Design and synthesis of novel quinazoline nitrogen mustard derivatives as potential therapeutic agents for cancer

Article abstract of DOI:10.1016/j.ejmech.2013.06.055

Thirteen novel quinazoline nitrogen mustard derivatives were designed, synthesized and evaluated for their anticancer activities in vitro and in vivo. Cytotoxicity assays were carried out in five cancer cell lines (HepG2, SH-SY5Y, DU145, MCF-7 and A549) and one normal human cell line (GES-1), in which compound 22b showed very low IC₅₀ to HepG2 (the IC₅₀ value is 3.06 μM), which was lower than Sorafenib. Compound 22b could inhibit cell cycle at S and G₂/M phase and induce cell apoptosis. In the HepG2 xenograft model, 22b exhibited significant cancer growth inhibition with low host toxicity in vivo.

Full text of DOI:10.1016/j.ejmech.2013.06.055

European Journal of Medicinal Chemistry 67 (2013) 293e301
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel quinazoline nitrogen mustard
derivatives as potential therapeutic agents for cancer
,
,
,
Shilei Li a 1, Xiao Wang a 1, Yong He a b, Mingxia Zhao a, Yurong Chen a, Jingli Xu a,
Man Feng a, Jin Chang a, Hongyu Ning a, Chuanmin Qi a
,
*
a Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
b Experimental Chemistry Center, Beijing Normal University, Beijing 100875, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen novel quinazoline nitrogen mustard derivatives were designed, synthesized and evaluated for
their anticancer activities in vitro and in vivo. Cytotoxicity assays were carried out in five cancer cell lines
(HepG2, SH-SY5Y, DU145, MCF-7 and A549) and one normal human cell line (GES-1), in which com-
Received 27 March 2013
Received in revised form
2 June 2013
Accepted 24 June 2013
Available online 4 July 2013
pound 22b showed very low IC50 to HepG2 (the IC50 value is 3.06 mM), which was lower than Sorafenib.
Compound 22b could inhibit cell cycle at S and G2/M phase and induce cell apoptosis. In the HepG2
xenograft model, 22b exhibited significant cancer growth inhibition with low host toxicity in vivo.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Quinazoline
Nitrogen mustard
Anticancer
Cell cycle
Apoptosis
Xenograft model
1. Introduction
developed in clinical application. Many of these drugs, such as
Chlorambucil [6e8], Melphalan [9e11], Cyclophosphamide [12e
As an important pharmacophore, quinazoline has a variety of
biological activities, such as anticancer [1,2], antibacterial [3], anti-
inflammatory [4] and so on. Anticancer has been widely used in
clinical therapy. FDA has approved several quinazoline derivatives
as anticancer drugs, such as Gefitinib, Erlotinib, Lapatinib and so on
(1, 2 and 3, Fig. 1). Based on the good performances of quinazoline
derivatives in anticancer application, development of novel qui-
nazoline derivatives as anticancer drugs is a promising field.
Nitrogen mustard was the earliest bifunctional alkylating agent
[5]. By interrupting DNA biosynthesis and in doing so caused DNA
damage, the nitrogen mustard derivatives possess huge genotox-
icity. Until now, a lot of nitrogen mustard derivatives have been
14] and so on (5, 6 and 7, Fig. 1), have revolutionized the treat-
ment of cancers. However, these drugs still have many drawbacks,
the most severe shortcoming is the host toxicity, which induces
many side effects to the patients [15]. To conquer the problem,
researchers have made lots of attempts. Currently, the research of
nitrogen mustard derivatives is mainly concentrating on targeting.
To achieve this goal, there are three major ways: (1) attaching ni-
trogen mustard to glucose, for example, D19575 (8, Fig. 1). Re-
searchers found that it was assimilated by glucose transportation
pharmacodynamics and the toxicity of the compound was small
[16e19]; (2) using amino acids as carriers of nitrogen mustard, for
example, Melphalan [20](6, Fig. 1); (3) affinity tumor heterocyclic
pharmacophore was used as transportor [21e25]. As a heterocyclic
pharmacophore, quinazoline and its derivatives are ideal carriers
for nitrogen mustard. Herein, we designed and synthesized a series
of quinazoline nitrogen mustard derivatives by introducing nitro-
gen mustard group to 4-aminoquinazoline directly. In order to seek
out novel potential anticancer agents, we conducted the cytotox-
icity experiment in vitro. Furthermore, we elucidated the possible
anticancer mechanism of the newly synthesized compounds by
conducting cell cycle analysis and cellular apoptosis experiments.
Abbreviations: DMAP, 4-dimethylaminopyridine; MTT, (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide; HepG2, human liver carcinoma cell line;
SH-SY5Y, human neuroblastoma cell line; MCF-7, human breast cancer cell line;
A549, human alveolar adenocarcinoma cell line; DU145, human prostate cancer cell
lines; PI, propidium iodide; HCC, hepatic carcinoma; FBS, fetal bovine serum; RNase
A, ribonuclease A; PBS, phosphate buffer solution.
* Corresponding author. Tel.: þ86 13681380097; fax: þ86 10 58802075.
E-mail address: qicmin@sohu.com (C. Qi).
These authors contributed equally.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.055

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S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
O
F
HN
N
Cl
HN
O
HN
Cl
HN
N
Br
H
N
O
S
O
F
O
O
N
O
O
O
O
O
N
O
O
N
N
N
• HCl
N
N
3. Lapatinib
Cl
1. Erlotinib
2. Gefitinib
4. PD153035
Cl
Cl
Cl
OH
Cl
N
P
O
O
HN
P
HN
N
HO
N
Cl
NH2
COOH
O
O
Cl
HO
HN
O
OH
COOH
Cl
6. Melphalan
5. Chlorambucil
7. Cyclophosphamide
8. D19575
Fig. 1. Several aminoquinazoline and nitrogen mustard derivatives.
