Synthesis and in vitro antiproliferative evaluation of pyrimido[5,4-c] quinoline-4-(3H)-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.10.044

A series of pyrimido[5,4-c]quinoline-4-(3H)-one derivatives variously substituted at positions 2 and 3 were synthesized and evaluated for their in vitro antiproliferative activities against a panel of six human cancer cell lines. Biological evaluation rev

Full text of DOI:10.1016/j.ejmech.2011.10.044

European Journal of Medicinal Chemistry 47 (2012) 206e213
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antiproliferative evaluation of pyrimido[5,4-c]quinoline-4-
(3H)-one derivatives
,
Yong Ai a, Yong-Ju Liang b, Jian-Chao Liu c, Hong-Wu He c, Yu Chen a, Chu Tang a, Guang-Zhong Yang a
,
*
,
Li-Wu Fu b
*
a Laboratory for Natural Product Chemistry, College of Pharmacy, South Central University for Nationalities, 708 Minyuan Road, Wuhan 430074, PR China
b State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou 510060, PR China
c College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2011
Received in revised form
20 September 2011
Accepted 23 October 2011
Available online 25 October 2011
A series of pyrimido[5,4-c]quinoline-4-(3H)-one derivatives variously substituted at positions 2 and 3
were synthesized and evaluated for their in vitro antiproliferative activities against a panel of six human
cancer cell lines. Biological evaluation revealed that the vast majority of derivatives exhibited moderate
tumor growth inhibitory activities. In particular, compound 7e showed effective anti-tumor activity with
broad-spectrum toward numerous cell lines and the most active member in this study. This derivative
displaying significant activity against KB (IC50: 4.9
GLC-82 (IC50: 7.88 M), MDA-MB-453 (IC50: 18.2
m
M), CNE2 (IC50: 13.8
m
M), MGC-803 (IC50: 4.8
mM),
m
m
M) and MCF-7 (IC50: 10.1
m
M) cell lines could be
Keywords:
Synthesis
Pyrimido[5,4-c]quinoline-4-(3H)-one
Antiproliferative activities
Antitumor agent(s)
considered as the most promising and useful template for future development to obtain more potent
anti-tumor agent(s).
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
kinase C (PKC) exhibited effective growth inhibition against cancer
cell lines such as A 431 cells and HT 1080 cells [15]; 2-amino-pyr-
Although there has been great progress in the development of
treatment and prevention for cancer, it still remains an enormous
threat to people’s health in the 21st century, representing the
second primary cause of death in the world [1]. In the past years,
considerable efforts have been made to develop innovative strate-
gies for finding safe and effective methods of treating this disease.
With the increasing understanding of the biological process
involved in cancer cell survival and the discovering of new target,
more and more novel chemical therapeutic drugs have been
designed for treatment of cancer.
The derivatives of pyrimidoquinolines have been attracted great
interest over many years due to their broad bioactivities. A great
deal of investigations on the synthesis of pyrimidoquinolines was
carried out [2e9], and many derivatives with excellent anti-HIV
and anti-tumor activities have been obtained [10e14]. For
example, 5-deazaflavins {pyrimido[4,5-b]quinoline-2,4 (3H,10H)-
diones} and 2-deoxo-2-phenyl-5-deazaflavins {2-phenylpyrimido
[4,5-b]quinolin-4(10H)-ones} as selective inhibitors of protein
imido[4,5-c]quinolin-1(2H)-ones and 2,5-diaryl-3-methylpyrimido
[4,5-c]quinolin-1(2H)-one derivatives as a class of cytotoxic anti-
mitotic agents have been reported in the literatures [13,16]; in
addition, many attentions have also been focused on the synthesis
and bioactivities of pyrimido[5,4-c]quinoline derivatives [17e22].
Although previous researches revealed that they exhibited several
significant pharmacological activities (e.g. antioxidant [20], anti-
malarials [21], antiherpetic [22]), these reported pyrimido[5,4-c]
quinoline derivatives suffered from serious limitations such as the
rapid development of drug resistant. Thus, novel, potent, selective
and less toxic agents containing pyrimido[5,4-c]quinoline system
are still urgently required to triumph over the limitations.
In the present work, a novel series of pyrimido[5,4-c]quinoline-
4-(3H)-one derivatives bearing variously substituted at positions 2
and 3 were synthesized and tested for in vitro antiproliferative
activity. The preliminary bioassay showed that some of them have
antiproliferative activity and the most active compound 7e against
KB (IC50: 4.9
GLC-82 (IC50: 7.88
(IC50: 10.1 M) cell lines could be regarded as the most promising
m
M), CNE2 (IC50: 13.8
m
M), MGC-803 (IC50: 4.8
mM),
mM), MDA-MB-453 (IC50: 18.2
mM) and MCF-7
m
* Corresponding authors.
E-mail addresses: yanggz888@126.com (G.-Z. Yang), fulw@mail.sysu.edu.cn
(L.-W. Fu).
and useful template for future development to obtain more potent
anti-tumor agent(s).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.044

Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
207
2. Chemistry
MCF-7 breast adenocarcinoma cells by performing MTT assay.
These results were summarized in Table 2 and presented as the
concentration of drug inhibiting 50% cell growth (IC50). 5-FU which
is one of the most effective anticancer agents was used as the
reference drug in this study.
The synthetic methods of pyrimido[5,4-c]quinoine derivatives
using pyrimidine moiety [19,20] or quinoline moiety [21e23] as
sarting materials have appeared in the literature. Here, we present
three synthetic routes to novel pyrimido[5,4-c]quinoine derivatives
(Scheme 1).
4. Results and discussion
Firstly, a series of pyrimido[5,4-c]quinoine derivatives bearing
3-substituted groups have been synthesized. The general procedure
for the synthesis of derivatives 6aek was described in Scheme 1.
Ethyl 4-amino-2-methylquinoline-3-carboxylate (2) was prepared
in 68% yield from 2-Aminobenzonitrile (1) by treatment with ace-
toacetic ester and tin tetrachloride in refluxing toluene. It was
treated with triethyl orthoformate using acetic anhydride as
a catalyst to yield intermediate 5. Upon reaction with n-butylamine,
n-propylamine, isopropylamine, hydrazine hydrate, glycol, etha-
4.1. Nuclear magnetic resonance spectral studies
In this paper, IR, 1H-NMR spectra, 13C NMR spectra and mass
spectra were used for the identification and confirmation of the
newly assigned structures. Assignments of the signals are based on
the chemical shifts and intensity patterns.
The 1H-NMR spectra of all the target compounds, showed
a singlet at dH 2.97e3.12 ppm, integrating for 3 protons: this data
suggested that the signal belonged to 5-Me. Still, four downfield
aromatic protons on the quinoline ring were distinctly observed in
1H-NMR spectra. The structures of the compounds 6aek were
confirmed in particular by the presence of a proton resonance at the
2-position as a singlet signal at dH 8.21e9.03 ppm in 1H-NMR
spectra. The H NMR spectra of compounds 8bec showed singlets
for (NH2) proton at dH 5.75 and another singlets for one (NH) proton
at dH 9.81e9.98.
nolamine,
b
-phenylethylamine, benzylamine, ethylamine, (ꢀ)-sec-
butylamine and isobutylamine, yielded corresponding 3-
substituted pyrimido[5,4-c]quinoine-4-(3H)-one derivatives 6aek.
