Novel methyl indolinone-6-carboxylates containing an indole moiety as angiokinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.08.031

A novel series of methyl indolinone-6-carboxylates bearing an indole moiety were identified as potent angiokinase inhibitors. The most active compound, A8, potently targeted the kinase activities of vascular endothelial growth factor receptors 2 and 3, and platelet-derived growth factor receptors α and β, with IC₅₀ values in the nanomolar range. In addition, A8 effectively suppressed the proliferation of human umbilical vein endothelial cells, and HT-29 and MCF-7 cancer cells, by inducing apoptosis. Compound A8 is thus a promising candidate for further investigation.

Full text of DOI:10.1016/j.ejmech.2017.08.031

Accepted Manuscript
Novel methyl indolinone-6-carboxylates containing an indole moiety as angiokinase
inhibitors
Mingze Qin, Ye Tian, Xiaoqing Sun, Simiao Yu, Juanjuan Xia, Ping Gong, Haotian
Zhang, Yanfang Zhao
PII:
S0223-5234(17)30634-7
10.1016/j.ejmech.2017.08.031
EJMECH 9675
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 May 2017
Revised Date: 11 August 2017
Accepted Date: 11 August 2017
Please cite this article as: M. Qin, Y. Tian, X. Sun, S. Yu, J. Xia, P. Gong, H. Zhang, Y. Zhao, Novel
methyl indolinone-6-carboxylates containing an indole moiety as angiokinase inhibitors, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel methyl indolinone-6-carboxylates containing an indole moiety
as angiokinase inhibitors
Mingze Qina, Ye Tiana, Xiaoqing Suna, Simiao Yua, Juanjuan Xiaa, Ping Gonga, Haotian Zhangb,*,
Yanfang Zhaoa,*
a
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, 103 Wenhua Road, Shenyang 110016, PR China
b
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, PR China
Corresponding author.
Tel: +86 24 2398 6282; Fax: +86 24 2398 6282;
E-mail address: yanfangzhao@126.com (Y. Zhao); zhanghaotian2087@163.com (H. Zhang)
Abstract
A novel series of methyl indolinone-6-carboxylates bearing an indole moiety
were identified as potent angiokinase inhibitors. The most active compound, A8,
potently targeted the kinase activities of vascular endothelial growth factor receptors 2
and 3, and platelet-derived growth factor receptors α and β, with IC50 values in the
nanomolar range. In addition, A8 effectively suppressed the proliferation of human
umbilical vein endothelial cells, and HT-29 and MCF-7 cancer cells, by inducing
apoptosis. Compound A8 is thus a promising candidate for further investigation.
Keywords: methyl indolinone-6-carboxylates; ring-fused strategy; angiokinase
inhibitors; antitumour activity

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1. Introduction
Angiogenesis is critical to the growth of solid tumours, and is regulated by a
complex balance among several endogenous molecules [1,2]. Vascular endothelial
growth factor (VEGF) directly stimulates endothelial cell proliferation and vascular
permeability by binding to its cognate receptor (VEGFR). Thus, VEGF acts as a
highly potent proangiogenic factor [3,4]. In addition, several studies have elucidated
indispensable roles of platelet-derived growth factor (PDGF) and its cognate receptor
(PDGFR) during angiogenesis. These molecules regulate the recruitment of pericytes,
which are required for blood vessel formation and stabilization [5,6].
Inhibiting tumour angiogenesis by simultaneously targeting the VEGFR and
PDGFR has been proven to provide a successful therapeutic approach [7–9]. The
indolinone scaffold facilitates a suitable chemical interaction with the hinge region of
the VEGFR and PDGFR, whereby the lactam group interacts with the protein
backbone via two pivotal hydrogen bonds [10]. A large number of bioactive
indolinones have been reported by different groups [11–13]. Nintedanib, a potent
indolinone-based compound, has just been approved for clinical use in patients with
non-small cell lung cancer or idiopathic pulmonary fibrosis [14]. In addition to
targeting angiokinases within the VEGFR and PDGFR families with high affinity, this
compound also inhibits the activity of the fibroblast growth factor receptor (FGFR),
which is involved in the “angiogenic switch” response to sustained blockade of VEGF
signalling [15].
Here, we reported the discovery of a series of indolinones bearing an indole
moiety that act as potent angiokinase inhibitors (Fig. 1). Detailed structure-activity
relationship studies were conducted and led to the identification of compound A8.
The results of our biological evaluations clearly demonstrated that A8 potently
inhibited the activities of angiokinases including VEGFR-2, VEGFR-3, PDGFRα and
PDGFRβ. In addition, A8 inhibited the proliferation of human umbilical vein
endothelial cells (HUVECs), as well as cancer cell lines such as HT-29 and MCF-7,
by inducing apoptosis.
(Insert Figure 1 here)
2. Results and discussion
2.1. Chemistry
The preparation of compounds A1–A4 is described in Scheme 1. Intermolecular
cyclisation of 2-amino-5-nitrophenol (1) with chloroacetyl chloride afforded
benzoxazole intermediate 2 [18], which was readily converted to amines 3a and 3b
via sequential nucleophilic substitution and hydrogenation reactions in a one-pot
manner. The benzimidazole intermediates 6a and 6b were prepared using a similar
method, except that N1-methyl-4-nitrobenzene-1,2-diamine (4) was used as the
starting material. Compounds A1–A4 were generated by condensation of the
corresponding amine (3a, 3b, 6a and 6b) with commercially available methyl
(E)-3-(methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate
addition-elimination sequence.
(15)
in
an
(Insert Scheme 1 here)
The synthesis of compounds A5–A17 is outlined in Scheme 2. Amidation of

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5-nitro-1H-indole-2-carboxylic acid (7) with secondary amines gave rise to the
desired intermediates (8a and 8b). Intermediate 8b underwent an N-alkylation
reaction and readily converted to 8c and 8d, respectively, via a SN2 mechanism [19].
The selective reduction of the amide groups of 8a and 8b using borane afforded 9a
and 9b, which were further hydrogenated to provide amines 10a and 10b, respectively.
Alternatively, intermediates 8a–d were readily converted to the corresponding amines
11a–d under standard hydrogenation conditions. On the other hand, 5-nitro-1H-indole
(12) was converted to Mannich bases 13a, 13e and 13g via a classical Mannich
reaction mechanism [16,20]. The 13b, 13c, 13d and 13f intermediates were obtained
when 13a and 13e were reacted with suitable alkylating reagents. Intermediates 14a–g
were generated when 13a–g were exposed to hydrogen. Reaction of these amines
(10a, 10b, 11a–d and 14a–g) with intermediate 15 generated the desired compounds,
A5–A17.
(Insert Scheme 2 here)
2.2. Biological evaluation
2.2.1. Discovery of indole-containing compounds A5 and A6
The binding mode of nintedanib at the ATP-binding site of VEGFR-2 has been
confirmed[10]. The methyl indolinone-6-carboxylate scaffold formed three hydrogen
bonds to Cys919, Glu917 and Lys868 in the hinge region, providing high-affinity binding
to this kinase. Furthermore, the methyl piperazinyl group was oriented to the solvent
region, and the nitrogen atom of this moiety interacted with Glu850 via a bidentate
ionic bond. Based on these observations, we conducted a programme of
structure-based modifications of the chemical structure of nintedanib, in an attempt to
identify potent analogues. At the beginning of our study, a ring-fused strategy was
applied to build a bicyclic fragment. Furthermore, previous reports by Roth et al. and
ourselves [14,17] indicated that dimethylamine and methyl piperazinyl groups might
provide optimal substitutions, and compounds A1–A6, which contained benzoxazole,
benzimidazole and indole moieties, respectively, were therefore synthesized as
proof-of-concept compounds.
First, the effects of compounds A1–A6 on the enzymatic activities of VEGFR-2
and PDGFRβ were evaluated. As shown in Table 1, the benzoxazole derivative A2
exhibited moderate inhibitory activity, whereas A1 was completely inactive at a
concentration of 1 µM, indicating that the benzoxazole fragment was not well
tolerated. In contrast, the benzimidazole and indole derivatives (A3–A6) displayed
promising effects on enzymatic activity. Benzimidazoles A3 and A4 produced
effective inhibition of PDGFRβ, with 50% inhibitory concentrations (IC50) of 45.5
and 61 nM, respectively. However, both compounds were less active against
VEGFR-2 (IC50: 384.7 and 257.2 nM, respectively). Compounds with an indole
moiety (A5 and A6) exhibited significantly greater inhibition of VEGFR-2 (IC50:
138.3 and 106.1 nM, respectively), and provided suitable starting points for further
optimization.
(Insert Table 1 here)
To explore the potential of these compounds further, they were screened for
antiproliferative activity in selected human cancer cell lines, which included A549

