Synthesis and evaluation of anticancer, antiphospholipases, antiproteases, and antimetabolic syndrome activities of some 3H-quinazolin-4-one derivatives

Article abstract of DOI:10.1080/14756366.2019.1574780

Some new 3H-quinazolin-4-one derivatives were synthesised and screened for anticancer, antiphospholipases, antiproteases, and antimetabolic syndrome activities. Compound 15d was more potent in reducing the cell viabilities of HT-29 and SW620 cells lines to 38%, 36.7%, compared to 5-FU which demonstrated cell viabilities of 65.9 and 42.7% respectively. The IC₅₀ values of 15d were ～20?μg/ml. Assessment of apoptotic activity revealed that 15d decreased the cell viability by down regulating Bcl2 and BclxL. Moreover, compounds, 8j, 8d/15a/15e, 5b, and 8f displayed lowered IC₅₀ values than oleanolic acid against proinflammatory isoforms of hGV, hG-X, NmPLA_2, and AmPLA₂. In addition, 8d, 8h, 8j, 15a, 15b, 15e, and 15f showed better anti-α-amylase than quercetin, whereas 8g, 8h, and 8i showed higher anti-α-glucosidase activity than allopurinol. Thus, these compounds can be considered as potential antidiabetic agents. Finally, none of the compounds showed higher antiproteases or xanthine oxidase activities than the used reference drugs.

Full text of DOI:10.1080/14756366.2019.1574780

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and evaluation of anticancer,
antiphospholipases, antiproteases, and
antimetabolic syndrome activities of some
3H-quinazolin-4-one derivatives
Nahed N. E. El-Sayed, Norah M. Almaneai, Abir Ben Bacha, Omar Al-Obeed,
Rehan Ahmad, Maha Abdulla & Ahmed M. Alafeefy
To cite this article: Nahed N. E. El-Sayed, Norah M. Almaneai, Abir Ben Bacha, Omar Al-Obeed,
Rehan Ahmad, Maha Abdulla & Ahmed M. Alafeefy (2019) Synthesis and evaluation of anticancer,
antiphospholipases, antiproteases, and antimetabolic syndrome activities of some 3H-quinazolin-4-
one derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 672-683, DOI:
10.1080/14756366.2019.1574780
To link to this article: https://doi.org/10.1080/14756366.2019.1574780
©
2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Published online: 01 Mar 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 672–683
2
https://doi.org/10.1080/14756366.2019.1574780
RESEARCH PAPER
Synthesis and evaluation of anticancer, antiphospholipases, antiproteases, and
antimetabolic syndrome activities of some 3H-quinazolin-4-one derivatives
a,b
a
c
d
d
d
Nahed N. E. El-Sayed , Norah M. Almaneai , Abir Ben Bacha , Omar Al-Obeed , Rehan Ahmad , Maha Abdulla
e
and Ahmed M. Alafeefy
a
b
Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia; National Organization for Drug Control and
c
d
Research, Giza, Egypt; Biochemistry Department, College of Sciences, King Saud University, Riyadh, Saudi Arabia; Department of Surgery,
College of Medicine, King Saud University, Riyadh, Saudi Arabia; Department of Chemistry, Kulliyyah of Science, International Islamic University
Malaysia, Kuantan, Pahang Darul Makmur, Malaysia
e
ABSTRACT
ARTICLE HISTORY
Some new 3H-quinazolin-4-one derivatives were synthesised and screened for anticancer, antiphospholi-
pases, antiproteases, and antimetabolic syndrome activities. Compound 15d was more potent in reducing
the cell viabilities of HT-29 and SW620 cells lines to 38%, 36.7%, compared to 5-FU which demonstrated
cell viabilities of 65.9 and 42.7% respectively. The IC50 values of 15d were ꢀ20 mg/ml. Assessment of
apoptotic activity revealed that 15d decreased the cell viability by down regulating Bcl2 and BclxL.
Moreover, compounds, 8j, 8d/15a/15e, 5b, and 8f displayed lowered IC50 values than oleanolic acid
Received 12 December 2018
Revised 17 January 2019
Accepted 21 January 2019
KEYWORDS
3
H-quinazolin-4-one;
colorectal cancer;
phospholipases; proteases;
metabolic syndrome
against proinflammatory isoforms of hGV, hG-X, NmPLA2, and AmPLA . In addition, 8d, 8h, 8j, 15a, 15b,
2
15e, and 15f showed better anti-a-amylase than quercetin, whereas 8g, 8h, and 8i showed higher anti-
a-glucosidase activity than allopurinol. Thus, these compounds can be considered as potential antidiabetic
agents. Finally, none of the compounds showed higher antiproteases or xanthine oxidase activities than
the used reference drugs.
Introduction
Several in vitro and in vivo clinical studies have suggested a posi-
Colorectal cancer (CRC) is the third-most common cancer and the tive correlation between the activity of certain proteases, such as tryp-
1
5
6
7
8
9
fourth-most common cause of cancer-related death worldwide . sin , cathepsin B , collagenases , thrombin , and neutrophil elastase
One of the key features of cancer is deregulation of apoptosis of and colorectal carcinogenesis, invasion, proliferation, and metastasis.
2
the altered cells . Apoptosis is mediated by a complex network of
2
Furthermore, three phospholipases, namely (hsPLA -IIA), group
1
0
BCL-2 family of proteins, comprising two types of proteins, namely V, and group X are found to paly indirect pro-tumorigenic roles
anti-apoptotic proteins, which include Bcl2, BclxL, and Mcl-1, and by promoting chronic inflammatory bowel diseases (IBD) that are
pro-apoptotic proteins, which include Bax and Bak . Hence, inhib- common risk factors associated with CRC .
3
11
iting the anti-apoptotic BCL-2 proteins can be established as a
mechanism of anticancer defence for the treatment of CRC .
In addition, recent studies have linked the incidence of CRC to
certain metabolic syndrome components, especially elevated
4
CONTACT Nahed N. E. El-Sayed
2452, Riyadh 11451, Saudi Arabia
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
nelsayed@ksu.edu.sa, nahednas@yahoo.com
Department of Chemistry, College of Sciences, King Saud University, P.O. Box
2
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
673
12
plasma glucose (type 2 diabetes mellitus) . Several enzymes are ionisation mode: EI (Micro Analytical Center, Cairo University,
reported to be involved in glucose homeostasis, among them Egypt). Elemental analyses were obtained from the Microanalytical
1
3
14
a-amylase, a-glucosidase , and xanthine oxidase . Hence, target- Data Unit at Cairo University, Giza, Egypt.
2
6
27
28
29
30
31
ing these enzymes has high potential in preventing CRC.
Compounds 3a,b , 3c , 3d , 6a , 6b , 13a, 13b, and 15a
The 3H-quinazolin-4-one scaffold is known to be an import- were reported previously.
1
5
ant building block of various bioactive natural products . In
addition to this, many marketed drugs or experimental drug
candidates incorporate this pharmacophore as part of the over-
16
32,33
all topography of the molecules such as ispinesib, nolatrexed, General procedure for the preparation of compounds 5a–c
and raltitrexed (the anticancer agents); afloqualone with seda-
A mixture of the appropriate 2-methyl-benzo[d][1,3]oxazin-4-one
tive and muscle relaxant effects, balaglitazone with antidiabetic
activities, proquazone (as non-steroidal anti-inflammatory agent),
and elinogrel (as reversible P2Y12 receptor antagonist) which is
used for the treatment of acute coronary syndrome. Moreover,
several new methodologies have been developed for the syn-
thesis of 4-quinazolinone derivatives either using one pot multi-
derivative 3a-c (0.003 mol) and 3,4,5-trimethoxyaniline
4
ꢁ
(
0.003 mol, 0.55 g) was fused in oil bath at 200 C for 2 h. After
ꢁ
cooling down the temperature to 100 C, 37% HCl (Conc) (3 ml)
was added till all the material was dissolved completely. The reac-
tion mixture was cooled down to room temperature and was
made alkaline with diluted NaOH solution (25%). The crude pre-
cipitated solid in each case, was filtered, washed with water, dried,
and recrystallised from ethanol to yield compounds 5a–c.
17
component approaches
or via multistep routes for
investigating their pharmacological potential as dual EGFR/HER2
1
8
19
inhibitors , phosphoinositide 3-kinase inhibitors , and as anti-
2
0
21
22
hypertensive , antioxidant , anticonvulsant , and anti-inflam-
matory agents.
2
3
Based on these observations, and in continuation to our
ongoing research programme aims to identify new bioactive mol-
2
-Methyl-3-(3,4,5-trimethoxy-phenyl)-3H-quinazolin-4-one (5a)
2
4,25
ecules
herein, we report the synthesis, characterisation, and
ꢁ
ꢂ1
Black powder, yield (53%), mp 88–90 C; ꢀmax (KBr)/cm
3080
cytotoxicity screening (including evaluation of apoptotic activity),
for a new series of 3H-quinazolin-4-one derivatives against HT-29
and SW620 cell lines derived from CRC. Furthermore, the inhibi-
tory potentials of the synthesised compounds against certain
phospholipases and proteases implicated in cancer and inflamma-
tory disorders, in addition to a-amylase, a-glucosidase, and xan-
thine oxidase, managing metabolic syndrome are investigated
with the aim of eradicating the risk factors for CRC or combatting
the disease.
(
1
7
CH-Ar), 2935 & 2837 (CH-aliphatic), 2368, 1682 (C ¼ O), 1600 &
503 (C ¼ C), 1465, 1422, 1376, 1321, 1233, 1124, 1002, 936, 835,
5
71, 700, 653, 524; d (850 MHz; CDCl ) 8.26 (1H, d, J= 7.6, CH -
H
3
6
quinazolin-4-one), 7.77–7.75 (1H, m, CH -quinazolin-4-one), 7.66
(
8
7
1H, dd, J= 3.4, 8.5, CH -quinazolin-4-one), 7.47–7.45 (1H, m, CH -
’
2
6’
quinazolin-4-one), 6.47 (2H, s, H and H -trimethoxyphenyl), 3.90
3H, s, OCH ), 2.32 (3H, s, CH ); d (213 MHz;
), 3.84 (6H, s, 2 ꢃ OCH
(
3
3
3
C
CDCl ) 162.36 (C ¼ O), 154.41 (C ¼ N), 154.22, 147.41, 138.46,
3
1
6
34.70, 133.29, 127.04, 126.79, 126.73, 120.69, 105.19 (6 ꢃ CH-Ar &
ꢃ C -Ar), 60.96 (OCH ), 56.27 (2 ꢃ OCH ), 24.04 (CH ); MS (EI) m/z
q
3
3
3
þ
þ
þ
Materials and methods
(%) [M þ 2] 328.10 (2.50), [M þ 1] 327.10 (16.69), [M ] 326.10
(77.52), 311.10 (46.20), 297.10 (3.85), 279.05 (2.13), 265.05 (1.90),
Chemistry
1
83.05 (4.87), 168.10 (7.32), 147.65 (8.78), 144.10 (14.54), 143.10
The reactions were monitored and the purity of the final products (100.00), 134.10 (4.69), 116.10 (6.24), 102.05 (4.96), 77.00 (6.38),
was checked using thin layer chromatography (TLC) on pre-coated 57.05 (3.38). Anal. calcd. for C H N O (326.13): C, 66.25; H, 5.56;
1
8 18 2 4
silica gel sheets (60 F254, Merck, Kenilworth, NJ). The developed N, 8.58. Found: C, 66.23; H, 5.46; N, 8.66.
