Synthesis and evaluation of novel 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives as potential multi-target tyrosine kinase receptor inhibitors

Article abstract of DOI:10.3390/molecules22060913

Signaling pathways of VEGFs and PDGFs are crucial in tumor angiogenesis, which is essential in solid tumor progression and metastasis. This study reports our strategy for designing and synthesizing a series of novel 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives as potential multi-target tyrosine kinase receptor inhibitors. The target compounds were obtained by condensation of 5-substituted oxindoles with N-substituted 2-pyrrolidone aldehyde 7 in satisfactory yields. Of these, 11 and 12 had the highest potency and, compared to sunitinib, showed: (1) significant increase in anti-proliferation of various cancer cells with a favorable selective index (SI); (2) higher inhibitory potency against both VEGFR-2 and PDGFRβ. The molecular modeling results showed that, in terms of VEGFR-2 binding, the synthesized products had a similar binding mode to sunitinib but with tighter interaction.

Full text of DOI:10.3390/molecules22060913

molecules
Article
Synthesis and Evaluation of Novel 2-Pyrrolidone-Fused
2-Oxoindolin-3-ylidene)methylpyrrole Derivatives
(
as Potential Multi-Target Tyrosine Kinase
Receptor Inhibitors
Ting-Hsuan Yang , Chun-I Lee , Wen-Hsin Huang and An-Rong Lee 1,2,
1
2
2
*
1
Graduate Institute of Medical Sciences, National Defense Medical Center, No. 161, Section 6,
Mingchuan East Road, Taipei 11490, Taiwan; taffw@gmail.com
School of Pharmacy, National Defense Medical Center, No. 161, Section 6, Mingchuan East Road,
Taipei 11490, Taiwan; acirlee@gmail.com (C.-I.L.); wenhsin@ndmctsgh.edu.tw (W.-H.H.)
Correspondence: 416806@gmail.com; Tel.: +886-2-8792-3100 (ext. 18873)
2
*
Academic Editor: Pierre Koch
Received: 29 March 2017; Accepted: 29 May 2017; Published: 31 May 2017
Abstract: Signaling pathways of VEGFs and PDGFs are crucial in tumor angiogenesis, which is
essential in solid tumor progression and metastasis. This study reports our strategy for designing and
synthesizing a series of novel 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives
as potential multi-target tyrosine kinase receptor inhibitors. The target compounds were obtained by
condensation of 5-substituted oxindoles with N-substituted 2-pyrrolidone aldehyde
7 in satisfactory
yields. Of these, 11 and 12 had the highest potency and, compared to sunitinib, showed: (1) significant
increase in anti-proliferation of various cancer cells with a favorable selective index (SI); (2) higher
inhibitory potency against both VEGFR-2 and PDGFRβ. The molecular modeling results showed
that, in terms of VEGFR-2 binding, the synthesized products had a similar binding mode to sunitinib
but with tighter interaction.
Keywords: multi-target kinase inhibitor; VEGFR-2 inhibitor; PDGFR
2-oxoindolin-3-ylidene)methylpyrrole
β inhibitor angiogenesis;
(
1
. Introduction
Angiogenesis, a process of new blood vessel formation from pre-existing vessels, is pivotal
in both physiological, such as wound healing, reproduction and embryonic development, and
pathophysiological, such as rheumatoid arthritis, atherosclerosis and cancer, conditions [ ]. Of the six
distinguishing characteristics of cancer, tumor angiogenesis is considered among the most important
hallmarks of cancer because it exacerbates malignant cancer growth and metastatic dissemination [ ].
1
2
To excrete tumor metabolic waste and supply oxygen and nutrients for tumor growth, new blood
3
vessels are essentially formed if the tumor size is beyond 2 mm . To avoid apoptosis, necrosis and/or
provide paths for metastasis to survive, tumor cells secrete several pro-angiogenesis factors inducing
new blood vessel formation near tumor tissues [
factors (VEGF), platelet-derived growth factors (PDGF), and epidermal growth factors (EGF) [
3
]. These factors include vascular endothelial growth
].
3
Restricting new blood vessel formation around tumor tissues may also reduce tumor size and/or
avoid metastasis and thus bring beneficial effects of targeted therapy.
Many factors, including VEGFs, PDGFs and their receptor tyrosine kinases (RTKs), VEGF
receptors 1–3 (VEGFR1–3), and PDGF receptor-
angiogenesis signaling pathways in the tumor microenviroment [
key receptors in tumor-induced angiogenic signal transduction, promote endothelial cell proliferation,
α
and -
β
(PDGFR-
α
/β), participate in tumor-induced
4]. Among them, VEGFR-1 and -2,
Molecules 2017, 22, 913; doi:10.3390/molecules22060913
www.mdpi.com/journal/molecules

Molecules 2017, 22, 913
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migration, and survival [
3
–5
]. Moreover, PDGFs and PDGFRs support tumor-induced angiogenesis by
stimulating proliferation of both fibroblasts and vascular smooth muscle cells, both of which stabilize
newly formed blood vessels [ ]. Thus, targeting both VEGFs and PDGFs signaling is considered
5
a promising approach to developing anti-angiogenesis drugs.
In 1971, Folkman proposed that anti-angiogenesis might be an effective strategy for cancer
treatment [
as dasatinib, sorafenib, semaxanib (SU5416), orantinib (SU6668), and sunitinib (Figure 1), have
been developed and some of them have been approved [ ]. Semaxanib was the first of these
to be clinically tested as a receptor tyrosine kinase (RTK) inhibitor for VEGFRs [ ]. The lipophilic
2-oxoindolin-3-ylidene)methylpyrrole moiety in semaxanib was then identified as a prerequisite for
3]. Sice then, various potent small-molecule ATP-competitive VEGFR inhibitors, such
6–9
9
(
the inhibitory activity against ATP-competitive RTKs. Further structural modification of semaxanib
successfully introduced a substituted pyrrole indolinone derivative sunitinib as a very potent and orally
effective anti-angiogenesis agent inhibiting cellular signaling by targeting multiple RTKs, including
VEGFRs, PDGFRs, aurora kinases (A, B and C), RET, Flt-3, CSF-1R, and c-Kit [10–13]. The USFDA
approved the use of sunitinib for treating advanced renal cell carcinoma (RCC), gastrointestinal stromal
tumors (GISTs), and imatinib-intolerant and -refractory GISTs. Sunitinib was also the first cancer drug
simultaneously approved for different indications because of its inhibiting effects in signal pathways
that have important roles in both tumor angiogenesis and tumor cell proliferation [14]. Afterwards,
multitudinous (2-oxoindolin-3-ylidene)methylpyrrole derivatives have been designed and synthesized
as potent small-molecule RTK inhibitors [15–19].
Figure 1. The structures of some multi-targeted kinase inhibitors. Some inhibitors, such as semaxanib,
orantinib, and sunitinib, share a common structure of (2-oxindolin-3-ylidene)methylpyrrole nucleus.
Molecular modeling has indicated that the oxindole moiety of sunitinib, crucial for binding to
the ATP binding site of RTKs, occupies the adenine pocket and supplies two hydrogen bonds for
binding VEGFR2 (Glu917 and Cys919) [20,21]. Additionally, both the pyrrole functionality and the
C(5)-F element of (2-oxoindolin-3-ylidene)methylpyrrole in sunitinib structure enhance affinity by
inducing further hydrophobic interactions [20]. In contrast, the water-soluble diethylaminoethyl
tail of sunitinib lies on the protein surface [19,22,23]. The RTK inhibitory activity and selectivity of
(2-oxoindolin-3-ylidene)methylpyrrole derivatives revealed a strong relationships with structural
modifications. Jun changed the methylpyrrole part of sunitinib into a bicyclic N-substituted
pyrrolo-fused six-, seven-, and eight-membered heterocycles to obtain a series of more rigid
analogs. They discovered certain products as potent inhibitors of VEGFR-2, PDGFR
β and c-kit
both enzymatically and cellularly [19]. This study reports the design, synthesis, and structure-activity

