Synthesis and antitumor activities of 4H-Pyrano[3,2-h]quinoline-3- carbonitrile, 7H-Pyrimido[4',5':6,5]pyrano[3,2-h]quinoline, and 14HPyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]quinoline derivatives

Article abstract of DOI:10.2174/157018012800389331

Several 4H-pyrano [3,2-h] quinoline 3,4,7-9, 7H-pyrimido[4',5':6,5] pyrano[3,2-h]quinoline 10a,b and 14Hpyrimido[4',5':6,5]pyrano[3,2-h][1,2,4] triazolo[1,5-c]quinoline 11a-c derivatives were obtained by treatment of (E) 2-(4-chlorostyryl)-8-hydroxyquinoline 1, (E) 2-amino-4-(4-chlorophenyl)-9-(4- chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitr-ile 3 or (E) 9-amino-7-(4-chlorophenyl)-2-(4-chlorostyryl)-8-imino-8,9-dihydro-7H-pyrimido- [4',5':6,5]pyrano [3,2-h]quinoline 10b with different electrophiles followed by nucleophilic reagents. Structures of these compounds were established on the basis of IR, ¹H NMR, ¹³C NMR, ¹³C NMR-DEPT, ¹³C NMR-APT and MS data. The antitumor activity of the synthesized compounds was investigated and compared with that of the standard drug vinblastine, a well-known anticancer drug, using MTT colorimetric assay. Among them, compounds 10b and 3 showed the most potent activity against the human breast tumor cells (MCF-7) and the human lung carcinoma cells (HCT), while compound 10b exhibited the most potent activity against the human hepatocellular carcinoma cells (HepG-2). The structure-activity relationships are discussed.

Full text of DOI:10.2174/157018012800389331

Letters in Drug Design & Discovery, 2012, 9, 459-470
459
Synthesis and Antitumor Activities of 4H-Pyrano[3,2-h]quinoline-3-car-
bonitrile, 7H-Pyrimido[4',5':6,5]pyrano[3,2-h]quinoline, and 14H-
Pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]quinoline Derivatives
Abdullah Megbas Al-Ghamdi1, Ashraf Hassan Fekry Abd El-Wahab2, Hany Mostafa Mohamed2 and
Ahmed Mohamed El-Agrody*,1
1Chemistry Department, Faculty of Science, King Khalid University, 9004, Abha, Saudi Arabia
2Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo, 11884, Egypt
Received December 09, 2011: Revised January 12, 2012: Accepted January 30, 2012
Abstract: Several 4H-pyrano[3,2-h]quinoline 3,4,7-9, 7H-pyrimido[4',5':6,5]pyrano[3,2-h]quinoline 10a,b and 14H-
pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]quinoline 11a-c derivatives were obtained by treatment of (E) 2-(4-
chlorostyryl)-8-hydroxyquinoline 1, (E) 2-amino-4-(4-chlorophenyl)-9-(4-chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-
carbonitr-ile 3 or (E) 9-amino-7-(4-chlorophenyl)-2-(4-chlorostyryl)-8-imino-8,9-dihydro-7H-pyrimido-[4',5':6,5]pyrano
[3,2-h]quinoline 10b with different electrophiles followed by nucleophilic reagents. Structures of these compounds were
established on the basis of IR, 1H NMR, 13C NMR, 13C NMR-DEPT, 13C NMR-APT and MS data. The antitumor activity
of the synthesized compounds was investigated and compared with that of the standard drug vinblastine, a well-known
anticancer drug, using MTT colorimetric assay. Among them, compounds 10b and 3 showed the most potent activity
against the human breast tumor cells (MCF-7) and the human lung carcinoma cells (HCT), while compound 10b exhibited
the most potent activity against the human hepatocellular carcinoma cells (HepG-2). The structure-activity relationships
are discussed.
Keywords: Antitumor, (E) 2-(4-chlorostyryl)-8-hydroxyquinoline, 4H-pyrano[3,2-h]quinoline, 7H-pyrimido[4',5':6,5]pyrano
[3,2-h]quinoline, SAR.
INTRODUCTION
chlorophenyl)-9-(4-chlorostyryl)-4H-pyrano[3,2-h]quinoline-
3-carbonitrile 3 (Scheme 1).
The quinoline nucleus is frequently found in bioactive
compounds and plays an important role in biochemical
processes [1-8]. In particular, styrylquinolines having
inhibitory activity against HIV integrase have been reported
recently [9-17]. In addition, the anti-proliferative effects of
styrylquinolines derivatives on tumor cell lines have been
Compound 3 was subjected for further reactions to
produce fused heterotetracyclic or heteropentacyclic systems
incorporating pyrimidine or pyrimido[1,2,4]triazolo nuclei in
addition to pyranoquinoline moiety. Thus, condensation of 3
with benzaldehyde in ethanol and piperidine under reflux
observed
and
recently
reported
[18-24].
Other
gave
the
2-benzylideneamino-4-(4-chlorophenyl)-9-(4-
styrylquinoline derivatives have also gained strong attention
recently due to their extensive biological activities [25-27].
chlorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (4)
(Scheme 2).
In view of the above observations and in continuation of
our program on the chemistry of 4H-pyran derivatives [28-
43], we report herein the synthesis of a variety of 4H-
When
2-benzylideneamino-4-(4-chlorophenyl)-9-(4-
chlorostyryl)-4H-pyrano[3,2-h]-quinoline-3-carbonitrile (4)
was treated with hydrazine hydrate or phenyl hydrazine in
ethanol at room temperature or under reflux, the addition
product 5 was formed (R = H or Ph, respectively). From the
intermediate 5, benzaldehyde hydrazone or benzaldehyde
phenylhydrazone was eliminated to give β-enaminonitrile 3
[37] instead of the pyrimidopyranoquinoline derivative 6
(Scheme 2).
pyrano[3,2-h]quinoline-3-carbonitrile,
7H-pyrimido[4',5':
6,5]pyrano[3,2-h]quinoline, and 14H-pyrimido[4',5':6,5]
pyrano[3,2-h][1,2,4]triazolo[1,5-c]quinoline derivatives. A
selection of these compounds was investigated for their
antitumor activities. The chemical structures of the studied
compounds and structure-activity relationships (SAR) are
discussed in this work.
Structure 4 was established on the basis of spectral data.
The IR spectrum showed the presence of a CN stretch at υ
2212 cm-1. The 1H and 13C NMR spectra of 4 showed signals
at δ 9.42 (s, 1H, N=CH) and 158.2 ppm (=CH) respectively.
The mass spectrum of 4 showed m/z (%) peaks at 561
(M++4, 6), 559 (M++2, 40), 557 (M+, 60) with the base peak
at 89 (100). The (E) configuration of compound 4 was
established from the coupling constant values (J = 16.5 Hz).
RESULTS AND DISCUSSION
The reaction of (E) 2-(4-chlorostyryl)-8-hydroxyquino-
line (1) with α-cyano-p-chlorocinnamonitrile (2) in eth-
anolic piperidine under reflux afforded (E) 2-amino-4-(4-
Treatment of 3 with triethyl orthoformate in acetic
anhydride at reflux gave the corresponding (E) 2-
*Address correspondence to this author at the Chemistry Department,
Faculty of Science, King Khalid University, 9004, Abha, Saudi Arabia; Tel:
+966 530175825; Fax: +966 072417251; E-mail: elagrody_am@yahoo.com
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

460 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Al-Ghamdi et al.
