Pyrimido[4,5-c]quinolin-1(2H)-ones as a novel class of antimitotic agents: Synthesis and in vitro cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2006.10.008

Several 2-amino-pyrimido[4,5-c]quinolin-1(2H)-ones variously substituted at positions 3, 5, and 9 were prepared from their corresponding lactones. The target compounds were investigated for in vitro cytotoxic activity against a panel of human cancer cell

Full text of DOI:10.1016/j.ejmech.2006.10.008

European Journal of Medicinal Chemistry 42 (2007) 344e350
http://www.elsevier.com/locate/ejmech
Original article
Pyrimido[4,5-c]quinolin-1(2H )-ones as a novel class of antimitotic
agents: Synthesis and in vitro cytotoxic activity
b
b
Kamel Metwally a, , Harris Pratsinis , Dimitris Kletsas
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
b Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre of Scientific Research ‘‘Demokritos’’, Athens, Greece
Received 2 August 2006; received in revised form 12 October 2006; accepted 14 October 2006
Available online 4 December 2006
Abstract
Several 2-amino-pyrimido[4,5-c]quinolin-1(2H )-ones variously substituted at positions 3, 5, and 9 were prepared from their corresponding
lactones. The target compounds were investigated for in vitro cytotoxic activity against a panel of human cancer cell lines, namely, lung fibro-
sarcoma HT-1080, colon adenocarcinoma HT-29, and breast carcinoma MDA-MB-231. Analysis of data revealed that the presence of chloro at
position 9 has a major positive impact on cytotoxic activity. Additional halogen substitution at the para position of the 3-phenyl group further
enhances activity. Furthermore, compound (25) was found to dose-dependently inhibit tubulin polymerization. In accordance, flow cytometric
analysis of the most potent compounds (23e26) indicated that the tested compounds induce cell cycle arrest in the G2/M phase. The obtained
results introduce the rarely described pyrimido[4,5-c]quinolin-1(2H )-one ring system as a new scaffold for promising antimitotic agents.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Cytotoxic agents; Antimitotic; Pyrimido[4,5-c]quinolin-1(2H )-ones
1. Introduction
function, microtubules are required to be highly dynamic. In
other words, microtubules should have the ability to lengthen
and shorten through a reversible GTP-mediated process of as-
sociation and dissociation of the a/b-tubulin heterodimers at
both ends [3]. Microtubule-targeting drugs alter the dynamic
behavior of microtubules through binding to tubulin thereby
blocking mitotic cell progression and subsequently leading
to apoptotic cell death [4e6]. Evidences indicate that even mi-
nor alteration of microtubule dynamics can have major conse-
quences on cell cycle progression at mitosis [7e9].
Noteworthy, it has been postulated that mitosis can be blocked
by abnormally rapid microtubule dynamics as well as by in-
hibited dynamics [10]. Two distinct categories of microtu-
bule-targeting drugs are currently identified: agents that
inhibit tubulin polymerization or ‘‘microtubule-destabilizing
agents’’ such as the vinca alkaloids and colchicines, and agents
that promote tubulin polymerization or ‘‘microtubule-stabiliz-
ing agents’’ such as the taxanes exemplified by paclitaxel and
docetaxil [11]. For these reasons, tubulin became an attractive
Recent revolutionary advances in the field of molecular bi-
ology and cancer cell biology have created new targets for an-
titumor compounds. Microtubules are hollow cylindrical
protein polymers composed of a- and b-tubulin heterodimers.
The tubulin heterodimers assemble to form the protofilaments
which in turn associate longitudinally to form a microtubule as
a long tube of 25 nm diameter [1]. During cell division, the
microtubules are arranged in a unique manner to form the mi-
totic spindle which is the key cellular machinery driving mito-
sis in the metaphase where the replicated chromosomes are
congressed to the equator, and the anaphase where chromo-
somes are segregated towards the spindle poles to generate
two new daughter cells [2]. To perform this highly dynamic
* Corresponding author. Tel.: þ20 5523 08021; fax: þ20 5523 03266.
E-mail address: kametwally@hotmail.com (K. Metwally).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.10.008

K. Metwally et al. / European Journal of Medicinal Chemistry 42 (2007) 344e350
345
target in anticancer drug development. However, development
of resistance and neurological, hematological, and other side
effects significantly detracts from the therapeutic effectiveness
of the currently available agents [12e14]. These findings
sparked a worldwide search for new classes of drugs acting by
the same mechanism yet enjoying better pharmacological
profile.