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(human non-small cell lung cancer), MCF-7 (human breast cancer) and HT-29
(human colon cancer) cells. These findings demonstrated that both benzoxazoles (A1
and A2) were inactive at the cellular level, which was consistent with their weak
inhibition of receptor kinase activity. Similarly, the benzimidazoles (A3 and A4)
exhibited weak antiproliferative activity, thereby limiting their suitability for further
investigations. The indole derivatives (A5 and A6) produced effective inhibition of
the proliferation of the tested cancer cell lines, especially in HT-29 and MCF-7 cells.
Taken together, these findings indicated that the indole moiety may be suitable for this
region, and detailed modifications could be conducted to increase potency further.
2.2.2. Modifications of indole precursors
Previous findings [10] indicated that a rational orientation of the amino group,
which facilitated an ionic interaction with Glu850, was crucial for activity. Compounds
A11, A15 and A17 were prepared by shifting the aminomethyl group at the 2-position
of indole to the 3-position, with the aim of evaluating the influence of this structural
change on activity. However, these compounds exhibited sharply reduced inhibition of
VEGFR-2. Notably, replacing the dimethylamino and piperazinyl groups with a
morpholino fragment resulted in a loss of potency. Compounds A11 and A15
suppressed PDGFRβ activity with IC50 values of 60.7 and 135.1 nM, respectively,
whereas the corresponding value for A17 was 241.6 nM. These results were
consistent with our previous findings [17], which indicated that a dimethylamino or
piperazinyl group was preferred.
(Insert Table 2 here)
The promising activity of A11 as an inhibitor of PDGFRβ led to the preparation
of more derivatives via N-alkylation of the indole, with the intention of improving
potency. Although this approach generally failed to identify potent compounds, some
valuable results were obtained. As compared with the A11 precursor (IC50 for
VEGFR-2 > 1000 nM; IC50 for PDGFRβ = 60.7 nM), the introduction of methyl (A12;
IC50 for VEGFR-2 = 808.5 nM; IC50 for PDGFRβ = 64.6 nM), ethyl (A13; IC50 for
VEGFR-2 = 431.2 nM; IC50 for PDGFRβ = 34.3 nM) or isopropyl (A14; IC50 for
VEGFR-2 = 646.5 nM; IC50 for PDGFRβ = 49.2 nM) groups did not offer any
significant advantage with respect to the inhibition of enzymatic activity. Notably,
some of these derivatives (A11, A12 and A14) effectively suppressed the proliferation
of HT-29 cells with IC50 values of 0.13, 0.46 and 0.25 µM, respectively, which
suggested the involvement of other biological targets beyond those examined in the
current study. The biological results demonstrated that the amino group linked to the
3-position of indole was not well tolerated. As a consequence, research focus returned
to the 2-substituted indole scaffold.
The methylene linker between the amino group and indole was replaced by an
amide unit, and this generated compounds A7 and A8. Interestingly, these two
compounds produced different results. Compound A7, with a dimethylamino group,
was completely inactive in the enzymatic assay. However, compound A8, bearing a
piperazinyl group, was very active, with IC50 values of 69.1 and 22 nM for VEGFR-2
and PDGFRβ activities, respectively. Based on these observations, minor
modifications were made at the indole nitrogen atom, which produced the alkylated

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derivatives, A9 and A10. Both of these compounds exhibited slightly reduced potency
with respect to VEGFR-2 and PDGFRβ inhibition, as compared to A8. Interestingly,
the effective inhibition of enzymatic activity by A8 translated to potent
antiproliferative activity against cancer cell lines. This compound suppressed the
growth of A549, MCF-7 and HT-29 cells, with IC50 values of 2.99, 1.56 and 1.17 µM,
respectively. To exclude the effect of cytotoxicity, A8 was evaluated for inhibitory
activity against normal cells derived from human embryonic kidney (HEK293T).
However, no significant inhibition of proliferation was observed for treatment with 10
µM A8 (Figure S1). Taken together, these findings identified compound A8 as a
promising candidate for subsequent biological assessment.
2.2.3. Analysis of apoptosis
To determine whether compound A8 inhibited the proliferation of cancer cells by
inducing apoptosis, a biparametric cytofluorimetric analysis using propidium iodide
and annexin V-fluorescein isothiocyanate staining was conducted in HT-29 and
MCF-7 cells. In addition, human umbilical vein endothelial cells (HUVECs) were
employed to further evaluate the effect of A8 on angiogenesis. These cell lines were
treated with A8 at different concentrations (0, 0.3, 1 and 3 µM) for 48 h prior to
staining and analysis by flow cytometry. As shown in Figure 2, the apoptosis rate in
HUVECs was significantly elevated from 7.57% (control) to 81.57% in the presence
of 1 µM A8. When the cells were treated with 3 µM A8, the apoptosis rate reached
95.28%, which indicated that A8 induced apoptosis of HUVECs in a
concentration-dependent manner. Severe A8-triggered apoptosis was also observed in
HT-29 and MCF-7 cells (Figure 2).
(Insert Figure 2 here)
2.2.4. Inhibitory activity against selected angiokinases
In an attempt to investigate the effects of compound A8 on a range of
angiokinases, we evaluated its inhibitory activities against VEGFR-3, PDGFRα and
FGFR1. As shown in Table 3, A8 produced strong inhibition of VEGFR-3 (IC50
=
18.2 nM) and PDGFRα (IC50 = 4.4 nM), and moderate activity against FGFR1 (IC50 =
1507 nM). These biological results clearly demonstrated that compound A8 was a
potent inhibitor of several angiokinases.
(Insert Table 3 here)
2.3. Molecular docking study
To elucidate the favorable VEGFR-2 inhibition of A8, a detailed docking
analysis was conducted using AutoDock 4.2. The binding conformations were
analyzed using Discovery Studio Visualizer 4.0. The X-ray structure of the kinase
domain of VEGFR-2 was obtained from the Protein Data Bank (PDB code: 3C7Q).
Figure 3 showed the binding model overlay of compound A8 with nintedanib, which
suggested that the two compounds acted in a similar way. Also as shown in Figure 3,
A8 formed three hydrogen bonds with Cys919, Glu917 and Lys868 in the hinge region.
Furthermore, the nitrogen atom of the piperazinyl group interacted with Glu850 via a
bidentate ionic bond. The docking results indicated that compound A8 fitted well to
the kinase domain of VEGFR-2.
(Insert Figure 3 here)

