Synthesis, in-vitro cytotoxicity, and a preliminary structure-activity relationship investigation of pyrimido[4,5-c]quinolin-1(2H)-ones

Article abstract of DOI:10.1002/ardp.200900281

As part of our ongoing research effort to develop new antimitotic agents based on the recently reported pyrimido[4,5-c]quinoline-1(2H)-one ring skeleton, we were interested in identifying structural elements that contribute to the cytotoxicity of this cla

Full text of DOI:10.1002/ardp.200900281

Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
465
Full Paper
Synthesis, in-vitro Cytotoxicity, and a Preliminary
Structure-Activity Relationship Investigation of
Pyrimido[4,5-c]quinolin-1(2H)-ones
Kamel Metwally1, Ashraf Khalil1, Harris Pratsinis2, and Dimitris Kletsas2
1 Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa,
Kingdom of Saudi Arabia
2 Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre of Scientific Research
‘‘Demokritos’’, Athens, Greece
As part of our ongoing research effort to develop new antimitotic agents based on the recently
reported pyrimido[4,5-c]quinoline-1(2H)-one ring skeleton, we were interested in identifying
structural elements that contribute to the cytotoxicity of this class of compounds. The effect of
several quinoline-ring substituents was examined and the new compounds were evaluated in vitro for
cytotoxicity against three human cancer cell lines namely, lung fibrosarcoma HT-1080, colon
adenocarcinoma HT-29, and breast carcinoma MDA-MB-231. Most of the compounds showed
cytotoxic activity in the low micromolar and sub-micromolar range. Structure-activity
relationship information revealed that a combination of electronic and steric factors may be
involved. Flow cytometric cell cycle analysis performed on HT-1080 cells revealed that the most
cytotoxic compounds 48, 50, 54, 59, and 63 inhibit the S-phase and arrest the cells in the G2/M phase of
the cell cycle suggesting an antimitotic action of these compounds.
Keywords: Antimitotic agents / Pyrimido[4,5-c]quinolin-1(2H)-ones / Structure-activity relationship (SAR)
Received: November 17, 2009; Accepted: February 9, 2010
DOI 10.1002/ardp.200900281
Introduction
such as vinblastine and vincristine, inhibit tubulin polymeri-
zation thereby blocking microtubule assembly and are there-
fore called ‘‘microtubule-destabilizing agents’’ [3, 4].
Whether microtubule-stabilizing or -destabilizing, the
ultimate biological consequence is the alteration of spindle
dynamics and cell-cycle arrest during mitosis [5].
Furthermore, it has been recently established that some of
these agents, particularly those binding to the colchicine
domain, act as vascular-disrupting agents through selectively
targeting microtubules of endothelial cells of the tumor-
associated microvasculature system resulting in inhibition
of blood supply to the growing tumor [6–10]. Some vascular-
disrupting agents are currently in ongoing clinical trials to
be used alone or in combination with other cancer chemo-
therapeutic agents [8, 10]. Noteworthy in this respect is that
vascular-disrupting agents are different from anti-angiogenic
agents, which inhibit tumor neovascularization rather than
affecting already established tumor blood vessels [11–13].
With these unique mechanistic aspects, antimitotic agents
comprise a good alternative to DNA-interacting drugs in
cancer chemotherapy. Although some of these agents are
Interfering with microtubule dynamics has been proved to be
a successful strategy for enriching the clinical oncology
arsenal with new chemotherapeutic drug candidates [1]. As
was the case with most major drug discoveries, lead anti-
mitotic agents, also called spindle poisons, were inspired by
nature [2]. Antimitotic agents were categorized depending
on their ability to polymerize tubulin or depolymerize
microtubules into two major classes. Agents which promote
tubulin polymerization thereby blocking microtubule disas-
sembly are referred to as ‘‘microtubule-stabilizing agents’’
and are typified by the taxoids such as paclitaxel and the
epothilones. On the other hand, agents such as colchicine,
podophyllotoxin, combretastatin A-4, and the vinca alkaloids
Correspondence: Kamel Metwally, Department of Pharmaceutical
Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa,
Kingdom of Saudi Arabia.
E-mail: kametwally@hotmail.com
Fax: þ966 3 581-7174
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466
K. Metwally et al.
Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
currently used in the treatment of certain human malignan-
cies, their narrow therapeutic window, limited bioavailabil-
ity, drug resistance, and complex syntheses or isolation
and their mechanism of action was investigated through cell
cycle analysis.
procedures significantly detract from their clinical success Chemistry
[14]. Therefore, there is a need for the development of new
antimitotic agents with improved pharmacological profile.
In this regard, we have previously reported the synthesis
and in-vitro cytotoxic activity of 2-amino-3,5,9-trisubstituted-
pyrimido[4,5-c]quinolin-1(2H)-ones (A) as a novel class of anti-
mitotic agents (Fig. 1). It was generally concluded that
the presence of a chloro-substituent at C-9 has a major
positive impact on cytotoxic activity and the presence of a
4-chlorophenyl substituent at C-3 further enhances activity.
With respect to C-5 substituents, a 4-chlorophenyl was
slightly better than a 4-bromophenyl in terms of cytotoxicity.
The most potent compounds dose-dependently inhibited
tubulin polymerization and arrested the cell cycle in the
G2/M phase [15]. In another study, we examined the effect
of replacing the 2-amino group by hydrophobic aryl residues.
