N′-[(E)-Arylidene]-2-(2,3-dihydro-3-oxo-4H-1,4-benzoxazin-4-yl)-acetohydrazides: Synthesis and Evaluation of Caspase Activation Activity and Cytotoxicity

Article abstract of DOI:10.1002/cbdv.201800322

In our search for novel small cytotoxic molecules potentially activating procaspase-3, we have designed and synthesized a series of novel N′-[(E)-arylidene]-2-(2,3-dihydro-3-oxo-4H-1,4-benzoxazin-4-yl)acetohydrazides (5, 6). Biological evaluation revealed that seven compounds, including 5h, 5j, 5k, 5l, 5n, 6a, and 6b, exhibited moderate to strong cytotoxicity against three human cancer cell lines (SW620, colon cancer; PC-3, prostate cancer; NCI-H23, lung cancer). Among these compounds, two most cytotoxic compounds (5h and 5j) displayed from 3- up to 10-fold higher potency than PAC-1 and 5-FU in three cancer cell lines tested. Three compounds 5j, 5k, and 5n were also found to display better caspases activation activity in comparison to PAC-1. Especially, compound 5k activated the level of caspases activity by 200% higher than that of PAC-1. From this study, three compounds 5j, 5k, and 5n could be considered as potential leads for further design and development of caspase activators and anticancer agents.

Full text of DOI:10.1002/cbdv.201800322

Article Type: Full Paper
(E)-N'-Arylidene-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-
yl)acetohydrazides: Synthesis and Evaluation of Caspase
Activation Activity and Cytotoxicity
Le Cong Huan,a Le Cong Truc,a Cao Viet Phuong,a Pham-The Hai,a Le-Thi-Thu Huong,b Nguyen Tran
Phuong Linh,a Nguyen Thi Thuan,a Eun Jae Park,c YeoJin Choi,c Jong Soon Kang,d Sang-Bae Han,*,c
Nguyen-Hai Nam,*,a Phuong-Thao Tran,*,a
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam
bSchool of Medicine and Pharmacy, Vietnam National University, 144 XuanThuy, Hanoi, VietNam
cCollege of Pharmacy, Chungbuk National University, 194-31, Osongsaengmyung-1, Heungdeok, Cheongju, Chungbuk, 28160, Republic
of Korea
dBio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungbuk, Republic of Korea
*Corresponding authors:
Tel.: +84-4-39330531;
Fax: +84-4-39332332;
Emails: namnh@hup.edu.vn (N.H. Nam); thaotp119@gmail.com (T.P. Thao); shan@chungbuk.ac.kr (S.B. Han)
In our search for novel small cytotoxic molecules potentially activating procaspase-3, we have designed and synthesized a
series of novel (E)-N'-arylidene-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazides (5, 6). Biological
evaluation revealed that seven compounds, including 5h, 5j, 5k, 5l, 5n and 6a-b, exhibited moderate to strong cytotoxicity
against three human cancer cell lines (SW620 (colorectal adenocarcinoma), PC3 (prostate adenocarcinoma), and NCI-H23
(adenocarcinoma, non-small cell lung cancer)). Of these, two most cytotoxic compounds (5h and 5j) displayed from 3- up
to 10-fold higher potency than PAC-1 and 5-FU in three cancer cell lines tested. Three compounds 5j, 5k, and 5n were also
found to display better caspases activation activity in comparison to PAC-1. Especially, compound 5k activated the level of
caspases activity by 200% higher than that of PAC-1. From this study, three compounds 5j, 5k, and 5n could be considered
as potential leads for further design and development of caspase activators and anticancer agents.
Keywords: Acetohydrazides • benzo[b][1,4]oxazine • cytotoxicity • caspase activation
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Introduction
Dysregulation of apoptosis is one of the primary causes leading to the development and progression of cancer [1].
Therefore, targeting apoptosis has become an attractive approach for the development of new anticancer agents [2]. A
diversity of compounds, such as tenovin-1 (a p53 disruptors) [3], GDC-0152 (an inhibitor of XIAP) [4], or ABT-199 (an
inhibitor of Bcl-2) [5] have been demonstrated to act directly on different proteins involved in the apoptotic pathway, thus
inducing apoptosis and leading to the death of cancer cells.
Caspases are a family of cysteine proteases enzymes which play very important role in the initiation and execution
of apoptosis [6]. This enzyme family has many different members (caspases 1-14). Among these, caspase-3 is considered
as one of key enzymes important for the apoptotic process [6]. In the cell, caspase-3 exists as a low-activity zymogen,
procaspase-3, which has been found to be overexpressed in various human cancers, including colon cancer, lung cancer,
melanoma, hepatoma, breast cancer, lymphoma, and neuroblastoma [6-12]. Activation of procaspase-3 by appropriate
small molecules has been demonstrated to restore apoptosis in cancer cells and this approach proves to be more
advantageous over the direct apoptosis induction by the above mentioned compounds [6, 13].
PAC-1 (Figure 1) is one of the first procaspase activating compounds reported recently [6, 14]. This caspase activator
showed very promising in vivo antitumor activity profile in several xenografted models [6, 14]. Detailed investigation into
structure-activity relationships of PAC-1 and their derivatives revealed that the acylhydrazone functionality (B-region,
Figure 1) plays very important role for its procaspase activating and anticancer activity, due to the strong complex
formation of this acylhydrazone moiety with zinc ion [15].
2H-Benzo[b][1,4]oxazin-3(4H)-one is an important scaffold commonly employed in the design of diverse biological
compounds, especially anticancer agents [16, 17]. Based on the biological importance of 2H-benzo[b][1,4]oxazin-3(4H)-
one, as well as the pharmacophoric structure of PAC-1 and related acylhydrazone procaspase activators, we have
designed a novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one-incorporating acylhydrazones (Figure 1). In these newly
designed compounds, the 4-benzylpiperazin-1-yl part of PAC-1 (region A) was replaced by the 2H-benzo[b][1,4]oxazin-
3(4H)-one moiety. The phenyl ring of PAC-1 (region C) was replaced by different substituted phenyl or isatins. The
acylhydrazone moiety (region B) linking two regions A and C were kept unchanged due to its important role in interaction
with zinc ion of procaspase-3. This paper describes the results from synthesis and biological evaluation of the designed
2H-benzo[b][1,4]oxazin-3(4H)-one-incorporating
benzo[b][1,4]oxazin-4-yl)acetohydrazides).
acylhydrazones
((E)-N'-arylidene-2-(3-oxo-2,3-dihydro-4H-
Figure 1. Structure of PAC-1 and rational design of novel (E)-N'-arylidene-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-
yl)acetohydrazides
Results and Discussion
Chemistry
The target acetohydrazides incorporating 2H-benzo[b][1,4]oxazin-3(4H)-one (5a-n, 6a-d) were synthesized using a four-
step pathway in moderate overall yields (Scheme 1). The key intermediate 4 was prepared from 2-aminophenol (1) via
three steps. In the first step, 2-aminophenol was refluxed with an excess amount of chloroacetyl chloride in a mixture of
isobutylmethyl ketone/water to afford 1,4-benzoxazine-3(4H)-one (2)in a quantitative yield (93%). In this reaction, a small
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amount of water was added to quench the excess of chloroacetyl chloride. The second step was a nucleophilic substitution
of the benzoxazine 2 with ethyl chloroacetate in acetone under basic conditions (K2CO3) with a catalytic amount of KI to
give the ester 3. Further acyl nucleophilic substitution of the ester 3 with hydrazine gave acetohydrazide 4 in a
quantitative yield. Finally, a condensation reaction of the intermediate 4 with different benzaldehydes or isatin derivatives
to form the target compounds 5a-n and 6a-d, respectively.
