Design, synthesis and biological evaluation of novel 1,5-disubstituted isatin derivatives as antitumor agents

Article abstract of DOI:10.1007/s00044-020-02627-z

Isatin (1H-indole-2,3-dione) was reported to possess anticancer activities through its effect on tumor proliferation, apoptosis, and metastasis in vitro and in vivo. Here, we described the synthesis of a novel series of 1,5-disubstituted isatin derivative

Full text of DOI:10.1007/s00044-020-02627-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02627-z
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of novel
1,5-disubstituted isatin derivatives as antitumor agents
Huijun Zhuo1 Zhen Zhang1 Yang Liu2 Jingya Zhang1 Guisen Zhao1
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Received: 10 July 2020 / Accepted: 29 August 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Isatin (1H-indole-2,3-dione) was reported to possess anticancer activities through its effect on tumor proliferation, apoptosis,
and metastasis in vitro and in vivo. Here, we described the synthesis of a novel series of 1,5-disubstituted isatin derivatives
with 2-indolinone scaffold as antitumor agents. Most of the synthesized compounds revealed potent antiproliferative effects
in mantle cell lymphoma (MCL) cell lines, among which 7l possessed promising activities with IC50 values ranging from 0.4
to 1.3 μM. Following flow cytometric analysis, compound 7l efficiently arrested the cell cycle at G2/M phase, and induced
apoptosis. Thus, this study shows promise in therapeutics of 1,5-disubstituted isatin derivatives in MCL and provides novel
potential and efficient antitumor agents.
Graphical Abstract
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Keywords Apoptosis Isatin derivatives Mantle cell lymphoma Antitumor
Introduction
machinery is a hallmark of cancer [7, 8]. Mutagenic inac-
tivation of apoptotic proteins and overexpression of anti-
apoptotic proteins have been found in a variety of cancers,
which are the cause of cell proliferation [5, 7, 9].
Apoptosis, an evolutionary highly conserved form of pro-
grammed cell death, refers to the orderly death of cells
controlled by genes in order to maintain a stable internal
environment [1, 2]. Apoptosis can be triggered by extrinsic
and intrinsic death receptors, and the intrinsic pathway is
closely regulated by the B-cell lymphoma 2 (Bcl-2) family
of intracellular proteins [3–6]. Escaping apoptotic cell death
Mantle cell lymphoma (MCL) is a highly aggressive
form of non-Hodgkin-lymphoma (NHL) with a median
survival of ~3–5 years and accounts for 6–8% of NHL
[10–12]. In spite of the low incidence rate, MCL is con-
sidered to be incurable in clinic due to poor prognosis and
limited survival [13]. Increased expression of antiapoptotic
proteins BCL-2, MCL-1, and Bcl-xL, and loss of proa-
poptotic proteins BIM is common in MCL [14–16].
Accumulating evidence suggests that the pathogenetic
processes of MCL is related to impaired apoptosis reg-
ulation [14, 17–19]. Targeting antiapoptotic molecules to
induce apoptosis is emerging as a promising therapeutic
strategy in MCL.
* Guisen Zhao
guisenzhao@sdu.edu.cn
1
Department of Medicinal Chemistry, Key Laboratory of Chemical
Biology (Ministry of Education), School of Pharmaceutical
Sciences, Cheeloo College of Medicine, Shandong University,
Jinan, 250012 Shandong, PR China
2
Isatin (1H-indole-2,3-dione, Fig. 1) and its derivatives
can participate in a variety of biological activities, which
Department of Lymphoma and Myeloma, The University of Texas
MD Anderson Cancer Center, Houston, TX 77030, USA

Medicinal Chemistry Research
Fig. 1 Design of the target
compounds
Scheme 1 Reagents and
conditions: a fuming HNO3,
concentrated H2SO4, 0 °C 1 h;
b 2,2-dimethyl-1,3-propanediol,
p-toluenesulfonic acid,
cyclohexane, 85 °C, 16 h; c 1,2-
dichloro-4-(chloromethyl)
benzene, K2CO3, DMF, 85 °C,
1.5 h; d H2, 10% Pd/C, ethyl
acetate, r.t., 12 h; e substituted
aromatic formic acid, HBTU,
DIEPA, DMF, r.t., 12 h; f acetic
acid, concentrated hydrochloric
acid, r.t., 12 h
have demonstrated anticancer [20, 21], antiviral [22],
antibacterial [23], anti-HIV [24] efficacy, etc. Compound
apoptosis activator 2 (AA2, Fig. 1), a benzyl substituted
isatin derivative, strongly induces caspase-3 activation,
poly(ADP-ribose) polymerase cleavage, and DNA frag-
mentation, leading to the destruction of cells with IC50 of
4–9 μΜ [25, 26]. Notably, compound 8l (Fig. 1) is another
isatin analog with favorable in vitro antiproliferation
activity that was reported in our previous article [27]. In
this paper, we used 1H-indole-2,3-dione as the scaffold,
modified the substituents at N-1 and C-5 with 3,4-
dichlorobenzyl group and different substituted side chain
respectively to design the novel 1,5-disubstituted isatin
derivatives (Fig. 1). The biological effects of the designed
compounds were investigated for their antiproliferative
activity against MCL cells, as well as efficacy in cell
apoptosis and cell cycle.
Results and discussion
Chemistry
The series of 1,5-disubstituted isatin derivatives were syn-
thesized according to Scheme 1. Commercially available
isatin (1) was treated with fuming nitric acid under ice bath
conditions to give the nitrated indoledione (2). The
carbonyl-protection of intermediate (3) was generated by
2,2-dimethyl-1,3-propanediol and p-toluenesulfonic acid as
catalyst in cyclohexane. Then, deprotonation at N-1 under
alkaline conditions, followed by the addition of the 1,2-
dichloro-4-(chloromethyl)benzene to give the intermediate
(4). Subsequently, the nitro group was reduced under mild
conditions with Palladium on carbon (Pd/C) under hydro-
gen atmosphere to corresponding intermediate (5). Acyla-
tion of aniline (5) was performed using standard amide

Medicinal Chemistry Research
coupling conditions with 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) and N,
N-diisopropylethylamine (DIPEA) in dimethylformamide
(DMF). Final deprotection with acetic acid yielded target
compounds (7a–7u) in good overall yields.
0.5 to 5 µM. The results showed that compound 7l was
capable of increasing apoptosis in a dose-dependent manner.
(Fig. 2a). It was demonstrated that 7l could induce an
increase in the late and early cellular apoptosis, which was in
accord with its antiproliferative capacity that Rec-1 was
more sensitive to 7l than Z138 (IC50 = 0.4 μM and 1.1 μM,
respectively). The results showed a shift of the 7l-treated
cells from the normal to the apoptotic phase. Next, to
address the mechanism responsible for MCL cell cycle
progression, the Rec-1 and Z138 cell lines were stained and
analysed by flow cytometry. As shown in Fig. 2b, 7l sig-
nificantly blocked the cell cycle at the G2/M phase in a dose-
dependent manner in Rec-1 cell after 24 h treatment. Z138
cells with 7l were also arrested at G2/M phase, preventing
cell cycle progression and promoting cell death (Fig. 2c).