Ar
9
N10
8
7
1
O
NH2
CN
2
3
CN
EtOH / pip.
reflux
6a
6
+
Ar
-CH
=C
1
CN
N
Ar
4a
4
5
OH
1
2
Ar1
3
Ar = Ar1 = p-ClC6H4
Scheme 1. Synthetic protocol of compound 3.
Ar
N
Ar
N
O
NH2
CN
O
N=CHPh
CN
PhCHO
pip.
EtOH /
reflux
Ar1
3
Ar1
4
H2
N
H
N
R
Ar
N
Ar
H
H
NNHR
CHPh
N
O
N
O
N
Ph
-PhCH=NNHR
N
CN
NHR
3
Ar1
R = H or Ph
NH
5
Ar1
6
Ar = p-Clstyryl Ar1 = p-ClC6H4
Scheme 2. Synthetic protocol of compound 4.
Ar
Ar
N
Ar
N
N
O
N=CH-N(Me)2
CN
O
NH2
CN
O
N=CHOEt
HC(OEt)3
DMF-DPA
Ac2O / reflux
CN
benzene / reflux
Ar1
8
3
Ar1
Ar1
7
Ar = p-Clstyryl Ar1 = p-ClC6H4
Scheme 3. Preparation of compounds 7 and 8.
ethoxymethyleneamino-4-(4-chlorophenyl)-9-(4-chlorostyryl)-
4H-pyrano[3,2-h]quinoline-3-carbonitrile (7), while reaction
of 3 with dimethylformamide-dipentylacetal (DMF-DPA) in
dry benzene under reflux gave (E) 4-(4-chlorophenyl)-9-(4-
chlorostyryl)-2-dimethylaminomethyleneamino-4H-pyrano-
[3,2-h]quinoline-3-carbonitrile (8) (Scheme 3).
at δ 161.8 (N=CH), 64.2 (CH2), 13.9 (CH3), respectively.
1
Meanwhile, the H NMR and 13C NMR spectra of 8 showed
signals at δ 8.71 (N=CH), 3.12 (s, 6H, 2CH3) and at δ 155.1
(N=CH), 34.4 ppm (CH3), respectively. The 13C NMR-DEPT
spectra at 45°, 90°, 135° and 13C NMR-APT for compounds
7 and 8 gave additional evidence for the proposed structures,
in addition to the mass spectra. The relative (E)
configurations of compounds 7 and 8 were established from
the coupling constant values (J = 16-16.5 Hz).
Structures of 7 and 8 were established on the basis of IR,
1H NMR, 13C NMR 13C NMR-DEPT, 13C NMR-APT and
MS data. The IR spectra of compounds 7 and 8 showed the
presence of CN stretches at υ 2207 and at υ 2199 cm-1,
respectively. The 1H NMR and 13C NMR spectra of
compound 7 showed characteristic signals at δ 8.93 (N=CH),
4.54 (q, 2H, CH2, J = 7 Hz), 1.46 (t, 3H, CH3, J = 7 Hz) and
Treatment of the imidate 7 with NH3 gas in methanol at
room temperature for 1h gave the open chain product, (E) 2-
aminomethyleneamino-4-(4-chlorophenyl)-9-(4-chlorostyryl)-
4H-pyrano[3,2-h]quinoline-3-carbonitrile (9), while reacton

Synthesis and Antitumor Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 461
Ar
N
Ar
Ar
N
N
O
N=CH-N(Me)2
O
N=CHOEt
CN
O
N=CHNH2
CN
(Me)2NH
NH3 gas
CN
MeOH / R.T.
MeOH / R.T.
Ar1
8
Ar1
7
Ar1
9
MeNH2 or
NH2NH2.H2O
EtOH/R.T./1 h
DMF-DPA
Ar
2
benzene / reflux
1
N
3
a
b
3
c
O
N
d
4
10
9
R
3'
1
4'
4a
6
5
2
3
h
2'
1'
e
f
5'
6'
N
g
4
5
7a
8
6
7
Ar1
NH
10a,b
a; R = CH3
b; R = NH2
Ar = p-Clstyryl Ar1 = p-ClC6H4
Scheme 4. Synthetic protocol of compounds 8-10.
with methylamine or hydrazine hydrate gave the cyclic
addition product (E) 9-methyl/amino-7-(4-chlorophenyl)-2-
(4-chlorostyryl)-8-imino-8,9-dihydro-7H-pyrimido[4',5':6,5]
pyrano[3,2-h]quinoline (10a,b), respectively (Scheme 4).
and 144.3 ppm (C-10) for 10b. The 13C NMR-DEPT spectra
at 45°, 90°, 135° and 13C NMR-APT for compound 9 and
10a gave additional evidences for the proposed structures, in
addition to the mass spectrum. The (E) configurations of
compounds 9 and 10 were established from the coupling
constant values (J = 16-16.5 Hz).
Reaction of the imidate 7 with dimethylamine in
methanol at room temperature for 1h afforded the amidine
derivative 8 (Scheme 4), which can be obtained as described
before from the reaction of 3 and dimethylformamide-
dipentylacetal (DMF-DPA) (m.p. and mixed m.p.) (Scheme
3).
The imino compound 10b proved to be a useful
intermediate for the synthesis of a variety of 2-substituted-
14H-pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]
quinoline derivatives. Thus, treatment of 10b with ethyl
cyanoacetate and with diethyl oxalate in refluxing absolute
ethanol afforded 14-(4-chlorophenyl)-9-(4-chlorotyryl)-2-
cyanomethyl-14H-pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]tri-
azolo[1,5-c]quinoline (11a) and ethyl 14-(4-chlorophenyl)-9-
(4-chlorotyryl)-14H-pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]
triazolo[1,5-c]-quinoline-2-carboxylate (11b), respectively.
Aroylation of 10b with benzoyl chloride in refluxing dry
benzene proceeded readily to give the 2-phenyl derivative
11c (Scheme 5).
The structures of 9 and 10 were supported by IR
spectroscopy, which showed the presence of NH2
absorptions at υ 3451, 3330 and a CN stretch at υ 2198 cm-1
for 9, a NH absorption at υ 3257 for 10a and a NH & NH2 at
υ 3331, 3275, 3184 cm-1 for 10b, respectively. The 1H NMR
and 13C NMR spectra of 9 showed signals at δ 8.69 (s, 1H,
N=CH) and at δ 155.1 (N=CH). Characteristic resonances
were observed at δ 7.78 (s, 1H, H-10), 3.31 (s, 3H, CH3) and
136.5 (C-10), 35.3 (CH3) for 10a and at δ 7.83 (s, 1H, H-10)
Ar
Ar
N
9
8
N
10
11
7
6
N
O
O
N
5
CNCH2CO2Et, (CO2Et)2
or PhCOCl
a
b
4
N
c
12
N
1
N
3
5
NH2
14
13
N
Ar1
NH
1
Ar1
2
11a-c
10b
R
a; R = CH2CN
b; R = CO2Et
c: R = Ph
Ar = p-Clstyryl Ar1 = p-ClC6H4
Scheme 5. Synthetic protocol of compounds 11a-c.

462 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Al-Ghamdi et al.
Structures 11a-c were established on the basis of spectral
data and in conjunction with our previous work [29–35, 42].