In the past two decades, there has been a growing interest in
2-phenyl-4-quinolones (A), the aza bioisosteric analogues of
flavones (B), as potent cytotoxic antimitotic agents interacting
with microtubules [15e21]. Likewise, natural flavonoids have
been reported to display potent cytotoxic activity through inhi-
bition of tubulin polymerization [22,23]. Meanwhile, 2-phenyl-
4-anilinoquinolines (C) and related condensed heterocyclic
compounds were extensively reported to have appreciable cy-
totoxic activities [24e29]. It was generally concluded that
not only the coplanar polycyclic core but also peripheral sub-
stituents have considerable impact on cytotoxic activity. In
the same context, we report herein the synthesis and the in vitro
cytotoxic activity of pyrimidio[4,5-c]quinolin-1(2H )-ones
(D) as a new class of antimitotic agents (Chart 1). The mecha-
nism of action of the new agents was explored through cell cy-
cle analysis performed by a flow cytometric assay and a tubulin
polymerization assay.
hydrochlorides (1 and 2) under basic conditions [30,31]. Sub-
sequently, the acids were cyclized with acetic anhydride under
reflux conditions to afford the key lactones 3-methyl-5-aryl-
1H-[1,3]oxazino[4,5-c]quinolin-1-ones (7 and 8) in good
yields. Alternatively, reacting the starting acids (3e6) with
benzoyl chloride or 4-chlorobenzoyl chloride at room temper-
ature followed by cyclization of the resulting intermediate
using acetic anhydride gave the lactones (9e15) in moderate
overall yields. Finally, the requisite pyrimido[4,5-c]quinolin-
1(2H )-ones (16e26) were obtained via hydrazinolysis of the
intermediate lactones (7e15) through heating with hydrazine
hydrate in 2-ethoxyethanol as a high boiling solvent.
Structure of the target pyrimido[4,5-c]quinolin-1(2H )-ones
(16e26) as well as the intermediate lactones (7e15) was con-
1
firmed by means of IR, H NMR, mass spectral data and mi-
croanalysis. IR spectra of (7e15) showed a characteristic
lactone carbonyl stretching frequency around 1765 cmꢀ1
.
Mass spectra revealed the appearance of isotopic peaks owing
to the presence of halogens. Unfortunately, these intermediate
1
lactones could not be characterized by H NMR spectrometry
1
due to lack of solubility in the available H NMR solvents
such as CDCl3, DMSO-d6, CD3OD, CD3CD2OD and
1
CD3COCD3. In the H NMR spectra of the target compounds
(16e26), a singlet peak in the region d 6.0e6.3 integrating for
two protons was characteristic of the NH2 group. The methyl
protons of compounds (16), (17) and (22) appeared as a sharp
singlet at d 2.7. Aromatic protons appeared at their expected
chemical shifts. All the new compounds were microanalyzed
satisfactorily for C, H, N.
2. Chemistry
Synthetic route to the target pyrimido[4,5-c]quinolin-
1(2H )-ones (16e26) is illustrated in Scheme 1. Phenacylamine
hydrochlorides (1 and 2) were prepared according to a standard
method, as reported previously [30]. The starting 3-amino-
2-arylquinoline-4-carboxylic acids (3e6) were synthesized
following a modified Pfitzinger procedure by reacting isatin
or 5-chloroisatin with 4-chloro- or 4-bromophenacylamine
3. Results and discussion
The widely accepted MTT assay was used for the in vitro
cytotoxic evaluation of the target compounds (16e26) against
O
O
R1
R1
O
N
H
R2
R2
(A)
(B)
NH2
N
R2
R3
O
HN
R1
N
R1
N
N
R2
(C)
(D)
Chart 1.

346
K. Metwally et al. / European Journal of Medicinal Chemistry 42 (2007) 344e350
COOH
NH2.HCl
O
R1
NH2
R1
a)
O
O
+
N
N
H
R2
(3-6)
(1,2)
R2
b)
NH2
N
O
R3
O
O
R3
R1
N
R1
N
c)
N
N
(16-26)
(7-15)
R2
R2
R1 = H, Cl
R2 = Cl, Br
R3 = CH3, C6H5, 4-Cl-C6H4
Scheme 1. Reagents and conditions: (a) NaOH, EtOH, H2O, THF, reflux, 30 min. (b) For (7) and (8): (CH3CO)2O, reflux 5 h; for (9e15): C6H5COCl or 4-Cle
C6H4COCl, pyridine, room temperature, 12 h followed by (CH3CO)2O, reflux 5 h. (c) NH2NH2$H2O, 2-ethoxyethanol, reflux, 24 h.
a panel of three human cancer cell lines, i.e., lung fibrosar-
coma HT-1080, colon adenocarcinoma HT-29, and breast car-
cinoma MDA-MB-231. Close inspection of cytotoxic data
revealed that a 9-chloro substituent is the critical determinant
of cytotoxic activity in the 3-aryl series (Table 1). Evidently,
removal of the 9-chloro of (23), (24), (25), and (26) to give
(18), (19), (20), and (21), respectively, resulted in a remarkable
decrease of cytotoxicity against the three cell lines used in the
assay. In general, compounds (22e26) were more potent than
their 9-unsubstituted counterparts. Moreover, introduction of
a chloro at the para position of the 3-phenyl ring enhances cy-
totoxic activity, particularly in the 9-unsubstituted type of
compounds as evidenced by the appreciably higher potency
of (20) and (21) compared to (18) and (19), respectively. On
the other hand, the type of halogen at the 5-phenyl ring
appeared to have variable influence on cytotoxic activity.
