Synthesis and biological evaluation of 3-substituted-indolin-2-one derivatives containing chloropyrrole moieties

Article abstract of DOI:10.3390/molecules16119368

Eighteen novel 3-substituted-indolin-2-ones containing chloropyrroles were synthesized and their biological activities were evaluated. The presence of a chlorine atom on the pyrrole ring was crucial to reduce cardiotoxicity. The presence of a 2-(ethylamino) ethylcarbamoyl group as a substituent at the C-4' position of the pyrrole enhanced the antitumor activities notably. IC ₅₀ values as low as 0.32, 0.67, 1.19 and 1.22 μM were achieved against non-small cell lung cancer (A549), oral epithelial (KB), melanoma (K111) and large cell lung cancer cell lines (NCI-H460), respectively.

Full text of DOI:10.3390/molecules16119368

Molecules 2011, 16, 9368-9385; doi:10.3390/molecules16119368
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of 3-Substituted-indolin-2-
one Derivatives Containing Chloropyrrole Moieties
Yun-Zhou Jin 1, Da-Xu Fu 1, Nan Ma 1, Zhan-Cheng Li 1, Quan-Hai Liu 2, Lin Xiao 2 and
Rong-Hua Zhang 1,*
1
Department of Chemistry, Tongji University, Shanghai, 200092, China
2
Department of Pharmacology, Shanghai Institute of Pharmaceutical Industry, Shanghai, 200434,
China
* Author to whom correspondence should be addressed; E-Mail: tj_zrh@163.com;
Tel.: +86-021-65988570-8542.
Received: 10 October2011; in revised form: 3 November 2011 / Accepted: 3 November 2011 /
Published: 8 November 2011
Abstract: Eighteen novel 3-substituted-indolin-2-ones containing chloropyrroles were
synthesized and their biological activities were evaluated. The presence of a chlorine atom
on the pyrrole ring was crucial to reduce cardiotoxicity. The presence of a 2-(ethyl-
amino)ethylcarbamoyl group as a substituent at the C-4′ position of the pyrrole enhanced
the antitumor activities notably. IC50 values as low as 0.32, 0.67, 1.19 and 1.22 µM were
achieved against non-small cell lung cancer (A549), oral epithelial (KB), melanoma (K111)
and large cell lung cancer cell lines (NCI-H460), respectively.
Keywords: antitumor activities; indolin-2-one; chloropyrrole; synthesis
1. Introduction
Angiogenesis, an important physiological process of new capillary blood vessel formation, is essential
for the survival, growth and metastasis of tumors [1,2]. Vascular endothelial growth factor (VEGF),
platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and their receptor tyrosine
kinases (RTKs) play key roles in angiogenesis and vascular maintenance via the autocrine and
paracrine loops. Among these growth factors, by binding to its receptor VEGF is currently the most
potent and clinically relevant angiogenic factor [3-5]. The vascular endothelial growth factor receptor
(VEGFR) belongs to the RTKs superfamily and mainly comprises fms-like tyrosine kinase-1 (FLT-1,

Molecules 2011, 16
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VEGFR1), kinase domain-containing receptor (KDR, VEGFR2) and fms-like tyrosine kinase-4 (FLT4,
VEGFR3), which contribute to tumor progression via mediation of tumor angiogenesis and
lymphangiogenesis [6,7]. Therefore, inhibition of the RTK pathway, which results in inhibition of
tumor angiogenesis, has become an important strategy for discovering new antitumor drugs [8-11].
In recent years, indolin-2-one derivatives, especially those with heterocyclic methylene substituents
attached to the C-3 position of the indolin-2-one ring, have been disclosed as potent inhibitors of RTK
in vitro and demonstrated antiangiogenic properties in vivo. The (Z)-3-((3,5-dimethyl-1H-pyrrol-2-
yl)methylene) indolin-2-one structure is found in many antitumor drugs [12,13]. SU5416 (Figure 1) is
the first selective KDR inhibitor in clinical trials. Its derivative SU11248 (sunitinib, Figure 1) was the
first RTK oral inhibitor approved by the US Food and Drug Administration (FDA) for the treatment of
advanced renal cell carcinoma and gastrointestinal stromal tumors [14-16]. In SU6668 (Figure 1) and
sunitinib, 2-carboxyethyl and 2-diethylaminoethylcarbamoyl groups were introduced into the C-4′ position
of the pyrrole ring, respectively. Tang et al. reported that pyrrolo-fused-heterocycle-indolin-2-one
analogues showed potent inhibition against VEGFR and good efficacies against HT-29 cell tumor
xenografts in nude mice [17]. Khanwelkar et al. found that introduction of a ureido group into the C-6
position of 3-pyrrolemethylideneindolin-2-one enhances the potency of inhibition against VEGFR,
PDGFR [18].
Figure 1. Structures of SU5416, SU6668 and sunitinib.
H3C
H3C
C2H4COOH
CH3
H3C
CONH(CH2)2NEt2
CH3
'
4
'
3
5'
'
CH3
2
4
7
N
H
N
H
'
N
H
3a
7a
1
F
3
5
6
O
O
O
2
N
H
N
H
N
H
1
SU6668
SU5416
Sunitinib
The introduction of halogen atoms, mainly fluorine or chlorine, has been used as a tool to enhance
the potency of many pharmaceutical lead compounds [19,20]. The presence of halogen atom changes
the volumetric and conformational properties, as also increases membrane permeability leading to
improved absorption [21]. The importance of chlorine atoms in drug design is well documented.
Besides having a larger size than fluorine, chlorine is a moderate halogen bond acceptor. Compared
with bromine and iodine, the chlorine-carbon bond is stable enough to be inserted into diverse
heterocycles of pharmacological value. In some drugs, subunits bearing chlorine can be accommodated
in hydrophobic pockets of the biological targets [22]. Hrib et al. reported that the introduction of
chlorine into nemonapride constituted a ubiquitous function-determining domain, while the removal of
the chlorine was deleterious to the selectivity among the dopaminergic receptors [23]. Among
leishmanicidal N-benzylcytisine derivatives, the chloro derivative demonstrated 10-fold stronger
inhibition ability than others [24]. On the aromatic ring of arvanil, the replacement of a 3-methoxy
group with a chlorine atom increased the capability to inhibit FAAH [25].
In previous studies, we introduced halogen atoms onto the pyrrole rings attached to an indolin-2-
one framework and found that those with bromine atoms exhibited low antitumor activities, while

Molecules 2011, 16
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those with chlorine atoms exhibited good antitumor activities and low cardiotoxicity. Crystallographic
study of indolin-2-one with FGFR indicates that C-4′ position on the pyrrole is positioned close to the
opening of the binding pocket and could be exposed to solvents [26]. We envisioned that modification
of the substituted indolin-2-ones by adjusting its hydrophilicity might further enhance their biological
activities. Accordingly, we introduced different N-substituents to a pyrrole-4′-formamide moiety
attached to the C-3 position of indolin-2-one. We were also curious about the effects of changing the
substituents on the phenyl ring on the inhibitory activities. Herein, we wish to report the synthesis of
eighteen novel substituted indolin-2-ones and the in vitro biological evaluation of their activity against
four cancer cell lines (A549, KB, K111, NCI-H460), VEGFR2 and cardiotoxicity.
2. Results and Discussion
2.1. Chemistry
4-Chloro-5-formyl-2-methyl-1H-pyrrole-3-carboxylic acid (6) was prepared from vinyl acetate (1)
in five steps (Scheme 1). Bromination of vinyl acetate (1), followed by reaction with ethyl acetoacetate,
afforded pyrrole derivative 3. Treatment of 3 with freshly prepared Vilsmeier reagent gave aldehyde 4,
which was chlorinated with sulfuryl chloride to afford 5. Compound 6 was obtained after hydrolysis of
compound 5 with aqueous sodium hydroxide.
Scheme 1. Synthesis of 4-chloro-5-formyl-2-methyl-1H-pyrrole-3-carboxylic acid (6).
Reagents and conditions: (a) Br2CCl4, 0–5 °C, 98%; (b) ethyl acetoacetate, 17% ammonia,
0–10 °C, 50%; (c) POCl3, DMF, 0–10 °C, 90%; (d) SO2Cl2, HOAc, 0–5 °C, 44%; (e) 10 N aq.
NaOH, 80 °C, 75%.
As shown in Scheme 2, the key intermediates 12a–d, analogues of 6, were prepared from ylide 7
in five steps. Wittig reaction of ylide 7 with aldehydes produced 3-alkyl/H-acrylates 8a–c. Ethyl
4-alkyl/H-pyrrole-3-caboxylates 9a–c were prepared by the cyclization of 8a–c with tosylmethyl
isocyanide (TosMIC). Formylation of 9a–c afforded 10a–c. Chlorination of substituted pyrrole 10–c
and hydrolysis of the produced chloro-substituted esters 11a–d yielded the pyrrolecarboxylic acids 12a–d.

