Synthesis and antitumor activity of novel quinazoline derivatives containing thiosemicarbazide moiety

Article abstract of DOI:10.1016/j.ejmech.2012.06.003

Series of novel derivatives of quinazoline containing thiosemicarbazide moiety 5 and 9 have been synthesized and tested for their antitumor activities in vitro against a panel of five human cancer cell lines. Bioassay results indicated that most of the prepared compounds exhibited cytotoxicity against various cancer cells. From the structure-activity relationships it was found that unsubstituted quinazoline ring and benzene ring or halogen substituted benzene ring in quinazoline derivatives 5 and 9 would be the most favorable for their antitumor activity.

Full text of DOI:10.1016/j.ejmech.2012.06.003

European Journal of Medicinal Chemistry 54 (2012) 925e930
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antitumor activity of novel quinazoline derivatives containing
thiosemicarbazide moiety
,
,
,
b,
Junbo He a 1, Xiaoguo Wang a 1, Xiaoqin Zhao b, YongJu Liang b, Hongwu He a , Liwu Fu
*
*
a Key Laboratory of Pesiticide & Chemistry Biology, Ministry of Education, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, PR China
b State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou 510060, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of novel derivatives of quinazoline containing thiosemicarbazide moiety 5 and 9 have been
synthesized and tested for their antitumor activities in vitro against a panel of five human cancer cell
lines. Bioassay results indicated that most of the prepared compounds exhibited cytotoxicity against
various cancer cells. From the structureeactivity relationships it was found that unsubstituted quina-
zoline ring and benzene ring or halogen substituted benzene ring in quinazoline derivatives 5 and 9
would be the most favorable for their antitumor activity.
Received 22 September 2011
Received in revised form
1 June 2012
Accepted 2 June 2012
Available online 12 June 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Synthesis
Quinazoline
Thiosemicarbazide
Antitumor activity
1. Introduction
transduction [16]. Some anilinoquinazoline compounds have been
approved for the treatment of breast cancer, for example, Gefitinib
With the acknowledgment that cytotoxic chemotherapeutic
agents could be used to target cancers exhibiting high proliferative
rates [1,2], great attention has been attracted and noticeable prog-
ress has been made in the discovery and development of modern
anticancer drug over the years. However, despite advances that have
led to the development of new therapies, cancer continues to be
a major health problem in the worldwide due to various factors
[3e5]. Therefore, discovering newer and safer anticancer agents
remains critically important and continues to be a challenge [6].
In the course of finding novel chemical substances that may
serve as innovative antitumor agents, quinazoline derivatives are of
particular interest. These quinazoline derivatives have been iden-
tified as new class of cancer chemotherapeutic agents against solid
tumors [7e12] and are known to be potent inhibitors of epidermal
growth factor receptor (EGFR) [13e15] composed of the cellular
trans-membrane tyrosine kinases. The epidermal growth factor
receptor (EGFR) is over-expressed in many human tumors. 4-
Anilinoquinazoline derivatives exert their antitumor activity
through the inhibition of the EGFR over-expression by obstructing
the EGFR autophosphorylation and EGF-stimulated signal
(IressaTM) [17], Erlotinib (TarcevaTM) [18]. Based on the importance
of these molecules, the synthesis of novel quinazoline derivatives
and their functionalized derivatives is of high interest.
On the other hand, thiosemicarbazide also exhibits significant
biological activities, ranging from antitumor, fungicidal, bacteri-
cidal, anti-inflammatory and antiviral activities [19e21]; therefore,
the thiosemicarbazide is a highly efficient pharmacophore in drug
molecular design. In order to find novel potent quinazoline anti-
tumor agents, which may act on the same enzyme target where the
4-anilinoquinazoline derivatives act, this research focuses on
designing some new quinazoline derivatives, wherein the active
pharmacophores thiosemicarbazide have been attached at the 4th
position of the quinazoline ring, and the antitumor activity of these
new compounds was evaluated. This reasearch reports the
synthesis of these novel quinazoline derivatives containing thio-
semicarbazide moiety and the evaluation of their in vitro antitumor
activity against various human cancer cell lines.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel./fax: þ86 (0) 27 67867960.
E-mail addresses: he1208@mail.ccnu.edu.cn (H. He), fulw@mail.sysu.edu.cn
(L. Fu).
2.1.1. Synthesis of compounds 5ae5j
The reaction sequence employed for synthesis of the title
compounds 5ae5j is shown in Scheme 1. The starting material 2-
1
Co-first authors.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.003

926
J. He et al. / European Journal of Medicinal Chemistry 54 (2012) 925e930
Table 1
2-amino-4,5-dimethoxybenzoic acid (1) was conveniently cyclized
The preparation of compounds 5ae5j and 9ae9j.
to 6,7-dimethoxyquinazolin-4(3H)-one (6) by heating it with
guanidine acetate at 140 ꢀC in N2 atmosphere, and then compound
6 yielded the corresponding 4-chloro derivative 7 upon refluxing
with freshly distilled sulfurous dichloride as described in the
literature [9]. The 4-chloro derivative 7 reacted with hydrazine
hydrate in tetrahydrofuran medium to give 4-hydrazinyl-
6,7-dimethoxyquinazoline (8) in good yield [26]. The key inter-
mediate 4-hydrazinyl-6,7-dimethoxyquinazoline (8) was readily
converted to the title compounds 9ae9j (Table 1) by reacting it
with different substituted benzoyl isothiocyanates. All the synthe-
sized compounds 9ae9j were confirmed by 1H NMR, 13C NMR and
element analysis data, explained in the experimental part.
