New Acetohydrazides Incorporating 2-Oxoindoline and 4-Oxoquinazoline: Synthesis and Evaluation of Cytotoxicity and Caspase Activation Activity

Article abstract of DOI:10.1002/cbdv.201900670

In our search for new small molecules activating procaspase-3, we have designed and synthesized a series of new acetohydrazides incorporating both 2-oxoindoline and 4-oxoquinazoline scaffolds. Biological evaluation showed that a number of these acetohydrazides were comparably or even more cytotoxic against three human cancer cell lines (SW620, colon cancer; PC-3, prostate cancer; NCI?H23, lung cancer) in comparison to PAC-1, a first procaspase-3 activating compound, which was used as a positive control. One of those new compounds, 2-(6-chloro-4-oxoquinazolin-3(4H)-yl)-N′-[(3Z)-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]acetohydrazide activated the caspase-3 activity in U937 human lymphoma cells by 5-fold higher than the untreated control. Three of the new compounds significantly induced necrosis and apoptosis in U937 cells.

Full text of DOI:10.1002/cbdv.201900670

Title: New Acetohydrazides Incorporating 2-Oxoindoline and 4-
Oxoquinazoline: Synthesis and Evaluation of Cytotoxicity and
Caspase Activation Activity
Authors: Nguyen-Hai Nam, Le Cong Huan, Duong Tien Anh, Bui Xuan
Truong, Phan Huy Duc, Pham-The Hai, Lai Duc-Anh, Le-Thi-
Thu Huong, Eun Jae Park, Hye Jin Lee, Jong Soon Kang,
Phuong-Thao Tran, Dao Thi Kim Oanh, Sang-Bae Han, and
Dinh Thi Thanh Hai
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201900670
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900670

10.1002/cbdv.201900670
Chemistry & Biodiversity
Chem. Biodiversity
New Acetohydrazides Incorporating 2-Oxoindoline and 4-
Oxoquinazoline: Synthesis and Evaluation of Cytotoxicity and
Caspase Activation Activity
Le Cong Huan,a Duong Tien Anh,a Bui Xuan Truong,a Phan Huy Duc,a Pham-The Hai,a Lai Duc-Anh,a
Le-Thi-Thu Huong,b Eun Jae Park,c Hye Jin Lee,c Jong Soon Kang,d Phuong-Thao Tran,a Dinh Thi
Thanh Hai,a Dao Thi Kim Oanh,*,a Sang-Bae Han,*,c Nguyen-Hai Nam*,a
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, 10000, Vietnam
bSchool of Medicine and Pharmacy, Hanoi National University, 144 Xuan Thuy, Hanoi, 10000, 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, 28116, Republic of Korea
*Corresponding authors: Tel.: +84-4-39330531; Fax: +84-4-39332332; Emails: namnh@hup.edu.vn (N.H. Nam).
In our search for new small molecules activating procaspase-3, we have designed and synthesized a series of new acetohydrazides
incorporating both 2-oxoindoline and 4-oxoquinazoline scaffolds (5a-t). Biological evaluation showed that
a number of these
acetohydrazides were comparably or even more cytotoxic against three human cancer cell lines (SW620, colon cancer; PC-3, prostate
cancer; NCI-H23, lung cancer) in comparison to PAC-1, a first procaspase-3 activating compound, which was used as a positive control.
Compound 5k activated the caspase-3 activity in U937 human lymphoma cells by 5-fold higher than the untreated control. Three
compounds, including 5k, 5n and 5q, significantly induced necrosis and apoptosis in U937 cells.
Keywords: Acetohydrazides • quinazolin-4(3H)-one• cytotoxicity • caspase activation
Introduction
Defects in the apoptotic pathway often lead to dysregulation of apoptosis, the primary cause of malignancy initiation and cancer
development.[1] Various chemotherapeutic agents currently used in clinical settings exert their anticancer activity by blocking the action of
antiapoptotic proteins or apoptotic regulators.[1] Today, targeting apoptosis signalling pathway continues to be one of the attractive
approaches for the development of new anticancer agents.[2]
There are several ways currently employed by medicinal chemists to restore apoptosis in cancer cells. One common approach is to directly
target apoptotic proteins by small molecules.[1] Hitherto, diverse small molecules, such as tenovin-1 (an inhibitor of p53 protein), GDC-
0152 (an inhibitor of XIAP protein), or ABT-199 (an inhibitor of Bcl-2), have been developed and demonstrated to act directly on different
proteins involved in the apoptotic pathway, thereby inducing apoptosis and leading to the death of cancer cells.[3-5]
The second effective approach to induce apoptosis is through caspases pathway. Caspases are a class of cysteine-aspartic proteases
enzymes which play very important role in the initiation and execution of apoptosis.[6] This enzyme family has fourteen members
(caspases 1-14), but three caspases, including caspase-3, caspase-7, and caspase-9, are considered as the most important enzymes for the
apoptotic process.[6] In particular, caspase-3, which, in the cells, exists as a low-activity zymogen, procaspase-3, plays key role in both
intrinsic and extrinsic apoptosis pathways.[7] Procaspase-3 has been found to be overexpressed in various human cancers, including lung
cancer, melanoma, colon cancer, breast cancer, hepatoma, lymphoma, and neuroblastoma.[8-13] Activation of procaspase-3 has been
demonstrated to effectively induce apoptosis in cancer cells. [8,13]
PAC-1 (Figure 1) is one of the first procaspase activating compounds developed by Hergenrother’s.[8,14] This compound showed very
promising in vivo antitumor activity profile in several human xenografted models.[8,14] It has been granted orphan drug status for the
1
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10.1002/cbdv.201900670
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treatment of glioblastoma multiforme, a deadly brain cancer, by the U.S. Food and Drug Administration. Currently, it is undergoing phase
1 clinical trials for brain cancer.[14] Structurally PAC-1 comprises of three distinct regions, including regions A, B, and C (Figure 1). Of these,
the acetohydrazide functionality (region B) plays important role for its procaspase activating activity due to its strong complexation with
zinc ion located at the active binding site of the enzyme.[15]
Based on the pharmacophoric feature of PAC-1 we have designed a new series of acetohydrazides (Figure 1). In the designed compounds,
the 4-benzylpiperazin-1-yl part of PAC-1 (A-region) was replaced by the quinazoline heterocyclic system, meanwhile the phenyl ring of
PAC-1 (C-region) was replaced by different substituted 2-oxoindolines. Both quinazolines and 2-oxoindolines are common biological
scaffolds found in diverse bioactive compounds, especially anticancer agents.[16,17] The acylhydrazide moiety (B-region) serving as a linker
between A- and C-regions was kept unchanged. This paper describes the results from the synthesis, biological evaluation and docking
studies of the designed compounds.
