Design, synthesis, and bioevaluation of novel oxoindolin-2-one derivatives incorporating 1-benzyl-1H-1,2,3-triazole

Article abstract of DOI:10.1007/s00044-019-02488-1

In our search for novel bioactive molecules, three series of indolin-2-one derivatives incorporating 1-benzyl-1H-1,2,3-triazole moiety were synthesized. The compounds were initially designed as acetylcholine esterase (AChE) inhibitors based on the structu

Full text of DOI:10.1007/s00044-019-02488-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02488-1
ORIGINAL RESEARCH
Design, synthesis, and bioevaluation of novel oxoindolin-2-one
derivatives incorporating 1-benzyl-1H-1,2,3-triazole
Ta Thu Lan1 Duong Tien Anh Pham-The Hai Do Thi Mai Dung Le Thi Thu Huong Eun Jae Park
1 ●
1 ●
1 ●
2 ●
3 ●
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4 ●
1 ●
3 ●
1
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Hye Won Jeon Jong Soon Kang Nguyen Thi Thuan Sang-Bae Han Nguyen-Hai Nam
Received: 19 September 2019 / Accepted: 29 November 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In our search for novel bioactive molecules, three series of indolin-2-one derivatives incorporating 1-benzyl-1H-1,2,3-
triazole moiety were synthesized. The compounds were initially designed as acetylcholine esterase (AChE) inhibitors based
on the structural feature of donepezil, a known AChE inhibitor which is currently used clinically to treat Alzheimer’s disease
(
AD). Two compounds 4g and 3a were found to be the most potent in inhibition of AChE with inhibition percentages of 51
and 50% when tested at the concentration of 100 μM. Docking assays were carried out in order to explain the
structure–activity relationships of these compounds compared with Donepezil against AChE enzyme. In DPPH free radical-
scavenging assay, most compounds showed only weak scavenging activity. Noteworthy, additional cytotoxic evaluation of
the compounds against three human cancer cell lines (SW620, human colon cancer; PC3, prostate cancer; NCI-H23, lung
cancer) revealed that five compounds, including 3c, 3e, 5c, 5e, and 5g, exhibited strong cytotoxicity (IC50 values in the range
of 0.65–7.17 µM). Compound 5g was the most potent one with IC50 values as low as 0.65 μM, even more potent than
adriamycin, a positive control. Thus, compound 5g would be promising for further development as an anticancer agent.
●
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Keywords Acetylcholine esterase inhibitors Oxoindolin-2-one Cytotoxicity Docking simulation
Introduction
and other cognitive impairments. It is the most common
form of dementia (Huang and Mucke 2012; Kumar et al.
2015). Currently, the disease affect nearly 40 million indi-
viduals of people over 65 and accounts for ~80% of
dementia patients worldwide (Kumar et al. 2015). It is
known that many factors contribute to the initiation and
progression of the disease, however, due to the complexity
of the AD’s etiology and pathologenesis, the principal
causes of the disease remain unclear. To date, the disease is
still one of the incurable neurological disorders. The clinical
management of the disease relies mostly on the sympto-
matic treatment (Huang and Mucke 2012).
Recently, with the enormous efforts of scientists devoted
in studying the AD’s pathology, a number of critical factors
causing the disease have been indicated, such as abnormal
posttranslational modifications of tau protein, which result
in neurofibrillary tangles; deposition of amyloid β-protein
Alzheimer’s disease (AD) is a progressive and neurode-
generative disease characterized by gradual loss of memory
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02488-1) contains supplementary
material, which is available to authorized users.
*
*
Sang-Bae Han
shan@cbnu.ac.kr
Nguyen-Hai Nam
namnh@hup.edu.vn
1
2
3
Hanoi University of Pharmacy, 13-15 Le Thanh Tong,
Hanoi, Vietnam
School of Medicine and Pharmacy, Hanoi National University,
(Aβ) plagues; and a decrease in the level of the neuro-
1
44 Xuan Thuy, Hanoi, Vietnam
transmitter acetylcholine (Olivero et al. 2014; Yiannopou-
lou and Papageorgiou 2013). Among these, the
acetylcholine decrease in the brain is considered as one
the most underlying factors. In fact, all drugs approved for
the treatment of AD currently, including donepezil, tacrine,
College of Pharmacy, Chungbuk National University, 194-31,
Osongsaengmyung-1, Heungdeok, Cheongju, Chungbuk 28160,
Republic of Korea
4
Bio-Evaluation Center, Korea Research Institute of Bioscience and
Biotechnology, Cheongju, Chungbuk, Republic of Korea

Medicinal Chemistry Research
galantamine, and rivastigmine (Fig. 1), are acetylcholine
esterase inhibitors (Rodda and Carter 2012).
picrylhydrazyl (DPPH) free radical-scavenging and cyto-
toxic assays is also presented.
Acetylcholine esterase (AChE; EC 3.1.1.7) is a member
of the serine hydrolase enzyme family. These enzymes
catalyze the hydrolysis of a neurotransmitter acetylcholine,
resulting in the breakdown of acetylcholine into choline and
acetic acid. Consequently, the concentration of acetylcho-
line is decreased and the cholinergic neurotransmission is
terminated (Massoulié et al. 1993). Thus, inhibition of
acetylcholine esterase would prevent the hydrolysis of
acetylcholine and subsequently elevate the cholinergic
neurotransmission, resulting in clinical improvement of the
disease. Due to the therapeutic importance of AChE in AD
pathology, many medicinal chemists worldwide have
devoted intensive efforts in developing novel AChE inhi-
bitors. As a result, structurally diverse AChE inhibitors both
naturally and synthetic, have been reported in the past
decades (Singh et al. 2013). Joined in that efforts we have
designed several series of donepezil analogs which incor-
porate oxoindolin-2-one core imbedded with 1-benzyl-4H-
Materials and methods
Chemistry
Thin layer chromatography, which was performed using
Whatman® 250 μm Silica Gel GF Uniplates and visualized
under UV light at 254 nm, was used to check the progress of
reactions and preliminary evaluation of compounds’
homogeneity. Melting points were measured using a Gal-
lenkamp 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 (240–400 mesh) as sta-
1
tionary phase. Nuclear magnetic resonance spectra ( H and
1
3
C NMR) were recorded on a Bruker 500 MHz spectro-
1
,2,3-triazole ring (Fig. 2). It is envisioned that the incor-
meter with dimethyl sulfoxide (DMSO)-d as solvent unless
6
poration of the triazole moeity not only retains a nitrogen
atom similar to that in donepezil but also introduces a
heterocycle with more possible interactions with the
enzyme binding site. This paper describes the synthesis and
AChE inhibitory activity of these compounds series.
Additional evaluation of the compounds in 1,1-diphenyl-2-
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, Electrospray ionization (ESI), were recorded
using PE Biosystems API2000 (Perkin Elmer, Palo Alto,
Fig. 1 Structures of some AChE
inhibitors
Fig. 2 Design of novel AChE
inhibitors

