Synthesis and biological evaluation of 3-(substituted-benzylidene)-1,3- dihydro-indolin derivatives as human protein kinase CK2 and p60c-Src tyrosine kinase inhibitors

Article abstract of DOI:10.1248/bpb.30.715

Human protein kinase CK2 is an ubiquitous serine/threonine kinase that is typically found in tetrameric complexes consisting of two catalytic (α and/or α′) and two regulatory β subunits. Although there is growing evidence that besides the participation of CK2 in a complex series of cellular functions, this protein kinase is involved in cell viability, cell proliferation, and neoplastic transformation. In the present study, a series of 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thione derivatives and the corresponding indolin-2-one congeners were tested for their inhibition of human recombinant protein kinase CK2 in vitro. The efficacy of these compounds was compared with their inhibitory results of p60^c-Src tyrosine kinase. It was found that 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thione derivatives are more effective than indolin-2-one congeners for the inhibition of CK2 and p60^c-Src tyrosine kinase.

Full text of DOI:10.1248/bpb.30.715

April 2007
Biol. Pharm. Bull. 30(4) 715—718 (2007)
715
Synthesis and Biological Evaluation of 3-(Substituted-benzylidene)-1,3-
dihydro-indolin Derivatives as Human Protein Kinase CK2 and p60c-Src
Tyrosine Kinase Inhibitors
c
Süreyya ÖLGEN, ,a Claudia GÖTZ,b and Joachim JOSE
*
a University of Ankara, Faculty of Pharmacy, Department of Pharmaceutical Chemistry; Tandogan/Ankara 06100, Turkey:
b Medicinal Biochemistry and Molecular Biology, Saarland University; D-66424 Homburg-Saarbrucken, Germany: and
c Bioanalytics, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf;
Universitätsstr. 1, D-40225 Düsseldorf, Germany. Received October 27, 2006; accepted November 28, 2006
Human protein kinase CK2 is an ubiquitous serine/threonine kinase that is typically found in tetrameric
complexes consisting of two catalytic (a and/or aꢀ) and two regulatory b subunits. Although there is growing evi-
dence that besides the participation of CK2 in a complex series of cellular functions, this protein kinase is in-
volved in cell viability, cell proliferation, and neoplastic transformation. In the present study, a series of 3-(substi-
tuted-benzylidene)-1,3-dihydro-indolin-2-thione derivatives and the corresponding indolin-2-one congeners were
tested for their inhibition of human recombinant protein kinase CK2 in vitro. The efficacy of these compounds
was compared with their inhibitory results of p60c-Src tyrosine kinase. It was found that 3-(substituted-benzyl-
idene)-1,3-dihydro-indolin-2-thione derivatives are more effective than indolin-2-one congeners for the inhibition
of CK2 and p60c-Src tyrosine kinase.
Key words neoplastic transformation; human protein kinase; casein kinase-2 (CK2); p60c-Src; cancer
Protein kinases have been thoroughly investigated as im- only few inhibitors of this enzyme are available.14) For long
portant targets for therapeutic intervention.1) Casein kinase-2 the best known inhibitors were emodin (IC50ꢁ2.0 mM)15) and
(CK2) probably is the most pleiotropic member of the pro- 4,5,6,7-tetrabromo-1-H-benzotriazole (TBB, 0.9 mM).16) More
tein kinase family, with ꢀ200 substrates known to date.2) It recently, a 7-substituted indoquinazoline compound was
now seems unlikely that this enzyme plays any role in the in identified out of a 400000-compound library with an IC50
vivo phosphorylation of casein, the protein from which its value of 0.08 mM towards the rat enzyme by using a virtual
name originally derived.3) Many investigations suggest that screening approach.17) Moreover, some polyhalogeno benz-
CK2 plays an essential role at various stages during the cell imidazole compounds have considerable inhibitory activity