Compound
R
Appearance
Yield(%)a
Mp(oC)
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
2-Cl
3-Cl
4-Cl
2-F
4-F
4-CF3
2-CH3O
2-CH3
4-CH3
H
2-Cl
3-Cl
4-Cl
2-F
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
Yellow solid
70
57
64
68
76
80
69
65
86
88
56
77
68
72
92
63
80
71
66
82
213e216
215e216
205e207
>260
216e218
>260
227e229
205e207
218e220
216e218
186e188
212e214
193e194
224e226
197e199
199e201
223e225
206e208
215e216
198e199
5j
9a
9b
9c
9d
9e
9f
9g
9h
9i
2.2. Biological evaluation
4-F
2.2.1. In vitro cytotoxicity and structureeactivity relationship
4-CF3
2-CH3O
2-CH3
4-CH3
The in vitro antitumor activity of the newly synthesized
compounds 5ae5j, 9ae9j, and a series of mixtures of thio-
semicarbazide (T) and quinazoline intermediates (2, 3, 4, 6, 7, 8)
was evaluated against a panel of five human cancer cell lines,
including KB (oral carcinoma cell), CNE2 (nasopharyngeal carci-
noma cell), MGC-803 (gastric carcinoma cell), MCF-7 (breast
adenocarcinoma cell), and GLC-82 (human lung cancer cell) by
MTT method [27]. 5-FU was chosen as a reference drug [28,29] due
to its availability and widespread use. The inhibitory activities
(IC50) are summarized in Table 2. As depicted in Table 2, the
mixtures of thiosemicarbazide (T) and quinazoline intermediates
9j
a
Isolated yield.
aminobenzoic acid was conveniently cyclized to quinazolin-4(3H)-
one (2) by heating it with formamide at 125e130 ꢀC as described in
the literature [22]. Upon refluxing with freshly distilled phosphorus
oxychloride and pentachlorophosphorane, compound 2 yielded the
corresponding 4-chloro derivative 3 [23], which produced 4-
hydrazinylquinazoline (4) in moderate yield when subjected to
condensation with hydrazine hydrate in alcoholic medium [24].
The key intermediate 4-hydrazinylquinazoline (4) was readily
converted to the title compounds 5ae5j (Table 1) by reacting it
with different substituted benzoyl isothiocyanates, which were
prepared by reacting substituted benzoyl chloride with potassium
thiocyanate in dry acetonitrile [25].
2e4, 6e8 displayed no cytotoxic activities (IC50 > 50
most of the synthesized compounds exhibited more potent cyto-
toxic activities (IC50 < 10 M) against the three or five human
mM), however,
m
cancer cell lines in comparison with 5-FU. Special compounds 5a,
5b 5c, 5d, 5e, 5f, 9a, 9b, 9c, 9d, and 9e displayed higher activity
than 5-FU against all five human cancer cell lines. Although most
compounds showed potent antitumor activity (IC50 < 10
mM),
The structures of all the newly synthesized compounds 5ae5j
were confirmed by 1H NMR, 13C NMR and element analysis data,
explained in the experimental part. The 1H NMR spectrum of 5a,
some compounds displayed selectivity between the five human
cancer cell lines. For KB cell line, the compounds 5a, 5e, and 5f
showed the more potent inhibitory activity with IC50
the appearance of broad signals at
the presence of eNHCO, eNHC
respectively.
d
14.34, 12.08, and 11.21 revealed
N, and eNHCS protons,
2.33 ꢁ 0.62
For CNE2 cell line, the compounds 5a and 5d showed the best
M,
m
M, 2.51 ꢁ 0.81
m
M and 1.87 ꢁ 0.40
mM respectively.
¼
inhibitory activity with IC50 1.49 ꢁ 0.38
mM and 1.53 ꢁ 0.68
m
respectively. For MGC-803 cell line, the compounds 5d and 9h
2.1.2. Synthesis of compounds 9ae9j
exhibited more potent inhibitory activity with corresponding IC50
The reaction sequence employed for synthesis of the title
compounds 9ae9j is also shown in Scheme 1. The starting material
1.86 ꢁ 0.80
m
M and 1.59 ꢁ 0.13
mM. However, for MCF-7 cell line
and GLC-82 cell line, only compounds 5c showed the better
Scheme 1. Reagents and conditions: (a) HCONH2, 125e127 ꢀC, 5 h; (b) POCl3, PCl5; (c) NH2NH2ꢂH2O (85%), EtOH, reflux; (d) ArCONCS, dioxane, rt; (e) guanidine acetate, 140 ꢀC, N2,
10 h; (f) SOCl2, 78 ꢀC; (g) NH2NH2ꢂH2O (85%), THF, rt; (h) ArCONCS, dioxane, rt.

J. He et al. / European Journal of Medicinal Chemistry 54 (2012) 925e930
927
Table 2
In vitro cytotoxicity of compounds 5ae5j, 9ae9j, and the mixtures of thiosemicarbazide (T) and quinazoline intermediates 2e4, 6e8.
a
Compound
R
In vitro cytotoxicity IC50
KBb
(mM)
CNE2b
MGC-803b
MCF-7b
GLC-82b
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
2-Cl
3-Cl
4-Cl
2-F
4-F
4-CF3
2-CH3O
2-CH3
4-CH3
H
2.33 ꢁ 0.62
7.71 ꢁ 1.34
4.74 ꢁ 0.75
3.94 ꢁ 1.59
2.51 ꢁ 0.81
1.87 ꢁ 0.40
7.92 ꢁ 3.01
>50
2.77 ꢁ 0.76
10.1 ꢁ 2.10
3.77 ꢁ 1.31
3.56 ꢁ 0.80
4.83 ꢁ 0.65
4.59 ꢁ 1.22
4.07 ꢁ 1.24
4.53 ꢁ 1.00
8.84 ꢁ 1.97
3.85 ꢁ 0.69
11.4 ꢁ 2.45
24.2 ꢁ 7.80
>50
1.49 ꢁ 0.38
6.09 ꢁ 1.63
2.92 ꢁ 0.59
1.53 ꢁ 0.68
4.61 ꢁ 2.02
2.84 ꢁ 0.80
22.5 ꢁ 8.75
15.7 ꢁ 3.90
5.46 ꢁ 2.04
17.2 ꢁ 3.30
8.18 ꢁ 0.78
9.67 ꢁ 1.07
7.46 ꢁ 3.22
7.53 ꢁ 2.48
12.7 ꢁ 5.40
16.1 ꢁ 3.11
16.5 ꢁ 6.63
8.41 ꢁ 1.80
10.5 ꢁ 2.22
33.3 ꢁ 9.81
>50
6.99 ꢁ 1.38
9.14 ꢁ 2.07
4.54 ꢁ 1.03
1.86 ꢁ 0.80
5.01 ꢁ 2.01
3.60 ꢁ 0.59
10.6 ꢁ 2.80
14.3 ꢁ 4.00
4.24 ꢁ 1.63