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3. Conclusions
In summary, a novel series of indolinone derivatives containing an indole moiety
were identified as potent angiokinase inhibitors. Biological optimization led to the
identification of A8, which potently inhibited the activities of VEGFR-2, VEGFR-3,
PDGFRα and PDGFRβ with IC50 values of 69.1, 18.2, 4.4 and 22 nM, respectively.
Although the overall inhibitory activity of A8 was slightly less than that of nintedanib,
its novel chemical structure provides a promising candidate for further development.
In addition, compound A8 effectively suppressed the proliferation of HUVECs and
HT-29 and MCF-7 cancer cells by inducing apoptosis. Further investigations on the
pharmacokinetics and in vivo activity of A8 are underway.
4. Experimental section
4.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without
further purification. All the reactions were monitored by TLC using silica gel GF/UV
254. Flash chromatography was performed using silica gel (300–400 mesh). The
purity of the synthesized compounds was measured by high performance liquid
chromatography (HPLC, Agilent, USA), and was confirmed to be higher than 95%.
Melting points were determined on a Büchi Melting Point B-540 apparatus (Büchi
1
13
Labortechnik, Flawil, Switzerland). The H and C NMR spectra were recorded on
Bruker AV-400 spectrometer, with TMS as an internal standard. The low resolution of
ESI-MS was recorded on an Agilent 1100 LC-MS spectrometer, and high resolution
mass spectrometry was performed on an Agilent Accurate-Mass Q-TOF 6530 in ESI
mode.
4.1.1. 2-(Chloromethyl)-6-nitrobenzo[d]oxazole (2)
To a solution of intermediate 1 (4.93 g, 0.032 mol) in chlorobenzene (30 mL)
was added chloroacetyl chloride (4.03 g, 0.036 mol) dropwise at 0 °C, followed by
addition of pyridine (0.13 g, 1.6 mmol). Stirring was continued for 1 h at room
temperature. Subsequently, p-toluenesulfonic acid (0.55 g, 3.2 mmol) was added, the
mixture was stirred for another 8 h under reflux. The precipitate was filtered off, the
filtrate was washed with brine. The solvent was concentrated to give a residue, which
was triturated with ethanol to give intermediate 2 (4.76 g, 70.2%) as a yellow solid.
1H NMR (400 MHz, DMSO-d6) δ 6.48 (d, J = 8.3 Hz,1H, ArH), 6.27 (d, J = 2.5 Hz,
1H, ArH), 6.04 (dd, J = 8.3, 2.6 Hz, 1H, ArH), 4.24 (s, 2H, CH2). ESI-MS m/z: 213.0
[M+H]+.
4.1.2. 2-(Chloromethyl)-1-methyl-5-nitro-1H-benzo[d]imidazole (5)
To a solution of intermediate 4 (2 g, 12 mmol) and triethylamine (1.34 g, 13.2
mmol) in ethyl acetate (10 mL) was added chloroacetyl chloride (1.48 g, 13.2 mmol)
dropwise at 0 °C. The mixture was stirred for 30 min at room temperature, then
acetic acid (0.5 mL) was added. Stirring was maintained for 20 h under reflux. After
that time, the solution was cooled down. The precipitate was filtered off to give a
crude product, which was recrystallized from ethyl acetate to give 5 (1.85 g, 68.5%)
as a yellow solid. ESI-MS m/z: 226.1 [M+H]+.
4.1.3. General procedure for preparation of intermediates 3a, 3b, 6a, and 6b
To a solution of an intermediate (2 or 5, 4.7 mmol) and appropriate amine (4.7

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mmol) in dichloromethane (5 mL) was added N,N-diisopropylethylamine (5.5 mmol).
The mixture was stirred at 30 °C for 8–12 h until TLC showed the completion of the
reaction. The solution was diluted with ethanol (20 mL), and palladium on charcoal
(10% w/w) was added. The reaction mixture was hydrogenated at room temperature
for 5 h. After that time, the catalyst was filtered off and the filtrate was evaporated.
The residue was purified by column chromatography (CH
2
Cl /MeOH, 100/1 to 30/1)
2
to generate the desired intermediates.
4.1.3.1. 2-((Dimethylamino)methyl)benzo[d]oxazol-6-amine (3a). Gray solid. Yield:
67%. 1H NMR (400 MHz, DMSO-d6) δ 6.61 (d, J = 8.1 Hz, 1H, ArH), 6.14–6.00 (m,
2H, ArH), 4.72 (s, 2H, NH2), 4.60 (s, 2H, CH2), 2.90 (s, 6H, CH3). ESI-MS m/z:
192.1 [M+H]+.
4.1.3.2. 2-((4-Methylpiperazin-1-yl)methyl)benzo[d]oxazol-6-amine (3b). Gray solid.
Yield: 70%. ESI-MS m/z: 247.2 [M+H]+.
4.1.3.3. 2-((Dimethylamino)methyl)-1-methyl-1H-benzo[d]imidazol-5-amine (6a).
1
Gray solid. Yield: 62%. H NMR (400 MHz, DMSO-d6) δ 7.15 (d, J = 8.5 Hz, 1H,
ArH), 6.72 (d, J = 1.8 Hz, 1H, ArH), 6.58 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 4.86 (s, 2H,
NH2), 3.70 (s, 3H, NCH3), 3.61 (s, 2H, CH2), 2.20 (s, 6H, N(CH3)2). ESI-MS m/z:
205.1 [M+H]+.
4.1.3.4. 1-Methyl-2-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-5-amine
1
(6b). Gray solid. Yield: 77%. H NMR (400 MHz, DMSO-d6) δ 7.17 (d, J = 8.5 Hz,
1H, ArH), 6.72 (s, 1H, ArH), 6.59 (dd, J = 8.5, 1.6 Hz, 1H, ArH), 3.88 (s, 2H, NH2),
3.73 (s, 2H, CH2), 3.71 (s, 3H, CH3), 2.78 (s, 4H, piperazinyl), 2.57 (s, 4H,
piperazinyl), 2.48 (s, 3H, CH3). ESI-MS m/z: 260.2 [M+H]+.
4.1.4. General procedure for preparation of intermediates 8a and 8b
To a solution of intermediate 7 (10 mmol), appropriate amine (30 mmol), and
HATU (30 mmol) in DMF (15 mL) was added N,N-diisopropylethylamine (30 mmol)
slowly. The mixture was stirred at room temperature for 12 h. When TLC showed the
completion of the reaction, the solution was poured into water. The precipitate was
filtered off to give the desired intermediates.
4.1.4.1. N,N-dimethyl-5-nitro-1H-indole-2-carboxamide (8a). Orange solid. Yield:
72%. ESI-MS m/z: 234.1 [M+H]+.
4.1.4.2. (4-Methylpiperazin-1-yl)(5-nitro-1H-indol-2-yl)methanone (8b). Yellow solid.
Yield: 63%. ESI-MS m/z: 289.1 [M+H]+.
4.1.5. General procedure for preparation of intermediates 8c and 8d
To a solution of intermediate 8b (1 mmol) in dichloromethane was added
sodium hydride (2 mmol) at 0 °C. Stirring was continued for 30 min. After that time,
appropriate alkyl halide (1.5 mmol) in dichloromethane was added dropwise. The
mixture was stirred at room temperature for 3–6 h until TLC indicated the completion
of the reaction. The reaction was quenched with aqueous NH4Cl. The organic phase
was washed with water, and was concentrated to give a residue, which was purified
by column chromatography (CH
2
Cl /MeOH, 100/1 to 75/1) to afford the target
2
compounds.
4.1.5.1. (1-Methyl-5-nitro-1H-indol-2-yl)(4-methylpiperazin-1-yl)methanone (8c).
Light yellow solid. Yield: 57%. ESI-MS m/z: 303.1 [M+H]+.

