Concise synthesis and structure-activity relationships of combretastatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes of potent antitubulin agents

Article abstract of DOI:10.1021/jm049802l

The synthesis and study of the structure-activity relationships of two new classes of synthetic antitubulin compounds based on 1-aroylindole and 3-aroylindole skeletons are described. Lead compounds 3, 10, and 14 displayed potent cytotoxicities with IC

Full text of DOI:10.1021/jm049802l

J . Med. Chem. 2004, 47, 4247-4257
4247
Con cise Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of Com br eta sta tin A-4
An a logu es, 1-Ar oylin d oles a n d 3-Ar oylin d oles, a s Novel Cla sses of P oten t
An titu bu lin Agen ts
J ing-Ping Liou,† Yi-Ling Chang,‡ Fu-Ming Kuo,† Chun-Wei Chang,† Huan-Yi Tseng,‡ Chiung-Chiu Wang,†
Yung-Ning Yang,‡ J ang-Yang Chang,§ Shiow-J u Lee,*,‡ and Hsing-Pang Hsieh*,†
Medicinal Synthetic Laboratory and High Throughput Screening Laboratory, Division of Biotechnology and Pharmaceutical
Research, and Division of Cancer Research, National Health Research Institutes, Taipei 114, Taiwan, Republic of China
Received March 9, 2004
The synthesis and study of the structure-activity relationships of two new classes of synthetic
antitubulin compounds based on 1-aroylindole and 3-aroylindole skeletons are described. Lead
compounds 3, 10, and 14 displayed potent cytotoxicities with IC50 ) 0.9-26 nM against human
NUGC3 stomach, MKN45 stomach, MESSA uterine, A549 lung, and MCF-7 breast carcinoma
cell lines. The inhibition of proliferation correlated with in vitro polymerization inhibitory
activities. Structure-activity relationships revealed that 6-methoxy substitution of 3-aroylin-
doles and 5-methoxy substitution of 1-aroylindoles contribute to a significant extent for maximal
activity by mimicking the para substitution of the methoxy group to the carbonyl group in the
case of aminobenzophenones. Addition of a methyl group at the C-2 position on the indole ring
exerts an increased potency. The 3,4,5-trimethoxybenzoyl moiety was necessary for better
activity but not essential and can be replaced by 3,5-dimethoxybenzoyl and 3,4,5-trimethoxy-
benzyl moieties. We conclude that 1- and 3-aroylindoles constitute an interesting new class of
antitubulin agents with the potential to be clinically developed for cancer treatment.
In tr od u ction
(MDR) cancer cell lines. CA4P, a disodium phosphate
prodrug, is undergoing phase II clinical trials as a tumor
vascular targeting agent.3 It displays potent antitumor
effects in a wide variety of preclinical tumor models as
well as substantial antivascular activity in tumor blood
flow while causing no significant blood flow retention
in normal tissues.5 The encouraging antivascular/
anticancer activity of CA4P has stimulated significant
interest in a number of diverse ligands designed to
mimic CA4 (Chart 2).6
Microtubules are hollow tubes consisting of R- and
â-tubulin heterodimers that polymerize parallel to a
cylindrical axis. Tubulin-binding molecules interfere
with the dynamic instability of microtubules and thereby
disrupt microtubules inducing cell cycle arrest in the
M-phase, forming abnormal mitotic spindles, and finally
leading to apoptotic cell death.1 A variety of natural
compounds, such as paclitaxel, epothilone A, vinblas-
tine, combretastatin A-4 (CA4), dolastatin 10, and
colchicine, attack microtubules by interfering with the
dynamics of tubulin polymerization and depolymeriza-
tion, resulting in mitotic arrest.2 More recently, it has
been established that some tubulin binders, CA4, com-
bretastatin A-1, AC-7700, and ZD6126, selectively target
the vascular system of tumors (Chart 1).3 These agents
induce morphological changes in the endoethelial cells
of the tumor’s blood vessels so as to irreversibly shut
down the blood flow to neoplastic cells while leaving the
blood supply to healthy cells intact.
We have been actively engaged in searching novel
anticancer agents that target tubulin and have earlier
reported 2- and 3-aminobenzophenones that show strong
growth inhibitory activities against a panel of cancer
cell lines including MDR cell lines as compared to CA4
(Chart 3).7,8 Our work on aminobenzopheneones re-
vealed that the carbonyl moiety located between two
aromatic rings should be fixed as a basic motif along
with a methoxy group at the para position for maximal
cytotoxicity.7,8 An amino group in the B ring plays an
integral role for maximal cytotoxicity although its
position appears to be more flexible as compared to the
methoxy group and can be at either the C-2 or the C-3
position. Aminobenzophenones 1 and 2 strongly inhib-
ited cancer cellular growth and tubulin polymerization
and caused significant arrest of mitosis. In continuation
of our earlier work, we have designed two new series of
compounds, namely, 1-aroylindoles and 3-aroylindoles,
based on bioisosteric replacement of the olefinic linker
and the B-ring in the CA4 skeleton. We herein describe
the rationale for the design, concise synthesis, and
structure-activity relationships (SAR) of 1- and 3-aroyl-
indoles as potent antitubulin agents in continuation of
our search for promising anticancer agents.
CA4, a natural product isolated by Pettit and co-
workers in 1988 from the South African bush willow
tree Combretum caffrum, strongly inhibits the polym-
erization of tubulin by binding to the colchicine-binding
site.4 CA4 exerts a potent cytotoxicity against a variety
of human cancer cells including multidrug resistant
* To whom correspondence should be addressed. (S.-J .L.) Tel: 886-
2-2653-4401 ext. 6583. Fax: 886-2-2792-9703. E-mail: slee@nhri.org.tw.
(H.-P.H.) Tel: 886-2-2653-4401 ext. 6577. Fax: 886-2-2792-9703.
E-mail: hphsieh@nhri.org.tw.
† Medicinal Synthetic Laboratory, Division of Biotechnology and
Pharmaceutical Research.
‡ High Throughput Screening Laboratory, Division of Biotechnology
and Pharmaceutical Research.
§ Division of Cancer Research.
10.1021/jm049802l CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/17/2004

4248 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Liou et al.
Ch a r t 1
Ch a r t 2
Ch a r t 3
enamine and enamide groups by introducing 1-aroylin-
doles and 3-aroylindoles, respectively. On the other
hand, we exploited the ketoenamine and enamide
groups as bioisosteric replacement in order to examine
the effect on cytotoxicity and tubulin polymerization by
novel CA4 analogues. Furthermore, utilizing an indole
as a model structure, various commercially available
scaffolds could be exploited to optimize the activity by
manipulating the substituents on the indole ring. Most
importantly, we also envisaged that the methoxy group
at the C-6 position of 3-aroylindoles and the C-5 position
of 1-aroylindoles would mimic the methoxy group present
at the para position to the olefinic bond/carbonyl moiety
in CA4 and aminobenzophenones, respectively, thus
imparting maximum cytotoxicity (Chart 4).
Design
The cis double bond acting as a linker between the A
and the B rings of CA4 is the most important factor for
the inhibition of cancer cell growth.6a-c Many linkers
that constrain the two phenyl rings to a similar cis-
restricted conformation have been reported to increase
the activity and improve the pharmacokinetic profiles
of the designed compounds in comparison to CA4.6b This
intrigued us with an idea to design a series of heterocycle-
based CA4 analogues by adding five-membered hetero-
cycles to the B ring of the CA4 structure; i.e., to replace
the olefinic moiety and B ring of CA4 with the keto-
Thirty-two derivatives with 3-aroylindole (3-18) and
1-aroylindole (19-34) as basic skeletons were system-
atically prepared via a concise synthesis from substi-
tuted indoles for biological evaluations. The SAR were
elucidated with different substitutions at the C-2, C-4,
C-5, C-6, or C-7 position on the indole ring, a carbonyl
or methylene group at the N-1 or C-3 position, and
various substitutions on the phenyl ring (Chart 5).

Combretastatin A-4 Analogues: 1- and 3-Aroylindoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4249
Ch a r t 4
scribed above, gave 3-aroylindole 37, which was desi-
lylated with the tetrabutylammonium floride (TBAF) to
the desired 3-aroyl-6-hydroxyindole 9. Compound 9 was
further reacted with the corresponding alkyl halides in
the presence of K2CO3 in anhydrous acetone to afford
6-alkoxy-3-aroylindoles (10-12).
An alkyl group was introduced at the C-2 position of
6-methoxyindole to synthesize compound 14 as outlined
in Scheme 3. The N-sulfonylation of 6-methoxyindole
with benzylsulfonyl chloride and NaOH in the presence
of tetra-n-butylammonium bromide (TBAB) as a phase
transfer catalyst yielded sulfonamide 38. The lithiated
species of 38 with t-BuLi in tetrahydrofuran (THF) at
-20 °C reacted with CH3I to give the corresponding
2-methylindole 39, which on deprotection with aqueous
NaOH afforded 6-methoxy-2-methylindole 40. Once the
desired indole 40 was obtained, electrophilic substitu-
tion at the C-3 position was performed as described
above to afford 6-methoxy-2-methyl-3-aroylindole 14.