It was found that the 2-aryl analogues (B) retain cytotoxicity
of their 2-amino counterparts [16]. In continuation of this
research effort, we were interested in exploring the influence
of quinoline-ring substituents on the cytotoxic activity
Novel pyrimido[4,5-c]quinolin-1(2H)-ones 46–63 were synthe-
sized as shown in the general reaction sequence depicted in
Scheme 1. The effect of various electron-withdrawing 7- and/
or 9-halo substituents was studied through synthesizing
analogues 47–52 and 56–61 whereas the synthesis of 53,
54, 62, and 63 allowed us to investigate electron-donating
9-methyl and 9-methoxy substituents. Preliminarily, the 3-
aminoquinoline-4-carboxylic acids 10–27 were obtained in
good yields following a modified Pfitzinger procedure
through reaction of isatins 1–9 with 4-chloro or 4-bromophen-
acylamine hydrochloride in the presence of sodium hydrox-
ide. Unfortunately, our attempts to prepare 3-amino-2-(4-
chlorophenyl)-6-nitroquinoline-4-carboxylic acid and 3-
amino-2-(4-bromophenyl)-6-nitroquinoline-4-carboxylic acid
starting from 5-nitroisatin under different reaction con-
ditions were unsuccessful. Treatment of the acids 10–27 with
4-chlorobenzoyl chloride in pyridine at room temperature
afforded the corresponding lactones 28–45 in 51–63% yields.
IR spectra showed the appearance of characteristic absorp-
tion bands at 1754–1776 cmꢀ1 corresponding to the lactone
carbonyl group. Unfortunately, 1H-NMR spectral analysis
could not be performed for all the lactones due to lack of
adequate solubility in the available deuterated solvents.
The desired target compounds 46–63 were synthesized
by reacting the lactones 28–45 with hydrazine hydrate in
2-ethoxyethanol under reflux conditions. 1H-NMR spectra
showed a characteristic singlet at d ¼ 6.01–6.09 ppm inte-
grating for two protons corresponding to the amino group.
All the synthesized compounds were characterized by spec-
tral data analysis that confirmed the assigned structures.
In addition, the new compounds were microanalyzed
satisfactorily for C, H, N.
through formulating
a
preliminary structure-activity
relationship for this new class of antimitotic agents. In the
present work, pyrimido[4,5-c]quinolin-1(2H)-ones having sub-
stituents with different electronic and steric characteristics
(C) were synthesized and evaluated for in-vitro cytotoxicity
B)
R2
A)
NH2
O
N
R3
O
N
CH3
R1
N
N
N
N
R2
Results and discussion
R1
Cl
C)
The antiproliferative activity of the new pyrimido[4,5-c]-
quinolin-1(2H)-ones 47, 49–54, 56, and 58–63 was determined
in vitro against three human cancer cell lines, namely, lung
fibrosarcoma HT-1080, colon adenocarcinoma HT-29, and
breast carcinoma MDA-MB-231 using the widely accepted
MTT assay. Related compounds from our previous investi-
gation [15], namely 46, 48, 55, and 57 were co-tested with
the new compounds under the same assay conditions for
comparison purposes. Doxorubicin hydrochloride was
included in the experiments as a reference cytotoxic com-
pound. The results were expressed as IC50 values which
represent the compound concentrations required to produce
NH2
N
2
O
3
1
10
7
N
4
9
8
R1
5
N
6
R2
Figure 1. Structures of 2-amino-3,5,9-trisubstituted-pyrimido[4,5-c]
quinolin-1(2H)-ones (A), 2-aryl analogues (B), and pyrimido[4,5-c]
quinolin-1(2H)-ones having substituents with different electronic and
steric characteristics (C).
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Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
Pyrimido[4,5-c]quinolin-1(2H)-ones
467
COOH
O
NH2
a
R1
R1
O
N
H
N
1-9
10-27
R2
b
Cl
Cl
NH2
O
N
O
O
c
N
N
R1
R1
N
N
46-63
R2
R2
28-45
46: R1= H, R2= Cl
47: R1= 9-F, R2= Cl
48: R1= 9-Cl, R2= Cl
49: R1= 9-Br, R2= Cl
50: R1= 9-I, R2= Cl
55: R1= H, R2= Br
56: R1= 9-F, R2= Br
57: R1= 9-Cl, R2= Br
58: R1= 9-Br, R2= Br
59: R1= 9-I, R2= Br
51: R1= 7-Cl, R2= Cl
52: R1= 7,9-di-Cl, R2= Cl
53: R1= 9-Me, R2= Cl
54: R1= 9-OMe, R2= Cl
60: R1= 7-Cl, R2= Br
61: R1= 7,9-di-Cl, R2= Br
62: R1= 9-Me, R2= Br
63: R1= 9-OMe, R2= Br
Reagents and conditions:
(a) 4-Chloro- or 4-bromophenacylamine hydrochlorides, NaOH, H2O/EtOH/THF, 85°C, then reflux;
(b) 4-chlorobenzoyl chloride, pyridine, room temp.; (c) hydrazine hydrate 99%, 2-ethoxyethanol,
reflux.
Scheme 1. Synthesis of compounds 10–63.
a 50% inhibition of cell growth after three days of incubation
compared to untreated controls (Table 1).
which demonstrated an activity intermediate between 48
and 51, indicating that the 9-position is the best position
for chloro substitution. A similar behavior was observed in
the 5-(4-bromophenyl) series. Compound 61 was more active
than 60 but exhibited lower cytotoxic activity when com-
pared to 57 against HT-1080 and MDA-MB-231 cell lines.
These initial results urged us to focus on the 9-position of
the pyrimido[4,5-c]quinolin-1(2H)-one ring. Investigating
other halogens was our next approach in the present study.
Introduction of a 9-fluoro substituent resulted in compounds
47 and 56, which were generally found to be the least active
among the 9-halo substituted analogues in both sets of
compounds against all cell lines tested. Compared to the
unsubstituted parent compounds 46 and 55, however, 9-
fluoro substitution generally appears to contribute positively
to cytotoxic activity.