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4.1.5.2. (1-Ethyl-5-nitro-1H-indol-2-yl)(4-methylpiperazin-1-yl)methanone (8d). Light
yellow solid. Yield: 60%. ESI-MS m/z: 317.1 [M+H]+.
4.1.6. General procedure for preparation of intermediates 9a and 9b
To a solution of intermediate (8a and 8b, 6.4 mmol) in THF (10 mL) was added
borane dimethyl sulphide complex (2M solution in THF, 19 mL) at room temperature.
The reaction mixture was heated at reflux for 1 h. To the mixture was added 6M HCl
dropwise and stirred under reflux for a further 30 minutes. The reaction mixture was
cooled to room temperature, when 4N NaOH solution was added. The mixture was
extracted with dichloromethane, and washed with brine. The organic layer was
concentrated to give the crude product, which was purified by column
chromatography (CH
2
Cl /MeOH, 100/1 to 80/1) to afford the desired intermediate.
2
4.1.6.1. N,N-dimethyl-1-(5-nitro-1H-indol-2-yl)methanamine (9a). Yellow solid. Yield:
63%. ESI-MS m/z: 220.1 [M+H]+.
4.1.6.2. 2-((4-Methylpiperazin-1-yl)methyl)-5-nitro-1H-indole (9b). Yellow solid.
Yield: 72%. ESI-MS m/z: 275.2 [M+H]+.
4.1.7. General procedure for preparation of intermediates 10a and 10b
At room temperature, palladium on charcoal (10% w/w) was added to a solution
of intermediates (9a and 9b, 1.8 mmol) in ethanol. The reaction mixture was
hydrogenated for 6 h. When TLC showed the completion of the reaction, the catalyst
was filtered off. The filtrate was evaporated to give the desired intermediates.
4.1.7.1. 2-((Dimethylamino)methyl)-1H-indol-5-amine (10a). Yellow solid. Yield:
1
81%. H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H, NH), 7.01 (d, J = 8.4 Hz, 1H,
ArH), 6.61 (d, J = 1.0 Hz, 1H, ArH), 6.44 (dd, J = 8.5, 1.8 Hz, 1H, ArH), 6.02 (s, 1H,
ArH), 5.10 (s, 2H, NH2), 3.57 (s, 2H, CH2), 2.24 (s, 6H, CH3). ESI-MS m/z: 190.1
[M+H]+.
4.1.7.2. 2-((4-Methylpiperazin-1-yl)methyl)-1H-indol-5-amine (10b). Yellow solid.
Yield: 66%. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H, NH), 6.99 (d, J = 8.4 Hz,
1H, ArH), 6.58 (s, 1H, ArH), 6.41 (d, J = 8.0 Hz, 1H, ArH), 5.96 (s, 1H, ArH), 4.64 (s,
2H, NH2), 3.48 (s, 2H, CH2), 2.33 (br, 8H, piperazinyl), 2.14 (s, 3H, CH3). ESI-MS
m/z: 245.2 [M+H]+.
4.1.8. General procedure for preparation of intermediates 11a–11d
Intermediates 11a–11d were prepared according to the synthetic method as
described for intermediates 10a and 10b by using intermediate 8a–8d as the reactants.
4.1.8.1. 5-Amino-N,N-dimethyl-1H-indole-2-carboxamide (11a). Yellow solid. Yield:
42%. ESI-MS m/z: 204.1 [M+H]+.
4.1.8.2. (5-Amino-1H-indol-2-yl)(4-methylpiperazin-1-yl)methanone (11b). Yellow
solid. Yield: 53%. ESI-MS m/z: 259.2 [M+H]+.
4.1.8.3. (5-Amino-1-methyl-1H-indol-2-yl)(4-methylpiperazin-1-yl)methanone (11c).
Yellow solid. Yield: 68%. ESI-MS m/z: 273.2 [M+H]+.
4.1.8.4. (5-Amino-1-ethyl-1H-indol-2-yl)(4-methylpiperazin-1-yl)methanone (11d).
Yellow solid. Yield:70%. ESI-MS m/z: 287.2 [M+H]+.
4.1.9. General procedure for preparation of intermediates 13a, 13e, and 13g
To a mixture of intermediate 12 (6.2 mmol), paraformaldehyde (9.3 mmol) in
acetic acid (5 mL) was added appropriate secondary amine (9.3 mmol). The mixture

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was stirred at 35 °C for 5–8 h and was monitored by TLC. The solution was alkalized
with aqueous NaOH to pH 8–9, and was extracted with dichloromethane. The organic
phase was concentrated to give a residue, which was purified by column
chromatography (CH
2
Cl /MeOH, 100/1 to 75/1) to provide the desired intermediates.
2
4.1.9.1. N,N-dimethyl-1-(5-nitro-1H-indol-3-yl)methanamine (13a). Yellow solid.
Yield: 42%. ESI-MS m/z: 220.1 [M+H]+.
4.1.9.2. 3-((4-Methylpiperazin-1-yl)methyl)-5-nitro-1H-indole (13e). Yellow solid.
Yield: 51%. ESI-MS m/z: 275.2 [M+H]+.
4.1.9.3. 4-((5-Nitro-1H-indol-3-yl)methyl)morpholine (13g). Yellow solid. Yield: 56%.
ESI-MS m/z: 262.1 [M+H]+.
4.1.10. General procedure for preparation of intermediates 13b–13d and 13f
Intermediates 13b–13d and 13f were prepared according to the synthetic method
as described for intermediates 8c and 8d by using intermediate 13a and 13e as the
reactants. The crude product was purified by silica gel column chromatography eluting
with CH
2
Cl2
/MeOH (120/1 to 70/1).
4.1.10.1.
N,N-dimethyl-1-(1-methyl-5-nitro-1H-indol-3-yl)methanamine
(13b).
Yellow solid. Yield: 67%. ESI-MS m/z: 234.1 [M+H]+.
4.1.10.2. 1-(1-Ethyl-5-nitro-1H-indol-3-yl)-N,N-dimethylmethanamine (13c). Yellow
solid. Yield: 62%. ESI-MS m/z: 248.1 [M+H]+.
4.1.10.3. 1-(1-Isopropyl-5-nitro-1H-indol-3-yl)-N,N-dimethylmethanamine (13d).
Yellow solid. Yield: 76%. ESI-MS m/z: 262.1 [M+H]+.
4.1.10.4.
1-Methyl-3-((4-methylpiperazin-1-yl)methyl)-5-nitro-1H-indole
(13f).
Yellow solid. Yield: 57%. ESI-MS m/z: 289.2 [M+H]+.
4.1.11. General procedure for preparation of intermediates 14a–14g
Intermediates 14a–14g were prepared according to the synthetic method as
described for intermediates 10a and 10b by using intermediate 13a–13g as the
reactants. The crude product was purified by silica gel column chromatography eluting
with CH
2
Cl /MeOH (100/1 to 60/1) to afford 14a–14g, respectively.
2
4.1.11.1. 3-((Dimethylamino)methyl)-1H-indol-5-amine (14a). Brown solid. Yield:
1
46%. H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H, NH), 7.02 (d, J = 9.4 Hz, 2H,
ArH), 6.75 (s, 1H, ArH), 6.46 (dd, J = 8.3, 1.5 Hz, 1H, ArH), 4.48 (s, 2H, NH2), 3.42
(s, 2H, CH2), 2.13 (s, 6H, CH3). ESI-MS m/z: 190.1 [M+H]+.
4.1.11.2. 3-((Dimethylamino)methyl)-1-methyl-1H-indol-5-amine (14b). Brown solid.
Yield: 69%. ESI-MS m/z: 204.1 [M+H]+.
4.1.11.3. 3-((Dimethylamino)methyl)-1-ethyl-1H-indol-5-amine (14c). Brown oil.
Yield: 72%. ESI-MS m/z: 218.1 [M+H]+.
4.1.11.4. 3-((Dimethylamino)methyl)-1-isopropyl-1H-indol-5-amine (14d). Yellow
solid. Yield: 57%. ESI-MS m/z: 232.2 [M+H]+.
4.1.11.5. 3-((4-Methylpiperazin-1-yl)methyl)-1H-indol-5-amine (14e). Brown solid.
Yield: 57%. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H, NH), 7.03 (d, J = 8.2 Hz,
2H, ArH), 6.74 (d, J = 1.8 Hz, 1H, ArH), 6.47 (dd, J = 8.4, 1.7 Hz, 1H, ArH), 3.53 (s,
2H, CH2), 2.36 (m, J = 23.9 Hz, 7H, piperazinyl, CH3), 2.19 (s, 4H, piperazinyl).
ESI-MS m/z: 245.2 [M+H]+.
4.1.11.6.
1-Methyl-3-((4-methylpiperazin-1-yl)methyl)-1H-indol-5-amine
(14f).

