Structural modifications of indolinones bearing a pyrrole moiety and discovery of a multi-kinase inhibitor with potent antitumor activity

Article abstract of DOI:10.1016/j.bmc.2020.115486

Structural modifications of compound 2, an angiokinase inhibitor reported by our group were performed, which led to the discovery of methyl (Z)-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7h). Compound 7h exhibited excellent inhibitory activity against angiokinases including VEGFR-1/2/3, PDGFRα/β, and FGFR-1, as well as LYN and c-KIT kinases. At the cellular level, compound 7h significantly attenuated phosphorylation of AKT and ERK proteins, potently inhibited colony formation of HT-29, MKN74, and HepG2 cancer cells, and induced cell apoptosis. Upon incubation with human liver microsome, 7h exhibited comparable metabolic stability to nintedanib. Compound 7h has emerged as a promising lead compound for future drug design.

Full text of DOI:10.1016/j.bmc.2020.115486

Journal Pre-proofs
Structural modifications of indolinones bearing a pyrrole moiety and discov‐
ery of a multi-kinase inhibitor with potent antitumor activity
Mingze Qin, Ye Tian, Xiao Han, Qi Cao, Shuaishuai Zheng, Chunyang Liu,
Xia Wu, Lei Liu, Yangyang Meng, Xiaobo Wang, Haotian Zhang, Yunlei
Hou
PII:
DOI:
Reference:
S0968-0896(20)30307-2
https://doi.org/10.1016/j.bmc.2020.115486
BMC 115486
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
26 January 2020
20 March 2020
30 March 2020
Please cite this article as: Qin, M., Tian, Y., Han, X., Cao, Q., Zheng, S., Liu, C., Wu, X., Liu, L., Meng, Y.,
Wang, X., Zhang, H., Hou, Y., Structural modifications of indolinones bearing a pyrrole moiety and discovery of
a multi-kinase inhibitor with potent antitumor activity, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115486
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Structural modifications of indolinones bearing a pyrrole moiety and
discovery of a multi-kinase inhibitor with potent antitumor activity
Mingze Qin a,d, Ye Tian a, Xiao Han b, Qi Cao a, Shuaishuai Zheng a, Chunyang Liu a,
a
a
a
Xia Wu , Lei Liu , Yangyang Meng , Xiaobo Wang d,*, Haotian Zhang c,*, Yunlei
Hou a,*
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China
b
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, PR
China
c
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang
110016, PR China
d Chinese People’s Liberation Army Logistics Support Force No. 967 Hospital, Dalian
116021, PR China
Corresponding authors.
Tel: +86 24 2398 6282
E-mail address: houyunlei901202@163.com (Y. Hou); zhanghaotian2087@163.com
(H. Zhang); wxbbenson0653@sina.com (X. Wang).
Abstract
Structural modifications of compound 2, an angiokinase inhibitor reported by our
group were performed, which led to the discovery of methyl
(Z)-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amin
o)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7h). Compound 7h exhibited
excellent inhibitory activity against angiokinases including VEGFR-1/2/3,
PDGFRα/β, and FGFR-1, as well as LYN and c-KIT kinases. At the cellular level,
compound 7h significantly attenuated phosphorylation of AKT and ERK proteins,
potently inhibited colony formation of HT-29, MKN74, and HepG2 cancer cells, and
induced cell apoptosis. Upon incubation with human liver microsome, 7h exhibited
comparable metabolic stability to nintedanib. Compound 7h has emerged as a
promising lead compound for future drug design.
Keywords: structural modification; multi-kinase inhibitor; antitumor activity

1. Introduction
Angiogenesis
is
an
indispensable
physiological
process
during
embryonic development and wound healing.1–3 However, considerable studies
demonstrated that the progression of primary tumor is angiogenesis-dependent, which
provide necessary nutrient for further growth and metastasis of tumor cells.4,5
Angiogenesis is regulated by several endogenous molecules. Vascular
endothelial growth factor receptors (VEGFRs) are transmembrane tyrosine kinases
consisting of three structurally related isoforms. The binding of VEGFR with its
cognate ligand activates several downstream signal cascades, which are responsible
for cell proliferation and survival.2 VEGFR-2 is considered the major mediator, and is
a valid target for drug discovery.6–8 In addition, increasing studies indicate that
VEGFR-1 and VEGFR-3 are independently up-regulated in several human
malignancies, such as prostate, lung, and colorectal tumors. Higher levels of these
receptors are commonly correlated with formation of metastasis.9–11 These findings
highlighted the importance of VEGFR family in tumorigenesis. Platelet-derived
growth factor (PDGF) and its cognate receptor mediate an essential signaling for
growth of perivascular smooth muscle and pericytes, which are important for
neovascularization.12 Aberrant PDGFRs activation has been documented in clinical
samples of tumor such as glioma and prostate cancer, making PDGFRs as druggable
therapeutic targets.13 Upon sustained blockade of VEGF-driven pathway, several
other molecules may involve, such as fibroblast growth factor receptor (FGFR),
providing a compensatory mechanism to facilitate tumor angiogenesis.14
Considering the complicated regulation of tumor angiogenesis, combination
therapy and multi-target inhibitors are considered promising strategies to inhibit
tumor growth and circumvent drug resistance. On the other hand, the high degree of
structural similarities between VEGFR, PDGFR, and FGFR kinases creates
possibility to identify inhibitors with synergistic activity. Nintedanib is an authorized
inhibitor of VEGFR, PDGFR, and FGFR, with excellent in vivo activity in various
tumor models.15 It contains an indolinone scaffold, where by the lactam group forms
pivotal hydrogen bonds with the amino acid residues of target proteins.16,17
In our previous study, we have disclosed two 6-methoxycarbonyl indolinones (1
and 2) as angiokinases inhibitors.18 Here, our recent work regarding the structural
optimizations of these precursors is reported (Fig. 1), which ultimately leads to the
discovery
of
methyl
(Z)-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amin
o)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7h), a more potent multi-kinase
inhibitor with excellent antitumor activity.