The IR spectra showed the presence of a CN band at υ 2257
cm -1 for 11a and a CO band at υ 1745 cm-1 for 11b. The 1H
and 13C NMR spectra of 11a showed signals at δ 3.60 (s, 2H,
CH2) and 23.8 ppm (CH2). Characteristic resonances were
observed at 4.36 (q, 2H, CH2, J = 7.2 Hz) and 1.07 ppm (t,
3H, CH3, J = 7.2 Hz) with the respective signals in the 13C
spectrum at 56.0 and 18.5 ppm for 11b. The mass spectra of
compounds 11a-c gave additional evidence for the proposed
structures. The (E) configurations of compounds 11a-c were
established from the coupling constant values (J = 16-16.5
Hz).
activities [1-27]. Compounds 3,4, and 7-10 were evaluated
for their human tumor cell growth inhibitory activity against
three cell lines: breast adenocarcinoma (MCF-7), lung
carcinoma (HCT) and hepatocellular carcinoma (HepG-2).
The measurement of cell growth and viability were
determined as described in the literature [44]. In vitro cytoto-
xicity evaluation using viability assays were performed by a
Regional Center for Mycology & Biotechnology (RCMP),
Al-Azhar University (Egypt), and using vinblastine as a
standard drug. The inhibitory activity of the preceding
compounds are given in Table 1 and Fig. (1).
The results from (Table 1) indicated that compounds 10b
and 3 were the most active compounds against MCF-7 and
HCT, while the remaining compounds exhibited lower
activities as compared with the standard drug vinblastine.
Finally, compound 10b was the most active compound
against HepG-2. It is worth mentioning that 10b consistently
produced low IC50 values much like vinblastine.
Condensation of 10b with benzaldehyde under reflux in
ethanolic piperidine afforded the open chain product 9-
benzylideneamino-7-(4-chlorophenyl)-2-(4-chlorostyryl)-8-
imino-8,9-dihydro-7H-pyrimido[4',5':6,5]pyrano[3,2-h]qui-
noline (12) [42] (Scheme 6).
Ar
STRUCTURE-ACTIVITY RELATIONSHIP (SAR)
STUDIES
N
O
N
A panel of three different human cancer cell lines: MCF-
7, HCT and HepG-2 was used for these experiments. The
inhibitory concerntration (IC50) of compound 3 and its
analogs are summarized in (Table 1).
PhCHO
10b
N
EtOH / pip.
N
NH
CHPh
Ar1
12
PhCOCl
The results from (Table 1) indicated that compound 3 has
the higher potent antitumor activity against MCF-7 than its
analogs 9, 8, 7, and 4. By maintaining the p-chlorophenyl-4,
p-chlorostyryl-9 and cyano-3 groups (electron-withdrawing
groups) in 4H-pyrano[3,2-h]quinoline moiety, the SAR at
the position-2 was explored. Replacing the amino-2 group
with other electron-donating group such as (-N=CHNH2/-
N=CHNMe2/-N=CHOEt/-N=CHPh) (more increasing in
size) in compounds 4, and 7-9 respectively, resulted in
reduction of potency, suggesting that there might be a size
limited pocket at position C-2. In addition, incorporating a
pyrimidine nucleus at the position-2,3 with -NH-8/-NH2-9
(electron-withdrawing groups) for compound 10b, enhances
potency than compound 3 and its analogs, while replacement
of the -NH2-9 group with the -CH3-9 resulted in a large
reduction of potency of compound 10a, suggesting that a
strong electron-donating group might be preferred at position
C-9.
Dioxane/pip
Ar
11c
-H2
N
O
N
H
N
N
H
N
Ar1
Ar = p-Clstyryl Ar1 = p-ClC6H4
Scheme 6. Synthetic protocol of compounds 11c and 12.
Ph
Cyclization of 12 in 1,4-dioxane-piperdine solution under
reflux [42] afforded the cycloaddition product 11c (Scheme
6), which can also be obtained as described above from the
aroylation of 10b with benzoyl chloride (m.p. and mixed
m.p.) (Scheme 5). The structure of 12 was established on the
basis of its IR spectrum, which showed the presence of NH
In the case of HCT, an investigation of SAR revealed that
compound 3 (p-chlorophenyl-4, p-chlorostyryl-9, cyano-3
and amino-2 groups) has the most potent activity against
HCT compared to its analogs compounds 9, 7, 8, and 4.
Replacing the amino-2 group with other electron-donating
groups such as (-N=CHNH2/-N=CHOEt/ -N=CHNMe2/-
N=CHPh) (more increasing in size) in compounds 9, 7, 8 and
4 respectively, resulted in reduction of potency, suggesting
that there might be a size limited pocket at position C-2.
Incorporating a pyrimidine nucleus at the position-2,3 with
the electron-donating groups -NH-8/-NH2-9 for compound
10b, improve the antitumor activity over that of compound 3
and its analogs, while replacement of the -NH2-9 group with
the -CH3-9 group resulted in a large reduction of the potency
of compound 10a, suggesting that a strong electron-donating
group might be preferred at position C-9. Finally, compound
absorption at υ 3211 cm-1. The H and 13C NMR spectra of
1
12 showed signals at δ 10.22 (s, 1H, N=CH) and 155.6
(N=CH). The mass data of compound 12 gave additional
evidence for the proposed structure. The (E) configuration of
compound 12 was established from the coupling constant
values (J = 16.5 Hz).
ANTITUMOR ASSAYS
The quinoline derivatives were chosen for this study
because it is known that quinoline and fused quinoline
derivatives are important families of active compounds with
a wide range of the biological and the pharmacological

Synthesis and Antitumor Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 463
Table 1. Cytotoxicity of Various Concentrations of 3,4, and 7-10 on MCF-7, HCT, and HepG-2 Cellsa
Compounds
MCF-7 Cell
viability %
HCT Cell
viability %
IC50
(µmol/l)
HepG-2 Cell
viability %
IC50
(µmol/l)
Conc. (µmol/l)
IC50 (µmol/l)
Vinblastine
55.01
27.50
13.75
6.88
3.44
1.72
0
7.82
16.27
21.68
28.2
14.38
16.13
24.25
45.13
55.00
72.13
100
15.18
29.6
6.7
2.9
5.1
48.75
60.35
76.24
100
38.06
47.54
53.42
100
3
4
7
8
9
106.61
53.31
26.65
13.33
6.66
3.33
0
29.56
33.82
39.71
45.44
61.91
86.62
100
24.71
28.97
33.82
42.65
58.97
69.12
100
48.40
68.20
74.00
79.20
85.80
95.40
100
11.9
67.3
63.2
40.5
38.7
10.2
67.9
59.4
71.3
57.7
102.8
89.77
44.88
22.44
11.22
5.61
2.81
0
41.85
58.23
76.44
88.87
97.02
100
42.36
57.64
70.80
81.56
90.02
98.68
100
38.16
53.24
67.56
79.08
88.44
97.52
100
53
100
95.24
47.62
23.81
11.91
5.95
2.97
0
32.50
59.75
76.38
84.75
91.00
100
25.56
59.13
75.71
88.10
97.94
100
40.56
53.24
70.48
81.16
96.22
100
59.8
100
100
100
95.42
47.71
23.85
11.93
5.96
2.98
0
20.50
44.25
66.13
79.00
94.75
99.88
100
34.29
65.95
89.13
97.86
100
29.44
41.60
64.82
97.54
88.66
97.12
100
39.1
100
100
100.81
50.40
25.20
12.60
6.30
3.15
0
38.17
42.83
54.67
72.83
87.17
96.50
100
35.63
52.06
83.33
95.08
98.65
100
25.64
39.72
56.24
73.86
86.22
94.18
100
34.7
100

464 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Al-Ghamdi et al.
