Synthesis of substituted benzylamino- and heterocyclylmethylamino carbodithioate derivatives of 4-(3H)-quinazolinone and their cytotoxic activity

Article abstract of DOI:10.1002/ardp.200500264

A new series of substituted benzylamino- and heterocyclylmethylamino carbodithioate derivatives of 4-(3H)-quinazolinone were synthesized via four steps starting from 2-amino-5-methyl-benzoic acid and initially screened against A-549 (human non-small cell

Full text of DOI:10.1002/ardp.200500264

250
Arch. Pharm. Chem. Life Sci. 2006, 339, 250–254
Full Paper
Synthesis of Substituted Benzylamino- and
Heterocyclylmethylamino Carbodithioate Derivatives of
-(3H)-Quinazolinone and their Cytotoxic Activity
4
1
2
1
2,3
1
1
Sheng-Li Cao , Yu-Ping Feng , Xiao-Lin Zheng , Yu-Yang Jiang , Mei Zhang , Yue Wang and
Meng Xu
1
1
Department of Chemistry, Capital Normal University, Beijing, China
The Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry,
2
Tsinghua University, Beijing, China
The Key Laboratory of Chemical Biology, Tsinghua University, Shenzhen, China
3
A new series of substituted benzylamino- and heterocyclylmethylamino carbodithioate deriv-
atives of 4-(3H)-quinazolinone were synthesized via four steps starting from 2-amino-5-methyl-
benzoic acid and initially screened against A-549 (human non-small cell lung cancer), HCT-8
(
5
human colon cancer), and Bel-7402 (human liver cancer) cell lines at the single concentration of
lg/mL using the colorimetric MTT assay. The IC50 values were determined for the compounds
reaching F70% inhibition in primary screening by serial dilution. Among the newly synthesized
compounds, 9n exhibited potent in vitro cytotoxicity against A-549, HCT-8, and Bel-7402 cell
lines with the IC50 values of 1.65, 0.93, and 1.43 lM, respectively.
Keywords: 4-(3H)-Qquinazolinone / Dithiocarbamate / Cytotoxicity / MTT assay /
Received: December 30, 2005; Accepted: February 7, 2006
DOI 10.1002/ardp.200500264
Introduction
Recently, classical antifolates such as raltitrexed 1 [1–3]
(
Fig. 1) and pemetrexed [4, 5], structurally analogous to
natural folic acid, have been successfully used in cancer
chemotherapy. However, many researchers are presently
devoted to developing nonclassical antifolates in order to
overcome the problems associated with classical antifo-
lates [6–10]. One strategy commonly used is to replace
the benzoyl L-glutamic acid moieties in classical antifo-
lates with lipophilic side chains, the successful example
being AG337 2 [11–13] (Fig. 1) which is currently under
clinical trials. In our research program on 4-(3H)-quinazo-
linone derivatives acting as antitumor agents, we have
incorporated dithiocarbamate moieties with 4-(3H)-qui-
nazolinone to synthesize a series of 4-(3H)-quinazolinone
derivatives bearing various dithiocarbamate side chains.
Figure 1. Structures of Raltitrexed 1 and AG337 (2).
The in vitro evaluation of these compounds as inhibitors
of the growth of K562 (human myelogenous leukaemia)
cells showed that compound 3 (Fig. 2) containing a benzy-
lamine moiety in the dithiocarbamate side chain was
active with an IC50 value of 4.0 lM [14]. These results pro-
Correspondence: Dr. Sheng-Li Cao, Capital Normal University, Depart-
ment of Chemistry, Beijing, 100037, China.
E-mail: sl_cao@sohu.com
Fax: +86 10 689-03040
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Arch. Pharm. Chem. Life Sci. 2006, 339, 250–254
New 4-Quinazolinone Derivatives
251
Table 1. In vitro cytotoxicity of compounds 9a–p against A-549, HCT-8, and Bel-7402 cell lines.
