Synthesis, antimicrobial activity and molecular modeling study of substituted 5-aryl-pyrimido[5,4-c] quinoline-2,4-diones

Article abstract of DOI:10.3109/14756366.2011.654113

A series of pyrimido[5,4-c]quinoline-2,4-dione derivatives 5a-k were synthesized in moderate yields via a thermolysis reaction of equimolar ratio of 5-arylidine-1,3-dimethylbarbituric acid derivatives 3a-d with aniline derivatives 4a-d at 150-180 °C for 1

Full text of DOI:10.3109/14756366.2011.654113

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 530–538
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.654113
RESEARCH ARTICLE
Synthesis, antimicrobial activity and molecular
modeling study of substituted 5-aryl-pyrimido[5,4-c]
quinoline-2,4-diones
Mohamed A. Ismail1,2, Shar Al-Shihry1, Reem K. Arafa3, and Usama El-Ayaan1,2
1Department of Chemistry, College of Science, King Faisal University, Hofuf, Saudi Arabia, 2Department of Chemistry,
Faculty of Science, Mansoura University, Mansoura, Egypt, and 3Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Cairo University, Cairo, Egypt
Abstract
A series of pyrimido[5,4-c]quinoline-2,4-dione derivatives 5a–k were synthesized in moderate yields via a thermolysis
reaction of equimolar ratio of 5-arylidine-1,3-dimethylbarbituric acid derivatives 3a–d with aniline derivatives 4a–d
at 150–180 °C for 1–2h. Eight of the synthesized compounds were chosen for a primary in vitro one-dose anticancer
assay performed using the full NCI 60 cell panel. Only compound 5b showed moderate GI% at the used dose
(10 μM) against four of the tested cell lines corresponding to leukemia SR (GI%: 51), non small-cell lung cancer
HOP-92 (GI%: 63), melanoma UACC-62 (GI%: 53) and renal cancer UO-31 (GI%: 69). On the other hand, antimicrobial
screening of the whole set of the synthesized compounds was performed against three Gram +ve and two Gram −ve
bacterial strains. Results of the antimicrobial screening showed that compounds 5d, 5e, 5f, 5h and 5k have broad-
spectrum antibacterial efficacy being moderately active against all the tested Gram +ve and two Gram −ve bacteria.
Also, compound 5a showed interesting results being only active against Streptococcus faecalis and both tested Gram
−ve strains viz. E. coli and P. aeruginosa. In order to compare the binding mode of the most active compounds 5e and
5f along with the inactive compound 5c we docked these compounds into the empty binding site of topoisomerase
II DNA gyrase (PDB ID: 1KZN), and results were compared with the bound inhibitor Clorobiocin.
Keywords: Antimicrobial, anticancer, 5-arylidine-1,3-dimethylbarbituric acid, pyrimido[5,4-c]quinoline-2,4-dione,
molecular modeling
Introduction
being of significant importance as synthetic antimalari-
Pyrimidines and their annelated derivatives are of par-
ticular importance being widely spread in nature chiefly
in the nucleobases found in nucleic acids viz the three
pyrimidines cytosine, thymine, and uracil as well as the
purines adenine and guanine1. Consequently, derivatives
thereof have been extensively studied and many have
been found useful in the field of chemotherapy. Among
the pyrimidine-containing chemotherapeutics currently
in use are the anticancer 5-fluorouracil, the antiviral
zidovudine and the antifungal flucytosine in addition to
als, a disease that claims 1 to 3 million lives annually, for
example chloroquine8,9. Also, many antibiotics and UT
antiinfectives are based on the quinoline scaffold, like
norfloxacin, levofloxacin and others10,11
.
In DNA replication, one group of enzymes has proved
to be effective target for therapeutic agents, which is
topoisomerase enzyme. DNA gyrase is a type II topoi-
somerase found in all bacteria and controls the topologi-
cal state of DNA12. DNA gyrase consists of two subunits
GyrA (875 amino acids) and GyrB (804 amino acids)
with the active species being a heterotetramer A2B213.
Mechanistic studies have revealed the steps involved in
the gyrase supercoiling reaction14. is process involves
many other antimicrobial agents2–7
.
On the other hand, quinolines play a fairly important
role as chemotherapeutics, many derivatives of which
Address for Correspondence: Usama El-Ayaan, Department of Chemistry, College of Science, King Faisal University, P.O. Box 380, Hofuf
31982, Saudi Arabia. Tel: +966 553901011. E-mail: uelayaan@kfu.edu.sa
(Received 27 July 2011; revised 23 December 2011; accepted 23 December 2011)
530

Substituted 5-aryl-pyrimido[5,4-c]quinoline-2,4-diones 531
the wrapping of DNA around the A2B2 complex, cleav-
age of this DNA on both strands, and the passage of
a segment of DNA through the double strand break.
