Novel 3,4-dihydro-4-oxoquinazoline-based acetohydrazides: Design, synthesis and evaluation of antitumor cytotoxicity and caspase activation activity

Article abstract of DOI:10.1016/j.bioorg.2019.103202

In search for novel small molecules with antitumor cytotoxicity via activating procaspase-3, we have designed and synthesized three series of novel (E)-N′-benzylidene-4-oxoquinazolin-3(4H)-yl)acetohydrazides (5a-j, 6a-h, and 7a-h). On the phenyl ring ò the benzylidene part, three different substituents, including 2-OH-4-OCH₃, 4-OCH₃, and 4-N(CH₃)₂, were introduced, respectively. Biological evaluation showed that the acetohydrazides in series 5a-j, in which the phenyl ring of the benzylidene part was substituted by 2-OH-4-OCH₃ substituent, exhibited potent cytotoxicity against three human cancer cell lines (SW620, colon; PC-3, prostate; NCI-H23, lung). Most of the compounds, in this series, especially compounds 5c, 5b and 5h, also significantly activated caspase-3 activity. Among these, compound 5c displayed 1.61-fold more potent than PAC-1 as caspase-3 activator. Cell cycle analysis showed that compounds 5b, 5c, and 5h significantly arrested the cell cycle in G1 phase. Further apoptotic studies also demonstrated compounds 5b, 5c, and 5h as strong apoptotic cell death inducers. The docking simulation studies showed that these compounds could activate procaspase via chelating Zn²⁺ ion bound to the allosteric site of the zymogen.

Full text of DOI:10.1016/j.bioorg.2019.103202

BioorganicChemistry92(2019)103202
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel 3,4-dihydro-4-oxoquinazoline-based acetohydrazides: Design,
synthesis and evaluation of antitumor cytotoxicity and caspase activation
activity
Le Cong Huana,e, Phuong-Thao Trana, Cao Viet Phuonga, Phan Huy Duca, Duong Tien Anha,
Pham The Haia, Le Thi Thu Huongb, Nguyen Thi Thuana, Hye Jin Leec, Eun Jae Parkc,
⁎
Jong Soon Kangd, Nguyen Phuong Linhf, Tran Trung Hieuf, Dao Thi Kim Oanha,
,
⁎
⁎
Sang-Bae Hanc, , Nguyen-Hai Nama,
a Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Viet Nam
b School of Medicine and Pharmacy, Hanoi National University, 144 Xuan Thuy, Hanoi, Viet Nam
c College of Pharmacy, Chungbuk National University, 194-31, Osongsaengmyung-1, Heungdeok, Cheongju, Chungbuk 28160, Republic of Korea
d Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungbuk 28116, Republic of Korea
e Thai Binh University of Medicine and Pharmacy, Thai Binh City, Viet Nam
f Hanoi Amsterdam High School, Hanoi, Viet Nam
A R T I C L E I N F O
A B S T R A C T
Keywords:
In search for novel small molecules with antitumor cytotoxicity via activating procaspase-3, we have designed
and synthesized three series of novel (E)-N′-benzylidene-4-oxoquinazolin-3(4H)-yl)acetohydrazides (5a-j, 6a-h,
and 7a-h). On the phenyl ring ò the benzylidene part, three different substituents, including 2-OH-4-OCH3, 4-
OCH3, and 4-N(CH3)2, were introduced, respectively. Biological evaluation showed that the acetohydrazides in
series 5a-j, in which the phenyl ring of the benzylidene part was substituted by 2-OH-4-OCH3 substituent,
exhibited potent cytotoxicity against three human cancer cell lines (SW620, colon; PC-3, prostate; NCI-H23,
lung). Most of the compounds, in this series, especially compounds 5c, 5b and 5h, also significantly activated
caspase-3 activity. Among these, compound 5c displayed 1.61-fold more potent than PAC-1 as caspase-3 acti-
vator. Cell cycle analysis showed that compounds 5b, 5c, and 5h significantly arrested the cell cycle in G1 phase.
Further apoptotic studies also demonstrated compounds 5b, 5c, and 5h as strong apoptotic cell death inducers.
The docking simulation studies showed that these compounds could activate procaspase via chelating Zn2+ ion
bound to the allosteric site of the zymogen.
Acetohydrazides
Quinazolin-4(3H)-one
Cytotoxicity
Caspase activation
Apoptotic inducers
1. Introduction
In addition, different enzymes are also involved in regulation of
cellular apoptosis. Among these, caspases with at least fourteen mem-
Cancer is characterized by uncontrol growth and rapid proliferation
of abnormal cells, which are able to surpass apoptotic response by
different mechanisms [1,2]. Targeting apoptosis, has therefore, become
one of the attractive approaches nowadays for the design and devel-
opment of new anticancer agents [3]. With the advances in cancer
molecular biology, various proteins involving cellular apoptotic
pawthway, such as p53, XIAP, Bcl-2, BAX, BIM, to name a few, have
been identified [2,3]. A number of small molecules targeting these
proteins (e.g. tenovin-1, a p53 disruptor; GDC-0152, an inhibitor of
XIAP; or ABT-199, a Bcl-2′s inhibitor) have been shown to effectively
induce apoptosis, thus leading to the death of cancer cells [4–6].
bers (caspases 1–14), constitute a large family of cysteine proteases
enzymes with important role in the initiation and execution of apop-
tosis [7]. Among these, caspase-3, which exists as a low activity zy-
mogen in cells, known as procaspase-3, is one of the key enzymes in-
volved in the apoptotic process [7]. Procaspase-3 is found
overexpressed in various human cancers (e.g. colon cancer, lung cancer,
melanoma, hepatoma, breast cancer, lymphoma, and neuroblastoma)
[7–12]. It has been demonstrated that activation of procaspase-3 by
appropriate small molecules effectively restores apoptosis in cancer
cells [13–16]. Since procaspase-3 is overexpressed in various cancer
cells, it is expected that targeting procaspase-3 would be more
⁎ Corresponding authors.
E-mail addresses: shan@cbnu.ac.kr (S.-B. Han), namnh@hup.edu.vn (N.-H. Nam).
https://doi.org/10.1016/j.bioorg.2019.103202
Received 23 April 2019; Received in revised form 11 August 2019; Accepted 13 August 2019
Availableonline14August2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Fig. 1. Structures of PAC-1 and some previously designed 4-oxoquinazoline-based acetohydrazides.
advantageous in comparison to targeting direct apoptotic proteins.
PAC-1, the first procaspase activating compound (Fig. 1) reported
recently, has been shown to have very promising in vivo antitumor
activity profile in several xenografted models [7,14]. Studies on the
structure-activity relationships of PAC-1 indicated that the ortho-hy-
droxy-N-acylhydrazone functionality (B-region, Fig. 1) formed a strong
complex with zinc ion and played vital role for its procaspase activating
and anticancer activity [15,16]. In our previous research to develop
novel small molecules activating caspases we have designed, synthe-
sized and evaluated a series of quinazoline-4(3H)-one-based acetohy-
drazides (Fig. 1) [17,18]. Structure-activity relationships regarding the
substituents on the phenyl part of the structure has been investigated
and three substituents, including 2-OH-4-OCH3, 4-OCH3, and 4-N
(CH3)2 (Fig. 2), were found to be the most favorable for bioactivity. In
this study we decided to further optimize the substituents on the qui-
nazoline-4(3H)-one moiety (Fig. 2) in an attempt to obtain more potent
candidates for further development.
2. Materials and methods
2.1. Chemistry
Thin layer chromatography which was performed using Whatman®
250 μm Silica Gel GF Uniplates and visualized under UV light at
254 nm, was used to check the progress of reactions and preliminary
evaluation of compounds’ homogeneity. Melting points were measured
using a Gallenkamp Melting Point Apparatus (LabMerchant, London,
United Kingdom) and are uncorrected. Purification of compounds was
carried out using crystallization methods and/or open silica gel column
flash chromatography employing Merck silica gel 60 (240 to 400 mesh)
as stationary phase. Nuclear magnetic resonance spectra (1H NMR)
were recorded on a Bruker 500 MHz spectrometer with DMSO‑d6 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
ionization modes including electron ionization (EI), Electrospray ioni-
zation (ESI), were recorded using PE Biosystems 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
Fig. 2. Cytotoxicity of some previously designed 4-oxoquinazoline-based acetohydrazides and further design of 4-oxoquinazoline-based acetohydrazides.
2

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Scheme 1. Synthesis of acetohydrazides incorporating quinazolin-4(3H)-one (5a-j, 6a-h, 7a-h). Reagents and conditions: (a) H2N-CHO, 120 °C, 3 h; (b) ethyl
chloroacetate, KI, K2CO3, acetone, 60 °C, 3.5 h; (c) N2H4·H2O, EtOH, reflux; (d) Ar-CHO or isatin der., AcOH conc., EtOH, reflux.
analyzer. All reagents and solvents were purchased from Aldrich or
Fluka Chemical Corp. (Milwaukee, WI, USA) or Merck unless noted
otherwise. Solvents were used directly as purchased unless otherwise
indicated.