Structure-activity relationships (SAR)
Since it was concluded from our previous work [15] that the 5-
(4-chlorophenyl) and 5-(4-bromophenyl) analogues showed
only a slight difference in cytotoxic potency, it seemed
relevant to conduct the present SAR study on two separate
sets of compounds: a set with a 5-(4-chlorophenyl) residue and
another with a 5-(4-bromophenyl) residue for additional con-
firmation. As might be expected, the two sets of compounds
displayed a similar pattern of structure-activity relationship
in the three cell lines. Because previously [15], a 9-chloro
substituent was found to be a critical determinant of cyto-
toxic activity, we were interested initially in examining the
influence of chloro substitution on other positions of the
ring. The 7-chloro analogue 51 was substantially less
active than the 9-chloro analogue 48 exhibiting 9- to 17-fold
reduction in cytotoxicity against the three cell lines tested.
Double chloro substitution gave the 7,9-dichloro analogue 52
Interestingly, the 9-iodo analogue 59 showed higher cyto-
toxicity as compared to the 9-chloro analogue 57 against the
three cancer cell lines. Similarly, compound 50 was nearly
as potent as 48 against colon adenocarcinoma and breast
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Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
Table 1. In-vitro cytotoxicity of the target pyrimido[4,5-c]quinolin-1(2H)-ones using the MTT assay.
Compound
R1
R2
Cell line
HT-29
HT-1080
MDA-MB-231
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Doxo
H
9-F
9-Cl
9-Br
9-I
7-Cl
7,9-di-Cl
9-Me
9-OMe
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
1.82 ꢁ 0.41
1.99 ꢁ 0.03
1.06 ꢁ 0.33
1.11 ꢁ 0.02
0.64 ꢁ 0.04
9.12 ꢁ 0.57
1.15 ꢁ 0.08
1.47 ꢁ 0.18
0.32 ꢁ 0.14
7.12 ꢁ 0.71
2.07 ꢁ 0.31
1.84 ꢁ 0.19
0.97 ꢁ 0.27
1.04 ꢁ 6.2
23.42 ꢁ 9.69
7.61 ꢁ 1.18
1.96 ꢁ 0.32
3.89 ꢁ 1.58
1.86 ꢁ 0.37
33.00 ꢁ 8.37
9.58 ꢁ 2.65
>100
10.10 ꢁ 4.18
60.41 ꢁ 24.91
6.26 ꢁ 2.49
10.76 ꢁ 4.12
2.91 ꢁ 0.57
3.92 ꢁ 1.09
24.11 ꢁ 5.58
2.99 ꢁ 0.86
>100
50.12 ꢁ 5.90
3.37 ꢁ 0.74
1.47 ꢁ 0.42
2.06 ꢁ 0.16
1.34 ꢁ 0.30
17.77 ꢁ 3.64
5.51 ꢁ 1.70
>100
2.06 ꢁ 0.21
37.27 ꢁ 6.52
4.76 ꢁ 0.98
3.99 ꢁ 0.70
3.04 ꢁ 1.07
2.32 ꢁ 0.37
10.45 ꢁ 0.33
9.93 ꢁ 1.77
>100
9-F
9-Cl
9-Br
9-I
7-Cl
8.21 ꢁ 0.22
2.43 ꢁ 0.03
0.86 ꢁ 0.14
0.49 ꢁ 0.22
0.022 ꢁ 0.08
7,9-di-Cl
9-Me
9-OMe
6.74 ꢁ 3.23
1.69 ꢁ 0.47
0.35 ꢁ 0.13
0.030 ꢁ 0.02
IC50 values in mM.
carcinoma cells and slightly more active in the lung fibro-
sarcoma cell line. In the 5-(4-chlorophenyl) series, the 9-halo
substituents contributed to cytotoxicity in the order of:
I > Cl > Br > F whereas, in the 5-(4-bromophenyl) series,
the halogens ranked differently in the three cell lines tested.
These results imply that a combination of electronic, steric,
and hydrophobic factors may be involved.
exhibiting IC50 values of 0.32 and 0.49 mM, respectively, but
showed only modest cytotoxic activity against other cell
lines tested. In terms of selectivity, the tested compounds
demonstrated generally higher selectivity to lung fibrosar-
coma HT-1080 cell line as compared to other cell lines used
in the assay with colon adenocarcinoma cells being the least
susceptible.
The effect of electron-donating groups was also studied
through synthesizing and testing compounds 53, 54, 62,
and 63. Methylation of the 9-position to give compounds 53
and 62 resulted in complete loss of activity against colon
adenocarcinoma and breast carcinoma cell lines. In the lung
fibrosarcoma screen, however, compound 53 was almost
equipotent to the unsubstituted parent compound 46, while
compound 62 displayed about 8-fold higher cytotoxicity as
compared to its unsubstituted counterpart 55. The 9-methoxy
analogues 54 and 63 were the most potent among the
compounds tested against the lung fibrosarcoma cell line
Effect on cell-cycle progression
Compounds that had the highest activity in cytotoxicity
assays, namely, 48, 50, 54, 59, and 63 were selected for cell
cycle studies in human lung fibrosarcoma HT-1080 cells
through flow cytometry after staining with propidium io-
dide. As shown in Table 2, all the compounds tested inhibited
the S-phase and caused a significant accumulation of cells at
the G2/M phase of the cell cycle relative to the untreated
control, which is in agreement with the microtubule-
disrupting effect of 48 shown previously [15].
Table 2. Cell-cycle phase distribution (%). §
Conclusion
Compound G0/G1
S
G2/M
We have formulated
a preliminary structure-activity
relationship for pyrimido[4,5-c]quinoline-1(2H)-ones recently
identified as cytotoxic antimitotic agents. Several
substituents with different electronic and steric properties
at different positions of the quinoline ring were investigated
for their contribution to cytotoxicity of this class of
compounds. Among the substituents examined, chloro,
iodo, and methoxy groups at C-9 were generally shown
48
50
54
59
63
Control
44.0
42.3
48.8
44.7
45.4
45.0
10.2
15.7
17.2
16.7
5.7
45.8
42.0
34.1
38.7
49.0
27.6
27.4
§
One out of two similar experiments is depicted.