Within this set of compounds, it was found that compounds
of the 3-aryl series are generally more potent than their 3-
methyl analogues. Compound (16) which has neither 9-chloro
nor 3-aryl was found to be devoid of cytotoxicity against the
three cell lines tested. In general, the tested compounds were
more active against fibrosarcoma cells HT-1080 with breast
carcinoma MDA-MB-231 cells being the least sensitive.
The mechanistic aspects of the synthesized compounds
were investigated through flow cytometry and tubulin poly-
merization assay. To this end, after incubation of human fibro-
sarcoma cells for 36 h with the most active compound (25) or
with control compounds (colcemide and paclitaxel), polymer-
ized tubulin was separated from soluble tubulin and the two
fractions were assessed by Western analysis, as described in
Table 1
In vitro cytotoxicity of the target pyrimido[4,5-c]quinolin-1(2H )-ones using the MTT assay (IC50 values in mM)a
Compound
R1
R2
R3
Cell line
HT-1080
HT-29
MDA-MB-231
16
17
18
19
20
21
22
23
24
25
26
H
Cl
Br
Cl
Br
Cl
Br
Cl
Cl
Br
Cl
Br
CH3
CH3
>100
>100
77.8 (ꢁ11.1)
>100
>100
>100
H
44.8 (ꢁ6.2)
23.0 (ꢁ0.8)
47.4 (ꢁ3.7)
6.84 (ꢁ1.60)
10.8 (ꢁ0.8)
34.3 (ꢁ7.2)
2.90 (ꢁ0.58)
1.48 (ꢁ0.06)
1.23 (ꢁ0.22)
1.84 (ꢁ0.40)
H
C6H5
C6H5
>100
>100
H
89.9 (ꢁ5.0)
18.1 (ꢁ7.1)
23.2 (ꢁ9.1)
52.0 (ꢁ24.0)
4.68 (ꢁ0.79)
2.14 (ꢁ0.62)
1.97 (ꢁ0.07)
2.82 (ꢁ0.28)
H
4-CleC6H4
4-CleC6H4
CH3
55.3 (ꢁ21.6)
34.2 (ꢁ20.1)
>100
H
Cl
Cl
Cl
Cl
Cl
C6H5
C6H5
15.8 (ꢁ5.1)
13.7 (ꢁ4.4)
1.82 (ꢁ0.53)
2.89 (ꢁ0.27)
4-CleC6H4
4-CleC6H4
Doxorubicin
0.022 (ꢁ0.08)
0.35 (ꢁ0.13)
0.030 (ꢁ0.020)
a
The results represent the mean (ꢁstandard deviation) of three independent experiments and are expressed as IC50, the concentration that reduced by 50% the
optical density of treated cells with respect to untreated controls.

K. Metwally et al. / European Journal of Medicinal Chemistry 42 (2007) 344e350
347
Table 2
Cell cycle phase distribution (%)a
detail in Section 4. In this assay, colcemide, which was used as
a known tubulin polymerization inhibitor, reduced the amount
of polymerized tubulin detected (Fig. 1), while paclitaxel,
known to stabilize microtubule assembly, increased the frac-
tion of polymerized tubulin, in accordance with the data ap-
pearing in the literature [32]. As shown in Fig. 1, compound
(25) dose-dependently inhibited tubulin polymerization in
comparison with the untreated culture, showing a mode of ac-
tion similar to colcemide. Furthermore, cell cycle analysis us-
ing staining with the DNA binding dye propidium iodide,
followed by flow cytometry, showed that incubation with com-
pound (25) for 36 h caused arrest of HT-1080 cells in the G2/M
phase of the cell cycle and suppression of the S-phase, in
agreement with the observed inhibition of tubulin polymeriza-
tion (Table 2). Moreover, compounds (23), (24) and (26) also
block cell proliferation by arresting the G2/M phase of the cell
cycle. Finally, the tested compounds seem to provoke cell ap-
optosis, as estimated by the appearance of a sub-diploid peak
in the DNA-content histograms (percentage of total events col-
lected by the flow cytometer: 2.21% for (23), 5.51% for (24),
7.62% for (25), 9.14% for (26) compared to 1.27% for
control).
Compound
G0/G1
S
G2/M
23
24
50.68
45.78
57.25
49.76
51.53
26.86
28.00
11.26
22.48
29.82
22.46
26.22
31.49
27.75
18.65
25
26
Control
a
One out of two similar experiments is depicted.
4.1.1. General procedure for 3-methyl-5-aryl-1H-[1,3]
oxazino[4,5-c]quinolin-1-ones (7 and 8)
A mixture of the appropriate 3-amino-2-aryl-4-quinoline-
carboxylic acid (10 mmol) and acetic anhydride (10 mL)
was heated under reflux for 5 h. The reaction mixture was
cooled and the obtained solid was filtered, washed with cold
ethanol, dried, and recrystallized from DMF/EtOH.
4.1.1.1. 5-(4-Chlorophenyl)-3-methyl-1H-[1,3]oxazino[4,5-c]
quinolin-1-one (7). Yield 79%, mp 194e195 ꢂC (DMF/
EtOH); IR (KBr, cmꢀ1): 1763 (lactone C]O). MS (m/z):
323 (Mþ, 13.3%), 251 (base peak). Anal. calcd for
C18H11ClN2O2: C, 66.99; H, 3.44; N, 8.68. Found: C, 67.16;
H, 3.67; N, 8.76.