2.1.1. (E)-N′-(2-Hydroxy-4-methoxybenzylidene)-2-(7-methyl-4-
oxoquinazolin-3(4H)-yl)acetohydrazide (5b)
White solid; Yield: 65%. mp: 184–185 °C. Rf = 0.59 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3271 (NH); 3181 (OH); 3066 (CH
aren); 2968, 2897, 2839 (CH, CH2); 1682 (C]O); 1663 (C]N); 1612
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.99, 11.62 (2s, 0.5H,
0.5H, OH); 11.27, 10.21 (2s, 0.5H, 0.5H, CONH); 8.37–8.29 (m, 2H,
N = CH, H2); 8.05 (dd, J = 8.0 Hz, J′ = 2.5 Hz, 1H, H5); 7.67 (d,
J = 8.5 Hz, 1H, H6); 7.54 (d, J = 4.5 Hz, 1H, H8); 7.40 (d, J = 8.0 Hz,
1H, H6′); 6.54–6.50 (m, 2H, H3′, H5′); 5.17, 4.78 (2s, 1.0H, 1.0H, N-CH2-
CO); 3.77 (d, J = 3.0 Hz, 3H, OCH3); 2.49 (s, 3H, CH3).13C NMR
(125 MHz, DMSO‑d6, ppm): δ 168.1, 163.5, 162.7; 162.5; 160.6; 159.7;
158.5; 149.2, 149.0, 148.7, 148.7, 148.5, 145.6, 145.5, 142.5, 131.4,
128.2, 127.4, 127.3, 126.4, 126.3, 119.6, 119.5, 113.5, 112.1, 107.0,
101.6, 101.4, 55.9, 55.8, 55.7, 47.8, 47.2, 21.8. HR-MS (ESI) m/z
365.1232 [M-H]−. Anal. Calcd. For C19H18N4O4 (366.1328): C, 62.29;
H, 4.95; N, 15.29. Found: C, 62.38; H, 5.11; N, 15.18.
The synthesis of acetohydrazides incorporating quinazolin-4(3H)-
one (5–7) was carried out as illustrated in Scheme 1. Details are de-
scribed below.
The mixtures of anthranilic acid or respective 5-substituted-2-ami-
nobenzoic acid (1) (1 mmol) and an excess of formamide (10 mmol) in a
round-bottom flask were heated at 120 °C with stirring for 3–5 h. The
reaction was checked by TLC. After the starting materials completely
disappeared, the resulting mixtures were cooled to room temperature
and then poured into ice-cold water. The light or dark brown pre-
cipitates were formed. The precipitates were filtered and washed three
times with water (20 mL each) and dried to give quinazoline-4(3H)-one
derivatives (2). These intermediates were used for the next step without
further purification.
To a solution of each respective quinazoline-4(3H)-one intermediate
(2) (1 mmol) in acetone (10 mL) were added K2CO3 (207 mg,
1.5 mmol). The resulting mixture was heated at 80 °C with stirring for
30 min. KI (16.6 mg, 0.1 mmol) was added and after stirring for further
15 min, ethyl chloroacetate (0.13 mL, 1.2 mmol) diluted with acetone
(1 mL) was dropwise added into the mixture. The reaction mixture was
further stirred at 60 °C for 3 h. After the reaction completed, the re-
sulting mixture was cooled, poured into ice-cold water. The brown
solids were formed, filtered and dried to give the corresponding ethyl 2-
(4-oxoquinazolin-3-(4H)-yl)acetate derivatives 3.
2.1.2. (E)-N′-(2-Hydroxy-4-methoxybenzylidene)-2-(6-methyl-4-
oxoquinazolin-3(4H)-yl)acetohydrazide (5c)
White solid; Yield: 60%. mp: 188–189 °C. Rf = 0.57 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3181 (OH); 3069 (CH aren); 2986,
2918, 2851 (CH, CH2); 1663 (C]N); 1624, 1605, 1566 (C]C). 1H NMR
(500 MHz, DMSO‑d6, ppm): δ 11.97, 11.60 (2s, 0.5H, 0.5H, OH); 11.26,
10.19 (2s, 0.5H, 0.5H, CONH); 8.37, 8.31 (2s, 0.5H, 0.5H, N = CH);
8.29 (d, J = 8.0 Hz, 1H, H2); 7.96 (s, 1H, H5); 7.70 (dd, J = 5.5 Hz,
J′ = 3.0 Hz, 1H, H7); 7.68 (d, J = 2.5 Hz, 1H, H8); 7.63 (dd, J = 8.5 Hz,
J′ = 5.5 Hz, 1H, H6′); 6.54–6.48 (m, 2H, H3′, H5′); 5.17, 4.78 (2s, 1.0H,
1.0H, CH2); 3.76 (s, 3H, OCH3); 2.47 (s, 3H, CH3). 13C NMR (125 MHz,
DMSO‑d6, ppm): δ 168.1, 163.5, 162.7, 162.5, 160.7, 159.7, 158.5,
148.5, 148.3, 148.1, 146.6, 142.5, 138.4, 137.3, 136.3, 136.2, 131.4,
128.2, 127.6, 125.8, 125.7, 113.5, 112.1, 107.0, 101.6, 101.4, 55.8,
55.7, 47.9, 47.3, 21.3. HR-MS (ESI) m/z 365.1255 [M-H]−. Anal. Calcd.
For C19H18N4O4 (366.1328): C, 62.29; H, 4.95; N, 15.29. Found: C,
62.18; H, 4.83; N, 15.37.
To a respective solution of the ester 3 (0.5 mmol) in ethanol (10 mL)
was added slowly 0.12 mL of hydrazine monohydrate (2.5 mmol). The
mixture was stirred at room until the starting material was completely
consumed. The white precipitates were formed, filtered, and washed
with cold-ethanol (3 times). The white solids (the hydrazides 4) were
collected, dried under vacuum and used for the next step without fur-
ther purification.
Each of the hydrazides 4 (0.5 mmol) was dissolved in ethanol
(20 mL), then 2 drops of concentrated acetic acid, followed by benzal-
dehyde or isatine derivatives (1.0 mmol) were added. The mixture was
refluxed until the reaction completed. The precipitates were formed,
filtered, and washed with ethanol (3 times). The white solids were
collected, dried under vacuum and re-crystalised in ethanol or column
chromatography (DCM/MeOH) to obtain the desired products (5–7).
The physicochemical and spectral data for compounds 5a, 6a, 6c, 6h,
and 7c had been reported previously [17].
2.1.3. (E)-2-(6,7-Dimethoxy-4-oxoquinazolin-3(4H)-yl)-N′-(2-hydroxy-4-
methoxybenzylidene)acetohydrazide (5d)
White solid; Yield: 58%. mp: 197–198 °C. Rf = 0.59 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3161 (OH); 3084 (CH aren); 2963,
2901, 2841 (CH, CH2); 1692 (C]O); 1645 (C]N); 1603, 1501 (C]C).
1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.97, 11.60 (2s, 0.5H, 0.5H,
OH); 11.28, 10.19 (2s, 0.5H, 0.5H, CONH); 8.37–8.25 (m, 2H, N = CH,
H2); 7.67, 7.47 (2d, J = 8.5 Hz, 0.5H, 0.5H, H6′); 7.47 (s, 1H, H5); 7.20
(s, 1H, H8); 6.52 (s, 1H, H5′); 6.48 (s, 1H, H3′); 5.16, 4.77 (2s, 0.5H,
0.5H, NCH2CO); 3.94, 3.89, 3.77 (3 s, 9H, OCH3). 13C NMR (125 MHz,
3

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
DMSO‑d6, ppm): δ 168.2, 163.6, 162.6, 162.5, 160.1, 159.7, 158.4,
155.1, 155.0, 149.3, 149.2, 148.5 ,146.5, 147.5, 144.7, 142.5, 131.4,
128.2, 115.1, 115.0, 113.5, 112.1, 108.5, 107.0, 105.6, 105.5, 101.6,
101.4, 56.5, 56.2, 55.8, 47.8, 47.2. HR-MS (ESI) m/z 454.1714
[M + H + CH3CN]+. Anal. Calcd. For C20H20N4O6 (412.1383): C,
58.25; H, 4.89; N, 13.59. Found: C, 58.37; H, 4.95; N, 13.48.
