Synthesis, biological evaluation and molecular docking studies of 2-amino-3,4,5-trimethoxyaroylindole derivatives as novel anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.03.081

A series of novel 2-amino-3,4,5 trimethoxyaroylindole derivatives was synthesized and evaluated against selected human cancer cell lines of breast (MCF-7) and colon (HT-29). Introduction of an amino group at the C-2 position on ring A of 3,4,5-trimethoxyaroylindole derivatives resulted in novel compounds, i.e., 2-amino-3,4,5-trimethoxyaroylindole derivatives exhibiting excellent cytotoxic activity against human cancer cell lines. Substitution with methoxy group at R⁶ in 2-amino-3,4,5-trimethoxyaroylindole 5d exhibited excellent cytotoxic activity against MCF-7 (0.013 μM) and colon HT-29 (0.143 μM) indicating slightly higher potency than Combretastatin A-4. Molecular modeling studies of 2-amino-3,4,5-trimethoxyaroylindole derivatives have similar structural alignment as colchicine in protein (PDB code: 1SA0) and exhibited hydrogen bond interaction between para position of 3,4,5-trimethoxyphenyl ring with CYS 241 and N-H molecule of indole ring with Val 315 of receptor molecule.

Full text of DOI:10.1016/j.bmcl.2016.03.081

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2115–2118
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and molecular docking studies
of 2-amino-3,4,5-trimethoxyaroylindole derivatives as novel
anticancer agents
⇑
Vijay K. Patel, Harish Rajak
Medicinal Chemistry Research Laboratory, SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur 495 009, C.G., India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2-amino-3,4,5 trimethoxyaroylindole derivatives was synthesized and evaluated against
selected human cancer cell lines of breast (MCF-7) and colon (HT-29). Introduction of an amino group at
the C-2 position on ring A of 3,4,5-trimethoxyaroylindole derivatives resulted in novel compounds, i.e., 2-
amino-3,4,5-trimethoxyaroylindole derivatives exhibiting excellent cytotoxic activity against human
cancer cell lines. Substitution with methoxy group at R6 in 2-amino-3,4,5-trimethoxyaroylindole 5d
Received 25 December 2015
Revised 4 March 2016
Accepted 24 March 2016
Available online 24 March 2016
exhibited excellent cytotoxic activity against MCF-7 (0.013 lM) and colon HT-29 (0.143 lM) indicating
Keywords:
slightly higher potency than Combretastatin A-4. Molecular modeling studies of 2-amino-3,4,5-
trimethoxyaroylindole derivatives have similar structural alignment as colchicine in protein (PDB code:
1SA0) and exhibited hydrogen bond interaction between para position of 3,4,5-trimethoxyphenyl ring
with CYS 241 and N–H molecule of indole ring with Val 315 of receptor molecule.
Ó 2016 Elsevier Ltd. All rights reserved.
2-Amino-3,4,5-trimethoxyaroylindole
Combretastatin A-4
Synthesis
Anticancer
Microtubules has been identified as an attractive and well
established molecular targets for anticancer therapy because
microtubule influences the crucial processes of cell, such as divi-
sion, motility, shape maintenance and intracellular transport.1,2
Combretastatin A-4 (CA-4) is a low molecular weight compound
that binds to colchicine binding site causing impediment with
microtubule assembly by prevention of tubulin polymerization.3,4
A number of CA-4 analogues such as CA-4P, EPC2407 (Crolibulin),
AVE8062 (ombrabulin), ABT-751 (E7010), OXi4503, T138067, BNC-
105P, MPC-6827, etc. are in different phases of clinical trials.5–7
CA-4 and its analogs have been divided into three major structural
elements, i.e., ring A (trimethoxyphenyl ring), ring B (substituted
phenyl ring) and the bridgehead linker. The substitution of
ethylene bridge of CA-4 with a carbonyl group furnished a
benzophenone type CA-4 analogue, named phenstatin. It possess
significant anticancer activity in a variety of tumor models, indicat-
ing that carbonyl group as linker retain the non-planar character as
CA-4.6 Introduction of an amino group at the C-2 position on ring A
of CA-4 and phenstatin analogues exhibited excellent cytotoxic
activity in variety of human cell lines (Fig. 1) and tubulin polymer-
ization inhibition. This introduction of amino group causes
increased polarity leading to enhancement of the aqueous solubil-
ity. Structure–activity relationships (SAR) information indicated
that the introduction of an amino group at the C-2 position on ring
A of CA-4 and benzophenone analogues plays an important role in
maximizing activity.8–10
Replacement of ring B to indole derivative prompted to synthe-
size a series of aroylindole based CA-4 analogs. A number of
aroylindole derivatives have been designed and synthesized which
exhibit cytotoxic activity on several cancer cell lines due to its
excellent antitumor and antivascular activities.11–16 BPR0L075 (6-
methoxy-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)methanone have
exhibit excellent cytotoxic activity against a panel of cell lines then
CA-4 (Fig. 1).11–13 Introduction of hydroxy group at the C-2 posi-
tion on ring A of aroylindole derivative exhibits marked antiprolif-
erative activity against KB and MKN45 cells with IC50 values of 8.8
and 10.5 nM, respectively, binds strongly to the colchicine binding
site and leads to inhibition of tubulin polymerization (Fig. 1).17 An
excellent anticancer activity of aroylindole based CA-4 analogs
attracted considerable interest of medicinal chemists in the design
and preparation of analogs as novel antitumor agents. Introduction
of an amino group at the C-2 position on ring A of 3,4,5-trimethox-
yaroylindole derivatives to synthesized 2-amino-3,4,5-trimethox-
yaroylindole derivatives which exhibited excellent cytotoxic
activity.
