(E)-N'-Arylidene-2-(4-oxoquinazolin-4(3H)-yl) acetohydrazides: Synthesis and evaluation of antitumor cytotoxicity and caspase activation activity

Article abstract of DOI:10.1080/14756366.2018.1555536

In our search for novel small molecules activating procaspase-3, we have designed and synthesised a series of novel acetohydrazides incorporating quinazolin-4(3H)-ones (5, 6, 7). Biological evaluation revealed eight compounds with significant cytotoxicity against three human cancer cell lines (SW620, colon cancer; PC-3, prostate cancer; NCI-H23, lung cancer). The most potent compound 5t displayed cytotoxicity up to 5-fold more potent than 5-FU. Analysis of structure-activity relationships showed that the introduction of different substituents at C-6 position on the quinazolin-4(3H)-4-one moiety, such as 6-chloro or 6-methoxy potentially increased the cytotoxicity of the compounds. In term of caspase activation activity, several compounds were found to exhibit potent effects, (e.g. compounds 7 b, 5n, and 5l). Especially, compound 7 b activated caspases activity by almost 200% in comparison to that of PAC-1. Further docking simulation also revealed that this compound potentially is a potent allosteric inhibitor of procaspase-3.

Full text of DOI:10.1080/14756366.2018.1555536

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
(E)-N'-Arylidene-2-(4-oxoquinazolin-4(3H)-yl)
acetohydrazides: Synthesis and evaluation of
antitumor cytotoxicity and caspase activation
activity
Le Cong Huan, Cao Viet Phuong, Le Cong Truc, Vo Nguyen Thanh, Hai Pham-
The, Le-Thi-Thu Huong, Nguyen Thi Thuan, Eun Jae Park, A Young Ji, Jong
Soon Kang, Sang-Bae Han, Phuong-Thao Tran & Nguyen-Hai Nam
To cite this article: Le Cong Huan, Cao Viet Phuong, Le Cong Truc, Vo Nguyen Thanh, Hai
Pham-The, Le-Thi-Thu Huong, Nguyen Thi Thuan, Eun Jae Park, A Young Ji, Jong Soon
Kang, Sang-Bae Han, Phuong-Thao Tran & Nguyen-Hai Nam (2019) (E)-N'-Arylidene-2-(4-
oxoquinazolin-4(3H)-yl) acetohydrazides: Synthesis and evaluation of antitumor cytotoxicity and
caspase activation activity, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 465-478,
DOI: 10.1080/14756366.2018.1555536
To link to this article: https://doi.org/10.1080/14756366.2018.1555536
©
2019 The Author(s). Published by Informa
View supplementary material
Submit your article to this journal
View Crossmark data
UK Limited, trading as Taylor & Francis
Group.
Published online: 17 Jan 2019.
Article views: 150
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 465–478
2
https://doi.org/10.1080/14756366.2018.1555536
RESEARCH PAPER
(E)-N’-Arylidene-2-(4-oxoquinazolin-4(3H)-yl) acetohydrazides: Synthesis and
evaluation of antitumor cytotoxicity and caspase activation activity
a
a
a
a
a
b
Le Cong Huan , Cao Viet Phuong , Le Cong Truc , Vo Nguyen Thanh , Hai Pham-The
, Le-Thi-Thu Huong ,
a
c
c
d
c
a
Nguyen Thi Thuan , Eun Jae Park , A Young Ji , Jong Soon Kang , Sang-Bae Han , Phuong-Thao Tran
Nguyen-Hai Nam
and
a
a
b
Pharmaceutical Chemistry, Hanoi University of Pharmacy, Hanoi, Vietnam; School of Medicine and Pharmacy, Vietnam National University,
c
d
Hanoi, Vietnam; College of Pharmacy, Chungbuk National University, Cheongju, Republic of Korea; Bio-Evaluation Center, Korea Research
Institute of Bioscience and Biotechnology, Cheongju, Republic of Korea
ABSTRACT
ARTICLE HISTORY
In our search for novel small molecules activating procaspase-3, we have designed and synthesised a ser-
ies of novel acetohydrazides incorporating quinazolin-4(3H)-ones (5, 6, 7). Biological evaluation revealed
eight compounds with significant cytotoxicity against three human cancer cell lines (SW620, colon cancer;
PC-3, prostate cancer; NCI-H23, lung cancer). The most potent compound 5t displayed cytotoxicity up to
Received 14 September 2018
Revised 29 November 2018
Accepted 30 November 2018
KEYWORDS
5
-fold more potent than 5-FU. Analysis of structure-activity relationships showed that the introduction of
Acetohydrazides; quinazo-
lin-4(3H)-one; cytotoxicity;
caspase activation
different substituents at C-6 position on the quinazolin-4(3H)-4-one moiety, such as 6-chloro or 6-methoxy
potentially increased the cytotoxicity of the compounds. In term of caspase activation activity, several
compounds were found to exhibit potent effects, (e.g. compounds 7 b, 5n, and 5l). Especially, compound
7
b activated caspases activity by almost 200% in comparison to that of PAC-1. Further docking simulation
also revealed that this compound potentially is a potent allosteric inhibitor of procaspase-3.
GRAPHICAL ABSTRACT
1
. Introduction
Despite the significant potential role of caspases in cancer
1
2
Cancer is one of two leading killers worldwide currently. The primary pathology , very few caspase-specific activators have been devel-
cause of cancer development and progression is the dysregulation oped thus far
. PAC-1, the first procaspase activating com-
of apoptosis . Compounds such as p53 disruptors (tenovin-1) and pound (Figure 1), is among the first procaspase activating
inhibitors of XIAP (GDC-0152) or Bcl-2 (ABT-199) act directly on compounds reported in recent years which shows promising in
proteins in the apoptotic pathway to induce apoptosis and lead to vivo antitumor activity profile . Studies on the structure-activity
5
,13,14
1
2
3
4
5
the death of cancer cells. Caspases are a family of cysteine proteases relationships of PAC-1 and other related compounds pointed out
enzymes important for maintaining homeostasis through regulating that the acyl hydrazone moiety (B-region, Figure 1) plays an
cell death. A number of studies have demonstrated that direct acti- important role for their broad bioactivity, owing to their ability to
1
5,16
vation of procaspase-3 by small molecules could have advantages form stable complexes with zinc
over the above apoptosis-inducing compounds, because procas-
Quinazolin-4(3H)-one is a common scaffold found in many
pase-3 is found overexpressed in various human tumours, including diverse biological compounds (e.g. luotonin A, rutaecarpine,
.
5
6
7
8
those of colon cancer , lung cancer , melanoma , hepatoma , breast tryptanthrin, chloroqualone, and alloqualone). The quinazoline
9
10
11
cancer , lymphoma , and neuroblastoma .
heterocyclic system, in particular, is also present as a core
CONTACT Sang-Bae Han
Chungbuk, 28160, Republic of Korea; Nguyen-Hai Nam
shan@cbnu.ac.kr
College of Pharmacy, Chungbuk National University, 194-31, Osongsaengmyung-1, Heungdeok, Cheongju,
namnh@hup.edu.vn; Phuong-Thao Tran
thaotp119@gmail.com
Hanoi University of Pharmacy,
Pharmaceutical Chemistry, 13-15 Le Thanh Tong, Hanoi, Vietnam
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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.

4
66
L. C. HUAN ET AL.
structure in a number of tyrosine kinase inhibitors and anti- light at 254 nm, was used to check the progress of reactions and
1
7
cancer agents (e.g. gefitinib, erlotinib) . Based on the pharma- preliminary evaluation of compounds’ homogeneity. Melting
cophoric structure of PAC-1 and related acylhydrazone points were measured using Gallenkamp Melting Point
procaspase activators, and the biological importance of quina- Apparatus (LabMerchant, London, United Kingdom) and are uncor-
a
rected. Purification of compounds was carried out using crystal-
lization methods and/or open silica gel column flash
chromatography employing Merck silica gel 60 (240 to 400 mesh)
zolin-4(3H)-one scaffold, we have designed three series of novel
acyl hydrazones (Figure 1). In the designed compounds, the 4-
benzylpiperazin-1-yl part of PAC-1 (A-region) was replaced by
the quinazolin-4(3H)-one heterocycle system. The phenyl ring
of PAC-1 (C-region) was replaced by different substituted phe-
nyl, heteroaryl rings or isatins. The acylhydrazone moiety
1
as stationary phase. Nuclear magnetic resonance spectra ( H NMR)
were recorded on a Bruker 500 MHz spectrometer with DMSO-d
6
as solvent unless otherwise indicated. Tetramethylsilane was used
as an internal standard. Chemical shifts are reported in parts per
million (ppm), downfield from tetramethylsilane. Mass spectra
with different ionisation modes including electron ionisation (EI),
Electrospray ionisation (ESI), were recorded using PE Biosystems
(
B-region) serves as a linker between A- and C-regions was
kept unchanged. This paper describes the results from synthe-
sis, biological evaluation and docking studies of the
designed compounds.
V
R
API2000 (Perkin Elmer, Palo Alto, CA, USA) and Mariner (Azco
Biotech, Inc. Oceanside, CA, USA) mass spectrometers, respectively.
The elemental (C, H, N) analyses were performed on a Perkin
Elmer model 2400 elemental analyser. All reagents and solvents
were purchased from Aldrich or Fluka Chemical Corp. (Milwaukee,
WI, USA) or Merck unless noted otherwise. Solvents were used dir-
ectly as purchased unless otherwise indicated.
2
. Material and methods
2.1. Chemistry
Thin layer chromatography which was performed using
V
R
Whatman 250 mm Silica Gel GF Uniplates and visualised under UV
The synthesis of acetohydrazides incorporating quinazolin-
4
(3H)-one (5, 6, 7) was carried out as illustrated in Scheme 1.
Details are described below.
The mixtures of anthranilic acid (1a) or 5-substituted-2-amino-
benzoic acid (1b–d) (1 mmol) and formamide (3 mmol) were
ꢀ
stirred at 120 C for 3 h. Upon completion, the resulting mixtures
were cooled, poured into ice-cold water, the light brown solids
were formed, filtered, and washed with water (3 times) and dried
to give quinazoline-4(3H)-on derivatives (2a–d), which were used
for the next step without further purification.
To a respective solution of quinazoline-4(3H)-on derivatives
(
1
2a–d) (1 mmol) in acetone (10 ml) were added K
.5 mmol). The resulting mixtures were stirred at 80 C for 30 min,
2
CO
3
(206.9 mg,
ꢀ
then KI (16.6 mg, 0.1 mmol) was added. After stirring for further
5 min, 0.13 ml of ethyl chloroacetate (1.2 mmol) were dropped
1
Figure 1. Structure of PAC-1 and rational design of novel (E)-N’-arylidene-2–(4-
oxoquinazolin-4(3H)-yl)acetohydrazides.
slowly into the mixtures. The reaction mixtures were again stirred
ꢀ
at 60 C for 3 h. After completion of the reaction, the resulting
ꢀ
Scheme 1. Synthesis of acetohydrazides incorporating quinazolin-4(3H)-one (5, 6, 7). Reagents and conditions: (a) H
2
N-CHO, 120 C, 3 h; (b) ethyl chloroacetate, KI,
ꢀ
2 3 2 4 2
K CO , acetone, 60 C, 3.5h; (c) N H .H O, EtOH, reflux; (d) Ar-CHO or isatin der., AcOH conc., EtOH, reflux.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
467
mixtures were cooled, poured into ice-cold water. The brown sol- 8.03 (ꢂ78%, 22%) (dd, J¼ 8.5 Hz, 1.5 Hz, 1H, H
ids were formed, filtered and dried to give the ethyl 2–(4-oxoqui- 1.5 Hz, 1H, H ); 7.72 (d, J¼ 8.0 Hz, 1H, H
); 7.82 (t, J¼ 7.5 Hz, 1H, H
nazolin-3-(4H)-yl)acetate derivatives 3a–d.
7.69 (td, J¼ 7.5 Hz, 1.5 Hz, 1H, H ); 7.58 (td, J¼ 8.0 Hz, 1.0 Hz, 1H, H
To a respective solution of compounds 3a–d (0.5 mmol) in 5.23, 4.81 (ꢂ78%, 22%) (s, 2H, NCH CO). 13C NMR (125 MHz, DMSO-d
ethanol (10 ml) was added 0.12 ml of hydrazine monohydrate ppm): d 168.5 (CONH), 160.2 (C ¼ O), 148.5 (C ¼C–N¼ C ), 148.0 (C
2.5 mmol) slowly. The mixture was stirred at room until the start- 143.0 (C ), 133.6 (C ), 130.7 (C ), 128.2 (C
), 139.9 (N¼CH), 134.5 (C
ing material consumed. The white precipitates formed were fil- 128.0 (C ), 127.2 (C ), 127.1 (C ), 126.0 (C ), 124.6 (C
tered and washed with cold ethanol (3 times), the off-white solids (C CO). Anal. Calcd. For C17 (351.0968):
¼C–C¼ O), 46.9 (NCH
4a–d) were collected, dried under vacuum and carried on to the C, 58.12; H, 3.73; N, 19.93. Found: C, 58.16; H, 3.70; N, 19.96.
3
0
); 7.87 (td, J¼ 8.5 Hz,
);
);
0
5
7
8
0
4
6
,
6
2
),
),
8
2
2
(
2
0
7
5
0
4
0
0
6
0
0
), 121.4
1
6
8
5
3
H N O
13 5 4
5
2
(
next step without further purification.
The acetohydrazides 4a–d (0.5 mmol) were dissolved in ethanol
20 ml), 2 drops of concentrated acetic acid, followed by benzalde-
(
E)-N’-(3-Chlorobenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5d)
White solid; Yield: 58%. mp: 184.0–185.0 C. R
(
hyde or isatine derivatives (1.0 mmol) were added. The mixtures
were refluxed until the reaction completed. The precipitates
formed were filtered and washed with ethanol (3 times), the off-
white solid products were collected, dried under vacuum and re-
crystalised in ethanol or column chromatography (MeOH:DCM) to
obtain the desired product 5a–z, 6a–g, 7a–b.
ꢀ
f
¼0.55 (DCM: MeOH
, ppm): d 11.91 (s, 1H, CONH);
); 8.16–8.15 (m, 1H, H
); 8.05 (s, 1H, N ¼ CH);
.85–7.82 (m, 2H, H , H ); 7.71–7.69 (m, 2H, H , H ); 7.57–7.49 (m,
H, H , H , H CO). 13C NMR
); 5.25, 4.80 (ꢂ80%, 20%) (s, 2H, NCH
1
¼
14: 1). H-NMR (500 MHz, DMSO-d
6
8
7
3
.37 (s, 1H, H
2
5
0
0
7
2
6
8
0
0
4
6
5
2
(125 MHz, DMSO-d
6
, ppm): d 168.4 (CONH), 160.3 (C ¼ O), 148.5
(C ¼C–N ¼ C ), 148.0 (C ), 145.7 (N¼CH), 142.7 (C
0
), 136.1 (C
0
3
),
),
8
2
2
1
(
E)-N’-Benzylidene-2–(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5a) 134.5 (C
7
), 133.7 (C
4
0
), 130.7 (C
0
5
0
), 129.7 (C
¼C–C ¼ O), 47.1 (NCH
(340.0727): C, 59.92; H, 3.85; N, 16.44.