Secondly, the iminophosphorane 3 was obtained in a sat-
isfactory yield when 2 was treated with triphenylphosphine,
hexachloroethane and Et3N. As shown in Scheme 1, the imino-
phosphorane 3 reacted with substituted phenyl isocyanate to give
carbodiimide 4 by using aza-Wittig reaction in a mild condition.
The reaction of carbodiimide 4 with substituted phenols yielded
7aej in high yields under the condition of heating for 4e6 h in the
presence of a catalytic amount of K2CO3.
13C NMR spectra of all the compounds were taken and the signal
obtained further confirmed the proposed structures. All the
compounds showed a signal at 23.0e27.5ppm due to methyl
carbon. The characteristic peaks observed within the 13C NMR
spectra of synthesized derivatives are given in Section 6.
Finally, carbodiimide 4 was treated with 2-methylpropan-2-
amine or hydrazine hydrate to yield 8aec in satisfactory yield at
room temperature for 12-13h using EtONa as a catalyst. Meanwhile,
compound 8b was reacted with triethyl orthoformate to afford the
cyclized compound 9. The preparations are summarized in Table 1.
4.2. In vitro antiproliferative activity
The present results demonstrate that some compounds exhibi-
ted significant activity against certain cancer cell lines in compar-
ison with 5-FU. Among them, compound 7e showed the best
3. Pharmacology
To evaluate the anticancer potencies of these newly synthesized
inhibitory activity with IC50 values 4.9
mM,13.8 mM, 4.8 mM, 7.88 mM,
pyrimido[5,4-c]quinoline-4(3H)-one
derivatives,
the
anti-
18.2 M, 10.1 M against KB, CNE2, MGC-803, GLC-82, MDA-MB-
m
m
proliferative activities of compounds 6aek, 7aej, 8aec and 9 were
tested against six human cancer cell lines including KB human oral
carcinoma cells, CNE2 human nasopharyngeal carcinoma cells,
MGC-803 human gastric carcinoma cells, GLC-82 human lung
carcinoma cells, MDA-MB-453 human breast carcinoma cells and
453 and MCF-7, respectively.
Compounds 7a, 7c and 7e displayed more potent inhibitory
activity than compounds 7b and 7d, indicating that introduction of
bulky and electron-withdrawing substituent such as groups
bearing naphthalene, bromo and nitryl would benefit the potency.
R2
NH
R1
PPh3
N
N
NH2
N
N
N
R1
O
N
N
CO2Et
CN
CO2Et
Me
CO2Et
i
iii
vii
ii
N
Me
NH2
Me
N
Me
4
1
3
2
8a-c
viii
PhN
vi
iv
R2
O
N
R1
O
R1
N
EtOHC
N
N
N
N
N
N
N
CO2Et
Me
v
O
O
Me
N
Me
N
Me
N
5
6a-k
7a-j
9
Scheme 1. Synthesis of derivatives 6aek, 7aej, 8aec and 9. Reagents and conditions: (i) CH3COCH2COOCH2CH3, SnCl4, 130 ꢁC, 6 h; (ii) PPh3, C2Cl6, Et3N, CH3CN, rt, 24 h; (iii) R1NCO,
CH2Cl2, 45 ꢁC, 12 h; (iv) CH(OEt)3/Ac2O, reflux, 6 h; (v) RNH2/anhydrous acetonitrile, 40 ꢁC, 8 h; (vi) R2OH, K2CO3, 75 ꢁC, 4e6 h; (vii) R2NH2, rt, 0.5e1 h, CH3CH2ONa, CH3CH2OH, rt,
12 h; (viii) CH(OEt)3, reflux, 4 h.

208
Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
Table 1
The preparation of derivatives 6aek, 7aej, 8aec and 9.
Compound
R1
R2
Yield (%)
Mp. (ꢁC)
6a
6b
6c
6d
6e
6f
n-Bu
n-Pr
i-Pr
NH2
e(CH)2OH
e(CH)2 NH2
e
e
e
e
e
e
50.0
50.0
46.0
43.0
53.0
51.9
123.4e125.1
129.9e131.0
119.4e121.4
291.0e292.0
248.6e249.2
232.8e235.4
6g
e
36.7
167.6e168.8
6h
6i
Bn
Et
e
e
34.7
30.7
160.0e161.2
128.1e129.0
6j
e
e
40.7
50.2
33.8
134.2e135.0
138.3e140.1
208.4e210.4
6k
7a
Cl
Cl
Me
O
7b
32.2
209.2e210.3
7c
30.2
29.5
208.3e210.2
270.6e272.4
Cl
Cl
Br
O
O
7d
Cl
7e
34.6
248.7e251.2
Cl
NO2
7f
30.5
34.5
196.0e197.5
193.8e194.9
F
F
F
Cl
7g
Me
Cl
7h
7i
33.6
35.5
207.7e208.9
216.8e219.1
F
Me
O

Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
209
Table 1 (continued )
Compound
R1
R2
Yield (%)
30.9
Mp. (ꢁC)
Me
O
7j
225.6e226.9
8a
8b
8c
30.6
40.0
42.1
240.7e242.6 ꢁC
285.7e287.3
286.8e290.0
eNH2
eNH2
Cl
PhN
9
37.7
335.0e336.0
N
N
Compared compound 8b (MGC-803 IC50: >50
mM) with 8c (MGC-
5. Conclusion
803 IC50: 14.7 M), it can be found that the 4-chloro at 2 position
m
would enhance antiproliferative activity. Meanwhile, the cyclized
compound 9 showed weak antiproliferative activity.
In summary, we have synthesized a novel series of pyrimido
[5,4-c]quinoline-4-(3H)-one derivatives. Compounds 7a, 7c and 7e
showed potential antiproliferative activity with broad-spectrum
against several human cancer cell lines. Compound 7e which was
the most active members in this study displayed more or similar
potent antiproliferative activities against cancer cell lines in
comparison with 5-FU. These findings have encouraged us to
continue the development and testing of novel pyrimidoquinoline
derivatives and to conduct further studies to investigate SAR and
their mechanisms of action.
