The antimitotic pyrimido[4,5-c]quinolin-1(2H)-one scaffold: Probing substituents at position 3

Article abstract of DOI:10.1007/s00044-015-1321-0

As a continuation of our efforts to optimize the antimitotic effect of our pyrimido[4,5-c]quinolin-1(2H)-one scaffold, we were interested in exploring the SAR of substituents at position 3 of this fused ring system. Target compounds 2a-r having substituen

Full text of DOI:10.1007/s00044-015-1321-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1321-0
ORIGINAL RESEARCH
The antimitotic pyrimido[4,5-c]quinolin-1(2H)-one scaffold:
probing substituents at position 3
•
Kamel Metwally Harris Pratsinis
•
Dimitris Kletsas
Received: 22 August 2014 / Accepted: 29 December 2014
Ó Springer Science+Business Media New York 2015
Abstract As a continuation of our efforts to optimize the
antimitotic effect of our pyrimido[4,5-c]quinolin-1(2H)-
one scaffold, we were interested in exploring the SAR of
substituents at position 3 of this fused ring system. Target
compounds 2a–r having substituents with diverse elec-
tronic and steric characteristics at the 3-phenyl ring were
synthesized and tested for in vitro cytotoxicity against lung
fibrosarcoma HT-1080 and colon adenocarcinoma HT-29
human cancer cell lines using the widely accepted MTT
assay. Most of the compounds displayed cytotoxic effects
in the low micromolar range. Clear-cut SAR conclusions
were drawn from the available biological data. Generally,
o-substitution results in a marked decrease or complete loss
of cytotoxicity. Multimethoxy substitution in this particular
position does not contribute to the cytotoxic effect but
leads to cytotoxic selectivity for HT-1080 versus HT-29
cells. Interestingly, the best position for cytotoxicity of a
halogen was the p-position, whereas m-substitution was the
most favourable within the methyl/methoxy series. To
confirm the mechanism of action previously identified for
our closely related analogues, the most cytotoxic com-
pounds were evaluated for their effects on cell cycle pro-
gression and tubulin polymerization. All compounds tested
were found to arrest HT-1080 cells in the G2/M phase of
the cell cycle and inhibit tubulin polymerization in a sim-
ilar fashion to colcemid which is a known tubulin poly-
merization inhibitor.
Graphical Abstract
Halogen is optimal
NH2
O
N
Alkyl/alkoxy is optimal
Weak or no activity
Cl
N
N
Cl
µ
IC50; HT-1080 cells: 3-CH3 = 1.0
M
3-OCH3 = 1.0
µ
M
4-I = 1.1
µ
M
Keywords Cytotoxic agents Á Anticancer Á Antimitotic Á
Pyrimido[4,5-c]quinolin-1(2H)-ones
Introduction
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1321-0) contains supplementary
material, which is available to authorized users.
Microtubules, simply polymers of alternating a- and b-
tubulin subunits, are prominent components in the cyto-
skeleton (Zhou and Giannakakou, 2005). Not only micro-
tubules play a crucial role in mitosis and cell replication, but
they are also involved in other important cellular activities
such as cell movement, cell shape, and transport of organ-
elles inside the cell (Nogales, 2001; Downing, 2000, Sorger
et al., 1997; Honore et al., 2005). Chemical substances,
natural or synthetic, which have the ability to bind tubulin,
K. Metwally (&)
Department of Medicinal Chemistry, Faculty of Pharmacy,
Zagazig University, Zagazig, Egypt
e-mail: kametwally@hotmail.com
H. Pratsinis Á D. Kletsas
Laboratory of Cell Proliferation and Ageing, Institute of
Biology, National Centre of Scientific Research ‘‘Demokritos’’,
Athens, Greece
123

Med Chem Res
interfere with microtubule dynamics by either promoting or
inhibiting tubulin polymerization and hence microtubule
assembly (Zhou and Giannakakou, 2005; Jordan and Wilson,
2004). Drugs which interfere with microtubule dynamics are
called antimitotic agents. These drugs can be broadly cate-
gorized into two groups, namely tubulin polymerization
promoters and inhibitors. The taxanes such as paclitaxel and
docetaxel typify tubulin polymerization promoters, whereas
colchicine, combretastatins, and the vinca alkaloids typify
tubulin polymerization inhibitors. It is well documented that
tubulin-binding drugs induce mitotic catastrophe followed
by apoptotic cell death by arresting cells in the G2/M phase
of cell cycle (Honore et al., 2005; Dumontet and Jordan,
2010; Amos, 2011). In addition, these drugs were reported to
have clinically relevant antiangiogenic and vascular-dis-
rupting properties, which were attributed to their ability to
inhibit endothelial cell proliferation, migration, and tube
formation (Schwartz, 2009). Given such importance in
cancer pathophysiology, tubulin remains one of the most
attractive targets in cancer chemotherapy.
selectivity for certain cancer cell lines but were generally less
potent than their 2-amino counterparts (Metwally et al., 2007b,
2013). This directed us to focus on studying 2-aminopyrimi-
do[4,5-c]quinolin-1(2H)-ones. Among quinoline benzenoid
ring substituents tested, chloro, iodo, or methoxy groups at C-9
were generally shown to give the most potent cytotoxic
compounds. Substantially lower cytotoxic activity was dis-
played by 7-chloro versus their 9-chloro counterparts in all cell
lines tested (Metwally et al., 2010).
