Synthesis and biological evaluation of 1-(4′-Indolyl and 6′-Quinolinyl) indoles as a new class of potent anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.04.065

A novel series of the biheterocycles-based compounds with core structure distinguished from combretastatin A-4 (1) and colchicine (5) have been synthesized and evaluated as potent anti-mitotic agents. Compound 1-(4′-Indolyl and 6′-quinolinyl)-4,5,6-trimethoxyindoles 13 and 19 showed substantial anti-proliferative activity against various human cancer cell lines, regardless to the tissue origin and the expression of multiple-drug resistance MDR1, with a mean IC₅₀ value of 38 and 24 nM respectively. Compound 13 (IC₅₀ = 1.7 μM) also exhibited similar anti-tubulin activities to 1 (IC₅₀ = 1.8 μM) and displayed strong binding property to the colchicine binding site on the microtubules. Computational modeling analysis revealed that the binding mechanism of compound 13 is similar to that of CA4.

Full text of DOI:10.1016/j.ejmech.2011.04.065

European Journal of Medicinal Chemistry 46 (2011) 3623e3629
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 1-(40-Indolyl and 60-Quinolinyl) indoles as
a new class of potent anticancer agents
,
,d,
Mei-Jung Lai a 1, Jang-Yang Chang b 1, Hsueh-Yun Lee a, Ching-Chuan Kuo b, Mei-Hsiang Lin a,
Hsing-Pang Hsieh c, Chi-Yen Chang b, Jian-Sung Wu c, Su-Ying Wu c, Kuang-Shing Sheye, Jing-Ping Liou a
,
*
a School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wuxing Street, Taipei 11031, Taiwan, ROC
b National Institute of Cancer Research, National Health Research Institutes, Tainan 70456, Taiwan, ROC
c Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 350, Taiwan, ROC
d Division of Hematology and Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan 70456, Taiwan, ROC
e Department of Obstetrics and Gynecology, Cheng Hsin General Hospital, Taipei, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of the biheterocycles-based compounds with core structure distinguished from com-
bretastatin A-4 (1) and colchicine (5) have been synthesized and evaluated as potent anti-mitotic agents.
Compound 1-(40-Indolyl and 60-quinolinyl)-4,5,6-trimethoxyindoles 13 and 19 showed substantial anti-
proliferative activity against various human cancer cell lines, regardless to the tissue origin and the
expression of multiple-drug resistance MDR1, with a mean IC50 value of 38 and 24 nM respectively.
Received 1 March 2011
Received in revised form
20 April 2011
Accepted 21 April 2011
Available online 20 May 2011
Compound 13 (IC50 ¼ 1.7
m
M) also exhibited similar anti-tubulin activities to 1 (IC50 ¼ 1.8
mM) and
displayed strong binding property to the colchicine binding site on the microtubules. Computational
modeling analysis revealed that the binding mechanism of compound 13 is similar to that of CA4.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Inhibition of tubulin polymerization
Anti-mitotic agent
1. Introduction
structural rigidity, have all been reported as important for its bio-
logical activity [4]. Colchicine (5) is another naturally occurred anti-
A number of natural products, such as paclitaxel, vincristine,
combretastatin A-4 (1) and colchicine (5), can induce cancer cell
apoptosis through interference with tubulin polymerization and
depolymerization, and the causation of subsequent mitotic arrest
[1]. Recently, it has been demonstrated that these microtubule-
targeted anticancer compounds can also function as vascular-
disrupting agents (VDA) that cause rapid and selective disruption
of the established tumor vasculature [2]. Since anti-mitotic
compounds can induce anti-tumor and anti-angiogenic effects,
the usefulness of compounds such as combretastatin A-4P (2, fos-
bretabulin disodium) and AVE8062 (4, ombrabulin) have currently
been evaluated in various clinical trials (Fig. 1).
Combretastatin A-4 (1) is the most potent compound isolated
from the bark of the South African tree Combretum caffrum, extracts
of which have been used as anti-mitotic preparations [3]. At the
structural level, the trimethoxy moiety of A-ring, C30-hydroxy and
C40-methoxy substituents of B-ring, and the cis-related double
bond between two aromatic rings that provides necessary
mitotic compound that exists in similar dihedral relationship to the
plane of trimethoxybenzene ring and other oxygen-substituted
ring, which resembles the structure of the cis-stilbene in 1, and
acts at the same binding site of the microtubule as 1 [5,6]. However,
Cushman et al. reported that the phenanthrene 6, a conformation-
ally rigid (planar) analog of 1, which contains a covalent bond
between two aromatic rings (A- and B-ring) that are [6,6,6]-fused,
displayed dramatic loss of cytotoxicity (ED50 ¼ 1.1 to > 25
mM)
and tubulin polymerization inhibition (IC50 > 40 M). Thus, we
m
synthesized a series of compounds that consist a novel scaffold,
[6,5]-bicyclic aromatic (indole) with N1-arylation, in an attempt to
mimic the cis-related and nonplanar configuration of compounds
1 and 5. Here, we describe the structure-activity relationship of
1-aryl- and 1-heteroaryl-4,5,6-trimethoxyindoles as a new class of
tubulin depolymerization inhibitors (Fig. 2).
2. Results and discussion
2.1. Chemistry
The syntheses of 1-aryl and 1-heteroarylindoles (7-20) are
depicted in the Scheme 1. The starting 4,5,6-trimethoxyindole (21)
was synthesized in advance based on the Dr. Shannon’s
* Corresponding author. Tel.: þ886 2 27361661x6130; fax: þ866 2 27369558.