The general method for the synthesis of 1-aroylindoles
(19-34) is depicted in Scheme 4. The preparation
involved a one step electrophilic substitution of mono-,
di-, or trimethoxybenzoyl chlorides at the N-1 position
of various commercially available substituted indoles in
58-88% yields. The typical experiment included the
treatment of appropriate indole with NaOBut in THF
followed by stirring with the corresponding benzoyl
chloride at room temperature for 2 h to obtain the
desired 1-aroylindole. Compound 34, with a methylene
group in place of the carbonyl group, was prepared by
treating 5-methoxyindole with 3,4,5-trimethoxybenzyl
chloride in the presence of NaOBut in 72% yield.
To unequivocally confirm the molecular structures of
1- and 3-aroylindole series, 3 and 19 were subjected to
single-crystal X-ray analysis as shown in Figure 1. The
dihedral angles between indole and 3,4,5-trimethoxy-
phenyl rings for 3 and 19 are similar, 53.2 and 52.7°,
respectively. It indicates that indole and 3,4,5-tri-
methoxyphenyl rings are not coplanar as is the case in
the dihedral angles between the two phenyl rings of
CA1, which is 66.0°.10
Resu lts a n d Discu ssion
Biologica l Eva lu a tion . The two series of synthe-
sized indoles (3-34) were evaluated for their cytotoxic
activities against five human cancer cell lines, two
stomach carcinomas (NUGC3 and MKN45), one uterine
carcinoma (MESSA), one lung carcinoma (A549), and
one breast carcinoma (MCF7). The results are shown
in Tables 1 and 2.
3-Ar oylin d oles. We first evaluated the effect of the
methoxy substitution on the indole ring in the 3-aroylin-
dole series for cytotoxic activity. Compounds 3, 4, 5, and
6 with a methoxy group at the C-6, C-4, C-5, and C-7
positions, respectively, on the indole ring were evaluated
for cytotoxic activity. The SAR information indicates
that in the 3-aroylindole series a methoxy group located
at the C-6 position results in the best activity, shifting
to the C-5 or C-7 positions results in moderate activity,
while shifting to the C-4 position decreases the activity
drastically. Notably, compound 3 showed IC50 values of
1-23 nM against five different human cancer cell lines,
which was 50-500-fold more potent than compounds
4-6. Furthermore, this finding also confirms our ratio-
nale that the methoxy group should be related at the
C-6 position of the indole ring in 3-aroylindoles for the
Ch em istr y. The general method for the synthesis of
3-aroylindoles 3-8, 13, and 15-18 is shown in Scheme
1. The preparation involved a one step synthesis with
52-80% yields from di- or trimethoxy-substituted ben-
zoyl chloride and various commercially available sub-
stituted indoles. It is well-known that the indoles readily
undergo electrophilic reactions particularly at the C-3
position in the presence of Grignard reagents as base
and metal chlorides as Lewis acids.9 The typical experi-
ment included the treatment of substituted indole with
EtMgBr, ZnCl2, and AlCl3 followed by stirring with the
appropriate benzoyl chloride at room temperature for
5 h to obtain the desired 3-aroylindole. The conversion
of a carbonyl group into a methylene was achieved
through an efficient synthesis utilizing NaBH4 reduction
of 3 at room temperature to afford 18 in 95% yield.
To study the steric effect of the substituent at the C-6
position, a series of 6-alkoxy-3-aroylindoles were pre-
pared as shown in Scheme 2. Demethylation of 6-meth-
oxyindole with BBr3 followed by silyl ether protection
with TBDMSCl gave 36. Electrophilic substitution of
indole with 3,4,5-trimethoxybenzoyl chloride, as de-

4250 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Liou et al.
Ch a r t 5
Sch em e 1a
a
Reagents and conditions: (a) EtMgBr, ZnCl2, AlCl3, CH2Cl2, 52-80%. (b) NaBH4, CH3OH, 95%.
Sch em e 2a
a
Reagents and conditions: (a) BBr3, CH2Cl2, 0 °C, 72%. (b) Imidazole, TBDMSCl, THF. (c) 3,4,5-Trimethoxybenzoyl chloride, EtMgBr,
ZnCl2, AlCl3, CH2Cl2. (d) TBAF, THF, 54% from 35. (e) K2CO3, C2H5I or C3H7I, or i-C3H7Br, acetone, reflux, 72-82%.
maximal activity to mimic the carbonyl moiety and the
methoxy group located at the para position to each other
in 2- and 3-aminobenzophenones.7,8
Further investigation of electronic effects on the
indole ring revealed that 7 with an additional methoxy
group at the C-5 position, 8 with a fluoro substitution

Combretastatin A-4 Analogues: 1- and 3-Aroylindoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4251
Sch em e 3a
a
Reagents and conditions: (a) 50% NaOH, TBAB, PhSO2Cl, THF. (b) t-BuLi, CH3I, THF, -20 °C, 77% in two steps. (c) NaOH, EtOH,
reflux, 95%. (d) 3,4,5-Trimethoxybenzoyl chloride, EtMgBr, ZnCl2, AlCl3, CH2Cl2, 76%.
Sch em e 4a
a
Reagents and conditions: (a) NaOBut, THF, 58-88%. (b) NaOBut, 3,4,5-trimethoxybenzyl chloride, CH3CN, reflux, 72%.
of alkoxy substituents at the C-6 position, ethoxy-,
propyloxy-, and i-propyloxy-substituted compounds 10,
11, and 12, respectively, were prepared. Compound 10,
with an ethoxy group, retains substantial cytotoxicity
in comparison with 3, but a further increase in the
bulkiness of the substituent resulted in a slight decrease
in potency thus revealing that the steric effect of the
substitutions at the crucial C-6 position of indole ring
influences cytotoxic activities.
The introduction of a methyl group at the C-2 position
of 3-aroylindoles 5 and 3, gave 3-aroyl-2-methylindoles
13 and 14, respectively, with an apparently increased
cell growth inhibition as compared to the parent com-
pounds. The moderate activity of 5 was dramatically
increased in the case of 13 by an order of magnitude
for cytotoxicity against NUGC3, MESSA, and MCF-7
cell lines. A similar phenomenon was also observed in
the cases of 3 and 14, where th e introduction of a
methyl group at the C-2 position resulted in improve-
ment of the IC50 values to single digit nanomolar values
in all five cell lines.
A report by Cushman et al.11 that the removal of
Figu r e 1. Crystal structures of 3-aroylindole (3) and 1-aroylin-
dole (19).
either the C-4 or the C-5 methoxy group on the A ring
causes a substantial loss of cytotoxicity in the CA4
system sparked us to investigate the effect of the 3,4,5-
trimethoxybenzoyl group in the 3-aroylindole series.
Compound 15, with a 3,4-dimethoxy-benzoyl group, and
compound 17, with a benzoyl group, exhibited dramati-
cally diminished activities. A surprising observation,
however, was that 16, with a 3,5-dimethoxybenzoyl
at the C-6 position, and 9 with a hydroxyl group at the
C-6 position exhibited a drastic loss of activities. It was
also noteworthy that the more water soluble 3-aroyl-6-
hydroxyindole 9 maintained a moderate cytotoxicity due
to the weaker electron-donating ability of the hydroxyl
group. In an effort to further understand the steric effect

4252 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Ta ble 1. IC50 Values (nM ( SDa) of 3-Aroylindoles (3-18) and CA4
Liou et al.
stomach
NUGC3
stomach
MKN45
uterine
MESSA
lung
A549
breast
MCF-7
compd
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
CA4
1 ( 0
4 ( 2
487 ( 148
326 ( 84
323 ( 28
3708 ( 953
669 ( 108
43 ( 7
4 ( 1
23 ( 6
7 ( 1
3359 ( 1263
360 ( 140
474 ( 134
9393 ( 1052
884 ( 246
3524 ( 805
6499 ( 230
614 ( 282
729 ( 157
9057 ( 1633
639 ( 46
4772 ( 1697
9242 ( 1313
>10000
>10000
>10000
585 ( 180
>10000
>10000
889 ( 268
>10000
>10000
815 ( 368
728 ( 74
1 ( 0
52 ( 18
40 ( 1
50 ( 18
0.9 ( 0.1
1 ( 0
47 ( 1
41 ( 14
42 ( 5
4 ( 1
2 ( 2
3 ( 3
56 ( 15
10 ( 3
160 ( 93
4 ( 1
26 ( 21
230 ( 103
121 ( 15
596 ( 294
6 ( 2
11 ( 8
3 ( 2
111 ( 54
3 ( 2
>10000
>10000
>10000
2 ( 2
>10000
>10000
>10000
>10000
>10000
20 ( 8
1 ( 0
3939 ( 689
38 ( 4
24 ( 9
8064 ( 2037
50 ( 13
23 ( 7
8562 ( 2491
52 ( 14
>10000
6 ( 0
8 ( 1
82 ( 8
13 ( 14
117 ( 86
a
SD, standard deviation; all experiments were independently performed at least three times.