3.76 ꢁ 0.67
3.05 ꢁ 1.33
6.44 ꢁ 1.12
4.06 ꢁ 0.38
3.71 ꢁ 0.71
2.39 ꢁ 0.43
8.30 ꢁ 0.85
9.28 ꢁ 3.88
1.59 ꢁ 0.13
11.1 ꢁ 1.30
19.7 ꢁ 7.20
NTd
5.96 ꢁ 1.43
10.5 ꢁ 3.30
3.54 ꢁ 1.03
5.96 ꢁ 2.56
5.02 ꢁ 0.85
4.59 ꢁ 1.06
8.05 ꢁ 0.94
8.7 ꢁ 3.00
16.5 ꢁ 6.70
7.82 ꢁ 2.17
6.99 ꢁ 1.85
23.1 ꢁ 5.60
11.5 ꢁ 1.60
8.94 ꢁ 3.82
8.50 ꢁ 1.54
25.8 ꢁ 5.00
16.8 ꢁ 7.90
8.80 ꢁ 1.70
19.3 ꢁ 1.90
22.3 ꢁ 5.20
NT
8.37 ꢁ 3.45
10.8 ꢁ 4.20
3.24 ꢁ 0.53
6.68 ꢁ 3.13
6.46 ꢁ 2.54
6.07 ꢁ 1.77
12.2 ꢁ 2.1
9.5 ꢁ 4.40
13.2 ꢁ 4.30
11.2 ꢁ 0.50
6.55 ꢁ 1.32
10.0 ꢁ 1.96
8.37 ꢁ 1.38
9.62 ꢁ 1.90
8.64 ꢁ 1.52
15.3 ꢁ 4.60
16.5 ꢁ 2.90
16.0 ꢁ 3.00
21.5 ꢁ 4.1
29.9 ꢁ 3.90
NT
5j
9a
9b
9c
9d
9e
9f
9g
9h
9i
2-Cl
3-Cl
4-Cl
2-F
4-F
4-CF3
2-CH3O
2-CH3
4-CH3
9j
T
T
T
c and 2
c and 3
c and 4
>50
>50
>50
>50
>50
>50
>50
>50
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
Tc and 6
T
T
c and 7
c and 8
>50
>50
NT
NT
NT
5-FU
8.28 ꢁ 1.50
15.1 ꢁ 6.40
18.5 ꢁ 4.17
14.2 ꢁ 7.70
17.4 ꢁ 1.40
a
IC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control.
b
KB cells were the human oral carcinoma cells and CNE2 cells were the human nasopharyngeal carcinoma cells, MGC-803 cells were the human gastric carcinoma cells,
MCF-7 cells were the human breast adenocarcinoma cells and GLC-82 cells were the human lung cancer cells.
c
T means the thiosemicarbazide, the mass ratio of the thiosemicarbazide and quinazoline intermediates 2-4, 6-8 is 1:1, respectively.
NT means not tested.
d
inhibitory activity with IC50 3.54 ꢁ 1.03
m
M and 3.24 ꢁ 0.53
m
M,
with unsubstituted quinazoline ring and benzene ring or chloro or
fluoro substituted benzene ring were found to be the most favor-
able for the antitumor activity, which will help us to further design
more potent antitumor agents. These findings also have encour-
aged us to further investigate their mechanisms of action.
respectively.
From the antitumor activity against five human cancer cell
lines, preliminary structureeactivity relationships of the
synthesized compounds were obtained. In general, compared
with compounds 5ae5j, the introduction of methoxyl on position
of 6 and 7 of the quinazoline ring (compounds 9ae9j) led to
equivalent or decreased cytotoxicity, for example, the inhibitory
activity 5a > 9a, 5d > 9d, 5f > 9f. On the other hand, substitutions
on the benzene ring had great effect on the cytotoxicity. More-
over, compounds with no substitution (5a) or halogen substituent
(5d and 5f) on the benzene ring showed higher inhibitory
activity. In comparison to compounds 5d and 5f, the compound
5g (R ¼ 4-CF3), with bulky and electron-withdrawing group on
the benzene ring, exhibited greater reduction in the inhibitory
activity. Furthermore, the introduction of methyl and methyoxy
groups on the benzene ring (5he5j and 9he9j) also lowered the
inhibitory activity. From the above-mentioned analysis, it can be
concluded that the compounds with unsubstituted quinazoline
ring and benzene ring or chloro or fluoro substituted benzene
ring were found to be the most favorable for the antitumor
activity.
4. Experimental
4.1. General procedures
1H NMR spectra were recorded in DMSO-d6 solution on a Varian
Mercury-Plus 600 spectrometer at 600 MHz, 13C NMR spectra were
recorded in DMSO-d6 solution on a Varian Mercury-Plus 400
spectrometer at 400 MHz, or a Varian Mercury-Plus 600 spec-
trometer at 600 MHz and chemical shifts were recorded in parts per
million (ppm) with TMS as the internal reference. Elemental anal-
yses were performed by a Vario EL III elemental analyzer. Melting
points were measured on an electrothermal melting point appa-
ratus and were uncorrected. Chemicals and reagents were obtained
from commercial sources, and all of the solvents were anhydrous.
Column chromatography was carried out with Merch silica gel
(230e400 mesh).
3. Conclusion
4.2. Preparation of 4-hydrazinylquinazoline (4) [24]
In summary, we have designed and synthesized novel series of
quinazoline derivatives containing thiosemicarbazide moiety. Five
human cancer cell lines were used to measure cytotoxic activity of
the synthesized quinazoline derivatives. The preliminary investi-
gation showed some compounds displayed higher activity against
all five human cancer cell lines than 5-FU. The results of the
structureeactivity relationships indicated that the compounds
Compound 3 (10 mmol) and hydrazine hydrate 85% (30 mmol)
in 50 mL of ethanol was heated under reflux for 4 h. The reaction
mixture was concentrated and allowed to cool. The solid product
obtained was filtered, washed with water and dried. Recrystalli-
zation with ethanol afforded the desired solid compound 4, mp.
163e164 ꢀC in 64% yield. 1H NMR (DMSO-d6, 600 MHz)
d 4.73 (s, 2H,

928
J. He et al. / European Journal of Medicinal Chemistry 54 (2012) 925e930
NH2), 7.45 (s, 1H, AreH), 7.69 (d, 2H, J ¼ 31.2 Hz, AreH), 8.16 (s, 1H,
AreH), 8.47 (s, 1H, CH), 9.64 (s, 1H, NH).