E-mail address: jpl@tmu.edu.tw (J.-P. Liou).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.065

3624
M.-J. Lai et al. / European Journal of Medicinal Chemistry 46 (2011) 3623e3629
trimethoxyindoles (12-20), and the reference compounds (1
H3CO
H3CO
H3CO
H3CO
and 5) were evaluated against five human cancer cell lines,
including cervical carcinoma KB cells, colorectal carcinoma HT29
cells, non small cell lung carcinoma H460 cells, and stomach
carcinoma MKN45 cells, as well as the MDR1-positive KB-VIN10
cells which over-expresses P-gp 170/MDR1, a relevant causing
factor of drug resistance.(Table 1)
A
NHCOCH3
O
A
B
CH3O
B
OCH3
R
OCH3
OCH3
First, we evaluated the anti-proliferative effect of the substitu-
tion of phenyl group at the N1-position of 4,5,6-trimethoxyindole
core. As the p-methoxy group in the B-ring of 1 is important for
its activity [9], compound 7 was synthesized and evaluated for the
anti-proliferative activity. Compound 7 demonstrated substantial
cytotoxicity against five human cancer cell lines with a mean IC50
value of 168 nM. In an effort to increase its potency, the 30-amino
and 30-hydroxy substituted 1-arylindoles 9 and 10 were prepared to
mimic the structure of 3 and 1 with an amino and hydroxyl group at
the C30-position of B-ring of cis-stilbene, respectively. Interestingly,
growth inhibition activity of the compound 9, namely 1-(30-amino-
40-methoxyphenyl)-4,5,6-trimethoxyindolewas reduced by >10-
fold in five cancer cell lines as compared to the compound 7 with
IC50 values ranging from 1378 to 4146 nM (9 vs 7). A similar
phenomenon was also observed in the case of 10, namely 1-(30-
hydroxy-40-methoxyphenyl)-4,5,6-trimethoxyindole, which dis-
played a decreased cytotoxicity against H460, HT29, and MKN45
human cancer cell lines as compared to 7. It is worth noting that 10
was more effective in targeting oral KB and KB-VIN10 cancer cells as
compared to other cell lines with IC50 values of 131 and 68 nM,
respectively. Pervious SAR studies of the combretastatin-derivative
20-aminocombretastatins [10] and the benzophenonedrelated
compound 20-aminobenzophenones [11] revealed that the amino
group located at the C-20 position of B-ring plays a pivotal role for
their activity. Therefore, we prepared the compound 8 with an
amino group located at the 20-position of 1-arylindole. Compound
8 displayed around one fold elevation in potency as compared to
7 with a mean IC50 value of 80 nM (8 vs 7) Thus, the introduction of
5: Colchicine
1: Combretastatin A-4 (CA4), R = OH
2: Combretastatin A-4P (CA4P), R = OPO3Na2
3: R = NH2
4: AVE-8062, R = NH-serine
Fig. 1. Tubulin polymerization inhibitors target the colchicine-binding domain of tubulin.
methodologies [7]. In addition, Ullmann-type arylation utilizing CuO
as a catalyst was applied in this study to carry out the N-1 substitution
of 4,5,6-trimethoxyindole. The 4,5,6-trimethoxyindole (21) was
reacted with the appropriated halogen-substituted benzenes and
heterocycles, such as N-methylindoles, benzofurans, quinolines and
quinazolines, in the presence of CuO and K2CO3 or Cs2CO3 in DMF to
give the desired 1-phenyl-4,5,6-trimethoxyindoles (7-11) and
1-heteroaryl-4,5,6-trimethoxyindoles (12-20). Notably, the reaction
of 4,5,6-trimethoxyindole (21) with 4-bromoquinoline yielded
compound 17 in the highest yield (64%) compared with other aryla-
tion reactions in this study. The reduction of nitro group in compound
22, 23, and 24 with Fe/NH4Cl in isopropanol/H2O gave the corre-
sponding amines 8, 9, and 25, respectively. Compound 11 bearing
N,N-dimethylamino group was obtained by treating 25 with methyl
iodide in the presence of KOtBu in THF.
2.2. Biological evaluations
2.2.1. Anti-proliferative activity in vitro
The anti-proliferative activity of the synthesized compounds
1-aryl-4,5,6-trimethoxyindoles (7-11) and 1-heteroaryl-4,5,6-
H3CO
H3CO
H3CO
H3CO
3
NHCOCH3
A
A
A
4
H3CO
H3CO
B
5
B
3'
OH
OCH3
CH3O
B
OCH3
OCH3
4'
O
OCH3
OCH3
6
5
1
1
N
4
H3CO
B-ring
A
5
H3CO
6
OCH3
1-phenyl, 1-benzofuranyl, 1-indolyl, 1-quinolinyl, and
1-quinazolinyl-4,5,6-trimethoxyindoles
B-ring = R
14: R = 5'-(1'-methylindolyl)
7: R = 4'-methoxyphenyl
15: R = 5'-benzofuranyl
8: R = 2'-amino-4'-methoxyphenyl
9: R = 3'-amino-4'-methoxyphenyl
10: R = 3'-hydroxy-4'-methoxyphenyl
11: R = 4'-N,N-dimethylaminophenyl
12: R = 3'-(1'-methyl-6'-methoxyindolyl)
13: R = 4'-(1'-methylindolyl)
16: R = 2'-(6'-methoxyquinolinyl)
17: R = 4'-quinolinyl
18: R = 5'-quinolinyl
19: R = 6'-quinolinyl
20: R = 6'-qinazolinyl
Fig. 2. Design of the 1-aryl and 1-heteroarylindoles as anti-mitotic compounds.