Cell antiproliferation assay
This series of compounds were investigated for their effect
against MCL cell lines (Mino, Maver-1, Jeko-1, Z138, Rec-
1, JVM-2 and Jeko-R). Ibrutinib (IBN), which has received
FDA-approval in MCL treatment [28], and aforementioned
AA2 served as the positive control. The results were
expressed as the IC50 (50% maximun inhibitory concentra-
tion). As shown in Table 1, tested compounds were gen-
erally more potent than the reference IBN and AA2 in most
of tested cancer cell lines, specifically compounds 7c–7i, 7k,
7l, 7n–7q and 7s–7u with IC50 values ranged from
0.4–0.8 μM, about 5 to10-fold more potent than IBN (IC50
= 3.9 μM) against IBN-sensitive Mino cells. Furthermore,
compounds 7b, 7j and 7r showed moderate activities (IC50
= 3.7 μM, 2.2 μM and 2.2 μM, respectively) against Mino
cancer cell compared to IBN. The IC50 values of tested
compounds ranged from 0.4 to 4.3 μM, 0.7 to 18.1 μM, and
0.4 to 6.3 μM in comparison to IBN (IC50 = 12.7 μM,
24.2 μM and 11.7 μM, respectively) among the three dif-
ferent IBN-resistant cell lines (Maver-1, Z138, Jeko-R).
Furthermore, replacing amide side chain with the amino
group (7a) weakened the antiproliferation activity. As for
7b–7u that possess different amide chain at C-5, substituted
amide side chain were benzoyl or picolinoyl moiety exert
more potent antiproliferation activity than phenylacetyl.
Among them, the antiproliferation activity of 7n was
stronger than that of 7c, 7e, 7i–7j and 7m, which can be
inferred that the contribution of meta-position substituted
benzoyl moiety for activity is electron donating > with-
drawing groups. Besides, the antiproliferation activity of
picolinoyl moiety substituted compounds (7s–7u) gradually
increased (4-picolinoyl > 3-picolinoyl > 2-picolinoyl) for
IBN-resistant cell lines. All compounds with dual sub-
stituents (7o, 7p, 7q, 7r) exhibited better antiproliferative
activities in IBN-sensitive cell with twofold potent than in
IBN-resistant cell. Five remarkable compounds (7g, 7l, 7n,
7t, 7u) showed no bias between IBN-sensitive cell and IBN-
resistant cell with similar IC50 = 0.4–1.4 μM.
Experimental
General procedures
Unless otherwise stated, all starting materials, solvents, and
reagents were purchased and used from commercial sour-
ces. All reagents were weighed out under ambient condi-
tions. Flash column chromatography was performed using
silica gel 60 (200–300 mesh). Thin layer chromatography
(TLC) was carried out on silica gel plates and visualized by
UV. Melting points using melting point apparatus (Buchi
Labortechnik AG, Switzerland) were measured in open
capillary tubes. Nuclear magnetic resonance spectra were
recorded on Bruker Avance DRX - 400 spectrometers, at
400 MHz 1H NMR frequency, 101 MHz 13C NMR fre-
quency. H and C chemical shifts are reported in ppm.
Multiplicities are reported as follows: s (singlet), br (broad),
d (doublet), dd (doublet of doublets), dt (doublet of triplets),
t (triplet), q (quartet), and m (multiplet). Coupling constants
(J) are reported in hertz. The mass spectra (MS) were
measured with LCQ FLEET (ThermoFisher, USA).
General procedure for the synthesis of 5-nitroindoline-2,3-
dione (2)
To a solution of isatin 1 (200 mg, 1.36 mmol) in con-
centrated H2SO4 (3 mL) was added dropwise fuming nitric
acid (124 mg, 1.77 mmol) at 0 °C. The mixture was stirred
for an additional 1 h in ice bath conditions. Finally, the
reaction mixture was poured into ice/water (50 mL) of
crushed ice, the formed precipitate was filtered, washed
several times with water and dried to afford 5-nitroisatin 2.
Yellow solid; yield 85%; mp: 257–259 °C; 1H NMR
(400 MHz, DMSO-d6): δ 11.66 (s, 1H, NH), 8.45 (dd,
J = 8.7, 2.4 Hz, 1H, Ph-H), 8.22 (d, J = 2.4 Hz, 1H, Ph-H),
7.09 (d, J = 8.7 Hz, 1H, Ph-H).
Cell apoptosis and cell cycle assay
In order to elucidate the potential antitumor mechanism, the
compound (7l) was further evaluated for cell apoptosis assay
with Rec-1 and Z138 cells by an Annexin V-FITC/PI dual
staining assay, for which compound 7l was diluted to
achieve four increasing drug concentrations ranging from

Medicinal Chemistry Research
Table 1 Cell antiproliferation
assay assessing the effects of
novel 1,5-disubstituted isatin
derivatives on MCL cell lines
Code
R1
Cell viability assay, IC50 /μM
Minoa
Maver-1b
Jeko-1a
Z138b
Rec-1a
JVM-2a
Jeko-Rb
IBN
AA2
7a
---
---
H
3.9
12.7
3.2
5.9
24.2
32.5
14.6
0.5
1.1
ND
17.0
10.7
ND
11.7
5.4
2.1
0.9
NDc
19.5
19.9
21.8
7b
7c
7d
7e
7f
3.7
0.8
0.8
0.8
0.4
0.4
0.5
0.7
2.2
0.4
0.4
4.3
0.4
0.5
0.6
0.5
2.2
0.4
0.4
0.4
2.0
0.4
0.4
0.4
0.5
0.4
0.7
1.0
2.5
0.4
0.4
4.3
0.4
0.8
0.7
0.9
2.7
0.5
0.4
0.4
ND
ND
ND
ND
0.4
0.4
0.4
0.4
1.0
0.4
0.4
1.5
0.4
0.4
0.4
0.4
0.5
0.4
0.4
0.4
1.2
0.7
0.7
0.7
2.4
1.0
4.8
3.8
7.4
1.7
1.1
18.1
1.4
3.6
4.0
4.2
9.8
3.2
1.4
1.3
ND
ND
ND
ND
0.4
0.4
0.4
0.9
1.8
0.4
0.4
2.7
0.4
0.5
0.5
0.5
0.9
0.4
0.4
0.4
ND
ND
ND
ND
2.2
1.4
2.2
2.0
4.1
1.7
1.3
8.5
1.4
2.2
2.3
2.5
9.2
1.9
1.2
1.0
1.9
0.3
0.3
0.5
0.5
0.4
0.7
1.1
4.1
0.4
0.4
5.4
0.4
0.9
1.0
1.4
6.3
0.9
0.4
0.4
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
aIBN-sensitive cell lines
bIBN-resistance cell lines
cND not detected

Medicinal Chemistry Research
Fig. 2 Cell apoptosis and cell cycle assay with 7l treatment in Rec-1
and Z138 cells. a Apoptosis assay of 7l at the indicated concentrations
in Rec-1 and Z138 cells for 24 h; b Cell cycle analysis of 7l at the
indicated concentrations in Rec-1 for 24 h; c Cell cycle analysis of 7l at
the indicated concentrations in Z138 cells for 24 h
General procedure for the synthesis of 5′,5′-dimethyl-5-
nitrospiro[indoline-3,2′-[1,3]dioxan]-2-one (3)
J = 8.7, 2.4 Hz, 1H, Ph-H), 8.08 (d, J = 2.4 Hz, 1H, Ph-H),
7.04 (d, J = 8.7 Hz, 1H, Ph-H), 4.49 (d, J = 11.0 Hz,
2H, -OCH2), 3.55 (d, J = 11.2 Hz, 2H, -OCH2), 1.34 (s, 3H,
C-CH3), 0.84 (s, 3H, C-CH3).
