Design, synthesis and preliminary biological evaluation of novel benzyl sulfoxide 2-indolinone derivatives as anticancer agents

Article abstract of DOI:10.3390/molecules22111979

In this work, a series of novel benzyl sulfoxide 2-indolinone derivatives was designed and synthesized as potent anticancer agents. Tyrosine kinase inhibitory activity assay indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was further investigated against five human cancer cell lines (HeLa, HepG2, MCF-7, SCC-15, and A549). Several compounds exhibited evident activities. Among them, (Z)-3-(((4-bromobenzyl)sulfinyl)methylene)indolin-2-one (6j) and (Z)-3-((benzylsulfinyl) methylene)-5-bromoindolin-2-one (6o) were found to be effective tyrosine kinase inhibitors (IC₅₀ = 1.34 and 2.69 μM, respectively) in addition to having noteworthy antitumor potential (the average IC₅₀ value of 6j or 6o was less than 40 μM). This class of novel derivatives has promising potential for further development as anticancer agents.

Full text of DOI:10.3390/molecules22111979

molecules
Article
Design, Synthesis and Preliminary Biological
Evaluation of Novel Benzyl Sulfoxide 2-Indolinone
Derivatives as Anticancer Agents
Lin Tang, Tao Peng, Gang Wang, Xiaoxue Wen, Yunbo Sun, Shouguo Zhang *, Shuchen Liu *
and Lin Wang *
Beijing Institute of Radiation Medicine, Beijing 100850, China; adamtl@126.com (L.T.); peng-tao@sohu.com (T.P.);
wgang85@126.com (G.W.); crystalcat@vip.sina.com (X.W.); sunyunbo0919@126.com (Y.S.)
* Correspondence: zhangshouguo1409@sina.com (S.Z.); liusc118@sohu.com (S.L.);
Wanglin@bmi.ac.cn or wanglin07@sina.com (L.W.); Tel.: +86-010-6693-2239 (L.W.)
Received: 12 October 2017; Accepted: 14 November 2017; Published: 16 November 2017
Abstract: In this work, a series of novel benzyl sulfoxide 2-indolinone derivatives was designed
and synthesized as potent anticancer agents. Tyrosine kinase inhibitory activity assay indicated
that most of the compounds showed significant activity. The in vitro antiproliferative activity
of these compounds was further investigated against five human cancer cell lines (HeLa,
HepG2, MCF-7, SCC-15, and A549). Several compounds exhibited evident activities. Among
them, (Z)-3-(((4-bromobenzyl)sulfinyl)methylene)indolin-2-one (6j) and (Z)-3-((benzylsulfinyl)
methylene)-5-bromoindolin-2-one (6o) were found to be effective tyrosine kinase inhibitors
(
IC50 = 1.34 and 2.69
µM, respectively) in addition to having noteworthy antitumor potential
(the average IC50 value of 6j or 6o was less than 40
µM). This class of novel derivatives has promising
potential for further development as anticancer agents.
Keywords: sulfoxide; 2-indolinone derivatives; tyrosine kinase inhibitor; anticancer
1. Introduction
Malignant tumors threaten human health and life [1]. Conventional treatments for cancer, including
surgery, chemotherapy, and radiation, have significantly improved the prognosis and survival of cancer
patients. However, several disadvantages, such as severe surgical trauma and severe damage to normal
tissue cells, have stunted their therapeutic uses. Therefore, development of more specific drugs than
existing ones is becoming a hot research topic supported by many governments [2–4].
Targeted therapy is a new approach for cancer treatment. It has some advantages, such as
specificity, and few side effects, which traditional methods do not have. For targeted cancer therapies,
small-molecule inhibitors of tyrosine kinase activity are ideal candidates that target tumor cells via
binding to intracellular molecules [5–9]. In recent years, 28 small-molecule kinase inhibitors, which
have been actively pursued as promising targeted therapeutics, have been approved by the US Food
and Drug Administration (FDA) [10,11].
2-Indolone, also known as oxindole, is a kind of aromatic heterocyclic compound. 2-Indolone
structures can be found in many natural alkaloids. For instance, indirubin, an effective component
of antileukemic drugs isolated from the traditional Chinese medicine qingdai (indigo naturalis), is a
bisindole antitumor drug that can inhibit the growth of multiple transplanted animal tumors [12]. After
years of research, a large number of synthesized 2-indolinone derivatives have been found to show
widespread biological activities, such as anti-inflammatory, antimicrobial, and antitumor effects [13,14].