plates were examined by exposure to ultraviolet light using VL-
6
.LC (254, 365 nm, 50/60 Hz). All the melting points were deter-
mined in open capillary tubes with a Gallenkamp melting point
ꢁ
apparatus ( C). The infra-red (IR) spectra were recorded on a 6-Fluoro-2-methyl-3–(3,4,5-trimethoxy-phenyl)-3H-quinazolin-4-
Perkin Elmer Fourier transform infra-red (FTIR) spectrophotometer one (5b)
ꢂ1
(
Spectrum BX 1000) in wave numbers (cm ) with potassium
ꢁ
ꢂ1
Black crystals, yield (55%), mp 193–195 C; ꢀ
(
(KBr)/cm 3079
max
bromide (KBr) discs. Nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker NMR spectrometer: 1-Ascend 850 MHz
for H and 213 MHz for C, or on 2-Avance 600 MHz for H and
CH-Ar), 2931 & 2835 (CH-aliphatic), 2375, 2278, 1673 (C ¼ O), 1601
1
13
1
& 1484 (C ¼ C), 1425, 1374, 1339, 1313, 1235, 1183, 1125 (C–F),
1
3
1065, 1008, 966, 878, 843, 778, 752, 723, 674, 626, 558, 526, 458;
1
50 MHz for
C (Nuclear Magnetic Resonance Center, KAU,
5
d
H
(500 MHz; CDCl
3
) 7.84–7.82 (1H, m, CH -quinazolin-4-one),
7.67–7.65 (1H, m, CH -quinazolin-4-one), 7.49–7.45 (1H, m, CH -
quinazolin-4-one), 6.52 (2H, s, H and H -trimethoxyphenyl), 3.91
Jeddah, KSA), or on a Bruker Avance 500 spectrometer operating
7
8
1
13
ꢁ
at 500 MHz for H and 125 MHz for C at 25 C (Research Unit,
College of Pharmacy, Prince Sattam Bin Abdulaziz University,
AlKharj, KSA), or on an Eclipse 300 FT NMR spectrometer operating
’
2
6’
(
3H, s, OCH ), 3.86 (6H s, 2 ꢃ OCH ), 2.32 (3H, s, CH ); d (125 MHz;
3
3
3
C
1
13
ꢁ
CDCl
3
) 161.62 (C ¼ O), 159.65 (C -F), 154.20 (C ¼ N), 153.65,
q
at 300 MHz spectrometer for H and at 75 MHz for C at 25 C
KSU, Riyadh, KSA). The chemical shifts were expressed in (ppm)
using tetramethylsilane as the internal standard; coupling con- (5 ꢃ CH-Ar & 6 ꢃ C
stants (J) were expressed in Hz. Deuterochloroform (CDCl
) was (CH
1
44.06, 138.48, 133.03, 129.23, 123.18, 122.99, 121.87, 105.12
(
q
-Ar), 60.89 (OCH
3
), 56.24 (2 ꢃ OCH
3
), 23.87
þ
þ
3
1
3
); MS (EI) m/z (%) [M þ 1] 345.10 (21.69), [M ] 344.10 (100)
used as solvent. The splitting patterns (multiplicities) in HNMR 330.10 (11.87), 329.10 (57.89), 313.10 (3.99), 297.05 (2.71), 161.05
were designated as: singlet (s), broad singlet (br. s), doublet (d), (86.15), 162.05 (11.84), 134.10 (7.80), 107.05 (4.64), 94.05 (7.76),
double of doublet (dd), triplet (t), and multiplet (m). Mass spectra 77.00 (1.91), 57.00 (3.40). Anal. calcd. for C18
H17FN O (344.12): C,
2 4
were recorded on Shimadzu Qp-2010 Plus mass spectrometer in 62.79; H, 4.98; N, 8.14. Found: C, 62.68; H, 4.63; N, 8.02.

6
74
N. N. E. EL-SAYED ET AL.
6
-Chloro-2-methyl-3–(3,4,5-trimethoxy-phenyl)-3H-quinazolin-4-
(1H, s, CH-Ar), 8.15 (1H, s, CH-Ar), 8.12 (1H, d, J= 8.5, CH-Ar), 7.92
(2H, d, J= 8.2, 2 ꢃ CH-Ar), 7.88 (1H, d, J= 7.9, CH-Ar), 7.79 (1H, dd,
one (5c)
ꢂ1
J= 8.5, 1.8, CH-Ar), 7.61–7.53 (3H, m, 3 ꢃ CH-Ar), 2.68 (3H, s, CH );
3
ꢁ
Black powder, yield (60%), mp 188–190 C; ꢀmax (KBr)/cm 3075
d
(125 MHz; CDCl ) 166.60 (C ¼ O), 157.65 (HC ¼ N), 154.66
C
3
(
1
8
CH-Ar), 2934 & 2839 (CH-aliphatic), 2369, 1680 (C ¼ O), 1598 &
(C
q
=N), 145.31, 137.46, 135.46, 132.90, 132.56, 130.26, 129.69,
504 (C ¼ C), 1467, 1423, 1375, 1312, 1236, 1126, 998, 948, 892,
34 (C–Cl), 771, 738, 692, 613, 524, 452; d (500 MHz; CDCl ) 8.18
1
28.98, 128.80, 128.28, 128.01, 126.98, 122.98, 122.81, 119.86
H
3
þ
81
5
7
(10 ꢃ CH-Ar & 6 ꢃ C
q 3
-Ar), 22.98 (CH ); MS (EI) m/z (%) [M , Br]
(1H, d, J= 2.4, CH -quinazolin-4-one), 7.69 (1H, dd, J= 8.7, 2.4, CH -
þ
79
8
393.05 (2.94), [M , Br] 391.00 (2.85), 239.95 (100.00), 237.95
(98.69), 197.90 (6.54), 195.95 (4.69), 155.95 (5.37), 153.05 (22.90),
quinazolin-4-one), 7.60 (1H, d, J= 8.7, CH -quinazolin-4-one), 6.51
2’
6’
(2H, s, H and H -trimethoxyphenyl), 3.92 (3H, s, OCH ), 3.88 (6H,
3
1
(
1
27.10 (21.27), 100.05 (3.19), 75.00 (18.97), 63.00 (3.47), 57.05
1.26). Anal. calcd. for C20 14BrN O (391.03): C, 61.24; H, 3.60; N,
0.71. Found: C, 61.08; H, 3.39; N, 10.65.
s, 2 ꢃ OCH
3
), 2.32 (3H, s, CH
54.75 (C ¼ N), 154.25, 145.91, 138.56, 135.02, 132.95, 132.36,
28.52, 126.23, 121.73, 105.09 (5 ꢃ CH-Ar & 7 ꢃ C -Ar), 60.93
3
); d
C
(125 MHz; CDCl
3
) 161.28 (C ¼ O),
H
3
1
1
q
þ
37
(OCH ), 56.28 (2 ꢃ OCH ), 24.02 (CH ); MS (EI) m/z (%) [M , Cl ]
3
3
35
3
þ
3
62.10 (34.88), [M , Cl ] 360.10 (100.00), 347.05 (22.20), 345.10
(E)-2-Methyl-3-[(4-pyridin-2-yl-benzylidene)-amino]-3H-
(
3
(
60.80), 331.05 (4.80), 329.05 (4.53), 315.05 (1.90), 313.05 (3.09),
01.05 (1.04), 299.10 (1.56), 179.00 (27.46), 177.00 (84.58), 152.05
3.81), 150.05 (6.54), 136.05 (5.31), 123.05 (3.29), 110.00 (6.17),
quinazolin-4-one (8c)
ꢁ
Beige powder (benzene), yield (85%), mp 188–190 C; ꢀ
(KBr)/
max
ꢂ1
7
5
5.00 (6.71), 57.10 (3.05). Anal. calcd. for C H ClN O (360.09): C, cm 3042 (CH-Ar), 2940 (CH-aliphatic), 1675 (C ¼ O), 1600 & 1468
18
17
2 4
9.92; H, 4.75; N, 7.76. Found: C, 59.88; H, 4.62; N, 7.59.
(C ¼ C), 1436, 1378, 1327, 1230, 1181, 1154, 1108, 987, 939, 875,
47, 772, 718, 688, 663, 626, 589, 479; d (500 MHz; CDCl ) 9.12
1H, s, CH ¼ N), 8.74 (1H, d, J= 4.7, CH-Ar), 8.30 (1H, d, J= 7.9, CH-
Ar), 8.15 (2H, d, J= 8.2, 2 ꢃ CH-Ar), 8.00 (2H, d, J= 8.2, 2 ꢃ CH-Ar),
.80–7.73 (3H, m, 3 ꢃ CH-Ar), 7.68 (1H, d, J= 8.1, CH-Ar), 7.48 (1H,
t, J= 7.5, CH-Ar), 7.36–7.28 (1H, m, CH-Ar), 2.69 (3H, s, CH ); d
125 MHz; CDCl ) 165.64 (C ¼ O), 158.80 (HC ¼ N), 156.12 (HC ¼ N),
8
H
3
(
3
4
Synthesis of Schiff’s base derivatives (8a–j)
7
A mixture of 3-amino-2-methyl-3H-quinazolin-4-one derivatives
a,b (0.002 mol) and the appropriate aromatic aldehyde 7a–e,
3
C
6
(
3
namely, 2-naphthaldehyde, 4-(pyridin-2-yl) benzaldehyde, 3-
methoxy-2-nitrobenzaldehyde, 4-formylbenzonitrile, and 1,1 -
biphenyl]-4-carbaldehyde (0.002 mol) in absolute ethanol (20 ml)
containing (3 ml) glacial acetic acid was heated under reflux for
0
154.18 (C =N), 149.93, 146.46, 143.05, 136.92, 134.34, 133.19,
q
1
29.24, 127.35, 127.27, 126.93 126.43, 122.87, 121.56, 120.88
þ
(
3
11 ꢃ CH-Ar & 5 ꢃ C
q
-Ar), 22.94 (CH
3
); MS (EI) m/z (%) [M þ 1]
þ
41.10 (0.69), [M ] 340.10 (1.39), 181.05 (4.88), 161.05 (10.51),
60.10 (100.00), 145.10 (2.83), 132.10 (3.91), 118.10 (12.08), 90.00
1
0 h. The excess solvent was removed under reduced pressure.
1
The obtained solid in each case was washed with water, air dried,
and recrystallised from the appropriate solvent to obtain the cor-
responding Schiff’s base.
(
5.53), 76.00 (16.01), 50.00 (3.71). Anal. calcd. for C21
16 4
H N O
(340.13): C, 74.10; H, 4.74; N, 16.46. Found: C, 73.92; H, 4.63;
N, 16.28.
