Synthesis and biological evaluation of 3-amino-3-hydroxymethyloxindoles as potential anti-cancer agents

Article abstract of DOI:10.1039/c6ra27536b

A series of substituted 3-amino-3-hydroxymethyloxindoles (4a-4i) synthesized through a multi-component approach showed anticancer potency via in vitro cytotoxicity screening. Further, to develop the efficiency, derivatives (5a-5m) were synthesized and evaluated for their anti-proliferation activity. The most potent compound 5m showed cytotoxic effect toward SJSA-1 cells (IC₅₀ = 3.14 μM) as well as inhibiting the growth of other cancer cell lines (HCT-116, Jurkat, KB and Bel7402). Further investigation revealed that 5m induced a significant G2/M cell cycle arrest and time- and dose-dependent cellular apoptosis in SJSA-1 cells. These results suggested that new compound 5m has anti-proliferating and pro-apoptotic effects, which might be a candidate for cancer therapies.

Full text of DOI:10.1039/c6ra27536b

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PAPER
Synthesis and biological evaluation of 3-amino-3-
hydroxymethyloxindoles as potential anti-cancer
agents†
Cite this: RSC Adv., 2017, 7, 23265
Kaili Jia,‡ Xinxin Lv,‡ Dong Xing, Jiuwei Che, Donglan Liu, Nilesh J. Thumar,
*
*
Suzhen Dong and Wenhao Hu
A
series of substituted 3-amino-3-hydroxymethyloxindoles (4a–4i) synthesized through a multi-
component approach showed anticancer potency via in vitro cytotoxicity screening. Further, to develop
the efficiency, derivatives (5a–5m) were synthesized and evaluated for their anti-proliferation activity.
The most potent compound 5m showed cytotoxic effect toward SJSA-1 cells (IC50 ¼ 3.14 mM) as well as
inhibiting the growth of other cancer cell lines (HCT-116, Jurkat, KB and Bel7402). Further investigation
revealed that 5m induced a significant G2/M cell cycle arrest and time- and dose-dependent cellular
apoptosis in SJSA-1 cells. These results suggested that new compound 5m has anti-proliferating and
pro-apoptotic effects, which might be a candidate for cancer therapies.
Received 29th November 2016
Accepted 15th April 2017
DOI: 10.1039/c6ra27536b
rsc.li/rsc-advances
amino-3-hydroxymethyloxindoles type scaffolds.11 Further,
screening of synthesized compounds has revealed their anti-
Introduction
cancer activity. Inspired by the primary results, we further
synthesized a series of oxindole derivatives to evaluate their
anti-proliferation activity for advance study. The compounds
with the best activity were assessed for wide-spectrum anti-
cancer studies and analyzed for its effects on cell cycle and
apoptosis.
Although tremendous efforts and resources have been invested
for many years in the development of novel anti-cancer thera-
pies, cancer remains the second leading cause of mortality in
the world. It has been reported that a total of 1 658 370 new
cancer cases and 589 430 cancer deaths are projected to occur in
the United States in 2015.1 New approaches to treat cancers are
urgently needed. The paradigm of novel anticancer drug
discovery has been shied to molecularly-targeted therapeutics,
but the conventional approach to drug discovery based on
identication of cytotoxic agents on multiple human tumor cell
lines, one kind of phenotypic screening, remains the single
most successful route.2
Compared to target-based screening, phenotypic screening
without particular target in drug discovery highly depends on
the quality of new molecule entities with molecular complexity
and diversity.3,4 Fortunately, such screening library of complex
and diverse compounds simultaneously with new scaffolds can
be quickly and easily built through multi-component reactions
(MCRs).5 Our group has developed a series of ylides or zwitter-
ionic intermediates “trapping” MCRs which have been veried
as an efficient approach to form heterocycle.6–10 Rh(II) catalyzed
MCR of 3-diazooxindoles, anilines and formalin synthesized 3-
Results and discussion
Chemistry
Herein, oxindole derivatives (4a–4i, 5a–5m) were synthesized by
three-component “trapping” reaction (Scheme 1) as following:
in ethyl acetate (EA) solution of catalyst Rh2(OAc)4, (1 mol%),
aniline 2 and formalin 3 was added sequentially. The suspen-
sion was stirred at 60 ꢀC for several minutes. Aerwards, 3-
diazooxindoles 1 in EA was added to suspension over 1 hour via
a syringe pump at the same temperature. Upon completion of
the addition, the reaction mixture was le on stirring for an
additional hour until no further transformation was monitored
by TLC test. Solvent was evaporated and the residue was puri-
ed by ash chromatography on silica gel to afford the products
in moderate yield (53–89%).
As marked in the Scheme 2, the benzyl, methyl and phenyl
group which is represented by R1, R2 and R3 in Table 1 were
chemically modied. According to primary biological test
results mentioned in Table 1, we realized that the benzyl group
at R1 position and the methyl group at R2 position were more
efficient at respective positions for the antitumor activity of
these compound analogues. These results diverted our atten-
tion modication at R3 position. In order to compare the effect
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, School of Chemistry and Molecular Engineering, East China Normal
University, 3663 North Zhongshan Road, Shanghai 200062, China. E-mail: szdong@
brain.ecnu.edu.cn; whu@chem.ecnu.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra27536b
‡ These authors contribute to this paper equally.