8.03 (d, J = 8.5 Hz, 1H, H7); 7.71–7.66 (m, 1H, H8); 7.46 (d,
J = 9.0 Hz, 1H, H6′); 6.54–6.50 (m, 2H, H3′, H5′); 5.19, 4.80 (2s, 1.2H,
0.8H, NCH2CO); 3.77 (s, 3H, OCH3). 13C NMR (125 MHz, DMSO‑d6,
ppm): δ 167.8, 163.2, 162.7, 162.5, 159.7, 158.5, 149.7, 149.6, 148.6,
147.6, 147.5, 142.5, 137.9, 131.3, 130.2, 128.6, 128.1, 123.5, 120.1,
113.5, 112.1, 107.0, 101.6, 101.4, 55.8, 55.7, 48.1, 47.6. MS (ESI) m/z
428.97 [M-H]−. Anal. Calcd. For C18H15BrN4O4 (330.0277): C, 50.13;
H, 3.51; N, 12.99. Found: C, 50.20; H, 3.59; N, 12.88.
2.1.4. (E)-2-(7-Fluoro-4-oxoquinazolin-3(4H)-yl)-N′-(2-hydroxy-4-
methoxybenzylidene)acetohydrazide (5e)
White solid; Yield: 64%. mp: 186–187 °C. Rf = 0.54 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3176 (OH); 3076 (CH aren); 2976,
2905, 2839 (CH, CH2); 1694 (C]O); 1667 (C]N); 1630, 1603, 1570
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.97, 11.63 (2s, 0.4H,
0.6H, CONH); 11.24, 10.19 (2s, 0.4H, 0.6H, OH); 8.88 (s, 1H, H2); 8.43,
8.42 (2s, 0.4H, 0.6H, N = CH); 8.23 (s, 1H, H5); 7.67, 7.46 (2d,
J = 8.5 Hz, 0.4H, 0.6H, H6′); 7.55–7.53 (m, 2H, H8, H6); 6.53–6.48 (m,
2H, H3′, H5′); 5.18, 4.79 (2s, 1.2H, 0.8H, NCH2CO); 3.77 (s, 3H, OCH3).
13C NMR (125 MHz, DMSO‑d6, ppm): δ 162.7, 162.5, 161.1, 160.1,
159.7, 158.5, 150.8, 150.7, 150.5, 150.4, 148.6, 142.6, 133.2, 131.4,
129.8, 129.7, 128.1, 119.0, 116.4, 116.3, 116.2, 116.1, 113.5, 113.01,
113.0, 112.8, 112.1, 111.9, 107.6, 107.0, 101.6, 101.4, 56.0, 55.8,
55.7, 47.9, 47.4. HR-MS (ESI) m/z 369.0990 [M-H]−. Anal. Calcd. For
2.1.8. (E)-N′-(2-Hydroxy-4-methoxybenzylidene)-2-(6-nitro-4-
oxoquinazolin-3(4H)-yl)acetohydrazide (5i)
White solid; Yield: 55%. mp: 184–185 °C. Rf = 0.58 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3179 (OH); 3069 (CH aren); 2982,
2903 (CH, CH2); 1697 (C]O); 1668 (C]N); 1630, 1603, 1522 (C]C).
1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.98, 11.65 (2s, 0.4H, 0.6H,
OH); 11.19, 10.18 (2s, 0.4H, 0.6H, CONH); 8.53 (d, J = 5.0 Hz, 1H,
H2); 8.44–8.43 (m, 1H, N = CH); 8.39–8.27 (m,3H, H5, H7, H8); 7.65,
7.44 (2d, J = 8.5 Hz, 0.4H, 0.6H, H6′); 6.51–6.45 (m, 2H, H3′, H5′);
5.21, 4.82 (2s, 1.2H, 0.8H, NCH2CO); 3.74 (s, 3H, OCH3). 13C NMR
(125 MHz, DMSO‑d6, ppm): δ 163.0, 162.7, 162.5, 159.9, 159.7, 158.5,
151.7, 151.3, 151.1, 148.9, 148.6, 142.6, 131.3, 128.9, 128.1, 126.1,
122.9, 121.4, 121.3, 113.4, 112.1, 107.0, 101.6, 101.4, 55.8, 55.7,
48.2, 47.8. HR-MS (ESI) m/z 396.0969 [M-H]−. Anal. Calcd. For
C
18H15FN4O4 (370.1077): C, 59.38; H, 4.08; N, 15.13. Found: C, 59.47;
H, 4.19; N, 15.09.
C18H15N5O6 (397.1022): C, 54.41; H, 3.81; N, 17.63. Found: C, 54.53;
H, 3.75; N, 17.74.
2.1.5. (E)-2-(6-Fluoro-4-oxoquinazolin-3(4H)-yl)-N′-(2-hydroxy-4-
methoxybenzylidene)acetohydrazide (5f)
2.1.9. (E)-N′-(2-Hydroxy-4-methoxybenzylidene)-2-(7-nitro-4-
White solid; Yield: 62%. mp: 186–187 °C. Rf = 0.53 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3198 (OH); 3063 (CH aren); 2899,
2839 (CH, CH2); 1691 (C]O); 1663 (C]N); 1630, 1601, 1570 (C]C).
1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.96, 11.62 (2s, 0.4H, 0.6H,
OH); 11.24, 10.18 (2s, 0.4H, 0.6H, CONH); 8.38, 8.29 (2s, 0.4H, 0.6H,
N = CH); 8.37 (s, 1H, H2); 7.85–7.76 (m, 3H, H5, H7, H8); 7.67, 7.46
(2d, J = 9.0 Hz, 0.4H, 0.6H, H6′); 6.54–6.47 (m, 2H, H3′, H5′); 5.19,
4.80 (2s, 1.2H, 0.8H, NCH2CO); 3.77 (s, 3H, OCH3).13C NMR (125 MHz,
DMSO‑d6, ppm): δ 167.9, 163.3, 162.7, 162.5, 160.1, 159.7, 158.5,
148.6, 148.4, 145.5, 142.6, 131.4, 130.7, 128.1, 123.5, 123.1, 113.5,
112.1, 111.3, 111.1, 107.0, 101.6, 101.4, 55.8, 55.7, 48.0, 47.5. HR-MS
(ESI) m/z 369.0990 [M-H]−. Anal. Calcd. For C18H15FN4O4 (370.1077):
C, 59.38; H, 4.08; N, 15.13. Found: C, 59.27; H, 4.17; N, 15.21.
oxoquinazolin-3(4H)-yl)acetohydrazide (5j)
White solid; Yield: 58%. mp: 185–186 °C. Rf = 0.57 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3265 (NH); 3098 (CH aren); 3034,
2945, 2841 (CH, CH2); 1722 (C]O); 1668 (C]N); 1629, 1609, 1570
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.98, 11.60 (2s, 0.4H,
0.6H, OH); 11.12, 10.11 (2s, 0.4H, 0.6H, CONH); 8.78–8.76 (m, 1H,
H2); 8.53–8.50 (m, 2H, N = CH, H5); 8.28, 8.20 (2s, 0.4H, 0.6H, H8);