cycle. Genetic studies in yeast indicate that CK2 is required against CK2 (IC50 in the 0.49—0.93 mM range).18)
through both G1/S and G2/M transitions.3) Furthermore, cell
3-(Substituted-benzylidene)-1,3-dihydro indolin-2-one de-
cycle progression in mammalian cells can be inhibited by rivatives have been reported as potent and selective inhibitors
blocking CK2 activity.4,5) These studies suggest that CK2 is of different receptor tyrosine kinases. Among them, SU541619)
required at multiple transitions in the cell cycle and could and SU666820) (Fig. 1) were reported as the most active
provide a promising target to interrupt the progress of prolif- compounds against VEGF-R (Flk-1/KDR). Another com-
eration in eukaryotic cells.6) Beyond the importance of CK2 pound, Sunitinib Maleate (Fig. 1), was found an inhibitor of
in the context of cell survival and cell proliferation, there is a multiple receptor tyrosine kinases involved in cancer includ-
large body of evidence that CK2 is involved in neoplastic ing VEGF and platelet-derived growth factor receptors.21)
transformation and cancer.7,8) In a number of different can- More recently, 1-benzyl-indole-2-piperidinoethyl carboxyl-
cers such as prostate,9) mammary gland,10) lung11) and oth- ate was found a potent inhibitor of p60c-Src tyrosine kinase
ers,12) abnormally high levels of CK2 have been observed. with an IC50 value of 1.37 mM.22) In our previous study, sever-
The results of these investigations and others suggest that al 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-one and
CK2 is an attractive and promising target for anti-neoplastic thione derivatives were reported as p60c-Src protein tyrosine
therapeutics. CK2 is exploited by viruses to phosphorylate kinase inhibitors.23)
proteins essential to their lifecycle and may also play a role
As the part of our interest in the area of protein kinase
in viral infections. This makes CK2 additionally an attractive inhibitors, we synthesized four new 3-(substituted-benzyl-
target for antiviral drugs.13) Despite the growing evidence on idene)-1,3-dihydro-indolin-2-one and thione derivatives.
CK2 participation in malignant transformation and cancer, These compounds and previously reported 3-(substituted-
Fig. 1. Structures of the Indole Derivatives Sunitinib Maleate, SU 5416, and SU 6668, Which Are Known as Inhibitors of Human Receptor Tyrosine Kinases
∗ To whom correspondence should be addressed. e-mail: olgen@pharmacy.ankara.edu.tr
© 2007 Pharmaceutical Society of Japan

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Vol. 30, No. 4
benzylidene)-1,3-dihydro-indolin-2-one and thione deriva- 6ꢃ), 7.43 (d, 1H, Jꢁ7.59 Hz, H-4), 7.31 (t, 1H, H-6), 7.23 (t,
tives23) were tested for their inhibitory activity towards 1H, H-5), 6.87 (d, 1H, Jꢁ7.72 Hz, H-7). MS m/z: 239.55
human recombinant protein kinase CK2. Our results indicate (Mꢄ).
that substituted 3-benzylidene-1,3-dihydro-indolin-2-thiones
(E)-3-(4ꢃ-Chloro-benzylidene)-1,3-dihydro-indolin-2-
could provide a new molecular scaffold for the rational de- thione (10): 1H-NMR (400 MHz, DMSO-d6) d: 11.56 (s, 1H,
sign of selective human CK2 inhibitors.
MATERIAL AND METHODS
NH), 7.69 (d, 2H, Jꢁ7.23 Hz, H-2ꢃ, 6ꢃ), 7.48—7.12 (m, 3H,
H-vin, H-4, H-6), 6.95 (t, 1H, H-5), 6.77—6.63 (m, 3H, H-
3ꢃ, 5ꢃ, H-7). MS m/z: 272.34 (MꢄH).
(Z)-3-(3ꢃ-Fluoro-benzylidene)-1,3-dihydro-indolin-2-
General Experimental Procedure Melting points were thione (14): 1H-NMR (400 MHz, DMSO-d6) d: 11.59 (s, 1H,
measured by capillary melting point apparatus (Buchi SMP NH), 7.72 (t, 1H, H-4ꢃ), 7.33 (s, 1H, H-vin), 7.30—7.24 (m,
20). Thin-layer chromatography was carried out on alu- 3H, H-2ꢃ, H-5ꢃ, H-6ꢃ), 7.10 (d, 1H, Jꢁ7.92 Hz, H-4), 7.04—
minum-backed silica gel plates (Merck 60 F254). Flash col- 6.46 (m, 2H, H-5, H-6), 6.10 (d, 1H, Jꢁ7.55 , H-7). MS m/z:
umn chromatography was performed on Merck silica gel 256.46 (Mꢄ).