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Scheme 1 Synthesis of 3-amino-3-hydroxymethyloxindoles 4 & 5.
Scheme 2 Library optimization strategy for 3-amino-3-hydroxymethyloxindoles to improve SJSA-1 inhibition activity.
on bioactivity of different abundant groups at R3 position, we
Biological activities
have prepared a series of analogues having groups such as –F,
Anti-proliferative activity evaluation. Our random screening
had found some of 3-amino-3-hydroxymethyloxindoles had
antitumor activity. To obtain new compounds with anticancer
activity, CCK-8 assay was used to measure the cytotoxicity of all
the known and newly-synthesized substituted 3-amino-3-
hydroxymethyloxindoles in SJSA-1 cells. The results are
summarized in Table 1. A total of seven compounds had good
growth inhibitory activity when used at 10 mM. These
compounds were further measured their IC50 values of cytotoxic
effects. The IC50 value ranged from 2.94 to 10.81 mM. Among
them, compounds 4h and 5m showed the most potent inhibi-
tory effects with IC50 values of about 3 mM.
Regarding the structure–activity relationship, several corre-
lations can be made from these data. Compound 4c with 5-Br
substituent on oxindole ring has shown more inhibition activity
than 4b. Through the evaluation of R1 group, with benzyl (4b)
moiety replaced by methyl group (4a), a previous suppression
on the inhibition percentage has been observed. As the affection
of R2 group was concerned, we attempted to rene the hydrogen
(5e, 5f) with uorine atom (5g, 5l) and methyl group (5h, 5m).
We were pleased to nd a considering increase on the cytotoxic
activity when methyl group (5h, 5m) appears on R1 position. So,
with this optimal condition in hand, we introduced a series of
substitute group on R3 phenyl group. Aer comparison of the
bioactivity data, the 2,4-diuorine-phenyl group was considered
competent in the R3 position. Another noteworthy point is 4-Cl
(4f) and 4-I (4h) were expelled from preferable list due to its high
IC50 value which failed to satisfy our basic requirement even
though they show off signicantly valuable activity on SJSA-1
inhibition percentage test.
–Cl, –Br, –I and –NO2, on the phenyl ring at R3 position and
studied subsequently.
Table 1 Inhibitory activity of compounds 4a–4i and 5a–5m on SJSA-1
cell proliferation present as inhibition (%) and IC50 (mM)a
Compound R1
R2
R3
Inhibition (%) IC50 (mM)
b
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
Me
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
H
2,6-Di-Cl
2,6-Di-Cl
16.14
32.69
90.78
ꢁ1.73
41.59
89.23
41.46
69.46
97.12
7.51
—
—
5.74
—
—
3.5
—
2.94
10.81
—
—
—
5-Br 2,6-Di-Cl
H
H
H
H
H
H
H
H
H
H
H
H
5-F
2,4-Di-Cl
2-I
4-Cl
4-Br
4-I
4-NO2
2-F
3,5-Di-CF3 37.90
3,4-Di-F
2-Cl-4-F
2,6-Di-F
2,6-Di-F
2,6-Di-F
23.66
25.83
ꢁ4.67
7.29
19.86
38.56
90.08
81.03
25.31
48.41
93.65
—
—
—
—
Bn
Me
Me
Bn
4-Me–Bn
4-Me–Bn
Bn
5-Me 2,6-Di-F
H
H
H
—
2,6-Di-F
2,4-Di-F
2,4-Di-F
2,4-Di-F
6.99
6.19
—
—
3.14
5j
5k
5l
Bn
Bn
5-F
5m
5-Me 2,4-Di-F
a
Inhibition of SJSA cell proliferation produced by the tested
b
compounds at 10 mM. The IC50 of compounds were not determined
since the inhibition rate at 10 mM was lower than 50%.
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Table 2 In vitro cytotoxicity of compound 5m on five human cancer
cell lines and two normal cell lines
analyzed their cell cycle distribution by ow cytometry. The
results showed that 57.46% of cells were in the G0/G1 phrase,
7.56% in the S phrase, and 28.22% in the G2/M phrase in the
control group (Fig. 1A and B). On the contrast, a signicant
decreased percentage of cells were in G0/G1 phrase 48 h
following compound 5m treatment at higher concentration
(51.76%, 42.79% and 25.1% for 5 mM, 10 mM and 20 mM,
respectively). Meanwhile, the proportion of cells in G2/M phrase
were signicantly increased (30.65%, 35.67% and 44.88% for 5
mM, 10 mM and 20 mM, respectively) (Fig. 1A and B). These
results suggested that compound 5m treatment induced cell
cycle arrest at G2/M in a dose-dependent manner, which might
be one of the primary mechanisms responsible for the anti-
cancer activity of the compound.
Apoptosis induction. In the experiment of cell cycle analysis
by ow cytometry, we observed that more and more compound-
treated cells entered subG0 phrase (3.27%, 7.68% and 15.1% for
5 mM, 10 mM and 20 mM, respectively; compared with control,
0.47%) (Fig. 1), a phrase related to cell apoptosis, which sug-
gested that compound 5m could also induced cell apoptosis. In
order to further conrm the effect, SJSA-1 cells were treated with
5m at different concentrations (0 mM, 2 mM, 5 mM or 10 mM) and
Hoechst 33 342 staining using microscopy were carried out
24 h, 48 h or 72 h aer treatment. In the control cells (treated
with only the vehicle DMSO), the cells kept growing without
visible apoptosis. On the contrast, shrunken nucleus and
peripherally clumped and fragmented chromatin were observed
following compound treatment at higher concentration (5 mM
and 10 mM), a typical characteristic of cells undergoing
apoptosis (Fig. 2).