The 2-amino-3,4,5-trimethoxyaroylindole derivatives were
synthesized by six step reaction sequence, starting with commer-
cially available methyl 3,4,5-trimethoxy benzoate (1). The indole
substituted 2-nitro-3,4,5-trimethoxyaroylindole derivatives (4)
⇑
Corresponding author. Tel.: +91 9827911824; fax: +91 7752260140.
E-mail address: harishdops@yahoo.co.in (H. Rajak).
http://dx.doi.org/10.1016/j.bmcl.2016.03.081
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

2116
V. K. Patel, H. Rajak / Bioorg. Med. Chem. Lett. 26 (2016) 2115–2118
R2
R2
A
O
H3CO
H3CO
R3'
OH
H3CO
H3CO
A
B
B
OCH3
OCH3
OCH3
OCH3
R2=OH, Combretastatin A-4;
IC50=57nM (HT-29).
R2=NH2, R3'=OH, Phenstatin;
IC50=560nM (HT-29).
R2=NH2, 2-amino Combretastatin A-4;
IC50 = 36nM (HT-29).
R2=NH2, R3'=NH2, 2-amino Phenstatin derivetive;
IC50 = 16nM (HT-29).
R
OCH3
O
X
NH
N
H3CO
H3CO
R1
R2
H3CO
H3CO
OCH3
OCH3
BPR0L075
Aroylindole derivatives
IC50 nM = 7 (MCF-7)/23 (A549)/4 (MESSA)/4
(MKN45)/1 (NUGC)/4 (KB)/7 (H460)/1.1 (HT-29).
CA-4 IC50 nM = 8 (MCF-7)/117 (A549)/13 (MESSA)/
82 (MKN45)/>10000 (NUGC)/6.2 (KB)/17 (H460).
X=C=O/CH2;
R=subtitution at different position of indole ring;
R1=H/CH3/C2H5/CH2COOH etc;
R2=H/CH3/COOCH3/COOC2H5 etc.
OCH3
O
HO
NH
H3CO
H3CO
OCH3
(2-Hydroxy-3,4,5-trimethoxyphenyl)(6-methoxy-1
Hindol-3-yl)methanone
IC50 nM = 8.8 (KB)/10.5 (MKN45).
CA-4 IC50 nM = 2.5 (KB)/3.2 (MKN45).
Figure 1. Structure and biological activity of Combretastatin A-4, phenstatin and aroylindole derivatives.