Table 2
Antiproliferative activities of compounds 6a-k, 7a-j, 8a-c and 9 against KB, CNE2,
MGC-803, GLC-82, MDA-MB-453, and MCF-7 cells.
a
Compound
In vitro antiproliferative IC50
(
m
M)
KB
CNE2
MGC-803
GLC-82
MDA-MB-453
MCF-7
6a
6b
6c
6d
6e
6f
6g
6h
6i
>50
>50
>50
>50
>50
39.8
>50
>50
>50
>50
>50
33.9
39.2
29.5
30.9
4.9
>50
>50
>50
>50
>50
>50
>50
26.5
>50
>50
>50
47.0
>50
28.7
47.7
13.8
nt
>50
>50
>50
42.9
>50
>50
>50
>50
17.2
>50
>50
>50
>50
>50
39.9
>50
>50
>50
>50
>50
18.5
>50
15.3
>50
4.8
>50
>50
>50
>50
>50
>50
35.2
>50
>50
>50
>50
42.1
>50
>50
>50
7.88
nt
>50
>50
>50
>50
>50
>50
>50
>50
15.5
>50
>50
>50
>50
>50
45.7
49.5
>50
>50
>50
>50
>50
49.7
26.6
>50
18.2
nt
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
43.5
>50
>50
>50
>50
>50
37.9
34
35.7
>50
10.1
nt
>50
>50
35.5
>50
>50
>50
>50
>50
9.67
6. Experimental protocols
6.1. Chemistry
6.1.1. General
Melting points were measured on an electrothermal melting
point apparatus and are uncorrected. IR spectra were recorded on
an NE XUS-470 infrared spectrometer as KBr pellets with absorp-
tion in cmꢂ1. 1H-NMR spectra were recorded in CDCl3, DMSO-d6 or
CD3COCD3 as solvent on a Varian Mercury 400 (or 600) spec-
trometer and resonances are given in ppm (d) relative to TMS. MS
spectra were measured with a Finnigan MS spectrometer. All of the
solvents and materials were reagent grade and purified as required.
All compounds were routinely checked by thin-layer chromatog-
raphy (TLC) on pre-coated silica gel GF254 plates (Qingdao Haiyang
Chemical Co., Ltd., P. R. China). Column chromatography was per-
formed using silica gel (200e300 mesh) from Qingdao Haiyang
Chemical Group Co., China.
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
ntb
nt
>50
>50
>50
22.8
>50
>50
30.3
>50
9.85
41.4
>50
>50
>50
>50
>50
14.7
>50
16.6
7j
8a
8b
8c
9
6.1.2. General procedure for the preparation of 3-substituted 5-
methylpyrimido[5,4-c]- quinolin-4(3H)-ones (6aek)
2-Aminobenzonitrile (1; 2.36 g, 20 mmol) and SnCl4 (3.7 ml,
32 mmol) were add to a stirred solution of ethyl acetoacetate
(2.6 ml, 20 mmol) in anhydrous toluene (50 ml). The reaction
mixture was stirred under nitrogen at room temperature for 0.5 h,
5-FU
Antiproliferative activity was determined by MTT assay as shown in Experimental
6.2 part. All data are presented as mean values of three independent experiments.
Coefficients of variation were <10%.
a
IC50: concentration of the tested compound that inhibits 50% of cell growth.
nt: not test.
b

210
Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
and then heated at reflux for 6 h (130 ꢁC). The mixture was added to
sat.aq. Na2CO3 solution (150 ml, pH 10), and the resulting suspen-
sion was extracted with AcOEt (3 ꢃ 50 ml). The combined extracts
were dried (Na2SO4) and concentrated under reduced pressure, the
obtained residue was subjected to column chromatography (silica
gel, eluent: petroleum ether-acetone, 7:3) to afford 2 in 68% yield.
To a solution of ethyl 4-amino-2-methylquinoline-3-carboxylate
2 (0.46 g, 2 mmol) in 20 ml triethyl orthoformate, 1 ml of acetic
anhydride was added under nitrogen at temperature. The solution
was stirred and refluxed for 6 h. At which time the reaction was
completed, the solution was concentrated under vacuum. Removal
of the solvent gave intermediate 5, which was used directly without
further purification.
To the solution of 5 prepared above in anhydrous acetonitrile
(10 ml) was added substituted primary amine (3 ml). The mixture
was stirred for 8 h at 40 ꢁC and filtered, the filtrate was condensed
and the residue was recrystallized from dichloromethane/petro-
leum ether to give pure 3-substituted-5-methylpyrimido[5,4-c]
quinolin-4(3H)-ones 6aek.
IR(KBr): 1681 cmꢂ1 (C]O), 3409 cmꢂ1 (eOH); 1H NMR (400 MHz,
DMSO-d6): 3.03 (s, 3H, Me), 3.72 (br s, 2H, CH2), 4.11 (br s, 2H, CH2),
5.01 (s,1H, OH), 7.69 (t, J ¼ 8.0,1H, Ar-H), 7.90 (t, J ¼ 8.0,1H, Ar-H), 7.99
(d, J ¼ 8.0, 1H, Ar-H), 8.73 (d, J ¼ 8.0, 1H, Ar-H), 8.67 (s, 1H, CH); 13C
NMR (125 MHz, CDCl3): dC 159.8, 159.0, 153.2, 153.1, 147.8, 128.1, 132.1,
126.5,122.7,124.2,112.2, 49.2, 57.9, 27.2; EI MS: m/z 256 ([Mþ1]þ,10),
255 ([M]þ, 63), 212 (64), 211 (100),194 (19),183 (40),169 (5),167 (10);
HREIMS m/z 255.1013 (calcd for C14H13N3O2, 255.1012).
6.1.2.6. 3-(2-aminoethyl)-5-methylpyrimido[5,4-c]quinolin-4(3H)-
one (6f). Pale yellow powder, yield 51.9%, m.p. 232.8e235.4 ꢁC;
IR(KBr): 1669 cmꢂ1 (C]O), 3356 cmꢂ1 (NH2); 1H NMR (400 MHz,
DMSO-d6): 3.02 (s, 3H, Me), 2.89 (br s, 2H, CH2), 4.00 (br s, 2H, CH2),
7.68 (t, J ¼ 8.0,1H, Ar-H), 7.89 (t, J ¼ 8.0,1H, Ar-H), 7.98 (d, J ¼ 8.0,1H,
Ar-H), 8.74 (d, J ¼ 8.0, 2H, Ar-H); 13C NMR (125 MHz, CDCl3): dC 153.3,
159.9, 159.1, 128.1, 132.0, 126.5, 124.2, 122.8, 147.8, 153.1, 112.4, 49.5,
39.7, 27.3; EI MS: m/z 254 ([M]þ, 5), 253 (2), 226 (6), 225 (43), 212
(100),197 (23); HREIMS m/z 254.1167 (calcd for C14H14N4O, 254.1167).
6.1.2.7. 5-Methyl-3-phenethylpyrimido[5,4-c]quinolin-4(3H)-one
(6g). White solid, yield 36.7%, m.p. 167.6e168.8 ꢁC; IR(KBr):
1690 cmꢂ1 (C]O); 1H NMR (400 MHz, DMSO-d6): 3.04 (s, 3H,
Me), 3.07 (t, J ¼ 6.8, 2H, CH2), 4.27 (t, J ¼ 6.4, 2H, CH2), 7.23e7.31
(m, 5H, Ar-H), 7.66 (t, J ¼ 8.0, 1H, Ar-H), 7.89 (t, J ¼ 8.0, 1H, Ar-H),
7.98 (d, J ¼ 8.0, 1H, Ar-H), 8.56 (s, 1H, Ar-H), 8.66 (d, J ¼ 8.0, 1H, Ar-
H); 13C NMR (100 MHz, DMSO-d6): dC 26.5, 33.8, 47.5, 111.8, 122.4,
123.8, 125.9, 126.2, 127.7, 128.1, 128.4, 128.4, 131.5, 137.5, 147.6,
151.8, 151.8, 152.8, 158.7, 159.4; EI MS: m/z 316 ([Mþ1]þ, 5), 315
(23), 212 (15), 211 (100), 183 (17), 104 (24); HREIMS m/z 315.1380
(calcd for C20H17N3O, 315.1379).