Molecules 2011, 16
Scheme 2. Synthesis of substituted 2-chloro-5-formyl-1H-pyrrole-3-carboxylic acids 12a–d.
9371
Reagents and conditions: (a) aldehydes, benzene, reflux, 87–92%; (b) TosMIC, Et2O/DMSO, NaH,
r.t., 88–90%; (c) POCl3, DMF, 0–10 °C, 85–93%; (d) SO2Cl2, HOAc, 0–5 °C, 33–50%; (e) 10 N aq.
NaOH, 80 °C, 75–92%.
The synthetic route to target compounds 14a–r is shown in Scheme 3 (the substituents of these
compounds are listed in Table 1). Compounds 6 and 12a–d were transformed into amides 13a–n by
the reaction with different amines. Knoevenagel condensations of 13a–n with 5-substituted indolin-2-ones
produced the target compounds 14a–r.
Scheme 3. Synthesis of 3-substituted-indolin-2-one derivatives 14a–r.
O
R2
R4
R3
O
O
R4
R3
OH
R3
R2
R2
a
R4 = substituted NH2
R5 = H, F, Cl, Br, CH3
N
H
b
R5
O
OHC
OHC
N
H
N
H
N
H
6: R2 = Cl; R3 = CH3
12a-d: R2 = H, CH3, C2H5, Cl; R3 = Cl
13a-n
14a-r
Reagents and conditions: (a) amines, HOBt, EDCL, Et3N, DMF, r.t., 70–80%; (b) 5-substituted
indolin-2-ones, EtOH, Et3N, 40–55%.
2.2. Biological Evaluation
The antitumor activities of 14a–r were evaluated in vitro on four tumor cell lines, including non-
small cell lung cancer (A549), oral epithelial (KB), melanoma (K111) and large cell lung cancer cell
lines (NCI-H460) by the MTT assay, and the results are summarized in Table 1.

Molecules 2011, 16
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Table 1. Structures of compounds 14a–r and their antitumor activities on four tumor cell lines.
IC50 SD (µM)
Compd.
14a
R2
R3
R4
R5
A549
KB
K111
NCI-H460
NHCH2CH2N
O
Cl
CH3
F
1.47 0.13
52.91 7.93
>100
>100
N
N CH3
NH
Cl
Cl
CH3
CH3
F
F
>100
>100
>100
>100
14b
14c
HN
2.43 0.26
1.35 0.12
3.41 0.52 1.41 0.17
14d
14e
14f
Cl
CH3 NH(CH2)2CH3
F
>100
>100
>100
>100
>100
>100
Cl
CH3 NH(CH2)3N(CH3)2
CH3 NH(CH2)2N(CH3)2
CH3 NH(CH2)2NHC2H5
CH3 NH(CH2)2N(C2H5)2
CH3 NH(CH2)2N(C2H5)2
CH3 NH(CH2)2N(C2H5)2
CH3 NH(CH2)2N(C2H5)2
CH3 NH(CH2)2N(C2H5)2
F
2.33 0.31
1.69 0.17
1.03 0.09
1.87 0.26
0.32 0.03
>100
2.88 0.33
1.56 0.08
0.67 0.08
1.25 0.10
1.15 0.20
34.21 2.96
5.09 1.01
64.42 7.51
>100
Cl
F
1.35 0.28 1.67 0.18
1.19 0.21 1.41 0.09
1.79 0.12 32.1 3.83
14g
14h
14i
Cl
F
Cl
F
Cl
Cl
Br
H
CH3
F
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14j
Cl
14k
14l
Cl
>100
Cl
>100
14m
14n
14o
14p
14q
14r
Cl
Cl
Cl
Cl
Cl
Cl
Cl
NH(CH2)2N(C2H5)2
NH(CH2)2N(C2H5)2
NH(CH2)2N(C2H5)2
NH(CH2)2NHC2H5
NH(CH2)2N(C2H5)2
NH(CH2)2NHC2H5
>100
H
F
3.37 0.27
2.14 0.26
1.41 0.09
16.71 2.7
1.49 0.11
2.93 0.25
>100
4.23 0.65
1.51 0.11
0.69 0.07
34.39 2.90
1.43 0.21
2.60 0.18
83.39 9.76
CH3
CH3
C2H5
C2H5
F
F
1.24 0.16 1.66 0.23
>100 >100
F
F
3.72 0.46 1.22 0.10
3.83 0.43 4.79 0.62
Sunitinib CH3
control Br
CH3 NH(CH2)2N(C2H5)2
CH3 NH(CH2)2N(C2H5)2
F
F
>100
>100
First, compound 14a with a morpholine ring was examined. It was found to be effective towards
A549, but ineffective on KB, K111 and NCI-H460. Compound 14b, a nitrogen analogue of 14a, and
N-propyl amide 14d had IC50s above 100 µM towards all four cell lines. However, the use of
hydrophilic N-substituents as the basic side chain resulted in increased antitumor activities of the
amides. The IC50s were on the µM order of magnitude. Pleasantly, 14c, 14f, 14g and 14h were
effective on the four different cell lines. It should be noticed that the IC50 values of 14g with a
2-(ethylamino)ethylcarbamoyl group was just 1/4–1/2 of those of sunitinib, indicating the
effectiveness of changing N-substituents. By comparison with N,N-(2-(diethylamino)ethyl)amides,
N-(2-(ethylamino)-ethyl)amides were much more active against the four cell lines, in particular against
K111 and NCI-H460 (14g vs. 14h, 14o vs. 14p, 14q vs. 14r). On the basis of the crystallographic
structure study [24], the functional group at C-4′ position of pyrrole should be exposed to water.
Perhaps the secondary amine structure and low steric hindrance of the terminal ethylamino group of
N-(2-(ethylamino)ethyl)amides are favourable for the formation of hydrogen-bonds with water to
improve the solubility.
In order to examine the effects of chlorine atom on antitumor activities, two compounds 14h and
14o were synthesized by replacing one methyl group on the pyrrole ring of sunitinib with a chloro
group. It was found that compounds 14h and 14o showed higher antitumor activities against A549 and