Compound
R
A-549
Inhibition
HCT-8
% Inhibition
Bel-7402
% Inhibition IC50 (lM)
%
IC50 (lM)
IC50 (lM)
at 5 lg/mL
at 5 lg/mL
at 5 lg/mL
a)
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
(Ph)
4-CH
2
CH-
PhCH
45.9
62.2
71.5
69.6
–0.7
7.5
80.8
82.5
68.2
68.2
4.1
nt
53.0
88.5
82.7
75.9
22.3
55.1
88.4
89.6
88.5
87.1
11.1
11.9
–5. 5
88.6
88.6
89.2
89.4
78.4
76.3
nt
34.8
86.9
84.7
nt
3
2
–
nt
2.83
nt
6.60
>10
>10
nt
2.81
2.67
>10
nt
4-(CH
3
O)PhCH
O)PhCH
O)
3,4,5-(CH O)
3,4-methylenedioxyPhCH
2
–
–
b)
2-(CH
3
2
68.5
2,4-(CH
3
2
PhCH
2
–
2
nt
31.8
35.9
86.3
86.6
82.1
85.7
10.2
12.0
7.8
86.6
69.6
61.6
88.0
74.7
84.4
3
3
PhCH
–
nt
nt
nt
2
–
>10
>10
nt
nt
nt
>10
>10
>10
>10
nt
2.15
1.45
6.29
1.49
nt
4-FPhCH
2-FPhCH
4-ClPhCH
2,4-Cl PhCH
4-HOOCPhCH
2-PyridinylCH
3-PyridinylCH
2-ThiophenylCH
2-TetrahydrofurylCH
PhCH
2
–
–
2
2
–
2
2
–
2
–
0
nt
nt
nt
nt
nt
2
–
–
–4.4
81.8
70.4
81.9
81.7
71.0
76.7
nt
2
1.65
>10
4.01
3.43
0.044
1.77
0.93
>10
4.12
0.56
0.20
2.61
1.43
2.88
7.90
1.94
0.13
2.61
b)
2
–
b)
2
–
2
–
Raltitrexed 1
5
-Fluorouracil
a)
b)
nt – not tested.
For the Bel-7402 cell line, the IC50 values of the compounds effecting F60% inhibition at the concentration of 5 lg/mL were also
tested.
Scheme 1. Synthetic route to compounds 9a–p.
a) Ac
bromosuccinimide, (PhCO)
h; d) CS , K PO , DMF, rt, 2 h.
2
O, reflux 1.5 h; b) NH
3
/ H
2
O, rt, 48 h; c) N-
, reflux 3
2
O
2
, CHCl
3
2
3
4
Results and discussion
Chemistry
The preparation of intermediate 6, 2,6-dimethyl-4-(3H)-
quinazolinone, by heating 2-amino-5-methylbenzoic acid
4
[
together with thioacetamide has already been reported
15]. Due to the high reaction temperature and being free
of solvent, the product was frequently contaminated
Figure 2. Structure of compound 3.
moted us to prepare a new series of 4-(3H)-quinazolinone with a black resin-like substance and needed to be recrys-
derivatives 9a–p (Scheme 1 and Table 1), in which various tallized repeatedly to obtain a pure product, usually lead-
substituted benzyl groups or heterocycles such as pyri- ing to a lower yield. Therefore, an alternative method
dine, thiophene or tetrahydrofuran were used to replace was utilized herein to overcome the above-mentioned
the benzyl group in compound 3, to search for more problem. The starting compound 4 was cyclized with
effective antitumor agents acting as possible nonclassical acetic anhydride to give 2,6-dimethylbenzoxazin-4-one 5,
antifolates and to investigate the structure-activity rela- which was converted into 6 in high yield by reaction
tionships of this type of compounds.
with aqueous ammonia at room temperature. Bromina-
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252
S.-L. Cao et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 250–254
tion of 6 with N-bromosuccinimide (NBS) to yield 6-bro- We are grateful to the Natural Science Foundation of Beijing
momethyl-2-methyl-4-(3H)-quinazolinone 7, which was City (Project No. 7042006) and the special fund for training of
reacted with the mixture of carbon disulfide, anhydrous outstanding talents of Beijing City (Project No.