OCH3
R4
OCH3
OCH3
OCH3
H3CO
OCH3
R5
R6
H3CO
H3CO
OCH3
ii,iii
R2
i
+
NO2
NO2
COOCH3
N
H
COCl
R7
COOCH3
2
1
3
(a-r)
a: R2=R4=R5=R6=R7=H;
b: R2=R5=R6=R7=H, R4=OCH3;
c: R2=R4=R6=R7=H, R5=OCH3;
d: R2=R4=R5=R7=H, R6=OCH3;
R =R =R =R =H, R =OCH3;
R =R =R =H, R =R =OCH3;
R =R =R =R =H, R =F;
R =R =R =R =H, R =Cl;
R =R =R =R =H, R =Br;
R =R =R =R =H, R =CH3;
iv
2
4
5
6
7
e:
f:
g:
h:
i:
R7
R6
R5
2
4
7
5
6
2
4
5
7
6
NH
2
4
6
7
5
R2
2
4
6
7
5
R4
2
4
6
7
5
H3CO
H3CO
j:
O
4
5
6
7
2
k:
R =R =R =R =H, R =COOCH3;
l: R4=R6=R7=H, R2=COOCH3, R5= Cl;
m: R4=R6=R7=H, R2=COOCH3, R5=Br;
n: R4=R6=R7=H, R2=COOCH3, R5=OCH3;
o: R4=R5=R6=R7=H, R2=COOC2H5;
NO2
OCH3
4 (a-r)
p: R4=R6=R7=H, R2 = COOC2H5, R5 =Cl;
q: R4=R6=R7= H, R2=COOC2H5, R5=Br;
r: R4=R6=R7=H, R2=COOC2H5, R5=OCH3;
v
R7
R7
R6
R5
R6
R5
NH
NH
R2
R2
vi
R4
R4
H3CO
H3CO
H3CO
H3CO
O
NH2
OCH3
6 (a-j)
NH2
OCH3
5 (a-r)
Scheme 1. Synthesis of 2-amino-3,4,5-trimethoxyaroylindole derivatives. Reagents and conditions: (i) HNO3, 10–30 °C, stirring for 1–2 h, 26%; (ii) NaOH, EtOH, stirring for
2 h at 45–50 °C, 90%; (iii) CH2Cl2, SOCl2, reflux for 1 h at 60 °C; (iv) AlCl3, reflux for 3–5 h, 80–90 °C (Friedel–Craft acylation), 58–73%; (v) Sn, HCl, reflux at 70 °C, 10% K2CO3,
42–62%; (vi) NaBH4, EtOH, reflux for 3 h, 32–54%.

V. K. Patel, H. Rajak / Bioorg. Med. Chem. Lett. 26 (2016) 2115–2118
2117
Table 1
Structure, In vitro cell cytotoxic of compounds 2-amino-3,4,5-trimethoxyaroylindole derivatives (5 (a–r) and 6 (a–j)) against MCF-7 and colon HT-29 cell lines
R5
R6
R7
R4
H2N
H3CO
X
NH
R2
OCH3
H3CO
Compd
R2
R4
R5
R6
R7
X
MCF-7 (GI50
l
Ma)
HT-29 (GI50 l
Ma)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
—
—
H
H
OMe
H
H
OMe
H
Cl
Br
Me
H
Cl
Br
OMe
H
Cl
Br
OMe
H
H
OMe
H
H
H
H
OMe
H
OMe
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
OMe
F
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
—
—
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
C@O
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
—
0.900
>100
0.528
0.013
9.963
38.45
42.83
0.321
0.360
12.35
0.193
0.171
0.124
0.102
0.480
0.590
0.425
0.540
45.0
NA
32.8
1.83
NA
NA
NA
NA
NA
9.8
>100
0.95
0.143
48.6
24.4
64.24
4.85
3.83
>100
2.38
2.14
1.94
1.24
5.63
6.84
5.38
5.15
>100
NA
>100
74.8
NA
NA
NA
NA
NA
NA
H
H
COOMe
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
COOEt
H
H
H
H
H
H
H
H
H
H
OMe
H
Cl
Br
Me
—
—
6j
H
—
—
NA
CA-4
BPR0L075
—
—
0.025
0.260
—
0.007b
0.001c
a
b
c
GI50 = compound concentration required to inhibit 50% growth and data are expressed as the mean from the dose–response curves of at least three experiments.
IC50 values taken from the literature for comparison.11
IC50 values taken from the literature for comparison.13
were synthesized with 2-nitro-3,4,5-trimethoxy benzoyl chloride
and various commercially available substituted indoles using Frie-
del–Craft acylation. It is well-known that indole ring readily under-
goes electrophilic reactions particularly at the C-3 position with
Lewis acids by Friedel–Craft acylation. The steps involved in the
synthesis of 2-amino-3,4,5-trimethoxyaroylindole derivative
(Scheme 1) includes (i) nitration of methyl 3,4,5-trimethoxy ben-
zoate for the synthesis of 2, (ii) base hydrolysis of ester for the syn-
thesis of 3, (iii) chlorination of acid with thionyl chloride, (iv)
Friedel–Crafts acylation for the synthesis of 2-nitro-3,4,5-
trimethoxyaroylindole for the synthesis of 4 (a–r), (v) Reduction
of the nitro group for the synthesis of 5 (a–r), (vi) Reduction of car-
bonyl group for the synthesis of 6 (a–j).
substitution with the methoxy group causes significant increase
in activity. Substitution at R6 with methoxy group 5d exhibited
excellent cytotoxic activity against MCF-7 (0.013
l
M), colon HT-
29 (0.143
l
M) indicating slight higher potency than CA-4 while
any substitution at R4 and R7 significantly decrease the activity.