M.-J. Lai et al. / European Journal of Medicinal Chemistry 46 (2011) 3623e3629
3625
H
OC
H
OC
3
3
4
3
H
H
3CO
H
H
3CO
a
5
N
R
N
3CO
6
3CO
H
-
7 25
21
2'
3'
5'
4'
N
=
R
3'
N
6'
N
C
H
H
O
4'
OC
4'
OC
H
C
3
H
3CO
H
N
H
O
3
3
3
H
3C
12
1
6
7
10
1
3
14
15
H
OC
3
6'
5'
4'
N
O2
2'
3'
N
O2
N
N
4'
N
H
OC
N
N
H
OC
O2
N
3
3
18
17
19
20
22
23
24
NH2
2'
3'
NH2
4'
N
NH2
25
H
OC
3
H
OC
3
8
9
11
Scheme 1. Reagents and conditions: a. (1) various aryl or heteroaryl halides, K2CO3 or Cs2CO3 (K2CO3 for iodine-substituted compounds; Cs2CO3 for bromine or chlorine-substituted
compounds), CuO, DMF, reflux; (2) for 22-23, Fe, NH4Cl, Isopropanol/H2O, reflux; (3) for 25, CH3I, KOtBu, THF, rt.
an amino group at the C-20 position of 1-aryl-4,5,6-trimethoxyin-
doles, in addition to a methoxy group at C-40 position, plays
important role in the enhancement of cytotoxicity. The fact that
40-methoxy in the B-ring of 1 can be replaced with a 40-N,N-
dimethylamino group inspired us to synthesize compound 11 with
a C-40 dimethylamino group in the 1-arylindole series. As envis-
aged, 11 exhibited substantial anti-proliferative activity against five
cell lines with a mean IC50 value of 127 nM, which is comparable to
that of 7.
As the introduction of an aryl group (phenyl) at the N1-position
of 4,5,6-trimethoxyindoles resulted with a substantial potency of
compounds 7, 8, and 11, we decided to determine whether the
1-heteroarylindoles would also exhibit this anti-proliferative effect
in cells. The six-five fused (indole and benzofuran) and sixesix
fused (quinoline and quinazoline) heterocycles were introduced at
the N1-position of 4,5,6-trimethoxyindole core and the bioactivity
of these compounds have been evaluated. In the N1-indolylindole
series, compounds 12, 13, and 14 with 30-(10-methyl-60-methox-
yindolyl), 40-(10-methylindolyl), and 50-(10-methylindolyl) groups
on the 4,5,6-trimethoxyindole ring, revealed strong anti-
proliferative activity with mean IC50 values of 69, 38, and 63 nM,
respectively. Results of SAR study revealed that in the 1-
indolylindole series, placement of the 4,5,6-trimethoxyindolyl
group at the C-40 position of indole ring showed good bioactivity.
In contrast, re-placement of the 4,5,6-trimethoxyindolyl group to
the C-30 or C-50 position resulted in a slightly reduced activity.
Notably, 1-(40-indolyl)-4,5,6-trimethoxyindole (13) showed anti-
proliferative activity with IC50 values of 25e49 nM against five
cancer cell lines, and this compound was 2e4 fold more potent than
the 1-aryl(phenyl)-4,5,6-trimethoxyindoles 7, 8, and 11. The
1-benzofuraylindole, compound 15 exhibited decreased growth
inhibition activity as compared to 1-indolyl series (compounds
12-14), indicating that the benzofuran substitution at the N1-
position of 4,5,6-trimethoxyindole was not preferred. In the
1-heteroarylindole series with the sixesix fused heterocycle
substitution (quinoline), evaluation of the position effect of 4,5,6-
trimethoxyindolyl moiety on the quinoline ring was performed.
The regioisomers 1-(20-,40-,50-,60)-quinolinylindoles (compounds
16, 17, 18, and 19) were evaluated for anti-proliferative activity. The
1-(60-quinolinyl)-4,5,6-trimethoxyindole (19) showed excellent
bioactivity with IC50 values in a range of 11e34 nM. Re-placement
of the 4,5,6-trimethoxyindolyl group to the C-40 position (17)
resulted in moderate activity, while the C-50 position analog (18)
showed weak cytotoxicity. Furthermore, re-placement of the indole
group to the C-20 position, as in 16, reduced the bioactivity drasti-
cally. As the 1-(60-quinolinyl)indole (19) demonstrated strong anti-
proliferative activity and exhibited higher potency than other
1-(quinolinyl)indoles (16-18), the 1-(60-quinazolinyl)-4,5,6-
trimethoxyindole (20) was synthesized. However, re-placement
of the quinoline with quinazoline resulted in drastic decrease in
bioactivity by at least 100-fold (19 vs 20).
2.2.2. Inhibition of tubulin polymerization through binding to the
colchicine binding site
To investigate whether these biaromatic system compounds
(1-arylindoles and 1-heteroarylindoles) could interfere with the

3626
M.-J. Lai et al. / European Journal of Medicinal Chemistry 46 (2011) 3623e3629
Table 1
The IC50 values (nM ꢂ SDa) of compounds 7-20, and reference compounds (1 and 5) in various cancer cell lines.
