3-Substitued indoles: One-pot synthesis and evaluation of anticancer and Src kinase inhibitory activities

Article abstract of DOI:10.1016/j.bmcl.2011.05.010

An efficient and economical method was developed for the synthesis of 3-substituted indoles by one-pot three-component coupling reaction of a substituted or unsubstituted benzaldehyde, N-methylaniline, and indole or N-methylindole using Yb(OTf)₃-SiO₂ as a catalyst. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), human ovarian adenocarcinoma (SK-OV-3), and c-Src kinase activity. The 4-methylphenyl (4o and 4p) and 4-methoxyphenyl (4q) indole derivatives inhibited the cell proliferation of SK-OV-3 and HT-29 cells by 70-77% at a concentration of 50 μM. The unsubstituted phenyl (4d) and 3-nitrophenyl (4l) derivatives showed the inhibition of c-Src kinase with IC₅₀ values of 50.6 and 58.3 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2011.05.010

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3511–3514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Substitued indoles: One-pot synthesis and evaluation of anticancer and
Src kinase inhibitory activities
V. Kameshwara Rao a, Bhupender S. Chhikara b, Amir Nasrolahi Shirazi b, Rakesh Tiwari b,
a,
Keykavous Parang b, , Anil Kumar
⇑
⇑
a Department of Chemistry, Birla Institute of Technology and Science, Pilani 333 031, Rajasthan, India
b Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and economical method was developed for the synthesis of 3-substituted indoles by one-pot
three-component coupling reaction of a substituted or unsubstituted benzaldehyde, N-methylaniline,
and indole or N-methylindole using Yb(OTf)3–SiO2 as a catalyst. All the synthesized compounds were
evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), human ovarian adeno-
carcinoma (SK-OV-3), and c-Src kinase activity. The 4-methylphenyl (4o and 4p) and 4-methoxyphenyl
(4q) indole derivatives inhibited the cell proliferation of SK-OV-3 and HT-29 cells by 70–77% at a concen-
Received 7 April 2011
Accepted 2 May 2011
Available online 7 May 2011
Keywords:
Anticancer
Cancer
tration of 50
l
M. The unsubstituted phenyl (4d) and 3-nitrophenyl (4l) derivatives showed the inhibition
M, respectively.
Ó 2011 Elsevier Ltd. All rights reserved.
of c-Src kinase with IC50 values of 50.6 and 58.3
l
Indole
One-pot
Src kinase
Synthesis
The 3-substituted indoles are structural units of many natural
and biologically interesting compounds, which possess various
pharmacological activities.1–4 The indole derivatives serve as a
scaffold in a number of antibacterial,5 antiviral,6 and protein kinase
inhibitors.7 Indole-based derivatives have been investigated for
anticancer activities. Indole-3-carbinols have been previously re-
ported to exhibit anticancer activities against a number of human
cancers through acting on different cellular signaling pathways.8
1-Aroylindoles and 3-aroylindoles have shown potent cytotoxicity
against different human cancer cell lines.9.
Several indole derivatives have shown tyrosine kinase inhibi-
tion in low micromolar range.7,10 3-Substituted 2,20-dithiobis(1H-
indoles) have been reported to show inhibition against protein
tyrosine kinases (PTKs), such as EGFR and non receptor v-Src tyro-
sine kinases.11 SU5416 (Fig. 1) is an indole-based FIK-1/KDR inhib-
itor, and is currently in clinical trials against ovarian cancer.12–14
The Src family of tyrosine kinases (SFKs) is comprised of nine
tyrosine kinases viz., Src, Lck, Fyn, Yes, Hck, Blk, Fgr, Lyn, and
Yrk. SFKs have critical roles in multiple signaling pathways that
breast, prostate, and pancreas when compared with the normal
tissues. c-Src serves as a key modulator of cancer cell invasion
and metastasis through reducing cell adhesion and facilitating
motility.17–19 Thus, considerable interest has been evolved
around the design of Src kinase inhibitors for the treatment of
cancer and anti-invasion therapy.20 A number of small molecule
inhibitors21,22 have shown potential biological activity as both
CH3
HN
CH3
O
N
H
Figure 1. Chemical structure of SU5416.