7.86 (dd, J = 9.0 Hz, J′ = 6.0 Hz, 1H, H6); 7.59, 7.37 (2d, J = 8.5 Hz,
0.4H, 0.6H, H6′); 6.44–6.38 (m, 2H, H3′, H5′); 5.15, 4.76 (2s, 1.2H,
0.8H, NCH2CO); 3.67 (s, 3H, OCH3). 13C NMR (125 MHz, DMSO‑d6,
ppm): δ 167.6, 163.0, 162.7, 162.5, 160.1, 158.5, 152.7, 152.4, 148.7,
145.9, 142.7, 129.7, 129.0, 128.1, 122.7, 122.6, 121.9, 113.5, 107.0,
101.6, 101.4, 55.8, 55.7, 48.2, 47.8. HR-MS (ESI) m/z 396.0916 [M-
H]−. Anal. Calcd. For C18H15N5O6 (397.1022): C, 54.41; H, 3.81; N,
17.63. Found: C, 54.49; H, 3.91; N, 17.55.
2.1.6. (E)-2-(7-Chloro-4-oxoquinazolin-3(4H)-yl)-N′-(2-hydroxy-4-
methoxybenzylidene)acetohydrazide (5g)
White solid; Yield: 68%. mp: 189–190 °C. Rf = 0.57 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3179 (OH); 3069 (CH aren); 2970,
2911, 2841 (CH, CH2); 1697 (C]O); 1667 (C]N); 1632, 1603, 1570
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 8.43, 8.27 (2s, 0.4H,
0.6H, CONH); 8.43, 8.37 (2s, 0.4H, 0.6H, N = CH); 8.41 (s, 1H, H2);
8.29 (s, 1H, H5); 7.93–7.90 (m, 1H, H7); 7.79–7.76 (m, 1H, H8);
7.67–7.46 (2d, J = 8.5 Hz, 0.6H, 0.4H, H6′); 6.53–6.50 (m, 2H, H3′,
H5′); 5.20, 4.81 (2s, 1.2H, 0.8H, NCH2CO); 3.77 (s, 3H, OCH3). 13C NMR
(125 MHz, DMSO‑d6, ppm): δ 167.8, 163.3, 162.7, 162.5, 159.8, 158.6,
149.6, 149.4, 148.6, 147.3 , 142.6, 135.2, 135.1, 132.0, 131.9, 131.3,
130.1, 128.1, 125.5, 125.4, 123.1, 113.5, 112.1, 106.9, 101.6, 101.4
55.8, 55.7, 48.1, 47.5. MS (ESI) m/z 386.8 [M + H]+. Anal. Calcd. For
2.1.10. (E)-N′-(4-Methoxybenzylidene)-2-(7-methyl-4-oxoquinazolin-
3(4H)-yl)acetohydrazide (6b)
White solid; Yield: 64%. mp: 172–173 °C. Rf = 0.64 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3175 (OH); 3063 (CH aren); 2982,
2939 (CH, CH2); 1670 (C]O); 1670 (C]N); 1601, 1516 (C]C). 1H
NMR (500 MHz, DMSO‑d6, ppm): δ 11.73, 11.67 (2s, 0.25H, 0.75H,
CONH); 8.33 (2s, 1H, H2); 8.18, 8.01 (2s, 0.25H, 0.75H, N = CH); 8.05
(dd, J = 8.0; J′ = 3.5 Hz, 1H, H6); 7.65–7.70 (m, 2H, H2′, H6′); 7.53 (s,
1H, H8); 7.40 (d, J = 8.0 Hz, H5); 7.01–7.04 (m, 2H, H3′, H5′); 3.82 (s,
3H, OCH3); 5.19, 4.75 (2s, 1.5H, 0.5H, NCH2CO); 2.49 (s, 1H, CH3). 13
C
NMR (125 MHz, DMSO‑d6, ppm): δ 168.5, 163.7, 161.4, 161.3, 160.6,
149.2, 149.1, 148.7, 147.6, 145.6, 145.5, 144.6, 129.2 129.0, 127.3,
127.1, 127.0, 126.4, 126.3, 119.6, 119.5, 114.8, 55.8, 47.9, 47.3. HR-
C
18H15ClN4O4 (386.0782): C, 55.89; H, 3.91; N, 14.49. Found: C, 55.97;
H, 3.46; N, 14.40.
MS (ESI) m/z 349.1272 [M-H]−
. Anal. Calcd. For C19H18N4O3
(350.1379): C, 65.13; H, 5.18; N, 15.99. Found: C, 65.25; H, 5.11; N,
16.10.
2.1.7. (E)-2-(7-Bromo-4-oxoquinazolin-3(4H)-yl)-N′-(2-hydroxy-4-
methoxybenzylidene)acetohydrazide (5h)
White solid; Yield: 67%. mp: 211–212 °C. Rf = 0.61 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3190 (OH); 3065 (CH aren); 2984,
2911, 2837 (CH, CH2); 1697 (C]O); 1670 (C]N); 1630, 1607, 1570
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.98, 11.65 (2s, 0.4H,
0.6H, OH); 11.24, 10.20 (2s, 0.4H, 0.6H, CONH); 8.43, 8.42 (2s, 0.4H,
0.6H, H2); 8.36, 8.28 (2s, 0.4H, 0.6H, N = CH); 8.25–8.24 (m, 1H, H5);
2.1.11. (E)-2-(6,7-Dimethoxy-4-oxoquinazolin-3(4H)-yl)-N′-(4-
methoxybenzylidene)acetohydrazide (6d)
White solid; Yield: 62%. mp: 187–188 °C. Rf = 0.65 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3179 (OH); 3082 (CH aren); 3007,
2943, 2833 (CH, CH2); 1678 (C]O); 1661 (C]N); 1504 (C]C). 1H
NMR (500 MHz, DMSO‑d6, ppm): δ 11.71, 11.65 (2s, 0.3H, 0.7H,
4

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
CONH); 8.26 (s, 1H, H2); 8.18, 8.01 (2s, 1.4H, 0.6H, N = CH);
7.70–7.66 (m, 2H, H2′, H6′); 7.47 (s, 1H, H8); 7.07–7.02 (m, 2H, H3′,
H5′); 5.19, 4.75 (2s, 1.4H, 0.6H, NCH2CO); 3.94–3.81 (m, 9H, OCH3).
13C NMR (125 MHz, DMSO‑d6, ppm): δ 168.6 162.2 161.3, 160.1, 155.0,
149.3, 147.7, 144.8, 129.2, 129.0, 127.0, 114.9 108.5, 105.6, 56.5,
56.2, 55.8, 47.3. HR-MS (ESI) m/z 395.1345 [M-H]−. Anal. Calcd. For
135.0, 134.9, 129.0, 128.7, 127.7, 127.6, 127.5, 126.5, 122.0, 121.7,
112.3, 48.0, 47.4. HR-MS (ESI) m/z 348.1456 [M-H]−. Anal. Calcd. For
C
19H19N5O2 (349.1539): C, 65.32; H, 5.48; N, 20.04. Found: C, 65.41;
H, 5.37; N, 20.13.
2.1.16. (E)-N′-(4-(Dimethylamino)benzylidene)-2-(7-methyl-4-
C
20H20N4O5 (396.1434): C, 60.60; H, 5.09; N, 14.13. Found: C, 60.71;
oxoquinazolin-3(4H)-yl)acetohydrazide (7b)
H, 5.18; N, 14.21.
White solid; Yield: 63%. mp: 197–198 °C. Rf = 0.63 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3177 (OH); 3063 (CH aren); 2963,
2907 (CH, CH2); 1668 (C]N); 1603, 1533 (C]C). 1H NMR (500 MHz,
DMSO‑d6, ppm): δ 11.56, 11.51 (2s, 0.25H, 0.75H, CO-NH); 8.33(s, 1H,
H2); 8.33, 8.09 (2s, 0.25H, 0.75H, N = CH); 8.09–8.03 (m, 1H, H5);
7.55–7.51 (m, 3H, H2′, H6′, H8); 7.40 (d, J = 8.0 Hz, 1H, H6); 6.76–6.74
(m, 2H, H3′, H5′); 5.17, 4.73 (2s, 1.5H, 0.5H, NCH2CO), 2.98 (m, 6H, N
(CH3)2); 2.49 (m, 3H, CH3). 13C NMR (125 MHz, DMSO‑d6, ppm): δ
168.1, 160.6, 152.0, 149.2, 148.7, 145.5, 145.4, 130.0, 129.0, 128.9,
128.7 127.3, 126.4, 121.7, 119.6, 112.3, 112.2 47.8, 47.3, 21.8. HR-MS
(ESI) m/z 364.1780 [M-H]−. Anal. Calcd. For C20H21N5O2 (363.1695):
C, 66.10; H, 5.82; N, 19.27. Found: C, 66.17; H, 5.95; N, 19.19.
2.1.12. (E)-2-(7-Fluoro-4-oxoquinazolin-3(4H)-yl)-N′-(4-
methoxybenzylidene)acetohydrazide (6e)
White solid; Yield: 67%. mp: 180–181 °C. Rf = 0.66 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3179 (OH); 3065 (CH aren); 2967,
2907, 2841 (CH, CH2); 1674 (C]O); 1603 (C]N); 1568 (C]C). 1H
NMR (500 MHz, DMSO‑d6, ppm): δ 11.74, 11.70 (2s, 0.2H, 0.8H,
CONH); 8.43 (2s, 1H, H2); 8.25–8.18 (m, 1H, H8); 8.02 (s, 1H, N = CH);
7.70–7.65 (m, 2H, H2′, H6′); 7.54–7.52 (m, 1H, H6); 7.47–7.04 (m, 1H,
H5); 7.02, 7.01 (2s, 2H, H3′, H5′); 5.21, 4.75 (2s, 1.6H, 0.4H, NCH2CO);
3.82 (s, 3H, OCH3). 13C NMR (125 MHz, DMSO‑d6, ppm): δ 168.3,
167.1, 165.1, 163.5, 161.4, 161.3, 160.1, 150.8, 150.7, 150.5, 150.4,
147.7, 144.7, 130.5 129.8, 129.7, 129.2, 129.0, 127.0, 126.9, 119.0,
116.3, 116.1, 114.8, 113.0, 112.8, 55.8, 48.0, 47.5, 47.4. HR-MS (ESI)
m/z 355.1201 [M-H]−. Anal. Calcd. For C18H15FN4O3 (354.1128): C,
61.01; H, 4.27; N, 15.81. Found: C, 61.17; H, 4.35; N, 15.75.
