Synthesis and structure-activity relationships of N-methyl-5,6,7- trimethoxylindoles as novel antimitotic and vascular disrupting agents

Article abstract of DOI:10.1021/jm3014663

Several new series of 5,6,7-trimethoxyindole derivatives were synthesized and their structure-activity relationships (SARs) were studied. Some of these compounds exhibited strong antiproliferative activities in the submicromolar range. N-Methyl-5,6,7-trim

Full text of DOI:10.1021/jm3014663

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of N‑Methyl-5,6,7-
trimethoxylindoles as Novel Antimitotic and Vascular Disrupting
Agents
Deng-Gao Zhao,† JianJun Chen,† Ya-Rong Du,† Yan-Yan Ma,† Ya-Xiong Chen,‡ Kun Gao,*,†
and Bu-Rong Hu*,‡
†State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou
730000, People’s Republic of China
‡Key Laboratory of Heavy Ion Radiation Biology and Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou
730000, People’s Republic of China
S
* Supporting Information
ABSTRACT: Several new series of 5,6,7-trimethoxyindole derivatives were
synthesized and their structure−activity relationships (SARs) were studied.
Some of these compounds exhibited strong antiproliferative activities in the
submicromolar range. N-Methyl-5,6,7-trimethoxylindoles 21 and 31 displayed
the highest antiproliferative activities, with IC50 values ranging from 22 to 125
nM in four human cancer cell lines and activated human umbilical vein
endothelial cells (HUVECs). In addition to vascular disrupting activity verified
by in vitro assays, compounds 21 and 31 displayed much higher selectivity for
activated HUVECs versus quiescent HUVECs than those of colchicine and
combretastatinA-4. The polymerization of cancer cell tubulin was inhibited and
the cell cycle was arrested in the G2/M phase after treatment with 21 and 31. It
was showed that 21 disrupted tumor vasculature by use of in vivo assay. Our
results suggest that these two new compounds we synthesized may become the
promising leads for the development of vascular disrupting agents.
INTRODUCTION
■
The vasculature has a pivotal role in growth and survival of
solid tumors.1 Given its inherent differences with normal
vasculature, the tumor vasculature has become an attractive
target for anticancer drug discovery.2 Vascular-disrupting agents
(VDAs) selectively target established tumor vasculature to
cause a significant shutdown in blood flow to solid tumors,
resulting in extensive tumor-cell necrosis while the blood flow
in normal tissues remains relatively intact.3 VDAs have many
advantages over other cancer therapies.4 Currently, two major
groups of VDAs, small-molecule VDAs and ligand-directed
VDAs, are constructed for cancer treatment.
A number of antimitotic agents, such as colchicine 3 (Figure
1), vincristine, and vinblastine, show potent vascular disrupting
properties.5 More recently, many agents binding tubulin at or
near the colchicine binding site, including combretastatinA-4
(CA-4) 1,6 2 (Oxi4503),7 4 (ZD6126),8 and 5 (BNC105),9
among others, are currently in clinical trials or undergoing
preclinical testing.
Figure 1. Some anitmitotic agents with vascular-disrupting activities.
cytotoxicity against tumor endothelial cells.10,11 Tumor
endothelial cells are constantly exposed to proangiogenic
It is well-known that the damage of antimitotic agents on
tumor vasculature has been ascribed to a direct effect on
endothelial cells.3,10 To discover new antimitotic agents which
growth factors and, as a consequence, are in a constant state
selectively disrupt tumor vasculature, it is important to evaluate
these analogues not only regarding their cytotoxicity and
antitubulin activity but also regarding their ability to selective
Received: July 16, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Reagents and conditions: (a) THF, t-BuOK, 61−65%; (b) EtMgBr, ZnCl2, AlCl3, CH2Cl2, 37−62%; (c) CH3I, THF, t-BuOK, 93−98%; (d) acetyl
chloride, THF, t-BuOK, 52%; (e) benzenesulfonyl chloride, THF, t-BuOK, 46%; (f) 10% Pd/C, NH4COOH, CH3OH, reflux, 90−99%.
of activation and angiogenesis.12 Therefore, human umbilical
vein endothelial cells (HUVECs) grown in the presence
(activated) and absence (quiescent) of growth factors may
model the endothelial cells of tumor and normal vasculature,
respectively.7,8,11,13 Evaluating the selectivity of tested com-
pounds for activated HUVECs compared with quiescent
HUVECs may be useful for identifying their selectivity toward
tumor vasculature. Indeed, this approach has been applied to
the discover process of 5, which selectively disrupts tumor
vasculature and displays a wider therapeutic margin.9,11 Herein,
we also applied this approach to discover new antimitotic
agents that display potent and selective toxicity toward tumor
vasculature.
Compounds 1 and 4 are thought to be the lead compounds
among the class both as antimitotic agents and VDAs.4
Hundreds of compounds, mimicking 1 and 4, have been
synthesized.14 Through structure−activity relationships (SARs)
studies, it has been established that these compounds bear two
polyoxygenated aromatic rings, and the trimethoxybenzene
moiety is crucial to obtain antiproliferative and antimitotic
activities.14 However, it should be noted that most efforts have
been so far concentrated on increasing their cytotoxic and
capacity to inhibit tubulin, while little efforts were paid in the
antivascular effects.10,14 With respect to antivascular activity, it
is difficult to derive meaningful structure−activity relationships
from the data obtained, and more extensive structural
modifications need to be exploited.10
degree of antivascular selectivity, following the SAR-guided
discovery process, we replaced the trimethoxybenzene moiety
with a more hindered trimethoxyindole skeleton, and synthesiz-
ing series of 5,6,7-trimethoxyindole derivatives: (1) 1-
aroylindoles and 1-benzylindoles, (2) 3-aroylindoles and 3-
benzylindoles, and (3) N1-substituted-3-aroylindoles and N1-
substituted-3-benzylindoles. The screening results of our series
biological experiments demonstrated that both 21 and 31
among our synthesized compounds had high degree of
selectivity for activated HUVECs and are promising leads for
the development of vascular-disrupting agents.
RESCULT AND DISCUSSION
■
Chemistry. The synthetic route followed for the synthesis
of the desired aroylindoles 6−23 is outlined in Scheme 1. The
5,6,7-trimethoxylindole and 5,6,7-trimethoxy-2-methylindole
was prepared according to the procedure previously reported.16
The direct electrophilic substitution of 5,6,7-trimethoxylindole
and with corresponding benzoyl chloride in the presence of
EtMgBr, ZnCl2, and AlCl3 gave the desired 3-aroylindoles 8,
12−14, 15a, 16−18, and 19a in 37−62% yields.17 The N1-
aroylindoles 6 and 7a were synthesized in 61−65% yields from
corresponding benzoyl chloride by allowing it to react with the
5,6,7-trimethoxylindole at room temperature in the presence of
t-BuOk. In a similar manner, compounds 10 and 11 were
prepared from 8 using acetyl chloride and benzenesulfonyl
chloride, respectively. When 3-aroylindoles 8, 15a, and 19a
reacted with iodomethane, compounds 20, 21a, and 23a were
obtained in excellent yields, respectively. Transfer hydro-
genation of 7a, 15a, 19a, 21a, and 23a with ammonium
formate and palladium on charcoal yielded the desired 7, 15,
19, 21, and 23.
The replacement of the trimethoxybenzene ring by
benzoheterocyclic structures has received little attention so
far.14 Recently, some trimethoxyindole derivatives have been
reported as potent antimitotic agents with antivascular
activity.15 To discover potential antitumor agents with a high
B
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
The benzylindoles 24−31 were synthesized as shown in
Scheme 2. The N1-benzylindoles 24 and 25a were synthesized
Table 1. Antiproliferative Activity of the Synthesized
Compounds against A-549 Human Lung Adenocarcinoma
b
b
compd
IC50 = (nM SDs)
compd
20
IC50 = (nM SDs)
a
Scheme 2
6
>10000
>10000
481.7 7.47
28.30 6.36
146.15 6.00
107.8 18.9
3088 426
3192.4 185.5
2987 448
>10000
7
21
8
8289 397
>10000
22
9
23
10
11
12
13
14
15
16
17
18
19
5274 267
>10000
24
25
>10000
26
>10000
27
>10000
28
387.4 28.5
117.99 10.88
144.3 23.42
71.72 7.22
20.00 2.04
166.08 8.68
>10000
29
30
>10000
31
>10000
colchicine
2623 153
1
3.64 0.54
a
b
Antiproliferative activity was determined by CCK-8 assay. IC50
=
compound concentration required to inhibit tumor cell proliferation
by 50% after cells were treated with compounds for 72 h. Data are
expressed as means SDs (standard deviations) from at least three
independent experiments.
8 and 12−18 and 3-benzyltrimethoxyindole derivatives 26−29
demonstrated that the 3′-hydroxy-4′-methoxy substitution (15,
29) resulted in best activity.
a
Reagents and conditions: (a) DMF, KOH, KI, 39−47%; (b)
NH4HCO3, 80% aqueous acetone (v/v), 44−55%; (c) CH3I, THF,
t-BuOK, 93−98%; (d) 10% Pd/C, NH4COOH, CH3OH, reflux, 90−
99%.