Ta ble 2. IC50 Values (nM ( SDa) of 1-Aroylindoles (19-34) and CA4
stomach
NUGC3
stomach
MKN45
uterine
MESSA
lung
A549
breast
MCF-7
compd
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
CA4
368 ( 210
34 ( 11
1687 ( 151
671 ( 326
590 ( 154
>10000
3924 ( 1219
3615 ( 859
4074 ( 1100
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
75 ( 17
>10000
9625 ( 867
>10000
>10000
>10000
>10000
>10000
2907 ( 185
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
62 ( 25
7533 ( 2932
>10000
4486 ( 1043
>10000
>10000
>10000
>10000
>10000
>10000
28 ( 28
7908 ( 1087
>10000
9429 ( 990
>10000
>10000
5050 ( 1709
>10000
39 ( 40
432 ( 101
4488 ( 528
319 ( 73
102 ( 72
>10000
>10000
9279 ( 1248
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
7610 ( 2368
649 ( 128
82 ( 8
9297 ( 1217
13 ( 15
9212 ( 1365
178 ( 140
8723 ( 2212
8 ( 1
a
SD, standard deviation; all experiments were independently performed at least three times.
group, maintained a substantial cytotoxicity against five
different cell lines except NUGC3 in comparison with
3. The replacement of the carbonyl linker (3) by a
methylene (18) resulted in a similar or slightly reduced
bioactivity, indicating that sp2 center of the carbonyl
moiety is critical but not essential in the 3-aroylindole
series.
the C-5 position in the 1-aroylindole series is as critical
for a strong activity as a methoxy group at the C-6
position in the 3-aroylindole series. Further investiga-
tion of the electronic effect revealed that the methoxy
group plays a critical role with regard to potency, as
23, 24, and 25 with cyano, fluoro, and nitro electron-
withdrawing groups, respectively, were completely inac-
tive.
In comparison with CA4, five lead compounds (3, 10,
14, 16, and 18) displayed similar or greater growth
inhibitory activities against five human cancer cell lines.
It is worthy to pinpoint that the cytotoxicities of
3-aroylindoles showed more sensitivity against stomach
carcinoma MKN45 cells as compared to other cell lines.
For instance, lesser compounds 4, 7, 8, 9, 11, 12, and
17 exhibited 3-15-fold more potent activity against
MKN45 cells than the other four human cancers cells.
1-Ar oylin d oles. As shown in Table 2, 16 1-aroylin-
doles were also evaluated for their cytotoxic activities
against five human cancer cell lines. Similar to the
study on 3-aroylindoles, we first systemically evaluated
the position of the methoxy substitution on the indole
ring in 1-aroylindoles for cytotoxic activity. Compound
19, with a methoxy group at the C-5 position, exhibited
a 10-fold stronger activity than 20, 21, and 22 with a
methoxy group at the C-4, C-6, and C-7 positions,
respectively. It is postulated that a methoxy group at
The potency improvement by adding a methyl group
at the C-2 position observed in the 3-aroylindole series
intrigued us to explore whether 1-aroylindoles also
exhibit this effect. A resemblance in the 1-aroylindole
series showed that compound 26, with an additional
methyl group as compared to 19, showed an increased
growth inhibition by an order of magnitude in all five
cell lines. However, the potency improvement in 27,
with an additional methyl group as compared to 21, was
not apparent, but interestingly, it did show a 10-fold
elevation in potency against MKN45 cells.
The unexpected strong potency of the 3,5-dimethoxy-
benzoyl group (16) inspired us to scrutinize whether a
similar tendency can be observed in 1-aroylindoles.
Replacing the 3,4,5-trimethoxybenzoyl functionality
with 3,4-dimethoxybenzoyl, 3,5-dimethoxybenzoyl, 3,4-
methylenedioxobenzoyl, 3-methoxybenzoyl, 4-methoxy-
benzoyl, or benzoyl group (28-33) yielded inactive

Combretastatin A-4 Analogues: 1- and 3-Aroylindoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4253
Ta ble 3. Inhibition of Tubulin Polymerization by 3, 14, 16, 19,
26, and CA4
out via a concise and efficient synthesis starting from
substituted indoles and that most of the active com-
pounds were prepared by a one step synthesis. Certain
1- and 3-aroylindoles 3, 10, 14, 16, 18, and 26, especially
compounds 3 (BPR0L075) and 14, have the potential
as promising anticancer agents for further studies in
terms of potent cytotoxic and antitubulin activities.12
inhibition of tubulin
compd
polymerization IC50 (µM ( SD)a
3
2.18 ( 0.16
0.90 ( 0.10
2.69 ( 0.43
3.76 ( 0.51
3.31 ( 0.09
2.03 ( 0.41
14
16
19
26
CA4
Exp er im en ta l Section
Melting points were determined on a Yanaco (MP-500D)
melting point apparatus and are uncorrected. NMR (1H NMR
and 13C NMR) spectra were obtained with a Varian Mercury-
300 spectrometer operating at 300 and 75 MHz, respectively,
with chemical shifts in parts per million (ppm, δ) downfield
from tetramethylsilane as an internal standard. High-resolu-
tion mass spectra (HRMS) were measured with a Finnigan
(MAT-95XL) electron impact (EI) mass spectrometer. Elemen-
tal analyses were performed on a Heraeus CHN-O Rapid
microanalyzer. Flash column chromatography was done using
silica gel (Merck Kieselgel 60, no. 9385, 230-400 mesh ASTM).
The reactions were monitored by thin-layer chromatography
(TLC) using Merck 60 F254 silica gel glass-backed plates (5 cm
× 10 cm); zones were detected visually under ultraviolet
irradiation (254 nm) or by spraying with phosphomolybdic acid
reagent (Aldrich) followed by heating at 80 °C. All solvents
were dried according to standard procedures. All reagents were
used as purchased without further treatment unless otherwise
stated. All reactions were carried out under an atmosphere of
dry nitrogen.
a
SD, standard deviation; all experiments were independently
performed at least three times.
compounds, except for 29, with a 3,5-dimethoxybenzoyl
group, which still showed a moderate activity against
MKN45 cells. Finally, replacing a carbonyl linker (19)
with methylene (34) resulted in a potency decrease by
10-20-fold.
Notably, the consistent tendency of the position effect
from both series fully supports our rationale that the
methoxy group at the C-6 position of 3-aroylindoles and
the C-5 position of 1-aroylindoles would mimic the
methoxy group present at the para position to the
carbonyl moiety in aminobenzophenones, thus impart-
ing maximum cytotoxicity. The addition of a methyl
substituent at the C-2 position resulted in a significant
potency increase in both series.
To investigate whether the activities of these indole
compounds were related to interactions with the micro-
tubule system, their in vitro polymerization inhibitory
activities were measured (Table 3). The results demon-
strated that the drug cytotoxicity correlated with the
inhibition of tubulin polymerization. For instance, com-
pound 14 clearly displayed a more potent cytotoxicity
as well as inhibition of GTP-induced polymerization of
MAP-rich tubulin as compared to CA4 and 3. Compound
19, which was 100-fold less potent than compounds 3
and 14 and 10-fold less potent than compounds 16 and
26, weakly inhibited tubulin polymerization. The order
of tubulin polymerization activity was 14 > CA4 ∼ 3 >
16 > 26 > 19.
Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum were purchased from HyClone, and nonessential
amino acids were purchased from Biological Industries. MTS
assay kits were obtained from Promega. Propidium iodide,
PIPES, and GTP were purchased from Sigma. MAP-rich
tubulin was purchased from Cytoskeleton Inc.
6-Meth oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (3). To
a mixture of 6-methoxyindole (0.3 g, 2.03 mmol) and anhydrous
zinc chloride (0.56 g, 4.07 mmol) in dry dichloromethane (10
mL), ethylmagnesium bromide (0.9 mL, 3.0 M solution in
diethyl ether) was added over 10 min at room temperature.
The suspension was stirred for 1 h, and then, the solution of
3,4,5-trimethoxybenzoyl chloride (0.7 g, 3.05 mmol) in dry
dichloromethane (10 mL) was added dropwise over 5 min. The
reaction mixture was stirred for another 1 h followed by the
addition of aluminum chloride (0.27 g, 2.03 mmol). The
resultant thick mixture was vigorously stirred for 5 h while
monitoring by TLC (EtOAc: n-hexane ) 1:1). The reaction was
quenched with water (10 mL) and extracted with CH2Cl2 (20
mL × 3). The combined organic layer was dried over anhydrous
MgSO4 and evaporated to give a brown oil, which was
chromatographed over silica gel (EtOAc: n-hexane ) 1:1) and
recrystallized (CH2Cl2/EtOAc) to afford compound 3 (0.5 g,
72%) as a white solid; mp 185-187 °C. 1H NMR (300 MHz,
CDCl3): δ 3.84 (s, 3H), 3.87 (s, 6H), 3.92 (s, 3H), 6.89 (d, J )
2.1 Hz, 1H), 6.96 (dd, J ) 8.9, 2.4 Hz, 1H), 7.09 (s, 2H), 7.59
(d, J ) 3 Hz, 1H), 8.23 (d, J ) 8.7 Hz, 1H), 9.10 (br, 1H, NH).