O
O
NH2
OC2H5
NH
N
OH
O
O
O
ClCH2COOC2H5
Chloroacetyl chloride
K2CO3, KI, acetone
isobutylmethyl ketone:H2O
NaHCO3, 0 oC-rfx
1
3
2
O
N
H
N
N
R
R
Ar-CHO
O
O
O
EtOH, rfx
NHNH2
N
H2N-NH2.H2O
EtOH, rfx
5a-n
O
O
O
O
O
R
H
N
O
N
4
H
N
N
N
H
EtOH, rfx
O
O
6a-d
Scheme 1.Synthesis of acetohydrazides incorporating benzizoxazole moiety (5a-n, 6a-d)
The structures of the synthesized compounds were determined based on the elemental analysis and analysis of
spectroscopic data, including IR, MS, 1H and 13C NMR. In the 1H and 13C NMR spectra, the peaks attributing to the
methylene group on the 2H-benzo[b][1,4]oxazin-3(4H)-one heterocycle and acetohydrazide moiety appeared as doubles.
These doubles were resulted from anti-E and syn-E isomers of the acetohydrazide functional group, as has been discussed
thoroughly previously [18]. All MS, 1H and 13C NMR spectra can be found in the Supporting Information.
Bioactivity
Next, the synthesized compounds were evaluated for their cytotoxicity assay using SRB method as described previously
[19] with slight modifications [18]. We firstly evaluated the inhibitory effects of the synthesized compounds on the growth
of three human cancer cell lines, including SW620 (colorectal adenocarcinoma), PC3 (prostate adenocarcinoma), and NCI-
H23 (adenocarcinoma, non-small cell lung cancer), in the initial screening. In this initial screening, the compounds were
evaluated at the concentrations of 30 μg/mL. The growth percentages (CGP) of the SW620, PC-3 and NCI-H23 cells in the
presence of respective compounds at these concentrations were measured. The results are shown in Table 1 and Figure 2.
The results were averages of triplicate experiments. 5-Fluorouracil was used as a positive control. As can be seen from
Table 1, compounds 5a-f with either no substituent or halogen substituents at either position 2 or 4 on the phenyl ring
exhibited insignificant inhibitory effects on the growth of SW620, PC-3 and NCI-H23 cells (CGP, 60.85-79.99%), even at
the concentrations of 30 μg/mL. Compounds 5g and 5i with 4-methoxy or 4-hydroxy substituents on the phenyl moiety
also insignificantly inhibited the growth of three cancer cell lines tested (CGP, 66.23-77.74%). Interestingly, when the
hydroxy group was substituted at position 2, the resulting compound 5h completely inhibited the growth of all three
cancer cell lines (Table 1). The addition of an extra hydroxy substituent at either position 3 or 5 retained strong cell growth
inhibition effects, as manifested by the cell growth percentages of three cancer cell lines under the presence of
compounds 5j and 5l at 30 μg/mL, respectively (Table 1). It was, however, the additional hydroxy or methoxy substituent
at position 4 significantly decreased the cell inhibitory effects of the resulting compounds 5k and 5n. Meanwhile, the
addition of the two more hydroxy groups at both position 3 and 4 practically led to the loss of cytotoxicity of the resulting
compound 5m. Thus, it seemed that, for compounds of series 5a-n, only compound with 2-OH substituent (5h) and
compounds with 2-OH substituents plus either -OH or -OCH3 substituents at position 3, 4 or 5 (5j-l, 5n) were favorable for
cytotoxicity.
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Regarding the isatin-incorporating compounds 6a-d, only compounds 6a and 6b exhibited significant cancer cell
growth inhibition (Table 1). Compound 6c (with 5’-CH3 substituent) and compound 6d (with 5’-OCH3 substituent) did not
affect the growth of cancer cells significantly. Though the number of compounds investigated was still limited, it would be
suggested that substitution of electron-releasing groups like -CH3 or -OCH3 at position 5 was not favorable, while electron-
withdrawing groups like –Cl at this position seemed to be more tolerable for cytotoxicity.
Table 1. Effects of the compounds on the growth of some human cancer cells
Cell growth percentage (CGP, %)1/Cell lines2
Compound
R
SW620
PC3
NCI-H23
70.70 ± 3.91
68.77 ± 4.64
63.49 ± 4.12
79.99 ± 1.42
75.62 ± 4.89
76.61 ± 2.87
66.19 ± 3.23
-16.00 ± 1.75
75.75 ± 3.19
-10.69 ± 5.87
13.30 ± 2.83
-17.11 ± 1.13
75.67 ± 3.73
13.93 ± 3.28
21.78 ± 4.93
14.88 ± 2.71
75.23 ± 4.63
73.94 ± 4.88
23.22 ± 2.35
75.48 ± 0.13
60.85 ± 4.93
70.88 ± 1.81
75.98 ± 3.42
61.92 ± 3.89
78.96 ± 7.29
63.63 ± 3.61
-15.32 ± 6.77
66.23 ± 4.61
-8.64 ± 0.79
13.51 ± 6.91
-13.66 ± 5.43
70.59 ± 2.38
11.35 ± 6.07
25.29 ± 2.92
23.79 ± 6.51
73.48 ± 2.59
76.36 ± 3.93
68.43 ± 6.06
71.46 ± 5.87
77.74 ± 7.06
77.65 ± 5.54
64.31 ± 5.69
78.21 ± 4.27
70.09 ± 5.10
-12.65 ± 5.47
77.74 ± 4.24
-17.20 ± 7.28
5.50 ± 2.90
-20.73 ± 4.45
77.29 ± 4.04
9.64 ± 6.78
16.52 ± 2.86
19.25 ± 1.11
77.85 ± 3.93
75.94 ± 3.50
5a
5b
H
2'-Cl
5c
5d
5e
5f
3'-Cl
4'-Cl
4'-F
4'-Br
5g
5h
5i
4'-OCH3
2'-OH
4'-OH
5j
2',3'-(OH)2
2',4'-(OH)2
5k
5l
5m
5n
6a
6b
6c
2',5'-(OH)2
2',3',4'-(OH)3
2'-OH-4'-OCH3
H
5'-Cl
5'-CH3
6d
5'-OCH3
5-FU3
17.35 ± 9.01
25.45 ± 1.21
1Cell growth percentage, compounds were assayed at 30 μg/mL. Data represent the averages of triplicate experiments; 2Cell lines:
3
SW620, colorectal adenocarcinoma; PC3, prostate adenocarcinoma; NCI-H23, adenocarcinoma, non-small cell lung cancer; 5-
FU: 5-Fluorouracil, a positive control.