ACCEPTED MANUSCRIPT
Yellow solid. Yield: 66%. ESI-MS m/z: 259.2 [M+H]+.
4.1.11.7. 3-(Morpholinomethyl)-1H-indol-5-amine (14g). Yellow solid. Yield: 57%.
1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H, NH), 7.03 (d, J = 8.4 Hz, 2H, ArH),
6.76 (d, J = 1.8 Hz, 1H, ArH), 6.46 (d, J = 10.5 Hz, 1H, ArH), 4.45 (s, 2H, NH2),
3.60–3.50 (t, 4H, morpholinyl), 3.47 (s, 2H, CH2), 2.34 (s, 4H, morpholinyl). ESI-MS
m/z: 232.1 [M+H]+.
4.1.12. General procedure for preparation of compounds A1–A17.
A mixture of intermediate (3a, 3b, 6a, 6b, 10a, 10b, 11a–d, and 14a–g, 1.5
mmol) and methyl (E)-3-(methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate
(1.5 mmol) in methanol (10 mL) was stirred under reflux for 5–10 h. When TLC
showed the completion of the reaction, the solution was cooled to room temperature.
The precipitate was filtered off to give a crude product, which was recrystallized from
methanol to provide the target compounds.
4.1.12.1.
Methyl
(Z)-3-(((2-((dimethylamino)methyl)benzo[d]oxazol-6-yl)amino)(phenyl)methylene)-2-
1
oxoindoline-6-carboxylate (A1). Yellow solid. Yield: 42%. Mp: 298.2–301.7 °C. H
NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H, NH), 10.92 (s, 1H, NH), 7.63–7.54 (m,
3H, ArH), 7.48 (dd, J = 7.7, 1.4 Hz, 2H, ArH), 7.41 (d, J = 1.3 Hz, 1H, ArH), 7.18 (dd,
J = 8.2, 1.5 Hz, 1H, ArH), 6.67 (d, J = 8.3 Hz, 1H, ArH), 6.41 (dd, J = 8.4, 2.4 Hz, 1H,
ArH), 6.27 (d, J = 2.4 Hz, 1H, ArH), 5.78 (d, J = 8.2 Hz, 1H, ArH), 4.66 (s, 2H, CH2),
13
3.76 (s, 3H, OCH3), 2.95 (s, 6H, CH3). C NMR (101 MHz, DMSO-d6) δ 170.60,
166.93, 159.24, 156.11, 144.40, 136.30, 133.69, 132.77, 132.51, 130.74, 129.86,
129.67, 128.80, 123.94, 123.38, 121.84, 117.91, 117.36, 110.33, 109.83, 96.95, 60.48,
56.49, 52.21. HRMS (ESI) for C27H24N4O4 [M + H]+, calcd: 469.1870, found:
469.1873.
4.1.12.2.
Methyl
(Z)-3-(((2-((4-methylpiperazin-1-yl)methyl)benzo[d]oxazol-6-yl)amino)(phenyl)methy
lene)-2-oxoindoline-6-carboxylate (A2). Yellow solid. Yield: 53%. Mp:
1
310.8–314.0 °C. H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H, NH), 10.92 (s, 1H,
NH), 7.56 (m, J = 11.0, 4.8 Hz, 3H, ArH), 7.48 (d, J = 6.5 Hz, 2H, ArH), 7.41 (d, J =
1.0 Hz, 1H, ArH), 7.18 (dd, J = 8.3, 1.3 Hz, 1H, ArH), 6.72 (d, J = 8.3 Hz, 1H, ArH),
6.45 (dd, J = 8.4, 2.2 Hz, 1H, ArH), 6.31 (d, J = 2.2 Hz, 1H, ArH), 5.79 (d, J = 8.2 Hz,
1H, ArH), 4.69 (s, 2H, CH2), 3.77 (s, 3H, OCH3), 3.44 (m, J = 6.7, 3.8 Hz, 4H,
piperazinyl), 2.90 (m, J = 12.8 Hz, 4H, piperazinyl), 2.59 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6) δ 170.61, 166.91, 159.14, 154.88, 144.58, 136.41, 133.76,
132.71, 132.31, 130.79, 129.88, 129.59, 128.83, 124.12, 123.96, 121.88, 118.00,
117.47, 110.49, 109.87, 97.24, 60.41, 56.49, 52.86, 52.24, 41.78, 19.02. HRMS (ESI)
for C30H29N5O4 [M + H]+, calcd: 524.2292, found: 524.2287.
4.1.12.3.
Methyl
(Z)-3-(((2-((dimethylamino)methyl)-1-methyl-1H-benzo[d]imidazol-5-yl)amino)(phen
yl)methylene)-2-oxoindoline-6-carboxylate (A3). Yellow solid. Yield: 37%. Mp:
1
304.3–307.1 °C. H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H, NH), 10.93 (s, 1H,
NH), 7.54–7.47 (m, 5H, ArH), 7.42 (d, J = 1.2 Hz, 1H, ArH), 7.35 (d, J = 8.6 Hz, 1H,
ArH), 7.18 (dd, J = 8.2, 1.4 Hz, 1H, ArH), 7.15 (d, J = 1.6 Hz, 1H, ArH), 6.93 (dd, J =

ACCEPTED MANUSCRIPT
8.6, 1.8 Hz, 1H, ArH), 5.76 (d, J = 8.2 Hz, 1H, ArH), 3.77 (s, 3H, OCH3), 3.74 (s, 3H,
CH3), 3.60 (s, 2H, CH2), 2.16 (s, 6H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 170.69,
166.95, 160.41, 153.68, 142.17, 136.28, 134.51, 132.78, 132.36, 130.57, 129.83,
129.74, 128.94, 123.86, 121.83, 120.05, 117.32, 115.02, 110.44, 109.80, 96.79, 56.49,
52.21, 45.64, 30.44. HRMS (ESI) for C28H27N5O3 [M + H]+, calcd: 482.2187, found:
482.2181.
4.1.12.4.
Methyl
(Z)-3-(((1-methyl-2-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-5-yl)amin
o)(phenyl)methylene)-2-oxoindoline-6-carboxylate (A4). Yellow solid. Yield: 60%.
1
Mp: 188.3–192.8 °C. H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H, NH), 10.93 (s,
1H, NH), 7.51 (m, J = 7.1, 4.3 Hz, 5H, ArH), 7.43 (s, 1H, ArH), 7.35 (d, J = 8.6 Hz,
1H, ArH), 7.18 (dd, J = 8.2, 1.0 Hz, 1H, ArH), 7.14 (d, J = 1.1 Hz, 1H, ArH), 6.93 (dd,
J = 8.5, 1.5 Hz, 1H, ArH), 5.76 (d, J = 8.2 Hz, 1H, ArH), 3.77 (s, 3H, OCH3), 3.75 (s,
3H, CH3), 3.68 (s, 2H, CH2), 2.35 (m, J = 36.5 Hz, 8H, piperazinyl), 2.14 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO-d6) δ 170.69, 166.95, 160.39, 153.15, 142.17,
136.28, 134.52, 132.78, 132.37, 130.58, 129.82, 129.75, 128.92, 123.87, 121.84,
120.04, 117.32, 114.94, 110.46, 109.81, 96.80, 55.02, 54.89, 53.01, 52.21, 49.06,
46.05. HRMS (ESI) for C31H32N6O3 [M + H]+, calcd: 537.2609, found: 537.2615.
4.1.12.5.
Methyl
(Z)-3-(((2-((dimethylamino)methyl)-1H-indol-5-yl)amino)(phenyl)methylene)-2-oxoin
doline-6-carboxylate (A5). Yellow solid. Yield: 57%. Mp: 296.2.1–299.7 °C. 1H NMR
(600 MHz, DMSO-d6) δ 12.28 (s, 1H, NH), 11.05 (s, 1H, NH), 10.88 (s, 1H, NH),
7.50 (m, J = 6.0 Hz, 2H, ArH), 7.46 (dd, J = 7.8, 1.8 Hz, 2H, ArH), 7.43 (d, J = 1.4 Hz,
1H, ArH), 7.17 (dd, J = 8.2, 1.6 Hz, 1H, ArH), 7.10 (d, J = 8.6 Hz, 1H, ArH), 7.07 (d,
J = 1.7 Hz, 1H, ArH), 6.68 (dd, J = 8.6, 2.1 Hz, 1H, ArH), 6.11 (s, 1H, ArH), 5.76 (s,
13
2H, ArH), 3.77 (s, 3H, OCH3), 3.47 (s, 2H, CH2), 2.15 (s, 6H, CH3). C NMR (151
MHz, DMSO-d6) δ 170.60, 170.60, 166.91, 166.91, 160.74, 160.74, 138.54, 135.97,
134.54, 132.88, 130.29, 129.89, 129.68, 129.50, 128.82, 128.09, 123.45, 121.67,
118.63, 117.01, 115.77, 111.46, 109.68, 100.89, 96.07, 56.40, 55.28, 45.24. HRMS
(ESI) for C28H26N4O3 [M + H]+, calcd: 467.2078, found: 467.2083.
4.1.12.6.
Methyl
(Z)-3-(((2-((4-methylpiperazin-1-yl)methyl)-1H-indol-5-yl)amino)(phenyl)methylene)-
2-oxoindoline-6-carboxylate (A6). Yellow solid. Yield: 42%. Mp: 245.3–248.7 °C. 1H
NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H, NH), 11.03 (s, 1H, NH), 10.91 (s, 1H,
NH), 7.47 (dd, J = 20.4, 8.9 Hz, 6H, ArH), 7.17 (d, J = 8.0 Hz, 1H, ArH), 7.13–7.04
(m, 2H, ArH), 6.68 (d, J = 8.3 Hz, 1H, ArH), 6.12 (s, 1H, ArH), 5.78–5.69 (m, 1H,
ArH), 3.77 (s, 3H, OCH3), 3.51 (s, 2H, CH2), 2.33 (m, 8H, piperazinyl), 2.14 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO-d6) δ 170.69, 167.01, 160.86, 138.14, 136.05,
134.65, 132.95, 130.40, 129.99, 129.75, 129.61, 128.91, 128.17, 123.54, 121.76,
118.75, 117.10, 115.86, 111.58, 109.77, 101.25, 96.15, 55.49, 55.07, 53.07, 52.18,
46.18. HRMS (ESI) for C31H31N5O3 [M + H]+, calcd: 522.2500, found: 522.2507.
4.1.12.7.
Methyl
(Z)-3-(((2-(dimethylcarbamoyl)-1H-indol-5-yl)amino)(phenyl)methylene)-2-oxoindoli
ne-6-carboxylate (A7). Yellow solid. Yield: 37%. Mp: 343.2–346.0 °C. 1H NMR (400