(
E)-2-Methyl-3-[(naphthalen-2-ylmethylene)-amino]-3H-
(
E)-6-Bromo-2-methyl-3-[(4-pyridin-2-yl-benzylidene)-amino]-3H-
quinazolin-4-one (8a)
quinazolin-4-one (8d)
ꢁ
White powder (ethanol), yield (74%), mp 163–165 C; ꢀ
(KBr)/
max
ꢂ1
ꢁ
Beige powder (benzene), yield (77%), mp 209–210 C; ꢀ
(KBr)/
max
cm ; 3062 (CH-Ar), 2929 (CH-alipahtic), 2371, 2340, 1667 (C ¼ O),
ꢂ1
cm 3149 & 3060 (CH-Ar), 2952 (CH-aliphatic), 1670 (C ¼ O), 1604
1
1
596 & 1468 (C ¼ C), 1426, 1377, 1346, 1320, 1268, 1227, 1156,
121, 1031, 934, 898, 866, 831, 778, 755, 693, 660, 621, 477; d
&
1
1465 (C ¼ C), 1375, 1319, 1278, 1240, 1208, 1158, 1124, 1961,
H
035, 987, 876, 833, 775, 727, 674 (C-Br), 645, 564; d (850 MHz;
H
(
8
600 MHz; CDCl ) 9.18 (1H, s, CH ¼ N), 8.30 (1H, d, J= 8.4, CH-Ar),
3
CDCl ) 9.10 (1H, s, CH ¼ N), 8.77 (1H, d, J= 4.3, CH-Ar), 8.41 (1H, d,
.16–8.14 (2H, m, 2 ꢃ CH-Ar), 7.93–7.92 (2H, m, 2 ꢃ CH-Ar), 7.89 (1H,
3
J= 2.6, CH-Ar), 8.17 (2H, d, J= 7.7, 2 ꢃ CH-Ar), 8.01 (2H, d, J= 7.7,
d, J= 7.8, CH-Ar), 7.75 (1H, d, J= 7.2, CH-Ar), 7.67 (1H, d, J= 8.4, CH-
Ar), 7.59 (1H, t, J= 7.2, CH-Ar), 7.55 (1H, t, J= 7.8, CH-Ar), 7.47 (1H, t,
2
ꢃ CH-Ar), 7.85–7.79 (3H, m, 3 ꢃ CH-Ar), 7.55 (1H, d, J= 8.5, CH-
J= 7.8, CH-Ar), 2.70 (3H, s, CH ); d (150 MHz; CDCl ) 166.32 (C ¼ O), Ar), 7.36–7.32 (1H, m, CH-Ar), 2.68 (3H, s, CH
3 C 3
); d (213 MHz; CDCl )
3
C
3
1
1
1
58.84 (HC ¼ N), 154.21 (C =N), 146.49, 135.41, 134.37, 132.93, 165.98 (C ¼ O), 157.67 (HC ¼ N), 155.79 (HC ¼ N), 154.69 (C
q
=N),
q
32.42, 130.46, 128.97, 128.95, 128.20, 128.02, 127.30, 126.95, 149.51, 145.26, 137.56, 133.23, 129.74, 129.40, 128.79, 127.54,
26.94, 126.46, 122.88, 121.58 (11 ꢃ CH-Ar & 5 ꢃ C
q
-Ar), 23.00 (CH
3
); 123.08, 122.95, 121.23, 119.94 (10 ꢃ CH-Ar & 6 ꢃ C
q
-Ar), 22.95
); MS (EI) m/z (%) [M þ 1] 419.05 (1.37), [M ] 418.00 (2.03),
10.24), 160.10 (100.00), 153.10 (14.99), 139.10 (4.10), 127.10 (8.79), 240.00 (11.07), 239.95 (100.00%), 237.95 (98.10), 199.90 (2.69),
þ
þ
þ
þ
MS (EI) m/z (%) [M þ 1] 314.10 (0.44), [M ] 313.10 (1.41), 161.05 (CH
3
(
1
18.10 (11.47), 90.05 (11.47), 76.00 (13.65), 63.00 (2.80), 50.00 (3.12). 197.90 (6.83), 181.05 (6.55), 179.05 (4.60), 154.05 (13.02), 152.05
O (313.12): C, 76.66; H, 4.82; N, 13.41. (1.88), 139.10 (3.04), 127.10 (6.15), 116.10 (3.42), 90.05 (4.15), 78.05
Anal. calcd. for C20
15 3
H N
7
9
Found: C, 76.62; H, 4.77; N, 13.29.
4
(3.66), 75.00 (17.16). Anal. calcd. for C21H15 BrN O (418.04): C,
6
0.16; H, 3.61; N, 13.36. Found: C, 60.03; H, 3.43; N, 13.11.
(
E)-6-Bromo-2-methyl-3-[(naphthalen-2-ylmethylene)-amino]-3H-
quinazolin-4-one (8b)
(E)-3-[(3-Methoxy-2-nitro-benzylidene)-amino]-2-methyl-3H-
quinazolin-4-one (8e)
ꢁ
ꢂ1
White powder (ethanol), yield (65%), mp 200 C; ꢀmax (KBr)/cm
3
(
ꢁ
056 (CH-Ar), 2926 (CH-aliphatic), 1932, 1675 (C ¼ O), 1602 & 1465 Beige powder (ethanol), yield (55%), mp 178–180 C; ꢀmax (KBr)/
ꢂ1
C ¼ C), 1371, 1347, 1313, 1272, 1235, 1208, 1180, 1153, 1122, cm 3017 (CH-Ar), 2975 & 2935 (CH-aliphatic), 1680 (C ¼ O), 1615
1
5
072, 1037, 954, 890, 860, 831, 812, 777, 740, 700, 672 (C-Br), 629, & 1571 (C ¼ C), 1537, 1464, 1428, 1376, 1283, 1227, 1156, 1107,
69, 539, 514, 473; d (500 MHz; CDCl
) 9.15 (1H, s, CH ¼ N), 8.39 1055, 1029, 964, 933, 875, 850, 772, 743, 691, 651, 589, 471, 419;
H
3

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
675
d
H
(850 MHz; CDCl
3
) 9.41 (1H, s, CH ¼ N), 8.26 (1H, d, J= 7.7, CH- (E)-4-[(6-Bromo-2-methyl-4-oxo-4H-quinazolin-3-ylimino)-
Ar), 7.75 (1H, t, J= 7.7, CH-Ar), 7.67 (1H, d, J= 7.7, CH-Ar), 7.56 (1H, methyl]-benzonitrile (8h)
t, J= 7.7, CH-Ar), 7.49–7.46 (2H, m, 2 ꢃ CH-Ar), 7.23 (1H, d, J= 8.5,
ꢁ
White powder (acetone), yield (75%), mp 228–230 C; ꢀ
(KBr)/
max
CH-Ar), 3.96 (3H, s, OCH
3
), 2.6 (3H, s, CH
3
); d
C
(213 MHz; CDCl
3
)
ꢂ1
cm 3059 (CH-Ar), 2927 (CH-aliphatic), 2230 (CN), 1680 (C ¼ O),
1
1
59.22 (C ¼ O), 158.52 (HC ¼ N), 154.87 (C =N), 151.44, 139.74,
q
1
1
d
602 & 1467 (C ¼ C), 1420, 1376, 1328, 1277, 1242, 1211, 1156,
34.76, 131.30, 127.39, 126.77, 126.14, 122.03, 121.38, 115.92
123, 1071, 981, 899, 876, 831, 778, 723, 675 (C-Br), 644, 555, 433;
(
7 ꢃ CH-Ar & 5 ꢃ C -Ar), 56.78 (OCH ), 22.65 (CH ); MS (EI) m/z (%)
5
q
3
3
H
(500 MHz; CDCl
3
) 9.32 (1H, s, CH ¼ N), 8.40 (1H, s, CH -quinazo-
þ
[
M ] 338.10 (4.91), 321.10 (8.62), 290.10 (8.60), 261.10 (2.25),
86.05 (2.04), 160.10 (100.00), 143.10 (3.47), 132.10 (4.81), 117.10
18.04), 104.05 (3.96), 76.00 (26.95), 63.00 (4.85), 50.00 (6.06). Anal.
calcd. for C H N O (338.10): C, 60.35; H, 4.17; N, 16.56. Found:
lin-4-one ring), 8.00 (2H, d, J= 7.7, 2 ꢃ CH-benzonitrile ring), 7.84
1
(
7
(
1H, d, J= 8.5, CH -quinazolin-4-one ring), 7.81 (2H, d, J= 7.7,
8
2
ꢃ CH-benzonitrile ring), 7.55 (1H, d, J= 8.5, CH -quinazolin-4-one
1
7 14 4 4
ring), 2.64 (3H, s, CH ); d (125 MHz; CDCl ) 162.65 (C ¼ O), 157.83
3
C
3
C, 60.03; H, 3.94; N, 16.37.
(
HC ¼ N), 154.53 (C =N), 145.027, 137.87, 136.93, 132.67, 129.78,
q
1
29.01, 128.9, 122.81, 120.14, 118.04, 115.49 (7 ꢃ CH-Ar, 5 ꢃ C
q
-Ar
þ
81
þ
&
CꢄN), 22.98 (CH
3
); MS (EI) m/z (%) [M , Br] 368.01 (2.60), [M ,
7
9
Br] 366.01 (2.56), 354.30 (3.01), 313.30 (12.92), 297.30 (5.50),
39.20 (12.54), 227.10 (2.29), 213.15 (2.33), 185.10 (2.94), 171.10
2.88), 143.15 (3.48), 129.10 (12.26), 111.15 (14.46), 97.15 (21.74),
(
E)-6-Bromo-3-[(3-methoxy-2-nitro-benzylidene)-amino]-2-
2
(
methyl-3H-quinazolin-4-one (8f)
ꢁ
Beige powder (ethanol), yield (66%), mp 234–235 C; ꢀmax (KBr)/ 83.05 (26.49), 71.05 (43.57), 69.05 (42.50), 59.05 (56.18), 57.05
ꢂ1
79
cm
C ¼ O), 1601 & 1533 (C ¼ C), 1464, 1436, 1373, 1303, 1207, 1153, 15.26. Found: C, 55.49; H, 2.88; N, 15.19.