Relegation of the break results in the introduction of two
negative supercoils. ese processes require the binding
and hydrolysis of ATP15. Inhibition of DNA gyrase blocks
relaxation of supercoiled DNA, relaxation being a require-
ment for transcription and replication. DNA gyrase is a
selective target for antibacterial agents, such as the most
studied quinolone and coumarin antibiotics. Quinolone
drugs (e.g. ciprofloxacin) affect the protein subunit GyrA
and coumarins (1-benzopyran-2-ones) (e.g. novobiocin,
clorobiocin) act on GyrB16. Computer docking technique
plays an important role in the drug design and discovery,
as well as in the mechanistic study by placing a molecule
into the binding site of the target macromolecule in a non-
covalent fashion17,18, and predicting the binding geometry
and hydrogen bonds formed with the surrounding amino
acids as well as the mode of the ligand to the receptor,
which all contribute to the docking score. MOE as a flexi-
ble docking program facilitates the prediction of favorable
protein–ligand complex structures with reasonable accu-
racy and speed (MOE 2008.10 of Chemical Computing
Group. Inc.).
dissolved in DMSO and diluted with saline to the appro-
priate volume. e compounds under test were added to
the cell monolayer. Monolayer cells were incubated with
the compound(s) for 48 h at 37 °C and in atmosphere of
5% CO2. After 48 h, cells were fixed, washed, and stained
for 30 min with 0.4% (wt/vol) SRB dissolved in 1% acetic
acid. Excess unbound dye was removed by four washes
with 1% acetic acid and attached stain was recovered
with Tris–EDTA buffer. Color intensity was measured in
an ELISA reader at a wave length of 570 nm.
Antimicrobial screening of pyrimido[5,4-c]quinoline-2,4-dione
derivatives
Experimental design An extensive assessment of the
antimicrobial activity of the 11 newly synthesized
pyrimido[5,4-c]quinoline derivatives 5a–k was per-
formed. e screening was carried out against 5 different
microbial strains; three organisms representing Gram
+ve bacteria viz. Bacillus subtilis, Staphylococcus Aureus
and Streptomyces faecalis; two Gram −ve bacterial strains
Escherichia coli and Pseudomonas aeruginosa. e activ-
ity of the tested compounds was evaluated using disc dif-
fusion method comparing the diameter of the inhibition
zone produced by the new derivatives with those of the
standard antibacterial agent Tetracycline.
Given the promising properties of pyrimidines and
quinolines and in continuation of our interest in prepar-
ing biologically active heterocycles, we decided to study
the effect of combining the pyrimidine and quinoline
scaffolds in one molecule on the biological activity of such
a ring system. erefore, this study covers the synthesis
and molecular modeling study of a series of substituted
5-aryl-pyrimido[5,4-c]quinoline-2,4-diones and evalua-
tion of their anticancer and antimicrobial activities.
Experimental procedure Antimicrobial activity of the
tested samples was determined using a modified Kirby-
Bauer disc diffusion method22–25
.
Blank paper discs with a diameter of 8.0mm were
impregnated with 10 µL of the stock solutions of the
tested compounds and placed on agar where the chemi-
cal diffused from the disc into the agar only around the
disc. Organisms susceptible to the tested compound do
not grow in the area around the disc in what is known as
a “Zone of inhibition” whose diameters were measured
(in mm) with slipping calipers.
Experimental section
Biology
Anticancer screening of pyrimido[5,4-c]quinoline-2,4-dione
derivatives
Standard discs of Tetracycline (Antibacterial agent)
served as positive controls for antimicrobial activity while
filter discs impregnated with 10 µL of the solvent (DMSO)
were used as a negative control.
Experimental design e chosen compounds were sub-
jected to the in vitro disease-oriented NCI 60 human
cells screening panel assay to be evaluated for their in
vitro anticancer activity. In this protocol, all compounds
submitted to the screen are tested initially at a single
high dose (10 μM) in the full NCI 60 cell panel including
leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast cancer cell lines. A
48 h continuous drug exposure protocol was used, and a
sulphorhodamine B (SRB) protein assay was employed
to estimate cell viability or growth19–21. e data obtained
are a mean graph of the percent growth of treated cells,
and presented as percentage growth inhibition (GI %)
caused by the tested compounds.
