Synthesis and in vitro antitumor activity of new quinoline, pyrimido[4,5-b]quinoline, [1,2,3]triazino[4,5-b]quinoline, and [1,2,4]triazolo[2′,3′:3,4]pyrimido[6,5-b]quinoline analogs

Article abstract of DOI:10.1007/s00044-013-0519-2

New series of quinoline, pyrimido[4,5-b]quinoline, [1,2,3]triazino[4,5-b] quinoline, and [1,2,4]triazolo[2′,3′:3,4]pyrimido[6,5-b]quinoline analogs have been synthesized and characterized by analytical and spectrometrical methods (IR, ¹H NMR, <

Full text of DOI:10.1007/s00044-013-0519-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0519-2
ORIGINAL RESEARCH
Synthesis and in vitro antitumor activity of new quinoline,
pyrimido[4,5-b]quinoline, [1,2,3]triazino[4,5-b]quinoline,
and [1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinoline analogs
N. S. El-Gohary
Received: 6 October 2012 / Accepted: 29 January 2013
Ó Springer Science+Business Media New York 2013
Abstract New series of quinoline, pyrimido[4,5-b]quin-
oline, [1,2,3]triazino[4,5-b]quinoline, and [1,2,4]triazol-
o[20,30:3,4]pyrimido[6,5-b]quinoline analogs have been
synthesized and characterized by analytical and spectro-
metrical methods (IR, 1H NMR, 13C NMR, MS). Fifteen of
the newly synthesized compounds; namely, 3a, b, 4b, 6a,
b, 10a–f, and 14a–d were evaluated for their in vitro
antitumor activity at the National Cancer Institute (NCI) 60
cell lines panel assay. Compounds 4b and 10f are the most
active members in this study, demonstrating significant
broad spectrum antitumor activity against most of the tes-
ted sub-panel tumor cell lines. The detailed synthesis,
spectroscopic, and biological data are described.
In the past few years, several studies have been devoted
to the synthesis and biological activities of quinoline and
pyrimidoquinoline derivatives. Some of them showed
antitumor activity (Abbas et al., 2011; Abouzid and
Shouman, 2008; Abu-Hasem and Aly, 2012; Ali et al.,
2007, 2008; Al-Said et al., 2011; Alqasoumi et al., 2010;
Ghorab et al., 2009, 2010, 2011; Shrestha et al., 2008;
Yong et al., 2012), while other derivatives have been used
as antimicrobial agents (El-Gazzar et al., 2008; Selvi et al.,
2006; Suresh et al., 2003). Also, some of these derivatives
displayed antimalarial activity (Joshi et al., 2005), anal-
gesic properties (El-Gazzar et al., 2008; Kidwai and Negi,
1997) as well as anti-inflammatory activity (El-Gazzar
et al., 2008). In continuation to our previous work (El-
Ashmaway et al., 2012) directed toward facile synthesis of
pyrimidoquinolines of potential antitumor activity, I
thought to explore the utility of compounds 1a, b as key
precursors for the synthesis of new annulated quinoline
derivatives to examine their antitumor activity.
Keywords Synthesis ꢀ Quinolines ꢀ Pyrimidoquinolines ꢀ
Triazinoquinolines ꢀ Triazolopyrimidoquinolines ꢀ
Antitumor activity
Introduction
Although there have been great advances in the detection
and treatment of cancer, it remains one of the greatest
medical challenges, with the incidence of some malig-
nancies continuing to increase (Jemal et al., 2007).
Therefore, development of novel chemotherapeutic agents
is an important and challenging task for the medicinal
chemists. Hence, many research programs are directed
toward the design and synthesis of new drugs for their
chemotherapeutic usage.
Results and discussion
Chemistry
A general approach to synthesize the designed compounds is
outlined in Schemes 1, 2, 3. 2-Amino-1,4,5,6,7,8-hexahy-
dro-4-(substituted phenyl)quinoline-3-carbonitriles (1a, b),
the key starting materials necessary for this study were
previously prepared (El-Ashmaway et al., 2012).
Condensation of compounds 1a, b with dimethyl acety-
lenedicarboxylate (DMAD) in refluxing dimethyl sulfoxide
(DMSO) and in the presence of potassium carbonate gave
4-amino-6,7,8,9-tetrahydro-2,3-bis(methoxycarbonyl)-5-
(substituted phenyl)-5H, 10H-pyrimido[4,5-b]quinolines
N. S. El-Gohary (&)
Department of Medicinal Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
e-mail: dr.nadiaelgohary@yahoo.com
123

Med Chem Res
R
Scheme 1 Synthesis of the
target compounds 2a, b–7a, b
R
NHCSNH2
N
CN
N
S
N
N
H
COC2H5
CH3
N
H
H
5a,b
4a,b
C
H
3
C
/
(
N
O
C
2
C
S
4
H
5
H
OH
N
c
)
3
A
R
R
R
O
N
NH2
CN
COOCH3
NH
N
H3COOC
C
C
COOCH3
NaNO2
N
H
1a,b
NH2
K2CO3 /DMSO
N
H
3a,b
COOCH3
HCl/AcOH
rt
N
N
H
2a,b
4
O
NC
S
2
0
0
H
O
c
n
6
/
O
Co
R
R
O
N
NH
CONH2
NH2
CN
N
H
6a,b
N
H
7a,b
R= 4-Cl; 3,4-(OCH3)2
R
Scheme 2 Synthesis of the
target compounds 8a–d and
10a–f
R
R
R2
R1
O
N
O
N
CONH2
NH2
R1C(OC2H5)3
Xylene
9a-c
NH
NH
R1
AcOH
N
N
H
N
R1
N
H
H
8a-d
10a-f
7a,b
9a: R1= H; R2= 4-NO2
R1= H ; CH3
R= 4-Cl; 3,4-(OCH3)2
9b: R1= 4-N(CH3)2; R2= 4-CH3
9c: R1= 3,4-(OCH3)2; R2= 4-OCH3
(2a, b) (Scheme 1). The IR spectra of these compounds
showed characteristic carbonyl absorption band at 1679 and
1681 cm-1, also it showed the absence of (C:N) absorption
band confirming the formation of the desired cyclic struc-
tures. The 1H NMR spectrum of 2a exhibited two significant
singlets at d 3.83 and 4.91 ppm corresponding to (2OCH3)
and (NH2), respectively. Moreover, the signals for (4CH2),
(C5–H) and aromatic protons were observed at the expected
regions. 13C NMR spectrum of the same compound dis-
played two characteristic signals at d 52.1 and 52.2 ppm
corresponding to (2OCH3).
bands for (2NH) and (C=O) groups at 3423, 3301, and
1643 cm-1, respectively. In addition, the mass spectrum of
3b showed a molecular ion peak at m/z 340 which is in
agreement with its molecular formula C18H20N4O3.
The ortho aminonitriles 1a, b were further utilized for
another cyclocondensation reaction using ammonium thio-
cyanate in refluxing glacial acetic acid to afford the thiourea
derivatives 4a, b (Scheme 1). 1H NMR spectrum of 4a dis-
played two significant singlets at d 10.52 and 11.16 ppm for
(2NH), the remaining protons appeared at the expected
regions. 13C NMR spectrum of the same compound showed a
characteristic signal at d 183.2 ppm for (2C=S). The mass
spectrum of 4b showed a molecular ion peak at m/z 429
which is in agreement with the expected structure.
The triazinoquinolines 3a, b were prepared by diazoti-
zation of the ortho aminonitriles 1a, b via treatment with
aqueous sodium nitrite and a mixture of concentrated
hydrochloric/glacial acetic acids (3:1) (Scheme 1). IR
spectrum of 3a demonstrated the characteristic absorption
Condensation of compounds 1a, b with triethyl ort-
hoacetate yielded the ethyl acetimidate derivatives 5a,
123

Med Chem Res
R1
Scheme 3 Synthesis of the
target compounds 12a, b, 14a–
d, and 15a, b
R
R
R
NH
N
N
R1
CONHNH2
13a,b
N
CN
N
NHNH2
C2H5OH
N
N
NH
DMF
CHOC2H5
N
H
N
H
N
H
N
11a,b
14a-d
12a,b
R1= H; OH
NH2CSNHNH2
C2H5OH
R
NH
H
N
N
NH2
N
H
N
S
15a,b
R= 4-Cl; 3,4-(OCH3)2
b (Scheme 1). The mass spectrum of 5a showed a molec-
ular ion peak at m/z 356 corresponding to its molecular
ones (El-Ashmaway et al., 2012) with respect to their
melting points, mixed melting points, and TLC analysis
using chloroform/methanol (9:1) as an elution solvent.