104, 1063, 973, 937, 897, 836, 779, 739, 670 (C-Br), 639, 589; d
3070 & 3012 (CH-Ar), 2977 & 2926 (CH-aliphatic), 1677 (100.00). Anal. calcd. for C17H11 BrN O (366.01): C55.61; H, 3.02; N,
4
(
1
H
(
7
(
OCH
1
1
850 MHz; CDCl ) 9.35 (1H, s, CH ¼ N), 8.38 (1H, d, J= 2.6, CH-Ar),
3
(
E)-3-[(Biphenyl-4-ylmethylene)-amino]-6-bromo-2-methyl-3H-
.82 (1H, dd, J= 8.5, 2.6, CH-Ar), 7.58–7.56 (2H, m, 2 ꢃ CH-Ar), 7.48
quinazolin-4-one (8j)
1H, d, J= 8.5, CH-Ar), 7.25 (1H, d, J= 8.5, CH-Ar), 3.97 (3H, s,
ꢁ
3
), 2.63 (3H, s, CH
57.93 (HC ¼ N), 155.38 (C
3
); d
C
(213 MHz; CDCl
3
) 159.28 (C ¼ O), White powder (ethanol), yield (97%), mp 208–210 C; ꢀmax (KBr)/
ꢂ1
=N), 151.47, 144.56, 139.68, 137.94, cm 3058 (CH-Ar), 2920 (CH-aliphatic), 1676 (C ¼ O), 1604 & 1467
q
31.36, 129.85, 128.57, 125.86, 122.76, 122.14, 120.31, 116.16 (C ¼ C), 1373, 1319, 1276, 1236, 1206, 1180, 1154, 1074, 1007, 907,
8
9
2
2
75, 831, 757, 722, 689 (C-Br), 644, 555, 467; d (500 MHz; CDCl )
(
6 ꢃ CH-Ar & 6 ꢃ C -Ar), 56.80 (OCH ), 22.63 (CH ); MS (EI) m/z (%)
H 3
q
3
3
þ
81
þ
79
.03 (1H, s, CH ¼ N), 8.41 (1H, d, J= 2.3, CH-Ar), 7.96 (2H, d, J= 8.3,
ꢃ CH-Ar), 7.82 (1H, dd, J= 8.7, 2.3, CH-Ar), 7.73 (2H, d, J= 8.3,
ꢃ CH-Ar), 7.66 (2H, d, J= 7.3, 2 ꢃ CH-Ar), 7.54 (1H, d, J= 8.7, CH-
[
3
(
1
M , Br] 418.00 (4.65), [M , Br] 416.00 (4.58), 401.00 (6.90),
99.00 (6.63), 370.00 (5.89), 368.00 (5.13), 341.00 (1.54), 334.00
1.46), 265.95 (2.41), 263.95 (2.42), 239.95 (97.78), 237.95 (100.00),
97.90 (8.61), 195.95 (6.33), 169.95 (3.24), 167.95 (2.54), 155.95
7.13), 153.95 (7.22), 133.10 (1.53), 131.10 (3.71), 117.10 (7.38),
Ar), 7.49 (2H, t, J= 7.3, 2 ꢃ CH-Ar), 7.41 (1H, t, J= 7.3 CH-Ar), 2.63
(3H, s, CH
3
); d
C
(125 MHz; CDCl
3
) 166.41 (C ¼ O), 157.64 (HC ¼ N),
(
1
54.61 (C
q
=N), 145.45, 145.34, 139.92, 137.49, 131.44, 129.71,
1
5
3
16.10 (5.63), 101.10 (4.34), 89.00 (4.73), 76.00 (9.56), 59.05 (13.18),
79
13
129.43, 129.02, 128.81, 128.25, 127.66, 127.23, 122.97, 119.87
7.05 (4.20). Anal. calcd. for C H
BrN O (416.01): C, 48.94; H,
4 4
17
þ
81
(
4
12 ꢃ CH-Ar & 6 ꢃ C
q 3
-Ar), 22.94 (CH ); MS (EI) m/z (%) [M , Br]
.14; N, 19.15. Found: C, 48.83; H, 2.97; N, 18.93.
þ
79
19.05 (1.70), [M , Br] 417.05 (1.67), 239.95 (100.00), 237.95
(
1
98.77), 197.90 (6.94), 195.95 (4.46), 179.05 (24.10), 165.05 (5.04),
52.10 (13.04), 131.10 (2.31), 116.10 (3.58), 116.10 (3.58), 90.05
(3.62), 75.00 (15.91), 63.00 (3.20), 57.05 (2.43). Anal. calcd. for
(
E)-4-[(2-Methyl-4-oxo-4H-quinazolin-3-ylimino)-methyl]-
7
9
C H
BrN O (417.05): C, 63.17; H, 3.86; N, 10.05. Found: C, 62.97;
3
benzonitrile (8g)
22 16
H, 3.63; N, 9.89.
ꢁ
White powder (acetone), yield (55%), mp 222–224 C; ꢀmax (KBr)/
ꢂ1
cm 3043 (CH-Ar), 2960 (CH-aliphatic), 2226 (CN), 1674 (C ¼ O),
1
9
599 & 1467 (C ¼ C), 1406, 1376, 1320, 1235, 1145, 1111, 1034, Synthesis of (2-methyl-4-oxo-4H-quinazolin-3-yl)-carbamic acid
44, 868, 836, 766, 687, 660, 555; d (500 MHz; CDCl ) 9.37 (1H, s, isobutyl ester (10)
H
3
5
CH ¼ N), 8.28 (1H, d, J= 8.0, CH -quinazolin-4-one), 8.00 (2H, d,
J= 8.2, 2 ꢃ CH-benzonitrile ring), 7.80 (2H, d, J= 8.2, 2 ꢃ CH-benzo-
A
mixture
of
3-amino-2-methyl-3H-quinazolin-4-one
6a
(0.0028 mol, 0.5 g) and isobutyl chloroformate 9 (13.6 equiv.,
7
nitrile ring), 7.77 (1H, app. t, J= 7.1, CH -quinazolin-4-one), 7.68
35
0
.038 mol, 5.2 g, 4.94 ml) was refluxed for 10 h . The excess
8
6
(1H, d, J= 8.0, CH -quinazolin-4-one), 7.49 (1H, app. t, J= 7.6, CH -
reagent was evaporated under reduced pressure and the remain-
ing residue was treated with diethyl ether. The precipitated solid
was filtered, washed with water, and purified by recrystallisation
from benzene to obtain the title compound as a beige powder
quinazolin-4-one), 2.73 (3H, s, CH ); d (125 MHz; CDCl ) 162.10
3
C
3
(
1
C ¼ O), 159.10 (HC ¼ N), 154.14 (C =N), 146.23, 137.25, 134.71,
q
32.69, 128.98, 127.34, 127.07, 126.75, 121.48, 118.16, 115.36
þ
(
2
(
8 ꢃ CH-Ar, 4 ꢃ C -Ar & CꢄN), 23.02 (CH ); (EI) m/z (%) [M þ 2]
ꢁ
ꢂ1
q
3
with 50% yield, mp 168–170 C; v
(KBr)/cm 3223 (NH), 3080
max
þ
þ
90.10 (3.21), [M þ 1] 289.10 (1.58), [M ] 288.10 (5.65), 273.10
(
1
9
CH-Ar), 2963 & 2875 (CH-aliphatic), 1755 (C ¼ O), 1694 (C ¼ O),
3.00), 272.10 (6.19), 260.10 (1.60), 186.05 (3.69), 161.10 (10.65),
651 (C ¼ N), 1606 & 1508 (C ¼ C), 1468, 1380, 1232, 1108, 1046,
1
60.10 (100.00), 145.10 (4.68), 143.10 (3.86), 131.10 (14.47), 130.10
88, 942, 872, 772, 719, 695, 633, 520, 462; d (850 MHz; CDCl )
H
3
(17.06), 119.10 (11.52), 117.10 (19.54), 102.10 (18.65), 101.10 (5.04), 8.19 (1H, d, J= 7.7, CH-Ar), 7.75 (1H, app. t, J= 7.7, CH-Ar), 7.64
9
5
2.05 (5.14), 90.05 (11.61), 77.00 (20.32), 76.00 (37.53), 60.00 (4.25), (1H, d, J= 8.5, CH-Ar), 7.62 (1H, br. s, NH), 7.44 (1H, t, J= 7.7, CH-
9.05 (19.07), 51.00 (6.85), 50.00 (14.71). Anal. calcd. for C H N O Ar), 4.00 (2H, apparent s, O– CH – CH–(CH ) ), 2.60 (3H, s, CH ),
1
7
12
4
2
3 2
3
(
288.10): C, 70.82; H, 4.20; N, 19.43. Found: C, 70.69; H, 4.13; 1.96–1.93 (1H, m, O-CH -CH-(CH ) ), 0.94 (6H, d, J= 6.0, O–
2 3 2
N, 19.27.
CH
2
–CH–(CH
3 2
) );
d
C
(125 MHz; CDCl
3
)
160.78 (C ¼ O), 156.28

6
76
N. N. E. EL-SAYED ET AL.
(
C ¼ O), 156.15 (C ¼ N), 146.83, 134.89, 126.94, 126.85, 126.58, 361.10 (35.50), 359.10 (100.00), 324.10 (11.09), 301.00 (12.62),
1
1
2
20.60 (4 ꢃ CH-Ar, 2 ꢃ C
q
-Ar), 72.71 (OCH
2
), 27.78 (CH), 21.52 (CH
3
), 283.05 (37.48), 267.05 (81.50), 249.10 (51.70), 235.05 (33.05), 201.00
þ
þ
8.63 (2 ꢃ CH ); MS (EI) m/z (%) [M þ 1] 276.10 (21.00), [M ] (89.80), 193.05 (3.31), 180.05 (4.87), 166.05 (14.31), 150.05 (8.84),
3
75.10 (100.00), 260.10 (1.46), 220.05 (4.41), 202.00 (11.98), 175.05 133.10 (77.88), 104.10 (28.46), 89.00 (25.12), 77.00 (63.94), 63.00
3
5
(95.02), 160.05 (9.43), 146.10 (85.56), 131.10 (4.53), 131.10 (4.53), (10.37), 51.00 (11.84). Anal. calcd. for C22
H
17 ClN
17.10 (27.22), 90.00 (23.15), 76.00 (30.46), 63.00 (8.24), 57.05 67.25; H, 4.36; N, 7.13. Found: C, 67.20; H, 4.32; N, 7.06.