The 2,2-dimethyl-1,3-propanediol (108 mg, 1.04 mmol)
and a catalytic amount of p-toluene sulfonic acid (25 mg,
145 μmol) was added to a suspension of 5-nitroisatin 2
(200 mg, 1.04 mmol) in cyclohexane (10 mL). The
resulting reaction mixture then was stirred at reflux tem-
perature for 12 h. Upon completion, the mixture was
cooled. Then, the precipitate was filtered, washed with
water and dried. Purification by chromatography on silica
gel (petroleum ether: ethyl acetate = 5: 1) to give the ketal
of 5-nitroisatin 3.
General procedure for the synthesis of 1-(3,4-
dichlorobenzyl)-5′,5′-dimethyl-5-nitrospiro[indoline-3,2′-
[1,3]dioxan]-2-one (4)
To a solution of intermediate 3 (200 mg, 0.72 mmol) and
anhydrous K2CO3 (199 mg, 1.44 mmol) in dried DMF
(2 mL) was added 1,2-dichloro-4-(chloromethyl) benzene
(169 mg, 0.86 mmol). The reaction mixture was heated to
85 °C and stirred for 1.5 h. After completion of the reaction,
the reaction mixture was cooled to ambient temperature and
White solid; yield 84%; mp: 201–203 °C; 1H NMR
(400 MHz, DMSO-d6): δ 11.20 (s, 1H, NH), 8.28 (dd,

Medicinal Chemistry Research
subsequently poured into ice/water (50 mL). The precipitate
was filtered, washed with water, and then recrystallized with
petroleum ether and ethyl acetate to obtain the desired
product 4.
General procedure for the synthesis of target compounds
(7a-7u)
The appropriate intermediates 6 (300 μmol) was dissolved
in acetic acid (20 mL) and treated with the concentrated
hydrochloric acid (2 mL). the reaction mixture was stirred
overnight at room temperature and then pour into water
(100 mL). The flocculent precipitate formed after cooling
was separated by filtration and purified by recrystallization
in methanol to get title compounds series 7a–7u.
White solid; yield 80%; mp: 137–141 °C; 1H NMR
(400 MHz, DMSO-d6): δ 8.34 (dd, J = 8.7, 1.6 Hz, 1H,
Ph-H), 8.14(d, J = 2.2 Hz, 1H, Ph-H), 7.64 (d, J = 8.7 Hz,
2H, aromatic), 7.31 (d, J = 8.8 Hz, 1H, Bn-H), 7.22 (dd,
J = 8.3, 2.0 Hz, 1H, Bn-H), 4.98 (s, 2H, -NCH2), 4.50 (d,
J = 11.0 Hz, 2H, -OCH2), 3.61 (d, J = 10.9 Hz, 2H,
-OCH2), 1.37 (s, 3H, C-CH3), 0.87 (s, 3H, C-CH3).
5-Amino-1-(3,4-dichlorobenzyl)indoline-2,3-dione
(7a)
General procedure for the synthesis of 5-amino-1-(3,4-
dichlorobenzyl)-5′,5′-dimethylspiro[indoline-3,2′-[1,3]
dioxan]-2-one (5)
Black solid; yield 47%; mp: 222–224 °C; 1H NMR
(400 MHz, DMSO-d6): δ 7.72 (d, J = 1.9 Hz, 1H, Ph-H),
7.60 (d, J = 8.3 Hz, 1H, Ph-H), 7.40 (dd, J = 8.3, 2.0 Hz,
1H, Bn-H), 6.84–6.73 (m, 2H, aromatic), 6.66 (d, J =
8.2 Hz, 1H, Bn-H), 5.15 (s, 2H, -NCH2), 4.82 (s, 2H, -NH2).
13C NMR (101 MHz, DMSO-d6): δ 184.37 (s, C=O),
158.76 (s, –NC=O), 145.98 (s, Ar–C), 140.35 (s, Ar–C),
137.68 (s, Ar–C), 131.71 (s, Ar–C), 131.18 (s, Ar–C),
130.52 (s, Ar–C), 129.85 (s, Ar–C), 128.17 (s, Ar–C),
122.58 (s, Ar–C), 118.77 (s, Ar–C), 111.97 (s, Ar–C),
109.89 (s, Ar–C), 42.19 (s, -CH2). MS(ESI): calcd for
C15H10Cl2N2O2 [M − 2H]− 318.01, found 318.28.
To a mixture of the intermediate 4 (280 mg, 640 μmol) in
ethyl acetate (25 mL) was added palladium on carbon
(140 mg, 10 wt %). The reaction mixture was stirred at
ambient temperature for 12 h under a hydrogen atmosphere.
After completion of the reaction, the mixture was filtered
through diatomite powder, and the residue was washed with
ethyl acetate. The filtrate was evaporated under reduced
pressure to give the crude residue. Purification was achieved
by recrystallized with ethyl acetate/petroleum ether.
1
Light yellow solid; yield 77%; and mp: 171–174 °C; H
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-2-(4-fluoro-
phenyl)acetamide (7b) Red solid; yield 40%; mp:
NMR (400 MHz, DMSO-d6): δ 7.63 (d, J = 8.3 Hz, 1H,
Bn-H), 7.55 (d, J = 2.0 Hz,1H, Bn-H), 7.21 (dd, J = 8.3,
2.0 Hz, 1H, Bn-H), 6.76 (d, J = 2.2 Hz, 1H, Ph-H), 6.64 (d,
J = 8.3 Hz, 1H, Ph-H),6.48 (dd, J = 8.3, 2.3 Hz, 1H, Ph-H),
4.97 (s, 2H, -NCH2), 4.75 (s, 2H, -NH2), 4.50 (d, J =
10.9 Hz, 2H -OCH2), 3.51 (d, J = 11.0 Hz, 2H -OCH2),
1.30 (s, 3H, C-CH3), 0.84 (s, 3H, C-CH3).
1
198–200 °C; H NMR (400 MHz, DMSO-d6): δ 10.29 (s,
1H, -CONH), 7.88 (d, J = 2.1 Hz, 1H, aromatic), 7.76 (d,
J = 2.0 Hz, 1H, aromatic), 7.66–7.58 (m, 2H, phenylacetyl),
7.44 (dd, J = 8.3, 2.0 Hz, 1H, aromatic), 7.35 (dd, J = 8.6,
5.6 Hz, 2H, aromatic), 7.14 (t, J = 8.9 Hz, 2H, phenylace-
tyl), 6.89 (d, J = 8.5 Hz,1H, aromatic), 4.89 (s, 2H, -NCH2),
3.62 (s, 2H, -NH2). 13C NMR (101 MHz, DMSO-d6): δ
1
General procedure for the synthesis of intermediates (6)
183.34 (s, –C=O), 169.59 (s, -NHC=O),161.60 (d, J =
240.8 Hz, -C6H5F), 159.11 (s, -NC=O), 145.80 (s, Ar–C),
HBTU (257 mg, 678 μmol), and DIEA (109 mg, 847 μmol)
were added to a mixture of appropriate commercially
available acid (678 μmol) in DMF (6 mL). The mixture was
stirred under ice bath for about 40 min, then amine inter-
mediate 5 (230 mg, 565 μmol) was added. The mixture was
stirred at ambient temperature for overnight. TLC analysis
showed that starting material was consumed. Water
(100 mL) was added to the reaction, the precipitate was
filtered off and washed with water. Water (100 mL) and
ethyl acetate (50 mL) were added to the filtrate, the organic
layer was collected, and the aqueous layer was extracted
with ethyl acetate (×3). The combined organic layers were
washed with brine, dried over anhydrous anhydrous mag-
nesium sulfate, and concentrated to give the crude product
which was purified by column chromatography on silica gel
(petroleum ether: ethyl acetate = 5: 1) to afford the desired
intermediates 6.