Semaxanib (SU5416, II in Figure 1), first reported in 1996, is a tyrosine kinase inhibitor drug designed by
Sugen as a cancer therapeutic. Semaxanib is a potent and selective synthetic inhibitor of the Flk-1/KDR
Molecules 2017, 22, 1979; doi:10.3390/molecules22111979
www.mdpi.com/journal/molecules

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vascular endothelial growth factor (VEGF) receptor tyrosine kinase. It targets the VEGF pathway,
and both in vivo and in vitro studies have demonstrated its antiangiogenic potential [15]. A related
compound, SU11248 was further developed by Sugen and then by Pfizer and was FDA approved as
sunitinib (Sutent) for the treatment of renal carcinoma in January 2006 [16]. Moreover, many other
tyrosine kinase inhibitors, such as PHA-665752, SU11274, TSU-68 (orantinib), and BIBF-1000, were
developed as antitumor drugs [17–19]. The majority of compounds with tyrosine kinase inhibitory
activity are 3-methylene-substituted indolinones or substituted at C-3 with Schiff base structures [15–23].
Figure 1. Chemical structures of I, II, III and IV.
Recently, our laboratory reported a new class of 2-indolinone derivatives (I in Figure 1) based
on structure features derived from SU5416 (II in Figure 1). This new class of 2-indolinone derivatives
exhibited excellent tyrosine kinaseinhibitory activity and anticancer activity. Among them, Indo 5
(III in Figure 1) was the most active compound that exhibited excellent biological activities both in vitro
and in vivo [24]. Moreover, Indo 5 has become a preclinical anticancer drug candidate in our institution.
To overcome the disadvantage of Indo 5 and obtain more effective and ideal antitumor agents, another
series of novel compounds (IV in Figure 1) bearing 3-methylene-substituted indolinones were designed
based on these findings. The basic tricyclic structure (including the structure of 2-indolinone and a
benzene ring) was reserved because at least three rings are considered necessary for the anticancer
activity. The sulfoxide group was introduced to this parent nucleus structure for the first time, due to
its broad-spectrum biological activities, such as insecticidal, fungicidal, and antitumor effects.
2. Results and Discussion
2.1. Chemistry
The target compounds were synthesized via a four-step synthetic route from indolin-2-ones (
as shown in Scheme 1. Compounds 2a 2b were synthesized from indolin-2-ones and DMA-DMF as
described in the literature [25]. Thiourea was employed to convert benzylic halides (3a 3n) to the
corresponding benzylic thiols (4a 4n) in a one-pot reaction via the basic hydrolysis of isothiouronium
salt [26]. Compounds 5a 2p were synthesized from the reaction of 2a 2b with 4a 4o in the presence
of TEA [25]. Finally, the sulfides 5a 5p were oxidized to obtain the corresponding sulfoxides (6a 6p)
1)
–
–
–
–
–
–
–
–
with H2O2 in acetic acid [27]. All the synthesized compounds were purified using recrystallization or
silica gel column chromatography. The structures of the target compounds were characterized using
1H-NMR, 13C-NMR, and HRMS spectral analyses.
2.2. Biological Activity
The 16 designed compounds were first evaluated for tyrosine kinase inhibitory activity using
ELISA assay. Indo 5 was used as a reference compound. The kit was self-made and is a very effective
and economical method for preliminary tyrosine kinase inhibitory activity evaluation [28]. The IC50
values for each compound are summarized in Table 1. Apparently, most of the compounds showed
significant tyrosine kinase inhibitory activity. The IC50 values of 15 compounds (6a, 6b, 6g–6k, 6m–6o)
were much lower than that of Indo 5. Compound 6j exhibited the most potent activity with an IC50
value of 1.34 µM.

