Synthesis and anti-tyrosine kinase activity of 3-(substituted-benzylidene)- 1, 3-dihydro-indolin derivatives: Investigation of their role against p60 c-Src receptor tyrosine kinase with the application of receptor docking studies

Article abstract of DOI:10.1016/j.farmac.2005.01.015

A series of 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thione derivatives were synthesized as modified congeners of 3-(substituted- benzylidene)-1, 3-dihydro-indolin-2-one series. All the synthesized compounds were examined for their in vitro anti-tyrosine kinase activity against p60 ^c-Src. The activity results revealed that compounds (Z)-3-(4′-Dimethylamino-benzylidene)-1, 3-dihydro-indolin-2-thione (12) (E)-3-(2′, 6′-Dichloro-benzylidene)-1, 3-dihydro-indolin-2-thione (13) and (E)-3-(3′-Hydroxy-4′-methoxy-benzylidene)-1, 3-dihydro-indolin-2-thione (19) exhibited anti-tyrosine kinase activity with IC₅₀ value of 21.91, 21.20 and 30.92 μM, respectively. These results are comparable to PP1 [1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3, 4-d]pyrimidine-4-yl-amine] (IC₅₀ = 0.17 μM), which is reported as a potent and selective p60^c-Src tyrosine kinase inhibitor. Some thio congeners are found to be more potent than oxo derivatives; however, no significant correlation was observed between the activity profiles of these two series. Docking program was used to investigate the docking mode of each compound at the active site. Among all of the compounds, only (Z)-3-(2′-Chloro-benzylidene)-1, 3-dihydro-indolin-2-one (8) and (E)-3-(3′-Nitro-benzylidene)-1, 3-dihydro-indolin-2-thione (16) were docked at the active site where the PP1 was embedded.

Full text of DOI:10.1016/j.farmac.2005.01.015

Il Farmaco 60 (2005) 497–506
http://france.elsevier.com/direct/FARMAC/
Synthesis and anti-tyrosine kinase activity
of 3-(substituted-benzylidene)-1, 3-dihydro-indolin derivatives:
investigation of their role against p60c-Src receptor tyrosine kinase
with the application of receptor docking studies
Sureyya Olgen a,b,*, Eiichi Akaho b, Dogu Nebioglu a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara, Tandogan/Ankara 06100, Turkey
b Faculty of Pharmaceutical Sciences and High Technology Research Institute, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishiku,
Kobe 651 2180, Japan
Received 15 September 2004; received in revised form 17 January 2005; accepted 21 January 2005
Available online 31 May 2005
Abstract
A series of 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thione derivatives were synthesized as modified congeners of 3-(substituted-
benzylidene)-1, 3-dihydro-indolin-2-one series. All the synthesized compounds were examined for their in vitro anti-tyrosine kinase activity
against p60c-Src. The activity results revealed that compounds (Z)-3-(4′-Dimethylamino-benzylidene)-1, 3-dihydro-indolin-2-thione (12) (E)-
3-(2′, 6′-Dichloro-benzylidene)-1, 3-dihydro-indolin-2-thione (13) and (E)-3-(3′-Hydroxy-4′-methoxy-benzylidene)-1, 3-dihydro-indolin-2-
thione (19) exhibited anti-tyrosine kinase activity with IC50 value of 21.91, 21.20 and 30.92 µM, respectively. These results are comparable to
PP1 [1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3, 4-d]pyrimidine-4-yl-amine] (IC50 = 0.17 µM), which is reported as a potent and selective p60c-Src
tyrosine kinase inhibitor. Some thio congeners are found to be more potent than oxo derivatives; however, no significant correlation was
observed between the activity profiles of these two series. Docking program was used to investigate the docking mode of each compound at
the active site. Among all of the compounds, only (Z)-3-(2′-Chloro-benzylidene)-1, 3-dihydro-indolin-2-one (8) and (E)-3-(3′-Nitro-
benzylidene)-1, 3-dihydro-indolin-2-thione (16) were docked at the active site where the PP1 was embedded.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 3-Substitued indolin-2-ones; Thio congeners; Tyrosine kinase inhibition; DOCK 4.0; Enzyme interaction
1. Introduction
leukemia, breast, and colon cancer. For these reasons, c-Src
has been suggested as an important anticancer target [3]. The
inhibition of p60c-Src tyrosine kinase could be beneficial for
cancer therapy, such as inhibition of uncontrolled tumor cell
growth, inhibition of metastasis, inhibition of tumor angio-
genesis via reducing VEGF levels with low toxicity [4]. In
addition, the inhibitors of p60c-Src tyrosine kinase particu-
larly have been identified as potential therapeutics for
osteoporosis [5]. In the last decades, some 3-(substituted-
benzylidene)-1, 3-dihydro indolin-2-one derivatives were
reported as potent and selective inhibitor against different
RTKs, which have been involved in tumor growth, metasta-
sis and angiogenesis. Among them, SU5416 and SU6668
(Fig. 1.) have been utilized as specific inhibitor of VEGF-R
(Flk-1/KDR and Flt-1) in in vitro studies [6–8]. These recep-
tors are known to be involved in angiogenesis, and direct evi-
The normal cellular homologue (c-src) of the Rous sar-
coma virus oncogene (v-src), the first molecularly defined
proto-oncogene, encodes the p60c-Src PTK (Protein Tyrosine
Kinase). The p60c-Src (2ptk) belongs to non-receptor-linked
membrane-anchored PTKs (src-family PTKs) [1]. This pro-
tein is also involved in oncogenic signal transduction by the
receptor tyrosine kinases (RTKs) EGFR/HER1, HER2 and
PDGFR [2]. It has been implicated in the development of
* Corresponding author. Present address: Research Fellow at University
of Michigan, Department of Medicinal Chemistry, College of Pharmacy,
CCL 4550, Ann Arbor, MI 48109-1065 USA. Tel.: +1 734 647 3239; fax:
+1 734 763 5633.