The versatility of arylideneanilines as aldehyde source-
cum-dehydrating agents in heterocyclization reactions
(Reddy et al., 2005) was earlier demonstrated in benz-
imidazole (Sarojini et al., 1972) and quinazolinone
(Hanumanthu et al., 1976) chemistry. Therefore, I assumed
that benzylideneanilines 9a–c may as well react with 7a,
b to provide 10a–f in one step. Thus, reaction of 7a, b with
the benzylideneanilines 9a–c (Singh et al., 2010) in (1:2)
molar ratio in refluxing glacial acetic acid afforded py-
rimido[4,5-b]quinolin-5-one derivatives 10a–f. Obviously,
the second mole of benzylideneaniline acts as a dehydrat-
ing agent (Reddy et al., 2005) (Scheme 2). Mass spectrum
of compound 10a showed a molecular ion peak at m/z 390
1
weight. H NMR spectrum of 5b showed three significant
singlets at d 2.69, 6.45, and 8.48 ppm for (CH3), (C4–H),
and (NH), respectively, in addition to the triplet-quartet
pattern characteristic for the ethoxy protons at d 1.34 and
4.32–4.39 ppm, respectively. 13C NMR spectrum of the
same compound displayed two characteristic signals for the
ethyl carbons at d 14.5 and 62.3 ppm.
Heating compounds 1a, b with excess ethyl cyanoace-
tate yielded the 2-(cyanomethyl)pyrimido[4,5-b]quinoline
derivatives 6a, b (Scheme 1). The IR spectra of these
compounds revealed the presence of the absorption bands
characteristic for (C=O) and (C:N) groups at the expected
1
regions. Moreover, H NMR spectra showed a character-
istic singlet at d 4.14 ppm for (CH2CN). In addition, mass
spectrum of compound 6b showed a molecular ion peak at
m/z 378 corresponding to its molecular weight along with a
base peak at m/z 109.
1
and a base peak at m/z 312. H NMR spectrum of 10e
displayed three significant singlets at d 3.04, 3.85, and
6.71 ppm for (2CH3), (2OCH3), and (C5–H), respectively,
in addition to two doublets at d 6.76 and 7.68 ppm for (C2-
aromatic protons).
Hydrolysis of compounds 1a, b using 70 % sulfuric acid
at 60 °C afforded 2-amino-1,4,5,6,7,8-hexahydro-4-
(substituted phenyl)quinoline-3-carboxamides (7a, b) in 70
and 55 % yield, respectively, (Scheme 1). The IR spectra
of these compounds showed the disappearance of (C:N)
absorption band with concomitant appearance of (C=O)
absorption band confirming the formation of the desired
structures. In addition, 13C NMR spectrum of 7a displayed
significant signal at d 169.0 ppm for (C=O).
Reaction of quinoline-3-carboxamides 7a, b with tri-
ethyl orthesters in refluxing xylene yielded the pyrimi-
do[4,5-b]quinolin-5-one derivatives 8a–d (Scheme 2).
These compounds were identical to the previously prepared
In conclusion, a facile general method for the synthesis
of pyrimido[4,5-b]quinolin-5-one derivatives 8a–d and
10a–f from quinoline-3-carboxamides 7a, b was developed
by inserting one-carbon unit derived from suitable triethyl
orthoesters and benzylidene anilines, respectively (Reddy
et al., 2005).
Treatment of ethyl formimidates 11a, b (El-Ashmaway
et al., 2012) with phenylhydrazine in refluxing ethanol
yielded 3,4,6,7,8,9-hexahydro-4-imino-3-phenylamino-5-
(substituted phenyl)-5H, 10H-pyrimido[4,5-b]quinolines
(12a, b) (Scheme 3). The disappearance of the (C:N)
123

Med Chem Res
Table 1 Percentage growth inhibition (GI %) of in vitro sub-panel tumor cell lines at 10 lM concentration of compounds 3–14
Subpanel tumor cell lines % Growth inhibition (GI %)a
3a
3b
4b
6a
6b
10a
10b
10c
10d
10e
10f
14a
14b
14c
14d
Leukemia
CCRF-CEM
HL-60(TB)
K-562
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11.7
–
13.2
13.5
–
–
–
–
–
–
–
–
–
–
–
–
–
17.6
–
11.2
13.6
–
–
MOLT-4
11.0
–
19.8 12.3
RPMI-8226
SR
–
10.8 12.3 13.2 10.5
–
–
40.6
–
21.4
–
–
–
Non-small lung cancer
A549/ATCC
EKVX
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.3
11.0
–
–
–
–
10.9
–
13.0
HOP-62
15.7 25.4 12.5
13.3 25.8 11.2
12.8 10.2 15.7
13.3
12.3 16.0 16.6
HOP-92
–
12.0 35.9
–
26.3 14.0 42.1 36.8 12.2
19.6 15.3
NCI-H226
NCI-H23
10.3 15.9 18.6
–
11.3 12.3
–
11.0 15.9 13.6 13.3 15.2
–
10.4
–
–
–
–
15.3 12.1
–
–
–
–
–
12.4
–
–
–
–
–
–
–
–
–
11.7 12.9 10.7
–
–
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
–
–
–
–
–
–
13.5
43.4 11.4
20.2 15.8 10.1 14.5 14.4 25.8 23.5
31.4 16.4
13.5 15.9 12.8 10.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.2
–
–
–
–
–
–
–
–
15.9
–
–
–
–
–
–
–
–
10.3 11.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.2
–
–
–
10.7 10.2
13.1
–
HT29
–
–
–
–
–
14.4
–
KM 12
21.3
–
–
–
–
SW-620
–
–
–
–
CNS Cancer
SF-268
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.2
–
–
–
–
–
–
–
–
–
–
–
SF-295
14.4
21.1
–
29.6
–
SF-539
–
14.5
–
–
–
–
–
SNB-19
10.7 10.4
10.7
–
SNB-75
28.3 20.3 20.4
15.5 12.0 11.2 28.4
22.6 10.5 20.5
19.1
Melanoma
LOX IMVI
MALME-3 M
M14
–
–
–
–
–
–
–
–
–
–
–
–
16.1
10.9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11.2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
21.1 15.1 14.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
13.0
–
–
–
–
–
–
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGORV1
70.7
–
–
–
–
–
12.6 10.0
–
10.0
–
–
–
–
–
–
–
–
–
–
13.0 10.9 25.5
–
17.8
–
–
13.0 10.9
18.6
–
–
23.2
–
–
–
–
–
–
–
–
–
–
nt
14.3
nt
–
nt
nt
14.4 nt
–
13.4 17.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
–
–
11.1
–
–
–
–
–
–
–
–
–
–
10.8
–
–
12.6
–
–
12.6
–
11.7
14.7
–
–
12.1
–
–
11.7
–
123

Med Chem Res
Table 1 continued
Subpanel tumor cell lines % Growth inhibition (GI %)a
3a
3b
4b
6a
6b
10a
10b
10c
10d
10e
10f
14a
14b
14c
14d
SK-OV-3
Renal cancer
786-0
–
–
18.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
A498
28.3 13.2
13.0
11.4
–
–
13.2 21.7
18.0
–
20.6 25.1 53.6
20.0
ACHN
–
16.0 11.4 13.8
13.1
–
–
28.8
–
–
CAKI-1
16.1 23.4 10.0
–
–
23.4 10.5 13.5 12.4 24.6 13.1
RXF 393
SN12C
–
–
–
14.4 14.3
–
–
–
14.4
–
10.3
–
–
–
–
–
–
19.9
–
10.8
–
–
–
15.5
–
–
14.6 12.0 10.8 12.9 15.0
TK-10
–
–
–
–
–
–
–
UO-31
43.4 48.6 43.5 37.7 26.9 43.3 47.4 41.7 48.6 29.4 66.3 40.0 29.0 38.7 29.3
Prostate cancer
PC-3
10.2 13.4 23.5
–
–
–
–
–
–
–
–
12.5 13.5 11.4 17.1 19.7 10.4 19.8
–
–
DU-145
–
–
–
–
–
–
–
–
–
–
Breast cancer
MCF-7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
13.5
–
–
–
–
–
–
–
–
34.3
–
–
–
–
–
MDA-MB-231/ATCC
HS 578T
BT-549
17.4
17.7
–
12.0 12.6
11.1 20.5 12.6 12.8
–
–
–
–
–
–
–
–
–
14.1
–
–
–
–
–
nt
T-47D
13.3 21.1 27.5 14.1
13.3 21.1 14.2
15.5 17.6
21.1 14.9 28.3 14.7 15.8 26.2 18.8
21.2 10.8 28.5 23.1 23.2 21.4
MDA-MB-468
–
–
–
–
Bold values used to point out the active compounds and lethal effect
nt not tested
a
-, GI \10 %
absorption band in IR spectra, disappearance of triplet–
1
compounds 11a, b with thiosemicarbazide in refluxing etha-
nol. Mass spectrum of 15a showed a molecular ion peak at m/
z 387 corresponding to its molecular weight along with a base
quartet pattern characteristic for ethyl protons in H NMR
spectra, and disappearance of ethyl carbons signals in 13C
NMR spectra confirmed the formation of the desired cyclic
structures. Mass spectrum of compound 12a showed a
molecular ion peak at m/z 404 which is in agreement with
its molecular formula C23H22ClN5 along with a base peak
at m/z 283. 1H NMR spectrum of the same compound
showed two significant singlets at d 7.99 and 9.56 ppm for
(2NH), the remaining protons appeared at the expected
regions.