Compound
Cell type (IC50 nM ꢂ SDa)
KB
KB-VIN10
H460
HT29
MKN45
145 ꢂ 7
7
8
9
186 ꢂ 14
102 ꢂ 12
3250 ꢂ 212
131 ꢂ 53
148 ꢂ 33
85 ꢂ 35
135 ꢂ 22
50 ꢂ 2
198 ꢂ 19
96 ꢂ 10
179 ꢂ 12
101 ꢂ 16
4146 ꢂ 76
6395 ꢂ 1160
148 ꢂ 33
81 ꢂ 26
49 ꢂ 15
1544 ꢂ 19
5899 ꢂ 760
105 ꢂ 21
59 ꢂ 21
1378 ꢂ 180
68 ꢂ 11
63 ꢂ 8
2393 ꢂ 13
737 ꢂ 64
172 ꢂ 27
81 ꢂ 13
10
11
12
13
14
15
16
17
18
19
20
5[8]
1
40 ꢂ 7
32 ꢂ 5
25 ꢂ 5
49 ꢂ 26
44 ꢂ 23
40 ꢂ 32
73 ꢂ 20
53 ꢂ 9
76 ꢂ 4
74 ꢂ 2
40 ꢂ 9
537 ꢂ 50
7369 ꢂ 820
216 ꢂ 23
900 ꢂ 8
265 ꢂ 24
9114 ꢂ 824
130 ꢂ 9
535 ꢂ 71
11 ꢂ 4
690 ꢂ 106
6170 ꢂ 472
318 ꢂ 99
955 ꢂ 101
31 ꢂ 12
646 ꢂ 110
3652 ꢂ 1247
329 ꢂ 63
921 ꢂ 108
34 ꢂ 5
322 ꢂ 106
11482 ꢂ 1076
197 ꢂ 44
682 ꢂ 223
24 ꢂ 7
18 ꢂ 2
7950 ꢂ 636
10 ꢂ 1
3449 ꢂ 1153
121 ꢂ 10
0.7 ꢂ 0.4
7515 ꢂ 200
20 ꢂ 0.1
2.3 ꢂ 0.9
8136 ꢂ 371
15 ꢂ 0.5
4309 ꢂ 144
12 ꢂ 2
2 ꢂ 0.4
94 ꢂ 26
4.8 ꢂ 0.8
a
SD: standard deviation. All experiments were independently performed for at least three times.
polymerization of microtubule, competitive binding ability of
compounds 8,13,14,19 and the reference compounds 1 and 5 to the
colchicine-binding site were evaluated (Table 2). Here, results of
the tubulin polymerization inhibition assay were consistent with
the results of the cell proliferation assay. The 1-heteroaryl-4,5,6-tri-
methoxyindoles 13, 14, and 19 were efficacious in inhibiting the
interactions with Asn258, Met259, Lys352 and Val181. Results of
the computational modeling analysis further support the results of
the colchicine binding assay that these compounds do bind to the
colchicine-binding site of microtubules (Fig. 3).
3. Conclusion
assembly of microtubule with IC50 values of 1.7, 2.7, and 2.7 mM,
respectively. The 1-aryl (phenyl)-4,5,6-trimethoxyindoles 8 was
less potent as compared to 1, but similar to the reference 5. The
order of inhibition of tubulin polymerization by 1-aryl(heteroaryl)
indoles is 13 > 14 z 19 > 8, indicating that a indolyl (40-, 50-) or
quinolinyl (60-) substituent at the 1-position of 4,5,6-trimethoxyin-
dole plays an important role in determining the level of both
anti-tubulin and anti-proliferative activities. Results of the
colchicine-binding competition assay revealed that 1-(40-indolyl)-
4,5,6-trimethoxyindole (13) strongly bound to the colchicine-
binding domain of tubulin. In addition, both 1-(50-indolyl)- and
1-(60-quinolinyl)-4,5,6-trimethoxyindoles (14 and 19) exhibited
moderate binding activity.
In this study, we have identified the biheterocycles (1-
indolylindole and 1-quinolinylindole) as a new class of potent
microtubule destabilizing agents, acting through the binding with
the colchicine binding site on the microtubule. The lead compounds
1-(40-indolyl)-4,5,6-trimethoxyindoles (13) and 1-(60-quinolinyl)-
4,5,6-trimethoxyindole (19) exhibited anti-proliferative activity
with IC50 values ranging from 11 to 49 nM in a diverse set of human
cancer cell lines from different tissue origins, including MDR-
expressing cell line KB-VIN10. These two compounds also demon-
strated potent tubulin polymerization inhibitory activity with IC50
values of 1.7 and 2.7 mM, respectively. SAR analysis of the 4,5,6-
trimethoxyindole substitution pattern indicated that the 20-
amino-40-methoxyphenyl substitution of 1-arylindoles (8), and the
40- and 50-indolyl, and 60-quinolinyl substitution of 1-heteroarylin-
doles (13, 14, and 19) all contribute to the increased bioactivity. A
comparison between structures of 1 with cis-stilbene, 5 with [6,7,7]-
tricyclic ring, and inactive phenanthrene 6 with [6,6,6]-fused
aromatic revealed that the introduction of [6,5]-bicyclic heterocy-
cles, the 4,5,6-trimethoxyindole, with the N1-aryl or heteroaryl
moiety is able to convert the inactive compound 6 into active
compounds such as 7, 8, 11, 12, 13, 14, and 19. This information is
important for the design of structurally-related tubulin inhibitors or
combretastatins analogs in the future.
2.3. Molecular modeling study
To further understand the underlying structure that give rise to
the strong microtubule binding ability of 13, molecular modeling
studies of 13 and 1 were carried out. Compound 13 and 1 were
docked onto the colchicine binding site of tubulin (PDB 1SA0) using
Gold (version 4.0) in default mode. Results revealed that the
binding mode of 13 is similar to that of 1. The A-rings of 13 and 1 are
located at the similar area and made hydrophobic interactions with
Cys241, Leu248, Ala250 and Leu255. The B-rings of both
compounds also occupied the same pocket and made hydrophobic
4. Experimental section
Table 2
Inhibition of Tubulin Polymerization and Colchicine Binding by Compounds 8, 13, 14,
19, and the reference compounds (1 and 5).