The introduction of a methyl group at the N-1 position of 3-
aroylindoles (20−22) and 3-benzylindoles (30, 31) resulted in
apparently increased antiproliferative activities as compared to
its parent compounds, but, in contrast, the introduction of an
acetyl (10) or a sulfonyl group (11) at the N-1 position and the
introduction of a methyl group at the C-2 position of 3-
aroylindoles (23) resulted in reduced activities. In these series,
the replacement of 3′-OH group with a halogen atom (16−18,
22, 28, 30) caused loss of antiproliferative activities.
in 39−47% yields from corresponding benzyl bromide by
allowing it to react with the 5,6,7-trimethoxylindole at room
temperature in the presence of KI and KOH.18 Because our
attempts to convert 3-aroylindoles into 3-benzylindoles have
failed, the synthesis of 3-benzylindoles was accomplished via
Friedel−Crafts acylation.19 The 3-benzylindoles 26−29a and its
isomer 2-benzylindoles were obtained by a one-step procedure
applied to 5,6,7-trimethoxylindole and the appropriate benzyl
bromide with ammonium hydrogencarbonate as base in 80%
aqueous acetone (v/v). The 5:1 mixture of the 3-benzylindoles
and its isomer were finally separated by reversed-phase silica gel
column chromatography. When 3-benzylindoles 28 and 29a
reacted with iodomethane, compounds 30 and 31a were
obtained in excellent yields, respectively. In the manner
described above, transfer hydrogenation of 25a, 29a, and 31a
yielded the desired 25, 29, and 31, respectively.
Antiproliferative Activity. All the synthesized trimethox-
yindole derivatives and reference compounds colchicine and 1
were initially screened for their antiproliferative activity against
the human lung arcinoma cell line A549 (Table 1). Cell
proliferation was determined by CCK-8 assay after a treatment
period of 72 h. Compounds 21 and 31 displayed potent
antiproliferative activities in A549 cells, with IC50 values of 28
and 72 nM, respectively. Compounds 23 and 29 also showed
considerable antiproliferative activities, with IC50 values of 108
and 118 nM, respectively. A preliminary comparison between
antiproliferative activities of synthesized trimethoxyindole
derivatives suggested that 3-aroyltrimethoxyindole and 3-
benzyltrimethoxyindole derivatives had a higher antiprolifer-
ative activity than 1-aroyltrimethoxyindole (6, 7) and 1-
benzyltrimethoxyindole derivatives (24, 25). The antiprolifer-
ative activities of 3-benzyltrimethoxyindole derivatives were
comparable to those of 3-aroyltrimethoxyindole derivatives.
The comparison among the 3-aroyltrimethoxyindole derivatives
The antiproliferative property of the trimethoxyindole
derivatives 21, 23, 29, and 31 were further confirmed in
other three human cancer cell lines (Table 2). The
a
Table 2. Antiproliferative Activity of Selected Compounds
against Different Human Cancer Cell Lines
b
IC50 = (nM SDs)
compd
HeLa
HepG2
HT1080
colchicine
37.38 9.58
125.37 4.66
632.2 68.5
148.51 1.81
40.68 8.20
46.74 2.63
44.45 1.88
91.63 12.17
72.17 14.20
45.49 0.48
15.38 0.18
26.32 3.98
573.94 86.0
117.86 11.65
21
23
29
31
42.18 10.39
a
b
Antiproliferative activity was determined by CCK-8 assay. IC50
=
compound concentration required to inhibit tumor cell proliferation
by 50% after cells were treated with compounds for 72 h. Data are
expressed as means SDs (standard deviations) from at least three
independent experiments.
antiproliferative activities of compounds 21 and 31 were higher
than the 23 and 29, with IC50 values ranging from 26 to 125
nM. These result were quiet coincident with SRAs described
above. Although less potent than 1, the antiproliferative
activities of 21 and 31 are comparable to those of colchicine.
Endothelial Cell Proliferation Assay. According to the
method of Flynn et al.,11 key compounds were evaluated for
C
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
antiproliferative activity in activated HUVECs and quiescent
HUVECs. As shown in Table 3, compounds 21 and 31
a
Table 3. Antiproliferative Activity of Selected Compounds
against HUVECs
b
c
actived HUVEC IC50
=
quesicent HUVEC
selective
d
compd
(nM SDs)
IC50 = (nM SDs)
ratio
colchicine
8.91 0.52
3.34 0.45
327 17.35
25.3 3.13
96.4 11.8
43.7 5.03
228 21.3
27 4.58
7.60 0.44
8.90 0.62
2975 218
5240 280
150.7 8.62
160.7 22.1
2453 225
6296 790
0.85
2.66
9.10
207
1
15
21
22
23
29
31
1.56
3.68
10.8
233
a
These procedures were modified from previously described
protocols. Antiproliferative activity was determined by CCK-8 assay.
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50% after cells were treated with compounds for 48 h.
Data are expressed as means SDs (standard deviations) from at least
b
three independent experiments For activated growth conditions,
HUVEC cells were cultured in endothelial cell medium (M&C Gene
Technology, China) containing 0.03 mg/mL endothelial cell growth
c
supplement. For quiescent growth conditions, HUVEC cells were
seeded at 15000 cells/well in endothelial cell basal medium (M&C
Gene Technology, China) containing 0.5% fetal calf serum.
d
Selectivity ratio = (IC50 quiescent)/(IC50 activated).
exhibited 207- and 233-fold selectivity (ratio) for activated
HUVECs verse quiescent HUVECs, respectively. In contrast, 1
and colchicine only exhibited 2.66- and 0.85-fold selectivity,
respectively. In addition, 15, 22, 23, and 29 showed relatively
low selectivity (9.10-, 1.56-, 3.68-, and 10.8-fold, respectively).
Capillary Formation and Disruption Assays. To further
verify the selectivity observed in 21 and 31 and their vascular-
disrupting capability, their activities on formation of capillary-
like assay (activated HUVECs engaged in) as well as capillary-
like structure disruption assay (quiescent HUVECs engaged in)
were carried out as described.11 HUVECs were plated on
Matrigel and allowed to form capillary tubes in the presence or
absence of compound from the beginning. We found that 21,
31, and reference compound colchicine inhibited the formation
of capillary tubes at 0.1 μM (Figure 2). It suggested that
compounds 21 and 31 were highly potent against activated
endothelial cells that are engaged in capillary-like structures
formation.
Subsequently, tested compounds and control was added to
capillary tubes formed by endothelial cells on Matrigel 16 h
after seeding (Figure 3).20 Cultures were photographed
immediately following compound addition and 4 h after
exposure to test compounds. Total capillary length was
determined by measuring with ImageJ software. Compared to
the total capillary length at the 0 h, the length at the 4 h time
point decreased, by 11% in the DMSO treatment control
group, 13% in the treatment of 0.1 μM 21, 10% in the
treatment of 0.1 μM 31, and 56% in the treatment of 0.1 μM
colchicine group, respectively. It showed that both 21 and 31
did not disrupt preformed capillaries at 0.1 μM, while they were
able to inhibit the formation of capillary tubes. In contrast,
colchicine not only inhibited the formation of capillary tubes
but also disrupted formed capillaries at the concentration of 0.1
μM. These results consisted with the endothelial cell
proliferation assay results, which further demonstrated that
Figure 2. Effects of tested compounds on the formation of capillary-
like structures by endothelial cells. Human umbilical vein endothelial
cells (HUVECs) were plated onto a thick layer of Matrigel in the
presence of vehicle (control) or tested compounds at the indicated
concentrations. Images were taken 22 h after plating. Magnification
×40.
compounds 21 and 31 have higher selectivity for activated
endothelial cells.
Moreover, after the addition of 1 μM of 21 and 31, there
were 49% and 51% decreases at the 4 h time point, respectively.
These results showed that both 21 and 31 have vascular-
disrupting properties.
Immunocytochemistry, Tubulin Polymerization Assay
and Cell Cycle Analysis. To confirm the mechanism of action
of these compounds, we performed immunocytochemistry,
tubulin polymerization assay, and flow cytometry analysis. First,
A549 cells were treated with two potent compounds (21 and
31) and reference compound 1, and microtubule structure was
detected using immunocytochemistry staining. As shown in
Figure 4A, the microtubule structures in control cells were slim
and fibrous. After treatment with the compounds, microtubules
became short and wrapped around the nucleus.
Subsequently, we performed a tubulin polymerization assay
as described previously.21,22 A549 cells were exposed to 1, 21,
or 31 for 6 h and then harvested in lysis buffer containing
paclitaxel, which prevents further tubulin rearrangements. The
lysates were then centrifuged to separate the polymerized
(pellet) and soluble (supernatant) tubulin. Both the pellet and
the supernatant were collected and Western blotting was
conducted to determine levels of the polymerized (pellet)
tubulin and soluble form (supernatant). As shown in parts B
and C of Figure 4, while in control cells the amount of
polymerized tubulin (46% of total tubulin) was almost equal to
D
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Effects of tested compounds on performed capillary-like structures. Control (0.1% DMSO) or tested compounds at the indicated
concentrations were added to cords formed by endothelial cells on Matrigel 16 h after seeding. Images were taken 0 and 4 h after tested compounds
addition. Magnification ×40.
the soluble form (54% of total tubulin), the cellular
polymerized tubulin decreased dramatically when cells were
treated with the tested compounds. Total tubulin values of
21%, 27% and 31% in the polymerized state were found after
cells were treated with 1, 21, and 31, respectively. These data
showed that 21 and 31 inhibited tubulin polymerization which
were similar to 1.