1
peak at m/z 60. In addition, H NMR spectrum of 15b dis-
played five characteristic singlets at d 4.50, 6.51, 7.51, 7.61,
and 8.56 ppm indicating (NH2), (C5–H) and (3NH), respec-
tively. The 13C NMR spectrum of the same compound
exhibited a significant signal at d 181.0 ppm for (C=S).
Preliminary in vitro antitumor screening
The triazolo[20,30:3,4]pyrimido[6,5-b]quinoline deriva-
tives 14a–d could be successfully obtained via the reaction
of compounds 11a, b with benzhydrazides 13a, b (Deep
et al., 2010) in refluxing N,N-dimethylformamide (DMF)
(Scheme 3). The IR spectrum of 14b showed the disap-
pearance of the (C:N) absorption band. 1H NMR spectrum
of the same compound showed a characteristic singlet at d
10.03 ppm for (OH), the remaining protons appeared at the
expected regions. Mass spectrum of 14c showed a molecular
ion peak at m/z 439 corresponding to its molecular weight.
Finally, the {pyrimido[4,5-b]quinolin-3-yl}thiourea deriv-
atives 15a, b were synthesized through condensation of
Out of the newly synthesized compounds, fifteen derivatives;
namely, 3a, b, 4b, 6a, b, 10a–f, and 14a–d were selected by
the National Cancer Institute (NCI) in vitro disease-oriented
human cells screening panel assay to be evaluated for their
antitumor activity. A single dose (10 lM) of the test com-
pounds was used in the full NCI 60 cell lines panel assay
which includes nine tumor subpanels, namely; leukemia,
non-small cell lung, colon, CNS, melanoma, ovarian, renal,
prostate, and breast cancer cells (Boyd and Paull, 1995;
Grever et al., 1992; Monks et al., 1991; Skahan et al., 1990).
The data reported as mean-graph of the percent growth of the
treated cells, and presented as percentage growth inhibition
123

Med Chem Res
(GI %). The obtained results of the tested compounds
(Table 1) showed distinctive potential pattern of selectivity,
as well as broad-spectrum antitumor activity.
Conclusion
Compounds 4b and 10f are broad-spectrum antitumor
agents showing effectiveness toward numerous cell lines
belonging to different tumor subpanels. Compound 4b is
the most active member in this study, while compound 6b
is almost inactive. From these results, I can conclude that
not only 5-(3,4-dimethoxyphenyl) moiety is essential for
antitumor activity but also other substituents on the
pyrimidine nucleus play an important role on the activity of
these compounds, i.e., the presence of 2-thioxo group and
2-(3,4-dimethoxyphenyl) moiety enhanced the antitumor
activity as in compounds 4b and 10f, respectively. On the
other hand, incorporation of 2-cyanomethyl moiety abol-
ished the activity as in compound 6b.
Regarding the activity toward individual cell lines, all
tested compounds showed selective activity against renal
cancer cell line (UO-31). Furthermore, compounds 3a, 4b,
6a, 10a–f, 14a, c, d exhibited cytotoxic activity against non-
small cell lung cancer cell line (NCI-H522). In addition, the
compounds 3a, b, 4b, 6a, 10a, b, d–f, and 14a–d were proved
to be cytotoxic toward breast cancer cell line (T-47D).
Meanwhile, melanoma cell line (MDA-MB-435) and leu-
kemia cell line (SR) were proved to be selectively sensitive
to 4b with GI values of 70.7 and 40.6 %, respectively.
Moreover, compound 10f displayed promising activity
against renal cancer cell line (UO-31) with GI value 66.3 %.
In addition, the same compound exhibited moderate activity
against breast cancer cell line (MCF-7) with GI value
34.3 %. Compound 14c was proved to be the most active
member in this study toward renal cancer cell line (A498)
with GI value of 53.6 %. With regard to broad-spectrum
antitumor activity; close examination of the data presented in
Table 1, revealed that compounds 4b and 10f are the most
active members in this study, showing effectiveness toward
numerous cell lines belonging to different tumor subpanels.
Experimental
Chemistry
All melting points (°C) were recorded on Fisher-Johns
melting point apparatus and are uncorrected. The infrared
spectra were recorded in KBr disk using a Unicam SP 1000
IR spectrometer (v in cm-1) at Faculty of Science, Mansoura
University. Nuclear magnetic resonance (1H and 13C NMR)
spectra were obtained on 300 MHz FT-NMR spectrometer
at Faculty of Science, Cairo University. The chemical shifts
are expressed in d ppm using tetramethylsilane (TMS) as
internal reference and DMSO-d6 as solvent. Mass spectra
were recorded on JEOL JMS-600H spectrometer using
electron impact technique at 70 eV at Microanalytical Unit,
Cairo University. Microanalyses (C, H, N) were performed
at Microanalytical Unit, Cairo University, and were in
agreement with the proposed structures within 0.4 of the
calculated values. Reaction times were monitored using TLC
plates, Silica gel 60 F254 precoated (E. Merck) and the spots
were visualized by UV (366 nm). Chloroform: methanol
(9:1) was adopted as elution solvent. Compounds 1a, b and
11a, b were prepared according to the reported procedures
(El-Ashmaway et al., 2012), compounds 9a–c were prepared
adopting the described procedure (Singh et al., 2010).
Compounds 13a, b were prepared following the literature
procedure (Deep et al., 2010).
Structure–activity correlation
Structure–activity correlation, based on the number of cell
lines that were proved to be sensitive toward each of the
tested compounds, revealed that, 5-(3,4-dimethoxyphenyl)-
[1,2,3]triazino[4,5-b]quinoline 3b and 5-(3,4-dimethoxy-
phenyl)pyrimido[4,5-b]quinoline derivatives 4b, 10d, 10f,
and 14c are more active antitumor agents than their 5-(4-
chlorophenyl) counterparts, 3a, 10a–c, and 14a. The intro-
duction of thioureido group at position 4- of 5-(3,4-dime-
thoxyphenyl)pyrimido[4,5-b]quinoline nucleus produced
4b with the broadest spectrum antitumor activity and
improved selectivity toward non-small cell lung, colon,
CNS, melanoma, renal, and breast cancer cell lines. Mean-
while, the introduction of 3,4-dimethoxyphenyl moiety at
position 2- of 5-(3,4-dimethoxyphenyl)pyrimido[4,5-
b]quinoline nucleus produced 10f with dramatic increase in
antitumor activity and enhanced selectivity toward leuke-
mia, non-small cell lung, CNS, renal, and breast cancer
cell lines. On the other hand, introduction of cyanomethyl
at position 2- of 5-(3,4-dimethoxyphenyl)pyrimido[4,5-b]quin-
oline nucleus produced 6b with nearly abolished activity.