Figure 1. Design strategy of compounds 7a–7l and 8–10.
2. Results and discussion
2.1. Chemistry
The synthetic route for preparation of compounds 7a–7l is outlined in Scheme 1.
Briefly, the fluoro benzene or heterocycle 3 underwent a nucleophilic coupling with
pyrrole or 2-methyl-1H-pyrrole to generate intermediates 4a–4e, which were
converted to intermediates 5a–5l via Mannich reaction followed by hydrogenation
reaction as previously reported.18 Finally, Michael addition reactions were employed
by reacting intermediates 5a–5l with commercially available intermediate methyl
(E)-3-(methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate (6), to yield the
target compounds 7a–7l.
Scheme 1. Reagents and conditions: (a) pyrrole or 2-methyl-1H-pyrrole, NaH, DMF,
80 °C, 8–11 h; (b) (i) paraformaldehyde, appropriate amine, HOAc, 35 °C, 6–10 h;
(ii) Pd/C, EtOH, 25 °C, 4–6 h; (c) 6, MeOH, reflux, 4–12 h.
The synthesis of compounds 8–10 is described in Scheme 2. Hydrolysis of ester
group of 7h provided compound 8, which was reacted with methylamine or
dimethylamine to yield compounds 9 and 10, respectively.

Scheme 2. Reagents and conditions: (a) NaOH, MeOH, reflux, 0.5 h, then HCl; (b)
TBTU, HOBT, DIPEA, appropriate amine, DMF, 25 °C, 5 h.
2.2. Biological evaluation
2.2.1. Modifications of precursors and discovery of compound 7h
With the intention of discovering more potent angiokinases inhibitors, we
undertook a chemistry campaign based on the indolinone precursors. Regarding
inhibitors 1 and 2, we speculated that the dihedral angle between pyrrole and phenyl
ring A is crucial for high activity. Thus, from the viewpoint of structure-based drug
design, we modified the pyrrole moiety and ring A of them to adjust the dihedral
angle, and further to promote their binding with the kinase (Fig. 1). Accordingly,
compounds 7a–7l were prepared and screened for inhibitory activity against
VEGFR-2 and PDGFRβ. The parent compounds, 1 and 2, showed reproducible
activity in inhibition of VEGFR-2 and PDGFRβ, with IC50 values of 82.3 and 11, 58.3
and 55 nM, respectively.
Initially, we investigated the influence of modified aromatic ring A on activity.
Encouraged by the magic methyl group of nintedanib, we first introduced a methyl
group and prepared compounds 7a and 7b. To our disappointment, as shown in Table
1, both compounds exhibited decreased activity against VEGFR-2 and PDGFRβ than
their parent compounds (7a, IC50 = 492 and 31 nM, respectively; 7b, IC50 = 248 and
206 nM, respectively). Similarly, the replacement of the phenyl ring with heterocyclic
bioisostere, pyridine, led to a sharp loss in activity (7c, IC50 = 472 and 26 nM,
respectively; 7d, IC50 = 584 and 114 nM, respectively). Unsurprisingly, combination
of the two variations remarkably eroded the enzymatic activity, as shown for 7e and
7f. These biological results clearly demonstrated that further modification of aromatic
ring A was undesirable. Subsequently, research focus was shifted to the pyrrole
moiety, and compounds 7g and 7h were readily obtained. Much to our delight, while
7g showed moderate inhibition of VEGFR-2 and PDGFRβ (IC50 = 147.6 and 45.3
nM, respectively), compound 7h displayed very potent activity, demonstrating IC50
values of 6.5 and 9.9 nM, respectively. We speculated that the introduction of the
methyl group dramatically tuned the dihedral angle between phenyl ring and pyrrole,
which facilitated the formation of ionic interaction between the piperazinyl group
with Glu850. Previously, we reported that a dimethylamine or methyl piperazinyl
group might represent the optimal amino groups attached to pyrrole.17,18 To further
confirm this speculation, compounds 7i and 7j bearing a diethyl amino and
morpholinyl group, respectively, were synthesized and evaluated. Both two
compounds exhibited promising activity in inhibition of selected kinases,
demonstrating IC50 values ranging 23.8–227.7 nM, but were less activity as compared
to compound 7h. Furthermore, the replacement of phenyl ring of compound 7h with
pyridine also failed to improve enzymatic inhibitory activity (7l, VEGFR-2, IC50
288 nM; PDGFRβ, IC50 = 123 nM).
=
It was reported that the cleavage of ester was a major metabolic pathway of
nintedanib.15,19 To simulate the metabolic process of 7h, we prepared its hydrolysis
product, 8. Interestingly, compound 8 also showed excellent activity in inhibition of
VEGFR-2 (IC50 = 14 nM). The replacement of the ester group with amide fragment

led to compounds 9 and 10, both of which showed reduced activity.
Table 1. Inhibitory activities of compounds against VEGFR-2 and PDGFRβ.
N
R2
R1
N
N
N
N
R3
N
X
NH
O
NH
O
NH
O
O
O
N
N
H
N
H
R4
H
O
O
1
2
7a-7l, 8-10
IC50 (nM)a
Compound
X
R1
R2
R3
R4
VEGFR-2
82.3 ± 6.3
PDGFRβ
1
2
11 ± 0.6
58.3 ± 5.9
492 ± 37.8
55 ± 2.6
31 ± 2.2
O
O
O
O
O
O
O
O
O
O
O
N
7a
7b
7c
7d
7e
7f
C
C
N
N
N
N
C
C
C
C
N
methyl
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
N
N
methyl
248 ± 35.6
472 ± 22.7
584 ± 27.6
>1000
206 ± 11.3
26 ± 3.9
114 ± 5.8
71 ± 3.1
>300
N
H
N
N
H
methyl
methyl
H
N
N
N
>1000
N
7g
7h
7i
methyl
methyl
methyl
methyl
methyl
147.6 ± 9.7
6.5 ± 0.9
45.3 ± 6.9
9.9 ± 0.7
N
N
H
H
100.8 ± 17.6 23.8 ± 3.3
N
O
N
7j
H
227.7 ± 16
700 ± 55.6
37 ± 1.9
32 ± 2.7
N
7k
H