2.1.17. (E)-2-(6,7-Dimethoxy-4-oxoquinazolin-3(4H)-yl)-N′-(4-
(dimethylamino)benzylidene)acetohydrazide (7d)
White solid; Yield: 57%. mp: 193–194 °C. Rf = 0.65 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3173 (OH); 3082 (CH aren); 2968,
2805 (CH, CH2); 1670 (C]N); 1605, 1531, 1504 (C]C). 1H NMR
(500 MHz, DMSO‑d6, ppm): δ 11.55, 11.50 (2s, 0.3H, 0.7H, CONH); 8.33
(s, 1H, H2); 7.93 (s, 1H, N = CH); 7.55, 7.51 (2s, 1.4H, 0.6H, H2′, H6′);
7.16 (d, J = 8.0 Hz, 2H, H5, H8); 6.77, 6.76 (2d, 1.4, 0.6H, H3′, H5′);
5.11, 4.72 (2s, 0.6H, 1.4H, NCH2CO); 3.93 (s, 6H, OCH3); 2.98 (s, 6H, N
(CH3)2).13C NMR (125 MHz, DMSO‑d6, ppm): δ 168.2, 164.5, 163.3
160.3 152.1, 152.0, 150.9, 149.8, 149.7, 148.5, 145.5, 128.9, 128.7,
128.2, 121.7, 117.1, 115.4, 112.3, 108.8, 56.3, 47.7, 31.2. HR-MS (ESI)
m/z 408.1663 [M-H]−. Anal. Calcd. For C21H23N5O4 (409.1750): C,
61.60; H, 5.66; N, 17.10. Found: C, 61.71; H, 5.55; N, 17.23.
2.1.13. (E)-2-(6-Fluoro-4-oxoquinazolin-3(4H)-yl)-N′-(4-
methoxybenzylidene)acetohydrazide (6f)
White solid; Yield: 66%. mp: 181–182 °C. Rf = 0.67 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3283 (NH); 3159 (OH); 3019(CH
aren); 2963, 2843 (CH, CH2); 1676 (C]O); 1653 (C]N); 1603, 1541
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.74, 11.71 (2s, 0.2H,
0.8H, CONH); 8.39 (s, 1H, H2); 8.19, 8.02 (2s, 0.2H, 0.8H, N = CH);
7.84–7.69 (m, 5H, H2′, H6′, H5, H7, H8); 7.04, 7.03 (2s, 1.6H, 0.4H, H3′,
H5′); 5.23, 4.79 (2s, 1.6H, 0.4H, NCH2CO); 3.83 (m, 3H, OCH3). 13C
NMR (125 MHz, DMSO‑d6, ppm): δ 168.3, 161.3, 160.1, 159.8, 148.6,
145.5, 144.7, 130.6, 130.4, 129.3, 129.0 127.1, 127.0, 123.6, 123.2,
2.1.18. (E)-N′-(4-(Dimethylamino)benzylidene)-2-(7-fluoro-4-
114.8, 111.3, 111.1, 55.8, 47.6. HR-MS (ESI) m/z 355.1203 [M-H]−
.
oxoquinazolin-3(4H)-yl)acetohydrazide (7e)
Anal. Calcd. For C18H15FN4O3 (354.1128): C, 61.01; H, 4.27; N, 15.81.
Found: C, 61.12; H, 4.35; N, 15.73.
White solid; Yield: 65%. mp: 190–191 °C. Rf = 0.67 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3123 (OH); 3067 (CH aren); 2968,
2897, 2814 (CH, CH2); 1668 (C]O); 1605, 1504 (C]C). 1H NMR
(500 MHz, DMSO‑d6, ppm): δ 11.56, 11.52 (2s, 0.25H, 0.75H, CONH);
8.42 (s, 1H, H2); 8.25–8.22 (m, 1H, H8); 8.09, 7.94 (2s, 0.25H, 0.75H,
N = CH); 7.56–7.43 (m, 4H, H2′, H6′, H5, H6); 6.77–6.74 (m, 2H, H3′,
H5′); 5.19, 4.75 (2s, 1.5H, 0.5H, NCH2CO); 2.98 (s, 6H, N(CH3)2). 13C
NMR (125 MHz, DMSO‑d6, ppm): δ 167.9 167.1, 165.1, 163.1, 160.1,
152.1, 152.0, 150.8, 150.7, 150.6, 150.5, 148.6, 145.6, 129.8, 129.7,
129.0, 128.7, 121.7, 119.0, 116.3, 116.1, 112.9, 112.8, 112.3, 47.9,
2.1.14. (E)-2-(6-Bromo-4-oxoquinazolin-3(4H)-yl)-N′-(4-
methoxybenzylidene)acetohydrazide (6h)
White solid; Yield: 66%. mp: 186–187 °C. Rf = 0.69 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3206 (NH); 3156 (OH); 3064 (CH
aren); 2899, 2826 (CH, CH2); 1705 (C]O); 1663 (C]N); 1605, 1518
(C]C). 1H NMR (500 MHz, DMSO‑d6, ppm): δ 11.72 (s, 1H, CONH);
8.43 (s, 1H, H2); 8.25, 8.15 (2s, 0.6H, 0.4H, N = CH); 8.05–8.00 (m,
2H, H7, H5); 7.70–7.60 (m, 3H, H2′, H6′, H8); 7.03, 7.02 (2s, 1.2H, 0.8H,
47.4. HR-MS (ESI) m/z 366.1359 [M-H]−
. Anal. Calcd. For
H3′, H5′); 5.22, 4.79 (2s, 1.6H, 0.4H, NCH2CO); 3.82 (s, 3H, OCH3). 13
C
C19H18FN5O2 (367.11445): C, 62.12; H, 4.94; N, 19.06. Found: C,
NMR (125 MHz, DMSO‑d6, ppm): δ 168.2, 163.4, 161.4, 161.3, 159.7,
149.7, 149.6, 147.7, 147.6, 144.7, 137.9, 137.9, 130.2, 129.3, 129.0,
128.6, 128.6, 127.0, 126.9, 123.5, 120.1, 114.8, 55.8, 48.1, 47.6. HR-
62.23; H, 5.05; N, 18.96.
2.1.19. (E)-N′-(4-(Dimethylamino)benzylidene)-2-(6-fluoro-4-
oxoquinazolin-3(4H)-yl)acetohydrazide (7f)
MS (ESI) m/z 415.0399 [M-H]−
.
White solid; Yield: 63%. mp: 191–192 °C. Rf = 0.66 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3175 (OH); 3067 (CH aren); 2965,
2901 (CH, CH2); 1667 (C]N); 1535 (C]C). 1H NMR (500 MHz,
DMSO‑d6, ppm): δ 11.57, 11.54 (2s, 0.2H, 0.8H, CONH); 8.38, 8.37 (2s,
0.2H, 0.8H, H2); 8.09, 7.93 (s, 0.2H, 0.8H, N = CH); 7.85–7.75 (m, 3H,
H5, H6, H8); 7.56, 7.51 (2s, 0.4H, 1.6H, H2′, H6′); 6.77, 6.74 (2s, 0.4H,
1.6H, H3′, H5′); 5.20, 4.76 (2s, 1.6H, 0.4H, NCH2CO); 2.98 (s, 6H, N
(CH3)2). 13C NMR (125 MHz, DMSO‑d6, ppm): δ 167.9, 163.0, 161.7,
160.1, 159.7, 152.1, 152.0, 148.6, 148.6, 145.6, 145.5, 130.7, 130.6,
129.0, 128.7, 123.5, 123.3, 123.2, 123.1, 121.7, 112.3, 112.2, 111.3,
111.1, 48.0, 47.5. HR-MS (ESI) m/z 368.1520 [M + H]+. Anal. Calcd.
For C19H18FN5O2 (367.11445): C, 62.12; H, 4.94; N, 19.06. Found: C,
62.19; H, 4.83; N, 19.17.
2.1.15. (E)-N′-(4-(Dimethylamino)benzylidene)-2-(4-oxoquinazolin-
3(4H)-yl)acetohydrazide (7a)
White solid; Yield: 63%. mp: 190–191 °C. Rf = 0.62 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3210 (NH); 3069 (CH aren); 2972,
2899, 2816 (CH, CH2); 1668 (C]N); 1603 (C]N); 1504 (C]C). 1H
NMR (500 MHz, DMSO‑d6, ppm): δ 11.57, 11.53 (2s, 0.25H, 0.75H,
CONH); 8.38, 8.10 (2s, 0.25H, 0.75H, N = CH); 8.18 (d, J = 7.0 Hz, 1H,
H5); 7.94 (s, 1H, H2); 7.87 (t, J = 7.0 Hz, 1H, H6); 7.73 (d, J = 8.0 Hz,
2H, H2′, H6′); 6.76 (d, J = 8.0 Hz, 2H, H3′, H5′); 7.59 (d, J = 7.5 Hz, 1H,
H7); 7.55 (d, J = 8.5 Hz, 1H, H8); 5.20, 4.77 (2s, 0.5H, 1.5H, NCH2CO);
3.35 (s, 3H, CH3); 2.99 (s, 6H, N(CH3)2). 13C NMR (125 MHz, DMSO‑d6,
ppm): δ 168.1, 163.2, 160.8, 152.1, 152.0, 149.2, 149.1, 148.6, 145.6,
5

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Table 1
Cytotoxicity of the compounds against some human cancer cell lines.