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1a, 2a: R1 = H
1b, 2b: R1 = Br
3a, 4a: R2 = H, R3 = H, R4 = H
3b, 4b: R2 = F, R3 = H, R4 = H
3c, 4c: R2 = H, R3 = F, R4 = H
3d, 4d: R2 = H, R3 = H, R4 = F
3e, 4e: R2 = Cl, R3 = H, R4 = H
3f, 4f: R2 = H, R3 = Cl, R4 = H
3g, 4g: R2 = H, R3 = H, R4 = Cl
3h, 4h: R2 = Br, R3 = H, R4 = H
3i, 4i: R2 = H, R3 = Br, R4 = H
3j, 4j: R2 = H, R3 = H, R4 = Br
5a, 6a: R1 = H, R2 = H, R3 = H, R4 = H
5b, 6b: R1 = H, R2 = F, R3 = H, R4 = H
5c, 6c: R1 = H, R2 = H, R3 = F, R4 = H
5d, 6d: R1 = H, R2 = H, R3 = H, R4 = F
5e, 6e: R1 = H, R2 = Cl, R3 = H, R4 = H
5f, 6f: R1 = H, R2 = H, R3 = Cl, R4 = H
5g, 6g: R1 = H, R2 = H, R3 = H, R4 = Cl
5h, 6h: R1 = H, R2 = Br, R3 = H, R4 = H
5i, 6i: R1 = H, R2 = H, R3 = Br, R4 = H
5j, 6j: R1 = H, R2 = H, R3 = H, R4 = Br
3k, 4k: R2 = H, R3 = OCH3, R4 = H 5k, 6k: R1 = H, R2 = H, R3 = OCH3, R4 = H
3l, 4l: R2 = CH3, R3 = H, R4 = H
3m, 4m: R2 = H, R3 = CH3, R4 = H
3n, 4n: R2 = H, R3 = H, R4 = CH3
5l, 6l: R1 = H, R2 = CH3, R3 = H, R4 = H
5m, 6m: R1 = H, R2 = H, R3 = CH3, R4 = H
5n, 6n: R1 = H, R2 = H, R3 = H, R4 = CH3
5o, 6o: R1 = Br, R2 = H, R3 = H, R4 = H
5p, 6p: R1 = Br, R2 = F, R3 = H, R4 = H
Scheme 1. Reagents and conditions: (a) DMF-DMA, toluene, room temperature, 2~4 h; (b) (i) thiourea,
ethanol, reflux, 10 min, (ii) sodium hydroxide solution, reflux, 1 h; (c) TEA, methanol, room temperature,
◦
12 h; (d) H2O2, acetic acid, 40 C, 4~5 h.
Table 1. The tyrosine kinase inhibitory activity of compounds 6a~6p.
Tyrosine Kinase Inhibitory Activity
Compound
IC50 (µM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
3.16 ± 0.31
3.74 ± 0.19
9.32 ± 0.66
9.30 ± 0.47
5.09 ± 0.12
5.96 ± 0.14
3.30 ± 0.03
3.52 ± 0.15
3.32 ± 0.19
1.34 ± 0.03
2.23 ± 0.14
16.02 ± 0.05
3.85 ± 0.09
4.39 ± 0.23
2.69 ± 0.29
7.93 ± 0.20
11.60 ± 0.79
6p
Indo 5
To investigate the relationship between anticancer activity and tyrosine kinase inhibitory activity,
these compounds were further evaluated for in vitro cytotoxicity against five cancer cell lines (human
cervical carcinoma cell line HeLa, human breast carcinoma cell line MCF-7, human liver carcinoma cell
line HepG2, human tongue carcinoma cell line SCC-15, and human lung carcinoma A549) using MTS

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assay [29]. Indo 5 was also used as a reference compound. The IC50 values for each compound are
summarized in Table 2.
Table 2. The anti-cancer activity of compounds 6a~6p.
HeLa
MCF-7
HepG2
SCC-15
A549
Compound
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
11.17 ± 0.42
13.99 ± 0.27
24.37 ± 0.54
25.02 ± 0.68
70.88 ± 1.17
60.27 ± 1.98
40.92 ± 0.88
79.87 ± 1.57
48.42 ± 0.14
24.05 ± 0.52
22.67 ± 0.64
>100
27.03 ± 1.20
27.76 ± 0.78
18.22 ± 0.66
19.83 ± 0.25
19.64 ± 0.46
35.18 ± 1.16
49.12 ± 1.08
31.54 ± 0.82
49.12 ± 0.29
>100
93.02 ± 1.50
92.63 ± 0.97
>100
75.06 ± 1.93
30.77 ± 0.29
59.08 ± 037
81.57 ± 2.62
41.43 ± 2.20
55.09 ± 1.51
32.34 ± 0.77
76.42 ± 1.50
24.19 ± 0.49
52.08 ± 0.94
47.82 ± 1.47
42.02 ± 0.70
36.25 ± 0.53
96.83 ± 0.95
86.50 ± 1.15
75.35 ± 0.72
98.29 ± 2.04
71.77 ± 2.18
36.90 ± 0.49
49.36 ± 1.33
>100
58.85 ± 0.75
>100
>100
>100
>100
49.87 ± 0.93
64.97 ± 1.47
>100
90.28 ± 2.14
>100
96.78 ± 3.76
>100
>100
>100
>100
>100
82.14 ± 3.34
34.90 ± 0.34
86.72 ± 5.16
>100
50.33 ± 1.51
90.49 ± 2.32
28.63 ± 1.58
>100
64.91 ± 1.57
44.01 ± 1.89
62.34 ± 2.00
>100
79.32 ± 0.79
59.56 ± 0.97
54.863 ± 0.66
>100
36.04 ± 2.13
58.99 ± 0.97
54.44 ± 0.88
>100
6p
Indo 5
22.15 ± 0.41
28.16 ± 1.08
29.16 ± 0.29
In the cytotoxic assay, 10 compounds (6a
activity against the HeLa cell line. In particular, compounds 6a
than Indo 5. Four compounds (6a 6c 6j and 6o) showed good antitumor activity against the MCF-7