N
N
O
7l
8
N
C
C
C
H
H
H
H
methyl
methyl
methyl
methyl
288 ± 30.5
14 ± 2
123 ± 15.2
NT
O
N
N
HO
O
H
N
N
N
9
485 ± 37.2
NT
O
O
N
N
N
10
65 ± 6.7
4.7 ± 0.3
NT
nintedanib
3.8 ± 0.4
NT: not test.
a The biological data are generated from at least two independent experiments.
2.2.2. Kinase inhibition profile of 7h
Through the structural modification strategy, compound 7h was identified as a
promising inhibitor of angiokinases, although the potency was slightly less active than
nintedanib. To further confirm the biological targets of 7h, we evaluated it in a panel
of kinases correlated with tumor growth (Table 2). In addition to VEGFR-2 and
PDGFRβ, 7h displayed strong inhibition of VEGFR-1, VEGFR-3, PDGFRα, and
FGFR1 with IC50 values ranging 6.3–31 nM, confirming its potent efficacy against
angiokinases. It could be noted that 7h demonstrated moderate activity against SRC
(IC50 = 178 nM), while showed an excellent inhibition of LYN kinase (IC50 = 16 nM).
Recent studies have shown that Src-family members played an important role for
proliferation of cancer stem cells.20 Synergistic inhibition of Src-family by 7h might
help for treating cancer. Also, 7h exhibited excellent inhibitory activity against c-KIT
kinase (IC50 = 17 nM), which was considered an oncogenic driver in a variety of
human cancers such as melanomas.21 Selected kinases including CDK2, CDK4, CHK,
and B-Raf were not significantly inhibited at 1 µM concentration, which indicated
that compound 7h possessed favorable kinase selectivity to a certain degree.
Table 2. Inhibitory activities of compound 7h against selected kinases.
IC50 (nM)a
Enzyme
VEGFR-1
VEGFR-2
VEGFR-3
PDGFRα
PDGFRβ
FGFR-1
SRC
7h
Nintedanib
23 ± 0.8
4.7 ± 0.3
3.0 ± 0.2
1.9 ± 0.3
3.8 ± 0.4
99 ± 8.9
16 ± 2.1
55 ± 2.8
NT
31 ± 2.1
6.5 ± 0.9
6.3 ± 0.5
7.0 ± 0.1
9.9 ± 0.7
23 ± 2.5
178 ± 15
16 ± 1.3
17 ± 0.5
LYN
c-KIT

CDK2
CDK4
>1000
>1000
>1000
>1000
>1000
NT
CHK
>1000
NT
B-Raf
NT: not test.
a The biological data are generated from at least two independent experiments.
2.2.3. Western blotting analysis of 7h
Considering that compound 7h was a potent multi-kinase inhibitor, we analyzed
its effects on the phosphorylation of AKT and ERK proteins, which were important
downstream signaling effectors of receptor tyrosine kinases. Representative cancer
cell lines (HT-29 and HepG2) were treated with 7h at different concentrations. As
shown in Figure 2, 7h suppressed the phosphorylation of AKT (Ser473) in a
dose-dependent manner with a significant inhibition of ERK (Thr202/Tyr204)
phosphorylation at 3 μM in both tested cancer cell lines.
Figure 2. The suppressive effects of 7h on p-AKT (Ser473) and p-ERK
(Thr202/Tyr204) in HT-29 (A) and HepG2 (B) cells.
2.2.4. Inhibition of colony formation by 7h
To elucidate the impact of 7h on proliferation of cancer cells, colony formation
assays were performed. HT-29, MKN74, and HepG2 cancer cells were treated with
7h at indicated concentrations, respectively. As shown in Figure 3, 7h exhibited
excellent activity in inhibition of colony formations in a dose-dependent manner, with
remarkable activity at 1 µM concentration for all tested cancer cells. With respect to
MKN74 cancer cells, treatment with 1 μM 7h almost completely suppressed the
colony formation, and was more active than nintedanib.

Figure 3. Colony formation of HT-29 (A), HepG2 (B), and MKN74 cells (C) treated
with 7h and nintedanib. Relative colony formation rate is shown as mean ± SEM. *P
< 0.05, **P < 0.01, and ***P < 0.001 versus vehicle group; P-values were calculated
using an unpaired two-tailed Student’s t-test.
2.2.5. Induction of cell apoptosis by 7h
A biparametric cytofluorimetric analysis was conducted to evaluate the effects of
7h on cancer cells apoptosis. As shown in Figure 4, 7h triggered severe apoptosis of
HT-29, MKN74, and HepG2 cells in a dose-dependent manner. Human umbilical vein
endothelial cells (HUVECs) were used to evaluate the effects of 7h on endothelial
cells, the proliferation of which was considered a critical process of angiogenesis.
Significantly, the apoptotic rate of HUVECs was increased from 7.11% (control) to
64.69% in the 3 μM group. In contrast, no significant induction of apoptosis was
observed in normal cells HEK293T (Fig. S1).
Figure 4. Effects of 7h on cell apoptosis in HT-29 (A), HepG2 (B), MKN74 (C), and
HUVEC (D) cells.
2.2.6. In vitro metabolic stability of 7h
Compound 7h was incubated with human and mouse liver microsomes,
respectively, to evaluate the metabolic stability in vitro. The results in Table 3
revealed that 7h (Clint = 81.97 mL/min/kg) suffered slightly higher clearance upon
incubation with human microsome than that of nintedanib (Clint = 63.5 mL/min/kg),
and thus possessed a relatively shorter T1/2 (21.21 min vs 27.38 min). Detailed
pharmacokinetic profile of 7h should be further assessed in in vivo experiments.
Table 3. Liver microsomal stability of compound 7h.
Human
Mouse
Compound
T1/2 (min) Clint (mL/min/kg)
T1/2 (min) Clint (mL/min/kg)
7h
21.21
81.97
12.53
435.65