137.30 (s, Ar–C), 135.51 (s, Ar–C), 132.35 (d, 4J = 3.1 Hz,
-C6H5F), 131.74 (s, Ar–C), 131.46 (d, J = 8.0 Hz, 2 C,
3
-C6H5F), 131.15 (s, Ar–C), 130.57 (s, Ar–C), 129.79 (s,
Ar–C), 128.38 (s, Ar–C), 128.19 (s, Ar–C), 118.43 (s,
Ar–C), 115.75 (s, Ar–C), 115.49 (d, 2J = 21.1 Hz, 2 C,
-C6H5F), 111.56 (s, Ar–C), 42.57 (s, -NCH2), 42.28 (s,
-COCH2). MS (ESI): calcd for C23H15Cl2FN2O3 [M − H]−
455.04, found 455.15.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-4-fluoro-
benzamide (7c) Red solid; yield 33%; mp: 285–286 °C;
1H NMR (400 MHz, DMSO-d6): 10.36 (s, 1H, -CONH),
8.08–7.96 (m, 3H, aromatic), 7.85 (dd, J = 8.5, 2.1 Hz, 1H,
Ph-H), 7.79 (d, J = 1.7 Hz, 1H, aromatic), 7.62 (d, J =
8.3 Hz, 1H, aromatic), 7.46 (dd, J = 8.3, 1.8 Hz, 1H, Bn-H),
7.38 (t, J = 8.8 Hz, 2H, benzoyl), 6.96 (d, J = 8.5 Hz, 1H,
aromatic), 4.92 (s, 2H, -NCH2). 13C NMR (101 MHz,

Medicinal Chemistry Research
DMSO-d6): δ 183.34 (s, –C=O), 164.85 (s, –NHC=O),
164.63 (d, J = 247.8 Hz, -C6H5F), 159.18 (s, –NC=O),
aromatic), 7.47 (dd, J = 8.3, 1.8 Hz, 1H, aromatic), 6.96 (d,
J = 8.5 Hz, 1H, aromatic), 4.92 (s, 2H, -NCH2). 13C NMR
(101 MHz, DMSO-d6): δ 183.31 (s, –C=O), 164.98 (s,
–NHC = O), 159.18 (s, –NC=O), 146.25 (s, Ar–C), 137.32
(s, Ar–C), 135.23 (s, Ar–C), 133.97 (s, Ar–C), 131.96 (s,
2 C, benzoyl), 131.75 (s, Ar–C), 131.17 (s, Ar–C), 130.58
(s, Ar–C), 130.22 (s, 2 C, benzoyl), 129.82 (s, Ar–C),
129.76 (s, Ar–C), 128.22 (s, Ar–C), 126.02 (s, Ar–C),
118.43 (s, Ar–C), 117.10 (s, Ar–C), 111.45 (s, Ar–C), 42.32
(s, -CH2). MS (ESI): calcd for C22H13BrCl2N2O3 [M-H + 2]−
502.95, found 503.05.
1
146.17 (s, Ar–C), 137.32 (s, Ar–C), 135.37 (s, Ar–C),
4
131.76 (s, Ar–C), 131.35 (d, J = 2.8 Hz, -C6H5F), 131.17
3
(s, Ar–C), 130.85 (d, J = 9.0 Hz, 2 C, -C6H5F), 130.59 (s,
Ar–C), 129.82 (s, Ar–C), 129.73 (s, Ar–C), 128.21 (s,
Ar–C), 118.41 (s, Ar–C), 117.08 (s, Ar–C), 115.89 (d, 2J =
21.8 Hz, 2C, -C6H5F), 111.44 (s, Ar–C), 42.32 (s, -NCH2).
MS (ESI): calcd for C22H13Cl2FN2O3 [M − H]− 441.03,
found 441.03.
4-Chloro-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
benzamide (7d) Red solid; yield 18%; mp: 247–250 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.42 (s, 1H, -CONH),
8.05–8.01 (m, 1H, aromatic), 7.98 (dd, J = 8.6, 1.8 Hz, 2H,
Ph-H), 7.85 (dt, J = 8.5, 2.1 Hz, 1H, benzoyl), 7.62 (d, J =
8.4 Hz, 2H), 7.48–7.28 (m, 3H, aromatic), 6.98 (dd, J =
12.8, 8.5 Hz, 1H, Bn-H), 4.92 (s, 2H, –NCH2). 13C NMR
(101 MHz, DMSO-d6): δ 183.32 (s, –C=O), 164.85 (s,
–NHC=O), 159.18 (s, -NC=O), 146.23 (s, Ar–C), 137.32
(s, Ar–C), 137.05 (s, Ar–C), 135.28 (s, Ar–C), 133.58 (s,
Ar–C), 131.76 (s, Ar–C), 131.17 (s, Ar–C), 130.09 (s, 2 C,
benzoyl), 129.82 (s, Ar–C), 129.13 (s, Ar–C), 129.00 (s,
2C, benzoyl), 128.22 (s, Ar–C), 127.83 (s, Ar–C), 118.40
(s, Ar–C), 117.13 (s, Ar–C), 111.44 (s, Ar–C), 42.32 (s,
-CH2). MS (ESI): calcd for C22H13Cl3N2O3 [M − H]−
458.00, found 458.10.
4-Cyano-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
benzamide (7g) Red solid; yield 74%; mp: 295–297 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.58 (s, 1H, -CONH),
8.10 (d, J = 8.3 Hz, 2H, aromatic), 8.04 (d, J = 7.8 Hz, 3H,
aromatic), 7.85 (dd, J = 8.5, 2.0 Hz, 1H, aromatic), 7.79 (d,
J = 1.5 Hz, 1H, aromatic), 7.62 (d, J = 8.3 Hz, 1H, aro-
matic), 7.47 (dd, J = 8.3, 1.6 Hz, 1H, aromatic), 6.96 (t, J =
8.6 Hz, 1H, aromatic), 4.92 (s, 2H, -NCH2). 13C NMR
(101 MHz, DMSO-d6): δ 183.26 (s, –C=O), 164.55 (s,
–NHC = O), 159.19 (s, –NC=O), 146.43 (s, Ar–C), 138.93
(s, Ar–C), 137.30 (s, Ar–C), 135.00 (s, Ar–C), 133.01 (s,
2 C, Ar–C), 131.76 (s, Ar–C), 131.17 (s, Ar–C), 130.58 (s,
Ar–C), 129.81 (s, Ar–C), 128.96 (s, 2 C, Ar–C), 128.21 (s,
Ar–C), 118.76 (s, Ar–C), 118.46 (s, Ar–C), 117.12 (s,
Ar–C), 114.47 (s, Ar–C), 111.50 (s, Ar–C), 42.33 (s,
-NCH2). MS (ESI): calcd for C23H13Cl2N3O3 [M − H]−
448.03, found 448.11.