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Figure 2. Effects of the compounds at 30 µg/mL on the growth of three human cancer cell lines. Cell lines: SW620, colorectal
adenocarcinoma; PC3, prostate adenocarcinoma; NCI-H23, adenocarcinoma, non-small cell lung cancer
From the initial screening, seven compounds including 5h, j, k, l, n and 6a-b were further evaluated at 5 different
concentrations to get the IC50 values using the same assay protocols. 5-FU and PAC-1 were used as positive controls. The
results are presented in Table 2. Two compounds 6a and 6b showed only moderate cytotoxicity against three human
cancer cell lines with IC50 values ranging from 14.84 to 31.92 μM. These values for 5-FU and PAC-1 were 8.84-13.61 μM and
4.16-5.82 μM, respectively. Compounds 5k and 5n displayed about similar cytotoxic potency compared to 5-FU, while
compound 5l was approximately twice more potent than 5-FU and slightly more potent than PAC-1 against three human
cancer cell lines tested (Table 2). Two compounds 5h and 5j were found to be the most cytotoxic with similar potency. The
IC50 values of these two compounds for three cancer cell lines were between 1.38-1.67 μM. Against SW620 cells,
compounds 5h and 5j were found to be 5-fold more potent than 5-FU and nearly 4-fold more potent than PAC-1. Against
PC-3 and NCI-H23 cells, these two compounds were found to be approximately 10-fold more potent than 5-FU and nearly
3-fold more potent than PAC-1.
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Table 2. Cytotoxicity of selected compounds against some human cancer cell lines
4'
5'R
O
3'
O
3
O
3
6'
H
H
2'
2'
1'
N
N
1'
7'
3R'
4'
2
N4
2
N
N
4
N
N
H
1O
O
1
O
O
6'
5
6
5
6
5'
6a-d
5a-n
8
8
7
7
Cytotoxicity (IC50,2 μM)/Cell lines3
Compound
R
MW
LogP1
SW620
PC3
NCI-H23
5h
5j
2'-OH
325.32
341.32
341.32
341.32
355.35
350.33
384.77
130.08
392.49
0.67
0.19
0.19
0.19
0.50
0.32
0.97
0.81
3.43
1.66±0.18
1.64±0.09
9.96±0.85
4.86±0.18
1.38±0.12
1.67±0.26
6.39±0.01
3.07±0.64
10.55±0.98
14.84±1.20
25.68±1.95
13.61±0.46
4.16±0.52
1.41±0.34
1.58±0.03
7.30±0.70
2.96±0.09
11.82±2.08
18.50±0.09
31.92±0.46
13.45±3.92
5.32±0.21
2',3'-(OH)2
2',4'-(OH)2
2',5'-(OH)2
5k
5l
5n
6a
6b
2'-OH-4'-OCH3
11.30±1.29
20.61±4.34
26.95±3.80
8.84±1.92
5.82±0.20
H
5'-Cl
5-FU4
PAC-14
1Calculated by EPI 320 software; 2The concentration (μM) of compounds that produces a 50% reduction in enzyme activity or cell growth, the
3
numbers represent the averages from triplicate experiments; Cell lines: SW620, colorectal adenocarcinoma; PC3, prostate adenocarcinoma;
NCI-H23, adenocarcinoma, non-small cell lung cancer; 45-FU: 5-Fluorouracil, PAC-1 (Figure 1), positive controls.
Next, to probe the ability of the compounds to activate caspases activity, the compounds in series 5 and 6 were
preliminarily evaluated in the caspases activation assay using U937 cells. The compounds (at the final concentrations of 50
µM) were incubated with U937 cells (5 × 105 cells/well) for 24 h. The cells were then harvested and lysed. The cell lysates
(200 μg/100 μL/well) were mixed with Ac-DEVD-pNA (a substrate of caspases 3, 6 and 7). The OD values from each well
were then measured at 405 nm every 30 min for 12 h. The slope of the linear portion for each well was determined as the
enzyme activity and presented in Figure 3(A). In this assay, PAC-1 was used as a positive control. It was very interesting to
find that compounds 5a-g, 5i, 5m, 6c-d, which were not cytotoxic in the cytotoxic assay against three human cancer cell
lines, showed no or only minimal caspases activation activity. Two compounds 6a and 6b, which were only moderately
cytotoxic with IC50 values ranging from 14.84 to 31.92 μM, were also found to be not active in the caspases activation
assay. Noteworthy, however, five compounds including 5k, 5j, 5n, 5l, and 5h caused significant elevation in caspases
activity. Especially, compound 5k activated caspases activity by more than 200% in comparison to that of PAC-1 at a time
point of 720 min (Figure 3(B)). The caspases activation activities of compound 5j and 5n were approximately 174 and 118%
higher than that of PAC-1, respectively. Compound 5l was slight less potent than PAC-1 in term of caspases activation.
Compound 5h also caused activation ofcaspases activity, but its potency was just about 40% of PAC-1’s potency. Thus, the
five most cytotoxic compounds, including 5k, 5j, 5n, 5l, and 5h, were the compounds which caused activation in the
caspases activity in U937 cells.
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In this study, however, the potency of the compounds’s caspases activation was not finely correlated with their
cytotoxicity. Compound 5h and 5j were similarly cytotoxic in all three human cancer cell-lines; however, the caspases
activation activity of compound 5h was much lower than that of compound 5j. Compound 5k was the most potent in term
of caspases activator, however, it was approximately 3- to 7-fold weaker than compounds 5l and 5n in term of cytotoxicity.
Thus, more indepth studies need to be carried out to fully understand the cytotoxic mechanisms of these compounds.
Nevertheless, from this investigation, three compounds 5j, 5k, and 5n could be considered as potential leads for further
design and development of caspases activators and anticancer agents.
Figure 3. (A) Caspases inducing activity of the compounds 5a-n and 6a-d. Compounds were tested at 50 µM. (B) Relative caspases
activation activity of some compounds in comparison to PAC-1 (at a time point of 720 min). Compounds were tested at 50 µM. Caspases
activation activity of PAC-1 is considered as 100.
Conclusions
In this study we have designed and synthesized
a series of 18 novel (E)-N'-arylidene-2-(3-oxo-2,3-dihydro-4H-
benzo[b][1,4]oxazin-4-yl)acetohydrazides (5, 6). Biological evaluation revealed that seven compounds, including 5h, 5j, 5k,
5l, 5n and 6a-b, exhibited moderate to strong cytotoxicity against three human cancer cell lines (SW620 (colorectal
adenocarcinoma), PC3 (prostate adenocarcinoma), and NCI-H23 (adenocarcinoma, non-small cell lung cancer)). Two
compounds 5h and 5j were found to be the most cytotoxic with the potency from 3- up to 10-fold higher than that of PAC-
1 and 5-FU in three cancer cell lines tested. Three compounds 5j, 5k, and 5n were also found to display better caspases
activation activity in comparison to PAC-1. Especially, compound 5k activated the level of caspases activity by 200%
higher than that of PAC-1. From this study, three compounds 5j, 5k, and 5n could be considered as potential leads for
further design and development of caspases activators and anticancer agents.
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Experimental Section
Chemistry
Thin layer chromatography which was performed using Whatman® 250 μm Silica Gel GF Uniplates and visualized under
UV light at 254 and 365 nm, was used to check the progress of reactions and preliminary evaluation of compounds’
homogeneity. In all cases, the compounds achieved purity of 97% or above, as determined by HPLC. 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
ionization (ESI), were recorded using PE Biosystems API2000 (Perkin Elmer, Palo Alto, CA, USA) and Mariner® (Azco
Biotech, Inc.Oceanside, CA, USA) mass spectrometers, respectively. 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.