13C NMR (75 MHz, CDCl3): δ 55.8, 56.5, 61.2, 95.1, 106.4,
112.2, 116.9, 120.4, 122.9, 132.7, 135.8, 137.3, 140.8, 152.8,
157.4, 190.3. MS (EI) m/z: 341 (M+, 100%), 298 (16%), 174
(21%). HRMS (EI) for C19H19NO5 (M+): calcd, 341.1263; found,
341.1278. Anal. (C19H19NO5) C, H, N.
Con clu sion s
We have identified two new series of compounds, the
1-aroyl-5-methoxyindoles and 3-aroyl-6-methoxyindoles,
as novel classes of highly potent antitubulin agents
based on the bioisosterism concept utilizing CA4 as a
template. The lead compounds 3, 10, 14, 16, and 18
showed a 5-10-fold increase in cytotoxicity as compared
to CA4 against several human cancer cell lines. The
SAR information of the indole substitution pattern
revealed that 6-methoxy substitution of 3-aroylindoles
and 5-methoxy substitution of 1-aroylindoles contribute
to a significant extent for maximal activity by mimicking
the para substitution of the methoxy group to the
carbonyl group in the case of aminobenzophenones.
Adding a methyl group at the C-2 position in both series
exerts a potency increase. The 3,4,5-trimethoxybenzoyl
functionality can be simplified to the 3,5-dimethoxy-
benzoyl moiety without a substantial activity loss in the
3-aroylindoles. A slight lowering of the potency by
replacing the carbonyl group with the methylene group
indicates that the sp2 center of a carbonyl moiety is
critical but not essential. Another noteworthy point is
that the preparation of all of the analogues was carried
4-Meth oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (4). The
title compound was obtained as a pale yellow crystalline solid
(recrystallized from CH2Cl2/EtOAc) in 70% yield from 3,4,5-
trimethoxybenzoyl chloride and 4-methoxyindole in a similar
manner as described for the preparation of 3; mp 232-233
1
°C. H NMR (300 MHz, CDCl3): δ 3.79 (s, 3H), 3.84 (s, 6H),
3.94 (s, 3H), 6.65 (d, J ) 7.8 Hz, 1H), 7.07 (d, J ) 8.1 Hz, 1H),
7.18 (s, 2H), 7.23 (d, J ) 8.1 Hz, 1H), 7.53 (d, J ) 3.0 Hz, 1H),
8.7 (br, 1H). 13C NMR (75 MHz, CDCl3): δ 55.6, 56.5, 61.2,
100.1, 102.5, 104.6, 107.5, 116.1, 117.8, 124.8, 130.1, 135.5,
137.9, 141.4, 152.4, 154.1, 189.9. MS (EI) m/z: 341 (M+, 100%),
311 (17%), 195 (13%). HRMS (EI) for C19H19NO5 (M+): calcd,
341.1263; found, 341.1271.

4254 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Liou et al.
5-Meth oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (5). The
title compound was obtained as a pale yellow solid (recrystal-
lized from CH2Cl2/EtOAc) in 71% yield from 3,4,5-trimethoxy-
benzoyl chloride and 5-methoxyindole in a similar manner as
(d, 8.4 Hz, 1H). 13C NMR (75 MHz, CD3OD): δ 56.8, 61.2, 98.2,
107.3, 113.1, 116.9, 121.0, 123.3, 135.9, 137.3, 139.5, 141.7,
154.0, 155.5, 192.3. MS (EI) m/z: 327 (M+, 100%), 284 (16%),
161 (49%). HRMS (EI) for C18H17NO5 (M+): calcd, 327.1107;
found, 327.1087. Anal. (C18H17NO5) C, H, N.
1
described for the preparation of 3; mp 194-195 °C. H NMR
(300 MHz, CDCl3): δ 3.86 (s, 9H), 3.92 (s, 3H), 6.94 (dd, J )
9.0, 2.7 Hz, 1H), 7.08 (s, 2H), 7.32 (d, J ) 9.0 Hz, 1H), 7.66 (d,
J ) 3.3 Hz, 1H), 7.91 (d, J ) 2.7 Hz, 1H), 9.31 (br, 1H). 13C
NMR (75 MHz, CDCl3): δ 55.9, 56.5, 61.1, 103.7, 106.5, 112.5,
114.7, 116.8, 127.5, 131.5, 134.0, 136.2, 141.0, 153.2, 156.6,
190.8. MS (EI) m/z: 341 (M+, 100%), 297 (16%), 173 (21%).
6-Eth oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (10). To
a solution of 9 (0.20 g, 0.61 mmol) in acetone (15 mL),
potassium carbonate (0.17 g, 1.22 mmol) and iodoethane (0.10
mL, 1.22 mmol) were added. The reaction mixture was heated
under reflux for 12 h and quenched with cold water. The
solution was extracted with EtOAc (15 mL × 3), dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
further purified by flash chromatography (EtOAc:n-hexane )
1:1) and recrystallized (CH2Cl2/EtOAc) to yield the desired 10
HRMS (EI) for
341.1264. Anal. (C19H19NO5‚0.5H2O) C, H, N.
C
19H19NO5 (M+): calcd, 341.1263; found,
7-Meth oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (6). The
title compound was obtained as a yellow solid in 70% yield
from 3,4,5-trimethoxybenzoyl chloride and 7-methoxyindole in
a similar manner as described for the preparation of 3; mp
1
(0.18 g, 82%) as a white solid; mp 180-181 °C. H NMR (300
MHz, CDCl3): δ 1.44 (t, J ) 6.9 Hz, 3H), 3.87 (s, 6H), 3.92 (s,
3H), 4.05 (q, J ) 6.9 Hz, 2H), 6.88 (d, J ) 2.1 Hz, 1H), 6.95
(dd, J ) 8.7, 2.4 Hz, 1H), 7.06 (s, 2H), 7.58 (d, J ) 3.0 Hz,
1H), 8.21 (d, J ) 9 Hz, 1H), 8.89 (br, 1H, NH). 13C NMR (75
MHz, CDCl3): δ 15.2, 56.5, 61.2, 64.0, 95.9, 106.3, 112.7, 116.8,
120.4, 122.8, 132.8, 135.9, 137.4, 140.7, 152.8, 156.6, 190.4.
MS (EI) m/z: 355 (M+, 100%), 326 (14%). HRMS (EI) for
1
245-246 °C. H NMR (300 MHz, CDCl3): δ 3.90 (s, 6H), 3.94
(s, 3H), 3.99 (s, 3H), 6.78 (d, J ) 7.8 Hz, 1H), 7.12 (s, 2H),
7.25 (t, J ) 8.1 Hz, 1H), 7.71 (d, J ) 3.0 Hz, 1H), 7.93 (d, J )
8.1 Hz, 1H), 8.95 (br, 1H). 13C NMR (75 MHz, CDCl3): δ 55.7,
56.5, 61.2, 100.2, 103.9, 106.5, 114.9, 117.6, 123.4, 127.0, 127.8,
132.3, 136.0, 141.0, 146.0, 152.9, 190.4. MS (EI) m/z: 341 (M+,
100%), 298 (12%), 174 (28%). HRMS (EI) for C19H19NO5 (M+):
calcd, 341.1263; found, 341.1245.
C
20H21NO5 (M+): calcd, 355.1420; found, 355.1404. Anal.
(C20H21NO5) C, H, N.
6-P r opoxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in dole (11). The
title compound was obtained as a pale yellow solid in 78% yield
in a manner similar for the preparation of 10 by use of propyl
iodide; mp 141-143 °C. 1H NMR (300 MHz, CDCl3): δ 1.03
(t, J ) 7.5 Hz, 3H), 1.81 (m, J ) 7.2 Hz, 2H), 3.83 (s, 6H), 3.84
(m, 2H), 3.91 (m, 3H), 6.85 (d, J ) 2.1 Hz, 1H), 6.94 (dd, J )
8.7, 2.4 Hz, 1H), 7.07 (s, 2H), 7.57 (d, J ) 3.0 Hz, 1H), 8.21 (d,
J ) 8.7 Hz, 1H), 9.61 (s, 1H). 13C NMR (75 MHz, CDCl3): δ
11.1, 23.1, 56.6, 61.3, 70.2, 96.1, 106.5, 112.9, 116.8, 120.4,
122.9, 133.2, 136.0, 137.6, 140.8, 152.9, 156.9, 190.6. MS (EI)
m/z: 369 (M+, 100%), 370 (M+1, 21%), 326 (26%). HRMS (EI)
for C21H23NO5 (M+): calcd, 369.1576; found, 369.1591.
5,6-Dim eth oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (7).