Nintedanib
27.38
63.5
-
-
2.2.7. In vivo antitumor activity of 7h
Considering high levels of biological activity and moderate metabolic stability,
compound 7h was evaluated for in vivo activity in a HT-29 human colon cancer
xenograft model. Nude mice bearing established HT-29 tumor xenografts were dosed
orally for 18 days. Compound 7h (100 mg/kg, qd) displayed mild inhibition of tumor
growth, with tumor growth inhibition (TGI) value of 13.39% while that of nintedanib
(100 mg/kg, qd) was 44.27% (Figure 5). The tested compound was well-tolerated, no
significant loss of body weight was observed during treatment.
Figure 5. In vivo effect of compound 7h on tumor volume (A) and body weight (B) in
xenograft model of HT-29. Mice were orally dosed once daily for 18 days at
100mg/kg. Results are expressed as the mean ± SD. (n = 6 per group).
2.3. Molecular docking study
Docking study of compound 7h at the ATP-binding site of VEGFR-2 was
performed to explore its probable binding mode. The crystal structure of VEGFR-2
kinase domain was obtained from the Protein Data Bank (PDB code: 3C7Q). As
shown in Figure 6A, 7h matched well with this binding site, accounting for its high in
vitro activity in inhibition of VEGFR-2. Three hydrogen bonds were formed between
the 6-methoxycarbonyl indolinone moiety of 7h with Glu917, Cys919, and Lys868 in
the hinge region. Particularly, the piperazinyl group interacted with Glu850 via an
ionic interaction as well as a carbon hydrogen bond, which further improved inhibitor
binding. Figure 6B showed the high-degree overlay of 7h and nintedanib at this
binding site.

Figure 6. (A) Probable interactions between compound 7h and VEGFR-2. Dashed
lines indicated the contacts between the inhibitor and selected residues. (B) Binding
model overlap of compound 7h (grey) and nintedanib (yellow) at the binding site.
3. Conclusions
In summary, structural modifications of compound 2, an angiokinase inhibitor
reported previously were performed in this study, which generated a potent
multi-kinase inhibitor, 7h, for the treatment of tumor. Compound 7h exhibited
excellent potency against angiokinases, with IC50 values ranging 6.3–31 nM. Also, it
displayed potent activity against other kinases correlated with tumor growth, such as
LYN and c-KIT kinases. In cells, 7h effectively attenuated the phosphorylation of
AKT and ERK proteins. Significant anti-proliferative activity was also exhibited by
7h, which potently inhibited colony formation of HT-29, HepG2, and MKN74 cancer
cells, and induced cell apoptosis. Molecular docking analysis demonstrated that 7h
matched well with the active site of VEGFR-2, and the ionic interaction between
piperazinyl group and Glu850 was crucial for high kinase affinity, which contributed
to excellent in vitro activity of 7h. Nonetheless, in our initial in vivo study with 7h in
a HT-29 xenograft model, effects on growth of tumor were only mild. The results
might be ascribed to its relatively high hepatic clearance as exhibited in an in vitro
assay.
The structure-activity relationships of the newly synthesized compounds were
explored, providing valuable information for the future work. For example,
modifications of aromatic ring A were unsuitable, but further investigations on
6-position of indolinone moiety were deserved. In future studies, structural
modifications of 7h are warranted to improve its pharmacokinetic property, and thus,
excellent in vivo antitumor activity can be expected.
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. General procedure for preparation of compounds 4a–4e
To a solution of pyrrole or 2-methyl-1H-pyrrole (0.15 mol) in DMF (100 mL),
NaH (0.195 mol, 60%) was added in portions, then appropriate intermediate 3 was
added dropwise. The mixture was stirred at 80 °C for 8–11 h, and was monitored by
TLC. The mixture was poured into ice water, and stirred for 15 min. The precipitate
was filtered off to give a crude product, which was purified by column

chromatography to give the desired compound.
1
4.1.1.1. 1-(2-Methyl-4-nitrophenyl)-1H-pyrrole (4a). Yellow solid. Yield: 27%. H
NMR (600 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.14 (s, 1H), 7.52 (s, 1H), 7.09 (s, 2H),
6.31 (s, 2H), 2.37 (s, 3H). ESI-MS m/z: 203.1 [M+H]+.
1
4.1.1.2. 5-Nitro-2-(1H-pyrrol-1-yl)pyridine (4b). Yellow solid. Yield: 41%. H NMR
(600 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.68 (dd, J = 9.1, 2.7 Hz, 1H), 7.96 (d, J = 9.1
Hz, 1H), 7.81 (s, 2H), 6.42 (m, 2H). ESI-MS m/z: 190.1 [M+H]+.
4.1.1.3. 3-Methyl-5-nitro-2-(1H-pyrrol-1-yl)pyridine (4c). Yellow solid. Yield: 56%.
1H NMR (600 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.70 (s, 1H), 7.43 (s, 2H), 6.35 (s,
2H), 2.50 (s, 3H). ESI-MS m/z: 204.1 [M+H]+.
1
4.1.1.4. 2-Methyl-1-(4-nitrophenyl)-1H-pyrrole (4d). Yellow solid. Yield: 36%. H
NMR (600 MHz, DMSO-d6) δ 8.34 (m, 1H), 8.33 (m, 1H), 7.70 (m, 1H), 7.69 (m,
1H), 7.02 (s, 1H), 6.19 (s, 1H), 6.08 (s, 1H), 2.27 (s, 3H). ESI-MS m/z: 203.1
[M+H]+.
4.1.1.5. 2-(2-Methyl-1H-pyrrol-1-yl)-5-nitropyridine (4e). Yellow solid. Yield: 53%.
1H NMR (600 MHz, DMSO-d6) δ 9.28 (s, 1H), 8.66 (s, 1H), 7.82 (s, 1H), 7.41 (s,
1H), 6.23 (s, 1H), 6.11 (s, 1H), 2.49 (s, 3H). ESI-MS m/z: 204.1 [M+H]+.
4.1.2. General procedure for preparation of compounds 5a–5l
To a mixture of paraformaldehyde (15 mmol) and appropriate secondary amine
(15 mmol) in acetic acid (5 mL) was added intermediate 4a–4e (10 mmol). The
solution was stirred at 35 °C for 6–10 h, and was monitored by TLC. After that time,
the acetic acid was concentrated. The residue was diluted with ethanol (15 mL), and
Pd/C (10%) was added. The reaction mixture was hydrogenated for 4–6 h. When TLC
showed the completion of the reaction, the catalyst was filtered off, then the solvent
was evaporated. The residue was purified by column chromatography to afford the
desired intermediate.
4.1.2.1. 4-(2-((Dimethylamino)methyl)-1H-pyrrol-1-yl)-3-methylaniline (5a). Yellow
solid. Yield: 16%. ESI-MS m/z: 230.1 [M+H]+.
4.1.2.2. 3-Methyl-4-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)aniline (5b).
Yellow solid. Yield: 22%. ESI-MS m/z: 285.2 [M+H]+.
4.1.2.3. 6-(2-((Dimethylamino)methyl)-1H-pyrrol-1-yl)pyridin-3-amine (5c). Yellow
solid. Yield: 37%. ESI-MS m/z: 217.1 [M+H]+.
4.1.2.4. 6-(2-((4-Methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)pyridin-3-amine (5d).
Yellow solid. Yield: 26%. ESI-MS m/z: 272.2 [M+H]+.
4.1.2.5. 6-(2-((Dimethylamino)methyl)-1H-pyrrol-1-yl)-5-methylpyridin-3-amine (5e).
Yellow solid. Yield: 19%. ESI-MS m/z: 231.2 [M+H]+.
4.1.2.6.
5-Methyl-6-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)pyridin-3-amine (5f).
Yellow solid. Yield: 23%. ESI-MS m/z: 286.2 [M+H]+.
4.1.2.7. 4-(2-((Dimethylamino)methyl)-5-methyl-1H-pyrrol-1-yl)aniline (5g). Yellow
solid. Yield: 39%. 1H NMR (400 MHz, DMSO-d6) δ 6.90 (d, J = 8.3 Hz, 2H), 6.64 (d,
J = 8.3 Hz, 2H), 6.35 (d, J = 2.5 Hz, 1H), 5.92 (d, J = 2.6 Hz,1H), 5.42 (s, 2H), 3.79
(s, 2H), 2.37 (s, 6H), 1.93 (s, 3H). ESI-MS m/z: 230.2 [M+H]+.
4.1.2.8. 4-(2-Methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)aniline (5h).