Cell
SJSA-1 HCT116 Jurkat KB
Bel7402 HEK293 HL7702
11.83 39.19
IC50 (mM) 3.14
5.1 5.38 18.35 25.04
To ensure if these compounds had broad-spectrum anti-
cancer activity and preference of killing cancer cells over
normal cells, the IC50 of proliferation inhibition was also
measured in other four cancer cell lines and two normal cell
lines. Compound 5m also showed some extent of anti-
proliferation activities in all the four cells lines (IC50 ¼ 5.1
mM, 5.38 mM, 18.35 mM, 25.04 mM for HCT116, Jurkat, KB,
Bel7402 cells, respectively, Table 2). The IC50 of cytotoxicity in
HL7702 and HEK293 cells is 39.19 mM and 11.83 mM, respec-
tively. Therefore, 5m showed better effects of growth inhibition
in SJSA-1, HCT116 and Jurkat cells than other cancer and
normal cells.
Cell cycle analysis. It is well known that inhibitors of cell
proliferation work by interfering with cell cycle or inducing cell
death. In order to identify whether compound 5m has any
effects on cell cycle, SJSA-1 cells were treated with compound
5m at different concentration (0 mM, 2 mM, 5 mM, 10 mM or 20
mM) for 48 h, the cells were collected, stained with PI, and
To further quantify the effects of 5m-induced apoptosis,
Annexin V–FITC and PI dual staining were performed using ow
cytometry. The analysis of stained cells distinguished between
four groups: (1) viable cells (Annexin Vꢁ, PIꢁ); (2) early apoptotic
cells (Annexin V+, PIꢁ); (3) late apoptotic cells (Annexin V+, PI+);
(4) necrotic cells (Annexin Vꢁ, PI+). Firstly, we detected the time-
dependent apoptosis. SJSA-1 cells were treated by 5m at 10 mM
Fig. 1 Effect of compound 5m on cell cycle progression in SJSA-1
cells. Cells were treated with compound 5m at 0, 2, 5, 10 or 20 mM for Fig. 2 Compound 5m induced apparent cell apoptosis. Cells were
48 h, stained with PI. Then cell cycle was analyzed by flow cytometry. treated with compound 5m at 0, 2, 5, or 10 mM for 24 h, 48 h or 72 h
(A) Representative photographs from three independent experiments and nuclear morphology was checked by Hoechst 33 342 staining by
were displayed. (B) Cell cycle proportion in (A) was quantitated. Data microscopy at 200ꢃ and representative photographs were captured.
presented are the mean ꢂ SD of three independent experiments.
The yellow arrows stand for nucleus of apoptotic cells.
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or 20 mM for 24 h, 48 h or 72 h and then were subject to
apoptosis evaluation. The results showed that the percentage of
apoptotic cells (including early apoptotic and late apoptotic
population) at both concentrations was elevated as the treat-
ment time was extended (Fig. 3). In another experiment, 5m at
different concentration were added into SJSA-1 cells and its
effects were analyzed aer 48 h. Similarly, 5m at high concen-
tration induced more apoptotic cells than lower concentration
(2 mM, 16.67%; 5 mM, 30.66%; 10 mM, 38.66%; 20 mM, 58.02%)
(Fig. 4), suggesting
a dose-dependent effect. Therefore,
compound 5m induced apoptosis of SJSA-1 cells in a time- and
dose-dependent manner. Moreover, western blotting experi-
ments were performed to study the underlying mechanism of
5m-induced apoptosis. Our preliminary data (refer ESI†)
showed that 5m caused the ratio of Bax/Bcl-2, two apoptosis-
related proteins, to be increased, suggesting that the intrinsic
mitochondrial apoptosis pathway, rather than the extrinsic
pathway, was involved in 5m-induced apoptosis. Further
investigations are essential to illuminate the accurate
mechanism.
Conclusion
Fig. 3 Time-dependent cellular apoptosis. Cells were treated with
compound 5m at 10 or 20 mM for 24 h, 48 h or 72 h and apoptosis was
evaluated using Annexin V–FITC/PI dual staining kit by flow cytometry.
(A) Representative photographs from three independent experiments
were displayed. (B) Cell apoptosis rates were calculated and plotted in
response to different time.
In this study, we synthesized a potent series of oxindole
compounds (4a–4i, 5a–5m) using MCR approach.11 These
compounds were screened for anticancer activity using the
CCK-8 assay. Among them, compound 4h and 5m displayed the
most potent anti-proliferating effect against SJSA-1 cells (IC50 z
3.0 mM). Besides, 5m showed broad-spectrum anti-cancer
activity because it can inhibit the proliferation of other cancer
cells. The ow cytometry results showed that 5m inhibited the
cell growth and caused the cell cycle to be arrested at the G2/M
phase. Meanwhile, 5m induced apoptosis of SJSA-1 cells in
a dose- and time-dependent manner. These data proved the
potent cell proliferation inhibitory abilities and pro-apoptotic
activities of compound 5m in SJSA-1 cells. To sum up, we
regarded compound 5m as a promising, potent anti-cancer
candidate among the 3-amino-3-hydroxymethyloxindoles
studied. Further study on the in vivo anticancer effects of 5m
compound and its underlying mechanism is in progress.