Cpd
R
R′
MW
LogPa
Cytotoxicity (IC50
SW620
,
b μM)/Cell linesc
PC3
NCI-H23
5a
5b
5c
H
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
2-OH-4-OCH3
4-OCH3
352.34
366.37
366.37
412.40
370.34
370.34
386.79
431.24
397.34
397.34
336.35
350.37
350.37
396.40
354.34
354.34
370.79
415.25
349.39
363.41
363.41
409.44
367.38
367.38
383.83
428.28
130.08
392.49
1.19
1.74
1.74
0.84
1.40
1.40
1.84
2.08
1.01
1.01
0.95
1.50
1.50
0.60
1.16
1.16
1.60
1.84
1.05
1.60
1.60
0.70
1.25
1.25
1.70
1.94
−0.81
3.43
6.10
2.16
2.55
> 10
1.63
1.54
1.56
0.98
3.75
> 10
> 10
> 10
4.99
> 10
> 10
> 10
2.45
> 10
5.91
> 10
4.68
> 10
> 10
> 10
> 10
> 10
8.84
4.32
0.68
0.28
0.21
8.46
1.28
0.18
0.10
9.85
1.89
1.74
> 10
1.14
1.35
1.75
1.19
3.58
> 10
> 10
> 10
4.05
> 10
> 10
> 10
3.34
> 10
3.01
> 10
3.58
> 10
> 10
> 10
> 10
> 10
13.45
4.09
1.28
7-CH3
6-CH3
6,7-(OCH3)2
7-F
1.48
0.13
0.18
1.90
5d
5e
5f
> 10
1.31
0.09
0.29
0.03
0.10
0.58
0.06
0.12
0.04
0.01
0.31
0.03
0.03
0.08
0.07
0.24
6-F
1.51
5g
5h
5i
6-Cl
1.25
6-Br
1.30
6-NO2
7-NO2
H
4.18
5j
> 10
> 10
> 10
4.22
6a
6b
6c
7-CH3
6-CH3
6,7-(OCH3)2
7-F
4-OCH3
4-OCH3
0.37
0.63
0.31
6d
6e
6f
4-OCH3
> 10
> 10
> 10
3.10
4-OCH3
6-F
4-OCH3
6g
6h
7a
7b
7c
6-Cl
4-OCH3
0.03
1.41
0.30
0.32
0.83
0.33
0.32
0.11
0.11
6-Br
4-OCH3
> 10
4.73
H
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
7-CH3
6-CH3
6,7-(OCH3)2
7-F
> 10
4.60
7d
7e
7f
> 10
> 10
> 10
> 10
> 10
13.61
3.98
6-F
7g
7h
5-FUe
PAC-1
6-Cl
6-Br
1.92
0.34
0.46
0.46
3.92
0.10
dNot tested.
a
b
Calculated by ChemDraw 9.0 software.
The concentration (μM) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent the averaged results from
triplicate experiments with deviation of less than 10%. Data for 5a, 6c, 6h, 7c were reported previously [17].
c
e
Cell lines: SW620, colon cancer; PC3, prostate cancer; NCI-H23, lung cancer.
5-FU: 5-Fluorouracil, a positive control.
2899, 2818 (CH, CH2); 1690 (C]O); 166 (C]N); 1533 (C]C). 1H NMR
(500 MHz, DMSO‑d6, ppm): δ 11.55, 11.53 (2s, 0.2H, 0.8H, CONH);
8.42, 8.41 (2s, 0.2H, 0.8H, H2); 8.11, 8.09 (2s, 0.2H, 0.8H, N = CH);
7.93–7.90 (m, 2H, H5, H8); 7.77 (d, J = 8.0 Hz, 1H, H6); 7.56–7.51 (m,
2H, H2′, H6′); 6.77–6.74 (m, 2H, H3′, H5′); 5.20, 4.76 (2s, 1.6H, 0.4H,
NCH2CO); 2.99 (s, 6H, N(CH3)2). 13C NMR (125 MHz, DMSO‑d6, ppm): δ
167.8, 159.8, 152.0, 149.6, 147.3, 145.7, 135.1, 131.9, 130.1, 129.0,
128.7, 125.5, 123.2, 121.7, 112.3, 47.6. HR-MS (ESI) m/z 382.1069 [M-
H]−. Anal. Calcd. For C19H18ClN5O2 (383.1149): C, 59.45; H, 4.73; N,
18.25. Found: C, 59.53; H, 4.88; N, 18.14.
2.1.21. (E)-2-(6-Bromo-4-oxoquinazolin-3(4H)-yl)-N′-(4-
(dimethylamino)benzylidene)acetohydrazide (7h)
White solid; Yield: 66%. mp: 207–208 °C. Rf = 0.63 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3198 (NH); 3121 (OH); 2976, 2899,
2820 (CH, CH2); 1690 (C]O); 1672 (C]N); 1607, 1535 (C]C). 1H
NMR (500 MHz, DMSO‑d6, ppm): δ 11.56, 11.54 (2s, 0.2H, 0.8H,
CONH); 8.43, 8.42 (2s, 0.2H, 0.8H, H2); 8.25–8.08 (m, 1H, N = CH);
8.08–8.01 (m, 1H, H5); 7.93 (s, 1H, H8); 7.69 (d, J = 8.5 Hz, 1H, H6);
7.55–7.51 (m, 2H, H2′, H6′); 6.77–6.74 (m, 2H, H3′, H5′); 5.20, 4.76 (2s,
1.6H, 0.4H, NCH2CO); 2.98 (s, 6H, N(CH3)2). 13C NMR (125 MHz,
DMSO‑d6, ppm): δ 167.8, 162.9, 159.7, 152.1, 152.0, 149.8, 149.7,
148.6, 147.6, 145.7, 137.8, 130.2, 129.0, 128.7 128.6, 128.5, 123.5,
Fig. 3. Caspase-3 activation activity of some representative compounds. U937
human lymphoma cells cells were treated with representative compound
(50 µM) for 24 h. Cell lysate was used to detect caspase-3 activation by caspase-
3 assay kit. UN: untreated, VH: vehicle (DMSO. 0.05%).
2.1.20. (E)-2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N′-(4-
(dimethylamino)benzylidene)acetohydrazide (7g)
White solid; Yield: 68%. mp: 198–199 °C. Rf = 0.65 (DCM:
MeOH = 14: 1). IR (KBr, cm−1): 3196 (OH); 3125 (CH aren); 2978,
6

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BioorganicChemistry92(2019)103202
Fig. 4. Relative caspase-3 activation activity of some compounds in comparison to PAC-1. Compounds were tested at 50 µM.
121.6, 120.1, 120.0, 112.3, 48.1, 47.6. MS (ESI) m/z 427.9 [M + H]+
.
2.4. Cell cycle analysis
Anal. Calcd. For C19H18BrN5O2 (427.0644): C, 53.28; H, 4.24; N, 16.35.
Found: C, 53.37; H, 4.33; N, 16.28.
U937 human lymphoma cells (5 × 105/ml per well) were plated in
6-well culture plates and allowed to grow for 24 h. The cells were
treated with compounds (50 μM) for 24 h, and then harvested. The
harvested cells were washed twice with ice-cold PBS, fixed in 75% ice-
cold ethanol, and stained with propidium iodide (PI) in the presence of
RNase at room temperature for 30 min. The stained cells were analyzed
for DNA content using a FACScalibur flow cytometer (BD Biosciences,
San Jose, CA, USA) and the data were processed using Cell Quest Pro
software (BD Biosciences).
2.2. Cytotoxicity assay
The cytotoxicity of the synthesized compounds was evaluated
against three human cancer cell lines, including SW620 (colon cancer),
PC3 (prostate cancer), and NCI-H23 (lung cancer). The cell lines were
purchased from a Cancer Cell Bank at the Korea Research Institute of
Bioscience and Biotechnology (KRIBB). The media, sera and other re-
agents that were used for cell culture in this assay were obtained from
GIBCO Co. Ltd. (Grand Island, New York, USA). The cells were culture
in DMEM (Dulbecco’s Modified Eagle Medium) until confluence. The
cells were then trypsinized and suspended at 3 × 104 cells/mL of cell
culture medium. On day 0, each well of the 96-well plates was seeded
with 180 μl of cell suspension. The plates were then incubated in a 5%
CO2 incubator at 37 °C for 24 h. Compounds were initially dissolved in
dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations
by culture medium. Then 20 μl of each compounds’ samples, which
were prepared as described above, were added to 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 compounds was measured by the colori-
metric method, as described previously [18] with slight modifications
[20–23]. The IC50 values were calculated using a Probits method and
were averages of three independent determinations (SD ≤ 10%) [24].