AreH), 8.03 (d, 1H, J ¼ 7.2 Hz, AreH), 8.11 (s, 1H, CH), 11.27 (s, 1H,
NHCS), 12.08 (s, 1H, NH), 14.13 (s, 1H, NHCO); 13C NMR (DMSO-d6,
100 MHz) d 116.0, 116.2, 116.7, 117.0, 122.2, 124.0, 124.6, 126.1, 130.4,
4.3. General procedure for the synthesis of title compounds
(5ae5j) [30]
131.9, 133.9, 136.1, 147.6, 149.3, 158.0, 160.5, 164.1, 172.2; Anal. Calcd.
For C16H12FN5OS: C, 56.30; H, 3.54; N, 20.52. Found: C, 56.51; H,
3.75; N, 20.69.
To the mixture of compound 4 (2 mmol) in 30 mL dry 1,4-
dioxane was added substituted benzoyl isothiocyanate. The reac-
tion mixture was stirred at room temperature overnight. The
formation of yellow solid was filtered, washed with water and
dried. Recrystallization with DMF and water afforded the desired
compounds.
4.3.6. 4-Fluoro-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5f)
Yellow solid, yield: 80%, mp. > 260 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d
7.26 (d, 1H, J ¼ 7.8 Hz, AreH), 7.33e7.39 (m, 3H, AreH),
7.58 (t, 1H, J ¼ 7.8 Hz, AreH), 8.02e8.07 (m, 3H, AreH), 8.10 (s, 1H,
CH), 11.25 (s, 1H, NHCS), 12.05 (s, 1H, NH), 14.29 (s, 1H, NHCO); 13C
4.3.1. N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5a)
NMR (DMSO-d6, 150 MHz) d 115.4, 115.5, 116.7, 117.1, 124.1, 126.2,
128.9, 131.6, 131.6, 132.0, 136.1, 147.7, 149.2, 163.9, 166.2, 172.9; Anal.
Calcd. For C16H12FN5OS: C, 56.30; H, 3.54; N, 20.52. Found: C, 56.15;
H, 3.57; N, 20.68.
Yellow solid, yield: 70%, mp. 213e216 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d
7.26 (d, 1H, J ¼ 7.8 Hz, AreH), 7.39 (t, 1H, J ¼ 7.8 Hz,
AreH), 7.52 (t, 2H, J ¼ 7.8 Hz, AreH), 7.59 (t, 1H, J ¼ 7.8 Hz, AreH),
7.64 (t, 1H, J ¼ 7.2 Hz, AreH), 7.98 (d, 1H, J ¼ 7.2 Hz, AreH), 8.03 (d,
1H, J ¼ 7.8 Hz, AreH), 8.12 (s, 1H, CH), 11.21 (s, 1H, NHCS), 12.08 (s,
4.3.7. N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)-4-
(trifluoromethyl)benzamide (5g)
1H, NH), 14.34 (s, 1H, NHCO); 13C NMR (DMSO-d6, 150 MHz)
d
116.7,
Yellow solid, yield: 69%, mp. 227e229 ꢀC; 1H NMR (DMSO-d6,
117.1, 124.1, 126.2, 128.4, 128.6, 131.9, 132.3, 132.8, 136.1, 147.7, 149.2,
167.2, 172.9; Anal. Calcd. For C16H13N5OS: C, 59.43; H, 4.05; N, 21.66.
Found: C, 59.06; H, 4.11; N, 21.23.
600 MHz)
d
7.26 (d, 1H, J ¼ 7.8 Hz, AreH), 7.39 (t, 1H, J ¼ 7.2 Hz,
AreH), 7.59 (t, 1H, J ¼ 7.2 Hz, AreH), 7.88 (d, 2H, J ¼ 8.4 Hz, AreH),
8.04 (d, 1H, J ¼ 7.2 Hz, AreH), 8.11 (s, 1H, CH), 8.14 (d, 2H, J ¼ 8.4 Hz,
AreH), 11.52 (s, 1H, NHCS), 12.09 (s, 1H, NH), 14.27 (s, 1H, NHCO);
4.3.2. 2-Chloro-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5b)
13C NMR (DMSO-d6,150 MHz)
d 116.7,117.1,122.9,124.1,124.7,125.3,
126.2, 129.6, 132.0, 136.2, 136.4, 147.7, 149.4, 166.3, 172.6; Anal.
Calcd. For C17H12F3N5OS: C, 52.17; H, 3.09; N, 17.89. Found: C, 51.71;
H, 2.98; N, 17.71.
Yellow solid, yield: 57%, mp. 215e216 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d
7.25 (d, 1H, J ¼ 7.8 Hz, AreH), 7.38 (d, 1H, J ¼ 7.2 Hz,
AreH), 7.42 (d, 1H, J ¼ 7.8 Hz, AreH), 7.50e7.53 (m, 2H, AreH), 7.57
(d, 2H, J ¼ 6.6 Hz, AreH), 8.02 (d, 1H, J ¼ 7.8 Hz, AreH), 8.09 (s, 1H,
CH), 11.62 (s, 1H, NHCS), 12.06 (s, 1H, NH), 14.09 (s, 1H, NHCO); 13C
4.3.8. 2-Methoxy-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5h)
NMR (DMSO-d6, 100 MHz)
d
116.7, 117.1, 125.2, 126.1, 129.5, 132.0,
Yellow solid, yield: 65%, mp. 205e207 ꢀC; 1H NMR (DMSO-d6,
136.1, 136.4, 149.3, 166.2, 172.6; Anal. Calcd. For C16H12ClN5OS: C,
53.71; H, 3.38; N, 19.57. Found: C, 54.10; H, 3.47; N, 19.71.
600 MHz) d 7.16 (s, 1H, AreH), 7.26e7.30 (m, 2H, AreH), 7.39 (s, 1H,
AreH), 7.59 (s, 1H, AreH), 7.65 (s, 1H, AreH), 7.98 (d, 1H, J ¼ 7.2 Hz,
AreH), 8.02 (d, 1H, J ¼ 7.8 Hz AreH), 8.13 (s, 1H, CH), 10.98 (s, 1H,
NHCS), 12.11 (s, 1H, NH), 14.17 (s, 1H, NHCO); 13C NMR (DMSO-d6,
4.3.3. 3-Chloro-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5c)
100 MHz)
d 56.7, 112.8, 116.6, 117.0, 119.3, 121.3, 124.0, 126.1, 131.3,
Yellow solid, yield: 64%, mp. 205e207 ꢀC; 1H NMR (DMSO-d6,
131.9, 134.8, 136.1, 147.6, 149.3, 157.4, 163.6, 171.8; Anal. Calcd. For
C17H15N5O2S: C, 57.78; H, 4.28; N, 19.82. Found: C, 57.73; H, 4.25; N,
19.97.