(275.13): C, 61.08; H, 6.22; N,
2
OS (392.08): C,
1
(86.01). Anal. calcd. for C14
H
N
17 3
O
3
1
5.26. Found: C, 61.00; H, 6.01; N, 15.17.
N-(2,6-Dimethyl-phenyl)-2-(4-oxo-3-phenyl-3,4-dihydro-
quinazolin-2-ylsulfanyl)-acetamide (15c)
0
Synthesis of (E)-N,N-dimethyl-N -(2-methyl-4-oxo-4H-quinazolin-
ꢂ1
ꢁ
White crystals, yield (55%), mp 218–220 C; ꢀmax (KBr)/cm 3331
3
-yl)-formamidine (12)
mixture of 3-amino-2-methyl-3H-quinazolin-4-one
0.0028 mol, 0.5 g) and N,N-dimethyl formamide dimethyl acetal 11 1205, 1112, 1030, 962, 881, 768, 695, 641, 595, 568, 523, 468; d
5.39 equiv. 0.0151 mol, 1.79 g, 2 ml) in ethanol (15 ml) was (850 MHz; CDCl ) 8.66 (1H, br. s, NH), 8.27 (1H, d, J= 7.7, CH-Ar),
(
NH), 3065 (CH-Ar), 2971 & 2917 (CH-aliphatic), 2366, 1664 (C ¼ O),
A
(
(
6a 1601 (C ¼ O), 1552 & 1493 (C ¼ C), 1467, 1362, 1335, 1299, 1264,
H
3
3
6
refluxed for 2 h . The excess solvent was removed under reduced 7.76 (1H, t, J= 7.7, CH-Ar), 7.62 (1H, d, J= 7.7, CH-Ar), 7.59–7.58
pressure. The remaining solid was washed with n-hexane and (2H, m, 2 ꢃ CH-Ar), 7.47 (1H, t, J= 7.7, CH-Ar), 7.33–7.32 (2H, m,
recrystallised from ethanol to obtain the title compound as a yel- 2 ꢃ CH-Ar), 7.11–7.07 (2H, m, 2 ꢃ CH-Ar), 7.04 (2H, d, J= 6.8,
ꢁ
ꢂ1
low powder with 50% yield, mp 100–102 C; ꢀmax (KBr)/cm 3010 2 ꢃ CH-Ar), 3.99 (2H, s, CH
2
), 2.18 (6H, s, 2 ꢃ CH
3 C
); d (312 MHz;
(
(
1
CH-Ar), 2924 & 2807 (CH-aliphatic), 2145, 1660 (C ¼ O), 1621 CDCl ) 166.94 (C ¼ O), 161.33 (C ¼ O), 157.78 (C ¼ N), 146.99,
3
C ¼ N), 1609 & 1589 (C ¼ C), 1475, 1418, 1366, 1323, 1237, 1174, 135.26, 135.23, 135.19, 133.55, 130.55, 130.05, 129.02, 128.27,
120, 1069, 944, 874, 783, 701, 660, 626, 551, 509; dH (500 MHz; 127.66, 127.38, 126.75, 125.73, 119.91 (12 ꢃ CH-Ar, 6 ꢃ C -Ar),
q
þ
CDCl
3
) 8.23 (1H, dd, J= 7.7, 1.7, CH-Ar), 7.74 (1H, s, HC ¼ N), 35.49 (CH
2
þ
), 18.33 (2 ꢃ CH
3
); MS (EI) m/z (%) [M þ 1] 416.15
7
7
2
.69–7.67 (1H, m, CH-Ar), 7.62 (1H, dd, J= 8.5, 2.6, CH-Ar), (1.05), [M ] 415.15 (2.86), 400.10 (9.45), 296.05 (20.13), 295.05
.41–7.39 (1H, m, CH-Ar), 3.08 (3H, s, N-CH ), 3.04 (3H, s, N-CH ), (100.00), 268.05 (28.58), 253.05 (10.77), 235.10 (30.01), 221.05
3
3
.52 (3H, s, CH ); d (125 MHz; CDCl ) 162.22 (C ¼ O), 160.08 (5.97), 177.05 (7.25), 162.00 (2.47), 148.05 (3.93), 132.10 (20.59),
3
C
3
(
(
HC ¼ N), 155.08 (C
q
=N), 146.39, 133.39, 126.41, 125.61, 121.04 119.05 (11.21), 105.10 (2.87), 91.05 (12.43). Anal. calcd. for
4 ꢃ CH-Ar & 2 ꢃ C
q
-Ar), 40.88, 34.53 (N(CH
3
)
2
), 22.56 (CH
3
); MS (EI)
24 21 3 2
C H N O S (415.14): C, 69.37; H, 5.09; N, 10.11. Found: C, 69.18;
þ
þ
m/z (%) [M þ 1] 231.10 (5.52), [M ] 230.10 (34.52), 215.05 (1.48), H, 4.92; N, 10.00.
1
86.00 (10.30), 161.10 (11.53), 160.10 (100.00), 145.10 (5.33), 132.10
(
6
6
7.42), 118.10 (21.02), 104.10 (1.60), 90.00 (9.13), 76.00 (16.94),
N-(2,6-Dimethyl-phenyl)-2-(6-methyl-4-oxo-3-phenyl-3,4-dihydro-
quinazolin-2-ylsulfanyl) -acetamide (15d)
14 4
3.00 (5.01), 50.00 (6.27). Anal. calcd. for C12H N O (230.12): C,
2.59; H, 6.13; N, 24.33. Found: C, 62.47; H, 5.98.; N, 24.18.
ꢁ
ꢂ1
White powder, yield (57%), mp 229–230 C; ꢀ
(KBr)/cm 3269
max
(
(
1
5
NH), 3024 (CH-Ar), 2961 & 2919 (CH-aliphatic), 2372, 2341, 1688
Synthesis of 2-(4-chloro-benzylsulfanyl)-6-methyl-3-phenyl-3H-
quinazolin-4-one (15a , 15b) and 2-(4-oxo-3-phenyl-3,4-
dihydro-quinazolin-2-ylsulfanyl)-acetamide derivatives (15c–f)
C ¼ O), 1651 (C ¼ O), 1574 & 1547 (C ¼ C), 1486, 1408, 1336, 1296,
3
1
260, 1199, 1103, 1032, 963, 912, 836, 774, 748, 692, 648, 604,
43, 504; dH (500 MHz; CDCl ) 8.70 (1H, br. s, NH), 8.05 (1H, s, CH-
3
A mixture of the appropriate derivative of 2-mercapto-3-phenyl- Ar), 7.57–7.49 (5H, m, 5 ꢃ CH-Ar), 7.32–7.30 (2H, m, 2 ꢃ CH-Ar),
3
H-quinazolin-4-one (13a’,b’) (0.0018 mol) and 4-chlorobenzyl 7.07–7.02 (3H, m, 3 ꢃ CH-Ar), 3.96 (2H, s, CH
2
), 2.46 (3H, s, CH
) 166.99 (C ¼ O), 161.39
3
),
37,38
chloride 14a or chloroacetamide derivatives 14b,c
0
0
(1 equiv., 2.16 (6H, s, 2 ꢃ CH
3 C 3
); d (125 MHz; CDCl
.0018 mol) and potassium carbonate (2.5 equiv., 0.0045 mol, (C ¼ O), 156.66 (C ¼ N), 145.07, 136.98, 136.57, 135.42, 135.18,
39
.62 g) in dry acetone (20 ml) was refluxed for 6 h . The hot reac- 133.64, 130.46, 130.01, 129.08, 128.25, 127.32, 127.05, 125.59,
tion mixture was filtered and the filtrate was concentrated under 119.64 (11 ꢃ CH-Ar, 7 ꢃ C
q
-Ar), 35.49 (CH
); MS (EI) m/z (%) [M þ 1] 430.10 (1.24), [M ] 429.15
2
), 21.30 (CH
3
), 18.35
þ
þ
reduced pressure. The resulting solid in each case was washed (2 ꢃ CH
3
with water, air dried, and recrystallised from ethanol to obtain (3.40), 414.10 (9.75), 309.05 (100.00), 282.05 (35.11), 267.05 (10.35),
compounds 15a–f. 249.10 (38.79), 235.05 (5.16), 205.05 (1.86), 177.05 (5.60), 160.05
2.87), 146.10 (16.74), 133.10 (10.36), 121.10 (4.97), 104.05 (10.15),
1.05 (11.20), 77.00 (21.32), 59.00 (3.71). Anal. calcd. for
S (429.15): C, 69.91; H, 5.40; N, 9.78. Found: C, 69.76; H,
.32; N, 9.64.
(
9
2
-(4-Chloro-benzylsulfanyl)-6-methyl-3-phenyl-3H-quinazolin-4-
25 23 3 2
C H N O
5
one (15b)
ꢁ
ꢂ1
White powder, yield (96%), mp 164–165 C; ꢀmax (KBr)/cm 3058
(
1
9
(
7
(
1
1
(
1
CH-Ar), 2915 (CH-aliphatic), 1689 (C ¼ O), 1617 & 1575 (C ¼ C),
N-(2,3-Dimethyl-phenyl)-2-(6-methyl-4-oxo-3-phenyl-3,4-dihydro-
quinazolin-2-ylsulfanyl) -acetamide (15f)
548, 1487, 1401, 1399, 1335, 1299, 1259, 1204, 1128, 1089, 1014,
63, 824 (C–Cl), 742, 692, 641, 539, 506; d (500 MHz; CDCl ) 8.01
H
3
5
ꢁ
ꢂ1
1H, s, CH -quinazolin-4-one), 7.54–7.48 (5H, m, 5 ꢃ CH-Ar), White powder, yield (76%), mp 218–220 C; ꢀ
(KBr/cm 3259
max
.29–7.18 (6H, m, 6 ꢃ CH-Ar), 4.32 (2H, s, CH
125 MHz; CDCl
) 161.83 (C ¼ O), 155.60 (C ¼ N), 145.82, 136.17, 1648 (C ¼ O), 1574 & 1545 (C ¼ C), 1487, 1300, 1263, 1204, 1147,
36.12, 135.90, 135.38, 133.28, 130.77, 130.00, 129.72, 129.26, 963, 834, 787, 750, 694, 651; d (500 MHz; CDCl ) 8.90 (1H, s, NH),
2
), 2.44 (3H, s, CH
); d
3 C
(NH), 3000 (CH-Ar), 2925 (CH-aliphatic), 2371, 2341, 1692 (C ¼ O),
3
H
3
28.68, 126.76, 126.05, 119.72 (12 ꢃ CH-Ar & 6 ꢃ C -Ar), 36.22 8.06 (1H, s, CH-Ar), 7.57–7.53 (5H, m, 5 ꢃ CH-Ar), 7.49 (1H, d,
q
þ
37
þ
CH
,
2
), 21.33 (CH
3
); MS (EI) m/z (%) [M , Cl] 394.05 (27.71), [M
Cl] 393.10 (19.45), [M , Cl] 392.10 (69.22), 375.05 (1.96), J= 7.3, CH-Ar), 6.97 (1H, d, J= 6.9, CH-Ar), 3.97 (2H, s, CH ), 2.48
þ
J= 7.4, CH-Ar), 7.32–7.25 (2H, m, 2 ꢃ CH-Ar), 7.08 (1H, apparent t,
3
5
þ
35
2

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
677
(3H, s, CH
3
), 2.23 (3H, s, CH
3
), 2.01 (3H, s, CH
); d
3 C
(125 MHz; In vitro evaluation of antiphospholipases activities
CDCl ) 167.11 (C ¼ O), 161.38 (C ¼ O), 156.74 (C ¼ N), 145.11, The inhibitory activities of the synthesised 3H-quinazolin-4-one
3
1
1
37.41, 136.94, 136.50, 135.39, 135.31, 130.46, 129.96, 129.10, derivatives against a group of phospholipases were investigated
42
28.95, 127.31, 127.05, 125.93, 125.67, 121.93, 119.66 (11 ꢃ CH-Ar according to the method of De Aranjo and Radvany using five
&
7 ꢃ C
q
-Ar), 36.14 (CH
2
), 21.30 (CH
3
), 20.55 (CH
3
), 13.82 (CH
); MS sPLA
3
2
s: human group IIA (hG- IIA), human group V (hG-V), human
) and Naja mos-
). Ten microliters of each
solution (20 lg/ml) was mixed with 10 ll of each compound
01.10 (4.81), 77.00 (13.43), 59.00 (21.59). Anal. calcd. for at different concentrations (0–50 lg/ml) and the mixture was incu-
S (429.15): C, 69.91; H, 5.40; N, 9.78. Found: C, 69.85; H, bated for 20 min at room temperature. Then, 1 ml of the PLA sub-
.35; N, 9.75.
strate (3.5 mM lecithin suspended in 100 mM NaCl, 10 mM CaCl
mM NaTDC, and 0.055 mM red phenol, pH 7.6) was added. The
þ
(
2
(
1
EI) m/z (%) [M ] 429.15 (0.15), 414.10 (0.65), 309.05 (100.00), group X (hG-X), Apis mellifera bee venom (AmPLA
2
82.05 (5.52), 269.10 (6.32), 249.10 (13.83), 235.05 (3.23), 189.05 sambica mossambica sPLA
2 2
(NmPLA
1.52), 161.10 (1.76), 146.10 (10.05), 133.10 (6.23), 113.10 (7.77), sPLA
2
C
25
H
23 3
N O
2
2
5
,
2
3
hydrolysis kinetics was followed spectrophotometrically for 5 min
at 558 nm. The results were reported as the inhibition percentage
that was calculated by comparison with a control experiment
Biological evaluation
Cell culture
(absence of compound) and the IC50 values were determined
Human CRC cell lines, HT-29 and SW620 cells were grown in RPMI
from the curve. Oleanolic acid (Sigma) was used as the posi-
tive control.