(Table 1). Contd…..
Compounds
MCF-7 Cell
viability %
HCT Cell
viability %
IC50
(µmol/l)
HepG-2 Cell
viability %
IC50
(µmol/l)
Conc. (µmol/l)
IC50 (µmol/l)
10a
98.04
49.02
24.51
12.26
6.13
3.06
0
42.00
50.17
74.00
82.33
95.17
99.67
100
40.32
72.70
80.95
87.46
95.40
99.29
100
40.88
56.16
72.64
89.35
96.28
100
48.2
82
68.8
100
10b
97.85
48.92
24.46
12.23
6.12
3.06
0
16.14
23.00
27.93
41.43
79.57
93.21
100
18.57
23.93
30.60
36.55
47.86
61.43
100
17.98
23.72
31.18
40.36
46.48
64.32
100
9.6
5.9
5.3
aIC50 values expressed in µmol/l as the mean values of triplicate wells from at least three experiments.
Fig. (1). IC50 values of some 4H-pyrano[3,2-h]quinoline and 7H-pyrimido[4',5':6,5]pyrano[3,2-h]quinoline derivatives aganist MCF-7, HCT,
and HepG-2 tumor cells..
9
(p-chlorophenyl-4, p-chlorostyryl-9, cyano-3 and
resulted in a large reduction of potency of compound 10a,
suggesting that a strong electron-donating group might be
preferred at position C-9.
N=CHNH2-2 groups) showed moderate antitumor activity
against HepG-2 as compared to that of the standard drug
vinblastine, while more decrease is shown in the antitumor
activity with other electron-donating groups such as -
N=CHNMe2-2/-N=CHPh-2/-N=CHOEt-2/-NH2-2 in compo-
unds 8, 4, 7 and 3 respectively. In addition, incorporating a
pyrimidine nucleus at the position-2,3 with the electron-
donating groups -NH-8/-NH2-9 for compound 10b, rendered
it more potent activity than compound 3 and its analogs.
Thus compound 10b exhibited higher antitumor activity
against HepG-2 than the standard drug vinblastine, while
replacement of the -NH2-9 group with the -CH3-9 group
CONCLUSION
Eleven compounds of 4H-pyrano[3,2-h]quinoline, 7H-
pyrimido[4',5':6,5]pyrano[3,2-h]quinoline and 14H-pyri-
mido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]quinoline
derivatives were prepared. Structures of the synthesized
compounds were elucidated on the basis of IR, 1H NMR, 13
C
NMR, 13C NMR-DEPT, 13C NMR-APT, and MS data. Some
of the newly synthesized compounds (3,4, 7-10) were tested

Synthesis and Antitumor Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 465
against three tumor cell lines: MCF-7, HCT, and HepG-2.
Compounds 10b and 3 had the most potent antitumor
activity against MCF-7 and HCT, while compound 10b has
the most potent antitumor activity against HepG2. This
potency could be attributed to the incorporation of a
pyrimidine nucleus with pyranoquinoline moiety in the
presence of the electron-donating groups (-NH-8/-NH2-9), in
combination with the electron-withdrawing groups (p-
ClC6H4-7; p-Clstyryl-2) or the presence of the electron-
withdrawing groups (p-ClC6H4-4; p-Clstyryl-9; -CN-3) and
the strong activating electron-donating group (-NH2-2) in
4H-pyrano[3,2-h]quinoline moiety. A more extensive study
is also warranted to determine additional antitumor
parameters to give a deeper insight into structure activity
relationship and to optimize the effectiveness of this series of
molecules, which can then be used in bigger scenario such as
drug design or development of antitumor therapeutics.
Calcd for C34H21Cl2N3O: C, 73.12; H, 3.79 N, 7.52. Found:
C, 73.15; H, 3.82; N, 7.55 %.
Reaction of 4 with Hydrazine Derivatives
A mixture of 2-benzylideneamino-4-(4-chlorophenyl)-9-
(4-chlorostyryl)-4H-pyrano-[3,2-h]quinoline-3-carbonitrile 4
(0.557 g, 10 mmol), hydrazine hydrate or phenyl hydrazine
(0.01 mmol) in EtOH (20 mL) was stirred at room
temperature or refluxed for 2 h to give 3 (m.p. and mixed
m.p.) yield (83%).
Reaction of β-Enaminonitrile 3 with Triethyl Orthoformate
General Procedure
A mixture of β-enaminonitrile 3 (0.469 g, 10 mmol),
triethyl orthoformate (10 mmol) and acetic anhydride (30
mL) was refluxed for 3 h. The solvent was removed under
reduced pressure and the resulting solid was recrystallized
from proper solvent to give 7. The physical and spectral data
of the compound 7 was as follows.
EXPERIMENTAL SECTION
Melting points were determined with a Stuart Scientific
Co. Ltd apparatus. IR spectra were determined as KBr
pellets on a Jasco FT/IR 460 plus spectrophotometer. 1H
NMR and 13C NMR spectra were recorded using a Bruker
AV 500 MHz spectrometer. 13C NMR spectra were obtained
using distortionless enhancement by polarization transfer
(DEPT), with this technique, the signals of CH & CH3
carbon atoms appears normal (up) and the signal of carbon
atoms in CH2 environments appears negative (down). 13C
NMR spectra were obtained using attached proton test
(APT), with this technique, the signals of CH and CH3
carbon atoms appear normal (up) and the signals of CH2 and
Cq environments appear negative (down).The MS was
measured on a Shimadzu GC/MS-QP5050A spectrometer.
Elemental analyses were performed on a Perkin-Elmer 240
microanalyser.