4. Experimental protocols
4.1.1.2. 5-(4-Bromophenyl)-3-methyl-1H-[1,3]oxazino[4,5-c]
quinolin-1-one (8). Yield 74%, mp 187e190 ꢂC (DMF/
EtOH); IR (KBr, cmꢀ1): 1763 (lactone C]O). MS (m/z):
367 (Mþ, 34.7%), 295 (base peak). Anal. calcd for
C18H11BrN2O2: C, 58.88; H, 3.02; N, 7.63. Found: C, 58.75;
H, 2.83; N, 7.53.
4.1. Synthesis
Melting points were determined on a Gallenkamp melting
point apparatus and are uncorrected. IR spectra were recorded
on a Pye Unicam SP 1000 IR spectrophotometer using KBr
1
pellets. H NMR spectra were recorded on a Varian-Mercury
200 MHz spectrometer in DMSO-d6. Chemical shifts were ex-
pressed in parts per million (ppm) with tetramethylsilane
(TMS) as an internal standard. MS spectra were measured
with an HP 5995 instrument. Elemental analyses (C, H, N)
were performed at the Microanalytical Unit, Cairo University
and the National Research Centre, Cairo, Egypt. All com-
pounds were routinely checked by thin-layer chromatography
(TLC) on aluminum-backed silica gel plates. All solvents were
dried by standard methods. Compounds (1e6) were reported
previously [30].
4.1.2. General procedure for 3,5-diaryl-1H-[1,3]oxazino
[4,5-c]quinolin-1-ones (9e15)
A mixture of the appropriate 3-amino-2-aryl-4-quinoline-
carboxylic acids (3e6; 10 mmol) and benzoyl chloride or 4-
chlorobenzoyl chloride (11 mmol) in dry pyridine (20 mL)
was allowed to stir at room temperature for 12 h. The reaction
mixture was poured into ice water and the obtained solid was
filtered, washed with ethanol, and dried. The solid was sus-
pended in acetic anhydride (10 mL) and the mixture was
heated at reflux for 5 h. After cooling, the crude product was
filtered, washed with ethanol, dried, and recrystallized from
the appropriate solvent.
4.1.2.1. 5-(4-Chlorophenyl)-3-phenyl-1H-[1,3]oxazino[4,5-c]
quinolin-1-one (9). Yield 64%, mp 257e258 ꢂC (DMF/
EtOH); IR (KBr, cmꢀ1): 1766 (lactone C]O). MS (m/z):
429 (Mþ1, 16.1%), 104 (base peak). Anal. calcd for
C23H13ClN2O2: C, 71.79; H, 3.40; N, 7.28. Found: C, 71.65;
H, 3.91; N, 7.36.
4.1.2.2. 5-(4-Bromophenyl)-3-phenyl-1H-[1,3]oxazino[4,5-c]
quinolin-1-one (10). Yield 61%, mp 255e257 ꢂC (DMF/
EtOH); IR (KBr, cmꢀ1): 1766 (lactone C]O). MS (m/z):
429 (Mþ, 28.3%), 76 (base peak). Anal. calcd for
Fig. 1. Inhibition of cellular tubulin polymerization by compound (25). Cells
were treated with the indicated concentrations of compound (25) or control
compounds and tubulin polymerization was assessed by Western analysis as
described in Section 4.

348
K. Metwally et al. / European Journal of Medicinal Chemistry 42 (2007) 344e350
C23H13BrN2O2: C, 64.35; H, 3.05; N, 6.53. Found: C, 64.55;
H, 3.03; N, 6.59.
9.67e9.72 (m, 1H, Ar-H). Anal. calcd for C18H13BrN4O: C,
56.71; H, 3.44; N, 14.70. Found: C, 56.26; H, 3.44; N, 14.58.
4.1.2.3. 5-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-[1,3]oxa-
zino[4,5-c]quinolin-1-one (11). Yield 59%, mp 268e270 ꢂC
(DMF); IR (KBr, cmꢀ1): 1763 (lactone C]O). MS (m/z):
465 (Mþ1, 19.2%), 139 (base peak). Anal. calcd for
C23H12BrClN2O2: C, 59.57; H, 2.61; N, 6.04. Found: C,
59.80; H, 2.47; N, 6.14.
4.1.3.3.
2-Amino-5-(4-chlorophenyl)-3-phenylpyrimido[4,5-
c]quinolin-1(2H )-one (18). Yield 59%, mp 235e237 ꢂC
1
(DMF); H NMR (DMSO-d6) d: 6.05 (s, 2H, NH2), 7.51e
7.58 (m, 5H, Ar-H), 7.84e7.86 (m, 2H, Ar-H), 7.93e7.97
(m, 2H, Ar-H), 8.17e8.20 (m, 3H, Ar-H), 9.68e9.72 (m,
1H, Ar-H). Anal. calcd for C23H15ClN4O: C, 69.26; H, 3.79;
N, 14.05. Found: C, 69.30; H, 3.20; N, 13.91.
