Synthesis and pp60c-Src tyrosine kinase inhibitory activities of novel indole-3-imine and amine derivatives substituted at N1 and C5

Article abstract of DOI:10.1002/ardp.200800216

A series of novel 1,3,5-trisubstituted indole derivatives, namely, N-benzyl 5-phenyl indole-3-imine, N-benzyl-5-(p-fluorophenyl)indole-3-imine and their corresponding amine congeners, were designed and synthesized as pp60 ^c-Src tyrosine kinase

Full text of DOI:10.1002/ardp.200800216

Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
Z. Kılıc˛ et al.
333
Full Paper
Synthesis and pp60c-Src Tyrosine Kinase Inhibitory Activities
of Novel Indole-3-Imine and Amine Derivatives Substituted at
N1 and C5
Zuhal Kiliꢀ1, Yasemin G. Isgꢁr1, 2, and Sꢂreyya ꢃlgen1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara, Tandogan-Ankara,
Turkey
2 Nanomedicine Research Center, Gazi University, Gꢀlbasi, Ankara, Turkey
A series of novel 1,3,5-trisubstituted indole derivatives, namely, N-benzyl 5-phenyl indole-3-
imine, N-benzyl-5-(p-fluorophenyl)indole-3-imine and their corresponding amine congeners,
were designed and synthesized as pp60c-Src tyrosine kinase inhibitors, and their inhibitory activ-
ities toward pp60c-Src tyrosine kinase were evaluated by in-vitro kinase assay. Pre-screening at two
doses of compounds against kinase target revealed that, except for the N-benzyl-5-phenyl indole
imine derivatives 7a–7d, all indole derivatives show the target inhibition at varying levels. Con-
sequently, the compounds, 8c, 8f, 8g, and 8h, were selected for prescreening tests. The dose-
response curves for up to six concentrations (250 to 7.8 lM) of the active compounds were
obtained by tyrosine kinase assay and the four-parameter logistic analysis of these data resulted
in the IC50s of 4.69, 74.79, 75.06, and 84.23 lM for compounds 8c, 8f, 8g, and 8h, respectively.
Therefore, compound 8c, 1-(1-benzyl-5-phenyl-1H-indole-3-yl)-N-(4-fluorobenzyl)methanami-
ne N HCl, was the promising inhibitor for pp60c-Src, followed by compounds 8g and 8h. Under the
same conditions, compound 8f did not provide any reasonable inhibition pattern to be consid-
ered as active compound. Therefore, among all four active compounds, compound 8f was not
found suitable for further analysis.
Keywords: Amine derivatives / Enzyme inhibition / Indole-3-imine / Tyrosine kinase pp60c-Src
Received: November 26, 2008; accepted: January 9, 2009
DOI 10.1002/ardp.200800216
/
regulation and promotion of diverse cellular processes,
including cell growth and differentiation, motility, pro-
liferation, adhesion, migration, and immune cell func-
tion [3, 4]. Although Src itself is weakly oncogenic, recent
advances have implicated its role in the pathophysiology
of cancer, showing that the constitutive oncogenic acti-
vation in cancer cells can be blocked by selective tyrosine
kinase inhibitors [5]. Src tyrosine kinase activity has been
shown to be an important component in the epithelial to
mesenchymal transition that occurs in the early stages of
invasion of carcinoma cells [6], and elevated protein lev-
els and catalytic activity of Src have also been detected in
many cancers including breast, colon, lung, pancreatic,
skin, and head and neck [7, 8]. Src activity is also known
to be essential in the turnover of focal adhesion, a critical
cell motility component [9, 10]. Evidence from the clini-
Introduction
The c-Src (pp60c-Src) protein tyrosine kinase (PTK) is a mem-
ber of the largest family of nonreceptor PTKs, namely the
Src family kinases (SFKs), which is composed of enzymes
homologous to c-Src PTK [1]. Src tyrosine kinase was dis-
covered as the first proto-oncogene product that pos-
sesses intrinsic protein kinase activity [2]. The members
of SFK, and especially c-Src, have been implicated in the
Correspondence: Prof. Sꢁreyya ꢂlgen, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Ankara University, 06100, Tandogan/
Ankara, Turkey.
E-mail: olgen@pharmacy.ankara.edu.tr
Fax: +90 312 213 1081
Abbreviations: Protein tyrosine kinase (PTK); Src family kinases (SFKs)
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Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
Figure 1. Potent inhibitors of pp60c-Src tyrosine kinase.
cal study also supports a link between deregulated Src of Src-mediated tyrosine phosphorylation [17, 18].
activity with increased invasive potential of tumor cells Toward the development of more effective SFK inhibi-
[11]. Therefore, with an emphasis on combination thera- tors, it is important to draw attention to the pyrazolopyr-
pies with standard chemotherapeutic agents, Src kinase imidines PP1[1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyri-
inhibitors may enhance their therapeutic value by midine-4-yl-amine] and PP2[1-tert-Butyl-3-p-chloro-1H-pyr-
increasing the sensitivity of tumors to the chemothera- azolo[3,4-d] pyrimidine-4-yl-amine], which were found as
peutic agents, and also by preventing metastasis to selective inhibitors of Src-family member pp60c-Src with
improve the disease prognosis [12]. Consequently, consid- IC50 in the nanomolar range (Fig. 1) [19, 20]. More
ering their ability to participate and control the many recently, several pyrrolopyrimidine compounds (7-pyrro-
phases of tumor progress and metastasis, targeting Src lidinyl- and 7-piperidinyl-5-pyrrolo[2,3-d]-pyrimidines
and many members of SFKs is a promising way of discov- and 7-alkyl- and 7-cycloalkyl-5-aryl-pyrrolo[2,3-d]pyrimi-
ering new anticancer therapeutics for metastatic dis- dines) were reported as potent tyrosine kinase pp60c-Src
eases. Inhibition of Src by small molecule compounds inhibitors, which are useful in the treatment of osteopo-
can be achieved at several sites, including protein-pro- rosis [21, 22]. 2-Amino-4-oxo-pyrrolo[2,3-d] pyrimidine
tein interactions between the Src SH2 and SH3 domains (LY231514, Pemetrexed) (Fig. 1) is also a pyrrolopyrimi-
and their substrates, interaction between protein sub- dine compound, which was reported as a strong anti-
strates and the substrate-binding site, and ATP-binding cancer agent [23].