6.1.2.1. 3-Butyl-5-methylpyrimido[5,4-c]quinolin-4(3H)-one (6a). Pale
yellow powder, yield 50%, m.p. 123.4e125.1 ꢁC; IR(KBr): 1681 cmꢂ1
(C]O); 1H NMR (400 MHz, CDCl3): 0.95 (t, J ¼ 6.8, 3H, Me), 1.40 (m,
2H, CH2), 1.76 (m, 2H, CH2), 3.11 (s, 3H, Me), 3.95 (t, J ¼ 7.2, 2H, CH2),
7.55 (t, J ¼ 8.0,1H, Ar-H), 7.77 (t, J ¼ 8.0,1H, Ar-H), 7.99 (d, J ¼ 8.0,1H,
Ar-H), 8.21 (s,1H, CH), 8.68 (d, J ¼ 8.0,1H, Ar-H); 13C NMR (100 MHz,
CDCl3): dC 13.6, 19.9, 27.5, 31.2, 47.4, 112.7, 123.1, 124.4, 126.4, 128.3,
132.1, 148.4,150.4,153.8,160.0,160.3; EI MS: m/z 267 ([M]þ, 72), 266
(17), 250 (5), 238 (11), 225 (51), 211 (100), 194 (12), 183 (23);
HREIMS m/z 267.1373 (calcd for C16H17N3O, 267.1373).
6.1.2.2. 5-Methyl-3-propylpyrimido[5,4-c]quinolin-4(3H)-one(6b). Pale
yellow powder, yield 50%, m.p. 129.9e131.0 ꢁC; IR(KBr): 1676 cmꢂ1
(C]O); 1H NMR (400 MHz, DMSO-d6): 0.93 (t, J ¼ 6.8, 3H, Me),1.77 (m,
2H, CH2), 3.00 (s, 3H, Me), 4.00 (t, J ¼ 7.6, 2H, CH2), 7.68 (t, J ¼ 8.0, 1H,
Ar-H), 7.89 (t, J ¼ 8.0, 1H, Ar-H), 7.96 (d, J ¼ 8.0, 1H, Ar-H), 8.70 (d,
J ¼ 8.0, 1H, Ar-H), 8.78 (s, 1H, CH); 13C NMR (100 MHz, DMSO-d6): dC
10.5, 21.4, 26.8, 47.7, 111.7, 122.3, 123.8, 126.1, 127.7, 131.6, 147.4, 152.2,
152.7, 158.6, 159.3; EI MS: m/z 254 ([Mþ1]þ, 13), 253 (75), 225 (6), 212
(14), 211 (100), 194 (8), 183 (29); HREIMS m/z 253.1218 (calcd for
C15H15N3O, 253.1218).
6.1.2.8. 3-Benzyl-5-methylpyrimido[5,4-c]quinolin-4(3H)-one
(6h).
Pale yellow powder, yield 34.7%, m.p. 160.0e161.2 ꢁC; IR(KBr):
1680 cmꢂ1 (C]O); 1H NMR (400 MHz, DMSO-d6): 3.01 (s, 3H, Me),
5.27 (s, 2H, CH2), 7.31e7.42 (m, 5H, Ar-H), 7.70 (t, J ¼ 8.0, 1H, Ar-H),
7.90 (t, J ¼ 8.0, 1H, Ar-H), 7.99 (d, J ¼ 8.0, 1H, Ar-H), 9.03 (s, 1H, Ar-
H), 8.76 (d, J ¼ 8.0, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6): dC
26.4, 49.1, 112.0, 122.4, 123.9, 126.0, 127.4, 127.4, 127.8, 128.3, 128.3,
131.6, 136.0, 147.7, 152.0, 152.0, 152.9, 158.7, 159.4; EI MS: m/z 302
([Mþ1]þ, 23), 301 ([M]þ, 100), 224 (11), 211 (14), 91 (90); HREIMS m/z
301.1210 (calcd for C19H15N3O, 301.1211).
6.1.2.3. 3-Isopropyl-5-methylpyrimido[5,4-c]quinolin-4(3H)-one
(6c). Pale yellow powder, yield 46.0%, m.p. 119.4e121.4 ꢁC;
IR(KBr): 1673 cmꢂ1 (C]O); 1H NMR (400 MHz, DMSO-d6): 1.49
(d, J ¼ 6.8, 6H, 2Me), 3.01 (s, 3H, Me), 5.05 (m, 1H, CH), 7.68 (t,
J ¼ 8.0, 1H, Ar-H), 7.87 (t, J ¼ 8.0, 1H, Ar-H), 7.95 (d, J ¼ 8.0, 1H, Ar-
H), 8.70 (d, J ¼ 8.0, 1H, Ar-H), 8.85 (s, 1H, CH); 13C NMR (100 MHz,
DMSO-d6): dC 20.9, 20.9, 26.9, 46.2, 111.5, 122.2, 123.7, 126.1,
127.7, 131.6, 147.4, 149.7, 152.0, 158.8, 159.0; EI MS: m/z 254
([Mþ1]þ, 12), 253 (76), 225 (4), 212 (15), 211 (100), 194 (5), 183
(36); HREIMS m/z 253.1214 (calcd for C15H15N3O, 253.1214).
6.1.2.9. 3-Ethyl-5-methylpyrimido[5,4-c]quinolin-4(3H)-one (6i). Pale
yellow crystal, yield 30.7%, m.p. 128.1e129.0 ꢁC; IR(KBr): 1684 cmꢂ1
(C]O); 1H NMR (400 MHz, DMSO-d6): 1.35 (t, J ¼ 6.0, 3H, Me), 3.03 (s,
3H, Me), 4.09 (q, J ¼ 6.8, 2H, CH2), 7.68 (t, J ¼ 8.0, 1H, Ar-H), 7.90 (t,
J ¼ 8.0, 1H, Ar-H), 7.99 (d, J ¼ 8.0, 1H, Ar-H), 8.83 (s, 1H, CH), 8.74 (d,
J ¼ 8.0, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6): dC 14.0, 26.7, 41.6,
111.9, 122.5, 123.9, 126.1, 127.8, 131.7, 147.7, 152.0, 152.9, 158.8, 159.4; EI
MS: m/z 240 ([Mþ1]þ, 17), 239 ([M]þ, 100), 210 (8), 211 (61), 183 (26);
HREIMS m/z 239.1064 (calcd for C14H13N3O, 239.1063).