Molecules 2017, 22, 913
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relationships (SARs) of a series of novel (2-oxoindolin-3-ylidene)methylpyrrole derivatives, bearing at
C(5) of oxindole various halogens, fused with an N-substituted 2-pyrrolidone which might result in
a smaller, flatter, and more rigid conformation (Figure 2), and thus restrict the ability of the hydrophilic
diethylaminoethyl tail to rotate in the hope of facilitating the binding affinity. The target molecules were
prepared by condensation of 5-substituted oxindoles with N-substituted 2-pyrrolidone aldehyde
Several of the resulting compounds proved to be potent small-molecule kinase inhibitors. Among them,
and 12 consistently displayed the most potent activities and favorable selective indexes (SI) and
7.
1
1
thus were chosen as the candidates for further preclinical development.
Figure 2. Drug design, and retrosynthetic approach of the target compounds.
2
. Results and Discussion
2
.1. Chemistry
Scheme 1 outlines the synthetic sequence that our laboratory used to prepare targets 8–12.
The starting material 2-tert-butyl 4-ethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate (
via the Paal-Knorr pyrrole synthesis [24]. Selective oxidation on -methyl of with ceric ammonium
nitrate (CAN) at room temperature for 1 h quantitatively formed , which bore an -formyl functionality
at the pyrrole ring. Compound was then coupled with N,N-diethylethylenediamine, and then,
without isolation, reduced directly with activated sodium borohydride/p-toluenesulfonic acid in
1) was synthesized
α
1
2
α
2
ethanol to obtain
Compound was transformed into
Hydrolytic decarboxylation of
was used directly for selective formylation at C(5) to yield
a Vilsmeier rfeagent. Condensation of with 5-substitued oxindoles in the presence of piperidine at
room temperature afforded target molecules.
The key intermediate was initially synthesized using methods described in the literature [25].
First, reductive amination was performed to transform the carbonyl group directly to its
corresponding amine derivative by treating aldehyde with N,N-diethylethylenediamine followed
by p-toluenesulfonic acid monohydrate-activated sodium borohydride under solvent-free conditions;
however, compound was obtained in only 43% yield, along with a string of unidentifiable
impurities. In our modified experiments, ethanol was used as the solvent for reductive amination
of . The resulting yield substantially increased to 90%. We hypothesized that the use of ethanol in
3
in a satisfactory yield [25]. Ester
3
was hydrolyzed in methanol into carboxylic acid
4.
0
4
5
by lactamization promoted by 1,1 -carbonyldiimidazole (CDI).
5
with sulfuric acid in methanol could provide
6. Without purification,
6
7, achieved smoothly by treatment with
7
3
3
2
3
2
this reaction vastly improved the yield of 3 because the protic polar solvent ethanol might effectively
be involved in the formation of an appropriate transition state required for the subsequent reductive
amination and thus greatly facilitated the reaction. The unreacted
were then easily removed by column chromatography.
2 and all the impurities generated

Molecules 2017, 22, 913
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Scheme 1. Synthesis of N-substituted 2-pyrrolidone fused (2-oxoindolin-3-ylidene)methylpyrrole
derivatives. Reagents and conditions: (a) CAN, THF, AcOH, H O, 1 h, 98%; (b) N,N-diethylethylenediamine,
2
◦
EtOH, r.t., 4 h; (c) NaBH , TsOH H O, 6 h, 90%; (d) 1N NaOH , MeOH, 65 C, 6 h, 96%; (e) CDI, dry
4
2
(aq)
THF, 6 h, 56%; (f) H SO , MeOH, reflux, 4.5 h; (g) Vilsmeier reagent, DCM, r.t., 5 h, 91%; (h) piperidine,
2
4
indoline-2-ones, EtOH, r.t., 5 h, 43–56%.
In the synthesis of the first bicyclic compound
5, Jun revealed that an elegant direct
lactamization that formed lactams with medium size (six-, seven-, and eight-membered ring)
could be used either by treating with lithium hydroxide or with trimethylaluminium, followed
I
by treating with 2 M hydrochloric acid if decarboxylation products were desired, to give
pyrrolo-fused-heterocycles-2-indolinones II in the yield of 27–97% (Scheme 2) [19]. Their experimental
results showed that lactamization yields correlated with the ring size of the products. The marked
success of Jun in synthesizing medium-sized lactams with simultaneous decarboxylation prompted
us to prepare the smaller but desired lactam
our synthesis of would have been achieved in a single step starting from
attempt lactamization of 3, based on this strategy, to obtain desired 5 under various conditions failed
and culminated in producing the hydrolyzed product . Reactions at higher temperatures only gave
trace amount of the product. Variation in solvents and use of microwave irradiation usually obtained
6. By applying this seemingly promising approach,
6
3
. Unfortunately, all our
4
yields approximating 10%. Even the modified method using DABAL-Me or AlMe /DABAL-Me or
3
3
3
all other similar approaches under various conditions proved to be fruitless [25–29]. The likely culprit
is the significant difference in the ring strain between
6 and II (n = 1, 2, 3). These experimental results
clearly indicate that the Jun’s method of lactamization excludes the preparation of N-substituted
2
-pyrrolidone derivatives to be used in our synthesis.
Scheme 2. Synthesis of pyrrolo-fused-heterocycles-2-indolinones II [19]. Reagents and conditions:
(
a) LiOH/ethylene glycol; (b) AlMe , toluene, then HCl.
3