site within the tyrosine kinase domain of Src [13, 14]. In
Studies on indole-derived tyrosine kinase inhibitors
terms of selectivity, cellular potency, and possible thera- revealed that several active indole derivatives have the
peutic applications, Src kinase inhibitors appear to be ability to inhibit several PTKs. On the other hand, com-
the highly promising therapeutic agents. However, the pounds utilizing an indole scaffold were reported with
only dual src/abl (Abelson-Murine Leukemia) inhibitor moderate activity for Src kinase as in the studies with
that has been approved as cancer therapeutics by the hydroxyindole derivatives [24]. Moreover, recently
FDA is dasatinib, a derivative of aminothiazole to be used reported substituted 3-[3-(aminopropyl)-4,5,6,7-tetrahy-
for chronic myelogenous leukemia (CML) [15, 16]. Several dro-1H-indole-2-yl-methylene]-1,3-dihydro-indole-2-one
classes of small molecules which are structurally classi- derivatives were shown as potent and selective inhibitors
fied as pyrazolo[3,4-d]pyrimidines, pyrrolo[2,3-d]pyrimi- of the PTKs, namely Src and Yes. Among these derivatives,
dines, pyrido[2,3-d] pyrimidines, quinolines, and indoli- in particular, 3-[3-(dimethylaminopropyl)-4,5,6,7-tetrahy-
nones have been reported as ATP-competitive inhibitors dro-1H-indole-2-yl-methylene]-2-oxo-2,3-dihydro-1H-in-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
Novel Indole-3-Imine and Amine Derivatives
335
Reagents and conditions: (i) Pd(PPh3)4, NaCO3, arylboronic acids, anhydrous toluene; (ii) NaH, BnBr, DMF, 08C to room temper-
ature; (iii) DMF, POCl3, – 158C to room temperature; (iv) substituted benzylamines, MgSO4, CH2Cl2, reflux; (v) NaBH4, 508C.
Scheme 1. Synthesis of 1,3,5-trisubstituted indole derivatives.
dole-5-sulfonic acid methylamide was reported as prom- tuted indole derivatives, their inhibitory activity on Src
ising inhibitor of Src kinase with IC50 of 0.1 lM (Fig. 1) kinase, and the impact of the substituents on the indole
[25].
ring in terms of their activity on the kinase target. The
Our previous studies on the indole scaffold bearing new inhibitors were identified and their structure-activ-
inhibitor research revealed that 1-benzyl-indole-2-piperi- ity relationship with respect to the imine and amine sub-
dinoethyl carboxylate has the ability to inhibit Src with stitution at third position of indole was discussed as
IC50 of 1.34 lM [26]. Our continuing interest in indole described in the following section.
derivatives expanded with a study on a series of 3-(substi-
tuted-benzylidene)-1,3-dihydro-indolin-2-thione
deriv-
atives and their corresponding indolin-2-one congeners
as Src PTK inhibitors. Of the derivatives analyzed in this
Results and discussion
study, we reported that (Z)-3-(49-dimethylamino-benzyli- The synthetic routes of N-benzyl-5-phenyl-indole-3-imine,
dene)-1,3-dihydro-indolin-2-thione and (Z)-3-(29,69-di- N-benzyl-5-(p-fluorophenyl) indole-3-imine and their
chloro-benzylidene)-1,3-dihydro-indolin-2-thione are amine congeners are described in Scheme 1. Palladium-
moderately active Src PTK inhibitors, with IC50 of 21.91 catalyzed cross-coupling reaction between bromoindole
and 21.20 lM, respectively [27]. With regard to previous and arylboronic acids provided 5-phenyl indole 1 and 5-
studies, we focused on the design, synthesis, and activity (p-fluorophenyl)indole 2 [28, 29]. Organoboron reagents
testing of 20 novel 1,3,5-trisubstituted indole derivatives exhibit greater functional group compatibility than
as pp60c-Src tyrosine kinase inhibitors. With the guidance organozinc or Grignard reagents and they are nontoxic
of results from the literature, here, we particularly and thermally, air- and moisture-stable, and inexpensive.
focused on substituents at 1,3,5-positions on indole ring Therefore, they were chosen for the biaryl synthesis [30].
to improve the inhibitory potency of molecules against For employing the arylboronic acids, Pd(Ph3)4 was used as
the Src kinase target. Therefore, in this study, we report the catalyst, Na2CO3 as the base, and a mixture of toluene
the synthesis and biological activity of 1,3,5-trisubsti- / EtOH / H2O as solvent. The benzylation of 5-phenyl
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Data shown here is the prescreening result of compounds at two different concentrations, each in duplicate (high dose,
250 lM and low dose 50 lM).