Medicinal Chemistry Research
®
CA, USA) and Mariner (Azco Biotech, Inc. Oceanside,
CA, USA) mass spectrometers, respectively. The elemental
(
the reaction completed (12–24 h). The corresponding
resulting mixtures were evaporated under reduced pressure
to give the residues, which were re-dissolved in 50 ml of
DCM. The mixtures were filtered and the DCM layers were
evaporated under reduced pressure to give the targeting
compounds 3a–g.
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.
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)indoline-2,3-dione
The synthesis of novel oxoindolin-2-ones imbedded with
-benzyl-4H-1,2,3-triazole scaffold (3–5) was carried out as
illustrated in Scheme 1. Details are described below.
(3a) Orange solid; Yield: 63%. mp: 173–174 °C. R = 0.65
f
−
1
1
(DCM: MeOH = 20: 1). IR (KBr, cm ): 3134 (CH, arene);
1
2970 (CH, CH ); 1728 (C=O); 1611, 1468 (C=C). H-
2
NMR (500 MHz, DMSO-d , ppm): δ 8.23 (1H, s, H-6’); 7.63
(1H, t, J = 7.75 Hz, H-4), 7.57 (1H, d, J = 7.50 Hz, H-7,
6
General procedures for the synthesis of compounds 3a–g
7
.38–7.32 (3H, m, H-4”, H-5”, H-6”), 7.27 (2H, d, J =
8.00 Hz, H-3”, H-7”), 7.16–7.12 (2H, m, H-5, H-6), 5.57
(2H, s, H-1”a, H-1”b), and 4.97 (2H, s, H-1’a, H-1’b). 13
To a respective solution of isatins 1a–g (1 mmol) in
dimethylformamide (DMF) (3 mL) were added K CO
C
2
3
(
165.5 mg, 1.2 mmol). The mixtures were stirred at 80 °C
NMR (125 MHz, DMSO-d , ppm): δ 183.51, 158.30,
6
for 1 h, then then a catalytic amount of KI (8.3 mg,
150.62, 138.48, 136.35, 129.22, 128.63, 128.38, 124.91,
124.28, 123.82, 118.12, 111.62, 53.37, and 35.59. ESI-MS
m/z: 319.20 [M + H]+
0
0
.05 mmol) was added. After stirring for further 15 min,
.15 ml of a solution of propargyl bromide 80% in toluene
was dropped slowly into the mixtures. The reaction mix-
tures were again stirred at 60 °C for 3 h. The reaction was
checked by TLC. After the reaction completed, the
resulting mixtures were cooled, poured into ice-cold
water, and acidified to pH ~ 4. The orange solids formed
were filtered and dried to give the propargylated isatins 2,
which were used for the next step without further
purification.
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-fluoroindoline-
2,3-dione (3b) Orange solid; Yield: 67%. mp: 185–186 °C.
−
1
R = 0.67 (DCM: MeOH = 20: 1). IR (KBr, cm ): 3055
f
(CH, arene); 2901 (CH, CH ); 1728 (C=O); 1616, 1468
2
1
(C=C). H-NMR (500 MHz, DMSO-d , ppm): 8.23 (1H, s,
6
H-6’), 7.53–7.47 (2H, m, H-4’, H-7’), 7.38–7.32 (3H, m,
H-4”, H-5”, H-6”), 7.28 (2H, d, J = 8.00 Hz, H-3”, H-7”),
A respective solution of compounds 2a–g and (azido-
methyl)benzene (1 mmol) in acetonitrile (10 mL) were
stirred at room temperature for 10 min, then CuI (19.1 mg,
7.19–7.17 (1H, m, H ), 5.57 (2H, s, H-1”a, H-1”b), and
6
1
3
4.97 (2H, s, H-1’a, H-1’b). C NMR (125 MHz, DMSO-d6,
ppm): δ 182.91, 159.97, 158.33, 158.05, 146.87, 142.19,
136.38, 129.22, 129.17, 128.62, 128.38, 124.48, 124.29,
0
.1 mmol) was added. The mixture was stirred at 50 °C until
Scheme 1 Synthesis of oxoindolin-2-one derivatives imbedded with 1-benzyl-4H-1,2,3-triazole scaffold (3–5)