Docking methodology
Docking studies were performed using MOE software
Version 2008.10. e X-ray crystal structure of the anti-
microbial agent Clorobiocin bound to topoisomerase II
DNA gyrase was obtained from the RCSB Protein Data
Bank (PDB ID: 1KZN). e enzyme was prepared for
docking as follows: (a) Clorobiocin interactions were
determined to reveal the different types of interaction and
a validation for the docking was performed (Figure 1).
(b) Clorobiocin and solvent molecules were removed
from the binding site. (c) e hydrogens were added
with their standard geometry. (d) e newly synthesized
compounds were constructed and were energy mini-
mized for 1000 iterations reaching a convergence of 0.01
kcal/molA. (e) Docking of the energy minimized ligand
molecules was carried out using the default MOE-dock
Experimental procedure Cells were plated in 96-mul-
tiwell microtiter plate (104 cells/well) for 24 h before
treatment with the test compound to allow attachment
of cells to the wall of the plate. Tested compounds were
© 2013 Informa UK, Ltd.

532 M. A. Ismail et al.
parameters. e docking aims at searching for favorable
binding conformations. e quality of the docked struc-
tures was evaluated by measuring the intermolecular
energy of the ligand-DNA gyrase-B assembly dG calcu-
recrystallized from ethanol/EtOAc to afford compound 3b in
79% yield, mp 154–155 °C; Lit.29 mp 149–151 °C.
5 - ( 3 , 4 - D i m e t h o x y b e n z y l i d e n e ) - 1 , 3 -
dimethylpyrimidine-2,4,6-trione (3c): Adopting the
same methodology used for preparation of 3b, starting with
3,4-dimethoxybenzaldehyde instead of 4-methoxybenz-
aldehyde. Yield 89%, mp 229–231 °C; Lit.29 mp 226–228 °C;
Lit.30 mp 208–210 °C. IR (KBr) ν’ 3122, 3004 (aromatic CH),
2947, 2906, 2839 (CH3), 1720, 1651 (CO), 1598, 1556, 1502
(C=C) cm−1. 1H NMR (CDCl3); δ 3.39, 3.40 (2s, 6H; 2 × CH3),
3.97, 3.98 (2s, 6H; 2 × OCH3), 6.94 (d, J=8.7 Hz, 1H), 7.78 (dd,
J=8.7 Hz, 2.1 Hz, 1H), 8.38 (d, J=2.1 Hz, 1H), 8.48 (s, 1H).
5-(2-Ethoxybenzylidene)-1,3-dimethylpyrimidine-
2,4,6-trione (3d): Adopting the same methodology used
for preparation of 3b, starting with 2-ethoxybenzaldehyde
instead of 4-methoxybenzaldehyde. Yield 85%, mp 165–
166 °C; Lit.31 melting point not reported. IR (KBr) ν’ 3114,
3095, 2987, 2950, 2900 (CH), 1730, 1670 (CO), 1577, 1541
(C=C) cm−1. 1H NMR (CDCl3); δ 1.37 (t, J=7.2 Hz, 3H), 3.28
(s, 3H), 3.35 (s, 3H), 4.07 (q, J=7.2 Hz, 2H), 6.84 (d, J=8.4
Hz, 1H), 6.92 (t, J=7.2 Hz, 1H), 7.39 (t, J=7.2 Hz, 1H), 7.98
(d, J=8.4 Hz, 1H), 8.85 (s, 1H). MS (EI) m/e (rel.int.); 288
(M+, 24), 243 (100). Anal. Calc. for C15H16N2O4 (288.30): C,
62.49; H, 5.59; N, 9.72. Found. C, 62.21; H, 5.73; N, 9.48.
lated in kcal/molA (S value in MOE)26,27
.
Chemistry
Melting points were recorded using a Gallenkamp
melting-point apparatus and are uncorrected. in-layer
chromatography (TLC) analysis was carried out on silica
gel60F254 precoatedaluminumsheetsanddetectedunder
ultraviolet (UV) light. Infrared (IR) spectra were recorded
using KBr wafer technique on a Shimadzu 5800 Fourier
transform FT-IR Spectrometer at College of Science,
1
King Faisal University. H NMR spectra were recorded
employing a Varian Unity Plus 300 spectrometer and
chemical shifts (δ) are in parts per million (ppm) relative
to tetramethylsilane (TMS) as internal standard. Mass
spectra were recorded on a GC-MS (Schimadzu QP-1000
EX) spectrometer. Elemental analyses were performed
on Perkin-Elmer 2400 analyser at the microanalytical
laboratories of the Faculty of Science, Cairo University
and are within 0.4 of the theoretical values. All chemi-
cals and solvents were purchased from Aldrich Chemical
Co., Fisher Scientific. All solvents were reagent grade.