E-mail address: solgen@umich.edu (S. Olgen).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.01.015

498
S. Olgen et al. / Il Farmaco 60 (2005) 497–506
Fig. 1
.
Indole derivatives SU 5416, SU 6668 and 1-Benzyl-indole-2-piperidinoethyl carboxylate as TKIs inhibitor.
dence from studies show that SU5416 and SU6668 inhibit
tumor-dependent angiogenesis in vivo. SU5416 has advanced
into clinical trials as an antiangiogenic agent [9]. Since it was
demonstrated that tumor growth depends on angiogenesis,
the tyrosine kinase inhibitors have become an important group
of compounds to investigate new anti-cancer agents.
In our previous paper, it was shown that some 2-substituted
indole esters were potent inhibitor of the p60c-Src PTK enzyme
[10]. Among them, 1-benzyl-indole-2-piperidinoethyl car-
boxylate (Fig. 1.) was found to be a highly potent p60c-Src
PTK enzyme inhibitor (IC50 = 1.34 µM) comparable to PP1
[1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3, 4-d]pyrimidine-4-yl-
amine] (IC50 = 0.17 µM), which had been previously reported
as a potent and selective p60c-Src PTK enzyme inhibitor [2].
As a part of our interest in the area of p60c-Src inhibitors, we
disclose our synthetic efforts and molecular modeling stud-
ies that have led to the evaluation of a new group of
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one and of
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thiones as
tyrosine kinase inhibitors.
In the attempt to broaden our understanding the role of the
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one
derivatives on tyrosine kinase inhibition, we decided to syn-
thesize both 3-(substituted-benzylidene)-1, 3-dihydro-indolin-
2-ones and a closely related group of congeners, namely
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thiones
(Scheme 1). We anticipated that replacement of the oxo with
the thio group would affect the binding and the kinase inhibi-
tory properties of compounds. The docking of derivatives was
studied using the DOCK 4.0 program to investigate the role
of substituents for the same purpose.
2. Experimental procedures
2.1. Chemistry
Melting points were measured with a capillary melting
point apparatus (Buchi SMP 20). Thin-layer chromatogra-
phy was carried out on aluminum-backed silica gel plates
Scheme 1. Preparation of compounds 1–21.

S. Olgen et al. / Il Farmaco 60 (2005) 497–506
499
(Merck 60 F254). Flash column chromatography was carried
out on Merck silica gel (230–400 mesh). The 1H-NMR spec-
tra were recorded on Bruker 400 AMX spectrometer for
400 MHz and are referenced to Me4Si for organic solutions.
Mass spectra were recorded on a Micromass Autospec high-
resolution mass spectrometer. 37 °C incubator (Masuda
SA-30) was used for incubation reaction. Micro TAITA plate
reader (450 nm neighboring filter, BioRad 550) was used to
measure of absorbance of phoshorylation reaction.
2.1.3. General procedure for 3-(substituted-benzylidene)-1,
3-dihydro-indolin-2-one and 2-thione derivatives (3–21)
A reaction mixture of oxindole or thioindole (1 equiv.),
aldehyde (1.2 equiv.), and piperidine (0.1 equiv.) in ethanol
(1–2 ml/1 mmol) was stirred at 90 °C for 3–5 h. It was cooled
and the solvent was removed under vacuum. The precipitate
or oily compounds were purified by silicagel column chro-
matography (Hexanes/Ethylacetate, 50:50). The isomers,
obtained as a mixture of both E and Z, were separated by
using the same solvent system on the silicagel column chro-
matography. The data for minor isomers are not reported. The
physical and analytical data of compounds are reported in
Table 1 and below.
Takara Universal Tyrosine Assay Kit (Takara-bio co.) was
used to test our synthesized compounds. The contents of a
kit: PTK substrate immobilized microplate (8 well × 12),
kinase reacting solution (11.0 ml), 40 mMATP-2Na (0.55 ml),
extraction buffer (11.0 ml), PTK control (0.50 ml), anti
phoshotyrosine (PY20-HRP, for 5.5 ml/H2O), blocking solu-
tion (11.0 ml), HRP coloring solution (TMBZ = Tetra methyl
benzidine, 12.0 ml).
Reagents: N, N-Dimethylformamide, sulfuric acid, 0.05%
tween in PBS were purchased from Wako Pure Chemical
Industries Ltd. 2-nitro-5-chlorobenzaldehyde, 3-nitro-
benzaldehyde, o-chlorobenzaldehyde, 2-chloro-5-nitro-
benzaldehyde, 3, 4-dichloro benzaldehyde, 2, 6-dichloro-
2.1.3.1. (Z)-3-(4′-Dimethylamino-benzylidene)-1, 3-dihydro-
indolin-2-one (3). 1H-NMR (400 MHz, DMSO-d6), d 10.42
(s, 1H, NH), 8.44 (d, 2H, J = 9.24, H-2′, 6′), 7.60 (s, 1H,
H-vin), 7.59 (d, 1H, J = 7.70, H-4), 7.18 (t, 1H, H-6), 7.09 (t,
1H, H-5), 6.83 (d, 1H, J = 7.83, H-7), 6.76 (d, 2H, J = 9.24,
H-3′, 5′), 3.01 (s, 6H, N(CH3)2). Anal. C17H16N2O. 264.10
(M+).
