Synthesis, Biological, and Computational Evaluation of Novel 1,3,5-Substituted Indolin-2-one Derivatives as Inhibitors of Src Tyrosine Kinase

Article abstract of DOI:10.1002/ardp.201500109

Several substituted indolin-2-one derivatives were synthesized and evaluated for their activities against Src kinase. Several compounds showed activity against Src, with IC₅₀ values in the low micromolar range. Among them, compound 2f showed the most significant activity with an IC₅₀ value of 1.02 μM. Molecular docking studies have been performed for evaluation of the binding modes of compound 2f into the Src active site. The docking structure of compound 2f disclosed that the indole NH forms a hydrogen bond with the carbonyl of Met341. These results suggest that our novel compound 2f is a promising compound for the further development of indole-based drugs targeting Src kinase. A novel series of indole-2-one derivatives were synthesized and evaluated for their Src inhibitory activities. Compound 2f was found as the most promising compound with an IC₅₀ value of 1.02 μM. Molecular docking studies were performed to disclose the binding mode for compound 2f.

Full text of DOI:10.1002/ardp.201500109

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Arch. Pharm. Chem. Life Sci. 2015, 348, 715–729
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Full Paper
Synthesis, Biological, and Computational Evaluation of Novel
1,3,5-Substituted Indolin-2-one Derivatives as Inhibitors of Src
Tyrosine Kinase
1
2
1,3
€
€
€
Zuhal Kilic-Kurt , Filiz Bakar , and Sureyya Olgen
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara,
Turkey
Department of Biochemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul Kemerburgaz University,
Bagcılar, Istanbul, Turkey
2
3
Several substituted indolin-2-one derivatives were synthesized and evaluated for their activities
against Src kinase. Several compounds showed activity against Src, with IC50 values in the low
micromolar range. Among them, compound 2f showed the most significant activity with an IC50 value
of 1.02 mM. Molecular docking studies have been performed for evaluation of the binding modes of
compound 2f into the Src active site. The docking structure of compound 2f disclosed that the indole
NH forms a hydrogen bond with the carbonyl of Met341. These results suggest that our novel
compound 2f is a promising compound for the further development of indole-based drugs targeting
Src kinase.
Keywords: Cancer / Indolin-2-one / Molecular docking / Src kinase / Tyrosine kinase activity
Received: March 23, 2015; Revised: July 19, 2015; Accepted: July 21, 2015
DOI 10.1002/ardp.201500109
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
addition, the Src proto-oncogene has frequently been
implicated in the initiation and progression of human
Introduction
The last two decades have witnessed the developments on Src
kinase research and selective small molecule inhibitors [1].
Increased Src protein kinase activity in numerous human
cancer cell lines has been shown as the possible reason of
several types of tumors [2, 3]. Src kinase activity is usually 4- to
20-fold higher in mammary carcinomas compared to normal
tissues [4–7]. The Src kinases have also been implicated in
other common human cancers such as lung [8], neural
(neuroblastomas, retinoblastomas) [9], ovarian [10], esoph-
ageal [11], gastric cancers [12, 13], and melanoma [14]. In
colorectal cancer [15, 16]. Src activity has recently been
studied in pancreatic cancer. Elevated levels of Src protein and
kinase activity were found in pancreatic ductal carcinomas as
well as pancreatic cell lines [17]. Src has also been implicated in
breast cancer [18]. Additionally, antisense Src expressed in
ovarian and colon tumor cells inhibited tumor growth in nude
mice and reduced vascularization [19–21] suggesting the
direct relationship between Src kinase activity and cancer. On
the basis of these findings, Src has been a candidate
therapeutic target in the treatment of cancer.
In the last two decades, a number of heteroaromatic small-
molecule inhibitor templates have been studied as Src kinase
inhibitors. Several purine, pyrrolopyrimidine, pyridopyrimi-
dine, naphthyridone, quinazoline, oxindoles, and quinoline-
based inhibitors have been reported [22, 23]. The majority of
these inhibitors exploit ATP-binding site competing with ATP
and present one to three hydrogen bonds with the critical
amino acids located in the active site of Src like adenine ring of
€
€
Correspondence: Prof. Sureyya Olgen, Department of Pharma-
ceutical Chemistry, Faculty of Pharmacy, Istanbul Kemerburgaz
University, Bagcılar, Istanbul 34217, Turkey.
E-mail: sureyya.olgen@kemerburgaz.edu.tr
Fax: þ90-212-4458171
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ATP [24]. In general, ATP-binding site inhibitors of Src kinase
contain a largely planar rigid structure, heteroaromatic ring
system that occupies the adenine binding site and exhibit
hydrophobic interactions with hydrophobic region of ATP
binding site. Additionally, these compounds have a hydrogen
bond donor/acceptor pair and form similar hydrogen bonds as
ATP at the ATP-binding site of Src kinase [25].
AutoDock Vina to evaluate the binding properties and
structure–activity relationships of the compounds.
Results and discussion
Chemistry
Indole-based derivatives showed promising inhibitory
The target compounds (2a–h, 6a–h, and 7a–h) were prepared
following the procedure reported in Scheme 1. For the
synthesis of 3-(substituted-benzylidene)-5-(4-fluorophenyl)
indolin-2-one derivatives (2a–h), 5-iodo indolin-2-one (1)
was obtained by a Wolff–Kishner reduction of 5-iodoisatin
in 50% yield [31]. Synthesis of 5-(4-fluorophenyl)indolin-
2-one (2) was achieved by palladium-catalyzed Suzuki
cross-coupling reaction of 5-iodo indolin-2-one (1) with
p-fluorophenylboronic acid [32]. For the preparation of
1-(3-substituted-benzylidene-2-oxoindolin-5-yl)-3-ethylurea
(6a–h) and 1-benzyl-3-(3-substituted-benzylidene-2-oxoindo-
lin-5-yl)thiourea (7a–h) derivatives, indolin-2-one (3) was
synthesized by Wolff–Kishner reduction of isatine with
hydrazine hydrate [31]. Then, 5-nitroindolin-2-one (4) was
prepared by stirring of indolin-2-one (3) with potassium
nitrate in concentrated sulfuric acid at 0–5°C for 30 min [33].
5-Aminoindolin-2-one (5) was generated from nitro compound
(4) by catalytic hydrogenation in moderate yield [34]. Reaction
of compound 5 with ethylisocyanate or benzylisothiocyanate
afforded the corresponding ureidoindolin-2-one derivatives
(compounds 6 or 7) [35]. Finally, the target compounds (2a–h,
6a–h, and 7a–h) were prepared by condensation of 2, 6, and 7
with substituted benzaldehydes in ethanol at reflux [36].
The synthesis of the compounds 13a–d was carried out as
outlined in Scheme 2. At first, 5-nitroisatine (8) was prepared
by stirring of isatine with NaNO3 and sulfuric acid at 0°C for 1 h
[37]. Compound 9 was synthesized by p-toluene sulfonic acid
properties of Src kinase. In the literature,
a series of
pyrrolyllactone indolinones was reported as potent Src
inhibitors; among them compound I (Fig. 1) exhibits potent
inhibitory activity against Src kinase (IC50 ¼ 1.1 mM) [26].
Recently, various substituted indolin-2-one derivatives were
reported as KDR, Flt-1, PDGFR, and FGFR1 inhibitors.
Compound II (Fig. 1) bearing amino piperidinyl at 5-position
of oxindole ring also exhibited inhibitory activity for Src
kinase at low concentration (IC50 ¼ 33 nM), when screened
against a wider panel of kinases [27]. We previously found
indole-based compound (III, Fig. 1) as potent Src inhibitor with
IC50 of 1.34 mM [28]. Compounds IV and V (Fig. 1) of a series
of 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thione
derivatives and the corresponding indolin-2-one congeners
were identified as moderately active Src inhibitors, with
IC50 of 21.91 and 21.20 mM, respectively [29]. As part of our
continuing efforts to develop potent Src inhibitors, we
designed a series of some indole-3-imines and their corre-
sponding amine derivatives, bearing indole as heterocyclic
ring system, hydrophobic side chain, and hydrogen bond
donor/acceptor group as in other ATP-competitive Src
inhibitors. Among them, compound VI (Fig. 1) was found as
a promising Src inhibitor with IC50 of 4.69 mM [30]. In the
current study, 1,3,5-substituted-indolin-2-one derivatives
were synthesized and evaluated for the effects on Src activity.
In addition, the molecular docking study was carried out by
Figure 1. Indole-based Src inhibitors.
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Scheme 1. Synthesis of 5-fluorophenyl-3-(substituted-benzylidene)indolin-2-one (2a–h), 1-(3-substituted-benzylidene-2-oxoindo-
lin-5-yl)-3-ethylurea (6a–h) and 1-benzyl-3-(3-substituted-benzylidene-2-oxoindolin-5-yl)thiourea (7a–h) derivatives. Reagents and
conditions: (a) hydrazine hydrate, 140°C, 4 h, (b) p-fluorophenyl boronic acid, Pd(PPh3)4, Na2CO3 (sat.)/DME, 85°C, 5 h, (c) substituted
benzaldehydes, piperidine, ethanol, reflux, (d) KNO3/H2SO4, 0–5°C, (e) Pd/C 10%, H2/MeOH, 1 atm, 4 h, rt, (f) Et-NCO or Ph–CH2–NCS,
ethylacetate, rt.
catalyzed cyclocondensation reaction of 8 with ethylene
glycol in toluene at 110°C [38]. Mixture of 9 and NaH was
treated with benzyl bromide in DMF to give the desired
N-benzylated compound 10, which was reduced to compound
11 by catalytic hydrogenation [37, 38]. Compound 11 was
treated with p-fluorobenzaldehyde in toluene at room
temperature in the presence of molecular sieves 4 A to afford
̊
imine derivatives of 11. Without any work-up, the reaction of
Scheme 2. Synthesis of 1-benzyl-5-(4-fluorobenzylamino)-3-(substituted-phenylimino)indolin-2-one derivatives (13a–d). Reagents
and conditions: (a) NaNO3, H2SO4, 0°C, (b) p-toluenesulfonic acid, ethylene glycol, toluene, (c) NaH, DMF, benzyl bromide, (d) Pd/C
̊
10%, H2, MeOH, 1 atm, 4 h, rt, (e) p-fluorobenzaldehyde, molecular sieves 4 A, toluene, 24 h, rt, (f) NaBH4, MeOH, 3 h, rt, (g) conc. HCl/
CH3COOH (4:1), reflux, 1 h, (h) anilin derivatives, glacial acetic acid, ethanol, 90°C.
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this product with NaBH4 in methanol afforded its amine
congener 12 [39]. Compound 13 was synthesized by hydrolysis
of 12 with a mixture of hydrochloric and acetic acid (4:1) [40].
