Synthesis, antitumor and antimicrobial activity of some new 6-methyl-3-phenyl-4(3H)-quinazolinone analogues: in silico studies

Article abstract of DOI:10.3109/14756366.2015.1060482

Some new derivatives of substituted-4(3H)-quinazolinones were synthesized and evaluated for their in vitro antitumor and antimicrobial activities. The results of this study demonstrated that compound 5 yielded selective activities toward NSC Lung Cancer EKVX cell line, Colon Cancer HCT-15 cell line and Breast Cancer MDA-MB-231/ATCC cell line, while NSC Lung Cancer EKVX cell line and CNS Cancer SF-295 cell line were sensitive to compound 8. Additionally, compounds 12 and 13 showed moderate effectiveness toward numerous cell lines belonging to different tumor subpanels. On the other hand, the results of antimicrobial screening revealed that compounds 1, 9 and 14 are the most active against Staphylococcus aureus ATCC 29213 with minimum inhibitory concentration (MIC) of 16, 32 and 32 μg/mL respectively, while compound 14 possessed antimicrobial activities against all tested strains with the lowest MIC compared with other tested compounds. In silico study, ADME-Tox prediction and molecular docking methodology were used to study the antitumor activity and to identify the structural features required for antitumor activity.

Full text of DOI:10.3109/14756366.2015.1060482

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–15
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1060482
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RESEARCH ARTICLE
Synthesis, antitumor and antimicrobial activity of some new
6-methyl-3-phenyl-4(3H)-quinazolinone analogues: in silico studies
Amer M. Alanazi1, Alaa A.-M. Abdel-Aziz1,2, Taghreed Z. Shawer3, Rezk R. Ayyad3, Abdulrahman M. Al-Obaid1,
Mohamed H. M. Al-Agamy4, Azza R. Maarouf2, and Adel S. El-Azab1,5
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 2Department of Medicinal Chemistry,
3
Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar
4
University, Cairo, Egypt, Department of Pharmaceutics and Microbiology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, and
5Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
Abstract
Keywords
Some new derivatives of substituted-4(3H)-quinazolinones were synthesized and evaluated for
their in vitro antitumor and antimicrobial activities. The results of this study demonstrated that
compound 5 yielded selective activities toward NSC Lung Cancer EKVX cell line, Colon Cancer
HCT-15 cell line and Breast Cancer MDA-MB-231/ATCC cell line, while NSC Lung Cancer EKVX
cell line and CNS Cancer SF-295 cell line were sensitive to compound 8. Additionally,
compounds 12 and 13 showed moderate effectiveness toward numerous cell lines belonging
to different tumor subpanels. On the other hand, the results of antimicrobial screening revealed
that compounds 1, 9 and 14 are the most active against Staphylococcus aureus ATCC 29213
with minimum inhibitory concentration (MIC) of 16, 32 and 32 mg/mL respectively, while
compound 14 possessed antimicrobial activities against all tested strains with the lowest MIC
compared with other tested compounds. In silico study, ADME-Tox prediction and molecular
docking methodology were used to study the antitumor activity and to identify the structural
features required for antitumor activity.
Antimicrobial, in silico study, in vitro
antitumor, molecular docking,
quinazolinones
History
Received 12 March 2015
Revised 22 May 2015
Accepted 26 May 2015
Published online 10 July 2015
Introduction
novel antibacterial agents other than the analogs of existing
antibiotics7.
Cancer, a continuing to be a major health problem worldwide, is
the leading cause of human mortality exceeded only by cardio-
vascular diseases1. Cancer is a disease characterized by a shift in
the controlled mechanisms of cell proliferation and differenti-
ation2. Malignancy is caused by abnormalities in cells, which
might be due to inherited genes or outside exposure of the body to
chemicals, radiation or even infectious agents3,4. Several tech-
niques have been adopted for the treatment and eradication of
cancerous cells. These techniques involved surgery, radiation,
immunotherapy, chemotherapy and chemoprevention. Ideal antic-
ancer drugs would eradicate cancer cells without harming normal
tissues. Unfortunately, no currently available agents meet this
criterion and the clinical use of drugs involves a weighing of
benefits against toxicity in a search of favourable therapeutic
index5. On the other hand, the spread of antibiotic resistance
among pathogenic bacteria has become a major problem for the
clinical management of infectious diseases6. Such medical health
problems have encouraged many medicinal chemists to search for
Quinazolines are frequently used in medicine because of their
wide spectrum of biological activities8–26. Based on the good
performances of quinazoline derivatives in anticancer application,
development of novel quinazoline derivatives as anticancer drugs
is a promising field9. FDA has approved several quinazoline
derivatives as anticancer drugs, such as Gefitinib, Erlotinib,
Lapatinib and Vandetanib9. Moreover, a series of triazol-4-yl-
substituted quinazolines and 2-thio-[1,2,4]triazolo[1,5-c]quinazo-
line derivatives have been identified as potent antimicrobial
agents27. Therefore, the development of a novel and efficient
anticancer agent that can be used for prophylaxis as well as the
treatment of bacterial infections in cancer patients remains an
important goal in medicinal chemistry.
Based on aforementioned rational and our studies on quinazo-
line derivatives as attractive candidates for antitumor9,23–25 and
antimicrobial agents10,11,21, we have designed a number of new
quinazoline derivatives containing a 3-phenyl substituent attached
with different fragments at 2-position to identify the dual
antitumor and antimicrobial activities. Moreover, an in silico
study, ADME-T prediction and molecular docking studies were
used to identify the structural features required for the antitumor
properties of the designed compounds.
Address for correspondence: Adel S. El-Azab, Pharmaceutical Chemistry
Department, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia. Tel: +966 14679096. Fax: +966 1 467
6383. E-mail: adelazaba@yahoo.com