6 6 8
0
), 127.2 (C ), 127.1 (C
ꢀ
White solid; Yield: 60%. mp: 179.7–181.5 C. R ¼0.53 (DCM: MeOH 126.0 (C
), 125.8 (C
13ClN
), 121.4 (C
CO). Anal.
2
f
5
2
5
ꢁ
1
¼
14: 1). IR (KBr, cm ): 3506 (NH); 3236 (N ¼ C–H aromatic); 3060, Calcd. For C17
H
4
O
2
2
990 (CH, aren); 2830 (CH ); 1719, 1680, 1531 (C ¼ O, C ¼ N); 1608 Found: C, 59.95; H, 3.83; N, 16.41.
2
1
(
7
C ¼ C). H-NMR (500 MHz, DMSO-d , ppm): d 11.90, 11.84 (ꢂ26%,
6
4%) (s, 1H, CONH); 8.39 (s, 1H, H ); 8.24, 8.07 (ꢂ22%, 78%) (s, 1H,
2
(
E)-N’-(4-Chlorobenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy-
N ¼ CH); 8.17 (d, J ¼ 7.5 Hz, 1H, H ); 7.87 (t, J ¼ 7.5 Hz, 1H, H );
5
7
drazide (5e)
7
(
.76–7.71 (m, 3H, H
0
, H
0
, H ); 7.58 (t, J ¼ 7.5 Hz, 1H, H ); 7.48–7.45
2
6
8
6
ꢀ
White solid; Yield: 31%. mp: 184.2–185.4 C. R ¼0.56 (DCM: MeOH
f
m, 3H, H3
0
, H4
0
, H5
0
); 5.24, 4.80 (ꢂ23%, 77%) (s, 2H, NCH CO). 13C
2
1
¼
14: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.96, 11.89 (ꢂ26%,
6
NMR (125 MHz, DMSO-d , ppm): d 168.3 (CONH), 160.3 (C ¼ O),
6
7
4%) (s, 1H, CONH); 8.34 (s, 1H, H ); 8.24, 8.07 (ꢂ22%, 78%) (s, 1H,
2
1
(
C
3
48.6 (C
), 130.1 (C
), 126.1 (C
07.9 [M þ H] . Anal. Calcd. For C17
8
¼C–N ¼ C
), 128.9 (C
), 121.5 (C
¼C–C ¼ O), 47.0 (NCH
(306.1117): C, 66.66;
H, 4.61; N, 18.29. Found: C, 66.63; H, 4.64; N, 18.32.
2
), 148.1 (C
2
), 144.3 (N¼CH), 134.5 (C
), 127.2 (C ), 127.1 (C ), 126.9 (C
CO). MS (ESI) m/z
7
), 133.9
N ¼ CH); 8.17 (d, J ¼ 7.5 Hz, 1H, H
.78 (d, J ¼ 8.5 Hz, 1H, H ); 7.59 (t, J ¼ 7.5 Hz, 1H, H
J ¼ 8.5 Hz, 2H, H3’, H4’);5.24, 4.81 (ꢂ23%, 77%) (s, 2H, NCH
NMR (125 MHz, DMSO-d
), 148.6 (C
), 130.0 (C , C
, C ), 126.5 (C
5
); 7.86–7.92 (m, 3H, H
2
0 0
, H
); 7.53 (d,
CO). 13C
6 7
, H );
C
1
0
4
0
2
0
, C
6
0
6
8
3
0
,
7
8
6
0
5
5
5
2
þ
2
14 4 2
H N O
6
, ppm): d 168.8 (CONH), 161.1 (C¼ O), 149.0
(C
8
¼C–N ¼ C
33.5 (C
29.0 (C
2
2
), 144.3 (N¼CH), 136.5 (C4’), 135.0 (C
), 129.5 (C , C ), 127.7 (C ), 127.6 (C
), 121.9 (C
¼C–C ¼ O), 47.4 (NCH
13ClN (340.0727): C,
59.92; H, 3.85; N, 16.44. Found: C, 59.89; H, 3.88; N, 16.47.
7
),
),
1
1
1
0
2
0
6
0
3
0
5
0
6
8
0
0
2
CO). MS
(
E)-N’-(2-Chlorobenzylidene)-2–(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5b)
White solid; Yield: 44%. mp: 180.0–182.0 C. R
3
5
5
5
ꢁ
(ESI) m/z 339.1 [M-H] . Anal. Calcd. For C17
H
4 2
O
ꢀ
f
¼0.55 (DCM: MeOH
14: 1). IR (KBr, cm ): 3506 (NH); 3240 (N ¼ C–H aromatic); 3059,
987 (CH, aren); 1707, 1685, 1558 (C ¼ O, C ¼ N); 1608 (C ¼ C); 777
ꢁ
1
¼
2
(
E)-N’-(4-Fluorobenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5f)
White solid; Yield: 49%. mp: 181.0–182.0 C. R
1
(
7
8
(
C–Cl). H-NMR (500 MHz, DMSO-d , ppm): d 12.09, 11.97 (ꢂ22%,
6
8%) (s, 1H, CONH); 8.63, 8.45 (ꢂ22%, 78%) (s, 1H, N ¼ CH); 8.38,
.37 (s, 1H, H ); 8.16 (dd, J ¼ 8.0 Hz, 1.5 Hz, 1H, H ); 8.04, 7.94
ꢀ
f
¼0.59 (DCM: MeOH ¼
1
2
5
1
7
4: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.89, 11.83 (ꢂ22%,
6
ꢂ77%, 23%) (dd, J ¼ 7.0 Hz, 2.0 Hz, 1H, H
0
), 7.85 (td, J ¼ 8.0 Hz,
6
8%) (s, 1H, CONH); 8.38, 8,25 (ꢂ18%, 82%) (s, 1H, H ); 8.17, 7.97
2
1
.5 Hz, 1H, H ); 7.72 (d, J ¼ 8.0 Hz, 1H, H ); 7.56 (t, J ¼ 7.5 Hz, 1H,
0 0
7
8
(
ꢂ81%, 19%) (dd, J ¼ 9.0 Hz, 1.0 Hz, 1H, H ); 8.73, 8.25 (ꢂ13%, 87%)
5
H ); 7.53, 7.52 (dd, J ¼ 7.0 Hz, 1.5 Hz, 1H, H
); 7.47–7.42 (m, 2H, H ,
3 4
6
(s, 1H, N ¼ CH); 7.86 (td, J ¼ 8.5 Hz, 1.5 Hz, 2H, H ); 7.84, 7.80 (ꢂ78%,
7
H
0
); 5.24, 4.81 (ꢂ80%, 20%) (s, 2H, NCH CO). 13C NMR (125 MHz,
5
2
0
0
6
2
2%) (dd, J ¼ 3.5 Hz, 3.0 Hz, 2H, H , H ); 7.74 (d, J ¼ 8.0 Hz, 1H, H );
2
8
DMSO-d , ppm):
d
168.4 (CONH), 160.2 (C ¼ O), 148.5
6
7.58 (td, J ¼ 8.0 Hz, 1.0 Hz, 1H, H ); 7.38, 7.31 (ꢂ11%, 81%) (t,
0 0
6
(
1
1
C ¼C–N ¼ C ), 148.0 (C ), 140.3 (N¼CH), 134.4 (C ), 133.0 (C
0
),
8
2
2
7
1
J ¼ 9.0 Hz, 2H, H
, H
); 5.24, 4.80 (ꢂ79%, 21%) (s, 2H, NCH CO). 13C
0
3
5
2
31.4 (C
26.8 (C5
0
0
), 128.9 (C3 ), 127.6 (C6
), 131.1 (C ), 127.2 (C ), 127.0 (C ),
0 0 0
4 6 8
2
NMR (125 MHz, DMSO-d , ppm): d 168.7 (CONH), 162.6 (C ), 160.8
6 4
), 125.9 (C ), 121.4 (C ¼C–C ¼ O), 46.9 (NCH CO). MS (ESI)
5
þ
5
2
(C ¼ O), 149.1 (C ), 148.6 (C ¼C–N ¼ C ), 143.6 (N¼CH), 135.0 (C ),
2
8
2
7
m/z 340.9 [M þ H] . Anal. Calcd. For C H ClN O (340.0727): C,
1
7
13
4
2
131.1 (C
0
), 129.64 (C
0
), 129.57 (C
0
), 127.7 (C ), 127.6 (C ), 126.5 (C ),
1
2
6
6
8
5
5
9.92; H, 3.85; N, 16.44. Found: C, 59.90; H, 3.87; N, 16.46.
1
21.9 (C
m/z 323.2 [M-H] . Anal. Calcd. For C17
H, 4.04; N, 17.28. Found: C, 62.93; H, 4.07; N, 17.31.
5
¼C–C ¼ O), 116.6 (C
3
0
), 116.5 (C
5
0
2
), 47.5 (NCH CO). MS (ESI)
ꢁ
H
4 2
13FN O (324.1023): C, 62.96;
(
E)-N’-(2-Nitrobenzylidene)-2–(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5c)
White solid; Yield: 56%. mp: 182.1–183.3 C. R
ꢀ
¼0.60 (DCM: MeOH ¼ (E)-N’-(4-Bromobenzylidene)-2–(4-oxoquinazolin-3(4H)-yl)acetohy-
6
, ppm): d 11.93 (s, 1H, CONH); 8.65, drazide (5g)
f
1
1
8
4: 1). H-NMR (500 MHz, DMSO-d
.46 (ꢂ23%, 77%) (s, 1H, N ¼ CH); 8.38, 8.37 (s, 1H, H
); 8.13, 8.10 (dd, J¼ 8.0 Hz, 1.5 Hz, 1H, H
ꢀ
2
); 8.16 (dd, White solid; Yield: 33%. mp: 181.2–182.4 C. R ¼0.57 (DCM: MeOH
f
1
J¼ 7.5 Hz, 1.0 Hz, 1H, H
5
6
0
); 8.08, ¼ 14: 1). H-NMR (500 MHz, DMSO-d
6
, ppm): d 11.93, 11.85 (ꢂ22%,

4
68
L. C. HUAN ET AL.
7
8%) (mestnova) (s, 1H, CONH); 8.36 (s, 1H, H
2
); 8.22, 8.05 (ꢂ24%, (E)-N’-(2,3-Dihydroxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)ace-
); 7.86 (td, tohydrazide (5k)
7
6%) (s, 1H, N ¼ CH); 8.16 (dd, J ¼ 7.5 Hz, 1.0 Hz, 1H, H
5
ꢀ
J ¼ 7.0 Hz, 1.0 Hz, H ); 7.82, 7.66 (ꢂ14%, 86%) (d, J ¼ 7.5 Hz, 2H, H
0
,
White solid; Yield: 36%. mp: 172.1-173.6 C. R ¼0.57 (DCM: MeOH
7
4
f
ꢁ1
H
6
0
); 7.72 (d, J ¼ 8.5 Hz, 1H, H
8
). 7.70 (d, J ¼ 7.0 Hz, 2H, H
3
0
, H
5
0
); ¼ 14: 1). IR (KBr, cm ): 3564 (NH); 3452 (OH); 3259 (CH, aren);
1
7
1
1
.57 (t, J ¼ 7.5 Hz, 1H, H ); 5.23, 4.79 (ꢂ78%, 22%) (s, 2H, NCH CO). 2890 (CH, CH ); 1710 (C ¼ O); 1614, 1566 (C ¼ C). H-NMR (500 MHz,
6
2
2
0
3C NMR (125 MHz, DMSO-d
6
, ppm): d 168.3 (CONH), 160.2 (C ¼ O), DMSO-d
6
, ppm): d 12.20, 10.08 (ꢂ50%, 50%) (s, 1H, 2 -OH); 10.80,
48.5 (C ¼C–N ¼ C ), 148.0 (C ), 143.1 (N¼CH), 134.5 (C ), 133.2 11.70 (ꢂ26%, 74%) (s, 1H, CONH); 9.30, 9.50 (ꢂ40%, 60%) (s, 1H,
8
2
2
7
0
(
(
C
1
0
), 132.0 (C
3
0
), 131.8 (C
5
0
), 130.2 (C
4
0
), 128.8 (C
6
0
), 127.2 (C
6
), 127.1 3 -OH); 8.39 (d, 1H, J ¼ 7.5 Hz, H4’); 8.37 (s, 1H, H
2
); 8.38 (s, 1H,
); 7.73 (m, 1H,
); 5.20,
CO). 13C NMR (125 MHz, DMSO-d
ppm): d d 167.9 (CONH); 161.1 (C
); 160.7 (C ¼ O); 158.6 (C
); 143.0 (C ); 134.9 (C ); 131.5
C ), 126.0 (C ), 123.3 (C
0
), 121.4 (C ¼C–C ¼ O), 46.9 (NCH CO). N ¼ CH); 8.17 (d, J ¼ 8.0 Hz, 1H, H
); 7.87 (m, 1H, H
5 7
8
5
4
5
2
H
); 7.26-7.31 (m, 1H, H
0
); 7.00 (m, 1H, H
); 6.72 (m, 1H, H5
0 0
Anal. Calcd. For C17
1
H
13BrN
O
4 2
(384.0222): C, 53.00; H, 3.40; N,
8
4
6
4.54. Found: C, 52.97; H, 3.43; N, 14.57.
4.82 (ꢂ22%, 78%) (s, 2H, NCH
2
6
,
0
0
);
2
4
1
49.1 (N ¼ CH); 148.6 (C
8
¼C–N ¼ C
2
2
7
(
E)-N’-(2-Hydroxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy- (C ); 127.7 (C ); 127.6 (C ); 126.5 (C ); 121.9 (C ¼C–C ¼ O); 112.0
6
1
5
6
8
5
drazide (5h)
(C
0
); 108.4 (C
5
0
); 102.9 (C
3
0
); 47.3 (NCH
2
CO). MS (ESI) m/z
ꢀ
þ
White solid; Yield: 37%. mp: 176.0–177.2 C. R ¼0.54 (DCM: MeOH 338.9 [M þ H] .