Nuclear magnetic resonance (1H NMR and 13C NMR) spectrawere
obtained with the Bruker DRX-500 and Bruker DRX-400 spec-
trometers, with chemical shift in parts per million (ppm, d) down-
Cmpd
Tubulina IC50 ꢂ SD (
mM)
Colchicine binding abilityb
(% ꢂ SD)
field fromTMS as an internal standard. High-resolution mass spectra
(HRMS) were measured with a JEOL (JMS-700) electron impact (EI)
mass spectrometer. Purity of the final compounds were determined
using an Hitachi 2000 series HPLC system using C-18 column (Agi-
8
4.7 ꢂ 0.5
1.7 ꢂ 0.1
2.7 ꢂ 0.5
2.7 ꢂ 0.2
4.1 ꢂ 0.5
1.8 ꢂ 0.2
44 ꢂ 6
83 ꢂ 1
65 ꢂ 4
72 ꢂ 2
13
14
19
5 [8]
1
lent ZORBAX Eclipse XDB-C18 5
mm. 4.6 mm ꢀ 150 mm) and were
found to be ꢁ95%. Flash column chromatography was performed
using silica gel (Merck Kieselgel 60, No. 9385, 230e400 mesh ASTM).
All reactions were carried out under dry nitrogen surrounded
atmospheric conditions.
97 ꢂ 2
a
Inhibition of tubulin polymerization [12].
Inhibition of [3H]colchicine binding [13]. Tubulin was at 1
m
M; [3H]colchicine
b
and inhibitors were both at 5 M.
m

M.-J. Lai et al. / European Journal of Medicinal Chemistry 46 (2011) 3623e3629
3627
Fig. 3. Superimposition of 13 (cyan) and 1 (yellow) in the colchicine binding site of tubule (PDB 1SA0) using Gold 4.0 software. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
4.1. General procedures for the preparation of 1-aryl and
1-heteroarylindoles
ammonium chloride (0.02 g, 0.28 mmol) in isopropanol (5 mL) and
water (1 ml) were refluxed for 16 h. The reaction mixture was fil-
trated and then evaporated to give a residue that was purified by
silica gel flash column chromatography (EtOAc : n-hexane ¼ 1 : 3 :
1% NH3(aq)) to afford the desired 8 as an orange solid (0.02 g, 49%);
4.1.1. 4,5,6-Trimethoxy-10-methyl-1’H-[1,40]biindolyl (13)
To a stirred mixture of 21 (0.25 g, 1.21 mmol), 4-bromo-1-
methylindole (0.31 g, 1.45 mmol), cesium carbonate (0.79 g,
2.41 mmol) and copper oxide (0.01 g, 0.12 mmol) in DMF (2 mL)
was refluxed for 24 h. After cooling, the reaction was quenched
with water and extracted with EtOAc. The combined organic layer
was dried over anhydrous MgSO4, concentrated under reduced
pressure and purified by silica gel flash column chromatography
(EtOAc : n-hexane ¼ 1 : 3) to afford compound 13 as a pale white
solid (0.12 g, 30%); mp 107e108 ꢃC; 1H NMR (500 MHz, CDCl3)
1H NMR (500 MHz, CDCl3):
d 3.58 (s, 2H), 3.79 (s, 3H), 3.83 (s, 3H),
3.88 (s, 3H), 4.14 (s, 3H), 6.32 (s, 1H), 6.39 (d, J ¼ 2.0 Hz, 1H), 6.40 (d,
J ¼ 9.0 Hz, 1H), 6.68 (d, J ¼ 3.0 Hz, 1H), 6.98 (d, J ¼ 3.0 Hz, 1H), 7.09
(dd, J ¼ 9.0, 2.0 Hz,1H); MS (EI): m/z (%): 313 (62), 328 (100); HRMS-
EI for C18H20N2O4 (Mþ): calcd, 328.1423; found, 328.1421.
4.1.4. 2-Methoxy-5-(4,5,6-trimethoxy-indol-1-yl)-phenylamine (9)
The title compound was obtained as a brown solid (0.02 g, 9.5%)
(two steps) from compound 21 and 1-iodo-4-methoxy-3-
nitrobenzene in a similar manner as described for the prepara-
d
3.75 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 4.17 (s, 3H), 6.39 (d,
J ¼ 3.0 Hz, 1H), 6.67 (s, 1H), 6.75 (d, J ¼ 3.0 Hz, 1H), 7.08 (d,
J ¼ 3.0 Hz, 1H), 7.21 (dd, J ¼ 1.0, 7.0 Hz, 1H), 7.30 (d, J ¼ 3.0 Hz, 1H),
7.33 (dd, J ¼ 7.0, 8.0 Hz, 1H), 7.37 (d, J ¼ 8.0, 1H). MS (EI) m/z: 336
(Mþ, 100%), 321 (78%). HRMS-EI for C20H20N2O3 (Mþ): calcd,
tion of 8; mp 175e177 ꢃC; 1H NMR (500 MHz, CDCl3):
d 3.83 (s, 3H),
3.88 (s, 3H), 4.12 (s, 3H), 6.64 (d, J ¼ 3.0 Hz, 1H), 6.68 (s, 1H), 6.74 (d,
J ¼ 7.0 Hz,1H), 6.82e6.84 (m, 2H), 7.06 (d, J ¼ 3.0 Hz,1H); MS (EI) m/
z (%): 297 (95), 313 (100); HRMS-EI for C18H20N2O4 (Mþ): calcd,
328.1423; found, 328.1422.
336.1474; found, 336.1474; 13C NMR (100 MHz, DMSO):
d 32.7, 55.8,
60.4, 60.8, 89.6, 98.3, 100.2, 109.0, 115.2, 115.3, 121.5, 123.4, 127.1,
130.4, 131.0, 132.5, 135.4, 137.9, 145.2, 150.5.