cell line. Three compounds (6d 6j and 6o) and two compounds (6j and 6o) exhibited considerable
–
6d
,
6j
,
6k and 6m–6p) showed potent antineoplastic
,
6b and 6o exhibited stronger activity
,
,
,
cytotoxic activity against the HepG2 cell line and the SCC-15 cell line, respectively. Nevertheless,
almost all compounds did not exhibit significant anticancer activity against the A549 cell line. Notably,
the more effective compounds 6j and 6o exhibited comparable antiproliferative activity against four
cancer cell lines compared with compound Indo 5. In general, most of the compounds with good
tyrosine kinase inhibitory activity also showed significant anticancer activity. 6j and 6o were two of
the most potent compounds. However, 6l and 6p exhibited both weak tyrosine inhibitory activity and
anticancer activity. This finding implied a significant correlation between tyrosine inhibitory activity
and anticancer activity.
A structure activity relationship study revealed that the introduction of methyl and halogen
groups at the 2,3,4-positions of the benzene ring has no evident effect on activity, whereas an increase
in activity was observed with para-bromo substitution. The presence of bromo at the 5-position of
the indole ring also enhanced activity to a great extent. Unfortunately, the biological activities were
simultaneously decreased due to the poor aqueous solubility of this class of sulfoxide derivatives.
3. Experimental Section
3.1. Chemistry
All chemicals and reagents were purchased from commercial suppliers, of reagent grade, and were
used without further purification. Melting points were recorded in an open capillary tube and were
uncorrected. Reactions were monitored using TLC and performed on silica gel glass plates containing 60
GF-254. Visualization was achieved by UV light (
λ
max = 254 or 365 nm). Purification of compounds was
performed using silica gel (200–300 mesh) column chromatography. H-NMR and 13C-NMR spectra
1
were recorded using Bruker (Palo Alto, CA, USA) AV-400 and AV-600 NMR spectrometers in DMSO-d6

Molecules 2017, 22, 1979
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with TMS as internal standard. Mass spectral data were obtained using electron spray ionization on a
Micromass ZabSpec high-resolution mass spectrometer (Karlsruhe, Germany).
Note: Only the synthesis and characterization of target compounds are presented in this article.
The intermediates mentioned in Scheme 1 are described in Supplementary Materials.
General Procedure for the Synthesis of Compounds (6a–6p)
To an ice cold solution of 5a–5p (1.0 mmol, 1 eq.) in 50 mL acetic acid, 30% H2O2 (1.2 mmol,
1.2 eq.) was added. Then, the reaction was heated to 40 ◦C for approximately 4–5 h. After completion
of the reaction (monitored using TLC), the mixture was poured into ice water. The formed yellow
precipitate was filtered, washed with water, and dried under vacuum. The crude product was mixed
with 20 mL of anhydrous ethyl acetate and stirred for 2 h at room temperature. The solid was filtered,
washed with anhydrous ethyl acetate and dried to obtaint the target compounds (6a–6p).
(Z)-3-((Benzylsulfinyl)methylene)indolin-2-one (6a). Obtained in 31.1% yield, yellow solid. m.p.
◦
191–192 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.20 (d, 1H, J = 12.6 Hz), 4.32 (d, 1H, J = 12.6 Hz),
6.88 (d, 1H, J = 7.9 Hz), 7.01 (t, 1H, J = 8.0 Hz), 7.30–7.41 (m, 6H), 7.60 (s, 1H), 7.72 (d, 1H, J = 7.6 Hz),
10.86 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.649, 145.054, 143.953, 132.652, 131.857,
δ
130.754, 128.768, 128.314, 123.012, 122.210, 121.056, 110.707, 60.339; HRMS-ESI (m/z) Calcd. for
C16H14NO2S [M + H]+: 284.0745, Found: 284.0738.