4.1.2.4. 3,5-Di(4-chlorophenyl)-1H-[1,3]oxazino[4,5-c]quino-
lin-1-one (12). Yield 58%, mp 269e272 ꢂC (DMF); IR
4.1.3.4.
2-Amino-5-(4-bromophenyl)-3-phenylpyrimido[4,5-
(KBr, cmꢀ1): 1766 (lactone C]O). MS (m/z): 421 (Mþ2
,
c]quinolin-1(2H )-one (19). Yield 52%, mp 220e221 ꢂC
(DMF/EtOH); 1H NMR (DMSO-d6) d: 6.11 (s, 2H, NH2),
7.55e7.59 (m, 3H, Ar-H), 7.74 (d, 2H, J ¼ 8.79 Hz, Ar-H),
7.88e7.93 (m, 2H, Ar-H), 7.97e8.02 (m, 2H, Ar-H), 8.11
(d, 2H, Ar-H), 8.21e8.26 (m, 1H, Ar-H), 9.73e9.77 (m, 1H,
Ar-H). Anal. calcd for C23H15BrN4O: C, 62.32; H, 3.41; N,
12.64. Found: C, 62.01; H, 3.58; N, 12.41.
13.1%), 138 (base peak). Anal. calcd for C23H12Cl2N2O2: C,
65.89; H, 2.88; N, 6.68. Found: C, 65.82; H, 2.73; N, 6.69.
4.1.2.5. 5-(4-Bromophenyl)-9-chloro-3-phenyl-1H-[1,3]oxa-
zino[4,5-c]quinolin-1-one (13). Yield 63%, mp 298e300 ꢂC
(DMF/EtOH); IR (KBr, cmꢀ1): 1766 (lactone C]O). MS
(m/z): 464 (Mþ, 19.0%), 76 (base peak). Anal. calcd for
C23H12BrClN2O2: C, 59.57; H, 2.61; N, 6.04. Found: C,
59.56; H, 2.19; N, 6.15.
4.1.3.5. 2-Amino-3,5-di(4-chlorophenyl)pyrimido[4,5-c]quino-
lin-1(2H )-one (20). Yield 57%, mp 280e282 ꢂC (DMF/
EtOH); H NMR (DMSO-d6) d: 6.03 (s, 2H, NH2), 7.54e
1
4.1.2.6. 5-(4-Bromophenyl)-9-chloro-3-(4-chlorophenyl)-1H-
[1,3]oxazino[4,5-c]quinolin-1-one (14). Yield 62%, mp >
300 ꢂC (DMF/EtOH); IR (KBr, cmꢀ1): 1766 (lactone C]O).
MS (m/z): 499 (Mþ, 22.4%), 138 (base peak). Anal. calcd
for C23H11BrCl2N2O2: C, 55.45; H, 2.23; N, 5.62. Found: C,
55.83; H, 1.56; N, 5.73.
7.60 (m, 4H, Ar-H), 7.82e7.88 (m, 2H, Ar-H), 7.95e7.98
(m, 2H, Ar-H), 8.11e8.19 (m, 3H, Ar-H), 9.66e9.69 (m,
1H, Ar-H). Anal. calcd for C23H14Cl2N4O: C, 63.79; H,
3.26; N, 12.93. Found: C, 63.83; H, 3.51; N, 12.66.
4.1.3.6. 2-Amino-5-(4-bromophenyl)-3-(4-chlorophenyl)pyri-
mido[4,5-c]quinolin-1(2H )-one (21). Yield 58%, mp 293e
1
4.1.2.7. 9-Chloro-3,5-di(4-chlorophenyl)-1H-[1,3]oxazino[4,
294 ꢂC (DMF/EtOH); H NMR (DMSO-d6) d: 6.04 (s, 2H,
5-c]quinolin-1-one (15). Yield 64%, mp > 300 ꢂC (DMF);
NH2), 7.58 (d, 2H, J ¼ 4.59 Hz, Ar-H), 7.69 (d, 2H,
J ¼ 5.39 Hz, Ar-H), 7.84e7.88 (m, 2H, Ar-H), 7.96 (d, 2H,
J ¼ 5.39 Hz, Ar-H), 8.06 (d, 2H, J ¼ 5.39 Hz, Ar-H), 8.17e
8.20 (m, 1H, Ar-H), 9.68e9.71 (m, 1H, Ar-H). Anal. calcd
for C23H14BrClN4O: C, 57.82; H, 2.95; N, 11.73. Found: C,
58.03; H, 2.45; N, 11.83.
IR (KBr, cmꢀ1): 1766 (lactone C]O). MS (m/z): 456 (Mþ2
,
22.4%), 139 (base peak). Anal. calcd for C23H11Cl3N2O2: C,
60.89; H, 2.44; N, 6.17. Found: C, 61.19; H, 2.17; N, 6.30.