4.1.5. 2-Methoxy-5-(4,5,6-trimethoxy-indol-1-yl)-phenol (10)
The title compound was obtained as a brown liquid (0.03 g, 19%)
from compound 21 and 5-Iodo-2-methoxy-phenol [14] in a similar
way as described for the preparation of 13; 1H NMR (500 MHz,
4.1.2. 4,5,6-Trimethoxy-1-(4-methoxy-phenyl)-1H-indole (7)
The title compound was obtained as a white solid (0.16 g, 43%)
from compound 21 and 1-iodo-4-methoxybenzene in a similar way
as described for the preparation of 13; mp 96e98 ꢃC; 1H NMR
CDCl3):
d 3.83 (s, 3H), 3.88 (s, 3H), 3.96 (s, 3H), 4.12 (s, 3H), 5.77 (s,
(500 MHz, CDCl3):
d
3.82 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H), 4.13 (s,
1H), 6.66 (d, J ¼ 3.0 Hz, 1H), 6.72 (s, 1H), 6.95e6.96 (m, 2H), 7.06 (d,
J ¼ 2.0 Hz, 1H), 7.08 (d, J ¼ 3.0 Hz, 1H); MS (EI) m/z (%): 314 (60), 329
(100); HRMS-EI for C18H19NO5 (Mþ): calcd, 329.1263; found,
329.1264.
3H), 6.64 (s, 1H), 6.67 (d, J ¼ 3.0 Hz, 1H), 7.03 (d, J ¼ 8.5 Hz, 2H), 7.09
(d, J ¼ 3.0 Hz, 1H), 7.37 (d, J ¼ 8.5 Hz, 2H); MS (EI): m/z (%): 298 (93),
313 (100); HRMS-EI for C18H19NO4 (Mþ): calcd, 313.1314; found,
313.1314.
4.1.6. Dimethyl-[4-(4,5,6-trimethoxy-indol-1-yl)-phenyl]-amine (11)
Compound 25 was obtained as a brown solid (0.06 g, 21%) (two
steps) from compound 21 and 1-iodo-4-nitrobenzene in a similar
way as described for the preparation of 8; mp 109e111 ꢃC; 1H NMR
4.1.3. 5-Methoxy-2-(4,5,6-trimethoxy-indol-1-yl)-phenylamine (8)
Compound 22 was obtained as a yellow liquid (0.05 g, 14%) from
compound 21 and 1-iodo-4-methoxy-2-nitrobenzene in a similar
way as described for the preparation of 13; 1H NMR (500 MHz,
(500 MHz, CDCl3): d 3.79 (s, 2H), 3.81 (s, 3H), 3.87 (s, 3H), 4.12 (s, 3H),
CDCl3):
d
3.72 (s, 3H), 3.83 (s, 3H), 3.95 (s, 3H), 4.14 (s, 3H), 6.23 (s,
6.62 (s, 1H), 6.64 (d, J ¼ 3.0 Hz, 1H), 6.78 (d, J ¼ 8.5 Hz, 2H), 7.06 (d,
J ¼ 3.0 Hz,1H), 7.22 (d, J ¼ 8.5 Hz, 2H); MS (EI) m/z (%): 283 (91), 298
(100); HRMS-EI : m/z [M þ H]þ calcd for C17H18N2O3: 298.1317,
found: 298.1316. To a solution of 25 (0.14 g, 0.47 mmol) and
1H), 6.71 (d, J ¼ 3.4 Hz, 1H), 6.95 (d, J ¼ 3.4 Hz, 1H), 7.24e7.25 (m,
1H), 7.43 (d, J ¼ 8.7 Hz, 1H), 7.51 (d, J ¼ 2.9 Hz, 1H). Then, to a stirred
mixture of 22 (0.05 g, 0.14 mmol), iron (0.03 g, 0.42 mmol), and

3628
M.-J. Lai et al. / European Journal of Medicinal Chemistry 46 (2011) 3623e3629
potassium carbonate (0.13 g, 0.94 mmol) in DMF (2 mL) was stirred
for 10 min at room temperature. Iodomethane (0.12 ml, 1.88 mmol)
was added to the reaction mixture with continuous stirring for 2 h.
The reaction solvent was removed under reduced pressure then
quenched with water and extracted with CH2Cl2. The combined
organic layer was dried over anhydrous MgSO4, concentrated under
reduced pressure, and purified by silica gel flash column chroma-
tography (EtOAc : n-hexane ¼ 1 : 4) to afford the desired 11 as
a brown solid (0.09 g, 59%); mp 93e95 ꢃC; 1H NMR (500 MHz,
J ¼ 4.5 Hz, 1H), 7.55 (dd, J ¼ 7.5, 7.5 Hz, 1H), 7.76 (dd, J ¼ 7.5, 8.5 Hz,
1H), 7.78 (d, J ¼ 7.5 Hz, 1H), 8.23 (d, J ¼ 8.5 Hz, 1H), 9.02 (d,
J ¼ 4.5 Hz, 1H); MS (EI) m/z (%): 319 (85), 334 (100); HRMS-EI for
C20H18N2O3 (Mþ): calcd, 334.1317; found, 334.1319.
4.1.12. 5-(4,5,6-Trimethoxy-indol-1-yl)-quinoline (18)
The title compound was obtained as a brown solid (0.07 g, 43%)
from compound 21 and 5-bromoquinoline in a similar way as
described for the preparation of 13; mp 173e174 ꢃC; 1H NMR
CDCl3):
d
3.02 (s, 6H), 3.82 (s, 3H), 3.88 (s, 3H), 4.13 (s, 3H), 6.65 (d,
(500 MHz, CDCl3):
d
¼ 3.64 (s, 3H), 3.89 (s, 3H), 4.18 (s, 3H), 6.17 (s,
J ¼ 3.0 Hz,1H), 6.65 (s,1H), 6.83 (d, J ¼ 8.5 Hz, 2H), 7.08 (d, J ¼ 3.0 Hz,
1H), 7.31 (d, J ¼ 8.5 Hz, 2H); MS (EI) m/z (%):311 (42), 326 (100);
HRMS-EI for C19H22N2O3 (Mþ): calcd, 326.1630; found, 326.1628.