(Z)-3-(((2-Fluorobenzyl)sulfinyl)methylene)indolin-2-one (6b). Obtained in 23.6% yield, yellow solid. m.p.
◦
183–184 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.30 (d, 1H, J = 12.9 Hz), 4.37 (d, 1H, J = 12.9 Hz),
6.83 (d, 1H, J = 7.8 Hz), 6.96 (t, 1H, J = 7.6 Hz), 7.14–7.40 (m, 5H), 7.47 (s, 1H), 7.66 (d, 1H, J = 7.6 Hz),
10.80 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.580, 161.998, 160.392, 144.414, 143.975,
δ
133.266, 133.245, 133.083, 131.936, 130.620, 130.567, 124.651, 124.633, 123.054, 122.222, 121.024, 118.763,
118.662, 115.711, 115.567, 110.689, 52.692; HRMS-ESI (m/z) Calcd. for C16H13FNO2S [M + H]+: 302.0651,
Found: 302.0645.
(Z)-3-(((3-Fluorobenzyl)sulfinyl)methylene)indolin-2-one (6c). Obtained in 39.1% yield, yellow solid. m.p.
◦
186–187 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.25 (d, 1H, J = 12.8 Hz), 4.36 (d, 1H, J = 12.5 Hz),
6.88 (d, 1H, J = 7.6 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.16–7.45 (m, 5H), 7.56 (s, 1H), 7.72 (d, 1H, J = 7.4 Hz),
10.87 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.775, 166.595, 163.148, 161.529, 161.438,
δ
144.057, 143.941, 143.468, 138.142, 135.404, 134.226, 134.175, 133.327, 132.602, 132.530, 132.277, 130.868,
130.813, 130.783, 127.409, 127.226, 126.967, 123.089, 122.565, 122.437, 120.871, 119.687, 117.735, 117.590,
117.497, 117.355, 115.560, 115.456. 115.422, 115.319, 110.967, 110.935, 59.690, 57.929; HRMS-ESI (m/z)
Calcd. for C16H13FNO2S [M + H]+: 302.0651, Found: 302.0646.
(Z)-3-(((4-Fluor◦obenzyl)sulfinyl)methylene)indolin-2-one (6d). Obtained in 25.7% yield, yellow solid.
1
m.p. 193–194 C. H-NMR (400 MHz, DMSO-d6)
δ (ppm): 4.21 (d, 1H, J = 12.6 Hz), 4.32 (d, 1H,
J = 12.9 Hz), 6.88 (d, 1H, J = 7.8 Hz), 7.00 (t, 1H, J = 7.6 Hz), 7.19–7.38 (m, 5H), 7.54 (s, 1H), 7.71 (d,
1H, J = 7.6 Hz), 10.86 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
δ
(ppm): 166.235, 165.996, 162.940,
162.828, 161.207, 144.377, 143.747, 143.589, 138.367, 134.651, 132.623, 132.566, 132.390, 132.333, 132.153,
132.100, 131.845, 131.475, 130.926, 127.609, 127.081, 126.113, 122.640, 121.812, 121.703, 120.638, 119.415,
115.430, 115.385, 115.226, 115.124, 114.147, 114.007, 110.313, 58.850, 57.098, 56.608; HRMS-ESI (m/z)
Calcd. for C16H13FNO2S [M + H]+: 302.0651, Found: 302.0645.
(Z)-3-(((2-Chlorobenzyl)sulfinyl)methylene)indolin-2-one (6e). Obtained in 31.6% yield, yellow solid. m.p.
◦
202–203 C. 1H-NMR (400 MHz , DMSO-d6)
δ
(ppm): 4.43 (d, 1H, J = 12.6 Hz), 4.54 (d, 1H, J = 12.6 Hz),
6.85 (d, 1H, J = 7.8 Hz), 7.00 (t, 1H, J = 7.6 Hz), 7.28–7.48 (m, 5H), 7.55 (s, 1H), 7.69 (d, 1H, J = 7.8 Hz),
10.79 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.113, 143.561, 143.526, 133.967, 133.080,
δ
132.910, 131.566, 129.803, 129.346, 129.000, 126.969, 122.680, 121.802, 120.632, 110.258, 56.057; HRMS-ESI
(m/z) Calcd. for C16H13ClNO2S [M + H]+: 318.0356, Found: 318.0350.

Molecules 2017, 22, 1979
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(Z)-3-(((3-Chlorobenzyl)sulfinyl)methylene)indolin-2-one (6f). Obtained in 28.6% yield, yellow solid. m.p.