Figure 2. Activity comparison of N-benzyl-5-phenyl indole derivatives 7a–7e and 8a–8e
as%-inhibition exerted on pp60c-Src (650610– 7 units/lL) kinase target activity.
indole 1 and 5-(p-fluorophenyl)indole 2 was performed, singlet at 9.42–9.77 ppm. The CH2 protons of CH2NH-CH2-
following standard procedures using NaH 95% in dry Ph (or substituted Ph) were measured as triplet at 4.35–
DMF giving N-benzyl-5-phenyl indole 3 and N-benzyl-5-(p- 4.48 ppm and 4.17–4.28 ppm, respectively. The mass
fluorophenyl)indole 4 [31]. N-Benzyl-5-phenyl-indole-3- spectra of compounds were taken with ESI methods and
carbaldehyde 5 and N-benzyl-5-(p-fluorophenyl)-3-carbal- the mass values of compounds were monitored as [M + 1]
dehyde 6 were synthesized by using Vilsmaier formyla- and [M+]. The expected isotop peaks of Cl and Br atoms
tion [32]. Indole-3-carbaldehydes were then subjected to a were detected as a ratio of 3 : 1 and 1 : 1, respectively,
condensation with substituted benzylamines in CH2Cl2 and numbers of peaks were also detected depending on
resulting in the Schiff' bases as Z / E (syn / anti) isomer mix- the number of atoms. Elemental analysis of the com-
tures 7a-7j [33]. The reaction of imine compounds with pounds indicated purity in the range l 0.4%.
NaBH4 afforded their oily amine congeners [34, 35] which
The biological activity of the compounds was eval-
were converted into a solid form as HCl salts 8a–8j. uated by an in-vitro tyrosine-kinase assay that measures
Attempts to separate the Z / E (syn / anti) isomer mixtures the changes in the enzymatic activity of pp60c-Src tyrosine
by flash chromatography were not successful, because of kinase by virtue of following the alterations in the phos-
the decomposition of compounds during the procedure. phorylation level of the immobilized substrate with
All the synthesized compounds were characterized by 1H- respect to DMSO (vehicle) control [36]. The activity of the
NMR, IR, MS spectral methods, and the purity of final synthesized compounds was initially analyzed via pre-
compounds was determined by elemental analysis. All liminary screening (pre-screening) at two concentrations
data were consistent with the proposed structures. The of the compounds, reflecting the high (250 lM) and low
data are shown in experimental part; for the Physico- (50 lM) doses, to determine those capable of altering the
chemical properties and spectral data of the compounds substrate-phosphorylation level with reasonable inhibi-
see Table 1.
tion profile. The pre-screening results were reported as%-
N-Benzyl protons of indole were observed as a sharp inhibition of the Src activity at 50 lM and 250 lM of the
singlet at 5.14–5.34 ppm for imine and 5.46–5.50 ppm compounds, with respect to DMSO control. The pre-
for amine derivatives. The chemical shifts of all aro- screening of each compound was performed as two inde-
matics protons were detected at the 6.75–8.62 ppm for pendent experiments, each in duplicates, and the data is
imine and 7.16–8.13 ppm for amine compounds. The presented as bar graphs, as given in Figs. 2 and 3. Tyro-
characteristic CH=N protons of imine compounds were sine-kinase assay was performed at 350610– 7 unit/lL
detected at 8.42–8.62 ppm as a mixture of Z / E (syn / anti) pp60c-Src for both preliminary and dose-response screen-
isomers. The CH2 peaks of CH=N-CH2-Ph (or substituted ing of the compounds. Those compounds that yielded
Ph) were found at 4.64–4.83 ppm. The NH peaks of a minimum 50% inhibition at 50 lM and more than 70%
CH2NH-CH2-Ph (or substituted Ph) were obtained as broad at 250 lM, were reported as “active” from pre-screening
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Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
Novel Indole-3-Imine and Amine Derivatives
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Table 1. Physicochemical properties and spectral data of the compounds.
Comp. Isomers M.p.
% Yield
Molecular
Formula
Mass
ESI-MS: m/z
Elemental Analysis
(Calcd. / Found)
1H-NMR
8C
C
H
N
7a
7b
E/Z
E/Z
95
89
79.84
78.80
C29H24N2
401.26
[M + 1]
86.97
87.21
6.04
5.78