Figure 1.Structure of PAC-1 and rational design of new (Z)-N'-(2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazides.
Results and Discussion
Chemistry
The new (Z)-N'-(2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazides (5a-t) were synthesized as illustrated in Scheme 1.
The synthetic pathway consisted of 4 steps. The target compounds 5a-t were obtained in moderate to good yields. The first intermediates
(4-oxoquinazolines, 2a-e) were formed by employing a Niementowski condensation of anthranilic acid (1a) or 5-substituted-2-
aminobenzoic acid (1b-e) and formamide. The condensation was carried out at 120 oC. The second step involved a reaction of the
intermediates 2a-e with ethyl chloroacetate. This was a nucleophilic substitution reaction and best occurred in acetone as a solvent and
K2CO3 was used as a base.[18, 19] A small amount of KI was used to facilitate the reaction. The intermediate esters 3a-e were obtained in
very good yields.
The third step in the synthetic pathway was an acyl nucleophilic substitution of the intermediate esters 3a-e with hydrazine monohydrate.
The reaction was carried out in refluxing ethanol to afford the corresponding hydrazides 4a-ein quantitative yields. In the last step, the
Aldol condensation of the newly resulting hydrazides 4a-e with different isatins gave the target compounds 5a-t in good yields.
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1
1
All target compounds synthesized were structurally confirmed by spectroscopic evidences, including IR, MS, H and 13C NMR. All H NMR
spectra of compounds 5a-t showed typical peaks around 4.81-5.22 ppm, which were attributable for two methylene protons.[18, 19] The
methylene carbons, meanwhile, appeared around 44-46 ppm in the 13C NMR spectra. Also in the 13C NMR spectra of compounds 5a-t,
there were typical peaks appeared around 158-161 ppm, attributable for the carbon of the C4=O functional group in the 4-oxoquinazoline
ring. Additional peaks appeared more downfield, around 167-169 ppm, were typical for the carbon of the CONHN= functionality. The
carbonyl carbons on the 2-oxoindoline ring typically appeared around 162-165 ppm.
Scheme 1.Synthesis of acetohydrazides incorporating a quinazoline moiety (5)
Bioactivity
Cytotoxicity
The synthesized compounds were evaluated for their cytotoxicity against three human cancer cell lines, including SW620 (colon cancer),
PC3 (prostate cancer) and NCI-H23 (lung cancer), using SRB protocol. 5-FU (5-fluorouracil), PAC-1 and ADR (Adriamycin) were used as
positive controls. The results are summarized in Table 1.
Because numerous substituents could be substituted at different positions on the 4-oxoquinazoline system, and similarly, a number of
substituents could be substituted at different positions on the 2-oxoindoline part. So, we decided to firstly design 8 compounds 5a-h with
R being H and R’ being diverse substituents, including both electron-withdrawing (EW) and electron-releasing (ER) groups (-F, -Cl, -Br, -
NO2, CH3, -OCH3) at positions 5 and 7 of the 2-oxoindoline moiety. Further, we designed 5i-k, 5l-n, 5o-q, and 5r-t bearing 6-Cl, 6-Br, 6-I
and 6-CH3 substituents, respectively, on the 4-oxoquinazoline ring to investigate the substituents’s electron effects. With each substituent
on the 4-oxoquinazoline part, three compounds bearing no substituent or 5’-Cl (EW group) or 5’-CH3 (ER group) on the 2-oxoindoline
moiety were synthesized.
Among eight compounds 5a-h, it was found that compound 5e with R’ being 5-CH3 group showed the best cytotoxicity with the lowest
IC50 values of 11.12-15.25 M in three human cancer cell lines tested. Among compounds 5i-k, 5l-n, and 5r-t, which were designed with 6-
Cl, 6-Br, and 6-CH3 substituents, respectively, on the 4-oxoquinazoline ring, it was very interesting to find that only three compounds,
including 5k, 5n, and 5t, which were substituted with the 5’-CH3 group on the 2-oxoindoline system, exhibited the most potent
cytotoxicity. Of three compounds 5k, 5n, and 5t, compound 5t was found to be similarly potent to PAC-1, while two compounds 5k and
5n were around 3-fold more potent than PAC-1. Two compounds 5k and 5n were also the most potent in the series and their cytotoxicity
was almost comparable to that of ADR, a strong cytotoxic anticancer agent currently used widely to treat different cancers.
All three compounds 5r-t, which were 6-I substituted on the 4-oxoquinazoline ring, displayed relatively good cytotoxicity. Their potency
was also equipotent to that of PAC-1 but less potent than two compounds 5k and 5n. The results obtained suggested that the EW
3
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substituents on the 4-oxoquinazoline system like halogens (-Cl, -Br, -I) were more favorable than 6-CH3, an ER group. Recently,
compounds with similar structures and bioactivity have also been reported.[20] On the 2-oxoindoline moiety, substituents played very
delicate effects towards bioactivity, with 5’-CH3 group offering the most potent cytotoxicity for the compounds synthesized in most cases.