5-Benzylidene-1,3-dimethylpyrimidine-2,4,6-trione (3a)
was prepared according to the reported literature28.
General Procedure for preparation of pyrimido[5,4-c]
quinoline-2,4-diones 5a–k
A mixture of 5-arylidene-1,3-dimethylbarbituric acid
derivatives 3a–d (10 mmol) and aniline derivatives
4a–d (10 mmol) was fused in an oil bath at 150–180 °C
for 1–2 h. e reaction mixture was cooled and triturated
with ethanol, the precipitated products were filtered off
and purified by either recrystallization from EtOH/DMF
or column chromatography on silica gel using petroleum
ether (35–60 °C)/EtOAc (7:3) as an eluent.
Preparation of 5-arylidine-1,3-dimethylbarbituric acids 3b–d
5-(4-Methoxybenzylidene)-1,3-dimethylpyrimidine-
2,4,6-trione(3b):Toasolutionof1,3-dimethylbarbituricacid
(15.6g, 100 mmol) and 4-methoxybenzaldehyde (13.62g, 100
mmol) in methanol (150mL) was added few drops of conc.
HCl. e reaction mixture was heated at reflux for 3h and
the precipitate was filtered off, washed with methanol and
Figure 1. Left panel: 2D sketch of the binding mode of Clorobiocin into its binding site of DNA gyrase-B, showing three hydrogen bonds as
green dotted lines (with residues Arg136, Asp73 and Asn46) and one Pi-cationic interaction with Arg76; right panel: Docking validation of
Clorobiocin with DNA gyrase-B: crystal structure ligand (red) docked ligand (yellow); RMSD 0.41 Å.
Journal of Enzyme Inhibition and Medicinal Chemistry

Substituted 5-aryl-pyrimido[5,4-c]quinoline-2,4-diones 533
1,3,9-Trimethyl-5-phenylpyrimido[5,4-c]quino-
line-2,4-dione (5a): adopting the reported methodol-
ogy32, mp >300 °C.
5-(4-Methoxyphenyl)-1,3,9-trimethylpyrimido
[5,4-c]quinoline-2,4-dione (5b): adopting the reported
methodology32, mp 251–252.5 °C.
316 (17). Anal. Calc. for C19H14ClN3O2 (351.79): C, 64.87;
H, 4.01; N, 11.94. Found. C, 64.55; H, 4.06; N, 12.03.
8 , 1 0 - D i m e t h o x y - 1 , 3 - d i m e t h y l - 5 -
phenylpyrimido[5,4-c]quinoline-2,4-dione (5h): Yield
40 %, mp >300 °C (EtOH/DMF). IR (KBr) ν’ 3067, 3006,
2952 (CH), 1708, 1664 (CO), 1618, 1585, 1556 (C=N, C=C)
cm−1. 1H NMR (DMSO-d6); δ 3.12 (s, 3H), 3.16 (s, 3H), 3.69
(s, 3H), 3.95 (s, 3H), 6.43 (s, 1H), 6.95–7.09 (m, 4H), 7.31
(s, 2H). MS (EI) m/e (rel.int.); 377 (M+, 100), 378 (M+ + 1,
71), 349 (45), 234 (40). Anal. Calc. for C21H19N3O4 (377.39):
C, 66.83; H, 5.07; N, 11.13. Found. C, 66.59; H, 5.27; N,
11.39.
5-(3,4-Dimethoxyphenyl)-1,3,9-trimethylpyrimido
[5,4-c]quinoline-2,4-dione (5c): Yield 53 %, mp 276–277
°C (EtOH/DMF). IR (KBr) ν’ 2991, 2935, 2837 (CH), 1708,
1
1668 (CO), 1635, 1606, 1569 (C=N, C=C) cm−1. H NMR
(DMSO-d6); δ2.35 (s, 3H), 3.18 (s, 3H), 3.69 (s, 3H), 3.70 (s,
3H), 3.87 (s, 3H), 6.72 (dd, J = 8.1 Hz, 2.1 Hz, 1H), 6.82 (d,
J = 2.1 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 7.16 (d, 2.1 Hz, 1H),
7.68 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H). MS
(EI) m/e (rel.int.); 391 (M+, 100), 392 (M+ + 1, 20), 376 (10),
363 (10). Anal. Calc. for C22H21N3O4 (391.42): C, 67.51; H,
5.41; N, 10.74. Found. C, 67.16; H, 5.62; N, 10.89.