Molecules 2017, 22, 913
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Our laboratory finally found a way to circumvent the predicament in a stepwise manner. First,
ester was hydrolyzed into carboxylic acid with 1 N sodium hydroxide in 96% yield. Performing
CDI-mediated lactamization of the resulting zwitterionic compound in dry tetrahydrofuran under
a nitrogen atmosphere proved effective for synthesizing the desired fused pyrrole in 56% yield.
Compound was then subjected to hydrolytic decarboxylation with sulfuric acid under a reflux
condition to provide . Without isolation and purification, crude was smoothly formylated at C(2) by
treatment with a Vilsmeier reagent in dichloromethane to furnish in 91% yield. Condensation of
with 5-substitued oxindoles afforded target molecules 8–12 in 43–56% yields.
3
4
4
5
5
6
6
7
7
All target compounds were isolated as free bases. In most cases, crystallization or column
chromatography was required for purification. The structures of most synthetic intermediates and
products were established by spectroscopy and high-resolution mass spectrometry.
2
.2. Anti-Proliferation Activity and Acute Cytotoxicity
Table 1 shows the in vitro anti-proliferation activity of the synthetic products 8–12 against human
colon cancer line HCT116, human non-small cell lung cancer cells NCI-H460, and human renal cell
carcinoma 786-O. For comparison, sunitinib was employed as a positive control. The preliminary
results showed that all synthetized compounds had significant inhibiting effects on HCT-116 cancer
cell proliferation, but for
were approximately equal to that of sunitinib (IC50 3.42
IC50 1.05 ± 0.18 M) and 12 (C(5)-I) (IC50 0.42 0.16 M) were much better. As to the anti-proliferation
effect on the NCI-H460 cells, 10 showed poor inhibitory activity (IC50 > 10 M); 11 was approximately
equal in potency to sunitinib (IC50 = 6.57 and 6.23 M, respectively). Notably, 12 revealed prominent
inhibitory activity (IC50 = 2.95 M). As to the anti-proliferation effect on the 786-O cells, both
and were once again void of activity (IC50 > 10 M). The potencies of compounds 10 12
IC50 approximately 7 µM) were comparable to that of sunitinib.
8
which bears C(5)-H. While the IC50 values of
9 (C(5)-F) and 10 (C(5)-Cl)
±
0.57 M), the values for 11 (C(5)-Br)
µ
(
±
µ
8–
µ
µ
µ
8
9
µ
–
(
Table 1. Cellular activities and selective indexes of 8–12 and sunitinib.
IC50 (µM)/Selective Index (SI)
Compd.
R
HCT116
NCI-H460
786-O
Detroit 551
3
2
.42 ± 0.57
6.23 ± 0.57
6.27 ± 0.67
-
9.48 ± 0.18
Sunitinib
.77
1.52
1.51
4
>
.25 ± 1.88
>10
nd
>10
nd
8
9
-H
-F
>10
2.35
2
>
.94 ± 0.66
3.4
>10
nd
>10
nd
>10
3
>
.09 ± 0.70
3.23
>10
nd
7.30 ± 1.20
1
1
1
0
1
2
-Cl
-Br
-I
>10
>1.37
1
>
.05 ± 0.18
9.52
6.57 ± 1.64
7.06 ± 0.30
>10
>1.52
>1.42
0
2
.42 ± 0.16
2.95 ± 0.83
7.76 ± 0.29
9.21 ± 1.61
1.93
3.12
1.19
nd: not detected.

Molecules 2017, 22, 913
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Potential anticancer drug candidates should have greater selectivity for tumor cells compared
with normal cells. Therefore, human normal fibroblast cells Detroit 551 were used for evaluation so as
to indicate the selective indexes of 8–12 and sunitinib. In Table 1, our synthetic compounds generally
showed much less toxicity to Detroit 551 cells than sunitinib. Even potent 12 had high selectivity
against HCT116 and NCI-H460 cancer cell lines.
To exclude the possibility that the inhibitory responses of sunitinib and compounds 8–12 were
resulted from the acute cytotoxicity of indicated compounds, this study performed a WST-8 assay to
assess their effects of acute cellular toxicity on Detroit 551, HCT116, NCI-H460 and 786-O cell lines.
Figure 3 shows the experimental results. The results clearly revealed that all the synthetic products
8
–12, together with sunitinib, were nearly free from acute cellular toxicity on all the tested cell lines.
Figure 3. Acute cytotoxicity assay of (
a) Detroit 551; (b) HCT116; (c) NCI-H460; (d) 786-O cells
incubated with DMSO (1%), sunitinib and 8–12 (10 µM).
In the (2-oxoindolin-3-ylidene)methylpyrrole system, the C(5) halogen substituent is known
to have critical roles in the cytotoxicity and anti-proliferation of cancer cells [16 18 19 30 31].
,
, , ,
Structural modification of sunitinib by replacing the fluorine atom on C(5) with an iodine led
to approximately 23-fold weaker potency against HT29 cells [30]. In our 2-pyrrolidone-fused
(
2-oxoindolin-3-ylidene)methylpyrrole system, compound
or slightly better potency in the anti-proliferation activity against HCT116 to that of sunitinib
IC50 2.94 M vs. 3.42 M). The corresponding 10 (C(5)-Cl), 11, (C(5)-Br) and 12 (C(5)-I) showed
9 (C(5)-F) had approximately equal
(
µ
µ
a similar trend. For example, compound 12 was strikingly potent, with approximately 8-fold stronger
than sunitinib and 10-fold stronger than 8 (C(5)-H).
Apoptosis of human colon cancer cells, including HCT116 and HT29 cells, can be induced by
VEGFR-2 inhibitors, such as regorafenib, crizotinib, sorafenib, and sunitinib, via p53 upregulated
modulator of apoptosis (PUMA) [32
transcriptionally activated via the NF-
–
35]. Recently, Zhang demonstrated that PUMA could be
κ
B pathway after aurora kinase inhibition in HCT116 cells [36].
Sunitinib has well recognized inhibitory activity against aurora kinase, and the six-membered
cycloalkanone fused (2-oxoindolin-3-ylidene)methylpyrroles exhibit further improvement in the
inhibition of aurora kinase [18]. We hypothesized that our synthetic derivatives might have a profound
effect on the binding affinity of aurora kinase. Therefore, an aurora A kinase inhibition percentage at

Molecules 2017, 22, 913
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1.0 µM of sunitinib and the synthetic compounds were thus assessed to validate our hypothesis. Table 1
shows the experimental results, which indicated that all our newly synthetic products showed excellent
but without noticeably differentiating inhibitory activity against aurora A (92.1–95.9%), comparing
to sunitinib (50.7%). Thus, these results suggested that, in binding to and inhibition of aurora A,
compared to the conformation of the bicycle pyrrole scaffold, the C(5) substitution effect might play
a less significant role.
Inhibition of aurora kinase leads to a polyploidy cell cycle profile [18
HCT116 cells incubated with compounds and 11 for 24 h exhibited an increase phase of polyploid
Figure 4c,f). Surprisingly, the cell cycle profile of HCT116 cells incubated with sunitinib, 10, and 12
for 24 h caused G0/G1 cell cycle arrest (Figure 4b,d,e,g). The best explanation is that compounds and
inhibited HCT116 cells proliferation via significant aurora kinase inhibition while sunitinib, 11
and 12 might be otherwise involved in different mechanisms.
,37]. The cell cycle profile of
8
(
9,
8
1
1
9,
In summary, the experiments in this study suggest that the structural skeleton constructed by
fusion of (2-oxoindolin-3-ylidene)methylpyrrole with N-substituted 2-pyrrolidone and introduction
of a proper halogen at C(5) of oxindole has a strong influence on both anti-proliferation activity and
selectivity. The C(5)-halogen substituents of the title compounds seemed to be advantageous in general
and had marked effect: the bulkier halogen the substituent is, the stronger anti-proliferation activity
it has.
Figure 4. Cell cycle profiles of HCT-116 cells treated with (
sunitinib; ( ) 5.0 M of ; ( ) 5.0 M of ; ( ) 5.0 M of 10; ( ) 1.0
M1: G0/G1phase, M2: S phase, M3: G2/M phase.
a
) 1% DMSO (control); (
b) 5.0 µM of
c
µ
8
d
µ
9
e
µ
f
µ
M of 11; ( ) 1.0 M of 12 for 24 h.
g
µ