Table 1. Cytotoxicity of the compounds against some human cancer cell lines
Cytotoxicity (IC50,
[b]M)/Cell lines[c]
Cpd
R
R’
MW
LogP[a]
SW620
PC3
NCI-H23
5a
5b
5c
5d
5e
5f
H
H
5-F
347.33
365.32
381.78
426.23
361.36
377.36
381.78
392.33
381.78
416.22
395.80
426.23
460.67
440.26
473.23
507.67
487.26
361.36
395.80
375.39
0.04
0.24
0.69
0.93
0.59
0.13
0.69
0.26
0.69
1.33
>30
27.32±3.55
17.58±1.98
16.23±1.36
27.34±3.01
13.32±0.03
18.87±1.98
22.87±1.97
21.98±1.78
26.33±1.87
11.54±1.56
1.82±0.05
17.32±1.47
20.56±1.86
1.59±0.21
3.25±0.15
29.21±4.86
18.23±1.77
19.22±2.01
23.33±2.07
11.12±0.72
19.29±2.00
23.76±2.01
26.33±2.41
28.37±2.10
10.34±0.99
2.71±0.18
18.44±1.74
19.09±1.16
2.10±0.16
2.81±0.17
H
19.32±2.35
17.21±1.67
25.34±2.42
15.25±0.16
17.23±1.78
21.00±1.09
27.54±2.65
29.11±2.05
12.24±1.45
1.42±0.13
19.21±1.41
23.24±1.87
1.73±0.30
3.97±0.32
4.03±0.53
3.18±0.12
21.19±1.99
17.54±3.21
5.43±1.06
H
5-Cl
5-Br
5-CH3
5-OCH3
7-Cl
H
H
H
5g
5h
5i
H
H
5-NO2
H
6-Cl
6-Cl
6-Cl
6-Br
6-Br
6-Br
6-I
5j
5-Cl
5-CH3
H
5k
5l
1.24
0.93
1.58
1.48
1.21
1.86
1.76
0.59
1.24
1.14
5m
5n
5o
5p
5q
5r
5-Cl
5-CH3
H
6-I
5-Cl
5-CH3
H
4.28±0.16
6.82±0.15
19.22±2.01
15.76±2.00
4.33±0.36
5.77±1.16
6-I
3.83±0.33
20.44±1.78
16.37±1.79
3.84±0.04
6-CH3
6-CH3
6-CH3
5s
5t
5-Cl
5-CH3
5-FU]d]
130.08
392.49
-0.81
3.43
8.84±1.92
5.82±0.20
13.61±0.46
4.16±0.52
13.45±3.92
5.32±0.21
PAC-1
ADR[e]
543.5
1.27
1.12±0.14
1.16±0.28
1.29±0.12
[a]Calculated by ChemDraw 9.0 software; [b]The concentration (M) 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%.; [c]Cell lines: SW620, colon cancer; PC3, prostate cancer; NCI-H23, lung
cancer; [d]5-FU: 5-Fluorouracil, a positive control. [e]ADR: Adriamycin, a positive control
Caspase activation activity
From the results in the SRB assay we selected 6 compounds, including 5e.5k, 5n, 5p, 5q, and 5t for further evaluation in a caspase-3
activation assay. U937 human lymphoma cells (5 × 105/ml per well) were cultured for 24 h, then treated with compounds (50 M) for
further 24 h, and then harvested. The substrate Ac-DEVD-pNA at the final concentration of 0.4 M was added to the cell lysate (100 g/50
l). Then, after one-hour incubation, the absorbance of each well was read at 405 nm. The results (OD values over time period of 12 hours)
were illustrated in Figure 2. The data obtained showed that PAC-1 at the concentration of 50 µM caused an 11-fold increase in caspase-3
activity, in comparison to the untreated control (UN). Compound 5q exhibited the most potent caspase-3 activating activity. At 50 µM
concentration of this compound, the caspase-3 activity increased by 5-fold. This activity was still substantially lower than that of PAC-1.
Other compounds, including 5e, 5k, 5n, 5p and 5t, showed only negligible effects on induction of caspase-3 activity in this assay. Given
that compounds 5q, 5t, 5k, and 5n displayed equipotent or even more potent cytotoxicity in three human cancer cell lines assayed in
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comparison to PAC-1, it is likely that these compounds might exert better activating activity on other caspases, or they might possess
differential activity on other targets, e.g. kinases.
Figure 2. Caspase-3 activation activity of some representative compounds. U937 human lymphoma cells were treated with representative compounds (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%).
Cell apoptosis analysis
SRB assay showed that two compounds 5k and 5n induced strong cytotoxicity against three human cancer cell lines. We next performed
an Annexin V-FITC/propidium iodide (PI) dual staining assay to confirm whether compounds 5k and 5n can induce apoptosis. For
comparison, compound 5q was also included. During early apoptosis, phosphatidylserine (PS) locates on the cytosolic (inner) side of the
cell membrane, and translocates to the extracellular (outer) surface. Because annexin V has a high affinity for PS, so when it is
fluorescently labelled with fluorescein isothiocyanate (FITC), it can be used to identify early-stage apoptotic 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. So, severe and late apoptotic cells are stained with both annexin V and PI.
Meanwhile, necrotic cells are stained with PI only. We treated U937 (lymphoma cancer) and SW620 (colon cancer) cells with the
compounds at 50 µM for 24 h and stained the cells with Annexin V-FITC and PI. In U937 cells compounds 5n and 5q moderately increased
the percentages of Annexin V-PI-double positive cells, namely latter stage of dead cells (Figure 3A). However, their effects were still much
weaker than that of PAC-1, which caused more than 60% of cells into late apoptosis. Instead, compounds 5q caused a larger pool of
necrotic cells (9.49%, Figure 3A). Compound 5k induced a similar percentage of necrotic cells (10.20%) (Figure 3A). In SW620 cells, all
three compounds 5k, 5n and 5q only slightly caused the cells become necrotic (Figure 3B). Morphologically, compound 5k caused SW620
cells flatten in almost a similar manner PAC-1 did (Figure 4).
Figure 3. Apoptosis (Annexin V/PI) analysis of some compounds. U937 (A) or SW620 (B) cells were treated with compounds (50 M) 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-positive
population.
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Figure 4. Morphology changes of cells treated with compound 5k, 5n and 5q or PAC-1. SW620 2.5x105 cells/ml seeding (500 µl in 24 well) were incubated for
24 hours, then compounds or PAC-1 (50 μM) were added and incubated further for 24 hours. Then, the cells were photographed using an Imaging Device
(Celldiscoverer 7) with 40X lens. Scale bar: 50 mm.
Molecular docking studies
From the experimental results, we found that compound 5q exhibited higher activation effect at concentration of 50 μM when compared
to PAC-1; meanwhile 5k, 5n, 5p, 5q, and 5t showed very low activity at that concentration. This compound was further investigated the
caspase activation mechanism via a molecular docking simulation.
Figure 5. Metal complex and geometries between PAC-1 and compound 5q with Zinc ion (extracted from locked caspase-6 PDB ID: 4FXO)
Compound 5q was designed by modifying the 4-benzylpiperazin-1-yl part of PAC-1 (A-region) by the quinazoline heterocyclic system,
meanwhile the phenyl ring of PAC-1 (C-region) was replaced by different substituted 2-oxoindolines (Figure 1). As widely proven, PAC-1
and its derivatives could activate procaspase-3 through the chelation of inhibitory zinc ion at the binding site of the enzyme.[21] Up to date,
however, it still lacks a zinc-bound crystal structure of procaspase-3. To address this issue, we followed the same protocol published
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recently.[18, 22] We selected an homologous structure of caspase-3, that is caspased-6 inhibited by zinc ion whose crystallographical
coordinates was determinated at acceptable resolution (2.85 Å).[23] Despite some diversity, this protein model is useful to study the
structure-activity relationship related to the zinc binding mode of 5q and to improve the activity of synthesized compounds.