The synthesis of novel (E)-N'-arylidene-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazides (5a-n,
6a-d) was carried out as illustrated in Scheme 1. Details are described below.
General procedures for the synthesis of compounds 5 and 6
To a solution of the 2-aminophenol (1) (1.0 mmol) in isobutyl methyl ketone (0.6 mL), was added NaHCO3 (200 mg, 2.39
mmol) and water (0.6 mL) and the resulting mixture was cooled in an ice-bath. Chloroacetyl chloride (130 mg, 1.15 mmol)
was added dropwise with stirring and the cold mixture was set aside to become ambient; it was then refluxed for 4 hours
and cooled to give the 1,4-benzoxazine-3(4H)-one (2) in 93% yields.
To the solution of 1,4-benzoxazine-3(4H)-one (2) (0.224g, 1.5mmol) in acetone (10 mL) was added K2CO3 (345 mg,
2.5 mmol). The resulting mixture was stirred at 60 °C for 30 minutes, then KI (166 mg, 1 mmol) was added. After stirring
for further 15 minutes, 0.50 mL of ethyl chloroacetate (4.5 mmol) was dropped slowly into the reaction mixture. The
reaction mixture was again stirred at 60 °C for 3 hours. When the reaction completed, the solvent was evaporated. The
residue was dispersed in 10 mL water, extracted 3 times with dichloromethane. The combined organic layer was then
evaporated to obtain the intermediate ethyl 2-(3-oxo-2,3-dihydro-4H- benzo[b][1,4]oxazin-4-yl)acetate (3), which was a
viscous yellow liquid.
To the solution of ethyl 2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetate (3) (1.0 mmol) in ethanol (10 mL)
was slowly added 3.0 ml of hydrazine monohydrate (3.0 mmol). The mixture was stirred at 0°C until the starting material
was completely consumed. The white precipitates formed were filtered and washed with cold-ethanol (3 times). The off-
white solid product (4, 2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide) was collected, dried under
vacuum and used for the next step without further purification.
The acetohydrazide (4) (0.7 mmol) was dissolved in ethanol (20 mL), 2 drops of concentrated acetic acid, followed by
benzaldehyde or isatin derivatives (1.0 mmol) were added. The mixture was refluxed until the reaction completed. The
precipitates formed was filtered and washed with ethanol (3 times). The off-white solid products were collected, dried
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under vacuum and re-crystalised in ethanol or colum chromatography (MeOH : DCM) to obtain the desired products (5a-n,
6a-d).
(E)-N'-Benzylidene-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5a)
White solid; Yield: 43%. mp: 177.3-178.5 oC. Rf = 0.68 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3442 (NH); 3203 (N=C-H
1
aromatic); 3091, 2949 (CH, arene); 2854 (CH2); 1701, 1668, 1504 (C=O, C=N); 1618 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.75, 11.73 (~25%, 75%) (s, 1H, CONH); 8.24, 8.06 (~25%, 75%) (s, 1H, N=CH); 7.75, 7.71 (~75%, 25%) (dd, J = 8.0 Hz,
2.0 Hz, 2H, H2’,H6’); 7.47-7.43 (m, 3H, H3’, H4’, H5’); 7.06-7.00 (m, 4H, H5, H6, H7, H8); 5.09, 4.68 (~75%, 25%) (s, 2H, NCH2CO);
4.73, 4.72 (~25%, 75%) (s, 2H, OCH2CO). 13C -NMR (125 MHz, DMSO-d6, ppm): δ 168.4, 165.0 (CONH); 165.1, 163.8 (C3=O);
147.7, 144.5 (N=CH); 145.1 (C8-C-O); 134.5 (C1 ); 130.6, 130.5 (C4 ); 129.6, 129.5 (C5=C-N); 129.3 (C3’, C5’), 127.6, 127.5 (C2’,
C6’), 124.2, 124.0 (C7), 123.2 (C6), 117.0 (C5), 116.0, 115.9 (C8), 67.5 (C1), 43.4, 42.8 (NCH2CO). MS (ESI) m/z 309.9 [M+H]+.
Anal. Calcd. For C17H15N3O3 (309.1113): C, 66.01; H, 4.89; N, 13.58. Found: C, 66.05; H, 4.92; N, 13.61.
(E)-N'-(2-Chlorobenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5b)
White solid; Yield: 32%. mp: 178.7-180.5 oC. Rf = 0.69 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3564 (NH); 3269 (N=C-H
1
aromatic); 3115, 2991 (CH, arene); 2829 (CH2); 1687, 1504 (C=O, C=N); 1608 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ
11.98, 11.91 (~25%, 75%) (s, 1H, CONH); 8.63, 8.44 (~25%, 75%) (s, 1H, N=CH); 8.09, 7.95 (~75%, 25%) (dd, J1 = 7.5 Hz, J2 =
2.0 Hz, 1H, H6’); 7.56 (dd, J1 = 9.0 Hz, J2 = 1.0 Hz, 1H, H3’); 7.48-7.42 (m, 2H, H4’, H5’); 7.03-7.07 (m, 4H, H5, H6, H7, H8); 5.11,
4.69 (~75%, 25%) (s, 2H, NCH2CO); 4.73, 4.72 (~25%, 75%) (s, 2H, OCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 168.5,
165.0 (CONH); 165.1, 163.9 (C3=O), 145.0 (C8=C-O), 140.6 (N=CH), 133.6, 133.1 (C1′); 132.0, 131.9 (C2′); 130.4 (C4′); 129.5,
129.4 (C5=C-N); 128.1, 128.0 (C3’), 127.5, 127.3 (C5′, C6′); 124.1, 123.9 (C7), 123.2 (C6), 117.0 (C5), 115.9 (C8), 67.4 (C1); 43.4,
42.8 (NCH2CO). MS (ESI) m/z 343.9 [M+H]+. Anal. Calcd. For C17H14ClN3O3 (343.0724): C, 59.40; H, 4.10; N, 12.22. Found: C,
59.37; H, 4.12; N, 12.25.
(E)-N'-(3-Chlorobenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5c)
White solid; Yield: 48%. mp: 179.7-181.5 oC. Rf = 0.69 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3456 (NH); 3184 (N=C-H
1
aromatic); 3066, 2949 (CH, arene); 2850 (CH2); 1699, 1668, 1504 (C=O, C=N); 1595 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.89, 11.84 (~25%, 75%) (s, 1H, CONH); 8.22, 8.04 (~25%, 75%) (s, 1H, N=CH); 7.86, 7.77 (~75%, 25%) (s, 1H, H2’);
7.71-7.68 (m, 1H, H6’); 7.50-7.48 (m, 2H, H4’, H5’); 7.03-7.02 (m, 4H, H5, H6, H7, H8); 5.11, 4.69 (~75%, 25%) (s, 2H, OCH2CO);
4.73 (s, 2H, NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 168.6, 165.1 (CONH); 166.5, 165.0 (C3=O); 145.0 (C8=C-O);
145.9, 142.9 (N=CH); 136.8, 136.7 (C1′); 134.1, 134.0 (C3′); 131.2, 131.1 (C4′); 130.2, 130.1 (C5′); 129.5, 129,4 (C5=C-N); 126.9,
126.4 (C2′), 126.5, 126.1 (C6′), 124.1, 123.96 (C7); 123.1 (C6), 117.0, 116.9 (C5); 115.97, 115.90 (C8); 67.5 (OCH2CO); 43.3, 42.9
(NCH2CO). MS (ESI) m/z 343.9 [M+H]+. Anal. Calcd. For C17H14ClN3O3 (343.0724): C, 59.40; H, 4.10; N, 12.22. Found: C,
59.43; H, 4.08; N, 12.18.