2.5. Apoptosis assay
The Annexin V-FITC/PI dual staining assay was used to determine
the percentage of apoptotic cells. U937 cells (5 × 105/ml per well)
were plated in 6-well culture plates and allowed to grow for 24 h. The
cells were treated with compounds (50 μM) for 24 h, and then har-
vested. The harvested cells were washed twice with ice-cold PBS and
incubated in the dark at room temperature in 100 mL of 1 × binding
buffer containing 1 µl Annexin V-FITC and 12.5 mL PI. After 15 min
incubation, cells were analyzed for percentage undergoing apoptosis
using a FACScalibur flow cytometer (BD Biosciences). The data were
processed using Cell Quest Pro software (BD Biosciences).
2.6. Docking studies
2.3. Caspase-3 activation assay
Molecular Operating Environment version MOE 2009.10 [25] was
used to perform the docking studies of synthesized compounds in cat-
alytic domain site of caspase-6 (PDB entry 4FXO) [26]. The docking
procedures was similar to those reported recently [22,27], that include
removing co-crystal ligand, adding hydrogen atoms, parameter setting,
minimizing structure, and fixing charge. The London dG (default) was
used as first rescoring function with force field (MMFF94x) refinement,
and GBVI/WSA as a second one to estimate the negative binding free
energy profile of the complex (kCal/mol) [22]. It is emphasized that for
docking caspases, water molecules must be conserved as they are an
integral part of allosteric mechanisms [26]. The results of docking ex-
periments could be visualized using Chimera 1.13.1 [27].
Caspase activity was measured by using caspase 3 assay kit ac-
cording to the manufacturer’s instructions (abcam, MA, USA). U937
human lymphoma cells (5 × 105/ml per well) were plated in 6-well
culture plates and allowed to grow for 24 h. The cells were treated with
compounds (50 μM) for 24 h, and then harvested. The harvested cells
were washed twice with ice-cold PBS and treated with lysis buffer in-
cluded in the kit. Cell lysate (100 μg/50 μl) was mixed with 50 μl of 2x
reaction buffer and 5 μl of DEVD-p-NA substrate as the instruction of
caspase-3 assay kit (Abcam, cat. N. ab39401). Fluorescence was mea-
sured after one-hour incubation.
7

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Fig. 5. Cell cycle analysis of some compounds. U937 human lymphoma cells were treated with compounds (50 μM) for 24 h. The harvested cells were stained with
propidium iodide (PI) in the presence of RNase and then were analyzed for DNA content. UN: untreated, VH: vehicle (DMSO. 0.05%). Data was represented as
histograms (A) and bar graphs (B).
3. Results and discussions
straightforwardly based on analysis of spectroscopic data, including IR,
MS, 1H and 13C NMR. Theoretically, due to enamine tautomerization,
the quinazoline-4(3H)-ones (2a-j) could react with ethyl chloroacetate
to give either or both N3- and O-alkylated products. However, it has
been shown previously that when acetone was used as the reaction
solvent, the alkylation reaction gave exclusively N3-alkylated products
(3a-j) [17,28]. The formation of N3-alkylated products 3a-j was de-
monstrated clearly from 1H and 13C NMR spectroscopic data. In the 1H
NMR spectra of the final products (5–7), the singlet peaks attributable
for two methylene protons generally appeared at around
4.80–5.20 ppm, corresponding to the methylene protons of N3-alky-
lated compounds [17,28]. For the O-alkylated products, these methy-
3.1. Chemistry
The designed quinazolin-4(3H)-one-based acetohydrazides (5a-j,
6a-g, and 7a-h) were obtained via four step synthetic pathway, as de-
picted in scheme 1. The first step involved a Niementowski condensa-
tion of anthranilic acid (1a) or its substituted derivatives (1b-j) with an
excess of formamide at 120 °C to give the quinazoline-4(3H)-ones (2a-j)
in quantitative yield (89–97%). In the next step, the quinazoline-4(3H)-
ones (2a-j) were reacted with ethyl chloroacetate in acetone with a
catalytic amount of KI under basic conditions (K2CO3) to give the in-
termediate esters 3a-j. Further acyl nucleophilic substitution of the
esters 3a-j with hydrazine monohydrate in ethanol under refluxing
conditions afforded the hydrazides 4a-j at the third step. Finally, an
Aldol condensation of the hydrazids 4a-j with (2-hydroxy-4-methoxy)
benzaldehyde, 4-methoxybenzaldehyde or 4-dimethylaminobenzalde-
hyde furnished the desired products 5a-j, 6a-h, and 7a-h, respectively.
The target compounds 5a-j, 6a-h, and 7a-h were generally obtained in
good overall yields.
lene protons normally appear more downfield (5.6–5.7 ppm). In the 13
C
NMR spectra, one peak appeared at around 167–168 ppm was attribu-
table for C]O of the CONHN = functionality. Other peak appeared at
around 160–163 ppm was attributable for the carbon of the C4]O
functional group. For the O-alkylated products, the peak attributed for a
carbon of C4eO moiety normally appear more downfield than 169 ppm
[17,28]. Also, in the O-alkylated products, the carbon of the methylene
group was often more downfield, at around 73–77 ppm [17,28], while
in the present cases, the methylene carbons generally appeared around
The structures of the synthesized compounds were determined
8

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Fig. 6. Apoptosis (Annexin V/PI) analysis of some compounds. U937 cells were treated with compounds (50 μM) for 24 h. The harvested cells were incubated with
Annexin V-FITC and PI. UN: untreated, VH: vehicle (DMSO. 0.05%). Area 1 = PI positive population, Area 2: Annexin V-postivie population. Data was represented as
histograms (A) and bar graphs (B).
46 ppm, which was typical of the methylene carbon attached to N3.
compounds in series 5a-j. Although compound 5a, with no substituent
on the quinazolinone part, was found with only moderate cytotoxicity
(IC50, 6.10–9.85 μM), most other compounds with 6- or 7-substituted
quinazoline rings displayed potent cytotoxicity in all three human
cancer cell lines. Both electron releasing groups (such as 6- or 7-methyl
substituents) or electron withdrawing groups (e.g. 6- or 7-F, -Cl, -Br)
were found to enhance cytotoxicity. In general, electron withdrawing
groups seemed to be more favorable for cytoxicity (as seened with IC50
values of compounds 5e-h vs. IC50 values of compounds 5b and 5c).
Regarding the position of the substituents on the quinazoline ring,
substitution at position 6 appeared to be more favorable than sub-
stitution at position 7, but only in some cases (compound 5i vs. com-
pound 5j). The effects of substituent’s position was more profoundly
noted in series 6 and 7 (compounds 6c vs. 6b; compounds 7c vs. 7b). A
bulky substituent as 6,7-dimethoxy group substantially reduced the
compounds’s cytotoxicity. Compound 5j with 7-nitro substituent on the
quinazoline moiety was an exception. This compound did not show
significant cytotoxicity in all three cancer cell lines up to 10 μM.
In summary, among the three substituents on the phenyl ring of the
hydrazone moiety, the 2-OH-4-OCH3 group was found to be the best
substituent for cytotoxicity. This (2-OH-4-OCH3)-substituted phenyl, in
combination with the quinazolinones which were appropriated
3.2. Bioactivity
Three series of compounds, 5a-j, 6a-h, and 7a-h, were firstly
evaluated for their cytotoxicity in three human cancer cell lines, in-
cluding SW620 (human colon cancer); PC3 (human prostate cancer),
and NCI-H23 (human lung cancer). 5-Fluorouracil was used as a posi-
tive control. PAC-1 was also included in the assay. The results are
presented in Table 1.
As can be seen from Table 1, in series 6, no compound others than
compounds 6c and 6g, showed significant cytotoxicity in three cancer
cell lines tested, up to the concentration of 10 μM. Similarly, in series 7,
only two compounds 7a and 7c exhibited moderate cytotoxicity with
IC50 values in the range between 3.01 and 5.91 μM. Other compounds
in the series did not show significant cytotoxicity up to 10 μM against
any human cancer cell line assayed. These results indicate that the 4-
methoxy and 4-dimethylamino substituents on the phenyl moiety were
actually not potential for bioactivity. Only delicate combinations with
some appropriate subtituent on the quinazolinone part could afford
moderate cytotoxicity, as seen with compounds 6c, 6g, 7a and 7c.