f
ꢁ
1
¼
14: 1). IR (KBr, cm ): 3460 (NH); 3066 (OH); 3021 (CH, aren);
1
2
912 (CH, CH ); 1697 (C ¼ O); 1608, 1570 (C ¼ C). H-NMR (500 MHz,
2
(
E)-N’-(2,4-Dihydroxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)ace-
tohydrazide (5l)
White solid; Yield: 42%. mp: 175.1–176.8 C. R
DMSO-d
1
6
, ppm): d 12.09, 11.75 (ꢂ34%, 66%) (s, 1H, CONH); 10.94,
0
0.10 (ꢂ37%, 63%) (s, 1H, 2 -OH); 8.47, 8.39 (s, 1H, N ¼ CH); 8.38
ꢀ
f
¼0.64 (DCM: MeOH
(
(
H
4
s, 1H, H
d, J ¼ 7.5 Hz, 1H, H
, H ); 7.32 – 7.26 (m, 1H, H
0
4
.81 (ꢂ65%, 35%) (s, 2H, NCH
2
); 8.18 (d, J ¼ 8 Hz, 1H, H
); 7.50 – 7.72 (m, 1H, H
); 6.94 – 6.88 (m, 2H, H3
5
); 7.89 (t, J ¼ 7 Hz, 1H, H
); 7.60 – 7.57 (m, 2H,
, H5 ); 5.22,
CO). 13C-NMR (125 MHz, DMSO), d
68.3 (CONH); 160.7 (C ¼ O); 156.9 (C ); 149.1 (N ¼ CH); 148.6
7
); 7.78
ꢁ
1
¼
14: 1). IR (KBr, cm ): 3450 (NH); 3182 (OH); 3057 (CH, aren);
0
6
8
1
2
978 (CH, CH
2
); 1732 (C ¼ O); 1631, 1539 (C ¼ C). H-NMR (500MHz,
0
0
0
6
6
DMSO-d
1
1
6
, ppm): d 11.89, 11.54 (ꢂ26%, 74%) (s, 1H, CONH); 11.09 (s,
2
0
0
H, 2 -OH); 9.83 (s, 1H, 4 -OH); 8.39 (d, 1H, J ¼ 7.5 Hz, H4’); 8.37 (s,
1
0
2
H, H ); 8.24 (s, 1H, N ¼ CH); 8.16 (d, J ¼ 8.0 Hz, 1H, H ); 7.86 (t, 1H,
2
5
(
(
(
(
C
8
¼C–N ¼ C
2
); 142.0 (C ); 134.9 (C4’); 131.8 (C
2
6
0
); 129.6 (C
7
); 127.8
0
); 119.9
5
J ¼ 8.0 Hz, H ); 7.73 (t, 1H, J ¼ 8.0 Hz, H ); 7.55–7.59 (m, 3H, H , H ,
7
8
6
3’
C ); 127.6 (C ); 126.5 (C ); 121.9 (C ¼C–C ¼ O); 120.5 (C
5
6
8
5
H ); 6.33 (m, 1H, H ); 5.16, 4.77 (ꢂ22%, 78%) (s, 2H, NCH CO). 13C-
5’
5’
2
C
1
0
); 116.8 (C
3
0
); 47.4 (NCH
2 14 4 3
CO). Anal. Calcd. For C17H N O
NMR (125MHz, DMSO-d , ppm): d 168.3 (CONH), 163.3 (C ), 161.0
6
2’
322.1066): C, 63.35; H, 4.38; N, 17.38. Found: C, 63.38; H, 4.35;
(
1
(
(
6
C
4’), 160.7 (C ¼ O), 148.9 (C
8
¼C–N ¼ C
), 127.5 (C
), 103.0 (C
ESI) m/z 338.9 [M þ H] . Anal. Calcd. For C17
0.35; H, 4.17; N, 16.56. Found: C, 60.32; H, 4.14; N, 16.58.
2
), 148.5 (C
2
), 142.9 (N¼CH),
N, 17.41.
34.9 (C
7
), 131.6 (C6’), 127.7 (C
6
8
), 126.5 (C
5
), 121.9
CO). MS
C
5
¼C–C ¼ O), 112.0 (C
1
0
), 108.3 (C
5
0
3
0
), 47.3 (NCH
2
þ
14 4 4
H N O (338.1015): C,
(
E)-N’-(4-Hydroxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5i)
White solid; Yield: 43%. mp: 178.2–179.3 C. R
ꢀ
f
¼0.55 (DCM: MeOH
1
¼
14: 1). H-NMR (500MHz, DMSO-d , ppm): d 11.64, 11.58 (s, 1H,
6
(
E)-N’-(2,5-Dihydroxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)ace-
CONH); 9.90 (s, 1H, OH); 8.37, 8.36 (s, 1H, H
2
); 8.16 (dd, J ¼ 8.0,
tohydrazide (5m)
1
1
6
.0 Hz, 1H, H ); 8.13, 7.97 (s, 1H, N ¼ CH); 7.86 (td, J ¼ 7.5 Hz, 1.0 Hz,
5
White solid; Yield: 38%. mp: 173.2–174.6 R ¼0.56 (DCM: MeOH ¼
f
H, H
7
); 7.72 (d, J ¼ 8.0 Hz, 1H, H
8
); 7.59–7.53 (m, 3H, H
6
, H
2
0
, H
6
0
);
1
1
7
1
1
1
4: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.92, 11.78 (ꢂ26%,
6
.84, 6.82 (d, J ¼ 8.5 Hz, 2H, H
3
0
, H
5
0
); 5.20, 4.76 (ꢂ78%, 22%) (s, 2H,
0
0
4%) (s, 1H, CONH); 11.78 (s, 1H, 2 -OH); 9.34 (s, 1H, 5 -OH); 8.38 (s,
H, H
); 8.31, 8.39 (ꢂ22%, 78%) (s, 1H, N ¼ CH); 8.16 (d, J ¼ 7.5 Hz,
H, H ); 7.73 (t, 1H, J ¼ 8.0 Hz, H ); 7.59 (t,
); 7.88 (t, 1H, J ¼ 7.5 Hz, H
H, J ¼ 7.5 Hz, H ); 7.18 (s, 1H, H6’); 6.71–6.76 (m, 2H, H3’, H4’); 6.33
CO). 13C-NMR
NCH CO). 13C NMR (125MHz, DMSO-d , ppm): d 167.8 (CONH),
2
6
2
1
60.2 (C¼ O), 159.4 (C–OH), 148.6 (C
8
¼C–N ¼ C
2 2
), 148.1 (C ), 144.5
5
7
8
(
1
N¼CH), 134.4 (C ), 128.6 (C
0
, C
0
), 127.2 (C ), 127.0 (C ), 126.0 (C ),
7
2
6
6
8
5
6
24.9 (C
1
0
), 121.4 (C
5
¼C–C ¼ O), 115.7 (C
3
0
, C
5
0
), 46.9 (NCH
2
CO). MS
(
(
m, 1H, H5’); 5.20, 4.79 (ꢂ22%, 78%) (s, 2H, NCH
2
þ
(
6
ESI) m/z 323.2 [Mþ H] . Anal. Calcd. For C H N O (322.1066): C,
1
7 14 4 3
125MHz, DMSO-d , ppm): d 168.3 (CONH), 160.7 (C¼ O), 150.5 (C ),
6
2’
3.35; H, 4.38; N, 17.38. Found: C, 63.33; H, 4.40; N, 17.35.
1
50.3 (C ), 148.9 (C ¼C-N ¼ C ), 148.5 (C ), 142.0 (N¼CH), 135.0
2’
8
2
2
(
C ), 127.6 (C ), 127.5 (C ), 127.4 (C ), 119.6 (C
0
), 119.4 (C ), 117.5
4
0
7
6
8
5
1
(
C6
0
), 120.7 (C3
0
), 121.9 (C ¼C-C ¼ O), 47.2 (NCH CO).
5
2
(
E)-N’-(4-Methoxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5j)
White solid; Yield: 41%. mp: 185.0–186.0 C. R
ꢀ
f
¼0.56 (DCM: MeOH
14: 1). H-NMR (500MHz, DMSO-d , ppm): d 11.97, 11.73 (ꢂ23%,
(
E)-N’-(2-Hydroxy-4-methoxybenzylidene)-2-(4-oxoquinazolin-3(4H)-
yl)acetohydrazide (5n)
White solid; Yield: 30%. mp: 177.1–178.3 C. R
1
¼
6
7
7%) (s, 1H, CONH); 8.57, 8.56 (s, 1H, H
2
); 8.23, 8.04 (ꢂ23%, 77%) (s,
ꢀ
f
¼0.63 (DCM: MeOH
¼ 9: 1). IR (KBr, cm ): 3466 (NH); 3226 (OH); 3057 (CH, aren); 2964
ꢁ
1
1
H, N ¼ CH); 8.18, 8.17 (dd, J ¼ 8.0 Hz, 1.0 Hz, H ); 7.90 (td,
5
1
J ¼ 8.0 Hz, 1.0 Hz, 1H, H
7
); 7.76 (d, J ¼ 8.5 Hz, 1H, H
8
); 7.69, 7.65 (d,
(
CH, CH ); 1745 (C ¼ O); 1612, 1562 (C ¼ C). H-NMR (500 MHz,
2
J ¼ 8.0 Hz, 2H, H , H ); 7.61 (t, J ¼ 8.0 Hz, 1H, H ); 7.02, 7.01 (d,
0
0
2
6
6
DMSO-d , ppm): d 11.90, 11.62 (ꢂ26%, 74%) (s, 1H, CONH); 10.27
6
J ¼ 8.5 Hz, 2H, H
3
0
, H
5
0
); 5.24, 4.82 (ꢂ78%, 22%) (s, 2H, NCH
2
CO); (s, 1H, 2’-OH); 8.38 (s, 1H, N ¼ CH); 8.37 (s, 1H, H ); 8.16 (d,
2
3
.81, 3.80 (s, 3H, OCH
3
). 13C NMR (125MHz, DMSO-d
6
, ppm): d 167.7 J ¼ 8.0 Hz, 1H, H ); 7.89 (t, 1H, J ¼ 8.0 Hz, H ); 7.73 (t, 1H, J ¼ 8.0 Hz,
5
7
(
(
CONH), 160.9 (C¼ O), 159.9 (C
0
–OCH ), 149.0 (C ¼C–N ¼ C ), 147.2 H );7.55 (t, 1H, J ¼ 8.0 Hz, H ); 7.45 (d, 1H, J ¼ 9.0 Hz, H ); 6.47–6.52
4
3
8
2
8
6
6’
C
2
), 144.3 (N¼CH), 134.7 (C
7
), 128.7 (C
6
), 128.5 (C
2
0
, C
6
0
), 127.4 (C
8
), (m, 2H, H , H ); 5.20, 4.79 (ꢂ22%, 78%) (s, 2H, NCH CO); 3.77 (s,
3’
5’
2
1
26.4 (C
0
), 126.2 (C ), 121.2 (C ¼C–C ¼ O), 114.3 (C
0
, C5
0
), 55.3 3H, 4’-OCH ). 13C-NMR (125 MHz, DMSO-d , ppm):d 168.0 (CONH);
1
5
5
3
3
6
þ
(OCH
3
), 47.1 (NCH
2
CO). MS (ESI) m/z 337.1 [M þ H] .
162.5 (C
4
0
); 160.7 (C ¼ O); 158.5 (C
2
0
); 149.1 (N ¼ CH); 148.6 (C ¼C-
8

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
469
N ¼ C
2
); 142.5 (C
2
); 135.0 (C
¼C–C ¼ O); 113.5 (C
); 47.3 (NCH
7
); 131.4 (C
6
0
0
); 127.7 (C
); 107.0 (C
5
); 127.6 (C
6
); N ¼ CH); 7.77 (s, 1H, H
5
); 7.92 (d, 1H, J ¼ 7.0 Hz, H
7
); 7.72–7.74 (m,
1
5
26.5 (C ); 121.9 (C
8
5
1
5
0
); 101.4 (C
3
0
); 3H, H , H , H ); 7.45–7.48 (m, 2H, H , H ); 5.25, 4.79 (ꢂ22%, 78%)
8
2’
6’
4’
5’
0
þ
5.7 (4 -OCH
Calcd. For C18
Found: C, 61.32; H, 4.61; N, 15.93.
3
2
CO). MS (ESI) m/z 353.0 [M þ H] . Anal. (s, 2H, NCH CO). 13C-NMR (125 MHz, DMSO-d , ppm): d 168.5
2
6
H
16
4
N O
4
(352.1172): C, 61.36; H, 4.58; N, 15.90. (CONH), 159.7 (C ¼ O), 149.5 (C ¼C–N ¼ C ), 147.3 (C ), 144.8
0
8
2
2
(N¼CH), 135.1 (C ), 134.3 (C
), 131.9 (C ), 130.7 (C ), 130.0 (C ),
1 7 4’ 5
6
1
29.3 (C , C ), 127.3 (C , C ), 125.4 (C ), 123.1 (C ¼C–C ¼ O), 47.6
2
’
6’
3’
5’
8
5
þ
(NCH CO). MS (ESI) m/z 340.9 [M þ H] .
2
(
E)-N’-(3-Hydroxy-4-methoxybenzylidene)-2-(4-oxoquinazolin-3(4H)-
yl)acetohydrazide (5o)
ꢀ
White solid; Yield: 31%. mp: 175.2–176.5 C. R ¼0.58 (DCM: MeOH
f
1
(E)-2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N’-(4-fluorobenzylide-
¼
14: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.91, 11.62 (ꢂ26%,
6
ne)acetohydrazide (5s)
7
4%) (s, 1H, CONH); 9.26 (s, 1H, 3’-OH);8.36 (s, 1H, H ); 8.15 (d,
2
ꢀ
White solid; Yield: 38%. mp: 185.0-186.0 C. R ¼0.67 (DCM: MeOH
J ¼ 7.5 Hz, 1H, H ); 8.06, 7.91 (ꢂ25%, 75%) (s, 1H, N ¼ CH); 7.85 (t,
f
5
1
¼
14: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.89, 11.83 (ꢂ26%,
1
H, J ¼ 7.5 Hz, H ); 7.71 (t, 1H, J ¼ 7.5 Hz, H );7.56 (t, 1H, J ¼ 7.5 Hz,
6
7
8
7
4%) (s, 1H, CONH); 8.40 (s, 1H, H
2
); 8.05 (s, 1H, H
5
); 8.01 (s, 1H,
H ); 7.22 (s, 1H, H ); 7.05 (d, 1H, J ¼ 8.0 Hz, H ); 6.90 (d, 1H,
6
2’
6’
N ¼ CH); 7.89 (d, 1H, J ¼ 7.0 Hz, H
7
); 7.80 (d, 2H, J ¼ 8.0 Hz, H2’, H6’);
J ¼ 8.0 Hz, H ); 5.19, 4.79 (ꢂ22%, 78%) (s, 2H, NCH CO); 3.80 (s,
5’
2
7
.57 (d, 1H, J ¼ 7.0 Hz, H ); 7.28 (d, 2H, J ¼ 8.0 Hz, H3’, H5’); 5.25 (s,
8
3
H, 4’-OCH ); 13C-NMR (125 MHz, DMSO-d , ppm): d 168.5 (CONH),
3
6
1
(
60.2 (C ¼ O), 149.7 (C
0
), 148.7 (C3
0
), 148.6 (C ¼C–N ¼ C ), 148.1 2H, NCH
2 6
CO). 13C-NMR (125 MHz, DMSO-d , ppm): d 167.9 (CONH),
4
8
2
C ), 144.4 (N¼CH), 134.5 (C ), 127.2 (C ), 127.0 (C ), 126.0 (C
0
), 164.0 (C 0
4
), 159.3 (C ¼ O), 149.0 (C ¼C–N ¼ C ), 146.8 (N¼CH),
8
2
2
7
6
8
1
0
), 129.5 (C ), 129.1 (C
0
),
),
1
5
21.7 (C
0
), 121.4 (C ¼C–C ¼ O), 112.2 (C
0
), 111.9 (C6
0
), 126.1 (C ), 143.2 (C
2
), 134.6 (C
), 124.9 (C
7.1 (NCH CO). MS (ESI) m/z 357.1 [M-H] .