Materials and methods
Chemicals
Diazocompound 1 was prepared according to the literature
methods.12–14 All isolated compounds were characterized on the
1
basis of H NMR and 13C NMR spectroscopic data and HRMS
(TOF-Q) data. 1H NMR and 13C NMR chemical shis are re-
ported in ppm using tetramethylsilane (TMS) as an internal
standard.
General procedure for the synthesis of 3-amino-3-
Fig.
4 Concentration-dependent cellular apoptosis. Cells were
treated with compound 5m at 0, 2, 5, 10 or 20 mM for 48 h and
apoptosis was evaluated using Annexin V–FITC/PI dual staining kit by
flow cytometry. (A) Representative photographs from three indepen-
dent experiments were displayed. (B) Cell apoptosis rates were
calculated and plotted in response to different concentrations.
hydroxymethyloxindoles (4a–4i and 5a–5m)
To a mixture of Rh2(OAc)4 (0.001 mmol), aniline 2 (0.10 mmol),
formalin 3 (37% aqueous solution of formaldehyde, 0.60 mmol)
in EtOAc (1.0 mL) at 60 ꢀC and diazo compound 1 (0.1 mmol) in
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EtOAc (1.0 mL) were added via a syringe pump for 1 h. Aer the
completion of the addition, the reaction mixture was stirred for
additional 1 h. Solvents were removed under reduced pressure
to give the crude products. The crude products were puried by
ash chromatography on silica gel (ethyl acetate/petroleum
ether ¼ 1 : 10 to 1 : 1) to give pure products. The compounds
(4a–4i) were prepared according to literature methods.11
Hoechst staining
Nuclear morphology was detected by staining nuclei with
Hoechst 33 342. SJSA-1 cells (1 ꢃ 104) were seeded in twenty-
four-well plates for 24 h. Thereaer, cells were treated with
compound 5m at different concentration for 24 h, 48 h or 72 h.
Cells were washed with PBS and xed with 4% para-
formaldehyde. Cells were stained with Hoechst 33 342 and
images were captured using uorescence microscope (Leica).
Cell culture
Five human cancer cell lines and two human normal cells were Annexin V–FITC/PI dual staining
used in the study, including SJSA-1 (osteosarcoma), HCT116
To determine effect of compound 5m on apoptosis in SJSA-1
(colon cancer), Bel7402 (liver cancer), KB (Oral epithelial carci-
noma), Jurkat (human T lymphocyte leukaemia), HL7702
(normal liver) and HEK293 cells (embryonic kidney). They were
purchased from Cell Bank of China Science Academy
(Shanghai, China), and cultured aseptically using the corre-
sponding media supplemented with 10% (v/v) fetal bovine
serum and 100 units per mL each of penicillin and strepto-
cells, Annexin V–FITC/PI dual staining was carried out by ow
cytometry. SJSA-1 cells (1 ꢃ 105 cells per well) were seeded in six-
well plates and allowed to grow to 70 to 80% conuence. Then
the cells were treated with compound 5m at concentrations of 0,
2, 5, 10 or 20 mM for 48 h, respectively (dose–response experi-
ment) or 10 mM and 20 mM for 24 h, 48 h or 72 h, respectively
(time–response experiment). At the end of the incubation, the
cells were harvested by trypsinization and washed twice with
cold PBS. Then the cells were resuspended in 400 mL of 1ꢃ
binding buffer and stained with Annexin V–FITC and PI using
the apoptosis detection kit from Beyotime. Cells were measured
using an easyCyte 6HT-2L (Millipore) and analyzed by FlowJo
soware. The percentage of cells for the lower and upper right
quadrant was used for statistical analysis using Graphpad
Prism 5. All experiments were conducted in triplicate, and for
each measurement, at least 1 ꢃ 104 cells were counted.
ꢀ
mycin, pH 7.2 and 5% CO2 humidied atmosphere at 37 C.
Cell viability assay (cck-8 assay)
In vitro cytotoxicity of the compounds was evaluated by the CCK-
8 assay. Briey, SJSA-1 cells were respectively trypsinized and
seeded in 96-well plates at a proper density the day before
compound administration. Each tested compound was dis-
solved in DMSO (10 mM) and diluted in media. Then the
compound was added to the cells at 10 mM. The control cells
were treated with the vehicle DMSO. Aer 72 h incubation, 10
mL of CCK8 solution (5 g Lꢁ1; Yeasen) in the media was added to
each well and incubated for additional 4 h. Finally, the optical
density (OD) was measured at 450 nm using a microplate reader
(spectraMax M5/M5e, Sunnyvale, CA, USA) and a reference
wavelength at 620 nm. If one compound inhibits cell prolifer-
ation with percentage inhibition higher than 50%, the IC50
value was determined by testing the inhibitory effects of the
compound with 10 gradient-dilution concentrations with at
least three replicates per concentration. As for the compound
with the best growth-inhibitory activity, its IC50 value in other
six cell lines (HCT116, Bel7402, KB, Jurkat, HL7702 and
HEK293) were further determined.