600 MHz)
d
7.26 (d, 1H, J ¼ 8.4 Hz, AreH), 7.39 (d, 1H, J ¼ 7.2 Hz,
AreH), 7.53e7.58 (m, 2H, AreH), 7.69 (d, 1H, J ¼ 7.2 Hz, AreH), 7.90
(d, 1H, J ¼ 8.4 Hz, AreH), 8.03 (d, 2H, J ¼ 6.0 Hz, AreH), 8.10 (s, 1H,
CH), 11.40 (s, 1H, NHCS), 12.07 (s, 1H, NH), 14.25 (s, 1H, NHCO); 13C
4.3.9. 2-Methyl-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5i)
NMR (DMSO-d6, 100 MHz)
d 116.6, 117.1, 124.0, 126.1, 127.3, 128.4,
130.3, 131.9, 132.4, 133.0, 134.4, 136.1, 149.2, 165.9, 172.7; Anal.
Calcd. For C16H12ClN5OS: C, 53.71; H, 3.38; N,19.57. Found: C, 53.49;
H, 3.39; N, 19.93.
Yellow solid, yield: 86%, mp. 218e220 ꢀC; 1H NMR (DMSO-d6,
600 MHz) d 2.39 (s, 3H, CH3), 7.25e7.30 (m, 3H, AreH), 7.38-7.42 (m,
2H, AreH), 7.46 (d, 1H, J ¼ 7.2 Hz, AreH), 7.59 (t, 1H, J ¼ 7.8 Hz,
AreH), 8.04 (d, 1H, J ¼ 7.8 Hz, AreH), 8.10 (s, 1H, CH), 11.32 (s, 1H,
NHCS), 12.06 (s, 1H, NH), 14.19 (s, 1H, NHCO); 13C NMR (DMSO-d6,
4.3.4. 4-Chloro-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5d)
150 MHz)
d 19.5, 116.7, 117.2, 124.1, 125.5, 126.1, 128.0, 130.5, 130.6,
Yellow solid, yield: 68%, mp. > 260 ꢀC; 1H NMR (DMSO-d6,
131.9, 134.5, 135.7, 136.1, 147.7, 149.1, 169.6, 173.0; Anal. Calcd. For
C17H15N5OS: C, 60.52; H, 4.48; N, 20.76. Found: C, 60.25; H, 4.40; N,
20.46.
600 MHz)
d
7.25 (d, 1H, J ¼ 8.4 Hz, AreH), 7.38 (d, 1H, J ¼ 7.8 Hz,
AreH), 7.57 (d, 3H, J ¼ 8.4 Hz, AreH), 7.98 (d, 2H, J ¼ 8.4 Hz,
AreH), 8.02 (d, 1H, J ¼ 8.4 Hz, AreH), 8.10 (s, 1H, CH), 11.31 (s,
1H, NHCS), 12.05 (s, 1H, NH), 14.27 (s, 1H, NHCO); 13C NMR
4.3.10. 4-Methyl-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5j)
(DMSO-d6, 150 MHz)
d 116.7, 117.1, 126.2, 128.5, 130.6, 131.2,
132.0, 136.1, 137.6, 147.7, 149.2, 166.3, 172.8; Anal. Calcd. For
C16H12ClN5OS: C, 53.71; H, 3.38; N, 19.57. Found: C, 53.47; H,
3.17; N, 19.79.
Yellow solid, yield: 88%, mp. 216e218 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d
2.38 (s, 3H, CH3), 7.25 (d, 1H, J ¼ 8.4 Hz, AreH), 7.32 (d,
2H, J ¼ 7.8 Hz, AreH), 7.38 (t, 1H, J ¼ 7.8 Hz, AreH), 7.58 (t, 1H,
J ¼ 7.2 Hz, AreH), 7.90 (d, 2H, J ¼ 7.8 Hz, AreH), 8.03 (d, 1H,
J ¼ 8.4 Hz, AreH), 8.10 (s, 1H, CH), 11.05 (s, 1H, NHCS), 12.04 (s, 1H,
4.3.5. 2-Fluoro-N-(2-(quinazolin-4-yl)hydrazinecarbonothioyl)
benzamide (5e)
NH),14.33 (s,1H, NHCO); 13C NMR (DMSO-d6,100 MHz)
d 21.1,116.6,
Yellow solid, yield: 76%, mp. 216e218 ꢀC; 1H NMR (DMSO-d6,
117.1, 124.0, 126.1, 128.6, 128.9, 129.4, 131.9, 136.1, 143.2, 147.6, 149.0,
166.9,173.0; Anal. Calcd. For C17H15N5OS: C, 60.52; H, 4.48; N, 20.76.
Found: C, 60.39; H, 4.62; N, 21.17.
600 MHz)
d
7.27 (d, 1H, J ¼ 7.8 Hz, AreH), 7.33 (d, 1H, J ¼ 8.4 Hz,
AreH), 7.39 (s, 2H, AreH), 7.59e7.62 (m, 2H, AreH), 7.71 (s, 1H,

J. He et al. / European Journal of Medicinal Chemistry 54 (2012) 925e930
929
4.4. Preparation of 4-hydrazinyl-6,7-dimethoxyquinazoline (8) [26]
For C18H16ClN5O3S: C, 51.74; H, 3.86; N, 16.76. Found: C, 51.90; H,
3.84; N, 16.82.
Compound 7 (10 mmol) and hydrazine hydrate 85% (30 mmol)
in 50 mL of THF was stirred for 16 h at room temperature. The
reaction mixture was concentrated and allowed to cool. The solid
product obtained was filtered, washed with water and dried.