Medicinal Chemistry Research
1
3
1
5
24.26, 119.07, 119.01, 113.06, 113.00, 112.01, 111.81,
3.34, and 35.62. ESI-MS m/z: 337.10 [M + H]
C NMR (125 MHz, DMSO-d , ppm): δ 185.42, 183.76,
6
+
138.79, 136.70, 133.24, 129.33, 129.21, 128.92, 128.62,
1
28.58, 128.37, 125.13, 111.49, 53.68, 53.35, and 20.51.
+
1
2
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-chloroindoline-
ESI-MS m/z: 333.20 [M + H]
,3-dione (3c) Orange solid; Yield: 66%. mp: 191–192 °C.
−
1
R = 0.68 (DCM: MeOH = 20: 1). IR (KBr, cm ): 3078
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-methoxyindo-
f
(
CH, arene); 2968 (CH, CH ); 1724 (C=O); 1605, 1470
line-2,3-dione (3g) Orange solid; Yield: 55%. mp:
2
1
(
C=C). H-NMR (500 MHz, DMSO-d , ppm): 8.20 (1H, s,
196–197 °C. R = 0.65 (DCM: MeOH = 20: 1). IR (KBr,
6
f
−
1
H-6’), 7.69 (1H, dd, J = 8.50 Hz, J’ = 2.00 Hz, H-7), 7.63
1H, d; J = 2.00 Hz, H-4), 7.38–7.32 (3H, m, H-4”, H-5”,
cm ): 3069 (CH, arene); 2901 (CH, CH ); 1721 (C=O);
1588, 1487 (C=C). H-NMR (500 MHz, DMSO-d , ppm):
2
1
(
6
H-6”), 7.27 (2H, d, J = 8.00 Hz, H-3”, H-7”), 7.15 (1H, d,
J = 8.50 Hz, H-6), 5.57 (2H, s, H-1”a, H-1”b), and 4.97
(
8.20 (1H, s, H ’), 7.36–7.34 (3H, m, H-4”, H-5”, H-6”),
6
7.27 (2H, d, J = 6.50 Hz, H-3”, H-7”), 7.24–7.21 (1H, m,
H-7), 7.16 (1H, d, J = 3.00 Hz, H-4), 7.08 (1H, d, J =
8.50 Hz, H-6), 5.56 (2H, s, H-1”a, H-1”b), 4.94 (2H, s,
2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6,
ppm): δ 182.40, 158.07, 149.13, 142.09, 137.32, 136.37,
1
3
1
1
29.22, 128.63, 128.38, 128.04, 124.40, 124.23, 119.54,
H-1’a, H-1’b), and 3.76 (3H, s, 5-OCH3). C NMR (125
13.34, 53.33, and 35.66. ESI-MS m/z: 353.10 [M + H]+
MHz, DMSO-d , ppm): δ 183.80, 158.33, 156.29, 144.45,
6
136.39, 129.22, 128.62, 128.37, 124.29, 124.21, 118.56,
1
2
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-chloroindoline-
112.71, 109.68, 56.38, 53.33, and 35.54. ESI-MS m/z:
349.10 [M + H]+
,3-dione (3d) Orange solid; Yield: 60%. mp: 193–194 °C.
−
1
R = 0.68 (DCM: MeOH = 20: 1). IR (KBr, cm ): 3080
f
(
CH, arene); 2986 (CH, CH ); 1726 (C=O); 1605, 1470
General procedures for the synthesis of compounds 4
2
1
(
C=C). H-NMR (500 MHz, DMSO-d , ppm): 8.15 (1H, s,
6
H-6’), 7.95 (1H, d, J = 7.50 Hz, H-4), 7.44 (1H, d, J =
.00 Hz, H-6), 7.37–7.31 (3H, m, H-4”, H-5”, H-6”), 7.25
2H, d, J = 7.00 Hz, H-3”, H-7”), 7.12 (1H, t, J = 7.50 Hz,
H-5), 5.55 (2H, s, H-1”a, H-1”b), and 4.26 (2H, s, H-1’a,
Each of the compounds 3 was dissolved in MeOH, then
hydroxylamine hydrochloride (685 mg, 10 mmol) was
added, followed by dropwise addition of a solution of
NaOH (400 mg in 1 mL of water). The mixtures were stirred
at room temperature for 2 h. At the end of this reaction, the
resulting reaction mixtures were poured into ice-cold water,
neutralized to pH ~ 7 and acidified by dropwise addition of
a solution of HCl 5% to induce maximum precipitation. The
precipitates were filtered, dried, and re-crystalised in
methanol to give the targeting compounds 4.
8
(
1
3
H-1’b). C NMR (125 MHz, DMSO-d , ppm): δ 163.34,
6
1
1
3
44.02, 142.46, 139.54, 136.54, 135.14, 129.18, 128.54,
28.20, 126.84, 124.84, 123.33, 118.49, 115.49, 53.26, and
+
7.70. ESI-MS m/z: 353.00 [M + H]
1
2
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-bromoindoline-
,3-dione (3e) Orange solid; Yield: 57%. mp: 203–204 °C.
−
1
R = 0.65 (DCM: MeOH = 20: 1). IR (KBr, cm ): 2985
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(hydroxyimino)
indolin-2-one (4a) Yellow solid; Yield: 60%. mp:
f
1
(
CH, CH ); 1728 (C=O); 1601, 1470 (C=C). H-NMR
2
(
500 MHz, DMSO-d , ppm): 8.20 (1H, s, H-6’), 7.81 (1H,
190–191 °C. R = 0.50 (DCM: MeOH = 20: 1). IR (KBr,
6
f
−
1
dd, J = 8.50 Hz, J’ = 2.00 Hz, H-7), 7.73 (1H, d, J =
.00 Hz, H-4), 7.39–7.32 (3H, m, H-4”, H-5”, H-6”), 7.28
2H, d, J = 7.00 Hz, H-3”, H-7”), 7.13 (1H, d, J = 8.00 Hz,
H-6), 5.56 (2H, s, H-1”a, H-1”b), and 4.97 (2H, s, H-1’a,
cm ): 3119 (OH); 3007 (CH, arene); 2777 (CH, CH );
2
1
2
(
1717 (C=O); 1605, 1456 (C=C). H-NMR (500 MHz,
DMSO-d , ppm): 13.55 (1H, s, N–OH), 8.17 (1H, s, H-6’),
6
8.00 (1H, d, J = 7.00 Hz, H-4), 7.41–7.27 (6H, m, H-7, H-
3”, H-4”, H-5”, H-6”, H-7”), 7.12–7.08 (2H, m, H-5, H-6),
5.55 (2H, s, H-1”a, H-1”b), and 4.99 (2H, s, H-1’a, H-1’b).
1
3
H-1’b). C NMR (125 MHz, DMSO-d , ppm): δ 182.45,
6
1
1
3
57.91, 149.49, 140.13, 136.36, 129.22, 128.92, 128.63,
1
3
28.38, 127.13, 124.26, 119.92, 115.56, 113.77, 53.34, and
C NMR (125 MHz, DMSO-d , ppm): δ 163.31, 142.96,
6
79
81
+
5.66. ESI-MS m/z: 397.00 ( Br), 399.00 ( Br) [M + H]
142.74, 136.45, 132.23, 129.20, 128.59, 128.36, 127.17,
1
24.02, 123.20, 110.06, 53.27, and 35.13. ESI-MS m/z:
+
1
2
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-methylindoline-
334.20 [M + H]
,3-dione (3f) Orange solid; Yield: 70%. mp: 194–195 °C.
−
1
R = 0.67 (DCM: MeOH = 20: 1). IR (KBr, cm ): 3069
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-fluoro-3-(hydro-
xyimino)indolin-2-one (4b) Yellow solid; Yield: 56%. mp:
f
(
CH, arene); 2922 (CH, CH ); 1717 (C=O); 1597, 1487
2
1
(
C=C). H-NMR (500 MHz, DMSO-d , ppm): 8.22 (1H, s,
197–198 °C. R = 0.53 (DCM: MeOH = 20: 1). IR (KBr,
6
f
−
1
H-6’), 7.44–7.28 (7H, m, H-4, H-7, H-4”, H-5”, H-6”, H-3”,
H-7”), 7.04 (1H, d, J = 7.5 Hz, H ), 5.56 (2H, s, H-1”a, H-
cm ): 3161 (OH); 3063 (CH, arene); 2814 (CH, CH );
1711 (C=O); 1614, 1472 (C=C). H-NMR (500 MHz,
2
1
6
1
”b), 4.95 (2H, s, H-1’a, H-1’b), and 2.27 (3H, s, 5-CH3).
DMSO-d , ppm): 13.78 (1H, s, N–OH), 8.17 (1H, s, H-6’),
6