control
a diverse spectrum of biological activities, such as
R''
growth factor signaling, cell growth, division, differentiation, sur-
vival, adhesion, migration, and invasion.16 c-Src tyrosine kinase
is the prototype of SFKs. c-Src upregulation has been observed
in a number of epithelial tumors, such as ovary, colon, lung,
CH3
N
H
H3C
R
H
O
N
R
Yb(OTf)3-SiO2
CH3CN
N
R'
N
R''
R'
3
1
2
4
⇑
Corresponding authors.
E-mail address: kparang@uri.edu (K. Parang).
Scheme 1. Synthesis of 3-substituted indoles.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.010

3512
V. K. Rao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3511–3514
Table 2
anti-proliferative and anti-invasive agents in preclinical studies
in different solid tumor types.23–26
Chemical structures of 3-substituted indoles (4a–r) and their Src kinase inhibitory
activity
In continuation of our efforts towards the development of or-
ganic transformations catalyzed by metal triflates27 and synthesis
of small molecules as anticancer agents and/or c-Src kinase inhib-
itors,15,28 herein we report an expeditious one-pot synthesis of 3-
substituted indoles by three component condensation catalyzed
by Yb(OTf)3–SiO2 (Scheme 1) and evaluation of their anticancer
and c-Src kinase inhibitory activity.
The 3-indole derivatives were synthesized by one-pot conden-
sation reaction of indole or N-methylindole, a substituted or
unsubstituted benzaldehyde, and N-methylaniline. The reaction
condition optimizations were performed by monitoring a model
reaction between indole, 4-chlorobenzaldehyde and N-methylani-
line. The model reaction was carried out in various solvents, such
as DCM, DMSO, DMF, THF, acetonitrile, and ionic liquid
[bmim][BF4], using Yb(OTf)3–SiO2 as a catalyst. Among these sol-
vents acetonitrile was found to be most efficient reaction media
to give 4a in good yield (88%) while the reaction yield in other sol-
vents was very poor.
Furthermore, catalyst conditions were optimized by using dif-
ferent variations of catalyst, catalyst loading, and time period of
reaction (Table 1). The yield of 4a was poor and required longer
time when reaction was performed with either Yb(OTf)3 or silica
gel alone. Among the screened catalysts Yb(OTf)3–SiO2 (5–
10 mol %), Ce(OTf)3-SiO2 (5 mol %), and Cu(OTf)2–SiO2 (5 mol %)
were found to give good yield of 4a. The Yb(OTf)3–SiO2 (5 mol %)
gave the highest yield (88%) of 4a and, therefore, further studies
were carried out using this as a catalyst of choice. In case of other
acidic catalysts supported on silica gel such as pTSA–SiO2 (71%),
FeCl3–SiO2 (59%) the yield of 4a was moderate but accompanied
with generation of bis(indolyl)methane (5–30%) as a side product.