(230—400 mesh). 1H-NMR spectra were recorded on Bruker
(E)-3-(4ꢃ-Methoxy-benzylidene)-1,3-dihydro-indolin-2-
400 AMX spectrometer at 400 MHz and are referenced to thione (15): 1H-NMR (400 MHz, DMSO-d6) d: 11.45 (s, 1H,
Me4Si for organic solutions. Mass spectra were taken on a NH), 7.69 (d, 2H, Jꢁ7.57 Hz, H-2ꢃ, 6ꢃ), 7.40—7.22 (m, 2H,
Micromass Autospec high-resolution mass spectrometer. El- H-vin, H-4), 7.15 (t, 1H, H-6), 6.92 (t, 1H, H-5), 6.77—6.58
emental analyses were obtained from a Leco-932 CHNS-O (m, 3H, H-3ꢃ, 5ꢃ, H-7), 3.64 (s, 3H, OCH3). MS m/z: 267.20
analyzer.
Reagents: p-Chloro-benzaldehyde, 3-fluoro-benzaldehyde,
(Mꢄ).
Preparation of Recombinant Human CK2 Enzyme
A
and 4-methoxy-benzaldehyde were purchased from Fluka. purification protocol was set up according to the method of
Hydrazine hydrate (Merck), hydrogen chloride (Merck), Grankowski et al.27) with modifications. For this purpose a-
sodium carbonate (Merck), anhydrous sodium sulfate subunit (CSNK2Al) and b-subunit (CSNK2B) of protein ki-
(Merck), ethyl acetate (Merck), hexane (Merck), isatine nase CK2 were expressed separately in a bacterial expression
(Aldrich), anhydrous tetrahydrofurane (Aldrich), piperidine system (pT7-7/BL2l(DE3).28) Freshly transformed bacterial
(BDH), and ethanol (Riedel) were used for reactions.
cells were grown overnight at 37 °C until they reached the
Synthesis of Oxoindole (1) 15 g (0.1 mol) isatine was stationary phase. With the seperate overnight culture for each
dissolved in 60 ml (1.2 mol) hydrazine hydrate and refluxed subunit, CSNK2Al and CSNK2B, 6 l of fresh medium was
at 140 °C for 4 h. The reaction mixture was poured into ice- inoculated and cultivated until an OD500 of 0.6 was reached.
cold water and acidified by 6 N HCl. After standing at room Protein expression was induced by adding IPTG to a final
temperature for 2 days, 7.5 g of pure oxo-indole crystals was concentration of 1 mM and was run at 30 °C for 5—6 h for
obtained. Yield, 55%. mp 127 °C (lit. 127—129 °C).24)
CSNK2Al and at 37 °C for 3 h for CSNK2B. Bacterial cells
Synthesis of Thioindole (2) 26.67 g (0.06 mol) of P2S5 were harvested by centrifugation (6000ꢂg at 4 °C for
and 6.36 g (0.06 mol) of Na2CO3 were suspended in 20 ml of 10 min) and disrupted by sonification (3ꢂ30 s on ice). Cell
anhydrous THF at 0 °C and the mixture was stirred for debris was removed by centrifugation and the bacterial ex-
20 min. 7 g (0.05 mol) oxoindole 1 was dissolved in 25 ml an- tracts by this strategy for both subunits were combined and
hydrous THF and added dropwise to this mixture. This was subjected to a three-column purification procedures as de-
stirred at 0 °C for 30 min, allowed to adapt to room tempera- scribed previously.28) The fractions containing active CK2
ture, and stirred overnight. The reaction mixture was poured holoenzyme were determined by activity measurement using
into ice-cold water and extracted with ethyl acetate (3ꢂ the synthetic peptide substrate RRRDDDSDDD. Fractions
100 ml). The organic layer was washed with brine (3ꢂ exhibiting CK2 enzymatic activity were combined and ana-
100 ml) and dried over anhydrous Na2SO4. Evaporation of lyzed by SDS page and western blot. They were stored in
the solvent gave the crude compound, which was purified by aliquots at 80 °C until used for testing.
silica gel column chromatography (hexane : ethylacetate, 70 :
30). By this procedure 6.5 g of pure compound was obtained. zyme (50 ngꢁ5 U, 1 U corresponds to the amount of kinase
Yield 83%. mp 149 °C (lit. 147—149 °C in MeOH).25)
catalyzing the transfer of 1 mmol phosphate/min to the spe-
Inhibition of Recombinant Human CK2 CK2 holoen-
General Synthesis of 3-(Substituted-benzylidene)-1,3- cific substrate) were pre-incubated at room temperature for
dihydro-indolin-2-one and 2-Thione Derivatives (3—16) 10 min with the inhibitor in a final concentration of 10 mM or
A reaction mixture of oxoindole or thioindole (1 eq), the DMSO as a control in a total volume of 20 ml in kinase
aldehyde (1.2 eq), and piperidine (0.1 eq) in ethanol (1— buffer (50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 10 mM
2 ml/1 mmol) was stirred at 90 °C for 3—5 h.26) This was MgCl2, 1 mM DTT). The inhibitors were routinely kept in a
cooled and the solvent removed under vacuum. The precipi- DMSO stock solution with a concentration of 5 mM. The re-
tate or oily compounds were purified by silicagel column action was started by adding 30 ml assay buffer (25 mM
chromatography (hexane : ethylacetate, 50 : 50). The isomers, Tris/HCl, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 mM
obtained as a mixture of E and Z, were separated by the same ATP, 0.19 mM substrate (synthetic peptide RRRDDDSDDD)
solvent system on silica gel column chromatography. The 0.6 mCi [g-32]ATP). The synthetic substrate peptide RRRD-
data for minor isomers are not reported.