(E) 2-Ethoxymethyleneamino-4-(4-chlorophenyl)-9-(4-ch-
lorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (7)
ο
Colorless needles from benzene; m.p. 272-273 C; 81%;
IR (KBr) ν (cm-1): 3050, 3035, 2984, 2960, 2900, 2865, 2850
(CH stretching), 2207 (CN); 1H NMR (500 MHz) (CDCl3) δ:
8.93 (s, 1H, N=CH), 8.14-7.07 (m, 12H, aromatic), 7.72 (d,
1H, =CH, J = 16.5 Hz), 7.31(d, 1H, =CH, J = 16.5 Hz), 5.04
(s, 1H, H-4), 4.54 (q, 2H, CH2, J = 7 Hz), 1.46 (t, 3H, CH3, J
= 7 Hz); 13C NMR (125 MHz) (CDCl3) δ: 161.8 (N=CH),
160.3 (C-2), 156.0 (C-9), 155.1 (C-10b), 144.6, 142.8 (C-
10a), 137.4, 136.6 (C-7), 135.2, 133.4 (=CH), 131.6, 129.6
(=CH), 129.5, 129.1, 129.0 (C-5), 128.9, 127.1 (C-6a), 126.3
(aromatic), 124.1 (C-4a), 123.4 (C-6), 120.8 (C-8), 120.4
(CN), 79.2 (C-3), 64.2 (CH2), 41.4 (C-4), 13.9 (CH3); 13C
NMR-DEPT spectrum at 135о CH, CH3 [positive (up)], CH2
[negative (down)], revealed the following signals at δ 161.8
(N=CH), 136.5 (C-7), 133.4 (=CH), 129.6 (=CH), 129.5
(aromatic), 129.1 (aromatic), 129.0 (C-5), 128.9 (aromatic),
126.4 (aromatic); 123.4 (C-6), 120.8 (C-8), 64.2 (CH2), 41.4
(C-4), 13.9 (CH3). In the DEPT spectrum at 90° only CH
signals are positive (up) and showed δ 161.8 (N=CH), 136.5
(C-7), 133.4 (=CH), 129.6 (=CH), 129.5 (aromatic), 129.1
(aromatic), 129.0 (C-5), 128.9 (aromatic), 126.3 (aromatic);
123.4 (C-6), 120.8 (C-8), 41.4 (C-4), 13.9 (CH3). In the
DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed
signals at δ 161.76 (N=CH), 136.5 (C-7), 133.4 (=CH),
129.6 (=CH), 129.5 (aromatic), 129.1 (aromatic), 129.0 (C-
5), 128.9 (aromatic), 126.3 (aromatic); 123.4 (C-6), 120.8
(C-8), 64.2 (CH2), 41.4(C-4), 13.9 (CH3). 13C NMR-APT
spectrum CH, CH3 [positive (up)], CH2, Cq [negative
(down)], revealed the following signals at δ 161.8 (N=CH),
160.3 (C-2), 155.0 (C-9 ), 155.1 (C-10b), 144.6 (aromatic),
142.8 (C-10a), 137.4 (aromatic), 136.5 (C-7), 135.2
(aromatic), 133.4 (=CH), 131.6 (aromatic), 129.6 (=CH),
129.5 (aromatic), 129.1 (aromatic ), 129.0 (C-5), 128.9
(aromatic), 127.1 (C-6a), 126.3 (aromatic), 124.1 (C-4a),
123.4 (C-6), 120.8 (C-8), 120.4 (CN), 79.2 (C-3), 64.1
(CH2), 41.4 (C-4), 13.9 (CH3); MS m/z (%): 529 (M++4, 1),
527 (M++2, 7), 525 (M+) (10), 358 (100); Anal. Calcd for
(E) 2-Amino-4-(4-chlorophenyl)-9-(4-chlorostyryl)-4H-
pyrano[3,2-h]quinoline-3-carbonitrile (3)
Prepared according to the literature procedure [43].
(E) 2-Benzylideneamino-4-(4-chlorophenyl)-9-(4-chloro-
styryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (4)
A mixture of 3 (0.469 g, 10 mmol), benzaldehyde (0.01
mmol), ethanol (20 mL) and piperidine (0.5 mL) was
refluxed for 2 h. The solid product was collected by filtration
and crystallized from ethanol give 4 as pale yellow crystals
ο
from benzene m.p. 281-282 C; yield 81%; IR (KBr) ν (cm-
1): 3067, 3060, 3022, 2970, 2915 (CH stretching), 2212
1
(CN); H NMR (500 MHz) (CDCl3) δ: 9.42 (s, 1H, N=CH),
8.39-7.17 (m, 17H, aromatic), 8.01 (d, 1H, =CH, J = 16.5
Hz), 7.71 (d, 1H, =CH, J = 16.5 Hz), 5.47 (s, 1H, H-4); 13C
NMR (125 MHz) (CDCl3) δ (ppm): 164.1 (C-2), 158.2
(=CH), 155.7 (C-9), 142.6 (C-10b), 142.4, 137.8 (C-10a),
136.6 (C-7), 135.12, 134.4 (=CH), 133.7, 133.6, 133.3,
132.5, 130.3 (=CH), 129.3 (C-5), 129.1, 129.1, 129.0, 127.2
(C-6a), 126.2 (aromatic), 124.3 (C-4a), 121.0 (C-8), 120.2
(C-6), 117.4 (CN), 86.6 (C-3), 41.9 (C-4); MS m/z (%): 561
(M++4, 6), 559 (M++2, 40), 557 (M+, 60), 89 (100); Anal.

466 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Al-Ghamdi et al.
C30H21Cl2N3O2: C, 68.45; H, 4.02; N, 7.98. Found: C, 68.35;
H, 3.64; N, 7.92 %.
and recrystallized from proper solvent to give 9. The
physical and spectral data of compound 9 was as follows.
Reaction of β-enaminonitrile 3 with Dimethylformamide-
Dipentylacetal (DMF-DPA)
(E) 2-Aminomethyleneamino-4-(4-chlorophenyl)-9-(4-ch-
lorostyryl)-4H-pyrano[3,2-h]quinoline-3-carbonitrile (9)
General Procedure
ο
Colorless needles from benzene; m.p. 290-291 C; 83%;
A mixture of β-enaminonitrile 3 (0.469 g, 10 mmol),
dimethylformamide-dipentylacetal (DMF-DPA) (0.231 g, 10
mmol) and benzene (30 mL) was refluxed for 3 h. The
solvent was removed under reduced pressure and the
resulting solid was recrystallized from proper solvent to give
8. The physical and spectral data of the compound 8 was as
follows.
IR (KBr) ν (cm-1): 3451, 3330 (NH2), 3055, 3030, 2950 (CH
stretching), 2198 (CN); 1H NMR (500 MHz) (CDCl3) δ: 8.69
(s, 1H, N=CH), 8.40-7.15 (m, 12H, aromatic), 8.09 (bs, 1H,
NH2, exchangeable by D2O), 7.88 (d, 1H, =CH, J = 16.5
Hz), 7.54 (d, 1H, =CH, J = 16.5 Hz), 5.14 (s, 1H, H-4); 13C
NMR (125 MHz) (CDCl3) δ: 155.1 (N=CH), 155.0 (C-9),
143.9 (C-10b), 143.1, 137.8 (C-10a), 136.5 (C-7), 135.1,
133.2, 133.0 (=CH), 131.8, 129.8 (=CH), 129.4, 129.1,
128.9, 128.8 (C-5), 127.0 (C-6a), 126.3 (aromatic), 121.2 (C-
4a), 123.4 (C-8), 121.0 (C-6), 120.0 (CN), 71.6 (C-3), 41.7
(C-4); 13C NMR-DEPT spectrum at 135о CH, CH3 [positive
(up)], CH2 [negative (down)], revealed the following signals
at δ 155.1 (N=CH), 136.5 (C-7), 133.0 (=CH), 129.8 (=CH),
129.4 (aromatic), 129.1 (aromatic), 128.9 (aromatic), 128.8
(C-5), 126.3 (aromatic) 123.4 (C-8), 121.0 (C-6), 41.7 (C-4).
In the DEPT spectrum at 90° only CH signals are positive
(up) and showed δ 155.1 (N=CH), 136.5 (C-7), 133.0 (=CH),
129.8 (=CH), 129.4 (aromatic), 129.1 (aromatic), 128.9
(aromatic), 128.8 (C-5), 126.3 (aromatic) 123.4 (C-8), 121.0
(C-6), 41.7(C-4). In the DEPT spectrum at 45° (CH, CH2 and
CH3 positive) revealed signals at δ 155.1 (N=CH), 136.5 (C-
7), 133.0 (=CH), 129.8 (=CH), 129.4 (aromatic),
129.1(aromatic), 128.9 (aromatic), 128.8 (C-5), 126.3
(aromatic) 123.4 (C-8), 121.0 (C-6), 41.7 (C-4). In the 13C
NMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq
[negative (down)], revealed the following signals at δ 160.5
(C-2), 155.1 (N=CH), 155.0 (C-9), 143.9 (C-10b), 143.1
(aromatic), 137.9 (C-10a), 136.5 (C-7), 135.1 (aromatic),
133.2 (aromatic), 133.0 (=CH), 131.8 (aromatic), 129.8
(=CH), 129.4 (aromatic), 129.1 (aromatic), 128.9 (aromatic),
128.8 (C-5), 127.0 (C-6a), 126.3 (aromatic) 121.2 (C-4a),
123.4 (C-8), 121.0 (C-6), 120.0 (CN), 71.6 (C-3), 41.7 (C-4);
MS m/z (%): 500 (M++4, 8), 498 (M++2, 66) with a base
peak at 496 (M+, 100); Anal. Calcd for C28H18Cl2N4O: C,
67.61; H, 3.65; N, 11.26. Found: C, 67.48; H, 3.30; N, 11.05.