1H), 6.80 (d, J ¼ 3.0 Hz, 1H), 7.13 (d, J ¼ 3.0 Hz, 1H), 7.37 (dd, J ¼ 4.0,
8.5 Hz, 1H), 7.62 (d, J ¼ 7.5 Hz, 1H), 7.83 (dd, J ¼ 1.5, 7.5 Hz, 1H), 7.85
(d, J ¼ 7.5 Hz, 1H), 8.24 (d, J ¼ 8.5 Hz, 1H), 8.98 (dd, J ¼ 1.5, 4.0 Hz,
1H); MS (EI) m/z (%): 319 (75), 334 (100); HRMS-EI for C20H18N2O3
(Mþ): calcd, 334.1317; found, 334.1316.
4.1.7. 4,5,6,60-tetramethoxy-10-methyl-1’H-[1,30]biindolyl (12)
The title compound was obtained as a brown solid (0.04 g, 11%)
from compound 21 and 3-iodo-1-methyl-6-methoxyindole in
4.1.13. 6-(4,5,6-Trimethoxy-indol-1-yl)-quinoline (19)
a
similar way as described for the preparation of 13; mp
The title compound as an orange solid (0.06 g,13%) was obtained
from compound 21 and 6-bromoquinoline in a similar way as
described for the preparation of 13; mp 95e97 ꢃC; 1H NMR
131e133 ꢃC; 1H NMR (500 MHz, CDCl3):
d
3.74 (s, 3H), 3.82 (s, 3H),
3.89 (s, 3H), 3.91 (s, 3H), 4.16 (s, 3H), 6.56 (s, 1H), 6.68 (d, J ¼ 3.0 Hz,
1H), 6.81 (dd, J ¼ 2.0, 8.5 Hz, 1H), 6.85 (d, J ¼ 2.0 Hz, 1H), 7.10 (s, 1H),
7.12 (d, J ¼ 3.0 Hz, 1H), 7.33 (d, J ¼ 8.5 Hz, 1H); MS (EI) m/z (%): 351
(83), 366 (100); HRMS-EI for C21H22N2O4 (Mþ): calcd, 366.1580;
found, 366.1580.
(500 MHz, CDCl3):
d 3.84 (s, 3H), 3.90 (s, 3H), 4.15 (s, 3H), 6.77 (d,
J ¼ 3.0 Hz, 1H), 6.83 (s, 1H), 7.25 (d, J ¼ 3.0 Hz, 1H), 7.48 (dd, J ¼ 4.5,
8.5 Hz, 1H), 7.87 (s, 1H), 7.89 (dd, J ¼ 2.5, 4.5 Hz, 1H), 8.20 (d,
J ¼ 8.5 Hz, 1H), 8.26 (d, J ¼ 8.5 Hz, 1H), 8.96 (dd, J ¼ 2.5, 4.5 Hz, 1H);
MS (EI) m/z (%): 319 (74), 334 (100); HRMS-EI for C20H18N2O3 (Mþ):
4.1.8. 4,5,6-Trimethoxy-10-methyl-1’H-[1,50]biindolyl (14)
calcd, 334.1317; found, 334.1316; 13C NMR (100 MHz, DMSO):
d 8.1,
The title compound as a white solid (0.20 g, 48%) was obtained
from compound 21 and 5-bromo-1-methylindole in a similar way
as described for the preparation of 13; mp 127e129 ꢃC; 1H NMR
56.0, 60.5, 60.8, 89.5, 101.5, 116.0, 121.1, 122.2, 126.2, 126.7, 128.5,
130.6, 132.0, 136.1, 136.9, 145.9, 150.5, 151.1.
(500 MHz, CDCl3):
d
3.78 (s, 3H), 3.87 (s, 3H), 3.89 (s, 3H), 4.15 (s,
4.1.14. 6-(4,5,6-Trimethoxy-indol-1-yl)-quinazoline (20)
3H), 6.55 (d, J ¼ 3.5 Hz, 1H), 6.68 (d, J ¼ 3.5 Hz, 1H), 6.69 (s, 1H), 7.17
(d, J ¼ 4.0 Hz, 1H), 7.18 (d, J ¼ 4.0 Hz, 1H), 7.31 (dd, J ¼ 2.0, 8.5 Hz,
1H), 7.43 (d, J ¼ 8.5 Hz, 1H), 7.68 (d, J ¼ 2.0 Hz, 1H). MS (EI) m/z (%):
321 (84), 336 (100) [M þ H]þ; HRMS-EI for C20H20N2O3 (Mþ): calcd,
The title compound was obtained as a brown solid (0.07 g, 22%)
from 22 and 6-iodo-quinazoline [16] in a similar way as described
for the preparation of 13; mp 141e143 ꢃC; 1H NMR (500 MHz,
CDCl3):
d
3.84 (s, 3H), 3.90 (s, 3H), 4.14 (s, 3H), 6.79 (d, J ¼ 3.5 Hz,
336.1474; found, 336.1472; 13C NMR (100 MHz, DMSO):
d
32.6, 55.8,
1H), 6.81 (s, 1H), 7.24 (d, J ¼ 3.5 Hz, 1H), 7.97 (d, J ¼ 2.5 Hz, 1H), 8.11
(dd, J ¼ 2.5, 8.5 Hz, 1H), 8.22 (d, J ¼ 8.5 Hz, 1H), 9.37 (s, 1H), 9.45 (s,
1H); MS (EI) m/z(%) : 320 (68) 335 (100); HRMS-EI for C19H17N3O3
(Mþ): calcd, 335.1270; found, 335.1269.