Last, we evaluated effects of 21 and 31 on cell cycle. A549
cells were treated with 21, 31, and 1 at indicated concentrations
for 24 h. After staining with propidium iodide, cells then were
analyzed by flow cytometry. As shown in Figure 5 and Table 4,
cells treated with 21 and 31 resulted in severe G2/M arrest
(exhibited 79% and 80% of cells arrest at the G2/M-phase,
respectively), while control cells were mainly in G1 phase.
These results demonstrate that these compounds act as
antimitotic agents.
Perfused Vascular Volume Assay in Vivo. The efficacy
of the representative compound 21 in disrupting tumor
vasculature was determined using perfused vascular volume
assay in vivo, as reported previously.6,22 The nude mice bearing
size-matched A549-derived tumors were treated with a single
administration of tested compounds (100 mg/kg) or vehicle.
After 16 h of administration, functional vascular volume of
tumors was assessed using Hoechst 33342, which acts as a
marker of blood perfusion. Perfused vessels were identified by
their fluorescent outlines, and the results for treated tumors
were expressed as a percentage of control values. Quantitative
analysis of the tumor sections showed that following treatment
with 21 and 1, functioning vascular volumes were reduced by
68% and 72%, respectively (Figure 6A,B). These data
demonstrate that 21 is a potential efficient vascular disrupting
agent in vivo.
aroyindoles and 3-benzylindoles had higher antiproliferative
activities than other series; adding a methyl group at the N-1
position in both series gave compounds 15, 21, 22, 29, and 31
and exerts a potency increase. The N1-methyl 5,6,7-
trimethoxyindole derivatives 21 and 31 showed potent
antiproliferative activities against several tumor cell lines.
Furthermore, two independent in vitro assay paradigms
confirmed that compounds 21 and 31 are far more potent
against activated HUVECs. This degree of selectivity was not
observed with colchicine and 1. In addition, 21 and 31
disrupted the network of capillary-like structures, indicating
potential vascular-disrupting activity. Immunocytochemistry
staining assay demonstrated that 21 and 31 disrupted the
microtubule structure. Tubulin polymerization assay showed
that 21 and 31 act as tubulin polymerization inhibitors. Similar
to other antitubulin agents, 21 and 31 caused arrest at G2/M
phase of the cell cycle. Finally, the representatively compound
21 significantly reduced vascular volume of A549 tumors in
vivo, which demonstrated the in vivo efficacy of 21 as a vascular
disrupting agent.
Taken together, these in vitro and in vivo data suggest that
compounds 21 and 31 may become promising leads for further
studies in terms of potential vascular-disrupting activity and
high degree of selectivity for activated endothelial cells. The
replacement of the trimethoxybenzene by N-methyl-5,6,7-
trimethoxyindole leads to a increase in the selectivity against
activated HUVECs. These results demonstrate that N-methyl-
5,6,7-trimethoxyindole could be considered to replace the 3,4,5-
trimethoxybenzene moiety of colchcine and 1, revealing
another avenue for the development of novel vascular disrupt
agents.
EXPERIMENTAL SECTION
Chemistry. All commercial chemicals and solvents are reagent
grade and were used without further treatment unless otherwise noted.
All reactions were carried out under an atmosphere of dry argon. H
■
CONCLUSION
■
1
We have synthesized several series of 5,6,7-trimethoxyindole
derivatives that mimick colchcine and 1. SARs observed for
members of these series revealed that the synthesized 3-
NMR spectra were obtained with a Varian Mercury-300BB NMR (300
MHz) and Varian Mercury Plus-400 (400 MHz) spectrometers with
E
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Effects of 21, 31, and 1 on cell cycle. A549 cells were
harvested after treatment with 0.1 μM 21, 31, and 0.01 μM 1 for 24 h.
DMSO-treated (0.1%) cells served as controls. The percentage of cells
in each phase of cell cycle was analyzed by the standard propidium
iodide procedure as described in the Experimental Section.
Figure 4. Effect of tested compounds on microtubules. (A) Control
cells (0.1% DMSO) or cells were treated for 16 h with 21 (0.05 μM),
31 (0.1 μM), or 1 (0.01 μM), then fixed and immunofluorescent
staining with an anti-β-tubulin antibody (red) and DIPA (blue, for
nuclear staining). Images were captured with Zeiss microscopy (63×
oil immersion objective). Results are representative of three separate
experiments which yielded comparable results. (B) Effect of tested
compounds on cellular tubulin polymerization. Control cells (0.1%
DMSO) or cells were treated for 6 h with indicated compounds (all at
0.1 μM), then lysed in the presence of paclitaxel and centrifuged to
separate polymerized fraction (P) from soluble fraction (S), then both
fractions were analyzed by Western blot. (C) Statistical chart of (B)
quantified by ImageJ software. The % polymerized was calculated by
dividing the β-tubulin in the polymerized fraction by the sum of the β-
tubulin in the polymerized and soluble fractions; bars, the mean SE
of at least three independent experiments.
Table 4. Cell Cycle Distribution of A549 Cell Line Treated
a
with 21, 31, and 1
% of cells in G1
phase
% of cells in S
phase
% of cells in G2/M
phase
compd
control
53.37
9.11
5.07
3.90
32.56
26.40
14.34
14.70
8.52
58.61
78.85
80.48
1
21
31
a
Cell cycle analysis was performed after treatment of cells with 21, 31,
and 1 using a FACSCalibur (BD Biosciences, Mountain View, CA).
A549 cells were treated with 0.1 μM 21 and 31, and 0.01 μM 1 for 24
h. Cell cycle distribution was analyzed by the standard propidium
iodide procedure as mentioned under the Experimental Section.
TMS as internal standard. HRESIMS data were measured on a Bruker
Daltonics APEX II 47e spectrometer. Column chromatography (CC):
YMC gel (ODS-A, 12 nm, S-50 μm, YMC Co., Kyoto, Japan); silica
gel (200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, PR
China). TLC: silica gel GF 254 (Qingdao Marine Chemical Factory,
Qingdao, PR China). Purity of the final compounds were determined
using an Waters 1525−2998 HPLC (Waters Co., USA) system using
C-18 column (Sun Fire, 5 μm, 4.6 mm × 150 mm) and were found to
be ≥95%, unless otherwise stated.
124.0, 113.8, 113.8, 106.9, 97.9, 61.1, 60.4, 56.2, 55.5. HRMS calcd for
C19H19NO5 [M + H]+, 342.1336; found, 342.1339.
(3-Hydroxy-4-methoxyphenyl)(5,6,7-trimethoxy-1H-indol-1-yl)-
methanone (7). The crude intermediate 7a was prepared as described
for 6 using 5,6,7-trimethoxy-1H-indole and 3-(benzyloxy)-4-methox-
ybenzoyl chloride. 7 was prepared as described for 21 using 7a. The
crude residue purified by column chromatography on silica gel (eluent,
petroleum/ethyl acetate 3:1) to afford compound 7 as an off-white
solid (58% yield, two steps). 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J
= 2.4 Hz, 1H), 7.42 (dd, J = 8.4 Hz, 2 Hz, 1H), 7.24 (d, J = 3.6 Hz,
1H), 6.92 (d, J = 8.4 Hz, 1H), 6.80 (s, 1H), 6.47 (d, J = 3.6 Hz, 1H),
3.97 (s, 3H), 3.92 (s, 3H), 3.91 (s, 6H), 3.89 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 166.8, 151.4, 150.5, 145.5, 142.3, 140.7, 129.1, 127.6,
127.1, 123.9, 123.6, 116.3, 110.0, 106.9, 97.9, 61.2, 60.4, 56.2, 56.1.
HRMS calcd for C19H19NO6[M + H]+, 358.1285; found, 358.1287.
(5,6,7-Trimethoxy-1H-indol-3-yl)(4-methoxyphenyl)methanone
(8). To a mixture of 5,6,7-trimethoxy-1H-indole (0.249 g, 1.2 mmol)
and anhydrous zinc chloride (0.327 g, 2.4 mmol) in dry dichloro-
methane (10 mL), ethylmagnesium bromide (0.54 mL, 3.0 M solution
in diethyl ether) was added at room temperature. At this temperature,
the reaction mixture was stirred for 1 h before the addition of 4-
methoxybenzoyl chloride (1.8 mmol) in dry dichloromethane (10
(5,6,7-Trimethoxy-1H-indol-1-yl)(4-methoxyphenyl)methanone
(6). To a solution of 5,6,7-trimethoxy-1H-indole (0.35 g, 1.69 mmol)
in THF (10 mL), potassium tert-butoxide (0.284 g, 2.54 mmol) was
added at room temperature. At this temperature, the reaction mixture
was stirred for 15 min before the addition of 4-methoxybenzoyl
chloride (2.54 mmol) in dry THF (5 mL). After stirring for another 6
h at room temperature, the reaction mixture was evaporated and then
water was added and the mixture was extracted with diethyl ether. The
combined organic extracts was dried over anhydrous MgSO4 and
concentrated in vacuo. The crude residue purified by column
chromatography on silica gel (eluent, petroleum/ethyl acetate 5:1)
1
to afford compound 6 (350 mg, 61%) as an off-white solid. H NMR
(400 MHz, CDCl3) δ 7.82 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 3.6 Hz,
1H), 6.97 (d, J = 8.8 Hz, 2H), 6.84 (s, 1H), 6.48 (d, J = 3.2 Hz, 1H),
3.92 (s, 3H), 3.89 (s, 3H), 3.88 (s, 6H). 13C NMR (100 MHz, CDCl3)
δ 166.9, 163.4, 151.3, 142.3, 140.7, 132.4, 132.4, 129.0, 127.5, 126.3,
F
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mixture was stirred for 15 min before the addition of benzenesulfonyl
chloride (0.7 mmol). After stirring for another 6 h at room
temperature, the reaction mixture was evaporated, and then water
was added and the mixture was extracted with diethyl ether. The
combined organic extracts was dried over anhydrous MgSO4 and
concentrated in vacuo. The crude residue purified by column
chromatography on silica gel (eluent, petroleum/ethyl acetate 5:1)
to afford compound 11 (64 mg, 46%) as a light-yellow solid. 1H NMR
(400 MHz, CDCl3) δ 8.20 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.87 (d, J
= 8.4 Hz, 2H), 7.63 (s, 1H), 7.58 (m, 1H), 7.49 (m, 2H), 7.03 (d, J = 2
Hz, 2H), 3.91 (s, 6H), 3.81 (s, 3H), 3.79 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 189.7, 163.2, 152.5, 141.4, 140.9, 138.8, 135.1, 133.8,
131.8, 131.3, 131.3, 129.1, 129.1, 127.4, 127.4, 125.9, 123.0, 118.9,
113.9, 113.9, 99.3, 61.1, 61.0, 56.2, 55.5. HRMS calcd for C25H23NO7S
[M + H]+, 482.1268; found, 482.1272.