Molecules 2017, 22, 913
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2
.3. Kinase Inhibitory Assays
Table 2 shows the results of kinase inhibition assays for compounds 8-12, compared to sunitinib.
The IC50 values for 10 11, and, 12 against VEGFR-2 were 4- to 6-fold higher, and their IC50 values
against PDGFR were approximately 2- to 4-fold higher and 11 was the most potent. Comparing
8,
,
β
with sunitinib, The results once again displayed the similar trend that different C(5) substitutions had
significant influence on the biochemical activities against VEGFR-2 and PDGFRβ.
Unexpectedly, comparisons with C(5)-H (
showed that compound (C(5)-F) had much lower inhibitory activity against both VEGFR-2 and
PDGFR and, comparing to sunitinib, only little increase in inhibitory activity against VEGFR-2 but
decrease against PDGFR . These results markedly differed from the results reported in experiments
using 2-piperidinone fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives [19]. The influence
of the difference in ATP binding sites between VEGFR-2 and PDGFR is apparent and complex yet
8) and other C(5)-halogen analogs (10, 11, and 12)
9
β
β
β
nevertheless not very clear. A possible explanation for the increased inhibitory activity of VEGFR-2
is a better accommodation to (2-oxoindolin-3-ylidene)methylpyrroles with C(5)-halides, except for
C(5)-F.
Table 2. Enzymatic activities and LibDock score of 8–12 and sunitinib.
%
Inhibition at 50 nM
IC50 (nM)
% Inhibition of
Aurora A at 1.0 µM
LibDock Score of
VEGFR-2 Binding
Compd.
R
VEGFR-2
PDGFRβ
VEGFR-2
PDGFRβ
Sunitinib
-
-H
-F
-Cl
-Br
-I
29
56
35
58
64
58
44
64
36
64
68
62
140.0
30.2
110.7
35.0
24.7
35.1
61.4
22.4
97.5
24.9
16.1
29.3
50.7
95.9
93.1
93.7
93.1
92.9
110.303
134.754
137.901
130.921
129.433
125.031
8
9
10
11
12
2
.4. In Vitro Tube Formation Assay
The in vitro inhibitory activity against tube formation of compounds
angiogenesis kit (Kurabo). Figure 5 shows that they markedly inhibited the VEGF-induced tube
formation at 0.50 M, 0.25 M, and 0.10 M. Enhancement of the inhibitory activity against tube
formation with C(5)-halogen substitutions on oxindole followed the order Br > Cl > I > F (Table 3).
Compound (C(5)-F) was slightly more potent than sunitinib (p < 0.05) and slightly less potent
than , C(5) free from halogen substitution; Compound 11 (C(5)-Br) proved to be the most potent with
IC50 approximately 2-fold stronger than sunitinib (IC50 179 29 nM vs., 387 16 nM). The results
8–12 was tested with an
µ
µ
µ
9
8
±
±
were consistent with those for VEGFR-2 kinase inhibitory assays, which suggest that our synthetic
compounds inhibit tube formation via inhibiting VEGFR-2 pathway. Therefore, we conclude that
compound 11 is a good example of how sunitinib was significantly improved both in inhibiting
biochemical activity (VEGFR-2 and PDGFRβ) and in in vitro tube formation via our drug design.

Molecules 2017, 22, 913
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(
a)
(b)
(e)
(h)
(k)
(c)
(f)
(i)
(l)
(o)
(
d)
(
g)
(
j)
(
m)
(n)
Figure 5. Cont.

Molecules 2017, 22, 913
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(
p)
(q)
(r)
(
s)
(t)
Figure 5. Compounds
tube formation ( ) solvent control; (
and 0.50 M of sunitinib; ( ) VEGF 10 ng/mL and 0.25
.10 M of sunitinib; ( ) VEGF 10 ng/mL and 0.50 M of
) VEGF 10 ng/mL and 0.10 M of ; ( ) VEGF 10 ng/mL and 0.50
and 0.25 M of ; ( ) VEGF 10 ng/mL and 0.10 M of ; ( ) VEGF 10 ng/mL and 0.50
) VEGF 10 ng/mL and 0.25 M of 10; ( ) VEGF 10 ng/mL and 0.10 M of 10; ( ) VEGF 10 ng/mL
and 0.50 M of 11; ( ) VEGF 10 ng/mL and 0.25 M of 11; ( ) VEGF 10 ng/mL and 0.10 M of 11
) VEGF 10 ng/mL and 0.50 M of 12; ( ) VEGF 10 ng/mL and 0.25 M of 12; ( )VEGF 10 ng/mL and
.10 µM of 12. Scale bar = 100 µm.
8
–
12 inhibited tube formation induced by VEGF. Reprehensive photographs of
) VEGFR 10 ng/mL and 70 M of suramin; ( ) VEGF 10 ng/mL
M of sunitinib; ( ) VEGF 10 ng/mL and
; ( ) VEGF 10 ng/mL and 0.25 M of
M of ; ( ) VEGF 10 ng/mL
M of 10;
a
b
µ
c
µ
d
µ
e
0
µ
f
µ
8
g
µ
8;
(
h
µ
8
i
µ
9 j
µ
9
k
µ
9
l
µ
(
m
µ
n
µ
o
µ
p
µ
q
µ
;
(
r
µ
s
µ
t
0
Table 3. IC50 of in vitro VEGF-induced tube formation by 8–12 and sunitinib.
Compd.
R
IC50 of Tube Area (nM)
Sunitinib
-
-H
-F
-Cl
-Br
-I
387 ± 16
310 ± 15
367 ± 16
299 ± 70
179 ± 29
313 ± 37
8
9
1
1
1
0
1
2
2
.5. Molecular Modeling
For a clear understanding of the interaction between the title compounds 8–12 together with
sunitinib and the ATP-binding site of VEGFR-2 at the molecular level, a molecular modeling was
constructed. Figure 6 shows how the test ligands were docked into the ATP-binding site of VEGFR-2