◦
192–194 C. 1H-NMR (400 MHz, DMSO-d6)
6.88 (d, 1H, J = 7.8 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.28–7.42 (m, 5H), 7.56 (s, 1H), 7.72 (d, 1H, J = 7.3 Hz),
10.87 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.239, 144.172, 143.636, 133.870, 132.849,
δ (ppm): 4.24 (d, 1H, J = 12.6 Hz), 4.35 (d, 1H, J = 12.3 Hz),
δ
132.550, 131.526, 130.155, 130.084, 129.048, 127.848, 122.680, 121.826, 120.600, 110.323, 59.132; HRMS-ESI
(m/z) Calcd. for C16H13ClNO2S [M + H]+: 318.0356, Found: 318.0349.
(Z)-3-(((4-Chlorobenzyl)sulfinyl)methylene)indolin-2-one (6g). Obtained in 23.8% yield, yellow solid. m.p.
◦
193–195 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.22 (d, 1H, J = 12.9 Hz), 4.34 (d, 1H, J = 12.6 Hz),
6.88 (d, 1H, J = 7.6 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.30–7.46 (m, 5H), 7.52 (s, 1H), 7.71 (d, 1H, J = 7.3 Hz),
10.84 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.235, 144.302, 143.609, 132.789, 132.463,
δ
132.177, 131.495, 130.376, 128.309, 122.684, 121.818, 120.620, 110.311, 58.852; HRMS-ESI (m/z) Calcd.
for C16H13ClNO2S [M + H]+: 318.0356, Found: 318.0349.
(Z)-3-(((2-Bromobenzyl)sulfinyl)methylene)indolin-2-one (6h). Obtained in 50.0% yield, yellow solid. m.p.
◦
198–199 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.46 (d, 1H, J = 12.9 Hz), 4.56 (d, 1H, J = 12.9 Hz),
6.86 (d, 1H, J = 7.8 Hz), 7.00 (t, 1H, J = 7.1 Hz), 7.25–7.49 (m, 4H), 7.56 (s, 1H), 7.64 (d, 1H, J = 7.8 Hz),
7.70 (d, 1H, J = 7.6 Hz), 10.78 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.083, 143.529,
δ
143.271, 133.056, 132.659, 131.582, 130.720, 129.989, 127.520, 124.781, 122.701, 121.794, 120.638, 110.256,
58.383; HRMS-ESI (m/z) Calcd. for C16H13BrNO2S [M + H]+: 363.9830, Found: 363.9825.
(Z)-3-(((3-Bromobenzyl)sulfinyl)methylene)indolin-2-one (6i). Obtained in 51.7% yield, yellow solid. m.p.
◦
193–194 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.25 (d, 1H, J = 12.6 Hz), 4.27 (d, 1H, J = 12.6 Hz),
6.90 (d, 1H, J = 7.9 Hz), 7.03 (t, 1H, J = 7.7 Hz), 7.32–7.58 (m, 6H), 7.74 (d, 1H, J = 7.3 Hz), 10.89 (s, 1H,
NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.239, 144.176, 143.637, 134.135, 132.955, 132.548,
δ
131.524, 130.722, 130.437, 129.414, 122.678, 121.824, 121.440, 120.595, 110.321, 59.105; HRMS-ESI (m/z)
Calcd. for C16H13BrNO2S [M + H]+: 363.9830, Found: 363.9826.
(Z)-3-(((4-Bromobenzyl)sulfinyl)methylene)indolin-2-one (6j). Obtained in 40.0% yield, yellow solid. m.p.
◦
195–196 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.21 (d, 1H, J = 12.6 Hz), 4.33 (d, 1H, J = 12.6 Hz),
6.88 (d, 1H, J = 7.7 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.27–7.60 (m, 6H), 7.72 (d, 1H, J = 7.6 Hz), 10.87 (s, 1H,
NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.237, 144.304, 143.616, 132.641, 132.507, 132.465,
δ
131.493, 131.234, 130.779, 122.690, 121.816, 121.401, 120.618, 110.313, 58.905; HRMS-ESI (m/z) Calcd.
for C16H13BrNO2S [M + H]+: 363.9830, Found: 363.9824.