Recrystallization with ethanol and water afforded the desired solid
compound 8, mp. 199e202 ꢀC in 70% yield. 1H NMR (DMSO-d6,
4.5.5. N-(2-(6,7-dimethoxyquinazolin-4-yl)
hydrazinecarbonothioyl)-2-fluorobenzamide (9e)
Yellow solid, yield: 92%, mp. 197e199 ꢀC; 1H NMR (DMSO-d6,
600 MHz) d 3.85 (s, 6H, CH3O), 6.81 (s, 1H, AreH), 7.32e7.36 (m, 2H,
AreH), 7.42 (s, 1H, AreH), 7.63 (d, 1H, J ¼ 6.6 Hz, AreH), 7.72 (t, 1H,
600 MHz) d 3.84 (s, 3H, CH3O), 3.88 (s, 3H, CH3O), 4.56 (s, 2H, NH2),
7.08 (s, 1H, AreH), 7.57 (s, 1H, AreH), 8.37 (s, 1H, CH), 9.19 (s,
J ¼ 7.2 Hz, AreH), 8.09 (s, 1H, CH), 11.16 (s, 1H, NHCS), 12.00 (s, 1H,
NH), 14.12 (s, 1H, NHCO); 13C NMR (DMSO-d6, 150 MHz)
d 55.7, 99.1,
1H, NH).
104.3,109.6,116.2,116.3,122.2,122.3,130.5,131.3,134.0,146.6,147.9,
149.7,153.0,158.5,160.2,164.0,171.1; Anal. Calcd. For C18H16FN5O3S:
C, 53.86; H, 4.02; N, 17.45. Found: C, 53.53; H, 4.11; N, 17.21.
4.5. General procedure for the synthesis of title compounds (9ae9j)
[30]
4.5.6. N-(2-(6,7-dimethoxyquinazolin-4-yl)
To the mixture of compound 8 (2 mmol) in 30 mL dry 1,4-
dioxane was added substituted benzoyl isothiocyanate. The reac-
tion mixture was stirred at room temperature overnight. The
formation of yellow solid was filtered, washed with water and
dried. Recrystallization with DMF and water afforded the desired
compounds.
hydrazinecarbonothioyl)-4-fluorobenzamide (9f)
Yellow solid, yield: 63%, mp. 199e201 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d 3.91 (s, 6H, CH3O), 6.86 (s, 1H, AreH), 7.41 (t, 2H,
J ¼ 7.8 Hz, AreH), 7.47 (s, 1H, AreH), 8.13 (s, 1H, CH), 8.14 (d, 2H,
J ¼ 7.2 Hz, AreH), 11.23 (s, 1H, NHCS), 12.03 (s, 1H, NH), 14.34 (s, 1H,
NHCO); 13C NMR (DMSO-d6, 150 MHz)
d 55.7, 99.9, 104.4, 109.7,
115.3,115.5, 128.9, 131.2, 131.5, 146.5, 147.8, 149.7, 152.6, 163.9, 165.6,
166.0, 171.7; Anal. Calcd. For C18H16FN5O3S: C, 53.86; H, 4.02; N,
17.45. Found: C, 53.60; H, 4.01; N, 17.26.
4.5.1. N-(2-(6,7-dimethoxyquinazolin-4-yl)
hydrazinecarbonothioyl)benzamide (9a)
Yellow solid, yield: 56%, mp. 186e188 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d
3.84 (s, 6H, CH3O), 6.81 (s, 1H, AreH), 7.43 (d, 1H,
4.5.7. N-(2-(6,7-dimethoxyquinazolin-4-yl)
J ¼ 6.6 Hz, AreH), 7.51 (dd, 2H, J ¼ 7.8 Hz, 15 Hz, AreH), 7.62 (q, 1H,
J ¼ 7.8 Hz, AreH), 7.97 (t, 2H, J ¼ 7.8 Hz, AreH), 8.10 (s, 1H, CH), 11.08
(s, 1H, NHCS), 11.97 (s, 1H, NH), 14.28 (s, 1H, NHCO); 13C NMR
hydrazinecarbonothioyl)-4-(trifluoromethyl)benzamide (9g)
Yellow solid, yield: 80%, mp. 223e225 ꢀC; 1H NMR (DMSO-d6,
600 MHz) d 3.85 (s, 6H, CH3O), 6.79 (s, 1H, AreH), 7.42 (s, 1H, AreH),
(DMSO-d6, 150 MHz)
d
55.8, 99.1, 104.4, 109.7, 128.4, 128.5, 131.2,
7.88 (d, 2H, J ¼ 7.8 Hz, AreH), 8.09 (s, 1H, CH), 8.14 (d, 2H, J ¼ 8.4 Hz,
132.4, 132.8, 146.6, 147.8, 149.5, 152.6, 167.0, 171.7; Anal. Calcd. For
C18H17N5O3S: C, 56.38; H, 4.47; N, 18.27. Found: C, 55.98; H, 4.23;
N, 17.92.
AreH),11.41 (s,1H, NHCS),11.99 (s,1H, NHC¼ N),14.25 (s,1H, NHCO);
13C NMR (DMSO-d6, 150 MHz)
d 55.8, 99.1, 99.1, 109.7, 125.3, 129.6,
131.2, 136.5, 147.9,152.7, 166.1, 171.4; Anal. Calcd. For C19H16F3N5O3S:
C, 50.55; H, 3.57; N, 15.51. Found: C, 50.47; H, 3.71; N, 15.90.
4.5.2. 2-Chloro-N-(2-(6,7-dimethoxyquinazolin-4-yl)
hydrazinecarbonothioyl)benzamide (9b)
4.5.8. N-(2-(6,7-dimethoxyquinazolin-4-yl)
Yellow solid, yield: 77%, mp. 212e214 ꢀC; 1H NMR (DMSO-d6,
hydrazinecarbonothioyl)-2-methoxybenzamide (9h)
600 MHz)
d 3.86 (s, 6H, CH3O), 6.81 (s, 1H, AreH), 7.44 (d, 2H,
Yellow solid, yield: 71%, mp. 206e208 ꢀC; 1H NMR (DMSO-d6,
J ¼ 11.4 Hz, AreH), 7.51-7.59 (m, 3H, AreH), 8.10 (s, 1H, CH), 11.56 (s,
600 MHz) d 3.85 (s, 6H, CH3O), 3.91 (s, 3H, OCH3), 6.80 (s,1H, AreH),
1H, NHCS), 12.01 (s, 1H, NH), 14.08 (s, 1H, NHCO); 13C NMR (DMSO-
7.26e7.30 (m, 2H, AreH), 7.42 (d, 1H, J ¼ 7.8 Hz, AreH), 8.03 (d, 2H,
d6, 150 MHz) d 55.7, 55.8, 99.1, 104.3, 109.6, 127.1, 129.2, 129.5, 130.0,
J ¼ 7.8 Hz, AreH), 8.08 (s, 1H, CH), 11.21 (s, 1H, NHCS), 11.96 (s, 1H,
131.8, 134.8, 134.9, 146.6, 147.8, 149.7, 152.7, 166.6, 171.4; Anal. Calcd.