6.99 (CDCl3) d: 8.62–8.60 (m, 1H, CH=N, syn / anti mix-
7.05 ture), 8.62 (m, 1H, H-b), 7.68 –7.15 (m, 18H, aromatic
protons), 5.33 (s, 2H, CH2Ph), 4.83 (s, 2H, =N-CH2-Ph)
6.38 (CDCl3) d: 8.52 (m, J = 1.6 Hz, 1H, H-b), 8.42 (s, 1H,
6.46 CH=N, syn / anti mixture), 7.56–6.99 (m, 17H, aro-
matic protons), 5.14 (s, 2H, CH2Ph), 4.64 (s, 2H, =N-
CH2-Ph)
C29H23ClN2
435.38
[M + 1]
79.42
79.27
5.37
5.60
N (0.2 H2O)
7c
E/Z
E/Z
75
67.33
C29H23FN2
419.28
[M + 1]
81.81
81.57
5.63
5.80
N (0.4 H2O)
4.82
5.02
N (0.4 H2O)
6.58 (CDCl3) d: 8.58 (m, 2H, CH=N, syn / anti mixture and
6.74 H-b), 7.67-6.99 (m, 17H, aromatic protons), 5.34 (s,
2H, CH2Ph), 4.78 (s, 2H, =N-CH2-Ph)
5.87 (CDCl3) d: 8.66 (d, J = 1.6 Hz, 1H, H-b), 8.53 (s, 1H,
5.99 CH=N, syn / anti mixture), 7.67–7.11 (m, 16H, aro-
matic protons), 5.27 (s, 2H, CH2Ph), 4.80 (s, 2H, =N-
CH2-Ph)
7d
105–107 77.52
C
29H22Cl2N2
469.28
[M+]
73.08
72.89
7e
7f
E/Z
E/Z
E/Z
E/Z
88–90
90–91
76.51
64.50
C29H22F2N2
C29H23FN2
437.33
[M + 1]
79.46
79.14
5.10
4.82
(0.1 H2O) N
6.39 (CDCl3) d: 8.60 (d, J = 1.2 Hz, 1H, H-b), 8.54 (s, 1H,
6.48 CH=N, syn / anti mixture), 7.66–6.75 (m, 16H, aro-
matic protons), 5.26 (s, 2H, CH2Ph), 4.77 (s, 2H, =N-
CH2-Ph)
6.66 (CDCl3) d: 8.56 (d, J = 1.2 Hz, 1H, H-b), 8.54 (s, 1H,
6.71 CH=N, syn / anti mixture), 7.58–7.05 (m, 17H, aro-
matic protons), 5.25 (s, 2H, CH2Ph), 4.80 (s, 2H, =N-
CH2-Ph)
6.18 (CDCl3) d: 8.55 (s, 1H, CH=N, syn / anti mixture), 8.53
6.21 (s, 1H, H-b), 7.45 (s, 1H, H-a), 7.60–7.07 (m, 15H, aro-
matic protons), 5.31 (s, 2H, CH2Ph), 4.76 (s, 2H, =N-
CH2-Ph)
6.42 (CDCl3) d: 8.55 (s, 1H, CH=N, syn / anti mixture), 8.55
6.50 (s, 1H, H-b), 7.44 (s, 1H, H-a), 7.59–6.98 (m, 15H, aro-
matic protons), 5.29 (s, 2H, CH2Ph), 4.76 (s, 2H, =N-
CH2-Ph)
5.72 (CDCl3) d: 8.59 (s, 1H, H-b), 8.57 (s, 1H, CH=N, syn / anti
5.75 mixture), 7.61–7.08 (m, 15H, aromatic protons), 5.33
(s, 2H, CH2Ph), 4.82 (s, 2H, =N-CH2-Ph)
6.16 (CDCl3) d: 8.57 (s, 1H, CH=N, syn / anti mixture), 8.53
6.30 (d, J = 1.6 Hz, 1H, H-b), 7.48 (s, 1H, H-a), 7.60 –6.77 (m,
14H, aromatic protons), 5.33 (s, 2H, CH2Ph), 4.78 (s,
2H, =N-CH2-Ph)
6.32 (DMSO-d6) d: 9.51 (bs, 2H, +NH2), 8.02 (d, J = 1.6 Hz, 1H,
6.56 H-b), 7.74 (s, 1H, H-a), 7.70 –7.22 (m, 17H, aromatic
protons), 5.46 (s, 2H, CH2Ph), 4.35 (t, 2H, CH2NH), 4.17
(t, 2H, NH-CH2-Ph)
419.35
[M + 1]
82.87
82.70
5.56
5.55
N (0.1 H2O)
7g
7h
99–101 55.00
100–102 87.48
101–103 83.03
C
29H22ClFN2 453.36
76.90
77.07
4.90
4.67
[M + 1]
C29H22F2N2
437.37
[M + 1]
79.80
79.55
5.08
4.87
7i
7j
E/Z
E/Z
C29H21Cl2FN2 487.31
[M+]
71.20
70.97
4.36
4.08
N (0.1 H2O)
4.66
81–82
186
66.61
78.12
C29H21F3N2
C29H27ClN2
C29H26Cl2N2
455.43
[M + 1]
76.64
76.54
4.70
8a
8b
8c
8d
8e
8f
–
–
–
–
–
–
403.00
[M + 1]
78.69
78.79
6.23
6.16
N (0.2 H2O)
214–216 91.80
204–205 85.91
196–198 93.50
195–197 87.95
437.29
[M + 1]
73.57
73.51
5.54
5.63
5.92 (DMSO-d6) d: 9.51 (bs, 2H, +NH2), 8.05 (d, J = 1.6 Hz, 1H,
5.92 H-b), 7.75 (s, 1H, H-a), 7.72 –7.24 (m, 16H, aromatic
protons), 5.49 (s, 2H, CH2Ph), 4.38 (t, 2H, CH2NH), 4.21
(t, 2H, NH-CH2-Ph)
6.10 (DMSO-d6) d: 9.55 (bs, 2H, +NH2), 8.05 (d, J = 1.6 Hz, 1H,
6.12 H-b), 7.76 (s, 1H, H-a), 7.73 –7.24 (m, 16H, aromatic
protons), 5.48 (s, 2H, CH2Ph), 4.37 (t, 2H, CH2NH), 4.20
(t, 2H, NH-CH2-Ph)
C29H26ClFN2 421.27
[M + 1]
75.92
75.90
5.75
6.01
N (0.1 H2O)
C29H25Cl3N2
473.00
[M + 2]
68.09
67.92
5.00
4.70
N (0.2 H2O)
5.47 (DMSO-d6) d: 9.77 (bs, 2H, +NH2), 8.13 (d, J = 1.6 Hz, 1H,
5.54 H-b), 7.81 (s, 1H, H-a), 7.83 –7.24 (m, 15H, aromatic
protons), 5.50 (s, 2H, CH2Ph), 4.48 (s, 2H, CH2NH), 4.28
(s, 2H, NH-CH2-Ph)
5.90 (DMSO-d6) d: 9.57 (bs, 2H, +NH2), 8.11 (d, J = 1.6 Hz, 1H,
5.98 H-b), 7.78 (s, 1H, H-a), 7.80 –7.18 (m, 15H, aromatic
protons), 5.49 (s, 2H, CH2Ph), 4.45 (t, 2H, CH2NH), 4.22
(t, 2H, NH-CH2-Ph)
C29H25ClF2N2 439.39
[M + 1]
73.33
73.54
5.31
5.59
198
86.49
C29H26ClFN2 421.41
[M + 1]
76.22
76.16
5.73
5.67
6.13 (DMSO-d6) d: 9.53 (bs, 2H, +NH2), 8.03 (d, J = 1.6 Hz, 1H,
6.30 H-b), 7.76 (s, 1H, H-a), 7.75 –7.24 (m, 16H, aromatic
protons), 5.49 (s, 2H, CH2Ph), 4.37 (t, 2H, CH2NH), 4.19
(t, 2H, NH-CH2-Ph)
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Table 1. Continued.