Medicinal Chemistry Research
7
3
.77 (1H, d, J = 7.00 Hz, H-4), 7.38–7.27 (6H, m, H-7, H-
”, H-4”, H-5”, H-6”, H-7”), 7.15–7.10 (1H, m, H-6), 5.55
H-1’a, H-1’b), and 2.29 (3H, s, 5-CH3). 13C NMR (125
MHz, DMSO-d , ppm): δ 136.43, 132.55, 129.20, 128.59,
6
1
3
(
2H, s, H-1”a, H-1”b), and 5.00 (2H, s, H-1’a, H-1’b).
C
128.36, 124.19, 110.05, 53.29, 35.27, and 20.92. ESI-MS
NMR (125 MHz, DMSO-d , ppm): δ 136.44, 129.21,
1
m/z: 348.10 [M + H]+
6
+
28.60, 128.39, and 53.30. ESI-MS m/z: 352.10 [M + H]
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(hydroxyimino)-
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-chloro-3-(hydro-
5-methoxyindolin-2-one (4g) Yellow solid; Yield: 53%.
xyimino)indolin-2-one (4c) Yellow solid; Yield: 55%. mp:
mp: 204–205 °C. R = 0.53 (DCM: MeOH = 20: 1). IR
f
−1
−1
2
13–214 °C. R = 0.55 (DCM: MeOH = 20: 1). IR (KBr, cm ):
(KBr, cm ): 3204 (OH); 2905 (CH, CH ); 1717 (C=O);
f
2
1
3
379 (OH); 3082 (CH, arene); 2805 (CH, CH ); 1715 (C=O);
1614, 1481 (C=C). H-NMR (500 MHz, DMSO-d , ppm):
2
6
1
1607, 1464 (C=C). H-NMR (500 MHz, DMSO-d , ppm): 8.18
13.54 (1H, s, N–OH), 8.15 (1H, s, H-6’), 7.59 (1H, d, J =
2.50 Hz, H-4), 7.36–7.33 (7H, m, H-4”, H-5”, H-6”), 7.27
(2H, d, J = 8.00 Hz, H-3”, H-7”), 7.03–7.00 (2H, m, H-6,
H-7), 5.55 (2H, s, H-1”a, H-1”b), 5.00 (2H, s, H-1’a,
6
(1H, s, H-6’), 7.94 (1H, s, H-4), 7.36–7.27 (7H, m, H-6, H-7, H-
3
5
”, H-4”, H-5”, H-6”, H-7”), 5.55 (2H, s, H-1”a, H-1”b), and
.01 (2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6,
ppm): δ 142.54, 136.40, 129.21, 128.61, 128.38, 124.08,
1
3
H-1’b), and 3.73 (3H, s, 5-OCH3). C NMR (125 MHz,
5
3.31, 36.26, 35.32, and 31.25. ESI-MS m/z: 368.10
DMSO-d , ppm): δ 163.10, 155.71, 144.16, 142.77, 136.70,
136.44, 129.20, 128.60, 128.36, 123.99, 117.34, 116.34,
6
+
[
M + H]
1
13.58, 110.73, 56.14, 53.27, and 35.18. ESI-MS m/z:
+
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-chloro-3-(hydro-
xyimino)indolin-2-one (4d) Yellow solid; Yield: 52%. mp:
19–220 °C. R = 0.55 (DCM: MeOH = 20: 1). IR (KBr,
364.10 [M + H]
2
General procedures for the synthesis of compounds 5
f
−
1
cm ): 3120 (OH); 3009 (CH, arene); 1722 (C=O); 1601,
1
1
439 (C=C). H-NMR (500 MHz, DMSO-d , ppm): 13.87
Compounds 5a–g were synthesized via a three-step pathway
as illustrated in Scheme 1. The procedures were similar to
that described for compound 4 with O-methylhydroxylamine
hydrochloride was used instead of hydroxylamine
hydrochloride.
6
(
1H, s, N–OH), 8.14 (1H, s, H-6’), 8.08 (1H, d, J = 7.00 Hz,
H-4), 7.51 (1H, d, J = 8.00 Hz, H-6), 7.42–7.28 (3H, m, H-
”, H-5”, H-6”), 7.25 (2H, d, J = 7.00 Hz, H-3”, H-7”), 7.12
1H, t, J = 7.75 Hz, H-5), 5.55 (2H, s, H-1”a, H-1”b), and
.33 (2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6,
ppm): δ 164.13, 144.19, 142.67, 138.93, 136.57, 134.25,
4
(
5
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(methoxyimino)
1
1
29.18, 128.54, 128.19, 126.35, 124.74, 123.31, 118.81,
indolin-2-one (5a) Yellow solid; Yield: 58%. mp:
15.27, 53.24, and 37.57. ESI-MS m/z: 368.10 [M + H]+
209–210 °C. R = 0.59 (DCM: MeOH = 20: 1). IR (KBr,
f
−
1
cm ): 3063 (CH, arene); 2903 (CH, CH ); 1717 (C=O);
1607, 1464 (C=C). H-NMR (500 MHz, DMSO-d , ppm):
2
1
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-bromo-3-
6
(
5
hydroxyimino)indolin-2-one (4e) Yellow solid; Yield:
5%. mp: 228–229 °C. R = 0.56 (DCM: MeOH = 20: 1).
8.18 (1H, s, H-6’), 7.89 (1H, d, J = 2.50 Hz, H-4), 7.44 (1H,
d, J = 7.50 Hz, H-6), 7.37–7.32 (3H, m, H-4”, H-5”, H-6”),
7.27 (2H, d, J = 8.50 Hz, H-3”, H-7”), 7.13 (1H, d, J =
8.00 Hz, H-7), 7.10 (1H, t, J = 7.50 Hz, H-5), 5.55 (2H, s,
H-1”a, H-1”b), 4.98 (2H, s, H-1’a, H-1’b), and 4.22 (3H, s,
f
−
1
IR (KBr, cm ): 3163 (OH); 3082 (CH, arene); 2855 (CH,
CH ); 1717 (C=O); 1603, 1466 (C=C). H-NMR (500
MHz, DMSO-d , ppm): 13.83 (1H, s, N–OH), 8.18 (1H, s,
H-6’), 8.11 (1H, s, H-4), 7.62–7.13 (7H, m, H-6, H-7, H-3”,
H-4”, H-5”, H-6”, H-7”), 5.55 (2H, s, H-1”a, H-1”b), and
1
2
6
1
3
NOCH3). C NMR (125 MHz, DMSO-d , ppm): δ 162.45,
6
143.72, 143.69, 142.53, 136.42, 133.33, 129.20, 128.60,
128.36, 127.79, 124.06, 123.38, 115.47, 110.41, 65.01,
53.29, and 35.26. ESI-MS m/z: 348.10 [M + H]+
5
.00 (2H, s, H-1’a, H-1’b). 13C NMR (125 MHz, DMSO-d6,
ppm): δ 142.20, 136.40, 134.61, 129.20, 128.60, 128.37,
24.09, 112.28, 53.31, and 35.31. ESI-MS m/z: 412.10
1
(
7
9
81
+
Br), 414.10 ( Br) [M + H]
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-fluoro-3-(meth-
oxyimino)indolin-2-one (5b) Yellow solid; Yield: 55%.
1
5
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(hydroxyimino)-
-methylindolin-2-one (4f) Yellow solid; Yield: 63%. mp:
mp: 215–216 °C. R = 0.60 (DCM: MeOH = 20: 1). IR
f
−
1
(KBr, cm ): 2984 (CH, arene); 2901 (CH, CH ); 1711
2
1
95–196 °C. R = 0.54 (DCM: MeOH = 20: 1). IR (KBr,
(C=O); 1599, 1476 (C=C). H-NMR (500 MHz, DMSO-d ,
f
6
−
1
cm ): 3123 (OH); 3082 (CH, arene); 2833 (CH, CH );
1
DMSO-d , ppm): 13.44 (1H, s, N–OH), 8.17 (1H, s, H-6’),
8
ppm): 8.18 (1H, s, H-6’), 7.70 (1H, dd, J = 8.25 Hz, J’ =
2.50 Hz, H-4), 7.44–7.31 (4H, m, H-4, H-4”, H-5”, H-6”),
7.27 (2H, d, J = 8.00 Hz, H-3”, H-7”), 7.15 (1H, dd, J =
8.50 Hz, J’ = 4.00 Hz, H-6), 5.55 (2H, s, H-1”a, H-1”b),
2
1
717 (C=O); 1616, 1472 (C=C). H-NMR (500 MHz,
6
.11 (1H, s, H-4), 7.34–7.03 (7H, m, H-6, H-7, H-3”, H-4”,
H-5”, H-6”, H-7”), 5.55 (2H, s, H-1”a, H-1”b), 5.00 (2H, s,
4.99 (2H, s, H-1’a, H-1’b), and 4.24 (3H, s, NOCH3). 13
C