Regarding the [1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quin-
oline derivatives, the introduction of unsubstituted phenyl
moiety at position 2- as in compounds 14a and 14c broaden
the spectrum of antitumor activity in comparison with the
corresponding 2-(4-hydroxyphenyl) analogs 14b and 14d.
General procedure for the preparation of 4-amino-6,7,8,9-
tetrahydro-2,3-bis(methoxycarbonyl)-5-(substituted
phenyl)-5H, 10H-pyrimido[4,5-b]quinolines (2a, b)
Dimethyl acetylenedicarboxylate (0.425 g, 0.003 mol) was
added dropwise to a solution of ortho aminonitrile 1a,
b (0.002 mol), and potassium carbonate (0.83 g, 0.006 mol)
123

Med Chem Res
in DMSO (5 mL). The reaction mixture was heated at reflux
temperature for 12 h then cooled, diluted with water
(30 mL), and extracted with ether (3 9 15). The combined
ether extract was evaporated and the remaining residue was
purified by crystallization from ethanol/water (3:1) to give
dark red crystals of compounds 2a, b.
(2NH), 1643 (C=O). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.59–3.04 (m, 8H, 4CH2), 6.58 (s, 1H, C5–H),
7.23–7.65 (m, 6H, Ar–H, 2NH, D2O-exchangeable). 13C
NMR spectrum: (DMSO-d6, d ppm): 22.3 (2C), 25.6, 28.3,
31.8, 109.3, 111.5, 125.8 (2C), 130.1 (2C), 132.7, 135.1,
137.3 (2C), 161.7. MS m/z (%): 316 (16.98, M??1), 81
(100.00). Anal. Calcd for C16H15ClN4O (314.77): C 61.05,
H 4.80, N 17.80. Found: C 61.25, H 4.97, N 17.63.
4-Amino-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-2,3-
bis(methoxycarbonyl)-5H, 10H-pyrimido[4,5-b]quinoline
(2a) Yield 50 %, m.p. 232–234 °C. IR spectrum (KBr, v,
6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)-[1,2,3]triaz-
ino[4,5-b]quinolin-4(3H, 5H, 10H)-one (3b) Yield 70 %,
m.p. 218–220 °C. IR spectrum (KBr, v, cm-1): 3423, 3301
(2NH), 1642 (C=O). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.60–2.09 (m, 4H, 2CH2), 2.30–2.87 (m, 4H, 2CH2),
3.76 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 6.62-7.43 (m, 6H,
Ar–H, C5-H, 2NH, D2O-exchangeable). 13C NMR spec-
trum: (DMSO-d6, d ppm): 22.1, 22.5, 25.8, 32.3 (2C), 55.5,
55.7, 109.3, 111.9, 116.8, 118.7, 128.5, 132.5, 136.4,
138.2, 148.4, 148.8, 160.7. MS m/z (%): 340 (0.03, M?),
309 (100.00). Anal. Calcd for C18H20N4O3 (340.38): C
63.52, H 5.92, N 16.46. Found: C 63.75, H 5.71, N 16.13.
1
cm-1): 3444, 3403 (NH2, NH), 1681 (2C=O). H NMR
spectrum: (DMSO-d6, d ppm): 1.83–2.30 (m, 4H, 2CH2),
2.65–2.99 (m, 4H, 2CH2), 3.83 (s, 6H, 2OCH3), 4.21 (s,
2H, NH2, D2O-exchangeable), 6.42 (s, 1H, C5–H),
7.06–7.46 (m, 5H, Ar–H, NH, D2O-exchangeable). 13C
NMR spectrum: (DMSO-d6, d ppm): 22.3, 22.8, 26.1, 26.6,
33.4, 52.1, 52.2, 103.2, 107.4, 111.3, 124.8 (2C), 129.4
(2C), 132.2, 135.8, 142.7, 147.6, 152.8, 155.3, 167.9 (2C).
Anal. Calcd for C22H22ClN3O4 (427.88): C 61.75, H 5.18,
N 9.82. Found: C 61.43, H 4.92, N 9.52.
4-Amino-6,7,8,9-tetrahydro-2,3-bis(methoxycarbonyl)-5-
(3,4-dimethoxyphenyl)-5H, 10H-pyrimido[4,5-b]quinoline
(2b) Yield 45 %, m.p. 220–222 °C. IR spectrum (KBr, v,
General procedure for the preparation of 1,2,5,6,7,
8,9,10-octahydro-N-{5-(substituted phenyl)-2-thioxo-
pyrimido[4,5-b]quinolin-4-yl}thioureas (4a, b)
1
cm-1): 3446, 3367 (NH2), 3212 (NH), 1679 (2C=O). H
NMR spectrum: (DMSO-d6, d ppm): 1.76–2.36 (m, 4H,
2CH2), 2.62–2.87 (m, 4H, 2CH2), 3.81 (s, 6H, 2OCH3),
3.87 (s, 6H, 2OCH3), 4.28 (s, 2H, NH2, D2O-exchange-
able), 6.78–7.33 (m, 5H, C5–H, Ar–H, NH, D2O-
exchangeable). 13C NMR spectrum: (DMSO-d6, d ppm):
22.4 (2C), 25.3, 26.8, 32.6, 51.8, 51.9, 55.6, 55.7, 103.6,
106.7, 111.9, 112.2, 113.3, 121.8, 129.7, 145.8 (2C), 148.6
(2C), 153.1, 157.3, 168.7 (2C). MS m/z (%): 453 (0.03,
M?), 112 (100.00). Anal. Calcd for C24H27N3O6 (453.49):
C 63.56, H 6.00, N 9.27. Found: C 63.87, H 5.78, N 9.57.
Ammonium thiocyanate (0.456 g, 0.006 mol) was added to
a solution of compound 1a, b (0.002 mol) in glacial acetic
acid (10 mL). The reaction mixture was heated at reflux
temperature for 18 h, cooled and diluted with water. The
separated solid was collected by filtration, dried, and
crystallized from ethanol.
N-{5-(4-Chlorophenyl)-1,2,5,6,7,8,9,10-octahydro-2-thiox-
opyrimido[4,5-b]quinolin-4-yl}thiourea (4a) Yield 87 %,
m.p. 249–250 °C. IR spectrum (KBr, v, cm-1): 3441
(broad band NH2, 3NH). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.55–2.23 (m, 4H, 2CH2), 2.69–2.86 (m, 4H, 2CH2),
6.56 (s, 1H, C5-H), 7.16–7.68 (m, 7H, Ar–H, NH, NH2,
D2O-exchangeable), 10.52 (s, 1H, NH, D2O-exchange-
able), 11.16 (s, 1H, NH, D2O-exchangeable). 13C NMR
spectrum: (DMSO-d6, d ppm): 22.1, 22.5, 26.1, 29.9 (2C),
81.6, 111.8, 126.9 (2C), 129.9 (2C), 132.1, 134.8, 137.1,
143.7, 166.3, 183.2 (2C). MS m/z (%): 405 (30.43, M??1),
404 (26.09, M?), 109 (100.00). Anal. Calcd for
C18H18ClN5S2 (403.95): C 53.52, H 4.49, N 17.34. Found:
C 53.18, H 4.70, N 17.11.
General procedure for the preparation of 6,7,8,9-
tetrahydro-5-(substituted phenyl)-[1,2,3]triazino[4,5-
b]quinolin-4(3H, 5H, 10H)-ones (3a, b)
A solution of sodium nitrite (0.21 g, 0.0025 mol) in water
(2 mL) was added dropwise to a well-stirred cold suspen-
sion (-5 °C) of ortho aminonitrile 1a, b (0.002 mol) in a
mixture of concentrated hydrochloric acid (12 mL) and
glacial acetic acid (4 mL). After complete addition, the ice
bath was removed and stirring was continued at room
temperature for 24 h. The precipitated solid was filtered,
washed several times with water, dried and crystallized
from dioxane/water (2:1).