), 131.4 (C
6
), 130.4 (C
7
1
5
2
6
5
2
5
5.5 (4’-OCH ), 47.8 (NCH CO).
129.0 (C
4
6
0
), 122.6 (C ¼C–C ¼ O), 116.0 (C3’), 115.9 (C
8 5
5
0
3
2
-
2
(
E)-N’-(2,3,4-Trimethoxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)a-
cetohydrazide (5p)
White solid; Yield: 42%. mp: 183.5–184.6 C. R
(
E)-2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N’-(4-methoxybenzylide-
ne)acetohydrazide (5t)
White solid; Yield: 40%. mp: 187.0–188.1 C. R ¼0.60 (DCM: MeOH
ꢀ
f
¼0.60 (DCM: MeOH
, ppm): d 11.77, 11.48 (s, 1H,
CONH); 8.40, 8.25 (ꢂ25%, 75%) (s, 1H, N ¼ CH); 8.36 (s, 1H, H );
.16 (d, J ¼ 8.0 Hz, 1H, H ); 7.86 (t, J ¼ 7.5 Hz, 1H, H ); 7.72 (d,
J ¼ 8.0 Hz, 1H, H ); 7.61, 7.54 (d, J ¼ 8.5 Hz, 1H, H ), 7.56 (t,
J ¼ 8.0 Hz, 1H, H ); 6.93, 6.91 (d, J ¼ 8.5 Hz, 1H, H ); 5.20, 4.76
ꢂ77%, 23%) (s, 2H, NCH CO); 3.85 (s, 3H, OCH ); 3.85, 3.84 (s, 3H,
OCH ); 3.79, 3.78 (s, 3H, OCH ). 13C NMR (125 MHz, DMSO-d
1
¼
14: 1). H-NMR (500 MHz, DMSO-d
6
ꢀ
f
2
ꢁ
1
¼
14: 1). IR (KBr, cm ): 3211 (N ¼ C ¼ H aromatic); 3159 (N ¼ C-H
8
5
7
hydrazon); 3061, 2993 (CH, aren); 2904, 2829 (CH, CH ); 1710,
2
0
6
8
1
1
672, 1660, 1610, 1519 (C ¼ C, C ¼ N, C ¼ O); 1242 (C-OCH
3
). H-
0
2
6
NMR (500 MHz, DMSO-d
6
, ppm): d 11.73, 11.70 (ꢂ26%, 74%) (s, 1H,
(
2
3
CONH); 8.40 (s, 1H, H ); 8.16 (s, 1H, N ¼ CH); 8.09 (s, 1H, H ); 7.88
2
5
3
3
6
,
(
d, 1H, J ¼ 8.5 Hz, H ); 7.75 (d, 1H, J ¼ 8.5 Hz, H ); 7.66 (d, 2H,
ppm): d 167.8 (CONH), 160.2 (C ¼ O), 155.1 (C
4
0
-OCH
), 141.6 (N¼CH), 140.2 (C
), 127.1 (C ), 126.0 (C
), 120.0 (C ), 108.7 (C ), 61.7 (C
-OCH ), 46.9 (NCH CO).
3
), 152.5 (C
2
0
-
-
7
8
J ¼ 9.0 Hz, H , H ); 7.01 (d, 1H, J ¼ 9.0 Hz, H ); 5.21, 4.77 (ꢂ77%,
OCH
OCH
3
), 148.6 (C
8
¼C–N ¼ C
2
), 148.1 (C
2
3
0
2’
6’
3’
2
3%) (s, 2H, NCH
2
CO); 3.84 (s, 3H, 4’-OCH
3
); 13C-NMR (125 MHz,
3 7
), 134.5 (C ), 127.2 (C
6
8
5
), 121.4
-OCH ),
DMSO-d
6
, ppm): d 168.5 (CONH), 160.8 (C ¼ O), 159.2 (C4’), 149.0
), 134.6 (C ), 131.4 (C ),
), 126.4 (C1 ), 124.9 (C8), 122.6
), 55.3 (4’-OCH ), 47.1 ((NCH CO). MS
(
6
C
5
¼C–C ¼ O), 120.5 (C
6
0
1
0
5
0
2
0
3
0
0
(
1
C
8
¼C–N ¼ C
2
), 146.2 (N¼CH), 144.2 (C
2
6
7
0.5 (C -OCH ), 56.0 (C
3
3
4
3
2
29.5 (C ), 128.4 (C
0
0
,
C
0
6
0
5
2
(
C ¼C–C ¼ O), 114.3 (C
, C5
0
5
3
3
2
þ
(
E)-N’-(3,4,5-Trimethoxybenzylidene)-2-(4-oxoquinazolin-3(4H)-yl)a- (ESI) m/z 370.9 [M þ H] . Anal. Calcd. For C H ClN O
1
8
15
4 3
cetohydrazide (5q)
(370.0833): C, 58.31; H, 4.08; N, 15.11. Found: C, 58.34; H, 4.11;
ꢀ
White solid; Yield: 48%. mp: 182.3–183.7 C. R ¼0.61 (DCM: MeOH N, 15.08.
f
1
¼
14: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.79 (s, 1H, CONH);
6
8
1
.65, 8.38 (s, 1H, H ); 8.16 (dd, J ¼ 6.8, 1.0 Hz, 1H, H ); 8.36, 7.97 (s,
2
5
H, N ¼ CH); 7.86 (td, J ¼ 7.5, 2.0 Hz, 1H, H ); 7.72 (d, J ¼ 8.0 Hz, 1H,
7
(
E)-N’-Benzylidene-2-(6-methyl-4-oxoquinazolin-3(4H)-yl)acetohy-
H ), 7.57 (td, J ¼ 8.0 Hz, 2.0 Hz, 1H, H ); 7.22, 7.05 (ꢂ24%, 76%) (s,
8
6
drazide (5u)
1
H, H
0
); 7.05, 7.02 (ꢂ77%, 23%) (s, H, H ); 5.24, 4.90 (ꢂ79%, 21%)
0
2
6
ꢀ
White solid; Yield: 34%. mp: 180.0–181.0 C. R ¼0.68 (DCM: MeOH
f
(
s, 2H, NCH CO); 3.84, 3.81 (ꢂ88%, 12%) (s, 6H, C
OCH , C OCH );
0 0
3 3 5 3
2
ꢁ1
¼
9: 1). IR (KBr, cm ): 3450 (NH); 3223 (OH); 3080 (CH, aren);
3
1
1
.73, 3.71 (s, 3H, C4
0
OCH ). 13C NMR (125 MHz, DMSO-d , ppm): d
3
6
1
2
956 (CH, CH
500 MHz, DMSO-d
CONH); 8.31 (s, 1H, H ); 8.24, 8.08 (ꢂ22%, 78%) (s, 1H, N ¼ CH);
2
); 1732 (C ¼ O); 1608, 1570 (C ¼ C). H-NMR
68.2 (CONH), 160.2 (C ¼ O), 153.2 (C
0
-OCH ), 153.1 (C
0
-OCH3),
3
3
5
(
6
, ppm): d 11.88, 11.80 (ꢂ26%, 74%) (s, 1H,
48.6 (C ¼C–N ¼ C ), 148.1 (C ), 144.1 (N¼CH), 139.2 (C
0
), 134.5
8
2
2
4
2
(
(
C ), 129.4 (C
C ¼C–C ¼ O), 104.4 (C
0
), 127.2 (C ), 127.1 (C ), 126.0 (C ), 121.4
7
1
6
8
5
7
.96 (s, 1H, H ); 7.69 (d, 1H, J ¼ 8.5 Hz, H ); 7.75 (d, 2H, J ¼ 8.0 Hz,
5
7
0
), 104.2 (C2
0
), 60.2 (C5
0
-OCH ), 60.1 (C
0
-
5
6
3
3
H , H ); 7.62 (d, 1H, J ¼ 8.5 Hz, H ); 7.43-7.49 (m, 3H, H , H , H );
OCH ), 55.9 (C -OCH ), 47.1 (NCH CO).
4 3 2
0
2
’
6’
8
3’
4’
5’
3
5
.23, 4.79 (ꢂ78%, 22%) (s, 2H, NCH
2
3
CO); 2.47 (s, 3H, 6-CH ). 13C-
NMR (125 MHz, DMSO-d
6
, ppm): d 168.3 (CONH), 160.2 (C ¼ O),
), 144.3 (N¼CH), 138.9 (C ), 135.7
), 128.9 (C , C ), 127.1 (C ), 126.9 (C
(
E)-N’-Benzylidene-2-(6-chloro-4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (5r)
White solid; Yield: 32%. mp: 184.0–185.0 C. R
147.8 (C
8
¼C–N ¼ C
2
), 146.1 (C
2
6
(C ), 133.9 (C
7
1
0
), 130.1 (C
4
0
2
0
6
0
5
0
3
,
ꢀ
¼0.55 (DCM: MeOH C 0
), 125.3 (C ), 121.2 (C ¼C–C ¼ O), 47.0 (NCH CO), 20.8 (6-CH ).
f
5
8
5
2
3
ꢁ
1
þ
¼
14: 1). IR (KBr, cm ): 3506 (NH); 3163 (OH); 3059 (CH, aren); MS (ESI) m/z 321.0 [M þ H] . Anal. Calcd. For
C
16
H
18
N
4
O
2
1
2
980 (CH, CH
2
); 1710 (C ¼ O); 1608, 1558 (C ¼ C). H-NMR (500 MHz, (320.1273): C, 67.49; H, 5.03; N, 17.49. Found: C, 58.34; H, 4.11;
DMSO-d
6
, ppm): d 11.85 (s, 1H, CONH); 8.40 (s, 1H, H ); 8.01 (s, 1H, N, 15.08.
2

4
70
L. C. HUAN ET AL.
(E)-2-(6-Methyl-4-oxoquinazolin-3(4H)-yl)-N’-(2-nitrobenzylidene)a-
(E)-N’-(4-(dimethylamino)benzylidene)-2-(6-methyl-4-oxoquinazo-
cetohydrazide (5v)
lin-3(4H)-yl)acetohydrazide (5z)
ꢀ
ꢀ
White solid; Yield: 37%. mp: 182.2–183.4 C. R ¼0.72 (DCM: MeOH Light brown solid; Yield: 26%. mp: 186.0-187.0 C. R
f
¼0.68 (DCM:
f
1
ꢁ1
¼
9: 1). H-NMR (500 MHz, DMSO-d , ppm): d 12.12, 12.06 (ꢂ26%, MeOH ¼ 9: 1). IR (KBr, cm ): 3439 (NH); 3348 (OH); 3122 (CH,
6
1
7
4%) (s, 1H, CONH); 8.63, 8.44 (ꢂ23%, 77%) (s, 1H, N ¼ CH); 8.30 aren); 2791 (CH, CH
2
); 1735 (C ¼ O); 1610, 1537 (C ¼ C). H-NMR
(
s, 1H, H
J ¼ 8.0 Hz, H3’); 7.80 (m, 1H, H5’); 7.69 (d, 1H, J ¼ 8.0 Hz, H
m, 1H, H4’); 7.62 (d, 1H, J ¼ 8.0 Hz, H ); 5.20, 4.79 (ꢂ77%, 23%) (s,
H, NCH CO); 2.50 (s, 3H, 6-CH ). 13C-NMR (125 MHz, DMSO-d
2
); 7.94 (s, 1H, H
5
); 8.12 (d, 1H, J ¼ 8.0 Hz, H6’); 8.08 (d, 1H, (500 MHz, DMSO-d
6
, ppm): d 11.54, 11.49 (ꢂ27%, 73%) (s, 1H,
CONH); 8.29 (s, 1H, H ); 8.07, 7.91 (ꢂ23%, 77%) (s, 1H, N ¼ CH);
7
); 7.67
2
7
.94 (s, 1H, H ); 7.67 (d, 1H, J ¼ 8.0 Hz, H ); 7.61 (d, 1H, J ¼ 8.0 Hz,
(
2
8
5
7
H ); 7.53 (d, 2H, J ¼ 8.5 Hz, H ,H ); 6.74 (d, 2H, J ¼ 8.5 Hz, H ,H );
8
2’ 6’
3’ 5’
2
3
6
,
5
2
.16, 4.72 (ꢂ77%, 23%) (s, 2H, NCH CO);3.97 (s, 6H, 4’-N(CH ) );
ppm): d 168.6 (CONH), 160.1 (C ¼ O), 148.1 (C ¼C–N ¼ C ), 147.6
2
3 2
8
2
.42 (s, 3H, 6-CH
3
). 13C-NMR (125 MHz, DMSO-d
6
, ppm): d 167.6
(
1
1
C ), 146.0 (C ), 143.0 (N¼CH), 136.6 (C ), 135.7 (C ), 133.6 (C
0
),
5
2
’
2
6
7
(
1
1
CONH), 160.2 (C ¼ O), 151.4 (C
4
0
), 148.8 (C
), 135.7 (C ), 128.5 (C
), 121.3 (C
¼C–C ¼ O), 111.8 (C
CO). 20.8 (6-CH , 4’-N(CH
8
¼C–N ¼ C
), 128.2 (C
, C
2
), 146.1 (C
2
),
),
30.7 (C
0
), 128.3 (C
0
), 128.0 (C1
0
), 127.1 (C ), 125.3 (C ), 124.6 (C ),
CO), 20.8 (6-CH ).
2 3
4
6
5
8
3’
45.0 (N¼CH), 136.8 (C
6
7
1
0
2
0
, C
6
0
21.2 (C
5
¼C–C ¼ O), 46.8 (NCH
27.0 (C ), 125.3 (C
5
8
5
3
0
5
0
), 46.8
þ
(NCH
2
3
3
)
2
). MS (ESI) m/z 364.0 [M þ H] .
Anal. Calcd. For C H N O (363.1695): C, 66.10; H, 5.82; N, 19.27.
2
0 21 5 2
(
E)-N’-(4-Fluorobenzylidene)-2-(6-methyl-4-oxoquinazolin-3(4H)-
yl)acetohydrazide (5w)
White solid; Yield: 42%. mp: 183.2–184.5 C. R
Found: C, 66.14; H, 5.80; N, 19.30.