(Invitrogen) containing 10% heat-inactivated foetal bovine serum,
1
00 mg/ml streptomycin, 100 units/ml penicillin and 2 mmol/l
L-glutamine.
In vitro evaluation of antiproteases activities
The inhibitory effects of 21 3H-quinazolin-4-one derivatives on
several available therapeutically important proteases such as cath-
epsin-B, collagenase, elastase, thrombin, and trypsin were investi-
gated. The inhibition of the enzymes in the presence of test
compounds was evaluated by adding different concentrations of
each compound (0–75 lg/ml) to the respective reaction mixture,
preincubating for 15 min and then measuring the remaining
Cell viability assay
Cell viability was determined using the 3-(4, 5-dimethylthiazolyl-
2
)–2, 5-diphenyltetrazolium bromide) MTT assay according to
4
0
Mosmann . 5-Fluorouracil (5-FU) was used as positive control.
Briefly, after culturing cells in 96 well plates for 24 h, they were
treated with different compounds (30 lg/ml) for 24 h. Freshly pre-
pared MTT (5 mM) solutions were added to the cells and were fur-
ther incubated for 2 h at 37 C in 5% CO . One hundred
43
enzyme activity as previously described . The protease inhibitory
ꢁ
2
activity was expressed as inhibition percentage, which was deter-
mined by comparison with a control experiment. The results were
expressed as IC50 values that were determined from the standard
curve. A protease inhibitor cocktail (Sigma) was used as the posi-
tive control.
microliters of dimethyl sulphoxide (DMSO) was added in each well
to dissolve the formazan crystal, formed in the reaction during
incubation by careful pipetting. The absorbance of the product
was measured at 540 nm using a microplate reader. The experi-
ments were performed in triplicate for each condition. The graph
illustrates the mean and standard deviation (SD) values of three
independent experiments.
In vitro inhibition of a-amylase activity
The a-amylase inhibitory activity of the tested compounds was
44
investigated according to the reported method by Subranian .
Briefly, 10 mL of a-amylase (3,3 U) (Sigma) was mixed with 10 ml of
different concentrations (20–200 mg/ml) of each compound, the
appropriate solvent, or quercetin (positive control), and incubated
at 37 C for 5 min. After adding 180 ml of Labtest (amylase sub-
strate), the samples were incubated for 8 min and the absorbance
Cytotoxicity assay using the xCELLigence system
The optimal seeding concentration for proliferation of HT-29 was
determined. HT-29 cells (5000 cell in 150 ml medium/well) and
SW620 cells (12,000 cell in 150 ml medium/well) were seeded in
ꢁ
1
6 well plates (E-plate 16-ACEA Biosciences Inc., San Diego, CA) as
described in the xCELLigence Real Time Cell Analyzer (RTCA) DP
instrument manual provided by the manufacturer. After 24 h, 20
and 40 mg/ml of compound 15d were added and the experiments
were allowed to run for another 3 d. Baseline cell indexes were
calculated for at least two measurements from three replicate
experiments.
of first reaction was measured at 620 nm. Then, 100 ml of the reac-
tion mixture was incubated for more five additional minutes at
ꢁ
3
7 C and the absorbance of the second reaction was measured.
Labtest was diluted in distilled water (1:1) before being added to
the microplate. The concentration of quercetin used was same as
those of the test compounds. The a-amylase inhibition percentage
was calculated as follows: % inhibition = 100 _ (X2 sample _ X1
sample/X2 control _ X1 control)ꢃ 100 where X1 is the absorbance
of the initial reading and X2 is the absorbance of the final reading.
The results were expressed as IC50 determined from the curve.
Western blotting
Whole cell lysates were prepared using radioimmunoprecipitation
assay (RIPA) lysis buffer as described by Al-Khayal and co-work-
4
1
ers . Total protein concentration was determined using Bradford
protein reagent (Bio-Rad, Hercules, CA). Soluble proteins were In vitro inhibition of a-glucosidase activity
loaded on precast TGX gels and were analysed using immunoblot- The a-glucosidase inhibitory activity of the tested compounds was
ting with anti-Bcl2, anti-BclxL (dilution 1:1000; Santa Cruz evaluated according to the method described by Andrade-Cetto
4
5
Biotechnology, Dallas, TX), and anti-b-actin (1:10,000 Sigma, St. and collaborators based on the release of 4-nitrophenol a-D-glu-
Louis, MO). Reactivity was detected using horseradish peroxidase- copyranoside (4 NPGP). Briefly, 20 ml of different concentrations
conjugated secondary antibodies and chemiluminescence by (ranging from 0 to 50 mg/ml) of each compound, the appropriate
Clarity Western ECL substrate (Bio-Rad). Membrane was developed solvent, or quercetin (positive control) was mixed with 180 ml of
using C-Digit Blot Scanner (LI-COR, Hamburg, Germany).
the a-glucosidase enzyme from Saccharomyces cerevisiae (Sigma)

6
78
N. N. E. EL-SAYED ET AL.
O
O
O
3'
H
2
N
O
2'
4
O
O
O
N
O
4
5
8
3 1'
N
2
R
COOH
NH2
R
4a
8a
R
6
O
4
5'
Ac
2
O
6'
7
(i)
CH3
N
1
CH
3
(ii)
2
2
2
2
a, R= H
3a, R= H
3
3
3
b, R= F
c, R= Cl
d, R= Br
b, R= F
c, R= Cl
d, R= Br
5a, R= H
5b, R= F
5c, R= Cl
N
2
H
4
.H
2
O
(iii)
O
N
R
NH
2
N
6
a, R=H
b, R=Br
6
(
v)
(vi)
OCH
3
O
Cl
O
(iv)
H
3
CO
N
H
Ar
a-e
O
1
1
7
9
O
O
O
CH3
N
H
R
N
Ar
N
O
N
N
N
N
CH
3
O
N
CH
3
N
CH
3
N
CH
3
8a-j
10
12
a, R=H; Ar=
b, R= Br; Ar=
d, R= Br; Ar=
f, R= Br; Ar=
N
N
c, R=H; Ar=
e, R=H; Ar=
O
2
N
OCH
3
O
2
N
OCH
3
g, R=H; Ar=
i, R=H; Ar=
C
N
h, R= Br; Ar=
j, R= Br; Ar=
C N
ꢁ
Scheme 1. (i) Heating in domestic microwave at 540 watt for 8 min, cooling, and washing with petroleum ether; (ii) Fusion in oil bath at 200 C, for 2 h, cooling to
1
ꢁ
00 C, treatment with 37% HCl (Conc) till dissolution of the reaction mixture; cooling to room temperature and alkalisation with dilute NaOH (25%); (iii) ethanol, reflux
for 8 h; (iv) ethanol and acetic acid catalytic, reflux for 10 h; (v) reflux for 10h; (vi) ethanol, reflux for 2 h.
ꢁ
and the obtained mixture was incubated at 37 C for 2 min, fol- uric acid from xanthine according to the reported method by
ꢁ
46
lowed by additional incubation for more than 15 min at 37 C after Bondet and co-workers . Forty microliters xanthine oxidase
the addition of 150 ml of the colour reagent NPGP. The colorimet- (667 mM) and 15 ml of each compound (0–150 mg/ml), allopurinol
ric assay included 2 U of a-glucosidase, 5 mM of 4-NPGP, and (positive control), or appropriate solvent (negative control) were
1
0 mM potassium phosphate buffer, pH 6.9. The concentration of added to each microplate well and preincubated for 5 min at
quercetin used was identical to that of the tested compounds. 37 C. Then, 95 ml of reagent 1 (mixture of hydroxylamine
ꢁ
The reading assay was performed using a microplate reader at (0.2 mM), EDTA (0.1 mM), and xanthine oxidase (667 mM) in 50 mM
4
05 nm. The a-glucosidase inhibition percentage was calculated as phosphate buffer solution (pH 7.5) were added to the reaction
follows: % inhibition = 100 _ (X2 sample _ X1 sample/X2 control _ mixture and incubated at the same temperature for 30 min.
Subsequently, the absorbance was measured at 295 nm using a
X1 control) ꢃ 100 where X1 is the absorbance of the initial reading
microplate reader. Finally, after addition of 150 ml of uric acid
reagent, the absorbance was measured again. The concentration
of allopurinol used was same as that of the tested compounds.
Xanthine oxidase inhibition was calculated as follows: % inhibition
and X2 is the absorbance of the final reading, control is the
absorbance of the assay with the appropriate solvent. The results
were expressed as IC50 determined from the curve.
=
100 _ (X2 sample _ X1 sample/X2 control _ X1 control) ꢃ 100
In vitro inhibition of xanthine oxidase
where X1 is the absorbance of the initial reading and X2 is the
The xanthine oxidase inhibitory activities of the examined chem- absorbance of the final reading. The results were expressed as
ical compounds were determined by following the formation of IC50 determined from the curve.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
679
Results and discussion
corresponding to (CH ¼ N) groups resonating at
d
C
1
59.10–156.46 ppm.
Chemistry
Furthermore, the amino group of 3-amino-2-methyl-3H-quina-
The main targets of this investigation were 3H-quinazolin-4-one zolin-4-one 6a was transformed to the corresponding carbamate
derivatives possessing general structure 1.
group via direct nucleophilic substitution reaction for isobutyl
chloroformate 9 to yield the new carbamate derivative 10. The
3
5
structure of the latter product was established on the basis of its
1
13
Two synthetic approaches were used for the synthesis of
these scaffolds.