1-Benzyl-3-((2-uorophenyl)amino)-3-(hydroxymethyl)indolin-
2-one (5a). A white solid (73% yield).1H NMR (400 MHz, CDCl3)
d 7.30 (qd, J ¼ 6.6, 3.3 Hz, 7H), 7.08 (t, J ¼ 7.5 Hz, 1H), 6.96 (ddd,
J ¼ 11.6, 8.0, 1.4 Hz, 1H), 6.88 (d, J ¼ 7.8 Hz, 1H), 6.65–6.58 (m,
1H), 6.53 (t, J ¼ 7.6 Hz, 1H), 5.78–5.71 (m, 1H), 5.47 (d, J ¼
3.2 Hz, 1H), 5.12 (d, J ¼ 15.4 Hz, 1H), 4.80 (d, J ¼ 15.4 Hz, 1H),
3.97 (t, J ¼ 11.4 Hz, 1H), 3.73 (dd, J ¼ 11.5, 2.5 Hz, 1H), 2.91 (dd,
J ¼ 11.3, 2.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) d 177.58,
153.55, 151.17, 141.86, 135.32, 134.03 (d, J ¼ 11.2 Hz), 129.77,
128.87, 127.98, 127.72, 127.25, 124.23 (d, J ¼ 3.7 Hz), 124.03,
123.64, 118.96 (d, J ¼ 7.2 Hz), 114.91, 114.73, 114.30 (d, J ¼ 2.4
Hz), 110.11, 99.99, 68.07, 64.38, 44.18. HRMS (ESI) calcd for
C
22H19FN2O2 [M + Na]+: 385.1328, found 385.1320.
1-Benzyl-3-((3,5-bis(triuoromethyl)phenyl)amino)-3-(hydroxy-
1
methyl)indolin-2-one (5b). A white solid (85% yield). H NMR
(400 MHz, CDCl3) d 7.38–7.26 (m, 7H), 7.12 (dd, J ¼ 14.3, 6.4 Hz,
Cell cycle analysis
Cell cycle analysis was performed to check the distribution of 2H), 6.91 (d, J ¼ 7.8 Hz, 1H), 6.61 (s, 2H), 5.63 (s, 1H), 5.06–4.88
SJSA-1 cells in different phases caused by compound 5m using (m, 2H), 3.95 (t, J ¼ 11.6 Hz, 1H), 3.75 (dd, J ¼ 11.6, 1.8 Hz, 1H),
propidium iodide (PI) staining method. SJSA-1 cells were plated 2.90 (dd, J ¼ 11.1, 1.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
in 6-well plates (1 ꢃ 105 cells per well), and allowed to grow to d 176.90, 146.36, 141.92, 134.95, 132.34 (d, J ¼ 32.9 Hz), 130.40,
about 70% to 80% conuence and then the cells were exposed 129.06, 128.03, 127.15, 125.60, 124.52, 123.94 (d, J ¼ 8.6 Hz),
to compound 5m at a concentration of 0, 2, 5, 10 or 20 mM, 121.81, 114.03 (q, J ¼ 3.8, 2.3 Hz), 112.26, 110.51, 67.95, 64.27,
respectively. Aer 48 h, cells were trypsinized, washed with cold 44.36. HRMS (ESI) calcd for C24H18F6N2O2 [M + Na]+: 503.1170,
PBS, and permeabilized with 70% ethanol overnight. On the found 503.1163.
next day, the cells were stained with PI (Beyotime) according to
1-Benzyl-3-((3,4-diuorophenyl)amino)-3-(hydroxymethyl)
the manufacturer's instructions. Then cells were measured indolin-2-one (5c). A white solid (67% yield). 1H NMR (400 MHz,
using an easyCyte 6HT-2L (Millipore) and analyzed by FlowJo CDCl3) d 7.40–7.27 (m, 5H), 7.21 (d, J ¼ 7.0 Hz, 2H), 7.10 (t, J ¼
soware. All experiments were conducted in triplicate, and for 7.4 Hz, 1H), 6.86 (d, J ¼ 7.8 Hz, 1H), 6.74 (dd, J ¼ 18.6, 9.2 Hz,
each measurement, at least 20 000 cells were counted.
1H), 6.13–5.93 (m, 2H), 5.12 (d, J ¼ 15.2 Hz, 2H), 4.75 (d, J ¼
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15.4 Hz, 1H), 3.91 (t, J ¼ 11.4 Hz, 1H), 3.69 (d, J ¼ 11.3 Hz, 1H), 3H). 13C NMR (100 MHz, CDCl3) d 177.23, 155.91 (d, J ¼ 6.7 Hz),
2.94 (d, J ¼ 11.1 Hz, 1H). 13C NMR (100 MHz, CDCl3) d 177.51, 153.50 (d, J ¼ 6.7 Hz), 140.14, 135.66, 132.44, 129.59, 128.73,
142.46 (dd, J ¼ 8.2, 2.0 Hz), 141.90, 135.12, 129.97, 128.93, 127.79, 127.64, 127.30, 124.46, 122.40 (t, J ¼ 15.3 Hz), 120.39 (t, J
128.02, 127.45, 126.87, 123.86 (d, J ¼ 24.2 Hz), 117.35 (d, J ¼ 18.5 ¼ 9.5 Hz), 111.30 (dd, J ¼ 17.1, 7.0 Hz), 109.32, 68.40, 65.88,
Hz), 111.31 (dd, J ¼ 5.6, 3.3 Hz), 110.30, 104.74 (d, J ¼ 20.6 Hz), 44.04, 21.01. HRMS (ESI) calcd for C23H20F2N2O2 [M + Na]+ ¼
67.88, 65.13, 44.13. HRMS (ESI) calcd for C22H18F2N2O2 [M + 417.1391, found 417.1376.