The title compound was obtained as a pale white crystalline
(recrystallized from CH2Cl2/EtOAc) in 75% yield from 3,4,5-
trimethoxybenzoyl chloride and 5,6-dimethoxyindole in
a
similar manner as described for the preparation of 3. 1H NMR
(300 MHz, CDCl3): δ 3.89 (s, 6H), 3.92 (s, 6H), 3.98 (s, 3H),
6.93 (s, 1H), 7.11 (s, 2H), 7.59 (d, J ) 2.7 Hz, 1H), 7.91 (s,
1H), 8.72 (br, 1H). 13C NMR (75 MHz, CDCl3): δ 56.3, 56.4,
56.5, 61.1, 94.5, 103.7, 106.5, 117.1, 119.8, 130.7, 131.7, 136.1,
141.1, 147.4, 148.3, 153.1, 190.7. MS (EI) m/z: 371 (M+).
6-F lu or o-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (8). The
title compound was obtained as a pale white solid in 52% yield
from 3,4,5-trimethoxybenzoyl chloride and 6-fluoroindole in a
similar manner as described for the preparation of 3; mp 214-
215 °C. 1H NMR (300 MHz, CDCl3/CD3OD): δ 3.86 (s, 6H),
3.87 (s, 3H), 7.00 (td, J ) 9.6, 2.4 Hz, 1H), 7.04 (s, 2H), 7.11
(dd, J ) 9.3, 2.1 Hz, 1H), 7.70 (s, 1H), 8.20 (d, J ) 8.7, 5.4 Hz,
1H). MS (EI) m/z: 329 (M+, 100%), 287 (20%). HRMS (EI) for
6-Isop r op oxy-3-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (12).
The title compound was obtained as a pale white crystal in
72% yield in a manner similar to the preparation of 10 by use
of isopropyl iodide; mp 164-166 °C. 1H NMR (300 MHz,
CDCl3): δ 1.28 (d, J ) 6 Hz, 6H), 3.80 (s, 6H), 3.93 (s, 3H),
4.48 (m, 1H), 6.87 (d, J ) 2.1 Hz, 1H), 6.92 (dd, J ) 8.7, 2.1
Hz, 1H), 7.07 (s, 2H), 7.59 (d, J ) 3 Hz, 1 H), 8.21 (d, J ) 8.7
Hz, 1H), 9.75 (br, 1H). 13C NMR (75 MHz, CDCl3): δ 22.5, 56.6,
61.3, 71.0, 98.4, 106.5, 114.1, 116.7, 120.7, 122.9, 133.4, 136.0,
137.7, 140.8, 152.9, 155.5, 190.6. MS (EI) m/z: 369 (M+, 92%),
327 (100%), 284 (17%). HRMS (EI) for C21H23NO5 (M+): calcd,
369.1576; found, 369.1571.
2-Met h yl-5-m et h oxy-3-(3′,4′,5′-t r im et h oxyb en zoyl)in -
d ole (13). The title compound was obtained as a white solid
in 73% yield from 3,4,5-trimethoxybenzoyl chloride and 5-meth-
oxy-2-methylindole in a similar manner as described for the
preparation of 3; mp 157-159 °C. 1H NMR (300 MHz,
CDCl3): δ 2.54 (s, 3H), 3.73 (s, 3H), 3.86 (s, 6H), 3.94 (s, 3H),
6.83 (dd, J ) 8.7, 2.7 Hz, 1H), 7.06 (s, 2H), 7.08 (d, J ) 2.4,
1H), 7.22 (d, J ) 8.7 Hz, 1H), 8.50 (br, 1H). 13C NMR (75 MHz,
CDCl3): δ 15.0, 55.8, 56.4, 61.1, 103.1, 106.2, 107.2, 111.6,
112.2, 128.3, 129.8, 136.4, 140.6, 144.4, 152.7, 155.2, 192.1.
MS (EI) m/z: 355 (M+, 100%), 340 (35%), 308 (24%). HRMS
(EI) for C20H21NO5 (M+): calcd, 355.1420; found, 355.1442.
C
18H16FNO4 (M+): calcd, 329.1063; found, 329.1053.
6-Hyd r oxy-3-(3′,4′,5′-t r im et h oxyb en zoyl)in d ole (9). A
solution of 35 (0.5 g, 3.75 mmol) and diisopropylethylamine
(1.31 mL, 7.51 mmol) in dry N,N-dimethylformamide (10 mL)
was stirred for 10 min at room temperature. tert-Butyldi-
methylsilyl chloride (0.62 g, 4.13 mmol) was added to the
reaction solution in portions with continuous stirring. After
15 h, it was extracted with EtOAc (20 mL × 3), and the
combined organic layer was evaporated under reduced pres-
sure. The residual DMF was removed by vacuum to give the
crude silyl 36 as a pale yellow oil, which was used for the next
step without further purification.
To a mixture of 36 and anhydrous zinc chloride (1.02 g, 7.50
mmol) in dry dichloromethane (30 mL), ethylmagnesium
bromide (1.87 mL, 3.0 M solution in diethyl ether) was added
over 10 min at room temperature. The suspension was stirred
for 1 h, and then, the solution of 3,4,5-trimethoxybenzoyl
chloride (1.30 g, 5.63 mmol) in dry dichloromethane (20 mL)
was added dropwise over 5 min. The reaction mixture was
stirred for another 1 h followed by the addition of aluminum
chloride (0.6 g, 4.50 mmol). After the reaction was quenched
with water and extracted with CH2Cl2, the combined organic
layer was evaporated to give a pale yellow residue 37, which
was instantly treated with 1.0 M TBAF (4.5 mL) in THF (15
mL) for 15 h at room temperature. This stirred mixture was
evaporated, chromatographed (CH2Cl2:CH3OH ) 15:1), and
recrystallized (EtOAc/CH3OH) to afford desired 9 (0.66 g, 54%
from 35) as a white solid; mp 231-232 °C. 1H NMR (300 MHz,
CD3OD): δ 3.83 (s, 3H), 3.85 (s, 6H), 6.79 (dd, J ) 8.4, 2.1 Hz,
1H), 6.87 (d, J ) 2.1 Hz, 1H), 7.09 (s, 2H), 7.73 (s, 1H), 8.03
2-Met h yl-6-Met h oxy-3-(3′,4′,5′-t r im et h oxyb en zoyl)in -
d ole (14). The title compound was obtained as a pale white
solid in 76% yield from 3,4,5-trimethoxybenzoyl chloride and
6-methoxy-2-methylindole 40 in a similar manner as described
1
for the preparation of 3; mp 182-184 °C. H NMR (300 MHz,
CDCl3): δ 2.52 (s, 3H), 3.80 (s, 9H), 3.91 (s, 3H), 6.73 (dd, J )
8.7, 2.1 Hz, 1H), 6.79 (d, J ) 2.4 Hz, 1H), 7.05 (s, 2H), 7.35 (d,
J ) 8.7 Hz, 1H), 8.50 (br, 1H). 13C NMR (75 MHz, CDCl3): δ
14.8, 55.8, 56.5, 61.2, 94.7, 106.6, 110.7, 113.5, 121.5, 135.5,
136.1, 140.9, 142.6, 152.8, 156.3, 191.8. MS (EI) m/z: 355 (M+,
100%), 340 (24%), 306 (12%). HRMS (EI) for C20H21NO5 (M+):
calcd, 355.1420; found, 355.1438. Anal. (C20H21NO5) C, H, N.
6-Meth oxy-3-(3′,4′-d im eth oxyben zoyl)in d ole (15). The
title compound was obtained as a yellow solid in 80% yield

Combretastatin A-4 Analogues: 1- and 3-Aroylindoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4255
6.97 (s, 2H), 7.27-7.33 (m, 2H), 7.94 (d, J ) 8.1 Hz, 1H). MS
from 3,4-dimethoxybenzoyl chloride and 6-methoxyindole in
a similar manner as described for the preparation of 3; mp
155 °C. 1H NMR (300 MHz, CDCl3): δ 3.84 (s, 3H), 3.96 (s,
3H), 3.97 (s, 3H), 6.81 (dd, J ) 8.7, 2.1 Hz, 1H), 6.87 (s, 1H),
6.95 (d, J ) 8.4 Hz, 1H), 7.11 (d, J ) 1.8 Hz, 1H), 7.54-7.57
(m, 2H), 7.69 (dd, J ) 8.4, 1.8 Hz, 1H). 13C NMR (75 MHz,
CDCl3): δ 55.8, 56.4, 56.4, 93.8, 110.2, 111.8, 112.8, 113.0,
122.3, 123.8, 124.0, 131.0, 133.9, 138.9, 149.0, 152.7, 159.5,
185.1. MS (EI) m/z: 311 (M+). HRMS (EI) for C18H17NO4
(M+): calcd, 311.1158; found, 311.1140.
(EI) m/z: 341 (M+, 68%), 195 (100%). HRMS (EI) for C19H19
-
NO5 (M+): calcd, 341.1264; found, 341.1245.