3-Chloro-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
benzamide (7e) Red solid; yield 14%; mp: 222–224 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.44 (s, 1H, -CONH),
8.05–7.98(m, 2H, aromatic), 7.94–7.88 (m, 1H, aromatic),
7.85 (dt, J = 8.5, 2.1 Hz, 1H, benzoyl), 7.79 (d, J = 2.0 Hz,
1H, aromatic),7.68 (ddd, J = 8.0, 2.0, 1.0 Hz, 2H, benzoyl),
7.65–7.55 (m, 1H, aromatic), 7.46 (dd, J = 8.4, 2.0 Hz, 1H,
aromatic), 6.97 (d, J = 8.5 Hz, 1H, aromatic), 4.92 (s, 2H,
-NCH2). 13C NMR (101 MHz, DMSO-d6): δ 183.30
(s, –C=O), 164.47 (s, –NHC = O), 159.18 (s, –NC=O),
146.30 (s, Ar–C), 137.31 (s, Ar–C), 136.88 (s, Ar–C),
135.16 (s, Ar–C), 133.73 (s, Ar–C), 132.05 (s, Ar–C),
131.76 (s, Ar–C), 131.17 (s, Ar–C), 130.96 (s, Ar–C),
130.59 (s, Ar–C), 129.82 (s, Ar–C), 129.13 (s, Ar–C),
128.21 (s, Ar–C), 127.82 (s, Ar–C), 126.95 (s, Ar–C),
118.43 (s, Ar–C), 117.12 (s, Ar–C), 111.46 (s, Ar–C), 42.33
(s, -CH2). MS (ESI): calcd for C22H13Cl3N2O3 [M − H]−
457.00, found 457.12.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-4-methoxy-
benzamide (7h) Red solid; yield 81%; mp: 276–277 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.20 (s, 1H, -CONH),
8.04 (d, J = 1.9 Hz, 1H, Ph-H), 7.95 (d, J = 8.8 Hz, 2H,
benzoyl), 7.86 (dd, J = 8.5, 2.0 Hz, 1H, Ph-H), 7.79 (d, J =
1.4 Hz, 1H, aromatic), 7.63 (d, J = 8.3 Hz, 1H, aromatic),
7.47 (dd, J = 8.3, 1.5 Hz, 1H, Bn-H), 7.07 (d, J = 8.8 Hz,
2H, benzoyl), 6.95 (d, J = 8.5 Hz, 1H, Bn-H), 4.92 (s, 2H,
-NCH2), 3.84 (s, 3H, -OCH3). 13C NMR (101 MHz,
DMSO-d6): δ 183.40 (s, –C=O), 165.33 (s, –NHC = O),
162.50 (s, Ar–C), 159.18 (s, –NC=O), 145.95 (s, Ar–C),
137.35 (s, Ar–C), 135.69 (s, Ar–C), 131.76 (s, Ar–C),
131.17 (s, Ar–C), 130.58 (s, Ar–C), 130.04 (s, 2 C, Ar–C),
129.83 (s, Ar–C), 129.64 (s, Ar–C), 128.21 (s, Ar–C),
126.92 (s, Ar–C), 118.37 (s, Ar–C), 117.02 (s, Ar–C),
114.14 (s, 2 C, Ar–C), 111.38 (s, Ar–C), 55.92 (s, -OCH3),
42.32 (s, -NCH2). MS (ESI): calcd for C23H16Cl2N2O4
[M − H]− 453.05, found 453.14.
4-Bromo-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
benzamide (7f) Red solid; yield 81%; mp: 285–287 °C; 1H
NMR (400 MHz, DMSO-d6): δ 10.43 (s, 1H, -CONH), 8.03
(d, J = 2.0 Hz, 1H, aromatic), 7.90 (d, J = 8.5 Hz, 2H,
benzoyl), 7.84 (dd, J = 8.5, 2.1 Hz, 1H, aromatic),
7.79–7.75 (m, 3H, aromatic), 7.62 (d, J = 8.3 Hz, 1H,
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-3-nitroben-
1
zamide (7i) Red solid; yield 73%; mp: 241–243 °C; H
NMR (400 MHz, DMSO-d6): δ 10.69 (s, 1H, -CONH), 8.80
(s, 1H, aromatic), 8.46 (dd, J = 8.2, 1.2 Hz, 1H, aromatic),

Medicinal Chemistry Research
8.40 (d, J = 7.8 Hz, 1H, benzoyl), 8.05 (d, J = 1.9 Hz, 1H,
aromatic), 7.86 (t, J = 8.1 Hz, 2H, benzoyl), 7.80 (d, J =
1.6 Hz, 1H, aromatic), 7.63 (d, J = 8.3 Hz, 1H, aromatic),
7.48 (dd, J = 8.3, 1.7 Hz, 1H, aromatic), 6.99 (d, J =
8.5 Hz, 1H, aromatic), 4.93 (s, 2H, -NCH2). 13C NMR
(101 MHz, DMSO-d6): δ 183.26 (s, –C=O), 163.76 (s,
–NHC = O), 159.19 (s, –NC=O), 148.25 (s, Ar–C), 146.48
(s, Ar–C), 137.31 (s, Ar–C), 136.28 (s, Ar–C), 134.93 (s,
Ar–C), 134.62 (s, Ar–C), 131.76 (s, Ar–C), 131.18 (s,
Ar–C), 130.77 (s, Ar–C), 130.59 (s, Ar–C), 129.95 (s,
Ar–C), 129.82 (s, Ar–C), 128.22 (s, Ar–C), 126.82 (s,
Ar–C), 122.81 (s, Ar–C), 118.48 (s, Ar–C), 117.30 (s,
Ar–C), 111.51 (s, Ar–C), 42.35 (s, -NCH2). MS (ESI): calcd
for C22H13Cl2N3O5 [M − H]− 468.02, found 468.13.
(s, Ar–C), 42.34 (s, -NCH2). MS (ESI): calcd for
C22H13BrCl2N2O3 [M − H + 2]− 502.95, found 503.14.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-3-fluoro-
benzamide (7l) Red solid; yield 75%; mp: 247–250 °C; 1H
NMR (400 MHz, DMSO-d6): δ 10.42 (s, 1H, -CONH), 8.03
(d, J = 1.8 Hz, 1H, aromatic), 7.85 (dd, J = 8.5, 1.8 Hz, 1H,
aromatic), 7.82–7.75 (m, 3H, aromatic), 7.64–7.58 (m, 2H,
aromatic), 7.48–7.44 (m, 2H, aromatic), 6.97 (d, J = 8.5 Hz,
1H, aromatic), 4.92 (s, 2H, -NCH2). 13C NMR (101 MHz,
DMSO-d6): δ 183.30 (s, –C=O), 164.56 (s, –NHC = O),
162.41 (d, 1J = 243 Hz, C6H5-F), 159.18 (s, –NC = O),
3
146.31 (s, Ar–C), 137.22 (d, J = 6.9 Hz, C6H5-F), 135.16
(s, Ar–C), 131.76 (s, Ar–C), 131.19 (s, Ar–C), 131.17 (s,
Ar–C), 131.11 (s, Ar–C), 130.59 (s, Ar–C), 129.82 (s,
3
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-3-(trifluoro-
methyl)benzamide (7j) Red solid; yield 81%; mp:
210–213 °C; H NMR (400 MHz, DMSO-d6): δ 10.57 (s,
Ar–C), 129.79 (s, Ar–C), 128.22 (s, Ar–C), 124.3(d, J =
2
3.7 Hz, C6H5-F), 119.139 (d, J = 21 Hz, C6H5-F), 118.43
(s, Ar–C), 117.12 (s, Ar–C), 114.92 (d, 2J = 22.8 Hz, C6H5-
F), 111.46 (s, Ar-C), 42.34 (s, -NCH2). MS (ESI): calcd for
C22H13Cl2FN2O3 [M − H]− 441.03, found 441.13.
