Domino approach to 2-aroyltrimethoxyindoles as novel heterocyclic combretastatin A4 analogues

Article abstract of DOI:10.1016/j.ejmech.2010.10.018

Two series of 2-aroyltrimethoxyindoles were designed to investigate the effects of the replacement of the trimethoxyphenyl ring of phenstatin with a trimethoxyindole moiety. These compounds were efficiently prepared through a domino palladium-catalyzed se

Full text of DOI:10.1016/j.ejmech.2010.10.018

European Journal of Medicinal Chemistry 46 (2011) 95e100
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Domino approach to 2-aroyltrimethoxyindoles as novel heterocyclic
combretastatin A4 analogues
,
,
b
,
c
,
,
,d
Martin Arthuis a b, Renée Pontikis a , Guy G. Chabot d, Lionel Quentin c d, Daniel Scherman c
,
*
Jean-Claude Florent a
,b,
*
a Institut Curie, Centre de Recherche, 26 rue d’Ulm, Paris F-75248 cedex 05, France
b CNRS, UMR 176, 26 rue d’Ulm, Paris F-75248 cedex 05, France
c Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’Observatoire, 75006 Paris, France
d Laboratoire de Pharmacologie Chimique, Génétique et Imagerie, INSERM U 1022-CNRS UMR 8151, 4 avenue de l’Observatoire, 75006 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of 2-aroyltrimethoxyindoles were designed to investigate the effects of the replacement of
the trimethoxyphenyl ring of phenstatin with a trimethoxyindole moiety. These compounds were effi-
ciently prepared through a domino palladium-catalyzed sequence from 2-gem-dibromovinylanilines
substituted by three methoxy groups and arylboronic acids under carbon monoxide atmosphere. These
novel heterocyclic combretastatin A4 analogues were evaluated for their cell growth inhibitory prop-
erties and their ability to inhibit the tubulin polymerization.
Received 13 July 2010
Received in revised form
15 October 2010
Accepted 18 October 2010
Available online 10 November 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Combretastatin
Phenstatin
Aroylindole
Tubulin
Cytotoxicity
1. Introduction
The encouraging antivascular and antitumour profile of CA4 has
greatly contributed to the current interest in the design and
Combretastatin A4 (CA4), a natural product isolated by Pettit
et al. from the South African willow tree Combretum caffrum,
strongly inhibits tubulin polymerization and the proliferation of
murine and human cancer cell lines [1]. By interfering with
microtubule dynamics, CA4 was shown to perturb several cell
signalling pathways involved in regulating and maintaining the
cytoskeleton of proliferating endothelial cells in tumour vascula-
ture [2]. As a consequence of this cytoskeleton disruption, CA4
causes a rapid tumour vasculature shutdown leading to central
tumour necrosis, while leaving normal vasculature intact [3].
synthesis of several CA4 analogues [4]. Through SAR studies, it has
been established that the cis orientation of both phenyl groups, and
the 3,4,5-trimethoxy system on the A-ring, are essential require-
ments for the interaction of combretastatin-type analogues with
the tubulin colchicine site and the inhibition of its assembly into
microtubules. Therefore, the olefinic linker and the B-ring of CA4
have received greater attention from medicinal chemists. The cis
double bond in the stilbene framework of CA4 was, for example,
efficiently replaced by a carbonyl group leading to benzophenone
derivatives, such as in phenstatin 1 or as in the 1,2 or 3-aroylindoles
2, which demonstrated strong antiproliferative and antitubulin
activities [5e7] (Fig. 1).
MeO
A
However, it is interesting to note that replacement of the tri-
methoxyphenyl ring by benzoheterocyclic structures has received
little attention so far [8]. We thus decided to explore the possibility
to replace the trimethoxyphenyl ring of phenstatin with a trime-
thoxyindole skeleton, by synthesizing 2-aroylindole derivatives
with general structures 3 and 4 (Fig. 1). Our goal was to determine,
for the benzophenone-type CA4 analogues, the influence of a more
hindered A-ring bearing three methoxy groups in different posi-
tions on the antiproliferative and antitubulin activities.
MeO
B
MeO
OH
OMe
Combretastatin A-4 (CA4).
* Corresponding authors. Institut Curie, Centre de Recherche, 26 rue d’Ulm, Paris
F-75248 cedex 05, France.
E-mail address: renee.pontikis@curie.fr (R. Pontikis).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.018