Molecules 2011, 16
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KB than aunitinib. To improve the antitumor activities of compounds bearing chloro-substituted
pyrroles, the effects of the number and position of chloro groups were studied. Antitumor agents 14h,
14n and 14o bearing a monochloro-substituted pyrrole ring showed antitumor activities, while
antitumor agent 14m bearing a dichloro-substituted pyrrole ring had no effects. The position of the
chloro group did not seem to affect the activities greatly (cf. 14g vs. 14p, 14h vs. 14o). To investigate
the effects of other functional groups on the chloropyrrole ring, several substituents (hydrogen, methyl
and ethyl) at the C-3′ position of pyrrole (compounds 14n, 14o and 14q) were investigated. It was
found that compound 14o with a methyl group exhibited higher activity than the others. Bromine atom
as the substituent on pyrrole control was found to show no antitumor activities.
Next, the C-5 position of indolin-2-one was modified. Replacing the fluoro in 14h with chloro (14i)
led to a large increase in the potency towards A549 (from 1.87 µM to 0.32 µM). Unfortunately, 14i
showed little potency towards K111 and NCI-H460. When bromo, hydrogen or methyl groups were
introduced to the C-5 position of indolin-2-one (14j, 14k, 14l), the potency decreased substantially
(IC50s above 100 µM).
In conclusion, compounds 14c, 14f, 14g, 14h, 14i, 14p and 14r were found to have potent antitumor
activities, with 14g showing 3-4-fold higher inhibitory activities against KB and K111 (IC50 = 0.67 µM,
1.19 µM) than sunitinib (IC50 = 2.60 µM, 3.83 µM), while compounds 14i and 14r showed much
higher inhibitory activities against A549 and NCI-H460 (IC50 = 0.32 µM, 1.22 µM) than sunitinib
(IC50 = 2.93 µM, 4.79 µM), respectively.
Seven compounds in the 14a–r series with low IC50 values on the tumor cell lines were evaluated
for their inhibition of VEGFR2 using an enzyme-linked immunosorbent assay (ELISA), and the data
are summarized in Table 2. Except for compounds 14i and 14r, the other compounds all exhibited
good inhibitory effects against VEGFR2. In the compounds 14c, 14f, 14g, 14h and 14p bearing
different N-pyrrole-4′-formamide substituents, 14g and 14p bearing N-2-(ethylamino)ethyl groups
showed greatly improved biological activities against VEGFR2. It was found that 14h which was
synthesized by replacing one methyl group of Su11248 with chloro showed similar potency as
Su11248 (sunitinib), but replacing the terminal N-diethyl of 14h with one ethyl group (compound 14g)
greatly improved the inhibition. The different positions of the chloro atoms on the pyrrole ring of 14g
and 14p affected the activities. Compound 14g with a 3′-substituted chloropyrrole was more active
than 14p with a 5′-substituted chloropyrrole. In conclusion, compared with Su11248 (IC50 = 6.5 3.0 nM),
14f, 14g and 14p exhibited higher activities against VEGFR2. Among the three compounds, 14g
(IC50 = 5.0 1.1 nM) showed the best potency. The data would suggest that the inhibition effects
against other tyrosine kinase targets deserve to be evaluated in future studies.
Table 2. Inhibition activities of some 3-substituted-indolin-2-ones on VEGFR2.
Compd.
14c
IC50 SD (nM)
6.8 1.8
Compd.
14i
IC50 SD (nM)
11.2 2.6
6.0 1.2
14f
6.2 0.8
14p
14g
5.0 1.1
14r
8.7 1.3
14h
6.8 0.8
Sunitinib
6.5 3.0

Molecules 2011, 16
9374
Cardiotoxicity is a known serious side effect of sunitinib. The pivotal phase III metastatic renal cell
carcinoma trial indicated that 21% of sunitinib-treated patients experienced a decline in left ventricular
ejection fraction (LVEF) to below normal [27]. The cardiotoxicity of the synthesized compounds was
examined by evaluating their inhibition against hERG potassium currents in HEK239 cells and
expressed as IC50, as shown in Figure 2. Compound 14h, which exhibited good in vitro antitumor
activities and has a chloro group at the pyrrole ring C-3′ position, was selected as a candidate with low
cardiotoxicity. Pleasantly, it was found that the cardiotoxicity of 14h (IC50 = 452.4 nM) was about
9-fold lower than that of sunitinib (IC50 = 50.3 nM). The result indicated that the introduction of
chlorine atom by replacing one methyl group on pyrrole ring of sunitinib with a chloro reduced the
cardiotoxicity markedly. Next, compound 14g with the best inhibition against KB, K111 and VEGFR2
was also examined and its cardiotoxicity (IC50 = 430.1 nM) was about 8.5-fold lower than that of
sunitinib (IC50 = 50.3 nM). Research into the cardiotoxicity of the other compounds is in progress, and
the results will be reported in the future.
Figure 2. Inhibition against hERG potassium currents in HEK239 cells (IC50 SD).
3. Experimental
3.1. General
1H-NMR and C-NMR were recorded on a Bruker ARX-400 spectrometer with tetramethylsilane
13
as the internal standard and CDCl3 or DMSO-d6 as the solvent. Melting points were determined on a
Kruss KSP α melting point apparatus and were uncorrected. Mass Spectra (MS) and High Resolution
Mass Spectra (HRMS) were measured on a Waters Q-T Micromass spectrometer. All compounds were
routinely checked by thin layer chromatography (TLC) using Huanghai silica gel HSGF-254 glass
plates. Reagents (analytical grade) were obtained from commercial suppliers and used without further
purification, unless otherwise noted.
3.2. Synthesis of 1,2-Dibromoethyl Acetate (2)
To a solution of vinyl acetate (1, 18.4 mL, 0.20 mol) in carbon tetrachloride (10 mL) was added
bromine (13.5 mL, 0.27 mol) dropwise at 0 °C. The mixture was stirred for 1 h, and concentrated to
give crude liquid product 2 which was used in the next reaction without purification. Yield: 48.4 g

Molecules 2011, 16
9375
1
(98%); H-NMR (CDCl3): δ 2.19 (s, 3H, CH3), 3.85–3.95 (m, 2H, CH2), 6.71–6.74 (m, 1H, CH);
13C-NMR (CDCl3): δ 20.69, 33.12, 70.62, 168.03; MS (ESI): 246.86 (M+1)+.
3.3. Synthesis of Ethyl 2-Methyl-1H-pyrrole-3-carboxylate (3)
To a solution of 1,2-dibromoethyl acetate (2, 12.90 g, 0.15 mol) and ethyl acetoacetate (25.50 mL,
0.2 mol) was added 17% ammonia water (100 mL) dropwise at 0–10 °C during 2 h. Then the mixture
was stirred at room temperature overnight. The precipitated pure 3 was collected by filtration, washed
with water and dried under vacuum. Light yellow solid; Yield: 11.5 g (50%); m.p. 78.5–79.2 °C; 1H-NMR
(CDCl3): δ 1.36 (t, J = 7.13 Hz, 3H, CH3), 2.54 (s, 3H, CH3), 4.29 (q, J = 7.13 Hz, 2H, CH2), 6.57–6.59
(m, 2H, pyrrole), 8.55 (s, 1H, NH); 13C-NMR (CDCl3): δ 13.23, 14.52, 59.40, 110.50, 111.82, 115.78,
135.25, 165.85; MS (ESI): 154.08 (M+1)+.
3.4. General Procedure for Synthesis of Ethyl 3-Substituted-acrylates 8a–c
A mixture of ylide 7 (10 g, 0.03 mol) and aldehydes (formaldehyde, acetaldehyde, propaldehyde)
(0.03 mol) in benzene (30 mL) was refluxed for 5–6 h. Then the mixture was cooled to room
temperature and filtrated. The filtrate was concentrated and purified by flash column chromatography
to obtain liquid product (PE/EA = 15/1).
Ethyl acrylate (8a). Yield: 2.46 g (87%); 1H-NMR (CDCl3): δ 1.22 (t, J = 7.41 Hz, 3H, CH3), 4.15 (q,
J = 7.41 Hz, 2H, CH2), 5.56–5.71 (m, 1H, CH), 6.06–6.12 (m, 1H, CH), 6.27–6.31 (m, 1H, CH);
13C-NMR (CDCl3): δ 13.82, 60.15, 127.21, 132.78, 166.80; MS (ESI): 101.10 (M+1)+.
1
Ethyl but-2-enoate (8b). Yield: 2.85 g (89%); H-NMR (CDCl3): δ 1.25 (t, J = 7.33 Hz, 3H, CH3),
2.01–2.06 (m, 3H, CH3), 4.25 (q, J = 7.33 Hz, 2H, CH2), 5.83–5.88 (m, 1H, CH), 6.78–6.84 (m, 1H, CH);
13C-NMR (CDCl3): δ 14.22, 18.25, 61.41, 143.23, 146.81, 166.52; MS (ESI): 115.12 (M+1)+.
1
Ethyl pent-2-enoate (8c). Yield: 3.31 g (92%); H-NMR (CDCl3): δ 1.04 (t, J = 7.44 Hz, 3H, CH3),
1.26 (t, J = 7.44 Hz, 3H, CH3), 2.16–2.24 (m, 2H, CH2), 4.16 (q, J = 7.44 Hz, 2H, CH2), 5.76–5.81
(m, 1H, CH), 6.96–7.03 (m, 1H, CH); 13C-NMR (CDCl3): δ 12.10, 14.21, 25.21, 60.02, 120.38, 150.50,
166.74; MS (ESI): 129.15 (M+1)+.
3.5. General Procedure for Synthesis of Ethyl 4-Alkyl/H-1H-pyrrole-3-carboxylates 9a–c
A suspension of TosMIC (10.5 g, 0.053 mol) and ethyl 3-substituted-acrylates 8a–c (0.056 mol) in
dry ethyl ether/DMSO (100 mL/50 mL) was added dropwise under argon to a suspension of NaH (3.75 g,
0.094 mol) in ethyl ether (50 mL) at room temperature. Then the mixture was stirred for 4–5 h.
Ice water (200 mL) was added into the mixture. The aqueous phase was extracted with ethyl ether
(3 × 100 mL). The organic phase was dried with anhydrous Na2SO4, concentrated under vacuum to get
light brown liquid compounds 9a–c.
1
Ethyl 1H-pyrrole-3-carboxylate (9a). Yield: 6.5 g (88%); H-NMR (CDCl3): δ 1.36 (t, J = 7.14 Hz,
3H, CH3), 4.31 (q, J = 7.14 Hz, 2H, CH2), 6.65–6.67 (m, 1H, CH), 6.76–6.79 (m, 1H, CH), 7.45–7.46