At last, we carried out anticancer therapy of compound 22b in
HepG2 xenograft model.
As shown in Scheme 2, beginning from compound ethyl 3,4-
dihydroxybenzoate (10), we synthesized the key intermediate 4-
chloro-6,7-bis(2-methoxyethoxy)quinazoline (15) after 5 steps re-
action by following literature [26]. Products were often purified by
column chromatography [26] in step a (Scheme 2), which way is
inefficient. In this study, we purified the residue by recrystallization
in petroleum ether in high yield. Abiding by the similar measures in
Scheme 1, we developed the compounds 16aec.
2. Chemistry
The synthesis procedure of the new aminoquinazoline de-
rivatives is shown in Schemes 1e3. We introduced nitrogen
mustard group to the ortho, meta or para position of 4-benzene
ring of the leading compounds. All the newly synthesized com-
pounds were characterized by NMR, IR and MS, and their structures
were confirmed. 9aec (6,7-dimethoxy-4-aminoquinazolin de-
rivatives) were synthesized as shown in Scheme 1. The key inter-
mediate 4-chloro-6,7-dimethoxyquinazoline (6, Scheme 1) was
synthesized from 3,4-dimethoxybenzoic acid (1, Scheme 1) in
accordance with the literature method [26]. The preparation of
N1,N1-bis(2-chloroethyl)benzene diamine (8aec, Scheme 1) were
started from 1-chloro-2-nitrobenzene [27], 3-nitroaniline [27] and
1-chloro-4-nitrobenzene [27], respectively. Finally, we successfully
prepared three new compounds 9aec (Scheme 1) in high yield by
reacting intermediate 6 with 8aec [26].
Scheme 3 shows the synthesis protocol of a series of novel 4-
aminoquinazoline nitrogen mustard derivative. In the first step,
the 6-methoxy group of intermediate 4 was unprotected by L-
methionine in Methansulfonic at 120 ꢀC; Next, the 6-hydroxyl
group was protected by acetyl group in pyridine and acetic anhy-
dride which was catalyzed by DMAP; Then, compound 18 was
treated with POCl3 in toluene under reflux condition to produce 4-
chloro-7-methoxyquinazolin-6-yl acetate (19); Fourthly, products
20aec were prepared by coupling of 19 with 8aec in Isopropanol
under reflux; In step f, hydrolysis of the acetyl group of 20b or 20c
was carried out by using sodium hydroxide reacting with 20b or
20c in the mixture of methanol and water at 40 ꢀC to give products
Scheme 1. Synthesis protocol of quinazoline nitrogen mustard derivatives (9aec).

S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
295
Scheme 2. Synthesis protocol of quinazoline nitrogen mustard derivatives (16aec).
21bec (Scheme 3). Ultimately, targeting compounds 22bec were
obtained by reacting 21bec with 4-(3-chloropropyl)morpholine in
DMF and in the presence of Potassium Carbonate at 80 ꢀC under
nitrogen environment.
Sorafenib and Gefitinib were used as positive control. The cyto-
toxicity of the new compounds was shown in Table 1 in form of IC50
that was calculated by linear regression analysis of the concentra-
tioneresponse curves of each compound. We can see that 9a, 9b
and 9c exhibited modest cytotoxicity to HepG2, MCF-7, A549 and
DU145, 9b possessed moderate SH-SY5Y cell line proliferation in-
3. Results and discussion
hibition with the IC50 value at 12.43 mM; 16a, 16b, 16c had small
cytotoxicity in MCF-7, A549 and DU145. For against SH-SY5Y, 16b
and 16c exhibited moderate cytotoxic effects with the IC50 value at
3.1. In vitro cytotoxicity
16.85 and 13.16
activity against HepG2 with the IC50 value at 11.33
m
M, respectively. 16a possessed stronger inhibitory
The effect of all the newly synthesized compounds on cancer cell
proliferation was measured by MTT assay [28,29], and clinical drugs
mM; Compound
Scheme 3. Synthesis protocol of quinazoline nitrogen mustard derivatives (20aec; 21b, c; 22b, c).

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S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
Table 1
The cell cytotoxicity of newly synthesized quinazoline nitrogen mustard derivatives.
Compound
IC50 (mM)
HepG2
SH-SY5Y
MCF-7
A549
DU145
9a
9b
9c
16a
16b
16c
20a
20b
20c
21b
21c
77.24
17.45
29.95
11.33
33.49
84.97
48.37
29.12
47.05
23.26
46.72
3.06
22.61
12.43
7.36
89.21
>100
51.11
97.72
>100
>100
57.06
39.88
28.85
67.40
41.86
10.54
54.95
11.34
NDa
81.54
>100
>100
33.14
77.35
>100
62.86
39.14
28.79
32.97
53.37
6.71
>100
>100
>100
>100
>100
>100
45.79
16.85
13.16
28.18
23.98
17.35
21.14
43.89
8.60
99.41
21.59
49.23
41.54
75.19
20.04
16.95
24.91
22b
22c
Sorafenib
Gefitinib
12.19
8.42
29.79
20.72
19.54
18.21
27.71
27.71
NDa
>100
a
Not determined.
20aec, 21b and 21c showed moderate cytotoxicity to the five tested
cell lines, however, after the acetyl or 6-hydroxy group at 6-position
was substituted by 4-(3-chloropropyl)morpholine, the cytotoxicity
increased enormously. Compound 22b possessed remarkable cell
proliferation inhibition against HepG2, SH-SY5Y and A549.
On the other side, Table 1 showed that some of the key com-
pounds have selective cytotoxicity. Compound 16a had high se-
lective cytotoxicity against HepG2 cell line, while there were weak
growth inhibition to the other cell lines; 9b targeted SH-SY5Y cell
line with a relatively high cytotoxicity.