2
A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
Materials and methods
7-Methyl-4-phenyl-1-styryl-[1,2,4]triazolo[4,3-a]quinazolin-
5(4H)-one (8). Yield 60%, m.p. 291–292 ꢁC; 1H NMR (C5D5N):
ꢁ 8.08–7.93 (m, 1H), 7.93–7.45 (m, 5H), 7.83–7.59 (m, 3H),
Chemistry
Melting points were recorded on Barnstead 9100 Electrothermal 7.56–7.55 (m, 4H), 7.41–7.10 (m, 6H), 2.42 (d, 3H, J ¼ 6.0 Hz).
melting apparatus. IR spectra (KBr) were recorded on a FT-IR Ms: [M+ 378].
Perkin-Elmer spectrometer (ꢀ cmꢀ1) at Research Center, King
Saud University, Saudi Arabia. Nuclear magnetic resonance (1H
and 13C NMR) spectra were recorded on Bruker 500 MHz
spectrometer (Japan) using DMSO-d6, C5D5N or CDCl3 as
1.1.8.2. 7-Methyl-1,4-diphenyl-[1,2,4]triazolo[4,3-a]quinazolin-
1
5(4H)-one (9). Yield 65%, m.p. 299–300 ꢁC; H NMR (CDCl3
& TFA): ꢁ 8.13 (s, 1H), 7.60–7.50 (m, 8H), 8.45 (t, 2H,
solvents at Research Center, King Saud University, Saudi
J ¼ 6.5 Hz), 7.38 (t, 2H, J ¼ 7.5 Hz), 2.46 (s, 3H, CH3). 13C NMR
Arabia. The chemical shifts are expressed in ꢁ ppm using TMS
as internal standard. Mass spectra were recorded on an Agilent
(CDCl3): ꢁ 156.5, 151.5, 147.0, 135.9, 133.7, 132.8, 131.5, 130.0,
129.7, 129.3, 128.7, 127.8, 126.6, 126.1, 124.7, 117.6, 21.0. Ms:
[M+ 352].
6320 Ion Trap mass spectrometers at Research Center, King Saud
University, Saudi Arabia. Elemental analysis was carried out for
C, H and N at the Research Centre of College of Pharmacy, King
Saud University, Saudi Arabia and the results are within 0.4% of
the theoretical values. Solvent evaporation was performed under
reduced pressure using Buchan Rotatory Evaporator (Germany).
Ethyl-2-[2-(ethoxycarbonyl)hydrazono]-6-methyl-4-oxo-3-phenyl-
3,4-dihydroquinazoline-1(2H)-carboxylate (10)
A mixture of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-
one (5) (265 mg, 1 mmol) and diethyloxalate (150 mg, 1 mmol)
was refluxed in ethanol (5 mL) for 12 h, and the reaction mixture
was cooled and solvent was evaporated under reduced pressure.
The solid obtained was filtered, washed with water, dried and
recrystallized from ethanol.
Yield 67%, m.p. 221–222 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1747, 1734,
1710 (C¼O), 3288 (NH); 1H NMR (CDCl3): ꢁ 8.14 (s, 1H), 7.51–
7.39 (m, 7H), 7.55 (t, 2H, J ¼ 7.5 Hz), 4.23 (t, 2H, J ¼ 7.0 Hz),
2.38 (s, 3H, CH3), 1.55 (t, 2H, J ¼ 7.5 Hz), 1.23 (q, 3H,
J ¼ 7.5 Hz), 0.82 (t, 3H, J ¼ 7.5 Hz). 13C NMR (CDCl3): ꢁ
156.2, 147.1, 146.5, 142.5, 136.4, 134.1, 132.4, 132.1, 130.1,
129.7, 127.4, 126.6, 126.3, 117.4, 67.3, 30.2, 21.0, 18.9, 13.5. Ms:
[M+ 410].
Thin layer chromatography was performed on precoated
(0.25 mm) silica gel GF254 plates (E. Merck, Darmstadt,
Germany); compounds were detected with 254 nm UV lamp.
Silica gel (60–230 mesh) was employed for routine column
chromatography separations. Compounds 1–3 and
5 were
prepared according to our previous report25, while compounds 4
and 17 were reported by Kennewell et al.26 and Zhao28.
Preparation of compounds 3, 4 and 5
A solution of 2-(ethoxycarbonylmethyl)-thio-3-phenyl-4-oxo-6-
methyl-3H-quinazoline (3.0 g, 0.01 mol) and hydrazine hydrate
(99%, 8 mL) was heated under reflux for 1 h. The obtained solid
was filtered, dried and chromatographed (silica gel, 100 g, elution
with EtOAc–Hexan 1:10 v/v) to give compounds 3, 4 and 5 in 6%,
81% and 10%, respectively.
2-Benzylidenehydrazono-6-methyl-3-phenyl-2,3-dihydroquinazo-
lin-4(1H)-one (12)
1-Substituted-7-methyl-4-phenyl-[1,2,4]triazolo[4,3-a]
quinazolin-5(4H)-one (6–7)
A mixture of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-
one (5) (265 mg, 1 mmol) and benzaldehyde (106 mg, 1 mmol)
was refluxed in methanol (5 mL) for 12 h, and the reaction
mixture was cooled and solvent was evaporated under reduced
pressure. The solid obtained was filtered, washed with water,
dried and recrystallized from ethanol. Yield 80%, m.p. 253–
2-Hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-one
(5)
(265 mg, 1 mmol) and triethylorthoformate or triethylorthoacetate
(2.5 mL) were refluxed for 7 h, and the reaction mixture was
cooled and poured into ice water. The solid obtained was filtered,
washed with water, dried and recrystallized from ethanol.
1
254 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1717 (C¼O), 3322 (NH); H NMR
(DMSO-d6): ꢁ 10.73 (s, 1H, NH, D2O exchangeable), 8.52 (s, 1H),
7.93 (s, 1H), 7.76–7.72 (m, 3H), 7.46–7.00 (m, 9H), 2.35 (s, 3H,
CH3). 13C NMR (DMSO-d6): ꢁ 161.6, 147.8, 146.8, 142.2, 139.0,
138.3, 135.7, 132.4, 129.2, 128.8, 128.5, 126.9, 125.2, 122.3,
119.1, 117.9, 115.2, 20.5. Ms: [M+ 354].
7-Methyl-4-phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one
(6). Yield 64%, m.p. 270–272 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1716
(C¼O); 1H NMR (DMSO-d6): ꢁ 8.54 (s, 1H), 7.52–8.31 (m, 3H),
7.64–6.92 (m, 5H), 2.46 (s, 3H, CH3). 13C NMR (DMSO-d6): ꢁ
154.9, 151.8, 146.8, 142.4, 135.8, 133.0, 132.9, 129.5, 128.2,
125.9, 125.6, 123.5, 20.6. Ms: [M+ 276].
7-Methyl-4-phenyl-1-(phenylamino)-[1,2,4]triazolo[4,3-a]
quinazolin-5(4H)-one (13)
1,7-Dimethyl-4-phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-
one (7). Yield 66%, m.p. 280–282 ꢁC; 1H NMR (C5D5N): ꢁ 9.06
(s, 1H), 8.98 (d, 1H, J ¼ 8.0 Hz), 7.93–7.45 (m, 5H), 7.20–7.19
(m, 1H), 2.34 (s, 3H, CH3), 2.20 (s, 3H, CH3). 13C NMR,
(C5D5N): ꢁ 156.7, 151.4, 148.3, 148.1, 133.6, 133.3, 130.7, 130.5,
128.5, 127.2, 126.8, 120.4, 118.9, 21.4, 12.3. Ms: [M+ 290].
A mixture of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-
one (5) (265 mg, 1 mmol) and phenylisothiocyanate (135 mg,
1 mmol) was refluxed in methanol (5 mL) for 12 h, and the
reaction mixture was cooled and solvent was evaporated under
reduced pressure. The solid obtained was filtered, washed with
water, dried and recrystallized from ethanol.
Yield 73%, m.p. 300–302 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1716 (C¼O),
3327 (NH); 1H NMR (C5D5N): ꢁ 10ꢂ41 (s, 1H, NH, D2O
exchangeable), 8.46 (s, 1H), 8.10 (d, 2H, J ¼ 7.0 Hz), 7.70 (d, 2H,
J ¼ 6.5 Hz), 7.60 (d, 1H, J ¼ 8.0 Hz), 7.40–7.31 (m, 5H), 7.20–
7.17 (m, 1H), 7.08–7.07 (m, 1H), 2.32 (s, 3H, CH3). 13C NMR
(C5D5N): ꢁ 156.5, 147.7, 147.1, 140.5, 133.5, 133.4, 132.2, 130.6,
130.5, 129.9, 129.5, 128.9, 127.0, 126.7, 123.6, 120.6, 119.5,
21.4. Ms: [M+ 367].
1-Substituted-7-methyl-4-phenyl-[1,2,4]triazolo[4,3-a]
quinazolin-5(4H)-one (8–9)
A mixture of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-
one (5) (265 mg, 1 mmol) and appropriate carboxylic acid
(1 mmol) was refluxed for 8 h in a mixture of DMF (5 mL) and
phosphorus oxychloride (5 mL), and the reaction mixture was
cooled and poured into ice water. The solid obtained was filtered,
washed with water, dried and recrystallized from ethanol.