Comp. Isomers M.p.
% Yield
Molecular
Formula
Mass
ESI-MS: m/z
Elemental Analysis
(Calcd. / Found)
1H-NMR
8C
C
H
N
8g
8h
8i
–
–
–
–
209
200
193
88.77
88.48
82.82
C29H25Cl2FN2 455.44
[M + 1]
70.88
71.05
5.13
5.35
5.70 (DMSO-d6) d: 9.42 (bs, 2H,+NH2), 8.03 (d, J = 1.2 Hz, 1H,
5.85 H-b), 7.75–7.24 (m, 16H, aromatic protons), 5.48 (s,
2H, CH2Ph), 4.38 (s, 2H, CH2NH), 4.21 (s, 2H, NH-CH2-
Ph)
5.90 (DMSO-d6) d: 9.42 (bs, 2H, +NH2), 8.03 (d, J = 1.6 Hz, 1H,
5.94 H-b), 7.74 (s, 1H, H-a), 7.76 –7.24 (m, 15H, aromatic
protons), 5.48 (s, 2H, CH2Ph), 4.37 (s, 2H, CH2NH), 4.20
(s, 2H, NH-CH2-Ph)
C29H25ClF2N2 439.51
[M + 1]
73.33
73.02
5.31
5.12
C29H24Cl3FN2 489.24
[M+]
66.00
65.73
4.62
4.28
N (0.1 H2O)
5.30 (DMSO-d6) d: 9.70 (bs, 2H,+NH2), 8.11 (d, J = 1.2 Hz, 1H,
5.42 H-b), 7.81–7.24 (m, 15H, aromatic protons), 5.50 (s,
2H, CH2Ph), 4.48 (s, 2H, CH2NH), 4.28 (s, 2H, NH-CH2-
Ph)
5.68 (DMSO-d6) d: 9.55 (bs, 2H, +NH2), 8.09 (d, J = 1.2 Hz, 1H,
5.77 H-b), 7.77 (s, 1H, H-a), 7.81 –7.16 (m, 14H, aromatic
protons), 5.49 (s, 2H, CH2Ph), 4.45 (s, 2H, CH2NH), 4.22
(s, 2H, NH-CH2-Ph)
8j
187–188 89.17
C29H24ClF3N2 457.36
[M + 1]
70.66
70.50
4.91
4.52
Data shown here is the prescreening result of compounds at two different concentrations, each in duplicate (high dose,
250 lM and low dose 50 lM).
Figure 3. Activity comparison of N-benzyl-5-(p-fluoro)phenyl indole derivatives 7f–7j and
8f–8j as%-inhibition exerted on pp60c-Src (650610-7 units/lL) kinase target activity.
where the compounds were dissolved in the maximum- activity against the kinase target (Figs. 2 and 3) at both
tolerated DMSO-containing assay buffer. The active com- high and low doses of the compounds. Among N-benzyl-
pounds were further analyzed to obtain dose-response 5-phenyl indole imine derivatives 7a–7e, reduced solubil-
curves with varying concentrations of compounds ity was observed only for compounds 7a–7d and these
(DMSO a 0.6% of the assay). Their potential to inhibit were eventually inactive. Of these, slightly active 7e was
enzyme activity was reported as IC50 and determined by only partially soluble at the tolerated levels of DMSO,
virtue of nonlinear regression analysis (four parameter whereas solubility of 7a–7d could not be improved
logistic equation, sigmoidal dose-response) performed under the same conditions (Fig. 2). At 250 lM, of all com-
using GraphPad Prism version 4.0 for Windows (Graph- pounds analyzed, more than 70% inhibition was
Pad Software, San Diego Ca, USA; www.graphpad.com). observed for the amine salts of the N-benzyl-5-phenyl 8a–
The pre-screening of N-benzyl-5-phenyl 7a–8e and N-ben- 8e and N-benzyl-5-(p-fluoro)phenyl indole 8f–8j deriv-
zyl-5-(p-fluoro)phenyl 7f–8j indole derivatives revealed atives. At 50 lM, however, the 50% activity criterion was
that, except for 7a–7d, all compounds showed some only fullfilled by five compounds, and these were N-ben-
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Novel Indole-3-Imine and Amine Derivatives
339
Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at six doses of
compound 8c. Data represents the results of three independent experiments each in
duplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.
Figure 4. The dose-response curve for the compound 8c with
IC50 4.69 l 1.23 lM.
Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at five doses of
compound 8g. Data represents the results of two independent experiments each in
duplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.
Figure 6. The dose-response curve for the compound 8g with
IC50 75.06 l 1.24 lM.
with the prescreening results. This was due to various lev-
els of DMSO content used in these two assays. To get all
possible active compounds throughout the prescreening
at two doses of the compounds, the solubility was
increased with DMSO using its maximum-tolerated lev-
els. Knowing that DMSO interferes with enzyme activity
to some level of the vehicle used in compound dilutions
(data not shown), compounds with up to six doses were
prepared in the lowest levels of DMSO and so interference
that could possibly arise from the vehicle was eliminated
while constructing the dose-response curves. The
observed reductions at%-inhibition of the maximum
doses were therefore observed with the dose-response
curves of all four active compounds. The analysis of dose-
response data using the four-parameter logistic analysis
method (GraphPad Prism 4.0) revealed that the com-
pound 8c, 1-(1-benzyl-5-phenyl-1H-indole-3-yl)-N-(4-fluoro-
benzyl)methanamine … HCl, is the most potent inhibitor
of pp60c-Src tyrosine kinase with IC50 of 4.69 l 1.23 lM
(Fig. 4), followed by compounds 8g and 8h with IC50s of
75.06 l 1.24 (Fig. 6) and 84.2 3l 1.19 lM (Fig. 7), respec-
tively. Under the same assay conditions, compound 8f
with IC50 of 74.79 l 1.43 could not provide a reasonably
high maximum inhibition, and therefore a narrow assay
window, to be considered as active compound (Fig. 5).