Molecules 2011, 16
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13
(m, 1H, CH), 9.86 (s, 1H, NH); C-NMR (CDCl3): δ 14.43, 59.97, 109.46, 115.98, 119.28, 123.97,
166.11; MS (ESI): 140.07 (M+1)+.
1
Ethyl 4-methyl-1H-pyrrole-3-carboxylate (9b). Yield: 7.3 g (90%); H-NMR (CDCl3): δ 1.28 (t,
J = 7.12 Hz, 3H, CH3), 2.30 (s, 3H, CH3), 4.21 (q, J = 7.12 Hz, 2H, CH2), 6.54–6.57 (m, 1H, CH),
13
7.37–7.39 (m, 1H, CH), 9.01 (s, 1H, NH); C-NMR (CDCl3): δ 12.66, 14.39, 50.78, 114.28, 117.54,
120.93, 124.61. 166.45; MS (ESI): 154.06 (M+1)+.
Ethyl 4-ethyl-1H-pyrrole-3-carboxylate (9c). Yield: 7.9 g (89%); 1H-NMR (CDCl3): δ 1.21 (t, J = 7.43 Hz,
3H, CH3), 1.34 (t, J = 7.43 Hz, 3H, CH3), 2.76 (q, J = 7.43 Hz, 2H, CH2), 4.27 (q, J = 7.43 Hz, 2H, CH2),
6.54–6.56 (m, 1H, CH), 7.38–7.40 (m, 1H, CH), 8.65 (s, 1H, NH); 13C-NMR (CDCl3): δ 14.47, 14.59,
19.52, 59.38, 114.12, 116.14, 124.64, 128.05, 165.68; MS (ESI): 168.08 (M+1)+.
3.6. General Procedure for Synthesis of Ethyl 5-Formyl-2/4-alkyl-1H-pyrrole-3-carboxylates 4,10a–c
The Vilsmeier reagent was prepared by treatment of dry DMF (4.5 mL, 0.057 mol) with POCl3 (5.4 mL,
0.057 mol) at 0 °C and stirred for another hour at room temperature. In a flask, a solution of ethyl
2/4-alkyl-1H-pyrrole-3-carboxylates 3,9a–c (0.039 mol) in DMF (10 mL) was treated with the freshly
prepared Vilsmeier reagent at 0 °C. The resulting mixture was stirred for another hour at room
temperature, then poured into ice (50 g), adjusted the pH to 7–8 with 10 N aqueous NaOH.
The resulting mixture was heated to 60 °C for two h. The precipitated light yellow solid compounds
4,10a–c were collected by filtration and dried under vacuum.
Ethyl 5-formyl-2-methyl-1H-pyrrole-3-carboxylate (4). Light yellow solid; Yield: 6.4 g (90%); m.p.
1
132.5–133.3 °C; H-NMR (CDCl3): δ 1.38 (t, J = 7.13 Hz, 3H, CH3), 2.66 (s, 3H, CH3), 4.32 (q,
J = 7.13 Hz, 2H, CH2), 7.40–7.41 (m, 1H, CH), 9.42 (s, 1H, CHO), 10.99 (s, 1H, NH); 13C-NMR
(CDCl3): δ 13.55, 14.41, 60.02, 115.49, 124.47, 130.44, 144.01, 164.30, 179.17; MS (ESI): 182.13 (M+1)+.
Ethyl 5-formyl-1H-pyrrole-3-carboxylate (10a). Light yellow solid; Yield: 5.6 g (85%); m.p. 84.1–85.1 °C;
1H-NMR (CDCl3): δ 1.29 (t, J = 7.13 Hz, 3H, CH3), 4.25 (q, J = 7.13 Hz, 2H, CH2), 7.34–7.35 (m, 1H,
CH), 7.66–7.67 (m, 1H, CH), 9.49 (s, 1H, CHO), 10.42 (s, 1H, NH); 13C-NMR (CDCl3): δ 14.37,
60.39, 119.18, 121.62, 129.97, 133.05, 163.63, 180.04; MS (ESI): 168.07 (M+1)+.
Ethyl 5-formyl-4-methyl-1H-pyrrole-3-carboxylate (10b). Light yellow solid; Yield: 6.6 g (93%); m.p.
1
123.5–125.2 °C; H-NMR (CDCl3): δ 1.25 (t, J = 7.15 Hz, 3H, CH3), 2.63 (s, 3H, CH3), 4.23 (q,
J = 7.15 Hz, 2H, CH2), 7.66–7.67 (m, 1H, CH), 9.73 (s, 1H, CHO), 10.05 (s, 1H, NH); 13C-NMR
(CDCl3): δ 9.88, 11.23, 51.15, 117.11, 130.13, 130.54, 133.88, 164.51, 178.36; MS (ESI): 182.09 (M+1)+.
Ethyl 4-ethyl-5-formyl-1H-pyrrole-3-carboxylate (10c). Light yellow solid; Yield: 6.6 g (86%); m.p.
84.5–86.0 °C; 1H-NMR (CDCl3): δ 1.22 (t, J = 7.47 Hz, 3H, CH3), 1.29 (t, J = 7.47 Hz, 3H, CH3), 3.01
(q, J = 7.47 Hz, 2H, CH2), 4.24 (q, J = 7.47 Hz, 2H, CH2), 7.56–7.57 (m, 1H, CH), 9.64 (s, 1H, CHO),
13
10.32 (s, 1H, NH); C-NMR (CDCl3): δ 14.36, 16.92, 17.59, 59.96, 116.58, 129.86, 130.36, 140.59,
163.83, 178.35; MS (ESI): 196.15 (M+1)+.