The result of compounds 9c, 16a, 22b, 22c against human gastric
epithelial cell line (GES-1) was shown in Table 2. Cytotoxicity of 22b
against GES-1 was almost similar to Sorafenib, and 16a, 22c
possessed smaller cytotoxicity than Sorafenib.
Most of the classical nitrogen mustard derivatives usually exhibit
high and broad-spectrum toxicity. In this research, our objective is to
decrease the toxicity and increase the targeting by introducing ni-
trogen mustard group to the 4-aminoquinazoline, the result
confirmed the designing. It’s very interesting to evaluate the anti-
cancer activities of the new compounds further. The cell viability in
various concentrations of 22b along with time is shown in Fig. 2.
3.2. Cell cycle inhibition
Reports showed that DNA alkylating agents could induce DNA
damage, lead to cell cycle delay and arrest the cell cycle progression
predominantly at the G2/M boundary [30]. Thus, we studied the
influence of 22b on cell cycle distribution (Fig. 3). At first, the HepG2
cells were treated with various concentrations of 22b for 48 h, after
that, the cells were harvested, fixed in 70% ice-cold ethanol, stained
with PI and analyzed by a flow cytometer. The percentages of HepG2
cells in G1, S, and G2/M phases were calculated and shown in Fig. 4.
Each value represents the percentage of cells in the noted cell cycle
phase and is the average value of three independent determinations.
Cell cycle analysis showed a decrease of G1 and increase of S and G2/
M phase after 22b treatment for 48 h in HepG2. Dose-dependent
accumulation of S and G2/M phase after treatment of 22b was
observed. Furthermore, dose-dependent increase in the “sub G1”
Fig. 2. The cell viability curves in different concentration of 22b.
phase, which is the indication of the late apoptotic or dead cells, was
also observed after the treatment (Fig. 3)
3.3. Cell apoptosis
To seek out whether compound 22b could induce cell apoptosis,
we conducted the cell apoptosis experiment. Annexin V-FITC&PI
double-staining assay was used to detect the proportion of HepG2
cell apoptosis after treatment of 22b for 48 h [31]. The result is
shown in Figs. 5 and 6, the upper right section represents the late
apoptotic or necrotic cells, the lower right section represents the
cells which were in the early or middle apoptosis. Compared with
control, 22b increased the apoptosis percentage of HepG2 what-
ever the late apoptotic or necrotic cells or the early or middle
apoptotic cells after treatment of 22b for 48 h. Results showed
that 22b increased cell apoptosis in a dose-dependent manner.
The experiment confirmed that compound 22b could induce cell
apoptosis.
Table 2
IC50 of some key compounds against GES-1 for 48 h.
Cell line
GES-1
9c
Compound
16a
22b
22c
Sorafenib
15.80
IC50
(m
M)
6.27
25.98
14.26
20.55

S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
297
Fig. 3. Influences of compounds 22b on cell cycle of HepG2 for 48 h.
3.4. In vivo therapeutic activity
cell lines and the introduction of quinazoline overall decreased the
toxicity of nitrogen mustard. Mechanism studies demonstrated
that compound 22b could induce cell cycle delay and arrests the
cell cycle progression predominantly at the S and G2/M boundary,
and induce cell apoptosis. On the basis of above studies, we suc-
cessfully established xenograft model bearing HepG2 HCC and
carried out in vivo therapy experiment. Compound 22b could
induce tumor growth regression rate at 26.50% and 47.29% in
comparison with negative control at dose levels of 10 mg/kg and
20 mg/kg body weight, respectively. The result is positive. The body
weight changing indicated that the host toxicity of 22b is small.
This research proved that using affinity tumor heterocyclic
quinazoline as transporter of nitrogen mustard is a practicable way
and might provide some effective clues for the development of
novel antitumor nitrogen mustard or quinazoline derivatives.
In order to evaluate the therapeutic effect and assess the toxicity
of 22b, we conducted the in vivo therapeutic experiment [32]. The
HepG2 xenograft models were generated by injecting HepG2 cell
suspension in the right flank of each female nude mouse. Eight days
later, the average tumor weight ranged from 150 to 160 mg with 6
mice per group, therapy was started. 22b was administered via
intravenous injection every other day for 7 times at dose levels of
10 mg/kg and 20 mg/kg body weight, respectively. Positive control
Sorafenib and negative control vehicle were administered in the
same way as 22b. The result is shown in Fig. 7, compound 22b
exhibited significant tumor growth inhibition. When the dosage of
22b was 10 mg/kg, it caused 26.50% tumor growth regression in
comparison with negative control; when the dosage was 20 mg/kg,
the inhibition rate reached 47.29%, it was even better than the
positive control Sorafenib at the same dose level. On the other
hand, 22b has a very slight toxicity in vivo. When the dosage of 22b
was 10 mg/kg, the mean body weight variation (Fig. 8) of mice was
as same as the negative control. When the dosage of 22b was
20 mg/kg, it only slightly decreased the mean body weight of mice
(Fig. 8), which was similar to the positive control Sorafenib.
Therefore, we could conclude that compound 22b has an effective
therapy for HepG2 HCC tumors and is equipped with slightly host
toxicity in vivo.
5. Experimental section
5.1. General remarks
All commercial reagents and solvents were used without further
purification. Melting points were determined in capillary tubes
using a RY-1 apparatus and are uncorrected. 1H NMR and 13C NMR
were performed on
100 MHz), and chemical shifts (
a
Brucker spectrometer (400 MHz and
values) were reported as parts per
d
million (ppm) downfield from TMS. Mass spectra were recorded
using a Brucker Apex IV FTM instrument at Mass Spectroscopy
Center in Beijing Normal University by using high-resolution ESI or
EI. IR spectra were recorded on a SHIMADZU Affinity-1 infrared
spectrometer. The purity of the target compounds (9aec, 16aec,
20aec, 21b, c and 22b, c) was determined by analytical HPLC, and
was more than 95%.