Molecules 2017, 22, 913
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(
PDB ID: 4AGD) using Discovery studio LibDock [38]. LibDock is a method that places the generated
ligand conformations into the protein active site based on polar and apolar interaction sites (hotspot),
and the LibDock score was scored by default settings including Ligscore, piecewise linear potential
(PLP), Jain, potential of mean force (PMF) and PMF04. The results showed the predicted binding
mode of all our synthetic compounds as well as sunitinib. All our synthetic products and sunitinib
formed two hydrogen bonds (important for the binding of (2-oxoindolin-3-ylidene)methylpyrrole
derivatives and ATP-binding site) to VEGFR-2, with Glu917 and Cys919, respectively (Figure 6a–f).
The pyrrole NH of
interaction also occurred between the pyrrole moieties of
VEGFR-2 (Figure 6b–f). Furthermore, the diethylamine tails in
the solvent without significant interaction with VEGFR-2. The interaction with VEGFR-2, compounds
12, as a whole, showed a similar binding mode to sunitinib but with higher LibDock score (Table 2).
8
–
12 formed a hydrogen bond with C=O of Cys919 in VEGFR-2. A hydrophobic
12 and the phenyl group of Phe918 in
12 and sunitinib were exposed to
8
–
8–
8
–
Although the LibDock score of
VEGFR-2, the LibDock score of
8
8
–
12 did not show consistent order with the results of
8–12 against
–12 were much higher than that of sunitinib (Table 2). However,
the interaction was tighter, which might explain why most of our synthetic products exhibited potent
activities in the biochemical assay.
Figure 6. Docked binding modes of
crystal structure of VEGFR-2 in complex with sunitinib. (
in VEGFR-2; ( in VEGFR-2; ( 10 in VEGFR-2; (
8
–
12 in ATP binding site of VEGFR-2 (PDB ID: 4AGD) and the
) sunitinib (from PDB ID: 4AGD) in VEGFR-2;
11 in VEGFR-2; ( 12 in VEGFR-2. Ligands
a
e
(
b
)
8
c)
9
d
)
)
f)
are shown in yellow sticks; hydrogen bonds are shown as dashed black lines; pi-pi interaction is shown
in red line.
3
. Materials and Methods
3
.1. Chemistry
All the chemicals were purchased from Aldrich-Sigma Chemical Company (St. Louis, MO, USA),
and Alfa-Aesar Chemical Company (Lancashire, Heysham, England) and used without further

Molecules 2017, 22, 913
12 of 19
purification. All reactions were routinely monitored by TLC on Merck F254 silica gel plates. Silica
1
13
gel (70–230 mesh, Silicacycle) was used for chromatography. The H- and C-NMR spectra were
determined on an Agilent Varian-400 NMR (Agilent Technologies, Santa Clara, CA, USA) instrument
in CDCl , methanol-d or acetic acid-d unless otherwise noted. Chemical shifts (
δ) were expressed as
3
4
6
parts per million (ppm) downfield from tetramethylsilane (TMS) as the internal standard ( 0.00), and
σ
coupling constants (J) were given in hertz (Hz). High-resolution mass spectra (HRMS) using a Bruker
Impact HD (ESI) were performed in the Instrument Center of the Ministry of Science and Technology
at the National Chiao-Tung University, Taiwan. The reagents 5-chlorooxindole, 5-bromooxindole, and
5
-iodiooxindole, used for the synthesis of the target products 10, 11, and 12, respectively, were prepared
by the modified methods [39]. Dry tetrahydrofuran was freshly distilled from lithium aluminum
hydride (LAH) before use. All the other solvents were obtained from commercial sources and purified
before use if necessary. Images were acquired with a Leica DM1000 microscope (Leica Microsystems,
Wetzlar, Hessen, Germany). UV-VIS spectra were recorded on a Thermo Multiskan Go Microplate
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). IR spectra were registered on
a Thermo Nicolet iS5 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with
attenuated total reflection (ATR) method. The purities of the final compounds were greater than
9
3
5
5% as determined by analytical reverse-phase HPLC.
.1.1. Synthesis of 2–7
-Formyl-3-methyl-1H-pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester 4-ethyl ester (2)
3
,5-Dimethyl-1H-pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester 4-ethyl ester (1) (30.0 g, 0.113 mol)
was dissolved in THF (300 mL). To this stirred mixture was added AcOH (360 mL) and water (300 mL)
sequentially, then ceric ammonium nitrate (CAN) (246 g, 0.45 mol) was added to the mixture in one
portion. After stirring for additional 1 h at room temperature, the reaction mixture was poured into
ice-water (1000 mL), and stirred for further 30 min. The precipitate was then filtered and dried in
vacuo to yield, after crystallization from EtOAc, 31.1 g of analytically pure
2
(98%) as pale yellow
◦
◦
−1
crystals. Mp: 127–128 C, lit. 120–121 C [40]. UV λmax (MeOH), nm (log
ε
): 238 (3.97). IR (ATR), cm
O), 9.79 (s, 1H, N
:
1
3
281, 2994, 1720, 1697, 1660. H-NMR (400 MHz, CDCl )
δ, ppm: 10.25 (s, 1H, C
H
H
),
3
4
.38 (q, 2H, J = 7.2 Hz, -COOCH2CH ), 2.57 (s, 3H, Ar-CH3), 1.59 (s, 9H, -COOC(C
H
3)3), 1.26 (t, 3H,
3
13
J = 7.2 Hz, -COOCH CH3). C-NMR (100 MHz, CDCl3)
δ: 182.6, 163.7, 159.8, 132.9, 130.4, 125.1, 120.8,
2
8
2.9, 60.8, 28.3, 14.3, 11.3.
2
-tert-Butyl-4-ethyl 5-(((2-(diethylamino)ethyl)amino)methyl)-3-methyl-1H-pyrrole-2,4-dicarboxylate (3)
To a stirred solution of 5-formyl-3-methyl-1H-pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester
4
-ethyl ester (2) (2.81 g, 10.0 mmol) in EtOH (20 mL) was added dropwise N,N-diethylethylenediamine
(
(
1.5 mL). The resulting mixture was stirred for 4 h at room temperature. Sodium borohydride
0.38 g 10.0 mmol) and toluenesulfonic acid monohydrate (18.0 g, 10.0 mmol) were then added
to the mixture, stirred for a further 6 h, quenched with water (150 mL), and extracted with ethyl
acetate (30 mL 3). The combined organic layer was dried over anhydrous Na SO and filtered.
The filtrate was evaporated in vacuo to yield, after column chromatography (silica gel, 90:10:1
×
2
4
EtOAc-MeOH-TEA), 3.4 g (90%) of
IR (ATR) cm : 3293, 3216, 1685, 1683. H-NMR (400 MHz, methanol-d )
3
as pale yellow liquid. UV λmax (MeOH), nm (log
ε
): 228 (3.35).
−
1
1
δ, ppm: 4.27 (q, 2H, J = 7.2 Hz,
4
-
COOCH2CH ), 4.05 (s, 2H, Ar-CH2N-), 2.68 (t, 2H, J = 6.0 Hz, -CH NCH2CH -), 2.55-2.49 (m, 9H,
3
2
2
Ar-CH3, -NCH2CH N(CH2CH ) ), 1.55 (s, 9H, -COOC(C
H
) ), 1.34 (t, 3H, J = 7.2 Hz, -COOCH CH3),
2
3 2
3 3
2
13
1
1
.00 (t, 6H, J = 7.2 Hz, -N(CH C
19.4, 112.0, 80.9, 59.5, 52.5, 47.4, 47.0, 46.2, 28.4, 14.4, 11.8, 11.5.
H
) ). C-NMR (100 MHz, CDCl3)
δ, ppm: 165.5, 161.0, 141.3, 130.2,
2
3 2