(E)-N'-(4-Chlorobenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5d)
White solid; Yield: 61%. mp: 177.8-179.3 oC. Rf = 0.69 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3427 (NH); 3107, 2949 (CH,
1
arene); 2846 (CH2); 1699, 1666, 1506 (C=O, C=N); 1554 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.82, 11.80 (~25%,
75%) (s, 1H, CONH); 8.24, 8.06 (~25%, 75%) (s, 1H, N=CH); 7.80, 7.75 (~75%, 25%) (d, J = 8.5 Hz, 2H, H2’,H6’); 7.61, 7.52
(~25%, 75%) (d, J = 8.5 Hz, 2H, H3’,H5’); 7.05-7.01 (m, 4H, H5, H6, H7, H8); 5.09, 4.68 (~75%, 25%) (s, 2H, OCH2CO); 4.73, 4.72
(~75%, 25%) (s, 2H, NCH2CO).13C-NMR (125 MHz, DMSO-d6, ppm): δ 168.4, 165.1 (CONH); 165.1, 163.9 (C3=O); 145.0 (C8=C-
O); 146.2, 143.2 (N=CH), 135.0, 134.9 (C4′); 133.5, 133.4 (C1 ); 130.5, 129.4 (C5=C-N); 129.6, 129.5 (C2′,C6′); 129.4, 129.3 (C3′);
129.2, 129.1 (C5′); 124.1, 123.9 (C7); 123.1 (C6); 117.0,116.9 (C5); 115.9, 115.8 (C8); 67.5 (OCH2CO); 43.7, 42.8 (NCH2CO). MS
(ESI) m/z 343.9 [M+H]+. Anal. Calcd. For C17H14ClN3O3 (343.0724): C, 59.40; H, 4.10; N, 12.22. Found: C, 59.42; H, 4.13; N,
12.26.
(E)-N'-(4-Florobenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5e)
White solid; Yield: 45%. mp: 180.1-181.6 oC. Rf = 0.67 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3471 (NH); 3190 (N=C-H
1
aromatic); 3080, 2949 (CH, arene); 2850 (CH2); 1699, 1668, 1506 (C=O, C=N); 1606 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.76, 11.74 (~25%, 75%) (s, 1H, CONH); 8.25, 8.06 (~25%, 75%) (s, 1H, N=CH); 7.84, 7.79 (~75%, 25%) (dd, J1= 9.0
Hz, J2= 5.0 Hz, 2H, H2’, H6’); 7.30 (t, J= 9.0 Hz, 2H, H3’, H5’); 7.06-7.01 (m, 4H, H5, H6, H7, H8); 5.09, 4.68 (~75%, 25%) (s, 2H,
OCH2CO); 4.73, 4.72 (~25%, 75%) (s, 2H, NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 168.3 (CONH); 165.1, 165.0
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(C3=O); 162.6 (C4′); 145.1 (C8=C-O); 146.5, 143.3 (N=CH); 133.1, 133.0 (C5=C-N); 129.8, 129.7 (C1′); 129.6, 129.5 (C2′,C6′); 124.1,
123.9 (C7); 123.1 (C6); 117.0, 116.9 (C5); 116.4, 116.2 (C3′,C5′); 115.95, 115.90 (C8); 67.4 (OCH2CO); 43.3, 42.8 (NCH2CO). MS
(ESI) m/z 327.9 [M+H]+. Anal. Calcd. For C17H14FN3O3 (327.1019): C, 62.38; H, 4.31; N, 12.84. Found: C, 62.41; H, 4.34; N,
12.87.
(E)-N'-(4-Bromobenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5f)
White solid; Yield: 33%. mp: 181.0-182.0oC. Rf = 0.65 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3448 (NH); 3346 (N=C-H
1
aromatic); 3082, 2949 (CH, arene); 2852 (CH2); 1701, 1666, 1504 (C=O, C=N); 1612 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.82, 11.80 (~25%, 75%) (s, 1H, CONH); 8.22, 8.03 (~25%, 75%) (s, 1H, N=CH); 7.75, 7.73 (~25%, 75%) (d, J= 8.5 Hz,
2H, H2’, H6’); 7.67 (d, J= 7.5 Hz, 2H, H3’, H5’); 7.05-7.03 (m, 4H, H5, H6, H7, H8); 5.09, 4.68 (~75%, 25%) (s, 2H, OCH2CO); 4.73,
4.72 (~25%, 75%) (s, 2H, NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 168.4 (CONH); 165.1, 165.0 (C3=O); 145.1 (C8=C-
O); 146.4, 143.3 (N=CH); 133.7 (C1′); 132.5, 132.2 (C3′,C5′); 130.7 (C5=C-N); 129.5, 129.4, 129.3 (C2′,C6′); 124.1, 123.9 (C7); 123.8,
123.7 (C4′); 123.1 (C6); 117.0, 116.9 (C5); 116.0, 115.9 (C8); 67.4 (OCH2CO); 43.3, 42.8 (NCH2CO). MS (ESI) m/z 389.8 [M+H]+.
Anal. Calcd. For C17H14BrN3O3 (for Br 81 isotope, 389.0198): C, 52.60; H, 3.63; N, 10.82. Found: C, 52.71; H, 3.72; N, 10.76.
(E)-N'-(4-Methoxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5g)
White solid; Yield: 39%. mp: 185.1-186.3 oC. Rf = 0.68 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3427 (NH); 3186 (N=C-H
1
aromatic); 3078, 2951 (CH, arene); 2839 (CH2); 1701, 1666, 1504 (C=O, C=N); 1608 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.60 (s, 1H, CONH); 8.18, 8.00 (~25%, 75%) (s, 1H, N=CH); 7.70, 7.66 (~75%, 25%) (d, J= 8.5 Hz, 2H, H2’, H6’); 7.05-
7.01 (m, 6H, H5, H6, H7, H8, H3’, H5’); 5.06, 4.66 (~75%, 25%) (s, 2H, OCH2CO); 4.72 (s, 2H, NCH2CO); 3.81 (s, 3H, 4′-OCH3).
13C-NMR (125 MHz, DMSO-d6, ppm): δ 168.0 (CONH); 165.0, 164.9 (C3=O); 161.3, 161.2 (C4′); 147.5, 145.1 (C8=C-O); 144.4
(N=CH); 129.6, 129.5 (C5=C-N); 129.0, 129.0 (C2′,C6′); 127.0 (C1′); 124.1, 123.9 (C7); 123.1 (C6); 117.0, 116.9 (C5); 115.9, 115.8
(C8); 114.8 (C3′,C5′); 67.4 (OCH2CO); 55.7 (4′-OCH3); 43.3, 42.8 (NCH2CO). MS (ESI) m/z 339.9 [M+H]+. Anal. Calcd. For
C18H17N3O4 (339.1219): C, 63.71; H, 5.05; N, 12.38. Found: C, 63.74; H, 5.02; N, 12.41.