(5,6,7-Trimethoxy-1H-indol-3-yl)(3-methoxyphenyl)methanone
(12). 12 was prepared as described for 8 using 5,6,7-trimethoxy-1H-
indole and 3-methoxybenzoyl chloride; off-white solid (49% yield). 1H
NMR (400 MHz, CDCl3) δ 8.65 (brs, 1H), 7.70 (s, 1H), 7.61 (d, J =
3.2 Hz, 1H), 7.39 (m, 3H), 7.10 (m, 1H), 4.10 (s, 3H), 3.97 (s, 3H),
3.92 (s, 3H), 3.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 191.0,
159.5, 151.6, 141.9, 138.8, 138.6, 132.6, 129.2, 124.6, 122.2, 121.1,
117.4, 117.3, 113.4, 98.7, 61.4, 61.2, 56.3, 55.4. HRMS calcd for
C19H19NO5 [M + H]+, 342.1336; found, 342.1327.
Figure 6. Effects of tested compounds on perfused vascular volume
assay. (A) Representative images of perfused A549 tumors vascular
(blue staining) following the administration of 21 or 1. After
intraperitoneal (ip) administration of 21 and 1 (100 mg/kg) for 16
h, the fluorescent dye Hoechst 33342 was injected iv at a dose of 10
mg/kg, then tumors were excised and frozen 1 min later, and sections
were imaged on an inverted Zeiss Imager Z2 microscope.
Magnification ×40. (B) Statistical chart of (A). Data are expressed
as a percentage of the perfusion measured in untreated tumors; bars,
the mean SE of six tumors.
(5,6,7-Trimethoxy-1H-indol-3-yl)(p-tolyl)methanone (13). 13 was
prepared as described for 8 using 5,6,7-trimethoxy-1H-indole and 4-
1
methybenzoyl chloride; off-white solid (55% yield). H NMR (400
mL). The reaction mixture was stirred for another 1 h, followed by the
addition of aluminum chloride (0.27 g, 2.03 mmol). After stirring for
another 5 h at room temperature, the reaction mixture quenched with
water (10 mL) and extracted with CH2Cl2. The combined organic
extracts was dried over anhydrous MgSO4 and concentrated in vacuo.
The crude residue purified by column chromatography on silica gel
(eluent, petroleum/ethyl acetate 2:1) to afford compound 8 (180 mg,
MHz, CDCl3) δ 8.93 (brs, 1H), 7.73 (d, J = 8 Hz, 2H), 7.70 (s, 1H),
7.56 (d, J = 2 Hz, 1H), 7.27 (d, J = 8 Hz, 2H), 4.08 (s, 3H), 3.95 (s,
3H), 3.92 (s, 3H), 2.43 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
191.2, 151.4, 141.7, 138.8, 138.6, 137.9, 132.5, 128.9, 128.8, 128.8,
124.6, 122.3, 117.3, 98.7, 61.4, 61.2, 56.3, 21.5. HRMS calcd for
C19H19NO4 [M + H]+, 326.1387; found, 326.1377.
(5,6,7-Trimethoxy-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-
methanone (14). 14 was prepared as described for 8 using 5,6,7-
trimethoxy-1H-indole and 3,4,5-trimethoxybenzoyl chloride; off-white
solid (62% yield). 1H NMR (400 MHz, CDCl3) δ 9.07 (brs, 1H), 7.67
(s, 1H), 7.63 (d, J = 2.8 Hz, 1H), 7.11 (s, 2H), 4.09 (s, 3H), 3.96 (s,
3H), 3.93 (s, 3H), 3.93 (s, 3H), 3.92 (s, 3H), 3.89 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 190.4, 152.9, 152.9, 151.5, 140.9, 138.9, 138.7,
135.9, 132.4, 124.7, 122.4, 117.0, 106.3, 106.3, 98.6, 61.4, 61.2, 61.0,
56.3, 56.2. HRMS calcd for C21H23NO7 [M + H]+, 402.1557; found,
402.1554.
1
44%) as an off-white solid. H NMR (400 MHz, CDCl3) δ 9.3 (brs,
1H), 7.82 (d, J = 8 Hz, 2H), 7.67 (s, 1H), 7.54 (d, J = 2 Hz, 1H), 6.95
(d, J = 8 Hz, 2H), 4.05 (s, 3H), 3.91 (s, 3H), 3.91 (s, 3H), 3.86 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 190.4, 162.2, 151.3, 138.7, 138.7,
133.1, 132.4, 130.8, 124.7, 122.5, 113.5, 113.5, 117.1, 98.6, 61.4, 61.1,
56.2, 55.3. HRMS calcd for C19H19NO5 [M + H]+, 342.1336; found,
342.1331.
(5,6,7-Trimethoxy-1H-indol-3-yl)(2-methoxyphenyl)methanone
(9). 9 was prepared as described for 8 using 5,6,7-trimethoxy-1H-
1
indole and 2-methoxybenzoyl chloride; white solid (41% yield). H
(3-(Benzyloxy)-4-methoxyphenyl)(5,6,7-trimethoxy-1H-indol-3-
yl)methanone (15a). 15a was prepared as described for 8 using 5,6,7-
trimethoxy-1H-indole and 3-(benzyloxy)-4-methoxybenzoyl chloride;
NMR (400 MHz, CDCl3) δ 8.99 (brs, 1H), 7.67 (s, 1H), 7.39 (m,
3H), 6.98 (m, 2H), 4.10 (s, 3H), 3.93 (s, 3H), 3.90 (s, 3H), 3.75 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 191.8, 156.7, 151.4, 138.7, 130.9,
130.7, 128.7, 124.7, 120.1, 118.8, 111.5, 98.8, 61.4, 61.2, 56.2, 55.7.
HRMS calcd for C19H19NO5 [M + H]+, 342.1336; found, 342.1328.
(1-Acetyl-5,6,7-trimethoxy-1H-indol-3-yl)(4-methoxyphenyl)-
methanone (10). To a solution of 8 (0.1 g, 0.29 mmol) in THF (5
mL), potassium tert-butoxide (0.049 g, 0.44 mmol) was added at room
temperature. At this temperature, the reaction mixture was stirred for
15 min before the addition of acetyl chloride (0.6 mmol). After stirring
for another 6 h at room temperature, the reaction mixture was
evaporated, and then water was added and the mixture was extracted
with diethyl ether. The combined organic extracts was dried over
anhydrous MgSO4 and concentrated in vacuo. The crude residue
purified by column chromatography on silica gel (eluent, petroleum/
ethyl acetate 5:1) to afford compound 10 (58 mg, 52%) as an off-white
solid. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.85 (d, J = 8.0 Hz,
2H), 7.63 (s, 1H), 6.97 (d, J = 8.0 Hz, 2H), 3.98 (s, 3H), 3.94 (s, 3H),
3.92 (s, 3H), 3.87 (s, 3H), 2.69 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 189.9, 169.5, 163.0, 152.4, 141.6, 141.4, 134.0, 131.9, 131.1, 131.1,
126.2, 123.0, 119.4, 113.7, 113.7, 99.6, 61.2, 61.2, 56.2, 55.4, 25.4.
HRMS calcd for C21H21NO6 [M + H]+, 384.1442; found, 384.1439.
(1-Benzenesulfonyl-5,6,7-trimethoxy-1H-indol-3-yl)(4-
methoxyphenyl)methanone (11). To a solution of 8 (0.1 g, 0.29
mmol) in THF (5 mL), potassium tert-butoxide (0.049 g, 0.44 mmol)
was added at room temperature. At this temperature, the reaction
1
yellow solid (57% yield). H NMR (300 MHz, CDCl3) δ 8.47 (brs,
1H), 7.65 (s, 1H), 7.5−7.3 (m, 7H), 7.21 (d, J = 2.0 Hz), 6.97 (d, J =
8.1 Hz, 1H), 5.23 (s, 2H), 4.10 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H) 3.92
(s, 3H).