2.1.3.2. (Z)-3-(2′, 6′-Dichloro-benzylidene)-1, 3-indolin-2-
benzaldehyde,
4-carboxybenzaldehyde,
3-hydroxy-4-
1
one (4). H-NMR (400 MHz, DMSO-d6), d 10.69 (s, 1H,
methoxy benzaldehyde, 2-hydroxybenzaldehyde, 4-(N,
N-dimethyl)benzaldehyde were purchased from Fluka. The
other compounds were purchased from their companies as
listed below: 2-mercaptoethanol (ICN Biomedicals Inc.),
hydrazine hydrate (Merck), hydrogen chloride (Merck),
sodium carbonate (Merck), anhydrous sodium sulfate
(Merck), ethyl acetate (Merck), hexanes (Merck), phospho-
rous pentasulfide (Aldrich), anhydrous tetrahydrofuran (Ald-
rich), isatin (Aldrich), piperidin (BDH), ethanol (Riedel).
NH), 7.65 (d, 2H, J = 8.01, H-3′, 5′), 7.54 (d, 1H, J = 9.35,
H-4), 7.42 (s, 1H, H-vin), 7.23 (t, 1H, H-4′), 6.86 (t, 1H, H-6),
6.79 (t, 1H, H-5), 6.48 (d, 1H, J = 7.52, H-7). Anal.
C15H9Cl2NO. 289.00 (M+).
2.1.3.3. (Z)-3-(3′, 4′-Dichloro-benzylidene)-1, 3-indolin-2-
1
one (5). H-NMR (400 MHz, DMSO-d6), d 10.63 (s, 1H,
NH), 8.81 (s, 1H, H-2′), 8.23 (d, 1H, J = 8.45, H-6′), 7.94 (s,
1H, H-vin), 7.79–7.68 (m, 2H, H-4, H-5′), 7.25 (t, 1H, H-6),
7.02–6.82 (m, 2H, H-5, H-7). Anal. C15H9Cl2NO. 289.10
(M+).
2.1.1. Oxindol (1)
Fifteen grams (0.1 mol) isatin was dissolved in 60 ml
(1.2 mol) hydrazine hydrate and refluxed at 140 °C for 4 h.
The reaction mixture was poured into ice–water and acidi-
fied by 6 N HCl. After standing at room temperature for
2 days, 7.5 g of pure oxindol crystals were obtained. Yield
55%. M.p. 127 °C. (lit. [11], 127 °C).
2.1.3.4. (Z)-3-(2′-Chloro-5′-nitro-benzylidene)-1, 3-dihydro-
indolin-2-one (6). 1H-NMR (400 MHz, DMSO-d6), d 10.73
(s, 1H, NH), 8.54 (s, 1H, H-6′), 8.32 (d, 1H, J = 8.73, H-3′),
7.95 (d, 1H, J = 8.73, H-4′), 7.53 (s, 1H, H-vin), 7.27 (t, 1H,
H-6), 7.14 (d, 1H, J = 7.53, H-4), 6.89 (d, 1H, J = 8.04, H-7),
6.80 (t, 1H, H-5). Anal. C15H9ClN2O3. 300.27 (M+).
2.1.2. Thioindol (2)
26.67 g (0.06 mol) of P2S5 and 6.36 g (0.06 mol) of
Na2CO3 were suspended in 20 ml anhydrous THF at 0 °C
and the mixture was stirred for 20 min. Seven gram (0.05 mol)
oxindole 1 was dissolved in 25 ml anhydrous THF and added
to this mixture dropwise. The mixture was stirred at the same
temperature for 30 min, warmed up to r.t., and stirred over-
night. The reaction was poured into cold water and extracted
with ethyl acetate (3 × 100 ml). The organic layer was washed
with brine (3 ×100 ml) and dried over anhydrous Na2SO4.
Evaporation of the solvent gave the crude compound, which
was purified by silica gel column chromatography (Hexanes/
Ethylacetate, 70:30). 6.5 g of pure compound was obtained.
Yield 83%. M.p.149 °C (lit. [12], 147–149 °C in MeOH).
2.1.3.5. (Z)-3-(3′-Nitro-benzylidene)-1, 3-dihydro-indolin-2-
one (7). H-NMR (400 MHz, DMSO-d6), d 10.69 (s, 1H,
NH), 9.37 (s, 1H, H-2′), 8.63 (d, 1H, J = 7.76, H-6′), 8.26 (d,
1H, J = 8.52, H-4′), 7.95 (s, 1H, H-vin), 7.81–7.42 (m, 2H,
H-4, H-5′), 7.25 (t, 1H, H-6), 7.01 (t, 1H, H-5), 6.78 (d, 1H,
J = 7.70, H-7). Anal. C15H10N2O3. 266.27 (M+).
1
2.1.3.6. (Z)-3-(2′-Chloro-benzylidene)-1, 3-dihydro-indolin-
2-one (8). 1H-NMR (400 MHz, DMSO-d6), d 10.65 (s, 1H,
NH), 7.75 (d, 1H, J = 6.92, H-6′), 7.69 (d, 1H, J = 7.37, H-3′),
7.57 (s, 1H, H-vin), 7.53–7.45 (m, 2H, H-4′, H-5′), 7.22 (t,
1H, H-6), 7.12 (d, 1H, J = 7.70, H-4), 6.86 (d, 1H, J = 7.83,
H-7), 6.79 (t, 1H, H-5). Anal. C15H10 ClNO. 255.00 (M+).