ꢀ
f
¼0.66 (DCM: MeOH
1
¼
14: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.87, 11.80 (ꢂ25%,
6
(
E)-N’-(furan-2-ylmethylene)-2-(4-oxoquinazolin-3(4H)-yl)acetohy-
drazide (6a)
Light brown solid; Yield: 44%. mp: 181.0–182.3 C. R
7
5%) (s, 1H, CONH); 8.31 (s, 1H, H ); 8.24, 8.07 (ꢂ22%, 78%) (s, 1H,
2
N ¼ CH); 7.95 (s, 1H, H ); 7.77–7.82 (m, 2H, H , H ); 7.68 (d, 1H,
ꢀ
5
2’
6’
f
¼0.68 (DCM:
, ppm): d 11.77 (s, 1H,
); 8.17 (dd, J ¼ 8.0 Hz, 1.0 Hz, 1H, H );
.14, 7.96 (s, 1H, N ¼ CH), 8.0 (dd, J ¼ 13.5 Hz, 1.5 Hz, 1H, H ); 7.87
td, J ¼ 8.5 Hz, 1.5 Hz, 1H, H ); 7.74 (d, J ¼ 8.0 Hz, 1H, H ), 7.58 (td,
J ¼ 8.0 Hz, H
7
); 7.62 (d, 1H, J ¼ 8.0 Hz, H
8
); 7.29 (t, J ¼ 8.0 Hz, 2H,
1
MeOH ¼ 9: 1). H-NMR (500 MHz, DMSO-d
6
H
3’, H5’); 5.22, 4.78 (ꢂ78%, 22%) (s, 2H, NCH
2
CO); 2.46 (s, 3H, 6-
CONH); 8.37, 8.36 (s, 1H, H
2
5
CH
3
). 13C-NMR (125 MHz, DMSO-d , ppm): d 168.2 (CONH), 164.1
), 160.2 (C ¼ O), 147.7 (C ¼C–N ¼ C ), 146.1 (C ), 143.1 (N¼CH),
), 129.1 (C2 , C6
0 0 0
36.8 (C ), 135.7 (C ), 130.5 (C ), 127.1 (C ), 125.3
1
6
8
(
0
5
(C
4
0
8
2
2
7
8
1
6
7
5
J ¼ 8.0 Hz, 1.0 Hz, 1H, H ); 6.96, 6.96 (d, J ¼ 3.5 Hz, 1H, H
0
); 6.65
6
3
(
C ), 121.2 (C ¼C–C ¼ O), 116.0 (C
0
, C5
0
), 46.9 (NCH CO), 20.8 (6-
0
8
5
3
2
(dd, J ¼ 3.5 Hz, 1.5 Hz, 1H, H ); 5.16, 4.79 (ꢂ77%, 23%) (s, 2H,
4
-
CH ). MS (ESI) m/z 337.1 [M-H] .
3
NCH CO). 13C NMR (125 MHz, DMSO-d , ppm): d 168.1 (CONH),
2
6
1
60.3 (C ¼ O), 148.9 (C
1
0
), 148.6 (C
8
¼C–N ¼ C
2
), 148.1 (C
), 126.0 (C
0), 112.2 (C 0
), 46.9 (NCH CO). Anal. Calcd.
4 2
2
), 145.2
), 121.4
(C
5
0
), 134.5 (C
7
), 134.4, 127.2 (C
6
), 127.1 (C
8
5
(
E)-N’-(4-Bromobenzylidene)-2-(6-methyl-4-oxoquinazolin-3(4H)-
yl)acetohydrazide (5y)
White solid; Yield: 28%. mp: 182.6–183.5 C. R
(C
5
¼C–C ¼ O), 113.9 (C
3
For C H N O (296.0909): C, 60.81; H, 4.08; N, 18.91. Found: C,
1
5 12 4 3
ꢀ
f
¼0.70 (DCM: MeOH 60.85; H, 4.11; N, 18.94.
, ppm): d 11.92, 11.85 (ꢂ22%,
8%) (s, 1H, CONH); 8.29 (s, 1H, H ); 8.20, 8.03 (ꢂ23%, 77%) (s, 1H,
1
¼
9: 1). H-NMR (500 MHz, DMSO-d
6
7
2
(
E)-2-(4-Oxoquinazolin-3(4H)-yl)-N’-(thiophen-2-ylmethylene)aceto-
N ¼ CH); 7.94 (s, 1H, H ); 7.69 (d, 1H, J ¼ 8.5 Hz, H ); 7.65 (d,
5
7
hydrazide (6b)
J ¼ 8.5 Hz, 4H, H , H H , H ); 7.60 (d, 1H, J ¼ 8.5 Hz, H ); 5.20,
3
’
5’,
2’
6’
8
ꢀ
White solid; Yield: 26%. mp: 182.1–183.6 C. R ¼0.72 (DCM: MeOH
4
(
.76 (ꢂ77%, 23%) (s, 2H, NCH
125 MHz, DMSO-d
), 146.1 (C
), 131.8 (C , C
), 121.2 (C ¼C–C ¼ O), 46.9 (NCH CO), 20.8 (6-CH ).
2
CO); 2.45(s, 3H, 6-CH
3
). 13C-NMR
f
1
¼
9: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.84, 11.81 (s, 1H,
, ppm): d 168.3 (CONH), 160.1 (C ¼ O), 147.7
6
6
CONH); 8.45, 8.26 (ꢂ25%, 75%) (s, 1H, N ¼ CH); 8.37 (s, 1H, H
.17 (dd, J ¼ 8.0 Hz, 1.5 Hz, 1H, H ); 7.89 (td, J ¼ 8.0 Hz, 1.5 Hz, 1H,
); 7.74 (d, J ¼ 8.0 Hz, 1H, H ); 7.69 (d, J ¼ 5.5 Hz, 1H, H ); 7.60 (td,
J ¼ 8.0 Hz, 1.0 Hz, 1H, H ); 7.50 (dd, J ¼ 8.5 Hz, 1.0 Hz, 1H, H ); 7.17
dd, J ¼ 5.0 Hz, 3.5 Hz, 1H, H ); 5.15, 4.78 (ꢂ75%, 25%) (s, 2H,
NCH CO). 13C NMR (125 MHz, DMSO-d , ppm): d 167.9 (CONH);
2
);
(C
8
¼C–N ¼ C
33.2 (C
23.3 (C4
2
2
), 143.0 (N¼CH), 136.8 (C ), 135.7 (C
5
), 128.8 (C , C ), 127.1 (C ), 125.3 (C
6
7
),
),
8
5
1
1
1
0
3
0
5
0
2
0
6
0
8
H
7
8
0
5
0
5
2
3
0
3
6
(
0
4
2
6
(
E)-N’-(4-Methoxybenzylidene)-2-(6-methyl-4-oxoquinazolin-3(4H)-
yl)acetohydrazide (5x)
White solid; Yield: 40%. mp: 186.5–187.5 C. R ¼0.70 (DCM: MeOH
1
1
1
60.3 (C ¼ O); 148.6 (C ¼C–N ¼ C ); 148.1 (C ); 139.5 (N ¼ CH);
8
2
2
38.5 (C
0
); 134.5 (C ); 130.9 (C
0
); 128.8 (C
0
); 128.0 (C ); 127.2 (C );
4 6
0
ꢀ
2
7
3
5
f
27.1 (C ); 126.0 (C ); 121.5 (C ¼C–C ¼ O); 46.8 (NCH CO). Anal.
ꢁ
1
8
5
5
2
¼
9: 1). IR (KBr, cm ): 3460 (NH); 3188 (OH); 3093 (CH, aren); 2839
12 4 2
Calcd. For C15H N O S (312.0681): C, 57.68; H, 3.87; N, 17.94.
1
(CH, CH
2
); 1772 (C ¼ O); 1608, 1519 (C ¼ C). H-NMR (500 MHz,
, ppm): d 11.74, 11.67 (ꢂ22%, 78%) (s, 1H, CONH); 8.31 (s,
Found: C, 57.64; H, 3.91; N, 17.97.
DMSO-d
1
7
6
H, H ); 8.18, 8.01 (ꢂ22%, 78%) (s, 1H, N ¼ CH); 7.96 (s, 1H, H );
2
5
.01 (d, J ¼ 8.5 Hz, 2H, H , H ); 7.69 (d, 1H, J ¼ 8.5 Hz, H ); 7.67 (d,
3’
5’
7
(E)-N’-((1-Methyl-1H-pyrrol-2-yl)methylene)-2–(4-oxoquinazolin-
J ¼ 8.5 Hz, 2H, H , H );7.62 (d, 1H, J ¼ 8.5 Hz, H ); 5.20, 4.77 (ꢂ77%,
2’
6’
8
3(4H)-yl)acetohydrazide (6c)
2
1
1
3%) (s, 2H, NCH
2
CO); 3.81 (s, 3H, 4’-OCH
3
); 2.51 (s, 3H, 6-CH
3
).
),
ꢀ
White solid; Yield: 58%. mp: 184.5–185.2 C. R ¼0.70 (DCM: MeOH
f
0
1
3C-NMR (125 MHz, DMSO-d
60.2 (C ¼ O), 147.8 (C ¼C-N ¼ C
), 135.7 (C ), 128.5 (C , C ), 127.1 (C
21.2 (C ¼C-C ¼ O), 114.3 (C , C5
6
, ppm): d 168.0 (CONH), 160.8 (C
), 146.1 (C
), 126.5 (C
4
¼
9: 1). H-NMR (500 MHz, DMSO-d , ppm): d 11.54, 11.48 (ꢂ18%,
6
8
2
2
), 144.1 (N¼CH), 136.8
8
2%) (s, 1H, CONH); 8.36 (s, 1H, H ); 8.17 (dd, J ¼ 8.0 Hz, 1.0 Hz, 1H,
2
(C
6
7
2
0
6
0
5
1
0
), 125.3 (C
8
), H ); 7.97 (s, 1H, N ¼ CH); 7.88 (td, J ¼ 8.0 Hz, 1.5 Hz, 1H, H ); 7.73
5
7
1
2
0
0
), 55.3 (4’-OCH ), 46.9 (NCH CO), (d, J ¼ 8.0 Hz, 1H, H ); 7.58 (td, J ¼ 8.0 Hz, 1.0 Hz, 1H, H ); 7.00 (s,
5
3
3
2
8
6
þ
0.8 (6-CH ). MS (ESI) m/z 351.0 [M þ H] . Anal. Calcd. For 1H, H
0
); 6.51 (dd, J ¼ 3.5 Hz, 1.5 Hz, 1H, H
0
3
), 6.11 (dt, J ¼ 3.5 Hz,
3
5
C H N O (350.1379): C, 65.13; H, 5.18; N, 15.99. Found: C, 65.15; 2.5 Hz, 1H, H
H, 5.15; N, 15.62.
0
); 5.15, 4.76 (ꢂ88%, 12%) (s, 2H, NCH CO); 3.89, 3.82
1
9
18
4
2
4
2
(ꢂ82%, 18%) (s, 3H, CH
3 6
). 13C NMR (125 MHz, DMSO-d , ppm): d

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
471
1
68.0 (CONH); 160.8 (C ¼ O); 149.1 (C
); 128.8 (C ); 127.7 (C
); 121.9 (C
¼C–C ¼ O); 115.8 (C
CO); 37.0 (CH ). Anal. Calcd. For C16
2.13; H, 4.89; N, 22.64. Found: C, 62.09; H, 4.92; N, 22.67.
8
¼C–N ¼ C
); 127.6 (C
); 108.6 (C
(309.1226): C, (s, 2H, NCH
ppm):
2
); 148.6 (C
); 127.2 (C
); 47.4 J ¼ 3.5 Hz, H4’); 6.27 (d, 1H, J ¼ 3.5 Hz, H3’); 5.14, 4.76 (ꢂ75%, 25%)
CO); 2.36 (s, 3H, 2’-CH ). 13C-NMR (125 MHz, DMSO-d
), 148.6
2
); 138.1 8.01, 7.86 (ꢂ22%, 78%) (s, 1H, N ¼ CH); 7.86 (t, 1H, J ¼ 8.0 Hz, H
); 7.72 (d, 1H, J ¼ 8.0 Hz, H ); 7.54 (t, 1H, J ¼ 8.0 Hz, H ); 6.83 (d, 1H,
0
4
7
);
(
1
N ¼ CH); 134.9 (C
7
2
0
5
0
6
8
8
6
26.5 (C
5
5
3
0
(NCH
2
3
H
15
N
5
O
2
2
3
6
,
6
d
167.9 (CONH), 160.2 (C ¼ O), 154.7 (C
0
3
(
1
1
C ¼C–N ¼ C ), 148.1 (C ), 147.5 (C
0
), 136.9 (N¼CH), 134.5 (C ),
8
2
2
1
7
27.2 (C
6
), 127.1 (C
8
), 126.0 (C
5
), 121.4 (C
5
¼C–C ¼ O), 115.7 (C
0
),
5
0
(E)-2–(4-Oxoquinazolin-3(4H)-yl)-N’-(pyridin-2-ylmethylene)aceto-
08.6 (C 0
4
), 46.8 (NCH
2
CO), 13.5 (3 -CH
3
). Anal. Calcd. For
hydrazide (6d)
C H N O (310.1066): C, 61.93; H, 4.55; N, 18.06. Found: C, 61.97;
1
6 14 4 3
ꢀ
White solid; Yield: 36%. mp: 183.0–184.0 C. R ¼0.34 (DCM: MeOH H, 4.59; N, 18.09.
f
1
¼
9: 1). H-NMR (500 MHz, DMSO-d , ppm): d 12.11, 12.03 (ꢂ20%,
6
8
8
8
0%) (s, 1H, CONH); 8.64 (d, J ¼ 8.0 Hz, 1H, H
0
); 8.40 (s, 1H, H );
6 2
(
Z)-N’-(2-Oxoindolin-3-ylidene)-2–(4-oxoquinazolin-3(4H)-yl)aceto-
.18 (d, J ¼ 8.0 Hz, 1H, H ); 8.26, 8.12 (ꢂ20%, 80%) (s, 1H, N ¼ CH);
5
.03 (d, J ¼ 8.0 Hz, 1H, H
0
); 7.93-7.86 (m, 2H, H , H
0
); 7.74 (d, hydrazide (7a)
3
7
4
ꢀ
J ¼ 8.0 Hz, 1H, H ); 7.60 (td, J ¼ 7.5 Hz, 2.0 Hz, 1H, H ); 7.46 (td, Light yellow solid; Yield: 44%. mp: 183.3–184.0 C. R
¼0.72 (DCM:
, ppm): d 12.71 (s, 1H,
); 8.16 (d, 1H,
); 7.73 (d, 1H, J ¼ 8.0 Hz,
); 7.59 (d, 1H, J ¼ 7.5 Hz, H7’); 7.57 (t, 1H, J ¼ 8.0 Hz, H ); 7.41 (t,
); 47.4 (NCH CO). Anal. 1H, J ¼ 7.5 Hz, H5’); 7.12 (t, 1H, J ¼ 7.0 Hz, H6’); 6.97 (d, 1H,
8
6
f
1
J ¼ 5.0 Hz, 1.0 Hz, 1H, H
0
), 5.28, 4.83 (ꢂ81%, 19%) (s, 2H, NCH CO). MeOH ¼ 9: 1). H-NMR (500 MHz, DMSO-d
6
5
2
1
1
3C NMR (125 MHz, DMSO-d , ppm): d 169.0 (CONH); 160.8 (C ¼ O); NH-isatin); 11.30 (s, 1H, CONH); 8.40 (s, 1H, H
2
6
53.2 (C
0
); 150.1 (C6
0
); 149.0 (C ¼C–N ¼ C ); 148.6 (C ); 145.2 J ¼ 8.0 Hz, H
5
); 7.87 (t, 1H, J ¼ 8.0 Hz, H
7
2
8
2
2
(
1
N ¼ CH); 137.4 (C
0
); 135.1 (C ); 127.7 (C ); 127.6 (C ); 126.5 (C );
H
8
6
4
7
6
8
5
25.0 (C
0
); 121.9 (C ¼C–C ¼ O); 120.3 (C
0
5
5
3
2
J ¼ 7.5 Hz, H4’); 5.37 (s, 2H, NCH
2 6
CO); 13C-NMR (125 MHz, DMSO-d ,
Calcd. For C H N O (307.1069): C, 62.53; H, 4.26; N, 22.79.