5-(3, 4-Dimethoxyphenyl)-9-methoxy-1, 3-
dimethylpyrimido[5,4-c]quinoline-2,4-dione
(5i):
Yield 43 %, mp 253–255 °C(EtOH/DMF). IR (KBr) ν’ 3010,
2950 (CH), 1709, 1668 (CO), 1652, 1622, 1569, 1558, 1506
(C=N, C=C) cm−1. 1H NMR (CDCl3); δ 3.40 (s, 3H), 3.69 (s,
3H), 3.87 (s, 3H), 3.93 (s, 3H), 4.00 (s, 3H), 6.71–6.81 (m,
3H), 7.06 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 9.0 Hz, 2.7 Hz,
1H), 8.05 (d, J = 9.0 Hz, 1H). MS (EI) m/e (rel.int.); 407 (M+,
100), 392 (7). Anal. Calc. for C22H21N3O5 (407.42): C, 64.86;
H, 5.20; N, 10.31. Found. C, 64.73; H, 5.18; N, 10.25.
9 - M e t h o x y - 5 - ( 4 - m e t h o x y p h e n y l ) - 1 , 3 -
9-Chloro-5-(3,4-dimethoxyphenyl)-1,3-dimethyl
pyrimido[5,4-c]quinoline-2,4-dione (5d): Yield 47 %,
mp 276–278 °C (EtOH/DMF). IR (KBr) ν’ 3080, 3008, 2958,
2927, 2837 (CH), 1712, 1672 (CO), 1602, 1568, 1517 (C=N,
1
C=C) cm−1. H NMR (DMSO-d6); δ 3.17 (s, 3H), 3.67 (s,
3H), 3.70 (s, 3H), 3.87 (s, 3H), 6.75 (dd, J= 8.1 Hz, 1.8 Hz,
1H), 6.85 (d, J = 1.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.29
(d, J = 2.4 Hz, 1H), 7.83 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 7.95
(d, J = 9.0 Hz, 1H). MS (EI) m/e (rel.int.); 411 (M+, 100),
412 (M+ + 1, 12), 396 (49). Anal. Calc. for C21H18ClN3O4
(411.84): C, 61.24; H, 4.41; N, 10.20. Found. C, 61.19; H,
4.49; N, 10.13.
dimethylpyrimido[5,4-c]quinoline-2,4-dione
(5j):
purified by column chromatography on silica gel using
petroleum ether (35–60 °C)/EtOAc (7:3) as an eluent,
Rf = 0.40. Yield 55 %, mp 268–269.5 °C. IR (KBr) ν’ 3001,
2962, 2941 (CH), 1709, 1664 (CO), 1635, 1622, 1569 (C=N,
1
C=C) cm−1. H NMR (CDCl3); δ 3.40 (s, 3H), 3.69 (s, 3H),
5-(3,4-Dimethoxyphenyl)-8,10-dimethoxy-1,3-
3.90 (s, 3H), 3.93 (s, 3H), 6.71 (d, J = 2.7 Hz, 1H), 7.10 (d,
J = 8.7 Hz, 2H), 7.17 (d, J = 8.7 Hz, 2H), 7.47 (dd, J = 9.0 Hz,
2.7 Hz, 1H), 8.03 (d, J = 9.0 Hz, 1H). MS (EI) m/e (rel.int.);
377 (M+, 100), 362 (6). Anal. Calc. for C21H19N3O4 (377.39):
C, 66.83; H, 5.07; N, 11.13. Found. C, 66.95; H, 5.12; N,
10.88.
dimethylpyrimido[5,4-c]quinoline-2,4-dione
(5e):
Yield 40 %, mp 302–303.5 °C (EtOH/DMF). IR (KBr) ν’
2997, 2943, 2837 (CH), 1708, 1668 (CO), 1618, 1585, 1552
1
(C=N, C=C) cm−1. H NMR (DMSO-d6); δ 3.11 (s, 3H),
3.38 (s, 3H), 3.63 (s, 3H), 3.68 (s, 3H), 3.81 (s, 3H), 3.91
(s, 3H), 6.41 (d, J = 2.1 Hz, 1H), 6.57 (dd, J = 8.4 Hz, 2.1 Hz,
1H), 6.74 (d, J = 2.1 Hz, 1H), 6.88–6.91 (m, 2H). MS (EI)
m/e (rel.int.); 437 (M+, 100), 438 (M+ + 1, 32), 422 (26), 407
(11). Anal. Calc. for C23H23N3O6 (437.45): C, 63.15; H, 5.30;
N, 9.61. Found. C, 63.23; H, 5.46; N, 9.72.