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ 12.27 (s, 1H, NH), 11.57 (s, 1H, NH), 10.92 (s, 1H, NH),
7.56–7.44 (m, 5H, ArH), 7.42 (s, 1H, ArH), 7.22 (d, J = 8.5 Hz, 2H, ArH), 7.18 (d, J =
8.1 Hz, 1H, ArH), 6.85 (dd, J = 8.9, 1.1 Hz, 1H, ArH), 6.72 (s, 1H, ArH), 5.77 (d, J =
8.2 Hz, 1H, ArH), 3.77 (s, 3H, OCH3), 3.26 (s, 3H, CH3), 3.03 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6) δ 170.71, 166.97, 162.67, 160.69, 136.18, 134.04, 132.81,
131.92, 130.65, 130.49, 130.23, 129.88, 129.65, 128.95, 127.34, 123.73, 121.80,
117.49, 117.22, 112.72, 109.79, 105.06, 96.47, 52.20, 41.43. HRMS (ESI) for
C28H24N4O4 [M + H]+, calcd: 481.1870, found: 481.1868.
4.1.12.8.
Methyl
(Z)-3-(((2-(4-methylpiperazine-1-carbonyl)-1H-indol-5-yl)amino)(phenyl)methylene)-
2-oxoindoline-6-carboxylate (A8). Yellow solid. Yield: 50%. Mp: 260.7–264.8 °C. 1H
NMR (600 MHz, DMSO-d6) δ 12.27 (s, 1H, NH), 11.63 (s, 1H, NH), 10.90 (s, 1H,
NH), 7.58–7.45 (m, 5H, ArH), 7.43 (s, 1H, ArH), 7.22 (d, J = 8.7 Hz, 2H, ArH), 7.18
(d, J = 8.0 Hz, 1H, ArH), 6.87 (d, J = 8.6 Hz, 1H, ArH), 6.68 (s, 1H, ArH), 5.77 (d, J
= 8.3 Hz, 1H, ArH), 3.77 (s, 7H, OCH3, piperazinyl), 2.66 (s, 4H, piperazinyl), 2.44 (s,
3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 170.62, 166.88, 161.96, 160.55, 136.11,
134.21, 132.71, 130.98, 130.75, 130.41, 129.77, 129.57, 128.86, 126.98, 123.69,
121.88, 121.73, 117.34, 117.15, 112.69, 109.72, 104.75, 96.47, 53.95, 52.12, 48.97,
44.37. HRMS (ESI) for C31H29N5O4 [M + H]+, calcd: 536.2292, found: 536.2286.
4.1.12.9.
Methyl
(Z)-3-(((1-methyl-2-(4-methylpiperazine-1-carbonyl)-1H-indol-5-yl)amino)(phenyl)m
ethylene)-2-oxoindoline-6-carboxylate (A9). Yellow solid. Yield: 50%. Mp:
1
245.8–248.4 °C. H NMR (600 MHz, DMSO-d6) δ 12.28 (s, 1H, NH), 10.92 (s, 1H,
NH), 7.55–7.45 (m, 5H, ArH), 7.43 (s, 1H, ArH), 7.33 (d, J = 8.8 Hz, 1H, ArH), 7.25
(s, 1H, ArH), 7.18 (d, J = 8.3 Hz, 1H, ArH), 6.87 (s, 1H, ArH), 6.49 (s, 1H, ArH), 5.77
(d, J = 8.2 Hz, 1H, ArH), 3.77 (s, 3H, OCH3), 3.66 (s, 3H, CH3), 3.59 (m, 4H,
piperazinyl), 2.33 (m, 4H, piperazinyl), 2.20 (s, 3H, CH3). HRMS (ESI) for
C32H31N5O4 [M + H]+, calcd: 550.2449, found: 550.2450.
4.1.12.10.
Methyl
(Z)-3-(((1-ethyl-2-(4-methylpiperazine-1-carbonyl)-1H-indol-5-yl)amino)(phenyl)met
hylene)-2-oxoindoline-6-carboxylate (A10). Yellow solid. Yield: 50%. Mp:
1
329.3–331.2 °C. H NMR (600 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 10.98 (s, 1H,
NH), 7.60–7.53 (m, 5H, ArH), 7.49 (s, 1H, ArH), 7.43 (d, J = 8.8 Hz, 1H, ArH), 7.28
(s, 1H, ArH), 7.24 (d, J = 8.0 Hz, 1H, ArH), 6.93 (d, J = 8.4 Hz, 1H, ArH), 6.53 (s, 1H,
ArH), 5.82 (s, 1H, ArH), 4.23 (q, J = 6.9 Hz, 2H, CH2CH3), 3.83 (s, 3H, OCH3), 3.65
(s, 4H, piperazinyl), 2.39 (s, 4H, piperazinyl), 2.26 (s, 3H, CH3), 1.26 (t, J = 6.8 Hz,
3H, CH2CH3). HRMS (ESI) for C33H33N5O4 [M + H]+, calcd: 564.2605, found:
564.2611.
4.1.12.11.
Methyl
(Z)-3-(((3-((dimethylamino)methyl)-1H-indol-5-yl)amino)(phenyl)methylene)-2-oxoin
doline-6-carboxylate (A11). Yellow solid. Yield: 50%. Mp: 241.2–244.3 °C. 1H NMR
(400 MHz, DMSO-d6) δ 12.35 (s, 1H, NH), 11.13 (s, 1H, NH), 10.92 (s, 1H, NH),
7.54–7.44 (m, 5H, ArH), 7.43 (d, J = 1.1 Hz, 1H, ArH), 7.30 (s, 1H, ArH), 7.25–7.15
(m, 3H, ArH), 6.77 (dd, J = 8.7, 1.4 Hz, 1H, ArH), 5.78 (d, J = 8.2 Hz, 1H, ArH), 3.77