1
6 13 5 2
ppm):
d
168.9 (CONH), 162.4 (C ¼O),160.3 (C ¼O), 148.3
Found: C, 62.57; H, 4.23; N, 22.82.
2’
4
(
(
C ¼C–N ¼ C ), 148.0 (C ), 142.7 (C –C–NH), 135.4 (N¼CH), 134.6
8
2
2
7’
C
7
, C2’), 131.9 (C4’), 127.3 (C
6
, C6’), 126.0 (C
5
, C
8
), 122.6 (C5’), 121.3
(
E)-2–(4-Oxoquinazolin-3(4H)-yl)-N’-(pyridin-3-ylmethylene)aceto-
hydrazide (6e)
White solid; Yield: 39%. mp: 182.5–183.6 C. R
(C ¼C–C ¼ O), 120.8 (C –C–C ), 111.2 (C ), 46.5 (NCH CO). Anal.
5
3’
4’
7’
2
Calcd. For C18H13IN O (347.1018): C, 62.24; H, 3.77; N, 20.16.
5 3
ꢀ
¼0.36 (DCM: MeOH Found: C, 62.29; H, 3.81; N, 20.19.
, ppm): d 12.00 (s, 1H, CONH);
.91, 8.86 (ꢂ80%, 20%) (d, J ¼ 2.0 Hz, 1H, H ); 8.63, 8.61 (ꢂ88%,
2%) (dd, J ¼ 5.0 Hz, 1.5 Hz, 1H, H ); 8.39, 8.31 (ꢂ83%, 17%) (s, 1H,
); 8.18 (dd, J ¼ 8.0 Hz, 1.5 Hz, 2H, H , H ); 8.12 (s, 1H, N ¼ CH);
.89 (td, J ¼ 8.0 Hz, 1.5 Hz, 1H, H ); 7.74 (d, J ¼ 8.0 Hz, 1H, H ); 7.60
); 7.51 (dd, J ¼ 8.0 Hz, 4.5 Hz, 1H, H ),
.27, 4.82 (ꢂ79%, 21%) (s, 2H, NCH CO). 13C NMR (125 MHz,
, ppm): d 168.9 (CONH); 160.7 (C ¼ O); 151.1 (C ); 149.3
¼C–N ¼ C ); 148.6 (C ); 142.0 (N ¼ CH); 135.0 (C6’); 133.9 C );
30.3 (C1’); 127.7(C ); 127.6 (C ); 126.5 (C ); 124.4 (C ); 121.9
¼C–C ¼ O); 47.5 (NCH CO). Anal. Calcd. For
307.1069): C, 62.53; H, 4.26; N, 22.79. Found: C, 62.50; H, 4.27;
f
1
¼
9: 1). H-NMR (500 MHz, DMSO-d
6
8
1
0
2
(
Z)-2-(6-Iodo-4-oxoquinazolin-3(4H)-yl)-N’-(2-oxoindolin-3-ylide-
ne)acetohydrazide (7b)
Light yellow solid; Yield: 57%. mp: 183.7–184.5 C. R ¼0.73 (DCM:
0
6
H
7
2
5
0
4
ꢀ
f
7
8
ꢁ
1
MeOH ¼ 9: 1). IR (KBr, cm ): 3450 (NH); 3184 (OH); 3049 (CH,
(
5
td, J ¼ 8.0 Hz, 2.0 Hz, 1H, H
6
0
5
1
aren); 2902 (CH
500 MHz, DMSO-d
2
); 1724 (C ¼ O); 1685, 1523 (C ¼ C). H-NMR
6
, ppm): d 11.71 (s, 1H, NH-isatin); 10.87 (s, 1H,
2
(
DMSO-d
, C
6
0
4
CONH); 8.43 (s, 2H, H , H ); 8.17 (d, 2H, J ¼ 8.25 Hz, H , H ); 7.53 (d,
(C
2
0
8
2
2
7
2
5
7
8
1
6
H, J ¼ 8.5 Hz, H7’); 7.40 (d, 1H, J ¼ 6.85 Hz, H5’); 7.04 (br, 1H, H6’);
.92 (d, 1H, J ¼ 7.95 Hz, H4’); 5.30 (s, 2H, NCH CO); 13C-NMR
1
6
8
5
0
5
(
(
C
5
2
16 13 5 2
C H N O
2
(
(
(
125 MHz, DMSO-d , ppm): d 170.8 (CONH), 164.3 (C ¼O),158.9
6
2’
C ¼O), 149.1 (C ¼C–N ¼ C ), 147.3 (C ), 144.0 (C ), 143.0
4
8
2
2
7
N, 22.74.
C
7’–C–NH); 134.3 (N¼CH, C
23.1 (C5’), 121.8 (C
(C ), 47.5 (NCH CO). Anal. Calcd. For C16
6.10; H, 5.82; N, 19.27. Found: C, 66.14; H, 5.80; N, 19.30.
5 8 5
), 132.9 (C6’), 129.5 (C4’, C ), 126.2 (C ),
1
5
¼C–C ¼ O), 115.1 (C3’–C–C4’), 110.7 (C7’), 92.3
(
E)-2–(4-Oxoquinazolin-3(4H)-yl)-N’-(pyridin-4-ylmethylene)aceto-
6
2
H
12IN (363.1695): C,
O
5 3
6
hydrazide (6f)
ꢀ
White solid; Yield: 27%. mp: 181.2–182.5 C. R ¼0.40 (DCM: MeOH
f
1
¼
9: 1). H-NMR (500 MHz, DMSO-d , ppm): d 12.14, 12.08 (ꢂ25%,
6
2
.2. Cytotoxicity assay
H ); 8.15 (d, 1H, J ¼ 8.0 Hz, H ); 8.05 (s, 1H, N ¼ CH); 7.87 (t, 1H, The cytotoxicity of the synthesised compounds was evaluated
7
5%) (s, 1H, CONH); 8.64 (d, 2H, J ¼ 5.0 Hz, H , H ); 8.30 (s, 1H,
2’
6’
2
5
J ¼ 8.0 Hz, H ); 7.72 (d, 1H, J ¼ 8.0 Hz, H ); 7.69 (d, 2H, J ¼ 5.0 Hz, against three human cancer cell lines, including SW620 (colon
7
8
H , H ); 7.57 (t, 1H, J ¼ 8.0 Hz, H ); 5.26, 4.81 (ꢂ75%, 25%) (s, 2H, cancer), PC3 (prostate cancer), and NCI-H23 (lung cancer). The cell
2’
6’
6
NCH CO). 13CN-MR (125 MHz, DMSO-d , ppm): d 168.6 (CONH), lines were purchased from a Cancer Cell Bank at the Korea
2
6
1
1
(
60.2 (C ¼ O), 150.2 (C
0
, C5
0
), 148.5 (C ¼C–N ¼ C ), 148.4 (C ), Research Institute of Bioscience and Biotechnology (KRIBB). The
3
8
2
2
44.4 (N¼CH), 141.9 (C ), 134.5 (C ), 127.2 (C ), 127.1 (C ), 126.0 media, sera and other reagents that were used for cell culture in
1’
7
6
8
C ), 121.4 (C ¼C–C ¼ O), 120.8 (C
0
, C
0
), 46.9 (NCH CO). Anal. this assay were obtained from GIBCO Co. Ltd. (Grand Island, New
5
5
2
6 2
Calcd. For C H N O (307.1069): C, 62.53; H, 4.26; N, 22.79. York, USA). The cells were culture in DMEM (Dulbecco’s Modified
1
6 13 5 2
Found: C, 62.51; H, 4.28; N, 22.76.
Eagle Medium) until confluence. The cells were then trypsinized
4
and suspended at 3 ꢃ 10 cells/mL of cell culture medium. On day
0
, each well of the 96-well plates was seeded with 180 lL of cell
(
E)-N’-((5-Methylfuran-2-yl)methylene)-2–(4-oxoquinazolin-3(4H)-
yl)acetohydrazide (6g)
White solid; Yield: 39%. mp: 180.9–181.7 C. R
suspension. The plates were then incubated in a 5% CO incuba-
tor at 37 C for 24 h. Compounds were initially dissolved in
¼0.70 (DCM: MeOH dimethyl sulfoxide (DMSO) and diluted to appropriate concentra-
2
ꢀ
ꢀ
f
1
¼
9: 1). H-NMR (500 MHz, DMSO-d
6
, ppm): d 11.72, 11.66 (ꢂ25%, tions by culture medium. Then 20 lL of each compounds’ sam-
7
2
5%) (s, 1H, CONH); 8.35 (s, 1H, H ); 8.15 (d, 1H, J ¼ 8.0 Hz, H
5
); ples, which were prepared as described above, were added to

4
72
L. C. HUAN ET AL.
each well of the 96-well plates, which had been seeded with cell
suspension and incubated for 24-h, at various concentrations. The
plates were further incubated for 48 h. Cytotoxicity of the com-
pounds was measured by the colorimetric method, as described
1
8
19–22
previously with slight modifications
. The IC50 values were
23
calculated using a Probits method and were averages of three
independent determinations (the standard deviation: SD ꢄ 10%).
Figure 2. Structure of E/Z isomers of acetohydrazides.
2.3. Caspase-3 activation assay
5
U937 cells (5 ꢃ 10 cells/well) were plated in 6-well plate, and
allowed to be stabilised overnight. The cells were treated with
PAC-1 or compounds (50 lM in 0.1% dimethyl sulfoxide). After
2
4 h incubation, cells were harvested and washed twice with PBS.
The cells were lysed by 50 lL of chilled cell lysis buffer for 10 min
ꢀ
on 4 C. Cell lysate (200 lg/100 lL/well) were mixed with Ac-
DEVD-pNA (200 lM). ODs at 405 nm were measured every 30 min
for 12 h. The slope of the linear portion for each well was deter-
mined as the enzyme activity.
2.4. Docking studies
The docking simulations were carried out using the Molecular Figure 3. Structure of anti-E/syn-E isomers of acetohydrazides.
24
Operating Environment version MOE 2009.10 . The 3 D structure
of PAC-1 was directly obtained from Pubchem (https://pubchem.
ncbi.nlm.nih.gov/compound/pac-1) and the chemical structures of
the other compounds were constructed using the Builder module
in ethanol under refluxing condition. The final step in the pathway
involved an aldol condensation of hydrazids 4a–d with benzalde-
hydes or isatins. The desired products 5a–z, 6a–g, 7a–b were
obtained in moderate overall yields.
2
in MOE. All compounds were solvated with H O as solvent layer
width to 10 Å. The potential energy was minimised up to 0.0001
gradients using the MMFF94x force field cut-off. For protein prep-
aration, the crystal structures of pseudo-activated procaspase-3
The structures of the synthesised compounds were determined
straightforwardly based on analysis of spectroscopic data, includ-
1
13
ing IR, MS, H and C NMR. Full NMR and MS spectra can be
2
5
26
(3ITN)
and zinc-bound caspase-6 (4FXO)
enzymes were
found in Supporting Information. Quinazoline-4(3H)-ones (2a–d)
could react with ethyl chloroacetate to form both N - and O-alky-
3
lated products, depending on the reaction conditions. However, it
has been demonstrated that, when acetone was used as the reac-
retrieved from the Protein Data Bank (www.rcsb.org) and prepared
by employing the QuickPrep module. The binding site was deter-
mined on the basis of the PLB (Propensity for Ligand Binding)
24
score in the Site Finder module of MOE . All the water molecules
in the active site were kept for docking. Docking assays were per-
formed by MOE Dock, using the default flexible ligand/rigid recep-
3
tion solvent, the alkylation reaction gave only N -alkylated prod-
2
9
ucts . The formation of N -alkylated products was evidenced by
3
1
2
7
NMR spectroscopic data. Generally, in the H NMR spectra of the
tor protocol, Triangle Matcher placement, as previously reported .
We used London dG as first rescoring function with force field
final products (5a–z, 6a–g, 7a–b), the singlet peaks attributable
for two methylene protons appeared at around 4.8–5.4 ppm, cor-
(MMFF94x) refinement, and GBVI/WSA as a second one to esti-
responding to the methylene protons of N -alkylated com-
3
mate the negative binding free energy profile of the complex (S
value, kCal/mol). The best pose with the highest negative S value
was selected for each ligand. Biovia DS Visualiser can be utilised
as a visualisation tool to show up the potential interactions of the
2
9
pounds . For the O-alkylated products, these methylene protons
normally appear more downfield (5.6–5.7 ppm) in the H NMR
1
1
3
spectra. In the C NMR spectra, one peak appeared at around
167–169 ppm was attributable for C ¼ O of the CONHN ¼ group.
Other peak appeared at around 160–161 ppm was attributable for
C ¼O of the N -alkylated products. For the O-alkylated products,
2
8
ligands to the residues in the binding sites of caspase enzymes .
4
3
3
. Results and discussions
4
the carbon of C -O functionality should appear more downfield at
1
3
29
around 167–168 ppm in the C NMR spectra . It means that, if
the products were O-alkylated, there should be two peaks at
3.1. Chemistry
1
3
The target acetohydrazides incorporating quinazolin-4(3H)-one around 167–169 ppm in the C NMR spectra. Additionally, in the
(
5a–z, 6a–g, 7a–b) were synthesised via four step pathway, as in the C NMR spectra of all final products, there was a peak
illustrated in scheme 1. The first step was a Niementowski con- around 47 ppm, which was also typical of the methylene carbon
1
3
densation of anthranilic acid (1a) or 5-substituted-2-aminobenzoic from the -NCH
acid (1b–d) and formamide at 120 C to obtain quinazoline-4(3H)- for the carbon of this moiety should appear at around 77 ppm .