The synthesis of the compounds 15a–d and 16a–d is
described in Scheme 3. N-Benzylation of 5-iodoisatin with
benzyl bromide using NaH in DMF afforded compound 14
[41]. Synthesis of compounds 15 and 16 was achieved by
palladium-catalyzed Suzuki cross-coupling reaction of 5-
iodoisatin and N-benzyl-5-iodoisatin (14) with p-fluorophe-
nylboronic acid [32]. Finally, the target compounds (13a–d,
15a–d, and 16a–d) were generated from 13, 15, or 16 by
condensation reaction with various anilin derivatives in
ethanol in the presence of glacial acetic acid [42].
The benzylidene derivatives (2a–h, 6a–h, and 7a–h) of
target compounds were obtained as the E- (2a–c, 2f, 2h, 6a,
6b, 6d, 6g, 7a–d, and 7g) or Z-isomer (2e and 7f) or as mixture
of both isomers (2d, 2g, 6c, 6e, 6f, 6h, 7e, and 7h) while the
imine derivatives (13a–d, 15a–d, and 16a–d) exist as mixture of
E- and Z-isomer due to the exocyclic double bond. Due to
the rapid interconversion in solution at room temperature,
the mixture of isomers could not be separated by column
chromatography.
The configurations of compounds were determined by a
nuclear Overhauser effect (NOE) experiment and 1H NMR
spectra. In benzylidene derivatives, E-isomers have a NOESY
correlation between H-4 and H-20/H-60, whereas the Z-isomers
exhibit a NOESY correlation as follows: vinyl protons and H-4.
Among the compounds obtained as isomer mixture, E/Z ratios
of compounds 2g and 6c were determined as 2:1 using the
corresponding chemical shifts and integrals in the 1H NMR
spectra. H-20 and H-60 of 2g display a down-field shift
(8.51 ppm) in the Z-isomer than E-isomer (7.79 ppm) due to
the deshielding effect of the carbonyl group at the 2-position
of indole ring. The signals from H-4 of E-isomers of 2g and 6c
(7.87 and 7.60 ppm) were markedly shifted upfield relative to
the H-4 signals of Z-isomers 2g and 6c (8.02 and 7.74 ppm) due
to the shielding effect of H-20 and H-60 of benzylidene
substituent [43, 44]. For the imine derivatives, the E-isomer
was assigned to the major isomer because of the chemical
shifts of H-4 and NOE correlation between H-4 and H-20/H-60.
The 1H NMR chemical shifts of all compounds obtained as a
mixture of isomers have been reported here to prove the
major E-isomers.
Biological evaluation
The substituted benzylidene (2a–h, 6a–h, and 7a–h) and
substituted phenylimino (13a–d, 15a–d, and 16a–d) deriva-
tives of indolin-2-one are depicted in Table 1. Compounds
were evaluated for their inhibitory activity against Src kinase
by universal tyrosine kinase assay that measures the changes
in the enzymatic activity of Src kinase by virtue of following
the alterations in the phosphorylation level of the immobi-
lized substrate that was used for this analysis [45]. The activity
of the prepared compounds was analyzed at five concen-
trations (0.1, 1, 10, 100, and 1000 mM) and IC50 values were
calculated. Imatinib was tested as reference compound in this
assay and PP1 (known as Src inhibitor) was used as reference
compound, which was previously tested in same assay method
[28]. The IC50 values of the compounds are shown in Table 1.
Benzylidene derivative 2f containing rigid p-fluorophenyl
group at 5-position of indolin-2-one ring exhibited the best
inhibitory activity with IC50 value of 1.02 mM. Compound 2f
was found 1.4- and 6-fold less potent than imatinib (IC50
¼ 0.717 mM) and PP1 (IC50 ¼ 0.17 mM), respectively. Replace-
ment of the dimethylamino group of 2f with methoxy (2g)
and chloro substituent (2a) decreased the activity to IC50
values of 1.98 and 10.08 mM. Structural comparison between
2a and 2d indicated that introduction of chloro at 30-position
of phenyl ring of benzylidene side chain in 2a resulted in
complete loss of inhibition. Compound 2b, bearing fluoro
substituent at 40-position of benzylidene moiety did not show
inhibition of Src compared with 4-dimethylaminobenzylidene
derivative 2f.
To further investigate the influence of flexible and polar
group at the 5-position of indole scaffold, we kept the side
chain at 3-position of indole as substituted benzylidene group
Scheme 3. Synthesis of 5-(4-fluorophenyl)-3-(substituted-phenylimino)indolin-2-one (15a–d) and 1-benzyl-5-(4-fluorophenyl)-3-
(substituted-phenylimino)indolin-2-one (16a–d) derivatives. Reagents and conditions: (a) p-fluorophenyl boronic acid, Pd(PPh3)4,
Na2CO3 (sat), DME, 85°C, 5 h, (b) anilin derivatives, glacial acetic acid, ethanol, 90°C, (c) NaH, DMF, benzyl bromide.
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and changed the 4-fluorophenyl moiety at 5-position of
indolin-2-one ring (2a–h) as ethylurea (6a–h) and benzylth-
iourea (7a–h). Compound 6b was found 2.8- and 12.1-fold less
active than the imatinib and PP1, respectively. The insertion of
one fluoro in the ortho position of benzylidene moiety of 6b
leading to 6e abolished the activity. The replacement of the
fluoro in 6b with a chloro (6a), dimethylamino (6f), methoxy
(6g), and carboxy (6h) resulted in either complete loss or
dramatically reduction of activity. Of the benzylthiourea (7a–h)
derivatives, 2,4-difluorophenyl substituted compound 7e
(IC50 ¼ 1.24 mM) exhibited 1.7- and 7.2-fold less activity than
the imatinib and PP1, respectively. Exchanging the 20-fluoro
substituent with hydrogen was well tolerated when com-
pounds 7e (IC50 ¼ 1.24 mM) and 7b (IC50 ¼ 1.38 mM) were
compared. The carboxy substitution at 40-position of the 7h
(IC50 ¼ 2.01 mM) slightly decreased Src inhibition compared
with 40-fluorobenzylidene substituted compound 7b (IC50
¼ 1.38 mM). Compounds, bearing chloro (7a), dimethylamino
(7f), and methoxy (7g) substituents at the 40-position of
benzylidene moiety did not show inhibition of Src. Moving of
fluoro substituent from40- to the 30-position on the phenyl ring
of 6b and 7b, leading to 6c and 7c, resulted in complete loss of
inhibiton. FromSrcactivityresultsofbenzylidenederivatives, it
hasbeensuggestedthathydrophobicandrigidp-fluorophenyl
group at 5-position of indolin-2-one ring significantly
increased the potency in the case of 2a, 2f, and 2g relative
to their ethylurea (6a, 6f, and 6g) and benzylthiourea
derivatives (7a, 7f, and 7g). On the contrary, the polar and
flexible ethylurea and benzylthiourea substituents at the 5-
position of indole such as the cases of 6b and 7b significantly
improved the activity than p-fluorophenyl substiuted com-
pound 2b. Although the presence of ethylurea or benzylth-
iourea substitution at 5-position of indole ring has no
significant impact on activity when compounds 6b and 7b
are compared, benzylthiourea moiety increased the activity of
7e and 7h relative to ethylurea congeners 6e and 6h.
Among the imine derivatives, 16d exhibited the moderate
inhibitory activity against Src with IC50 values of 4.04 mM.
Replacement of indole hydrogen of 15d (IC50 ¼ 7.48 mM) with
benzyl group, 16d, brought approximately twofold increase
of Src inhibitory activity. Introduction of chloro substituent in
30 positions of 15a and 16a, leading to 15d and 16d,
significantly increased potency, while the activity was not
effected by similar modification of 13a to corresponding 13d.
Changing the rigid p-fluorophenyl group of 16d to flexible
benzylamino as in 13d resulted in dramatic reduction of Src
inhibitory activity. On the contrary, 13b exhibited weak
inhibition against Src compared with 16b. Compounds 13a–d,
15a–c, and 16a–c did not inhibit Src kinase.
In general, the hydrophobic compounds with logP value
ranging from 4 to 7 (Table 1) were found more active than
hydrophilic compounds (logP ¼ 1.4–2.95). This indicates that
lipophilicity of compounds would help improve inhibitory
activity and binding affinity with hydrophobic interactions.
When imine and benzylidene derivatives are structurally
compared, it was observed that introduction of nitrogen
instead of CH of benzylidene derivatives resulted in less Src
inhibitory activity.
Molecular docking
In this study, the active compounds (2f, 2g, 6b, 7b, 7e, 7h,
15d, and 16d) were docked into the active site of Src
tyrosine kinase to understand their binding modes by the
docking program AutoDock Vina 1.0.2 software package. At
the first step, the validation of the scoring function
implemented in AutoDock Vina was done by docking the
native ligand (PP2) into its binding site. The docked results
were compared to the crystal structure of the bound PP2–Src
complex (PDB code: 3GEQ). The obtained success rate is
excellent as the docked PP2 appeared to be superimposed
almost exactly on the native ligand. The RMSD of docked
̊
ligand (PP2) was 0.104 A. Binding mode of PP2 in Src kinase
has been extensively studied. Two highly conserved hydro-
gen bonds are created by N5 of PP2 and amino group of C4
of PP2 with NH group of Met 341 and carbonyl of Glu 339,
respectively [46]. The docked PP2 exhibited one hydrogen
bond between N5 of PP2 and NH of Met 341, whereas the
native ligand exhibited two hydrogen bonds with Met 341
and Glu 339.
Molecular insights based on molecular docking indicated
that favorable binding interactions of the active compounds
with the binding site of Src were visually analyzed. The best
active compound 2f located in somewhat similar position to
the PP2 with overlapping p-dimethylaminobenzylidene at 3-
position of indole ring and p-chlorophenyl group of PP2 and
showed a hydrogen bond with Met341 of critical amino acids
for the binding to the Src active site. Dimethylaminobenzyl-
idene moiety of 2f inserted into the hydrophobic pocket
surrounded by Val281, Lys295, Ile336, Ala403, and Phe405.
Moreover, p-fluorophenyl group at 5-position of indole ring
was directed toward the solvent accessible region as tert-
butyl group of PP2 (Fig. 2). In addition to hydrophobic
interactions, hydrogen bond interaction with critical amino
acid Met341 may account for the inhibitory activity of 2f at
low micromolar level comparable with PP1. Compound 2g
occupied the ATP-binding site with no hydrogen bond being
formed with backbone of the hinge region residues Glu339
and Met341. Compound 6b formed
a hydrogen bond
between carbonyl of ethylurea moiety and NH of Lys295.
Compounds 7b and 7h located in the active site of Src kinase
by forming hydrogen bonding interactions between their
side chain NH group at 5-position of indole ring and
backbone carbonyl groups of Leu273. Compound 7e showed
a hydrogen bond contact between its indole carbonyl and
hydroxy of Thr338. Among the active compounds, imine
derivatives 15d and 16d were not involved in direct hydrogen
bonding interactions with Src kinase. 4-Fluoro-3-chloro-
phenylimino side chain of these compounds overlapped
p-chlorophenyl group of PP2 and fitted into the hydrophobic
pocket like 2f. When conformations of 15d and 16d were
compared with 2f, the slight difference was observed in
position of p-fluorophenyl group of 15d and 16d, lying in the
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nitrate, 5-iodoisatin, ethylisocyanate, benzylisothiocyanate,
5-iodoisatin, dichlorometane, p-fluoroanilin, p-chloroanilin,
toluene, Pd/C 10%, and NaBH4 were purchased from Aldrich.