4.1.3. General procedure for 2-amino-5-aryl-
pyrimidino[4,5-c]quinolin-1(2H )-ones (16e26)
A mixture of the appropriate 1H-[1,3]oxazino[4,5-c]quino-
lin-1-one (7e15; 10 mmol) and hydrazine hydrate (85%;
4 mL) in 2-ethoxyethanol (20 mL) was heated at reflux for
24 h. The precipitated solid was filtered, washed with ethanol,
dried, and recrystallized from the appropriate solvent.
4.1.3.7. 2-Amino-9-chloro-5-(4-chlorophenyl)-3-methylpyri-
mido[4,5-c]quinolin-1(2H )-one (22). Yield 54%, mp 264e
1
265 ꢂC (DMF/EtOH); H NMR (DMSO-d6) d: 2.66 (s, 3H,
CH3), 6.23 (s, 2H, NH2), 7.55e7.59 (m, 2H, Ar-H), 7.78e
8.08 (m, 1H, Ar-H), 8.08e8.11 (m, 3H, Ar-H), 9.60 (d, 1H,
J ¼ 1.59 Hz, Ar-H). Anal. calcd for C18H12Cl2N4O: C,
58.24; H, 3.26; N, 15.09. Found: C, 57.92; H, 3.39; N, 14.88.
4.1.3.1.
2-Amino-5-(4-chlorophenyl)-3-methylpyrimido[4,5-
c]quinolin-1(2H )-one (16). Yield 56%, mp 291e293 ꢂC
1
(DMF); H NMR (DMSO-d6) d: 2.71 (s, 3H, CH3), 6.27 (s,
4.1.3.8. 2-Amino-9-chloro-5-(4-chlorophenyl)-3-phenylpyri-
2H, NH2), 7.61e7.65 (m, 2H, Ar-H), 7.85e7.89 (m, 2H, Ar-
H), 8.13e8.22 (m, 3H, Ar-H), 9.68e9.73 (m, 1H, Ar-H).
Anal. calcd for C18H13ClN4O: C, 64.20; H, 3.89; N, 16.64.
Found: C, 63.76; H, 3.89; N, 16.49.
mido[4,5-c]quinolin-1(2H )-one (23). Yield 61%, mp 281e
1
282 ꢂC (DMF/EtOH); H NMR (DMSO-d6) d: 6.14 (s, 2H,
NH2), 7.56e7.64 (m, 5H, Ar-H), 7.93e8.02 (m, 3H, Ar-H),
8.16e8.24 (m, 3H, Ar-H), 9.73 (d, 1H, J ¼ 1.99 Hz, Ar-H).
Anal. calcd for C23H14Cl2N4O: C, 63.76; H, 3.26; N, 12.93.
Found: C, 63.46; H, 3.54; N, 12.63.
4.1.3.2.
2-Amino-5-(4-bromophenyl)-3-methylpyrimido[4,5-
c]quinolin-1(2H )-one (17). Yield 60%, mp 294e296 ꢂC
(DMF/EtOH); 1H NMR (DMSO-d6) d: 2.71 (s, 3H, CH3), 6.27
(s, 2H, NH2), 7.75e7.79 (m, 2H, Ar-H), 7.84e7.89 (m, 2H,
Ar-H), 8.06e8.10 (m, 2H, Ar-H), 8.1e8.2 (m, 1H, Ar-H),
4.1.3.9. 2-Amino-5-(4-bromophenyl)-9-chloro-3-phenylpyri-
mido[4,5-c]quinolin-1(2H )-one (24). Yield 55%, mp 269e
1
270 ꢂC (DMF/EtOH); H NMR (DMSO-d6) d: 6.12 (s, 2H,

K. Metwally et al. / European Journal of Medicinal Chemistry 42 (2007) 344e350
349
NH2), 7.54e7.57 (m, 2H, Ar-H), 7.72e7.76 (m, 2H, Ar-H),
4.2.2. Cell cycle analysis
7.86e7.92 (dd, 1H, J ¼ 2.39, 2.39 Hz, Ar-H), 7.95e7.99 (m,
2H, Ar-H), 8.06e8.12 (dd, 2H, J ¼ 1.39, 1.79 Hz, Ar-H),
8.17 (d, 2H, J ¼ 8.79 Hz, Ar-H), 9.71 (d, 1H, J ¼ 2.39 Hz,
Ar-H). Anal. calcd for C23H14BrClN4O: C, 57.82; H, 2.95;
N, 11.73. Found: C, 57.30; H, 2.95; N, 11.66.
Cell cycle analysis was performed following incubation of
exponentially growing HT-1080 cells with the test substances
(5 mM) for 36 h. Treated cultures were then trypsinized,
washed in PBS, fixed in 50% ethanol, and stained with an RN-
ase-containing propidium iodide solution [34]. DNA content
was analyzed on a FACS Calibur (Becton Dickinson, San
Jose, CA, USA) flow cytometer using the ModFit software
(Verity Software House, Topsham, ME, USA).
4.1.3.10.