DOI: 10.3109/14756366.2015.1060482
New 6-methyl-3-phenyl-4(3H)-quinazolinone analogues
3
1-Thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-
one (14)
(1 M) trifluoroborane (2 mmol, 136 mg) in (10 mL) dry THF for
12 h. The solvent was removed under reduced pressure, and the
solid obtained was dried and chromatographed (SiO2, 20 g,
elution with CHCl3–AcOEt, 10:1 v/v) afforded compound 19.
Yield 88%, m.p. 230–232 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1704 (C¼O);
1H NMR (CDCl3): ꢁ 8.23 (s, 1H), 7.67–7.59 (m, 6H), 7.50 (d, 1H,
J ¼ 8.5 Hz), 4.62 (s, 2H), 2.48 (s, 3H, CH3).13C NMR (CDCl3): ꢁ
156.3, 149.5, 147.0, 146.5, 136.2, 134.0, 131.2, 130.6, 130.3,
127.6, 126.5, 126.1, 117.5, 33.6, 21.0. Ms: [M+ +1 325].
To a solution of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-
4(3H)-one (5) (2 mmol, 530 mg) in absolute ethanol (20 mL)
containing potassium hydroxide (5 mmol, 280 mg), carbon disul-
fide (760 mg, 10 mmol) was added and the reaction mixture was
stirred at room temperature for 3 h followed by heating under
reflux for 6 h. The reaction mixture was cooled; the solvent was
evaporated under reduced pressure. The residual solid obtained
was dissolved in water, acidified with dilute hydrochloric acid;
and the precipitated solid was filtered, washed with water and
crystallized from ethanol.
Antitumor screening
A primary anticancer assay was performed for an approximately
Yield 81%, m.p. 294–296 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1707 (C¼O), 60 human tumor cell lines panel derived from nine neoplastic
3330 (NH); 1H NMR (DMSO-d6): ꢁ 12ꢂ99 (s, 1H, SH, D2O diseases, in accordance with the protocol of the Drug Evaluation
exchangeable), 7.75 (s, 1H), 7.61 (d, 1H, J ¼ 7.0 Hz), 7.50–7.47 Branch, National Cancer Institute, Bethesda, MD29–33
(m, 2H), 7.42 (d, 1H, J ¼ 7.5 Hz), 7.36 (d, 1H, J ¼ 8.5 Hz), 7.27
.
(d, 2H, J ¼ 7.5 Hz), 2.37 (s, 3H, CH3). 13C NMR (DMSO-d6): ꢁ Antimicrobial screening
175.5, 159.8, 150.2, 139.3, 137.6, 136.6, 133.9, 129.0, 128.9,
The initial screening of antimicrobial activity and determination
of minimum inhibitory concentration (MIC) for the tested
compounds were performed by cup plate and broth dilution
methods, respectively with different strains34. Eighteen different
synthesized compounds were screened for their antimicrobial
128.0, 126.7, 125.6, 116.0, 115.7, 20.4. Ms: [M+ 308].
2-Chloro-N0-(6-methyl-4-oxo-3-phenyl-3,4-dihydroquinazolin-
2(1H)-ylidene)acetohydrazide (15)
A solution of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)- activity against four bacterial standard strains (Staphylococcus
one (5) (2 mmol, 530 mg) was stirred at room temperature with aureus ATCC 29213, Bacillus subtilis ATCC 10 400 and
chloroacetylchloride (2 mmol, 225 mg) in dichloromethane Escherichia coli ATCC 25922) and one yeast standard strain,
(10 mL) containing triethylamine (5 mmol, 505 mg) for 12 h and Candida albicans ATCC 2091. The tested compounds were
the reaction mixture was heated under reflux for 6 h. The reaction dissolved in dimethyl sulfoxide (DMSO) to obtain stock solution
mixture was diluted with water and extracted with chloroform, the (5120 mg/mL).
extract was washed successively with water and brine, dried over
anhydrous MgSO4, evaporated under reduced pressure and solid Cup plate method
obtained was crystallized from ethanol. Yield 88%, m.p. 215–
The tested organisms were grown in Cation Adjustment Mueller-
216 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1712, 1684 (C¼O), 3343, 3318 (NH);
Hinton (CAMH) broth (MerckÕ, Darmstadt, Germany) to mid-log
1H NMR (DMSO-d6): ꢁ 11ꢂ49 (s, 1H, NH, D2O exchangeable),
phase. The bacterial suspension was measured spectroscopically
9.80 (s, 1H, NH, D2O exchangeable), 7.80 (s, 1H), 7.68 (d, 2H,
using Spectrophotometer (LKBÕ Ultrospec) at 625 nm to give
J ¼ 7.5 Hz), 7.59–7.52 (m, 2H), 7.43 (t, 1H, J ¼ 7.5 Hz), 7.37 (d,
absorbance 0.1–0.14 (1 ꢃ 108 CFU/mL). The bacterial suspension
1H, J ¼ 8.0 Hz), 7.23 (t, 1H, J ¼ 8.0 Hz), 4.25 (s, 2H), 2.40 (s, 3H,
was diluted 1:100 in CAMH broth to obtain 1 ꢃ 106 CFU/mL.
CH3).13C NMR (DMSO-d6): ꢁ 166.7, 158.1, 147.8, 137.0, 136.6,
This suspension was swabbed on a CAMH agar plate (MerckÕ,
133.4, 129.6, 128.8, 126.0, 125.0, 123.5, 123.2, 115.3, 41.6, 20.4,
Darmstadt, Germany) and allowed to dry completely. Then six
Ms: [M + 1 343].
cups per plate were made in agar plate using cork borer. A 1 mL
of stock solution (5120 mg/mL) was one 2-fold diluted in 1 mL
2-Amino-6-methyl-3-phenylquinazolin-4(3H)-one (16), 6-methyl-
3-phenylquinazolin-4(3H)-one (17)
DMSO to obtain 2560 mg/mL. A 100 mL (256 mg) of the tested
compound was added into the cup. The plates were kept in
refrigerator at 4 ꢁC for 30 min for diffusion. Then, the plates were
incubated at 37 ꢁC for 24 h. After the incubation period, the
diameter of the inhibition zone (including the diameter cup) was
measured and recorded in mm. Ciprofloxacin, Ampicillin (10 mg/
cup) Flucytosine and Fluconazole (10 mg/mL) were used as
positive controls for antibacterial and antifungal, respectively. The
experiment was carried out in duplicate.
A solution of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-
one (5) (2 mmol, 530 mg) in (10 mL) absolute methanol containing
triethylamine (4 mmol, 405 mg) was refluxed for 12 h. The reaction
mixture was allowed to cool, the solvent was removed under
reduced pressure, and the solid obtained was dried and chromato-
graphed (SiO2, 20 g, elution with CHCl3–AcOEt, 10:1 v/v)
afforded compounds 16 and 17 in 32% and 31% yield, respectively.
Determination of MIC
2-Amino-6-methyl-3-phenylquinazolin-4(3H)-one
(16). Yield
32%, m.p. 228–230 ꢁC; IR (KBr, cmꢀ1) ꢀ: 1706 (C¼O), 3337,
3314 (NH); 1H NMR (DMSO-d6): ꢁ 10.80 (s, 1H, NH, D2O
exchangeable), 8.69 (s, 1H, NH, D2O exchangeable), 7.76 (s, 1H),
7.74 (d, 2H, J ¼ 7.5 Hz), 7.47 (d, 1H, J ¼ 7.0 Hz), 7.36–7.31 (m,
3H), 7.03 (t, 1H, J ¼ 7.0 Hz), 2.36 (s, 3H). 13C NMR (DMSO-d6):
ꢁ 161.6, 147.6, 147.8, 146.8, 139.0, 135.7, 132.3, 128.8, 126.4,
122.3, 120.6, 119.1, 118.0, 20.5. Ms: [M+ 251].
MIC was determined for the compounds that show antimicrobial
activity by cup plate method. Briefly, 2 mL of CAMH broth
(Oxoid Chemical Co., UK) was dispensed into screw glass tubes
(100 mm ꢃ 760 mm). Then the tubes were autoclaved. For each
compound, 14 tubes were used. Tubes number 13 and 14 were
used as positive growth control (no tested compound) and
negative control for medium sterility (no microorganism),
respectively. A 1 mL of stock solution (5120 mg/mL) was 10-
fold diluted in 9 mL CAMH to obtain 512 mg/mL. A 2 mL of the
tested compounds (512 mg/mL) was pipetted into the first tube and
mixed well. Then 2 mL was withdrawn from the 1st tube and
1-(Chloromethyl)-7-methyl-4-phenyl-[1,2,4]triazolo[4,3-a]
quinazolin-5(4H)-one (19)
A solution of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)- added to the 2nd tube to make a 2-fold dilution. This procedure
one (5) (2 mmol, 530 mg) was stirred at room temperature with was repeated down to 12th tube to reach the concentration of