A few substituents at position 3 were preliminarily
explored in a previous study (Metwally et al., 2007a). It was
concluded that a 3-phenyl ring significantly enhanced cyto-
toxicity when compared to a 3-methyl group. Moreover,
p-chloro substitution was found to further enhance cytotoxic
activity (Fig. 1). In this context, the aim of this investigation
was primarily toexamine, indepth, the effectofsubstitutionat
position 3 of our pyrimido[4,5-c]quinolin-1(2H)-one antimi-
totic scaffold for the purpose of formulating a comprehensive
structure–activity relationship. To achieve this goal, the
3-phenyl ringwas substituted atdifferent positions with atoms
and groups of diverse electronic and steric properties. Guided
by our previous SAR data, we fixed substituents at positions 5
and 9 as 4-chlorophenyl and chloro, respectively.
Our interest in pursuing anti-tubulin drugs had previously
led us to the discovery of pyrimido[4,5-c]quinolin-1(2H)-ones
as an interesting scaffold for antimitotic anticancer agents
(Metwally et al., 2007a). The first set of target compounds with
this fused ring system and having a 2-amino substituent
showed in vitro cytotoxic activity at the low micromolar level
against a panel of human cancer cell lines. Initial mechanistic
studies led to the identification of tubulin as the biological
target of this class of compounds (Metwally et al., 2007a).
Subsequently, we started a systematic structure–activity rela-
tionship study for the sake of having deep insight into struc-
tural determinants of cytotoxic activity and fine tuning the
antimitotic effect. Some important conclusions were drawn
from our previous SAR investigations. At position 2, substi-
tuted phenyl analogues were cytotoxic and showed cytotoxic
Materials and methods
Chemistry
Melting points were determined on a Barnstead Electro-
thermal 9100 melting point apparatus and are uncorrected.
1H-NMR spectra were recorded in DMSO-d6 on a Varian
Mercury 300-MHz spectrometer. Chemical shifts were
expressed in parts per million (ppm) downfield from tet-
ramethylsilane (TMS). 13C-NMR spectra were recorded at
75 MHz in DMSO-d6 on a Bruker 400-MHz spectrometer.
Elemental analyses (C, H, N) were performed at the
Microanalytical Unit, Cairo University, Cairo, Egypt. All
compounds were routinely checked by thin-layer chroma-
tography (TLC) on aluminium-backed silica gel plates. All
solvents were dried by standard methods. Compounds 1
and 2d were reported previously (Metwally et al., 2007a).
Synthetic route to the target compounds is outlined in
Scheme 1. The starting 3-amino-6-chloro-2-(4-chloro-
phenyl)quinoline-4-carboxylic acid (1) was synthesized from
5-chloroisatin and 4-chlorophenacylamine hydrochloride
under the basic conditions of the Pfitzinger reaction as pre-
viously reported (Metwally et al., 2007a). Condensation of
the starting acid with the appropriate benzoyl chlorides in
pyridine at room temperature afforded the intermediate lac-
tones which were used directly for the synthesis of the desired
target compounds (2a–r) in fair yields through condensation
with hydrazine hydrate in 2-methoxyethanol under reflux
NH2
O
N
R
Cl
N
N
Cl
IC50 (µM)
HT-1080
HT-29
CH3
C6H5
4-Cl-C6H4
34.3 ( 7.2)
2.90 ( 0.58)
1.23 ( 0.22)
52.0 ( 24.0)
4.68 ( 0.79)
1.97 ( 0.007)
Fig. 1 Previous cytotoxicity data of pilot position 3 probes
123

Med Chem Res
NH2
N
COOH
N
R
O
Cl
NH2
O
a,b
Cl
N
+
Cl
R
N
1
Cl
2a-r
Cl
R = 4-F, 3-F, 2-F, 4-Cl, 3-Cl, 2-Cl, 4-Br, 3-Br, 4-I
4-CH3, 3-CH3, 2-CH3, 4-OCH3, 3-OCH3, 2-OCH3
3,4-di-OCH3, 3,5-di-OCH3, 3,4,5-tri-OCH3
Scheme 1 Reagents and conditions: a pyridine, 0 °C to room temperature, 16 h and b hydrazine hydrate 99 %, 2-methoxyethanol, reflux, 24 h
conditions. All the target compounds in the present investi-
gation were structurally elucidated by 1H-NMR, EIMS
spectral data, and elemental analysis.
(m, 1H, C10-H). EIMS m/z 450 (M?). Anal. calcd for
C23H13Cl2FN4O: C, 61.21; H, 2.90; N, 12.42. Found: C,
60.98; H, 2.96; N, 12.07.
General procedure for 2-amino-9-chloro-5-
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(2-fluorophenyl)
substitutedarylpyrimido[4,5-c]quinolin-1(2H)-ones (2a–r)
pyrimido[4,5-c]quinolin-1(2H)-one (2c)
A solution of the 3-amino-6-chloro-2-(4-chlorophenyl)quin-
olone-4-carboxylic acid (1; 10 mmol) in pyridine was cooled
in an ice bath and the appropriate benzoyl chloride (25 mmol)
was added in a dropwise manner while stirring. After addition
was complete, the reaction mixture was allowed to stir at
room temperature for 16 h. Cold ethanol was added to the
reaction mixture, and the precipitated solid was filtered,
washed with water and ethanol, and dried. The solid was
suspended in 2-methoxyethanol (40 mL), hydrazine hydrate
(99 %; 4 mL) was added, and the mixture was heated at reflux
for 24 h. The precipitated solid was filtered, washed with
ethanol, dried, and recrystallized from the appropriate solvent.