For C18H16ClN5O3S: C, 51.74; H, 3.86; N, 16.76. Found: C, 51.88; H,
4.09; N, 16.29.
NHC ¼ N),14.17 (s, 1H, NHCO); 13C NMR (DMSO-d6,150 MHz)
d 55.5,
55.7, 99.0, 104.4, 109.5, 113.7, 124.2, 129.9, 130.1, 130.7, 131.2, 146.5,
147.8, 149.3, 152.7, 162.9, 166.3, 171.9; Anal. Calcd. For C19H19N5O4S:
C, 55.19; H, 4.63; N, 16.94. Found: C, 55.05; H, 4.38; N, 17.12.
4.5.3. 3-Chloro-N-(2-(6,7-dimethoxyquinazolin-4-yl)
hydrazinecarbonothioyl)benzamide (9c)
4.5.9. N-(2-(6,7-dimethoxyquinazolin-4-yl)
Yellow solid, yield: 68%, mp. 193e194 ꢀC; 1H NMR (DMSO-d6,
hydrazinecarbonothioyl)-2-methylbenzamide (9i)
600 MHz)
d
3.85 (s, 6H, CH3O), 6.80 (d,1H, J ¼ 4.2 Hz, AreH), 7.42 (d,
Yellow solid, yield: 66%, mp. 215e216 ꢀC; 1H NMR (DMSO-d6,
1H, J ¼ 3.6 Hz, AreH), 7.54 (d, 1H, J ¼ 5.4 Hz, AreH), 7.69 (s, 1H,
AreH), 7.91 (s, 1H, AreH), 8.02 (s, 1H, AreH), 8.08 (s, 1H, CH), 11.29
(s, 1H, NHCS), 11.97 (s, 1H, NH), 14.23 (s, 1H, NHCO); 13C NMR
600 MHz) d 2.39 (s, 3H, OCH3), 3.85 (s, 6H, CH3O), 6.80 (s,1H, AreH),
7.26e7.30 (m, 2H, AreH), 7.41 (t, 2H, J ¼ 7.8 Hz, AreH), 7.46 (d, 1H,
J ¼ 7.2 Hz, AreH), 8.08 (s, 1H, CH), 11.87 (s, 1H, NHCS), 11.95 (s, 1H,
(DMSO-d6, 150 MHz)
d
55.7, 55.8, 99.1, 104.4, 109.7, 127.3, 128.4,
NHC ¼ N),14.33 (s,1H, NHCO); 13C NMR (DMSO-d6,150 MHz)
d 19.5,
130.3, 131.2, 132.4, 133.1, 134.5, 146.6, 147.8, 152.6, 165.8, 171.5; Anal.
Calcd. For C18H16ClN5O3S: C, 51.74; H, 3.86; N, 16.76. Found: C,
51.62; H, 4.03; N, 16.46.
55.7, 99.0, 104.3, 109.7, 25.5, 127.9, 130.5, 131.2, 134.6, 135.7, 146.5,
147.8, 149.4, 152.5, 169.4, 171.8; Anal. Calcd. For C19H19N5O3S: C,
57.42; H, 4.82; N, 17.62. Found: C, 57.22; H, 4.36; N, 17.25.
4.5.4. 4-Chloro-N-(2-(6,7-dimethoxyquinazolin-4-yl)
hydrazinecarbonothioyl)benzamide (9d)
4.5.10. N-(2-(6,7-dimethoxyquinazolin-4-yl)
hydrazinecarbonothioyl)-4-methylbenzamide (9j)
Yellow solid, yield: 72%, mp. 224e226 ꢀC; 1H NMR (DMSO-d6,
Yellow solid, yield: 82%, mp. 198e199 ꢀC; 1H NMR (DMSO-d6,
600 MHz)
d
3.85 (s, 6H, CH3O), 6.79 (s, 1H, AreH), 7.41 (s,1H, AreH),
600 MHz) d 2.38 (s, 3H, CH3), 3.85 (s, 6H, CH3O), 6.79 (s, 1H, AreH),
7.58 (d, 2H, J ¼ 7.8 Hz, AreH), 7.99 (d, 2H, J ¼ 8.4 Hz, AreH), 8.09 (s,
7.32 (d, 2H, J ¼ 7.8 Hz, AreH), 7.42 (s, 1H, AreH), 7.90 (d, 2H,
1H, CH), 11.23 (s, 1H, NHCS), 11.98 (s, 1H, NH), 14.26 (s, 1H, NHCO);
J ¼ 7.8 Hz, AreH), 8.08 (s, 1H, CH), 10.97 (s, 1H, NHCS), 11.94 (s, 1H,
13C NMR (DMSO-d6, 150 MHz)
d
55.7, 99.0, 104.3, 109.7, 128.5, 130.5,
NHC ¼ N),14.31 (s, 1H, NHCO); 13C NMR (DMSO-d6,150 MHz)
d 21.2,
131.2, 137.6, 146.6, 147.8, 149.6, 152.6, 154.2, 166.1, 171.5; Anal. Calcd.
55.8, 99.1, 104.5, 109.7, 122.7, 128.6, 129.1, 129.5, 131.3, 143.3, 146.6,

930
J. He et al. / European Journal of Medicinal Chemistry 54 (2012) 925e930
147.9, 149.7, 152.7, 166.9, 171.8; Anal. Calcd. For C19H19N5O3S: C,
57.42; H, 4.82; N, 17.62. Found: C, 57.44; H, 5.11; N, 17.83.
References
[1] A. Gilman, Am. J. Surg. 105 (1963) 574e578.
[2] K.W. Kohn, Cancer Res. 56 (1996) 5533e5546.
[3] S. Eckhardt, Curr. Med. Chem. Anti-Cancer Agents 2 (2002) 419e439.
[4] A.A.-M. Abdel-Aziz, Eur. J. Med. Chem. 42 (2007) 614e626.
[5] H.-Y.P. Choo, M. Kim, S.K. Lee, S.W. Kim, S.W. Chung, Bioorg. Med. Chem. 10
(2002) 517e523.
[6] B.B.S. Zhou, H. Huang, M. Damelin, K.G. Geles, J.C. Grindley, P.B. Dirks, Nat. Rev.
Drug Discov. 8 (2009) 506e523.