potassium phosphate, and various amines 8 according to 20041D0501601) for financial support. We also thank the
a method described previously [14] to afford the desired National Center for Pharmaceutical Screening, Institute of
compounds 9a–p. The structures of the synthesized com- Materia Medica, Chinese Academy of Medical Sciences and Pek-
1
pounds were confirmed by ESI-MS, H-MNR, and elemen- ing Union Medical College, for performing the antitumor
tal analyses, and the data are given in Section 3, Experi- screening of our compounds.
mental.
Assay for cytotoxic activities
The newly synthesized compounds 9a–p were initially
Experimental
screened at the single concentration of 5 lg/mL using the
Melting points were determined on a XT4A microscopic melting
colorimetric MTT (3-(4,5-dimethylthiazo-2-yl)-2,5-diphe-
nyl-tetrazolium bromide) assay to test their in vitro cyto-
toxicity against A-549 (human non-small cell lung can-
cer), HCT-8 (human colon cancer), and Bel-7402 (human
liver cancer) cell lines. Compound 3, the clinically used
anticancer drug 5-fluorouracil, and raltitrexed 1, which
was synthesized according to our previously reported
method [16], were used for comparative purposes. The
cytotoxicity of tested compounds was estimated in terms
of percent growth inhibition compared with untreated
control cells, and the data are given in Table 1. Then, the
compounds effecting F70% inhibition in one-dose pre-
screening were retested by serial dilution from 5 to
point apparatus (Keyi Electrooptical, Beijing, China) or a WRS-1B
digital melting point apparatus (Precision Instruments, Shang-
hai, China) and are uncorrected. H-NMR spectra were recorded
1
on a Bruker AC-200P spectrometer at 200 MHz (Bruker, Zurich,
Switzerland) or on a JNM-ECA300 spectrometer at 300 MHz (Jeol,
Tokyo, Japan) using tetramethylsilane (TMS) as internal stan-
dard. Electrospray ionization (ESI) mass spectra were recorded
on an Esquire-LC 00136 mass spectrometer (Bruker Daltonics,
Bremen, Germany). Elemental analyses (C, H and N) were per-
formed by the Institute of Chemistry, Chinese Academy of
Science, on a Flash EA 1112 elemental analyzer (Thermo Elec-
tron, Waltham, MA, USA). Column chromatography was carried
out on silica gel (200–300 mesh, Haiyang Chemical, Qingdao,
China). 2-Amino-5-methylbenzoic acid 4 was prepared according
to the reported method [15], and other reagents are commercial
materials which were used without further purification.
0
.005 lg/mL. The results were expressed as IC50 (inhibi-
tory concentration 50%), the concentration of the com-
pound which inhibits the tumor cell growth by 50%, and
are also reported in Table 1.
Based on the results assembled in Table 1, it can be
seen that introduction of either the electron-donating
Synthesis of the compounds
2
,6-Dimethylbenzoxazin-4-one 5
A mixture of 1.5 g (0.01 mol) of 2-amino-5-methylbenzoic acid 4
and acetic anhydride 7.5 mL was heated at reflux under stirring
for 1.5 h. After cooling in a refrigerator, the precipitate was col-
lected by filtration and recrystallized from trichloromethane/
groups (–CH
3
, –OCH ) or the electron-withdrawing
3
groups (–F, –Cl, and –COOH) at the 49-position of the phe-
nyl ring did not cause the increase in cytotoxicity com-
pared with the parent compound 3. Moreover, the
change of the substituent position from 49 to 29 (compare
c, 9d and 9h, 9i) and the increase of the number of sub-
stituents (compare 9c, 9e, 9f and 9j, 9k) led to the
decrease in activity. Replacement of the phenyl group in 2,6-Dimethyl-4-(3H)-quinazolinone 6
compound 3 by 2-pyridinyl, 2-thiophenyl, and 2-tetrahy-
drofuryl, resulting in compounds 9m, 9o, and 9p, also
led to the decrease in activity. However, the cytotoxicity
of compound 9n, obtained by replacing of phenyl in com-
pound 3 by 3-pyridinyl, was at least comparable to that of
compound 3. Although the IC50 values of compound 9n
were obviously higher than those of raltitrexed 1, it
exhibited a higher cytotoxicity than 5-fluorouracil
against A-549, HCT-8, and Bel-7402 cell lines. Considering
that 9n can still be subjected to modification by convert-
ing it into its salts or quaternary ammonium salts, it is
worthy of being investigated further.