PAC-1 was firstly docked into the allosteric site of caspase-6 as reference compound. The result highlighted the role of ortho-hydroxy N-
acyl hydrazone as well as acylhydrazine moieties in chelating zinc ion, with the GBVI/WSA binding free energy (S) of -5.03 kCal/mol (Figure
5). This value is implemented in MOE using a force-field-based free-energy function with empirical terms to account for solvation and
hydrophobic effect of the complex. At the locked state, zinc ion established a tetrahedral coordination with Lys36, Glu244, His287, and
water molecule (number 402).[23] After docking assays, PAC-1 could penetrate into the allosteric pocket of caspase-6 and formed a stable
complex with zinc ion and a water molecule similar to caspase-zinc complex. The results of PAC-1 are in line with previous experiments.[14,
22, 24]
In comparison with PAC-1, 5q showed a slight difference in the zinc chelating mode. Two oxygen atoms of the quinazoline and
oxoindoline systems strictly bound to zinc ion with close distances of 1.4-1.5Å. Meanwhile the acylhydrazine linker bound to zinc ion via
>NH but not =N- as PAC-1 did. The GBVI/WSA binding free energy was -5.14 kCal/mol which is comparable with PAC-1. By comparing the
electrostatic potential maps and atom charge distributions of the bound conformations of 5q and PAC-1 (Figure 6), it can revealed that
the two ligands showed similar potential and charge distributions, especially at the N-acyl hydrazine motif (B-region). Many studies have
demonstrated the role of 2-hydroxyl group of the ortho-hydroxy N-acyl hydrazine motif in chelating zinc ion.[14, 25] In this regards, it can be
observed that the strained acetohydrazine of 5q is less favourable than PAC-1 in forming the tetrahedral coordination with zinc ion. In
addition, a more flexible side chain than the quinazoline could be more favored for the metal binding. We therefore expect that in the
future studies, these findings would be useful to design new oxoindoline derivatives with improved the chelating ability.
Figure 6. Comparison of the electrostatic potential maps and atom charge distribution of the bound conformations of PAC-1 and 5q in the docking complex.
Conclusions
In conclusion, we have designed and synthesized a series of new acetohydrazidesincorporating both 2-oxoindoline and 4-oxoquinazoline
heterocyclic systems (5a-t). Biological evaluation showed that a number of these acetohydrazides were comparably or even more
cytotoxic against three human cancer cell lines (SW620, colon cancer; PC-3, prostate cancer; NCI-H23, lung cancer) in comparison to PAC-
1, a first procaspase-3 activating compound used as a positive control. The 5-methyl group on the 2-oxoindoline ring appeared to be the
best substituent for the antitumor activity. Compound 5k activated the caspase-3 activity in U937 human lymphoma cells by 5-fold higher
than the untreated control. Three compounds, including 5k, 5n and 5q, significantly induced necrosis and apoptosis in U937 cells.
Experimental Section
Chemistry
Thin layer chromatography which was performed using Whatman® 250 m 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. Melting points were
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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. The elemental (C, H, N) analyses were performed on
a Perkin Elmer model 2400 elemental 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.
The synthesis of acetohydrazides incorporating 4-oxoquinazoline and 2-oxoindoline moieties (5a-t) was carried out as illustrated in
Scheme 1. Details are described below.
General procedures for the synthesis of compounds 5
The mixtures of anthranilic acid or 2-amino-4/5-substitutedbenzoic acid and formamide were stirred at 120 C, for 3 hours. Upon
completion, the resulting mixtures were cooled, then poured into ice-cold water, light grey solids appeared, and then were filtered,
washed with water (3 times) and dried to give quinazoline-4(3H)-on derivatives, which were used for the next reaction without further
purification.
To a respective solution of quinazoline-4(3H)-on derivatives in acetone were added K2CO3. The mixtures were stirred at 80C for 30
minutes, then KI was added. After stirring for further 15 minutes, ethyl chloroacetate were dropped slowly into the mixtures. The reaction
mixtures were again stirred at 60C for 3 hours. At the end of this reaction, the final mixtures were cooled, poured into ice-cold water.
Brown solids were formed, filtered and dried to give ethyl 2–(4-oxoquinazolin-3-(4H)-yl)acetate derivatives.
To a respective solution of compounds in ethanol was added hydrazine monohydrate, drop by drop. The mixture was stirred at room
temperature until the starting material reacted completely. The white precipitates formed were filtered and washed with cold ethanol (3
times), which were collected, dried under vacuum and carried on to the final step without further purification.
The above acetohydrazides were dissolved in hot methanol, then 10 drops of concentrated acetic acid were added, followed by the
addition of isatine derivatives into the mixtures. The mixtures were refluxed until the reaction finished. The precipitates formed were
filtered and washed with ethanol (3 times), the yellow-white solids were collected, dried under vacuum and recrystalised in ethanol or
column chromatography (MeOH:DCM) to obtain the desired products.
Yields, physical properties and spectroscopic data of the compounds are described below. The data for compounds 5a have been reported
previously.[18]
N'-(5-Fluoro-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5b)
Yellow solid; Yield: 54%. mp: 184.5-186.2 oC. Rf = 0.65 (DCM:MeOH = 14:1). IR (KBr, cm-1): 3296 (N-H); 3055, 2970 (CH, CH2); 1717 (C=O);
1
1663 (C=N); 1599 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.85 (s, 1H, NH-isatin); 10.88 (s, 1H, CONH); 8.41 (s, H2, 1H); 8.24-8.22 (m,
H4', 1H); 8.18 (d, J = 7.50, H5, 1H); 7.89 (t, J = 7.75, H7, 1H); 7.74 (d, J = 8.00, H8, 1H); 7.60 (t, J = 7.50, H6, 1H); 7.28 (d, J = 7.50, H7', 1H); 6.92 (dd,
J = 9.00, J’ = 4.50, H6', 1H); 5.32 (s, NCH2CO, 2H).13C NMR (125 MHz, DMSO-d6, ppm): δ 171.57, 164.86, 160.73, 159.01, 157.13, 148.93, 148.57,
140.81, 135.07, 127.73, 126.54, 121.89, 119.58, 119.39, 115.94, 115.87, 113.78, 111.95, 47.87. HR-MS (ESI) m/z calculated for C18H12FN5O3,
[M+H]+ 366.0924. Found, 366.0998.
N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5c)
Yellow solid; Yield: 58%. mp: 183.3-184.6 oC. Rf = 0.62 (DCM:MeOH = 14:1). IR (KBr, cm-1): 3163 (N-H); 3065, 3019, 2936 (CH, CH2); 1718
(C=O); 1694 (C=N); 1605 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.92 (s, NH-isatin, 1H); 10.99 (s, CONH, 1H); 8.41 (s, H2, H4', 2H); 8.18
(d, J = 7.50, H5, 1H); 7.90-7.87 (m, H7, 1H); 7.74 (d, J = 8.00, H8, 1H); 7.60 (t, J = 7.50, H6, 1H); 7.46 (d, J = 8.00, H7', 1H); 6.94 (d, J = 8.00, H6',
1H); 5.32 (s, NCH2CO, 2H).13C NMR (125 MHz, DMSO-d6, ppm): δ 164.63, 160.73, 148.94, 148.57, 143.17, 135.07, 132.60, 127.78, 127.69,
126.54, 126.42, 126.24, 121.89, 116.69, 112.49. HR-MS (ESI) m/z calculated for C18H12ClN5O3, {[M+H]+, 35Cl} 382.0628, {[M+H]+, 37Cl}