5 - ( 2 - E t h o x y p h e n y l ) - 9 - m e t h o x y - 1 , 3 -
dimethylpyrimido[5,4-c]quinoline-2,4-dione
(5k):
purified by column chromatography on silica gel using
petroleum ether (35-60 °C)/EtOAc (7:3) as an eluent,
Rf = 0.46. Yield 46 %, mp 255–257 °C. IR (KBr) ν’ 3060,
2979, 2954 (CH), 1710, 1660 (CO), 1622, 1568 (C=N, C=C)
8,10-Dimethoxy-5-(4-methoxyphenyl)-1,3-
1
dimethylpyrimido[5,4-c]quinoline-2,4-dione
(5f):
cm−1. H NMR (CDCl3); δ 1.05 (t, J = 7.2 Hz, 3H), 3.40 (s,
Yield 38 %, mp >300 °C (EtOH/DMF). IR (KBr) ν’ 2954,
3H), 3.68 (s, 3H), 3.88 (s, 3H), 4.00 (q, J = 7.2 Hz, 2H), 6.70
(d, J = 2.7 Hz, 1H), 7.07–7.14 (m, 3H), 7.43–7.50 (m, 2H),
7.94 (d, J = 9.0 Hz, 1H). MS (EI) m/e (rel.int.); 391 (M+, 58),
346 (100). Anal. Calc. for C22H21N3O4 (391.42): C, 67.51; H,
5.41; N, 10.74. Found. C, 67.39; H, 5.54; N, 10.46.
2912, 1835 (CH), 1708, 1668 (CO), 1635, 1616, 1560 (C=N,
1
C=C) cm−1. H NMR (DMSO-d6); δ 3.13 (s, 3H), 3.33 (s,
3H), 3.68 (s, 3H), 3.83 (s, 3H), 3.94 (s, 3H), 6.44 (s, 1H),
6.89 (d, J = 8.4 Hz, 2H), 6.94 (s, 1H), 6.99 (d, J = 8.4 Hz, 2H).
MS (EI) m/e (rel.int.); 407 (M+, 100), 408 (M+ + 1, 20), 379
(20). Anal. Calc. for C22H21N3O5 (407.42): C, 64.86; H, 5.20;
N, 10.31. Found. C, 64.49; H, 5.41; N, 10.32.
Results and discussion
9-Chloro-1,3-dimethyl-5-phenylpyrimido[5,4-c]
quinoline-2,4-dione (5g): Yield 45 %, mp 263-265 °C
(EtOH/DMF). IR (KBr) ν’ 3078, 3026, 2956, 2866 (CH),
Chemistry
In view of the significant therapeutic value of quinolines,
it was considered worthwhile to incorporate quinoline
moieties into the C5–C6 position of the oxypyrimidine
nucleus. us, a series of the pyrimido[5,4-c]quinoline-
2,4-dione derivatives 5a–k were synthesized in mod-
erate yields via a thermolysis reaction of equimolar
ratio of benzalbarbituric acid derivatives 3a–d with the
1
1716, 1676 (CO), 1652, 1602, 1566 (C=N, C=C) cm−1. H
NMR (DMSO-d6); δ 3.18 (s, 3H), 3.71 (s, 3H), 7.14 (d,
J = 2.4 Hz, 1H), 7.22–7.26 (m, 2H), 7.52–7.55 (m, 3H), 7.88
(dd, J = 9.0 Hz, 2.4 Hz, 1H), 8.01 (d, J = 9.0 Hz, 1H). MS (EI)
m/e (rel.int.); 351, 353 (M+, 100, 36: chlorine isotopes),
© 2013 Informa UK, Ltd.

534 M. A. Ismail et al.
Figure 2. Synthesis of pyrimido[5,4-c]quinoline-2,4-dione derivatives 5a–k.
respective aniline derivatives 4a–d at 150–180 °C for
1–2 h. e structures of pyrimido[5,4-c]quinolines 5a–k
were confirmed from their elemental analyses as well as
for 1,2,4-trisubstituted benzene moiety indicated by
the presence of three protons signals attributed to H-2;
H-5 and H-6; and singlet signal at δ 8.48 attributed for a
methine proton, in addition to four singlet signals attrib-
uted for protons of four methyl groups.
1
their spectral data. us, H NMR spectrum of 5c does
not show the signals corresponding to AA’-BB’ system for
a p-substituted benzene ring, however, it showed a new
ABX system attributed for 1,2,4-trisubstituted benzene.