1,2,5,6,7,8,9,10-Octahydro-N-{5-(3,4-dimethoxyphenyl)-2-
thioxopyrimido[4,5-b]quinolin-4-yl}thiourea (4b) Yield
83 %, m.p. 231–232 °C. IR spectrum (KBr, v, cm-1):
3423, 3301 (NH2, 3NH). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.75–2.29 (m, 4H, 2CH2), 2.60–2.89 (m, 4H, 2CH2),
5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-[1,2,3]triazino[4,5-
b]quinolin-4(3H, 5H, 10H)-one (3a) Yield 75 %, m.p.
decomp. 202 °C. IR spectrum (KBr, v, cm-1): 3423, 3301
123

Med Chem Res
General procedure for the preparation of 2-(cyanomethyl)-
6,7,8,9-tetrahydro-5-(substituted phenyl)pyrimido[4,5-
b]quinolin-4(3H, 5H, 10H)-ones (6a, b)
3.79 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.75–7.24 (m, 7H,
Ar–H, C5-H, NH, NH2–D2O-exchangeable), 9.38 (s, 1H,
NH, D2O-exchangeable), 10.55 (s, 1H, NH, D2O-
exchangeable). 13C NMR spectrum: (DMSO-d6, d ppm):
22.3, 22.8, 26.1, 30.0 (2C), 55.6, 55.7, 81.3, 111.2, 112.6,
113.1, 122.7, 130.5, 135.9, 142.1, 144.3, 148.6, 167.6,
183.6 (2C). MS m/z (%): 429 (71.93, M?), 428 (50.00,
M?-1), 246 (100.00). Anal. Calcd for C20H23N5O2S2
(429.56): C 55.92, H 5.40, N 16.30. Found: C 55.64, H
5.11, N 15.96.
A mixture of compound 1a, b (0.002 mol) and ethyl cya-
noacetate (5 mL) was heated at reflux temperature for 12 h.
The solvent was evaporated to dryness in vacuo and the
solid obtained was crystallized from dioxane.
5-(4-Chlorophenyl)-2-(cyanomethyl)-6,7,8,9-tetrahydropy-
rimido[4,5-b]quinolin-4(3H, 5H, 10H)-one (6a) Yield
65 %, m.p. 185–187 °C. IR spectrum (KBr, v, cm-1): 3353
(2NH), 2268 (C:N), 1689 (C=O). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.58–1.73 (m, 4H, 2CH2), 2.19 (t, 2H,
CH2), 2.70 (t, 2H, CH2), 4.14 (s, 2H, CH2CN), 6.57 (s, 1H,
2H, C5–H), 7.33 (d, 2H, Ar–H), 7.56 (d, 2H, Ar–H), 7.69
(s, 2H, 2NH, D2O-exchangeable). 13C NMR spectrum:
(DMSO-d6, d ppm): 22.1, 22.4, 25.8 (2C), 32.8 (2C), 87.8,
116.5, 118.2, 121.0 (2C), 128.7 (2C), 130.1, 133.4, 135.2,
152.7, 157.8, 161.9. MS m/z (%): 355 (0.36, M??2), 354
(0.51, M??1), 353 (1.47, M?), 283 (100.00). Anal. Calcd
for C19H17ClN4O (352.82): C 64.68, H 4.86, N 15.88.
Found: C 64.44, H 5.03, N 15.53.
General procedure for the preparation of ethyl N-[3-
cyano-1,4,5,6,7,8-hexahydro-4-(substituted
phenyl)quinolin-2-yl]acetimidates (5a, b)
A mixture of compound 1a, b (0.002 mol) and triethyl
orthoacetate (5 mL) was heated at reflux temperature for
6–8 h. Excess reagent was evaporated in vacuo and the
solid obtained was triturated with ice-water, filtered,
washed with water, dried and crystallized from ethanol.
Ethyl N-[4-(4-chlorophenyl)-3-cyano-1,4,5,6,7,8-hexahy-
droquinolin-2-yl]acetimidate (5a) Yield 55 %, m.p. de-
1
comp. 235 °C. H NMR spectrum: (DMSO-d6, d ppm):
2-(Cyanomethyl)-6,7,8,9-tetrahydro-5-(3,4-dimethoxy-
phenyl)pyrimido[4,5-b]quinolin-4(3H, 5H, 10H)-one (6b)
Yield 50 %, m.p. 173–175 °C. IR spectrum (KBr, v,
1.34 (t, 3H, OCH2CH3), 1.78–1.83 (m, 4H, 2CH2), 2.36 (t,
2H, CH2), 2.56 (s, 3H, CH3), 2.80 (t, 2H, CH2), 4.34–4.37
(q, 2H, OCH2CH3), 6.60 (s, 1H, C4-H), 7.11–7.36 (m, 4H,
Ar–H), 8.50 (s, 1H, NH, D2O-exchangeable). 13C NMR
spectrum: (DMSO-d6, d ppm): 14.3, 15.1, 22.2 (2C), 25.6,
26.2, 33.6, 62.2, 84.2, 112.1, 116.8, 123.8 (2C), 129.6 (2C),
131.7, 133.4, 138.1, 153.6, 168.3. MS m/z (%): 358 (0.20,
M??2), 357 (0.33, M??1), 356 (0.89, M?), 283 (100.00).
Anal. Calcd for C20H22ClN3O (355.86): C 67.50, H 6.23, N
11.81. Found: C 67.82, H 6.46, N 11.63.
1
cm-1): 3409, 3312 (2NH), 2270 (C:N), 1688 (C=O). H
NMR spectrum: (DMSO-d6, d ppm): 1.60–2.22 (m, 4H,
2CH2), 2.69–3.01 (m, 4H, 2CH2), 3.81 (s, 3H, OCH3), 3.87
(s, 3H, OCH3), 4.14 (s, 2H, CH2CN), 6.46 (s, 1H, C5-H),
6.88–7.63 (m, 5H, Ar–H, 2NH, D2O-exchangeable). 13C
NMR spectrum: (DMSO-d6, d ppm): 22.3, 22.5, 25.6, 26.3,
32.4 (2C), 55.7, 55.9, 88.3, 112.2 (2C), 115.5, 117.1, 121.9,
136.2 (2C), 145.3, 149.1, 153.3, 156.9, 163.4. MS m/z (%):
379 (39.63, M??1), 378 (31.71, M?), 109 (100.00). Anal.
Calcd for C21H22N4O3 (378.42): C 66.65, H 5.86, N 14.81.
Found: C 66.39, H 5.57, N 14.61.
Ethyl N-[3-cyano-1,4,5,6,7,8-hexahydro-4-(3,4-dimethoxy-
phenyl)quinolin-2-yl]acetimidate (5b) Yield 68 %, m.p.
1
163–164 °C. H NMR spectrum: (DMSO-d6, d ppm): 1.34
(t, 3H, OCH2CH3), 1.78–1.83 (m, 4H, 2CH2), 2.43 (t, 2H,
CH2), 2.69 (s, 3H, CH3), 2.85 (t, 2H, CH2), 3.77 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 4.32–4.39 (q, 2H, OCH2CH3),
6.45 (s, 1H, C4-H), 6.86–7.11 (m, 3H, Ar–H), 8.48 (s, 1H,
NH, D2O-exchangeable). 13C NMR spectrum: (DMSO-d6,
d ppm): 14.5, 15.1, 21.9, 22.3, 26.8 (2C), 33.9, 55.6, 55.7,
62.3, 83.9, 111.7, 112.3, 112.8, 116.5, 122.7, 135.2 (2C),
145.7, 148.5, 152.6, 167.9. MS m/z (%): 382 (1.08,
M??1), 381 (2.30, M?), 309 (100.00). Anal. Calcd for
C22H27N3O3 (381.47): C 69.27, H 7.13, N 11.02. Found: C
68.96, H 7.36, N 11.37.