384.0628. Found, 382.0702, 384.0702.
8
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Chem. Biodiversity
N'-(5-Bromo-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5d)
Yellow solid; Yield: 60%. mp: 182.7-183.oC. Rf = 0.60 (DCM:MeOH = 14:1). IR (KBr, cm-1):3186 (N-H); 3065, 2934, 2882 (CH, CH2); 1717 (C=O);
1688 (C=N); 1601 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.93 (s, NH-isatin, 1H); 10.99 (s, CONH, 1H); 8.53 (s, H4’, 1H); 8.43 (s, H2, 1H);
8.18 (d, J = 7.50, H5, 1H); 7.89 (t, J = 7.75, H7, 1H); 7.74 (d, J = 7.50, H8, 1H); 7.59 (t, J = 7.50, H6, H7’, 2H); 6.89 (d, J = 8.50, H6’, 1H); 5.32 (s,
NCH2CO, 2H).13C NMR (125 MHz, DMSO-d6, ppm): δ 171.68, 164.46, 160.73, 148.93, 148.57, 143.53, 135.43, 135.07, 128.85, 127.77, 127.69,
126.54, 123.75, 121.89, 121.80, 117.14, 114.87, 114.04, 112.97, 47.82. HR-MS (ESI) m/z calculated for C18H12BrN5O3, {[M+H]+, 79Br} 426.0123,
{[M+H]+, 81Br} 428.0123. Found, 426.1094, 428.1094.
N'-(5-Methyl-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5e)
Yellow solid. Yield: 51%. mp: 186.3-197.8 oC. Rf = 0.55 (DCM:MeOH = 14:1).IR (KBr, cm-1): 3308 (N-H); 3055, 2926 (CH, CH2); 1717 (C=O);
1
1668 (C=N); 1603 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.63 (s, NH-isatin, 1H); 10.75 (s, CONH, 1H); 8.41 (s, H2, 1H); 8.18 (d, J =
7.50 , H5, 1H); 8.10 (s, H4’, 1H); 7.89 (td, J = 8.25, J’ = 1.0, H7, 1H); 7.75 (d, J = 8.00, H8, 1H); 7.60 (t, J = 7.50, H6, 1H); 7.23 (d, J = 8.00, H7’, 1H);
6.82 (d, J = 8.00, H6’, 1H); 5.32 (s, NCH2CO, 2H); 2.31 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 164.89, 160.74, 148.97, 148.58,
142.15, 135.06, 133.56, 131.30, 127.77, 127.68, 127.18, 126.54, 121.90, 115.63, 110.84, 20.92. HR-MS (ESI) m/z calculated for C19H15N5O3,
[M+H]+ 362.1175. Found, 362.1252.
N'-(5-Methoxy-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5f)
Yellow solid. Yield: 68%. mp: 187.4-188.2 oC. Rf = 0.58 (DCM:MeOH = 14:1). IR (KBr, cm-1):3163 (N-H); 3061, 2951, 2835 (CH, CH2); 1676
(C=N); 1609 (C=C).1H-NMR (500 MHz, DMSO-d6, ppm): δ 12.77 (s, NH-isatin, 1H); 11.15 (s, CONH, 1H); 8.43 (s, H2, 1H); 8.18 (dd, J = 8.00, J’ =
1.00, H5, 1H); 7.89 (td, J = 8.50, J’ = 1.50, H7, 1H); 7.75 (d, J = 8.00, H8, 1H); 7.60 (td, J = 8.00, J’ = 1.00, H6, 1H); 7.21 (s, H4’, 1H); 7.00 (d, J = 7.00,
H7’, 1H); 6.90 (d, J = 8.50, H6’, 1H); 5.40 (s, NCH2CO, 2H); 3.79 (s, OCH3, 3H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 163.11, 160.72, 155.92,
148.82, 148.51, 136.8, 135.14, 127.81, 126.53, 121.81, 120.73, 118.66, 112.62, 106.37, 56.16, 47.19. HR-MS (ESI) m/z calculated for C19H15N5O4,
[M+H]+ 378.1124. Found, 378.1201.
N'-(7-Chloro-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5g)
Yellow solid; Yield: 52%. mp: 184.3-185.2 oC. Rf = 0.70 (DCM:MeOH = 14:1). IR (KBr, cm-1):3167 (N-H); 3059, 2951 (CH, CH2); 1711 (C=O);
1659 (C=N); 1599 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.82 (s, NH-isatin, 1H); 11.31 (s, CONH, 1H); 8.41 (s, H2, 1H); 8.19 (d, J = 6.00,
H4’, 1H); 8.18 (d, J = 7.00, H5, 1H); 7.89 (t, J = 7.50, H7, 1H); 7.74 (d, J = 8.00, H8, 1H); 7.60 (t, J = 7.50, H6, 1H); 7.50 (d, J = 8.00, H6’, 1H); 7.10 (s,
H5’, 1H); 5.32 (s, NCH2CO, 2H).13C NMR (125 MHz, DMSO-d6, ppm): δ 164.82, 160.74, 148.93, 148.56, 141.84, 135.08, 132.78, 125.21, 124.35.
HR-MS (ESI) m/z calculated for C18H12ClN5O3, {[M+H]+, 35Cl} 382.0628, {[M+H]+, 37Cl} 384.0628. Found, 382.0702, 384.0702.
N'-(5-Nitro-2-oxoindolin-3-ylidene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazide (5h)
Yellow solid; Yield: 70%. mp: 186.1-187.9 oC. Rf = 0.63 (DCM:MeOH = 14:1). IR (KBr, cm-1): 3379 (N-H); 1663 (C=N); 1611 (C=C), 1589, 1377
(NO2). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.27 (s, 1H, NH-isatin); 9.05, 8.90 (s, CONH, 0.34H, 0.66H); 8.38 (s, H2, 1H); 8.20-8.12 (m, H5,
H4’, H6’, 3H); 7.86 (td, J = 7.00, J’ = 1.50, H7, 1H); 7.73 (d, J = 8.50, H8, 1H); 7.56 (t, J = 7.50, H6, 1H); 7.06 (d, J = 9.0, H7’, 1H); 5.19 (s, NCH2CO,
2H).13C NMR (125 MHz, DMSO-d6, ppm): δ 160.79, 149.56, 149.41, 148.68, 142.73, 142.41, 134.68, 134.61, 127.69, 127.64, 127.36, 127.28,
126.47, 123.58, 122.12, 119.86, 119.28, 118.12, 112.66, 110.54, 110.16, 48.97. HR-MS (ESI) m/z calculated for C18H12N6O5, [M+H]+ 393.0869.
Found, 393.0944.