4
A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
0.125 mg/mL. A 2 mL was discarded from the 12th tube. A volume GBVI/WSA dG scoring function which estimates the free
of 2 mL of inoculums (1 ꢃ106 CFU/mL) was added to all tubes energy of binding of the ligand from a given pose was used to
except tube number 14 to give final 1 ꢃ 106 CFU/mL. Ampicillin rank the final poses. The ligand–enzyme complex with lowest
and Fluconazole were used as positive controls for antibacterial S score was selected.
and antifungal, respectively. The inoculated tubes were incubated
at 37 ꢁC for 20 h. After the incubation period, the results of MIC Results and discussion
were recorded manually and interpreted according to the guide-
lines of EUCAST.
Chemistry
Synthesis of 2-mercapto-6-methyl-3-phenylquinazolin-4(3H)-one
Docking methodology
as starting material in 90% yield was achieved by the reaction of
5-methylanthranilic acid with 2-phenylisothiocyanate in absolute
ethanol25. 2-Mercapto-6-methyl-3-phenylquinazolin-4(3H)-one
was reacted with ethyl 2-bromoacetate in dry acetone in the
presence of potassium carbonate at room temperature to afford
ethyl 2-[(6-methyl-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)
thio]acetate (1) in 95% yield, and the latter compound was
Molecular modeling studies were performed with MOE 2008.10,
software available from Chemical Computing Group Inc., 1010
Sherbrooke Street West, Suite 910, Montreal, Canada H3A2R7.
Selection of protein crystal structures
Ligand-bound crystallographic structure of epidermal growth heated with hydrazine hydrate in ethanol to furnish 2-[(6-methyl-
factor receptor (EGFR) kinase is available in the Protein Data 4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio]acetohydrazid
Bank. In this study, EGFR kinase crystal structure 1M17 was (2) in 90% yield. Furthermore, heating of the ester 1 with
evaluated and selected for docking. The errors of the protein were hydrazine hydrate gave 2-hydrazino-3-(2-methylphenyl)-3H-qui-
corrected by the structure preparation process in MOE. The first nazolin-4-one (5) as a key intermediate in 81% yield, in addition
step in the generation of suitable protein structures is the to 2-hydrazinyl-6-methylquinazolin-4(3H)-one (3) and 3-amino-
assignment of hydrogen positions on the basis of default rules. 2-hydrazinyl-6-methylquinazolin-4(3H)-one (4) as by-products in
Water molecules contained in the PDB file have been removed. 6% and 10% yield, respectively (Scheme 1).
Finally, partial charges (Gasteiger methodology) were calculated
and the active site of the ensemble has been defined as the was reacted with various one carbon donors, such as triethy-
2-Hydrazino-3-(2-methylphenyl)-3H-quinazolin-4-one
(5)
˚
collection of residues within 10.0 A of the bound inhibitor and lorthoformate, triethyortholacetate, cinnamic acid and benzoic
comprised the union of all ligands of the ensemble. All atoms acid, to produce 1-substituted-7-methyl-4-phenyl-[1,2,4]tria-
˚
located less than 10.0 A from any ligand atom were considered.
zolo[4,3-a]quinazolin-5(4H)-one derivatives (6–9) in 60–66%
yields. Ethyl 2-(2-(ethoxycarbonyl)hydrazono)-6-methyl-4-oxo-
3-phenyl-3,4-dihydroquinazoline-1(2H) carboxylate (10) was
obtained in 67% yield by the reaction of compound 5 with
diethyloxalate (Scheme 2).
Additionally, the synthesis of [(benzylidene)hydrazono]-6-
methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one (12) in 80%
yield and 7-methyl-4-phenyl-1-(phenylamino)-[1,2,4]triazolo[4,3-
a]quinazolin-5(4H)-one (13) in 73% yield were achieved by
boiling of compound 5 with benzaldehyde or phenylisothiocya-
nate in methanol. Moreover, compound 5 was treated with carbon
disulfide in ethanolic potassium hydroxide to afford 7-methyl-4-
Preparation of the ligand
The ligand coordinates were built using the builder tool of the
MOE program. Next, the correct atom types (including hybrid-
ization states) and correct bond types were defined, hydrogen
atoms were added, charges were assigned to each atom and finally
the structures were energy-minimized (MMFF94x, gradient:
0.01).
Docking experiment
The docking experiment on EGFR kinase was carried out by phenyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-
superimposing the energy minimized ligand on erlotinib in the 5(1H)-one (14) in 81% yield (Scheme 3).
PDB file 1M17, after which erlotinib was deleted. The default
triangle matcher placement method was used for docking. 3,4-dihydroquinazolin-2(1H)-ylidene)acetohydrazide (15) was
On the other hand, 2-chloro-N0-(6-methyl-4-oxo-3-phenyl-
O
N
O
N
O
N
N
N
H
N
N
BrCH COOEt
2
NH NH ,
O
2
2
NH
S
S
2
EtOH,
reflux
SH
O
O
1
2
NH NH , reflux
2
2
O
O
N
O
N
NH
2
NH
N
+
N
NH
+
NH
2
NH
2
2
N
N
H
N
H
N
H
3
4
5
Scheme 1. Synthesis of 2-hydrazinyl-6-methyl-3-phenyl-quinazolin-4(3H)-one (5) as a key intermediate.

DOI: 10.3109/14756366.2015.1060482
New 6-methyl-3-phenyl-4(3H)-quinazolinone analogues
5
obtained by heating compound 5 with chloroacetylchloride in Antitumor activity
dichloromethane. An attempt to cyclize compound 15 by boiling
In vitro antitumor evaluations of seven compounds indicated in
Table 1 were selected by National Cancer Institute, Bethesda,
Maryland, USA on the basis of degree of the structure variation
and computer modeling techniques for evaluation of their
anticancer activity.
The selected compounds 5–9, 12 and 13 were subjected to the
National Cancer Institute (NCI) in vitro disease-oriented human
cells screening panel assay for in vitro antitumor activity. A single
dose (10 mM) of the test compounds is used in the full NCI
60 cell lines panel assay including nine tumor subpanels namely;
in methanol containing triethylamine was unsuccessful and a
mixture of 2-amino-6-methyl-3-phenylquinazolin-4(3H)-one (16)
and 6-methyl-3-phenylquinazolin-4(3H)-one (17) was obtained
rather
than
1-(chloromethyl)-7-methyl-4-phenyl-[1,2,4]tria-
zolo[4,3-a]quinazolin-5(4H)-one (18) in 32% and 31% yields,
respectively. Finally, compound 15 was cyclized to the corres-
ponding 1-(chloromethyl)-7-methyl-4-phenyl-[1,2,4]triazolo[4,3-
a]quinazolin-5(4H)-one (19) by stirring the reaction mixture in
tetrahydrofuran containing Lewis acid, such as borontriflouride,
in 88% yield (Scheme 4).
O
O
N
N
orthoesters,
reflux
RCOOH,
5
POCl , reflux
3
N
N
N
N
N
N
R
R
diethyloxalate
EtOH, reflux
6; R= H
R= methyl
8; R= srtyryl
9;
7;
diethyloxalate
EtOH, reflux
R= Phenyl
X
O
N
O
N
N
N
N
N
N
H
O
O
NH
O
O
O
O
10
11
Scheme 2. Reactions of compound 5 with orthoesters, carboxylic acids and diethyloxalate.
O
N
O
N
O
N
N
NH
N
CS , EtOH
RCHO, MeOH
reflux
2
N
NH
2
N
KOH, reflux
N
N
H
NH
5
S
14
PhNCS
MeOH, reflux
12
O
N
N
N
N
HN
13
Scheme 3. Reactions of compound 5 with benzaldehyde, phenylisothiocyanate and carbondisulfide.