Yellow solid. Yield: 42%. 1H NMR (600 MHz, DMSO-d6) δ 8.37 (d, J = 8.8 Hz, 1H),
8.24 (d, J = 8.7 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 6.00 (dd, J
= 14.2, 3.3 Hz, 1H), 5.91 (dd, J = 13.8, 3.1 Hz, 1H), 3.18 (d, J = 6.4 Hz, 2H), 2.43–
1.92 (m, 14H). ESI-MS m/z: 285.2 [M+H]+.
4.1.2.9. 4-(2-((Diethylamino)methyl)-5-methyl-1H-pyrrol-1-yl)aniline (5i). Yellow
solid. Yield: 21%. ESI-MS m/z: 258.2 [M+H]+.
4.1.2.10. 4-(2-Methyl-5-(morpholinomethyl)-1H-pyrrol-1-yl)aniline (5j). Yellow
solid. Yield: 17%. ESI-MS m/z: 272.2 [M+H]+.
4.1.2.11.
6-(2-((Dimethylamino)methyl)-5-methyl-1H-pyrrol-1-yl)pyridin-3-amine
(5k). Yellow solid. Yield: 30%. ESI-MS m/z: 231.2 [M+H]+.
4.1.2.12.
6-(2-Methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)pyridin-3-amine (5l).
Yellow solid. Yield: 24%. ESI-MS m/z: 286.2 [M+H]+.
4.1.3. General procedure for preparation of compounds 7a–7l
At room temperature, to a solution of intermediate (5a–5l, 0.2 mmol) in
methanol
(7
mL)
was
added
intermediate
methyl
(E)-3-(methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate (0.2 mmol). The
mixture was stirred under reflux for 4–12 h and was monitored by TLC. Upon
completion of the reaction, the solution was cooled, the precipitate was filtered off to
give a crude product, which was recrystallized from methanol to afford the target
compound.
4.1.3.1.
Methyl
(Z)-3-(((4-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)-3-methylphenyl)amino)(phenyl
)methylene)-2-oxoindoline-6-carboxylate (7a). Yellow solid. Yield: 72%. Mp: 219.7–
221.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 12.26 (s, 1H), 11.01 (s, 1H), 7.62 (d, J =
7.0 Hz, 1H), 7.59 (t, J = 6.9 Hz, 2H), 7.53 (d, J = 6.4 Hz, 2H), 7.44 (s, 1H), 7.21 (d, J
= 7.9 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.91 (s, 1H), 6.68–6.62 (m, 2H), 6.08 (s, 1H),
6.04 (s, 1H), 5.87 (d, J = 8.1 Hz, 1H), 3.78 (s, 3H), 2.91 (s, 2H), 1.89 (s, 6H), 1.79 (s,
3H). HRMS (ESI) for C31H30N4O3 [M + H]+, calcd: 507.2391, found: 507.2397.
4.1.3.2.
Methyl
(Z)-3-(((3-methyl-4-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amino
)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7b). Yellow solid. Yield: 68%.
1
Mp: 262.2–264.7 °C. H NMR (600 MHz, DMSO-d6) δ 12.25 (s, 1H), 10.99 (s, 1H),
7.64–7.60 (m, 1H), 7.58 (t, J = 7.3 Hz, 2H), 7.53 (s, 2H), 7.43 (d, J = 1.4 Hz, 1H),
7.21 (dd, J = 8.2, 1.5 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 2.3 Hz, 1H), 6.65
(dt, J = 6.4, 3.0 Hz, 2H), 6.06 (t, J = 3.1 Hz, 1H), 6.02 (dd, J = 3.2, 1.6 Hz, 1H), 5.86
(d, J = 8.2 Hz, 1H), 3.78 (s, 3H), 2.97 (s, 2H), 2.10 (s, 3H), 2.04 (br, 8H), 1.81 (s,
3H). 13C NMR (151 MHz, DMSO-d6) δ 170.60, 166.77, 158.77, 138.04, 136.96,
136.65, 135.69, 132.46, 130.86, 129.85, 129.32, 128.97, 128.83, 128.78, 125.09,
124.47, 123.03, 121.90, 120.92, 117.71, 109.88, 109.52, 107.76, 98.02, 54.18, 52.84,
52.20, 50.90, 44.51, 17.41. HRMS (ESI) for C34H35N5O3 [M + H]+, calcd: 562.2813,
found: 562.2818.
4.1.3.3.
Methyl
(Z)-3-(((6-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)pyridin-3-yl)amino)(phenyl)me