General procedure for the preparation of 2-amino-
1,4,5,6,7,8-hexahydro-4-(substituted phenyl)quinoline-3-
carboxamides (7a, b)
Ortho aminonitrile 1a, b (0.01 mol) was heated at 60 °C
with 70 % sulfuric acid (12 mL) for 8 h. The reaction
mixture was cooled, diluted with cold water, and neutral-
ized with 10 % aqueous sodium hydroxide solution. The
precipitated solid was collected by filtration, washed with
water, dried, and crystallized from ethanol.
123

Med Chem Res
2-Amino-4-(4-chlorophenyl)-1,4,5,6,7,8-hexahydroquino-
line-3-carboxamide (7a) Yield 70 %, m.p. 188–189 °C.
IR spectrum (KBr, v, cm-1): 3461, 3442 (2NH2), 3338
(NH), 1664 (C=O). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.56–1.70 (m, 4H, 2CH2), 2.30 (s, 2H, NH2, D2O-
exchangeable), 2.48–2.63 (m, 4H, 2CH2), 5.96 (s, 2H, NH2,
D2O-exchangeable), 6.17 (s, 1H, C5-H), 7.12–7.43 (m, 5H,
Ar–H, NH, D2O-exchangeable). 13C NMR spectrum:
(DMSO-d6, d ppm): 22.4, 22.8, 25.7, 26.0, 32.2, 106.7,
115.2, 128.3 (2C), 130.0 (2C), 132.1, 136.5, 146.1, 156.3,
169.0. MS m/z (%): 304 (0.81, M?), 258 (100.00). Anal.
Calcd for C16H18ClN3O (303.79): C 63.26, H 5.97, N
13.83. Found: C 63.64, H 6.24, N 13.62.
5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-2-methylpyrimido
[4,5-b]quinolin-4(3H, 5H, 10H)-one (8c) (Crystallized
from ethanol/DMF (3:2)), yield 76 %, m.p. 223–225 °C (El-
Ashmaway et al., 2012).
6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)-2-methylpy-
rimido[4,5-b]quinolin-4(3H, 5H, 10H)-one (8d) (Crys-
tallized from DMF), yield 70 %, m.p. 210–212 °C
(El-Ashmaway et al., 2012).
General procedure for the preparation of 6,7,8,9-
tetrahydro-2-(substituted phenyl)-5-(substituted
phenyl)pyrimido[4,5-b]quinolin-4(3H, 5H, 10H)-ones
(10a–f)
2-Amino-1,4,5,6,7,8-hexahydro-4-(3,4-dimethoxy-
phenyl)quinoline-3-carboxamide (7b) Yield 55 %, m.p.
165–166 °C. IR spectrum (KBr, v, cm-1): 3460, 3440
(2NH2), 3336 (NH), 1663 (C=O). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.59-1.90 (m, 4H, 2CH2), 2.20 (s, 2H,
NH2, D2O-exchangeable), 2.64–2.75 (m, 4H, 2CH2), 3.82
(s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.65–7.05 (m, 6H, Ar–
H, C5–H, NH2, D2O-exchangeable), 7.75 (s, 1H, NH, D2O-
exchangeable). 13C NMR spectrum: (DMSO-d6, d ppm):
22.1, 22.6, 26.3, 26.9, 33.1, 55.5, 55.6, 104.6, 109.7, 112.2,
115.3, 122.6, 133.7 (2C), 144.9, 148.6, 158.1, 168.7. MS
m/z (%): 328 (4.30, M?-1), 296 (100.00). Anal. Calcd for
C18H23N3O3 (329.39): C 65.63, H 7.04, N 12.76. Found: C
65.31, H 7.36, N 12.48.
A mixture of compound 7a,b (0.002 mol) and the appro-
priate benzylideneaniline 9a–c (0.004 mol) in glacial ace-
tic acid (10 mL) was heated at reflux temperature for
6–8 h. The reaction mixture was diluted with water
(30 mL) and the separated solid was collected by filtration,
dried, and crystallized from ethanol/water (2:1).
5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-2-phenylpyr-
imido[4,5-b]quinolin-4(3H, 5H, 10H)-one (10a) Yield
80 %, m.p. 160–162 °C. IR spectrum (KBr, v, cm-1): 3388
(2NH), 1664 (C=O). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.58–1.77 (m, 4H, 2CH2), 2.32–2.80 (m, 4H, 2CH2),
6.18 (s, 1H, C5-H), 7.19–8.22 (m, 10H, Ar–H, NH, D2O-
exchangeable), 10.55 (s, 1H, NH, D2O-exchangeable). 13C
NMR spectrum: (DMSO-d6, d ppm): 22.6 (2C), 25.7, 26.2,
32.6, 105.3, 109.6, 127.1 (2C), 128.3, 128.7, 129.1 (2C),
130.3, 130.7, 131.2 (2C), 133.2 (2C), 138.6, 151.6, 153.4,
158.3. MS m/z (%): 391 (14.29, M??1), 390 (17.69, M?),
389 (37.69, M?-1), 312 (100.00). Anal. Calcd for
C23H20ClN3O (389.88): C 70.85, H 5.17, N 10.78. Found:
C 70.56, H 4.93, N 11.05.
General procedure for the preparation of 6,7,8,9-
tetrahydro-2-(unsubstituted or methyl)-5-(substituted
phenyl)pyrimido[4,5-b]quinolin-4(3H, 5H, 10H)-ones
(8a–d)
A mixture of compound 7a,b (0.002 mol) and the appro-
priate triethyl orthoester (0.002 mol) in xylene (10 mL)
was heated at reflux temperature for 6 hours. The reaction
mixture was cooled to room temperature and diluted with
n-hexane (20 mL). The precipitated solid was collected by
filtration, washed with n-hexane, dried, and crystallized
from the appropriate solvent.
5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-2-(4-dimethylamin-
ophenyl)pyrimido[4,5-b]quinolin-4(3H, 5H, 10H)-one
(10b) Yield 78 %, m.p. decomp. 183 °C. 1H NMR
spectrum: (DMSO-d6, d ppm): 1.78–2.79 (m, 8H, 4CH2),
3.06 (s, 6H, 2CH3), 6.60–7.46 (m, 9H, Ar–H, C5-H), 9.84
(s, 1H, NH, D2O-exchangeable), 10.47 (s, 1H, NH, D2O-
exchangeable). 13C NMR spectrum: (DMSO-d6, d ppm):
22.1, 22.3, 25.6, 26.8, 31.1, 40.7 (2C), 104.9, 111.2, 112.6
(2C), 122.6, 124.8 (2C), 128.3 (2C), 129.1 (2C), 130.2,
135.3, 137.6, 149.4, 151.7, 154.6, 158.7. MS m/z (%): 433
(64.52, M?), 429 (100.00). Anal. Calcd for C25H25ClN4O
(432.95): C 69.35, H 5.82, N 12.94. Found: C 68.98, H
5.65, N 12.73.
5-(4-Chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]
quinolin-4(3H, 5H, 10H)-one (8a) (Crystallized from
DMF), yield 78 %, m.p. decomp. 220 °C (El-Ashmaway
et al., 2012).
6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)pyrimido[4,5-
b]quinolin-4(3H, 5H, 10H))-one (8b) (Crystallized from
DMF), yield 65 %, m.p. 257–259 °C (El-Ashmaway et al.,
2012).
123

Med Chem Res
5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-2-(3,4-dimethoxy-
phenyl)pyrimido[4,5-b]quinolin-4(3H, 5H, 10H)-one
General procedure for the preparation of 3,4,6,7,8,9-
hexahydro-4-imino-3-phenylamino-5-(substituted phenyl)-
5H, 10H-pyrimido[4,5-b]quinolines (12a, b)
1
(10c) Yield 72 %, m.p. 167–169 °C. H NMR spectrum:
(DMSO-d6, d ppm): 1.78–2.85 (m, 8H, 4CH2), 3.70 (s, 6H,
2OCH3), 6.84–7.48 (m, 8H, Ar–H, C5-H), 9.77 (s, 1H, NH,
D2O-exchangeable), 10.45 (s, 1H, NH, D2O-exchange-
able). 13C NMR spectrum: (DMSO-d6, d ppm): 21.6, 22.4,
26.8 (2C), 32.3, 55.6, 55.8, 101.1, 109.8, 112.2, 112.7,
120.4, 122.6, 124.7 (2C), 128.1 (2C), 131.7, 132.3, 138.6,
146.7, 148.3, 152.1, 156.6, 159.3. MS m/z (%): 450 (35.37,
M?), 164 (100.00). Anal. Calcd for C25H24ClN3O3
(449.93): C 66.74, H 5.38, N 9.34. Found: C 67.11, H 5.10,
N 8.96.