Yield 50 %, mp 254–257 °C (DMF); 1H-NMR (DMSO-d6)
d: 5.93 (s, 2H, NH2), 7.35–7.39 (m, 2H, 3-phenyl-H), 7.55
(d, 2H, J = 6.33 Hz, 5-phenyl-H), 7.61–7.62 (m, 1H,
3-phenyl-H), 7.71–7.75 (m, 1H, 3-phenyl-H), 7.88–7.94
(m, 1H, C8-H), 8.09 (d, 2H, J = 6.33 Hz, 5-phenyl-H),
8.19 (d, 1H, J = 6.6 Hz, C7-H), 9.65–9.70 (m, 1H, C10-H).
EIMS m/z 450 (M?). 13C-NMR (DMSO-d6) d: 115.55,
115.76, 118.34, 123.07, 124.14, 124.39, 124.42, 124.85,
127.83, 130.05, 131.16, 131.19, 131.70, 132.51, 132.82,
133.95, 134.50, 136.39, 140.76, 143.01, 154.21, 157.04,
160.88. Anal. calcd for C23H13Cl2FN4O: C, 61.21; H, 2.90;
N, 12.42. Found: C, 60.96; H, 2.97; N, 12.71.
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(4-fluorophenyl)
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3-chlorophenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2a)
pyrimido[4,5-c]quinolin-1(2H)-one (2e)
Yield 55 %, mp 297–298 °C (DMF); 1H-NMR (DMSO-d6)
d: 6.10 (s, 2H, NH2), 7.35–7.40 (m, 2H, 3-Phenyl-H), 7.60
(d, 2H, J = 6.33 Hz, 5-phenyl-H), 7.92 (dd, 1H, J = 6.6,
1.65 Hz, C8-H), 8.02–8.05 (m, 2H, 3-phenyl-H), 8.15 (d,
2H, J = 6.33 Hz, 5-phenyl-H), 8.22–8.24 (m, 1H, C7-H),
9.74 (d, 1H, J = 1.92 Hz, C10-H). EIMS m/z 450 (M?).
Anal. calcd for C23H13Cl2FN4O: C, 61.21; H, 2.90; N,
12.42. Found: C, 60.99; H, 3.01; N, 12.68.
Yield 61 %, mp [ 300 °C (DMF); 1H-NMR (DMSO-d6) d:
6.09 (s, 2H, NH2), 7.54–7.63 (m, 3H, 3-phenyl-H, 5-phenyl-
H), 7.88 (d, 1H, J = 5.22 Hz, C8-H), 7.93–7.95 (m, 1H,
3-phenyl-H), 8.02–8.03 (m, 2H, 3-phenyl-H), 8.14 (d, 2H,
J = 6.33 Hz, 5-phenyl-H), 8.24 (d, 1H, J = 6.6 Hz, C7-H),
9.75 (s, 1H, C10-H). 13C-NMR (DMSO-d6) d: 117.66,
124.09, 124.69, 127.75, 128.74, 129.80, 129.90, 129.96,
130.30, 131.67, 132.49, 132.80, 133.87, 134.44, 136.03,
136.33, 140.56, 142.84, 155.55, 157.10, 160.81. EIMS m/z
466 (M?). Anal. calcd for C23H13Cl3N4O: C, 59.06; H, 2.80;
N, 11.98. Found: C, 58.82; H, 2.77; N, 12.09.
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3-fluorophenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2b)
1
Yield 58 %, mp 274–276 °C (DMF); H-NMR (DMSO-d6)
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(2-chlorophenyl)
d: 6.07 (s, 2H, NH2), 7.48–7.51 (m, 1H, Ar–H, 3-phenyl-H),
7.56–7.59 (m, 2H, 3-phenyl-H), 7.74–7.75 (m, 1H,
3-phenyl-H), 7.90–7.92 (m, 2H, C8-H, 5-phenyl-H),
8.11–8.27 (Complex m, 4H, 5-phenyl-H, C7-H), 9.72–9.73
pyrimido[4,5-c]quinolin-1(2H)-one (2f)
Yield 33 %, mp 223–225 °C (DMF); 1H-NMR (DMSO-d6)
d: 5.94 (s, 2H, NH2), 7.48–7.57 (m, 4H, 3-phenyl-H,
123

Med Chem Res
5-phenyl-H), 7.61–7.62 (d, 1H, J = 5.22 Hz, 3-phenyl-H),
7.68 (d, 1H, J = 5.49 Hz, 3-phenyl-H), 7.92 (dd, 1H,
J = 6.6, 1.92 Hz, C8-H), 8.06 (d, 2H, J = 6.33 Hz,
5-phenyl-H), 8.20–8.22 (m, 1H, C7-H), 9.73–9.74 (m, 1H,
C10-H). 13C-NMR (DMSO-d6) d: 118.71, 124.32, 125.08,
127.31, 128.04, 129.51, 130.25, 130.83, 131.46, 131.82,
132.00, 133.01, 134.24, 134.42, 134.80, 136.60, 140.88,
143.23, 155.92, 157.26, 160.88. EIMS m/z 466 (M?). Anal.
calcd for C23H13Cl3N4O: C, 59.06; H, 2.80; N, 11.98.
Found: C, 58.94; H, 2.85; N, 12.02.