DDSDDD has been identified in a study on the substrate
(Z)-3-(3ꢃ-Fluoro-benzylidene)-1,3-dihydro-indolin-2-one specificity determinants for CK2, which is the most effi-
1
(7): H-NMR (400 MHz, DMSO-d6) d: 10.62 (s, 1H, NH), ciently phosphorylated peptidic substrate.29) It is routinely
7.59 (s, 1H, H-vin), 7.57—7.49 (m, 4H, H-2ꢃ, H-4ꢃ, H-5ꢃ, H- used in studies on the enzyme activity of human CK2.30) The

April 2007
717
arginine residues at the N-terminal end can be used for pu-
rification of phosphorylated substrate by ion exchange chro-
matography and have no negative effect on enzyme activity.
Incubation was continued at 37 °C for 15 min then the mix-
ture was spotted onto a P81 ion exchange paper. After wash-
ing three times with excess phosphate (85 mM H3PO4) and
following with ethanol, the filter was dried and the appending
radioactivity determined by scintillation counter (Packard).
The radioactivity of the DMSO control was set as 100% CK2
activity and the values were obtained with the different in-
hibitors in 10 mM concentration. Each value was determined
at least three times in independent experiments.
RESULTS AND DISCUSSION
The 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-
thione and 2-one derivatives 3—16 were synthesized by con-
densing compounds 1, 2, and substituted aryl aldehydes in
the presence of different bases (Knovenagel reaction)31)
(Chart 1). Although knovenagel reaction was reported for the
synthesis of 3-(substituted-benzylidene)-1,3-dihydro-indolin-
Chart 1. Preparation of Compounds 1—16
2-one derivatives, it was successfully applied for the synthe- pounds was IC50 21.20 mM for compound 13 as shown in
sis of 3-(substituted-benzylidene)-1,3-dihydro indolin-2-thione Table 1. Compound 16 showed good activity at the 30.92 mM
derivatives in our laboratory.23) Synthesis of oxoindole (1) concentration. The 3-(2ꢃ-chloro-benzylidene)-1,3-dihydro-in-
was achieved by a Wolff–Kishner-like reduction of isatin dolin-2-one 4 (70.37 mM) also slightly inhibited the enzyme.
with hydrazine hydrate.32) The oxoindole was changed to the Interestingly, 3-(2ꢃ-chloro-benzylidene)-1,3-dihydro-indolin-
thioindole (2) by P2S5 and Na2CO3 in THF.33) All original 2-one 4 has a 4-fold greater potency than thio congener 11.
3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thione de- Only 3-(3ꢃ,4ꢃ-dichloro-benzylidene)-1,3-dihydro indolin de-
rivatives were first synthesized in our laboratory.23) The syn- rivatives of congeners 5 and 12 produced equal activity.
thesis and analysis data of newly synthesized original 3-(sub- Since some thio congeners were found more potent than the
stituted-benzylidene)-1,3-dihydro-indolin-2-thione
deriva- oxo, we suggest that the replacement of oxo with a thio
tives 10, 14, and 15 are summarized in the experimental sec- group could play a more important role in enhancement of
tion. Among the 3-(substituted-benzylidene)-1,3-dihydro-in- activity. The same substitutions to different congeners have
dolin-2-one derivatives, compounds 7 and 9 are original. The different impacts on activity. However, no clear relationships
synthesis and analysis data of compound 7 were reported in were observed between the activity profiles of identical
this paper and compound 9 was reported in our previous pub- congeners. The potency of this chemical series may also de-
lication.23) Data on other 3-(substituted-benzylidene)-1,3-di- pend, to a large extent, on the three-dimensional structure
hydro-indolin-2-one derivatives were previously reported by of R-substituents since a wide range of IC50 values (from
Daisley and Walker34) (compound 3), Neber and Rocker35) (4 ꢀ1000 mM to 20 mM) was observed depending on the nature
and 6), Howard et al.36) (5), and Sun et al.26) (compound 8).
of R-substituents as shown in Table 1.