Previously, we reported a suggested mechanism for the
formation of 5a32. In general, the suggested mechanism
for the formation of pyrimido[5,4-c]quinolines 5a–k,
involves addition reaction of aniline derivative 4a–d to
the 5-arylidine derivative 3a–d, followed by cyclization
to dihydropyrimido[5,4-c]quinoline intermediate which
was subsequently autooxidized (Figure 2). 5-Arylidine-
1,3-dimethylbarbituric acids 3a–d were prepared via a
direct acid-catalyzed condensation reaction between
equimolar ratios of 1,3-dimethylbarbituric acid and the
respective benzaldehyde derivatives 2a–d in metha-
nol as a solvent. e structures of compounds 3c and
3d were confirmed from their spectral data. us, 1H
NMR spectrum of 3c showed ABX system attributed
Biology
Anticancer screening of pyrimido[5,4-c]quinoline-2,4-dione
derivatives
Eight of the synthesized compounds 5a–h were chosen by
the National Cancer Institute (NCI, Betheseda, MD, USA)
for the in vitro disease-oriented human cells screening
panel assay to be evaluated for their in vitro cytotoxic
activity. A primary in vitro one-dose anticancer assay was
performed using the full NCI 60 cell panel in accordance
with the current protocol of the Drug Evaluation Branch,
NCI, Bethesda, MD, USA. ese cell lines were incubated
with one concentration (10 µM) for each tested com-
pound. A 48 h continuous drug exposure protocol was
used, and a sulphorhodamine B (SRB) protein assay was
employed to estimate cell viability or growth.
Journal of Enzyme Inhibition and Medicinal Chemistry

Substituted 5-aryl-pyrimido[5,4-c]quinoline-2,4-diones 535
Table 1. In vitro antimicrobial activity of pyrimido[5,4-c]quinoline-2,4-diones 5a–k.
Inhibition Zone Diameter (mm)
Compd #
Tetracycline
(5a)
(5b)
(5c)
B. subtilis (Gm +ve)
Staph. Aureus (Gm +ve)
Strept. faecalis (Gm +ve) E. coli (Gm –ve) P. aeruginosa (Gm –ve)
32
–
28
–
31
11
11
–
30
11
–
31
11
–
–
–
–
–
–
–
(5d)
(5e)
(5f)
(5g)
(5h)
(5i)
(5j)
(5k)
13
16
15
–
13
16
15
–
14
15
14
–
14
15
15
11
15
13
11
14
14
16
15
–
15
–
14
13
11
14
16
13
–
16
11
–
–
13
13
14
Data obtained from results of the anticancer screen-
ing of the eight tested quinoline derivatives can be
summarized as follows, while seven of the tested com-
pounds showed no growth inhibitory activity against all
the 60 tested cancer cell lines at the used dose (10 μM),
only compound 5b possessed some cytotoxic activity. It
showed moderate GI% against four of the tested cell lines
corresponding to leukemia SR (GI%: 51), non small-
cell lung cancer HOP-92 (GI%:63), melanoma UACC-
62 (GI%: 53) and renal cancer UO-31 (GI%: 69). It also
exhibited poor activity against seven cell lines compris-
ing three breast cancer cell lines (MCF, MDA-MB-231/
ATCC and HS 578T) with GI% 31, 38 and 33, respectively;
two non-small cell lung cancer cell lines HOP-62 (GI%:
30) and NCI-H522 (GI%: 37); one renal cancer cell line
(A498, GI%: 32) and one leukemia cell line (RPMI-8226,
GI%: 30).
drug Tetracycline. e following few lines display the
antibacterial activity of some pyrimidine and quino-
line derivatives either in the market or a few of those
recently reported for the sake of comparison. Whereas,
pyrimidine is pharmacologically inactive, its synthetic
derivatives possess an important role in modern medi-
cine33. Amongst the 2,4-diaminopyrimidine drugs, trime-
thoprim, an antibacterial drug is also a selective inhibitor
and selectively inhibits bacterial enzyme dihydrofolate
reductase (DHFR)34. Brodimoprim, is also found to be an
effective antibacterial compound35. Recently, some novel
reported pyrazolopyrmidine-diones showed highest
antimicrobial activity against Pseudomonas aeruginosa,
Enterococcus faecalis and Bacillus pumilus as compared
to the standard drug Streptomycin36.
On the other hand, quinolones represent a well-
known class of synthetic broad-spectrum antibacterial
agents including the market antibiotics levofloxacin and
ciprofloxacin37,38. ese drugs are active against a diverse
assembly of Gram −ve and Gram +ve bacteria. It is well
stated that the pharmacophore of this group of drugs is
the quinolone ring system which is responsible for the
biological activity of these drugs39. Furthermore, Holla
et al.40 described that their newly synthesized quinoline
substituted pyrazolo[3,4-d]pyrimidin-4-ones showed
maximum antibacterial activity against staphylococcus
aureus in comparison to the standard drug in that study
Streptomycin. Recently, a series of novel tetrahydroquin-
oline annulated heterocycles exhibited good antibacterial
activity against microorganisms of which one of them was
found to be as active as the antibiotic ciprofloxacin41.