4-Fluoro-3-chloroanilin and 4-methylthioanilin were pur-
chased from Acros and Lancaster, respectively. HCl, piperidine,
glacial acetic acid, and ethanol were purchased from Riedel
de Haen. 4-Methoxybenzaldehyde, 3-fluorobenzaldehyde
from Fluka were used for reactions. Solvents and reagents
were used without further purification. Analytical TLC was
carried out on Merck 0.2 mm pre-coated silica gel (60 F-254)
aluminum sheets (Merck), visualization by irradiation with an
UV lamp. Takara Universal Tyrosine Assay Kit (from Takara-Bio
Inc., Shiga, Japan) was used to test our compounds for
their Src kinase inhibition. The contents of a kit: PTK
substrate immobilized microplate (8 wells ꢀ 12), kinase react-
ing solution (11.0 mL), 40 mM ATP-2Na (0.55 mL), extraction
buffer (11.0 mL), PTK control (0.50 mL), anti-phoshotyrosine
(PY20-HRP, for 5.5 mL/H2O), blocking solution (11.0 mL),
horseradish peroxidase (HRP) coloring solution (tetramethyl-
benzidine, TMBZ,12.0 mL).
Melting points were measured with a capillary melting
point apparatus (BUCHI Melting Point B-540). The nuclear
magnetic resonance (1H NMR and 13C NMR) spectra were
recorded on Varian Mercury 400 NMR spectrometer 400 MHz
(Varian Inc., Palo Alto, CA, USA). The chemicals shift values
were expressed in parts per million (ppm) relative to
tetramethylsilane as an internal standard. Mass spectra were
recorded on a Waters ZQ Micromass LC-MS spectrometer
(Waters Corporation, Milford, MA, USA). Elemental analysis
was taken on a Leco-932 CHNS-O analyzer (Leco, St. Joseph,
MI, USA).
Figure 2. Comparison of PP2 (pink) and compound 2f (colored
by element) binding properties in the active site of Src. H-bond
is represented as yellow dashed lines.
vertical direction to the p-fluorophenyl of 2f. The decrease in
activity of 15d and 16d relative to 2f can be the result of
different conformations from 2f. In general, the binding
modes predicted by AutoDock modeling between the Src
and active compounds were correlated with inhibitory
activity.
Conclusion
In this study, we designed and synthesized novel 1,3,5-
substituted indolin-2-one derivatives as Src kinase inhibitors.
Some of the compounds (2f, 2g, 6b, 7b, 7e, 7h, 15d, and 16d)
exhibited single digit micromolar inhibition of Src. The best
activity was obtained by compound 2f with IC50 value of
1.02 mM. The compound 2f showed comparable activity with
PP1 and imatinib in Src kinase assay. In addition, the most
active compound 2f was docked into the Src active site and
favorable protein–ligand interactions were visually analyzed.
Results of a virtual docking study of compound 2f demon-
strated that it located into the active site of Src similar to PP2
and formed a hydrogen bond interaction with Met341
backbone amino acid of Src like PP2. The results reported
here provide a foundation for discovery of more active novel
indolin-2-one compounds as promising compounds for Src
kinase target in the future.
Synthesis of compounds 1–16
5-Iodoindolin-2-one (1)
A
stirred solution of 5-iodoisatin (0.01 mol; 3.06 g) in
hydrazine hydrate (15 mL) was heated to 140°C for 4 h. The
reaction was cooled to room temperature, poured into ice-
cold water, and acidified to pH 2 with 6 N hydrochloric acid.
After standing at room temperature for 2 days the precipitate
was collected by vacuum filtration, washed with water, and
dried in vacuo to give pure compound 1. Yield 50% (1.45 g),
mp 175–178°C (lit. [47], 170–172°C).
5-(4-Fluorophenyl)indolin-2-one (2)
5-Iodoindolin-2-one (1, 9.25 mmol; 2.39 g) was dissolved in
150 mL DME. Pd(PPh3)4 (0.46 mmol; 0.53 g), p-fluorophenyl-
boronic acid (0.01 mol; 1.55 g), and Na2CO3 solution (0.01 mol;
1.55 g in 30 mL H2O) were added, respectively. The mixture
was boiled at 80°C for 10 h. DME was removed under reduced
pressure, then the water layer was extracted by ethylacetate
(3 ꢀ 100 mL). The organic layer was washed with H2O
(2 ꢀ 10 mL), brine (10 mL), dried over anhydrous Na2SO4,
and concentrated at reduced pressure. The crude product was
purified by column chromatography (hexane/ethylacetate
¼ 6:3) to give pure compound 2. Yield: 26% (0.55 g), mp 211–
212°C [48].
Experimental
Chemistry
Pd(PPh3)4, p-fluorophenyl boronic acid, DME, hydrazine
hydrate, NaHCO3, ethylacetate, Na2SO4, molecular sieves
̊
4 A, anhydrous DMF, NaH, NaNO3, methanol, p-toluene-
sulfonic acid, and ethylene glycol were purchased from
Merck. 4-Fluorobenzaldehyde, 2,4-difluorobenzaldehyde,
4-chlorobenzaldehyde, N,N-dimethylaminobenzaldehyde,
3,4-dichlorobenzaldehyde, 4-carboxybenzaldehyde, potassium
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Indolin-2-one (3)
50-Nitrospiro[[1,3]dioxolane-2,30-indolin]-20-one (9)
Indolin-2-one was synthesized from isatin according to the
same procedure of compound 1. Yield: 41% (2.58 g), mp 127°C
(lit.[31], 127–129°C).
Ethylene glycol (0.093 mol; 5.20 mL) and a catalytic amount
of p-toluenesulfonic acid (0.63 mmol; 0.12 g) was added to
a
suspension of 5-nitroisatin (8, 0.024 mol; 4.77 g) in
toluene (300 mL). The reaction mixture then was refluxed
for 8 h using a Dean–Stark apparatus. The reaction mixture
was cooled, and the solution was concentrated in vacuo
and kept overnight at room temperature. The crude
product was recrystallized from toluene to give ketal of
5-nitroisatin (9). Yield: 81% (4.64 g), mp 216°C (lit. [38],
216–217°C).
5-Nitroindolin-2-one (4)
Isatin(0.03 mol;5 g)wasdissolvedin28 mLofcoldconcentrated
sulfuric acid at 0°C. After complete dissolution, potassium
nitrate (0.03 mol; 3.87 g) was added as
temperature of the mixture should not exceed 5°C. After
further stirring for 30min, the mixture was added to 200mL of
crushed ice. The precipitate was collected by filtration, washed
with water, and dried. The raw product was purified by
recrystallization from acetic acid (50%) to give pure compound
4. Yield: 35% (2.37 g), mp 240–243°C (lit. [33], 249–254°C).
a portion. The
10-Benzyl-50-nitrospiro[[1,3]dioxolane-2,30-indolin]-20-one
(10)
50-Nitrospiro[[1,3]dioxolane-2,30-indolin]-20-one (9, 0.019 mol;
4.64 g) and NaH (0.059 mol; 1.41 g) were stirred in anhydrous
dimethylformamide (10 mL) at 0°C, then stirred for 15 min at
room temperature. To the mixture, benzyl bromide
(0.019 mol; 2.35 mL) was added at 0°C. After being stirred
for 5 h at room temperature, the mixture was poured onto ice
and the product filtered. The crude product was washed with
cold water and dried to give 10. Yield: 72% (3.53 g), mp 164–
166°C. 1H NMR (DMSO-d6, 400 MHz): 4.42 (s, 4H, –(CH2)2–),
4.95 (s, 2H, –CH2–Ph), 7.25 (d, J ¼ 8.8 Hz, 1H, H-7), 7.29–7.31 (m,
3H, aromatic protons), 7.35–7.38 (m, 2H, aromatic protons),
8.25 (d, J ¼ 2.4 Hz, 1H, H-4), 8.32 (dd, Jo ¼ 8.0 Hz, Jm ¼ 2.4 Hz,
1H, H-6). MS (ESI): m/z 327.45 (MþH).
5-Aminoindolin-2-one (5)
Ten percent Pd/C (0.092 g) was added to a solution of 5-
nitroindolin-2-one (4, 2.07 mmol; 0.36 g) in 5 mL of methanol.
The mixture was hydrogenated (45 psi H2) for 3 h at room
temperature. Then the solution was filtered with celite and
filtrate was evaporated. The crude product was purified by
column chromatography (CH2Cl2/MeOH ¼ 9.5:0.5) to give
compound 5. Yield: 64% (0.19 g), mp 202–204°C (lit.[49],
213–214°C).
1-Ethyl-3-(2-oxoindolin-5-yl)urea (6)
Ethylisocyanate (3.24 mmol; 0.25 mL) was added to a solution
of 5-aminoindolin-2-one (5, 2.71 mmol; 0.4 g) in 5 mL of
ethylacetate. The mixture was stirred at the room tempera-
ture for 24 h. The precipitate was filtered, washed with
diethylether, and dried to give compound 6. Yield: 85%
(0.5 g), mp 183°C. 1H NMR (DMSO-d6) d: 0.99 (t, 3H, CH3), 3.02–
3.05 (m, 2H, –CH2–CH3), 3.37 (s, 2H, H-3), 5.93 (t, 1H,
NH–CO–NHC2H5), 6.62 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.05 (dd, 1H,
Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.27 (s, 1H, H-4), 8.14 (s, 1H,
NH–CO–NHC2H5), 10.13 (s, 1H, indole-NH). MS (ESI): m/z 220.6
(MþH).
50-Amino-10-benzylspiro[[1,3]dioxolane-2,30-indolin]-20-
one (11)
Compound 10 (3.11 mmol; 1.01 g) was dissolved in methanol
(20 mL) and palladium on activated carbon (0.14 g; % 10)
added to the solution at room temperature. The reaction
mixture was stirred under a hydrogen atmosphere for 4 h. The
catalyst was then removed by filtration over celite. The filtrate
was concentrated in vacuo to give crude product, which was
recrystallized from ethanol to give 11. Yield: 59% (0.55 g), mp
136–137°C. 1H NMR (DMSO-d6, 400 MHz): 4.24 (t, J ¼ 6.8 Hz,
2H, –CH2–), 4.39 (t, J ¼ 6.8 Hz, 2H, –CH2–), 4.73 (s, 2H, –CH2–Ph),
5.00 (s, 2H, –NH2), 6.50 (dd, Jo ¼ 8.0 Hz, Jm ¼ 2.4 Hz, 1H, H-6),
6.61 (d, J ¼ 8 Hz, 1H, H-7), 6.65 (d, Jm ¼ 2.4 Hz, 1H, H-4), 7.24–
7.27 (m, 3H, Ar-H), 7.31–7.35 (m, 2H, Ar–H). MS (ESI): m/z 297.6
(MþH).