An assay for inhibitor testing was set up in analogy to Yim
The introduction of 3ꢃ-OH, 4ꢃ-OCH3 substituents in the
et al.15) and Sarno et al.16) with modifications as described in aromatic ring (compound 16) caused higher activity of en-
Materials and Methods. The results of the test are summa- zyme CK2 and p60c-Src. In conclusion, the thio derivatives are
rized in Table 1. None of the indolin-2-one derivatives exhib- more effective for inhibition of both enzymes. This suggests
ited significant inhibition of human CK2. However, com- that 3-(substituted-benzylidene)-1,3-dihydro-indolin-2-thiones
pounds 8 and 9 showed a slight inhibition of the enzyme. To can provide a new structural principle for the rational design
our surprise, the potency of the identical thione congeners of selective human CK2 and p60c-Src tyrosine kinase in-
was higher for each example (inhibition in the 10—30% hibitors.
range). This indicates that the thione group plays an impor-
Although we have no experimental evidence as yet on this
tant role for binding of the inhibitor to the active site of CK2. issue, we propose that our compounds, based on structural
Since the enzyme inhibitory potency was found considerable similarities to SU5416 and SU6668, bind at the ATP-binding
for compounds 12 and 16, their IC50 values were calculated site in a competitive mode. At this stage, this would seem
and are reported as 24.6 and 65 mM, respectively, in Table 1.
true for both enzymes, and it appears interesting that com-
IC50 values were defined as the concentration of a com- pound 3 (2-one) showed weak potency on p60c-Src, but no in-
pound required to achieve 50% inhibition of tyrosine phos- hibition of CK2, whereas compound 10 (2-thione) showed
phorylation on the p60c-Src tyrosine kinase to ligand-stimu- some inhibition on CK2 but not on p60c-Src. These observa-
lated control reactions in the presence of vehicle alone (di- tions could be the clue for the design of selective inhibitors
methyl sulfoxide). Compounds with IC50 values ꢀ100 mM for both kinases. Such inhibitors could be helpful in the dis-
were considered inactive. Among the tested compounds, 4, section of the different roles of these enzymes in cellular
13, and 16 were found most active against the p60c-Src kinase. processes such as cell proliferation and signal transduction. It
The maximum inhibition attained among the tested com- is also known that some compounds considered to bind to the

718
Vol. 30, No. 4
Table 1. Inhibition of Human Protein Kinase CK2 by 3-(Substituted-benzylidene)-1,3-dihydro-indolin-2-one and Indolin-2-thione Derivatives
mp
(°C)
CK2 Inhib.
(%)
CK2
IC50 (mM)
p60c-Src PTK
IC50 (mM)
Compd. No.
R
Isomer
3
4
5
6
7
4ꢃ-Cl
2ꢃ-Cl
3ꢃ, 4ꢃ-Cl
2ꢃ, 6ꢃ-Cl
3ꢃ-F
4ꢃ-OCH3
3ꢃ-OH, 4ꢃ-OCH3
4ꢃ-Cl
E
Z
Z
Z
Z
E
Z
E
Z
Z
Z
Z
E
E
191—193
181
183—186
164—167
145—147
156
0
0
4
0
0
18
23
30
10
350.0
70.4
149.1
ꢀ1000
ꢀ1000
ꢀ1000
203.2
ꢀ1000
305.6
154.5
21.2
ꢀ1000
ꢀ1000
30.9
8
9
153
10
11
12
13
14
15
16
188—189
173—174
158—160
155—156
226—227
170—171
95—96
2ꢃ-Cl
3ꢃ, 4ꢃ-Cl
2ꢃ, 6ꢃ-Cl
3ꢃ-F
4ꢃ-OCH3
3ꢃ-OH, 4ꢃ-OCH3
24.6
65
19
21
30
(1999).
substrate site show considerable selectivity among different
enzymes. This gives rise to the possibility that highly specific
tyrosine kinase inhibitors can be developed. On the other
hand, dual inhibitors of both kinases, e.g. by compounds 12
16) Sarno S., Reddy H., Meggio F., Ruzzene M., Davies S. P., Donella-
Deana A., Shugar D., Pinna L. A., FEBS Lett., 496, 44—48 (2001).
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