2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N'-(2-oxoindolin-3-ylidene) acetohydrazide (5i)
Yellow solid; Yield: 66%. mp: 183.2-184.7 oC. Rf = 0.67 (DCM:MeOH = 14:1). IR (KBr, cm-1): 3345, 3244 (N-H); 2986, 2903 (CH, CH2); 1721
(C=O); 1674 (C=N); 1609 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.62 (s, NH-isatin, 1H); 10.78 (s, CONH, 1H); 8.36 (s, H2, 1H); 8.09 (d,
J = 7.00, H4’, 1H); 8.02 (s, H5, 1H); 8.83 (d, J = 8.50, H7, 1H); 7.69 (d, J = 9.00, H8, 1H); 7.32 (s, H6’, 1H); 6.97 (s, H7’, 1H); 6.84 (d, J = 8.00, H5’,
1H); 5.23 (s, NCH2CO, 2H).13C NMR (125 MHz, DMSO-d6, ppm): δ 164.80, 159.79, 149.44, 147.30, 144.49, 135.23, 133.35, 132.01, 130.10,
126.73, 125.51, 123.10, 122.24, 115.56, 111.18, 48.04. HR-MS (ESI) m/z calculated for C18H12ClN5O3, {[M-H]-, 35Cl} 380.0628, {[M-H]-, 37Cl}
382.0628. Found, 380.0551, 382.0551.
N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(6-chloro-4-oxoquinazolin-3(4H)-yl)acetohydrazide (5j)
9
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Chem. Biodiversity
Yellow solid; Yield: 67%. mp: 181.6-182.9 oC. Rf = 0.66 (DCM:MeOH = 14:1). IR (KBr, cm-1):3215 (N-H); 2986, 2903 (CH, CH2); 1720 (C=O);
1694 (C=N); 1611 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.84 (s, NH-isatin, 1H); 10.89 (s, CONH, 1H); 8.36 (s, H2, 1H); 8.32 (s, H4’, 1H);
8.02 (t, J = 2.00, H5, 1H); 7.82 (dd, J = 7.75, J’ = 2.25, H7, 1H); 7.69-7.67 (m, H8, 1H); 7.36 (d, J = 6.5, H7’, 1H); 6.83 (dd, J = 7.50, J’ = 1.50, H6’, 1H);
5.23 (s, NCH2CO, 2H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 171.56, 164.58, 159.78, 149.40, 147.29, 143.18, 135.23, 132.62, 132.02, 130.10,
127.30, 126.42, 126.25, 125.51, 123.08, 120.89, 116.66, 112.48, 48.02. HR-MS (ESI) m/z calculated for C18H11Cl2N5O3, {[M-H]-, (35Cl, 35Cl)}
414.0238, {[M-H]-, (37Cl, 35Cl)} 416.0238, {[M-H]-, (37Cl, 37Cl)} 418.0238. Found, 414.0164, 416.0164, 418.0164.
2-(6-Chloro-4-oxoquinazolin-3(4H)-yl)-N'-(5-methyl-2-oxoindolin-3-ylidene)acetohydrazide (5k)
Yellow solid; Yield: 69%. mp: 180.5-182.2 oC. Rf = 0.61 (DCM:MeOH = 14:1). IR (KBr, cm-1):3318, 3210 (N-H); 2970, 2905 (CH, CH2); 1721
1
(C=O); 1674 (C=N); 1607 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.53 (s, NH-isatin, 1H); 10.64 (s, CONH, 1H); 8.35 (s, H2, 1H); 8.02-
8.01 (m, H5, H4’, 2H); 7.82 (dd, J = 9.00, J’ = 2.50, H7, 1H); 7.68 (d, J = 8.50, H8, 1H); 7.12 (d, J = 8.00, H7’, 1H); 6.72 (d, J = 8.00, H6’, 1H); 5.21 (s,
NCH2CO, 2H); 2.20 (s, CH3, 3H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.79, 159.76, 149.46, 147.30, 142.10, 135.29, 135.21, 133.53, 132.87,
132.29, 132.09, 132.00, 131.27, 130.09, 127.15, 125.50 ,123.10 ,123.01, 115.66, 110.83, 20.92. HR-MS (ESI) m/z calculated for C19H14ClN5O3,
394.0785 {[M-H]-, 35Cl}, 396.0785 {[M-H]-, 37Cl}. Found, 394.0711, 396.0711.
2-(6-Bromo-4-oxoquinazolin-3(4H)-yl)-N'-(2-oxoindolin-3-ylidene)acetohydrazide (5l)
Yellow solid; Yield: 62%. mp: 182.1-183.0 oC. Rf = 0.62 (DCM:MeOH = 14:1). IR (KBr, cm-1):3343, 3244 (N-H); 2990, 2887 (CH, CH2); 1717
(C=O); 1668 (C=N); 1603 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.62 (s, NH-isatin, 1H); 10.77 (s, CONH, 1H); 8.37 (s, H2, 1H); 8.16 (d, J
= 2.50, H5, 1H); 8.09 (d, J = 7.50, H4’, 1H); 7.94 (dd, J = 8.75, J’ = 2.25, H7, 1H); 7.60 (d, J = 8.50, H8, 1H); 7.32 (t, J = 7.50, H6’, 1H); 6.96 (s, H7’,
1H); 6.83 (d, J = 7.50, H5’, 1H); 5.23 (s, NCH2CO, 2H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.65, 149.55, 147.56, 144.49, 137.95, 133.33,
130.22, 128.61, 126.70, 123.44, 123.16, 122.24, 120.17, 115.55, 111.17, 48.05. HR-MS (ESI) m/z calculated for C18H12BrN5O3, 424.0123 {[M-H]-,
79Br}, 426.0123 {[M-H]-, 81Br}. Found, 424.0042, 426.0042.
2-(6-Bromo-4-oxoquinazolin-3(4H)-yl)-N'-(5-chloro-2-oxoindolin-3-ylidene)acetohydrazide (5m)
Yellow solid; Yield: 65%. mp: 180.9-181.5 oC. Rf = 0.69 (DCM:MeOH = 14:1). IR (KBr, cm-1): 3325 (N-H); 3061, 2984, 2905 (CH, CH2); 1721
(C=O); 1670 (C=N); 1609 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.84 (s, NH-isatin, 1H); 10.89 (s, CONH, 1H); 8.38 (s, H4’, 1H); 8.36 (s,
H2, 1H); 8.16 (d, J = 2.50, H5, 1H); 7.94 (dd, J = 8.75, J’ = 2.25, H7, 1H); 7.61 (d, J = 9.00, H8, 1H); 7.37 (d, J = 7.00, H6’, 1H); 6.84 (d, J = 8.50, H7’,
1H); 5.23 (s, NCH2CO, 2H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 171.56, 164.58, 159.78, 149.40, 147.29, 143.18, 135.23, 132.62, 132.02,
130.10, 127.30, 126.42, 126.25, 125.51, 123.08, 120.89, 116.66, 112.48, 48.02. HR-MS (ESI) m/z calculated for C18H11BrClN5O3, 457.9733 {[M-
H]-, (35Cl, 79Br)}, 459.9733 {[M-H]-, (35Cl, 81Br), (37Cl, 79Br)}, 461.9733 {[M-H]-, (37Cl, 81Br)}. Found, 457.9650, 459.9630, 461.9616.