(Z)-3-(((3-Methoxybenzyl)sulfinyl)methylene)indolin-2-one (6k). Obtained in 32.1% yield, yellow solid.
m.p. 181–182 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 3.73 (s, 3H,), 4.17 (d, 1H, J = 12.6 Hz), 4.29
(d, 1H, J = 12.6 Hz), 6.87–6.93 (m, 4H), 7.01 (t, 1H, J = 7.6 Hz), 7.27–7.33 (m, 2H), 7.60 (s, 1H,), 7.72 (d,
1H, J = 7.3 Hz), 10.85 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.247, 159.128, 144.589,
δ
143.539, 132.843, 132.270, 131.451, 129.394, 122.597, 121.802, 120.668, 115.837, 113.495, 110.299, 60.004,
54.952; HRMS-ESI (m/z) Calcd. for C17H16NO3S [M + H]+: 314.0851, Found: 314.0845.
(Z)-3-(((2-Methylbenzyl)sulfinyl)methylene)indolin-2-one (6l). Obtained in 32.4% yield, yellow solid. m.p.
198–199 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 2.42 (s, 3H,), 4.28 (d, 1H, J = 12.6 Hz), 4.35 (d,
1H, J = 12.9 Hz), 6.88 (d, 1H, J = 7.6 Hz), 7.02 (t, 1H, J = 7.4 Hz), 7.18–7.34 (m, 5H), 7.72–7.75 (m, 2H),
10.80 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.223, 144.925, 143.470, 137.920, 132.198,
δ
131.673, 131.467, 130.167, 129.833, 128.093, 125.763, 122.614, 121.816, 120.676, 110.293, 57.551; HRMS-ESI
(m/z) Calcd. for C17H16NO2S [M + H]+: 298.0892, Found: 298.0894.
(Z)-3-(((3-Methylbenzyl)sulfinyl)methylene)indolin-2-one (6m). Obtained in 52.1% yield, yellow solid. m.p.
◦
174–175 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 2.32 (s, 3H,), 4.17 (d, 1H, J = 12.6 Hz), 4.29 (d, 1H,
J = 12.6 Hz), 6.91 (d, 1H, J = 7.8 Hz), 7.04 (t, 1H, J = 7.6 Hz), 7.15–7.34 (m, 5H), 7.64 (s, 1H), 7.75 (d,
1H, J = 7.6 Hz), 10.87 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.255, 144.804, 143.535,
δ

Molecules 2017, 22, 1979
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137.485, 132.200, 131.430, 131.388, 130.906, 128.568, 128.291, 127.429, 122.587, 121.806, 120.666, 110.201,
60.095; HRMS-ESI (m/z) Calcd. for C17H16NO2S [M + H]+: 298.0892, Found: 298.0894.
(Z)-3-(((4-Methylbenzyl)sulfinyl)methylene)indolin-2-one (6n). Obtained in 32.8% yield, yellow solid. m.p.
◦
186–188 C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 2.31 (s, 3H,), 4.15 (d, 1H, J = 12.6 Hz), 4.27 (d, 1H,
J = 12.6 Hz), 6.88 (d, 1H, J = 7.8 Hz), 7.01 (t, 1H, J = 6.7 Hz), 7.17–7.33 (m, 5H), 7.58 (s, 1H), 7.72 (d,
1H, J = 7.6 Hz), 10.86 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
(ppm): 166.249, 144.798, 143.524,
δ
137.219, 132.171, 131.412, 130.252, 128.951, 128.321, 122.591, 121.796, 120.676, 110.293, 59.638; HRMS-ESI
(m/z) Calcd. for C17H16NO2S [M + H]+: 298.0892, Found: 298.0894.
(Z)-3-((B◦enzylsulfinyl)methylene)-5-bromoindolin-2-one (6o). Obtained in 30.8% yield, yellow solid. m.p.
200–201 C. 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 4.21 (d, 1H, J = 12.6 Hz), 4.33 (d, 1H, J = 12.6 Hz),
6.88 (d, 1H, J = 7.9 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.27–7.34 (m, 3H), 7.53 (s, 1H), 7.59 (d, 1H, J = 8.4 Hz),
7.72 (d, 1H, J = 7.6 Hz), 10.87 (s, 1H, NH); 13C-NMR (150 MHz, DMSO-d6)
δ
(ppm): 166.237, 144.300,
143.618, 132.507, 132.467, 131.493,131.234, 130.779, 122.688, 121.816, 121.401, 120.618, 110.315, 58.905;
HRMS-ESI (m/z) Calcd. for C16H13BrNO2S [M + H]+: 363.9830, Found: 363.9826.