Finally, while our newly synthesized compounds are
less potent than the pyrimidines or quinolines in the
market they compare well with some reported ones.
Antimicrobial screening of pyrimido[5,4-c]quinoline-2,4-dione
derivatives
e newly synthesized compounds 5a-k were tested
against a panel of susceptible and resistant Gram +ve
bacteria (Bacillus subtilis, Staphylococcus Aureus and
Streptomyces faecalis) and Gram −ve (Escherichia coli and
Pseudomonas aeruginosa) bacteria. Results of the antimi-
crobial screening (Table 1) showed that compounds 5d,
5e, 5f, 5h and 5k have broad-spectrum antibacterial effi-
cacy being moderately active against all the tested Gram
+ve and Gram −ve bacteria. Compound 5i was active
against four of the tested bacterial strains. Interestingly,
compound 5b showed selectivity only towards
Streptococcus faecalis which is known to be a relatively
resistant strain of Gram +ve bacteria. Also, compound
5a showed interesting results being only active against
Streptococcus faecalis and both tested Gram −ve strains
viz. E. coli and P. aeruginosa. Finally, Compound 5j was
only slightly active against Staph. aureus and E.coli.
In conclusion, results of the antibacterial screening
showed that pyrimido[5,4-c]quinoline-2,4-diones 5d, 5e,
5f, 5h and 5k have broad-spectrum antibacterial efficacy
being moderately active against all the tested Gram +ve
and two Gram −ve bacteria as compared to the standard
Docking studies
In an attempt to explain the observed antimicrobial
data on a structural basis, automated docking studies
were carried out using MOE 10.2008 program installed
on 2.0G Core 2 Duo. e protein–ligand complex
based on the X-ray crystal structure of the topoisomer-
ase II DNA gyrase-B enzyme with its bound inhibitor
© 2013 Informa UK, Ltd.

536 M. A. Ismail et al.
Figure 3. 2D Ligand interaction of the 5-aryl-pyrimido[5,4-c]quinoline-2,4-diones with DNA gyrase-B; panel (1) compound 5c showing
only pi-cationic interaction with Arg76; panel (2) compound 5e showing pi-cationic interaction with Arg76 and H-bond with Arg136; panel
(3) compound 5f showing pi-cationic interaction with Arg76 and two H-bonds with Arg136 and Asn46.
Clorobiocin available through the RCSB Protein Data
Bank (PDB entry 1KZN) was employed for the dock-
ing study of the new compounds42. e active site of
the enzyme was defined to include residues within a
10.0Å radius to any of the inhibitor atoms. e binding
poses as well as the binding interaction modes of the
docked compounds with the surrounding amino acids
in the active pocket of DNA gyrase-B were used to try to
understand and rationalize the observed antimicrobial
activity of the most active compounds 5e and 5f along
with the inactive compound 5c. e binding mode of
topoisomerase II DNA gyrase-B (1KZN) with its bound
inhibitor Clorobiocin (Figure 1) showed that the bind-
ing complex involves 3 hydrogen bonds with residues
Arg136, Asp73 and Asn46 and one Pi-cationic interaction
with Arg76. Docking validation was performed with an
obtained RMSD value of 0.41Å ensuring precision and
reproducibility of the docking process (Figure 1). e
energy minimized structures of the most active com-
pounds 5e and 5f as well as of the inactive compound
5c with were docked in the active site of topoisomerase
II DNA gyrase-B. e 2D ligand interactions of these
3 derivatives with DNA gyrase-B displayed in Figure 3
shows that the inactive compound 5c possesses only a
pi–cationicinteractionwithArg76whichisalsoconserved
with the other two compounds 5e and 5f. Moreover, the
active compounds 5e and 5f demonstrated the presence
of extra H-bond interactions (one with Arg136 for 5e; two
with Arg136 and Asn46 for 5f) which can be regarded as
the reason behind their improved activity (Figure 3). For
further understanding, 3D overlay of Clorobiocin, 5c, 5e
and 5f in DNA-gyrase active pocket was performed which
revealed that the orientations of 5e and 5f are similar
with a slight displacement however 5c possessed a totally
Journal of Enzyme Inhibition and Medicinal Chemistry

Substituted 5-aryl-pyrimido[5,4-c]quinoline-2,4-diones 537
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is work was supported by Deanship of Scientific
Research, King Faisal University, Project No. 110010. e
in vitro anticancer screening was done by the National
Cancer Institute (NCI, Betheseda, MD, USA).
Declaration of interest
e authors declare no conflict of interest.
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