2-Amino-9-chloro-3,5-di(4-chlorophenyl)pyrimido
[4,5-c]quinolin-1(2H )-one (25). Yield 50%, mp 285e288 ꢂC
1
(DMF); H NMR (DMSO-d6) d: 6.07 (s, 2H, NH2), 7.54e
7.60 (m, 4H, Ar-H), 7.84 (d, 1H, J ¼ 1.59 Hz, Ar-H), 7.9e
8.0 (d, 2H, J ¼ 5.79 Hz, Ar-H), 8.09e8.15 (m, 3H, Ar-H),
9.63 (d, 1H, J ¼ 1.59 Hz, Ar-H). Anal. calcd for
C23H13Cl3N4O: C, 59.06; H, 2.80; N, 11.98. Found: C,
58.84; H, 2.96; N, 11.66.
4.2.3. In vitro inhibition of cellular tubulin polymerization
Exponentially growing HT-1080 cells (approximately
2 ꢃ 106 cells/dish) were treated with 5, 10, or 25 mM com-
pound (25) for 36 h. Negative control cultures were treated
with the corresponding concentrations of the vehicle
(DMSO) while positive controls with either 0.5 mM colcemide
(Biochrom) or 0.1 mM paclitaxel (GenexolÒ, Samyang Genex
Corporation, Daejeon, Korea e kindly provided by Dr. M.
Sagnou) for 18 h. Cultures were then trypsinized, washed in
PBS, and polymerized cellular tubulin was separated from sol-
uble tubulin dimers as described by Blagosklonny et al. [35].
Briefly, cells were lysed in a buffer containing 20 mM Trise
HCl (pH 6.8), 1 mM MgCl2, 2 mM EGTA, 0.5% Nonidet P-
40 and protease- and phosphatase-inhibitor cocktails (Sigma).
Supernatants were collected after centrifugation at 13,000 ꢃ g
for 10 min. The pellets were dissolved by heating for 5 min at
95 ꢂC in a SDS-polyacrylamide gel electrophoresis (PAGE)
sampling buffer and subjected to electrophoresis on a 10%
SDS-PAGE gel. After electrophoresis, the proteins were trans-
ferred to a PVDF membrane, and subjected to Western analy-
sis using an anti-a-tubulin monoclonal antibody and
peroxidase-conjugated anti-mouse antibody (Sigma). Detec-
tion of immunoreactive bands was performed by chemilumi-
nescence (ECL kit, Amersham Biosciences).
4.1.3.11. 2-Amino-5-(4-bromophenyl)-9-chloro-3-(4-chlorophe
nyl)pyrimido[4,5-c]quinolin-1(2H )-one (26). Yield 49%, mp
1
273e276 ꢂC (DMF/EtOH); H NMR (DMSO-d6) d: 6.07 (s,
2H, NH2), 7.58 (d, 2H, J ¼ 5.59 Hz, Ar-H), 7.69 (d, 2H,
J ¼ 3.79 Hz, Ar-H), 7.78 (d, 1H, Ar-H), 7.96 (d, 2H,
J ¼ 5.79 Hz, Ar-H), 8.04 (d, 2H, J ¼ 5.59 Hz, Ar-H), 8.15
(d, 1H, J ¼ 5.79 Hz, Ar-H), 9.67 (d, 1H, J ¼ 1.39 Hz, Ar-H).
Anal. calcd for C23H13BrCl2N4O: C, 53.94; H, 2.56; N,
10.94. Found: C, 53.67; H, 2.74; N, 10.67.
4.2. Biology
4.2.1. In vitro cytotoxicity
The new compounds were tested for their cytotoxic activity
on the following human solid tumor cell lines: lung fibrosar-
coma HT-1080 (American Type Culture Collection, Rockville,
MD), mammary adenocarcinoma MDA-MB-231 (ATCC) and
colorectal adenocarcinoma HT-29 (European Collection of
Cell Cultures, Salisbury, U.K.). All cells were routinely cul-
tured in Dulbecco’s minimal essential medium (DMEM;
Gibco BRL, Paisley, U.K.) supplemented with penicillin
(100 U/mL), streptomycin (100 mg/mL), and 10% fetal bovine
serum (media and antibiotics from Biochrom KG, Berlin, Ger-
many) in an environment of 5% CO2, 85% humidity, and
37 ꢂC, and they were subcultured using a trypsin 0.25%e
EDTA 0.02% solution. The cytotoxicity assay was performed
by a modification of the MTT method [33,34]. Briefly, the
cells were plated at a density of approximately 5000 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 with a micro-
plate reader at a wavelength of 550 nm (reference wavelength
690 nm). Doxorubicin hydrochloride was included in the ex-
periments 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.
References
[1] L. Amos, A. Klug, J. Cell Sci. 14 (1974) 523e549.
[2] J. McIntosh, E. Grishchunk, R. West, Annu. Rev. Cell Dev. Biol. 18
(2002) 193e219.
[3] A. Desai, T. Mitchison, Annu. Rev. Cell Dev. Biol. 13 (1997) 83e117.
[4] L. Wilson, M. Jordan, Chem. Biol. 2 (1995) 569e573.
[5] M. Jordan, K. Wendell, S. Gardiner, W. Derry, H. Copp, L. Wilson, Can-
cer Res. 56 (1996) 816e825.
[6] J. Chen, C. Yang, M. Cammer, S. Horwitz, Cancer Res. 63 (2003)
7891e7899.