Molecules 2011, 16
9377
3.7. General Procedure for Synthesis of Ethyl Chloro-substituted-5-formyl-1H-pyrrole-3-carboxylates
5, 11a–d
Sulfuryl chloride (1.66 mL, 0.025 mol, for the synthesis of 11a–c, 5, 4.00 mL, 0.060 mol, for
synthesis of 11d) was added dropwise within 10–15 min to the solution of ethyl 2/4-alkyl-5-formy-1H-
pyrrole-3-carboxylates 4, 10a–c (0.020 mol) dissolved in acetic acid (20 mL) cooled with an ice bath.
After addition, the mixture was stirred for 30–60 min at room temperature, and poured into ice water
(100 mL). The precipitated products 5, 11a–d were collected by filtration, dried under vacuum,
purified by flash column chromatography (PE/EA = 5/1).
Ethyl 4-chloro-5-formyl-2-methyl-1H-pyrrole-3-carboxylate (5). Light orange solid; Yield: 1.9 g (44%);
m.p. 189.3–190.0 °C; 1H-NMR (CDCl3): δ 1.36 (t, J = 7.13 Hz, 3H, CH3), 2.65 (s, 3H, CH3), 4.30 (q,
J = 7.13 Hz, 2H, CH2), 9.45 (s, 1H, CHO), 10.09 (s, 1H, NH); 13C-NMR (CDCl3): δ 12.55, 14.41,
59.02, 116.32, 124.87, 129.78, 143.01, 162.33, 179.08; MS (ESI): 216.04 (M+1)+.
Ethyl 2-chloro-5-formyl-1H-pyrrole-3-carboxylate (11a). Light yellow solid; Yield: 1.8 g (45%); m.p.
123.5–125.9 °C; 1H-NMR (CDCl3): δ 1.40 (t, J = 7.13 Hz, 3H, CH3), 4.36 (q, J = 7.13 Hz, 2H, CH2),
7.40–7.41 (m, 1H, CH), 9.48 (s, 1H, CHO), 9.96 (s, 1H, NH); 13C-NMR (CDCl3): δ 14.31, 60.70,
114.79, 122.26, 122.31, 130.40, 162.13, 178.67; MS (ESI): 202.09 (M+1)+.
Ethyl 2-chloro-5-formyl-4-methyl-1H-pyrrole-3-carboxylate (11b). Light yellow solid; Yield: 2.2 g
1
(50%); m.p. 173.5–174.3 °C; H-NMR (CDCl3): δ 1.22 (t, J = 7.13 Hz, 3H, CH3), 2.50 (s, 3H, CH3),
13
4.22 (q, J = 7.13 Hz, 2H, CH2), 9.58 (s, 1H, CHO), 9.90 (s, 1H, NH); C-NMR (CDCl3): δ 10.61,
11.25, 51.37, 113.35, 128.46, 129.17, 134.85, 163.36, 177.15; MS (ESI): 216.04 (M+1)+.
Ethyl 2-chloro-4-ethyl-5-formyl-1H-pyrrole-3-carboxylate (11c). Light yellow solid; Yield: 1.9 g
(43%); m.p. 109.5–111.8 °C; 1H-NMR (CDCl3): δ 1.29 (t, J = 7.47 Hz, 3H, CH3), 1.40 (t, J = 7.47 Hz,
3H, CH3), 3.07 (q, J = 7.47 Hz, 2H, CH2), 4.37 (q, J = 7.47 Hz, 2H, CH2), 9.66 (s, 1H, CHO), 10.51 (s,
1H, NH); 13C-NMR (CDCl3): δ 14.22, 16.89, 18.11, 60.39, 112.67, 127.72, 129.59, 141.85, 162.73,
177.28; MS (ESI): 230.06 (M+1)+.
Ethyl 2,4-dichloro-5-formyl-1H-pyrrole-3-carboxylate (11d). Light yellow solid; Yield: 1.6 g (33%);
1
m.p. 173.5–174.9 °C; H-NMR (CDCl3): δ 1.26 (t, J = 7.14 Hz, 3H, CH3), 4.30 (q, J = 7.14 Hz, 2H,
CH2), 9.65 (s, 1H, CHO), 9.99 (s, 1H, NH); 13C-NMR (CDCl3): δ 13.67, 58.35, 114.78, 122.36, 124.67,
129.38, 165.14, 177.29; MS (ESI): 237.03 (M+1)+.
3.8. General Procedure for Synthesis of Chloro-substituted-5-formyl-1H-pyrrole-3-carboxylic acids 6,
12a–d
Ethyl chloro-substituted-5-formyl-1H-pyrrole-3-carboxylates 5, 11a–d (0.014 mol) were dissolved
in a solution of NaOH (2.80 g, 0.070 mol) in MeOH/H2O (2 mL/30 mL), and the mixture was refluxed
for two hours, then extracted with dichloromethane (20 mL × 2). The aqueous layer was acidified with
18% hydrochloric acid to pH = 2–3 and the precipitated pure crystals 6, 12a–d were collected by
filtration, washed with water, and dried under vacuum.

Molecules 2011, 16
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4-Chloro-5-formyl-2-methyl-1H-pyrrole-3-carboxylic acid (6). Light yellow solid; Yield: 2.0 g (75%);
m.p. > 290 °C; 1H-NMR (DMSO-d6): δ 2.45 (s, 3H, CH3), 9.59 (s, 1H, CHO), 12.48 (s, 1H, NH), 13.21
13
(s, 1H, COOH); C-NMR (DMSO-d6): δ 14.25, 112.21, 127.11, 131.12, 143.03, 164.34, 177.54; MS
(ESI): 188.06 (M+1)+.
2-Chloro-5-formyl-1H-pyrrole-3-carboxylic acid (12a). Light yellow solid; Yield: 19.4 g (80%);
m.p. > 290 °C; 1H-NMR (DMSO-d6): δ 7.38–7.40 (m, 1H, CH), 9.45 (s, 1H, CHO), 12.67 (s, 1H, NH),
13
13.51 (s, 1H, COOH); C-NMR (DMSO-d6): δ 114.24, 122.88, 127.89, 131.48, 163.66, 180.11; MS
(ESI): 174.01 (M+1)+.
2-Chloro-5-formyl-4-methyl-1H-pyrrole-3-carboxylic acid (12b). Light yellow solid; Yield: 2.4 g (92%);
m.p. > 290 °C; 1H-NMR (DMSO-d6): δ 2.49 (s, 3H, CH3), 9.66 (s, 1H, CHO), 12.64 (s, 1H, NH), 13.21
13
(s, 1H, COOH); C-NMR (DMSO-d6): δ 10.90, 113.06, 128.09, 129.19, 133.76, 164.42, 178.80; MS
(ESI): 188.04 (M+1)+.
2-Chloro-4-ethyl-5-formyl-1H-pyrrole-3-carboxylic acid (12c). Light yellow solid; Yield: 2.5 g (89%);
m.p. 248.3–250.2 °C; 1H-NMR (DMSO-d6): δ 1.14 (t, J = 7.28 Hz, 3H, CH3), 2.99 (q, J = 7.28 Hz, 2H,
13
CH2), 9.65 (s, 1H, CHO), 12.63 (s, 1H, NH), 13.20 (s, 1H, COOH); C-NMR (DMSO-d6): δ 17.24,
17.82, 112.22, 128.07, 128.42, 140.35, 164.25, 178.70; MS (ESI): 202.10 (M+1)+.
2,4-Dichloro-5-formyl-1H-pyrrole-3-carboxylic acid (12d). Light yellow solid; Yield: 2.4 g (81%);
m.p. > 290 °C; 1H-NMR (DMSO-d6): δ 9.76 (s, 1H, CHO), 12.32 (s, 1H, NH), 13.15 (s, 1H, COOH);
13C-NMR (DMSO-d6): δ 114.28, 122.75, 124.37, 128.93, 165.09, 178.07; MS (ESI): 209.01 (M+1)+.
3.9. General Procedure for Synthesis of 3-Substituted-indolin-2-ones 14a–r
To a solution of compounds 6, 12a–d (0.006 mol) in DMF (10 mL) were added amines (0.0072 mol),
EDC·HCl (1.73 g, 0.009 mol), HOBt (1.22 g, 0.009 mol) and Et3N (1.22 g, 0.012 mol). The mixture
was stirred for 20 h at room temperature and poured into water (20 mL). The mixture was extracted
with dichloromethane (20 mL × 3). The organic phase was washed with saturated aqueous NaCl
(20 mL × 2), dried with anhydrous Na2SO4, and concentrated to get the crude products 13a–n which
were used in the next step without purification. To a solution of 13a–n dissolved in ethanol (8.0 mL)
were added indolin-2-ones (0.0066 mol) and Et3N (0.0010 mol). The mixture was heated to reflux for
2–4 h. The precipitated crystals were collected by filtration, purified by recrystallization from ethanol
or by flash column chromatography (DCM/MeOH = 15/1~12/1), and dried under vacuum at 80–90 °C
to get target compounds 14a–r.
(Z)-4-Chloro-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-N-(2-morpholinoethyl)-1H-pyrrole
-3-carboxamide (14a). Yellow solid; Yield: 0.8 g (32%); m.p. 268.5–269.7 °C; 1H-NMR (CDCl3): δ 2.56
(t, J = 7.12 Hz, 4H, CH2 × 2), 2.63 (t, J = 5.82 Hz, 2H, CH2), 2.71 (s, 3H, CH3), 3.55–3.59 (m, 2H,
CH2), 3.77 (t, J = 7.12 Hz, 4H, CH2 × 2), 6.83–7.70 (m, 6H, aromatic, vinyl, CONH, indolin-2-one
NH), 13.58 (s, 1H, pyrrole NH); 13C-NMR (CDCl3): δ 14.20, 36.30, 53.58, 57.30, 66.80, 107.32,
110.96, 117.99, 118.67, 118.82, 122.61, 124.46, 125.24, 126.53, 135.86, 137.29, 157.11, 162.50,
+
169.95; HRMS (ESI) m/z (M+H)+: calculated for C21H23ClFN4O3 : 433.1443, found 433.1440.