Molecules 2017, 22, 913
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2
-tert-Butyl 5-(((2-(diethylamino)ethyl)amino)methyl)-3-methyl-1H-pyrrole-2,4-dicarboxylic acid (4)
To a solution of (2.43 g, 6.40 mmol) in MeOH (20 mL) was added dropwise 1N NaOH (20 mL)
3
and the resulting mixture was then heated to reflux for 4 h. After cooling, the mixture was neutralized
with 1 N HCl and the solvent was then evaporated under reduced pressure. MeOH (20 mL) was added
to the residue and then filtered. The filtrate was evaporated in vacuo to get, after recrystallization
◦
from hot MeOH, 2.16 g of analytically pure
4
(96%) as pale yellow crystals. Mp: 119-120 C. IR (ATR),
−
1
ε
): 222 (2.98). 1H-NMR (400 MHz,
cm : 3208, 1568, 1475, 1471, 1137, UV λmax (MeOH), nm (log
methanol-d4) , ppm: 4.36 (s, 2H, Ar-CH2N-), 3.58 (t, 2H, J = 7.0 Hz, -CH NCH2CH -), 2.69 (t, 2H,
J = 7.0 Hz, -CH NCH CH2-), 2.61 (q, 4H, J = 7.2 Hz, -N(CH2CH ) ), 2.41 (s, 3H, Ar-CH3), 1.57 (s, 9H,
δ
2
2
2
2
3 2
13
-
COOC(CH3) ), 1.04 (t, 6H, J = 7.2 Hz, -N(CH CH ) ). C-NMR (100 MHz, methanol-d4) δ, ppm: 169.2,
3 2
3 2
1
62.5, 146.0, 126.5, 123.5, 121.1, 82.1, 52.1, 48.1, 47.2, 41.1, 28.7, 11.6, 10.9.
5
-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrole-2-carboxylic acid
tert-butyl ester (5)
Compound
4
(5.44 g, 16.0 mmol) and CDI (5.19 g, 32.0 mmol) were suspended in dry THF (300 mL)
◦
under a nitrogen atmosphere, and then heated to 65 C for 6 h. The reaction mixture was then filtered.
The filtrate was evaporated under reduced pressure and the residue was purified to give, after column
chromatography (silica gel, 90:10:1 EtOAc-MeOH-TEA), 3.0 g of the bicyclic compound
5
(56%) as pale
◦
−1
yellow solids. Mp: 176 C. UV λmax (MeOH), nm (log
ε): 270 (3.18), IR (ATR), cm : 3202, 1652, 1682.
1
H-NMR (400 MHz, methanol-d4) δ, ppm: 4.38 (s, 2H, ArCH2N-), 3.61 (t, 2H, J = 6.9 Hz, -NCH2CH -),
2
2
.72 (t, 2H, J = 6.9 Hz, -NCH CH2-), 2.67 (q, 4H, J = 7.2 Hz, -N(CH2CH ) ), 1.07 (t, 6H, J = 7.2 Hz,
2
3
2
13
-N(CH C
H
) ). C-NMR (100 MHz, CDCl3)
3 2
δ, ppm: 166.5, 161.7, 143.2, 125.3, 122.9, 121.7, 81.4, 52.0,
2
4
7.1, 46.4, 40.6, 28.4, 11.8, 10.6.
5
-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrole-2-carbaldehyde (7)
To a solution of (1.34 g, 4.00 mmol) in MeOH (100 mL) was added dropwise 15% H SO (10 mL).
The resulting mixture was then heated to reflux for 4.5 h. After the reaction mixture was cooled,
the solution was adjusted to pH 14 with 6 N NaOH and extracted with ethyl acetate (30 mL 3).
The organic layers were combined and then dried over anhydrous Na SO to obtain crude compound
5
2
4
×
2
4
6
which was used for the next step without further purification. To a solution of crude
6 (1.21 g,
5
.10 mmol) in dichloromethane (15 mL) was added Vilsmeier reagent (0.82 g, 6.1 mmol). The reaction
mixture was allowed to stir at room temperature for 5 h and then was adjusted to pH 14 by 1 N
NaOH and extracted with ethyl acetate (10 mL 3). The organic layers were combined and dried
over anhydrous Na SO and evaporated to give, after column chromatography (silica gel, 90:10:1
×
2
4
◦
7 (91%) as pale yellow solids. Mp: 170–171 C. UV λmax (MeOH), nm
EtOAc-MeOH-TEA), 1.1g of
−
1
1
(
(
log
s, 1H, C
ε
): 221 (2.92). IR (ATR), cm : 3093, 2702, 1639, 1562. H-NMR (400 MHz, CDCl )
δ
, ppm: 9.20
3
H
O), 4.36 (s, 2H, Ar-CH2-), 3.56 (t, 2H, J = 6.4 Hz, -CH NCH2CH -), 2.64 (t, 2H, J = 6.4 Hz,
2
2
-
-
CH NCH CH2-), 2.55 (q, 4H, J = 7.2 Hz, -N(CH2CH ) ), 2.47 (s, 3H, Ar-CH3), 1.00 (t, 6H, J = 7.2 Hz,
2 2 3 2
13
N(CH C
H
) ). C-NMR (100 MHz, CDCl3)
3 2
δ, ppm: 178.4, 165.8, 148.1, 134.1, 128.5, 121.9, 51.6, 46.6,
2
4
6.3, 40.4, 11.3, 8.9.
3
.1.2. Synthesis of 8–12
General procedure for the preparation of compounds 8–12.
To each stirred solution containing compound (263 mg, 1.0 mmol) in EtOH (15 mL) was added
7
dropwise a solution of 5-substituted oxindole (1.0 mmol) in EtOH (2 mL) and then piperidine (0.1 mL).
After stirring at room temperature for 6 h, the precipitate formed was filtrated, washed with EtOH,
and purified by column chromatography (silica gel, 90:10:1 EtOAc-MeOH-TEA).
(
2
Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)indolin-
◦
-one (
8
). Yield: 50%, pale orange solids. Mp: 167–168 C. UV λmax (MeOH), nm (log
ε
): 390 (4.12),