(3-Hydroxy-4-methoxyphenyl)(5,6,7-trimethoxy-1H-indol-3-yl)-
methanone (15). 15 was prepared as described for 21 using 7a; off-
1
white solid (93% yield). H NMR (400 MHz, CDCl3) δ 9.42 (brs,
1H), 7.67 (s, 1H), 7.54 (t, J = 2 Hz), 7.44 (d, J = 2 Hz, 1H), 7.38 (d, J
= 8 Hz, 1H), 6.88 (s, 1H), 4.04 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 190.4, 151.3, 149.5, 145.2, 138.7, 133.8,
133.0, 132.9, 124.7, 122.7, 121.9, 116.9, 115.3, 110.0, 98.7, 61.4, 61.2,
56.3, 56.0. HRMS calcd for C19H19NO6 [M + H]+, 358.1285; found,
358.1277.
(3-Chloro-4-methoxyphenyl)(5,6,7-trimethoxy-1H-indol-3-yl)-
methanone (16). 16 was prepared as described for 8 using 5,6,7-
trimethoxy-1H-indole and 3-chloro-4-methoxy benzoyl chloride; off-
1
white solid (45% yield). H NMR (400 MHz, CDCl3) δ 8.94 (brs,
1H), 7.91 (s, 1H), 7.76 (dd, J = 8.8 Hz, J = 2.8 Hz, 1H), 7.63 (s, 1H),
7.56 (d, J = 2.8 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 4.09 (s, 3H), 3.98
(s, 3H), 3.95 (s, 3H), 3.92 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
188.9, 157.5, 151.6, 138.9, 138.7, 133.8, 132.2, 131.0, 129.0, 124.7,
122.4, 122.3, 117.0, 111.4, 98.5, 61.4, 61.3, 56.3, 56.3. HRMS calcd for
C19H18ClNO5 [M + H]+, 376.0946; found, 376.0944.
G
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(3-Iodo-4-methoxyphenyl)(5,6,7-trimethoxy-1H-indol-3-yl)-
methanone (17). 17 was prepared as described for 8 using 5,6,7-
trimethoxy-1H-indole and 3-iodo-4-methoxy benzoyl chloride; off-
121.7, 115.2, 115.0, 110.0, 99.2, 62.0, 61.3, 56.2, 56.1, 36.5. HRMS
calcd for C20H21NO6 [M + H]+, 372.1442; found, 372.1443.
(3-Fluoro-4-methoxyphenyl)(5,6,7-trimethoxy-1-methyl-1H-
indol-3-yl)methanone (22). 22 was prepared from 18 as described for
1
white solid (37% yield). H NMR (400 MHz, CDCl3) δ 9.11 (brs,
1
1H), 8.29 (d, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H),
7.62 (s, 1H), 7.56 (d, J = 3.2 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 4.07
(s, 3H), 3.95 (s, 3H), 3.94 (s, 3H), 3.91 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 188.7, 160.5, 151.5, 140.3, 138.9, 138.7, 134.9, 132.3, 130.8,
124.7, 122.4, 117.0, 110.2, 98.6, 85.5, 61.5, 61.3, 56.6, 56.4. HRMS
calcd for C19H18INO5 [M + H]+, 468.0302; found, 468.0304.
(3-Fluoro-4-methoxyphenyl)(5,6,7-trimethoxy-1H-indol-3-yl)-
methanone (18). 18 was prepared as described for 8 using 5,6,7-
trimethoxy-1H-indole and 3-fluoro-4-methoxy benzoyl chloride; off-
white solid (41% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.12 (d, J
= 2.1 Hz 1H), 7.80 (d, J = 3.2 Hz, 1H), 7.65 (dd, J = 8.4 Hz, J = 1.2
Hz, 1H), 7.60 (dd, J = 12 Hz, J = 2.0 Hz, 1H), 7.30 (t, J = 8.4 Hz, 1H),
3.97 (s, 3H), 3.93 (s, 3H), 3.84 (s, 3H), 3.79 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 187.4, 152.2, 150.5, 149.7, 139.0, 138.4, 134.2,
133.1, 125.7, 124.6, 122.5, 115.8, 115.1, 113.2, 98.5, 61.2, 60.9, 56.2,
55.9. HRMS calcd for C19H18FNO5 [M + H]+, 360.1242; found,
360.1239.
20; off-white solid (93% yield). H NMR (400 MHz, CDCl3) δ 7.73
(s, 1H), 7.57−7.62 (m, 2H), 7.35 (s, 1H), 7.03 (t, J = 8.0 Hz, 1H),
4.04 (s, 3H), 4.02 (s, 3H), 3.96 (s, 6H), 3.95 (s, 3H), 3.92 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 188.3, 153.1, 151.4, 150.3, 140.5, 139.9,
137.8, 133.8, 125.5, 124.9, 124.3, 116.6, 114.8, 112.5, 99.1, 62.0, 61.3,
56.3, 56.2, 36.6. HRMS calcd for C20H20FNO5 [M + H]+, 374.1398;
found, 374.1395.
(3-Hydroxy-4-methoxyphenyl)(5,6,7-trimethoxy-1,2-dimethyl-
1H-indol-3-yl)methanone (23). 23 was prepared as described for 21
using 23a. The crude intermediate 23a was prepared from 19a as
described for 20. The crude residue purified by column chromatog-
raphy on silica gel (eluent, petroleum/ethyl acetate 3:1) to afford
1
compound 23 as an off-white solid (82% yield, two steps). H NMR
(400 MHz, CDCl3) δ 8.40 (brs, 1H), 7.38 (d, J = 2 Hz, 1H), 7.34 (dd,
J = 8.4 Hz, 2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.89 (s, 1H), 5.68 (brs,
1H), 4.07 (s, 3H), 3.97 (s, 3H), 3.89 (s, 6H), 3.77 (s, 3H), 2.45 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 191.8, 150.3, 149.5, 145.3, 141.4,
138.0, 137.8, 134.5, 123.6, 122.8, 122.3, 115.4, 114.6, 109.8, 98.2, 61.4,
61.2, 56.3, 56.1, 14.6. HRMS calcd for C20H23NO6 [M + H]+,
386.1598; found, 386.1598.
(3-Hydroxy-4-methoxyphenyl)(5,6,7-trimethoxy-2-methyl-1H-
indol-3-yl)methanone (19). 19 was prepared as described for 21 using
19a. The crude intermediate 19a was prepared as described for 8 using
5,6,7-trimethoxy-2-methyl-1H-indole and 3-(benzyloxy)-4-methoxy-
benzoyl chloride. The crude residue purified by column chromatog-
raphy on silica gel (eluent, petroleum/ethyl acetate 3:1) to afford
1-(4-Methoxybenzyl)-5,6,7-trimethoxy-1H-indole (24). To a sol-
ution of 5,6,7-trimethoxy-1H-indole (0.03 g, 0.14 mmol) in DMF (5
mL), KOH (0.012 g, 0.214 mmol) and KI (0.024 g, 0.14 mmol) were
added at room temperature. At this temperature, the reaction mixture
was stirred for 1 h. The reaction mixture was then cooled to 0 °C and
treated with 4-methoxybenzyl bromide (0.028 g, 0.14 mmol) with
continuous stirring at 0 °C for 1 h. The reaction mixture was then
quenched with water and extracted with diethyl ether. The combined
organic extracts was dried over anhydrous MgSO4 and concentrated in
vacuo. The crude residue purified by column chromatography on silica
gel (eluent, petroleum/ethyl acetate 12:1) to afford compound 24 (21
1
compound 19 as an off-white solid (52% yield, two steps). H NMR
(400 MHz, CDCl3) δ 8.40 (brs, 1H), 7.38 (d, J = 2 Hz, 1H), 7.34 (dd,
J = 8.4 Hz, 2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.89 (s, 1H), 5.68 (brs,
1H), 4.07 (s, 3H), 3.97 (s, 3H), 3.89 (s, 6H), 3.77 (s, 3H), 2.45 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 191.8, 150.3, 149.5, 145.3, 141.4,
138.0, 137.8, 134.5, 123.6, 122.8, 122.3, 115.4, 114.6, 109.8, 98.2, 61.4,
61.2, 56.3, 56.1, 14.6. HRMS calcd for C20H21NO6 [M + H]+,
372.1442; found, 372.1445.
(5,6,7-Trimethoxy-1-methyl-1H-indol-3-yl)(4-methoxyphenyl)-
methanone (20). To a solution of 8 (0.05 g, 0.145 mmol) in THF (5
mL), potassium tert-butoxide (0.025 g, 0.22 mmol) was added at room
temperature. At this temperature, the reaction mixture was stirred for
15 min before the addition of iodomethane (0.6 mmol). After stirring
for another 6 h at room temperature, the reaction mixture was
evaporated, and then water was added and the mixture was extracted
with diethyl ether. The combined organic extracts was dried over
anhydrous MgSO4 and concentrated in vacuo. The crude residue
purified by column chromatography on silica gel to afford compound
20 (50 mg, 97%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ
7.81 (d, J = 8.4 Hz, 2H), 7.73 (s, 1H), 7.35 (s, 1H), 6.97 (d, J = 8.4
Hz, 2H), 4.03 (s, 3H), 4.00 (s, 3H), 3.96 (s, 3H), 3.92 (s, 3H), 3.92 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 189.7, 162.1, 151.2, 140.4, 139.7,
137.9, 133.6, 130.8, 130.8, 124.9, 124.4, 115.1, 113.5, 113.5, 99.1, 62.0,
61.3, 56.2, 55.4, 36.5. HRMS calcd for C20H21NO5 [M + H]+,
356.1492; found, 356.1496.