Substitution at R2 position with methyl and ethyl carboxylate
results in enhancement of the activity, i.e., 5k–5r. Replacement
of keto group at bridge (X) with methyl group cause considerable
decline in activity i.e., 6 (a–r).
Molecular modeling studies were performed on Glide v5.818,19
(Schrodinger, LLC, New York, NY) to investigate the potential
interactions between synthesized 2-amino-3,4,5-trimethox-
yaroylindole derivative and protein (PDB code: 1SA0). All the 2-
amino-3,4,5-trimethoxyaroylindole derivatives were docked to
colchicines binding site of b tubulin (PDB 1SA0) for studying
the binding mode of compounds for antitumor activity. 2-
amino-3,4,5-trimethoxyaroylindole derivative (5d) possess simi-
lar structural alignment as colchicine in protein (PDB code:
1SA0) (Fig. 2a). 2-amino-3,4,5 trimethoxyaroylindole derivative
(5d) showed critical interactions between Cys241, Val318 and
meta, para-methoxy group at ring A. The hydrogen bond interac-
tion was found between para position of trimethoxyphenyl ring
with CYS 241 and N–H molecule of indole ring with Val 315 of
receptor molecule (Fig. 2b).
The synthesized 2-amino-3,4,5 trimethoxyaroylindole com-
pounds were evaluated for their anticancer activity against
selected human cancer cell lines of breast (MCF-7) and colon
(HT-29) using sulforhodamine B (SRB) method. The results of anti-
cancer activity are expressed in terms of growth inhibition fifty
(GI50
lM) values and are shown in Table 1. Unsubstituted 2-
amino-3,4,5-trimethoxyaroylindole 5a showed significant activity
against MCF-7 and colon HT-29. Substitution at R5 with electron
withdrawing group such as chloro and bromo, i.e., 5h, 5i, 5l, 5m,
5p, and 5q increases the cytotoxic activity but electron donor such
as methyl, i.e., 5j significantly decreases the activity, while

2118
V. K. Patel, H. Rajak / Bioorg. Med. Chem. Lett. 26 (2016) 2115–2118
Figure 2. Docking of compound 5d with protein (1SAO) on colchicine-binding site of b tubulin. (a) Docked pose alignments of 5d (colored by atom) with colchicine (green) in
the tubulin binding site. (b) XP Docking pose of 5d, pink dotted lines represents hydrogen binding between para position of trimethoxyphenyl ring with CYS 241 and N–H
molecule of indole ring with Val 315 of receptor molecule.
In conclusion,
a series of novel 2-amino-3,4,5-trimethox-
References and notes
yaroylindole derivatives were synthesized and evaluated for
their potential anticancer activity against selected human cancer
cell lines of breast (MCF-7) and colon (HT-29) using sulforho-
damine B (SRB) method. Introduction of an amino group at
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the C-2 position on ring
derivatives leads to the formation of novel compounds, i.e.,
2-amino-3,4,5-trimethoxyaroylindole derivatives possessing
excellent cytotoxic activity. Molecular modeling studies of
2-amino-3,4,5-trimethoxyaroylindole derivatives exhibited
A of 3,4,5-trimethoxyaroylindole
similar structural alignment as colchicine in protein (PDB code:
1SA0) and hydrogen bond interaction was also observed
between para position of trimethoxyphenyl ring with CYS 241
and N–H molecule of indole ring with Val 315 of receptor
molecule. These results represent a noteworthy step regarding
the design and development of novel 2-amino-3,4,5-trimethox-
yaroylindoles with anticancer activity.
Acknowledgements
We are highly thankful to Department of Science and Technol-
ogy-Science and Engineering Research Board (DST-SERB), New
Delhi, India and University Grants Commission (UGC), New Delhi,
India for providing financial assistance in the form of ‘Start up
Grant for Young Scientist’ [Grant No. SERB/LS-385/2013] and
‘Major Research Project’ [Grant No. 40-275/2011 (SR)], respec-
tively. We are grateful to Advanced Centre for Treatment, Research
and Education in Cancer (ACTREC), Navi Mumbai, India for cell line
studies.
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Supplementary data
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081.
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