Interestingly, more potent bioactivity was observed with
9

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Fig. 7. (A) 3D binding mode of PAC-1 (red molecule) in the allosteric site of caspase-6, and (B) main residues involved in the ligand-protein interactions. Protein is
shown as ribbon style, ligand shown as light blue ball and stick style.
arrested the cell cycle in G1 phase (Fig. 5). We next performed an
Annexin V-FITC/propidium iodide (PI) dual staining assay to confirm
whether series 5 can induce apoptosis. Phosphatidylserine locates on
the cytosolic (inner) side of the cell membrane, and translocates to the
extracellular (outer) surface during early apoptosis. Annexin V has a
high affinity for PS, and thus when fluorescently labelled with fluor-
escein isothiocyanate (FITC), can be used to identify early-stage apop-
totic cells. Propidium iodide (PI) is a fluorescent intercalating agent
that cannot cross the membrane of live cells. The membranes of cells in
the latter stages of apoptosis and dead cells are permeable to PI, and
their nuclei stain red. We treated U937 cancer cells with compounds at
50 µM for 24 h and stained the cells with Annexin V-FITC and PI.
Compounds 5b, 5c, and 5h significantly increased the percentages of
Annexin V-PI-double positive cells, namely latter stage of dead cells
(Fig. 6). Instead, Compounds 5e, 5f, 5g, and 5i significantly increased
the percentages of Annexin V-single positive cells, namely early stage of
dead cells (Fig. 6). These data demonstrate that compounds 5b, 5c, and
5h might be stronger apoptotic cell death inducers than compounds 5e,
5f, 5g, and 5i.
Fig. 8. (A) Molecular structure with atom numberings of docked complex be-
tween PAC-1 and Zinc ion (locker of caspase-6). (B) Tetragedral geometry of the
corresponding complex.
3.3. Docking studies
substituted by small, either electron releasing or electron withdrawing
groups at 6- or 7-positions, generally furnished compounds with potent
cytotoxicity in three human cancer cell lines (SW-620, PC-3, and NCI-
H23). These compounds were almost up to 10-fold more potent than 5-
FU and about 2–3 fold more potent than PAC-1. From these results we
selected 7 compounds, including 5b. 5c, and 5e-i for further evaluation
in a caspase-3 activation assay. The results illustrated in Figs. 3 and 4
show that compounds 5h and 5b were slightly more potent than PAC-1,
meanwhile compound 5f was equally potent as PAC-1. Three com-
pounds 5e, 5g and 5i were less potent than PAC-1 in caspase-3 acti-
vation. Very notheworthy, compound 5c was found to be approxi-
mately 1.6-fold more potent than PAC-1 in this assay. This compound
was the most potent caspase activator in the series.
As can be identified from the caspase activation assay, three com-
pounds (5b, 5c and 5h) were more potent than the reference PAC-1.
Their cytotoxicity was also impressive, especially 5h with IC50 of
0.98–1.3 μM in three cell lines, showing up to 3–5 times higher than
PAC-1. We therefore we decided to continue investigating the caspase
activation mechanism of these compound using molecular docking si-
mulation.
It is expected that our synthesized compounds, as derived from PAC-
1 structure, would activate procaspase-3 by chelating inhibitory zinc
ion through the key ortho-hydroxy-N-acylhydrazone functionality, thus
allowing procaspase-3 to autoactivate itself to caspase-3 [16]. Un-
fortunately, it is difficult to directly study this mechanism by docking
because up to date, there still lacks a zinc-bound crystal structure for
procaspase-3. Therefore, we performed the docking simulations with
another executioner caspase structurally homologous to caspase-3, that
is caspase-6 locked by Zn2+ whose crystal structure was reported by
Delgado and Hardy [26]. Although the results may not directly reflect
the catalytic mechanism of 5b, 5c and 5h in procaspase-3 activation,
SRB assay indicated that series 5, especially 5b, 5c, and 5h, might
induce strong cytotoxicity against cancer cells. We next used flow cy-
tometry to analyze the effects of series 5 on the cell cyles. We treated
U937 cancer cells with 50 μM of series 5 for 24 h and then analyzed
DNA content and observed that compounds 5b, 5c, and 5h significantly
10

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
Fig. 9. Metal complex and geometries between 5b, 5c, and 5h with Zinc (extracted from caspase-6 PDB structure ID:4FXO) showing the role of 2-hydroxyl group of
the ortho-hydroxy N-acyl hydrazone moiety in chelating zinc ion.
the docking study could be used, to some extent, to predict the struc-
ture-activity relationship related to the zinc binding mode of synthe-
sized compounds which would be useful for rational design and opti-
mization of novel acylhydrazone derivatives in future works.
three ligands, the highest possible affinity from a ligand towards the
metal, shown by the distance between chelating atoms to the metal and
the complexity of the geometry, can be observed in 5c, suggesting its
higher caspase activation ability compared to 5b and 5h. This result is
correlated with our experimental data given in Fig. 4. Last but not least,
in all cases, ortho-hydroxyl (O2) group consistently participated in the
chelation, reafirming once again the importance of this functionality in
sequesting zinc ion as well established in the literature [16,26].
As for so, all compounds were docked into the allosteric site of
caspase-6 for investigating the role of ortho-hydroxy N-acyl hydrazone
motif in chelating zinc ion, and the results were compared with those
obtained for PAC-1. As well analyzed, in the central cavity of this en-
zyme structure, there is a Zn2+ ion which exerts its inhibitory role by
establishing the tetrahedral geometry with Lys36, Glu244, His287 and
water [26,17,18]. The docking results, as shown in Fig. 7, clearly illu-
strated the chelating mode of PAC-1 to zinc. By penetrate in the allos-
teric pocket of caspase-6, PAC-1 was able to form a stable complex with
zinc ion, with similar coordination geometry as caspase did (Fig. 8). The
docking score was determined as −5.03 kCal/mol. It is highlighted the
role of ortho-hydroxyl group as well as acylhydrazine moiety for
forming metal chelation shown in this docking study which is in
agreement with those widely reported in literature [29–31].
4. Conclusions
In conclusion, we have reported three series of of novel (E)-N′-
benzylidene-4-oxoquinazolin-3(4H)-yl)acetohydrazides (5a-j, 6a-h,
and 7a-h), incorporating three different substituents, including 2-OH-4-
OCH3, 4-OCH3, and 4-N(CH3)2, respectively, on the phenyl ring of the
benzylidene moiety. On the quinazoline part different substituent were
introduced at positions 6 and 7. Biological evaluation showed that
series
(E)-N′-(2-hydroxy-4-methoxybenzylidene)-4-oxoquinazolin-
By applying the same docking procedure for tested compounds, si-
milar results of metal chelation as PAC-1 were observed for 5b,
meanwhile variations were observed for 5c and 5h (with a slight var-
iation). All compounds could pertubate the allosteric site in caspase 6
and formed stable tetrahedral coordination with Zn2+ and one water
molecules. As can be seen in Fig. 9, for compound 5c the carboxyl group
(O4) of 4-oxoquinazoline system played a role in chelating metal;
however for 5h exhibited a bidentate chelation with Zn2+ ion via ortho-
hydroxyl (O2) and amino (N4) groups of acylhydrazine moiety. Among
3(4H)-yl)acetohydrazides (5a-j) exhibited the most potent cytotoxicity
against three human cancer cell lines (SW620, colon; PC-3, prostate;
NCI-H23, lung), while in series 6a-h and 7a-h only some compounds
were moderately cytotoxic. The cytotoxic potency of compounds 5b, 5c,
5e-i was about 10-fold higher than 5-FU and 2- to 3-fold higher than
PAC-1. Small substituents at positions 6 or 7 on the quinazoline moiety,
either electron wihthdrawing or releasing, were found to enhance their
cytotoxicity. Most of the compounds in this series 5a-j, especially
compounds 5c, 5b and 5h, also significantly activated caspase-3
11

L.C. Huan, et al.
BioorganicChemistry92(2019)103202
activity. Of these, compound 5c displayed 1.61-fold more potent than
PAC-1 as caspase-3 activator. Cell cycle and apoptotic analyses showed
that 5b, 5c, and 5h significantly arrested the cell cycle in G1 phase and
these compounds act as as strong apoptotic cell death inducers. The
docking simulation studies showed that these compounds could acti-
vate procaspase via chelating Zn2+ ion bound to the allosteric site of the
zymogen. From this study we have demonstrated the 2-hydroxy-4-
methoxybenzylidene moiety in acetohydrazides appeared to be favor-
able scaffold for further development of caspase activators and antic-
ancer agents.
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We acknowledge the principal financial supports from the National
Foundation for Science and Technology of Vietnam (NAFOSTED, Grant
number 104.01-2018.01). The work was also partly supported by a
grant funded by the Korean Government (NRF, Grant number
2017R1A5A2015541).
[17] L.C. Huan, C.V. Phuong, L.C. Truc, V.N. Thanh, H. Pham-The, L.-T.-T. Huong,
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Declaration of Competing Interest
The authors report no conflict of interest.
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Further reading
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