Na]+: 403.1234, found 403.1233.
3-((2,6-Diuorophenyl)amino)-3-(hydroxymethyl)-1-(4-methyl-
1-Benzyl-3-((2-chloro-4-uorophenyl)amino)-3-(hydroxymethyl)- benzyl)indolin-2-one (5i). A white solid (67% yield). 1H NMR
indolin-2-one (5d). A white solid (53% yield). 1H NMR (400 MHz, (400 MHz, CDCl3) d 7.32 (dd, J ¼ 15.1, 7.6 Hz, 2H), 7.18–7.04 (m,
CDCl3) d 7.37–7.27 (m, 7H), 7.09 (t, J ¼ 7.5 Hz, 1H), 7.03 (dd, J ¼ 5H), 6.88 (d, J ¼ 7.8 Hz, 1H), 6.77 (ddd, J ¼ 11.1, 8.6, 2.7 Hz, 1H),
8.2, 2.5 Hz, 1H), 6.91 (d, J ¼ 7.7 Hz, 1H), 6.37 (td, J ¼ 8.6, 2.6 Hz, 6.28 (t, J ¼ 8.5 Hz, 1H), 5.70 (td, J ¼ 9.3, 5.5 Hz, 1H), 5.32 (d, J ¼
1H), 5.72 (s, 1H), 5.64 (dd, J ¼ 8.9, 5.1 Hz, 1H), 5.13 (d, J ¼ 2.8 Hz, 1H), 5.09 (d, J ¼ 15.3 Hz, 1H), 4.70 (d, J ¼ 15.3 Hz, 1H),
15.3 Hz, 1H), 4.77 (d, J ¼ 15.3 Hz, 1H), 3.98 (t, J ¼ 11.4 Hz, 1H), 3.96 (t, J ¼ 11.5 Hz, 1H), 3.72 (dd, J ¼ 11.5, 2.2 Hz, 1H), 2.90 (dd,
3.73 (dd, J ¼ 11.5, 1.7 Hz, 1H), 2.88 (dd, J ¼ 11.3, 1.8 Hz, 1H). 13
C
J ¼ 11.4, 2.2 Hz, 1H), 2.33 (s, 3H). 13C NMR (100 MHz, CDCl3)
NMR (100 MHz, CDCl3) d 177.27, 141.82, 138.47 (d, J ¼ 2.5 Hz), d 177.47, 141.92, 137.82, 132.17, 130.46 (dd, J ¼ 11.5, 2.6 Hz),
135.29, 129.90, 128.89, 128.10, 127.80, 126.94, 123.98, 123.75, 129.88, 129.52, 127.67, 127.13, 124.05, 123.64, 115.10 (dd, J ¼
116.76 (d, J ¼ 25.7 Hz), 113.93 (dd, J ¼ 20.8, 14.9 Hz), 110.16, 8.8, 3.5 Hz), 110.41 (dd, J ¼ 21.9, 3.8 Hz), 110.23, 103.80 (dd, J ¼
68.05, 64.78, 44.20. HRMS (ESI) calcd for C22H18ClFN2O2 [M + 26.5, 23.5 Hz), 67.86, 64.80, 43.89, 21.14. HRMS (ESI) calcd for
Na]+ ¼ 419.0939, found 419.0919.
C
22H20F2N2O2 [M + Na]+ ¼ 417.1391, found 417.1385.
1-Benzyl-3-((2,6-diuorophenyl)amino)-3-(hydroxymethyl)
3-((2,4-Diuorophenyl)amino)-3-(hydroxymethyl)-1-(4-methyl-
indolin-2-one (5e). A white solid (77% yield). 1H NMR (400 MHz, benzyl)indolin-2-one (5j). A white solid (83% yield). 1H NMR (400
CDCl3) d 7.30 (dd, J ¼ 12.0, 4.8 Hz, 3H), 7.25–7.12 (m, 4H), 6.97 MHz, CDCl3) d 7.31 (dd, J ¼ 15.6, 7.2 Hz, 2H), 7.21–6.98 (m, 5H),
(t, J ¼ 7.5 Hz, 1H), 6.82–6.57 (m, 4H), 5.02–4.82 (m, 3H), 3.95– 6.88 (d, J ¼ 7.8 Hz, 1H), 6.76 (ddd, J ¼ 11.2, 8.5, 2.8 Hz, 1H), 6.28
3.82 (m, 2H), 2.82 (dd, J ¼ 9.5, 4.0 Hz, 1H). 13C NMR (100 MHz, (ddd, J ¼ 10.6, 3.2, 1.9 Hz, 1H), 5.71 (td, J ¼ 9.3, 5.5 Hz, 1H), 5.31
CDCl3) d 177.34, 154.81 (dd, J ¼ 242.9, 6.7 Hz), 142.62, 135.56, (d, J ¼ 2.1 Hz, 1H), 5.08 (d, J ¼ 15.3 Hz, 1H), 4.70 (d, J ¼ 15.3 Hz,
129.36, 128.77, 127.70, 127.59, 127.31, 123.74, 122.75, 120.58 (t, 1H), 3.96 (t, J ¼ 11.4 Hz, 1H), 3.72 (dd, J ¼ 11.5, 2.4 Hz, 1H), 2.89
J ¼ 9.5 Hz), 111.28 (dd, J ¼ 17.2, 7.0 Hz), 109.58, 68.35, 65.92, (dd, J ¼ 11.3, 2.4 Hz, 1H), 2.33 (s, 3H). 13C NMR (100 MHz, CDCl3)
44.04. HRMS (ESI) calcd for C22H18F2N2O2 [M + Na]+ ¼ 403.1234, d 177.45, 141.97, 137.81, 132.18, 130.46 (dd, J ¼ 11.3, 4.1 Hz),
found 403.1224.