1
1H, -CONH), 8.29 (s, 1H, aromatic), 8.26 (d, J = 7.9 Hz,
1H, aromatic), 8.03 (d, J = 1.9 Hz, 1H, aromatic), 7.99 (d, J
= 7.8 Hz, 1H, aromatic), 7.86 (dd, J = 8.5, 2.0 Hz, 1H,
aromatic), 7.82–7.78 (m, 2H, aromatic), 7.63 (d, J = 8.3 Hz,
1H, aromatic), 7.48 (dd, J = 8.3, 1.6 Hz, 1H, aromatic),
3-Cyano-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
benzamide (7m) Red solid; yield 76%; mp 256–257 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.53 (s, 1H, -CONH),
8.40 (s, 1H, aromatic), 8.25 (d, J = 8.0 Hz, 1H, aromatic),
8.09 (d, J = 7.9 Hz, 1H, aromatic), 8.03 (d, J = 1.8 Hz, 1H,
aromatic), 7.85 (dd, J = 8.5, 2.0 Hz, 1H, aromatic), 7.79 (s,
1H, aromatic), 7.76 (d, J = 7.8 Hz, 1H, aromatic), 7.63 (d, J
= 8.3 Hz, 1H, aromatic), 7.47 (dd, J = 8.5, 1.4 Hz, 1H,
aromatic), 6.98 (d, J = 8.5 Hz, 1H, aromatic), 4.93 (s, 2H,
-NCH2). 13C NMR (101 MHz, DMSO-d6): δ 183.27 (s,
–C=O), 164.10 (s, -NHC = O), 159.18 (s, –NC = O),
146.41 (s, Ar–C), 137.30 (s, Ar–C), 135.94 (s, Ar–C),
135.01 (s, Ar–C), 132.96 (s, Ar–C), 131.76 (s, Ar–C),
131.70 (s, Ar–C), 131.18 (s, Ar–C), 130.59 (s, Ar–C),
130.41 (s, Ar–C), 129.81 (s, Ar–C), 129.76 (s, Ar–C),
128.21 (s, Ar–C), 118.75 (s, Ar–C), 118.47 (s, Ar–C),
117.08 (s, Ar–C), 112.05 (s, -CN), 111.51 (s, Ar–C), 42.34
(s, -NCH2). MS (ESI): calcd for C22H13Cl2N3O3 [M − H]−
448.03, found 448.03.
6.98 (d, J = 8.5 Hz, 1H, aromatic), 4.93 (s, 2H, -NCH2). 13
C
NMR (101 MHz, DMSO-d6): δ 183.28 (s, –C=O), 164.45
(s, –NHC = O), 159.19 (s, –NC=O), 146.40 (s, Ar–C),
137.31 (s, Ar–C), 135.80 (s, Ar–C), 135.05 (s, Ar–C),
132.29 (s, Ar–C), 131.76 (s, Ar–C), 131.17 (s, Ar–C),
130.59 (s, Ar–C), 130.30 (s, Ar–C), 129.82 (s, Ar–C),
129.69 (q, 2J = 32.2 Hz, -C6H5CF3), 128.77 (q, 3J = 3.4 Hz,
-C6H5CF3), 128.22 (s, Ar–C), 125.78 (s, Ar–C), 124.63 (q,
3J = 4.0 Hz, -C6H5CF3), 124.43 (q, 1J = 273.0 Hz,
-C6H5CF3), 118.46 (s, Ar–C), 117.28 (s, Ar–C), 111.48 (s,
Ar–C), 42.34 (s, -NCH2). MS (ESI): calcd for
C23H13Cl2F3N2O3 [M − H]− 491.03, found 491.13.
3-Bromo-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
benzamide (7k) Red solid; yield 84%; mp: 239–240 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.45 (s, 1H, -CONH),
8.14 (s, 1H, aromatic), 8.03 (d, J = 1.9 Hz, 1H, aromatic),
7.95 (d, J = 7.9 Hz, 1H, aromatic), 7.85 (dd, J = 8.6,
2.0 Hz, 1H, aromatic), 7.81 (d, J = 8.2 Hz, 1H, aromatic),
7.79 (d, J = 1.6 Hz, 1H, aromatic), 7.63 (d, J = 8.3 Hz, 1H,
aromatic), 7.52 (t, J = 7.9 Hz, 1H, aromatic), 7.47 (dd, J =
8.3, 1.5 Hz, 1H, aromatic), 6.97 (d, J = 8.5 Hz, 1H, aro-
matic), 4.92 (s, 2H, -NCH2). 13C NMR (101 MHz, DMSO-
d6): δ 183.30 (s, –C=O), 164.39 (s, –NHC = O), 159.18 (s,
–NC=O), 146.31 (s, Ar–C), 137.32 (s, Ar–C), 137.08
(s, Ar–C), 135.15 (s, Ar–C), 134.94 (s, Ar–C), 131.76 (s,
Ar–C), 131.21 (s, Ar–C), 131.17 (s, Ar–C), 130.64
(s, Ar–C), 130.59 (s, Ar–C), 129.82 (s, Ar–C), 129.79 (s,
Ar–C), 128.21 (s, Ar–C), 127.32 (s, Ar–C), 122.20 (s,
Ar–C), 118.43 (s, Ar–C), 117.13 (s, Ar–C), 111.46
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-3-methyl-
benzamide (7n) Red solid; yield 80%; mp: 218–221 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H, -CONH),
8.04 (d, J = 2.0 Hz, 1H, aromatic), 7.86 (dd, J = 8.5,
2.0 Hz, 1H, aromatic), 7.79–7.73 (m, 3H, aromatic), 7.62
(d, J = 8.3 Hz, 1H, aromatic), 7.48–7.41 (m, 3H, aromatic),
6.96 (d, J = 8.5 Hz, 1H, aromatic), 4.92 (s, 2H, -CH2), 2.40
(s, 3H, -CH3). 13C NMR (101 MHz, DMSO-d6): δ 183.37
(s, –C=O), 166.08 (s, –NHC = O), 159.18 (s, –NC = O),
146.08 (s, Ar–C), 138.26 (s, Ar–C), 137.34 (s, Ar–C),
135.52 (s, Ar–C), 134.94 (s, Ar–C), 132.80 (s, Ar–C),
131.75 (s, Ar–C), 131.17 (s, Ar–C), 130.58 (s, Ar–C),

Medicinal Chemistry Research
129.83 (s, Ar–C), 129.64 (s, Ar–C), 128.83 (s, Ar–C),
128.54 (s, Ar–C), 128.22 (s, Ar–C), 125.26 (s, Ar–C),
118.40 (s, Ar–C), 117.01 (, Ar–C s), 111.42 (s, Ar–C),
42.32 (s, -NCH2), 21.43 (s, -CH3). MS (ESI): calcd for
C23H16Cl2N2O3 [M − H]− 437.05, found 437.13.