2-(6-Bromo-4-oxoquinazolin-3(4H)-yl)-N'-(5-methyl-2-oxoindolin-3-ylidene)acetohydrazide(5n)
Yellow solid; Yield: 68%. mp: 179.3-180.1 oC. Rf = 0.57 (DCM:MeOH = 14:1). IR (KBr, cm-1): 3099, 2986, 2905 (CH, CH2); 1711 (C=O); 1682
(C=N); 1607 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.54 (s, NH-isatin, 1H); 10.65 (s, CONH, 1H); 8.37 (s, H2, 1H); 8.15 (d, J = 2.50, H5,
1H); 7.99 (s, H4’, 1H); 7.93 (dd, J = 8.75, J’ = 2.25, H7, 1H); 7.60 (d, J = 8.50, H8, 1H); 7.12 (d, J = 7.50, H7’, 1H); 6.72 (d, J = 8.00, H6’, 1H); 5.23 (s,
NCH2CO, 2H); 2.20 (s, CH3, 3H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 171.24, 164.83, 159.65, 149.54, 147.55, 142.15, 137.93, 133.56, 131.31,
130.21, 129.61, 127.15, 123.43, 120.17, 115.58, 110.84, 48.02, 20.91. HR-MS (ESI) m/z calculated for C19H14BrN5O3 , {[M+H]+, 79Br}, 440.0280,
{[M+H]+, 81Br}, 442.0280. Found, 440.0345, 442.0325.
2-(6-Iodo-4-oxoquinazolin-3(4H)-yl)-N'-(2-oxoindolin-3-ylidene)acetohydrazide (5o)
Yellow solid; Yield: 58%. mp: 179.7-180.5 oC. Rf = 0.62 (DCM:MeOH = 14:1). IR (KBr, cm-1):3262, 3148 (N-H); 3086, 2972, 2899, 2833 (CH,
CH2); 1724 (C=O); 1680 (C=N); 1603 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.62 (s, NH-isatin, 1H); 10.77 (s, CONH, 1H); 8.35 (s, H2, H5,
2H); 8.08 (dd, J = 8.50, J’ = 2.00, H4’, H7, 2H); 7.44 (d, J = 8.50, H8, 1H); 7.32 (t, J = 7.25, H6’, 1H); 6.97 (s, H7’, 1H); 6.84 (d, J = 8.00, H5’, 1H); 5.22
(s, NCH2CO, 2H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.46, 149.60, 147.84, 144.50, 143.46, 134.77, 133.36, 129.98, 126.72, 123.63,
122.25, 115.56, 111.18, 92.81, 48.09. HR-MS (ESI) m/z calculated for C18H12IN5O3, [M+H]+ 473.9984. Found, 474.1527
N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(6-iodo-4-oxoquinazolin-3(4H)-yl)acetohydrazide (5p)
Yellow solid; Yield: 53%. mp: 178.2-179.1 oC. Rf = 0.61 (DCM:MeOH = 14:1). IR (KBr, cm-1):3294, 3211 (N-H); 3084, 2986, 2903 (CH, CH2);
1
1719 (C=O); 1692 (C=N); 1609 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.83 (s, NH-isatin, 1H); 10.89 (s, CONH, 1H); 8.37 – 8.31 (m, H4’,
10
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10.1002/cbdv.201900670
Chemistry & Biodiversity
Chem. Biodiversity
H2, H5, 3H); 8.08 (td, J = 8.50, J’ = 4.00, H7, 1H); 7.44 (dd, J = 8.50, J’ = 1.00, H8, 1H); 7.37 (d, J = 8.00, H6’, 1H); 6.84 (d, J = 8.50, H7’, 1H); 5.22 (s,
NCH2CO, 2H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.46, 149.57, 147.84, 143.48, 143.17, 134.77, 132.59, 129.98, 126.42, 123.61, 116.68,
112.50, 92.83, 47.99. HR-MS (ESI) m/z calculated for C18H11ClIN5O3, {[M-H]-, 35Cl}, 505.9595, {[M-H]-, 37Cl}, 507.9595. Found, 505.9516,
507.9516
2-(6-Iodo-4-oxoquinazolin-3(4H)-yl)-N'-(5-methyl-2-oxoindolin-3-ylidene)acetohydrazide (5q)
Yellow solid; Yield: 62%. mp: 177.0-179.2 oC. Rf = 0.66 (DCM:MeOH = 14:1). IR (KBr, cm-1):3327 (N-H); 3065, 2986, 2903 (CH, CH2); 1715
(C=O); 1670 (C=N); 1609 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.54 (s, NH-isatin, 1H); 10.65 (s, CONH, 1H); 8.35 (s, H2, H5, 2H); 8.08
(dd, J = 8.75, J’ = 1.75, H7, 1H); 8.00 (s, H4’, 1H); 7.44 (d, J = 8.50, H8, 1H); 7.13 (d, J = 7.00, H7’, 1H); 6.72 (d, J = 8.00, H6’, 1H); 5.28 (s, NCH2CO,
2H); 2.21 (s, CH3, 3H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 159.46, 159.43, 149.60, 147.84, 143.46, 142.15, 134.76, 133.58, 132.88, 131.30,
129.97, 127.17, 123.63, 115.59, 110.85, 92.81, 47.99, 20.92. HR-MS (ESI) m/z calculated for C19H14IN5O3, [M+H]+ 488.0141. Found, 488.0207.