2
CO- moiety. In case of O-alkylated products, a peak
ꢀ
22
one derivatives (2a–d) in quantitative yield (93–97%). In the
Regarding the representation of the amide and imine func-
second step, an acylation between quinazoline-4(3H)-one deriva- tional groups, the acetohydrazides (5a–z, 6a–g, 7a–b) could form
tives (2a–d) and ethyl chloroacetate under basic conditions 4 isomers: anti-Z, anti-E, syn-Z, and syn-E. In the cases of aromatic
1
(
K CO ) in acetone with a catalytic amount of KI gave the select- aldehydes-condensed N-acylhydrazones, H-NMR spectra in DMSO-
2
3
29
ively N -alkylated intermediate esters 3a–d . These esters 3a–d
d showed 2 peaks of the -CH CO group at around 5.24 ppm and
6
2
3
participated in futher acyl nucleophile substitution with hydrazine 4.49 ppm with a ratio of 3:1. It could be attributed to the presence
monohydrate at the third step. This reaction proceeded smoothly of two possible syn-anti isomers of the amide bond or Z-E isomers

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
473
Table 1. Effects of the compounds on the growth of SW620 human colon cancer cells.
a
a
Cpd
R
R’/Ar
CGP (%)
Cpd
R
R’/Ar
CGP (%)
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
H
2-Cl
2-NO
3-Cl
4-Cl
4-F
4-Br
2-OH
4-OH
41.70 ± 4.89
3.88 ± 6.23
79.18 ± 3.46
83.23 ± 4.86
39.09 ± 4.17
65.77 ± 7.72
54.46 ± 3.97
35.50 ± 5.15
67.09 ± 8.29
91.40 ± 9.78
5r
5s
5t
5u
5v
5w
5y
5x
5z
6a
-Cl
-Cl
-Cl
-CH
-CH
-CH
-CH
-CH
-CH
-H
-H
4-F
4-OCH
-H
2-NO
4-F
4-Br
4-OCH
4-(NCH )
3 2
78.58 ± 0.81
86.71 ± 5.86
24.75 ± 1.51
9.91 ± 2.26
33.21 ± 7.89
85.41 ± 2.89
51.05 ± 7.38
213.24 ± 3.12
212.43 ± 4.66
83.73 ± 7.37
2
3
3
3
3
3
3
3
2
3
j
4-OCH
3
5
5
k
l
-H
-H
2,3-(OH)
2,4-(OH)
2
2
33.40 ± 4.15
6b
6c
-H
-H
84.82 ± 7.72
69.25 ± 4.17
10.38 ± 2.44
5
m
-H
2,5-(OH)
2
74.70 ± 6.61
6d
-H
82.31 ± 4.31
5
5
5
n
o
p
-H
-H
2-OH-4-OCH
3-OH-4-OCH
3
3
2.81 ± 3.61
78.68 ± 8.05
55.67 ± 6.37
37.76 ± 7.16
17.35 ± 9.01
b
6e
6f
-H
-H
-H
-H
-I
84.00 ± 6.92
77.79 ± 7.49
88.32 ± 5.28
85.58 ± 6.17
21.99 ± 5.14
-H
-H
2,3,4-(OCH
3,4,5-(OCH
3
)
)
3
3
6g
7a
7b
5
5
a
q
3
-H
-H
b
-FU
Cell growth percentage, compounds were assayed at 30 mg/mL; 5-FU: 5-Fluorouracil, a positive control.
Figure 4. Effects of the compounds at 30 mg/mL on the SW620 cells growth.
of the imine bond. To confirm the structures, we decided to pro- atom causes increasing density electron on the hydrogen and the
1
ceed with NOESY experiment. The NOESY spectrum indicated that proton is shifted upfield (Figure 3). Combining with H-NMR spec-
each diagonal signal from a conformer showed a cross-correlation tra, it was clearly shown that the major peak of NH in the upfield
with the corresponding signal of the other conformer. Thus, the was anti-isomer. This observation was in agreement with the
3
0
cross-correlation was caused by multi-conformers with a rotation study of Palla and coworkers , where upfield chemical shift of NH
bond (syn- and anti-isomers), not by a non-rotatable imine bond was anti-isomer. Thus, it could be concluded that the acetohydra-
(
Z and E isomers). Moreover, because of A (1,3) strain in Z-isomer zides (5a–z, 6a–g, 7a–b) formed anti-E and syn-E isomers with a
(
Figure 2), the E-isomer is more stable than Z-isomer. Therefore, ratio of about 3:1, respectively.
the hydrogen of NH amide and C ¼ O can be syn- or anti- position
in their relationship in an E imine configuration (Figure 3).
As it has been known, an amide bond is a partial double bond
3.2. Bioactivity
with a partial oxygen anion. Because the oxygen anion has sp2 The synthesised compounds were subjected to the cytotoxicity
1
8
hybridisation, the partial anion should be located in the sp2 assay using SRB method as described previously with slight
1
9–22
orbital with high s character for stabilisation. This orbital makes a modifications
sigma (d) bond with a carbon atom and will be stabled by anti- of the compounds on the growth of SW620 (colon cancer) cell
bond sigmaꢅ
. In the first screening, we evaluated the effects
ꢅ
(d ) at anti- position. When a hydrogen atom is at line. The cell growth percentages (CGP) of the cells in the pres-
anti- position, partial anion will donate electron to the hydrogen ence of respective compounds at 30 mg/mL were measured and

4
74
L. C. HUAN ET AL.
Table 2. Cytotoxicity of the selected compounds against some human cancer cell lines.
b
c
Cytotoxicity (IC50, lM)/Cell lines
a
Cpd code
R
R’/Ar
MW
LogP
SW620
PC3
NCI-H23
5
5
5
5
5
5
5
7
5
b
l
n
t
u
x
z
-H
-H
-H
-Cl
-CH
-CH
-CH
-I
2-Cl
2,4-(OH)
2-OH-4-OCH
3
340.76
338.32
352.34
370.79
320.35
350.37
363.41
473.22
130.08
392.49
1.52
0.63
1.19
1.60
1.42
1.50
1.60
1.21
ꢁ0.81
3.43
3.14 ± 0.56
9.43 ± 0.27
6.10 ± 0.68
2.45 ± 0.03
3.52 ± 0.03
4.99 ± 0.37
4.68 ± 0.30
3.97 ± 0.32
8.84 ± 1.92
5.82 ± 0.20
3.99 ± 0.15
16.52 ± 0.24
8.46 ± 1.28
3.10 ± 0.32
5.78 ± 0.06
4.22 ± 0.63
4.60 ± 0.33
3.25 ± 0.15
13.61 ± 0.46
4.16 ± 0.52
4.49 ± 0.24
18.97 ± 3.43
9.85 ± 1.28
3.34 ± 0.32
9.77 ± 0.31
4.05 ± 0.31
3.58 ± 0.11
2.81 ± 0.17
13.45 ± 3.92
5.32 ± 0.21
2
4-OCH
-H
3
3
3
3
4-OCH
3
3 2
4-(NCH )
b
-FU
-H
d
PAC-1
a
b
Calculated by EPI 320 software; The concentration (mM) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent
c
d
the averaged results from triplicate experiments with deviation of less than 10%.; Cell lines: SW620, colon cancer; PC3, prostate cancer; NCI-H23, lung cancer; 5-FU:
-Fluorouracil, a positive control.
5
presented in Table 1 and Figure 4. The results were averages of (OH)
three separate measurements. 5-Fluorouracil was used as a posi- cytotoxicity.
tive control.
Introduction of the 6-Cl substituent on the quinazolin-4(3H)-4-
Because this is our first investigation into the compounds of one part of compounds 5a and 5f did not enhance the cytotox-
2
, and 2-Cl, were found to be most potential for the
acetohydrazide type incorporating quinazolin-4(3H)-4-one moiety icity of these compounds (as seen with the high CGP values of
as potential anticancer agents, we initially designed 26 com- the resulting compounds 5r and 5 s, Table 1). However, when the
pounds (5a–5z) to get a glance on general structural features 6-Cl substituent was added on the quinazolin-4(3H)-4-one moiety
required for antitumor cytotoxicity. Seventeen compounds of compound 5j, the cytotoxicity of the resulting compound 5t
(5a–5q), bearing different substituents (R’) on the phenyl part and was substantially increased (as demonstrated by the CGP value of
no substituent on the 4-oxoquinazolin-4(3H) skeleton, were this compound (ꢁ4.75 ± 1.51%, Table 1) in comparison to that of
designed to investigate the effects of the substituents on the phe- compound 5j (91.40 ± 9.78%, Table 1). Interestingly, compound 5x
nyl moiety. Three compounds 5r–5t and five compounds 5u–5z which has the same 4-methoxy group on the phenyl ringside and
were designed to preliminarily investigate the effects of the elec- 6-methyl substituent on the 4-oxoquinazolin-4(3H) part was also
tron-withdrawing and electron-releasing substituents on the qui- found to be strongly inhibitory against the growth of SW620 cells
nazolin-4(3H)-4-one moiety towards the cytotoxicity of at the tested concentration of 30 mg/mL. Compound 5z with the
compounds in series 5. The synthesis of compound bearing 3- 4-dimethylamino group instead of 4-methoxy one was similarly
allyl-3-hydroxy substituent on the phenyl ring in series 5 was not potent. Introduction of the 6-methyl substituent on the quinazo-
attempted at this stage of investigation because structure-activity lin-4(3H)-4-one part of compound 5a was also found to enhance
relationships study of PAC-1 previously indicated that the 3-allyl the cytotoxicity of the resulting compound 5 u. Thus, collectively,
substituent on the phenyl ring of C-region in the structure of from the above results, it could be suggested that for compounds
7
,20
PAC-1 was not very important for PAC-1 bioactivity . In addition, of general structure 5, substituents like 2-chloro, 2-OH-4-CH
-allyl-2-hydroxybenzaldehyde was not readily accessible. (OH) or 4-methoxy and 4-dimethylamino on the phenyl part were
It can be seen from Table 1 that compounds 5a–5q, with few found to be favourable for cytotoxicity. Introduction of different
3
, 2,4-
3
2
exceptions, only moderately inhibited the growth of SW620 cells. substituents on the quinazolin-4(3H)-4-one moiety could also
In general, substitution at position 2 seemed to be more favour- potentially increase the cytotoxicity of the resulting compounds.
able for inhibition of cell growth. For example, compound 5 b at Replacement of the substituted phenyl moiety by isatin could also
3
0 mg/mL displayed very strong cellular inhibition towards the induce potential compounds (e.g. compound 7 b, which com-
growth of SW620 cells (CGP ¼ 3.88 ± 6.23%), while compounds 5d pletely inhibited the growth of SW620 cells when present at
and 5e exhibited much weaker effects (CGP ¼ 83.23 ± 4.86 and 30 mg/mL). However, replacement of the substituted phenyl moi-
3
9.09 ± 4.17%, respectively). Compound 5 h with 2-OH substituent ety by different heterocyclic rings like furane, thiophene, pyrrole,
was also significantly more potent (CGP ¼ 35.50 ± 5.15%) than or pyridine, all proved to be detrimental for cytotoxicity of the
compound 5i (CGP ¼ 67.09 ± 8.29%), which was 4-OH substituted. resulting compounds (6a–6g).
In case of compound 5 h, the addition of one extra OH group at
From the screening on SW620 cells, 8 compounds including
position 3 or 5 did not enhance the cytotoxicity. However, it was 5 b, 5 l, 5n, 5t, 5 u, 5x, 5z and 7 b were found to have potential
found that, addition of an extra OH group at position 4 signifi- cytotoxicity (Table 1, Figure 4). These compounds were subjected
cantly increased the cytotoxicity, as observed with compound 5 l to further evaluation against three cancer cell lines, including
(
CGP ¼ 10.38 ± 2.44%). Especially, addition of the methoxy group SW620, PC-3 (prostate cancer), and NCI-H23 (lung cancer), to get
at position 4 into compound 5 h resulted in compound 5n, which the IC50 values. The results are presented in Table 2. It was found
was the most potent among the compounds 5a–5q. that all these 8 compounds exhibited good cytotoxicity against
Multimethoxylated compounds 5p and 5q were not very active. three cell lines tested with IC50 values in low mM range. Except for
3
From these results, three substituents, including 2-OH-4-OCH , 2,4- compounds 5 l, all other 7 compounds were more potent than 5-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
475
FU, which was used as a positive control. Compound 5t was the compounds appeared to be more ideal than PAC-1 (logP, 3.43).
most potent in the series with IC50 values of 3- to 5-fold lower Our results clearly demonstrate the potentials of the quinazolin-
than that of 5-FU. Compared to PAC-1, compounds 5 l and 5n 4(3H)-4-one-based acetohydrazides as anticancer agents.
were less cytotoxic in three human cancer cell lines tested.
Eight compounds including 5 b, 5 l, 5n, 5t, 5 u, 5x, 5z and 7 b
Compounds 5x, 5z were equally potent compared to PAC-1 while were preliminarily evaluated for their ability to induce caspases
four compounds, including 5 b, 5t, 5 u, and 7 b, were more activity using U937 cells. The compounds or PAC-1, which was
potent. All compounds have logP values in a favourable range for used as a positive control, were incubated with U937 cells
5
oral administration (Table 2). In term of logP values, these (5 ꢃ 10 cells/well) for 24 h. The cells were then harvested and
lysed. The cell lysates (200 lg/100 lL/well) were mixed with Ac-
DEVD-pNA (a substrate of caspases 3, 6 and 7). The OD values
Figure 5. Caspases activation activity of some representative compounds. UN, Figure 6. Relative caspases activation activity of some compounds in comparison
untreated; VH, vehicle.
to PAC-1. Compounds were tested at 50 mM.
Figure 7. Representations of the reference activator PAC-1 docked into procaspase-3 (PDB: 3ITN) showing the (A) 3D conformation and (A1) 2D interactions in the
allosteric site, and PAC-1 docked into zinc-bound caspase-6 (PDB: 4FXO) showing (B) 3D orientation and (B1) 2D interactions in the allosteric inhibitory site. The zinc
ion is represented as a light blue sphere.