(d, J = 8.5 Hz, 1H, aromatic), 4.93 (s, 2H, -NCH2). 13C
NMR (101 MHz, DMSO-d6): δ 183.18 (s, –C=O), 161.62
(s, –NHC = O), 159.16 (s, –NC = O), 158.86 (d, 1J =
252.9 Hz, C6H5-F), 149.84 (d, 3J = 8.8 Hz, C6H5-F), 146.57
(s, Ar–C), 137.25 (s, Ar–C), 134.64 (s, Ar–C), 131.76 (s,
3
Ar–C), 131.65 (d, J = 3.3 Hz, C6H5-F), 131.17 (s, Ar–C),
3,4-Dichloro-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-
yl)benzamide (7o) Red solid; yield 82%; mp: 279–281 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.42 (s, 1H, -CONH),
8.14 (d, J = 1.8 Hz, 1H, aromatic), 7.94 (d, J = 1.9 Hz, 1H,
aromatic), 7.86 (dd, J = 8.4, 1.8 Hz, 1H, aromatic), 7.77 (br,
1H, aromatic), 7.75 (br, 1H, aromatic), 7.72 (d, J = 1.3 Hz,
1H, aromatic), 7.55 (d, J = 8.3 Hz, 1H, aromatic), 7.40 (dd,
J = 8.3, 1.5 Hz, 1H, aromatic), 6.90 (d, J = 8.5 Hz, 1H,
aromatic), 4.85 (s, 2H, -NCH2). 13C NMR (101 MHz,
DMSO-d6): δ 183.26 (s, –C=O), 163.59 (s, –NHC = O),
159.18 (s, –NC = O), 146.40 (s, Ar–C), 137.31 (s, Ar–C),
135.21 (s, Ar–C), 135.01 (s, Ar–C), 134.99 (s, Ar–C),
131.83 (s, Ar–C), 131.76 (s, Ar–C), 131.32 (s, Ar–C),
131.17 (s, Ar–C), 130.59 (s, Ar–C), 130.01 (s, Ar–C),
129.82 (s, 2 C, Ar–C), 128.49 (s, Ar–C), 128.22 (s, Ar–C),
118.46 (s, Ar–C), 117.15 (s, Ar–C), 111.49 (s, Ar–C), 42.34
(s). MS (ESI): calcd for C22H12Cl4N2O3 [M − H]− 492.96,
found 493.07.
130.86 (d, 2J = 15.8 Hz, C6H5-F), 130.59 (s, Ar–C), 129.80
(s, Ar–C), 129.19 (s, Ar–C), 128.21 (s, Ar–C), 120.26 (d,
4J = 3.4 Hz, C6H5-F), 118.58 (s, Ar–C), 116.46 (s, Ar–C),
2
112.67 (d, J = 27.2 Hz, C6H5-F), 111.69 (s, Ar–C), 42.34
(s, -NCH2). MS (ESI): calcd for C22H12Cl2FN2O5 [M − H]−
486.01, found 486.05.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-3,4-
dimethoxybenzamide (7r) Red solid; yield 82%; mp:
1
276–278 °C; H NMR (400 MHz, DMSO-d6): δ 10.18 (s,
1H, -CONH), 8.01 (d, J = 2.0 Hz, 1H, aromatic), 7.86 (dd,
J = 8.5, 2.1 Hz, 1H, aromatic), 7.79 (d, J = 1.7 Hz, 1H,
aromatic), 7.64–7.60 (m, 2H, aromatic), 7.52 (d, J = 1.8 Hz,
1H, aromatic), 7.47 (dd, J = 8.3, 1.8 Hz, 1H, aromatic),
7.09 (d, J = 8.5 Hz, 1H, aromatic), 6.95 (d, J = 8.5 Hz, 1H,
aromatic), 4.92 (s, 2H, -NCH2), 3.84 (s, 6H,-OCH3). 13C
NMR (100 MHz, DMSO-d6): δ 182.31 (s, –C=O), 164.26
(s, –NHC = O), 158.11 (s, –NC = O), 151.17 (s, Ar–C),
147.74 (s, Ar–C), 144.91 (s, Ar–C), 136.28 (s, Ar–C),
134.53 (s, Ar–C), 130.68 (s, Ar–C), 130.09 (s, Ar–C),
129.50 (s, Ar–C), 128.76 (s, Ar–C), 128.68 (s, Ar–C),
127.14 (s, Ar–C), 125.85 (s, Ar–C), 120.42 (s, Ar–C),
117.32 (s, Ar–C), 116.08 (s, Ar–C), 110.35 (s, Ar–C),
110.32 (s, Ar–C), 110.30 (s, Ar–C), 55.08 (s, -OCH3),
55.03 (s, -OCH3), 41.25 (s, -NCH2). MS (ESI): calcd for
C24H18Cl2N2O5 [M − H]− 483.06, found 483.16.
2,4-Dichloro-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-
yl)benzamide (7p) Red solid; yield 83%; mp: 237–238 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.67 (s, 1H, -CONH),
7.98 (d, J = 1.9 Hz, 1H, Ph-H), 7.78 (d, J = 1.4 Hz, 2H,
aromatic), 7.73 (dd, J = 8.5, 2.0 Hz, 1H, aromatic), 7.63
(dd, J = 10.0, 8.4 Hz, 2H, aromatic), 7.57 (dd, J = 8.2,
1.8 Hz, 1H, aromatic), 7.46 (dd, J = 8.3, 1.6 Hz, 1H, aro-
matic), 6.95 (d, J = 8.5 Hz, 1H, aromatic), 4.92 (s, 2H,
-NCH2). 13C NMR (101 MHz, DMSO-d6): δ 183.24 (s,
–C=O), 164.49 (s, –NHC = O), 159.16 (s, –NC=O),
146.31 (s, Ar–C), 137.26 (s, Ar–C), 135.81 (s, Ar–C),
135.51 (s, Ar–C), 134.99 (s, Ar–C), 131.75 (s, Ar–C),
131.73 (s, Ar–C), 131.16 (s, Ar–C), 130.83 (s, Ar–C),
130.58 (s, Ar–C), 129.79 (s, Ar–C), 129.75 (s, Ar–C),
128.83 (s, Ar–C), 128.20 (s, Ar–C), 128.00 (s, Ar–C),
118.54 (s, Ar–C), 116.14 (s, Ar–C), 111.66 (s, Ar–C), 42.31
(s, -NCH2). MS (ESI): calcd for C22H12Cl4N2O3 [M − H]−
492.96, found 493.18.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)picolina-
mide (7s) Red solid; yield 76%; mp: 241–243 °C; 1H
NMR (400 MHz, DMSO-d6): δ 10.88 (s, 1H, -CONH), 8.75
(d, J = 4.6 Hz, 1H, aromatic), 8.21 (d, J = 1.9 Hz, 1H,
aromatic), 8.16 (d, J = 7.7 Hz, 1H, aromatic), 8.08 (td, J =
7.7, 1.3 Hz, 1H, aromatic), 8.02 (dd, J = 8.5, 2.0 Hz, 1H,
aromatic), 7.79 (br, 1H, aromatic), 7.70–7.67 (m, 1H, aro-
matic), 7.63 (d, J = 8.3 Hz, 1H, aromatic), 7.47 (dd, J = 8.3,
1.4 Hz, 1H, aromatic), 6.97 (d, J = 8.6 Hz, 1H, aromatic),
4.92 (s, 2H, -NCH2). 13C NMR (100 MHz, DMSO-d6): δ
183.33 (s, –C=O), 163.16 (s, –NHC = O), 159.17 (s,
–NC=O), 150.09 (s, Cpyridyl-C = O), 148.92 (s, Ar–C),
146.33 (s, Ar–C), 138.64 (s, Ar–C), 137.34 (s, Ar–C),
134.83 (s, Ar–C), 131.75 (s, Ar–C), 131.17 (s, Ar–C),
130.60 (s, Ar–C), 129.96 (s, Ar–C), 129.89 (s, Ar–C),
128.27 (s, Ar–C), 127.51 (s, Ar–C), 122.98 (s, Ar–C),
118.39 (s, Ar–C), 117.12 (s, Ar–C), 111.41 (s, Ar–C), 42.33
(s, -NCH2). MS (ESI): calcd for C21H13Cl2N3O3 [M − H]−
424.03, found 424.16.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-2-fluoro-4-
nitrobenzamide (7q) Red solid; yield 89%; mp:
1
247–249 °C; H NMR (400 MHz, DMSO-d6): δ 10.83 (s,
1H, -CONH), 8.30 (dd, J = 9.6, 1.9 Hz, 1H, aromatic), 8.21
(dd, J = 8.6, 1.7 Hz, 1H, aromatic), 7.99–7.94 (m, 2H,
aromatic), 7.79 (d, J = 1.1 Hz, 1H, aromatic), 7.75 (dd, J =
8.6, 1.9 Hz, 1H, aromatic), 7.63 (d, J = 8.3 Hz, 1H, aro-
matic), 7.47 (dd, J = 8.4, 1.7 Hz, 1H, aromatic), 6.97

Medicinal Chemistry Research
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)nicotina-