(E) 4-(4-chlorophenyl)-9-(4-chlorostyryl)-2-dimethylami-
nomethyleneamino-4H-pyrano[3,2-h]quinoline-3-carbo-
nitrile (8)
Pale yellow crystals from benzene; m.p. 270-271 οC;
85%; IR (KBr) ν (cm-1): 3065, 3027, 2922, 2855, 2810 (CH
stretching), 2199 (CN); 1H NMR (500 MHz) (CDCl3) δ: 8.71
(s, 1H, N=CH), 8.34-7.15 (m, 12H, aromatic), 7.97 (d, 1H,
=CH, J = 16 Hz), 7.60 (d, 1H, =CH, J = 16 Hz), 5.16 (s, 1H,
H-4), 3.12 (s, 6H, 2CH3); 13C NMR (125 MHz) (CDCl3) δ:
159.9 (C-2), 155.1 (C-9), 155.1 (N=CH), 144.0 (C-10b),
143.1, 137.8 (C-10a), 136.5 (C-7), 135.2, 133.2 (=CH),
133.2, 131.8, 129.7 (=CH), 129.2, 129.0, 128.9, 128.8 (C-5),
127.0 (C-6a), 126.3 (aromatic), 123.4 (C-6), 121.2 (C-8),
121.2 (C-4a), 120.0 (CN), 71.5 (C-3), 41.7 (C-4), 34.4
(CH3),; 13C NMR-DEPT spectrum at 135о CH, CH3 [positive
(up)], CH2 [negative (down)], revealed the following signals
at δ 155.1(N=CH), 136.5 (C-7), 133.2 (=CH), 129.7 (=CH),
129.2 (aromatic), 129.0 (aromatic), 129.0 (aromatic), 128.8
(C-5), 126.3 (aromatic), 123.4 (C-6), 121.2 (C-8), 41.8 (C-
4), 34.4 (CH3). In the DEPT spectrum at 90° only CH signals
are positive (up) and showed δ 155.1 (N=CH), 136.5 (C-7),
133.2 (=CH), 129.7 (=CH), 129.2 (aromatic), 129.0
(aromatic), 129.0 (aromatic), 128.8 (C-5), 126.3 (aromatic),
123.4 (C-6), 121.2 (C-8), 41.7 (C-4). In the DEPT spectrum
at 45° (CH, CH2 and CH3 positive) revealed signals at δ
155.2 (N=CH), 136.5 (C-7), 133.2 (=CH), 129.7 (=CH),
129.2 (aromatic), 129.0 (aromatic), 129.0 (aromatic), 128.8
(C-5), 126.3 (aromatic), 123.4 (C-6), 121.2 (C-8), 41.7 (C-
4), 34.4 (CH3). 13C NMR-APT spectrum CH, CH3 [positive
(up)], CH2, Cq [negative (down)], revealed the following
signals at δ 159.9 (C-2), 155.1 (C-9), 155.1 (N=CH), 144.0
(C-10b), 143.1 (aromatic), 137.8 (C-10a), 136.5(C-7), 135.2
(aromatic), 133.2 (CH), 133.2 (aromatic), 131.8 (aromatic),
129.7 (=CH), 129.2 (aromatic), 129.0 (aromatic), 129.0
(aromatic), 128.8(C-5), 127.0 (C-6a), 126.3 (aromatic),
123.4 (C-6), 121.2 (C-8), 121.2 (C-4a ), 120.0 (CN), 71.5
(C-3), 41.7 (C-4), 34.4 (CH3); MS m/z (%): 524 (M+) (36),
50 (100); Anal. Calcd for C30H22Cl2N4O: C, 68.58; H, 4.22;
N, 10.66. Found: C, 68.68; H, 3.93; N, 10.53 %.
Reaction of 7 with Hydrazine Hydrate and Methylamine
General Procedure
A mixture of imadate 7 (0.525 g, 10 mmol) and
hydrazine hydrate or methylamine (0.031 g, 10 mmol) in
ethanol (30 mL) was stirred at room temperature for 1h. The
solid product was collected and recrystallized from proper
solvent to give 10a,b. The physical and spectra data of the
compounds 10a,b are as follows.
(E) 9-Methyl-7-(4-chlorophenyl)-2-(4-chlorostyryl)-8-imi-
no-8,9-dihydro-7H-pyrimido-[4',5':6,5]pyrano[3,2-h]qui-
noline (10a)
Reaction of 7 with Ammonia
General Procedure
Method (a)
ο
Colorless needles from benzene; m.p. 215-217 C; 85%;
IR (KBr) ν (cm-1): 3257 (NH), 3044, 2965 (CH stretching),
1
1647 (C=N); H NMR (500 MHz) (CDCl3) δ: 8.34-7.15 (m,
A mixture of imadate 7 (0.525 g, 10 mmol) and NH3 gas
bubbled in methanol (30 mL) was stirred for 1 h and then the
mixture was left overnight. The solid product was collected
12H, aromatic), 8.00 (d, 1H, =CH, J = 16 Hz), 7.78 (s, 1H,
H-10), 7.54 (d, 1H, =CH, J = 16 Hz), 6.60 (bs, 1H, NH,

Synthesis and Antitumor Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5 467
exchangeable by D2O), 5.02 (s, 1H, H-7), 3.31 (s, 3H, CH3);
13C NMR (125 MHz) (CDCl3) δ: 160.3 (C-8), 155.5 (C-11a),
155.2 (C-2), 144.6 (C-1b), 142.8, 137.4 (C-1a), 136.5 (C-
10), 136.5 (C-4), 135.2, 133.4 (=CH), 133.2, 131.6, 129.6
(=CH), 129.3, 129.0, 128.9, 128.7 (C-6), 126.9 (C-4a), 126.6
(aromatic), 121.7 (C-6a), 121.1 (C-5), 120.3 (C-3), 97.5 (C-
7a), 40.4 (C-4), 35.3 (CH3); 13C NMR-DEPT spectrum at
135о CH, CH3 [positive (up)], CH2 [negative (down)],
revealed the following signals at δ 136.5 (C-10), 136.5 (C-4),
133.4 (=CH), 129.6 (=CH), 129.0 (aromatic), 129.0
(aromatic), 128.9 (aromatic), 128.7 (C-6 ), 126.6 (aromatic),
121.0 (C-5), 120.3 (C-3), 40.4 (C-4), 35.3 (CH3). In the
DEPT spectrum at 90° only CH signals are positive (up) and
showed δ 136.5 (C-10), 136.5 (C-4), 133.4 (=CH), 129.6
(=CH), 129.0 (aromatic), 129.0 (aromatic), 128.9 (aromatic),
128.7 (C-6), 126.6 (aromatic), 121.0 (C-5), 120.3 (C-3), 40.4
(C-4). In the DEPT spectrum at 45° (CH, CH2 and CH3
positive) revealed signals at δ 136.5 (C-10), 136.5 (C-4),
133.4 (=CH), 129.6 (=CH), 129.0 (aromatic), 129.0
(aromatic), 128.9 (aromatic), 128.7 (C-6), 126.6 (aromatic),
121.0 (C-5), 120.3 (C-3), 40.4 (C-4), 35.3 (CH3). In the 13C
NMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq
[negative (down)], revealed the following signals at δ 160.3
(C-8), 155.5 (C-11a), 155.1 (C-2), 144.6 (C-1b), 142.8
(aromatic), 137.4 (C-1a), 136.5 (C-10), 136.5 (C-4), 135.2
(aromatic), 133.4 (=CH), 133.2 (aromatic), 131.6 (aromatic),
129.6 (=CH), 129.03 (aromatic), 129.0 (aromatic), 128.9
(aromatic), 128.7 (C-6), 126.9 (C-4a), 126.6 (aromatic),
121.7 (C-6a), 121.0 (C-5), 120.3 (C-3), 97.5 (C-7a), 40.4 (C-
4), 35.3 (CH3); MS m/z (%): 514 (M++4, 3), 512 (M++2, 15),
510 (M+, 25), 77 (100); Anal. Calcd for C29H20Cl2N4O: C,
68.11; H, 3.94; N, 10.96. Found: C, 67.72; H, 3.58; N, 10.63.