cyclohexane (1/3) to give 1.6 g (92%) of 5 as white solid, m. p.
1
1
23.3–123.78C; H-NMR (200 MHz, CDCl
3
) d: 2.26 (s, 3H, CH
–H), 7.92 (d, 1H, J =
-H). ESI-MS, m/z: 176.1
2
: C, 68.57; H, 5.14; N, 8.00. Found:
3
), 2.48
(
s, 3H, CH
3
), 7.37 (dd, 1H, J = 8.6, 2.1 Hz, C
-H), 8.58 (d, 1H, J = 8.6 Hz, C
NO
7
2
.1 Hz, C
5
8
+
[M+H] . Anal. calcd. for C
9
H
9
9
C, 68.50; H, 5.25; N, 7.97.
2,6-Dimethylbenzoxazin-4-one (5) (0.88 g, 5.0 mmol) was dis-
solved in 15 mL of ethanol and 20 mL of 25% aqueous ammonia
was added. The resulting mixture was stirred at room tempera-
ture for 48 h. After evaporation of ethanol, the aqueous solution
was cooled in the refrigerator overnight. The separated solid was
filtered off, washed with water, and recrystallized from acetic
acid to afford 0.77 g (89%) of 6 as white needles, m. p. 249.9–
2
50.08C (m. p. 249.2–250.68C [15]). All spectroscopic data were in
full accordance with [15].
6
-Bromomethyl-2-methyl-4-(3H)-quinazolinone 7
A mixture of 2,6-dimethyl-4-(3H)-quinazolinone (6) (2.44 g,
4.0 mmol), N-bromosuccinimide (2.75 g, 15.5 mmol), and ben-
1
zoyl peroxide (0.4 g, 1.7 mmol) in 300 mL of trichloromethane
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Arch. Pharm. Chem. Life Sci. 2006, 339, 250–254
New 4-Quinazolinone Derivatives
253
was stirred at reflux and under irradiation of a 100-W tungsten
lamp for 3 h. After cooling to room temperature, the resulting
precipitate was collected by filtration, washed with trichloro-
methane, and dried to give 2.69 g (76%) of 7 as white solid. m. p.
2 2
(s, 2H, CH S), 4.78 (s, 2H, CH Ph), 6.62 (s, 2H, Ph-H), 7.51 (d, 1H, J =
8.3 Hz, quinazolinone 8-H), 7.76 (dd, 1H, J = 8.3, 2.0 Hz, quinazo-
linone 7-H), 8.08 (d, 1H, J = 2.0 Hz, quinazolinone 5-H), 10.45 (s,
+
1H, NHCH
calcd. for C21
H, 5.28; N, 9.15.
2
), 12.21 (brs, 1H, NH). ESI-MS, m/z: 446.3 [M+H] . Anal.
>
3308C (m.p. >3308C [15]). All spectroscopic data were in full
H
23
N
3
O
4
S
2
: C, 56.61; H, 5.20; N, 9.43. Found: C, 56.28;
accordance with [15].