4
76
L. C. HUAN ET AL.
Figure 8. Topological interactions between 5b, 5n, 5t, and 7b with caspase-6 showing the role of the ortho-hydroxy N-acyl hydrazone moiety in chelating zinc in the
allosteric site.
were then measured at 405 nm every 30 min for 12 h. The slope of probe the binding ability of four-hit candidates 5 b, 5n, 5t, and
the linear portion for each well was determined as the enzyme 7 b towards procaspase-3 by using molecular docking simulation
activity and presented in Figure 5. Noteworthy, three compounds and then compare these results with those of PAC-1.
including 7 b, 5n, and 5 l caused significant elevation in caspases
According to the catalytic mechanism, these compounds, as
activity. Especially, compound 7 b activated caspases activity by derived from PAC-1, would activate procaspase-3 by chelating zinc
via the key ortho-hydroxy N-acyl hydrazone moiety, thus relieving
almost 200% in comparison to that of PAC-1 (Figure 6). The cas-
pases activation activity of compound 5n was approximately 1.4-
fold stronger than that of PAC-1. Compound 5 l was slightly less
potent than PAC-1 in term of caspases activation. From these
results, it was likely that the 2-hydroxy substituents on the phenyl
ring might play an important role for caspases activation of com-
pounds with a general structure 5.
In this study, however, it was difficult to delineate a correlation
between caspases activation and cytotoxicity of the compounds.
Compounds 7 b and 5t were found to be similarly potent in term
of cytotoxicity but compound 7 b was the most potent as a cas-
pases activator while the caspases activation activity of compound
1
6
the zinc-mediated inhibition . Unfortunately, a crystal structure of
entire zinc-bound procaspase-3 has not been determined so far,
then it is difficult to directly study the role of zinc binding in lig-
and-induced procaspase activation. Therefore, we decided to per-
form the docking assays using another executer caspase highly
homologous to caspase-3: caspase-6 locked by zinc whose crystal
26
complex structure was recently solved by Delgado and Hardy . In
addition, to confirm that all the compounds can bind to the allo-
steric site of procaspase-3 which also play a key role in the active
3
1,32
site formation at the dimer interface of the enzyme
, we
studied the interactions between 5 b, 5n, 5t, and 7 b towards pro-
caspase-3 by using the X-ray crystallographic structure of the
V266E procaspase-3 mutant (replacement of Val with Glu at pos-
5t was not very significant. Also, it was found that despite five
compounds 5 b, 5t, 5 u, 5x, and 5z exhibited potent cytotoxicity,
they showed only minimal caspases activation activity. Thus, more
in-depth studies need to be carried out to fully understand the
cytotoxic mechanisms of these compounds.
25
ition 266) reported by Walters et al.
Firstly, PAC-1 was docked into the allosteric site of procaspase-3.
The results showed that PAC-1 could strictly interact with numer-
ous residues at this site, including Ser205, Arg207, Asn208, Ser251,
and Phe256 (Figure 7(A, A1)). Analysing the H-bonding network
also revealed the significant contribution of water-mediated H-
bond between benzylpiperazine moiety and Arg207, Ser209, and
3.3. Molecular docking studies of 5 b, 5n, 5t, and 7b
As previously identified, compounds 5 b, 5t, and 7 b provided two Glu246. The binding affinity was ꢁ4.96kCal/mol. Subsequently, for
to five-fold increases in cytotoxicity against different cell-lines the first time, PAC-1 was docked into the zinc binding site of cas-
compared to reference compound 5-FU. Especially, two com- pase-6. In the crystal structure, the zinc is found in an exosite and
pounds 7 b and 5n have emerged as potential caspases activators was liganded by Lys36, Glu244, and His287 with a water molecule
and anticancer agents compared to PAC-1. It is of great interest to serving as the fourth ligand, forming a distorted tetrahedral ligation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
477
cytotoxicity against three human cancer cell lines, including
SW620 (human colon cancer), PC-3 (prostate cancer), and NCI-H23
(lung cancer). The most potent compound 5t displayed cytotox-
icity up to 5-fold more potent than 5-FU. Structure-activity rela-
tionship analysis revealed that when the aryl part was phenyl,
substituents like 2-chloro, 2-OH-4-CH , 2,4-(OH) or 4-methoxy and
3
2
4
-dimethylamino were favourable for cytotoxicity of the com-
pounds. In term of caspases activation activity, several compounds
were found to exhibit potent effects, (e.g. compounds 7 b, 5n,
and 5l). Especially, compound 7 b activated caspases activity by
almost 200% in comparison to that of PAC-1. Subsequent docking
simulation also revealed that this compound is a potent allosteric
inhibitor of procaspase-3, and the central role of the ortho-
hydroxy N-acyl hydrazone moiety in chelating zinc in the allosteric
site. Taking all above mentioned into account, several compounds,
such as 5n and 7 b, have emerged as potential hits for further
design and development of caspases activators and anticancer
agents. Further investigation of the substituents on the quinazo-
lin-4(3H)-4-one part is needed to optimise the bioactivity.
Figure 9. Conformations of 5n (green carbons) and 7b (purple carbons) docked
into the allosteric inhibitory site of caspase-6.
2
6
sphere . We sought to determine the ability of PAC-1 to chelate
zinc and relieve this ion from this allosteric inhibitory site of cas-
pase-6. As the results of docking, PAC-1 was able to chelate zinc
and more importantly perturbated the tetrahedral coordination
geometry of zinc to Lys36, Glu244, and His287 (Figure 7(B1)). The
carbonyl oxygens of PAC-1 can also form multiple H-bond interac-
tions with Lys36 and water molecule, suggesting the higher affinity
of PAC-1 to this allosteric site compared to zinc ion.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
We acknowledge the principal financial support from the National
The next steps involved in docking selected compounds 5 b, Foundation for Science and Technology of Vietnam (NAFOSTED,
n, 5t, and 7 b into the allosteric site of procaspase-3. The 3 D Grant number 104.01-2017.06). The work was also partly sup-
5
binding modes of these compounds were depicted in Figure 8. At ported by a grant funded by the Korean Government (NRF, Grant
first sight, all compounds 7 b displayed the highest affinity number 2017R1A5A2015541) and a small grant from Hanoi
(
5
ꢁ5.94 kCal/mol) among four analogues (S values of 5 b, 5n and University of Pharmacy. P.T. Hai acknowledges the support of
t were ꢁ4.46, ꢁ5.09 and ꢁ4.23 kCal/mol, respectively), and this NVIDIA Corporation with the donation of the Titan Xp GPU used
value is higher than that of PAC-1. In particular, compound 5n for this research.
and 7 b provided extensive interactions with residues in the active
site of procaspase-3, such as Arg208 and Phe250 (with 4-oxoqui-
nazoline), and Cys163, Tyr204, and Phe256 (with arylidene moi-
eties). We can also identify that the allosteric binding site
principally consists of hydrophobic as well as H-bond acceptor res-
idues , which in nature is favourable for the interaction with ace-
tihydrazide derivatives having substituents as complex aromatic
systems (e.g. quinazoline or oxoindoline) and H-bond donor
groups (e.g. -NH or -OH).
ORCID
Hai Pham-The
Phuong-Thao Tran
Nguyen-Hai Nam
http://orcid.org/0000-0003-4531-7223
http://orcid.org/0000-0003-4855-2544
http://orcid.org/0000-0001-8475-2530
3
2
We continued investigating the role of ortho-hydroxy N-acyl
hydrazone motif in chelating zinc ion that binds to the allosteric
inhibitory site of caspase-6. The topological docking representa-
tion in Figure 8 showed that all the compounds could bind to
zinc as anticipated. Two carbonyl and hydroxyl oxygens of com-
pounds 5 b and 7 b formed a bidentate chelation with the metal
ion, meanwhile, 5t and 5n interacted with zinc in the same man-
ner of PAC-1. The close distances from the carbonyl oxygens of
References
1
.
Storey S. Targeting apoptosis: selected anticancer strategies.
Nat Rev Drug Discov 2008;7:971–2.
2. Lain S, Hollick JJ, Campbell J, et al. Discovery, in vivo activity,
and mechanism of action of a small-molecule p53 activator.
Cancer Cell 2008;13:454–63.
3. Flygare JA, Beresini M, Budha N, et al. Discovery of a potent
small-molecule antagonist of inhibitor of apoptosis (IAP)
proteins and clinical candidate for the treatment of cancer
(GDC-0152). J Med Chem 2012;55:4101–13.
4. Souers AJ, Leverson JD, Boghaert ER, et al. ABT-199, a
potent and selective BCL-2 inhibitor, achieves antitumor
activity while sparing platelets. Nat Med 2013;19:202.
5n and 7 b to zinc ion (see Figure 9) are favourable for metal che-
lating. Compound 5n and 7 b also displayed more complex H-
bonding interaction network with the central residues in the allo-
steric site of caspase-6, such as Lys36, Tyr37, Glu244, Phe245,
Lys285 and His287, suggesting their higher binding affinity toward
caspase zymogens compared to 5 b, 5t and PAC-1.
5
.
Putt KS, Chen GW, Pearson JM, et al. Small-molecule activa-
tion of procaspase-3 to caspase-3 as a personalized anti-
cancer strategy. Nat Chem Biol 2006;2:543.
4
. Conclusions
In conclusion, we have reported a series of 35 (E)-N’-arylidene-
–(4-oxoquinazolin-4(3H)-yl)acetohydrazides with significant
6. Krepela E, Proch ꢀa zka J, Liu X, et al. Increased expression of
2
Apaf-1 and procaspase-3 and the functionality of intrinsic

4
78
L. C. HUAN ET AL.
apoptosis apparatus in non-small cell lung carcinoma. Bchm 19. Tung TT, Oanh DTK, Dung PTP, et al. New benzothiazole/thia-
2004;385:153–68.
zole-containing hydroxamic acids as potent histone deacetylase
7
.
Fink D, Schlagbauer-Wadl H, Selzer E, et al. Elevated procas-
inhibitors and antitumor agents. Med Chem 2013;9:1051–7.
pase levels in human melanoma. Melanoma Res 2001;11: 20. Nam NH, Huong TL, Dung DTM, et al. Synthesis, bioevalua-
3
85–93.
tion and docking study of 5-substitutedphenyl-1,3,4-thiadia-
zole-based hydroxamic acids as histone deacetylase
inhibitors and antitumor agents. J Enzyme Inhib Med Chem
8
9
.
.
Persad R, Liu C, Wu T-T, et al. Overexpression of caspase-3
in hepatocellular carcinomas. Mod Pathol 2004;17:861.
O’Donovan N, Crown J, Stunell H, et al. Caspase 3 in breast
cancer. Clin Cancer Res 2003;9:738.
2
014;29:611–8.
2
1. You YJ, Kim Y, Nam NH, et al. Antitumor activity of unsatur-
ated fatty acid esters of 4’-demethyldeoxypodophyllotoxin.
Bioorg Med Chem Lett 2003;13:2629–32.
2. Thuong PT, Na MK, Dang NH, et al. Antioxidant activities
of Vietnamese medicinal plants. Nat Prod Sci 2006;12:29–37.
3. Wu L, Smythe AM, Stinson SF, et al. Multidrug-resistant
phenotype of disease-oriented panels of human tumor cell
lines used for anticancer drug screening. Cancer Res 1992;
1
0. Keith FI, Tamara W-S, Eric DH, et al. Characterization of the
interleukin-1b-converting enzyme/ced-3-family protease, cas-
pase-3/CPP32, in hodgkin’s disease: lack of caspase-3 expres-
sion in nodular lymphocyte predominance hodgkin’s
disease. Am J Pathol 1999;154:1439–47.
2
2
1
1. Akira N, Yohko N, Hitoshi I, et al. High levels of expression
and nuclear localization of interleukin-1
enzyme (ICE) and CPP32 in favorable human neuroblasto-
mas. Cancer Res 1997;57:4578–84.
b converting
5
2:3029.
2
2
4. Molecular Operating Environment (MOE). 2009.10; Chemical
Computing Group ULC, 1010 Sherbooke St. West, Suite
1
1
1
2. Svingen PA, Loegering D, Rodriquez J, et al. Components of
the cell death machine and drug sensitivity of the national
cancer institute cell line panel. Clin Cancer Res 2004;10:6807.
3. Howard SR, Paul JH. Derivatives of procaspase-activating
compound 1 (PAC-1) and their anticancer activities. Curr
Med Chem 2016;23:201–41.
#910, Montreal, QC, Canada, H3A 2R7, 2009.
5. Walters J, Pop C, Scott Fiona L, et al. A constitutively active
and uninhibitable caspase-3 zymogen efficiently induces
apoptosis. Biochem J 2009;424:335.
6. Vel ꢀa zquez-Delgado EM, Hardy JA. Zinc-mediated allosteric
inhibition of caspase-6. J Biol Chem 2012;287:36000–11.
7. Huong TTL, Dung DTM, Huan NV, et al. Novel N-hydroxy-
benzamides incorporating 2-oxoindoline with unexpected
potent histone deacetylase inhibitory effects and antitumor
cytotoxicity. Bioorg Chem 2017;71:160–9.
2
0
4. Huan LC, Truc LC, Phuong CV, et al. N -[(E)-Arylidene]-2-(2,3-
2
dihydro-3-oxo-4H-1,4-benzoxazin-4-yl)-acetohydrazides: syn-
thesis and evaluation of caspase activation activity and cyto-
toxicity. Chem Biodivers 2018;15:e1800322.
1
1
5. Peterson QP, Goode DR, West DC, et al. PAC-1 activates pro-
caspase-3 in vitro through relief of zinc-mediated inhibition.
J Mol Biol 2009;388:144–58.
2
2
8. Dassault Syst ꢁe mes BIOVIA. Discovery Studio Modeling
Environment. San Diego, CA, USA: Accelrys Inc; 2016.
ꢂ
9. Spul ꢀa k M, Nov ꢀa k Z, Pal ꢀa t K, et al. The unambiguous synthe-
6. Peterson QP, Hsu DC, Goode DR, et al. Procaspase-3 activation
as an anti-cancer strategy: structure ꢁ activity relationship of
procaspase-activating compound 1 (PAC-1) and its cellular co-
localization with caspase-3. J Med Chem 2009;52:5721–31.
7. Asif M. Chemical characteristics, synthetic methods, and bio-
sis and NMR assignment of 4-alkoxy and 3-alkylquinazolines.
Tetrahedron 2013;69:1705–11.
0. Palla G, Predieri G, Domiano P, et al. Conformational behav-
iour and E/Z isomerization of N-acyl and N-aroylhydrazones.
Tetrahedron 1986;42:3649–54.
3
1
1
logical potential of quinazoline and quinazolinone deriva- 31. H €a cker H-G, Sisay MT, G u€ tschow M. Allosteric modulation of
tives. Int J Med Chem 2014;2014:1–27.
caspases. Pharmacol Ther 2011;132:180–95.
8. Skehan P, Storeng R, Scudiero D, et al. New colorimetric 32. Thomsen ND, Koerber JT, Wells JA. Structural snapshots
cytotoxicity assay for anticancer-drug screening. J Natl
Cancer Inst 1990;82:1107–12.
reveal distinct mechanisms of procaspase-3 and -7 activa-
tion. Proc Natl Acad Sci USA 2013;110:8477.