thylene)-2-oxoindoline-6-carboxylate (7c). Yellow solid. Yield: 53%. Mp: 216.7–
219.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 11.08 (s, 1H), 8.18 (s, 1H),
7.67–7.50 (m, 6H), 7.44 (t, J = 7.2 Hz, 1H), 7.38 (dd, J = 8.8, 2.5 Hz, 2H), 7.22 (dd, J
= 8.2, 1.4 Hz, 1H), 6.36 (s, 1H), 6.22 (s, 1H), 5.89 (d, J = 8.2 Hz, 1H), 3.93 (s, 2H),
3.78 (s, 3H), 2.42 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 170.68, 166.82, 158.60,
148.32, 143.58, 137.06, 134.39, 133.36, 132.07, 131.23, 131.15, 130.11, 129.18,
129.13, 124.94, 122.97, 122.07, 118.01, 116.11, 116.08, 110.69, 110.07, 98.96, 54.51,
52.31, 43.38. HRMS (ESI) for C29H27N5O3 [M + H]+, calcd: 494.2187, found:
494.2183.
4.1.3.4.
Methyl
(Z)-3-(((6-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)pyridin-3-yl)amino)(ph
enyl)methylene)-2-oxoindoline-6-carboxylate (7d). Yellow solid. Yield: 71%. Mp:
257.3–261.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 11.03 (s, 1H), 8.05
(d, J = 2.3 Hz, 1H), 7.63–7.51 (m, 6H), 7.48–7.38 (m, 2H), 7.22 (dd, J = 8.2, 1.5 Hz,
1H), 7.17 (s, 1H), 6.19–6.06 (m, 2H), 5.87 (d, J = 8.2 Hz, 1H), 3.78 (s, 3H), 3.53 (s,
13
3H), 3.17 (s, 2H), 2.31 (br, 8H). C NMR (101 MHz, DMSO-d6) δ 170.66, 166.83,
158.80, 148.92, 143.76, 136.99, 134.07, 133.28, 132.17, 131.07, 130.02, 129.25,
129.14, 124.84, 122.29, 122.23, 122.05, 117.96, 117.13, 117.11, 110.00, 108.99,
98.73, 54.37, 53.72, 52.31, 51.16, 49.06. HRMS (ESI) for C32H32N6O3 [M + H]+,
calcd: 549.2609, found: 549.2616.
4.1.3.5.
Methyl
(Z)-3-(((6-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)-5-methylpyridin-3-yl)amino)(p
henyl)methylene)-2-oxoindoline-6-carboxylate (7e). Yellow solid. Yield: 71%. Mp:
1
132.6.2–135.1 °C. H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.04 (s, 1H),
7.82 (s, 1H), 7.55 (m, 7H), 7.22 (d, J = 7.6 Hz, 1H), 6.87 (s, 1H), 6.11 (s, 2H), 5.90
13
(d, J = 8.1 Hz, 1H), 3.78 (s, 3H), 3.27 (s, 2H), 1.95 (br, 9H). C NMR (101 MHz,
DMSO-d6) δ 170.68, 166.80, 158.53, 150.29, 147.82, 141.02, 137.08, 134.89, 134.78,
132.17, 131.09, 130.11, 130.03, 129.17, 129.10, 124.98, 122.69, 122.08, 118.06,
110.03, 108.15, 99.00, 94.32, 54.23, 52.31, 44.40, 17.53. HRMS (ESI) for
C30H29N5O3 [M + H]+, calcd: 508.2343, found: 508.2350.
4.1.3.6.
Methyl
(Z)-3-(((5-methyl-6-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)pyridin-3-yl)a
mino)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7f). Yellow solid. Yield: 46%.
1
Mp: 134.3–137.0 °C. H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.08 (s, 1H),
7.81 (s, 1H), 7.62 (m, 3H), 7.55 (m, 2H), 7.45 (s, 1H), 7.41 (s, 1H), 7.22 (d, J = 8.2
Hz, 1H), 6.83 (s, 1H), 6.06 (s, 2H), 5.87 (d, J = 8.2 Hz, 1H), 3.78 (s, 3H), 3.42 (s,
3H), 3.33 (s, 2H), 2.33 (s, 4H), 2.14 (s, 4H), 1.93 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 170.65, 166.81, 158.54, 148.14, 140.90, 137.08, 134.90, 134.44, 132.23,
131.11, 130.62, 130.45, 130.04, 129.18, 129.04, 124.96, 122.47, 122.07, 118.03,
110.05, 109.88, 107.89, 98.99, 56.48, 54.00, 52.90, 52.31, 19.02. HRMS (ESI) for
C33H34N6O3 [M + H]+, calcd: 563.2765, found: 563.2761.
4.1.3.7.
Methyl
(Z)-3-(((4-(2-((dimethylamino)methyl)-5-methyl-1H-pyrrol-1-yl)phenyl)amino)(phenyl
)methylene)-2-oxoindoline-6-carboxylate (7g). Yellow solid. Yield: 58%. Mp: 216.7–