1
9
g: Yield 32%; m.p. 196.7–197.38C. H-NMR (200 MHz, DMSO-
d
6
) d: 2.34 (s, 3H, C
2 3 2 2
–CH ), 4.66 (s, 2H, CH S), 4.74 (s, 2H, CH Ph),
General procedure for the preparation of compounds
5
.99 (s, 2H, OCH O), 6.81 (m, 3H, Ph-H), 7.51 (d, 1H, J = 8.4 Hz, qui-
2
9
a–p
nazolinone 8-H), 7.76 (d, 1H, J = 8.4 Hz, quinazolinone 7-H), 8.07
(s, 1H, quinazolinone 5-H). ESI-MS, m/z: 400.1 [M+H] . Anal. calcd.
+
To a stirred mixture of amine 8 (1.5 mmol), anhydrous potas-
sium phosphate (0.32 g, 1.5 mmol) in 15 mL of N,N-dimethylfor-
mamide, carbon disulfide (0.57 g, 7.5 mmol) was added. After
stirring at room temperature for 30 min, 6-bromomethyl-2-
methyl-4-(3H)-quinazolinone 7 (0.25 g, 1 mmol) was added. Stir-
ring was continued at room temperature for 2 h, and the reac-
tion mixture was poured into 100 mL of water. The separated
precipitate was collected by filtration, and purified by column
chromatography on silica gel using dichloromethane/methanol
for C19
H
17
N
3
O
3
S
2
: C, 57.12; H, 4.29; N, 10.52. Found: C, 56.89; H,
4.58; N, 10.22.
1
9h: Yield 82%; m.p. 193–1948C. H-NMR (200 MHz, DMSO-d
6
) d:
2.34 (s, 3H, C
2
–CH
3
), 4.47 (s, 2H, CH
2
S), 4.83 (s, 2H, CH
2
Ph), 7.11–
7.37 (m, 4H, Ph-H), 7.52 (d, 1H, J = 8.4 Hz, quinazolinone 8-H),
7.75 (dd, 1H, J = 8.4, 2.1 Hz, quinazolinone 7-H), 8.07 (d, 1H, J =
2.1 Hz, quinazolinone 5-H) , 10.53 (s, 1H, NHCH
2
), 12.23 (s, 1H,
NH). ESI-MS, m/z: 374.2 [M+H] . Anal. calcd. for C18 OS : C,
57.89; H, 4.32; N, 11.25. Found: C, 57.93; H, 4.38; N, 11.20.
+
H16FN
3
2
(95/5) as eluent to afford the desired compounds 9.
1
1
9a: Yield 51%; m.p. 201.7–202.08C. H-NMR (300 MHz, DMSO-
9i: Yield 55%; m.p. 179–1808C. H-NMR (200 MHz, DMSO-d
6
) d:
d
6
) d: 2.34 (s, 3H, C –CH ), 4.68 (s, 2H, CH S), 7.02 (s, 2H, CHPh ),
2
3
2
2
2.33 (s, 3H, C –CH ), 4.66 (s, 2H, CH S), 4.85 (s, 2H, CH Ph), 7.23
2
3
2
2
7
8
=
.25–7.40 (m, 10H, Ph-H), 7.51 (d, 1H, J = 8.4 Hz, quinazolinone
-H), 7.75 (dd, 1H, J = 8.4, 2.1 Hz, quinazolinone 7-H), 8.08 (d, 1H, J
(m, 4H, Ph-H), 7.51 (d, 1H, J = 8.4 Hz, quinazolinone 8-H), 7.75 (d,
1H, J = 8.4 Hz, quinazolinone 7-H), 8.07 (s, 1H, quinazolinone 5-
2.1 Hz, quinazolinone 5-H), 10.94 (s, 1H, NHCH
2
), 12.19 (s, 1H,
OS
2
O: C, 66.13; H, 4.94; N, 9.64. Found: C, 65.96; H, 4.99; N,
H), 10.50 (s, 1H, NHCH
2
), 12.20 (s, 1H, NH). ESI-MS, m/z: 374.1
[M+H] . Anal. calcd. for C18 OS N CH OH: C, 56.29; H, 4.94;
N, 10.37. Found: C, 56.42; H, 4.74; N, 10.33.