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S. Olgen et al. / Il Farmaco 60 (2005) 497–506
Table 1
Analytical data and activity results of compounds 3–21
Compound number
R
Isomer Yield a (%) M.p. (°C)
Inhibition of cellular tyrosine kinase activity IC50,
µM b from free protein
290.00
>1000
149.13
>1000
>1000
70.37
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
PP1
4′-N(CH3)2
2′, 6′-Cl
Z
Z
Z
Z
Z
Z
Z
Z
E
Z
Z
Z
Z
E
Z
Z
E
Z
Z
49
85
44
65
88
56
88
85
82
67
44
85
46
48
62
59
67
40
43
230–233 (lit.234) c (18)
165–167 (lit.164) c (20)
186 (lit. 183–166) c (21)
265
258 (lit. 255–257) c (22)
178–181 (lit.178) c (22)
195–196 (lit. 195) c (23,24)
153
3′, 4′-Cl
2′-Cl, 5′-NO2
3′-NO2
2′-Cl
2’-OH
>1000
203.19
78.62
3′-OH, 4′-OCH3
2′-NO2, 5′-Cl
4′-N(CH3)2
2′, 6′-Cl
283–285
248–250
21.91
155–156
21.20
3′, 4′-Cl
158–160
154.54
560.60
322.27
305.56
>1000
30.92
2′-Cl, 5′-NO2
3′-NO2
164–165
190–192
2′-Cl
173–174
2′-OH
91–93
3′-OH, 4′-OCH3
2′-NO2, 5′-Cl
4′-COOH
95–96
195–196
124.46
ND
179–181
0.17
ND =Not determined.
a The number indicates the individual yields of separated isomers. The yields were not optimized.
b Values are obtained from at least three independent dose.
c The data of compounds were reported previously by other authors (Lit. [18,20–24]).
2.1.3.7. (Z)-3-(2′-Hydroxy-benzylidene)-1, 3-dihydro-indolin-
2-one (9). 1H-NMR (400 MHz, DMSO-d6), d 10.51 (s, 1H,
NH), 7.68 (s, 1H, H-vin), 7.61 (d, 1H, J = 7.72, H-6′), 7.48
(d, 1H, J = 7.61, H-3′), 7.30 (t, 1H, H-4′), 7.19 (t, 1H, H-5′),
6.96 (d, 1H, J = 8.24, H-4), 6.90 (t, 1H, H-6), 6.86–6.84 (m,
2H, H-5, H-7). Anal. C15H11NO2. 237.12 (M+).
2.1.3.10. (Z)-3-(4′-Dimethylamino-benzylidene)-1, 3-dihydro-
indolin-2-thione (12). H-NMR (400 MHz, DMSO-d6),
d 11.35 (s, 1H, NH), 7.66 (d, 2H, J = 6.96, H-2′, 6′), 7.29–
7.21 (m, 3H, H-vin, H-4, H-6), 7.12 (t, 1H, H-5), 6.90–6.49
(m, 3H, H-3′,5′, H-7), 3.29 (s, 6H, 4′-N(CH3)3). Anal.
C17H16N2S. 280.10 (M+).
1
2.1.3.8. (Z)-3-(3′-Hydroxy-4′-methoxy-benzylidene)-1,
3-dihydro-indolin-2-one (10). 1H-NMR (400 MHz, DMSO-
d6), d 10.37 (s, 1H, NH), 8.03 (s, 1H, H-2′), 7.68–7.35 (m,
3H, H-vin, H-5′, H-6′), 6.87 (d, 1H, J = 7.65, H-4), 6.81 (t,
1H, H-6), 6.71 (t, 1H, H-5), 6.65 (d, 1H, J = 7.55, H-7), 3.86
(s, 1H, OH), 3.83 (s, 3H, OCH3). Anal. C16H13NO3. 267.31
(M+).
2.1.3.11. (Z)-3-(2′, 6′-Dichloro-benzylidene)-1, 3-dihydro-
indolin-2-thione (13). H-NMR (400 MHz, DMSO-d6), d
1
11.43 (s, 1H, NH), 9.65 (s, 1H, H-vin), 8.63 (d, 1H, J = 7.62,
H-3′), 8.39 (d, 1H, J = 8.13, H-5′), 8.02 (d, 1H, J = 6.89,
H-4), 7.92 (t, 1H, H-4′), 7.80 (d, 1H, J = 7.70, H-7), 7.67 (t,
1H, H-6), 7.53 (t, 1H, H-5). Anal. C15H9Cl2NS. 307.95 (M +
H).
2.1.3.9. (E)-3-(2′-nitro-5′-chloro-benzylidene)-1, 3-dihydro-
indolin-2-one (11). 1H-NMR (400 MHz, DMSO-d6), d 10.52
(s, 1H, NH), 8.17 (d, 1H, J = 8.82, H-3′), 7.94 (s, 1H, H-vin),
7.92 (s, 1H, H-6′), 7.70 (d, 1H, J = 8.82, H-4′), 7.65 (d, 1H, J
= 7.49, H-4), 7.25 (t, 1H, H-6), 7.00 (t, 1H, H-5), 6.82 (d, 1H,
J = 7.74, H-7). Anal. C15H9ClN2O3. 300.16 (M+).
2.1.3.12. (Z)-3-(3′, 4′-Dichloro-benzylidene)-1, 3-dihydro-
indolin-2-thione (14). H-NMR (400 MHz, DMSO-d6), d
11.66 (s, 1H, NH), 7.72–7.67 (m, 3H, H-vin, H-2′, H-6′),
7.51–7.16 (m, 3H, H-5′, H-4, H-6), 7.01–6.70 (m, 2H, H-5,
H-7). Anal. C15H9Cl2NS. 307.49 (M + H).