A mixture of compound 11a,b (0.002 mol) and phen-
ylhydrazine (0.216 g, 0.002 mol) in absolute ethanol
(15 mL) was heated at reflux temperature for 10 h. The
reaction mixture was concentrated and the precipitated
solid was collected by filtration, washed with water, dried,
and crystallized from dioxane.
5-(4-Chlorophenyl)-3,4,6,7,8,9-hexahydro-4-imino-3-phe-
nylamino-5H, 10H-pyrimido[4,5-b]quinoline (12a) Yield
50 %, m.p. 222–223 °C. IR spectrum (KBr, v, cm-1): 3421
1
6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)-2-phenylpy-
rimido[4,5-b]quinolin-4(3H, 5H, 10H)-one (10d) Yield
65 %, m.p. 152–154 °C. IR spectrum (KBr, v, cm-1):
3299, 3197 (2NH), 1652 (C=O). MS m/z (%): 416 (32.98,
M??1), 415 (7.82, M?), 383 (100.00). Anal. Calcd for
C25H25N3O3 (415.48): C 72.27, H 6.06, N 10.11. Found: C
72.47, H 5.85, N 10.34.
(3NH). H NMR spectrum: (DMSO-d6, d ppm): 1.59–1.74
(m, 4H, 2CH2), 2.19 (t, 2H, CH2), 2.70 (t, 2H, CH2), 6.58
(s, 1H, C5–H), 7.32–7.58 (m, 11H, Ar–H, C2–H, NH, D2O-
exchangeable), 7.99 (s, 1H, NH, D2O-exchangeable), 9.56
(s, 1H, NH, D2O-exchangeable). 13C NMR spectrum:
(DMSO-d6, d ppm): 22.8, 25.7, 26.0, 31.4, 32.2, 87.9,
111.9, 115.1, 116.8, 120.7, 128.2 (2C), 129.9 (2C), 132.1
(2C), 132.5, 136.0, 138.2, 146, 150.1, 153.2, 168.0. MS m/
z (%): 404 (0.04, M?), 403 (0.03, M?-1), 283 (100.00).
Anal. Calcd for C23H22ClN5 (403.91): C 68.39, H 5.49, N
17.34. Found: C 68.11, H 5.21, N 17.60.
6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)-2-(4-dimeth-
ylaminophenyl)pyrimido[4,5-b]quinolin-4(3H, 5H, 10H)-
one (10e) Yield 60 %, m.p. 185–187 °C. 1H NMR
spectrum: (DMSO-d6, d ppm): 1.23–1.79 (m, 4H, 2CH2),
2.62–2.93 (m, 4H, 2CH2), 3.04 (s, 6H, 2CH3), 3.85 (s, 6H,
2OCH3), 6.71 (s, 1H, C5-H), 6.79 (d, 2H, Ar–H), 7.10–7.27
(m, 3H, Ar–H), 7.68 (d, 2H, Ar–H), 7.96 (s, 1H, NH, D2O-
exchangeable), 9.67 (s, 1H, NH, D2O-exchangeable). 13C
NMR spectrum: (DMSO-d6, d ppm): 22.1, 22.5, 26.6 (2C),
31.9, 41.1 (2C), 55.6, 55.7, 105.7, 111.5, 112.2 (2C), 112.7,
115.6, 122.1, 122.6, 128.3 (2C), 133.6, 135.2, 145.7, 147.3,
149.5, 152.3, 154.2, 158.6. MS m/z (%): 459 (1.57,
M??1), 458 (3.13, M?), 296 (100.00). Anal. Calcd for
C27H30N4O3 (458.55): C 70.72, H 6.59, N 12.22. Found: C
70.93, H 6.23, N 12.37.
3,4,6,7,8,9-Hexahydro-4-imino-5-(3,4-dimethoxyphenyl)-
3-phenylamino-5H, 10H-pyrimido[4,5-b]quinoline (12b)
Yield 55 %, m.p. 190–192 °C. IR spectrum (KBr, v,
cm-1): 3429 (3NH). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.59–1.86 (m, 4H, 2CH2), 2.32 (t, 2H, CH2), 2.67 (t,
2H, CH2), 3.75 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.52 (s,
1H, C5–H), 6.79–7.06 (m, 10H, Ar–H, C2–H, NH, D2O-
exchangeable), 8.70 (s, 1H, NH, D2O-exchangeable), 9.65
(s, 1H, NH, D2O-exchangeable). MS m/z (%): 430 (9.96,
M??1), 429 (17.57, M?), 92 (100.00). Anal. Calcd for
C25H27N5O2 (429.51): C 69.91, H 6.34, N 16.31. Found: C
69.64, H 6.58, N 15.97.
6,7,8,9-Tetrahydro-2,5-bis(3,4-dimethoxyphenyl)pyrimi-
do[4,5-b]quinolin-4(3H, 5H, 10H)-one (10f) Yield 60 %,
m.p. 161–162 °C. 1H NMR spectrum: (DMSO-d6, d ppm):
1.49–1.79 (m, 4H, 2CH2), 2.66–3.00 (m, 4H, 2CH2), 3.85
(s, 6H, 2OCH3), 3.87 (s, 6H, 2OCH3), 5.55 (s, 1H, C5-H),
6.98–7.57 (m, 7H, Ar–H, NH, D2O-exchangeable), 9.84 (s,
1H, NH, D2O-exchangeable). 13C NMR spectrum:
(DMSO-d6, d ppm): 22.0, 24.6, 25.3, 26.8, 32.4, 55.6, 55.7,
55.8, 55.9, 106.1, 111.3, 111.7, 113.0, 115.2, 115.5, 119.1,
120.8, 121.0, 129.3, 134.9, 146.5, 148.5, 148.8, 151.5,
151.7, 152.5, 156.9. MS m/z (%): 476 (8.03, M??1), 475
(3.55, M?), 84 (100.00). Anal. Calcd for C27H29N3O5
(475.54): C 68.19, H 6.15, N 8.84. Found: C 68.44, H 6.30,
N 8.67.
General procedure for the preparation of 7,8,9,10,
11,12-hexahydro-2,12-(substituted phenyl)-
[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinolines
(14a–d)
A mixture of compound 11a, b (0.002 mol) and the
appropriate benzhydrazide 13a, b (0.002 mol) in DMF
(10 mL) was heated at reflux temperature for 8–10 h. The
solvent was evaporated in vacuo and the remaining solid
was purified by crystallization from dioxane.
12-(4-Chlorophenyl)-7,8,9,10,11,12-hexahydro-2-phenyl-
[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinoline (14a)
Yield 57 %, m.p. 162–164 °C. 1H NMR spectrum:
123

Med Chem Res
General procedure for the preparation of 1-{6,7,8,9-
tetrahydro-4-imino-5-(substituted phenyl)pyrimido[4,5-
b]quinolin-3(4H, 5H, 10H)-yl}thiourea (15a, b)
(DMSO-d6, d ppm): 1.58–1.72 (m, 4H, 2CH2), 2.16 (t, 2H,
CH2), 2.71 (t, 2H, CH2), 6.63 (s, 1H, C12–H), 7.32–7.99
(m, 11H, Ar–H, C5-H, NH, D2O-exchangeable). 13C NMR
spectrum: (DMSO-d6, d ppm): 22.8 (2C), 26.2, 30.1, 33.7,
111.1, 113.5, 126.7 (2C), 127.2, 128.3, 128.9, 129.6 (2C),
130.1, 130.7, 131.3, 133.6, 136.8, 141.5, 148.2, 151.6,
157.3, 158.7. MS m/z (%): 415 (42.98, M??1), 414 (51.24,
M?), 413 (73.55, M?-1), 87 (100.00). Anal. Calcd for
C24H20ClN5 (413.90): C 69.64, H 4.87, N 16.92. Found: C
69.40, H 5.11, N 16.68.