+
+
NH). ESI-MS, m/z: 432.2 [M+H] . Anal. calcd. for C24
H
21
N
3
2
N
H16FN
3
2
3
1
9
/4H
.48.
b: Yield 74%; m.p. 208.6–209.68C. H-NMR (300 MHz, DMSO-
) d: 2.27 (s, 3H, Ph–CH ), 2.34 (s, 3H, C –CH ), 4.46 (s, 2H, CH S),
Ph), 7.12, 7.17 (2d, J = 8.2 Hz, 4H, Ph-H), 7.52 (d,
H, J = 8.3 Hz, quinazolinone 8-H), 7.75 (dd, 1H, J = 8.3, 2.1 Hz,
1
9j: Yield 46%; m.p. 199.6–200.68C. H-NMR (300 MHz, DMSO-d
6
)
1
9
d: 2.34 (s, 3H, C –CH ), 4.66 (s, 2H, CH S), 4.83 (s, 2H, CH Ph), 7.29
2
3
2
2
d
6
3
2
3
2
(d, 2H, J = 8.4 Hz, Ph-H), 7.39 (d, 2H, J = 8.4 Hz, Ph-H), 7.52 (d, 1H,
J = 8.4 Hz, quinazolinone 8-H), 7.75 (dd, 1H, J = 8.4, 2.0 Hz, quina-
4
1
.80 (s, 2H, CH
2
zolinone 7-H), 8.07 (d, 1H, J = 2.0 Hz, quinazolinone 5-H), 10.53 (s,
+
quinazolinone 7-H), 8.07 (d, 1H, J = 2.1 Hz, quinazolinone 5-H).
ESI-MS, m/z: 370.1 [M+H] . Anal. calcd. for C19
H, 5.14; N, 11.37. Found: C, 61.66; H, 5.22; N, 11.30.
1H, NHCH
calcd. for C18
55.50; H, 4.15; N, 10.76.
2
), 12.21 (brs, 1H, NH). ESI-MS, m/z: 390.3 [M+H] . Anal.
+
H
19
N
3
OS
2
: C, 61.79;
H16ClN
3
OS
2
: C, 55.42; H, 4.11; N, 10.78. Found: C,
1
1
9
c: Yield 45%; m.p. 203.1–203.48C. H-NMR (300 MHz, DMSO-
) d: 2.34 (s, 3H, C –CH ), 3.73 (s, 3H, OCH ), 4.66 (s, 2H, CH S),
.77 (s, 2H, CH Ph), 6.88 (d, 2H, J = 8.4 Hz, Ph-H), 7.21 (d, 2H, J =
.4 Hz, Ph-H), 7.51 (d, 1H, J = 8.2 Hz, quinazolinone 8-H), 7.75 (d,
H, J = 8.2 Hz, quinazolinone 7-H), 8.06 (s, 1H, quinazolinone 5-
9k: Yield 35%; m.p. 206–2088C. H-NMR (200 MHz, DMSO-d
6
) d:
d
6
2
3
3
2
2.35 (s, 3H, C
2
–CH
3
), 4.68 (s, 2H, CH
2
S), 4.84 (s, 2H, CH
2
Ph), 7.27 (d,
4
8
1
2
1H, J = 8.3 Hz, Ph 6-H), 7.43 (dd, 1H, J = 8.3, 1.9 Hz, Ph 5-H), 7.53 (d,
1H, J = 8.4 Hz, quinazolinone 8-H), 7.64 (d, 1H, J = 1.9 Hz, Ph 3-H),
7.75 (dd, 1H, J = 8.4, 1.8 Hz, quinazolinone 7-H), 8.08 (d, 1H, J =
H), 10.44 (s, 1H, NHCH
[
2
), 12.20 (brs, 1H, NH). ESI-MS, m/z: 386.2
M+H] . Anal. calcd. for C19 : C, 59.20; H, 4.97; N, 10.90.
Found: C, 58.84; H, 4.98; N, 10.58.