Medicinal Chemistry Research
NMR (125 MHz, DMSO-d , ppm): δ 162.32, 159.45,
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(methoxyi-
mino)-5-methylindolin-2-one (5f) Yellow solid; Yield:
6
1
1
1
57.55, 143.42, 142.40, 140.09, 136.41, 129.21, 128.61,
28.37, 124.08, 119.67, 119.48, 116.01, 115.93, 114.93,
62%. mp: 218–219 °C. R = 0.64 (DCM: MeOH = 20: 1).
f
−
1
14.72, 111.55, 111.48, 65.25, 53.30, and 35.39. ESI-MS
IR (KBr, cm ): 2986 (CH, arene); 2903 (CH, CH ); 1713
(C=O); 1614, 1483 (C=C). H-NMR (500 MHz, DMSO-d ,
2
+
1
m/z: 366.10 [M + H]
6
ppm): 8.16 (1H, s, H-6’), 7.73 (1H, s, H-4), 7.37–7.24 (6H,
m, H-7, H-3”, H-4”, H-5”, H-6”, H-7”), 7.01 (1H, d, J =
8.00 Hz, H-6), 5.55 (2H, s, H-1”a, H-1”b), 4.95 (2H, s, H-
1’a, H-1’b), 4.22 (3H, s, NOCH ), and 2.28 (3H, s, 5-CH ).
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-chloro-3-(meth-
oxyimino)indolin-2-one (5c) Yellow solid; Yield: 54%.
mp: 224–225 °C. R = 0.63 (DCM: MeOH = 20: 1). IR
f
3
3
−
1
13
(
KBr, cm ): 2986 (CH, arene); 2901 (CH, CH ); 1711
C NMR (125 MHz, DMSO-d , ppm): δ 162.46, 143.82,
2
6
1
(
C=O); 1605, 1468 (C=C). H-NMR (500 MHz, DMSO-d ,
142.62, 141.49, 136.43, 133.52, 132.47, 129.20, 128.60,
128.36, 128.24, 124.03, 115.52, 110.18, 64.96, 53.28,
35.26, and 20.93. ESI-MS m/z: 362.10 [M + H]+
6
ppm): 8.18 (1H, s, H-6’), 7.88 (1H, d, J = 2.00 Hz, H-4),
.53 (1H, dd, J = 8.50 Hz, J’ = 2.00 Hz, H-7), 7.37–7.32
3H, m, H-4”, H-5”, H-6”), 7.27 (2H, d, J = 8.00 Hz, H-3”,
7
(
H-7”), 7.17 (1H, d, J = 8.50 Hz, H-6), 5.55 (2H, s, H-1”a,
H-1”b), 5.00 (2H, s, H-1’a, H-1’b), and 4.25 (3H, s,
1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-methoxy-3-
(methoxyimino)indolin-2-one (5g) Yellow solid; Yield:
1
3
NOCH3). C NMR (125 MHz, DMSO-d , ppm): δ 162.12,
50%. mp: 224–225 °C. R = 0.62 (DCM: MeOH = 20: 1).
6
f
−
1
1
1
5
42.93, 142.50, 142.31, 136.39, 132.56, 129.21, 128.61,
IR (KBr, cm ): 3007 (CH, arene); 2812 (CH, CH ); 1719
(C=O); 1597, 1477 (C=C). H-NMR (500 MHz, DMSO-d ,
2
1
28.38, 112.21, 127.05, 124.08, 116.64, 112.02, 65.33,
6
+
3.31, and 35.42. ESI-MS m/z: 382.10 [M + H]
ppm): 8.16 (1H, s, H-6’), 7.88 (1H, s, H-4), 7.36–7.35 (3H,
m, H-4”, H-5”, H-6”), 7.27 (2H, d, J = 8.00 Hz, H-3”, H-
7”), 7.05–7.03 (2H, m, H-6, H-7), 5.55 (2H, s, H-1”a, H-
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-chloro-3-(meth-
oxyimino)indolin-2-one (5d) Yellow solid; Yield: 50%.
1”b), 4.95 (2H, s, H-1’a, H-1’b), 4.22 (3H, s, NOCH ), and
3
1
3
mp: 229–230 °C. R = 0.63 (DCM: MeOH = 20: 1). IR
3.74 (3H, s, 5-OCH3). C NMR (125 MHz, DMSO-d6,
ppm): δ 162.31, 155.77, 143.93, 142.60, 137.31, 136.43,
129.21, 128.61, 128.37, 124.02, 118.13, 116.02, 114.15,
111.04, 65.08, 56.50, 56.23, 53.27, and 35.29. ESI-MS m/z:
378.10 [M + H]+
f
−
1
(
(
KBr, cm ): 2986 (CH, arene); 2903 (CH, CH ); 1713
C=O); 1603, 1441 (C=C). H-NMR (500 MHz, DMSO-
2
1
d6, ppm): 8.15 (1H, s, H-6’), 7.96 (1H, dd, J = 7.50 Hz,
J’ = 1.00 Hz, H-4), 7.45 (1H, dd, J = 8.25 Hz, J’ =
1
.25 Hz, H-6), 7.37–7.32 (3H, m, H-4”, H-5”, H-6”), 7.25
(
2H, d, J = 8.00 Hz, H-3”, H-7”), 7.13 (1H, t, J =
AChE inhibition assay
7
1
.75 Hz, H-5), 5.55 (2H, s, H-1”a, H-1”b), 5.31 (2H, s, H-
’a, H-1’b), and 4.26 (3H, s, NOCH3). 13C NMR (125
Evaluation of the AChE inhibition activities was conducted
using the previously reported Ellman’s method (Ellman
et al. 1961) with slight modifications. In brief, to 60 μL of
50 mM NaHPO4 buffer (pH 7.7) were added 10 μL of
respective assayed sample (at stock solution of 0.5 mM).
Then, 10 μL of enzyme (0.005 unit enzyme per well) was
added. The resulting contents were mixed and preread at
405 nm, then the contents were preincubated for 10 min at
37 °C. The reaction in each well was initiated by the addi-
tion of 10 μL of 0.5 mM substrate (acetylthiocholine iodide
or butyrylthiocholine bromide) to each well, followed by
the addition of 10 μL DTNB (0.5 mM per well). The wells
were incubated for at 37 °C, then the absorbance of each
well was measured at 405 nm using 96-well plate reader
Synergy HT, Biotek, USA. All experiments were carried
out in triplicate. Eserine (0.5 mM) was used as a positive
control. The inhibition percentages were calculated by the
following formula:
MHz, DMSO-d , ppm): δ 163.35, 144.02, 142.48, 139.57,
1
1
6
36.55, 135.16, 129.18, 128.54, 128.20, 126.85, 124.86,
23.33, 118.51, 115.49, 65.41, 53.26, and 37.72. ESI-MS
+
m/z: 382.10 [M + H]
1
-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-bromo-3-(meth-
oxyimino)indolin-2-one (5e) Yellow solid; Yield: 54%.
mp: 234–235 °C. R = 0.65 (DCM: MeOH = 20: 1). IR
f
−
1
(
KBr, cm ): 2986 (CH, arene); 2901 (CH, CH ); 1719
2
1
(
C=O); 1603, 1465 (C=C). H-NMR (500 MHz, DMSO-d ,
6
ppm): 8.18 (1H, s, H-6’), 8.00 (1H, d, J = 1.50 Hz, H-4),
.53 (1H, dd, J = 8.50 Hz, J’ = 2.00 Hz, H-7), 7.37–7.32
3H, m, H-4”, H-5”, H-6”), 7.27 (2H, d, J = 8.00 Hz, H-3”,
7
(
H-7”), 7.12 (1H, d, J = 8.50 Hz, H-6), 5.55 (2H, s, H-1”a,
H-1”b), 4.99 (2H, s, H-1’a, H-1’b), and 4.25 (3H, s,
NOCH3). C NMR (125 MHz, DMSO-d , ppm): δ 162.01,
1
1
5
1
3
6
42.89, 142.80, 142.30, 136.39, 135.59, 129.72. 129.21,
28.61, 128.37, 124.07, 117.08, 114.84, 112.51, 65.35,
ControlAbs À SampleA
79
81
bs
3.31, and 35.40. ESI-MS m/z: 426.10 ( Br), 428.10 ( Br)
Inhibition Percentage ¼
 100
ControlAbs
+
[
M + H]