A mixture of compound 11a,b (0.002 mol) and thiosemi-
carbazide (0.182 g, 0.002 mol) in absolute ethanol
(15 mL) was heated at reflux temperature for 18 h. The
reaction mixture was concentrated and the precipitated
solid was collected by filtration, washed with aqueous
ethanol, dried, and crystallized from ethanol/water (2:1).
1-{5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-4-iminopyr-
imido[4,5-b]quinolin-3(4H, 5H, 10H)-yl}thiourea (15a)
Yield 78 %, m.p. 200–201 °C. 1H NMR spectrum:
(DMSO-d6, d ppm): 1.56–1.85 (m, 4H, 2CH2), 2.21 (t, 2H,
CH2), 2.70 (t, 2H, CH2), 4.50 (s, 2H, NH2, D2O-
exchangeable), 6.61 (s, 1H, C5–H), 7.11–7.58 (m, 7H, Ar–
H, C2–H, 2NH, D2O-exchangeable), 8.65 (s, H, NH, D2O-
exchangeable). 13C NMR spectrum: (DMSO-d6, d ppm):
22.3, 22.7, 26.9, 32.6 (2C), 89.1, 111.2, 125.1 (2C), 129.8
(2C), 130.6, 136.4, 138.7, 148.1, 156.9, 161.2, 181.6. MS
m/z (%): 387 (4.66, M?), 60 (100.00). Anal. Calcd for
C18H19ClN6S (386.9): C 55.88, H 4.95, N 21.72. Found: C
55.97, H 5.23, N 21.54.
12-(4-Chlorophenyl)-7,8,9,10,11,12-hexahydro-2-(4-
hydroxyphenyl)-[1,2,4]triazolo[20,30:3,4]pyrimido-[6,5-
b]quinoline (14b) Yield 52 %, m.p. 225–226 °C. IR
spectrum (KBr, v, cm-1): 3409 (OH), 3367 (NH). 1H NMR
spectrum: (DMSO-d6, d ppm): 1.60–1.89 (m, 4H, 2CH2),
2.32 (t, 2H, CH2), 2.87 (t, 2H, CH2), 6.57–6.93 (m, 5H, Ar–
H, C12–H), 7.27–7.95 (m, 6H, Ar–H, C5-H, NH, D2O-
exchangeable), 10.03 (s, 1H, OH, D2O-exchangeable). 13C
NMR spectrum: (DMSO-d6, d ppm): 22.7 (2C), 25.9, 30.4,
33.2, 111.3, 112.7, 117.1 (2C), 123.4, 127.1 (2C), 129.3
(2C), 130.1 (2C), 130.9, 136.4, 139.7, 148.7, 151.3, 153.8,
156.9, 159.2. MS m/z (%): 429 (0.01, M?-1), 283 (100.00).
Anal. Calcd for C24H20ClN5O (429.90): C 67.05, H 4.69, N
16.29. Found: C 67.37, H 4.43, N 15.97.
1-{6,7,8,9-Tetrahydro-4-imino-5-(3,4-dimethoxyphenyl)py-
rimido[4,5-b]quinolin-3(4H, 5H, 10H)-yl}thiourea (15b)
Yield 75 %, m.p. 198–200 °C. 1H NMR spectrum:
(DMSO-d6, d ppm): 1.59–1.86 (m, 4H, 2CH2), 2.32–2.86
(m, 4H, 2CH2), 3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
4.50 (s, 2H, NH2, D2O-exchangeable), 6.51 (s, 1H, C5–H),
6.78–7.20 (m, 4H, Ar–H, C2–H), 7.51 (s, 1H, NH, D2O-
exchangeable), 7.61 (s, 1H, NH, D2O-exchangeable), 8.56
(s, 1H, NH, D2O-exchangeable). 13C NMR spectrum:
(DMSO-d6, d ppm): 22.1, 22.5, 25.8, 32.8 (2C), 55.5, 55.7,
88.4, 111.6, 116.8, 118.7, 120.6, 128.5 (2C), 148.4 (2C),
148.8, 157.8, 160.7, 181.0. MS m/z (%): 412 (0.80, M?), 60
(100.00). Anal. Calcd for C20H24N6O2S (412.51): C 58.23,
H 5.86, N 20.37. Found: C 58.47, H 5.62, N 20.55.
7,8,9,10,11,12-Hexahydro-12-(3,4-dimethoxyphenyl)-2-
phenyl-[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]-quinoline
1
(14c) Yield 45 %, m.p. 158–160 °C. H NMR spectrum:
(DMSO-d6, d ppm): 1.59–2.27 (m, 4H, 2CH2), 2.49–2.88
(m, 4H, 2CH2), 3.80 (s, 3H, OCH3), 3.89 (s, 3H, OCH3),
6.51 (s, 1H, C12-H), 6.73–7.95 (m, 10H, Ar–H, C5–H, NH,
D2O-exchangeable). 13C NMR spectrum: (DMSO-d6, d
ppm): 22.3, 22.7, 26.1 (2C), 32.9, 55.7, 55.9, 111.9, 112.2
(2C), 113.3, 120.3, 125.9, 126.7, 128.1, 128.4, 129.7,
130.3, 130.8, 136.3, 147.9 (2C), 148.3, 148.7, 157.6, 158.9.
MS m/z (%): 441 (0.89, M??2), 440 (3.58, M??1), 439
(13.81, M?), 105 (100.00). Anal. Calcd for C26H25N5O2
(439.51): C 71.05, H 5.73, N 15.93. Found: C 71.32, H
5.51, N 15.63.
Antitumor screening
7,8,9,10,11,12-Hexahydro-2-(4-hydroxyphenyl)-12-(3,4-
dimethoxyphenyl)-[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]
Under sterile conditions, cell lines were grown in RPMI
1640 media (Gibco, NY, USA) supplemented with 10 %
fetal bovine serum (Biocell, CA, USA). The concentrations
of the compounds ranging from 0.01 to 100 lM were
prepared in phosphate buffer saline. Each compound was
initially solubilized in DMSO, however, each final dilution
contained less than 1% DMSO. Solutions of different
concentrations (0.2 mL) were pipetted into separate well of
96-multi-well microtiter plate, duplicate wells were pre-
pared for each individual dose. Cell culture (1.8 mL)
containing a cell population of 6 9 104 cells/mL was
1
quinoline (14d) Yield 40 %, m.p. 165–167 °C. H NMR
spectrum: (DMSO-d6, d ppm): 1.64–1.83 (m, 4H, 2CH2),
2.29 (t, 2H, CH2), 2.98 (t, 2H, CH2), 3.78 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 6.57–7.85 (m, 10H, Ar–H, C5–H, C12–
H, NH, D2O-exchangeable), 11.41 (s, 1H, OH, D2O-
exchangeable). MS m/z (%): 456 (6.40, M??1), 455 (4.19,
M?), 453 (100.00, M?-2). Anal. Calcd for C26H25N5O3
(455.51): C 68.56, H 5.53, N 15.37. Found: C 68.35, H
5.37, N 15.11.
123

Med Chem Res
for publication at Med Chem Res Oct 05, published online 17
Oct. doi:10.1007/s00044-012-0272-y
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pipetted into each well. Controls containing only phosphate
buffer saline and DMSO at identical dilutions, were also
prepared in the same manner. The cultures were incubated
with the compounds for 48 h in a humidified incubator at
37 °C and in atmosphere of 5 % CO2. After 48 h, cells in
each well were diluted 10 times with saline and counted
using a coulter counter (Boyd and Paull, 1995; Grever
et al., 1992; Monks et al., 1991; Skahan et al., 1990). The
data reported as mean-graph of the percent growth of the
treated cells, and presented as percentage growth inhibition
(GI %) (Table 1).
Acknowledgments The author is deeply thankful to the staff
members of the Department of Health and Human Services, National
Cancer Institute (NCI), Bethesda, Maryland, USA for carrying out the
antitumor screening of the newly synthesized compounds.
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