Molecules 2017, 22, 913
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IR (ATR), cm 1: 3172, 1651, 1568. H-NMR (400 MHz, acetic acid-d )
−
1
δ
, ppm: 7.75 (s, 1H, -C=C
), 7.03 (dd, 1H, J = 7.6, 7.6 Hz, Ar
), 4.59 (s, 2H, Ar-CH2-), 3.55 (t, 2H, J = 6.4 Hz, -NCH2CH -), 2.65 (t, 2H,
H
H
-),
),
4
7
6
.73 (d, 1H, J = 7.6 Hz, Ar
.98 (d, 1H, J = 7.6 Hz, Ar
H
H
), 7.18 (dd, 1H, J = 7.6, 7.6 Hz, Ar
H
2
J = 6.4 Hz, -NCH CH2-), 2.55 (q, 4H, J = 7.2 Hz, -N(CH2CH ) ), 2.47 (s, 3H, Ar-CH3), 0.99 (t, 6H,
2
3 2
13
J = 7.2 Hz, -N(CH C
H
) ), C-NMR (100 MHz, acetic acid-d4)
δ
, ppm: 171.5, 170.0, 149.6, 139.3, 133.6,
2
3 2
1
3
26.3, 126.0, 125.2, 123.4, 120.9, 119.6, 118.6, 111.4, 51.9, 48.6, 48.2, 39.6, 9.9, 8.8. HRMS m/z (ESI): calcd.,
79.2129 [M + H] , found, 379.2133 [M + H] .
+
+
(
Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-
◦
fluoroindolin-2-one (
(
(
9
). Yield: 56%, orange solids. Mp: 245–246 C, UV λmax (MeOH), nm (log
ε
): 291
, ppm: 7.62
), 6.96–6.90 (m, 2H, ArH × 2), 4.59 (s, 2H, Ar-CH2-),
.97 (t, 2H, J = 6.0 Hz, -NCH2CH -), 3.49 (t, 2H, J = 6.0 Hz, -NCH C -), 3.37 (q, 4H, J = 7.2 Hz,
− −1
4.33). IR (ATR), cm : 3534, 3400, 1668, 1572 cm . H-NMR (400 MHz, acetic acid-d ) δ
4
1
1
s, 1H, -C=CH-), 7.38 (dd, 1H, J = 9.0, 2.0 Hz, ArH
3
H
2
2
2
1
3
-N(CH2CH ) ), 2.47 (s, 3H, Ar-CH3), 1.31 (t, 6H, J = 7.2 Hz, -N(CH CH ) ). C-NMR (100 MHz, acetic
3
2
2
3 2
acid-d4)
δ, ppm: 169.8, 166.1, 158.6, 148.3, 135.5, 132.4, 127.1, 125.9, 125.0, 121.0, 117.1, 113.7, 110.7, 107.0,
+
+
4
9.0, 46.7, 46.6, 37.3, 10.0, 8.7. HRMS m/z (ESI): calcd., 397.2034 [M + H] ; found, 397.2038 [M + H] .
(
Z)-5-Chloro-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)
◦
methylene)indolin-2-one (10). Yield: 54%, orange solids. Mp: 252–253 C, UV λmax (MeOH), nm (log
3
(
Ar
ε
):
, ppm: 7.64
), 6.96 (d, 1H, J = 8.2 Hz,
), 4.59 (s, 2H, Ar-CH ), 3.97 (t, 2H, J = 6.0 Hz, NCH2CH -), 3.49 (t, 2H, J = 6.0 Hz, -NCH CH2-),
− −1
93 (4.37). IR (ATR), cm : 3162, 1634, 1582 cm . H-NMR (400 MHz, acetic acid -d ) δ
4
1
1
s, 1H, -C=CH-), 7.62 (d, 1H, J = 2.0 Hz, ArH), 7.16 (dd, 1H, J = 8.2, 2.0 Hz, ArH
H
2
2
2
3
.37 (q, 4H, J = 7.2 Hz, -N(CH2CH ) ), 2.47 (s, 3H, Ar-CH3), 1.31 (t, 6H, J = 7.2 Hz, -N(CH CH ) ).
3 2 2
3 2
13
C-NMR (100 MHz, acetic acid-d4)
δ
, ppm: 171.2, 169.7, 149.9, 137.7, 133.6, 128.6, 128.5, 127.9, 127.4,
+
1
26.6, 121.1, 119.6, 117.2, 112.5, 51.8, 48.6, 48.1, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 413.1744 [M + H] ;
+
found, 413.1743 [M + H] .
(
Z)-5-Bromo-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)
◦
methylene)indolin-2-one (11). Yield: 56%, orange solids. Mp: 266–267 C. UV λmax (MeOH), nm (log
3
(
Ar
ε
):
, ppm: 7.74
), 6.90 (d, 1H, J = 8.4 Hz,
), 4.55 (s, 2H, Ar-CH2-), 3.96 (t, 2H, J = 6.0 Hz, -NCH2CH -), 3.50 (t, 2H, J = 6.0 Hz, -NCH CH2-),
− −1
94 (4.56), IR (ATR), cm : 3161, 1634, 1581 cm . H-NMR (400 MHz, acetic acid -d ) δ
4
1
1
d, 1H, J = 1.6 Hz, ArH), 7.59 (s, 1H, -C=CH-), 7.30 (dd, 1H, J = 8.4, 1.6 Hz, ArH
H
2
2
3
.38 (q, 4H, J = 7.2 Hz, -N(CH2CH ) ), 2.46 (s, 3H, Ar-CH3), 1.33 (t, 6H, J = 7.2 Hz, -N(CH CH ) ).
3 2 2
3 2
13
C-NMR (100 MHz, acetic acid-d4)
δ
, ppm: 171.0, 169.6, 149.8, 138.0, 133.6, 130.7, 128.4, 127.3, 126.4,
+
1
22.4, 121.1, 117.0, 115.8, 112.9, 51.8, 48.5, 48.1, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 457.1239 [M + H] ;
+
found, 457.1236 [M + H] .
(
Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-
◦
iodoindolin-2-one (12). Yield: 43%, brown solids, Mp: 267–268 C. UV λmax (MeOH), nm (log
ε
): 394
, ppm: 7.94
), 6.81 (d, 1H, J = 8.0 Hz, Ar ), 4.59
s, 2H, Ar-CH2-), 3.97 (t, 2H, J = 6.0 Hz, -NCH2CH -), 3.49 (t, 2H, J = 6.0 Hz, -NCH CH2-), 3.37 (q, 4H,
−
1
−1
.
1H-NMR (400 MHz, acetic acid -d4)
δ
(
(
(
4.36), IR (ATR), cm : 3174, 1689, 1584 cm
s, 1H, -C=C ), 7.64 (s, 1H, Ar ), 7.50 (d, 1H, J = 8.0 Hz, Ar
H
H
H
H
2
2
13
J = 7.2 Hz, -N(CH2CH ) ), 2.47 (s, 3H, Ar-CH3), 1.31 (t, 6H, J = 7.2 Hz, -N(CH C
H ) ). C-NMR
3 2
3 2
2
(
1
[
100 MHz, acetic acid-d4)
δ
, ppm: 171.6, 169.8, 149.9, 149.9, 149.8, 133.5, 127.5, 126.5, 121.1, 116.1, 114.6,
+
12.2, 112.1, 106.7, 51.6, 49.4, 48.2, 39.5, 9.9, 8.8. HRMS m/z (ESI): 505.1100 [M + H] ; found, 505.1094
+
M + H] .
3
.2. Biology
3
.2.1. Cell Culture
The HCT116 (human colon cancer cells, BCRC 60349) and Detroit 551 (human normal fibroblast
cells, BCRC 60118) were maintained in DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS
HyClone, Logan, UT, USA). NCI-H460 (BCRC 60373) and 786-O (BCRC 60243) were maintained in
(