Hence, among all four actives, compound 8f was not suit-
able to be further developed as drug lead or biological
target probe.
Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at five doses of
compound 8f. Data represents the results of two independent experiments each in
duplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.
Figure 5. The dose-response curve for the compound 8f with
IC50 74.79 l 1.43 lM.
zyl-5-(p-fluoro)phenyl containing indole imine 7j, amine
salts of N-benzyl-5-(p-fluoro)phenyl indole compounds
8f–8h, and N-benzyl-5-phenyl containing indole amine
8c. Therefore, based on these prescreening results, only
four compounds, 8c, 8f, 8g, and 8h, were selected as
being active. The dose-response curves of these com-
pounds were obtained from two to three independent
tyrosine-kinase assays (each in duplicates, n = 3–6) for
five concentrations (250–15.62 lM) for compounds 8f–
8g and six concentrations (250–7.8 lM) for compound 8c
(Fig. 4, 5, 6, and 7). The dose-response curves of these com-
pounds were obtained using tyrosine-kinase assay for six
concentrations (250–7.8 lM) of compound 8c and five
concentrations (250–15.62 lM) of compounds 8f, 8g, and
8h. In the dose-response curves, 10–25% decrease in per-
cent inhibition was observed, as expected, compared
To understand the role of indole-ring substitutions
and the biological activity of derivatives, we evaluated
substituents on the indole scaffold and their activity
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Z. Kılıc˛ et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
analyzed, the four passing the prescreening criteria were
indoles having substitution on the third position with
either unsubstituted 8f or mono-halogen-substituted 8c,
8g, and 8h phenlyamines. As explained before, com-
pound 8f with a IC50 value of 74.79 l 1.43 was not consid-
ered as active compound. These different behaviors of
substitutions on activity might be due to their favorable
contribution to lipophilic and electronic factors of the
compounds. While no significant correlation was
observed, all active compounds were identified as those
possessing electron-withdrawing groups on the benzyl
ring at the third position of indole. This general consider-
ation may help to understand the concept of the SAR of
the derivatives analyzed here. The activity differences of
both imine and amine derivatives seem to be particularly
interesting, considering that the imine derivatives were
either inactive or less active compared with their corre-
sponding amine derivatives which were identified with
remarkable activity. One possible reason might be that
the amine compounds are more soluble in assay medium
and more efficiently reacted with enzyme.
Tyrosine kinase assay was performed at 350610 – 7 unit/lL pp60c-Src at five doses of
compound 8h. Data represents the results of two independent experiments each in
duplicate (n = 3 – 6) and the IC50 values obtained by non-linear regression analysis.
Figure 7. The dose-response curve for the compound 8h with
IC50 84.23 l 1.19 lM.
behavior on prescreening of compounds. Analyzing the
5-substitution on the indole ring, it was apparent that a
5-(4-fluoro)-phenyl substitution, regardless of any sub-
stituent at other positions of the indole ring, improved
the activity of the compounds compared with their corre-
sponding 5-phenyl-substituted compounds. This ten-
dency was also true for both imine and amine derivatives
of these compounds, and the remarkable difference was
seen from the screening at low dose (50 lM). In more
detailed analysis of screening results, compounds 8a–8e
from 5-phenyl-substituted indole amine series, were com-
pared with their corresponding members 8f–8j from 5-
(4-fluoro)-phenyl substituted indole amine series. At the
third position of the indole, substitutions were made on
the phenyl ring (Scheme 1, substitutions R2 and R3). Here,
the comparison of 3-substitution on the indole scaffold
revealed that except compounds 8c, 8d, and 8e, none of
the compounds from this series showed neither closer
nor better activities than their corresponding com-
pounds in the 5-(4-fluoro)-phenyl-substituted indole
amine series, when screening was performed at the low
dose (clear bars, Figs. 2 and 3). Compounds 8c and 8e
were the indole derivatives bearing 3-substitution with
mono- and di-fluoro-substituted phenlyamines, respec-
tively. Among the 5-(4-fluoro)-phenyl-indole amine deriv-
atives, the compounds' activities were highest when
unsubstituted or mono-halogen-substituted (8f, 8g, and
8h) compared to their corresponding derivatives with di-
halogen substituents (8i and 8j). Here, interestingly, the
active mono-halogen-bearing derivatives were those hav-
ing p-fluoro or p-chloro substituents in the benzyl ring at
third position of the indole scaffold. Of the compounds
In the light of these results, more advanced structural
optimizations with further biological analyses may facili-
tate efforts to investigate the role of substituents at the
1,3,5-positions of indoles to design and synthesize more
potent compounds as potential Src kinase inhibitors to
be develop them as drug leads or chemical probes for Src
kinase target in the future.
This work was partially supported by a grant from the Turkish
Scientific and Technical Research Institute (106S127 SBAG-HD-
141).
The authors have declared no conflict of interest.