129.96, 129.81, 129.64, 129.39, 127.78, 127.53, 127.14, 124.11,
3-((2,6-Diuorophenyl)amino)-3-(hydroxymethyl)-1-methyl- 123.97, 123.75, 123.51, 110.22 (d, J ¼ 32.0 Hz), 64.77, 43.90 (dd, J
indolin-2-one (5f). A white solid (89% yield). 1H NMR (400 MHz, ¼ 20.6, 13.2 Hz), 21.12 (d, J ¼ 18.8 Hz). HRMS (ESI) calcd for
CDCl3) d 7.41 (t, J ¼ 7.7 Hz, 1H), 7.33 (d, J ¼ 7.3 Hz, 1H), 7.14 (t, J C23H20F2N2O2 [M + Na]+ ¼ 417.1391, found 417.1382.
¼ 7.5 Hz, 1H), 6.97 (d, J ¼ 7.8 Hz, 1H), 6.75 (ddd, J ¼ 11.2, 8.5,
1-Benzyl-3-((2,4-diuorophenyl)amino)-3-(hydroxymethyl)
2.8 Hz, 1H), 6.43–6.33 (m, 1H), 5.73 (td, J ¼ 9.3, 5.4 Hz, 1H), 5.30 indolin-2-one (5k). A white solid (53% yield). 1H NMR (400 MHz,
(s, 1H), 5.27 (d, J ¼ 1.8 Hz, 1H), 3.92 (t, J ¼ 11.3 Hz, 1H), 3.68 (dd, CDCl3) d 7.39–7.27 (m, 5H), 7.24–7.17 (m, 2H), 7.10 (t, J ¼ 7.5 Hz,
J ¼ 11.5, 2.5 Hz, 1H), 3.28 (s, 3H), 2.82 (dd, J ¼ 11.2, 2.5 Hz, 1H). 1H), 6.87 (d, J ¼ 7.8 Hz, 1H), 6.82–6.70 (m, 1H), 6.27 (t, J ¼
13C NMR (100 MHz, CDCl3) d 177.31, 156.80, 142.77, 130.57, 8.4 Hz, 1H), 5.72 (td, J ¼ 9.2, 5.8 Hz, 1H), 5.31 (s, 1H), 5.11 (d, J ¼
130.00, 127.12, 124.00, 123.70, 114.29 (dd, J ¼ 8.9, 3.6 Hz), 15.4 Hz, 1H), 4.75 (d, J ¼ 15.4 Hz, 1H), 3.97 (t, J ¼ 11.4 Hz, 1H),
110.49 (dd, J ¼ 21.8, 3.9 Hz), 109.08, 103.76 (dd, J ¼ 26.6, 23.3 3.74 (d, J ¼ 11.5 Hz, 1H), 2.89 (d, J ¼ 11.1 Hz, 1H). 13C NMR (100
Hz), 67.84, 64.65, 53.50, 26.46. HRMS (ESI) calcd for MHz, CDCl3) d 177.48, 141.95, 135.24, 130.42 (dd, J ¼ 11.6, 3.2
C
16H14F2N2O2 [M + Na]+ ¼ 327.0921, found 327.0934.
Hz), 129.89, 128.86, 128.03, 127.66, 127.18, 124.11, 123.68,
115.31 (dd, J ¼ 8.8, 3.5 Hz), 110.41 (dd, J ¼ 21.6, 3.9 Hz), 110.14,
3-((2,6-Diuorophenyl)amino)-5-uoro-3-(hydroxymethyl)-1-
methylindolin-2-one (5g). A white solid (69% yield). 1H NMR 103.80 (dd, J ¼ 26.5, 23.4 Hz), 67.91, 64.87, 44.12. HRMS (ESI)
(400 MHz, CDCl3) d 7.00 (td, J ¼ 8.9, 2.6 Hz, 1H), 6.92 (dd, J ¼ 7.5, calcd for C22H18F2N2O2 [M + Na]+ ¼ 403.1234, found 403.1222.