Molecules 2017, 22, 913
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RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% FBS (HyClone, Logan, UT, USA). They were
◦
incubated at 37 C in a 5% CO atmosphere.
2
3
.2.2. Cell Proliferation Assay
The cells incubated as above were plated at a density of 2000 cells/well (cancer cells) [18,41,42] or
1
0,000 cells/well (Detroit 551) on a 96-well plate for 24 h; serial dilutions of indicated compounds were
added and incubated for additional 72 h. At the end of the incubation, cell viability was determined
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The MTT formazan crystals
were dissolved by DMSO, and the absorbance at 570 nm using a microplate spectrophotometer
(Thermo Fisher Scientific, Waltham, MA, USA) [43].
3
.2.3. Acute Cytotoxicity
The acute cytotoxicity effect of sunitinib and compounds
8–12 was determined by
Cell-Counting-Kit-8 (Dojindo, Rockville, MD, USA) assay on HCT116, NCI-460, 786-O and Detroit
51 with the manufacturer’s protocol. Cells were seeded at 5000 cells/well on a 96-well plate for 24 h.
The indicated compounds in different concentration (100 L) were added to cells. Post 6 h, old medium
was aspirated, and cells were washed three times with PBS. WST-8 (Dojindo, Rockville, MD, USA)
10 L) was added to each well, and the absorbance of the plate was recorded at 450 nm on a microplate
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
5
µ
(
µ
3
.2.4. Image Cytometry
Cell cycle profiles of HCT116 cells were determined with an NC-3000 image cytometer
(
ChemoMetec, Allerod, Denmark) in accordance with manufacturer’s protocol. Briefly, cells were
seeded at 200,000 cells/well on a 6-well plate for 24 h. 2 mL of indicated compounds (5.0 M for
sunitinib and 10, 1.0 M for 11 and 12) were added to cells. After incubation for 24 h, 100,000 cells
were harvested and centrifuged for 5 min at 400 g at room temperature, washed once with PBS (50 M)
and resuspended with 50 L of lysis buffer (ChemoMetec, Allerod, Denmark) containing 10 g/mL of
-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, ChemoMetec, Allerod, Denmark). The cells
µ
8–
µ
µ
µ
µ
2
◦
were incubated at 37 C for 5 min, 50 µL of stabilization buffer (ChemoMetec, Allerod, Denmark) was
added to the solution, and the cellular fluorescence was measured with an NC-3000 image cytometer
using NC-SlideA8 (ChemoMetec, Allerod, Denmark). The NC-3000 software (ChemoMetec, Allerod,
Denmark) was used for image acquisition, image analysis and quantification and data visualization.
3
.2.5. In Vitro Tube Formation Assay
Angiogenesis Kit (Kurabo, Neyagawa, Osaka, Japan) was used in accordance with the
manufacturer’s protocols. Briefly, HUVECs and fibroblasts were co-cultured in 24-well plates with
basal medium or solvent control medium (basal medium containing 1.0% DMSO) or solvent control
medium containing 9, 10, 11, and 12 (0.50 µM, 0.25 µM and 0.10 µM). The medium renewals with or
without agent in cultures were conducted every 3 days. After 11 days HUVECs were fixed and stained
with anti-CD31 antibody in line with the manufacturer’s protocols. The tube formation area were
measured using Angiogenesis Image Analyzer (Kurabo, Neyagawa, Osaka, Japan) with suaramin
(70 µM) as positive control.
3
.2.6. In Vitro Kinase Assay
The Reaction Biology Corporation (http://www.reactionbiology.com) HotSpot assay platform
was used to determine the inhibitory activity of compounds 8–12 and sunitinib against VEGFR-2,
PDGFR and aurora A, which was measured by quantifying the amount of 33P incorporated into the
substrate in the presence of the test compound [44]. Briefly, specific kinase and substrate and required
cofactors were prepared in reaction buffer. Test compounds were added into the reaction, after 20 min,

Molecules 2017, 22, 913
16 of 19
a mixture of ATP (Sigma, St. Louis, MO, USA) and 33P ATP (Perkin Elmer, Waltham, MA, USA) was
added to make a final concentration of 10.0 M. Reactions were stood at room temperature for 120 min,
and spot the reactions onto P81 ion exchange filter paper (Whatman Inc., Piscataway, NJ, USA)
µ
.
Unbound phosphate was removed by extensive washing of filters in 0.1% phosphoric acid. Kinase
activity data was reported as the percent remaining kinase activity in test compounds compared to
solvent control (DMSO).
3
.3. Molecular Modeling
Ligands-receptor docking calculation was carried out using the LibDock protocol. Briefly, receptor
active site and ligands were characterized into polar and apolar hotspots. The ligand poses were
placed into the receptor site by hotspots accordance. In this study CHARMm force field was used for
energy minimization of the ligand molecules and ligand-receptor binding. The binding sphere was
defined based on the PDB definition. Conformations of ligands were generated by the BEST method.
The LibDock score was scored by default settings including Ligscore, PLP, Jain, PMF and PMF04.
3
.4. Statistical Analysis
Statistical calculations were carried out with GraphPad Prism version 5. Results were reported as
the mean ± SD. Statistical significance was determined by the unpaired student’s t test.
. Conclusions
A series of novel N-substituted 2-pyrrolidone fused (2-oxoindolin-3-ylidene)methylpyrrole
4
derivatives 12 were successfully prepared and examined for their anti-tumor activities. Several
8–
of them significantly improved inhibitory activity against VEGFR-2 and PDGFR over sunitinib.
β
Compared to sunitinib, all our synthetic compounds showed a similar but tighter binding mode with
VEGFR-2. The nature of C(5) halogens and the rigid conformation of the scaffold obviously were
important determinants of both enzymatic and cellular anti-tumor activities. Among the products
obtained, 11 (C(5)-Br) and 12 (C(5)-I) consistently displayed the most potent and favorable selective
indexes and might be promising to be used as novel anti-angiogenic agents in the targeted therapy
of cancer. The results gleaned from this investigation have important potential applications in the
development of new anticancer drugs.
Acknowledgments: The authors would like to thank the Ministry of Science and Technology, R.O.C. for financially
supporting this research under Contract No. NSC-104-2320-B-016-004.
Author Contributions: T.-H.Y., C.-I.L., W.-H.H., and A.-R.L. conceived, designed, performed the experiments;
analyzed the data; contributed the reagents/materials/analysis tools; wrote; approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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