Medicinal Chemistry Research
IC50 values (in case measured) were calculated using
EZ–Fit Enzyme kinetics software (Perrella Scientific Inc.
Amherst, USA).
of synthesized compounds were built using the Builder
module of Molecular Operating Environment (MOE) 2019.01
(MOE 2019; Tung et al. 2013; Nam et al. 2014). They were
minimized using molecular mechanic force field (MM+)
option in order to remove all strain from the molecular
structures. Docking assays were performed based on the
search algorithm Monte-Carlo implemented into Internal
Coordinates Mechanics (ICM) package, keeping the protein
rigid and the ligands flexible (Arthur and Uzairu 2019). To
quantifying the binding affinity, ICM scoring function was
computed based on the parameters: (i) internal force-field
energy of the ligand, (ii) entropy loss of the ligand between
bound and unbound states, (iii) ligand-receptor hydrogen
bond interactions, (iv) polar and nonpolar solvation energy
differences between bound and unbound states, (v) electro-
static energy, (vi) hydrophobic energy, and (vii) hydrogen
bond donor or acceptor desolvation (Huang and Mucke 2012;
Neves et al. 2012). The lower the ICM score (kCal/mol), the
higher the chance the ligand is a binder. Biovia DS visualiser
was utilized to show up the potential interactions of the
ligands to the residues in the binding sites of AChE enzyme
(Dassault Systèmes BIOVIA 2016).
DPPH radical-scavenging assay
DPPH radical-scavenging activity was measured according to
a described method (Thuong et al. 2006). Briefly, 5 µl of each
sample dissolved in MeOH were added to 195 µl of 150 µM
methanolic DPPH in 96-well plates. The solution was mixed
for 1 min and incubated at room temperature in a dark place.
After 30 min, the absorbance of the reaction mixture was
measured at 520 nm on a microplate reader. The scavenging
activity was expressed as the degree of radical reduction of a
test group, in comparison with that of the control.
Cytotoxicity assay
Compounds were initially dissolved in dimethyl sulfoxide
(
DMSO) and diluted to appropriate concentrations by cul-
ture medium. 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 Dulbecco’s Modified Eagle Medium until
confluence. The cells were then trypsinized and suspended
Results and discussions
4
at 3 × 10 cells/mL of cell culture medium. On day 0, each
well of the 96-well plates was seeded with 180 μL of cell
suspension. The plates were then incubated in a 5% CO2
incubator at 37 °C for 24 h. 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 SRB colorimetric method, as described previously
Chemistry
The designed compounds 3a–g were synthesized by a two-
step pathway as illustrated in Scheme 1. A nucleophilic
substitution between propargyl bromide and isatins (1a–g)
was proceeded smoothly in dimethylformamide (DMF)
under basic conditions (K CO ). A catalytic amount of KI
and gentle heating (60 °C) afforded the intermediates 2a–g
in quantitative yields. In the next step, a Click reaction
between the propargyl intermediates 2a–g and azido-
methylbenzene resulted in the target compounds 3a–g in
relatively good yields (55–70%). Condensation of com-
pounds 3a–g with hydroxylamine.HCl or methoxylamine.
HCl in methanol (MeOH) at room temperature gave com-
pounds 4a–g and 5a–g, respectively, in moderate yields.
The structures of the synthesized compounds were
determined straightforwardly based on analysis of spectro-
2
3
(
Skehan et al. 1990) with slight modifications (Ye et al.
2
2
007; Thuong et al. 2006; Kim et al. 2002; Nam et al.
004). The IC50 values were calculated using a Probits
method (Wu et al. 1992) and were averages of three inde-
pendent determinations (SD ≤ 10%).
Docking studies
1
13
1
Molecular docking was carried out with the 3D X-ray crystal
structure of the target enzyme human AChE downloaded
from the Protein Data Bank (PDB ID: 4EY7) (Cheung et al.
scopic data, including IR, MS, H and C NMR. In the H-
NMR spectra of all compounds, there were always two
singlets, each interpreted for 2H, appeared around 5.00 and
5.50 ppm. These singlets were from two methylene groups
2012). The target structure was imported into the Molsoft.
1
icm-pro.v3.8.3 GUI (http://www.molsoft.com/icmpro/) (An
et al. 2005; Neves et al. 2012); then the co-ligand Donepezil
and water molecules were removed from the protein complex.
After that the hydrogens and other amino acids such as His,
Pro, Gly, Cys were optimized and the missing side chains
were also fixed (Arthur and Uzairu 2019). The 3D structures
in the structures. Also in the H-NMR spectra of all com-
pounds, there appeared a singlet at around 8.15–8.18 ppm,
interpreted for 1H, which was distinguished for the lone
aromatic proton on the triazole ring. All other evidences
from IR, MS, H, and C NMR spectra confirmed the
expected structures of compounds 3a–g, 4a–g, and 5a–g.
1
13

Medicinal Chemistry Research
Bioactivity
order of AChE inhibitory potency was found to be 4g (51%)
>
3g (40%) >5g (26%).
AChE inhibitory activity
When the assay concentration was lowered to 50 μM, it
was found that only compound 4g showed significant
inhibitory activity with an inhibition percentage of 35% in
relation to the positive control donepezil. Some other
compounds, such as 3a, 3b, 3e, 3f, 3g, and 4e exhibited
only minimal inhibition with inhibition percentages
of ~11–16%.
The synthesized compounds 3a–g, 4a–g, and 5a–g were
screened for their inhibitory effects on AChE at two con-
centrations of 50 and 100 μM. The acetylcholinesterase
inhibitor screening kit (colorimetric, BioVision (catalog
#
K197-100)) was used. The detection was kinetically
measured at 412 nm over 40 min. The enzyme inhibition
percentages in comparison with donepezil, which was used
as a positive control, at the final point were presented in Fig. 3.
It was found that at the concentrations of 100 μM, 12 out of
The obtained results above suggest that the designed
structures 3a–g, 4a–g, and 5a–g, were, though still not
strong AChE inhibitors, did exhibit AChE inhibition
potentials. Further structural manipulation on the benzyl
moiety or substitution patterns at positions 3 or 5 on the 2-
oxoindoline could potentially produce more novel and
potent AChE inhibitors.
2
1 compounds showed significant inhibition percentages.
Compounds 4g and 3a were the most potent in the inhibi-
tion of AChE with inhibition percentages of 51% and 50%,
respectively. Compounds 3e, 3f, and 3g were among the
next most potent with inhibition percentages of 38%, 43%,
and 40%, respectively. Compounds 3b and 3c were slightly
less potent with inhibition percentages of only 31% and
DPPH antioxidant activity
Since the antioxidant properties would be an added value to
treat AD, the free radical-scavenging abilities of the syn-
thesized compounds against DPPH were screened and the
results were summarized in Table 1. The compounds were
tested at the intial concentrations of 100 µg/mL. It was
found that only two compounds, including 3a and 3f, dis-
played moderate free radical quenching effects in DPPH
assay with the inhibition percentages of free radical for-
mation of 38.54 and 37.54%. Several other compounds,
including 3d, 3e, 3g, 4a, 4d, 4g, and 5d showed weak
activity. Other compounds exhibited no quenching ability at
the assayed concentrations. The results indicate that these
structure were not potential as free radical scavengers.
2
8%, respectively. In overall, the compounds in series 3a–g
appeared to be more potent than those in series 4a–g and
a–g. Introduction of the 3-oxime group on the 2-
5
oxoindoline moiety seemed to lower the AChE inhibitory
activity. Meanwhile the 3-methoxime functionality on this
2
-oxoindoline system greatly reduce the bioactivity of the
resulting compounds 5a–g. Exception was observed with
compounds 3g, 4g, and 5g, which were substituted with the
methoxy group at position 5 on the 2-oxoindoline ring. The
Cytotoxicity against some cancer cell lines
With the available in-house cytotoxic screening system, the
synthesized compounds were also screened for their cyto-
toxicity against three human cancer cell lines, which
included SW620 (colorectal adenocarcinoma), PC3 (pros-
tate adenocarcinoma), and NCI-H23 (adenocarcinoma, non-
small cell lung cancer) using SRB method. 5-FU (5-fluor-
ouracil), and ADR (adriamycin) were used as positive
controls. The results are presented in Table 2. It was very
interesting that we found five compounds, including 3c, 3e,
5
c, 5e, and 5g, with potent cytotoxicity against all three
cancer cell lines tested (IC50 values in the range of
.65–7.17 µM). Substitution of the chloro or bromo groups
0
at position 5 seemed to be favorable for cytotoxicity,
meanwhile the same chloro substitutent at position 7 led to
compounds (3d and 5d) with much weaker cytotoxicity
(IC50 values in the range of 13.56–21.11 µM). The oxime
Fig. 3 Relative inhibition of AChE by the compounds synthesized.
a At concentrations of 50 µM. b At concentrations of 100 µM
series (4a–g) were least cytotoxic among three series; even
two compounds 4c and 4e also displayed much higher IC50