6
A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
O
N
O
N
NH
2
N
16
O
N
H
N
N
+
ClCH COCl
2
TEA, toluene
reflux
NH
NH
2
N
N
H
CHCl , reflux
3
O
Cl
O
N
5
15
TEA, MeOH
reflux
X
N
BF , THF
3
17
O
N
O
N
O
N
N
N
N
N
Cl
N
19
18
Scheme 4. Synthesis of 1-(chloromethyl)-7-methyl-4-phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (19).
Table 1. Antitumor activity of quinazoline derivatives 5–9, 12 and 13 presented as growth inhibition percentages (GI%) over 60 subpanel tumor
cell lines.
60 cell lines assay in one dose 10.0 mM concentration: GI%
Compd No.
MGI%
3
PCE
9/55
Most sensitive cell lines
5
Leukemia (MOLT-4: 14.0%, SR: 22.0%), NSC Lung Cancer (EKVX 27.0%), Colon
Cancer (HCT-15: 25%), CNS Cancer (SF-268: 11%, SF-295: 21%), Melanoma
(MDA-MB-435: 13%), Breast Cancer (MDA-MB-231/ATCC: 60%, BT-549:
11%)
6
7
3
0
2/44
4/55
Leukemia (NCI-H522: 10%), Melanoma (UACC-62: 10%)
CNS Cancer (SF-295: 10%), Breast Cancer (MDA-MB-231/ATCC: 17%, BT-549:
10%, MDA-MB-468: 13%)
8
5
12/54
Leukemia (K-562: 16%, MOLT-4: 13%, SR: 22%), NSC Lung Cancer (EKVX: 52%),
Colon Cancer (HCT-15: 12%), CNS Cancer (SF-295: 36%, SF-539: 15%),
Melanoma (MDA-MB-435: 18%), Ovarian Cancer (OVCAR-5: 11%), Renal
Cancer (CAKI-1: 17%), Breast Cancer (BT-549: 17%, T-47D: 10%)
Leukemia (MOLT-4: 10%, SR: 26%), NSC Lung Cancer (NCI-H460: 11%), Colon
Cancer (HCT-116: 16%), CNS Cancer (SF-539: 12%), Melanoma (MDA-MB-
435: 14%), Ovarian Cancer (IGROV1: 19%, NCI/ADR-RES: 11%), Renal
Cancer (UO-31: 16%), Breast Cancer (MDA-MB-231/ATCC: 13%, BT-549:
26%)
9
4
11/55
12
13
12
11
23/55
25/55
Leukemia (CCRF-CEM: 32%, K-562: 43%, MOLT-4: 22%, SR: 51%), NSC Lung
Cancer (NCI-H226: 11%, NCI-H460: 44%), Colon Cancer (HCT-116: 35%,
HCT-15: 57%), CNS Cancer (SF-268: 12%, SF-295: 25%, SNB-75: 11%),
Melanoma (LOX IMVI: 30%, SK-MEL-5: 32%, UACC-62: 20%), Ovarian
Cancer (OVCAR-4: 21%, OVCAR-5: 11%, NCI/ADR-RES: 14%), Renal Cancer
(786-0: 12%, CAKI-1: 12%), Prostate Cancer (PC-3: 30%), Breast Cancer
(MCF7: 14%, MDA-MB-231/ATCC: 17%, MDA-MB-549: 11%)
Leukemia (K-562: 16%, MOLT-4: 30%, SR: 25%), NSC Lung Cancer (EKVX: 16%,
NCI-H226: 11%, NCI-H23: 16%), CNS Cancer (SF-295: 14%, SF-539: 15%,
SNB-19: 15%, SNB-75: 18%), Melanoma (MALME-3M: 17%, M14: 12%, MDA-
MB-435: 25%, SK-MEL-5: 13%, UACC-62: 10%), Ovarian Cancer (OVCAR-4:
21%, NCI/ADR-RES: 18%), Renal Cancer (ACHN: 13%, CAKI-1: 31%, RXF
393: 12%, UO-31: 37%), Prostate Cancer (PC-3: 16%), Breast Cancer (MDA-
MB-231/ATCC: 35%, BT-549: 30%, MDA-MB-468 19%)
PCE: positive cytotoxic effect, which is the ratio between number of cell lines with percentage growth inhibition410% and total number of cell lines.
MGI%: mean growth inhibition percentage.