(Z)-5-Bromo-3-(((2-fluorobenzyl)sulfinyl)methylene)indolin-2-one (6p). Obtained in 22.2% yield, yellow
solid. m.p. 208–209 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 4.34 (d, 1H, J = 12.9 Hz), 4.41 (d, 1H,
J = 12.9 Hz), 6.83 (d, 1H, J = 8.2 Hz), 7.19–7.49 (m, 5H), 7.71 (s, 1H), 8.00 (s, 1H), 10.97 (s, 1H, NH);
13C-NMR (150 MHz, DMSO-d6)
(ppm): 165.798, 161.598, 159.960, 146.583, 142.634, 133.716, 132.874,
δ
131.554, 130.273, 120.220, 125.429, 124.261, 122.688, 118.274, 118.173, 115.331, 115.189, 113.602, 112.212,
52.321; HRMS-ESI (m/z) Calcd. for C16H12BrFNO2S [M + H]+: 381.9736, Found: 381.9729.
3.2. Biological Evaluation
3.2.1. Tyrosine Kinase Inhibitory Activity Assay
The tyrosine kinase inhibitory activity assay was performed as described in the literature [28].
Details of the experimental procedures are presented in Supplementary Materials associated with this
article. The IC50 was calculated using GraphPad Prism Software (GraphPad Prism 5, La Jolla, CA, USA).
For each concentration, at least three wells were performed to calculate the average parameter.
3.2.2. Cytotoxic Activity Assays
Cell Culture
The five types of human cancer cell lines (HeLa, HepG2, MCF-7, SCC-15, and A549) were cultured
aseptically using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal
bovine serum (FBS) and penicillin (100 units mL−1)/streptomycin (100 mg mL−1), at pH 7.2 and 5%
CO2 humidified atmosphere at 37 ◦C. After attaining 80% confluence, the cells were trypsinized with
0.25 trypsin–EDTA and diluted with media to a fixed number of cells.
MTS Assay
Cytotoxic activity was assessed using the standard MTS method by using triplicate assay.
The cells were seeded into 96-well plates containing the medium at the density of 4000–6000 cells/mL
(100
in a culture medium to the concentrations needed. After 24 h, the cultured cells were treated with
concentrations of synthesized compounds (3.125 M to 100 M for tumor cells) for 48 h. After 48 h
of incubation, the supernatant was replaced by fresh medium (100 L/well) and 10 L MTS reagent
µL/well). The compounds were dissolved in DMSO to the concentration of 100 mM and diluted
µ
µ
µ
µ
([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt]) was added to each well. The plate was further incubated for 3 h at 37 ◦C in 5% CO2. The optical

Molecules 2017, 22, 1979
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absorbance in individual wells was determined at 492 nm using a microplate reader. The inhibition
rates were calculated by the following formula:
Inhibition rate (%) = (ODnegative control − ODsample)/(ODnegative control − ODblank) × 100%
IC50 was calculated using GraphPad Prism Software (GraphPad Prism 5, La Jolla, CA, USA).
For each concentration, at least three wells were performed to calculate the average parameter.
4. Conclusions
In summary, a series of novel benzyl sulfoxide 2-indolinone derivatives was synthesized and
evaluated in vitro for tyrosine kinase inhibitory activity and potential cytotoxic activity against five
types of cancer cell lines. Most of the compounds under investigation exhibited significant tyrosine
kinase inhibitory activity. Compounds 6j and 6o not only showed significant tyrosine kinase inhibitory
activity but also exhibited obvious antiproliferative activity against four tested cancer cell lines. Several
compounds showed encouraging activity in some cases with interesting selectivity against the HeLa
cell line relative to the other four cancer cell lines. The findings highlighted the potential of this class
of derivatives as new anticancer agents. Further detailed research will be conducted to evaluate the
molecular mechanism underlying the anticancer activity of these compounds. In addition, hydrophilic
groups will be introduced to the parent nucleus structure to improve the aqueous solubility of the
compounds to enhance their anti-tumor activity.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: The authors are grateful to the National Natural Science Foundation of China (Grant No.
81273431) for its financial support for this project. Tremendous thanks are owed to the Beijing Institute of
Pharmacology and Toxicology for helping with the 1H-NMR, 13C-NMR and HRMS spectral analyses.
Author Contributions: Lin Wang and Lin Tang conceived and designed the experiments. Lin Tang performed the
synthetic work and the biological activity assay, interpreted the results and wrote the paper. Shouguo Zhang and
Tao Peng edited the English language. Gang Wang, Xiaoxue Wen, Yunbo Sun, and Shuchen Liu contributed to the
data collection and analysis. All authors have approved the final version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 6a–6p are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).