Experimental
Chemistry
Anhydrous magnesium sulphate, sodium sulphate, hexane,
ethyl acetate, anhydrous dimethylformamide, sodium dihydro-
gen phosphate, potassium hydrogen phosphate, sodium chlor-
ide, dimethylsulfoxide, sulfuric acid, mercaptoethanol, phos-
phorous oxychloride, tetrakistriphenylphosphine palladium,
sodium carbonate, ethanol (from Merck, Darmstadt, Germany);
deutero dimethylsulfoxide, deutoro chloroform, benzylamine,
4-chlorobenzylamine, 4-fluorobenzylamine, 2,4-dichlorobenzyl-
amine, 2,4-difluorobenzylamine, indole, 5-bromoindole, phenyl-
boronic acid, 4-fluorophenylboronic acid (from Acros Organics,
Geel, Belgium); methanol, hydrochloric acid, dichloromethane,
anhydrous ethanol, sodium hydride, benzyl bromide, anhy-
drous toluene (from Sigma-Aldrich, St. Louis, MO, USA). Takara
Universal Tyrosine Assay Kit (from Takara-Bio Inc., Shiga, Japan)
was used to analyze the biological activity of the compounds syn-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
Novel Indole-3-Imine and Amine Derivatives
341
thesized in this study. The contents of a kit: PTK substrate immo-
bilized microplate (8 wells612), kinase reacting solution
(11.0 mL), 40 mM ATP-2 Na (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), HRP coloring solu-
tion (TMBZ = Tetra Methyl Benzidine, 12.0 mL). Melting points
were measured with a capillary melting point apparatus (Elec-
trothermal, Essex, UK) and are uncorrected. The Nuclear Mag-
netic Resonance (1H-NMR) spectra were recorded on Varian Mer-
cury 400 NMR spectrometer 400 MHz (Varian Inc., Palo Alto, CA,
USA). The chemicals shift values were expressed in parts per mil-
lion (ppm) relative to tetramethylsilane as an internal standard
and signals were reported as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet). Mass spectra were recorded on a Waters
ZQ Micromass LC-MS spectrometer (Waters Corporation, Mil-
ford, MA, USA) Electrosprey Ionization (ESI) method. Infrared
(IR) spectra were measured on Jasco FT/IR-420 (Jasco, Tokyo,
Japan). Elemental analysis was taken on a Leco-932 CHNS-O ana-
lyzer (Leco, St. Joseph, MI, USA). molecular Devices Spectra MAX
190 (from molecular Devices Corporation, Sunnyvale, CA, USA)
was used to measure of absorbance of phoshorylation reaction.
Analytical TLC was carried out on Merck 0.2 mm pre-coated
silica gel (60 F-254) aluminium sheets (Merck), visualization by
irradiation with an UV lamp. The flash column chromatography
was accomplished on silica gel 60 (230–400 mesh; Merck). The
dose-response curves of the compounds and non-linear regres-
sion analysis were performed using GraphPad Prism 4.0 (Graph-
Pad Software for Windows, San Diego, California, USA).
at room temperature. It was diluted with water and extracted
with EtOAc (3650 mL). The combined organic phase dried over
anhydrous Na2SO4. Evaporation of the solvent gave crude com-
pounds, which were purified by column chromatography (hex-
ane / ethylacetate = 9.5 : 0.5) and following EtOH crystallization.
2.81 g pure compound was obtained with 88.3% yield. M.p.: 99–
1008C [39].
N-Benzyl-5-(4-fluorophenyl) indole 4
5-(4-Fluoro)phenyl indole (2.88 g, 0.013 mol) was dissolved in
DMF (7 mL) and the solution was cooled to 08C. NaH (0.72 g,
0.03 g) was added and the mixture stirred for 15 min at 08C and
then 45 min at room temperature. Benzyl bromide (3.3 mL,
0.027 mol) was added dropwise to the reaction mixture and
stirred for 72 h at room temperature. It was diluted with water
and extracted with EtOAc (3650 mL). The combined organic
phase was dried over anhydrous Na2SO4. Evaporation of the sol-
vent gave crude the compounds, which were purified by column
chromatography (hexane / ethylacetate = 9.5 : 0.5) and following
EtOH crystallization, 3.15 g pure compound was obtained with
76.0% yield. M.p.: 7508C. 1H-NMR (CDCl3) d: 7.95 (s, 1H, H-c), 7.71–
7.21 (m, 12H, aromatic protons), 6.73 (d, 1H, H-b), 5.37 (s, 2H,
CH2Ph); ESI-MS: m/z 302.21 [M + 1].
N-Benzyl-5-phenyl indole-3-carbaldehyde 5
DMF (3.1 mL, 0.039 mol) and POCl3 (1.02 mL, 0.01 mol) were
stirred at –158C for 15 min and N-benzyl-5-phenyl indole (2.81 g,
0.01 mol) in 5 mL DMF was added. The mixture was stirred at
room temperature for 45 min. It was poured into cold water and
adjusted to basic pH by adding 10 mL of 2 M NaOH (1.91 g,
0.047 mol) solution. The collected crude solid was purified by
column chromatography (hexane / ethylacetate = 8 : 2) and fol-
lowed by crystallization with EtOH / H2O mixture. 2.08 g pure
5-Phenyl indole 1
5-Bromoindole (2.88 g, 0.014 mol) was dissolved in 20 mL anhy-
drous toluene and Pd(PPh3)4 (0.849 g, 0.735 mol were added and
then stirred at room temperature for 30 min. Na2CO3 (21 mL,
2 M) and phenylboronic acid (2.69 g, 0.022 mol) in 15 mL abso-
lute ethanol were added respectively. The mixture was boiled at
908C for 4 h and the layers were separated. The water layer was
extracted by ethylacetate (36100 mL) and dried over anhydrous
Na2SO4. The Crude product was purified by column chromatog-
raphy (hexane / ethylacetate = 9.5 : 0.5) and crystallized with
ethanol resulted in 1.59 g of pure compound with 56.0% yield.
M.p.: 7408C (lit. m.: 69–708C) [37].
1
compound was obtained with 67.2% yield. M.p.: 1018C. H-NMR
(CDCl3) d: 10.03 (s, 1H, CHO), 8.56 (s, 1H, H-b), 7.74 (s, 1H, H-a),
7.56–7.20 (m, 12H, aromatic protons), 5.39 (s, 2H, CH2Ph); IR
(KBr, cm– 1): 1656 (C=O); ESI-MS: m/z 312.14 [M + 1].