6.1.2.10. 3-sec-Butyl-5-methylpyrimido[5,4-c]quinolin-4(3H)-one
(6j). Red powder, yield 40.7%, m.p.134.2e135.0 ꢁC; IR(KBr):
1688 cmꢂ1 (C]O); 1H NMR (400 MHz, DMSO-d6): 0.82 (t, J ¼ 6.8,
3H, Me), 1.03 (d, J ¼ 6.4, 3H, Me), 1.33 (m, 2H, CH2), 4.34 (m, 1H,
CH), 7.43 (t, J ¼ 8.0, 1H, Ar-H), 7.67 (t, J ¼ 8.0, 1H, Ar-H), 7.77 (s, 1H,
Ar-H), 7.96 (s, 1H, Ar-H), 8.29 (d, J ¼ 8.0, 1H, Ar-H); 13C NMR
(100 MHz, DMSO-d6): dC 14.1, 26.8, 60.6, 102.2, 117.1, 122.9, 122.9,
124.5, 124.5, 128.1, 131.1, 131.1, 147.0, 153.1, 157.8, 168.5; EI MS: m/z
267 ([M]þ, 4), 230 (59), 185 (24), 184 (100); HREIMS m/z 267.1366
(calcd for C16H17N3O, 267.1367).
6.1.2.4. 5-Methyl-3-aminopyrimido[5,4-c]quinolin-4(3H)-one
(6d).
Pale yellow powder, yield 43.0%, m.p. 291.0e292.0 ꢁC; IR(KBr):
1682 cmꢂ1 (C]O), 3300 cmꢂ1, 3176 cmꢂ1 (NH2); 1H NMR (400 MHz,
DMSO-d6): 3.06 (s, 3H, Me), 6.04 (s, 2H, NH2), 7.70 (t, J ¼ 8.0,1H, Ar-H),
7.91 (t, J ¼ 8.0, 1H, Ar-H), 8.00 (d, J ¼ 8.0, 1H, Ar-H), 8.76 (d, J ¼ 8.0, 1H,
Ar-H), 8.83 (s, 1H, CH); 13C NMR (100 MHz, DMSO-d6): dC 26.3, 111.5,
122.5, 123.8, 126.1, 127.8, 131.5, 147.5, 151.3, 152.3, 158.6, 159.2; EI MS:
m/z 227 ([Mþ1]þ, 12), 226 (93), 211 (4), 198 (14), 197 (100), 169 (11),
142 (14); HREIMS m/z 226.0848 (calcd for C12H10N4O, 226.0849).
6.1.2.5. 3-(2-hydroxyethyl)-5-methylpyrimido[5,4-c]quinolin-4(3H)-
one (6e). Pale yellow powder, yield 53.0%, m.p. 248.6e249.2 ꢁC;
6.1.2.11. 3-Isobutyl-5-methylpyrimido[5,4-c]quinolin-4(3H)-one
(6k). Pale yellow crystal, yield 50.2%, m.p. 138.3e140.1 ꢁC;

Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
211
IR(KBr): 1679 cmꢂ1 (C]O); 1H NMR (400 MHz, DMSO-d6): 0.93
(d, J ¼ 6.0, 6H, 2Me); 2.15 (m, 1H, CH); 3.01 (s, 3H, Me); 3.87 (d,
J ¼ 6.4, 2H, CH2); 7.68 (t, J ¼ 8.0, 1H, Ar-H); 7.89 (t, J ¼ 8.0, 1H, Ar-
H); 7.98 (d, J ¼ 8.0, 1H, Ar-H); 8.73 (d, J ¼ 8.0, 1H); 8.77 (s, 1H); 13C
NMR (100 MHz, DMSO-d6): dC 19.2, 19.2, 26.8, 53.0, 111.7, 122.3,
123.8, 123.8, 126.1, 127.7, 131.6, 147.4, 152.3, 152.6, 158.7, 159.4; EI
MS: m/z 268 ([Mþ2]þ, 6), 267 (Mþ, 40), 210 (20), 211 (100), 183
(25); HREIMS m/z 267.1371 (calcd for C16H17N3O, 267.1372).
Ar-H), 7.40 (d, J ¼ 8.0, 2H, Ar-H), 7.51e7.64 (m, 3H, Ar-H), 7.82 (t,
J ¼ 8.0, 1H, Ar-H), 8.03 (d, J ¼ 8.0, 1H, Ar-H), 8.28 (d, J ¼ 8.0, 1H, Ar-
H); 13C NMR (100 MHz, CDCl3): dC 27.0, 109.7, 119.6, 122.7, 122.9,
123.4, 124.5, 126.0, 126.3, 128.3, 129.4, 129.8, 130.0, 132.3, 132.8,
133.0, 135.2, 135.4, 148.8, 153.7, 155.4, 159.9, 160.2, 162.0; EI MS: m/z
495 ([M þ 4]þ, 7), 493 ([M þ 2]þ, 29), 491 (Mþ, 21), 351 (67), 320
(38), 259 (100), 211 (14), 155 (85), 128 (31); HREIMS m/z 491.0042
(calcd for C24H15ClBrO2N3, 491.0041).
6.1.3. General procedure for the preparation of 3-substituted
phenyl-5-methyl-2- aryloxypyrimido[5,4-c]quinolin-4(3H)-one
(7aej)
6.1.3.4. 2-(benzo[d]
[1,3]dioxol-6-yloxy)-3-(4-chlorophenyl)-5-
mehylpyrimido[5,4-c]quinolin-4 (3H)-one (7d). White solid, yield
29.5%, m.p. 270.6e272.4 ꢁC; IR(KBr): 1697.8 cmꢂ1 (C]O); 1H NMR
(400 MHz, acetone-d6): 3.00 (s, 3H, Me), 6.12 (s, 2H, CH2), 6.83 (d,
J ¼ 8.0, 1H, Ar-H), 6.95 (d, J ¼ 8.0, 1H, Ar-H), 7.29 (d, J ¼ 8.0, 1H, Ar-
H), 7.54 (m, 1H, Ar-H), 7.69 (d, J ¼ 8.0, 2H, Ar-H), 7.72 (d, J ¼ 8.0, 2H,
Ar-H), 7.85 (t, J ¼ 8.0, 1H, Ar-H), 7.94 (d, J ¼ 8.0, 1H, Ar-H), 8.31 (d,
J ¼ 8.0, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6): dC 26.7, 102.0,
103.8, 108.0, 109.7, 114.2, 122.3, 124.0, 126.4, 128.1, 129.4, 130.5,
130.5, 132.4, 132.4133.7, 133.9, 145.3, 145.7, 147.7, 148.1, 152.9, 155.5,
159.0, 161.6; EI MS: m/z 459 ([Mþ2]þ, 13), 457 (Mþ, 29), 320 (23),
304 (100), 211 (3), 155 (43); HREIMS m/z 457.0827 (calcd for
C25H16ClO4N3, 457.0827).
To a solution of 2 (1.15 g, 5 mmol) in CH2Cl2 (60 ml) was added
Ph3P (2.62 g, 10 mmol), C2Cl6 (2.37 g, 10 mmol) and, in this order,
Et3N (5.0 ml). The mixture was stirred for 24 h at room tempera-
ture. Then, the solution was concentrated, and the obtained residue
was subjected to column chromatography (silica gel, eluent:
Petroleum ether-Acetone, 8:2) to afford the 3 in 70% yield.