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Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
Pyrimido[4,5-c]quinolin-1(2H)-ones
469
to give the most potent cytotoxic compounds. In the three
cell lines tested, substantially lower activity was displayed
by 7-chloro analogues as compared to their 9-chloro
counterparts. Interestingly, the 9-methyl analogues exhibited
remarkable selectivity for HT-1080 cells and were devoid of
activity against HT-29 and MDA-MB-231 cells. Cell cycle studies
indicated that the cytotoxic effects of these compounds are
most probably produced through disruption of mitosis.
3-Amino-2-(4-chlorophenyl)-6-methylquinoline-4-
carboxylic acid 17
Yield: 75%, m. p.: 241–2438C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
2.46 (s, 3H, CH3), 7.24–7.27 (dd, J ¼ 1.55, 1.34 Hz, 1H, Ar-H), 7.58–
7.61 (m, 2H, Ar-H), 7.68–7.74 (m, 3H, Ar-H), 8.13 (s, 1H, Ar-H). Anal.
calcd. for C17H13ClN2O2: C, 65.29; H, 4.19; N, 8.96. Found: C,
65.04; H, 4.02; N, 8.68.
3-Amino-2-(4-chlorophenyl)-6-methoxyquinoline-4-
carboxylic acid 18
Yield: 72%, m. p.: 234–2358C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
3.86 (s, 3H, OCH3), 7.06–7.10 (tt, J ¼ 4.67, 4.64 Hz, 1H, Ar-H),
7.57–7.61 (m, 2H, Ar-H), 7.67–7.70 (m, 2H, Ar-H), 7.73–7.77 (dd,
J ¼ 1.74, 1.75 Hz, 1H, Ar-H), 8.85–8.86 (t, J ¼ 4.36 Hz, 1H, Ar-H).
Anal. calcd. for C17H13ClN2O3: C, 62.11; H, 3.99; N, 8.52. Found: C,
62.19; H, 3.70; N, 8.16.
Experimental
Chemistry
Melting points were determined on a Barnstead Electrothermal
9100 melting point apparatus (Barnstead, Dubuque IA, USA) and
are uncorrected. IR spectra were recorded on a Shimadzu FTIR-
8400S spectrophotometer (Shimadzu, Tokyo, Japan) as KBr pel-
lets. 1H-NMR spectra were recorded at 300 MHz on a Varian-
Mercury 300BB spectrometer (Varian, Palo Alto, CA, USA) in
DMSO-d6. Chemical shifts were reported as parts per million
(ppm) downfield from tetramethylsilane (TMS), and coupling
constants (J) are given in Hertz (Hz). Elemental analyses (C, H,
N) were performed at the Microanalytical Unit, Cairo University,
Cairo, Egypt. All compounds were routinely checked by thin-
layer chromatography (TLC) on aluminum-backed silica gel
plates. All solvents were dried by standard methods.
Compounds 10–12, 15, 16, 19–21, 24, 25, 28, 30, 37, 39, 46, 48,
55, and 57 were reported previously [15, 17].
3-Amino-6-bromo-2-(4-bromophenyl)quinoline-4-
carboxylic acid 22
Yield: 73%, m. p.: 241–2448C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
7.51–7.52 (tt, J ¼ 3.69, 3.65 Hz, 1H, Ar-H), 7.61–7.64 (m, 2H, Ar-H),
7.74–7.78 (m, 3H, Ar-H), 8.67–8.68 (t, J ¼ 3.36 Hz, 1H, Ar-H). Anal.
calcd. for C16H10Br2N2O2: C, 45.53; H, 2.39; N, 6.64. Found: C,
45.38; H, 2.55; N, 6.53.
3-Amino-2-(4-bromophenyl)-6-iodoquinoline-4-carboxylic
acid 23
Yield: 72%, m. p.: 264–2668C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
7.57–7.70 (complex m, 4H, Ar-H), 7.73–7.77 (dd, J ¼ 2.10, 2.06 Hz,
2H, Ar-H), 8.85–8.86 (t, J ¼ 3.79 Hz, 1H, Ar-H). Anal. calcd.
for C16H10BrIN2O2: C, 40.97; H, 2.15; N, 5.97. Found: C, 40.78;
H, 1.99; N, 6.03.
General procedure for 3-amino-2-(4-halophenyl)-6-
substituted-quinoline-4-carboxylic acids 10–27
To a solution of the appropriate isatin (10 mmol) and sodium
hydroxide (80 mmol) in water (10 mL) at 858C a solution of the
appropriate phenacylamine hydrochloride (14 mmol) in a water/
ethanol/THF mixture (25 mL:25 mL:10 mL was added) in a drop-
wise manner over a period of 2 h and the mixture was then
heated at reflux for additional 30 min. After cooling, the reac-
tion mixture was concentrated under reduced pressure and
filtered through celite. The filtrate was acidified with acetic acid
and the solid obtained was filtered, washed with water, and
dried. The crude product was purified by recrystallization from
ethanol/ethyl acetate.
3-Amino-2-(4-bromophenyl)-6-methylquinoline-4-
carboxylic acid 26
Yield: 74%, m. p.: 245–2478C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
2.45 (s, 3H, CH3), 7.24–7.27 (dd, J ¼ 1.80, 1.77 Hz, 1H, Ar-H),
7.60–7.65 (m, 2H, Ar-H), 7.71–7.77 (m, 3H, Ar-H), 8.12 (s, 1H,
Ar-H). Anal. calcd. for C17H13BrN2O2: C, 57.16; H, 3.67; N, 7.84.
Found: C, 57.30; H, 3.52; N, 7.65.