1
mg, 47%) as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.00
(d, J = 8.8 Hz, 2H), 6.96 (d, J = 3.2 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H),
6.82 (d, J = 8.4 Hz, 2H), 6.39 (d, J = 2.8 Hz, 1H), 5.48 (s, 2H), 3.89
(s, 3H), 3.88 (s, 3H), 3.76 (s, 3H), 3.74 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 158.8, 148.9, 140.5, 138.8, 131.4, 129.3, 127.7, 127.7, 125.4,
123.7, 113.9, 113.9, 101.5, 97.5, 61.3, 61.2, 56.2, 55.2, 51.2. HRMS
calcd for C19H21NO4 [M + H]+, 328.1543; found, 328.1550.
2-Methoxy-5-(5,6,7-trimethoxy-1H-indol-1-yl)methyl)phenol
(25). The crude intermediate 25a was prepared as described for 24
using 5,6,7-trimethoxy-1H-indole and 3-(benzyloxy)-4-methoxybenzyl
bromide. 25 was prepared as described for 21 using 25a. The crude
residue purified by column chromatography on silica gel (eluent,
petroleum/ethyl acetate 5:1) to afford compound 25 as an off-white
solid (35% yield, two steps). 1H NMR (400 MHz, CDCl3) δ 6.95 (d, J
= 3.2 Hz, 1H), 6.82 (s, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 1.6
Hz, 1H), 5.68 (s, 1H), 5.43 (s, 2H), 3.87 (s, 3H), 3.87 (s, 3H), 3.79 (s,
3H), 3.74 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 148.7, 145.6,
145.6, 140.3, 138.6, 132.4, 129.2, 125.2, 123.5, 117.7, 112.8, 110.5,
101.4, 97.4, 61.2, 61.1, 56.1, 55.7, 51.1. HRMS calcd for C19H21NO5
[M + H]+, 344.1492; found, 344.1497.
3-(4-Methoxybenzyl)-5,6,7-trimethoxy-1H-indole (26). To a mix-
ture of 5,6,7-trimethoxy-1H-indole (0.65 g, 3.14 mmol) and
NH4HCO3 (0.1 g, 1.29 mmol) in 20 mL 80% aqueous acetone (v/
v), 4-methoxybenzyl bromide (0.13 g, 0.65 mmol) was added at room
temperature. After stirring for 2 h at room temperature, water was
added and the mixture was extracted with diethyl ether. The combined
organic extracts were dried over anhydrous MgSO4 and concentrated
in vacuo. The crude residue purified by column chromatography on
reversed-phase silica gel (eluent, methanol/water 1:1) to afford
compound 26 (94 mg, 44%) as a colorless liquid. 1H NMR (400 MHz,
CDCl3) δ 8.0q (brs, 1H), 7.21 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.8 Hz,
2H), 6.82 (d, J = 0.8 Hz, 1H), 6.68 (s, 1H), 4.06 (s, 3H), 4.00 (s, 2H),
3.90 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 157.8, 148.9, 138.7, 138.1, 133.2, 129.5, 129.5, 124.8, 123.1, 121.9,
(3-(Benzyloxy)-4-methoxyphenyl)(5,6,7-trimethoxy-1-methyl-1H-
indol-3-yl)methanone (21a). 21a was prepared from 19a as described
1
for 20; off-white solid (98% yield). H NMR (300 MHz, CDCl3) δ
7.73 (s, 1H), 7.47−7.31 (m, 7H), 7.11 (s, 1H), 6.98 (d, J = 8.8 Hz,
1H), 5.23 (s, 2H), 4.03 (s, 3H), 3.99 (s, 3H), 3.95 (s, 6H), 3.91 (s,
3H).
(3-Hydroxy-4-methoxyphenyl)(5,6,7-trimethoxy-1-methyl-1H-
indol-3-yl)methanone (21). A mixture of 21a (0.28 g, 0.63 mmol),
ammonium formate (0.400 g, 6.3 mmol), 10% palladium on carbon
(0.1 g), and 30 mL of methanol was heated under reflux for 1 h. After
it was cooled to room temperature, the solution was filtered and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (eluent, petroleum/ethyl acetate 3:1)
to give 0.202 g (90% yield) of 21 as an off-white solid. 1H NMR (400
MHz, CDCl3) δ 7.74 (s, 1H), 7.43 (d, J = 1.6 Hz, 1H), 7.39 (d, J = 8.8
Hz, 1H), 7.38 (s, 1H), 6.92 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 4.03 (s, 3H),
3.99 (s, 3H), 3.95 (s, 6H), 3.91(s, 3H). 13C NMR (100 MHz, CDCl3)
δ 189.6, 151.2, 149.2, 145.2, 140.4, 139.7, 138.1, 134.3, 124.9, 124.4,
H
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
116.5, 113.7, 113.7, 96.0, 61.4, 61.0, 56.4, 55.2, 30.8. HRMS calcd for
C19H21NO4 [M + H]+, 328.1543; found, 328.1550.
cultured in endothelial cell medium (M&C Gene Technology, China)
supplemented with 0.03 mg/mL endothelial cell growth supplement
(M&C Gene Technology, China), 10% fetal bovine serum, 0.1 mg/mL
heparin (M&C Gene Technology, China), and penicillium/
streptomycin. HUVECs at passages 2−6 were used from for all
experiments.
3-(3,4,5-Trimethoxybenzyl)-5,6,7-trimethoxy-1H-indole (27). 27
was prepared as described for 26 using 5,6,7-trimethoxy-1H-indole and
5,6,7-trimethoxybenzyl bromide. The crude residue purified by column
chromatography on reversed-phase silica gel (eluent, methanol/water
1
Cell Proliferation Assay. Cell proliferation was determined using
CCK-8 dye (Beyotime Inst Biotech, China) according to manufac-
ture’s instructions. Briefly, A549 (3 × 103 cells/well), HT1080 (1.5 ×
103 cells/well), HeLa (1.5 × 103 cells/well), or HepG2 (3 × 103 cells/
well) were plated in 96-well plate and allowed to adhere overnight.
Activated and quiescent HUVECs proliferation assay were carried out
in 96-well plates. To assess the effects of compounds on activated
endothelial cells, HUVECs plated at 2500 cells per well and cultured in
endothelial cell medium (M&C Gene Technology, China) supple-
mented with 0.03 mg/mL endothelial cell growth supplement (M&C
Gene Technology, China) and 10% fetal bovine serum.11 To assess the
effects of compounds on quiescent endothelial cells, HUVECs were
plated at 15000 cells per well in basal medium supplemented with
0.5% fetal calf serum. After incubation with tested compounds for 48
or 72 h, 10 μL of CCK-8 dye then was add to each well, and cells were
incubated at 37 °C for 2 h. The absorbance was finally determined at
450 nm using a microplate reader (TECAN Infinite M200).
Capillary Formation and Disruption Assays. Capillary
formation assays were conducted according to the modified protocols
previously described.11 HUVEC cells were plated on a thicker Matrigel
layer (BD Biosciences, 75 μLwell−1) in a density of 25000 cells per
well in 96-well plates and incubated for 22 h. Capillary disruption
assays were carried out when capillaries were allowed to form over a 16
h period and then added the test compound or control. Images were
captured by light microscopy (40× magnification) immediately
following compound addition and 4 h after exposure to test
compound. Tube formation was quantified by measuring the total
length of capillary structures using the software WCIF ImageJ. Two
representative fields were counted in each experiment.
Immunocytochemistry. A549 cells were plate on glass coverslips
and grown in the presence or absence of tested compounds for 24 h.
Cell cultures were then rinsed twice with PBS, fixed in 4%
paraformaldehyde for 20 min at room temperature, and permeabilized
with 0.5% Triton X-100 in PBS for 10 min on ice. Fixed cells were
rinsed in PBS and blocked with 5% normal goat serum (NGS) in PBS
for at least 60 min at room temperature. After a brief wash, the cells
were incubated with the anti-β-tubulin antibodies (Sigma-Aldrich, St
Louis, Mo) (1:2000) for 2 h at room temperature, followed by
incubated with the a goat antimouse secondary Texas Red-conjugated
antibody (ZSGB−Bio, China) for 1 h at room temperature. Finally,
the cells were incubated with DAPI (ZSGB−Bio, China) prior to
mounting. Images were captured with Zeiss microscopy under the 63×
oil immersion objective.
1:1) to afford compound 27 as an colorless liquid (55% yield). H
NMR (400 MHz, CDCl3) δ 8.07 (brs, 1H), 6.84 (d, J = 2.4 Hz, 1H),
6.71 (s, 1H), 6.53 (s, 2H), 4.06 (s, 3H), 3.99 (s, 2H), 3.96 (s, 3H),
3.90 (s, 3H), 3.83 (s, 3H), 3.80 (s, 6H). 13C NMR (100 MHz, CDCl3)
δ 153.1, 153.1, 149.0, 138.8, 138.1, 136.7, 136.2, 124.9, 123.1, 122.0,
115.8, 105.8, 105.8, 95.9, 61.4, 61.0, 60.8, 56.5, 56.1, 56.1, 32.1. HRMS
calcd for C21H25NO6 [M + H]+, 388.1755; found, 388.1759.