The first synthetic approach featured the preparation and
chemical transformations of the key intermediates benzo[d][1,3]ox-
azin-4-one derivatives 3a–d to various 3H-quinazolin-4-one deriva-
tives as depicted in Scheme 1.
IR, H NMR, C NMR, MS and HCN elemental analyses. The IR
spectrum displayed the characteristic stretching absorption bands
ꢂ1
at vmax 3223, 1755, and 1694 cm indicating the presence of NH
1
and two C ¼ O groups. The H NMR spectrum of compound 10
was characterised by the presence of a one-proton broad singlet
peak at d 7.62 ppm due to NH group, in addition to the presence
of a two-protons apparent singlet signal, a one-proton multiplet,
and a six-protons doublet signal with coupling constant value J of
The conventional methods available for the synthesis of com-
pounds 3a–d via cyclocondensation of anthranilic acid derivatives
26–28
2a–d with acetic anhydride
were time consuming, therefore,
6
.0 Hz at d 4.00, 1.96–1.93 and 0.94 ppm, respectively correspond-
1
3
these compounds were synthesised using the more efficient
2 3 2
ing to (O-CH -CH-(CH ) ) isobutoxy group. Moreover, the C NMR
microwave-assisted method. Fusion of oxazinones 3a–c with 3,4,5-
spectrum of carbamate 10 revealed the emergence of a new spec-
tral line at d
3
2,33
trimetrhoxyanline 4
yielded 2-methyl-3–(3,4,5-trimethoxy-phe-
C
156.28 ppm attributable to the C ¼ O of the carba-
nyl)-3H-quinazolin-4-one derivatives 5a–c. The assigned molecular
structures of these new quinazolin-4-one derivatives were in full
agreement with their spectroscopic and elemental analyses. Thus
the IR spectrum (KBr) of compound 5c, for example, displayed
mate functional group. The methylene, methine and two methyl
groups of isobutoxy moiety demonstrated three spectral lines at
d
C
72.71, 27.78, and 18.72 ppm, respectively. Finally, the mass
spectrum (EI) of compound 10 showed the anticipated molecular
þ
absorption bands at v
8
and C–Cl, respectively. The H NMR spectrum of compound 5c
3075, 2934; 2839, 1680, 1598; 1504 and
max
ion peaks [M þ 1] at m/z 276.10 (21.00%) for C14
17 3 3
H N O and
ꢂ1
þ
34 cm for aromatic-CH, aliphatic-CH, the carbonyl group, C ¼ C [M ] 275.10 (100.00%).
1
Next, 3-amino-2-methyl-3H-quinazolin-4-one 6a was trans-
revealed the presence of five aromatic protons, which were dis- formed to the corresponding N,N-dimethylformamidine derivative
played as; a one proton doublet signal at d 8.18 ppm with cou- 12 by refluxing with excess N,N-dimethyl formamide dimethyl ace-
5
36
pling constant J of 2.4 Hz corresponding to CH -quinazolin-4-one, tal (DMF-DMA) 11 in absolute ethanol . The structure of this new
1
13
a one-proton double-doublet signal at d 7.69 ppm with coupling compound was deduced from its IR, HNMR, CNMR, MS data,
constant J of 8.7, 2.4 Hz due to CH -quinazolin-4-one, a one-pro- and elemental analyses. The IR spectrum revealed the presence of
7
ton doublet signal at d 7.60 ppm with coupling constant J of absorption bands at v
3010, 2924; 2807, 1660, 1621, 1589;
max
8
ꢂ1
8
.7 Hz corresponding to CH -quinazolin-4-one, and a two-protons 1475 cm
which were attributed to CH-aromatic, CH-aliphatic,
1
singlet peak at d 6.51 ppm, which was attributed to the two C ¼ O, C ¼ N, and C ¼ C, respectively. In addition, the H NMR
’
6’
methine protons of 3,4,5-trimethoxyphenyl moiety (H2 and H ). spectrum of formamidine 12 was characterised by the presence of
The nine protons of the three methoxy groups were displayed as a new one-proton singlet signal at d 7.74 ppm attributed to azo-
a three-protons singlet at d 3.92, and a six-protons singlet at d methine proton (HC ¼ N) and two singlet signals, each integrating
3
.88 ppm. Finally, the three protons of the methyl group were to three protons resonating at d 3.08 and 3.04 ppm, corresponding
characterised by a three-protons singlet signal at d 2.32 ppm. to the two methyl groups in dimethylamino N(CH ) moiety.
3
2
1
3
13
Moreover, the C NMR spectrum also confirmed the assigned Furthermore, the C NMR spectrum of compound 12 demon-
structure based on the emergence of fifteen spectral lines at d
strated three new peaks at d 160.08, 40.88, and 34.53 ppm which
C
C
1
1
6
61.28 (CO), 154.75 (C ¼ N), 154.25, 145.91, 138.56, 135.02, 132.95, were attributed to HC=N-N(CH ) and, two carbons of the methyl
3
2
32.36, 128.52, 126.23, 121.73, 105.09 (5 ꢃ CH-Ar & 7 ꢃ C -Ar), groups in N(CH ) moiety, respectively. The mass spectrum (EI) of
q
3 2
þ
0.93 (OCH ), 56.28 (2 ꢃ OCH ) and 24.02 (CH -quinazolin-4-one). compound 12 showed the anticipated molecular ion peak [M ] at
3
3
3
The mass spectrum (EI) of compound 5c indicated the presence of m/z 230.00 (34.52%) for C H N O.
the expected molecular ion peak [M ] at m/z 360.10 (100%) for
C H17 ClN O .
18 2 4
1
2 14 4
þ
The second synthetic approach for preparation of 3H-quinazo-
3
5
lin-4-one derivatives involved the synthesis of compounds 15a–f
Refluxing of oxazinones 3a,d with excess hydrazine hydrate in via nucleophilic attack by 2-mercapto-3-phenyl-3H-quinazolin-4-
2
9,30
ethanol yielded 3-aminoquinazolinons 6a,b
verted to certain novel Schiff bases 8a–j via condensation with a cetamide derivatives 14b,c
series of aryl aldehydes 7a–e in absolute ethanol containing cata- adapting modified procedure to what was reported by Salman
, which were con- one derivatives 13a’,b’ on 4-chlorobenzyl chloride 14a or chloroa-
3
7,38
to yield compounds 15a-f by
34
39
lytic amount of glacial acetic acid . The structures of the new et al. as illustrated in Scheme 2.
Schiff’s bases were verified by their IR, H NMR, C NMR, mass
spectroscopic (MS) data, and CHN analyses. The IR spectra of com- were verified based on their respective IR, H NMR, C NMR, MS
pounds 8a–j indicated disappearance of the stretching bands of data, and elemental analyses. The IR spectra (KBr) of these products
the amino groups in 3-aminoquinazolin-4-one derivatives 6a,b. were characterised by the disappearance of the absorption bands
1
13
Structures of the new quinazolinone derivatives 15b and 15d,f
1
13
1
ꢂ1
The H NMR spectra were characterised by the disappearance of at vmax 3245 and, 3169 cm for the NH group (NH, SH exchange-
ꢂ1
the two-protons singlet signals corresponding to the amino pro- able) and at vmax 1195cm for the thiocarbonyl group (C ¼ S) of
1
tons of starting materials, which resonated at d 5.79 and starting substrates 13a,b. Furthermore, their H NMR spectra indi-
4
.89 ppm, accompanied by emergence of new one-proton singlet cated the disappearance of the signals at d 13.05 and 12.99 ppm
signals attributed to azomethine protons (CH ¼ N) at
due to the thiol protons of the starting materials. In addition, the
.41 8.70 ppm range. In addition, the C NMR spectra displayed spectrum of compound 15b showed characteristic two-protons
the expected spectral lines for each compound with the signals singlet signal at d 4.32 corresponding to the benzylic methylene
d
_
13
9

6
80
N. N. E. EL-SAYED ET AL.
O
N
R1
Cl
N
Cl
(
S
14a
i)
1
5a, R = H
1
Cl
1
5b, R = CH3
O
O
N
1
R1
R1
R3
CH O
3
N
N
Cl
N
H
(
i)
N
H
S
SH
R4
1
3a, R = H
13a', R = H
1
1
1
3b, R = CH
13b', R = CH3
1
14b,c
1
3
O
N
R1
N
CH3
H
N
R2
S
O
R3
1
1
1
1
5c, R = R = H; R = CH
1 2 3 3
5d, R = CH ; R = H; R = CH
1
3
2
3
3
5e, R = H; R = CH ; R = H
1
2
3
3
5f, R = R = CH ; R = H
1
2
3
3
2 3
Scheme 2. Reagents and conditions: (i) K CO and dry acetone, reflux for 6–8 h.
group. Analogously, the spectra of compounds 15d,f were charac- 8a, 8f, 8j, 15a, 15b, and 15d, were potently capable of inhibiting
terised by the emergence of new one-proton broad singlet signals cell viability of both human CRC cell lines. Compound 15d was
at d 8.70 and 8.90 ppm corresponding to NH groups, and two-pro- the most active in this set reducing the cell viabilities of HT-29
tons singlet signals at d 3.96 and, 3.97 belong corresponding to and SW620 cells lines to 38% and, 36.7%, compared to 5-FU which
methylene groups. In addition, the two methyl groups of the aceta- demonstrated cell viabilities of 65.9 and 42.7%, respectively.
mide moieties were represented by a six-protons singlet signal at d Hence, compound 15d was selected for further mechanistic stud-
2
.16 ppm for compound 15d, and two singlet signals each integrat- ies. The results indicated that compound 15d inhibited cell viabil-
ing to three-protons at d 2.23 and, 2.01ppm for compound 15f.
ity in a dose-dependent manner. The IC50 for 15d was ꢀ20 mg/ml
13
Furthermore, the
C NMR spectra of compounds 15b,d,f for HT-29 and SW620 cells (Supplementary Figures 2 and 3). These
revealed the absence of spectral lines at d
corresponding to the thiocarbonyl group in the starting materials which measures the cytotoxic effect of inhibitors in real time at
3a,b and the presence of new peaks at d
36.22, 35.49, and different time points and 15d was found to inhibit the cell prolif-
C
176.05 and 176.00 ppm results were again confirmed using the xCELLigence RTCA system,
1
3
C
6.14 ppm attributed to the new methylene groups. The carbonyl eration in human CRC HT-29 and SW620 cell lines in dose- and
time-dependent manner (Supplementary Figures 4 and 5).
and methyl groups of the acetamide moieties in compounds 15d,f
C
were observed at d 166.99, 167.11, 18.35, 20.55, and 13.82, respect-
ively. In addition, the mass spectra (EI) of compounds 15b,d,f dis-
played the anticipated molecular ion peaks.