60.3, 60.8, 88.9, 99.7, 100.7, 110.6, 115.3, 116.1, 118.2, 127.4, 128.4,
131.2, 131.2, 132.9, 135.0, 135.3, 145.2, 150.6.
4.1.9. 1-Benzofuran-5-yl-4,5,6-trimethoxy-1H-indole (15)
The title compound was obtained as a yellow liquid (0.01 g,
6.4%) from compound 21 and 5-bromo-benzofuran in a similar way
as described for the preparation of 13; 1H NMR (500 MHz, CDCl3):
4.2. Biology
Reagents for cell culture were obtained from Gibco-BRL Life
Technologies (Gaitherburg, MD). Microtubule-associated protein
(MAP)-rich tubulin was purchased from Cytoskeleton, Inc. (Denver,
CO). [3H]Colchicine (specific activity, 60e87 Ci/mmol) was
purchased from PerkinElmer Life Sciences (Boston, MA).
d
3.81 (s, 3H), 3.89 (s, 3H), 4.15 (s, 3H), 6.69 (s, 1H), 6.70 (d,
J ¼ 3.0 Hz, 1H), 6.85 (d, J ¼ 1.5 Hz, 1H), 7.16 (d, J ¼ 3.0 Hz, 1H), 7.39
(dd, J ¼ 1.5, 8.5 Hz, 1H), 7.63 (d, J ¼ 8.5 Hz, 1H), 7.66 (d, J ¼ 2.0 Hz,
1H), 7.73 (d, J ¼ 2.0 Hz, 1H); MS (EI) m/z (%): 308 (80), 323 (100);
HRMS-EI for C19H17NO4 (Mþ): calcd, 323.1158; found, 323.1158.
4.1.10. 6-Methoxy-2-(4,5,6-trimethoxy-indol-1-yl)-quinoline (16)
The title compound was obtained as a yellow solid (0.15 g, 43%)
from compound 21 and 2-chloro-6-methoxyquinoline [15] in
a similar way as described for the preparation of 13; mp 95e97 ꢃC;
4.2.1. Cell growth inhibitory assay
Human cervical cancer cells, non small cell lung carcinoma H460
cells, colorectal carcinoma HT29 cells, and stomach carcinoma
MKN45 cells were maintained in RPMI-1640 medium supplied with
5% fetal bovine serum. KB-VIN10 cells were generated from KB with
continuous exposure of increasing vincristine concentrations, and
displays over-expression of P-gp 170/MDR1. These cells were
maintained in growth medium supplemented with 10 nM vincris-
tine and. Cell in logarithmic phase were cultured at a density of 5000
cells/mL/well in a 24-well plate. KB-VIN10 cells were cultured in
drug-free medium for 3 days prior to use. In addition, all test
compounds were dissolved in DMSO at a 20 mM concentration, and
stored at ꢄ20 ꢃC before used. The cells were exposed to various
1H NMR (500 MHz, CDCl3):
d 3.92 (s, 3H), 3.93 (s, 3H), 3.98 (s, 3H),
4.11 (s, 3H), 6.77 (d, J ¼ 3.0 Hz, 1H), 7.10 (d, J ¼ 3.0 Hz, 1H), 7.39 (dd,
J ¼ 9.0, 3.0 Hz, 1H), 7.52 (d, J ¼ 8.5 Hz, 1H), 7.55 (d, J ¼ 3.0 Hz, 1H),
7.95 (d, J ¼ 9.0 Hz, 1H), 8.04 (s, 1H), 8.12 (d, J ¼ 8.5 Hz, 1H); MS (EI)
m/z (%): 349 (90), 364 (100); HRMS-EI for C21H20N2O4 (Mþ): calcd,
364.1423; found, 364.1422.
4.1.11. 4-(4,5,6-Trimethoxy-indol-1-yl)-quinoline (17)
The title compound was obtained as a yellow solid (0.11 g, 64%)
from compound 21 and 4-bromoquinoline in a similar way as
described for the preparation of 13; mp 153e155 ꢃC; 1H NMR
concentrations (ranging from 1 nM to 50 mM in serial 2-fold dilu-
tions) of the test drugs for 72 h. The methylene blue dye assay was
used to evaluate the effect of the test compounds on cell growth as
described previously [17]. The IC50 value resulting from 50%
(500 MHz, CDCl3):
d 3.68 (s, 3H), 3.89 (s, 3H), 4.16 (s, 3H), 6.37 (s,
1H), 6.83 (d, J ¼ 3.0 Hz, 1H), 7.18 (d, J ¼ 3.0 Hz, 1H), 7.45 (d,

M.-J. Lai et al. / European Journal of Medicinal Chemistry 46 (2011) 3623e3629
3629
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placed in 96-well microtiter plate in the presence of test
compounds or 2% (v/v) DMSO as control. Absorbance was measured
at 350 nm in a PowerWave X Microplate Reader (BIO-TEK Instru-
ments, Winooski, VT) at 37 ꢃC and recorded every 30 s for 30 min.
The area under the curve (AUC) used to determine the concentra-
tion that inhibited tubulin polymerization to 50% (IC50). The AUC of
the untreated control and 10 mM of colchicine was set to 100% and
0% polymerization, respectively, and the IC50 was calculated by
nonlinear regression in at least three independent experiments.
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Acknowledgments
This research was supported by the National Science Council of
Taiwan R.O.C. (grant no. NSC 99-2323-B-038-001, NSC 98-2113-M-
038-002-MY2, NSC99-2323-B-400-006, and NSC99-2323-B-038-
005-CC2) and the Department of Health of the Republic of Taiwan
R.O.C. (grant no. DOH99-TD-C-111-004).
Appendix. Supplementary material
Supplementary data related to this article can be found, in the
online version, at doi:doi:10.1016/j.ejmech.2011.04.065.
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