 nM, 200 nM, 1000 nM for 48 h, with β-actin as
a loading control. Control group indicates the vehicle control.
Figure 4. Effect of 9a on the organization of cellular microtubule network. SM-7721 cells
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were untreated (Control) and treated with 9a at concentration of 1.0 μM for 24 h.
Microtubules and unassembled tubulin are shown in green and the Nuclei in blue.
Figure 5. 9a inhibited tumor growth in SM-7721 xenograft model. The in vivo experiment
was done as described in the experimental section. (A) Tumor volume time curve. (B) Body
weight time curve (C) The picture of the stripping tumor from mice. (D) Bar chart of tumor
weight. *, P<0.05, **, P<0.01, and ***, P<0.001, significantly different compared with
vehicle control by one-way ANOVA.
Table 1. The antiproliferative activities of test compounds against four cancer cell lines.
a
IC50 (μM)
Compds
HepG2
0.025
0.129
>5.0
A549
0.056
0.147
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
MCF-7
0.032
0.099
>5.0
SW620
0.089
0.152
>5.0
9
a
9
b
9
c
9
d
>5.0
>5.0
>5.0
9
e
>5.0
>5.0
>5.0
9
f
>5.0
>5.0
>5.0
9
g
>5.0
>5.0
>5.0
9
h
>5.0
>5.0
>5.0
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9
i
1.25
0.88
0.92
2.36
1.37
1.90
1.12
1.05
0.99
3.56
4.12
3.65
9j
9
k
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%.
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Table 2. Activity of 9a against various human tumor cell lines.
Tumor type
Cell line
HCT116
COLO 205
HCT-8
IC50 (μM)
0.058
0.143
0.032
0.165
0.038
0.033
0.025
0.059
0.021
0.032
0.123
0.682
0.124
0.063
0.088
0.033
0.042
0.037
Colon
HCC827
H1993
Lung
Liver
Breast
H460
HepG2
BEL-7402
SM-7721
MCF-7
ZR-75-1
MDA-MB-231
SKOV-3
A2780S
PC-3
Ovarian
Prostate
DU145
A375
Melanoma
A2058
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