1
219.3 °C. H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 10.99 (s, 1H), 7.58 (m,
3H), 7.54–7.51 (m, 2H), 7.43 (d, J = 1.0 Hz, 1H), 7.21 (dd, J = 8.3, 1.3 Hz, 1H), 7.12
(d, J = 8.6 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 5.90 (dd, J = 13.7, 4.7 Hz, 2H), 5.82 (d,
J = 2.6 Hz, 1H), 3.78 (s, 3H), 2.95 (s, 2H), 1.92 (br, 9H). HRMS (ESI) for
C31H30N4O3 [M + H]+, calcd: 507.2391, found: 507.2396.
4.1.3.8.
Methyl
(Z)-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amino
)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7h). Yellow solid. Yield: 73%.
1
Mp: 200.2–203.2 °C. H NMR (600 MHz, DMSO-d6) δ 12.25 (s, 1H), 10.98 (s, 1H),
7.60 (dt, J = 2.7, 2.0 Hz,1H), 7.56 (m, 2H), 7.52 (dd, J = 8.0, 1.3 Hz, 2H), 7.43 (d, J =
1.4 Hz, 1H), 7.21 (dd, J = 8.2, 1.6 Hz, 1H), 7.13 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7
Hz, 2H), 5.88 (m, 2H), 5.80–5.78 (m, 1H), 3.78 (s, 3H), 2.94 (s, 2H), 2.18–2.11 (br,
11H), 1.91 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 170.61, 166.78, 158.94,
137.62, 136.65, 135.13, 132.40, 130.83, 129.76, 129.37, 129.28, 129.13, 129.06,
128.94, 124.43, 123.90, 121.88, 117.70, 109.85, 108.83, 106.31, 97.98, 55.13, 53.73,
52.19, 52.17, 46.08, 13.07. HRMS (ESI) for C34H35N5O3 [M + H]+, calcd: 562.2813,
found: 562.2817.
4.1.3.9.
Methyl
(Z)-3-(((4-(2-((diethylamino)methyl)-5-methyl-1H-pyrrol-1-yl)phenyl)amino)(phenyl)
methylene)-2-oxoindoline-6-carboxylate (7i). Yellow solid. Yield: 63%. Mp: 232.0–
1
235.3 °C. H NMR (600 MHz, DMSO-d6) δ 12.25 (s, 1H), 10.99 (s, 1H), 7.62–7.59
(m, 1H), 7.57 (m, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.44 (s, 1H), 7.21 (d, J = 8.2 Hz, 1H),
7.09 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 5.89 (d, J = 3.1 Hz, 1H), 5.86 (d, J
= 8.2 Hz, 1H), 5.79 (d, J = 2.9 Hz, 1H), 3.78 (s, 3H), 3.09 (s, 2H), 2.20 (q, J = 7.0 Hz,
4H), 1.88 (s, 3H), 0.65 (t, J = 7.0 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 170.61,
166.79, 158.92, 137.61, 136.64, 135.37, 132.34, 130.82, 130.14, 129.82, 129.36,
129.18, 128.98, 128.87, 124.42, 123.86, 121.87, 117.69, 109.86, 108.83, 106.22,
98.02, 52.19, 48.80, 45.80, 13.05, 11.77. HRMS (ESI) for C33H34N4O3 [M + H]+,
calcd: 535.2704, found: 535.2711.
4.1.3.10.
Methyl
(Z)-3-(((4-(2-methyl-5-(morpholinomethyl)-1H-pyrrol-1-yl)phenyl)amino)(phenyl)met
hylene)-2-oxoindoline-6-carboxylate (7j). Yellow solid. Yield: 70%. Mp: 241.0–242.7
1
°C. H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.99 (s, 1H), 7.61–7.54 (m,
3H), 7.52 (d, J = 6.9 Hz, 2H), 7.43 (d, J = 1.1 Hz, 1H), 7.21 (dd, J = 8.2, 1.3 Hz, 1H),
7.15 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 5.89 (m, 2H), 5.80 (d, J = 2.9 Hz,
1H), 3.78 (s, 3H), 3.38 (br, 4H), 2.97 (s, 2H), 2.06 (br, 4H), 1.91 (s, 3H). HRMS (ESI)
for C33H32N4O4 [M + H]+, calcd: 549.2496, found: 549.2500.
4.1.3.11.
Methyl
(Z)-3-(((6-(2-((dimethylamino)methyl)-5-methyl-1H-pyrrol-1-yl)pyridin-3-yl)amino)(p
henyl)methylene)-2-oxoindoline-6-carboxylate (7k). Yellow solid. Yield: 63%. Mp:
194.7–197.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 11.05 (s, 1H), 8.17
(d, J = 2.3 Hz, 1H), 7.63–7.53 (m, 5H), 7.47–7.42 (m, 2H), 7.38 (d, J = 8.6 Hz, 1H),
7.23 (dd, J = 8.2, 1.3 Hz, 1H), 6.16 (s, 1H), 5.93 (m, 2H), 3.78 (s, 3H), 3.54 (s, 2H),
2.21 (s, 6H), 2.04 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.67, 166.81, 158.49,

144.28, 137.14, 134.61, 133.57, 132.08, 131.14, 130.58, 130.56, 130.04, 129.27,
129.22, 129.16, 125.04, 122.08, 121.66, 118.11, 110.06, 108.65, 108.07, 99.15, 56.49,
52.32, 43.39, 13.45. HRMS (ESI) for C30H29N5O3 [M + H]+, calcd: 508.2343, found:
508.2341.
4.1.3.12.
Methyl
(Z)-3-(((6-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)pyridin-3-yl)a
mino)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7l). Yellow solid. Yield: 57%.
1
Mp: 217.5–220.1 °C. H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 11.05 (s, 1H),
8.15 (d, J = 2.3 Hz, 1H), 7.64–7.52 (m, 5H), 7.45 (s, 1H), 7.41–7.30 (m, 2H), 7.23 (d,
J = 8.2, 1H), 5.91 (d, J = 8.3 Hz, 2H), 5.81 (d, J = 2.5 Hz, 1H), 3.78 (s, 3H), 3.20 (s,
13
2H), 2.49–2.03 (br, 11H), 1.97 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 170.67,
166.82, 158.66, 148.04, 144.04, 137.08, 134.40, 133.17, 132.15, 131.08, 129.97,
129.73, 129.18, 128.77, 124.95, 122.13, 122.06, 118.04, 110.03, 109.79, 106.95,
99.99, 98.99, 54.33, 53.50, 52.31, 50.89, 44.81, 13.18. HRMS (ESI) for C33H34N6O3
[M + H]+, calcd: 563.2765, found: 563.2768.
4.1.4.
(Z)-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amino
)(phenyl)methylene)-2-oxoindoline-6-carboxylic acid (8).
To a solution of compound 7h (0.3 g, 0.53 mmol) in methanol (10 mL) was
added 1N NaOH solution (10 mL). The mixture was reflux for 0.5 h until TLC
showed the completion of the reaction. The methanol was evaporated, the solution
was acidized with 1N HCl solution to pH 6–7. The precipitate was filtered off to give
a crude product, which was washed with methanol to give compound 8 (0.15 g,
51.7%) as a yellow solid. Mp: 279.1–283.2 °C. ESI-MS m/z: 548.3 [M+H]+. 1H NMR
(400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.96 (s, 1H), 7.63–7.57 (m, 3H), 7.56–7.50
(m, 3H), 7.43 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 8.5 Hz, 2H), 6.95 (d, J =
8.4 Hz, 2H), 5.92 (d, J = 2.2 Hz, 1H), 5.88 (d, J = 8.3 Hz, 1H), 5.81 (d, J = 2.3 Hz,
1H), 3.00 (s, 2H), 2.28 (br, 11H), 1.91 (s, 3H). HRMS (ESI) for C33H33N5O3 [M +
H]+, calcd: 548.2656, found: 548.2651.
4.1.5. General procedure for preparation of compounds 9 and 10.
A mixture of compound 8 (0.6 mmol), TBTU (0.72 mmol), HOBT (0.72 mmol),
DIPEA, and appropriate amine (0.9 mmol) in DMF was stirred for 5 h at room
temperature. The mixture was poured into water, and stirred for 0.5 h. The precipitate
was filtered off to give a crude product, which was purified by column
chromatography (CH2Cl2/MeOH, 100/1 to 10/1) to give the desired compound.
4.1.5.1.
(Z)-N-methyl-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phe
nyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (9). Yellow solid. Yield:
46%. Mp: 230.7–234.0 °C. 1H NMR (600 MHz, DMSO-d6) δ 12.13 (s, 1H), 10.95 (s,
1H), 8.19 (m, J = 4.4 Hz, 1H), 7.62–7.55 (m, 3H), 7.54–7.50 (m, 2H), 7.36 (s, 1H),
7.12 (d, J = 8.7 Hz, 2H), 7.07 (s, 1H), 6.92 (d, J = 8.7 Hz, 2H), 5.88 (d, J = 3.2 Hz,
1H), 5.80 (t, J = 6.0 Hz, 2H), 2.95 (s, 2H), 2.72 (d, J = 4.5 Hz, 3H), 2.11 (br, 11H),
1.91 (s, 3H). HRMS (ESI) for C34H36N6O2 [M + H]+, calcd: 561.2973, found:
561.2976.