Molecules 2011, 16
9379
(Z)-3-((3-Chloro-5-methyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-yl)methylene)-5-fluoroindolin-
2-one (14b). Yellow solid; Yield: 0.8 g (32%); m.p. 276.7–278.5 °C; 1H-NMR (CDCl3): δ 2.36 (s, 3H,
CH3), 2.45 (s, 3H, CH3), 2.50 (t, J = 7.12 Hz, 4H, CH2 × 2), 3.10 (t, J = 7.12 Hz, 4H, CH2 × 2),
6.82–7.41 (m, 4H, aromatic, vinyl, CONH), 7.99 (s, 1H, indolin-2-one NH), 13.48 (s, 1H, pyrrole NH);
13C-NMR (CDCl3): δ 13.22, 46.08, 47.26, 54.88, 105.97, 110.20, 113.76, 116.87, 118.13, 119.85,
122.58, 124.48, 126.71, 133.75, 135.39, 158.02, 163.83, 169.76; HRMS (ESI) m/z (M+H)+: calculated
+
for C20H21ClFN4O2 : 403.1335, found 403.1337.
(Z)-N-(azetidin-3-yl)-4-chloro-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-1H-pyrrole-3-
1
carboxamide (14c). Yellow solid; Yield: 0.7 g (33%); m.p. 230.5–232.0 °C; H-NMR (DMSO-d6):
δ 1.23 (s, 1H, NH), 2.47 (s, 3H, CH3), 3.99–4.13 (m, 4H, CH2 × 2), 4.73–4.82 (m, 1H, CH), 6.84–8.26
(m, 5H, aromatic, vinyl, CONH), 10.95 (s, 1H, indolin-2-one NH), 13.75 (s, 1H, pyrrole NH);
13C-NMR (DMSO-d6): δ 11.02, 42.07, 53.04, 107.21, 110.30, 114.08, 117.98, 119.76, 120.01, 124.45,
126.32, 127.01, 131.43, 135.55, 158.42, 164.07, 170.12; HRMS (ESI) m/z (M+H)+: calculated for
+
C18H17ClFN4O2 : 375.1368, found 375.1369.
(Z)-4-Chloro-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-N-propyl-1H-pyrrole-3-carboxamide
(14d). Yellow solid; Yield: 1.0 g (43%); m.p. > 290 °C; 1H-NMR (DMSO-d6): δ 0.92 (t, J = 7.32 Hz,
3H, CH3), 1.49–1.58 (m, 2H, CH2), 2.46 (s, 3H, CH3), 3.18–3.23 (m, 2H, CH2), 6.87–7.84 (m, 5H,
aromatic, vinyl, CONH), 11.09 (s, 1H, indolin-2-one NH), 13.85 (s, 1H, pyrrole NH); 13C-NMR
(DMSO-d6): δ 11.94, 13.97, 22.89, 41.04, 107.12, 110.92, 114.09, 115.67, 118.55, 118.96, 121.82,
122.65, 124.38, 135.81, 136.56, 159.35, 162.66, 169.95; HRMS (ESI) m/z (M+H)+: calculated for
+
C18H18ClFN3O2 : 362.1072 (M+1), found 362.1073.
(Z)-4-Chloro-N-(3-(dimethylamino)propyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-1H-
1
pyrrole-3-carboxamide (14e). Yellow solid; Yield: 1.1 g (45%); m.p. 235.1–237.3 °C; H-NMR
(DMSO-d6): δ 0.87–0.91 (m, 2H, CH2), 2.20 (s, 6H, CH3 × 2), 2.41 (t, J = 6.76 Hz, 2H, CH2), 2.49
(s, 3H, CH3), 3.31–3.35 (m, 2H, CH2), 6.87–7.75 (m, 5H, aromatic, vinyl, CONH), 11.10 (s, 1H,
indolin-2-one NH), 13.87 (s, 1H, pyrrole NH); 13C-NMR (DMSO-d6): δ 13.90, 26.40, 34.37, 43.23,
56.60, 107.35 110.08, 112.33, 113.49, 118.03, 120.29, 123.80, 124.67, 125.17, 133.72, 139.49, 157.27,
+
164.55, 168.66; HRMS (ESI) m/z (M+H)+: calculated for C20H23ClFN4O2 : 405.1494, found 405.1491.
(Z)-4-Chloro-N-(2-(dimethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-1H-
1
pyrrole-3-carboxamide (14f). Yellow solid; Yield: 1.0 g (42%); m.p. 271.8–274.2 °C; H-NMR
(DMSO-d6): δ 2.21 (s, 6H, CH3 × 2), 2.41 (t, J = 6.76 Hz, 2H, CH2), 2.49 (s, 3H, CH3), 3.32 (q, J = 6.76 Hz,
2H, CH2), 6.87–7.75 (m, 5H, aromatic, vinyl, CONH), 11.09 (s, 1H, indolin-2-one NH), 13.87 (s, 1H,
pyrrole NH); 13C-NMR (DMSO-d6): δ 13.91, 34.42, 43.25, 56.66, 110.20, 112.47, 113.33, 115.58,
118.02, 118.13, 120.50, 123.89, 125.39, 133.81, 139.53, 157.34, 164.70, 168.81; HRMS (ESI) m/z
+
(M+H)+: calculated for C19H21ClFN4O2 : 391.1337, found 391.1338.
(Z)-4-Chloro-N-(2-(ethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-1H-pyrrole-
1
3-carboxamide (14g). Yellow solid; Yield: 0.8 g (33%); m.p. 211.0–211.8 °C; H-NMR (DMSO-d6):
δ 1.04 (t, J = 7.13 Hz, 3H, CH3), 1.21 (s, 1H, NH), 2.48 (s, 3H, CH3), 2.61 (q, J = 7.13 Hz, 2H, CH2),