4. Conclusion
In conclusion, we have successfully synthesized 13 new quina-
zoline nitrogen mustard derivatives, evaluated their antitumor
activities in vitro and in vivo, and studied their antitumor mecha-
nism. Most of them exhibited selective cytotoxicity toward certain
HepG2, SH-SY5Y, MCF-7, A549 and DU145 were obtained from
Cell Resource Center, Chinese Academy of Medicinal Sciences
(CAMS) and cultured in RPMI 1640 or DMEM medium containing
10% FBS in 5% CO2 at 37 ꢀC. Unless otherwise indicated, all medium
contained 100 U/mL penicillin and 100
mg/mL streptomycin. All
protocols requiring the use of mice were approved by the animal
care committee of Beijing Normal University.
5.2. Synthesis
5.2.1. N1,N1-Bis(2-chloroethyl)-N2-(6,7-dimethoxyquinazolin-4-yl)
benzene-1,2-diamine (9a)
N1,N1-Bis(2-chloroethyl)benzene-1,2-diamine (8a, 0.4 mmol,
93 mg) was added into a solution of 4-chloro-6,7-dimethox-
yquinazoline (6, 0.4 mmol, 90 mg) in Isopropanol (4 mL), the
mixture was stirred and heated at 80 ꢀC for 1 h under nitrogen
atmosphere. The reaction was stopped and stirred at room
Fig. 4. HepG2 Cell cycle distribution after the treatment of 22b for 48 h.

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S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
Fig. 5. Effects of compound 22b on cell apoptosis of HepG2 for 48 h.
temperature overnight, and then the reaction mixture was poured
into ice/water mixture under stirring, resulting in the precipitation
of solid, the precipitate was filtered and dried on the air to afford the
yellowish product 9a (140 mg, 76.5% yield). mp: 122 ꢀC. 1H NMR
100.51, 56.65 (d, J ¼ 10 Hz), 56.42 (d, J ¼ 10 Hz), 55.07, 42.34. MS
(ESIþ) m/z: 421.5 [M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1): 3413, 3252, 2924,
2850, 2353, 1626, 1561, 1509, 1433, 1351, 1276.5, 1235, 1159, 1068,
986, 850, 773, 661.
(400 MHz, CDCl3):
d
10.23 (s, 1H, eNH), 8.84 (d, 1H, J ¼ 8.2 Hz, ArH),
8.74 (s, 1H, ArH), 7.88 (s, 1H, ArH), 7.58 (s, 1H, ArH), 7.42 (m, 1H, ArH),
7.36 (m,1H, ArH), 7.27 (m,1H, ArH), 4.12 (s, 3H, -OCH3), 4.07 (s, 3H, e
OCH3), 3.55 (t, 4H, J ¼ 5.2 Hz, eCH2e), 3.45 (t, 4H, J ¼ 5.2 Hz, eCH2e).
5.2.2. N1,N1-Bis(2-chloroethyl)-N3-(6,7-dimethoxyquinazolin-4-yl)
benzene-1,3-diamine (9b)
N1,N1-Bis(2-chloroethyl)benzene-1,3-diamine (8b, 0.4 mmol,
93 mg) was added into a solution of 4-chloro-6,7-dimethoxyq-
uinazoline (6, 0.4 mmol, 90 mg) in Isopropanol (4 mL), the
mixture was stirred and heated at reflux for 1 h under nitrogen
atmosphere resulting in the precipitation of an orange solid. The
reaction was stopped and stirred at room temperature overnight,
13C NMR (100 MHz, DMSO-d6):
d 158.22, 156.11, 149.99, 148.96,
144.46, 136.30, 132.48, 127.68, 126.90, 124.67, 123.52, 107.21, 103.36,
Fig. 6. The apoptotic proportion of HepG2 after treatment with compound 22b for
Fig. 7. Effects of compound 22b in vivo against HepG2 HCC tumors in mice. Therapy
48 h.
was began from day 9 via intravenous injection every other day for 7 times.

S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
299
eOCH2e), 3.88 (d, J ¼ 4 Hz, 4H, eOCH2e), 3.50 (m, 14H, eCH2CH2e,
eCH3O). 13C NMR (100 MHz, CDCl3):
155.97,154.27,153.40,148.85,
d
147.38, 138.06, 137.19, 127.31, 123.82, 123.06, 120.79, 110.14, 108.74,
102.58, 70.83, 70.55, 68.92, 68.37, 59.37, 59.33, 58.03, 42.52. MS
(ESIþ) m/z: 508.8 [M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1): 3437, 3319,
2926, 2878, 2814, 1619, 1596, 1578, 1506, 1456, 1390, 1240, 1207,
1127, 923, 857, 748, 660.
5.2.5. N1-(6,7-Bis(2-methoxyethoxy)quinazolin-4-yl)-N3,N3-bis(2-
chloroethyl)benzene-1,3-diamine hydrochloride (16b)
N1,N1-Bis(2-chloroethyl)benzene-1,3-diamine (8b, 0.6 mmol,
140 mg) was added into
a solution of 4-chloro-6,7-bis(2-
methoxyethoxy)quinazoline (15, 0.6 mmol, 135 mg) in Iso-
propanol (6 mL), the mixture was stirred and heated at reflux for
1 h under nitrogen atmosphere resulting in the precipitation of an
orange solid. Then the reaction was stopped and stirred at room
temperature overnight, the precipitate was filtered, washed with
hot Isopropanol and dried on the air to afford the yellowish-green
product 16b (230 mg, 84% yield). mp: 214e216 ꢀC. 1H NMR
Fig. 8. The mean body weight variation of the bearing HCC mice during the process of
therapy.