3-Amino-2-(4-bromophenyl)-6-methoxyquinoline-4-
carboxylic acid 27
Yield: 70%, m. p.: 240–2418C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
3.85 (s, 3H, OCH3), 7.06–7.10 (dd, J ¼ 2.72, 2.70 Hz, 1H, Ar-H),
7.59–7.63 (m, 2H, Ar-H), 7.71–7.76 (m, 3H, Ar-H), 8.85–8.86
(d, J ¼ 2.73 Hz, 1H, Ar-H). Anal. calcd. for C17H13BrN2O3: C,
54.71; H, 3.51; N, 7.51. Found: C, 54.52; H, 3.33; N, 7.27.
3-Amino-6-bromo-2-(4-chlorophenyl)quinoline-4-
carboxylic acid 13
Yield: 76%, m. p.: 230–2338C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
7.51–7.55 (dd, J ¼ 2.21, 2.20 Hz, 1H, Ar-H), 7.59–7.63 (m, 2H,
Ar-H), 7.68–7.71 (m, 2H, Ar-H), 7.75–7.78 (d, J ¼ 8.76 Hz, 1H,
Ar-H), 8.66–8.67 (d, J ¼ 2.14 Hz, 1H, Ar-H). Anal. calcd. for
C16H10BrClN2O2: C, 50.89; H, 2.67; N, 7.42. Found: C, 50.67; H,
2.72; N, 7.32.
General procedure for 3-(4-chlorophenyl)-5-(4-chloro- or 4-
bromophenyl)-1H-[1,3]oxazino[4,5-c]quinolin-1-ones 28–45
To an ice-cooled solution of the appropriate 3-amino-2-aryl-4-
quinolinecarboxylic acid (10–27; 10 mmol) in dry pyridine
(20 mL) 4-chlorobenzoyl chloride (25 mmol) was added in a drop-
wise manner. After addition was completed, the mixture was
allowed to stir at room temperature for 36 h. The reaction
mixture was poured into ice water and the obtained solid was
filtered, washed with aqueous sodium bicarbonate, water then
3-Amino-2-(4-chlorophenyl)-6-iodoquinoline-4-carboxylic
acid 14
Yield: 71%, m. p.: 255–2588C (EtOH/EtOAc); 1H-NMR (DMSO-d6) d:
7.58–7.62 (m, 3H, Ar-H), 7.66–7.71 (m, 3H, Ar-H), 8.85–8.86 (d,
J ¼ 1.85 Hz, 1H, Ar-H). Anal. calcd. for C16H10ClIN2O2: C, 45.26; H,
2.37; N, 6.60. Found: C, 45.67; H, 2.51; N, 6.34.
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K. Metwally et al.
Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
ethanol, and dried. The crude product was purified by recrystal-
lization from the appropriate solvent.
9-Iodo-3-(4-chlorophenyl)-5-(4-bromophenyl)-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 41
Yield: 59%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1767 (lactone
–
C O). Anal. calcd. for C23H11BrClIN2O2: C, 46.85; H, 1.88; N, 4.75.
9-Fluoro-3,5-di(4-chlorophenyl)-1H-[1,3]oxazino[4,5-c]-
quinolin-1-one 29
–
Found: C, 47.00; H, 1.97; N, 5.00.
Yield: 62%, m. p.: 279–2828C (DMF); IR (KBr, cmꢀ1): 1772 (lactone
–
C O). Anal. calcd. for C23H11Cl2FN2O2: C, 63.18; H, 2.54; N, 6.41.
5-(4-Bromophenyl)-7-chloro-3-(4-chlorophenyl)-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 42
–
Found: C, 62.90; H, 2.85; N, 6.15.
Yield: 61%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1772 (lactone
–
C O). Anal. calcd. for C23H11BrCl2N2O2: C, 55.45; H, 2.23; N, 5.62.
–
Found: C, 55.52; H, 2.15; N, 5.79.
9-Bromo-3,5-di(4-chlorophenyl)-1H-[1,3]oxazino[4,5-c]-
quinolin-1-one 31
Yield: 53%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1767 (lactone
–
C O). Anal. calcd. for C23H11BrCl2N2O2: C, 55.45; H, 2.23; N, 5.62.
5-(4-Bromophenyl)-3-(4-chlorophenyl)-7,9-dichloro-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 43
–
Found: C, 55.71; H, 2.50; N, 5.80.
Yield: 58%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1771 (lactone
–
C O). Anal. calcd. for C23H10BrCl3N2O2: C, 51.87; H, 1.89; N, 5.26.
–
Found: C, 51.99; H, 2.10; N, 5.46.
3,5-Di(4-chlorophenyl)-9-iodo-1H-[1,3]oxazino[4,5-
c]quinolin-1-one 32
Yield: 51%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1770 (lactone
5-(4-Bromophenyl)-3-(4-chlorophenyl)-9-methyl-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 44
–
C O). Anal. calcd. for C23H11Cl2IN2O2: C, 50.67; H, 2.03; N, 5.14.
–
Found: C, 50.80; H, 2.35; N, 5.29.
Yield: 55%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1754 (lactone
–
C O). Anal. calcd. for C24H14BrClN2O2: C, 60.34; H, 2.95; N, 5.86.
–
Found: C, 60.62; H, 2.89; N, 6.12.
7-Chloro-3,5-di(4-chlorophenyl)-1H-[1,3]oxazino[4,5-
c]quinolin-1-one 33
Yield: 57%, m. p.: 298–3008C (DMF); IR (KBr, cmꢀ1): 1776 (lactone
5-(4-Bromophenyl)-9-methoxy-3-(4-chlorophenyl)-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 45
–
C O). Anal. calcd. for C23H11Cl3N2O2: C, 60.89; H, 2.44; N, 6.17.
–
Found: C, 60.69; H, 2.37; N, 6.34.
Yield: 56%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1759 (lactone
–
C O). Anal. calcd. for C24H14BrClN2O3: C, 58.38; H, 2.86; N, 5.67.
–
Found: C, 58.01; H, 2.68; N, 5.83.