mide (7t) Red solid; yield 49%; mp: 287–290 °C; 1H
NMR (400 MHz, DMSO-d6): δ 10.79 (s, 1H, -CONH), 9.24
(s, 1H, aromatic), 8.88 (d, J = 6.3 Hz, 1H, aromatic), 8.55
(d, J = 6.7 Hz, 1H, aromatic), 8.06 (d, J = 1.6 Hz, 1H,
aromatic), 7.88 (d, J = 8.5 Hz, 1H, aromatic), 7.80–7.77 (m,
2H, aromatic), 7.63 (d, J = 8.3 Hz, 1H, aromatic), 7.47 (dd,
J = 8.3, 1.6 Hz, 1H, aromatic), 6.98 (d, J = 8.5 Hz, 1H,
aromatic), 4.93 (s, 2H, -NCH2). 13C NMR (100 MHz,
DMSO-d6): δ 183.24 (s, –C=O), 162.90 (s, –NHC = O),
159.18 (s, –NC = O), 148.75 (s, Ar–C), 146.53 (s, Ar–C),
145.80 (s, Ar–C), 140.45 (, Ar–C s), 137.30 (s, Ar–C),
134.88 (s, Ar–C), 132.01 (s, Ar–C), 131.76 (s, Ar–C),
131.17 (s, Ar–C), 130.59 (s, Ar–C), 129.84 (s, Ar–C),
129.82 (s, Ar–C), 128.24 (s, Ar–C), 125.71 (s, Ar–C),
118.47 (s, Ar–C), 117.12 (s, Ar–C), 111.53 (s, Ar–C), 42.35
(s, -NCH2). HRMS (ESI): m/z [M + H]+ calcd for
C21H13Cl2N3O3: 426.0334, found: 426.0381.
Glo Luminecent cell viability assay kit (Promega, USA).
Briefly, the different kinds of cells were seeded indepen-
dently in a 96-well microtiter plate with containing 1 × 104
cells/well. The cells were exposed to different concentra-
tions (0.93–60 μmol/L) of compounds and incubated for
72 h at 37 °C. After treatment, CellTiter-Glo (30 μL) solu-
tion was added to each well. The absorbance of the samples
was determined at a wavelength of 570 nm with HTX
Multi-Mode Microplate Reader (BioTek, USA). Each
independent experiment was run three times and IC50 values
were calculated with Prism 6.0 Software (GraphPad, USA).
Flow cytometry assay (cell cycle and apoptosis)
Cells were plated on six-well culture plates at a density of 2 ×
105 cells/well and were treated with the indicated concentra-
tions (0.5, 1, 2.5, 5 μM) of 7l for 24 h after they adherence.
After culture, cells were centrifuged (1500 rpm, 5 min) and
washed twice in cold phosphate-buffered saline (PBS) before
exposure to staining buffer. To the cell suspension were
stained with 2 μL of FITC-Annexin V and 2 μL of PI,
according to the manufacturer’s protocol. After being gently
mixed, the flow tube should be incubated for 15 min at room
temperature in the dark. After addition of 200 μL of binding
buffer, apoptosis detection was performed by flow cytometry
using a Novocyte Flow Cytometer (ACEA Biosciences,
USA). Data were analyzed using Flowjo software. For cell
cycle assay, the treated cells were harvested by centrifugation
and washed twice with PBS, then fixed with ice-cold 70%
ethanol overnight at 4 °C and collected again. The fixed cells
were then washed twice with PBS and stained with 100 μL of
PI (100 μg/mL) in the presence of RNaseA (0.5 mg) at room
temperature, then they were analyzed by flow cytometry. The
results were analyzed by Novoexpress software.
N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)isonicotina-
mide (7u) Red solid; yield 82%; mp: 270–273 °C; 1H
NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H, -CONH), 8.98
(d, J = 5.9 Hz, 2H, pyridly), 8.22 (d, J = 5.8 Hz, 2H, pyr-
idly), 8.08 (d, J = 1.4 Hz, 1H, aromatic), 7.91 (dd, J = 8.5,
1.6 Hz, 1H, aromatic), 7.79 (s, 1H, aromatic), 7.62 (d, J =
8.3 Hz, 1H, aromatic), 7.47 (dd, J = 8.2, 0.7 Hz, 1H, aro-
matic), 7.00 (d, J = 8.5 Hz, 1H, aromatic), 4.93 (s, 2H,
-NCH2). 13C NMR (100 MHz, DMSO-d6): δ 183.19 (s,
–C=O), 163.14 (s, -NHC = O), 159.19 (s, –NC=O),
147.11 (s, 2 C, pyridly), 146.71 (s, Ar–C), 145.68 (s, Ar–C),
137.28 (s, Ar–C), 134.62 (s, Ar–C), 131.76 (s, Ar–C),
131.17 (s, Ar–C), 130.59 (s, Ar–C), 129.98 (s, Ar–C),
129.83 (s, Ar–C), 128.23 (s, Ar–C), 123.95 (s, 2 C, pyridly),
118.49 (s, Ar–C), 117.26 (s, Ar–C), 111.55 (s, Ar–C), 42.35
(s, -NCH2). MS (ESI): calcd for C21H13Cl2N3O3 [M − H]−
424.03, found 424.12.
Conclusion
Cell antiproliferation assay
In summary, we designed and synthesized a novel series of
1,5-disubstituted isatin derivatives. According to the biologi-
cal assay of compounds against MCL cell lines, most com-
pounds showed decreased proliferation ability compared with
IBN and AA2, among which compound 7l had demonstrated
potent efficacy with IC50 values from 0.4 up to 1.1 μM in
MCL cell lines. Moreover, in mechanistic studies, the repre-
sentative compound 7l also induced significant cell cycle
arrest in the G2/M phase and increased cell apoptosis. Col-
lectively, these structurally promise in discovery of new
anticancer therapeutics by inducing MCL cells apoptosis.
Cell culture
Experimental cells (MCL cell lines) were purchased from
Amercian Type Culture Collection (ATCC, USA) and cul-
tured in RPMI - 1640 (Gibco, USA) supplemented with 10%
fetal bovine serum (FBS, Gibco) at 37 °C with humidified
atomosphere containing 5% CO2 in a culture incubator. When
70% of the plate had been covered, cells were incubated,
harvested and used for proliferation and cycle analysis.
Cell viability assay
Acknowledgements This work was supported by the Key Research
and Development Project of Shandong Province, China (Nos.
2017CXGC1401 and 2019GSF108038).
The in vitro inhibitory effects of the compounds were
measured on a panel of MCL cell lines using the CellTiter-

Medicinal Chemistry Research
Compliance with ethical standards
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