6-Meth oxy-1-(3′,4′,5′-tr im eth oxyben zoyl)in dole (21). The
title compound was obtained as a white solid in 84% yield from
3,4,5-trimethoxybenzoyl chloride and 6-methoxyindole in a
similar manner as described for the preparation of 19; mp
1
128-129 °C. H NMR (300 MHz, CDCl3): δ 3.84 (s, 3H), 3.85
(s, 6H), 3.91 (s, 3H), 6.49 (d, J ) 3.6 Hz, 1H), 6.90 (dd, J )
8.4, 2.1 Hz, 1H), 6.95 (s, 2H), 7.21 (d, J ) 3.6 Hz, 1H), 7.41 (d,
J ) 8.4 Hz, 1H), 7.97 (d, J ) 2.4 Hz, 1H). 13C NMR (75 MHz,
CDCl3): δ 56.0, 56.9, 61.3, 100.5, 106.9, 108.5, 113.4, 121.3,
124.4, 126.4, 129.6, 137.0, 141.1, 153.1, 158.1, 168.3. MS (EI)
m/z: 341 (M+, 33%), 195 (100%). HRMS (EI) for C19H19NO5
(M+): calcd, 341.1263; found, 341.1248.
6-Meth oxy-3-(3′,5′-d im eth oxyben zoyl)in d ole (16). The
title compound was obtained as a white crystalline solid
(recrystallized from EtOAc) in 79% yield from 3,5-dimethoxy-
benzoyl chloride and 6-methoxyindole in a similar manner as
1
described for the preparation of 3; mp 163-164 °C. H NMR
7-Meth oxy-1-(3′,4′,5′-tr im eth oxyben zoyl)in dole (22). The
title compound was obtained as a pale yellow solid in 83% yield
from 3,4,5-trimethoxybenzoyl chloride and 7-methoxyindole in
a similar manner as described for the preparation of 19; mp
91-92 °C. 1H NMR (300 MHz, CDCl3): δ 3.67 (s, 3H), 3.73 (s,
6H), 3.86 (s, 3H), 6.51 (d, J ) 3.3 Hz, 1H), 6.69 (dd, J ) 6.3,
2.4 Hz, 1H), 7.03 (s, 2H), 7.09-7.15 (m, 2H), 7.30 (d, J ) 3.6
Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 55.8, 56.5, 61.2, 106.4,
107.4, 107.7, 113.7, 124.3, 125.9, 128.9, 129.3, 133.2, 142.1,
148.2, 152.9, 166.9. MS (EI) m/z: 341 (M+, 34%), 195 (100%).
(300 MHz, CDCl3): δ 3.84 (s, 6H), 3.87 (s, 3H), 6.64 (t, J ) 2.4
Hz, 1H), 6.90 (d, J ) 2.4 Hz, 1H), 6.97 (d, J ) 2.4 Hz, 2H),
7.00 (d, J ) 2.4 Hz, 1H), 7.62 (d, J ) 2.7 Hz, 1H), 8.31 (d, J )
9.0 Hz, 1H), 8.75 (br, 1H). 13C NMR (75 MHz, CDCl3): δ 55.6,
55.7, 94.8, 103.3, 106.5, 112.1, 116.8, 120.2, 123.0, 132.8, 137.0,
142.2, 157.2, 160.2, 190.4. MS (EI) m/z: 311 (M+, 100%), 296
(11%), 174 (50%), 148 (25%). HRMS (EI) for C18H17NO4 (M+):
calcd, 311.1157; found, 311.1143. Anal. (C18H17NO4) C, H, N.
6-Meth oxy-3-ben zoylin d ole (17). The title compound was
obtained as a pale gray solid in 53% yield from benzoyl chloride
and 6-methoxyindole in a similar manner as described for the
preparation of 3; mp 221-222 °C. 1H NMR (300 MHz,
CDCl3): δ 3.87 (s, 3H), 6.90 (d, J ) 2.1 Hz, 1H), 6.97 (dd, J )
8.7, 2.4 Hz, 1H), 7.43-7.59 (m, 4H), 7.80-7.82 (m, 2H), 8.28
(d, J ) 8.7 Hz, 1H), 8.52 (br, 1H). MS (EI) m/z: 251 (M+, 100%),
236 (24%), 175 (61%), 149 (40%). HRMS (EI) for C16H13NO2
(M+): calcd, 251.0946; found, 251.0943.
HRMS (EI) for
355.1262.
C
19H19NO5 (M+): calcd, 341.1263; found,
5-Cya n o-1-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (23). The
title compound was obtained as a pale gray solid in 85% yield
from 3,4,5-trimethoxybenzoyl chloride and 5-cyanoindole in a
similar manner as described for the preparation of 19; mp
1
167-168 °C. H NMR (300 MHz, CDCl3): δ 3.85 (s, 6H), 3.89
(s, 3H), 6.63 (d, J ) 3.6 Hz, 1H), 6.94 (s, 2H), 7.48 (d, J ) 3.6
Hz, 1H), 7.52 (d, J ) 1.5 Hz, 1H), 7.83 (s, 1H), 8.34 (d, J ) 8.4
Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 56.7, 61.3, 107.1, 108.0,
117.1, 119.6, 125.8, 127.8, 128.3, 129.8, 130.7, 137.8, 141.8,
153.2, 167.9. MS (EI) m/z: 336 (M+, 100%), 212 (2%). HRMS
(EI) for C19H16N2O4 (M+): calcd, 336.1111; found, 336.1115.
6-Met h oxy-3-(3′,4′,5′-t r im et h oxyb en zyl)in d ole (18). A
stirred solution of 3 (90 mg, 0.26 mmol) and sodium borohy-
dride (98 mg, 2.6 mmol) in ethanol (10 mL) was heated at
reflux for 12 h. The reaction was quenched with water at 0 °C
and extracted with CH2Cl2 (10 mL × 3). The combined organic
layer was dried over anhydrous MgSO4 and then evaporated
in vacuo to give a residue that was chromatographed over silica
gel (EtOAc:n-hexane ) 1:2) to afford 18 as a colorless solid in
5-F lu or o-1-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (24). The
title compound was obtained as a white solid in 62% yield from
3,4,5-trimethoxybenzoyl chloride and 5-fluoroindole in a simi-
lar manner as described for the preparation of 19; mp 106-
108 °C. 1H NMR (300 MHz, CDCl3): δ 3.88 (s, 6H), 3.93 (s,
3H), 6.57 (d, J ) 3.6 Hz, 1H), 6.95 (s, 2H), 7.09 (td, J ) 9.3,
2.7 Hz, 1H), 7.22-7.26 (m, 1H), 7.39 (d, J ) 3.9 Hz, 1H), 8.32
(dd, J ) 9.3, 4.8 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 56.4,
61.0, 106.1, 106.4, 106.6, 108.0, 112.3, 112.7, 117.1, 117.2,
128.8, 131.4, 131.5, 132.1, 141.0, 152.9, 157.8, 161.0, 167.6.
MS (EI) m/z: 329 (M+, 27%), 194 (100%). HRMS (EI) for
1
95% yield; mp 97-98 °C. H NMR (300 MHz, CDCl3): δ 3.79
(s, 9H), 3.83 (s, 3H), 4.02 (s, 2H), 6.52 (s, 2H), 6.77 (dd, J )
8.7, 2.1 Hz, 1H), 6.81 (s, 1H), 6.84 (d, J ) 2.1 Hz, 1H), 7.40 (d,
J ) 8.4 Hz, 1H), 8.03 (br, 1H). 13C NMR (75 MHz, CDCl3): δ
32.2, 55.8, 56.2, 61.0, 94.9, 105.9, 109.5, 115.6, 119.8, 121.4,
122.1, 136.3, 137.2, 137.4, 153.3, 156.7. MS (EI) m/z: 327 (M+,
100%), 279 (57%), 167 (78%). HRMS (EI) for C19H21NO4 (M+):
calcd, 355.1471; found, 355.1485. Anal. (C19H21NO4) C, H, N.
18H16FNO4 (M+): calcd, 329.1064; found, 329.1052.
5-Meth oxy-1-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (19). To
a solution of 5-methoxyindole (1 g, 6.79 mmol) in THF (30 mL),
sodium tert-butoxide (0.98 g, 10.19 mmol) was added and
stirred at room temperature for 15 min. 3,4,5-Trimethoxyben-
zoyl chloride (2.35 g, 10.19 mmol) was added to the reaction
mixture in one portion. After 15 h, the solvent was evaporated,
and the residue was extracted with CH2Cl2 (20 mL × 3). The
combined organic extracts were dried over anhydrous MgSO4
and evaporated to give a yellow oil, which was chromato-
graphed over silica gel (EtOAc:n-hexane ) 1:3) and recrystal-
lized (EtOAc/n-hexane) to afford 19 (2.03 g, 88%) as a pale
white solid; mp 98-100 °C. 1H NMR (300 MHz, CDCl3): δ 3.91
(s, 9H), 3.94 (s, 3H), 6.56 (d, J ) 3.6 Hz, 1H), 6.96 (s, 2H),
7.00 (m, 1H), 7.07 (d, J ) 2.4 Hz, 1H), 7.34 (d, J ) 3.6 Hz,
1H), 8.27 (d, J ) 9.0 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ
55.9, 56.5, 61.2, 103.6, 106.7, 108.5, 113.4, 117.1, 128.1, 129.4,
130.6, 131.6, 141.0, 153.0, 156.5, 167.7. MS (EI) m/z: 341 (M+,
14%), 195 (100%). HRMS (EI) for C19H19NO5 (M+): calcd,
341.1263; found, 341.1259. Anal. (C19H19NO5) C, H, N.