the precipitate was filtered, washed with hot i-PrOH and dried on
the air to afford the yellowish-green product 9b (143 mg, 78%
yield). mp: 250 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 11.40 (s, 1H, e
NH), 8.77 (s, 1H, ArH), 8.35 (s, 1H, ArH), 7.35 (s, 1H, ArH), 7.29 (t,
J ¼ 8.1 Hz, 1H, ArH), 7.10 (s, 1H, ArH), 7.00 (d, J ¼ 7.7 Hz, 1H, ArH),
6.71 (dd, J1 ¼1.8 Hz, J2 ¼ 8 Hz, 1H, ArH), 4.02 (s, 3H, eOCH3), 3.99 (s,
3H, eOCH3), 3.78 (m, 8H, eCH2CH2e). 13C NMR (100 MHz, DMSO-
(400 MHz, DMSO-d6): d 11.41 (s, 1H, eNH), 8.77 (s, 1H, ArH), 8.40 (s,
1H, ArH), 7.39 (s, 1H, ArH), 7.29 (t, J ¼ 7.7 Hz, 1H, ArH), 7.10 (s, 1H,
ArH), 6.99 (d, J ¼ 7. 6 Hz, 1H, ArH), 6.71 (d, J ¼ 7.8 Hz, 1H, ArH), 4.38
(s, 2H, eOCH2e), 4.32 (s, 2H, eOCH2e), 3.78 (s, 12H, eOCH2e, e
CH2CH2e), 3.38 (s, 6H, eCH3O). 13C NMR (100 MHz, DMSO-d6):
d6):
d 158.10, 156.09, 150.03, 148.51, 146.61, 137.90, 135.55, 129.47,
113.24, 109.90, 108.53, 107.18, 103.99, 99.79, 56.95 (d, J ¼ 7 Hz),
56.35 (d, J ¼ 7 Hz), 52.17, 41.09. MS (ESIþ) m/z: 421.4 [M ꢁ Clꢁ]þ. IR
(KBr pellet, cmꢁ1): 3422, 3210, 2937, 2837, 1634, 1572, 1513, 1407,
1213, 1157, 998.
d 158.06, 155.41, 149.21, 148.47, 146.60, 138.00, 135.49, 129.37,
113.22, 109.82, 108.56, 107.31, 105.37, 100.74, 69.92, 69.75, 69.11,
68.66, 58.38, 58.32, 52.25, 41.12. MS (ESIþ) m/z: 509.4 [M ꢁ Clꢁ]þ. IR
(KBr pellet, cmꢁ1): 3425, 2926, 2821, 2583, 1633, 1591, 1571, 1530,
1513, 1465, 1418, 1299, 1229, 1168.
5.2.3. N1,N1-Bis(2-chloroethyl)-N4-(6,7-dimethoxyquinazolin-4-yl)
benzene-1,4-diamine (9c)
5.2.6. N1-(6,7-Bis(2-methoxyethoxy)quinazolin-4-yl)-N4,N4-bis(2-
chloroethyl)benzene-1,4-diamine hydrochloride (16c)
N1,N1-Bis(2-chloroethyl)benzene-1,4-diamine (8c, 0.4 mmol,
93 mg) was added into a solution of 4-chloro-6,7-dimethoxyquin-
azoline (6, 0.4 mmol, 90 mg) in Isopropanol (4 mL), the mixture
was stirred and heated at reflux for 1 h under nitrogen atmosphere
resulting in the precipitation of an orange solid. The reaction was
stopped and stirred at room temperature overnight, the precipitate
was filtered, washed with hot Isopropanol and dried on the air to
afford the yellowish-green product 9c (132 mg, 72% yield). mp:
N1,N1-Bis(2-chloroethyl)benzene-1,4-diamine (8c, 0.6 mmol,
140 mg) was added into
a solution of 4-chloro-6,7-bis(2-
methoxyethoxy) quinazoline (15, 0.6 mmol, 135 mg) in Iso-
propanol (6 mL), the mixture was stirred and heated at reflux for
1 h under nitrogen atmosphere resulting in the precipitation of an
orange solid. Then the reaction was stopped and stirred at room
temperature overnight, the precipitate was filtered, washed with
hot Isopropanol and dried on the air to afford the yellowish-green
product 16c (222 mg, 81% yield). mp: 165 ꢀC. 1H NMR (400 MHz,
200 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 11.23 (s, 1H, -NH), 8.73 (s,
1H, ArH), 7.49 (d, J ¼ 8.7 Hz, 2H, ArH), 7.33 (s, 1H, ArH), 6.85 (d,
J ¼ 8.8 Hz, 2H, ArH), 4.00 (s, 3H, eOCH3), 3.98 (s, 3H, eOCH3), 3.78
DMSO-d6):
d 11.24 (s, 1H, eNH), 8.72 (s, 1H, ArH), 8.31 (s, 1H, ArH),
(m, 8H, eCH2CH2e). 13C NMR (100 MHz, DMSO-d6):
d
158.79,
7.49 (d, J ¼ 8.9 Hz, 2H, ArH), 7.36 (s, 1H, ArH), 6.85 (d, J ¼ 9.0 Hz, 2H,
157.60, 155.86, 149.92, 148.46, 144.79, 126.06, 111.55, 106.97, 104.01,
103.97, 99.65, 56.82, 56.28, 51.99, 41.04. MS (ESIþ) m/z: 421.4
[M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1): 3422, 2938, 1631, 1577, 1514, 1436,
1404, 1277, 1235, 1150, 1068.
ArH), 4.32 (m, 4H, eCH2O), 3.78 (s, 12H, eOCH2e, eCH2CH2e), 3.34
(s, 6H, eOCH3). 13C NMR (100 MHz, DMSO-d6):
d 157.54, 155.17,
149.11, 148.28, 144.77, 134.79, 126.35, 126.07, 111.56, 107.08, 105.40,
100.52, 69.94, 69.75, 69.07, 68.62, 58.38, 58.32, 52.12, 41.12. MS
(ESIþ) m/z: 509.4 [M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1): 3421, 2927,
2886, 1632, 1577, 1516, 1448, 1366, 1277, 1239, 1182.