J = 6.03 Hz, 3-phenyl-H), 7.59 (d, 2H, J = 6.6 Hz,
5-phenyl-H), 7.86–7.91 (m, 3H, 3-phenyl-H, C8-H), 8.15
(d, 2H, J = 6.57 Hz, 5-phenyl-H), 8.19–8.21 (d, 1H,
J = 6.6 Hz, C7-H), 9.72 (d, 1H, J = 1.65 Hz, C10-H). 13C-
NMR (DMSO-d6) d: 21.01, 117.00, 124.18, 124.58,
127.69, 128.33, 129.59, 130.19, 131.20, 131.57, 132.77,
133.69, 134.29, 136.36, 140.50, 140.67, 142.59, 156.65,
157.06, 160.75. EIMS m/z 446 (M?). Anal. calcd for
C24H16Cl2N4O: C, 64.44; H, 3.61; N, 12.53. Found: C,
64.47; H, 3.59; N, 12.80.
2-Amino-3-(4-bromophenyl)-9-chloro-5-(4-chlorophenyl)
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3-methylphenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2g)
pyrimido[4,5-c]quinolin-1(2H)-one (2k)
1
Yield 57 %, mp 288–290 °C (DMF); H-NMR (DMSO-d6)
Yield 51 %, mp 263–266 °C (DMF); 1H-NMR (DMSO-d6)
d: 2.40 (s, 3H, CH3), 6.10 (s, 2H, NH2), 7.35–7.43 (m, 2H,
3-phenyl-H), 7.59 (d, 2H, J = 6.6 Hz, 5-phenyl-H),
7.72–7.78 (m, 2H, 3-phenyl-H), 7.92 (dd, 1H, J = 6.57,
1.65 Hz, C8-H), 8.16 (d, 2H, J = 6.33 Hz, 5-phenyl-H),
8.22 (d, 1H, J = 6.6 Hz, C7-H), 9.74 (d, 1H, J = 1.92 Hz,
C10-H). EIMS m/z 446 (M?). Anal. calcd for
C24H16Cl2N4O: C, 64.44; H, 3.61; N, 12.53. Found: C,
64.23; H, 3.62; N, 12.36.
d: 6.09 (s, 2H, NH2), 7.56–7.61 (m, 2H, 5-phenyl-H),
7.71–7.77 (m, 2H, 3-phenyl-H), 7.87–7.90 (m, 3H, 3-phenyl-
H, C8-H), 8.11–8.13 (m, 2H, 5-phenyl-H), 8.18 (d, 1H,
J = 6.6 Hz, C7-H), 9.69 (d, 1H, J = 1.92 Hz, C10-H). 13C-
NMR (DMSO-d6) d: 117.52, 124.16, 124.38, 124.71, 127.82,
129.85, 130.92, 131.66, 132.30, 132.84, 133.33, 133.89,
134.47, 136.35, 140.64, 142.82, 155.98, 157.05, 160.86.
EIMS m/z 510 (M?). Anal. calcd for C23H13BrCl2N4O: C,
53.93; H, 2.56; N, 10.94. Found: C, 53.93; H, 2.60; N, 11.12.
2-Amino-3-(3-bromophenyl)-9-chloro-5-(4-chlorophenyl)
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(2-methylphenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2h)
pyrimido[4,5-c]quinolin-1(2H)-one (2l)
Yield 51 %, mp 275–276 °C (DMF); 1H-NMR (DMSO-d6)
d: 6.08 (s, 2H, NH2), 7.47–7.51 (m, 1H, 3-phenyl-H),
7.57–7.63 (m, 2H, 5-phenyl-H), 7.74–7.76 (m, 1H, 3-phe-
nyl-H), 7.91–7.93 (m, 2H, C8-H, 3-phenyl-H), 8.12–8.15
(m, 3H, 5-phenyl-H, 3-phenyl-H), 8.21–8.23 (d, 1H,
J = 6.6 Hz, C7-H), 9.73 (d, 1H, J = 1.65 Hz, C10-H).
EIMS m/z 510 (M?). Anal. calcd for C23H13BrCl2N4O: C,
53.93; H, 2.56; N, 10.94. Found: C, 53.95; H, 2.54; N, 11.09.
Yield 37 %, mp 219–221 °C (DMF); 1H-NMR (DMSO-d6)
d: 2.22 (s, 3H, CH3), 5.95 (s, 2H, NH2), 7.32–7.54 (m, 6H,
3-phenyl-H, 5-phenyl-H), 7.88–7.91 (m, 1H, C8-H), 8.02
(dd, 2H, J = 6.4, 1.8 Hz, 5-phenyl-H), 8.19 (dd, 1H,
J = 6.6, 1.92 Hz, C7-H), 9.70–9.74 (m, 1H, C10-H). 13C-
NMR (DMSO-d6) d: 19.42, 118.14, 124.48, 125.03,
125.62, 127.96, 129.02, 129.72, 129.95, 130.24, 131.70,
132.86, 134.06, 134.65, 134.80, 136.17, 136.78, 140.95,
142.97, 157.45, 157.61, 160.62. EIMS m/z 446 (M?). Anal.
calcd for C24H16Cl2N4O: C, 64.44; H, 3.61; N, 12.53.
Found: C, 64.19; H, 3.66; N, 12.35.
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(4-iodophenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2i)
Yield 46 %, mp 270–272 °C (DMF); 1H-NMR (DMSO-d6)
d: 6.08 (s, 2H, NH2), 7.56–7.58 (m, 2H, 5-phenyl-H),
7.72–7.74 (m, 2H, 3-phenyl-H), 7.87–7.91 (m, 3H, 3-phe-
nyl-H, C8-H), 8.11–8.17 (m, 3H, 5-phenyl-H, C7-H),
9.69–9.70 (m, 1H, C10-H). EIMS m/z 558 (M?). Anal.
calcd for C23H13Cl2IN4O: C, 49.40; H, 2.34; N, 10.02.