[7] A.S. El-Azab, M.A. Al-Omar, A.A.-M. Abdel-Aziz, N.I. Abdel-Aziz, M.A.-A. El-
Sayed, A.M. Aleisa, M.M. Sayed-Ahmed, S.G. Abdel-Hamide, Eur. J. Med. Chem.
45 (2010) 4188e4198.
[8] A.J. Bridges, H. Zhou, D.R. Cody, G.W. Rewcastle, A. McMichael,
H.D.H. Showalter, D.W. Fry, A.J. Kraker, W.A. Denny, J. Med. Chem. 39 (1996)
267e276.
[9] A. Lüth, W. Löwe, Eur. J. Med. Chem. 43 (2008) 1478e1488.
[10] C. Dietchfield, V.L. Johnson, A. Tighe, R. Ellston, C. Haworth, T. Johnson,
A. Mortlock, N. Keen, S.S. Taylor, J. Cell. Biol. 161 (2003) 267e280.
[11] F.H. Jung, G. Pasquet, C. Lamber-van der Brempt, J.M. Lohmann, N. Warin,
F. Renaud, H. Germain, C.D. Savi, N. Roberts, T. Johnson, C. Dousson, G.B. Hill,
A.A. Mortlock, N. Heron, R.W. Wilkinson, S.R. Wedge, S.P. Heaton, R. Odedra,
N.J. Keen, S. Green, E. Brown, K. Thompson, S. Brightwell, J. Med. Chem. 49
(2006) 955e970.
[12] L.T. Liu, T.T. Yuan, H.H. Liu, S.F. Chena, Y.T. Wu, Bioorg. Med. Chem. Lett. 17
(2007) 6373e6377.
[13] C.L. Arteaga, D.H. Johnson, Curr. Opin. Oncol. 13 (2001) 491e498.
[14] L. Kopper, Pathol. Oncol. Res. 14 (2008) 1e8.
4.6. Pharmacology
Cytotoxic activities were evaluated by standard MTT assay
according to the Mosmann’s method [27]. The MTT assay is based
on the reduction of the soluble 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) into a blueepurple for-
mazan product, mainly by mitochondrial reductase activity inside
living cells. The KB, CNE2, MGC-803, and GLC-82 cells used in
cytotoxicity assay were cultured in RPMI 1640 medium supple-
mented with 10% fetal calf serum and the MCF-7 cells were cultured
in DMEM medium supplemented with 10% fetal calf serum. Cells
were harvested during logarithmic growth phase and seeded in 96
well plates at a density of 1.5 ꢃ 104 cell/mL for KB, 2.0 ꢃ 104 cell/mL
for CNE2, MGC-803 and GLC-82, and 2.5 ꢃ 104 cell/mL for MCF-7 in
a final volume of 190
incubator. After 24 h, 10
plates and cultured at 37 ꢀC for 72 h. 20
m
L/well and incubated at 37 ꢀC in a 5% CO2
L tested compounds was added to 96-well
L of MTT (5 mg/mL stock
m
m
solution of saline) was added to each well and incubated for 3 h at
37 ꢀC. The supernatant was carefully removed from each well and
[15] E.R. Wood, A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson,
B. Ellis, C. Pennisi, E. Horne, K. Lackey, K.J. Alligood, D.W. Rusnak, T.M. Gilmer,
L. Shewchuk, Cancer Res. 64 (2004) 6652e6659.
100 mL of DMSO was added to each well to dissolve the formazan
crystals which were formed by the cellular reduction of MTT. The
absorbance of each well was detected in a microplate reader at
570 nm with 655 nm as reference. The results were expressed as
the IC50, which is the concentration of the drugs inducing a 50%
inhibition of cell growth of treated cells when compared to the
growth of control cells. Each experiment was performed at least 3
times. There was a good reproducibility between replicate wells
with standard errors below 10%.
[16] A.M.F. Kersemaekers, G.J. Fleuren, E.G. Kenter, L.J. Van den Broek, S.M. Uljee,
J. Hermans, M.J. Van de Vijver, Clin. Cancer Res. 5 (1999) 577e586.
[17] F. Barlesi, C. Tchouhadjian, C. Doddoli, P. Villani, L. Greillier, J.-P. Kleisbauer,
P. Thomas, P. Astoul, Fundam. Clin. Pharmacol. 19 (2005) 385e393.
[18] K.N. Ganjoo, H. Wakelee, Biologics: Targets Ther. 1 (2007) 335e346.
[19] A.K. Nandi, S. Chaudhri, S.K. Mazumdah, S. Ghosh, J. Chem. Soc. Perkin Trans. 2
(1984) 1729e1733.
[20] M.A. Ali, D.A. Chowdhary, M. Naziruddin, Polyhedron 3 (1984) 595e598.
[21] J.P. Scovill, D.L. Klayman, C.F. Franchino, J. Med. Chem. 25 (1982) 1261e1264.
[22] S.T. Oswald, V.P. Ghanshyam, J. Org. Chem. 41 (1976) 838e845.
[23] H. Schoenowsky, B.Z. Sachse, B. Naturforsh, Anorg. Chem. Org. Chem. 37
(1982) 907.
[24] M. Claesen, H. Vanderhaeghe, Bull. Soc. Chim. Belg. 68 (1959) 220e224.
[25] X.L. Yue, X.Q. Yu, Q. Zheng, Chin. J. Org. Chem. 29 (2009) 764e769.
[26] M.G. Hummersone, S. Gomez, K.A. Menear, X.L. Fan, G.C.M. Smith, WO 2006/
090167.
[27] T. Mossman, J. Immunol. Methods 65 (1983) 55e63.
[28] C.F. Jin, Y.J. Liang, H.W. He, L.W. Fu, Eur. J. Med. Chem. 46 (2011) 429e432.
[29] T.B. Cai, X. Tang, J. Nagorski, P.G. Brauschweiger, P.G. Wang, Bioorg. Med.
Chem. 11 (2003) 4971e4975.
Acknowledgments
The present work was supported by National Basic Research
Program of China (No. 2010CB126100), National Natural Science
Foundation of China (No. 20772042, 21172090). The research was
supported in part by the PCSIRT (No. IRT0953), Science and Tech-
nological Project of Guangzhou City (No. 12C32061587).
[30] A.S. Fouda, S.T. Shawagfeh, Bull. Soc. Chim. Fr. 1 (1991) 30e34.