(E)-N'-(2-Hydroxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5h)
White solid; Yield: 36%. mp: 175.2-177.0 oC. Rf = 0.56 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3487 (NH); 3431 (OH); 3078,
2949 (CH, arene); 2854 (CH2); 1669, 1666, 1506 (C=O, C=N); 1608 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.95, 11.66
(~43%, 57%) (s, 1H, CONH); 10.95, 10.06 (~43%, 57%) (s, 1H, 2’-OH); 8.46, 8.36 (~43%, 57%) (s, 1H, N=CH); 7.78, 7.57 (~57%,
43%) (dd, J1= 8.0 Hz, J2= 1.5 Hz, 1H, H6’); 7.31-7.24 (m, 1H, H4’); 7.08-7.03 (m, 4H, H5, H6, H7, H8); 6.93-6.86 (m, 2H, H3’, H5’);
5.06, 4.69 (~57%, 43%) (s, 2H, OCH2CO); 4.73, 4.72 (~43%, 57%) (s, 2H, NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ
168.0 (CONH), 165.0 (C3=O); 157.7, 156.8 (C2′); 147.7 (N=CH); 145.0 (C8=C-O); 131.9, 131.7 (C4′); 129.5, 129.4 (C5=C-N); 126.8
(C6′); 124.2, 123.9 (C7); 123.2 (C6); 120.6 (C5′); 119.9, 119.8 (C1′); 117.0, 116.8 (C3′); 116.9, 116.6 (C5); 116.0, 115.9 (C8); 67.4
(OCH2CO); 43.2, 42.7 (NCH2CO). MS (ESI) m/z 325.9 [M+H]+. Anal. Calcd. For C17H15N3O4 (325.1063): C, 62.76; H, 4.65; N,
12.92. Found: C, 62.79; H, 4.61; N, 12.95.
(E)-N'-(4-Hydroxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5i)
White solid; Yield: 65%. mp: 176.7-178.5 oC. Rf = 0.58 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3454 (NH); 3414 (OH); 2987 (CH,
1
arene); 2831 (CH2); 1685, 1658, 1504 (C=O, C=N); 1606 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.53 (s, 1H, CONH);
9.93, 9.92 (~25%, 75%) (s, 1H, 4′-OH); 8.13, 7.95 (~25%, 75%) (s, 1H, N=CH); 7.57, 7.53 (~75%, 25%) (d, J= 8.5 Hz, 2H, H2’,
H6’); 7.05-6.99 (m, 4H, H5, H6, H7, H8); 6.84 (d, J= 8.5 Hz, 2H, H3’, H5’); 5.04, 4.65 (~75%, 25%) (s, 2H, OCH2CO); 4.72, 4.71
(~25%, 75%) (s, 2H, NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 167.9, 164.9 (CONH); 165.0, 163.3 (C3=O); 159.9, 159.8
(C ); 145.0 (C =C-O); 144.8 (N=CH); 129.6, 129.5 (C =C-N); 129.3, 129.2 (C , C ); 125.5 (C ); 123.9 (C ); 123.1 (C ); 117.0,
′
1
4′
8
5
2’
6’
7
6
116.9 (C5); 116.1 (C3’, C5’); 115.9, 115.8 (C8); 67.4 (OCH2CO); 43.3, 42.7 (NCH2CO). MS (ESI) m/z 325.9 [M+H]+. Anal. Calcd.
For C17H15N3O4 (325.1063): C, 62.76; H, 4.65; N, 12.92. Found: C, 62.77; H, 4.63; N, 12.90.
(E)-N'-(2,3-Dihydroxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5j)
White solid; Yield: 42%. mp: 172.0-173.4 oC. Rf = 0.51 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3496 (NH); 3182 (N=C-H
1
aromatic); 3076, 2941 (CH, arene); 2850 (CH2); 1695, 1660, 1502 (C=O, C=N); 1610 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.96, 11.66 (~44%, 56%) (s, 1H, CONH); 10.71, 9.57 (~56%, 44%) (s, 1H, 2′-OH); 9.25, 9.17 (~56%, 44%) (s, 1H, 3′-
OH); 8.42, 8.36 (~44%, 56%) (s, 1H, N=CH); 7.22, 6.99 (d, J= 7.0 Hz, 1H, H6’); 7.22-6.98 (m, 4H, H5, H6, H7, H8); 6.87-6.84 (m,
1H, H4’); 6.84-6.68 (m, 1H, H5’); 5.05, 4.70 (~ 56%, 44%) (s, 2H, OCH2CO); 4.74, 4.72 (~44%, 56%) (s, 2H, NCH2CO). 13C-NMR
(125 MHz, DMSO-d6, ppm): δ 167.9 (CONH); 165.0 (C3=O), 163.6 (C2’), 148.6 (C3’); 146.1, 146.0 (C8=C-O); 142.6 (N=CH);
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129.6, 129.5 (C5=C-N); 123.9, 123.1 (C7); 124.2 (C6′), 123.1 (C6); 121.1 (C5′); 120.2, 119.6 (C1’); 119.7, 119.2 (C4’); 117.9, 117.2
(C5); 116.0; 115.9 (C8); 67.4 (OCH2CO); 43.3, 42.7 (NCH2CO). MS (ESI) m/z 341.9 [M+H]+. Anal. Calcd. For C17H15N3O5
(341.1012): C, 59.82; H, 4.43; N, 12.31. Found: C, 59.79; H, 4.45; N, 12.34.
(E)-N'-(2,4-Dihydroxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5k)
White solid; Yield: 47%. mp: 171.2-173.6 oC. Rf = 0.52 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3425 (NH); 3184 (OH); 3115
1
(N=C-H aromatic); 2953 (CH, arene); 2845 (CH2); 1687, 1660, 1502 (C=O, C=N); 1631 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.76, 11.47 (~49%, 51%) (s, 1H, CONH); 11.12, 9.99 (~49%, 51%) (s, 1H, 2′-OH); 9.96, 9.82 (~49%, 51%) (s, 1H, 4′-
OH); 8.32, 8.23 (~49%, 51%) (s, 1H, N=CH); 7.56, 7.34 (~49%, 51%) (d, J= 8.5 Hz, 1H, H6’); 7.06-7.00 (m, 4H, H5, H6, H7, H8);
6.36-6.33 (m, 2H, H3’, H5’); 5.01, 4.67 (~49%, 51%) (s, 2H, OCH2CO); 4.73, 4.71 (~51%, 49%) (s, 2H, NCH2CO). 13C-NMR (125
MHz, DMSO-d , ppm): δ 167.5 (CONH); 165.01 (C =O); 163.2 (C ); 161.2, 161.0 (C ); 145.1, 145.0 (C =C-O); 143.0 (N=CH);
′
4
6
3
2’
8
131.5 (C6’); 129.5, 129.4 (C5=C-N); 124.1, 123.9 (C7); 123.2 (C6); 117.0, 116.9 (C5); 115.97, 115.91 (C8); 110.8 (C1’); 108.3, 108.2
(C5’); 103.1, 102.8 (C3’); 67.4 (OCH2CO); 43.2, 42.7 (NCH2CO). MS (ESI) m/z 341.9 [M+H]+. Anal. Calcd. For C17H15N3O5
(341.1012): C, 59.82; H, 4.43; N, 12.31. Found: C, 59.85; H, 4.46; N, 12.29.