Evaluation of anti-apoptotic protein expression by compound 15d
SW620 cells were treated with various concentrations of 15d for
2
4 h. Subsequently, cell lysates were immunoblotted with the indi-
Biological evaluation
cated antibodies in order to investigate the mechanism via which
this compound inhibited cell viability of human CRC cells. Results
shown in Supplementary Figure 6 indicated that 15d inhibited the
anti-apoptotic proteins, Bcl2 and BclxL. These findings demon-
strate that compound 15d inhibited the cell viability of human
CRC cells by down regulating the expression of Bcl2 and BclxL
and triggering the apoptotic pathway.
Effects of the synthesised 3H-quinazolin-4-one derivatives on cell
viability of human colorectal cancer cell lines HT-29 and SW620
As CRC starts with initial benign stages advanced to late stage
leading to metastasis. Therefore, two human CRC cell lines which
belong to two different stages namely, HT-29 which is derived
from adenocarcinoma CRC and SW620 which is derived from
metastatic site were selected for in vitro evaluation of the cytotox-
icity (30 lg/ml) of twenty-one compounds incorporating the 3H- In vitro evaluation of antiphospholipase A
2 2
(PLA ) and antipro-
quinazolin-4-one scaffold.
teases activities of the synthesised 3H-quinazolin-4-one derivatives
Cell viability was monitored using the MTT assay and the As inflammation is associated with several types of cancers via
results are presented in Supplementary Figure 1. Compared to the influencing growth, apoptosis, and proliferation of cancer and
negative control, and the positive control (5-FU), the synthesised stromal cells, the synthesised 3H-quinazolin-4-one derivatives were
3H-quinazolin-4-one derivatives displayed varied degrees of cyto- further screened to evaluate their antiphospholipases activities
toxicity against the examined colorectal cell lines. Eight out of all against proinflammatory enzymes implicated in cancer namely,
1
0
the examined compounds showed significant decrease in cell via- hGIIA, hGV, and X . In addition, the inhibitory activity of these
bility. In one hand, compound 15c inhibited only HT-29 cell line compounds towards the allergen and inflammatory PLA from
2
4
7
(47.5% viability) whereas compound 5b inhibited only SW620 cell honey bee venom (Apis mellifera, AmPLA2) and proinflammatory,
line (46.6% viability). On the other hand, six compounds namely, heamolytic, myotoxic and neurotoxic Cobra (Naja mosambique

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
681
48
mosambique) venom PLA
2
(NmPLA
2
)
were evaluated by deter- the lowest IC50 compared to that of the reference drug (IC50;
mining their IC50 values. Results (Supplementary Table 1) revealed 0.215 ± 0.04 mg/ml).
that six compounds, namely, 5a, 8c, 8d, 8j, 15a, and 15d dis-
played the most promising potency in inhibiting the catalytic
In vitro evaluation of inhibitory activities of 3H-quinazolin-4-one
derivatives against enzymes implicated in metabolic syndrome
Considering an interactive relationship between diabetes and can-
cers, the in vitro inhibitory potential of the prepared 3H-quinazo-
lin-4-one derivative against a-amylase, a-glucosidase, and xanthine
oxidase (enzymes related to metabolic syndrome) was
investigated.
The IC50 values exhibited by the chemical compounds against
a-amylase activity are shown in Supplementary Table 3, which
indicated that compounds 8d, 8h, 8j, 15a, 15b, 15e, and 15f,
were more potent in suppressing the enzyme with IC50 values
ranging from 94.00 ± 2.83 mg/ml (compound 15b) to 121.00 ± 9.
activity of the studied hG-IIA, with IC50 values ranging from 5.
5 ± 1.06 to 8.60 ± 0.56 mg/ml compared to that recorded with ole-
7
anolic acid which was used as a standard drug (5.25 ± 0.35 mg/ml).
Among these compounds, 8d exhibited the lowest IC50 value (5.
7
5 ± 1.06 mg/ml).
The anti-PLA
and 8j inhibited the enzyme with IC50 values ranging from
.25 ± 0.78 to 9.60 ± 0.85 mg/ml compared to the value recorded
2
hGV data showed that three compounds; 5a, 8d,
6
with the standard drug (oleanolic acid, IC50; 7.50 ± 0.71 mg/ml).
Amongst them, compound 8j (IC ; 6.25 ± 0.78 mg/ml), was being
50
more potent than the reference drug.
The analysis of the inhibitory potency of the examined com-
9
1
0 mg/ml (compound 15e) than quercetin the reference drug (IC50
23.0 ± 2.83 mg/ml).
2
pounds against PLA hG-X showed that compounds; 5a, 5 b, 5c,
8d, 8j, 10, 15a, and 15e inhibited the enzymatic activity with IC50
In addition, compounds 8g (IC50 3.25 ± 0.35 mg/ml), 8h (IC50
.05 ± 0.21 mg/ml), and 8i (IC50 3.45 ± 0.64 mg/ml) were more
values ranging from 6.60 ± 0.85 to 9.50 ± 0.71 mg/ml. It worth to
note that compounds 8d, 15a, and 15e exhibited lowered IC50
values than the reference drug (IC50; 7.55 ± 0.64 mg/ml).
3
potent in inhibiting a-glucosidase activity than quercetin (IC50
3
.80 ± 0.28 mg/ml) (Supplementary Table 4).
Amongst the tested 3H-quinazolin-4-one derivatives which
Although none of the tested compounds was more effective
exhibited inhibitory activity against NmPLA
b, 5c, 8c, 8d, 8e, 8f, 15a, and 15d with IC50 values ranging from
.15 ± 0.50 to 8.50 ± 2.12 mg/ml compared to oleanolic acid (IC50
.90 ± 0.42 mg/ml). Interestingly, compound 5b demonstrated the
2
were compounds; 5a,
than allopurinol (reference drug) in inhibiting xanthine oxidase
XO) activity (Supplementary Table 5) (IC50 0.65 ± 0.07 mg/ml), com-
5
(
4
4
;
pound 8f (IC50 3.00 ± 1.41 mg/ml) was the most active among the
tested compounds.
lowest IC50 which is also lower than that of the reference
drug fusspot.
Finally, 13 compounds namely, 5a, 5b, 5c, 8b, 8c, 8d, 8e, 8f, Conclusions
i, 10, 15a, 15d, and 15e effectively suppressed AmPLA2 with
8
A series of 25 compounds incorporating the 3H-quinazolin-4-one
IC50 values ranging from 4.20 ± 0.57 to 9.40 ± 0.85 mg/ml compared
to the IC50 4.20 ± 0.42 mg/ml value of oleanolic acid. Based on
these results, AmPLA2 appears to be the maximally affected
phospholipase by the synthesised compounds with compound 8f
scaffold were synthesised , 20 of which were new and fully char-
acterised. Twenty-one compounds were screened for their cyto-
toxicity on human CRC cell lines HT-29 and SW620 using the MTT
assay. Six compounds namely, 8a, 8f, 8j, 15a, 15b, and 15d
potently reduced viability of both cell lines, with 15d the most
active in this set displaying residual viability 38%, 36.7% against
HT-29 and SW620 cells lines, respectively. Further mechanistic
studies using 15d revealed that this compound inhibited the cell
proliferation of HT-29 and SW620 cell lines in a dose- and time-
dependent manner with IC50 values ꢀ20 mg/ml. Evaluation of anti-
apoptotic protein expression by compound 15d revealed that it
reduced the viability of the used human CRC cells by down regu-
lating the expression of Bcl2 and BclxL.
Based on these results, 15d could be considered as an attract-
ive candidate for further investigations as a potential anticancer
agent. Furthermore, the prepared compounds were screened for
their inhibitory activities against certain phospholipases including,
hG-IIA, hG-V, hG-X, NmPLA , and AmPLA , and five proteases
namely, cathepsin-B, collagenase, thrombin, elastase, and trypsin,
in addition to three enzymes involved in metabolic syndrome
namely, a-amylase, a-glucosidase, and xanthine oxidase. Although
none of the tested compounds showed good antiprotease activity,
six compounds 8j, 8d/15a/15e, 5b, and 8f demonstrated higher
antiphospholipases potential against proinflammatory isoforms
hGV, hG-X, NmPLA2 and AmPLA2 than oleanolic acid, which was
(
IC ; 4.20 ± 0.57 mg/ml) demonstrating inhibitory activity equiva-
50
lent to that of the reference drug.
Similarly, the inhibitory potency of the selected 3H-quinazolin-
-one derivatives against five proteases namely, cathepsin-B, colla-
genase, thrombin, elastase, and trypsin was quantified by measur-
ing and comparing the IC50 values produced by these compounds
relative to the value produced by the protease inhibitor cocktail.
The results of anti-proteases shown in Supplementary Table 2
indicated that compounds 8j and 15e were being the most active
inhibitors against protease cathepsin-B with IC50 values of 1.40 ± 0.
4
0
0
6 and 1.60 ± 0.07 mg/ml respectively compared to cocktail (IC50
.175 ± 0.04 mg/ml).
;
Furthermore, compounds 8b, 8d, and 15e were found to be
the most active in suppressing the collagenase activity with IC50
2
2
values ranging from 3.00 ± 0.01 to 4.50 ± 0.71 mg/ml compared to
cocktail (IC ; 0.15 ± 0.07 mg/ml) with compound 8d exhibiting the
5
0
lowest IC50 value.
The highest anti-thrombin activity was demonstrated by com-
pounds 5b, 5c, 8e, 10, and 15b, with IC50 values ranging from
0
.90 ± 0.14 to 2.50±0.71 mg/ml, with 5b demonstrating the lowest
IC50 compared to cocktail (IC50; 0.25 ± 0.07 mg/ml).
The highest anti-elastase activity was demonstrated by com- used as reference drug.
pounds 5b, 5c, 8e, 10, and 15b, with IC50 values ranging from
Therefore, these compounds would be of interest as preclinical
.65 ± 0.02 mg/ml (compound 15b) to 1.75 ± 0.05 mg/ml (com- candidates in trials for anticancer and anti-inflammatory drugs.
pound 5c) compared to that of cocktail (IC50; 0.125 ± 0.04 mg/ml).
All the tested compounds were inactive against xanthine oxi-
0
Finally, the lowest IC50 values ranging from 0.50 ± 0.00 to dase, whereas compounds 8d, 8h, 8j, 15a, 15b, 15e, and 15f eli-
.25 ± 0.04 mg/ml against trypsin were demonstrated by com- cited higher a-amylase inhibition than the reference drug
2
pounds 5b, 5c, 8e, 10, and 15 b, with compound 15b displaying quercetin. Furthermore, compounds 8g, 8h, and 8i were more

6
82
N. N. E. EL-SAYED ET AL.
potent in inhibiting a-glucosidase activity than the reference drug 14. Li X, Meng X, Gao X, et al. Elevated serum xanthine oxidase
allopurinol. Consequently, these compounds can be considered as
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novel anti-diabetes mellitus (DM) drugs.
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The authors thank the RSSU at King Saud University for their tech-
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Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
19. Mekala N, Buddepu SR, Dehury SK, et al. A novel strategy
for the manufacture of idelalisib: controlling the formation
of an enantiomer. RSC Adv 2018;8:15863–9.
The authors extend their appreciation to the Deanship of
Scientific Research at King Saud University for funding this work
through research group number [RG-1435–083].
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