4.1.5.2.
(Z)-N,N-dimethyl-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl
)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (10). Yellow solid.
1
Yield: 46%. Mp: 221.4–223.6 °C. H NMR (600 MHz, DMSO-d6) δ 12.12 (s, 1H),
10.87 (s, 1H), 7.60–7.55 (m, 3H), 7.52 (d, J = 6.8 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H),
6.91 (s, 1H), 6.89 (d, J = 4.3 Hz, 2H), 6.63 (dd, J = 8.0, 1.3 Hz, 1H), 5.88 (d, J = 3.2
Hz, 1H), 5.84 (d, J = 8.0 Hz, 1H), 5.79 (d, J = 2.8 Hz, 1H), 2.95 (s, 2H), 2.90 (s, 6H),
2.14 (br, 11H), 1.91 (s, 3H). HRMS (ESI) for C35H38N6O2 [M + H]+, calcd: 575.3129,
found: 575.3122.
4.2. Pharmacology
4.2.1. Enzyme activity assay
The activities of compounds in inhibition of selected kinases were tested by
Shanghai ChemPartner Co., Ltd using a well-established mobility shift assay. Details
of the experiment have been described in reference.17,18
4.2.2. Western blotting analysis
HT-29 and HepG2 cells were cultured in 6-well plate over night and treated with
7h for 24h. About 1×106 cells were collected for immuno blotting analysis. The
primary antibody p-ERK, ERK, p-AKT, AKT (all from Cell Signaling Technology),
GAPDH (HangZhou GoodHere Technology), and secondary antibody were used. The
bands were visualized with chemiluminescence (ECL or Dura; Thermo Scientific).
Representative blots are shown from several experiments. Exposure analysis of
immunoblot strip was conducted by Gel imaging instrument (ChemiDoc XRS+,
Bio-Rad).
4.2.3. Colony formation assay
HT-29, MKN74, and HepG2 cancer cells were treated with 7h or nintedanib for
14 days. Details of the experiment have been described in reference.22
4.2.4. Flow cytometric assay
HUVECs were treated with different concentrations of compound 7h for 48
hours, while HT-29, MKN74, and HepG2 cells were treated for 72 hours,
respectively. Details of the experiment have been described in reference.22
4.2.5. In vitro metabolic stability assay
In vitro metabolic stability assay of compound 7h being incubated with human
and mouse liver microsomes was performed by Shanghai ChemPartner Co., Ltd.
More experiment details could be seen http://www.shangpharma.com/dmpk-2/.
4.2.6. Mouse tumor xenograft efficacy study
Female nu/nu mice (4–6 weeks old) were supplied by the Experimental Animal
Centre of Shenyang Pharmaceutical University and housed on particulate air-filtered
ventilated racks. All procedures and treatments were conducted in accordance with
the ethical regulations set by the Animal Experimentation Committee of Shenyang
Pharmaceutical University.
Tumor cells at a density of 2×106/mL were implanted subcutaneously into the
right flank of each nude mouse. When the tumor volume reached 200 mm3, the mice
were randomly assigned to control and treatment groups (n = 6 per group). The
treatment groups were treated with 7h or nintedanib via oral administration once daily.

The average tumor diameter was measured with vernier calipers twice per week. The
tumor volume (TV) was calculated as follows: TV = [length (mm) × width2 (mm2)]/2.
0
RTV (relative tumor volume) = TVDay N/TVDay ×100%. TGI (Tumor Growth
Inhibition value) = [1-RTV(treatment)/RTV(vehicle)] × 100%
4.3. Molecular docking
We used the co-crystal structure of VEGFR-2 kinase (PDB ID: 3C7Q) for the
preparation of molecular docking. Water molecules and original ligand were manually
removed before molecular docking. All docking calculations were performed using
Autodock 4.2. For the protein, the search space was defined as a cube 30 Å on each
side, centered on the protein (X, Y, Z coordinates: 21.331, 64.474, 30.745). All the
other parameters used in the docking possess were set to default values. The
molecular graphic manipulations and visualizations were performed using Discovery
Studio Visualizer 4.0. Finally, the model with the lowest energy was used for further
analysis.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
This work was supported by China Postdoctoral Science Foundation
(2018M633720) and Liaoning Revitalization Talents Program (XLYC1808037).
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