5.2.4. N1-(6,7-Bis(2-methoxyethoxy)quinazolin-4-yl)-N2,N2-bis(2-
chloroethyl)benzene-1,2-diamine hydrochloride (16a)
N1,N1-Bis(2-chloroethyl)benzene-1,2-diamine (8a, 0.6 mmol,
5.2.7. 4-(2-(Bis(2-chloroethyl)amino)phenylamino)-7-
140 mg) was added into
a
solution of 4-chloro-6,7-bis(2-
methoxyquinazolin-6-yl acetate (20a)
methoxyethoxy) quinazoline (15, 0.6 mmol, 135 mg) in Iso-
propanol (6 mL), the mixture was stirred and heated at 80 ꢀC for
40 min under nitrogen atmosphere. Then the reaction was cooled
down and added into 10 mL H2O, the mixture was extracted with
ethyl acetate (15 mL ꢂ 3), the organic layer was collected, dried
over Na2SO4 and evaporated by rotary evaporation under vacuum.
The resident was chromatographed over a column of silica gel
(A:P ¼ 1:2) to give a yellow solid. The solid was recrystallized in the
mixture of ethyl acetate and petroleum ether to afford the
yellowish product 16a (187.2 mg, 68.3% yield). mp: 102e103 ꢀC. 1H
A solution of 4-chloro-7-methoxyquinazolin-6-yl acetate (19,
0.33 mmol, 162 mg) and N1,N1-bis(2-chloroethyl)benzene-1,2-
diamine (8a, 0.33 mmol, 77 mg) in Isopropanol (7 mL) was stir-
red and heated to reflux under nitrogen atmosphere, reaction
progress was monitored by TLC and no starting materials were
detected after 30 min. The reaction was cooled down and added
into 10 mL H2O, the mixture was extracted with chloroform (CHCl3)
(17 mL ꢂ 3), the organic layer was collected, dried over Na2SO4 and
evaporated by rotary evaporation under vacuum. Then the resident
was chromatographed over a column of silica gel (A:P ¼ 1:5) to give
a yellowish solid. The solid was recrystallized in the mixture of
ethyl acetate and petroleum ether to afford the grayish white
product 20a (49 mg, 20.5% yield). mp: 166e168 ꢀC. 1H NMR
NMR (400 MHz, CDCl3):
ArH), 8.69 (s,1H, ArH), 7.48 (s,1H, ArH), 7.33 (t, J ¼ 7.72 Hz,1H, ArH),
d
9.28 (s, 1H, eNH), 8.87 (d, J ¼ 8.16 Hz, 1H,
7.26 (m, 1H, ArH), 7.08 (t, J ¼ 7.36 Hz, 1H, ArH), 4.30 (d, J ¼ 4 Hz, 4H,

300
S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
(400 MHz, CDCl3):
d
9.43 (s, 1H, eNH), 8.90 (d, J ¼ 8.3 Hz, 1H, ArH),
2H, ArH), 6.45 (d, J ¼ 7.6 Hz, 1H, ArH), 3.95 (s, 3H, eOCH3), 3.74 (m,
8H, eCH2CH2e). 13C NMR (100 MHz, DMSO-d6):
156.23, 153.70,
152.09, 146.51, 146.37, 146.01, 140.89, 129.16, 110.52, 109.72, 107.02,
106.78, 105.37, 55.82, 52.38, 40.98. MS (ESIþ) m/z: 406.9 [M þ H]þ.
IR (KBr pellet, cmꢁ1): 3389, 2963, 2359, 2335, 1616, 1576, 1501,
1429, 1352, 1232, 1148, 1009.
8.75 (s, 1H, ArH), 7.95 (s, 1H, ArH), 7.34 (t, J ¼ 8.0 Hz, 1H, ArH), 7.29
(d, J ¼ 8.0 Hz, 2H, ArH), 7.11 (t, J ¼ 8.0 Hz, 1H, ArH), 3.98 (s, 3H,
CH3COe), 3.53 (t, J ¼ 5.6 Hz, 4H, eCH2e), 3.43 (t, J ¼ 5.6 Hz, 4H, e
CH2e), 2.38 (s, 3H, eOCH3). 13C NMR (100 MHz, DMSO-d6):
d
d
168.90, 156.59, 155.75, 155.05, 139.85, 137.61, 137.37, 127.40,
124.02, 123.34, 120.52, 115.33, 109.75, 108.58, 57.78, 56.26, 42.20,
20.61. MS (ESIþ) m/z: 448.4 [M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1): 3447,
3314, 2972, 2371, 2345, 1767, 1628, 1597, 1524, 1506, 1389, 1206,
1163, 1132, 914.
5.2.11. 4-(4-(Bis(2-chloroethyl)amino)phenylamino)-7-
methoxyquinazolin-6-ol (21c)
Compound 20c (0.25 mmol, 120 mg) was added into CH3OH
(2.4 mL) and heated to 40 ꢀC, then the solution of NaOH (5 mol/L,
0.24 mL) was added dropwise. The reaction mixture was stirred at
40 ꢀC, reaction progress was monitored by TLC and no starting
materials were detected after 2 h. The reaction mixture was cooled
to room temperature and the PH was adjusted to 5e6 with 1 mol/L
HCl. The product was precipitated and filtered, washed with ice
water and dried on the air to give a yellow solid 21c (82 mg, 81%).