To a solution of iminophosphorane 3 (1.1 g, 2 mmol) in dry
methylene chloride (20 ml) was added isocyanatobenzene (0.24 g,
2 mmol), 1-fluoro-4-isocyanatobenzene (0.27 g, 2 mmol) or 1-
chloro-4-isocyanatobenzene (0.30 g, 2 mmol) under nitrogen at
45 ꢁC. After the reaction mixture was stirred for 12 h, the solvent
was removed under vacuum and Et2O/petroleum ether (1:2 30 ml)
was added to precipitate triphenylphosphine oxide. Removal of the
solvent gave carbodiimides 4, which were used directly without
further purification.
To the solution of 4 (2 mmol) in CH3CN (15 ml) was added
substituted phenol (2 mmol) and cat solid K2CO3 (0.024 g, 0.2 mmol).
The mixture was stirred for 4e6 hat75ꢁC and then filtered, the filtrate
was condensed and the residue was purified by silica gel column
chromatography using a 7:3 mixture of petroleum ether-acetone as
the eluent. The solvent was evaporated to dryness and the residue
recrystallised from ethanol, giving the expected compounds.
6.1.3.5. 2-(2-Chloro-4-nitrophenoxy)-3-(4-chlorophenyl)-5-methyl-
pyrimido[5,4-c]quinolin-4- (3H)-one (7e). White powder, yield 34.6%,
m.p. 248.7e251.2 ꢁC; IR(KBr): 1695 cmꢂ1 (C]O); 1H NMR (400 MHz,
CDCl3): 3.12 (s, 3H, Me), 7.48 (m, 3H, Ar-H), 7.61 (m, 3H, Ar-H), 7.81 (d,
J ¼ 8.0, 1H, Ar-H), 8.08 (d, J ¼ 8.0, 2H, Ar-H), 8.33 (d, J ¼ 8.0, 1H, Ar-H),
8.45 (s, 1H, Ar-H); 13C NMR (100 MHz, CDCl3): dC 26.4, 109.9, 120.0,
122.4, 123.3, 124.2, 124.7, 126.1, 126.6, 127.7, 128.3, 128.7, 129.4, 130.1,
132.2, 132.9, 135.9, 146.1, 148.4, 152.0, 153.0, 153.2, 160.0, 161.7; EI MS:
m/z 494 ([Mþ2]þ, 59), 492 ([M]þ, 92), 459 (20), 457 (60), 341 (20), 339
(63), 322 (32), 320 (100), 293 (14), 211 (10), 155 (99), 143 (14), 127
(53), 114 (11); HREIMS m/z 492.0388 (calcd for C24H14Cl2N4O4,
492.0388).
6.1.3.1. 3-(4-chlorophenyl)-5-methyl-2-(naphthalen-1-yloxy)pyrimido-
[5,4-c]quinolin-4(3H)- one (7a). Red solid, yield 33.8%, m.p.
208.4e210.4 ꢁC; IR(KBr): 1684.3 cmꢂ1 (C]O); 1H NMR (400 MHz,
DMSO-d6): 2.99 (s, 3H, Me), 7.40 (t, J ¼ 8.0, 1H, Ar-H), 7.52e7.82 (m,
12H, Ar-H), 7.97 (d, J ¼ 7.6, 1H, Ar-H), 8.04 (d, J ¼ 8.0, 1H, Ar-H); 13C
NMR (100 MHz, DMSO-d6): dC 26.1, 109.5, 118.1, 120.8, 121.9, 123.3,
125.2, 125.7, 125.8, 126.1, 126.3, 126.4, 126.4, 127.6, 127.7, 128.0, 129.1,
130.2,131.8,133.5,133.8, 133.9, 146.7, 147.7, 152.5,154.8, 158.5, 161.3; EI
MS: m/z 465 ([Mþ2]þ, 8), 463 (Mþ, 23), 320 (11), 310 (100), 211 (13),
155 (33), 128 (10), 75 (2); HREIMS m/z 463.1069 (calcd for
C28H18ClO2N3, 463.1071).
6.1.3.6. 2-(4-fluorophenoxy)-3-(4-fluorophenyl)-5-methylpyrimido
[5,4-c]quinolin-4(3H)-one (7f). Pale yellow powder, yield 30.5%,
m.p.196.0e197.5 ꢁC; IR(KBr): 1694 cmꢂ1 (C]O); 1H NMR (400 MHz,
acetone-d6): 2.97 (s, 3H, Me), 7.29e7.89 (m, 10H, Ar-H), 7.59 (t,
J ¼ 8.0, 1H, Ar-H), 8.24 (d, J ¼ 8.0, 1H, Ar-H); 13C NMR (100MHz,
DMSO-d6): dC 26.7, 109.7, 116.1, 116.2, 116.4, 122.3, 123.7, 123.8,
123.9, 126.4, 128.1, 130.7, 130.8, 131.1, 132.4, 147.5, 148.1, 152.7, 155.5,
158.7, 158.9, 160.9, 161.7, 163.3; EI MS: m/z 416 ([Mþ1]þ, 21), 415
(Mþ, 71), 350 (28), 349 (83), 322 (9), 321 (30), 305 (12), 304 (47),
279 (24), 278 (100), 227 (12), 211 (21),185 (15),184 (59),156 (8),155
(46), 143 (8), 128 (9); HREIMS m/z 415.1124 (calcd for C24H15F2N3O2,
415.1125).
6.1.3.2. 2-(2-methoxyphenoxy)-3-(4-chlorophenyl)-5-methylpyri-
mido[5,4-c]quinolin-4(3H)- one (7b). ꢂY1ellow solid, yield 32.2%, m.p.
209.2e210.3 ꢁC; IR(KBr): 1698.5 cm (C]O); 1H NMR (400 MHz,
CDCl3): 3.13 (s, 3H, Me), 3.79 (s, 3H, Me), 7.06 (d, J ¼ 8.0, 2H, Ar-H),
7.19 (d, J ¼ 8.0, 1H, Ar-H), 7.34 (t, J ¼ 8.0, 1H, Ar-H), 7.42e7.47 (m,
3H, Ar-H), 7.54e7.64 (m, 2H, Ar-H), 7.78 (t, J ¼ 8.0, 1H, Ar-H), 7.99
(d, J ¼ 8.0, 1H, Ar-H), 8.18 (d, J ¼ 8.0, 1H, Ar-H); 13C NMR
(100 MHz, CDCl3): dC 26.6, 56.0, 109.6, 112.7, 120.8, 122.6, 122.9,
124.6, 126.1, 127.4, 127.7, 129.5, 129.8, 132.5, 133.0, 135.2, 140.5,
148.4, 150.8, 153.9, 154.9, 160.1, 162.2; EI MS: m/z 445 ([Mþ2]þ, 9),
443 (Mþ, 22), 353 (35), 351 (100), 320 (20), 289 (31), 261 (13), 211
(13), 184 (13), 155 (43), 149 (27), 125 (20), 57 (9); HREIMS m/z
443.1032 (calcd for C25H18ClO3N3, 443.1033).