N-Benzyl-5-(4-fluorophenyl)indole-3-carbaldehyde 6
DMF (3.25 mL, 0.041 mol) and POCl3 (1.07 mL, 0.011 mol) were
stirred at –158C for 15 min and N-benzyl-5-(4-fluoro)phenyl
indole (3.15 g, 0.01 mol) in 5 mL DMF were added. The mixture
was stirred at room temperature for 45 min. It was poured into
cold water and adjusted to basic pH by adding 10 mL of 2 M
NaOH (2.0 g, 0.05 mol) solution. The collected crude solid was
purified by column chromatography (hexane / ethylacetate =
8 : 2) and followed by crystallization with EtOH / H2O mixture.
2.03 g pure compound was obtained with 59.2% yield. M.p.:
102–1048C. 1H-NMR (CDCl3) d: 10.01 (s, 1H, CHO), 8.51 (s, 1H, H-
b), 7.74 (s, 1H, H-a), 7.49-7.10 (m, 11H, aromatic protons), 5.38 (s,
2H, CH2Ph); IR (KBr, cm– 1): 1651 (C=O); ESI-MS: m/z 330 [M + 1].
5-(4-Fluorophenyl) indole 2
5-Bromoindole (5.02 g, 0.025 mol) was dissolved in 50 mL anhy-
drous toluene and Pd(PPh3)4 (1.48 g, 1.28 mmol) was added and
then stirred at room temperature for 30 min. Na2CO3 (37 mL,
2M) and 4-fluoro phenylboronic acid (5.37 g, 0.038 mol) in
10 mL absolute ethanol were added respectively. The mixture
was boiled at 908C for 4 h and the layers were separated. The
water layer was extracted by ethylacetate (36100 mL) and dried
over anhydrous Na2SO4. The Crude product was purified by col-
umn chromatography (hexane / ethylacetate = 9.5 : 0.5) and crys-
tallized with ethanol resulted in 2.88 g of pure compound with
53.4% yield. M.p.: 89–908C [38].
General procedure for N-[(1-benzyl-5-phenyl-1H-indole-
3-yl)methylene]-1-phenyl / substituted
phenylmethanamine 7a–7e and N-[(1-benzyl-5-(4-
fluorophenyl)-1H-indole-3-yl)methylene]-1-phenyl /
substituted phenylmethanamine 7f–7j
Compounds 5 or 6 (1 eq.) were dissolved in CH2Cl2 and MgSO4 (0.5
eq.) and corresponding amine derivatives (1 eq.) were added. The
mixture was refluxed for 40 h, MgSO4 was filtered off and the sol-
N-Benzyl-5-phenyl indole 3
5-Phenyl indole (2.17 g, 0.01 mol) was dissolved in DMF (6 mL)
and the solution was cooled to 08C. NaH (0.54 g, 0.02 g) was
added and the mixture stirred for 15 min at 08C and then
45 min at room temperature. Benzyl bromide (2.7 mL, 0.02 mol)
was added dropwise to the reaction mixture and stirred for 72 h
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342
Z. Kılıc˛ et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 333–343
vent was evaporated. The crude compounds were purified by
EtOH crystallization.
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General procedure for 1-(1-benzyl-5-phenyl-1H-indole-3-
yl)-N-(benzyl / substituted benzyl)methanamine N HCl
8a–8e and 1-(1-benzyl-5-(4-fluorophenyl)-1H-indole-3-
yl)-N-(benzyl / substituted benzyl)methanamine N HCl 8f–
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Compounds 7a–7j were dissolved in 5 mL MeOH and 5 eq.
NaBH4 was added. The mixture was heated 1 h at 508C. After the
solvent removed under reduced pressure, the residue was
diluted with water. The water layer was extracted with ethyl ace-
tate, and the organic layer was separated and dried over Na2SO4.
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In-vitro tyrosine-kinase (pp60c-Src) assay
The inhibition of the synthesized compounds was verified by vir-
tue of the ELISA-based in-vitro tyrosine-kinase assay where the
pp60c-Src tyrosine-kinase activity was measured by monitoring
the phosphorylation level of immobilized substrate (poly (Glu-
Tyr) peptide) using the classical ELISA-sandwich method (Takara
Universal Protein Tyrosine Kinase Assay, Tokyo, Japan). The kin-
ase assay was performed at 378C in a final assay volume of 50 lL.
The concentrations of the pp60c-Src used to construct the calibra-
tion curve were as follows: 1520, 760, 380, 190, 94.8, 47.4610– 7
units/lL for pp60c-Src. The kinase reactions were performed with
350610– 7 unit/lL pp60c-Src at 40 nM ATP. Following several
washing and incubation steps, the phosphorylation of the sub-
strate was probed with HRP-conjugated anti-phosphotyrosine
(PY20) antibody and the absorbance of the reaction mixture was
measured at 450 nm with a microplate reader. The inhibitory
activities of compounds against tyrosine kinase were monitored
by the diminished activity of kinase at 450 nm. The pp60c-Src tyro-
sine kinase activity (control) is measured as the difference
between the total activity in the absence and presence of vehicle
(DMSO), and the activity of enzyme in the presence of a com-
pound (in DMSO) is determined with respect to control. IC50
value was determined as the concentration of a compound
required to achieve 50% inhibition of pp60c-Src tyrosine kinase
activity with respect to control. Compounds to be tested were
prepared at final concentrations of 7 to 650 lM and all measure-
ments and 50% inhibitory concentration (IC50) determinations
from the dose-response curves were made within this range. The
IC50 values were determined by non-linear regression analysis,
the four-parameter logistic equation (Sigmoidal dose-response,
GraphPad Prism version 4.0 for Windows, GraphPad Software,
San Diego California USA).
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