1

S. Olgen et al. / Il Farmaco 60 (2005) 497–506
501
2.1.3.13. (Z)-3-(2′-Chloro-5′-nitro-benzylidene)-1, 3-dihydro-
indolin-2-thione (15). H-NMR (400 MHz, DMSO-d6), d
The phosphorylation assays were performed at 37 °C in a
final volume of 40 µl of tyrosine kinase. The concentrations
of the PTKs used to construct the calibration curve were as
follows: 600, 500, 341, 200, 100 × 10–7 units/µl for p60c-Src
PTK. The unit of 500 × 10–7/µl was used for each inhibitor.
Phosphorylation reactions were initiated by the addition of
40 nM ATP (10 µl) into each vessel, and the plate was subse-
quently incubated at 37 °C for 30 min. After the completion
of the reaction, the remaining liquid was decanted and the
vessels were washed with tween-PBS four times. The block-
ing solution (100 µl) was added to the vessels and incubated
at 37 °C for 30 min. After washing the plate with tween-PBS,
50 µl of anti-phosphotyrosine was added to the vessels and
incubated at 37 °C for 30 min. The reaction liquid was
decanted and the remaining solution was removed by rinsing
with tween-PBS four times. 100 µl of HRP coloring agents
was added and incubated at 37 °C for 15 min. Reaction is
terminated by the addition of 100 µl per well 1 N sulfuric
acid. The absorbance of the reaction mixture was measured
at 450 nm with a microplate reader.
1
11.78 (s, 1H, NH), 9.21 (s, 1H, H-6′), 8.77 (d, 1H, J = 7.28,
H-3′), 7.98 (d, 1H, J = 7.28, H-4′), 7.67 (d, 1H, J = 6.85,
H-4), 7.57 (s, 1H, H-vin), 7.32 (d, 1H, J = 8.21, H-7), 7.14 (t,
1H, H-6), 7.05 (t, 1H, H-5). Anal. C15H9ClN2O2S. 317.18
(M + H).
2.1.3.14. (E)-3-(3′-Nitro-benzylidene)-1, 3-dihydro-indolin-
1
2-thione (16). H-NMR (400 MHz, DMSO-d6), d 11.70 (s,
1H, NH), 8.35 (s, 1H, H-2′), 8.07–7.76 (m, 3H, H-vin, H-4′,
H-6′), 7.76–7.55 (m, 2H, H-5′, H-4), 7.35 (t, 1H, H-6), 7.32–
6.76 (m, 2H, H-5, H-7). Anal. C15H10N2O2S. 282.21 (M+).
2.1.3.15. (Z)-3-(2′-Chloro-benzylidene)-1, 3-dihydro-indolin-
1
2-thione (17). H-NMR (400 MHz, DMSO-d6), d 11.63 (s,
1H, NH), 8.24–7.47 (m, 2H, H-3′, H-6′), 7.28–7.17 (m, 3H,
H-vin, H-4′, H-5′), 6.93 (d 1H, J = 6.24, H-4), 6.86 (t, 1H,
H-6), 6.56 (t, 1H, H-5), 6.07 (d, 1H, J = 8.20, H-7). Anal.
C15H10ClNS. 271.86 (M+).
2.3. Molecular docking studies
2.1.3.16. (Z)-3-(2′-Hydroxy-benzylidene)-1, 3-dihydro-
indolin-2-thione (18). H-NMR (400 MHz, DMSO-d6), d
1
The DOCK [15] program is specifically designed for the
identification of small molecule inhibitors that are comple-
mentary to a targeted surface area or an active site. The dock-
ing experiments as well as receptor and ligand preparations
were performed on SGI Indigo Extreme (R4400) worksta-
tion. Insight II software (MSI) was used for drawing com-
pounds. To attach hydrogen atoms, molecules are converted
to the Sybyl (Tripos) mol2 files. The charge was assigned on
the drawn compound, which was optimized by Discover.
Empirical partial atomic charges were taken from the CVFF
force field with the assistance of Insight II software. The
enzyme 2ptk (p60c-Src), taken out from PDB (Protein Data
Bank) sum (URL: http://www.biochem.ucl.ac.uk/bsm/
pdbsum/), was placed into the Insight II where docking with
inhibitors were conducted. The tyrosine kinase p60c-Src used
for docking was chicken SRC tyrosine kinase. The three
dimensional structures were obtained by X-ray diffraction
analysis. The amino acid residues representing the active site
of tyrosine kinase p60c-Src, where the pocket is created, were
considered at Lys 295 and Glu 310. The secondary structure
of p60c-Src in the PDBsum database was shown in Fig. 2.
To rank each potential inhibitor, a pre-calculated contact-
scoring grid, based on distance between potential inhibitor
atoms and target area atoms, and a force field scoring grid,
based on molecular mechanics interaction energies consist-
ing of Van der Walls and electrostatic components were gen-
erated. The resulting output file for each screening, based on
distance or force field grids, contains the highest scoring com-
pounds ranked in order of their scores. PP1 and inhibitors
have been docked manually into the active site. DOCK
4.0 filled the pocket with spheres, moved the inhibitor to the
center of each sphere, and rotated it to score the docking
energy. This procedure of transforming and rotating the inhibi-
tor was repeated at each sphere center.
11.33 (s, 1H, NH), 9.08 (s, 1H, H-vin), 7.57 (d, 1H, J = 7.64,
H-6′), 7.40 (d, 1H, J = 8.02, H-3′), 7.34 (t, 1H, H-4′), 7.13 (t,
1H, H-5′), 6.92–6.72 (m, 2H, H-4, H-7), 6.48 (t, 1H, H-6),
6.28 (t, 1H, H-5). Anal. C15H11NOS. 253.06 (M+).