C
5-Nitr o-1-(3′,4′,5′-tr im eth oxyben zoyl)in d ole (25). The
title compound was obtained as a yellow solid in 58% yield
from 3,4,5-trimethoxybenzoyl chloride and 5-nitroindole in a
similar manner as described for the preparation of 19; mp
1
192-193 °C. H NMR (300 MHz, CDCl3): δ 3.90 (s, 6H), 3.95
(s, 3H), 6.77 (d, J ) 3.6 Hz, 1H), 6.98 (s, 2H), 7.56 (d, J ) 3.9
Hz, 1H), 8.26 (dd, J ) 9.0, 3.0 Hz, 1H), 8.43 (d, J ) 9.0 Hz,
1H), 8.53 (d, J ) 2.1 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ
56.5, 61.1, 106.9, 108.5, 116.2, 117.0, 119.9, 127.8, 130.1, 130.3,
138.8, 141.7, 144.1, 153.0, 167.6. MS (EI) m/z: 356 (M+, 28%),
195 (100%). HRMS (EI) for C18H16N2O6 (M+): calcd, 356.1008;
found, 356.1026.
2-Met h yl-5-m et h oxy-1-(3′,4′,5′-t r im et h oxyb en zoyl)in -
d ole (26). The title compound was obtained as a white solid
in 81% yield from 3,4,5-trimethoxybenzoyl chloride and 5-meth-
oxy-2-methylindole in a similar manner as described for the
1
preparation of 19; mp 97-98 °C. H NMR (300 MHz, CDCl3):
δ 2.41 (s, 3H), 3.80 (s, 9H), 3.93 (s, 3H), 6.34 (t, J ) 0.9 Hz,
1H), 6.65 (dd, J ) 9.0, 2.4 Hz, 1H), 6.92 (d, J ) 2.7 Hz, 1H),
6.95 (s, 2H), 6.97 (d, J ) 8.7 Hz, 1H). 13C NMR (75 MHz,
CDCl3): δ 15.9, 55.8, 56.5, 61.3, 102.6, 107.4, 108.8, 114.4,
115.3, 130.5, 132.0, 138.9, 142.3, 153.4, 156.0, 169.2. MS (EI)
m/z: 355 (M+). HRMS (EI) for C20H21NO5 (M+): calcd, 355.1420;
found, 355.1392. Anal. (C20H21NO5) C, H, N.
4-Meth oxy-1-(3′,4′,5′-tr im eth oxyben zoyl)in dole (20). The
title compound was obtained as a white solid in 83% yield from
3,4,5-trimethoxybenzoyl chloride and 4-methoxyindole in a
similar manner as described for the preparation of 19; mp
1
130-131 °C. H NMR (300 MHz, CDCl3): δ 3.89 (s, 6H), 3.94
(s, 3H), 3.97 (s, 3H), 6.74 (s, 1H), 6.76 (d, J ) 3.6 Hz, 1H),

4256 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Liou et al.
2-Met h yl-6-Met h oxy-1-(3′,4′,5′-t r im et h oxyb en zoyl)in -
d ole (27). The title compound was obtained as a pale white
solid in 80% yield from 3,4,5-trimethoxybenzoyl chloride and
6-methoxy-2-methylindole 40 in a similar manner as described
for the preparation of 19; mp 121-122 °C. 1H NMR (300 MHz,
CDCl3): δ 2.34 (s, 3H), 3.68 (s, 3H), 3.84 (s, 6H), 3.94 (s, 3H),
6.34 (d, J ) 0.9 Hz, 1H), 6.79 (d, J ) 2.4 Hz, 1H), 6.82 (s, 1H),
6.96 (s, 2H), 7.33 (dd, J ) 7.8, 1.2 Hz, 1H). 13C NMR (75 MHz,
CDCl3): δ 16.2, 55.9, 56.7, 61.4, 99.4, 107.3, 108.5, 111.6, 120.2,
123.4, 130.4, 136.3, 138.1, 142.1, 153.3, 156.6, 169.4. MS (EI)
m/z: 355 (M+, 100%), 226 (10%). HRMS (EI) for C20H21NO5
(M+): calcd, 355.1420; found, 355.1395.
(dd, J ) 8.7, 2.7 Hz, 1H), 7.04 (d, J ) 2.1 Hz, 1H), 7.24 (d, J
) 3.6 Hz, 1H), 7.47-7.72 (m, 5H), 8.29 (d, J ) 9.0 Hz, 1H).
MS (EI) m/z: 251 (M+, 31%), 122 (61%), 105 (100%). HRMS
(EI) for C16H13NO2 (M+): calcd, 251.0946; found, 251.0925.
5-Meth oxy-1-(3′,4′,5′-tr im eth oxyben zyl)in d ole (34). To
a stirred solution of 5-methoxyindole (0.5 g, 3.39 mmol) and
sodium tert-butoxide (0.65 g, 6.79 mmol) in anhydrous aceto-
nitrile (15 mL), the solution of 3,4,5-trimethoxybenzyl chloride
(0.96 g, 4.42 mmol) in anhydrous acetonitrile (15 mL) was
added dropwise at room temperature. The reaction mixture
was heated to reflux for 12 h. The solvent was evaporated,
and the residue was extracted with EtOAc (20 mL × 3). The
combined organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo to give a residue, which was further
purified by flash column over silica gel (EtOAc:n-hexane ) 1:3)
to provide pure 34 (0.8 g, 72%) as a white solid; mp 100-102
5-Meth oxy-1-(3′,4′-d im eth oxyben zoyl)in d ole (28). The
title compound was obtained as a pale white solid in 83% yield
from 3,4-dimethoxybenzoyl chloride and 5-methoxyindole in
a similar manner as described for the preparatitbon of 19; mp
1
1
153-154 °C. H NMR (300 MHz, CDCl3): δ 3.87 (s, 3H), 3.92
°C. H NMR (300 MHz, CDCl3): δ 3.73 (s, 6H), 3.79 (s, 3H),
(s, 3H), 3.96 (s, 3H), 6.54 (d, J ) 3.9 Hz, 1H), 6.94 (d, J ) 9.3
Hz, 1H), 6.98 (d, J ) 2.7 Hz, 1H), 7.05 (d, J ) 2.4 Hz, 1H),
7.31-7.35 (m, 2H), 8.23 (d, J ) 8.7 Hz, 1H). 13C NMR (75 MHz,
CDCl3): δ 55.9, 55.4, 103.0, 107.7, 109.9, 111.9, 112.9, 116.6,
122.9, 116.6, 122.9, 126.1, 127.9, 130.3, 131.2, 148.4, 151.7,
156.0, 167.2. MS (EI) m/z: 311 (M+, 76%), 165 (100%). HRMS
(EI) for C18H17NO4 (M+): calcd, 311.1157; found, 311.1154.
3.83 (s, 3H), 5.19 (s, 2H), 6.30 (s, 2H), 6.45 (dd, J ) 3.0, 0.6
Hz, 1H), 6.82 (dd, J ) 8.7, 2.4 Hz, 1H), 7.08 (m, 2H), 7.16 (d,
J ) 8.7 Hz). 13C NMR (75 MHz, CDCl3): δ 50.5, 55.8, 56.1,
60.8, 101.1, 102.4, 103.6, 110.2, 111.8, 128.4, 128.8, 131.4,
132.9, 137.0, 153.1, 153.7. MS (EI) m/z: 327 (M+, 100%), 284
(17%). HRMS (EI) for C19H21NO4 (M+): calcd, 327.1470; found,
327.1458.
5-Meth oxy-1-(3′,5′-d im eth oxyben zoyl)in d ole (29). The
title compound was obtained as a white solid in 86% yield from
3,5-dimethoxybenzoyl chloride and 5-methoxyindole in a simi-
lar manner as described for the preparation of 19; mp 95-97
6-Hyd r oxyin d ole (35). To a stirred solution of 6-methoxy-
indole (1 g, 6.79 mmol) in dry dichloromethane (30 mL), boron
tribromide (3.4 g, 13.60 mmol) was added dropwise at 0 °C.