1-Benzyl-3-(2-oxoindolin-5-yl)thiourea (7)
The compound was prepared from 5 (2.22 mmol; 0.33 g) and
benzylisothiocyanate (2.67 mmol; 0.35 mL) according to the
same procedure of compound 6. Yield: 67% (0.44 g), mp 248–
250°C. 1H NMR (DMSO-d6) d: 4.72 (d, 2H, J ¼ 4.4 Hz, CH2), 6.80
(d, 1H, Jo ¼ 8.8 Hz, H-7), 7.10 (dd, 1H, Jo ¼ 8.8 Hz, Jm ¼ 1.6 Hz,
H-6), 7.20–7.40 (m, 5H, aromatic benzyl protons), 7.60 (s, 1H,
10-Benzyl-50-(4-fluorobenzylamino)spiro[[1,3]dioxolane-
2,30-indolin]-20-one (12)
–
H-4), 8.01 (s, 1H, NH–CH –Ph), 9.45 (s, 1H, –NH–C S), 10.63
To a suspension of compound 11 (2.27 mmol; 0.67 g) and
–
2
̊
(s, 1H, indole-NH). MS (ESI): m/z 298.6 (MþH).
molecular sieves 4 A (1 g) in dry toluene (15 mL) was added
p-fluorobenzaldehyde (2.27 mmol; 0.24 mL), and then the
mixture was stirred at room temperature for 48 h. The
mixture was mixed with 10 mL of methanol and sodium
borohydride (4.55 mmol; 0.17 g) was added in portions at
room temperature. After completion of the reaction, the
mixture was filtered. Then, the filtrate was concentrated and
5% NaHCO3 was added. The organic material was extracted
with dichloromethane, the combined organic layer was
5-Nitroisatine (8)
A solution of NaNO3 (0.034 mol; 2.91 g) in H2SO4 (50 mL) at 0°C
was added dropwise over 1 h to a solution of isatin (0.034 mol;
5.04 g) in H2SO4 (58 mL). The reaction mixture then was
poured onto ice and the resulting precipitate was filtered
and washed with water to yield 5-nitroisatin (8). Yield: 72%
(4.77 g), mp 254–256°C (lit. [40], 252–254°C).
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washed with water and brine, then dried over anhydrous
Na2SO4. The solvent was evaporated and the residual solid
was crystallized from hot ethanol to afford compound 12.
of 1-benzyl-5-iodoindoline-2,3-dione (14, 1.93 mmol; 0,7 g) in
DME (50 mL) under nitrogen. The reaction mixture was
refluxed for 7 h. The organic solvent was removed under
reduced pressure. The residue was extracted with ethyl-
acetate and the combined organic layer was washed with
water and brine, then dried over anhydrous Na2SO4. The
solvent was evaporated and the crude product was purified by
column chromatography (hexane/ethylacetate ¼ 6:3) to af-
ford compound 16. Yield: 41% (0.26 g), mp 162–163°C. 1H
NMR (DMSO-d6, 400 MHz): 4.95 (s, 2H, –CH2), 7.04 (d, 1H,
Jo ¼ 8.4 Hz, H-7), 7.24–7.31 (m, 3H, aromatic protons), 7.35 (t,
2H, J ¼ 7.6 Hz, H-30, H-50), 7.46 (d, 2H, Jo ¼ 7.2 Hz, aromatic
protons), 7.68–7.73 (m, 2H, aromatic protons), 7.83 (d, 1H,
Jm ¼ 2.0 Hz, H-4), 7.87 (dd, 1H, Jo ¼ 8.0 Hz; Jm ¼ 2.0 Hz, H-6). MS
(ESI): m/z 332.8 (MþH).
1
Yield: 64% (0.59 g), mp 150°C. H NMR (DMSO-d6, 400 MHz):
4.16 (d, J ¼ 5.6 Hz, 2H, –NH–CH2–), 4.20 (t, J ¼ 6.8 Hz, 2H,
–CH2–), 4.35 (t, J ¼ 6.8 Hz, 2H, –CH2–), 4.68 (s, 2H, –CH2–Ph),
6.10 (t, J ¼ 6 Hz, 1H, –NH–), 6.42 (dd, Jo ¼ 8.0 Hz, Jm ¼ 2.4 Hz,
1H, H-6) 6.61 (d, J ¼ 8.8 Hz, 1H, H-7), 6.65 (d, J ¼ 2.4 Hz, 1H, H-
4), 7.08 (t, J ¼ 8.8 Hz, 2H, aromatic protons), 7.21–7.33 (m, 7H,
aromatic protons). MS (ESI): m/z 405.9 (MþH).
1-Benzyl-5-(4-fluorobenzylamino)indoline-2,3-dione (13)
Compound 12 (1.46 mmol; 0.59 g) was heated at reflux in a
mixture of concentrated hydrochloric acid (12 mL) and acetic
acid (3 mL) for 1 h. The reaction mixture was cooled to room
temperature. Removal of hydrochloric acid and acetic acid
under reduced pressure gave a crude product which was
recrystallized from ethanol to afford compound 13. Yield:
81% (0.43 g), mp 234°C. 1H NMR (DMSO-d6, 400 MHz): 4.25 (s,
2H, –CH2–NH–), 4.80 (s, 2H, –CH2–Ph), 6.07 (t, 1H, J ¼ 6.0 Hz,
–NH–), 6.72 (d, J ¼ 8 Hz, 1H, H-7), 6.84 (t, J ¼ 8.4 Hz, 2H,
aromatic protons), 7.14 (t, J ¼ 8.8 Hz, 2H, aromatic protons),
7.24–7.39 (m, 7H, aromatic protons). MS (ESI): m/z 361.7
(MþH).
General procedure for the synthesis of target compounds
2a–h, 6a–h, and 7a–h
A catalytic amount of piperidine (0.01 mmol) was added into a
solution of compounds 2, 6, and 7 (1 mmol) and substituted
benzaldehyde (1.1 mmol) in ethanol (5 mL). The mixture was
heated at reflux for 3–5 h. After cooling at room temperature,
the precipitate was filtered, washed with ethanol, and dried
at room temperature.
1-Benzyl-5-iodoindoline-2,3-dione (14)
(E)-3-(4-Chlorobenzylidene)-5-(4-fluorophenyl)indolin-2-
one (2a)
5-Iodoisatin (2.51 mmol; 0.68 g) was dissolved in anhydrous
dimethylformamide (3 mL) and NaH (7.54 mmol; 0.18 g) was
added at 0°C, then stirred for 15 min at room temperature.
Benzyl bromide (2.51 mmol; 0.3 mL) was added to this
mixture at 0°C. After being stirred for 2 h at room
temperature, the mixture was extracted with EtOAc. The
combined organic layers were washed with water and brine,
then dried over anhydrous Na2SO4. The solvent was
evaporated and the crude product was crystallized from
ethanol to afford compound 14. Yield: 41% (0.38 g), mp 150–
153°C (lit. [41], 149–152°C).
Yield: 80%, mp 190–192°C. 1H NMR (400 MHz, DMSO-d6) d: 6.97
(d, 1H, Jo ¼ 8.4 Hz, H-7), 7.25 (t, 2H, J ¼ 8.8 Hz, H-300, H-500), 7.48–
7.52 (m, 3H, H-6, H-200, H-600), 7.62 (d, 2H, Jo ¼ 8.4 Hz, H-20, H-60),
7.65 (s, 1H, H-vinyl), 7.69 (d, 1H, Jm ¼ 2.0 Hz, H-4), 7.83 (d, 2H,
Jo ¼ 8.4 Hz, H-30, H-50), 10.74 (s, 1H, NH). 13C NMR (400 MHz,
DMSO-d6) d: 168.54, 162.71, 160.29, 142.51, 136.73, 134.88,
134.31, 133.60, 133.23, 132.54, 131.27, 129.03, 128.80, 128.11,
128.03, 127.97, 121.30, 120.70, 115,80, 115.69, 110.55. MS (ESI):
m/z 350.8 (MþH). Elemental analysis calculated (%) for
C
21H13ClFNO ꢁ 0.04 H2O: C 71.96, H 3.76, N 3.99. Found: C
71.64, H 3.54, N 4.22.
5-(4-Fluorophenyl)indoline-2,3-dione (15)
Pd(PPh3)4 (0.42 mmol; 0.49 g), p-fluorophenyl boronic acid
(0.01 mol; 1.43 g), and sodium hydrogen carbonate
(0.017 mol; 143 g) in H2O (30 mL) were added into a solution
of 5-iodoisatin (8.5 mmol; 2.33 g) in DME (145 mL) under
nitrogen. The reaction mixture was refluxed for 5 h. The
organic solvent was removed under reduced pressure. The
residue was extracted with ethylacetate and the combined
organic layer was washed with water and brine, then dried
over anhydrous Na2SO4. The solvent was evaporated and the
crude product was purified by column chromatography
(hexane/ethylacetate ¼ 6:3) to afford compound 15. Yield
25% (0.5 g), mp 239–240°C [50].
(E)-3-(4-Fluorobenzylidene)-5-(4-fluorophenyl)indolin-2-
one (2b)
Yield: 72%, mp 249–252°C. 1H NMR (400 MHz, DMSO-d6) d:
6.97 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.24 (t, 2H, J ¼ 8.8 Hz, H-300, H-500),
7.40 (t, 2H, J ¼ 8.8 Hz, H-30, H-50), 7.48–7.52 (m, 3H, H-6, H-200,
H-600), 7.67 (s, 1H, H-vinyl), 7.71 (d, 1H, Jm ¼ 1.6 Hz, H-4), 7.84–
7.88 (m, 2H, H-20, H-60), 10.73 (s, 1H, NH). MS (ESI): m/z 334.6
(MþH). Elemental analysis calculated (%) for C21H13F2NO: C
75.67, H 3.93, N 4.20. Found: C 75.46, H 4.17, N 4.36.
(E)-3-(3-Fluorobenzylidene)-5-(4-fluorophenyl)indolin-2-
one (2c)
Yield: 75%, mp 230–234°C. 1H NMR (400 MHz, DMSO-d6) d:
6.95 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.22 (t, 2H, J ¼ 8.8 Hz, H-300, H-500),
7.32 (t, 1H, H-40 or H-50), 7.42–7.46 (m, 2H, H-200, H-600), 7.49 (dd,
1H, Jo ¼ 8.0 Hz, Jm ¼ 1.6 Hz, H-6), 7.55–7.62 (m, 3H, H-20, H-60,
H-40, or H-50), 7.64 (s, 1H, H-vinyl), 7.65 (d, 1H, Jm ¼ 1.6 Hz, H-4),
1-Benzyl-5-(4-fluorophenyl)indoline-2,3-dione (16)
Pd(PPh3)4 (9.66 mmol; 0.11 g), p-fluorophenyl boronic acid
(2.32 mmol; 0.32 g), and sodium hydrogen carbonate
(3.86 mmol; 0.32 g) in H2O (6.5 mL) were added into a solution
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10.74 (s, 1H, NH). MS (ESI): m/z 334.69 (MþH). Elemental
analysis calculated (%) for C21H13F2NO: C 75.67, H 3.93, N 4.20.