[7] J. Zhou, D. Panda, J. Landen, L. Wilson, H. Joshi, J. Biol. Chem. 277
(2002) 17200e17208.
[8] A. Yvon, P. Wadsworth, M. Jordan, Mol. Biol. Cell 10 (1999) 947e959.
[9] W. Derry, L. Wilson, M. Jordan, Biochemistry 34 (1995) 2203e2211.
[10] A. Gonclaves, D. Braguer, K. Kamath, L. Martello, C. Briand,
S. Horwitz, L. Wilson, M. Jordan, Proc. Natl. Acad. Sci. U.S.A. 98
(2001) 11737e11741.
[11] J. Zhou, P. Giannakakou, Curr. Med. Chem. 5 (2005) 65e71.
[12] S. Ambudkar, S. Dey, C. Hrycyna, M. Ramachandra, I. Pastan,
M. Gottesman, Annu. Rev. Pharmacol. Toxicol. 39 (1999) 361e398.
[13] E. Rowinsky, Annu. Rev. Med. 48 (1997) 353e374.
[14] K. Goa, D. Faulds, Drugs Aging 5 (1994) 200e234.
[15] Y. Lai, L. Huang, K. Lee, Z. Xiao, K. Bastow, T. Yamori, S. Kuo, Bioorg.
Med. Chem. 13 (2005) 265e275.

350
K. Metwally et al. / European Journal of Medicinal Chemistry 42 (2007) 344e350
[16] S. Kuo, H. Lee, J. Juang, Y. Lin, T. Wu, J. Chang, D. Lednicer, K. Paull,
C. Lin, E. Hamel, K. Lee, J. Med. Chem. 36 (1993) 1146e1156.
[17] L. Li, H. Wang, S. Kuo, T. Wu, A. Mauger, C. Lin, E. Hamel, K. Lee,
J. Med. Chem. 37 (1994) 3400.
[26] W. Peczynska-Czoch, F. Pognan, L. Kaczmarek, J. Boratynski, J. Med.
Chem. 37 (1994) 3503e3510.
[27] Y. Chen, H. Hung, C. Lu, K. Li, C. Tzeng, Bioorg. Med. Chem. 12 (2004)
6539e6546.
[18] M. Ferlin, G. Chiarelotto, V. Gasparotto, L. Via, V. Pezzi, L. Barzon,
G. Palu, I. Castagliuolo, J. Med. Chem. 48 (2005) 3417e3427.
[19] L. Li, H. Wang, S. Kuo, T. Wu, D. Lednicer, C. Lin, E. Hamel, K. Lee,
J. Med. Chem. 37 (1994) 1126e1135.
[20] Y. Xia, Z. Yang, P. Xia, K. Bastow, Y. Tachibana, S. Kuo, E. Hamel,
T. Hackl, K. Lee, J. Med. Chem. 41 (1998) 1155e1162.
[28] Y. Chen, C. Chung, I. Chen, P. Chen, H. Jeng, Bioorg. Med. Chem. 10
(2002) 2705e2712.
[29] S. Katoen-Chackal, M. Facompre, R. Houssin, N. Pommery, J. Goossens,
P. Colson, C. Bailly, J. Henichart, J. Med. Chem. 47 (2004) 3665e3673.
[30] J. Poletto, N. Westwood, D. Powell, N. Valley Cottage, D. Boschelli, M.
Novi, U.S. patent, 4,968,702, 1990.
[21] Y. Xia, Z. Yang, P. Xia, T. Hackl, E. Hamel, A. Mauger, J. Wu, K. Lee,
J. Med. Chem. 44 (2001) 3932e3936.
[22] K. Lee, K. Tagahara, H. Suzuki, R. Wu, M. Haruna, I. Hall, H. Huang,
K. Ito, T. Iida, J. Lai, J. Nat. Prod. 44 (1981) 830e835.
[31] W. Pfitzinger, J. Prakt. Chem. 33 (1886) 100e105.
[32] J. Chang, C. Chang, C. Kuo, L. Chen, Y. Wein, Y. Kuo, Mol. Pharmacol.
65 (2004) 77e84.
[33] F. Denizot, R. Lang, J. Immunol. Methods 89 (1986) 271e277.
[34] I. Kostakis, R. Tenta, N. Pouli, P. Marakos, A. Skaltsounis, H. Pratsinis,
D. Kletsas, Bioorg. Med. Chem. Lett. 15 (2005) 5057e5060.
[35] M. Blagosklonny, T. Schulte, P. Nguyen, E. Mimnaugh, J. Trepel,
L. Neckers, Cancer Res. 55 (1995) 4623e4626.
[23] Q. Shi, K. Chen, L. Li, J. Nat. Prod. 58 (1995) 475e482.
[24] Y. Zhao, Y. Chen, F. Chang, C. Tzeng, Eur. J. Med. Chem. 40(2005)792e797.
[25] Y. Chen, C. Huang, Z. Huang, C. Tseng, F. Chang, S. Yang, S. Lin,
C. Tzeng, Bioorg. Med. Chem. 14 (2006) 3098e3105.