Medicinal Chemistry Research
Table 1 DPPH scavenging activity of the synthesized compounds
Cpd
R
% inhibitiona
Cpd
R
% inhibition
Cpd
R
% inhibition
3
3
3
3
3
3
3
a
b
c
d
e
f
H
38.54
b
4a
H
14.95
4.32
4.65
13.29
5.98
–
5a
H
–
5-F
4b
5-F
5b
5-F
–
5-Cl
7-Cl
5-Br
5-CH3
5-OCH3
–
4c
5-Cl
7-Cl
5-Br
5-CH3
5-OCH3
5c
5-Cl
7-Cl
5-Br
5-CH3
5-OCH3
–
7.97
4d
5d
9.63
16.94
37.54
16.28
87.71
4e
5e
–
4f
5f
–
g
4g
18.27
87.71
5g
–
Quercetin
Quercetin
Quercetin
87.71
aInhibition percentages at the concentration of 100 µg/mL
bNegligible inhibition
values compared with that of compounds 3c, 3e and 5c, 5e.
Strikingly, compound 5g with 5-methoxy substituent
showed the most potent cytotoxicity with IC50 values
similar to or even lower than that of adriamycin. Surpris-
ingly, the corresponding compounds in series 3 and 4
PI, and their nuclei stain red. So, severe and late apop-
totic 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 compound at 50 µM for 24 h and
stained the cells with Annexin V-FITC and PI. PAC-1
(the first procaspase activating compound) was used as a
positive control. The results shown in Fig. 4 clearly
indicate that in U937 cells, compound 5g only moder-
ately increased late apoptotic cells but strongly induced
necrotic cells. The positive control, PAC-1, meanwhile
strongly induced U937 cells into late stage of apoptosis
(
compounds 3g and 4g) exhibited only moderate cytotoxi-
city. Thus, the substituents as well as substituent’s positions
seemed to play very delicate effects on the cytotoxicity. It
was very noteworthy to find a simple structure 5g with such
potent cytotoxicity and this compound would serve as an
important lead for further design of more potential antic-
ancer agents.
(
45.4%). In SW620 cells the effects of compound 5g was
seen differently. Compound 5g caused necrosis in only
1.22% of cell population. Morphologically, compound
Analysis of apoptosis of compound 5g
1
From the SRB assay 5g emerged as the most cytotoxic
compound three human cancer cell lines, even more
potent than ADR (adriamycin). With its interesting
cytotoxicity, we decided to perform an Annexin V-FITC/
propidium iodide (PI) dual staining assay to see whether
compounds 5g can induce apoptosis. During early
apoptosis, phosphatidylserine (PS) locates on the cyto-
solic (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
labeled with fluorescein isothiocyanate (FITC), it can be
used to identify early-stage apoptotic cells. PI is a
fluorescent intercalating agent that cannot cross the
membrane of live cells. The membranes of cells in the
later stages of apoptosis and dead cells are permeable to
5g caused SW620 cells flatten in almost a similar manner
PAC-1 did (Fig. 5).
Docking studies against AChE
From the synthesized compounds, 3a and 4g showed
acceptable inhibition activity against AChE enzyme.
Therefore we decided to use molecular docking simulations
to get more insight into structure–activity relationships of
these derivatives through the binding domain of AChE-
Donepezil complex (Cheung et al. 2012). Docking protocol
was validated by redocking the co-crystallized Donepezil
with the enzyme target. Root-mean square deviation
(RMSD) was used to evaluate how different the redocked
configuration is from the corresponding co-crystal

Medicinal Chemistry Research
Table 2 Cytotoxicity of the compounds against some human cancer cell lines
Cpd
R
MW
LogPa
Cytotoxicity (IC50, b μM)/Cell linesc
SW620
PC3
NCI-H23
3
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
a
b
c
H
318.2
336.1
352.1
352.1
398.1
332.1
348.1
333.2
351.1
367.1
367.1
413.1
347.1
363.1
347.1
365.1
381.1
381.1
427.1
361.1
377.1
130.08
543.5
392.49
1.66
1.86
2.31
2.31
2.55
2.21
1.74
3.74
3.94
4.39
4.39
4.63
4.29
3.82
3.71
3.92
4.36
4.36
4.61
4.26
3.80
−0.81
1.27
3.43
19.54 ± 1.78
13.32 ± 2.11
3.93 ± 0.52
17.43 ± 1.45
3.49 ± 0.06
25.25 ± 1.87
27.43 ± 2.09
25.46 ± 2.11
13.47 ± 1/56
10.55 ± 1.01
17.21 ± 1.90
9.29 ± 0.81
21.01 ± 1.90
15.93 ± 1.42
>30
>30
>30
5-F
15.85 ± 2.56
7.17 ± 0.14
14.22 ± 1.31
3.68 ± 0.07
28.43 ± 2.19
23.21 ± 2.00
>30
11.54 ± 1.98
5.35 ± 0.07
13.56 ± 1.76
4.30 ± 1.11
>30
5-Cl
7-Cl
5-Br
5-CH3
5-OCH3
H
d
e
f
g
a
b
c
20.14 ± 1.90
>30
5-F
15.32 ± 1.78
12.21 ± 1.19
19.20 ± 1.89
13.21 ± 1.22
23.54 ± 1.32
16.23 ± 1.45
25.31 ± 2.16
20.11 ± 1.95
2.62 ± 0.61
17.21 ± 1.67
1.59 ± 0.21
9.34 ± 0.74
1.11 ± 0.13
13.61 ± 0.46
1.16 ± 0.28
4.16 ± 0.52
19.32 ± 1.99
11.53 ± 1.13
23.33 ± 2.01
15.43 ± 1.31
20.12 ± 1.97
18.31 ± 1.75
27.33 ± 2.65
22.43 ± 2.03
2.87 ± 0.29
15.23 ± 1.35
1.40 ± 0.20
14.32 ± 1.21
0.99 ± 0.10
13.45 ± 3.92
1.29 ± 0.12
5.32 ± 0.21
5-Cl
7-Cl
5-Br
5-CH3
5-OCH3
H
d
e
f
g
a
b
c
5-F
18.31 ± 1.56
2.32 ± 0.55
21.11 ± 2.05
1.49 ± 0.02
10.31 ± 0.92
0.65 ± 0.03
8.84 ± 1.92
1.12 ± 0.14
5.82 ± 0.20
5-Cl
7-Cl
5-Br
5-CH3
5-OCH3
d
e
f
g
-FUd
ADRe
PAC-1f
aCalculated by ChemDraw 9.0 software
bThe concentration (μM) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent the averaged
results from triplicate experiments with deviation of <10%
cCell lines: SW620, colon cancer; PC3, prostate cancer; NCI-H23, lung cancer
d5-FU: 5-Fluorouracil, a positive control
e5-ADR: Adriamycin, a positive controle
fPAC-1: The first procaspase-3 activating compound, a positive control
Donepezil. As the results, redocked structure of this drug is
highly overlapped with the co-crystal ligand (RMSD =
(PAS) of AChE, such as π–π interactions with Trp286 and
H-bonds with Tyr72, Ser293, and Tyr341. The piperazine
ring interacts with acyl-binding pocket through multiple π–
π interactions with Tyr337 and Phe338 side chains. Mean-
while the phenyl moiety of Donepezil mainly interact with
the choline-binding site and forms strong π–π stacking
interactions against Trp86 aromatic side chain. These results
0
.22 Å) and docking score was −25.28 kCal/mol (Fig. 6). In
addition, the redocked and co-crystal Donepezil showed
similar interactions with the binding site of AChE. The 5,6-
dimethoxy-1-indanone moiety of the drug forms multiple
interactions with the residues at the peripheral anionic site