Found: C, 49.16; H, 2.39; N, 10.59.
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(4-methoxyphenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2m)
Yield 53 %, mp 292–294 °C (DMF); 1H-NMR (DMSO-d6)
d: 3.85 (s, 3H, OCH3), 6.13 (s, 2H, NH2), 7.07 (d, 2H,
J = 6.6 Hz, 3-phenyl-H), 7.61 (d, 2H, J = 6.33 Hz,
5-phenyl-H), 7.90 (dd, 1H, J = 6.6, 1.92 Hz, C8-H),
7.99–8.01 (m, 2H, 3-phenyl-H), 8.16 (d, 2H, J = 6.33 Hz,
5-phenyl-H), 8.21 (d, 1H, J = 6.57 Hz, C7-H), 9.73 (d, 1H,
J = 1.92 Hz, C10-H). EIMS m/z 462 (M?). Anal. calcd for
C24H16Cl2N4O2: C, 62.22; H, 3.48; N, 12.09. Found: C,
61.95; H, 3.54; N, 12.08.
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(4-methylphenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2j)
Yield 52 %, mp 276–277 °C (DMF); 1H-NMR (DMSO-d6)
d: 2.39 (s, 3H, CH3), 6.10 (s, 2H, NH2), 7.33 (d, 2H,
123

Med Chem Res
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3-methoxyphenyl)
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3,4,5-
pyrimido[4,5-c]quinolin-1(2H)-one (2n)
trimethoxyphenyl)pyrimido[4,5-c]quinolin-1(2H)-one (2r)
Yield 49 %, mp 237 °C, dec. (DMF/EtOH); 1H-NMR
(DMSO-d6) d: 3.82 (s, 3H, OCH3), 6.11 (s, 2H, NH2),
7.11–7.13 (m, 1H, 3-phenyl-H), 7.41–7.45 (m, 1H, 3-phe-
nyl-H), 7.51–7.53 (m, 2H, 3-phenyl-H), 7.56–7.59 (m, 2H,
5-phenyl-H), 7.87–7.90 (m, 1H, C8-H), 8.13–8.20 (m, 3H,
5-phenyl-H, C7-H), 9.70–9.71 (d, 1H, J = 1.65 Hz, C10-
H). EIMS m/z 462 (M?). Anal. calcd for C24H16Cl2N4O2:
C, 62.22; H, 3.48; N, 12.09. Found: C, 61.97; H, 3.39; N,
12.07.
Yield 51 %, mp 235–237 °C (DMF/EtOH); 1H-NMR
(DMSO-d6) d: 3.75 (s, 3H, 4-OCH3), 3.84 (s, 6H, 3,5-di-
OCH3), 6.21 (s, 2H, NH2), 7.36 (s, 2H, 3-phenyl-H), 7.60
(d, 2H, J = 6.57 Hz, 5-phenyl-H), 7.89 (d, 1H,
J = 6.87 Hz, C8-H), 8.17–8.22 (m, 3H, 5-phenyl-H, C7-H),
9.72–9.73 (m, 1H, C10-H). Anal. calcd for C26H20Cl2N4O4:
C, 59.67; H, 3.85; N, 10.71. Found: C, 59.60; H, 3.97; N,
11.02.
Biology
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(2-methoxyphenyl)
pyrimido[4,5-c]quinolin-1(2H)-one (2o)
In vitro cytotoxicity
Yield 38 %, mp 246–247 °C (DMF/EtOH); 1H-NMR
(DMSO-d6) d: 3.78 (s, 3H, OCH3), 5.95 (s, 2H, NH2),
7.11–7.19 (m, 2H, 3-phenyl-H), 7.54–7.56 (m, 4H, 3-phe-
nyl-H, 5-phenyl-H), 7.89 (d, 1H, J = 4.95 Hz, C8-H), 8.10
(d, 2H, J = 6.06 Hz, 5-phenyl-H), 8.19 (d, 1H,
J = 6.6 Hz, C7-H), 9.72 (s, 1H, C10-H). 13C-NMR
(DMSO-d6) d: 55.86, 111.28, 117.46, 120.36, 123.88,
124.11, 124.66, 127.69, 129.73, 129.95, 131.57, 131.71,
132.72, 133.67, 134.28, 136.35, 140.68, 142.67, 155.81,
156.84, 157.02, 160.05. EIMS m/z 462 (M?). Anal. calcd
for C24H16Cl2N4O2: C, 62.22; H, 3.48; N, 12.09. Found: C,
61.91; H, 3.40; N, 12.05.
The new compounds were tested for their cytotoxic activity
on the following human solid tumour cell lines: lung
fibrosarcoma HT-1080 (American Type Culture Collec-
tion, Rockville, MD, USA), mammary adenocarcinoma
MDA-MB-231 (ATCC), and colorectal adenocarcinoma
HT-29 (European Collection of Cell Cultures, Salisbury,
UK). All cells were routinely cultured in Dulbecco’s
minimal essential medium (DMEM) supplemented with
penicillin (100 U/mL), streptomycin (100 lg/mL), and
10 % foetal bovine serum (media and antibiotics from
Biochrom KG, Berlin, Germany) in an environment of 5 %
CO2, 85 % humidity, and 37 °C, and they were subcultured
using a trypsin 0.25 %–EDTA 0.02 % solution. The cyto-
toxicity assay was performed by a modification of the MTT
method (Metwally et al., 2007a). Briefly, the cells were
plated at a density of approximately 5,000 cells/well in
96-well flat-bottomed microplates, and after 24 h, the test
compounds were added, appropriately diluted with DMSO.