7,9-Dichloro-3,5-di(4-chlorophenyl)-1H-[1,3]oxazino[4,5-c]-
quinolin-1-one 34
Yield: 55%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1769 (lactone
General procedure for 2-amino-3-(4-chlorophenyl)-5-(4-
chloro- or 4-bromophenyl)pyrimido[4,5-c]quinolin-1(2H)-
ones 46–63
–
C O). Anal. calcd. for C23H10Cl4N2O2: C, 56.59; H, 2.06; N, 5.74.
–
Found: C, 56.39; H, 2.00; N, 5.94.
Hydrazine hydrate (99%, 1 mL) was added to a suspension of the
appropriate lactone (28–45; 10 mmol) in 2-ethoxyethanol
(10 mL) 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.
3,5-Di(4-chlorophenyl)-9-methyl-1H-[1,3]oxazino[4,5-c]-
quinolin-1-one 35
Yield: 51%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1771 (lactone
–
C O). Anal. calcd. for C24H14Cl2N2O2: C, 66.53; H, 3.26; N, 6.47.
–
Found: C, 66.84; H, 2.86; N, 6.67.
2-Amino-3,5-di(4-chlorophenyl)-9-fluoropyrimido[4,5-
c]quinolin-1(2H)-one 47
3,5-Di(4-chlorophenyl)-9-methoxy-1H-[1,3]oxazino[4,5-c]-
quinolin-1-one 36
Yield: 52%, m. p.: 274–2778C (DMF); IR (KBr, cmꢀ1): 3315, 3218
Yield: 54%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1755 (lactone
(NH ), 1674 (C O); 1H-NMR (DMSO-d6) d: 6.06 (s, 2H, NH2),
–
–
2
–
C O). Anal. calcd. for C24H14Cl2N2O3: C, 64.16; H, 3.14; N, 6.24.
–
Found: C, 63.88; H, 3.36; N, 6.43.
7.56–7.61 (m, 4H, Ar-H), 7.75–7.82 (ddd, J ¼ 3.03, 2.84, 2.99 Hz,
1H, Ar-H), 7.96–7.99 (d, J ¼ 8.52 Hz, 2H, Ar-H), 8.12–8.15 (d,
J ¼ 8.51 Hz, 2H, Ar-H), 8.24–8.29 (m, 1H, Ar-H), 9.35–9.40 (dd,
J ¼ 2.90, 2.89 Hz, 1H, Ar-H). Anal. calcd. for C23H13Cl2FN4O:
C, 61.21; H, 2.90; N, 12.42. Found: C, 61.30; H, 2.81; N, 12.46.
5-(4-Bromophenyl)-3-(4-chlorophenyl)-9-fluoro-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 38
Yield: 63%, m. p.: 268–2698C (DMF/EtOH); IR (KBr, cmꢀ1): 1771
–
(lactone C O). Anal. calcd. for C23H11BrClFN2O2: C, 57.35; H, 2.30;
–
N, 5.82. Found: C, 56.91; H, 2.39; N, 5.90.
2-Amino-9-bromo-3,5-di(4-chlorophenyl)pyrimido[4,5-
c]quinolin-1(2H)-one 49
Yield: 47%, m. p.: 295–2988C (DMF); IR (KBr, cmꢀ1): 3298, 3213
(NH ), 1671 (C O); 1H-NMR (DMSO-d6) d: 6.07 (s, 2H, NH2),
9-Bromo-5-(4-bromophenyl)-3-(4-chlorophenyl)-1H-
[1,3]oxazino[4,5-c]quinolin-1-one 40
–
–
2
7.56–7.62 (m, 4H, Ar-H), 7.96–8.03 (m, 3H, Ar-H), 8.11–8.16
(m, 3H, Ar-H), 9.88–9.89 (m, 1H, Ar-H). Anal. calcd. for
C23H13BrCl2N4O: C, 53.93; H, 2.56; N, 10.94. Found: C, 53.70;
H, 2.45; N, 11.13.
Yield: 61%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 1772 (lactone
–
C O). Anal. calcd. for C23H11Br2ClN2O2: C, 50.91; H, 2.04; N, 5.16.
–
Found: C, 50.88; H, 1.87; N, 5.31.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
Pyrimido[4,5-c]quinolin-1(2H)-ones
471
7.58–7.61 (d, J ¼ 8.53 Hz, 2H, Ar-H), 7.70–7.73 (d, J ¼ 8.51 Hz, 2H,
Ar-H), 7.96–8.02 (m, 3H, Ar-H), 8.05–8.13 (m, 3H, Ar-H), 9.86–9.87(d,
J ¼ 2.22 Hz, 1H, Ar-H). Anal. calcd. for C23H13Br2ClN4O: C, 49.63; H,
2.35; N, 10.07. Found: C, 49.70; H, 2.27; N, 10.10.
2-Amino-3,5-di(4-chlorophenyl)-9-iodopyrimido[4,5-
c]quinolin-1(2H)-one 50
Yield: 44%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 3288, 3199 (NH2),
1
–
–
1666 (C O); H-NMR (DMSO-d ) d: 6.07 (s, 2H, NH ), 7.57–7.62 (m,
6
2
4H, Ar-H), 7.95–8.00 (m, 3H, Ar-H), 8.14–8.18 (m, 3H, Ar-H), 10.12–
10.13 (m, 1H, Ar-H). Anal. calcd. for C23H13Cl2IN4O: C, 49.40; H,
2.34; N, 10.02. Found: C, 49.45; H, 2.11; N, 10.03.