1.8 Hz, quinazolinone 5-H), 10.53 (s, 1H, NHCH
NH). ESI-MS, m/z: 424.2 [M+H] . Anal. calcd. for C18
2
), 12.23 (s, 1H,
OS : C,
+
+
H
19
N
3
O
2
S
2
H
15Cl
2
N
3
2
50.94; H, 3.56; N, 9.90. Found: C, 50.77; H, 3.62; N, 9.78.
1
1
9
d: Yield 66%; m.p. 198–1998C. H-NMR (300 MHz, DMSO-d
.35 (s, 3H, C -CH ), 3.80(s, 3H, OCH ), 4.67 (s, 2H, CH S), 4.78 (s,
H, CH Ph), 6.86–7.29 (m, 4H, Ph-H), 7.52 (d, 1H, J = 8.6 Hz, quina-
6
) d:
9l: Yield 30%; m.p. 196.5–198.88C. H-NMR (200 MHz, DMSO-d
6
)
2
2
2
3
3
2
d: 2.33 (s, 3H, C –CH ), 4.67 (s, 2H, CH S), 4.92 (d, 2H, CH Ph), 7.35
2
3
2
2
2
(d, 2H, J = 8.2 Hz, Ph-H), 7.52 (d, 1H, J = 8.4, quinazolinone 8-H),
7.76 (dd, 1H, J = 8.4, 2.1 Hz, quinazolinone 7-H), 7.85 (d, 2H, J =
8.2 Hz, Ph-H), 8.08 (d, 1H, J = 2.1 Hz, quinazolinone 5-H), 10.58 (s,
zolinone 8-H), 7.76 (dd, 1H, J = 8.6, 2.1 Hz, quinazolinone 7-H),
8
.08 (d, 1H, J = 2.1 Hz, quinazolinone 5-H), 10.33 (s, 1H, NHCH
2
),
+
1
2.20 (s, 1H, NH). ESI-MS, m/z: 386.3 [M+H] . Anal. calcd. for
1H, NHCH
m/z: 400.2 [M+H] . Anal. calcd. for C19
55.86; H, 4.44; N, 10.29. Found: C, 55.92; H, 4.39; N, 10.03.
2
), 12.22 (brs, 1H, NH), 12.92 (brs, 1H, CO
2
H). ESI-MS,
+
C
19
H
19
N
3
O
2
S
2
: C, 59.20; H, 4.97; N, 10.90. Found: C, 58.91; H, 5.07;
H
17
N
3
O
3
S
2
N 1/2H O: C,
2
N, 10.70.
1
1
9
e: Yield 51%; m.p. 195–1978C. H-NMR (200 MHz, DMSO-d
6
) d:
S),
Ph), 6.45 (dd, 1H, J = 8.2, 2.0 Hz, Ph-H), 6.55 (d, 1H,
9m: Yield 58%; m.p. 214–2158C. H-NMR (300 MHz, DMSO-d
6
) d:
2
4
.33 (s, 3H, C
.68 (s, 2H, CH
2
–CH
3
), 3.74, 3.77 (2s, 6H, 2OCH
3
), 4.65 (s, 2H, CH
2
2.34 (s, 3H, C –CH ), 4.66 (s, 2H, CH S), 4.77 (s, 2H, CH Pyr), 7.22–
2
3
2
2
2
7.32 (m, 2H, Pyr-H), 7.52 (d, 1H, J = 8.6 Hz, quinazolinone 8-H),
7.72–7.81 (m, 2H, Pyr-H, quinazolinone 7-H), 8.08 (d, 1H, J =
2.0 Hz, quinazolinone 5-H), 8.52 (m, 1H, Pyr-H), 10.58 (s, 1H,
J = 2.0 Hz, Ph-H), 7.04 (d, 1H, J = 8.2 Hz, Ph-H), 7.52 (d, 1H, J =
.4 Hz, quinazolinone 8-H), 7.75 (dd, 1H, J = 8.4, 2.0 Hz, quinazo-
linone 7-H), 8.06 (d, 1H, J = 2.0 Hz, quinazolinone 5-H), 10.22 (s,
8
+
NHCH
2
), 12.20 (s, 1H, NH). ESI-MS, m/z: 357.2 [M+H] . Anal. calcd.