After a 72-h incubation, the medium was replaced with
MTT (Sigma) dissolved at a final concentration of 1 mg/
mL in serum-free, phenol red-free DMEM for a further 4-h
incubation. Then, the MTT formazan was solubilized in
2-propanol, and the optical density was measured using an
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3,4-
dimethoxyphenyl)pyrimido[4,5-c]quinolin-1(2H)-one (2p)
Yield 47 %, mp 282–283 °C (DMF/EtOH); 1H-NMR
(DMSO-d6) d: 3.81 (s, 3H, 4-OCH3), 3.84 (s, 3H, 3-OCH3),
6.18 (s, 2H, NH2), 7.09 (d, 1H, J = 6.06 Hz, 3-phenyl-H),
7.59–7.70 (m, 4H, 3-phenyl-H, 5-phenyl-H), 7.89–7.91 (m,
1H, C8-H), 8.16–8.21 (m, 3H, 5-phenyl-H, C7-H),
9.72–9.73 (m, 1H, C10-H). EIMS m/z 492 (M?). Anal.
calcd for C25H18Cl2N4O3: C, 60.86; H, 3.68; N, 11.36.
Found: C, 60.62; H, 3.72; N, 11.13.
InfiniteÒ M200 microplate reader (Tecan, Mannedorf,
¨
Switzerland) at a wavelength of 550 nm (reference wave-
length 690 nm). Doxorubicin hydrochloride (Sigma) was
included in the experiments as positive control. The results
represent the mean of three independent experiments and
are expressed as IC50, i.e. the concentration that reduced by
50 % the optical density of treated cells with respect to
untreated controls.
2-Amino-9-chloro-5-(4-chlorophenyl)-3-(3,5-
dimethoxyphenyl)pyrimido[4,5-c]quinolin-1(2H)-one (2q)
Yield 45 %, mp 294–295 °C (DMF/EtOH); 1H-NMR
(DMSO-d6) d: 3.80 (s, 6H, OCH3), 6.12 (s, 2H, NH2),
6.67–6.68 (m, 1H, 3-phenyl-H), 7.10–7.11 (m, 2H, 3-phe-
nyl-H), 7.59 (d, 2H, J = 6.03 Hz, 5-phenyl-H), 7.78–7.79
(m, 1H, C8-H), 8.16 (d, 2H, J = 6.33 Hz, 5-phenyl-H),
8.24 (d, 1H, J = 7.17 Hz, C7-H), 9.75 (d, 1H,
J = 1.11 Hz, C10-H). EIMS m/z 492 (M?). Anal. calcd for
C25H18Cl2N4O3: C, 60.86; H, 3.68; N, 11.36. Found: C,
60.60; H, 3.76; N, 11.70.
Cell cycle analysis
Cell cycle analysis was performed following incubation of
exponentially growing HT-1080 cells with the test sub-
stances (5 lM) for 36 h. Treated cultures were then
123

Med Chem Res
trypsinized, washed in phosphate-buffered saline (PBS),
fixed in 50 % ethanol, and stained with an RNAse-con-
taining propidium iodide solution (Metwally et al., 2007a).
DNA content was analysed on a FACS Calibur (Becton–
Dickinson, San Jose, CA, USA) flow cytometer using the
ModFit software (Verity Software House, Topsham, ME,
USA).
polymerization inhibition assay performed on a selected set
of the most cytotoxic compounds.
Overall, cytotoxicity data revealed that two major con-
clusions can be initially drawn. Firstly, o-substituents
bigger in size than a fluorine atom are not tolerated as
evidenced by the relatively low cytotoxic activity of
compounds 2c (o-fluoro; 6.4 1.8 lM) and 2o (o-meth-
oxy; 15.0 3.6 lM) and lack of cytotoxicity of 2f (o-
chloro) and 2l (o-methyl) up to 100 lM level when tested
on the HT-1080 cell lines. Secondly, methoxy substituents,
whatever was the number and pattern of substitution,
showed more than eightfold higher cytotoxic selectivity for
HT-1080 versus HT-29 cell lines. This finding was more
obvious with the multimethoxy-substituted compounds 2q
and 2r. While both compounds were inactive in the HT-29
cell line up to 100 lM level, they exhibited moderate
cytotoxic activity against HT-1080 cells. Multiple methoxy
substitution seemed to detract from cytotoxic activity in
both cell lines tested. In fact, HT-29 cells were almost
insensitive to multimethoxy-substituted compounds (2p–r).
Interestingly, the trimethoxy-substituted compound (2r)
had slightly lower cytotoxic activity (1.3 0.8 lM) when
compared to the m-methoxy analogue (2n; 1.0 0.3 lM)
against HT-1080 cell lines, but showed the highest cyto-
toxic selectivity for these cells versus HT-29 cell lines
among the target compounds tested (Table 1).
In vitro inhibition of cellular tubulin polymerization
Exponentially growing HT-1080 cells (approximately
2 9 106 cells/dish) were treated with the test compounds
(50 lM) for 24 h. Negative control cultures were treated
with the corresponding concentration of vehicle (DMSO),
while positive controls with either 1 lM colcemid (Bio-
chrom) or 0.2 lM paclitaxel (GenexolÒ, Samyang Genex
Corporation, Daejeon, Korea—kindly provided by Dr.