2-Amino-5-(4-bromophenyl)-3-(4-chlorophenyl)-9-
iodopyrimido[4,5-c]quinolin-1(2H)-one 59
Yield: 43%, m. p.: >3008C (DMF); IR (KBr, cmꢀ1): 3299, 3215 (NH2),
1
–
1666 (C O); H-NMR (DMSO-d ) d: 6.07 (s, 2H, NH ), 7.59–7.61 (d,
2-Amino-7-chloro-3,5-di(4-chlorophenyl)pyrimido[4,5-c]-
quinolin-1(2H)-one 51
–
6
2
J ¼ 8.47 Hz, 2H, Ar-H), 7.67–7.73 (d, J ¼ 8.48 Hz, 2H, Ar-H), 7.93–
7.97 (m, 3H, Ar-H), 8.06–8.09 (d, J ¼ 8.46 Hz, 2H, Ar-H), 8.13–8.16
(dd, J ¼ 1.82, 1.75 Hz, 1H, Ar-H), 10.10–10.11 (d, J ¼ 1.80 Hz, 1H,
Ar-H). Anal. calcd. for C23H13BrClIN4O: C, 45.76; H, 2.17; N, 9.28.
Found: C, 45.83; H, 2.37; N, 9.30.
Yield: 48%, m. p.: 258–2618C (DMF/EtOH); IR (KBr, cmꢀ1): 3321,
3221 (NH ), 1671 (C O); 1H-NMR (DMSO-d6) d: 6.06 (s, 2H, NH2),
–
–
2
7.59–7.62 (d, J ¼ 8.57 Hz, 4H, Ar-H), 7.78–7.84 (m, 1H, Ar-H), 7.98–
8.05 (m, 3H, Ar-H), 8.22–8.24 (d, J ¼ 8.52 Hz, 2H, Ar-H), 9.67–9.70
(m, 1H, Ar-H). Anal. calcd. for C23H13Cl3N4O: C, 59.06; H, 2.80; N,
11.98. Found: C, 59.30; H, 2.71; N, 11.81.
2-Amino-5-(4-bromophenyl)-7-chloro-3-(4-
chlorophenyl)pyrimido[4,5-c]quinolin-1(2H)-one 60
2-Amino-7,9-dichloro-3,5-di(4-chlorophenyl)pyrimido[4,5-c]-
quinolin-1(2H)-one 52
Yield: 49%, m. p.: 263–2658C (DMF/EtOH); IR (KBr, cmꢀ1): 3319,
3209 (NH ), 1663 (C O); 1H-NMR (DMSO-d6) d: 6.06 (s, 2H, NH2),
–
–
2
Yield: 43%, m. p.: 293–2948C (DMF); IR (KBr, cmꢀ1): 3306, 3213
7.59–7.62 (d, J ¼ 8.54 Hz, 2H, Ar-H), 7.73–7.75 (d, J ¼ 8.53 Hz, 2H,
Ar-H), 7.78–7.83 (m, 1H, Ar-H), 7.98–8.00 (d, J ¼ 8.53 Hz, 2H,
Ar-H), 8.02–8.05 (m, 1H, Ar-H), 8.14–8.17 (d, J ¼ 8.51 Hz, 2H,
Ar-H), 9.66–9.69 (d, J ¼ 8.50 Hz, 1H, Ar-H). Anal. calcd. for
C23H13BrCl2N4O: C, 53.93; H, 2.56; N, 10.94. Found: C, 53.58;
H, 2.69; N, 10.77.
1
–
(NH ), 1664 (C O); H-NMR (DMSO-d ) d: 6.09 (s, 2H, NH ), 7.55–7.61
–
2
6
2
(m, 4H, Ar-H), 7.96–7.99 (d, J ¼ 8.56 Hz, 2H, Ar-H), 8.07–8.08 (d,
J ¼ 2.31 Hz, 1H, Ar-H), 8.17–8.19 (d, J ¼ 8.55 Hz, 2H, Ar-H), 9.60–
9.61 (d, J ¼ 2.29 Hz, 1H, Ar-H). Anal. calcd. for C23H12Cl4N4O: C,
55.01; H, 2.41; N, 11.16. Found: C, 55.15; H, 2.20; N, 11.37.
2-Amino-5-(4-bromophenyl)-3-(4-chlorophenyl)-7,9-
dichloro-pyrimido[4,5-c]quinolin-1(2H)-one 61
2-Amino-3,5-di(4-chlorophenyl)-9-methylpyrimido[4,5-c]-
quinolin-1(2H)-one 53
Yield: 41%, m. p.: 297–2998C (DMF); IR (KBr, cmꢀ1): 3304, 3231
Yield: 43%, m. p.: 281–2848C (DMF); IR (KBr, cmꢀ1): 3271, 3182
1
1
–
–
(NH ), 1664 (C O); H-NMR (DMSO-d ) d: 6.09 (s, 2H, NH ), 7.59–7.62
–
–
(NH ), 1666 (C O); H-NMR (DMSO-d ) d: 2.60 (s, 3H, CH ), 6.03 (s,
2
6
2
2
6
3
(d, J ¼ 8.67 Hz, 2H, Ar-H), 7.72–7.75 (d, J ¼ 8.63 Hz, 2H, Ar-H), 7.97–
8.00 (d, J ¼ 8.62 Hz, 2H, Ar-H), 8.12–8.15 (m, 3H, Ar-H), 9.67–9.68 (d,
J ¼ 2.33 Hz, 1H, Ar-H). Anal. calcd. for C23H12BrCl3N4O: C, 50.54; H,
2.21; N, 10.25. Found: C, 50.63; H, 2.37; N, 10.03.
2H, NH2), 7.54–7.61 (m, 4H, Ar-H), 7.68–7.72 (dd, J ¼ 1.94,
1.93 Hz, 1H, Ar-H), 7.96–8.00 (m, 2H, Ar-H), 8.06–8.09 (d,
J ¼ 8.42 Hz, 1H, Ar-H), 8.10–8.15 (m, 2H, Ar-H), 9.49 (s, 1H, Ar-
H). Anal. calcd. for C24H16Cl2N4O: C, 64.44; H, 3.61; N, 12.53.
Found: C, 64.36; H, 3.28; N, 12.62.