Found: C 75.53, H 3.86, N 4.29.
(E)-3-(4-Carboxybenzylidene)-5-(4-fluorophenyl)indolin-
2-one (2h)
Yield: 81%, mp 275–279°C. 1H NMR (400 MHz, DMSO-d6) d:
6.98 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.22–7.26 (t, 2H, H-300, H-500), 7.47–
7.52 (m, 3H, H-200, H-600, H-6), 7.70 (s, 1H, H-vinyl), 7.75 (d, 1H,
Jm ¼ 1.6 Hz, H-4), 7.81 (d, 2H, Jo ¼ 7.6 Hz, H-20, H-60), 8.04 (d, 2H,
Jo ¼ 8.4 Hz, H-30, H-50), 10.78 (s, 1H, NH), 13.74 (s, 1H, COOH).
MS (ESI): m/z 360.8 (MþH). Elemental analysis calculated (%)
for C22H14FNO3 ꢁ 0.5 H2O: C 71.73, H 4.10, N 3.80. Found: C
71.60, H 4.12, N 3.82.
(E)-3-(3,4-Dichlorobenzylidene)-5-(4-fluorophenyl)-
indolin-2-one (2d)
Yield: 86%, mp 220–222°C. 1H NMR (400 MHz, DMSO-d6) d:
major E-isomer: 6.97 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.25 (t, 2H,
J ¼ 8.8 Hz, H-300, H-500), 7.50–7.54 (m, 3H, H-200, H-600, H-6), 7.61
(s, 1H, H-vinyl), 7.69 (d, 1H, Jm ¼ 1.2 Hz, H-4), 7.79 (d, 2H,
Jo ¼ 8.0 Hz, H-50, H-60), 8.10 (d, 1H, Jm ¼ 1.2 Hz, H-20), 10.76 (s,
1H, indole-NH). MS (ESI): m/z 385.79 (MþH). Elemental
analysis calculated (%) for C21H12Cl2FNO: C 65.64, H 3.15, N
3.65. Found: C 65.80, H 3.02, N 3.80.
(E)-1-(3-(4-Chlorobenzylidene)-2-oxoindolin-5-yl)-3-
ethylurea (6a)
Yield: 76%, mp 285–287°C. 1H NMR (400 MHz, DMSO-d6) d:
1.01 (t, 3H, CH3), 3.04–3.07 (m, 2H, CH2), 5.93 (t, 1H,
NH–CO–NHC2H5), 6.74 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.30 (dd, 1H,
Jo ¼ 8.4 Hz, Jm ¼ 1.6 Hz, H-6), 7.53 (s, 1H, H-vinyl), 7.56 (d, 2H,
Jo ¼ 8.4 Hz, H-20, H-60), 7.60 (d, 1H, Jm ¼ 2.0 Hz, H-4), 7.74 (d, 2H,
Jo ¼ 8.4 Hz, H-30, H-50), 8.18 (s, 1H, NH–CO–NHC2H5), 10.42 (s,
1H, indole-NH). 13C NMR (400 MHz, DMSO-d6) d: 168.52,
155.28, 137.36, 136.43, 134.47, 134.00, 133.91, 133.26, 131.15,
128.79, 128.65, 128.55, 120.61, 120.48, 113.40, 109.95, 33.95,
15.40. MS (ESI): m/z 342.5 (MþH), 344.9 (MþHþ2). Elemental
analysis calculated (%) for C18H16ClN3O2: C 63.25, H 4.72, N
12.29. Found: C 63.51, H 4.78, N 12.27.
(Z)-3-(2,4-Difluorobenzylidene)-5-(4-fluorophenyl)-
indolin-2-one (2e)
Yield: 24%, mp 245–247°C. 1H NMR (400 MHz, DMSO-d6) d:
6.90 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.19 (t, 1H, H-30 or H-50), 7.28 (t,
2H, J ¼ 8.4 Hz, H-300, H-500), 7.36 (t, 1H, H-30 or H-50), 7.53 (dd,
1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.72–7.76 (m, 2H, H-200, H-600),
7.92 (s, 1H, H-vinyl), 8.08 (d, 1H, Jm ¼ 1.6 Hz, H-4), 8.56–8.62 (m,
1H, H-60), 10.72 (s, 1H, NH). MS (ESI): m/z 352.69 (MþH).
Elemental analysis calculated (%) for C21H12F3NO ꢁ 0.9 H2O: C
68.62, H 3.78, N 3.81. Found: C 68.31, H 3.47, N 3.61.
(E)-3-(4-(Dimethylamino)benzylidene)-5-(4-fluorophenyl)-
indolin-2-one (2f)
(E)-1-(3-(4-Fluorobenzylidene)-2-oxoindolin-5-yl)-3-
ethylurea (6b)
Yield: 94%, mp 269–271°C. 1H NMR (400 MHz, DMSO-d6) d:
3.03 (s, 6H, N(CH3)2), 6.84 (d, 2H, Jo ¼ 9.2 Hz, H-30, H-50), 6.94 (d,
1H, Jo ¼ 8.0 Hz, H-7), 7.25 (t, 2H, H-300, H-500), 7.44 (dd, 1H,
Jo ¼ 8.0 Hz, Jm ¼ 1.6 Hz, H-6), 7.55–7.58 (m, 2H, H-200, H-600), 7.57
(s, 1H, H-vinyl), 7.72 (d, 2H, Jo ¼ 8.8 Hz, H-20, H-60), 8.00 (d, 1H,
Jm ¼ 1.6 Hz, H-4), 10.55 (s, 1H, NH). 13C NMR (400 MHz, DMSO-
d6) d: 169.46, 162.62, 160.20, 151.47, 141.48, 137.90, 137.21,
137.18, 132.19, 132.08, 132.02, 128.15, 128.07, 127.33, 122.51,
121.95, 120.95, 119.89, 115.77, 115.56, 111.41, 110.04, 39.65.
MS (ESI): m/z 359.7 (MþH). Elemental analysis calculated (%)
for C23H19FN2O: C 77.08, H 5.34, N 7.82. Found: C 77.40, H 5.43,
N 7.59.
Yield: 85%, mp 275–277°C. 1H NMR (400 MHz, DMSO-d6) d:
0.98 (t, 3H, CH3), 2.98–3.05 (m, 2H, CH2), 5.89 (t, 1H,
NH–CO–NHC2H5), 6.70 (d, 1H, Jo ¼ 8.8 Hz, H-7), 7.23 (dd, 1H,
Jo ¼ 8.4 Hz, Jm ¼ 1.6 Hz, H-6), 7.31 (t, 2H, J ¼ 8.8 Hz, H-30, H-50),
7.52 (s, 1H, H-vinyl), 7.60 (d, 1H, Jm ¼ 2.0 Hz, H-4), 7.72–7.76 (m,
2H, H-20, H-60), 8.13 (s, 1H, NH–CO–NHC2H5), 10.38 (s, 1H,
indole-NH). MS (ESI): m/z 326.8 (MþH). Elemental analysis
calculated (%) for C18H16FN3O2: C 66.45, H 4.96, N 12.92.
Found: C 66.35, H 5.08, N 12.93.
(E:Z ¼ 2:1)-1-(3-(3-Fluorobenzylidene)-2-oxoindolin-5-yl)-
3-ethylurea (6c)
Yield: 73%, mp 320–325°C. 1H NMR (400 MHz, DMSO-d6) d:
E-isomer: 0.97 (t, 3H, CH3), 2.99–3.02 (m, 2H, CH2), 5.89 (t, 1H,
NH–CO–NHC2H5), 6.71 (d, 1H, Jo ¼ 8.8 Hz, H-7), 7.22 (dd, 1H,
Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.25–7.31 (m, 1H, aromatic
proton), 7.51 (s, 1H, H-vinyl), 7.45–7.53 (m, 3H, aromatic
protons), 7.60 (d, 1H, Jm ¼ 2.0 Hz, H-4), 8.13 (s, 1H,
NH–CO–NHC2H5), 10.40 (s, 1H, indole-NH); Z-isomer: 1.02 (t,
3H, CH3), 3.02–3.10 (m, 2H, CH2), 6.02 (t, 1H, NH–CO–NHC2H5),
6.67 (d, 1H, Jo ¼ 8.8 Hz, H-7), 7.07 (dd, 1H, Jo ¼ 8.4 Hz,
Jm ¼ 2.0 Hz, H-6), 7.62 (s, 1H, H-vinyl), 7.45–7.53 (m, 2H, H-
40, H-50), 7.74 (d, 1H, Jm ¼ 2.0 Hz, H-4), 7.97 (d, 1H, Jo ¼ 8.0 Hz,
aromatic proton), 8.19 (s, 1H, NH–CO–NHC2H5), 8.50 (d, 1H,
Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, aromatic proton), 10.47 (s, 1H, indole-
NH). MS (ESI): m/z 326.9 (MþH). Elemental analysis calculated
(%) for C18H16FN3O2: C 66.45, H 4.96, N 12.92. Found: C 66.48,
H 5.13, N 12.85.
(E:Z ¼ 2:1)-3-(4-Methoxybenzylidene)-5-(4-fluorophenyl)-
indolin-2-one (2g)
Yield: 94%, mp 269–271°C. 1H NMR (400 MHz, DMSO-d6) d: E-
isomer: 3.80 (s, 3H, OCH3), 6.96 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.12
(d, 2H, Jo ¼ 8.8 Hz, H-30, H-50), 7.23–7.31 (m, 2H, H-300, H-500),
7.48–7.54 (m, 3H, H-200, H-600, H-6), 7.64 (s, 1H, H-vinyl), 7.79
(d, 2H, Jo ¼ 8.8 Hz, H-20, H-60), 7.87 (d, 1H, Jm ¼ 1.6 Hz, H-4),
10.66 (s, 1H, NH). Z-isomer: 3.80 (s, 3H, OCH3), 6.89 (d, 1H,
Jo ¼ 8.4 Hz, H-7), 7.06 (d, 2H, Jo ¼ 8.8 Hz, H-30, H-50), 7.23–7.31
(m, 2H, H-300, H-500), 7.48–7.54 (m, 1H, H-6), 7.71–7.74 (m, 2H,
H-200, H-600), 7.94 (s, 1H, H-vinyl), 8.02 (d, 1H, Jm ¼ 1.6 Hz, H-4),
8.51 (d, 2H, Jo ¼ 8.8 Hz, H-20, H-60), 10.66 (s, 1H, NH). MS (ESI):
m/z 346.8 (MþH). Elemental analysis calculated (%) for
C
22H16FNO2: C 76.51, H 4.67, N 4.06. Found: C 76.67, H 4.51,
N 4.05.