ACCEPTED MANUSCRIPT
(s, 3H, OCH3), 3.64 (s, 2H, CH2), 2.20 (s, 6H, CH3). 13C NMR (101 MHz, DMSO-d6)
δ 170.71, 166.99, 160.56, 136.10, 134.42, 132.94, 130.39, 129.96, 129.92, 129.66,
128.90, 127.94, 127.54, 127.48, 123.65, 121.76, 118.96, 117.20, 115.04, 112.18,
109.80, 96.32, 56.49, 53.57, 52.20, 44.22, 19.03. HRMS (ESI) for C28H26N4O3 [M +
H]+, calcd: 467.2078, found: 467.2085.
4.1.12.12.
Methyl
(Z)-3-(((3-((dimethylamino)methyl)-1-methyl-1H-indol-5-yl)amino)(phenyl)methylene
)-2-oxoindoline-6-carboxylate (A12). Yellow solid. Yield: 50%. Mp: 199.5–203.0 °C.
1H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H, NH), 10.92 (s, 1H, NH), 7.46 (m, 8H,
ArH), 7.31 (d, J = 8.7 Hz, 1H, ArH), 7.18 (dd, J = 8.3, 1.1 Hz, 1H, ArH), 6.85 (dd, J =
8.8, 1.4 Hz, 1H, ArH), 5.79 (d, J = 8.2 Hz, 1H, ArH), 4.08 (s, 2H, CH2), 3.77 (s, 3H,
OCH3), 3.74 (s, 3H, CH3), 2.47 (s, 6H, CH3). HRMS (ESI) for C28H26N4O3 [M + H]+,
calcd: 481.2234, found: 481.2241.
4.1.12.13.
Methyl
(Z)-3-(((3-((dimethylamino)methyl)-1-ethyl-1H-indol-5-yl)amino)(phenyl)methylene)-
2-oxoindoline-6-carboxylate (A13). Yellow solid. Yield: 50%. Mp: 233.3–236.1 °C.
1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H, NH), 10.91 (s, 1H, NH), 7.51 (ddd, J
= 12.0, 10.1, 5.9 Hz, 5H, ArH), 7.42 (d, J = 0.7 Hz, 1H, ArH), 7.31–7.22 (m, 2H,
ArH), 7.17 (dd, J = 8.3, 1.3 Hz, 1H, ArH), 7.11 (d, J = 1.2 Hz, 1H, ArH), 6.79 (dd, J =
8.7, 1.8 Hz, 1H, ArH), 5.76 (d, J = 8.2 Hz, 1H, ArH), 4.08 (q, J = 7.1 Hz, 2H,
CH2CH3),3.77(s, 3H, OCH3), 3.28 (s, 2H, CH2), 2.04 (s, 6H, CH3), 1.29 (t, J = 7.2 Hz,
3H, CH2CH3). 13C NMR (101 MHz, DMSO-d6) δ 170.69, 166.99, 160.30, 136.09,
133.93, 132.97, 130.45, 129.93, 129.83, 129.75, 128.88, 128.84, 128.33, 123.62,
121.79, 118.40, 117.21, 114.97, 111.44, 110.38, 109.79, 96.39, 54.24, 52.19, 45.17,
15.85. HRMS (ESI) for C30H30N4O3 [M + H]+, calcd: 495.2391, found: 495.2386.
4.1.12.14.
Methyl
(Z)-3-(((3-((dimethylamino)methyl)-1-isopropyl-1H-indol-5-yl)amino)(phenyl)methyle
ne)-2-oxoindoline-6-carboxylate (A14). Yellow solid. Yield: 50%. Mp:
1
219.9–222.0 °C. H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H, NH), 10.93 (s, 1H,
NH), 7.53 (d, J = 7.1 Hz, 3H, ArH), 7.48 (dd, J = 7.4, 1.4 Hz, 2H, ArH), 7.43 (d, J =
0.8 Hz, 1H, ArH), 7.35 (s, 1H, ArH), 7.32 (d, J = 8.8 Hz, 1H, ArH), 7.18 (dd, J = 8.2,
1.2 Hz, 1H, ArH), 7.12 (d, J = 0.6 Hz, 1H, ArH), 6.78 (dd, J = 8.7, 1.8 Hz, 1H, ArH),
5.76 (d, J = 8.2 Hz, 1H, ArH), 4.63 (dt, J = 13.3, 6.7 Hz, 1H, CH(CH3)2), 3.77 (s, 3H,
OCH3), 3.37 (s, 2H, CH2), 2.06 (s, 6H, CH3), 1.38 (d, J = 6.6 Hz, 6H, CH(CH3)2). 13C
NMR (101 MHz, DMSO-d6) δ 170.69, 166.99, 160.34, 136.11, 133.92, 133.01,
130.47, 129.93, 129.85, 129.73, 129.01, 128.84, 128.45, 123.65, 121.80, 118.53,
117.19, 114.96, 110.85, 110.39, 109.79, 96.41, 55.18, 53.04, 52.69, 52.18, 46.19,
15.84. HRMS (ESI) for C31H32N4O3 [M + H]+, calcd: 509.2547, found: 509.2554.
4.1.12.15.
Methyl
(Z)-3-(((3-((4-methylpiperazin-1-yl)methyl)-1H-indol-5-yl)amino)(phenyl)methylene)-
2-oxoindoline-6-carboxylate (A15). Yellow solid. Yield: 50%. Mp: 197.2–201.8 °C.
1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, NH), 11.00 (s, 1H, NH), 10.92 (s, 1H,
NH), 7.55–7.45 (m, 5H, ArH), 7.43 (d, J = 1.1 Hz, 1H, ArH), 7.18 (d, J = 8.7 Hz, 3H,
ArH), 7.13 (s, 1H, ArH), 6.77 (dd, J = 8.6, 1.8 Hz, 1H, ArH), 5.76 (d, J = 8.2 Hz, 1H,

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ArH), 3.77 (s, 3H, OCH3), 3.42 (s, 2H, CH2), 2.29 (m, 8H, piperazinyl), 2.18 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO-d6) δ 170.69, 166.99, 160.51, 136.07, 134.44,
133.00, 130.42, 129.95, 129.68, 128.85, 128.07, 126.90, 126.63, 123.59, 121.78,
118.74, 117.16, 114.85, 112.06, 109.78, 99.99, 96.28, 56.49, 54.99, 52.44, 52.19,
45.98. HRMS (ESI) for C31H31N5O3 [M + H]+, calcd: 522.2500, found: 522.2495.
4.1.12.16.
Methyl
(Z)-3-(((1-methyl-3-((4-methylpiperazin-1-yl)methyl)-1H-indol-5-yl)amino)(phenyl)m
ethylene)-2-oxoindoline-6-carboxylate (A16). Yellow solid. Yield: 50%. Mp:
1
188.7–190.8 °C. H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 10.92 (s, 1H,
NH), 7.50 (tt, J = 8.0, 4.0 Hz, 5H, ArH), 7.43 (d, J = 1.4 Hz, 1H, ArH), 7.24 (d, J =
8.7 Hz, 1H, ArH), 7.20–7.16 (m, 2H, ArH), 7.13 (d, J = 1.6 Hz, 1H, ArH), 6.83 (dd, J
= 8.7, 1.9 Hz, 1H, ArH), 5.77 (d, J = 8.2 Hz, 1H, ArH), 3.77 (s, 3H, OCH3), 3.68 (s,
13
3H, CH3), 3.39 (s, 2H, CH2), 2.42–2.17 (m, 8H, piperazinyl), 2.15 (s, 3H, CH3). C
NMR (101 MHz, DMSO-d6) δ 170.70, 166.99, 160.47, 136.11, 134.89, 132.98,
130.75, 130.45, 129.93, 129.86, 129.69, 128.86, 128.32, 123.66, 121.80, 118.75,
117.20, 115.02, 110.64, 110.40, 109.80, 96.38, 55.16, 52.85, 52.64, 52.18, 46.16,
32.84. HRMS (ESI) for C32H33N5O3 [M + H]+, calcd: 536.2656, found: 536.2660.
4.1.12.17.
Methyl
(Z)-3-(((3-(morpholinomethyl)-1H-indol-5-yl)amino)(phenyl)methylene)-2-oxoindolin
e-6-carboxylate (A17). Yellow solid. Yield: 50%. Mp: 257.0–259.2 °C. 1H NMR (400
MHz, DMSO-d6) δ 12.35 (s, 1H, NH), 11.25 (s, 1H, NH), 10.92 (s, 1H, NH),
7.55–7.45 (m, 5H, ArH), 7.43 (s, 1H, ArH), 7.37 (s, 1H, ArH), 7.32 (s, 1H, ArH), 7.21
(d, J = 8.6 Hz, 1H, ArH), 7.18 (dd, J = 8.3, 1.3 Hz, 1H, ArH), 6.78 (dd, J = 8.6, 1.6 Hz,
1H, ArH), 5.77 (d, J = 8.2 Hz, 1H, ArH), 3.77 (s, 3H, OCH3), 3.64 (s, 4H,
morpholinyl), 3.17 (s, 2H, CH2), 2.62 (s, 4H, morpholinyl). 13C NMR (101 MHz,
DMSO-d6) δ 170.71, 166.98, 160.61, 136.14, 134.36, 132.93, 130.44, 130.29, 130.25,
129.88, 129.64, 128.93, 128.02, 123.72, 121.81, 119.38, 119.36, 117.21, 115.01,
112.32, 109.81, 96.41, 65.35, 52.20, 52.05, 49.06. HRMS (ESI) for C30H28N4O4 [M +
H]+, calcd: 509.2183, found: 509.2177.
4.2. Pharmacology
4.2.1. Enzyme activity assay
The enzymatic activities of VEGFR-2 (Carna), VEGFR-3 (Invitrogen), PDGFRα
(BPS), PDGFRβ (Carna) and FGFR1 (Carna) were determined using well-established
mobility shift assays. The kinase reaction buffer consisted of 50 mM HEPES (pH
7.5), 0.0015% Brij35, 10 mM MgCl2 and 2 mM dithiothreitol. The stop buffer
contained 100 mM HEPES (pH 7.5), 0.015% Brij-35, 0.2% Coating Reagent and 50
mM EDTA.
Initially, the test compounds were diluted to 50-fold the highest concentration
tested using 100% dimethyl sulfoxide (DMSO). One hundred microliters of this
dilution was then transferred to a well of a 96-well plate. Control wells with no
compound or no enzyme were prepared by adding 100 µL of 100% DMSO to two
empty wells of the same 96-well plate, which was marked as the source plate. Then, 5
µL of the compound solution was transferred from the source plate to a new 96-well
plate (the intermediate plate). An additional 45 µL of kinase buffer was added to each