M. Sagnou). Cultures were then trypsinized and washed in
PBS, and polymerized cellular tubulin was separated from
soluble tubulin dimers as described previously (Metwally
et al., 2007a). Briefly, cells were lysed in a buffer con-
taining 20 mM Tris–HCl (pH 6.8), 1 mM MgCl2, 2 mM
EGTA, and 0.5 % Nonidet P-40 and protease and phos-
phatase inhibitor cocktails (Sigma) at room temperature
and under low ambient light (Blagosklonny et al., 1995).
Supernatants were collected after centrifugation at
13,0009g for 10 min. The pellets were dissolved by
heating for 5 min at 95 °C in a SDS–polyacrylamide gel
electrophoresis (PAGE) sampling buffer and subjected to
electrophoresis on a 10 % SDS-PAGE gel. After electro-
phoresis, the proteins were transferred to a PVDF mem-
brane and subjected to Western analysis using an anti-a-
tubulin monoclonal antibody and peroxidase-conjugated
anti-mouse antibody (Sigma). Detection of immunoreac-
tive bands was performed by chemiluminescence (ECL kit,
Amersham Biosciences) according to the manufacturer’s
instructions on a Fujifilm LAS-4000 luminescent image
analyser (Fujifilm Manufacturing Inc., Greenwood, SC,
USA).
For a more convenient discussion, the target compounds
can be categorized into two distinct series: the 3-halophe-
nyl and the 3-methyl/methoxyphenyl series. A clear-cut
distinction in cytotoxicity pattern was shown by the
3-halophenyl series versus the 3-methyl/methoxyphenyl
series. In the 3-halophenyl series, the p-halo substituents
consistently displayed higher cytotoxic activity when
compared to the m-halo substituents. For instance, the
p-chloro analogue (2d) was more cytotoxic than its
m-chloro counterpart (2e) in both cell lines tested. Inter-
estingly, the p-iodo compound (2i) was found to be the
most potent among the p-halo analogues displaying an IC50
of 1.1 lM against HT-1080 cell lines. In addition, this
particular compound exhibited the highest cytotoxic
selectivity for HT-1080 cells with an IC50 of 10.2 lM
against HT-29 cells. In the methyl/methoxyphenyl series,
however, m-substitution appeared to yield the most cyto-
toxic compounds. The m-methyl analogue (2k) was equi-
potent to its m-methoxy counterpart (2n) displaying
1.0 lM cytotoxic activity against HT-1080 cell lines. Both
compounds were found to be less potent in the HT-29 cell
lines with compound 2k showing slightly higher IC50 value
(5.7 0.5 lM) as compared to 2n (8.3 4.2 lM). In
both series, HT-1080 cells were generally more responsive
to the cytotoxic effect of the target compounds as com-
pared to HT-29 cells, in line with our previous observations
(Metwally et al., 2007a, 2013).
Results and discussion
All the target compounds were evaluated for in vitro
cytotoxicity against two human cancer cell lines, namely
lung fibrosarcoma HT-1080 and colon adenocarcinoma.
The data are represented in terms of IC50 values (lM)
which are the drug concentrations required for 50 % inhi-
bition of net cell proliferation in comparison with untreated
control cultures. Doxorubicin was used as a reference
cytotoxic compound. Furthermore, the mechanism of
action of the new compounds was investigated through
flow cytometric cell cycle analysis and tubulin
123

Med Chem Res
Table 1 In vitro cytotoxicity of the target pyrimido[4,5-c]quinolin-
1(2H)-ones using the MTT assay (IC50 values in lM)
Table 2 Cell cycle phase distribution (%)
Compound
G0/G1
S
G2/M
Compound
R
Cell line
HT-1080
2a
40.74
44.93
37.30
39.66
44.10
41.11
41.79
39.71
45.68
8.57
21.27
24.81
22.41
26.97
26.86
23.84
28.88
26.98
26.91
12.73
24.21
28.30
37.99
30.27
40.29
33.37
29.04
35.05
29.33
33.31
27.41
78.71
68.12
23.24
HT-29
2b
2d
2a
2b
2c
4-F
1.8 ( 0.2)
2.4 ( 0.1)
6.4 ( 1.8)
1.4 ( 0.3)
5.3 ( 0.8)
[100
2.0 ( 0.8)
5.8 ( 1.8)
32.0 ( 5.1)
2.4 ( 0.2)
3.9 ( 1.5)
NTa
2g
3-F
2i
2-F
2j
2d
2e
4-Cl
2k
3-Cl
2n
2f
2-Cl
2r
2g
2h
2i
4-Br
1.9 ( 0.2)
3.4 ( 0.4)
1.1 ( 0.5)
2.6 ( 1.0)
1.0 ( 0.2)
[100
3.2 ( 0.2)
7.7 ( 2.3)
10.2 ( 1.0)
4.9 ( 1.5)
5.7 ( 0.5)
NT
Colcemid
Paclitaxel
Control
3-Br
7.67
4-I
48.46
2j
4-CH3
3-CH3
2-CH3
4-OCH3
3-OCH3
2-OCH3
3,4-di-OCH3
3,5-di-OCH3
2k
2l
One out of two similar experiments is depicted
2m
2n
2o
2p
2q
2r
24.6 ( 9.2)
1.0 ( 0.3)
15.0 ( 3.6)
8.6 ( 5.3)
6.2 ( 1.4)
NT
8.3 ( 4.2)
NT
78.2 ( 5.7)
[100
3,4,5-tri-OCH3 1.3 ( 0.8)
[100
0.0035 ( 0.0001) 0.029 ( 0.01)
Doxo
–
The results represent the mean ( standard deviation) of three inde-
pendent experiments and are expressed as IC50, the concentration that
reduced by 50 % the optical density of treated cells with respect to
untreated controls
a
NT, not tested
Fig. 3 Induction of G2/M arrest by test compound 2d compared to
untreated cultures (control) as revealed by flow cytometric cell cycle
analysis of HT-1080 cells
Fig. 2 Inhibition of cellular tubulin polymerization by test com-