Molecules 2011, 16
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2.72 (t, J = 7.13 Hz, 2H, CH2), 3.32 (q, J = 7.13 Hz, 2H, CH2), 6.87–7.94 (m, 5H, aromatic, vinyl,
CONH), 11.27 (s, 1H, indolin-2-one NH), 13.87 (s, 1H, pyrrole NH); 13C-NMR (DMSO-d6):
δ 14.12, 15.31, 38.21, 43.51, 48.52, 107.02, 111.05, 114.15, 118.30, 118.78, 122.60, 124.43, 125.96,
126.63, 135.86, 137.11, 158.84, 162.76, 169.95; HRMS (ESI) m/z (M+H)+: calculated for
+
C19H21ClFN4O2 : 391.1337, found 391.1340.
(Z)-4-Chloro-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2-methyl-1H-
1
pyrrole-3-carboxamide (14h). Yellow solid; Yield: 1.1 g (44%); m.p. 245.6–246.9 °C; H-NMR
(DMSO-d6): δ 1.19 (t, J = 7.12 Hz, 6H, CH3 × 2), 2.39 (s, 3H, CH3), 2.69–2.77 (m, 6H, CH2 × 3),
3.31–3.43 (m, 2H, CH2), 6.78–7.77 (m, 5H, aromatic, vinyl, CONH), 11.07 (s, 1H, indolin-2-one NH),
13
14.12 (s, 1H, pyrrole NH); C-NMR (DMSO-d6): δ 8.42, 13.83, 34.21, 48.21, 50.52, 107.26, 109.98,
112.24, 117.99, 120.05, 120.19, 123.76, 124.97, 125.24, 133.66, 139.35, 156.86, 164.52, 168.55;
+
HRMS (ESI) m/z (M+H)+: calculated for C21H25ClFN4O2 : 419.1650, found 419.1653.
(Z)-4-Chloro-5-((5-chloro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2-methyl-1H-
1
pyrrole-3-carboxamide (14i). Yellow solid; Yield: 1.1 g (40%); m.p. 246.0–247.0 °C; H-NMR
(DMSO-d6): δ 1.14 (t, J = 7.26 Hz, 6H, CH3 × 2), 2.45 (s, 3H, CH3), 2.62–2.72 (m, 6H, CH2 × 3),
3.46–3.48 (m, 2H, CH2), 6.67–7.65 (m, 5H, aromatic, vinyl, CONH), 11.14 (s, 1H, indolin-2-one NH),
13
14.19 (s, 1H, pyrrole NH); C-NMR (DMSO-d6): δ 8.51, 14.04, 34.44, 48.31, 50.73, 113.28, 117.30,
117.77, 118.27, 120.34, 123.86, 125.44, 126.45, 126.47, 126.54, 136.06, 139.58, 164.81, 168.48;
+
HRMS (ESI) m/z (M+H)+: calculated for C21H25Cl2N4O2 : 435.1355, found 435.1354.
(Z)-5-((5-bromo-2-oxoindolin-3-ylidene)methyl)-2-chloro-N-(2-(diethylamino)ethyl)-4-methyl-1H-
1
pyrrole-3-carboxamide (14j). Yellow solid; Yield: 1.2 g (39%); m.p. 248.1–248.5 °C; H-NMR
(DMSO-d6): δ 1.01 (t, J = 7.10 Hz, 6H, CH3 × 2), 2.24 (s, 3H, CH3), 2.61–2.68 (m, 6H, CH2 × 3),
3.46–3.50 (m, 2H, CH2), 6.78–7.76 (m, 5H, aromatic, vinyl, CONH), 10.85 (s, 1H, indolin-2-one NH),
13
13.53 (s, 1H, pyrrole NH); C-NMR (DMSO-d6): δ 8.66, 13.94, 36.14, 50.31, 52.70, 113.26, 116.87,
117.27, 119.07, 121.15, 123.54, 125.24, 126.05, 126.49, 126.54, 136.06, 139.24, 162.56, 168.98;
2+
HRMS (ESI) m/z (M+H)+: calculated for C21H24BrClN4O2 and C21H26Cl2N4O2 : 478.0771 (M),
480.0742 (M+2), found 478.0773, 480.0743.
(Z)-4-Chloro-N-(2-(diethylamino)ethyl)-2-methyl-5-((2-oxoindolin-3-ylidene)methyl)-1H-pyrrole-3-
1
carboxamide (14k). Yellow solid; Yield: 1.1 g (43%); m.p. 238.5–240.1 °C; H-NMR (DMSO-d6):
δ 1.01 (t, J = 7.25 Hz, 6H, CH3 × 2), 2.43 (s, 3H, CH3), 2.52–2.57 (m, 6H, CH2 × 3), 3.32–3.36 (m, 2H,
CH2), 6.91–7.74 (m, 6H, aromatic, vinyl, CONH), 11.10 (s, 1H, indolin-2-one NH), 13.82 (s, 1H,
pyrrole NH); 13C-NMR (DMSO-d6): δ 8.45, 13.92, 34.37, 48.25, 50.70, 109.53, 112.17, 117.30,
118.24, 118.57, 119.42, 121.83, 124.12, 127.28, 136.62, 137.81, 138.93, 165.02, 168.83; HRMS (ESI)
+
m/z (M+H)+: calculated for C21H26ClN4O2 : 401.1744, found 401.1741.
(Z)-4-Chloro-N-(2-(diethylamino)ethyl)-2-methyl-5-((5-methyl-2-oxoindolin-3-ylidene)methyl)-1H-
1
pyrrole-3-carboxamide (14l). Yellow solid; Yield: 1.0 g (40%); m.p. 238.2–240.0 °C; H-NMR
(DMSO-d6): δ 0.99 (t, J = 7.09 Hz, 6H, CH3 × 2), 2.31 (s, 3H, CH3), 2.50 (s, 3H, CH3), 2.53–2.59 (m, 6H,
CH2 × 3), 3.39–3.43 (m, 2H, CH2), 6.79–7.64 (m, 5H, aromatic, vinyl, CONH), 10.98 (s, 1H, indolin-2-one

Molecules 2011, 16
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NH), 13.82 (s, 1H, pyrrole NH); 13C-NMR (DMSO-d6): δ 8.46, 13.89, 20.36, 34.41, 48.29, 50.81,
109.11, 112.05, 116.91, 118.57, 118.91, 119.01, 124.15, 127.71, 131.24, 135.58, 135.76, 138.68,
+
165.25, 168.88; HRMS (ESI) m/z (M+H)+: calculated for C22H28ClN4O2 : 415.1901, found 415.1903.
(Z)-2,4-Dichloro-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-1H-pyrrole-3-
1
carboxamide (14m). Yellow solid; Yield: 1.1 g (42%); m.p. 269.1–270.3 °C; H-NMR (DMSO-d6):
δ 1.03 (t, J = 7.17 Hz, 6H, CH3 × 2), 2.45–2.50 (m, 6H, CH3 × 2), 3.25–3.30 (m, 2H, CH2), 6.89–7.97
，
(m, 5H, aromatic, vinyl, CONH) 11.15 (s, 1H, indolin-2-one NH), 13.92 (s, 1H, pyrrole NH);
13C-NMR (DMSO-d6): δ 12.45, 35.15, 48.60, 55.12, 109.25, 112.34, 115.55, 118.03, 119.04, 120.11,
122.31, 126.36, 127.40, 128.11, 134.88, 158.57, 162.27, 169.66; HRMS (ESI) m/z (M+H)+: calculated
+
for C20H22Cl2FN4O2 : 439.1027, found 439.1024.
(Z)-2-Chloro-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-1H-pyrrole-3-
carboxamide (14n). Yellow solid; Yield: 1.0 g (39%); m.p. 233.5–234.7 °C; 1H-NMR (DMSO-d6): δ 0.98
(t, J = 7.16 Hz, 6H, 2 × CH3), 2.51–2.56 (m, 6H, 3 × CH2), 3.25–3.30 (m, 2H, CH2), 6.88–8.00 (m, 6H,
aromatic, vinyl, CONH, pyrrole), 11.17 (s, 1H, indolin-2-one NH), 14.32 (s, 1H, pyrrole NH);
13C-NMR (DMSO-d6): δ 12.37, 37.40, 47.21, 51.92, 107.15, 111.25, 114.55, 118.53, 119.64, 120.02,
121.39, 126.50, 127.37, 128.11, 135.96, 158.78, 161.77, 169.98; HRMS (ESI) m/z (M+H)+: calculated
+
for C20H23ClFN4O2 : 405.1494, found 405.1497.
(Z)-2-Chloro-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-4-methyl-1H-
1
pyrrole-3-carboxamide (14o). Yellow solid; Yield: 1.0 g (41%); m.p. 243.4–244.0 °C; H-NMR
(DMSO-d6): δ 0.99 (t, J = 7.05 Hz, 6H, CH3 × 2), 2.44 (s, 3H, CH3), 2.60–2.70 (m, 6H, CH2 × 3),
3.51–3.55 (m, 2H, CH2), 6.87–7.86 (m, 5H, aromatic, vinyl, CONH), 11.15 (s, 1H, indolin-2-one NH),
14.53 (s, 1H, pyrrole NH); 13C-NMR (DMSO-d6): δ 11.01, 12.36, 37.55, 47.02, 51.92, 107.16, 110.92,
113.92, 117.92, 119.64, 119.75, 125.11, 126.73, 127.01, 130.49, 135.42, 158.82, 162.68, 170.22;
+
HRMS (ESI) m/z (M+H)+: calculated for C21H25ClFN4O2 : 419.1650 , found 419.1652.
(Z)-2-Chloro-N-(2-(ethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-4-methyl-1H-pyrrole-
3-carboxamide (14p). Yellow solid; Yield: 0.8 g (34%); m.p. 215.0–216.8 °C; 1H-NMR (DMSO-d6): δ
1.03 (t, J = 7.13 Hz, 3H, CH3), 1.23 (s, 1H, NH), 2.43 (s, 3H, CH3), 2.60 (q, J = 7.13 Hz, 2H, CH2),
2.70 (t, J = 7.13 Hz, 2H, CH2), 3.32(q, J = 7.13 Hz, 2H, CH2), 6.87–7.89 (m, 5H, aromatic, vinyl,
CONH), 11.27 (s, 1H, indolin-2-one NH), 14.50 (s, 1H, pyrrole NH); 13C-NMR (DMSO-d6):
δ 10.97, 14.36, 38.37, 43.30, 48.06, 107.31, 110.96, 113.98, 118.01, 119.62, 119.90, 125.11, 126.76,
127.08, 130.45, 135.46, 157.67, 163.06, 170.23; HRMS (ESI) m/z (M+H)+: calculated for
+
C19H21ClFN4O2 : 391.1337, found 391.1335.
(Z)-2-Chloro-N-(2-(diethylamino)ethyl)-4-ethyl-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-1H-pyrrole-
1
3-carboxamide (14q). Yellow solid; Yield: 1.2 g (45%); m.p. 245.2–246.8 °C; H-NMR (DMSO-d6):
δ 0.98 (t, J = 7.09 Hz, 6H, CH3 × 2), 1.12 (t, J = 7.09 Hz, 3H, CH3), 2.44–2.62 (m, 6H, CH2 × 3), 2.92
(q, J = 7.09 Hz, 2H, CH2), 3.29–3.35 (m, 2H, CH2), 6.88–7.89 (m, 5H, aromatic, vinyl, CONH), 11.14
13
(s, 1H, indolin-2-one NH), 14.51 (s, 1H, pyrrole NH); C-NMR (DMSO-d6): δ 12.31, 17.29, 17.97,
37.53, 46.99, 51.93, 107.27, 110.93, 113.93, 118.08, 124.85, 125.93, 126.96, 127.06, 135.42, 137.26,