DOI: 10.3109/14756366.2015.1060482
New 6-methyl-3-phenyl-4(3H)-quinazolinone analogues
7
Table 2. Antitumor activity of compounds 5, 8, 12 and 13 with GI% over
the most sensitive tumour cell line.
Activity against colon cancer; compounds 5, 8 and 12 showed
GI values of 23%, 12% and 22% with colon HCT-15 cell line,
respectively, while compounds 9 and 12 demonstrated moderate
activities against HCT-116 cancer cell line with GI values of 16%
and 35%, respectively.
% Growth Inhibition (GI%)*
Subpanel tumor cell lines
5
8
12
13
5-Flu
Concerning CNS cancer; compounds 5, 7, 8, 9, 12 and 13
showed moderate potency against CNS cancer SF-295 cell line
with GI values of 21%, 10%, 36% and 25%, respectively.
Compounds 8, 9 and 13 showed GI values of 15%, 12% and
15% to CNS cancer SF-539 cell line, respectively. CNS cancer
SF-268 proved to be sensitive to compounds 5 and 12 with GI
values of 11% and 12%, while compounds 12 and 13 showed
certain activity to SNB-75 cancer cell with GI values of 11% and
18%, respectively. Additionally, compound 13 displayed modest
activity against SNB-19 cancer cell with GI value of 15%.
Regarding Melanoma; compounds 5, 9 and 13 were active
against MDA-MB-435 cell line with GI values of 13%, 14% and
25%, respectively. Melanoma UACC-62 and LOX IMVI cell lines
were sensitive to compounds 6 and 12 in 10% and 30%,
respectively.
Compound 13 showed moderate activities against MALME-
3 M and M14 cell lines in 17% and 12%, while compounds 12 and
13 possessed activities against UACC-62 and SK-MEL-5 cell
lines with GI values of 20%, 10%, 32% and 13%, respectively.
Investigation of activity toward ovarian cancer; compounds 12
and 13 showed moderate activities against Ovarian NCI/ADR-
RES and OVCAR-4 cell lines with GI values of 14%, 18%, 21%,
and 21%, respectively. OVCAR-5 cell line was perceptive to
compounds 8 and 12 with GI values of 11%, while Ovarian
IGROV1 and NCI/ADR-RES cell lines were responsive to
compound 9 with GI values of 19% and11%, respectively.
Relating to renal cancer; compounds 8 and 12 were active
against renal CAKI-1 cell line with GI values of 17% and 12%,
respectively, whereas ACHN, RXF 393, UO-31 and CAKI-1 cell
lines were sensitive to compound 13 with GI values of 13%, 12%,
37% and 31%, respectively. Renal UO-31and 786-0 cell lines were
responsive to compounds 9 and 12 with GI values of 16% and
12%, respectively. Prostate PC-3 cell line proved to be selectively
sensitive to compounds 12 and 8 with GI value of 30% and 16%,
respectively.
Leukemia
CCRF-CEM
K-562
SR
MOLT-4
NSC Lung Cancer
EKVX
NCI-H460
Colon Cancer
HCT-116
HCT-15
CNS Cancer
SF-295
Melanoma
LOX IMVI
MDA-MB-435
SK-MEL-5
Renal Cancer
CAKI-1
UO-31
Prostate Cancer
PC-3
Breast Cancer
MDA-MB 231/ATCC
BT-549
–
–
–
–
–
–
–
–
32
43
51
–
–
–
25
30
57.1
42.3
25
43
27
–
52
–
–
44
–
–
39.0
13
–
25
–
–
35
57
–
–
17.8
26
–
36
–
–
69.1
–
–
–
–
–
–
30
–
32
–
25
–
30.4
36.6
95.5
–
–
–
–
–
–
31
37
39.4
41.3
–
–
30
–
58.2
60
–
–
26
–
–
35
30
78.1
Nt
5-Flu: 5-fluorouracil, Nt: not tested.
*Only GI% higher than 25% are shown.
Leukemia, Non-small cell lung, Colon, CNS, Melanoma, Ovarian,
Renal, Prostate and Breast cancer cells29–33. The data are reported
as mean-graph of the percent growth of the treated cells, and
presented as percentage growth inhibition (GI%) caused by the
test compounds (Table 1).
The tested compounds 5–9, 12 and 13 displayed moderate
activities in the in vitro screening on the tested cell lines in 10 mM
concentration with positive cytotoxic effect (PCE) of 2/44, 4/55,
9/55, 12/54, 11/55, 23/55 and 25/55 respectively (Table 1).
Compounds 5, 8, 9, 12 and 13 showed a cytotoxic effect on some
of the cancer cell lines with mean growth inhibition percentages
(MGI%) of 3%, 5%, 4%, 12% and 11%, respectively (Table 1).
Comparison of the antitumor activities of compounds 5, 8, 12
and 13 with GI% over the most sensitive tumor cell line against 5-
flurouracil showed that compounds 5, 8, 12 and 13 (Table 2)
possess activities almost equal to or higher than those of 5-
flurouracil against most cell lines (Figure 1).
Pertaining to breast cancer; breast MDA-MB-231/ATCC and
BT-549 cell lines possessed convinced response to compounds 5,
7, 9, 12 and 13 with GI values of 60%, 17%, 13%, 17%, 35%, 11%,
10%, 26%, 11%, and 30%, respectively. Compounds 7 and 13
showed GI effectiveness against breast MDA-MB-468 cell line
with values of 13% and 19%, respectively. Additionally, com-
pounds 8 and 12 have selective activities against breast T-47D and
MCF7cell lines with GI values of 10% and 14%, respectively.
Regarding the activity toward individual cell lines; com-
pounds 5, 8, 9, 12 and 13 showed selective activities against
MOLT-4 and SR leukemia cell lines with GI values of 14%,
13%, 10%, 22%, 30%, 22%, 22%, 26%, 51% and 25%,
respectively. Additionally, compounds 8, 12 and 13 disclosed
activity against K-562 leukemia cell line with GI values of 16%,
43% and 16%, respectively, whereas NCI-H522 and CCRF-CEM
leukemia cell lines were sensitive to compounds 6 and 12,
respectively.
Non-small cell lung; EKVX cell line proved to be selectively
sensitive to compounds 5, 8 and 13 with GI values of 27%, 52%
and 16%, respectively. In addition, compounds 9 and 12 proved to
be susceptible to the NCI-H460 cell line with GI values of 11%
and 44%, respectively. Compounds 12 and 13 have dedicated
activity against NCI-H226 cell line with GI values of 11% of each;
meanwhile, compound 13 showed certain activity against
NCI-H322 cell line in 13%.
Antibacterial and antifungal activities
Compounds 1–19 in addition to the reference Ciprofloxacin and
Ampicillin were tested for their in vitro antibacterial activity
against Gram negative (E. coli ATCC 25922), Gram-positive (S.
aureus ATCC 29213 and B. subtilis ATCC 10400) microorgan-
isms. Also compounds 1–19 in addition to the references
Fluconazole and Flucytosine were tested for their in vitro
antifungal activity against Candida albicans ATCC2091. The
minimal inhibitory concentrations (MIC in mg/mL) or the lowest
drug concentrations that prevent visible growth of bacteria
(Table 3) were determined by a standard broth micro-dilution
technique using the European Committee for Antimicrobial
Susceptibility Testing (EUCAST) and Laboratory Standards
method34.
The results of MIC tests against Gram-positive and Gram-
negative bacteria revealed that compounds 2, 6, 8 and 17 were

8
A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
Figure 1. Antitumor activity of compounds 5, 8, 12 and 13 with GI% over the most sensitive tumour cell line.
Table 3. Antibacterial and antifungal activity of compounds 1–19 with MIC mg/mL.
Antimicrobial activities
Bacillus subtilis Escherichia coli
ATCC 10400 ATCC 25922
Staphylococcus aureus
ATCC 29213
Candida albicans
ATCC2091
Cup plate
(mm)
MIC
(mg/mL)
Cup plate
(mm)
MIC
(mg/mL)
Cup plate
(mm)
MIC
(mg/mL)
Cup plate
(mm)
MIC
(mg/mL)
Compound No.
1
2
3
4
5
6
7
8
19
Nil
Nil
11
Nil
Nil
Nil
Nil
17
12
Nil
10
17
11
10
16
ND
ND
128
ND
ND
ND
ND
32
128
ND
256
32
256
256
ND
128
1
16
ND
ND
Nil
Nil
15
14
12
Nil
14
Nil
15
11
12
Nil
15
Nil
Nil
Nil
12
25
18
ND
ND
128
128
256
ND
128
ND
128
256
256
ND
128
ND
ND
ND
128
1
12
Nil
Nil
11
Nil
Nil
13
Nil
Nil
Nil
13
15
19
Nil
Nil
Nil
Nil
29
22
ND
ND
64
ND
ND
64
ND
ND
64
ND
ND
ND
64
32
32
ND
ND
ND
ND
1
16
ND
ND
10
Nil
Nil
Nil
15
Nil
Nil
Nil
14
Nil
Nil
16
15
10
Nil
Nil
11
ND
ND
28
256
ND
ND
ND
64
ND
ND
ND
64
9
10
12
13
14
15
16
17
19
CI
AM
FLU
FL
ND
ND
64
64
256
ND
ND
256
ND
ND
16
Nil
12
27
20
ND
ND
16
ND
ND
ND
ND
18
16
FLU: Fluconazole; FL: Flucytosine; AM: Ampicillin; CI: Ciprofloxacin; MIC ¼ minimum inhibitory concentration; ND ¼ not determined.
inactive against all tested organisms either bacteria or fungi. On with MIC; 16, 128, 32, 128, 256, 32, 256, 256, 128 mg/mL,
the other hand, the tested compounds were more active against respectively.
Gram-positive bacteria (S. auerus, B. subtilis) than Gram-negative
bacteria (E. coli).
Additionally, compounds 3, 4, 5, 7, 9, 10, 12, 14 and 19
possessed activities against Gram-positive B. subtilis ATCC
Compounds 1, 4, 9, 10, 13, 14, 15, 16 and 19 exhibited 10400 with MIC; 128, 128, 256, 128, 128, 256, 256, 128, 128 mg/
selective activity against Gram-positive S. aureus ATCC 29213 mL, respectively. Compounds 4, 9, 10, 14 and 19 have broad