6.1.3.7. 2-(4-Chloro-3-methylphenoxy)-3-(4-fluorophenyl)-5-methyl-
pyrimido[5,4-c]quinolin-4 (3H)-one (7g). Pale yꢂel1low powder, yield
34.5%, m.p.193.8e194.9 ꢁC; IR(KBr): 1694 cm (C]O); 1H NMR
(400 MHz, DMSO-d6): 2.37 (s, 3H, Me), 2.97 (s, 3H, Me), 7.30 (d,
J ¼ 8.0, 1H, Ar-H), 7.36e7.61 (m, 5H, Ar-H), 7.72 (br s, 2H, Ar-H), 7.86
(t, J ¼ 8.0, 1H, Ar-H), 7.96 (d, J ¼ 8.0, 1H, Ar-H), 8.15 (d, J ¼ 8.0, 1H, Ar-
H); 13C NMR (100 MHz, DMSO-d6): dC 19.0, 25.9, 109.4, 115.6, 115.8,
120.5, 122.0, 123.5, 123.6, 125.9, 127.7, 129.3, 130.2, 130.4, 130.7, 131.8,
136.7, 147.9, 149.7, 152.4, 154.6, 158.5, 160.5, 161.2, 163.0; EI MS: m/z
445 ([M]þ, 50), 308 (100), 304 (47), 273 (52), 211 (12), 155 (75), 128
(20), 114 (8), 57 (4); HREIMS m/z 445.0997 (calcd for C25H17ClFN3O2,
445.0997).
6.1.3.3. 2-(4-bromophenoxy)-3-(4-chlorophenyl)-5-methylpyrimido
[5,4-c]quinolin-4(3H)-one (7c). Whꢂit1e solid, yield 30.2%, m.p.
208.3e210.2 ꢁC; IR(KBr): 1698.5 cm (C]O); 1H NMR (400 MHz,
CDCl3): 3.11 (s, 3H, Me), 7.17 (d, J ¼ 8.0, 2H, Ar-H), 7.25e7.28 (m, 2H,
6.1.3.8. 2-(2-chlorophenoxy)-3-(4-fluorophenyl)-5-methylpyrimido
[5,4-c]quinolin-4(3H)-one (7h). Pale yellow crystal, yield 33.6%,
m.p.207.7e208.9 ꢁC; IR(KBr): 1674 cmꢂ1 (C]O); 1H NMR (400 MHz,

212
Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
Acetone-d6): 3.01 (s, 3H, Me), 7.42e7.49 (m, 4H, Ar-H), 7.56 (m, 2H,
Ar-H), 7.66 (d, J ¼ 8.0, 1H, Ar-H), 7.78e7.85 (m, 3H, Ar-H), 7.97 (d,
J ¼ 8.0, 1H, Ar-H), 8.15 (d, J ¼ 8.0, 1H, Ar-H); 13C NMR (100 MHz,
Acetone-d6): dC 27.1, 110.8, 116.9, 117.2, 123.5, 124.9, 125.1, 126.8,
126.8, 127.3, 128.8, 129.2, 131.2, 131.6, 131.7, 132.1, 133.0,148.5,149.6,
153.8, 160.1, 162.4, 162.6, 164.9; EI MS: m/z 431 ([M]þ, 42), 396
(100), 321 (12), 304 (42), 294 (30), 259 (21), 201 (19), 155 (54), 128
(15), 77 (4); HREIMS m/z 431.0845 (calcd for C24H15ClFN3O2,
431.0844).
285.7e287.3 ꢁC; IR(KBr): 1675 cmꢂ1 (C]O), 3325 cmꢂ1 (NH2); 1H
NMR (400 MHz, DMSO-d6): 2.97 (s, 3H, Me), 5.75 (s, 2H, NH2),
7.51e7.61 (m, 3H, Ar-H), 7.79e8.00 (m, 4H, Ar-H), 8.53 (d, J ¼ 8.0,
1H, Ar-H), 9.98 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): dC 26.2,
107.2, 122.5, 123.4, 123.4, 124.2, 125.4, 127.6, 127.7, 128.2, 128.2,
131.3, 136.7, 147.9, 151.0, 152.7, 158.6, 160.4; EI MS: m/z 353
([M þ 2]þ, 30), 351 ([M]þ, 100), 322 (27), 320 (42), 212 (20), 155
(39), 128 (10); HREIMS m/z 351.0888 (calcd for C18H14Cl N5O,
351.0888).
6.1.3.9. 2-(2-methoxyphenoxy)-5-methyl-3-phenylpyrimido[5,4-c]
quinolin-4(3H)-one (7i). White powder, yield 35.5%, m.p.
216.8e219.1 ꢁC; IR(KBr): 1694 cmꢂ1 (C]O); 1H NMR (400 MHz,
Acetone-d6): 3.01 (s, 3H, Me), 3.82 (s, 3H, eOCH3)，7.08 (t, J ¼ 8.0,
1H, Ar-H), 7.24 (d, J ¼ 8.0,1H, Ar-H), 7.31 (d, J ¼ 8.0,1H, Ar-H), 7.37 (t,
J ¼ 8.0,1H, Ar-H), 7.47 (t, J ¼ 8.0,1H, Ar-H), 7.55 (s,1H, Ar-H), 7.65 (br s,
4H, Ar-H), 7.82 (t, J ¼ 8.0,1H, Ar-H), 7.95 (d, J ¼ 8.0,1H, Ar-H), 8.18 (d,
J ¼ 8.0,1H, Ar-H); 13C NMR (100 MHz, DMSO-d6): dC 26.7, 56.2,109.6,
113.4,118.2,120.8,122.3,122.7,123.7,126.3,127.6,128.1,128.5,128.8,
129.1,129.3,132.4,134.9,140.3,148.1,150.7,152.9,155.2,159.1,161.6;
EI MS: m/z 410 ([Mþ1]þ, 24), 409 ([M]þ, 85), 291 (11), 290 (62), 289
(100), 261 (45), 211 (13), 155 (74), 128 (20), 77 (13); HREIMS m/z
409.1426 (calcd for C25H19N3O2, 409.1426).
6.1.4.3. 2-(phenylamino)-5-methyl-3-aminopyrimido[5,4-c]quinolin-
4(3H)-one (8c). White solid, yield 42.1%, m.p. 286.8e290.0 ꢁC;
IR(KBr): 1659 cmꢂ1 (C]O), 3385, 3323 cmꢂ1 (NH2); 1H NMR
(400 MHz, DMSO-d6): 2.98 (s, 3H, Me), 5.75 (s, 2H, NH2), 7.19 (t,
J ¼ 8.0, 1H, Ar-H), 7.47 (t, J ¼ 8.0, 2H, Ar-H), 7.59 (t, J ¼ 8.0, 1H, Ar-H),
7.78e7.95 (m, 4H, Ar-H), 8.54 (d, J ¼ 8.0, 1H, Ar-H), 9.81 (s, 1H, NH);
13C NMR (100 MHz, DMSO-d6): dC 25.9, 107.0, 121.5, 122.4, 123.6,
123.9, 125.1, 125.1, 127.6, 128.2, 128.2, 131.0, 137.6, 147.8, 150.9, 152.7,
158.4, 160.2; EI MS: m/z 317 ([M]þ,100), 288 (16), 286 (49), 212 (16),
155 (26), 128 (7); HREIMS m/z 317.1274 (calcd for C18H15N5O,
317.1274).