pounds compared to untreated cultures (Ctrl) and cultures treated with
colcemid (Colc) or paclitaxel (Tax)
It is well documented that compounds interfering with
microtubule dynamics induce apoptosis in cancer cells
(Honore et al., 2005; Dumontet, 2010; Amos, 2011). To
examine the effect of our target compounds on cell cycle
progression, the most cytotoxic compounds (IC50 in the
range of 1.0–2.6 lM) were then tested for their effect on
cell cycle through a flow cytometric analysis. This method
highlights the effects of antimitotic compounds on the
distribution of cells in specific phases of the cell cycle. To
this end, the DNA content of HT-1080 cells after incu-
bation with 5 lM compound for 36 h was analysed,
through staining with propidium iodide followed by flow
cytometry. Colcemid and paclitaxel were included as
positive controls. All compounds tested were found to
123

Med Chem Res
References
arrest the cells in the G2/M phase of the cell cycle, in
agreement with the hypothesis of action through inter-
ference with tubulin polymerization (Fig. 2). Cell cycle
analysis data of the selected compounds are illustrated in
Table 2. This hypothesis was pursued further using a
tubulin polymerization assay, i.e. after incubation of HT-
1080 cells for 24 h with the test compounds (50 lM) or
with colcemid (1 lM) or paclitaxel (0.2 lM) used as
control compounds, polymerized tubulin was separated
from soluble tubulin and the two fractions were assessed
by Western analysis. As shown in Fig. 3, all the tested
compounds inhibited tubulin polymerization similarly to
colcemid, in contrast to paclitaxel, which stabilizes and
enhances tubulin polymerization.
Amos L (2011) What tubulin drugs tell us about microtubule structure
and dynamics. Semin Cell Dev Biol 22:916–926
Blagosklonny M, Schulte T, Nguyen P, Mimnaugh E, Trepel J,
Neckers L (1995) Taxol induction of p21WAF1 and p53 requires
c-raf-1. Cancer Res 55:4623–4626
Downing K (2000) Structural basis for the interaction of tubulin with
proteins and drugs that affect microtubule dynamics. Annu Rev
Cell Dev Biol 16:89–111
Dumontet C, Jordan M (2010) Microtubule-binding agents: a dynamic
field of cancer therapeutics. Nat Rev Drug Disc 9:790–803
Honore S, Pasquier E, Braguer D (2005) Understanding microtubule
dynamics for improved cancer therapy. Cell Mol Life Sci
62:3039–3056
Jordan M, Wilson L (2004) Microtubules as a target for anticancer
drugs. Nat Rev Cancer 4:253–265
Metwally K, Pratsinis H, Kletsas D (2007a) Pyrimido[4,5-c]quinolin-
1(2H)-ones as a novel class of antimitotic agents: synthesis and
in vitro cytotoxic activity. Eur J Med Chem 42:344–350
Metwally K, Aly O, Aly E, Banerjee A, Ravindra R, Bane S (2007b)
Synthesis and biological activity of 2,5-diaryl-3-methylpyri-
mido[4,5-c]quinolin-1(2H)-one derivatives. Bioorg Med Chem
15:2434–2440
Conclusion
We were able through the present investigation to shed
some light on the SAR pattern of substitution at position 3
of the antimitotic pyrimido[4,5-c]quinolin-1(2H)-ones.
Close inspection of biological results indicated that
o-substitution of the 3-phenyl ring generally decreases
cytotoxicity. On the other hand, p-substitution with a hal-
ogen or m-substitution with a methyl/methoxy group
enhances cytotoxic activity. Multimethoxy substitution was
not a positive contributor to cytotoxicity in both cell lines
tested. In general, the target compounds were more cyto-
toxic to HT-1080 cells as compared to HT-29 cells. The
most cytotoxic compounds arrested HT1080 cells in the
G2/M phase and inhibited tubulin polymerization. Syn-
thesis of new analogues with p-halophenyl and m-methyl/
methoxyphenyl substituents is currently underway.
Metwally K, Khalil A, Pratsinis H, Kletsas D (2010) Synthesis,
in vitro cytotoxicity, and
a preliminary structure-activity
relationship investigation of pyrimido[4,5-c]quinolin-1(2H)-
ones. Arch Pharm Chem Life Sci 8:465–472
Metwally K, Khalil A, Pratsinis H, Kletsas D, Sallam A, El-Sayed K
(2013) Structure–activity relationship investigation of methoxy
substitution on anticancer pyrimido[4,5-c]quinolin-1(2H)-ones.
Med Chem Res 22:4481–4491
Nogales E (2001) Structural insight into microtubule function. Annu
Rev Biophys Biomol Struct 30:397–420
Schwartz E (2009) Antivascular actions of microtubule-binding
drugs. Clin Cancer Res 15:2594–2601
Sorger P, Dobles M, Tournebize R, Hyman A (1997) Coupling cell
division and cell death to microtubule dynamics. Curr Opin Cell
Biol 9:807–814
Zhou J, Giannakakou P (2005) Targetting microtubules for cancer
chemotherapy. Curr Med Chem Anticancer Agents 5:65–71
Acknowledgments The authors are grateful to the Scientific
Research Administration, Zagazig University for providing the
financial support for this work (24, 2012–2013).
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