DOI: 10.3109/14756366.2015.1060482
New 6-methyl-3-phenyl-4(3H)-quinazolinone analogues
9
Figure 2. Antibacterial and antifungal activity of the active compounds against the reference drugs with MIC mg/mL.
Table 4. Calculated Lipinski’s rule of five for the designed antitumor compounds.
Parameter
Comp. No.
PCE*
MRy
Log Pz
PSAô
MWx
nOHNHjj
nON#
Nviolations**
5
6
7
8
9/55
2/44
4/55
12/54
11/55
23/55
25/55
–
76.67
80.77
85.19
115.09
105.87
107.52
108.93
25.9
1.46
2.9
3.13
5.0
4.54
4.68
4.8
70.72
50.50
51.02
51.02
51.02
57.06
62.53
58.2
266.3
276.29
290.32
378.43
352.39
354.4
367.4
130.0
393.0
3
0
0
0
0
1
1
2
1
5
5
5
5
5
5
6
4
7
0
0
0
0
0
0
0
0
0
9
12
13
5-Flu
Erlotinib
ꢀ0.84
–
110.06
3.07
74.73
*Data taken from Table 1.
yMolar refractometry.
zCalculated lipophilicity.
2
˚
ôPolar surface area (A ).
xMolecular weight.
jjNumber of hydrogen bond donor.
#Number of hydrogen bond acceptor.
**Number of violation from Lipinski’s rule of five.
In silico studies of antitumor activity
spectrum activities against both B. subtilis ATCC 10400 and
S. aureus ATCC 29213. Compounds 1, 4, 7, 12, 13 and 14 were
sensitive to Gram-negative E. coli ATCC 25922 in comparison
with MIC; 64, 64, 64, 64, 32 and 32 mg/mL, respectively.
Compounds 4 and 14 show extended spectrum activity against S.
aureus ATCC 29213, B. subtilis ATCC 10400 and E. coli ATCC
25922.
Regarding antifungal activities, compounds 1, 5, 9, 13, 14,
15 and 19 possessed activities against C. albicans ATCC 2091
with MIC; 256, 64, 64, 64, 64, 256 and 256 mg/mL, respect-
ively. The results revealed that compounds 9 and 14 were found
to be most active against S. auerus ATCC 29213 with MIC of
32 mg/mL and inhibition zone of 17 mm, while compound 14
had activity against all tested strains either bacterial or C.
albicans strain with lowest MIC against all the tested strains
(Figure 2).
Lipinski’s rule of five (the effect of lipophilic and
steric parameters)
As a part of our study, the compliance of compounds to the
Lipinski’s rule of five was evaluated35. In addition, the polar
surface area (PSA) of the tested compounds was also calculated
(Table 4), since it is another key property that has been linked to
drug bioavailability, where passively absorbed compounds with a
2
36
˚
PSA 4140 A are thought to have low oral bioavailability . The
results disclosed in Table 4 show that all of the synthesized
compounds comply with these rules (molecular weight ¼ 266–
378, Clog P ¼ 1.46–5.0, nON ¼ 5–6 and nOHNH ¼ 0–3). Hence,
theoretically, all of these compounds should present good passive
oral absorption and the differences in their bioactivity cannot be
attributed to this property24,35–37
.

10 A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15

DOI: 10.3109/14756366.2015.1060482
New 6-methyl-3-phenyl-4(3H)-quinazolinone analogues 11
Toxicity effects
Compd No.
M
T
I
R
5
Low
Low
Low
Low
Low
Low
Low
Low
High
High
Low
Low
Low
Low
Low
Low
Low
High
Low
Low
Low
Low
Low
Low
Low
Low
High
Low
Low
Low
Low
Low
Low
Low
Low
High
6
7
8
9
12
13
Erlotinib
5-Flu
Drug Score
Drug-Likeness
0.6
0.5
0.4
0.3
0.2
0.1
0
6
4
0.52
4.63
0.51
3.98
3.97
2.54
3.88
0.45
2.37
0.38
0.38
0.35
0.34
0.33
2
0
−0.4
0.06
−2
−4
−6
−8
−4.5
−6.73
Figure 3. In silico toxicity risks (upper panel), drug-Likeness (lower right panel) and drug-Score (lower left panel) of the active antitumor quinazoline
derivatives compared with reference drugs 5-Flu and Erlotinib (M, mutagenic; T, tumorigenic; I, irritant; R, reproductive).
Table 6. In silico predicted bioactivity of the designed antitumor compounds compared with reference drugs 5-Flu and Erlotinib.
Protease
inhibitor
Enzyme
inhibitor
GPCR
ligand
Kinase
inhibitor
Ion channel
modulator
Nuclear
receptor ligand
Compounds
5
6
7
8
ꢀ0.69
ꢀ0.91
ꢀ0.96
ꢀ0.71
ꢀ0.75
ꢀ0.72
ꢀ0.61
ꢀ0.16
ꢀ3.15
ꢀ0.25
ꢀ0.52
ꢀ0.65
ꢀ0.49
ꢀ0.56
ꢀ0.37
ꢀ0.47
0.18
ꢀ0.34
ꢀ0.52
ꢀ0.47
ꢀ0.30
ꢀ0.41
ꢀ0.40
ꢀ0.38
0.13
ꢀ0.22
ꢀ0.28
ꢀ0.33
ꢀ0.22
ꢀ0.18
ꢀ0.33
ꢀ0.04
0.68
ꢀ0.62
ꢀ0.48
ꢀ0.55
ꢀ0.39
ꢀ0.47
ꢀ0.66
ꢀ0.45
0.11
ꢀ0.80
ꢀ1.07
ꢀ1.09
ꢀ0.76
ꢀ0.82
ꢀ0.71
ꢀ0.94
ꢀ0.10
ꢀ3.04
9
12
13
Erlotinib
5ꢀFlu
ꢀ1.56
ꢀ2.60
ꢀ2.61
ꢀ1.95
The introduction of cyclic ring fragments incorporating the ADME-Tox evaluation
quinazoline core and the variation of the substituents on these
To estimate the prospect of the designed compounds as antitumor
agents compared with the reported antitumor agents erlotinib and
5-Flu; their drug-likeness were calculated according to absorp-
tion, distribution, metabolism, elimination and toxicity (ADME-
T) program (Table 5), and defined human intestinal absorption
(HIA) model37,38. It was predicted that the examined compounds
could be transported across the intestinal epithelium, and they can
cross the blood–brain barrier (BBB) and are of medium aqueous
soluble. The values of HIA, protein binding, BBB crossing and
solubility prediction for all compounds are presented in Table 5
using iLab2 online program (https://ilab.acdlabs.com/iLab2/
index.php). In general, all compounds presented some advantages
and disadvantages when compared to each other and the results
were compared with erlotinib and 5-Flu as reference drugs. No
marked differences, in human oral bioavailability (30%–470%),
human intestinal absorption (98–100%), human jejunum perme-
ability and health effects in rodent toxicity profiles, were observed
fragments have allowed us to evaluate the influence of
lipophilicity and steric parameters at the pharmacophoric part
of the molecules. Table 4 gathers cytotoxic effect as well as values
of Clog P (lipophilic factor), molar refractometry and PSA (steric
factors) for each compound, determined by using online
molinspiration program (http://www.molinspiration.com/cgi-bin/
properties) and iLab2 online program (https://ilab.acdlabs.com/
iLab2/index.php). It is clear that the cytotoxic effect increases
with the increase in molar refractometry from 76–85 cm3/mol
(5–7) to 105–115 cm3/mol (8, 9, 12 and 13). The optimal
refractometry for the active compounds was found to lie in the
range of 105.87–108.93 cm3/mol (Table 4). Although, lipophili-
city does not exert a significant effect on activity, an increase in
potency was observed in compounds 8, 9, 12 and 13 with log P
values 43.0 and ꢄ5.0. The results shown in Table 4 have almost
similar values with erlotinib which indicated the structural
similarity of the designed molecules with erlotinib.