2.6 Hz, 1H), 6.79 (dd, J ¼ 8.5, 4.0 Hz, 1H), 6.72–6.62 (m, 3H), 4.95
1-Benzyl-3-((2,4-diuorophenyl)amino)-5-uoro-3-(hydroxy-
(s, 1H), 3.88–3.75 (m, 2H), 3.22 (s, 3H), 2.93 (dd, J ¼ 8.6, 4.7 Hz, methyl)indolin-2-one (5l). A white solid (86% yield). 1H NMR (400
1H). 13C NMR (100 MHz, CDCl3) d 177.13 (d, J ¼ 0.9 Hz), 160.38, MHz, CDCl3) d 7.35–7.27 (m, 3H), 7.24–7.16 (m, 2H), 7.10 (dd, J ¼
157.98, 155.48 (d, J ¼ 6.8 Hz), 153.07 (d, J ¼ 6.6 Hz), 139.34, 7.4, 2.5 Hz, 1H), 6.98 (td, J ¼ 8.8, 2.6 Hz, 1H), 6.85–6.71 (m, 2H),
129.85–129.57 (m), 122.06 (t, J ¼ 15.0 Hz), 120.25 (t, J ¼ 9.6 Hz), 6.36–6.26 (m, 1H), 5.72 (td, J ¼ 9.2, 5.4 Hz, 1H), 5.28 (s, 1H), 5.11 (d,
115.71, 115.47, 112.06, 111.81, 111.45, 111.38, 111.28, 111.20 (d, J J ¼ 15.4 Hz, 1H), 4.73 (d, J ¼ 15.4 Hz, 1H), 3.94 (t, J ¼ 11.3 Hz, 1H),
¼ 1.7 Hz), 109.06 (d, J ¼ 8.0 Hz), 68.02, 66.01, 26.55. HRMS (ESI) 3.75 (dd, J ¼ 11.4, 2.4 Hz, 1H), 2.88 (dd, J ¼ 11.1, 2.4 Hz, 1H). 13
C
calcd for C16H13F3N2O2 [M + Na]+ ¼ 345.0827, found 345.0817.
NMR (100 MHz, CDCl3) d 177.20, 137.77, 134.91, 129.09, 129.02 (d,
1-Benzyl-3-((2,6-diuorophenyl)amino)-3-(hydroxymethyl)-5- J ¼ 1.0 Hz), 128.94 (d, J ¼ 3.0 Hz), 128.88, 128.26, 127.75, 127.54,
methylindolin-2-one (5h). A white solid (75% yield). 1H NMR 115.39 (d, J ¼ 3.5 Hz), 115.18, 112.59 (d, J ¼ 6.7 Hz), 112.34, 112.06,
(400 MHz, CDCl3) d 7.32–7.27 (m, 2H), 7.26–7.19 (m, 3H), 7.05– 111.07, 110.72 (dd, J ¼ 5.0, 1.9 Hz), 104.14 (d, J ¼ 2.0 Hz), 65.15 (d, J
6.94 (m, 2H), 6.80–6.64 (m, 3H), 6.62 (d, J ¼ 7.9 Hz, 1H), 5.01–4.80 ¼ 1.8 Hz), 44.31, 17.99. HRMS (ESI) calcd for C22H17F3N2O2 [M +
(m, 3H), 3.93–3.79 (m, 2H), 2.86 (dd, J ¼ 9.9, 3.7 Hz, 1H), 2.24 (s, Na]+ ¼ 421.1140, found 421.1142.
23270 | RSC Adv., 2017, 7, 23265–23271
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1-Benzyl-3-((2,4-diuorophenyl)amino)-3-(hydroxymethyl)-5-
methylindolin-2-one (5m). A white solid (75% yield). H NMR
1
(400 MHz, CDCl3) d 7.35–7.26 (m, 3H), 7.22 (dd, J ¼ 7.0, 2.3 Hz,
2H), 7.15 (s, 1H), 7.08 (d, J ¼ 8.0 Hz, 1H), 6.83–6.68 (m, 2H),
6.38–6.19 (m, 1H), 5.72 (td, J ¼ 9.3, 5.5 Hz, 1H), 5.30 (d, J ¼
6.6 Hz, 1H), 5.09 (d, J ¼ 15.4 Hz, 1H), 4.72 (d, J ¼ 15.4 Hz, 1H),
4.72 (d, J ¼ 15.4 Hz, 1H), 3.95 (t, J ¼ 11.4 Hz, 1H), 3.71 (dd, J ¼
11.5, 1.8 Hz, 1H), 2.96 (d, J ¼ 11.3 Hz, 1H), 2.29 (s, 3H). 13C NMR
(100 MHz, CDCl3) d 177.39, 156.78 (d, J ¼ 11.1 Hz), 154.40 (d, J ¼
11.0 Hz), 153.31 (d, J ¼ 11.6 Hz), 150.89 (d, J ¼ 11.7 Hz), 139.47,
135.36, 133.48, 130.54 (dd, J ¼ 11.5, 3.2 Hz), 130.19, 128.82,
127.98, 127.70, 127.13, 115.08 (dd, J ¼ 8.8, 3.6 Hz), 110.41 (dd, J
¼ 21.7, 3.9 Hz), 109.94, 103.77 (dd, J ¼ 26.6, 23.4 Hz), 67.93,
64.76, 44.14, 21.05. HRMS (ESI) calcd for C23H20F2N2O2 [M +
Na]+ ¼ 417.1391, found 417.1389.
Acknowledgements
We wish to thank the Natural Science Foundation of Shanghai
(16ZR1410500) for nancial support.
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Notes and references
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