-
1
H, NHCH
calcd. for C20
7.72; H, 5.24; N, 9.83.
2
), 12.20 (brs, 1H, NH). ESI-MS, m/z: 414.2 [M-H]. Anal.
for C17
56.64; H, 4.54; N, 15.23.
9n: Yield 55%; m.p. 149–1508C. H-NMR (200 MHz, DMSO-d
H
16
N
4
OS
2
N 1/4 H
2
O: C, 56.58; H, 4.58; N, 15.53. Found: C,
H
21
N
3
O
3
S
2
: C, 57.81; H, 5.09; N, 10.11. Found: C,
1
5
6
) d:
1
9f: Yield 22%; m.p. 173.3–173.88C. H-NMR (300 MHz, DMSO-d
6
)
2.34 (s, 3H, C
2
–CH
3
), 4.66 (s, 2H, CH
2
S), 4.86 (d, 2H, CH
2
Pyr), 7.36
d: 2.34 (s, 3H, C
2
–CH
3
), 3.63 (s, 3H, OCH
3
), 3.71 (s, 6H, 2OCH
3
), 4.68
(m, 1H, Pyr-H), 7.51 (d, 1H, J = 8.4 Hz, quinazolinone 8-H), 7.68
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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254
S.-L. Cao et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 250–254
(
m, 1H, Pyr-H), 7.75 (d, 1H, J = 8.4 Hz, quinazolinone 7-H), 8.06 (s,
% Inhibition = (Mean ODcontrol –Mean ODtest)/Mean ODcontrol6100%.
1
1
C
H, quinazolinone 5-H), 8.48 (m, 2H, Pyr-H), 10.55 (s, 1H, NHCH
2.20 (s, 1H, NH). ESI-MS, m/z: 357.2 [M+H] . Anal. calcd. for
2
),
+
The dose-response curves of the compounds effecting F70%
inhibition in one-dose prescreening for each cell line were meas-
ured with the concentrations of 5, 0.5, 0.05, and 0.005 lg/mL,
17
H
16
N
4
OS
2
N 5/4 H
2
O: C, 53.88; H, 4.92; N, 14.78. Found: C, 54.16;
H, 4.79; N, 14.38.
1
and the concentration causing 50% cell growth inhibition (IC50
was calculated. The results are given in Table 1.
)
9
o: Yield 49%; m.p. 194.2–195.38C. H-NMR (300 MHz, DMSO-
d
6
) d: 2.34 (s, 3H, C
2 3 2 2
–CH ), 4.66 (s, 2H, CH S), 5.01 (s, 2H, CH NH),
6.97 (dd, 1H, J = 4.8, 3.4 Hz, thiophene 4-H), 7.05 (d, J = 3.4 Hz, 1H,
thiophene 3-H), 7.42 (d, 1H, J = 4.8 Hz, thiophene 5-H), 7.51(d, 1H,
J = 8.6 Hz, quinazolinone 8-H), 7.75 (dd, 1H, J = 8.6, 2.1 Hz, quina-
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+
1
H, NHCH
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2
), 12.19 (brs, 1H, NH). ESI-MS, m/z: 362.3 [M+H] . Anal.
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N
3
OS
3
N 1/8 H
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[
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p: Yield 36%; m.p. 199–2008C. H-NMR (300 MHz, DMSO-d
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19 3 2 2
2
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[
[
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the above-mentioned medium at a density of 4610 cells per
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2
the cell medium in test wells was changed to new culture med-
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diphenyltetrazolium bromide, 0.5 mg/mL) in serum-free med-
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tional 4 h. Then, 200 lL of DMSO was added to each well and
mixed thoroughly to dissolve the resulting formazan product.
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i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com