stirred at room temperature for 1h. The solid product was
collected and recrystallized from proper solvent to give 8
(m.p. and mixed m.p.) yield (87%).
Method (b)
A mixture of β-enaminonitrile 3 (0.469 g, 10 mmol),
dimethylformamide-dipentylacetal (DMF-DPA) (0.231 g, 10
mmol) and benzene (30 mL) was refluxed for 3 h. The
solvent was removed under reduced pressure and the
resulting solid was recrystallized from proper solvent to give
8 (m.p. and mixed m.p.) yield (81%).
(E)
14-(4-Chlorophenyl)-9-(4-chlorotyryl)-2-cyanome-
thyl-14H-pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo-
[1,5-c]quinoline (11a)
A solution of 10b (0.511 g, 10 mmol) and ethyl
cyanoacetate (0.13 g, 10 mmol) in dry ethanol (30 mL) was
refluxed for 3 h to give 11a as colorless crystals which were
collected by filtration and recrystallized from benzene; m.p.
280–281 °C; 6%; IR (KBr) ν (cm-1): 3079, 2975, 2937, 2898
(CH stretching), 2257 (CN); 1H NMR (500 MHz) (CDCl3) δ:
9.23 (s, 1H, H-5), 8.34-7.15 (m, 12H, aromatic), 7.86 (d, 1H,
=CH, J = 16.5 Hz), 7.60 (d, 1H, =CH, J = 16.5 Hz), 5.73 (s,
1H, H-14), 3.60 (s, 2H, CH2), 23.81 (CH2); 13C NMR (125
MHz) (CDCl3) δ: 165.1 (C-6a), 155.7 (C-9), 155.5 (C-7a),
145.2 (C-14b), 142.5 (C-2), 142.2, 139.1, 136.5 (C-7b),
135.3 (C-5), 135.2 (C-11), 133.2, 132.5, 133.2 (=CH), 131.9,
130.5 (=CH), 130.3, 129.8 (C-13), 129.6, 128.9 (aromatic),
127.6 (C-11a), 124.6 (C-13a), 123.2 (C-12), 122.3 (C-10),
120.3 (C-14a), 117.5 (CN), 48.7 (C-14), 23.8 (CH2); MS m/z
(%): 564 (M++4, 1), 562 (M++2, 7), 560 (M+, 11), 351
(M++2, 2), 349 (M+, 6), 312 (30), 233 (8), 179 (17), 111
(100), 75 (49) Anal. Calcd for C31H18Cl2N6O: C, 66.32; H,
3.23; N, 14.97. Found: C, 66.42; H, 3.34; N, 15.06.
(E) 9-Amino-7-(4-chlorophenyl)-2-(4-chlorostyryl)-8-imi-
no-8,9-dihydro-7H-pyrimido[4',5':6,5]pyrano[3,2-h]qui-
noline (10b)
ο
Colorless needles from benzene; m.p. 260-261 C; 88%;
(E) Ethyl 14-(4-chlorophenyl)-9-(4-chlorotyryl)-14H-
pyrimido[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]
quinoline-2-carboxylate (11b)
IR (KBr) ν (cm-1): 3331, 3275, 3184 (NH & NH2), 3061,
1
3020, 2970, 2915 (CH stretching), 1656 (C=N); H NMR
(500 MHz) (CDCl3) δ: 8.34-7.33 (m, 12H, aromatic), 7.83 (s,
1H, H-10), 7.86 (d, 1H, =CH, J = 16.5 Hz), 7.60 (d, 1H,
=CH, J = 16.5 Hz), 6.76 (bs, 1H, NH, cancelled by D2O),
5.73 (bs, 2H, NH2, exchangeable by D2O), 5.42 (s, 1H, H-7);
13C NMR (125 MHz) (CDCl3) δ: 160.1 (C-8), 159.9 (C-11a),
157.1 (C-2), 155.5 (C-1b), 144.3 (C-10), 139.1, 137.9 (C-
1a), 136.5 (C-4), 135.2 (=CH), 133.2, 133.1, 130.5 (=CH),
130.3, 129.8, 129.6, 128.9 (C-6), 127.6 (C-4a), 126.9, 126.2
(C-6a), 123.5 (C-5), 122.3 (aromatic), 120.3 (C-3), 96.3 (C-
7a), 40.1 (C-7); MS m/z (%): 499 (M++4, - NH2, 1), 497
(M++2, - NH2, 6), 495 (M+, - NH2, 10), 406 (100); Anal.
Calcd for C28H19Cl2N5O: C, 65.63; H, 3.74; N, 13.67. Found:
C, 65.49; H, 3.57; N, 12.57.
A solution of 10b (0.511 g, 10 mmol) and diethyl oxalate
(0.146 g, 10 mmol) in absolute ethanol (30 mL) was refluxed
for 3 h to give 11b as colorless crystals which were collected
by filtration and recrystallized from benzene; m.p. 290–291
°C, 82%; IR (KBr) ν (cm-1): 3089, 2975, 2938, 2899 (CH
stretching), 1745 (CO); 1H NMR (500 MHz) (CDCl3) δ: 8.34
(s, 1H, H-5), 8.32-7.15 (m, 12H, aromatic), 7.89 (d, 1H,
=CH, J = 16 Hz), 7.54 (d, 1H, =CH, J = 16 Hz), 5.43 (s, 1H,
H-14), 4.36 (q, 2H, CH2, J = 7.2 Hz), 1.07 (t, 3H, CH3, J =
7.2 Hz); 13C NMR (125 MHz) (CDCl3) δ: 170.5 (C-6a),
160.3 (CO), 155.5 (C-2), 155.1 (C-9), 144.6 (C-7a), 142.8
(C-14b), 140.4, 137.4, 136.5 (C-7b), 135.5 (C-5), 135.2 (C-
11), 133.4 (=CH), 133.2, 131.6 (=CH), 129.6, 129.0, 129.0,
128.9, 128.7 (C-13), 126.9 (aromatic), 126.4 (C-11a), 123.4
(C-13a), 121.7 (C-12), 121.0 (C-14a), 120.3 (C-10), 56.0
(CH2), 40.4 (C-14), 18.5 (CH3); MS m/z (%): 597 (M++4, 1),
595 (M++2, 4), 593 (M+, 7), 412 (M++2, 7), 410 (M+, 10),
273 (9), 240 (8), 176 (7), 111 (69), 50 (100); Anal. Calcd for
C32H21Cl2N5O3: C, 64.66; H, 3.56; N, 11.78. Found: C,
64.48; H, 3.44; N, 11.58.