6.1.5. Synthesis of 10-methyl-3-phenyl- [1,2,4]triazolo[1,5-a]pyri-
midin[5,4-c]quinolin- 11(3H)-one (9)
6.1.3.10. 2-(2,4-dichlorophenoxy)-3-phenyl-5-methyl-3H-pyrimido
[5,4-c]quinolin-4-one (7j). White powder, yield 30.9%, m.p.
225.6e226.9 ꢁC; IR(KBr): 1698 cmꢂ1 (C]O); 1H NMR (600 MHz,
Acetone-d6): dH 3.11 (3H, s), 7.26e7.63 (9H, m, 9 ꢃ Ar-H), 7.76 (1H,
td, J ¼ 8.4,1.2Hz Ar-H), 7.98 (1H, d, J ¼ 9.0Hz, Ar-H), 8.16 (1H, d,
J ¼ 9.0Hz, Ar-H); 13C NMR (100 MHz, DMSO-d6): dC 26.7, 109.9,
122.2, 123.7, 125.7, 126.6, 127.0, 128.2, 128.5, 128.8, 129.3, 129.5,
129.9, 131.5, 132.6, 146.2, 148.1, 152.5, 154.2, 159.0, 161.5; EI MS: m/z
449 ([Mþ2]þ, 29), 448 ([Mþ1]þ, 11), 447 ([M]þ, 47), 414 (31), 413
(26), 412 (100), 330 (48), 329 (13), 328 (78), 293 (34), 286 (50), 211
(12), 155 (83), 143 (14), 128 (23), 114 (9), 101 (7), 77 (12); HREIMS m/
z 447.0533 (calcd for C24H15Cl2N3O2, 447.0534).
To a solution of 2-(phenylamino)-5-methyl-3-aminopyrimido
[5,4-c]quinolin-4(3H)-one 8c (0.99 g, 3mmol) prepared above,
triethyl orthoformate (8 ml) was added under vacuum. The solution
was stirred and refluxed for 4 h. At which time the reaction was
completed, the solution was concentrated,the residue was recrys-
tallized from petroleum ether/ethanol (8/2) to give pure compound
9. Pale solid, yield 37.7%, m.p. 335.0e336.0 ꢁC; IR(KBr): 1695 cmꢂ1
(C]O); 1H NMR (400 MHz, DMSO-d6): 3.08 (s, 3H, Me), 7.62 (d,
J ¼ 8.0, 2H, Ar-H), 7.74 (d, J ¼ 8.0, 2H, Ar-H), 7.83e7.92 (m, 2H, Ar-H),
8.08 (d, J ¼ 8.0, 2H, Ar-H), 8.66 (d, J ¼ 8.0, 1H, Ar-H), 9.49 (s, 1H, Ar-
H); 13C NMR (125 MHz, CDCl3): dC 163.5, 155.8, 151.3, 144.2, 138.3,
136.3, 133.1, 131.0, 130.1,127.0, 125.3, 124.8,120.7, 108.5, 23.0; EI MS:
m/z 328 ([Mþ1]þ, 20), 327 ([M]þ, 100), 301 (4), 300 (18), 271 (4),
128 (2), 77 (9); HREIMS m/z 327.1121 (calcd for C19H13N5O,
327.1121).
6.1.4. Synthesis of 2-(tert-butylamino)-3-phenyl-5-methyl-3H-
pyrimido[5,4-c]quinolin-4-one (8a), 2-(4-chlorophenylamino)-5-
methyl-3-aminopyrimido[5,4-c]quinolin-4(3H)-one (8b) and 2-
(phenylamino)-5-methyl-3-aminopyrimido[5,4-c]quinolin-4(3H)-
one (8c)
2-methylpropan-2-amine or hydrazine hydrate (2 mmol) was
added into the solution of 4 in CH2Cl2 (10 ml). After the reaction
mixture was stirred continuously for 0.5e1 h at room temperature,
the solvent was removed and 10 ml of anhydrous ethanol with
several drops of sodium ethoxide in ethanol were added. After
stirring for another 12 h at room temperature, the solution was
concentrated and the residue was purified by silica gel column
chromatography using a 7:3 mixture of petroleum ether-acetone as
the eluent to give pure compounds 8aec.
6.2. Antiproliferative activity
6.2.1. Cell culture
All the cell lines except MCF-7 used in antiproliferative assay
were cultured in RPMI 1640 medium containing 10% fetal calf
serum. MCF-7 were cultured in DMEM medium containing 10%
fetal calf serum.
6.2.2. Viability assay
The MTT assay was used to evaluate the in vitro antiproliferative
activity of these synthesized compounds. This method is based on
the reduction of the soluble 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) into a blue-purple for-
mazan product, mainly by mitochondrial reductase activity inside
living cells.
Cells were harvested during logarithmic growth phase and
seeded in 96 well plates at a density of 2 ꢃ 104/ml cells/ml in a final
volume of 190 ml/well and incubated at 37 ꢁC in a 5% CO2 incubator.
6.1.4.1. 2-(tert-butylamino)-3-phenyl-5-methyl-3H-pyrimido[5,4-c]
quinolin-4-one (8a). Light yellow powder, yield 30.6%,
m.p.240.7e242.6 ꢁC; IR(KBr): 1681 cmꢂ1 (C]O); 1H NMR (600 MHz,
CDCl3): dH 1.51 (9H, s), 3.04 (3H, s), 7.34 (2H, d, J ¼ 7.2 Hz, 2 ꢃ Ar-H),
7.59 (4H, m, 4 ꢃ Ar-H), 7.77 (1H, t, J ¼ 7.2 Hz, Ar-H), 7.97 (1H, d,
J ¼ 8.4 Hz, Ar-H), 8.70 (1H, d, J ¼ 7.8 Hz, Ar-H); 13C NMR (100 MHz,
CDCl3): dC 27.1, 29.0, 53.2,107.5,123.5,124.8,125.4,128.1,128.7,130.1,
130.9,131.7,134.6,151.5,155.3,160.5,162.2; EI MS: m/z 359 ([M þ 1]þ,
9), 358 ([M]þ, 35), 302 (60), 301 (100), 155 (10), 77(5); HREIMS
m/z 358.1799 (calcd for C22H22N4O, 358.1798).
After 24 h, 10
mL tested compounds was added to 96-well plates and
cultured at 37 ꢁC for 72 h. 20
mL of MTT (5 mg/ml stock 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 100 ml of
DMSO was added to each well to dissolve the formazan crystals
6.1.4.2. 2-(4-chlorophenylamino)-5-methyl-3-aminopyrimido[5,4-c]
quinolin-4(3H)-one (8b). White solid, yield 40.0%, m.p.

Y. Ai et al. / European Journal of Medicinal Chemistry 47 (2012) 206e213
213
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Acknowledgements
The Special Fund for Basic Scientific Research of Central
Colleges, South-Central University for Nationalities (ZZZ10006).
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