3-(3-Fluoro-4-methoxybenzyl)-5,6,7-trimethoxy-1H-indole (28).
28 was prepared as described for 26 using 5,6,7-trimethoxy-1H-indole
and 3-fluoro-4-methoxybenzyl bromide. The crude residue purified by
column chromatography on reversed-phase silica gel (eluent,
methanol/water 6:4) to afford compound 28 as an colorless liquid
(52% yield). 1H NMR (400 MHz, CDCl3) δ 8.02 (brs, 1H), 7.00 (d, J
= 2.0 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.85
(d, J = 2.0 Hz, 1H), 6.34 (s, 1H), 4.06 (s, 3H), 3.97 (s, 2H), 3.89 (s,
3H), 3.86 (s, 3H), 3.84 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 152.3
(d, J = 244 Hz), 149.1, 145.7, 138.8, 138.1, 134.3, 124.9, 123.9, 123.0,
122.0, 116.3, 115.6, 113.3, 95.8, 61.4, 61.0, 56.5, 56.4, 30.7. HRMS
calcd for C19H20FNO4 [M + H]+, 346.1449; found, 346.1445.
2-Methoxy-5-((5,6,7-trimethoxy-1H-indol-3-yl)methyl)phenol
(29). 29 was prepared as described for 21 using 29a. The crude
intermediate 29a was prepared as described for 26 using 5,6,7-
trimethoxy-1H-indole and 3-(benzyloxy)-4-methoxybenzyl bromide.
The crude intermediate 29a purified by column chromatography on
reversed-phase silica gel (eluent, methanol/water 6:4). Transfer
hydrogenation of 29a afforded compound 29 as an colorless liquid
(51% yield, two steps). 1H NMR (400 MHz, CDCl3) δ 7.98 (brs, 1H),
6.87 (d, J = 2.4 Hz, 2H), 6.78 (s, 2H), 6.70 (s, 1H), 5.56 (s, 1H), 4.06
(s, 3H), 3.97 (s, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 148.9, 145.5, 144.8, 138.7, 138.1, 134.5,
124.9, 123.2, 121.9, 119.9, 116.2, 114.9, 110.6, 96.0, 61.4, 61.0, 56.5,
56.0, 31.0. HRMS calcd for C19H21NO5 [M + H]+, 344.1492; found,
344.1495.
3-(3-Fluoro-4-methoxybenzyl)-5,6,7-trimethoxy-1-methyl-1H-in-
dole (30). 30 was prepared as described for 20 using 29; colorless
liquid (95% yield). 1H NMR (400 MHz, CDCl3) δ 6.99 (m, 1H), 6.97
(s, 1H), 6.87 (t, J = 8.8 Hz, 1H), 6.63 (s, 1H), 6.59 (s, 1H), 4.00 (s,
3H), 3.93 (s, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.86 (s, 3H), 3.84 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 152.3 (d, J = 243 Hz), 148.5,
145.7 (d, J = 11 Hz), 140.4, 139.0, 134.5 (d, J = 6 Hz), 128.2, 124.8,
124.4, 123.9 (d, J = 3 Hz), 116.3 (d, J = 18 Hz), 113.5, 113.4 (d, J = 4
Hz), 96.0, 61.9, 61.3, 56.4, 56.4, 35.2, 30.5. HRMS calcd for
C20H22FNO4 [M + H]+, 360.1606; found, 360.1602.
2-Methoxy-5-((5,6,7-trimethoxy-1-methyl-1H-indol-3-yl)methyl)-
phenol (31). 31 was prepared as described for 21 using 31a. The
crude intermediate 31a was prepared as described for 20 using 29a.
The crude residue purified by column chromatography on silica gel
(eluent, petroleum/ethyl acetate 3:1) to afford compound 31 as an
colorless liquid (84% yield, two steps). 1H NMR (400 MHz, CDCl3) δ
6.85 (d, J = 1.2 Hz, 1H), 6.77 (s, 1H), 6.76 (s, 1H), 6.67 (s, 1H), 6.59
(s, 1H), 4.00 (s, 3H), 3.91 (s, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.85 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 148.4, 145.4, 144.8, 140.3, 138.8,
134.6, 128.2, 124.7, 124.5, 119.9, 114.9, 114.1, 110.5, 96.0, 61.9, 61.3,
56.3, 56.0, 35.1, 30.8. HRMS calcd for C20H23NO5 [M + H]+,
358.1649; found, 358.1652.
Cell Culture. Human cervix epitheloid carcinoma HeLa, and
nonsmall cell lung cancer A549 were cultured in RPMI 1640 (Gibco)
supplemented with 10% fetal bovine serum (Gibco) and penicillium/
streptomycin at 37 °C in humidified 5% CO2 incubator.
Hepatocellular carcinoma HepG2 were cultured in Dulbecco’s
Modified Essential Medium (Gibco) with the same supplements as
above. Human fibrosarcoma HT1080 were cultured in Alpha-
Minimum Essential Medium with the same supplements as above.
HUVEC were purchased from PriCells (Wuhan, China) and routinely
Tubulin Polymerization Assay. Tubulin polymerization assay
was carried out essentially as described previously with some
modifications. Briefly, A549 cells were treated with tested compounds
or vehicle (0.1% DMSO) for 6 h, then trypsinized and washed once
with PBS. Cells were then resuspended in 75 μL of lysis buffer (20
mM Tris-HCl, pH 6.8, 1 mM MgCl2, 2 mM EGTA, protease
inhibitors, 0.5% Nonidet P-40) containing 1 μg/mL paclitaxel and
incubated for 15 min at 4 °C. Lysates were then centrifuged at 13000
rpm for 15 min at 4 °C. The pellet was then resuspended in 75 μL of
lysis buffer, and both the pellet and the supernatant were added 15 μL
of 5× SDS-loading buffer. Equal aliquots of pellet and supernatant
were loaded on SDS-PAGE, and tubulin was identified with an anti-β-
tubulin primary antibody (1:1000, Sigma-Aldrich). Western blots were
quantified using the WCIF Image J software, and the error bars
correspond to standard deviations (n ≥ 3).The percentage of
polymerized was determined by dividing the β-tubulin in the
polymerized fraction by the sum of the β-tubulin in the polymerized
and soluble fractions. Given the polymerized and soluble fractions are
equalized for each pair, the proportion of the polymerized to the
soluble tubulin fraction is irrelevant to the amount of total protein
loaded for each sample.22
I
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Cell Cycle Analysis. A549 cells treated with tested compounds for
24 h were washed once in PBS and resuspended in 1 mL of 70% ice
cold ethanol and stored at −20 °C. Fixed cells were washed twice in
PBS and then treated with 1 mL of 0.1 mg/mL of RNase A solution at
37 °C for 1 h. DNA was then stained with a PBS solution containing
0.1 mg/mL propidium iodide for 30 min at room temperature in the
dark. Cell cycle analysis was determined with FACSCalibur (BD
Biosciences, Mountain View, CA).
(2) Siemann, D. W.; Chaplin, D. J.; Horsman, M. R. Vascular
targeted therapies for treatment of malignant disease. Cancer 2004,
100, 2491−2499.
(3) Tozer, G. M.; Kanthou, C.; Baguley, B. C. Disrupting tumour
blood vessels. Nature. Rev. Cancer 2005, 5, 423−435.
(4) Thorpe, P. E.; Chaplin, D. J.; Blakey, D. C. The First
International Conference on Vascular Targeting: meeting overview.
Cancer Res. 2003, 63, 1144−1147.
Perfused Vascular Volume Assay. The use of animals was
approved by Institutional Animal Care and Use Committee of
Lanzhou University, with confirmed adherence to the ethical guide
lines for the care and use of animals. Female BALB/c nu/nu mice at
6−8 weeks (Beijing HFK Bioscience, Beijing, China) were sc
implanted with 1 × 107 A549 cells mixed with equal volume of
Matrigel (Becton Dickinson) in 0.2 mL at one flank per mouse. When
their tumors reached about 100 mm3, the xenograft tumor-bearing
nude mice were given a single ip injection of 21 and 1 (dissolved in a
mixture of DMSO/cremophor/saline (1:4:15)) at a dose of 100 mg/
kg. The control group was treated with vehicle mixture only. Sixteen
hours after administration, animals were iv injected with 10 mg/kg of
Hoechst 33342 (Invitrogen). After 1 min, tumors were excised and
cryostat sections were processed. Images of histologic sections were
captured with an inverted Zeiss Imager Z2 microscope. Quantification
of H33342 Hoechst staining was done using the software WCIF
ImageJ. Twenty representative fields were counted in each tumor.
(5) (a) Baguley, B. C.; Holdaway, K. M.; Thomsen, L. L.; Zhuang, L.;
Zwi, L. J. Inhibition of growth of colon 38 adenocarcinoma by
vinblastine and colchicine: evidence for a vascular mechanism. Eur. J.
Cancer 1991, 27, 482−487. (b) Hill, S. A.; Lonergan, S. J.; Denekamp,
J.; Chaplin, D. J. Vinca alkaloids: anti-vascular effects in a murine
tumour. Eur. J. Cancer 1993, 29A, 1320−1324. (c) Chaplin, D. J.;
Pettit, G. R.; Parkins, C. S.; Hill, S. A. Antivascular approaches to solid
tumour therapy: evaluation of tubulin binding agents. Br. J. Cancer
1996, 74 (Suppl), 86−88.