Preparation of (E) 4-(4-chlorophenyl)-9-(4-chlorostyryl)-2-
dimethylaminomethylene-amino-4H-pyrano[3,2-h]quino-
line-3-carbonitrile (8)
General Procedure
Method (a)
A mixture of imadate 7 (0.525 g, 10 mmol) and
dimethylamine (0.045 g, 10 mmol) in methanol (30 mL) was

468 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 5
Al-Ghamdi et al.
(E) 14-(4-Chlorophenyl)-9-(4-chlorotyryl)-2-phenyl-14H-
pyrimido-[4',5':6,5]pyrano[3,2-h][1,2,4]triazolo[1,5-c]
quinoline (11c)
mented with 10% heat-inactivated fetal calf serum, 1% L-
glutamine, HEPES buffer and 50 µmol/l gentamycin.
All cells were maintained at 37 оC in a humidified
atmosphere with 5% CO2 and were subcultures two to three
times a week.
Method (a)
A solution of 10b (0.511 g, 10 mmol) and benzoyl
chloride (0.13 g, 10 mmol) in dry benzene was refluxed for 6
h to give 11c as colorless crystals which were collected by
filtration and recrystallized from benzene; m.p. 393–394 °C,
78%; IR (KBr) υ (cm-1): 3057, 3030, 2977 (CH stretching),
1627 (C=N); 1H NMR (500 MHz) (CDCl3) δ: 9.77 (s, 1H, H-
10), 8.41-7.33 (m, 17H, aromatic), 7.84 (d, 1H, =CH, J = 16
Hz), 7.62 (d, 1H, =CH, J = 16 Hz), 6.30 (s, 1H, H-7); 13C
NMR (125 MHz) (CDCl3) δ: 170.0 (C-6a), 165.0 (C-2),
155.7 (C-9), 154.1 (C-7a), 153.0 (C-14b), 144.1, 143.0 (C-
7b), 140.5 (C-5), 137.7 (C-11), 136.7, 135.1 (=CH), 133.7,
133.3, 131.8, 131.0 (=CH), 130.3, 129.6, 129.3, 129.1, 129.0
(C-13), 128.9, 128.6 (aromatic) 127.2 (C-11a), 124.1 (C-
13a), 121.5 (C-12), 120.4 (C-10), 101.2 (C-14a), 40.0 (C-
14); MS m/z (%): 601 (M++4, 3), 599 (M++2, 14), 597 (M+,
28), 111 (100); Anal. Calcd for C35H21Cl2N5O: C, 70.24; H,
3.54; N, 11.70. Found: C, 70.33; H, 3.76; N, 11.87.
(b) Cytotoxicity Evaluation Using Viability Assay
The in-vitro cytotoxicity activity was studied against
three cell lines: MCF-7 (breast adenocarcinoma), HCT (lung
carcinoma) and HepG-2 (hepatocellular carcinoma) using the
colorimetric MTT assay as described and modified by Tim
Mossman [45]. The cells were seeded in 96-well microtitre
plate at a cell concentration of 1 x 104 cells per well in 100
µL of growth medium. Fresh medium containing different
concentrations of the test sample was added after 24 h of
seeding.
Serial two-fold dilutions of the metabolites were added
confluent cell monolayer. The microtitre plates were
о
incubated at 37 C in a humidified incubator with 5% CO2
for a period of 48 h. Three wells were used for each
concentration of the test sample.
Control cells were incubated without the test sample and
with or without DMSO. The little percentage of DMSO
present in the wells (maximal 0.1%) was found not to affect
the experiment.
Method (b)
Compound 12 (0.01 mmol) was heated under reflux in
1,4-dioxane (20 mL) and piperidine (0.5 mL) for 3 h to give
11c (m.p. and mixed m.p.) yield (68%).
о
After incubation of the cells for 24 h at 37 C, various
concentrations of sample were added, and the incubation was
continued for 48 h and viable cells yield was determined by
a colorimetric MTT method.
(E) 9-Benzylideneamino-7-(4-chlorophenyl)-2-(4-chloros-
tyryl)-8-imino-8,9-dihydro-7H-pyrimido[4',5':6,5]pyrano-
[3,2-h]quinoline (12)
In brief, after the end of the incubation period, the crystal
violet solution (1%) was added to each well for 30 minutes.
The stain was removed and the plates were rinsed using tap
water until all excess stain is removed. Glacial acetic acid
was then added to all wells and mixed thoroughly, and the
plates were read on ELISA reader, using a test wavelength of
490 nm. Treated samples were compared with the control in
the absence of the tested samples. All experiments were
carried out in triplicate. The cell cytotoxic effect of each
tested compound was calculated.
A mixture of 10b (0.511 g, 10 mmol), benzaldehyde
(0.106 g, 10 mmol), ethanol (20 mL) and piperidine (0.5 mL)
was refluxed for 2 h. The solid product, formed, was
collected by filtration and recrystallized from ethanol give 12
as yellow crystals; m.p. 395–396 °C; 87%; IR (KBr) ν (cm-
1): 3211 (NH), 3058, 2970, 2932, 2891 (CH stretching), 1639
(C=N); 1H NMR (500 MHz) (CDCl3) δ: 11.11 (bs, 1H, NH,
exchangeable by D2O), 10.22 (s, 1H, N=CH), 8.48 (s, 1H, H-
10), 8.38-7.29 (m, 17H, aromatic), 7.88 (d, 1H, =CH, J =
16.5 Hz), 7.62 (d, 1H, =CH, J = 16.5 Hz), 6.30 (s, 1H, H-7);
13C NMR (125 MHz) (CDCl3) δ: 164.0 (C-8), 160.0 (C-10),
158.5 (C-11a), 156.8 (C-2), 155.7 (N=CH), 144.6 (C-1b),
143.7, 137.9 (C-1a), 136.5 (C-4), 135.2, 134.2 (=CH), 133.4,
133.2, 131.7, 129.7 (=CH), 129.6, 129.1, 129.0, 128.9 (C-6),
128.8, 128.3, 127.0 (C-4a), 126.8, 126.2 (aromatic), 123.8
(C-6a), 123.4 (C-5), 120.3 (C-3), 96.8 (C-7a), 38.4 (C-7);
MS m/z (%): 603 (M++4, 2), 601 (M++2, 13), 599 (M+, 23),
55 (100); Anal. Calcd for C35H23Cl2N5O: C, 70.00; H, 3.86;
N, 11.66. Found: C, 69.49; H, 3.57; N, 11.47.
CONFLICT OF INTEREST
Declared none.
ACKNOWLEDGEMENTS
This study was supported by the King Abdulaziz City for
Science and Technology (KACST), No. A-S-11-0560. We
are deeply thankful to Mr. Ali Y. A. Alshahrani for making
the 1H NMR and 13C NMR samples.
PHARMACOLOGY
Antitumor Screening
(a) Cell Culture
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