The reaction mixture was warmed to room temperature. After
15 h, the reaction was quenched with water at 0 °C, followed
by extraction with CH2Cl2 (20 mL × 3). The combined extracts
were dried over anhydrous MgSO4. The solvent was evaporated
in vacuo, and the residue was chromatographed over silica gel
(CH2Cl2:CH3OH ) 10:1) to provide 35 (0.65 g, 72%) as a pale
1
°C. H NMR (300 MHz, CDCl3): δ 3.82 (s, 6H), 3.87 (s, 3H),
6.52 (dd, J ) 3.6, 0.6 Hz, 1H), 6.64 (t, J ) 2.4 Hz, 1H), 6.81 (d,
J ) 2.4 Hz, 2H), 6.97 (dd, J ) 9.0, 2.4 Hz, 1H), 7.04 (d, J )
2.1 Hz, 1H), 7.29 (d, J ) 3.6 Hz, 1H), 8.29 (d, J ) 9.0 Hz, 1H).
13C NMR (75 MHz, CDCl3): δ 55.70, 55.75, 103.4, 103.7, 106.7,
108.4, 113.2, 117.0, 128.0, 130.4, 131.6, 136.1, 156.2, 160.4,
167.8. MS (EI) m/z: 312 (M+1, 48%), 164 (100%). HRMS (EI)
for C18H17NO4 (M+): calcd, 311.1157; found, 311.1126.
1
yellow solid. H NMR (300 MHz, CD3OD): δ 6.30 (d, J ) 3.0
Hz, 1H), 6.57 (dd, J ) 8.4, 2.1 Hz, 1H), 6.79 (s, 1H), 7.03 (d, J
) 3.3 Hz, 1H), 7.31 (d, J ) 8.4 Hz, 1H).
5-Meth oxy-1-(3′,4′-m eth ylen ed ioxyben zoyl)in d ole (30).
The title compound was obtained as a pale yellow solid in 73%
yield from 3,4-(methylenedioxy)benzoyl chloride and 5-meth-
oxyindole in a similar manner as described for the preparation
1-Ben zen esu lfon yl-2-m eth yl-6-m eth oxylin d ole (39). A
suspension of 6-methoxyindole (3 g, 20.38 mmol), TBAB (0.7
g, 2.03 mmol), and 50% sodium hydroxide (25 mL) in THF (30
mL) and water (10 mL) was stirred vigorously for 20 min at
room temperature. The solution of benzenesulfonyl chloride
(5.2 mL, 40.76 mmol) in THF (20 mL) was added dropwise to
the reaction mixture. After 4 h, the reaction was extracted with
EtOAc (20 mL × 3). The combined organic layer was dried
over anhydrous MgSO4 and concentrated under vacuum to give
the residue 38, which was redissolved in THF (50 mL) at -20
∼ -30 °C and treated with a solution of tert-butyllithium (1.7
M in pentane, 16 mL, 26.5 mmol) dropwise. The resultant
mixture was continuously stirred at -30 °C for 30 min and
allowed to warm to 0 °C and stirred for another 20 min. The
reaction mixture was again cooled to -30 °C before the
addition of iodomethane (3.8 mL, 61.1 mmol) dropwise. After
continuous stirring at -30 °C for another 1 h, it was allowed
to warm to room temperature overnight. The solvent was
removed under reduced pressure. The residue was dissolved
in CH2Cl2 and washed with saturated NaHCO3. The aqueous
layer was extracted with CH2Cl2 (20 mL × 3). The combined
organic layer was dried over anhydrous MgSO4 and purified
by flash chromatography over silica gel (EtOAc:n-hexane )
1:8) to provide 39 (4.6 g, 77% from 6-methoxyindole) as a pale
1
of 19; mp 139-141 °C. H NMR (300 MHz, CDCl3): δ 3.84 (s,
3H), 6.04 (s, 2H), 6.51 (d, J ) 3.9 Hz, 1H), 6.87 (d, J ) 8.1 Hz,
1H), 6.95 (dd, J ) 9.0, 2.4 Hz, 1H), 7.02 (d, J ) 2.4 Hz, 1H),
7.19-7.26 (m, 2H), 7.29 (d, J ) 3.6 Hz, 1H), 8.23 (d, J ) 9.0
Hz, 1H). MS (EI) m/z: 295 (M+, 29%), 149 (100%). HRMS (EI)
for C17H13NO4 (M+): calcd, 295.0845; found, 295.0838.
5-Meth oxy-1-(3′-m eth oxyben zoyl)in d ole (31). The title
compound was obtained as an oil in 84% yield from 3-meth-
oxybenzoyl chloride and 5-methoxyindole in a similar manner
as described for the preparation of 19. 1H NMR (300 MHz,
CDCl3): δ 3.84 (s, 3H), 3.86 (s, 3H), 6.53 (d, J ) 3.6 Hz, 1H),
6.99 (dd, J ) 9.0, 2.7 Hz, 1H), 7.09-7.13 (m, 1H), 7.24-7.28
(m, 3H), 7.40 (t, J ) 8.1 Hz, 1H), 8.33 (d, J ) 9.0 Hz, 1H). 13C
NMR (75 MHz, CDCl3): δ 55.8, 56.0, 103.7, 108.7, 113.5, 114.2,
117.4, 118.0, 121.4, 128.4, 129.7, 130.7, 131.9, 135.8, 156.7,
159.6, 168.1. MS (EI) m/z: 281 (M+, 38%), 152 (75%), 147
(19%), 135 (100%). HRMS (EI) for C17H15NO3 (M+): calcd,
281.1052; found, 281.1061.
5-Meth oxy-1-(4′-m eth oxyben zoyl)in d ole (32). The title
compound was obtained as white solid in 81% yield from
4-methoxybenzoyl chloride and 5-methoxyindole in a similar
manner as described for the preparation of 19; mp 106-108
1
yellow solid. H NMR (300 MHz, CDCl3): δ 2.55 (s, 3H), 3.87
(s, 3H), 6.26 (s, 1H), 6.85 (dd, J ) 8.7, 2.4 Hz, 1H), 7.26 (d, J
) 8.7 Hz, 1H), 7.41 (m, 2H), 7.52 (m, 1H), 7.77 (m, 3H). 13C
NMR (75 MHz, CDCl3): δ 16.1, 56.0, 99.6, 109.6, 112.4, 120.5,
123.6, 126.3, 129.4, 133.8, 136.1, 138.1, 139.3, 157.4. MS (EI)
m/z: 301 (M+).
1
°C. H NMR (300 MHz, CDCl3): δ 3.80 (s, 3H), 3.82 (s, 3H),
6.49 (d, J ) 3.6 Hz, 1H), 6.92-6.96 (m, 3H), 7.01 (d, J ) 2.7
Hz, 1H), 7.27 (d, J ) 3.6 Hz, 1H), 7.66 (d, J ) 8.7 Hz, 1H). 13
C
NMR (75 MHz, CDCl3): δ 55.5, 55.7, 103.3, 107.8, 113.1, 113.6,
116.8, 126.3, 128.1, 130.6, 131.3, 156.2, 162.2, 167.5. MS (EI)
m/z: 281 (M+, 22%), 135 (100%). HRMS (EI) for C17H15NO3
(M+): calcd, 281.1052; found, 281.1066.
2-Meth yl-6-m eth oxyin d ole (40). A stirred solution of 39
(0.44 g, 1.46 mmol) and 3 M sodium hydroxide (14.6 mL, 43.8
mmol) in ethyl alcohol (30 mL) was heated at reflux for 16 h.
The solution was evaporated, and the residue was extracted
with CH2Cl2 (15 mL × 3). The combined organic layer was
dried over anhydrous MgSO4 and concentrated in vacuo. The
residue was purified by flash column over silica gel (EtOAc:
n-hexane ) 1:7) to afford 40 (0.22 g, 95%) as a pale gray solid.
5-Meth oxy-1-ben zoylin d ole (33). The title compound was
obtained as a pale white solid in 75% yield from benzoyl
chloride and 5-methoxyindole in a similar manner as described
for the preparation of 19; mp 104-107 °C. 1H NMR (300 MHz,
CDCl3): δ 3.87 (s, 3H), 6.53 (dd, J ) 3.9, 0.9 Hz, 1H), 6.97

Combretastatin A-4 Analogues: 1- and 3-Aroylindoles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4257
1H NMR (300 MHz, CDCl3): δ 2.36 (s, 3H), 3.82 (s, 3H), 6.15
(s, 1H), 6.70 (d, J ) 2.1 Hz, 1H), 6.78 (dd, J ) 8.4, 2.1 Hz,
1H), 7.40 (d, J ) 8.7 Hz, 1H), 7.78 (br, 1H). 13C NMR (75 MHz,
CDCl3): δ 13.9, 56.0, 94.8, 100.0, 109.2, 120.2, 123.4, 134.2,
136.9, 155.6. MS (EI) m/z: 161 (M+).
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seeded in 96 well plates and incubated in a CO2 incubator at
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Ack n ow led gm en t. We thank Dr. Chun-Chen Liao
of National Tsing Hua University for his valuable
suggestions concerning the manuscript. We are also
grateful to the National Health Research Institutes,
Taiwan, Republic of China, for financial support.
Su p p or tin g In for m a tion Ava ila ble: Characterization
(1H NMR, 13C NMR) of 3-40. This material is available free
of charge via the Internet at http://pubs.acs.org.
Refer en ces
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