2-Amino-5-(4-bromophenyl)-3-(4-chlorophenyl)-9-
methylpyrimido[4,5-c]quinolin-1(2H)-one 62
2-Amino-3,5-di(4-chlorophenyl)-9-methoxypyrimido[4,5-c]-
Yield: 48%, m. p.: 289–2918C (DMF/EtOH); IR (KBr, cmꢀ1): 3275, 3188
quinolin-1(2H)-one 54
Yield: 46%, m. p.: 271–2738C (DMF); 1H-NMR (DMSO-d6) d: 3.97
(s, 3H, OCH3), 6.01 (s, 2H, NH2), 7.48–7.60 (m, 5H, Ar-H), 7.96–8.00
(d, J ¼ 8.53 Hz, 2H, Ar-H), 8.08–8.13 (m, 3H, Ar-H), 9.17–9.18 (d,
J ¼ 2.83 Hz, 1H, Ar-H). Anal. calcd. for C24H16Cl2N4O2: C, 62.22; H,
3.48; N, 12.09. Found: C, 62.33; H, 3.37; N, 12.31.
1
–
(NH ), 1666 (C O); H-NMR (DMSO-d ) d: 2.59 (s, 3H, CH ), 6.02 (s, 2H,
–
2
6
3
NH2), 7.57–7.60 (d, J ¼ 8.49 Hz, 2H, Ar-H), 7.68–7.71 (d, J ¼ 8.42 Hz,
3H, Ar-H), 7.96–7.99 (d, J ¼ 8.47 Hz, 2H, Ar-H), 8.03–8.08 (m, 3H, Ar-
H), 9.48 (s, 1H, Ar-H). Anal. calcd. for C24H16BrClN4O: C, 58.62; H,
3.28; N, 11.39. Found: C, 58.33; H, 3.60; N, 11.09.
2-Amino-5-(4-bromophenyl)-3-(4-chlorophenyl)-9-
fluoropyrimido[4,5-c]quinolin-1(2H)-one 56
2-Amino-5-(4-bromophenyl)-3-(4-chlorophenyl)-9-
methoxy-pyrimido[4,5-c]quinolin-1(2H)-one 63
Yield: 54%, m. p.: 281–2848C (DMF/EtOH); IR (KBr, cmꢀ1): 3283,
Yield: 50%, m. p.: 280–2828C (DMF/EtOH); 1H-NMR (DMSO-d6) d:
3.99 (s, 3H, OCH3), 6.02 (s, 2H, NH2), 7.50–7.54 (dd, J ¼ 2.90,
2.90 Hz, 1H, Ar-H), 7.58–7.61 (d, J ¼ 8.58 Hz, 2H, Ar-H), 7.68–
7.71 (d, J ¼ 8.54 Hz, 2H, Ar-H), 7.96–7.99 (d, J ¼ 8.53 Hz, 2H,
Ar-H), 8.04–8.13 (m, 3H, Ar-H), 9.19–9.20 (d, J ¼ 2.83 Hz, 1H,
Ar-H). Anal. calcd. for C24H16BrClN4O2: C, 56.77; H, 3.18; N,
11.03. Found: C, 56.87; H, 3.39; N, 11.05.
3198 (NH ), 1665 (C O); 1H-NMR (DMSO-d6) d: 6.07 (s, 2H, NH2),
–
–
2
7.59–7.60 (d, J ¼ 8.52 Hz, 2H, Ar-H), 7.71–7.74 (d, J ¼ 8.48 Hz, 2H,
Ar-H), 7.76–7.83 (ddd, J ¼ 3.00, 2.98, 2.96 Hz, 1H, Ar-H), 7.97–7.99
(d, J ¼ 8.49 Hz, 2H, Ar-H), 8.06–8.09 (d, J ¼ 8.47 Hz, 2H, Ar-H),
8.25–8.30 (m, J ¼ 14.96 Hz, 1H, Ar-H), 9.36–9.41 (dd, J ¼ 2.97,
2.95 Hz, 1H, Ar-H). Anal. calcd. for C23H13BrClFN4O: C, 55.73;
H, 2.64; N, 11.30. Found: C, 56.00; H, 2.76; N, 11.26.
2-Amino-9-bromo-5-(4-bromophenyl)-3-(4-
chlorophenyl)pyrimido[4,5-c]quinolin-1(2H)-one 58
Biological screening
Cell culture and assessment of cytotoxicity
The new compounds were tested for their cytotoxic activity on
the following human solid tumor cell lines: lung fibrosarcoma
Yield: 50%, m. p.: 287–2908C (DMF/EtOH); IR (KBr, cmꢀ1): 3299,
3215 (NH ), 1672 (C O); 1H-NMR (DMSO-d6) d: 6.07 (s, 2H, NH2),
–
–
2
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

472
K. Metwally et al.
Arch. Pharm. Chem. Life Sci. 2010, 8, 465–472
HT-1080 (American Type Culture Collection (ATCC), Rockville,
MD, USA), mammary adenocarcinoma MDA-MB-231 (ATCC)
and colorectal adenocarcinoma HT-29 (European Collection of
Cell Cultures, Salisbury, U.K.). All cells were routinely cultured
in Dulbecco’s minimal essential medium (DMEM) supplemented
with penicillin (100 U/mL), streptomycin (100 mg/mL), and
10% fetal bovine serum (media and antibiotics were from
Biochrom KG, Berlin, Germany, while the serum was purchased
from Invitrogen Co., Carlsbad, CA, USA) in an environment
of 5% CO2, 85% humidity, and 378C, and they were subcultured
using a trypsin 0.25%-EDTA 0.02% solution. The cytotoxicity
assay was performed by a modification of the MTT method
[15]. 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
The authors are grateful to the Deanship of Scientific Research, King Faisal
University for providing the financial support for this work (Grant Number
10032/2009).
The authors have declared no conflict of interest.
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ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com