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(E)-1-(3-(3,4-Dichlorobenzylidene)-2-oxoindolin-5-yl)-3-
ethylurea (6d)
(E)-1-(3-(4-Carboxybenzylidene)-2-oxoindolin-5-yl)-3-
ethylurea (6h)
Yield: 73%, mp 335–337°C (decomp). 1H NMR (400 MHz,
DMSO-d6) d: 0.98 (t, 3H, CH3), 3.00–3.03 (m, 2H, CH2), 5.90
(t, 1H, NH–CO–NHC2H5), 6.71 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.25
(dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.47 (s, 1H, H-vinyl), 7.52
(d, 1H, Jm ¼ 2.0 Hz, H-4), 7.67 (dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 1.6 Hz,
H-60), 7.72 (d, 1H, Jo ¼ 8.0 Hz, H-50), 7.90 (d, 1H, Jm ¼ 1.6 Hz,
H-20), 8.14 (s, 1H, NH–CO–NHC2H5), 10.41 (s, 1H, indole-NH).
13C NMR (400 MHz, DMSO-d6) d: 168.31, 155.24, 137.48,
135.20, 134.59, 132.41, 131.77, 131.65, 131.13, 130.83,
129.55, 129.16, 120.83, 120.39, 113.39, 110.07, 33.94,
15.42. MS (ESI): m/z 376.7 (Mþ). Elemental analysis calculated
(%) for C18H15Cl2N3O2 ꢁ 0.07 H2O: C 57.27, H 4.04, N 11.13.
Found: C 56.90, H 4.04, N 11.12.
Yield: 81%, mp 290–295°C. 1H NMR (400 MHz, DMSO-d6) d:
major E-isomer 1.01 (t, 3H, CH3), 3.04–3.07 (m, 2H, CH2), 6.30
(t, 1H, NH–CO–NHC2H5), 6.74 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.45 (dd,
1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.58 (s, 1H, H-vinyl), 7.64 (d,
1H, Jm ¼ 2.0 Hz, H-4), 7.75 (d, 2H, Jo ¼ 8.4 Hz, H-20, H-60), 8.05 (d,
2H, Jo ¼ 8.4 Hz, H-30, H-50), 8.44 (s, 1H, NH–CO–NHC2H5), 10.42
(s, 1H, indole–NH), 13.70 (s, 1H, COOH). MS (ESI): m/z 352.7
(MþH). Elemental analysis calculated (%) for C19H17N3O4 ꢁ 0.5
H2O: C 63.32, H 5.03, N 11.66. Found: C 63.62, H 5.27, N 11.38.
(E)-1-Benzyl-3-(3-(4-chlorobenzylidene)-2-
oxoindolin-5-yl)thiourea (7a)
Yield: 75%, mp 227–228°C. 1H NMR (400 MHz, DMSO-d6) d:
4.70 (d, 2H, J ¼ 4.4 Hz, CH2), 6.84 (d, 1H, Jo ¼ 8.8 Hz, H-7), 7.13
(dd, 1H, Jo ¼ 8.8 Hz, Jm ¼ 1.6 Hz, H-6), 7.25–7.34 (m, 5H, benzyl
protons), 7.50 (d, 2H, Jo ¼ 8.4 Hz, H-20, H-60), 7.59 (s, 1H,
H-vinyl), 7.64 (s, 1H, H-4), 7.77 (d, 2H, Jo ¼ 8.4 Hz, H-30, H-50),
(E)-1-(3-(2,4-Difluorobenzylidene)-2-oxoindolin-5-yl)-3-
ethylurea (6e)
Yield: 76%, mp > 360°C. 1H NMR (400 MHz, DMSO-d6) d: major
E-isomer 0.98 (t, 3H, CH3), 3.01–3.06 (m, 2H, CH2), 5.90 (t, 1H,
NH–CO–NHC2H5), 6.72 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.22–7.28 (m,
2H, aromatic protons), 7.35 (s, 1H, H-vinyl), 7.41–7.47 (m, 2H,
H-4, aromatic proton), 7.79–7.82 (m, 1H, aromatic proton),
8.15 (s, 1H, NH–CO–NHC2H5), 10.45 (s, 1H, indole-NH). MS (ESI):
m/z 344.8 (MþH). Elemental analysis calculated (%) for
–
8.03 (s, 1H, –NH–CH –Ph), 9.44 (s, 1H, –NH–C S), 10.64 (s, 1H,
–
2
indole–NH). 13C NMR (400 MHz, DMSO-d6) d: 181.07, 168.60,
140.15, 139.10, 136.11, 135.30, 134.58, 134.21, 134.24, 133.63,
133.09, 132.42, 131.29, 128.80, 128.17, 127.95, 127.33, 126.80,
126.52, 120.56, 119.65, 110.02, 47.18. MS (ESI): m/z 420.2
(MþH); 422.1 (MþHþ2). Elemental analysis calculated (%) for
C
18H15F2N3O2 ꢁ 0.5 H2O: C 61.35, H 4.57, N 11.92. Found: C
C23H18ClN3OS: C 65.78, H 4.32, N 10.01, S 7.64. Found: C 65.43,
61.75, H 4.96, N 11.93.
H 4.44, N 9.91, S7.44.
(E)-1-(3-(4-Dimethylaminobenzylidene)-2-oxoindolin-5-
yl)-3-ethylurea (6f)
(E)-1-Benzyl-3-(3-(4-fluorobenzylidene)-2-
oxoindolin-5-yl)thiourea (7b)
Yield: 74%, mp 255–258°C. 1H NMR (400 MHz, DMSO-d6) d:
major E-isomer 1.03 (t, 3H, CH3), 3.03–3.09 (m, 8H, CH2, N
(CH3)2), 5.94 (t, 1H, NH–CO–NHC2H5), 6.72 (d, 1H, Jo ¼ 8.8 Hz,
H-7), 6.80 (d, 2H, Jo ¼ 8.4 Hz, H-30, H-50), 7.22 (dd, 1H,
Jo ¼ 8.0 Hz, Jm ¼ 2.0, H-6), 7.47 (s, 1H, H-vinyl), 7.66 (d, 2H,
Jo ¼ 9.2 Hz, H-20, H-60), 7.85 (s, 1H, H-4), 8.17 (s, 1H,
NH–CO–NHC2H5), 10.25 (s, 1H, indole-NH). MS (ESI): m/z
351.8 (MþH). Elemental analysis calculated (%) for
Yield: 76%, mp 220–222°C. 1H NMR (400 MHz, DMSO-d6) d:
4.70 (d, 2H, J ¼ 4.4 Hz, CH2), 6.84 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.12
(dd, 1H, Jo ¼ 8.4 Hz, J ¼ 2.0 Hz, H-6), 7.23–7.33 (m, 7H, benzyl
protons, H-30, H-50), 7.61 (s, 1H, H-vinyl), 7.66 (s, 1H, H-4), 7.79–
7.83 (m, 2H, H-20, H-60), 8.03 (s, 1H, –NH–CH2–Ph), 9.44 (s, 1H,
–
–
–NH–C S), 10.63 (s, 1H, indole NH). MS (ESI): m/z 404.1
–
–
(MþH). Elemental analysis calculated (%) for C23H18FN3OS:
C 68.47, H 4.50, N 10.41, S 7.95. Found: C 68.49, H 4.67, N 10.41,
S 7.74.
C
20H22N4O2: C 68.55, H, 6.33, N 15.99. Found: C 68.45, H
6.28, N 15.49.
(E)-1-Benzyl-3-(3-(3-fluorobenzylidene)-2-oxoindolin-
5-yl)thiourea (7c)
(E)-1-(3-(4-Methoxybenzylidene)-2-oxoindolin-5-yl)-3-
ethylurea (6g)
Yield: 70%, mp 191–193°C. 1H NMR (400 MHz, DMSO-d6) d:
4.69 (d, 2H, J ¼ 4.4 Hz, CH2), 6.85 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.12
(dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.22–7.33 (m, 6H,
aromatic protons), 7.47–7.58 (m, 3H, aromatic protons),
7.61 (s, 1H, H-vinyl), 7.65 (s, 1H, H-4), 8.01 (s, 1H, –NH–CH2–Ph),
Yield: 86%, mp 285–290°C. 1H NMR (400 MHz, DMSO-d6) d:
0.99 (t, 3H, CH3), 3.02–3.05 (m, 2H, CH2), 3.82 (s, 3H, OCH3),
5.90 (t, 1H, NH–CO–NHC2H5), 6.70 (d, 1H, Jo ¼ 8.8 Hz, H-7), 7.04
(d, 2H, Jo ¼ 8.8 Hz, H-30, H-50), 7.22 (dd, 1H, Jo ¼ 8.4 Hz,
Jm ¼ 2.0 Hz, H-6), 7.50 (s, 1H, H-vinyl), 7.69 (d, 2H, Jo ¼ 8.8 Hz,
H-20, H-60), 7.72 (d, 1H, Jm ¼ 2.0 Hz, H-4), 8.14 (s, 1H,
NH–CO–NHC2H5), 10.31 (s, 1H, indole–NH). 13C NMR
(400 MHz, DMSO-d6) d: 168.98, 168.87 160.41, 155.34,
137.02, 135.64, 134.31, 131.52, 126.87, 126.59, 125.98,
121.16, 120.04, 114.22, 113.36, 109.70, 55.35, 33.95, 15.47.
MS (ESI): m/z 338.8 (MþH). Elemental analysis calculated (%)
for C19H19N3O3: C 67.64, H 5.68, N 12.46. Found: C 67.70,
H 5.56, N 12.46.
–
–
9.44 (s, 1H, –NH–C S), 10.65 (s, 1H, indole NH). MS (ESI):
–
–
m/z 404.2 (MþH). Elemental analysis calculated (%) for
C
23H18FN3OS ꢁ 0.2H2O: C 67.86, H 4.55, N 10.32, S 7.87. Found:
C 67.94, H 4.59, N 10.28, S 7.73.
(E)-1-Benzyl-3-(3-(3,4-dichlorobenzylidene)-2-oxoindolin-
5-yl)thiourea (7d)
Yield: 78%, mp 225–227°C. 1H NMR (400 MHz, DMSO-d6) d:
4.70 (d, 2H, J ¼ 5.2 Hz, CH2), 6.85 (d, 1H, Jo ¼ 8.0 Hz, H-7), 7.14
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Z. Kilic-Kurt et al.