2.1.3.17. (E)-3-(3′-Hydroxy-4′-methoxy-benzylidene)-1,
3-dihydro-indolin-2-thione (19). 1H-NMR (400 MHz,
DMSO-d6), d 11.38 (s, 1H, NH), 8.69 (s, 1H, H-vin), 8.10–
7.23 (m, 3H, H-2′, H-5′, H-6′), 7.12–6.98 (m, 2H, H-4, H-6),
6.90 (t, 1H, H-5), 6.68 (d, 1H, J = 8.43, H-7), 3.87 (s, 1H,
OH), 3.65 (s, 3H, OCH3). Anal. C16H13NO2S. 284.52 (M +
H).
2.1.3.18. (Z)-3-(2′-Nitro-5′-chloro-benzylidene)-1, 3-dihydro-
1
indolin-2-thione (20). H-NMR (400 MHz, DMSO-d6), d
11.41 (s, 1H, NH), 8.96 (s, 1H, H-vin), 8.30 (s, 1H, H-6′),
8.19 (d, 1H, J = 7.07, H-3′), 8.14 (d, 1H, J = 8.70, H-4′), 7.81
(d, 1H, J = 732, H-4), 7.68 (d, 1H, J = 7.78, H-7), 7.59 (t, 1H,
H-6), 7.39 (t, 1H, H-5). Anal. C15H9ClN2O2S. 316.60 (M+).
2.1.3.19. (Z)-3-(4′-Carboxy-benzylidene)-1, 3-dihydro-
1
indolin-2-thione (21). H-NMR (400 MHz, DMSO-d6), d
13.18 (bs, 1H, COOH), 11.59 (s, 1H, NH), 7.76 (d, 2H, J
= 8.5, H-3′, H-5′), 7.31–7.18 (m, 4H, H-vin, H-2′, H-6′, H-4),
7.16 (t, 1H, H-6), 6.96 (t, 1H, H-5), 6.81 (d, 1H, J = 8.3,
H-7). Anal. C16H11NO2S. 281.15 (M+).
2.2. Biological assays
Takara Universal Tyrosine Assay Kit (Takara-bio co.) was
used to test the synthesized compounds. This assay deter-
mines the inhibitory activities of compounds against sub-
strate of tyrosine kinase [10,13,14].

502
S. Olgen et al. / Il Farmaco 60 (2005) 497–506
Fig. 2
.
Secondary representation of PTK p60c-Src in PDBsum datase.
Docking score of DOCK 4.0 is obtained by using Eq. 1. It
is a summation of Van der Waals interaction energy and elec-
trostatic energy both taking place between the ligand and the
enzyme. In the dock.in parameter file of DOCK 4.0, the ligand
flexibility was taken into account, while the protein was fixed
as rigid conformation. Therefore, docking energy scored can
be interpreted as one of the major score functions of the ligand,
although it is not as comprehensive as it should be. Based on
this theory, the lowest docking energy was taken into account
as one of the evaluation factors for strong inhibition.
sion and attraction parameters; D, the dielectric function; 332,
factor that converts the electrostatic energy into kilocalories
per mole; I; the atom of ligand; J, the atom of enzyme.
RMSD is computed and expressed in Å, and it is a struc-
tural comparison of two molecules in terms of distance.
Assume that the structure is defined in terms of the Cartesian
coordinates of the atoms and represented by an n × 3 coordi-
nate matrix, where n is the number of atoms in the molecule.
Structure translation can be done by adding a translation vec-
tor to each row of the coordinate matrix, and structure rota-
tion via multiplying the coordinate matrix by a rotation matrix.
X and Y are the coordinate matrices of two structures after
they are translated so that their centers of geometry coincide.
After docking of our proposed compounds against p60c-Src
tyrosine kinase, the top 25 docking results, which are arranged
in order from lowest to highest energy, were examined. The
lig rec
Aij
Bij
qiqj
Equation 1
−
+ 332
͚ ͚
12
6
ͫ
ͬ
rij
rij
Drij
i=1 j=1
rij, the distance between atoms i and j; qi, qj, the point
energy charge on atoms i and j; Aij, Bij, van der Waals repul-

S. Olgen et al. / Il Farmaco 60 (2005) 497–506
503
hydrogen bonds between compounds and enzyme were evalu-
ated by using Insight II software. The compound with the
highest number of hydrogen bond and the lowest binding
energy was noted for evaluation of its inhibitory potency and
selectivity for the enzyme active site; this compound should
be docked more firmly and inhibit the enzyme more strongly
than the other compounds.
kinase [13]. The main purpose of this assay is to block the
phosphorylation of the substrate with inhibitors, which are
competitive with substrate. The results were expressed as a
percentage of maximum activity and IC50 values were deter-
mined by graphic analysis. The results for tested compounds
are summarized in Table 1. IC50 values were defined as the
concentration of a compound required to achieve 50% inhi-
bition of tyrosine phosphorylation on the p60c-Src tyrosine
kinase to ligand-stimulated control reactions in the presence
of vehicle alone (dimethyl sulfoxide). The maximum inhibi-
tion attained among the tested compounds was IC50 21.91 µM.
This was observed for compounds 12 and 13 with IC50
21.91 and 21.20 µM, respectively as shown in Table 1. Com-
pound 19 also showed good activity at the 30.92 µM concen-
tration.Among the 3-(substituted-benzylidene)-1, 3-dihydro-
indolin-2-one derivatives, compound 8 (70.37 µM) and 11
(78.62 µM) were also found to be inhibitors against enzyme.
The potency of identical congeners was found different.