(6) Dark, G. G.; Hill, S. A.; Prise, V. E.; Tozer, G. M.; Pettit, G. R.;
Chaplin, D. J. Combretastatin A-4, an agent that displays potent and
selective toxicity toward tumor vasculature. Cancer Res. 1997, 1829−
1834.
(7) Hill, S. A.; Tozer, G. M.; Chaplin, D. J. Preclinical evaluation of
the antitumour activity of the novel vascular targeting agent Oxi 4503.
Anticancer Res. 2002, 22, 1453−1458.
(8) Davis, P. D.; Dougherty, G. J.; Blakey, D. C.; Galbraith, S. M.;
Tozer, G. M.; Holder, A. L.; Naylor, M. A.; Nolan, J.; Stratford, M. R.
L.; Chaplin, D. J.; Hill, S. A. ZD6126: a novel vascular-targeting agent
that causes selective destruction of tumor vasculature. Cancer Res.
2002, 62, 7427−7253.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
The HPLC results and H NMR and 13C NMR spectra of
(9) Flynn, B. L.; Gill, G. S.; Grobelny, D. W.; Chaplin, J. H.; Paul, D.;
Lesker, A. F.; Lavranos, T. C.; Chalmers, D. K.; Charman, S. A.;
Kostewicz, E.; Shackleford, D. M.; Morizzi, J.; Hamel, E.; Jung, M. K.;
Kremmidiotis, G. Discovery of 7-Hydroxy-6-methoxy-2-methyl-3-
(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105), a tubulin poly-
merization inhibitor with potent antiproliferative and tumor vascular
disrupting properties. J. Med. Chem. 2011, 54, 6014−6027.
(10) Schwartz, E. L. Antivascular Actions of Microtubule-Binding
Drugs. Clin. Cancer Res. 2009, 15, 2594−2601.
(11) Kremmidiotis, G.; Leske, A. F.; Lavranos, T. C.; Beaumont, D.;
Gasic, J.; Hall, A.; O’Callaghan, M.; Matthews, C. A.; Flynn, B. L.
BNC105: a novel tubulin polymerization inhibitor that selectively
disrupts tumor vasculature and displays single-agent antitumor efficacy.
Mol. Cancer Ther. 2010, 9, 1562−1573.
(12) Bussolati, B.; Deambrosis, I.; Russo, S.; Deregibus, M. C.;
Camussi, G. Altered angiogenesis and survival in human tumor-derived
endothelial cells. FASEB J. 2003, 17, 1159−116.
(13) Watts, M. E.; Woodcock, M.; Arnold, S.; Chaplin, D. J. Effects of
novel and conventional anti-cancer agents on human endothelial
permeability: influence of tumour secreted factors. Anticancer Res.
1997, 17, 71−75.
target compounds 8−31. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For K.G.: phone, +86-931-8912592; fax, +86-931-8912582; E-
mail, npchem@lzu.edu.cn. For B.-R.H.: E-mail, hubr@impcas.
ac.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Qiyun Zhu from the Lanzhou Veterinary
Research Institute for the animal feeding. This work was
supported by the National Natural Science Foundation of
China (no. 20972062) and the 111 Project of China to K.G.,
and One Hundred Talent program from CAS (Y063020BRO)
to B.H.
(14) (a) Li, Q.; Sham, H. L. Discovery and development of
antimitotic agents that inhibit tubulin polymerisation for the treatment
of cancer. Expert Opin. Ther. Pat. 2002, 12, 1663−1702. (b) Jordan, A.;
Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Tubulin as a target for
anticancer drugs: agents which interact with the mitotic spindle. Med.
Res. Rev. 1998, 18, 259−296. (c) Tron, G. C.; Pirali, T.; Sorba, G.;
Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal chemistry of
combretastatin A4: present and future directions. J. Med. Chem. 2006,
49, 3033−3044. (d) Hsieh, H. P.; Liou, J. P.; Mahindroo, N.
Pharmaceutical Design of Antimitotic Agents Based on Combretas-
tatins. Curr. Pharm. Des. 2005, 11, 1655−1677. (e) Nam, N.
Combretastatin A-4 Analogues as Antimitotic Antitumor Agents.
Curr. Med. Chem. 2003, 10, 1697−1722.
(15) (a) Arthuis, M.; Pontikis, R.; Chabot, G.; Quentin, L.;
Scherman, D.; Florent, J. C. Domino approach to 2-aroyltrimethox-
yindoles as novel heterocyclic combretastatin A4 analogues. Eur. J.
Med. Chem. 2011, 46, 95−100. (b) Lai, M. J.; Kuo, C. C.; Yeh, T. K.;
Hsieh, H.-P; Chen, L. T.; Pan, W.-Y.; Hsu, K.-Y.; Chang, J. Y.; Liou, J.
P. Synthesis and Structure−Activity Relationships of 1-Benzyl-4,5,6-
ABBREVIATIONS USED
■
CA4, combretastatin A-4; HUVECs, human umbilical vein
endothelial cells; SAR, structure−activity relationship; VDAs,
vascular-disrupting agents; THF, tetrahydrofuran; DMF, N,N-
dimethylformamide; IC50, half-maximum inhibitory concen-
tration; DMSO, dimethylsulfoxide; DAPI, 4′,6-diamidino-2-
phenylindole; TMS, tetramethylsilane; HPLC, high-perform-
ance liquid chromatography; TLC, thin-layer chromatography;
PBS, phosphate buffered saline; Tris, tris(hydroxymethyl)-
aminomethane; EGTA, ethylene glycol tetraacetic acid; SDS,
sodium dodecyl sulfate; PAGE, polyacrylamide gel electro-
phoresis
REFERENCES
■
(1) Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and
other disease. Nature Med. 1995, 1, 27−31.
J
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
trimethoxyindoles as a Novel Class of Potent Antimitotic Agents.
ChemMedChem 2009, 4, 588−593. (c) Lee, H. Y.; Chang, J. Y.; Chang,
L. Y.; Lai, W. Y.; Lai, M. J.; Shih, K. H.; Kuo, C. C.; Chang, C. C.;
Liou, J. P. Concise syntheses of N-aryl-5,6,7-trimethoxyindoles as
antimitotic and vascular disrupting agents: application of the copper-
mediated Ullmann-type arylation. Org. Biomol. Chem. 2011, 9, 3154−
3157.
(16) Morin, R.; Benington, F.; Clark, L. Synthsesis of 5,6,7-
trimethoxyindole possible intermediary metabolite of mescaline. J.
Org. Chem. 1957, 22, 331−332.
(17) (a) Yang, C. X.; Patel, H. H.; Ku, Y. Y.; Shah, R.; Sawick, D. The
use of Lewis acid in the reaction of zinc salts of indoles and acyl
chloride. Synth. Commun. 1997, 27, 2125−2132. (b) Liou, J. P.; Chang,
Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C. C.; Yang, Y.
N.; Chang, J. Y.; Lee, S. J.; Hsieh, H. P. Concise synthesis and
structure−activity relationships of combretastatin A-4 analogues, 1-
aroylindoles, and 3-aroylindoles, as novel classes of potent antitubulin
agents. J. Med. Chem. 2004, 47, 4247−4257.
(18) Liou, J. P.; Mahindroo, N.; Chang, C. W.; Guo, F. M.; Lee, S.
W.; Tan, U. K.; Yeh, T. K.; Kuo, C. C.; Chang, Y. W.; Lu, P. H.; Tung,
Y. S.; Lin, K. T.; Chang, J. Y.; Hsieh, H. P. Structure−activity
relationship studies of 3-aroylindoles as potent antimitotic agents.
ChemMedChem 2006, 1, 1106−1118.
(19) Hofmann, M.; Hampel, M.; Kanzian, T.; Mayr, H. Electrophilic
alkylations in neutral aqueous or alcoholic solutions. Angew. Chem., Int.
Ed. 2004, 43, 5402−5405.
(20) Kruczynski, A.; Poli, M.; Chazottes, R.; Berrichon, G.; Ricome,
C.; Giavazzi, R.; Hill, B.; Taraboletti, G. Anti-angiogenic, vascular-
disrupting and anti-metastatic activities of vinflunine, the latest vinca
alkaloid in clinical development. Eur. J. Cancer 2006, 42, 2821−2832.
(21) (a) Minotti, A. M.; Barlow, S. B.; Cabral, F. Resistance to
antimitotic drugs in Chinese hamster ovary cells correlates with
changes in the level of polymerized tubulin. J. Biol. Chem. 1991, 266,
3987−3994. (b) Theeramunkong, S.; Caldarelli, A.; Massarotti, A.;
Aprile, S.; Caprioglio, D.; Zaninetti, R.; Teruggi, A.; Pirali, P.; Grosa,
G.; Tron, G. C.; Genazzani, A. A. Dipartime regioselective Suzuki
coupling of dihaloheteroaromatic compounds as a rapid strategy to
synthesize potent rigid combretastatin analogues. J. Med. Chem. 2011,
54, 4977−4986.
(22) Deng, Z.-T.; Peng, T.; Wang, P.; Qi, X.; Chen, X.-H.; Li, Y.-X.;
Song, C.-L.; Geng, M.-Y.; Li, J. Effects of the novel vascular targeting
agent MDS-11P on tumor vascularity and its antitumor activity.
Biochem. Pharmacol. 2011, 82, 1832−1842.
K
dx.doi.org/10.1021/jm3014663 | J. Med. Chem. XXXX, XXX, XXX−XXX