(E)-N'-(2,5-Dihydroxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5l)
White solid; Yield: 63%. mp: 172.4-173.8 oC. Rf = 0.47 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3606 (OH); 3415 (NH); 3194
1
(N=C-H aromatic); 3011, 2949 (CH, arene); 2856 (CH2); 1687, 1653, 1502 (C=O, C=N); 1585 (C=C). H-NMR (500 MHz,
DMSO-d6, ppm): δ 11.83, 11.62 (~43%, 57%) (s, 1H, CONH); 10.08, 9.37 (~43%, 57%) (s, 1H, 2′-OH); 8.97, 8.90 (~43%, 57%) (s,
1H, 5′-OH); 8.39, 8.30 (~43%, 57%) (s, 1H, N=CH); 7.17-6.99 (m, 5H, H , H , H , H , H ); 6.76 - 6.70 (m, 2H, H , H ); 5.06,
′
6
5
6
7
8
3'
4'
4.68 (~ 57%, 43%) (s, 2H, OCH2CO); 4.73, 4.72 (~43%, 57%) (s, 2H, NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 167.9
(CONH); 165.1, 165.0 (C =O); 150.5, 150.4 (C ); 150.0, 149.9 (C ); 145.1, 145.0 (C =C-O); 142.0 (N=CH); 129.5, 129.4 (C =C-
′
3
2’
5
8
5
N); 124.1, 123.9 (C7); 123.2 (C6); 120.8 (C3’); 119.5, 119.3 (C4’); 117.5 (C6’); 117.0, 116.9 (C5); 116.0, 115.9 (C8); 113.7, 111.6 (C1’);
67.4 (OCH2CO); 43.3, 42.6 (NCH2CO). MS (ESI) m/z 341.9 [M+H]+. Anal. Calcd. For C17H15N3O5 (341.1012): C, 59.82; H, 4.43;
N, 12.31. Found: C, 59.80; H, 4.44; N, 12.30.
(E)-N'-(2,3,4-Trihydroxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5m)
White solid; Yield: 74%. mp: 171.8-172.6 oC. Rf = 0.45 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3417 (NH); 3101, 2981 (CH,
1
arene); 2845 (CH2); 1670, 1626, 1504 (C=O, C=N); 1607 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.80, 11.51 (~58%,
42%) (s, 1H, CONH); 11.10, 9.57 (~58%, 42%) (s, 1H, 2′-OH); 9.48, 9.38 (~58%, 42%) (s, 1H, 4′-OH); 8.56, 8.49 (~42%, 58%)
(s, 1H, 3′-OH); 8.27, 8.21 (~58%, 42%) (s, 1H, N=CH); 7.09-7.00 (m, 4H, H5, H6, H7, H8); 6.81, 6.40 (~42%, 58%) (d, J= 8.5 Hz,
1H, H6’); 6.39 (dd, J1 = 8.5 Hz, J2 = 2.0 Hz,1H, H5’); 5.02, 4.68 (~42%, 58%) (s, 2H, OCH2CO); 4.73, 4.72 (~58%, 42%) (s, 2H,
NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ 167.4 (CONH); 165.0 (C3=O); 149.9, 149.2 (C3’); 148.8, 147.8 (C2’); 145.0
(C8=C-O); 144.3 (N=CH); 133.2, 133.1 (C4’); 129.5, 129.4 (C5=C-N); 124.1, 123.9 (C7); 123.1, 121.5 (C6); 118.5 (C6’); 117.0, 116.9
(C5); 115.9, 115.3 (C8); 112.7, 111.1 (C1’); 108.2, 108.1 (C5’); 67.4 (OCH2CO); 43.1, 42.6 (NCH2CO). MS (ESI) m/z 357.9 [M+H]+.
Anal. Calcd. For C17H15N3O6 (357.0961): C, 57.14; H, 4.23; N, 11.76. Found: C, 57.11; H, 4.25; N, 11.79.
(E)-N'-(2-Hydroxy-4-methoxybenzylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (5n)
White solid; Yield: 83%. mp: 176.3-177.9 oC. Rf = 0.59 (DCM : MeOH = 14 : 1). IR (KBr, cm-1): 3458 (NH); 3186 (N=C-H
1
aromatic); 3080, 2945 (CH, arene); 2843 (CH2); 1699, 1664, 1504 (C=O, C=N); 1570 (C=C). H-NMR (500 MHz, DMSO-d6,
ppm): δ 11.84, 11.54 (~46%, 54%) (s, 1H, CONH); 11.28, 10.17 (~46%, 54%) (s, 1H, 2’-OH); 8.36, 8.26 (~46%, 54%) (s, 1H,
N=CH); 7.66, 7.46 (~46%, 54%) (d, J= 8.5 Hz, 1H, H6’); 7.08-7.01 (m, 4H, H5, H6, H7, H8); 6.54-6.51 (m, 2H, H3’,H5’); 5.03, 4.68
(~46%, 54%) (s, 2H, OCH2CO); 4.73, 4.72 (~54%, 46%) (s, 2H, NCH2CO); 3.77, 3.76 (~54%, 46%) (s, 4’-OCH3). 13C-NMR (125
MHz, DMSO-d6, ppm): δ 167.6 (CONH), 165.0 (C3=O); 163.4, 159.7 (C4’); 162.6, 158.4 (C2′); 145.1 (C8=C-O); 142.5 (N=CH);
131.3 (C6’); 129.5, 129.4 (C5=C-N); 124.1, 123.9 (C7); 123.2, 123.1 (C6); 117.0, 116.9 (C5); 116.0, 115.9 (C8), 112.1 (C1’); 106.9,
106.8 (C5’); 101.6, 101.3 (C3’); 67.4 (OCH2CO); 55.7, 55.6 (OCH3); 43.2, 42.7 (NCH2CO). MS (ESI) m/z 355.9 [M+H]+. Anal.
Calcd. For C18H17N3O5 (355.1168): C, 60.84; H, 4.82; N, 11.83. Found: C, 60.80; H, 4.79; N, 11.86.
(E)-2-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-N'-(3-oxoindolin-2-ylidene)acetohydrazide (6a)
Light yellow solid; Yield: 57%. mp: 179.7-181.5 oC. Rf = 0.67 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3460 (NH); 3234 (N=C-H
1
aromatic); 2985 (CH, arene); 2951 (CH2); 1703, 1668, 1504 (C=O, C=N); 1622 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ
13.22, 12.69 (~25%, 75%) ( (s, 1H, CONH); 11.30 (s, 1H, NH-isatin); 7.60 (s, 1H, H4’); 7.40 (t, J = 7.5 Hz, 1H, H6’); 7.14- 7.02 (m,
5H, H5’, H5, H6, H7, H8); 6.97 (d, J = 7.5 Hz, 1H, H3’); 5.23, 4.92 (~75%, 25%) (s, 2H, OCH2CO); 4.89, 4.75 (~25%, 75%) (s, 2H,
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NCH2CO). 13C-NMR (125 MHz, DMSO-d6, ppm): δ165.2 (CONH); 162.9 (C3=O); 145.1 (C8=C-O); 143.0 (C7’-C-NH); 132.3 (N-C-
C5, C6’, C2’); 124.1 (C7), 123.2 (C5’); 123.1 (C6); 121.3 (C4’); 120.1 (C5); 117.0 (O=C3’-C-C4’); 116.0 (C8); 111.6 (C7’); 67.4 (OCH2CO);
42.5 (NCH2CO). MS (ESI) m/z 350.9 [M+H]+. Anal. Calcd. For C18H14N4O4 (350.3282): C, 61.71; H, 4.03; N, 15.99. Found: C,
61.74; H, 4.06; N, 15.96.