For instance, the 3-(4′-Dimethylamino-benzylidene)-1,
3-dihydro-indolin-2-thione 12 demonstrated a 10-fold greater
potency than oxo congener 3. While 3-(2′, 6′-dichloro-
benzylidene)-1, 3-dihydro-indolin-2-thione 13, showed good
inhibition (21.20 µM), the oxo congeners 4, did not show any
3. Results and discussion
3.1. Chemistry
The 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-
one and 2-thione derivatives 3–21 were synthesized by con-
densing compounds 1, 2 and substituted aryl aldehydes in the
presence of different bases [16,17] (Scheme 1). Synthesis of
oxindole (1) was achieved by a Wolff–Kishner like reduction
of isatin with hydrazine hydrate [18]. The oxindole was
changed to the thioindole (2) by using P2S5 and Na2CO3 in
THF [19]. Among the 3-(substituted-benzylidene)-1,
3-dihydro-indolin-2-one derivatives 6, 10 and 11 are original
compounds. The data of other derivatives were previously
reported by Sun et al. [18] (compound 3), Villemin et al. [20]
(compound 4), Howard et al. [21] (compound 5), Neber and
Rocker [22] (compound 7, 8), Desimoni et al. [23] (com-
pound 9) and Wahl and Bagard [24] (compound 9). All
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thione
derivatives are original compounds, and the data from the syn-
thesized compounds are explained in the experimental sec-
tion and Table 1.
The 3-(substituted-benzylidene)-1, 3-dihydro-indolin
derivatives were obtained as either Z or E isomers and mix-
ture of Z/E isomers, depending on the characteristics of the
substitutions at the C-3 position (Table 1). The reason for a
majority of E isomerism of the compounds may be the result
of the steric or electronic interactions between the carbonyl
and thio groups at the C-2 position of the ring and the proton
at the C-2′(6′) position of the phenyl ring in their Z isomeric
form. The configuration of some compounds was determined
using NOE analysis. The E-configured compounds have a
NOE effect between the proton at the C-4 position and the
proton (s) in the C-3 substitution of the 3-(substituted-
benzylidene)-1, 3-dihydro-indolin-2-ones and 2-thiones. The
Z-configured compounds show a NOE effect between the pro-
ton at the C-4 position and the vinyl proton. These assign-
ments were also confirmed using 1H-NMR spectral data. The
vinyl protons display a slight down-field shift in the trans-
(E) isomers than cis-(Z) isomers due to the oxo and thio influ-
ence. The ortho-benzylidene protons were found more
deshielded in Z-isomers for the same reason. The results of
mass spectroscopic analysis also confirmed the structures
deduced.
activity.
Interestingly,
3-(2′-Chloro-benzylidene)-1,
3-dihydro-indolin-2-one 8 has a fourfold greater potency than
thio congener 17. Only 3′, 4′-dichloro benzylidenyl indolin
derivatives of both congeners 5 and 14 produced equal activ-
ity. Since some thio congeners were found more potent than
the oxo, we have suggested that the replacement of oxo with
a thio group could play a more important role in the enhance-
ment of activity. The same substitutions to different conge-
ners have different impacts on the activity. However, no clear
relationships were observed between the activity profiles of
identical congeners. The potency of this chemical series may
also depend, to a large extent, on the three dimensional struc-
ture of R-substituents since a wide range of IC50 values (from
over 1000 to 20 µM) was observed depending upon the nature
of R-substituents as shown in Table 1.
3.3. Molecular modeling studies
The DOCK 4.0 program [15], was utilized for the study of
binding mode of synthesized indole derivatives against pro-
tein tyrosine kinase enzyme p60c-Src. We have previously
reported the binding mode studies by using DOCK 4.0
[10,25]. In this study, the docking results of synthesized
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one and
2-thione derivatives were evaluated and are shown in Table 2.
Atoms responsible for H-bonds are shown in Fig. 3. The com-
pound with the highest number of hydrogen bonds, with the
lowest binding energy, and with the smallest RMSD is gen-
erally said to be a reasonable candidate for the inhibition of
the enzyme [26]. In some cases, a good correlation between
the potency and the number of hydrogen bonds is observed,
while other cases not. It is our understanding that our current
study belongs to the latter case. Hydrophobic interaction
3.2. Inhibition of tyrosine kinase (p60c-Src
)
Compounds were evaluated for their inhibitory activity
toward tyrosine phosphorylation for the p60c-Src tyrosine

504
S. Olgen et al. / Il Farmaco 60 (2005) 497–506
Table 2
Docking result of 3-(substituted-benzylidene)-1, 3-dihydro indolin-2-one and 2-thione derivatives
Compound number Energy
(kcal/mol)
01
Against p60c-Src
H
RMSD
bond (distance Ao)
3
6
–14.21
–13.78
12
01
N-3 with O-4 of Asp 404 (2.87)
N-3 with O-4 of Asp 404 (2.59)
O-4 with O-5 of Asp 404 (2.64)
N-3 with O-4 of Asp 404 (2.60)
O-4 with O-5 of Asp 404 (2.61)
N-2 with O-4 of Asp 404 (2.69)
N-2 with O-4 of Asp 404 (2.70)
O-1 with N-3 of Met 341 (2.72)
O-1 with N-3 of Met 341 (2.79)
N-2 with N-3 of Asp 404 (2.48)
N-2 with O-1 of Thr 338 (2.08)
O-1 with N-3 of Ile 294 (2.93)
O-1 with N-4 of Lys 295 (2.74)
O-3 with O-4 of Asp 404 (2.85)
O-3 with O-4 of Asp 404 (2.74)
O-4 with O-1 of Thr 338 (2.68)
O-4 with O-1 of Thr 338 (2.69)
N-2 with O-1 of Thr 338 (2.43)
N-2 with O-1 of Thr 338 (2.43)
N-2 with O-1 of Thr 338 (2.43)
O-4 with N-4 of Lys 295 (2.98)
O-4 with N-4 of Lys 295 (2.84)
N-3 with O-2 of Met 341 (2.48)
N-3 with O-2 of Gly 344 (2.59)
N-3 with O-2 of Val 394 (2.41)
O-4 with N-3 of Val 394 (2.39)
O-4 with N-3 of Gly 344 (2.61)
O-5 with N-3 of Gly 344 (0.58)
O-5 with N-4 of Lys 343 (2.67)
O-H 3 with O-5 of Asp 404 (2.95)
O-H 3 with O-4 of Asp 404 (2.31)
N-H 2 with O-5 of Asp 404 (2.39)
N-H 3 with O-5 of Asp 404 (2.39)
11.18
11.13
01
02
–13.77
02
11.13
7
01
02
03
04
05
06
–22.71
–22.18
–18.88
–18.73
–15.97
–13.30
01
02
08
09
13
15
11.71
11.72
12.61
12.63
11.14
11.33
8
9
01
01
02
01
02
03
04
05
01
02
03
–18.91
–19.36
–15.82
–19.63
–19.63
–18.20
–7.97
08
03
04
06
07
08
09
10
09
10
13
10.17
10.28
10.28
11.42
11.40
11.37
11.33
11.34
11.35
11.36
12.76
11
–1.87
16
–15.98
–15.89
–2.57
18
01
02
03
04
–18.68
–18.62
–9.34
03
04
10
11
10.63
10.63
6.80
–8.84
6.80
between an inhibitor and an enzyme is also an important fac-
tor to determine the interaction capability of inhibition against
the enzyme.