Molecules 2011, 16
9382
+
158.84, 162.82, 170.19; HRMS (ESI) m/z (M+H)+: calculated for C22H27ClFN4O2 : 433.1807, found
433.1809.
(Z)-2-Chloro-4-ethyl-N-(2-(ethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-1H-pyrrole-
1
3-carboxamide (14r). Yellow solid; Yield: 0.9 g (36%); m.p. 232.0–233.1 °C; H-NMR (DMSO-d6):
δ 1.07–1.14 (m, 6H, CH3 × 2), 1.22 (s, 1H, NH), 2.71 (q, J = 7.15 Hz, 2H, CH2), 2.80 (t, J = 7.15 Hz,
2H, CH2), 2.93 (q, J = 7.15 Hz, 2H, CH2), 3.44 (q, J = 7.15 Hz, 2H, CH2), 6.88–8.10 (m, 5H, aromatic,
vinyl, CONH), 11.20 (s, 1H, indolin-2-one NH), 14.58 (s, 1H, pyrrole NH); 13C-NMR (DMSO-d6):
δ 13.75, 17.26, 17.98, 37.87, 43.08, 47.57, 107.29, 110.98, 113.96, 118.19, 119.07, 119.57, 124.83,
125.96, 126.98, 135.48, 137.23, 158.84, 163.23, 170.18; HRMS (ESI) m/z (M+H)+: calculated for
+
C20H23ClFN4O2 : 405.1494, found 405.1497.
3.10. Cell Culture
Non-small cell lung cancer (A549), oral epithelial (KB), large cell lung cancer (NCI-H460) and
melanoma cell lines (K111) were cultured in Dulbecco’s modified Eagle medium (GIBCO) containing
10% (v/v) inactivated newborn calf serum (Boren Bio-Pharmaceutical Co., Ltd.: Guangzhou, China), 1 nM
L-glutamine (SANGON), 105 units/mL penicillin, 100 µg/mL streptomycin. Cells were grown in a 5%
CO2 incubator at 37 °C.
3.11. Cell Growth Inhibition Assay
The antitumor activities were determined by the MTT assay. Cells (2 × 103 cells/well) were
seeded in 96-well plates. After incubation overnight, each compound, which was dissolved in
dimethylsulfoxide (DMSO) and diluted with phosphate buffer solution (PBS), was added to each well
and the cells were cultured for another two days at 37 °C. 20 µL (5 mg/mL) MTT solution was added
to per well and the culture continued for another 3–4 h. The medium was removed and 100 µL DMSO
was added to dissolve formazan crystals. Absorbance was measured at 492 nm using an ELISA reader
(Thermo). The antitumor activities were expressed as IC50.
3.12. VEGFR2 Inhibition Assay
The VEGFR2 inhibition assay was performed by enzyme-linked immunosorbent assay (ELISA) in
96-well plates pre-coated with 2.5 µg/well Poly (Glu, Tyr)4:1 as a substrate. Each well was treated with
90 µL of 5.0 µM ATP (Amresco) solution and 10 µL of one of the seven compounds at varying
concentrations, which were diluted in reaction buffer (50 mM HEPES pH 7.4, 20 mM MgCl2, 0.1 mM
Na3VO4, 1 mM DTT). The known VEGFR2 inhibitor, Su11248 (sunitinib), was used as a positive
control, and 0.1% (v/v) DMSO was utilized as the negative control. The reaction was initiated by
adding 1 µL/well of VEGFR2 tyrosine kinase. After incubation for 1 h at 37 °C, the plates were
washed three times with phosphate buffered saline containing 0.1% Tween-20 (T-PBS). Next,
anti-phosphotyrosine (PY99; 1:1,000 dilution, 100 µL/well) antibody was added. After 1 h incubation
at room temperature, the plates were washed three times, and goat anti-mouse IgG horseradish
peroxidase (100 µL/well of 1:2,000 dilution) diluted in T-PBS was added. The plates were reincubated
at room temperature for 1 h, and washed as before. Finally, color development solution (0.03% H2O2 and

Molecules 2011, 16
9383
2 mg/mL o-phenylenediamine in 0.1 M citrate buffer, pH 5.5, 100 µL/well) was added and the plates
were incubated at room temperature until color emerged. The reaction was terminated by the addition
of 2 M H2SO4 (50 µL/well), and A492 was measured using a tunable wavelength microplate reader
(Molecular Devices SPECTRAMAX190). The inhibition rate was calculated using the equation:
[1 − (A492/A492 control)] × 100%.
4. Conclusions
In summary, we have prepared eighteen novel 3-substituted-indolin-2-one derivatives containing
different N-substituted-chloropyrroloformamides, and evaluated their biological activities. The antitumor
activities were affected by the N-substituents in the amides. Compounds 14c, 14f, 14g, 14h, 14i, 14p
and 14r exhibited potent antitumor activities against the four tested tumor cell lines. Compound 14g
showed the best inhibition against VEGFR2. Compounds 14g and 14h, with a chloro group attached to
the pyrrole moiety, showed low cardiotoxicity. Further experiments for the eighteen derivatives would
be required for evaluating the inhibition activities against other RTKs VEGFR3, PDGFRβ and FGFR1
to conclude a comprehensive structure-activity relationship.
Acknowledgments
Financial support was provided by Fund of Science and Technology Development, Shanghai, China
(No. 054319907 and No. 09431901400).
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Sample Availability: Samples of the compounds are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).