(E)-N'-(5-Chloro-3-oxoindolin-2-ylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (6b)
Light yellow solid; Yield: 39%. mp: 180.2-181.9 oC. Rf = 0.70 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3458 (NH); 3280 (N=C-H
1
aromatic); 3051 (CH, arene); 2854 (CH2); 1747, 1726, 1504 (C=O, C=N); 1602 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ
11.80 (s, 1H, CONH); 10.97 (s, 1H, NH-isatin); 8.38 (s, 1H, H6’); 7.46 (d, J = 8.5 Hz, 1H, H3’); 7.10-7.02 (m, 4H, H5, H6, H7, H8);
6.93 (d, J = 8.5 Hz, 1H, H4’); 5.13 (s, 2H, OCH2CO); 4.75 (s, 2H, NCH2CO). 13C NMR (125 MHz, DMSO-d6, ppm): δ 165.2
(CONH); 164.7 (C3=O); 145.0 (C8=C-O); 143.0 (C7’-C-NH); 132.5 (C2’, N-C-C5, C6’); 129.3 (C5’); 126.3 (C4’); 124.1 (C7); 123.1 (C5,
C6); 117.0 116.7 (C7’); 115.9 (C8); 112.4 (C3’-C-C4’); 67.4 (O-CH2-CO); 43.3 (NCH2CO). MS (ESI) m/z 384.8 [M+H]+. Anal. Calcd.
For C18H13ClN4O4 (384.7732): C, 56.19; H, 3.41; N, 14.56. Found: C, 56.15; H, 3.44; N, 14.58.
(E)-N'-(5-Methyl-3-oxoindolin-2-ylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (6c)
Light yellow solid; Yield: 44%. mp: 178.6-179.3 oC. Rf = 0.68 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3244 (N=C-H aromatic);
1
3149, 2983 (CH, arene); 2949 (CH2); 1705, 1668, 1504 (C=O, C=N); 1622 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.51
(s, 1H, CONH), 10.73 (s, 1H, NH-isatin); 8.06 (s, 1H, H4’); 7.22 (d, J = 8.0 Hz, 1H, H6’); 7.09-7.04 (m, 4H, H5, H6, H7, H8); 6.82 (d,
J = 8.0 Hz, 1H, H3’); 5.13 (s, 2H, OCH2CO); 4.76 (s, 2H, NCH2CO); 2.31 (s, 3H, 5′-CH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ
165.2 (CONH); 164.9 (C3=O); 145.0 (C8=C-O); 142.0 (C7’-C-NH); 133.5 (C2’, N-C-C5); 131.2 (C6’) ; 129.4 (C5’); 127.2 (C4’); 124.1
(C7); 123.1 (C5); 117.0 (C6); 116.7 (C7’); 115.9, 115.7 (C8); 110.8 (C3’-C-C4’); 67.4 (O-CH2-CO); 43.3 (NCH2CO); 20.9 (5′-CH3). MS
(ESI) m/z 365.0 [M+H]+. Anal. Calcd. For C19H16N4O4 (364.3547): C, 62.63; H, 4.43; N, 15.38. Found: C, 62.60; H, 4.45; N,
15.41.
(E)-N'-(5-Methoxy-3-oxoindolin-2-ylidene)-2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetohydrazide (6d)
Light yellow solid; Yield: 53%. mp: 180.1-181.7 oC. Rf = 0.72 (DCM : MeOH = 9 : 1). IR (KBr, cm-1): 3277 (N=C-H aromatic);
3136, 3051 (CH, arene); 2858 (CH2); 1660, 1533 (C=O, C=N); 1606 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 12.71 (s, 1H,
CONH), 11.12 (s, 1H, NH-isatin); 6.85-7.21 (m, 8H, H5, H6, H7, H8, H3′, H4′, H5′); 5.25 (s, 2H, OCH2CO); 4.75 (s, 2H, NCH2CO);
3.77 (s, 3H, 5′-OCH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 165.2 (CONH), 163.1 (C3=O), 155.9 (C6’), 145.1 (C8=C-O), 136.7
(C1’, C2’, N-C-C5), 123,2 (C5, C4’); 120.8 (C7), 118.5 (C7’-C-NH), 117.0 (C6), 116.0 (C8), 112.5 (C5’), 106.4 (C7’); 67.4 (OCH2CO);
56.1 (5′-OCH3). MS (ESI) m/z 381.0 [M+H]+. Anal. Calcd. For C18H13N4O5 (380.3541): C, 60.00; H, 4.24; N, 14.73. Found: C,
60.12; H, 4.13; N, 14.62.
Cytotoxicity assay
The synthesized compounds were evaluated for their cytotoxicity against three human cancer cell lines, including SW620
(colorectal adenocarcinoma), PC3 (prostate adenocarcinoma), and NCI-H23 (lung cancer). The cell lines were provide by a
Cancer Cell Bank at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). All media, sera and other
reagents that were used for cell culture in this assay were obtained from GIBCO Co. Ltd. (Grand Island, New York, USA).
Initially, the cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) to reach confluence. Then, the cells were
harvested by trypsinization using Tris base and suspended at 3 × 104 cells/mL using the same cell culture medium. On day
0, each well of the 96-well plates was seeded with 180 μL of cell suspension prepared above. The cells were then
incubated in a 5% CO2 incubator at 37 oC for 24 h. Stock solution of the tested compounds were prepared by dissolving in
dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations using the cell 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 colorimetric method, as described previously [19] with slight
modifications [18]. The IC50 values were calculated using a Probits method [20] and were averages of triplicate
experiments (SD ≤ 10%).
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Caspase-3 activation assay
U937 cells (5 × 105 cells/well) were plated in 6-well plate, and allowed to be stabilized overnight. The cells were treated
with PAC-1 or compounds (50 μM in 0.1% dimethyl sulfoxide). After 24 h incubation, cells were harvested and washed
twice with PBS. The cells were lysed by 50 μL of chilled cell lysis buffer for 10 min at 4 °C. Cell lysate (200 μg/100 μL/well)
were mixed with Ac-DEVD-pNA (200 μM). ODs at 405 nm were measured every 30 min for 12 h. The slope of the linear
portion for each well was determined as the enzyme activity.
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number.
Acknowledgements
We acknowledge the principal financial supports from the National Foundation for Science and Technology of Vietnam
(NAFOSTED, Grant number 104.01-2017.06). The work was also partly supported by a grant funded by the Korean
Government (NRF, Grant number 2017R1A5A2015541).
Author Contribution Statement
N-H.N, P-TT and S-B.H proposed the work. N-H.N, P-T.H, and L-T-T.H provided rational design. LCH, LCT, CVP, NTPL,
and NTT mainly developed the synthesis studies. S-B.H, EJP, YJC, and JSK performed the bioactivity testing assays. All
authors read and approved the final manuscript.
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