Medicinal Chemistry Research
Fig. 4 Apoptosis (Annexin V/PI) analysis of compound 5g. U937 (a)
or SW620 (b) cells were treated with compounds (50 μM) for 24 h.
The harvested cells were incubated with Annexin V-FITC and PI. UN
untreated, VH vehicle (DMSO. 0.05%). PAC-1 is a positive control
(the first procaspase activating compound). Area 1: PI positive popu-
lation, Area 2: Annexin V-positive population
Fig. 5 Morphology changes of
cells treated with compound 5g
or PAC-1. SW620 2.5 × 105
cells/ml seeding (500 ml in 24
well) were incubated for 24 h,
then compound 5g or PAC-1
(
50 μM) were added and
incubated further for 24 h. Then,
the cells were photographed
using an Imaging Device
(
celldiscoverer7) with ×40 lens.
Scale bar: 50 mm. PAC-1, the
first procaspase-3 activating
compound, was used as a
positive control

Medicinal Chemistry Research
are consistent with those previously published (Özdemir
et al. 2017; Semenov et al. 2015).
Applying the same docking procedures, we found that
compounds 3a and 4g could engage some characteristic
interactions previously determined for Donepezil with
AChE enzyme. The binding affinity of 3a and 4g was
Donepezil. This results match well with the experimental
values. From the docked orientations of these derivatives, as
shown in Fig. 7, with comparison with the isatin hetero-
cyclic systems in 3a, the 1-oxindole heterocyclic systems in
4g showed higher interactions with the residues in the PAS
and acyl pocket of AChE. Compound 4g provided multiple
stacking interactions with Trp286 and three H-bonds with
Phe295, Arg296, and Tyr341. The triazole ring of our
derivatives show extensive hydrophobic interactions against
Phe295 and Tyr341, similar to the interactions provided by
the piperazine moiety of Donepezil. In addition, strong H-
bond and van der Waals interactions were formed between
the 1-benzyl-1H-1,2,3-triazole moiety towards Tyr124,
suggesting the different conformations in the binding site of
synthesized derivatives from the Donepezil structure, as
shown in Fig. 7.
−
19.34 and −21.53 kCal/mol, slightly lower than that of
Conclusions
In conclusion, we have reported three series of indolin-2-
one derivatives incorporating 1-((1-benzyl-1H-1,2,3-tria-
zole moiety and evaluated their biological activities,
including AChE inhibition, DPPH free scavenging effects
and cytototoxicity against three human cancer cell lines
(SW620, human colon cancer; PC3, prostate cancer; NCI-
H23, lung cancer). Two compounds 4g and 3a were the
most potent in inhibition of AChE with inhibition percen-
tages of 51 and 50% when tested at the concentration of
Fig. 6 Superposition of co-crystal (green) and redocked (yellow)
Donepezil inside active site of AChE PDB ID 4EY7 structure
100 μM. Docking studies with the binding domain of
Fig. 7 Docked positions of compound 3a (a) and 4g (b) inside active site of AChE PDB ID 4EY7 structure

Medicinal Chemistry Research
AChE-Donepezil complex suggested that these compound
strictly bound to AChE but with lower affinity compared
with the Donepezil molecule. In DPPH free radical-
scavenging assay, most compounds showed weak activity.
Noteworthy, five compounds, including 3c, 3e, 5c, 5e, and
Molecular Operating Environment (MOE) 2019.01 chemical com-
puting group ULC (2019) 1010 Sherbooke St. West, Suite #910,
Montreal, QC, Canada, H3A 2R7
Nam NH, Huong TL, Dung DTM, Dung PTP, Oanh DTK, Park SH,
Kim K, Han BW, Yun J, Kang JS, Kim Y, Han SB (2014)
Synthesis, bioevaluation and docking study of 5-sub-
stitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as his-
tone deacetylase inhibitors and antitumor agents. J Enzym Inhib
Med Chem 29(5):611–618
Nam NH, Pitts RL, Sun S, Sardari S, Tiemo A, Xie M, Yan B, Parang
K (2004) Design of tetrapeptide ligands as inhibitors of the Src
SH2 domain Bioorg Med Chem 12(4):779–787
Neves MA, Totrov M, Abagyan R (2012) Docking and scoring with
ICM: the benchmarking results and strategies for improvement. J
Comput Aided Mol Des 26(6):675–686
5
g, exhibited strong cytotoxicity against three human cancer
cell lines. Compound 5g was the most potent one with IC50
values as low as 0.65 μM, even more potent than Adria-
mycin, a positive control. Compound 5g should be pro-
mising for further development as an anticancer agent.
Acknowledgements We acknowledge the principal financial supports
from the National Foundation for Science and Technology of Vietnam
Olivero G, Grilli M, Chen J, Preda S, Mura E, Govoni S, Marchi M
(
NAFOSTED, Grant number 104.01-2018.301). The work was also
(
2014) Effects of soluble β-amyloid on the release of neuro-
transmitters from rat brain synaptosomes. Front Aging Neurosci
:166
partly supported by a grant funded by the Korean Government (NRF,
Grant number 2017R1A5A2015541).
6
Özdemir Z, Yılmaz H, Sarı S, Karakurt A, Şenol FS, Uysal M (2017)
Design, synthesis, and molecular modeling of new 3(2H)-pyr-
idazinone derivatives as acetylcholinesterase/butyrylcholinester-
ase inhibitors. Med Chem Res 26(10):2293–2308
Rodda J, Carter J (2012) Cholinesterase inhibitors and memantine for
symptomatic treatment of dementia. BMJ 344:e2986
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Semenov VE, Zueva IV, Mukhamedyarov MA, Lushchekina SV,
Kharlamova AD, Petukhova EO, Mikhailov AS, Podyachev SN,
Saifina LF, Petrov KA, Minnekhanova OA, Zobov VV, Nikolsky
EE, Masson P, Reznik VS (2015) 6-Methyluracil derivatives as
bifunctional acetylcholinesterase inhibitors for the treatment of
Alzheimer’s disease. ChemMedChem 10(11):1863–1874
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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