The standardized reaction conditions were used further for the
synthesis of different 3-substituted indole derivatives.29 Indole or
N-methylindole, N-methylaniline, and a substituted or unsubsti-
tuted benzaldehyde were reacted to obtain 3-substituted indole
derivatives (4a–r). The products and their yields are summarized
in Table 2. All the compounds were characterized by 1H NMR,
13C NMR, and mass spectroscopy. The reaction is assumed to pro-
ceed through formation of imine after reaction of the benzalde-
hyde and N-methylaniline followed by nucleophilic attack of
indole to give a 3-substituted indole as shown in Scheme 2. The
structure of product is consistent with the synthesis of 3-substi-
tuted indoles via multicomponent condensation reaction of in-
doles, aldehyde, and amines.30
R''
CH3
N
R
N
R'
4
Yielda (%)
IC50 (lM)
b
Product
R
R0
R00
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Cl
88
83
86
78
75
84
82
81
79
81
82
51
72
88
85
78
80
83
>150
>150
>150
50.6
4-CH3
4-CH3O
H
4-OH
3-Br, 4-OH
3-CH3O
2,4-CH3O
H
>150
>150a
>150
>150
>150
98.3
60.5
58.3
71.6
100.0
>150
>150
106
CH3
CH3
CH3
H
4-Cl
4-CH3
3-NO2
H
4-OCH3
4-CH3
4-CH3
4-OCH3
4-Cl
5-Br
5-OCH3
5-OCH3
5-Br
5-Br
5-OCH3
H
H
H
H
H
H
87
a
Isolated yield.
b
The concentration at which 50% of enzyme activity is inhibited.
CH3
Yb(OTf)3-SIO2
H
NH
O
C
N
CH3
N
H
-H+
Yb(OTf)3-SIO2
H
H
N
O
N
H
N
CH3
-OH-
N
CH3
CH3
N
H
Table 1
Scheme 2. Plausible mechanism for synthesis of 4.
Optimization of reaction condition for model reaction generating 4a
Catalyst
Catalyst (mol %)
Time (h)
Yielda (%)
An array of 18 diversely substituted indoles was evaluated
SiO2
Yb(OTf)3
Zn(OTf)2
Ce(OTf)3
Cu(OTf)2
Ba(OTf)2
Yb(OTf)3–SiO2
Yb(OTf)3–SiO2
Yb(OTf)3–SiO2
Yb(OTf)3–SiO2
Yb(OTf)3–SiO2
Yb(OTf)3–SiO2
Zn(OTf)2–SiO2
Ce(OTf)3–SiO2
Cu(OTf)2–SiO2
Ba(OTf)2–SiO2
FeCl3–SiO2
pTSA–SiO2
—
5
5
5
5
5
1
2
3
4
5
10
5
5
5
5
5
5
6
4
4
4
4
4
2
2
2
2
2
2
2
2
2
2
2
3
15
72
52
71
68
50
47
58
67
74
88
84
62
81
81
67
59
71
against Src kinase. The results of Src kinase inhibitory activity of
compounds (4a–r) are shown in Table 2. Among all the screened
compounds, 4d, 4j, 4k, 4l, and 4r showed modest inhibition of
Src kinase with IC50 values of 50.6–98.3 lM. The data suggest that
the presence of either electron donating or electron withdrawing
groups on the phenyl ring (R00 position) was mostly less tolerated
as shown in compounds 4a–c, 4e–i, and 4n–p with IC50 value of
P100
value of 50.6
–NO2 at 3-position of phenyl ring (4l) exhibited comparable inhib-
lM. The unsubstituted indole derivative 4d showed an IC50
lM while introduction of electron withdrawing group
itory activity (IC50 = 58.3 lM).
DS visualizer docking studies31 were used to study the interac-
tions of 4d with the ATP binding site of the Src kinase. Compound
4d was superimposed on AZD05030, an anilinoquanzoline dual
specific c-Src/Abl kinase inhibitor32 in complex with the Src kinase
(PDB 2H8H).
a
Isolated yield.

V. K. Rao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3511–3514
3513
estimated inhibition constant at 3.67
lM while the reference li-
gand binding energy and estimated inhibition constant were
ꢀ7.36 kcal/mol and 4.01
lM, respectively. The RMSD from refer-
0
ence ligand is 1.230 ÅA.
The effect of the compounds on the cell proliferation of human
ovarian adenocarcinoma (SK-OV-3) and colon adenocarcinoma
(HT-29) cancer cells that also overexpress c-Src,33,34 was evaluated
at the concentration of 50 lM (Fig. 3). In general, most of the com-
pounds were more active against SK-OV-3 cells than HT-29 cells.