5.2.8. 4-(3-(Bis(2-chloroethyl)amino)phenylamino)-7-
methoxyquinazolin-6-yl acetate (20b)
A solution of 4-chloro-7-methoxyquinazolin-6-yl acetate (19,
0.46 mmol, 116 mg) and N1,N1-bis(2-chloroethyl)benzene-1,3-
diamine (8b, 0.46 mmol, 107 mg) in Isopropanol (5 mL) was stir-
red and heated to reflux under nitrogen atmosphere, reaction
progress was monitored by TLC and no starting materials were
detected after 1 h. The reaction mixture was cooled to room tem-
perature and the obtained precipitate was filtered through a glass
funnel, washed with hot isopropanol and dried on the air to afford
the yellow product 20b (113 mg, 51%). mp: 236e238 ꢀC. 1H NMR
mp: 196 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 9.63 (s, 1H, eOH), 9.32
(s, 1H, eNH), 8.34 (s, 1H, ArH), 7.78 (s, 1H, ArH), 7.57 (d, J ¼ 8.8 Hz,
2H, ArH), 7.15 (s,1H, ArH), 6.77 (d, J ¼ 8.8 Hz, 2H, ArH), 3.96 (s, 3H, e
OCH3), 3.74 (m, 8H, eCH2CH2e). 13C NMR (100 MHz, DMSO-d6):
d
156.50, 153.64, 152.00, 146.42, 144.80, 142.84, 129.44, 124.29,
(400 MHz, DMSO-d6):
d
11.24 (s, 1H, eNH), 8.84 (s, 1H, ArH), 8.69 (s,
111.89, 109.27, 106.41, 105.67, 55.81, 52.34, 41.24. MS (ESIþ) m/z:
406.8 [M þ H]þ. IR (KBr pellet, cmꢁ1): 3491, 3389, 3067, 2959, 2882,
2363, 1616, 1516, 1431, 1248, 1204, 1180, 1003, 920, 856, 814.
1H, ArH), 7.49 (s, 1H, ArH), 7.27 (t, J ¼ 7.6 Hz, 1H, ArH), 7.05 (m, 2H,
ArH), 6.69 (d, J ¼ 7.3 Hz, 1H, ArH), 3.99 (s, 3H, CH3COe), 3.75 (s, 8H,
eCH2CH2e), 2.37 (s, 3H, eOCH3). 13C NMR (100 MHz, DMSO-d6):
d
173.64, 163.94, 162.65, 155.77, 151.92, 145.43, 144.30, 142.96,
5.2.12. N1,N1-Bis(2-chloroethyl)-N3-(7-methoxy-6-(3-
134.75, 123.76, 118.14, 115.34, 113.37, 112.20, 106.32, 62.21, 57.47,
46.30, 25.36. MS (ESIþ) m/z: 448.7 [M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1):
3412, 3208, 3017, 2949, 2662, 2361, 2344, 1769, 1638, 1570, 1501,
1433, 1368, 1144.
morpholinopropoxy)quinazolin-4-yl)benzene-1,3-diamine (22b)
Compound 21b (0.11 mmol, 45 mg) and Potassium Carbonate
(0.24 mmol, 30 mg) was added into DMF (1.44 mL) and heated to
40 ꢀC for 20 min under nitrogen atmosphere, and then 4-(3-
chloropropyl) morpholine (20
mL, 17.7 mg) was added dropwise.
5.2.9. 4-(4-(Bis(2-chloroethyl)amino)phenylamino)-7-
methoxyquinazolin-6-yl acetate (20c)
The reaction mixture was stirred at 80 ꢀC for 5 h, reaction progress
was monitored by TLC and reaction found to be completed after this
time. The reaction was cooled down and added into 10 mL H2O, the
mixture was extracted with chloroform (CHCl3) (10 mL ꢂ 3), the
organic layer was collected, washed by water (15 mL ꢂ 3), dried over
Na2SO4 and evaporated by rotary evaporation under vacuum.
The resident was chromatographed over a column of silica gel
(A:P ¼ 1:2) to give a yellowish solid. The solid was recrystallized in
the ethyl acetate to afford the yellowish product 22b (27 mg, 46%
A solution of 4-chloro-7-methoxyquinazolin-6-yl acetate (19,
0.57 mmol, 144 mg) and N1,N1-bis(2-chloroethyl)benzene-1,4-
diamine (8b, 0.57 mmol, 133 mg) in Isopropanol (6 mL) was stir-
red and heated to reflux under nitrogen atmosphere, reaction
progress was monitored by TLC and no starting materials were
detected after 1 h. The reaction mixture was cooled to room tem-
perature and the obtained precipitate was filtered through a glass
funnel, washed with hot isopropanol and dried on the air to afford
the yellowish product 20c (196 mg, 71%). Carbonization tempera-
yield). mp: 178e179 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 9.37 (s, 1H,
eNH), 8.42 (s, 1H, eArH), 7.83 (s, 1H, ArH), 7.35 (s, 1H, ArH), 7.20 (m,
2H, ArH), 7.04 (d, J ¼ 8.0 Hz, 1H, ArH), 6.53 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 2.0 Hz, 1H, ArH), 4.19 (t, J ¼ 6.3 Hz, 2H, eCH2e), 3.93 (s, 3H, e
OCH3), 3.77 (m, 8H, eCH2CH2e), 3.58 (t, J ¼ 4.5 Hz, 4H, eCH2e), 2.50
(t, J ¼ 7.0 Hz, 2H, eCH2e), 2.40 (s, 4H, eCH2e),1.99 (q, J ¼ 6.4 Hz, 2H,
ture: 142 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 11.30 (s,1H, eNH), 8.81
(s, 1H, ArH), 8.71 (s, 1H, ArH), 7.52 (d, J ¼ 8.0 Hz, 3H, ArH), 6.84 (d,
J ¼ 8.0 Hz, 2H, ArH), 3.99 (s, 3H, CH3COe), 3.77 (s, 8H, eCH2CH2e),
2.38 (s, 3H, eOCH3). 13C NMR (100 MHz, DMSO-d6):
d 168.35,157.99,
157.14, 150.43, 144.94, 140.00, 138.66, 126.09, 125.74, 118.52, 111.61,
106.85, 100.91, 56.87, 52.06, 41.10, 20.11. MS (ESIþ) m/z: 448.7
[M ꢁ Clꢁ]þ. IR (KBr pellet, cmꢁ1): 3397, 3003, 2967, 2776, 2367, 2345,
1769, 1638, 1614, 1518, 1439, 1369, 1182, 1148, 1067, 1013, 918, 816.
eCH2e). 13C NMR (100 MHz, DMSO-d6):
d 156.48, 154.35, 152.75,
148.11, 146.92, 146.41, 140.53, 129.15, 111.25, 109.01, 107.27, 106.40,
103.02, 67.22, 66.13, 55.78, 54.93, 53.38, 52.35, 41.00, 25.86. MS
(ESIþ) m/z: 534 [M þ H]þ. IR (KBr pellet, cmꢁ1): 3341, 2949, 2810,
2384, 2291, 1611, 1578, 1510, 1431, 1240, 1152, 1009, 860, 750.