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well of the intermediate plate, which was then shaken for 10 min. The assay plate was
prepared by transferring 5 µL from each well of the 96-well intermediate plate to a
384-well plate, in duplicates. The prepared enzyme solution, containing the
appropriate kinase and kinase buffer, was added to each well of the 384-well assay
plate, which was then incubated at room temperature for 10 min prior to the addition
of 10 µL peptide solution; this contained a fluorescein amidite-labelled peptide
substrate and ATP, in kinase buffer. The mixture was incubated at 28°C for 1 h before
adding 25 µL stop buffer. The conversion data were copied from the Caliper program,
and the values were used to calculate kinase inhibition values using the following
equation: percentage inhibition = (max - conversion)/(max - min) × 100.
4.2.2. Cell viability assay
The viability of HEK293T, A549, HT-29 and MCF-7 cells was determined using
an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. These
cancer cell lines were cultured in minimum essential medium supplemented with 10%
foetal bovine serum.
Approximately 4 × 103 cells were suspended in cell culture medium, plated in a
96-well plate and incubated at 37°C in 5% CO2 for 24 h. The test compounds were
added to the culture medium and incubated for a further 72 h. Fresh MTT was then
added to each well at a final concentration of 5 µg/mL, and incubated with the cells at
37°C for 4 h. The formazan crystals in each well were dissolved in 100 µL DMSO,
and the absorbance at 492 nm (MTT formazan wavelength) and 630 nm (reference
wavelength) was measured by a microplate reader. The reported IC50 results
represented averages of at least three determinations and were calculated using the
Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
4.2.3. Flow cytometric assay
Apoptosis of HUVECs, HT-29 and MCF-7 cells was detected using a flow
cytometric assay. Briefly, cells were seeded in 6-well plates and incubated overnight.
The following day, cells were treated with different concentrations of compound A8
for 48 hours. The cells and supernatants were harvested and washed twice with cold
PBS and then resuspended in 100µl 1× Binding Buffer. 5 µl of FITC Annexin V and 5
µl PI were added in each tube and the cells were then gently vortexed incubated for
15 min at RT (25°C) in the dark. 400 µl of 1× Binding Buffer then added to each tube.
The stained cells were analyzed by a flow cytometer (FACS Calibur; BD).
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
This work was supported by grants from the National Natural Science
Foundation of China (81502924) and Innovation Training Project of Liaoning
Province (201610163013).
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Legends
Figure 1. Design strategy of the target compounds.
Figure 2. Effects of compound A8 on cell apoptosis in HUVECs (top), HT-29 (middle)
and MCF-7 (bottom) cells.
Figure 3. Docking model of compound A8 with VEGFR-2. Left: binding model
overlap of compound A8 (grey) and nintedanib (yellow) in the kinase domain (surface
representation). Right: potential interactions between A8 and VEGFR-2 depicted as a
stick model. Dashed lines depict contacts between the inhibitor and selected residues.
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, pyridine,
chlorobenzene, r.t., 1 h, then p-toluenesulfonic acid, reflux, 8 h; (b) appropriate amine,
DIPEA, CH2Cl2, 30 °C, 8–12 h, then H2, Pd/C, EtOH, r.t., 5 h; (c) methyl
(E)-3-(methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate, methanol, reflux,
5–10 h; (d) chloroacetyl chloride, Et3N, EtOAc, r.t., 30 min, then HOAc, reflux, 20 h.
Scheme 2. Reagents and conditions: (a) appropriate amine, HATU, DIPEA, DMF, r.t.,
12 h; (b) borane dimethyl sulphide, THF, reflux, 1 h, then HCl, reflux, 30 min; (c) H2,
Pd/C,
EtOH,
r.t.,
6
h;
(d)
methyl
(E)-3-(methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate, methanol, reflux,
5–10 h; (e) appropriate alkyl halide, NaH, CH2Cl2, r.t., 3–6 h; (f) CH2O, appropriate
amine, HOAc, 35 °C, 5–8 h.
Table 1. In vitro inhibitory profile of compounds A1–A6.
Table 2. In vitro inhibitory profile of compounds A7–A17.
Table 3. Inhibitory activity of compound A8 against selected angiokinases.

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Table 1. In vitro inhibitory profile of compounds A1–A6.
IC50 (nM)a
VEGFR-2
IC50 (µM)b
Compound
R
PDGFRβ
A549
>100
MCF-7
HT-29
NT
A1
A2
A3
A4
A5
A6
> 1000
79% inhibitionc
384.7
>500
NT
116.2
45.5
61.0
NT
>100
>100
NT
NT
>100
89.87 ± 2.31
0.70 ± 0.12
0.98 ± 0.26
0.32 ± 0.03
40.52 ± 1.62 22.54 ± 2.16
257.2
16.45 ± 0.88
39.14 ± 1.28
2.32 ± 0.23
3.27 ± 0.52
138.3
106.1
92.7
NT: not tested.
a The biological data are generated from at least two independent experiments.
b The biological data are generated from at least three independent experiments.
c Percent inhibition tested at 1 µM.

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Table 2. In vitro inhibitory profile of compounds A7–A17.
IC50 (nM)a
IC50 (µM)b
Compound
R1
R2
VEGFR-2
PDGFRβ
A549
>100
MCF-7
HT-29
>100
A7
A8
H
H
>1000
NT
NT
69.1
81.3
22.0
44.2
39.0
60.7
64.6
34.3
49.2
135.1
91.7
2.99 ± 0.22
75.19 ± 2.79
11.9 ± 1.17
2.27 ± 0.58
6.01 ± 0.33
7.63 ± 0.69
4.32 ± 0.06
7.40 ± 1.32
6.15 ± 1.07
NT
1.56 ± 0.53 1.17 ± 0.18
NT
NT
1.10 ± 0.02
2.19 ± 0.23
A9
methyl
ethyl
H
A10
A11
A12
A13
A14
A15
A16
104.6
3.94 ± 0.76 0.13 ± 0.03
2.28 ± 0.50 0.46 ± 0.05
5.53 ± 1.33 6.79 ± 2.07
5.67 ± 0.78 0.25 ± 0.05
>1000
808.5
methyl
ethyl
isopropyl
H
431.2
646.5
59%@1 µMc
>1000
NT
NT
4.01 ± 0.68 0.87 ± 0.12
methyl
H
NT
NT
A17
NT
8.5
241.6
3.5
22.62 ± 1.57 8.28 ± 0.79 0.83 ± 0.37
nintedanib
NT: not tested.
a The biological data are generated from at least two independent experiments.
b The biological data are generated from at least three independent experiments.
c Percent inhibition tested at 1 µM.

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Table 3. Activities of compound A8 in inhibition of selected angiokinases.
IC50 (nM)a
Compound
VEGFR-2
69.1
VEGFR-3
18.2
PDGFRα
4.4
PDGFRβ
22.0
FGFR-1
1507
A8
nintedanib
8.5
3.2
2.3
3.5
98.2
a The biological data are generated from at least two independent experiments.

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ò
ò
ò
An indolinone-based compound, A8, is identified as a potent angiokinase inhibitor.
A8 potently inhibits VEGFR-2, VEGFR-3, PDGFRα and PDGFRβ.
A8 induces apoptotic cell death in HUVECs, and HT-29 and MCF-7 cancer cells.