Archiv der Pharmazie
(dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.24–7.33 (m, 5H, benzyl
protons), 7.56 (s, 1H, H-vinyl), 7.61 (s, 1H, H-4), 7.66 (d, 1H,
Jo ¼ 8.4 Hz, H-50), 7.74 (dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-60),
7.97 (d, 1H, Jm ¼ 2.0 Hz, H-20), 8.02 (s, 1H, –NH–CH2–Ph), 9.44 (s,
(E)-1-Benzyl-3-(3-(4-carboxybenzylidene)-2-oxoindolin-5-
yl)thiourea (7h)
Yield: 72%, mp 170–173°C. 1H NMR (400 MHz, DMSO-d6) d:
major E isomer 4.74 (d, 2H, J ¼ 5.2 Hz, CH2), 6.81 (d, 1H,
Jo ¼ 8.4 Hz, H-7), 7.20 (dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 1.6 Hz, H-6),
7.28–7.33 (m, 5H, benzyl protons), 7.60 (s, 1H, H-vinyl), 7.63 (s,
1H, H-4), 7.72 (d, 2H, Jo ¼ 8.0 Hz, H-20, H-60), 8.06 (d, 2H,
Jo ¼ 8.0 Hz, H-30, H-50), 7.98 (s, 1H, –NH–CH2–Ph), 9.39 (s, 1H,
1H, –NH–C S), 10.67 (s, 1H, indole–NH). 13C NMR (400 MHz,
–
–
DMSO-d6) d: 181.07, 168.40, 167.16, 140.30, 138.85, 139.10,
135.03, 133.12, 132.95, 131.97, 131.68, 131.30, 130.81, 129.20,
128.94, 128.16, 127.29, 126.78, 120.32, 119.68, 110.14, 109.68,
47.16. MS (ESI): m/z 454.3 (MþH); 456.2 (MþHþ2). Elemental
analysis calculated (%) for C23H17Cl2N3OS: C 60.80, H 3.77,
N 9.25, S 7.06. Found: C 60.69, H 3.93, N 9.24, S 6.91.
–
–NH–C S), 10.54 (s, 1H, indole–NH), 13.50 (s, 1H, COOH). MS
–
(ESI): m/z 430.4 (MþH). Elemental analysis calculated (%) for
C
24H19N3O3S.0.5 H2O: C 65.73, H 4.59, N 9.58, S 7.31. Found:
C 65.55, H 4.55, N 10.33, S 7.70.
(E)-1-Benzyl-3-(3-(2,4-difluorobenzylidene)-2-oxoindolin-
5-yl)thiourea (7e)
General synthesis of 1-benzyl-5-(4-fluorobenzylamino)-3-
(substituted-phenylimino)indolin-2-one (13a–d), 5-(4-
fluorophenyl)-3-(substituted-phenylimino)indolin-2-one
(15a–d) and 1-benzyl-5-(4-fluorophenyl)-3-(substituted-
phenylimino)indolin-2-one (16a–d) derivatives
A mixture of compounds 13, 15, or 16 (0.01 mol) and anilin
derivatives (0.01 mol) in ethanol (5 mL) in the presence of
glacial acetic acid (0.2 mL) were refluxed for 12–24 h. On
cooling, the crystalline product was collected by filtration,
dried to afford 13a–d, 15a–d, and 16a–d. The analytical data
reported here are for the major isomer. The data for the
minor isomer are not reported.
Yield: 29%, mp 190–193°C. 1H NMR (400 MHz, DMSO-d6) d:
major E-isomer 4.68 (d, 2H, J ¼ 5.6 Hz, CH2), 6.85 (d, 1H,
Jo ¼ 8.0 Hz, H-7), 7.08–7.33 (m, 6H, aromatic protons), 7.42–
7.47 (m, 2H, aromatic protons), 7.49 (s, 1H, H-vinyl), 7.67 (s, 1H,
H-4), 7.84–7.90 (m, 1H, aromatic protons), 7.99 (s, 1H,
–
–NH–CH –Ph), 9.42 (s, 1H, –NH–C S), 10.67 (s, 1H, indole–NH).
–
2
MS (ESI): m/z 422.36 (MþH). Elemental analysis calculated (%)
for C23H17F2N3OS ꢁ 0.01 H2O: C 65.51, H 4.06, N 9.96, S 7.60.
Found: C 65.66, H 4.05, N 10.35, S 7.89.
(Z)-1-Benzyl-3-(3-(4-dimethylaminobenzylidene)-2-
oxoindolin-5-yl)thiourea (7f)
Yield: 78%, mp 226–228°C. 1H NMR (400 MHz, DMSO-d6) d:
3.04 (s, 6H, N(CH3)2), 4.73 (d, 2H, J ¼ 5.6 Hz, CH2), 6.75 (d, 2H,
Jo ¼ 8.0 Hz, H-30, H-50), 6.78 (d, 1H, Jo ¼ 8.8 Hz, H-7), 6.97
(dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2.0 Hz, H-6), 7.22–7.25 (m, 1H, H-400),
7.33 (d, 4H, J ¼ 4.4 Hz, H-200, H-300, H-500, H-600), 7.54 (d, 1H,
Jm ¼ 1.6 Hz, H-4), 7.61 (s, 1H, H-vinyl), 7.91 (s, 1H,
–NH–CH2–Ph), 8.46 (d, 2H, Jo ¼ 9.2 Hz, H-20, H-60), 9.42 (s, 1H,
(E)-1-Benzyl-5-(4-fluorobenzylamino)-3-(4-
fluorophenylimino)indolin-2-one (13a)
Yield: 68%, mp 202–204°C. 1H NMR (DMSO-d6, 400 MHz) d:
3.90 (d, 2H, J ¼ 6 Hz, –NH–CH2), 4.86 (s, 2H, –CH2Ph), 5.80 (d,
1H, J ¼ 2.4 Hz, H-4), 6.22 (t, 1H, J ¼ 6.0 Hz, –NH), 6.59 (dd, 1H,
Jo ¼ 8.0 Hz, Jm ¼ 2.4 Hz, H-6), 6.74 (d, 1H, Jo ¼ 8.4 Hz, H-7), 6.96–
6.99 (m, 2H, aromatic protons), 7.06–7.38 (11H, m, aromatic
protons). MS (ESI): m/z 454.9 (MþH). Elemental analysis
calculated (%) for C28H21F2N3O: C 74.16, H 4.67, N 9.27. Found:
C 73.97, H 4.55, N 9.15.
–NH–C S), 10.47 (s, 1H, indole-NH). 13C NMR (400 MHz, DMSO-
–
–
d6) d: 181.41, 167.79, 151.83, 139.88, 139.45, 138.62, 137.36,
134.85, 131.99, 131,87, 131.55, 128.13, 127.27, 126.66, 125.12,
124.89, 121.98, 119.95, 116.94, 116.72, 116.53, 111.05, 108.96,
108.85, 47.36. MS (ESI): m/z 429.3 (MþH). Elemental analysis
calculated (%) for C25H24N4OS: C 70.07, H 5.64, N 13.07, S 7.48.
Found: C 69.73, H 5.92, N 12.92, S 7.32.
(E)-1-Benzyl-3-(4-chlorophenylimino)-5-(4-
fluorobenzylamino)indolin-2-one (13b)
Yield: 77%, mp 213°C. 1H NMR (DMSO-d6, 400 MHz) d: 3.89 (d,
2H, J ¼ 6 Hz, –NH–CH2), 4.86 (s, 2H, –CH2Ph), 5.80 (d, 1H,
Jm ¼ 2.4 Hz, H-4), 6.27 (t, 1H, J ¼ 6.0 Hz, –NH), 6.59 (dd, 1H,
Jo ¼ 8.0 Hz, Jm ¼ 2.4 Hz, H-6), 6.74 (d, 1H, Jo ¼ 8.4 Hz, H-7), 6.97
(d, 2H, Jo ¼ 8.0 Hz, H-20 and H-60), 7.07 (d, 2H, Jo ¼ 8.0 Hz,
aromatic protons), 7.13 (t, 1H, J ¼ 8.8 Hz, aromatic proton),
7.25–7.36 (m, 6H, aromatic protons), 7.43 (d, 2H, J ¼ 8.4 Hz, H-
30 and H-50). MS (ESI): m/z 470 (MþH). Elemental analysis
calculated (%) for C28H21ClFN3O: C 71.56, H 4.50, N 8.94.
Found: C 71.36, H 4.40, N 8.92.
(E)-1-Benzyl-3-(3-(4-methoxybenzylidene)-2-oxoindolin-5-
yl)thiourea (7g)
Yield: 80%, mp 207–209°C. 1H NMR (400 MHz, DMSO-d6) d:
3.80 (s, 3H, OCH3), 4.66 (d, 2H, J ¼ 5.6 Hz, CH2), 6.80 (d, 1H,
Jo ¼ 8.0 Hz, H-7), 7.00 (d, 2H, Jo ¼ 8.8 Hz, H-30, H-50), 7.06 (dd,
1H, Jo ¼ 8.4 Hz, Jm ¼ 1.6 Hz, H-6), 7.17–7.30 (m, 5H, benzyl
protons), 7.55 (s, 1H, H-vinyl), 7.72 (d, 2H, Jo ¼ 8.8 Hz, H-20,
H-60), 7.73 (s, 1H, H-4), 7.98 (s, 1H, –NH–CH2–Ph), 9.39 (s, 1H,
–NH–C S), 10.52 (s, 1H, indole–NH). 13C NMR (400 MHz,
–
–
DMSO-d6) d: 181.13, 169.06, 160.60, 139.88, 139.25, 139.16,
136.34, 134.53, 132.20, 131.72, 128.15, 127.30, 126.99, 126.74,
126.45, 126.09, 125.31, 121.17, 119.53, 114.26, 113.98, 109.83,
55.38, 47.21. MS (ESI): m/z 416.3 (MþH). Elemental analysis
calculated (%) for C24H21N3O2S: C 69.37, H 5.09, N 10.11,
S 7.72. Found: C 69.27, H 5.03, N 10.06, S 7.58.
(E)-1-Benzyl-5-(4-fluorobenzylamino)-3-(4-
(methylthio)phenylimino)indolin-2-one (13c)
Yield: 49%, mp 221–222°C. 1H NMR (DMSO-d6, 400 MHz) d:
2.53 (s, 3H, –CH3), 3.87 (d, 2H, J ¼ 6 Hz, –NH–CH2), 4.86 (s, 2H,
–CH2Ph), 5.96 (d, 1H, Jm ¼ 2.4 Hz, H-4), 6.23 (t, 1H, J ¼ 6.4 Hz,
–NH), 6.58 (dd, 1H, Jo ¼ 8.0 Hz, Jm ¼ 2.4 Hz, H-6), 6.73 (d, 1H,
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Arch. Pharm. Chem. Life Sci. 2015, 348, 715–729
Novel Indole-2-one Derivatives as Src Inhibitors
Archiv der Pharmazie
Jo ¼ 8.0 Hz, H-7), 6.94 (d, 2H, Jo ¼ 8.0 Hz, H-20 and H-60), 7.06 (d,
2H, Jo ¼ 8 Hz, H-30 and H-50), 7.13 (t, 1H, J ¼ 8.8 Hz, aromatic
proton), 7.20–7.37 (m, 8H, aromatic protons). MS (ESI): m/z
482.9 (MþH). Elemental analysis calculated (%) for
1H, H-20), 7.23–7.30 (m, 4H, H-200, H-300, H-500, H-600), 7.42 (dd,
1H, Jo ¼ 8.0 Hz; Jm ¼ 2.4 Hz, H-60), 7.57 (t, 1H, J ¼ 8.0 Hz, H-50),
7.64 (dd, 1H, Jo ¼ 8.0; Jm ¼ 2.0 Hz, H-6), 11.10 (s, 1H, NH). MS
(ESI): m/z 369.7 (MþH). Elemental analysis calculated (%) for
C
29H24FN3OS: C 72.33, H 5.02, N 8.73, S 6.66. Found: C
C
20H11ClF2N2O ꢁ 0.2H2O: C 64.51, H 3.08, N 7.52. Found: C
72.68, H 4.99, N 8.78, S 6.78.