2-(6-Methyl-4-oxoquinazolin-3(4H)-yl)-N'-(2-oxoindolin-3-ylidene)acetohydrazide (5r)
Yellow solid; Yield: 60%. mp: 184.4 -186.6 oC. Rf = 0.58 (DCM:MeOH = 14:1).IR (KBr, cm-1): 3240, 3125 (N-H); 2984, 2905 (CH, CH2); 1722
(C=O); 1661 (C=N); 1601 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.68 (s, NH-isatin, 1H); 10.85 (s, CONH, 1H); 8.34 (s, H2, 1H); 8.17 (d, J
= 7.50, H4’, 1H); 7.96 (s, H5, 1H); 7.69 (d, J = 8.50, H7, 1H); 7.63 (d, J = 8.50, H8, 1H); 7.41 (t, J = 7.50, H6’, 1H); 7.05 (s, H7’, 1H); 6.92 (d, J = 7.50,
H5’, 1H); 5.30 (s, NCH2CO, 2H); 2.47 (s, CH3, 3H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 164.78, 160.68, 148.14, 146.59, 144.47, 137.43, 136.29,
133.31, 127.63, 126.71, 125.84, 123.16, 122.23, 121.67, 115.58, 111.16, 47.74, 21.29. HR-MS (ESI) m/z calculated for C19H15N5O3, [M-H]-
360.1174. Found, 360.1107.
N'-(5-Chloro-2-oxoindolin-3-ylidene)-2-(6-methyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide (5s)
Yellow solid; Yield: 58%. mp: 183.9 -184.7 oC. Rf = 0.57 (DCM:MeOH = 14:1). IR (KBr, cm-1):3198 (N-H); 2984, 2903 (CH, CH2); 1721 (C=O);
1
1686 (C=N); 1609 (C=C). H-NMR (500 MHz, DMSO-d6, ppm): δ 11.82 (s, NH-isatin, 1H); 10.89 (s, CONH, 1H); 8.32 (s, H2, 1H); 8.25 (s, H4’,
1H); 7.88 (s, H5, 1H); 7.61 (dd, J = 8.50, J’ = 1.50, H7, 1H); 7.55 (d, J = 8.50, H8, 1H); 7.37 (d, J = 7.50, H6’, 1H); 6.84 (d, J = 8.50, H7’, 1H); 5.21 (s,
NCH2CO, 2H); 2.38 (s, CH3, 3H). 13C NMR (125 MHz, DMSO-d6, ppm): δ 160.58, 148.11, 146.58, 143.17, 137.46, 136.31, 132.58, 127.63, 126.42,
125.85, 121.65, 116.70, 112.48, 21.30. HR-MS (ESI) m/z calculated for C19H14ClN5O3, {[M-H]-, 35Cl} 394.0785, {[M-H]-, 37Cl}. . 396.0785. Found,
394.0717, 396.0717.
N'-(5-Methyl-2-oxoindolin-3-ylidene)-2-(6-methyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide (5t)
Yellow solid; Yield: 70%; mp: 183.4-185.0 oC. Rf = 0.63 (DCM:MeOH = 14:1). IR (KBr, cm-1):3329 (N-H); 2984, 2920 (CH, CH2); 1719 (C=O);
1670 (C=N); 1603 (C=C). 1H-NMR (500 MHz, DMSO-d6, ppm): δ 11.60 (s, NH-isatin, 1H); 10.73 (s, CONH, 1H); 8.33 (s, H2, 1H); 8.09 (s, H4’, 1H);
7.96 (s, H5, 1H); 7.69 (dd, J = 8.50, J’ = 2.00, H7, 1H); 7.63 (d, J = 8.00, H8, 1H); 7.21 (d, J = 7.50, H7’, 1H); 6.81 (d, J = 8.00, H6’, 1H); 5.29 (s,
NCH2CO, 2H); 2.47-2.29 (m, 6H, CH3). 13C NMR (125 MHz, DMSO-d6, ppm): δ 160.68, 160.65, 148.15, 146.60, 142.14, 137.43, 136.36, 136.28,
133.55, 132.84, 132.28, 131.28, 127.63, 127.16, 125.84, 121.68, 121.60, 115.62, 110.83, 21.30. HR-MS (ESI) m/z calculated for C20H17N5O3, [M-
H]- 374.1331. Found, 374.1265.
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 reagents 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 L of cell suspension. The plates were then incubated in a 5% CO2 incubator at 37 oC for 24 h. Compounds
were initially dissolved in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations by culture medium. Then 20 L 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
[26]
compounds was measured by the colorimetric method, as described previously
with slight modifications.[27-30] The IC50 values were
calculated using a Probits method [31] and were averages of three independent determinations (SD ≤ 10%).
11
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10.1002/cbdv.201900670
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Caspase-3 activation assay
Caspase activity was measured by using caspase 3 assay kit according to the manufacturer’s instructions (abcam, MA, USA). Briefly, 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 M) for 24 h, and then harvested. The harvested cells were washed twice with ice-cold PBS and treated with lysis buffer
included in the kit. Cell lysate (100 g/50 l) was mixed with the substrate Ac-DEVD-pNA (40 L in buffer containing 50 mM HEPES pH 7.4,
100 mM NaCl, 10 mM DTT, 0.1 mM EDTA disodium salt, 0.01% CHAPS, 10% glycerol) was added to make the final concentration of 0.4
M as the instruction of caspase-3 assay kit (Abcam, cat. N. ab39401). After one-hour incubation, fluorescence was measured at 405 nm
every 30 min for 12 hours. The slope of the linear portion for each well was determined as the enzyme activity.
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 M) for 24 h, and then harvested.
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).
Docking studies
The 3D structures of synthesized compound 5q was constructed using the Builder module of MOE 2009.10;[32] then energy was minimized
based on the 94s variant of the Merck molecular force field (MMFF94s) in order to remove all strains from the molecular structure. The X-
[23]
ray crystal structure of caspase-6 was retrieved from the Protein Data Bank (PDB ID: 4FXO)
as the protein model for the docking
studies. Docking simulations were carried out by using the MOE Triangle Matcher placement method,[32] keeping the protein rigid and the
ligands flexible.[18, 22] The cofactor zinc ion and water molecules insight the binding site were conserved as they are an integral part of
allosteric mechanisms.[23] Binding site chosen was of 6.5Å surrounding the zinc ion. The binding site includes paramount amino acids in
triple catalysis Lys36, Glu244 and a water molecule. For estimate the negative binding free energy profile of the complex (S value,
kCal/mol), we adopted the London dG as first rescoring function with force field (MMFF94x) refinement, and GBVI/WSA as a second
one.[32] The electrostatic potential maps (EPM) and atom charge distributions of the bound conformations of each ligand were computed
using the Poisson-Boltzmann Equation (PBE) implemented in the MOE software. The maps were represented according to the levels of
the hydrophobic (Hyd), acceptor (Acc) and donor (Don) contours of the potentials as default (Hyd = -2.5, Acc = -1.0 and Don = -1.0
kCal/mol). The geometry of the resulting complexes was analyzed using Discovery Studio Visualizer 6.0(www.3dsbiovia.com).
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 104.01-2018.01). The work was also partly supported by a grant funded by the Korean Government (NRF, Grant number
2017R1A5A2015541).
Author Contribution Statement
D. T. K. O, N-H. N and S-B. H. proposed the work. N-H. N, L. C. H., D. T. A., B. X. T. and P. H. D. mainly developed the synthesis studies, S-
B. H, E. J. P., J. S. K., P. T. T., H. J. J. and D. T. K. O. performed the biological testing assays.P-T. H., L. D-A, L-T-T. H performed the
docking simulations and physicochemical computations.All authors read and approved the final manuscript.
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