result is generally considered to be fairy good in the area of
computer docking.
The hydrogen bond was formed between the oxygen of
compound 8 and the nitrogen of Lys 295 with its distance
being 2.74 Å. Compound 8, which bind active site amino acid
Lys 295 (Fig. 4.), has more activity than the other
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one
derivatives that bind the other amino acids around the active
site. For this reason, the goal of future studies should be
directed toward a search of the inhibitors, which have bind-
ing potency to the active site amino acid of protein kinase
p60c-Src. Compound 16 has two hydrogen bonds against Lys
295. The first hydrogen bond was formed between oxygen of
compound 16 and the nitrogen of Lys 295 with its distance
being 2.98 Å. Its lowest binding energy was found as
–15.98 kcal/mol. The other hydrogen bond was observed
between oxygen of compound and nitrogen of Lys 295 with
–15.89 kcal/mol binding energy and 2.84 Å distance. The
hydrogen bond is not the only factor, which makes the com-
pound active. There are other factors such as hydrophobic
Among the examined derivatives, compounds 8 and 16
showed hydrogen bonds against active site amino acid Lys
295 of protein tyrosine kinase p60c-Src (Table 2). Compound
8 exerted a good inhibitory capability against protein tyrosine
kinase p60c-Src with its IC50 being 70.37 µM. DOCK 4.0 is
the first docking program created by T. Kuntz group and it
generates spheres in the active site pocket around which
spheres inhibitors rotate [15]. Since the rotation takes place
only at the center of spheres, the entire calculation time is
shorter than the other docking system, while the docking pre-
cision is not as superior as it should be. Considering this fact,
our docking result showed a reasonable consistency with the
in vitro ELISA assay. Compound 8 showed a hydrogen bond
with the active site amino acid Lys 295 of protein tyrosine
kinase p60c-Src. This successful docking achievement is a ratio
of one (compound 8) out of two (compound 8 and compound
16, whose IC50 are not as bad as the most of others), and this

S. Olgen et al. / Il Farmaco 60 (2005) 497–506
505
the compounds are situated closer to the active site of the
enzyme and exert the better binding capacity of the inhibitor.
Compared to docking energy levels of both the thio and oxo
congeners, all compounds showed similar negative docking
energies low enough to be docked into the active site. As far
as the RMSD value is concerned, both the oxo and thio con-
geners have values ranging from 6 to 12, which represent the
fairly good superimposability between the compounds and
the enzyme active site.
In conclusion, we tested the inhibitory activity of
3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one and
thione derivatives by ELISA method in vitro and docking
search of inhibitors against p60c-Src tyrosine kinase. It was
found that inhibitory activity of some thio congeners was
higher than oxo congeners. Furthermore, some of the other
indole derivatives were found to be very active against p60c-
Src tyrosine kinase and several papers are intent in the litera-
ture. It was noteworthy to the evaluation of both series of
indole congeners to develop or find specific inhibitors for p60c-
Src tyrosine kinase.
Acknowledgements
This work was partially supported by a grant from the Turk-
ish Scientific and Technical Research Institute (SBAG-AYD-
400) and Research Grant of Ministry of Science and Educa-
tion (Grant No. 15590474), and also funded in part by the
Collaboration Grant A of Kobe Gakuin University. We thank
to Ms. Jyunko Hagiwara to helping tyrosine kinase activity.
Fig. 3. Atom responsible of compounds that is involved in hydrogen bon-
ding: superscripts indicate the atom responsible for hydrogen bonding with
amino acids.
interactions, steric interactions, and so on. Therefore, the
chemical modification of compound 16 adjusted to the struc-
ture of compound 8 may lead to the more active compound.
The docking energy levels and root mean square deviation
(RMSD) value of compounds are also shown in Table 2. The
RMSD value tells us how far the docked compound is situ-
ated from the ideal dock compound (the ligand), and it also
tells us the small RMSD value representing the superimpos-
ability of the two compounds. Both the lower energy levels
and the small RMSD values of compounds demonstrate that
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