64.18, H 3.02, N 7.38.
(E)-1-Benzyl-3-(3-chloro-4-fluorophenylimino)-5-(4-
fluorobenzylamino)indolin-2-one (13d)
(E)-1-Benzyl-5-(4-fluorophenyl)-3-(4-fluorophenylimino)-
indolin-2-one (16a)
Yield: 79%, mp 196–197°C. 1H NMR (DMSO-d6, 400 MHz) d:
3.94 (d, 2H, J ¼ 6 Hz, –NH–CH2), 4.86 (s, 2H, -CH2Ph), 5.80 (d,
1H, J ¼ 2.4 Hz, H-4), 6.26 (t, 1H, J ¼ 6.0 Hz, -NH), 6.60 (dd, 1H,
Jo¼ 8.0, Jm¼ 2.4 Hz, H-6), 6.68 (s, 1H, H-2’), 6.75 (d, 1H,
J ¼ 8.8 Hz, H-7), 6.94–7.11 (m, 5H, aromatic protons), 7.13 (t,
1H, J ¼ 8.8 Hz, aromatic proton), 7.24-7.42 (m, 5H, aromatic
protons). MS (ESI): m/z 488 (MþH). Elemental analysis
calculated (%) for C28H20ClF2N3O: C 68.92, H 4.13, N 8.61.
Found: C 69.19, H 3.91, N 8.68.
Yield: 64%, mp 194–195°C. 1H NMR (DMSO-d6, 400 MHz)
d: 5.00 (s, 2H, –CH2), 6.62 (d, 1H, J ¼ 2.0 Hz, H-4), 7.07
(d, 1H, J ¼ 8.0 Hz, H-7), 7.11–7.42 (m, 13H, aromatic pro-
tons), 7.61 (dd, 1H, Jo ¼ 8.0 Hz, Jm ¼ 2.0 Hz, H-6). MS (ESI):
m/z 425.9 (MþH). Elemental analysis calculated (%) for
C
27H18F2N2O ꢁ 0.3H2O: C 75.44, H 4.36, N 6.51. Found: C 75.21,
H 4.76, N 6.16.
(E)-1-Benzyl-3-(4-chlorophenylimino)-5-(4-fluorophenyl)-
indolin-2-one (16b)
(E)-5-(4-Fluorophenyl)-3-(4-fluorophenylimino)indolin-2-
one (15a)
Yield: 62%, mp 196–197°C. 1H NMR (DMSO-d6, 400 MHz)
d: 5.03 (s, 2H, –CH2), 6.57 (d, 1H, J ¼ 1.6 Hz, H-4), 7.11 (d, 1H,
J ¼ 8.0 Hz, H-7), 7.14–7.45 (m, 11H, aromatic protons), 7.56
(dd, 2H, Jo ¼ 8.8 Hz, H-30 and H-50), 7.65 (dd, 1H, Jo ¼ 8.2 Hz,
Jm ¼ 2.0 Hz, H-6). MS (ESI): m/z 441.8 (MþH). Elemental
analysis calculated (%) for C27H18ClFN2O ꢁ 0.6H2O: C 71.79,
H 4.28, N 6.20. Found: C 71.63, H 4.10, N 6.12.
Yield: 14%, mp 274–275°C. 1H NMR (DMSO-d6, 400 MHz) d:
6.65 (d, 1H, Jm ¼ 1.2 Hz, H-4), 7.05 (d, 1H, Jo ¼ 8.0 Hz, H-7),
7.15–7.35 (m, 6H, aromatic protons), 7.40 (t, 2H, J ¼ 8.8 Hz,
aromatic protons), 7.69 (dd, 1H, Jo ¼ 8.0, Jm ¼ 2.0 Hz, H-6),
11.20 (s, 1H, NH). MS (ESI): m/z 335.47 (MþH). Elemental
analysis calculated (%) for C20H12F2N2O ꢁ 2CH3COOH: C 63.43,
H 4,43, N 6,16. Found: C 63.50, H 4.17, N 6.56.
(E)-1-Benzyl-5-(4-fluorophenyl)-3-(4-(methylthio)-
phenylimino)indolin-2-one (16c)
(E)-3-(4-Chlorophenylimino)-5-(4-fluorophenyl)indolin-2-
one (15b)
Yield: 63%. mp 161°C. 1H NMR (DMSO-d6, 400 MHz) d: 2.52 (s,
3H, –CH3), 5.03 (s, 2H, –CH2), 6.67 (d, 1H, Jm ¼ 2.0 Hz, H-4), 7.10
(d, 1H, Jo ¼ 8.0 Hz, H-7), 7.16–7.46 (m, 13H, aromatic protons),
7.64 (dd, 1H, Jm ¼ 8.0 Hz, Jo ¼ 2.0 Hz, H-6). MS (ESI): m/z 453.9
(MþH). Elemental analysis calculated (%) for C28H21FN2OS
ꢁ 0.8H2O: C 72.01, H 4.87, N 5.99, S 6.86. Found: C 71.69, H 4.54,
N 5.90, S 7.00.
Yield: 16%, mp 280–282°C. 1H NMR (DMSO-d6, 400 MHz) d:
6.57 (d, 1H, Jm ¼ 2.0 Hz, H-4), 7.05 (d, 1H, J ¼ 8.0 Hz, H-7), 7.16
(d, 2H, Jo ¼ 8.0 Hz, H-20-60), 7.24–7.35 (m, 4H, H-200, H-300, H-500,
H-600), 7.62 (d, 2H, Jo ¼ 8.0 Hz, H-30, H-50), 7.70 (dd, 1H,
Jo ¼ 8.0 Hz, Jm ¼ 1.6 Hz, H-6), 11.30 (s, 1H, NH). MS (ESI): m/z
351.47 (MþH). Elemental analysis calculated (%) for
C
20H12ClFN2O ꢁ 1.8CH3COOH: C 61.77, H 4.21, N 6.10. Found:
(E)-1-Benzyl-3-(3-chloro-4-fluorophenylimino)-5-(4-
fluorophenyl)indolin-2-one (16d)
C 61.92, H 4.14, N 6.50.
Yield: 50%, mp 176–178°C. 1H NMR (DMSO-d6, 400 MHz) d:
5.03 (s, 2H, –CH2), 6.70 (d, 1H, Jm ¼ 1.6 Hz, H-4), 7.12 (d, 1H,
Jo ¼ 8.0 Hz, H-7), 7.17–7.49 (m, 11H, aromatic protons), 7.58 (t,
1H, J ¼ 8.8 Hz, H-30), 7.67 (dd, 1H, Jo ¼ 8.0 Hz, Jm ¼ 2.0 Hz, H-6).
MS (ESI): m/z 459.8 (MþH). Elemental analysis calculated (%)
for C27H17ClF2N2O ꢁ 1H2O: C 67.99, H 4.01, N 5.87. Found: C
67.89, H 3.95, N 5.72.
(E)-5-(4-Fluorophenyl)-3-(4-(methylthio)phenylimino)-
indolin-2-one (15c)
Yield: 12%, mp 279–283°C (decomp). 1H NMR (DMSO-d6,
400 MHz) d: 2.60 (s, 3H, SCH3), 6.60 (d, 1H, J ¼ 2.0 Hz, H-4), 6.98
(d, 1H, Jo ¼ 8.0 Hz, H-7), 7.04 (d, 2H, Jo ¼ 8.0 Hz, H-20 and H-60),
7.17–7.27 (m, 4H, H-200, H-300, H-500, H-600), 7.43 (dd, 2H,
Jo ¼ 8.0 Hz, H-30 and H-50), 7.63 (dd, 1H, Jo ¼ 8.0 Hz, Jm ¼ 1.6 Hz,
H-6), 11.09 (s, 1H, NH). MS (ESI): m/z 363.6 (MþH). Elemental
analysis calculated (%) for C21H15FN2OS ꢁ 1.6CH3COOH: C
63.39, H 4.70, N 6.10, S 6.99. Found: C 63.25, H 4.51, N 6.40,
S 7.31.
Src kinase assay
The activity of protein tyrosine kinase was determined by
enzyme-linked immunosorbent assay (ELISA) according to
the instructions of Universal Tyrosine Kinase Assay Kit
(Takara, MK410, Japan). This assay kit monitors the transfer
of g-phosphate residue from ATP to peptide substrates
immobilized on plate. Briefly, the test reagents were
applied on appropriate wells and phosphorylation of
tyrosine was started by ATP-2Na. The plate was incubated
(E)-3-(3-Chloro-4-fluorophenylimino)-5-(4-
fluorophenyl)indolin-2-one (15d)
Yield: 21%, mp 236°C. 1H NMR (DMSO-d6, 400 MHz) d: 6.63 (d,
1H, J ¼ 2.0 Hz, H-4), 7.00 (d, 1H, J ¼ 8.0 Hz, H-7), 7.05–7.08 (m,
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Z. Kilic-Kurt et al.
Archiv der Pharmazie
at 37°C for 30 min and the wells were blocked with blocking
solution. The phosphorylation of tyrosine was started by
ATP-2Na and the phosphorylation level of substrate was
probed with HRP-conjugated anti-phosphotyrosine (PY20)
antibody. The inhibitory activities of compounds against Src
tyrosine kinase were monitored by the diminished activity
of kinase at 450 nm. The kinase assay was performed at
37°C and concentrations of the Src used to construct the
calibration curve were as follows: 0.88, 0.44, 0.22, 0.11, 0.06,
0.03, and 0.015 U/mL for Src. The test compounds were
applied at a concentration range between 1 ꢀ 10ꢂ3 M and
1 ꢀ 10ꢂ7 M (0.1, 1, 10, 100, and 1000 mM). The Src tyrosine
kinase activity is measured as the difference between the
total activity of blank (DMSO) and the activity of enzyme in
the presence of test compounds. The measuring range of
the kit is from 32 fmol/well to 2 pmol/well (2.16 ꢀ 10ꢂ5 to
135 ꢀ 10ꢂ5 U/mL). To determine IC50 value, the increasing
concentrations of PTK control were applied to the wells and
a dose–response curve (linear regression) was constructed.
IC50 value was then estimated using this formula: y ¼ ax þ b,
IC50 ¼ (0.5 ꢂ b)/a. IC50 value was determined as the concen-
tration of a compound required to achieve 50% inhibition
of Src tyrosine kinase activity.
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