12 A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15

DOI: 10.3109/14756366.2015.1060482
New 6-methyl-3-phenyl-4(3H)-quinazolinone analogues 13
among the designed compounds. However, the solubility related predict if these compounds have analogous binding mode to the
parameters call for attention, since the promising compounds EGFR inhibitors24,41–43. Compounds 5, 12 and 13 were used for
were calculated to be at least as soluble as the reported docking study as representative examples of good predicted
compounds, and are predicted to have oral bioavailability and kinase inhibiting activity (Table 6) and promising antitumor
absorption significantly higher than that of the reported antitumor activity (Tables 1 and 2, and Figure 4). All the calculations were
agents 5-Flu and similar to erlotinib. It can be deduced from these performed using MOE 2008.10 software44 installed on 2.4G
results that the pharmacokinetic profile of the designed com- CoreÔ i7. The crystal structure of EGFR with erlotinib
pounds is affected and modified by the presence of cyclic triazole (TarcevaÔ) (PDB code: 1M17) was obtained from PDB45.
and the substituents connected to quinazoline scaffold.
The automated docking program of MOE 2008.10 was used to
dock compounds 5, 12, 13 and erlotinib into ATP binding site of
EGFR (Figure 4). The complexes were energy-minimized with a
MMFF94 force field46 till the gradient convergence 0.01 kcal/
mol was reached. The binding energies of ꢀ8.73, ꢀ15.21,
ꢀ12.89 and ꢀ27.03 kcal/mol were obtained for 5, 12, 13 and
erlotinib, respectively (Figure 4). These docking studies have
revealed that the quinazoline ring binds to a narrow hydrophobic
pocket in the N-terminal domain of EGFR-TK where N-1 of the
quinazoline ring interacts with the backbone NH of Met-769 via
a hydrogen bond, and similarly, a water (HOH-10) molecule-
mediated hydrogen bonding interaction with Thr-830 and Thr-
766 side chain. These interactions revealed the importance of
nitrogen atoms for binding and the subsequent inhibitory
capacity.
Drug score, drug-likeness and toxicities profiles
Currently there are many approaches that assess a compound
drug-likeness based on topological descriptors, fingerprints of
molecular drug-likeness structure keys or other properties39. In
the Osiris program (http://www.organic-chemistry.org/prog/peo)
the occurrence frequency of each fragment is determined within
the collection created by shredding 3300 traded drugs as well as
15 000 commercially available chemicals (Fluka) yielding a
complete list of all available fragments. In this work, we used
the Osiris program for calculating the fragment based drug-
likeness of the antitumor compounds also comparing them with 5-
Flu and erlotinib (Figure 3). Interestingly, the derivatives 5, 6, 7,
8, 9, 12 and 13 presented good drug-likeness values (ꢀ0.40 to
4.63) compared with 5-Flu and erlotinib (ꢀ4.50 and ꢀ6.73,
respectively). In this study, we also verified the drug-Score40 as
the theoretical data showed that compounds 5, 6, 7, 8, 9, 12 and 13
(0.33–0.52) presented values once again higher than 5-Flu and
erlotinib (0.06 and 0.38, respectively). Moreover, we used the
Osiris program to predict the overall toxicity of the most active
derivatives as it may point to the presence of some fragments
generally responsible for the irritant, mutagenic, tumorigenic or
reproductive effects in these molecules. Interestingly, all of the
active derivatives presented a low in silico toxicity risk profile,
better than 5-Flu and similar to erlotinib (Figure 3). These
theoretical data reinforced the cytotoxicity experimental data
described in this work pointing these compounds as lead
compounds for further study.
EGFR–TK complex with compounds 12 and 13 showed the
occurrence of three hydrogen bonds with Met-769 (3.22 and
˚
˚
3.26 A respectively), HOH-10 (3.49, 2.88, 3.61 and 3.09 A,
respectively) mediated hydrogen bonding interaction with Thr-
830 and Thr-766 side chain. Moreover, additional non-classical
hydrogen bonds are formed between aromatic moiety at 3-
position of quinazoline core and the side chain of Gln-767. The
lower interaction energy observed for 5 (Figure 4, upper right
panel) rationalizes the insufficient binding into the EGFR-TK
active site than that of compounds 12 and 13 (Figure 4, lower
panels). The insufficient binding can be explained in terms of the
occurrence of only two hydrogen bonds between the N-1 and
˚
carbonyl of quinazoline ring system with Met-769 (3.28 A) and
HOH-10 mediated hydrogen bonding interaction with Thr-830
˚
and Thr-766 side chain (2.98 A). In short, Figure 4 demonstrates
binding models of quinazoline in the ATP binding site and the
results of this molecular docking could support the postulation
Predicted bioactivity score (molinspiration calculations)
The molinspiration bioactivity prediction (http://www.molinspir- that our active compounds may act on the same enzyme target
ation.com/docu/miscreen/druglikeness.html) is fast (100 000 mol- where EGFR inhibitor acts confirming the molecular design of
ecules may be screened in about 30 min) and therefore allows the reported class of antitumor agents.
processing of very large molecular libraries. Validation tests
performed on various target classes (including of GPCR ligand,
Conclusion
ion channel modulator, nuclear receptor legend, kinase inhibitor,
New derivatives of substituted-4(3H)-quinazolinones were
synthesized and evaluated for their in vitro antitumor and
antimicrobial activity. A single dose (10 mM) of the test
compounds was used in the NCI 60 cell lines panel assay. The
results of this study demonstrated that compounds 5, 8, 12 and 13
yielded selective activities toward numerous cell lines belonging
to different tumor subpanels. They are compatible with Lipinski’s
rule of five (molecular weight, Clog P, hydrogen bond-donating
and accepting capabilities). Molecular docking studies further
supported the antitumor activity of compounds 5, 12, 13
compared with EGFR kinase inhibitors and further helped in
understanding the various interactions between the ligands and
enzyme active sites in detail and thereby helped to design novel
potent derivatives. On the other hand, the results of antimicrobial
screening revealed that compounds 1, 9 and 14 were found to be
protease inhibitor, enzyme inhibitor) show 10–20-fold increases
in hit rate in comparison with standard/random selection of
molecules for screening. Calculated drug likeness score of each
compounds were compared with the specific activity of other
compounds and the results were compared with standard drug.
For organic molecules, the probability is if the bioactivity score is
(40), then it is active, if (ꢀ5.0 to 0.0) then moderately active, if
(5ꢀ5.0) then inactive. The calculated value of the predicted
bioactivity for compounds 5, 6, 7, 8, 9, 12, 13, 5-Flu and erlotinib
is given in Table 6. The results indicated that the synthesized
quinazoline derivatives have a predicted kinase inhibiting activity
almost similar to erlotinib.
Molecular docking study
The level of the predicted activity against kinase enzyme and most active against S. auerus ATCC 29213 with MIC of 16, 32
antitumor activities of the proposed compounds over breast and 32 mg/mL and inhibition zone of 19, 17 and 17 mm,
cancer cell, colon cancer cell, lung cancer cell and renal cancer respectively, while compound 14 had activity against all tested
cell (EGFR is highly expressed) prompted us to perform strains either bacterial or C. albicans strain with lowest MIC
molecular docking into the ATP binding site of EGFR to against all the tested strains.

14 A. M. Alanazi et al.
J Enzyme Inhib Med Chem, Early Online: 1–15
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modeling study of some new 2,6-substituted-quinazolin-4-ones.
Bioorg Med Chem 2010;18:2849–63.
Declaration of interest
The authors extend their appreciation to the Deanship of Scientific
Research at King Saud University for funding the work through the
research group project No. RG-1435-046. The authors would like to
express their gratitude and thanks to the National Cancer Institute (NCI),
Bethesda Maryland, USA, http://dtp.cancer.gov/ for doing the antitumor
testing of the new compounds.
23. Al-Obaid AM, Abdel-Hamide SG, El-Kashef HA, et al. Synthesis,
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Supplementary Information