Indole-based SU5416 is currently in clinical trials against ovarian
cancer. Consistently compounds 4o–r inhibited the cell prolifera-
tion of both cancer cells significantly while 4a only inhibited
SK-OV-3. The 4-methylphenyl (4o and 4p) and 4-methoxyphenyl
(4q) indole derivatives inhibited the cell proliferation of SK-OV-3
and HT-29 cells by 70–77% whereas 4-cholorderivatives 4a and
4r inhibited the growth of ovarian cancer cells (SK-OV-3) by
approximately 72% and 77%, respectively (Fig. 3). Structure–
activity relationship studies suggest that the presence of bromo-
or methoxy-substituent at position 5 of indole ring (R) in addition
to methyl, methoxy, or chloro substitutent at as R00 is critical for
maximum anticancer activity as seen in compounds 4o–r. In gen-
eral, poor correlation was observed between Src kinase inhibitory
potency of the compounds and the inhibition of cell proliferation
in cancer cells, suggesting that differential cellular uptake and con-
tribution of other mechanisms in anticancer activities of these
compounds. Compounds 4d and 4r that showed modest Src kinase
inhibition, also inhibited the growth of SK-OV-3 by 55% and 77%,
respectively.
In conclusion, we have developed an ecofriendly and economi-
cal method for the synthesis of 3-substituted indoles by one-pot
three-component coupling reaction of a benzaldehyde, N-methy-
laniline, and indole or N-methylindole using Yb(OTf)3–SiO2 as cat-
alyst. To the best of our knowledge, this is the first report of the
synthesis and evaluation of 3-substituted indole derivatives as
Src kinase inhibitors and anticancer agents. The compounds 4d,
4j, 4k, 4l, and 4r showed modest inhibition of Src kinase while
compounds 4o–r inhibited the cell proliferation of ovarian and co-
lon cancer cells significantly. Structure–activity relationship
Figure 2. Comparison of interactions of 4d (grey) and AZD05030 (oxygens (red),
nitrogens (purple), carbons (light green), chlorine (green)) in ATP binding site of the
Src kinase based on molecular modeling. The compounds and side chains of amino
acids (yellow) are rendered in stick styles. Compounds are in the lowest energy
conformers predicted. The Figure is drawn using the Accelrys DS visualizer 2.5
system.
As can be seen from Figure 2, 4d interacts with the ATP binding
pocket in slightly different orientation when compared with refer-
ence quinazoline ligand. The chloro group in reference ligand ori-
ented towards and interacts with Ala403, whereas in 4d phenyl
rings lie in hydrophobic binding pocket. N1–H of indole ring in
compound 4d shows specific hydrogen bonding interaction with
Ile336. The hydrogen bonding interaction was not observed when
N1 was methylated in 4i. This interaction may have contributed to
higher inhibitory activity of unmethylated compounds 4d
(IC50 = 50.6 lM) versus N1-methylated analog 4i (IC50 >150 lM).
On the other hand, N1-methylated compounds 4j and 4k exhibited
improved inhibition activity versus 4a and 4b, respectively (Table
2), suggesting that hydrophobic interactions of methyl with
Ile336 may also partially contribute to modest inhibitory activity.
The binding energy for 4d was observed as ꢀ7.42 kcal/mol and
140
120
100
80
HT-29
SK-OV-3
60
40
20
0
Figure 3. Inhibition of HT-29 and SK-OV-3 cell proliferation by compounds 4a–r (50
lM) after 72 h incubation. The results are shown as the percentage of the control DMSO
that has no compound (set at 100%). All the experiments were performed in triplicate.

3514
V. K. Rao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3511–3514
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We acknowledge University Grant Commission (UGC), New
Delhi and the American Cancer Society Grant #RSG-07-290-01-
CDD for the financial support. We thank Mr. Vadiraj for his help
in molecular modeling studies.
Supplementary data
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Supplementary data (experimental procedures and character-
ization of compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.05.010.
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