5.2.10. 4-(3-(Bis(2-chloroethyl)amino)phenylamino)-7-
methoxyquinazolin-6-ol (21b)
5.2.13. N1,N1-Bis(2-chloroethyl)-N4-(7-methoxy-6-(3-
Compound 20b (0.21 mmol, 100 mg) was added into CH3OH
(2 mL) and heated to 40 ꢀC, then the solution of NaOH (5 mol/L,
0.2 mL) was added dropwise. The reaction mixture was stirred at
40 ꢀC, reaction progress was monitored by TLC and no starting
materials were detected after 2 h. The reaction mixture was cooled
to room temperature and the PH was adjusted to 5e6 with 1 mol/L
HCl. The product was precipitated and filtered, washed with ice
water and dried on the air to give a yellow solid 21b (77 mg, 91%).
Carbonization temperature: 231 ꢀC. 1H NMR (400 MHz, DMSO-d6):
morpholinopropoxy)quinazolin-4-yl)benzene-1,4-diamine (22c)
Compound 21c (0.12 mmol, 50 mg) and Potassium Carbonate
(0.24 mmol, 33.2 mg) was added into DMF (1.6 mL) and heated to
40 ꢀC for 20 min under nitrogen atmosphere, and then 4-(3-
chloropropyl) morpholine (20
mL, 17.7 mg) was added dropwise.
The reaction mixture was stirred at 80 ꢀC for 4.5 h, reaction progress
was monitored by TLC and reaction found to be completed after this
time. The reaction was cooled down and added 10 mL H2O, the
mixture was extracted with chloroform (CHCl3) (10 mL ꢂ 3), the
organic layer was collected, washed by water (15 mL ꢂ 3), dried over
Na2SO4 and evaporated by rotary evaporation under vacuum. The
d
9.68 (s, 1H, eNH), 9.19 (s, 1H, eOH), 8.39 (s, 1H, ArH), 7.77 (s, 1H,
ArH), 7.31 (s, 1H, ArH), 7.26 (d, J ¼ 7.6 Hz, 1H, ArH), 7.15 (t, J ¼ 7.6 Hz,

S. Li et al. / European Journal of Medicinal Chemistry 67 (2013) 293e301
301
resident was chromatographed over
a
column of silica gel
the first treatment. Tumor volumes were calculated using the
equation (l ꢂ w2)/2, where l and w refer to the larger and smaller
dimensions collected at each measurement. The density of the tu-
mor is equal to the water.
(A:P ¼ 1:2) to give a yellowish solid. The solid was recrystallized in
the Toluene to afford the yellowish product 22c (38 mg, 60% yield).
mp: 192 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 9.33 (s, 1H, eNH), 8.34
(s, 1H, ArH), 7.81 (s, 1H, ArH), 7.52 (d, J ¼ 8.4 Hz, 2H, ArH), 7.14 (s, 1H,
ArH), 6.80 (d, J ¼ 8.6 Hz, 2H, ArH), 4.17 (d, J ¼ 5.6 Hz, 2H, eCH2e),
3.92 (s, 3H, eOCH3), 3.75 (s, 8H, eCH2CH2e), 3.58 (s, 4H, eCH2e),
Acknowledgments
2.47 (m, 2H, eCH2e), 2.30 (s, 4H, eCH2e), 1.99 (s, 2H, eCH2e). 13
C
This work was supported by National Natural Science Founda-
tion of China (No. 21071022) and the Fundamental Research Funds
for the Central Universities. We especially thank Mr. Xiao Wang for
his excellent work and kindly help for what we have done in the
paper.
NMR (100 MHz, DMSO-d6):
d 156.61, 153.99, 153.01, 147.85, 146.49,
142.95, 129.05, 124.73, 111.76, 108.61, 107.09, 102.75, 67.00, 66.06,
55.66, 54.87, 53.31, 52.24, 41.15, 25.76. MS (ESIþ) m/z: 533.7.
[M þ H]þ. IR (KBr pellet, cmꢁ1): 3385, 3264, 3076, 2945, 2812, 2766,
2371, 1624, 1582, 1516, 1435, 1248, 1117, 993, 862, 814, 745, 619, 546.
Appendix A. Supplementary data
5.3. Cell viability assay
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.055.
Cell viability was determined by using the MTT assay. Cells were
planted at 3000 per well in 96-well plates and incubated 24 h in
RPMI 1640 or DMEM medium containing 10% FBS in 5% CO2 at
37 ꢀC. After that, various concentrations of compounds were added
into wells and cultured for another 72 h. Then, the MTT solution
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Female nude mouse bearing HepG2 HCC were prepared for the
in vivo research. The xenograft model was generated by subcu-
taneous injection of 1 ꢂ 106 HepG2 cells into the right frank of fe-
male nude mouse without anesthesia. The experiment was
performed after the tumor cells were inoculated for 7e14 days, at
which time the mice weight between 18 and 22 g and the tumor
weight between 140 and 160 mg. Compounds and vehicle was
administered via intravenous injection every other day, tumor
dimension and body weights were recorded every two days from