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M. Arthuis et al. / European Journal of Medicinal Chemistry 46 (2011) 95e100
Coupling reactions were next performed with arylboronic acids
O
O
bearing substituents known to favour interactions with tubulin:
a para-methoxy group [16] and, in place of the hydroxyl function of
CA4, a fluorine [16] or an amino group [17,18]. In agreement with
the pharmacophoric model established by Nguyen et al. [19], the
influence of a methyl group in the para-position was also examined,
as the hydrophobic center is considered as an essential feature for
activity. Thus, the target ketones 3be3d were obtained in 59, 73, 55
and 45% yields, respectively. It is noteworthy that the domino
reaction may be carried out with a boronic acid bearing an amino
reactive group giving aniline 3e in 45% yield. Introduction of
a pyridine ring, which might generate additional interactions with
tubulin amino acids, was also effective, albeit in modest yield, and
after an increased reaction time of 60 h (compound 3f, 45% yield).
To explore the influence of the substitution pattern of the indole
nucleus, domino reactions were performed with the regioisomeric
4,5,6-trimethoxyaniline 6. Under the same conditions, coupling
with phenylboronic acid generated the ketone 4a albeit in modest
yield (47%). Significant yield improvement was obtained by
increasing the reaction time to 60 h (61% yield). Therefore, reac-
tions of the aniline 6 with the five boronic partners previously used,
were conducted for 60 h, affording the desired aroylindoles 4be4f
in yields ranging from 46 to 63%.
MeO
MeO
OH
MeO
MeO
NH
OMe
MeO
MeO
MeO
MeO
R
1
2
MeO
MeO
O
MeO
MeO
O
N
H
N
H
R
3
4
R
Fig. 1. Structures of phenstatin, representative and designed aroylindole derivatives.
2. Chemistry
Relatively few methods for the synthesis of the 2-aroylindole
frameworks have been reported. The most common synthetic route
involves addition of a variety of acyl electrophiles on a N-protected-
2-lithioindole species [9]. However, this method is limited because
it requires a “de novo” construction of conveniently N-protected
indoles [10,11]. Alternatively, a 5-exo-dig iodocyclization of conve-
niently tethered dimethylanilines afforded 2-aroyl-N-methyl-
indoles [12], and some 2-acylindoles have been synthesized using
a Suzuki-type coupling reaction between the indole-2-carboxylic
acid chloride and boronic acids [13]. For our part, the synthesis of
the target 2-aroylindoles 3 and 4 was investigated according to our
reported procedure (Scheme 1), through a domino palladium-
catalyzed reaction involving a 2-gem-dibromovinylaniline and
a boronic acid under a pressure of carbon monoxide [14].
The required trimethoxyanilines 5 [14] and 6 were easily
prepared in a two-step sequence (Ramirez olefination followed by
reduction of the nitro group) from the o-nitrobenzaldehyde
derivatives 7 and 8 [15]. The domino reaction was first conducted
with the dibromoolefine 5 and phenylboronic acid under the
previously optimized conditions [Pd(PPh3)4, K2CO3, CO (12 bar),
dioxane]. By heating at 85 ꢀC for 24 h, the desired 2-aroylindole 3a
could be isolated in satisfactory yield (65%), opening the possibility
to get a novel series of CA4 analogues (Scheme 2).
Phenylvinylboronic acid also proved to be an efficient partner of
the domino reaction with anilines 5 and 6, affording the enones 9
and 10 in satisfactory yields (61 and 49%, respectively).
3. Biological results and discussion
The synthesized aroylindoles were evaluated (Table 1) for their
in vitro cytotoxicity against the murine B16 melanoma cell line
(MTT assay), and also for their ability to inhibit tubulin polymeri-
zation (fluorometric assay) [20], using CA4 as the reference
compound. Within the 2-aroyl-4,5,6-trimethoxyindole series,
compounds 3c and 3d presented a modest cytotoxic effect (IC50: 16
and 10
inhibition, only compound 3d, bearing a methyl group at the
4-position of the B-ring, displayed a fair activity (IC50: 4 M) but
mM, respectively). With regard to the tubulin polymerization
m
less than that of CA4. Within the subset of 5,6,7-trimethoxyindole
analogues, cytotoxic activity was nearly abolished except for the
p-toluyl derivative 4d (IC50: 10
mM), for which cytotoxicity and
antitubulin activity were not correlated, contrary to its isomeric
analogue 3d. The enones 9 and 10, designed as analogues of a dia-
rylbutadienic derivative that we have previously reported as
a potent tubulin polymerization inhibitor [21], were devoid of any
appreciable activity.
Selected compounds were next evaluated for their cell growth
inhibitory potency against three human colon adenocarcinoma cell
lines, HCT-116, the P-glycoprotein (P-gp)-expressing HCT-15 and
Br
O
R2 B(OH)2
CO, [Pd]
R1
R1
Br
NH2
R2
N
H
Scheme 1. Palladium-catalyzed 2-aroylindoles synthesis.
B(OH)2
R1
R1
R2
R1
R2
X
Br
MeO
MeO
CHO
NO2
MeO
MeO
MeO
MeO
O
i, ii
R3
CO (12 bar), iii
Br
NH2
N
H
X
R2
7,8
R3
5,6
3a-f,4a-f
7, 5, 3a-f: R1 = OMe, R2 = H
8, 6, 4a-f: R1 = H , R2 = OMe
X and R3 exemplified in Table 1
Scheme 2. Reagents and conditions: (i) CBr4 (1.5 equiv.), PPh3 (3 equiv.), CH2Cl2, 0 ꢀC to rt, 1 h; (ii) SnCl2.2H2O (5.5 equiv.), EtOH, reflux, 1.5 h; (iii) Pd(PPh3)4 (5 mol%), K2CO3
(5 equiv.), dioxane, 85 ꢀC, 24 h for adducts 3ae3e, 60 h for adducts 3f, 4ae4f.

M. Arthuis et al. / European Journal of Medicinal Chemistry 46 (2011) 95e100
97
Table 1
through a palladium-catalyzed domino reaction which proved to be
an efficient and convergent synthetic route to highly substituted 2-
aroylindoles.
Cytotoxicity and tubulin polymerization inhibition of synthesized aroylindole
derivatives.
R1
R1
With regard to the interaction with tubulin, this study indicates
that the 4,5,6-trimethoxy substitution pattern on the indole ring
and a small hydrophobic group in the para-position of the B-ring
are appropriate structural features, and therefore allows the iden-
tification of compound 3d as a frame for further investigations in
the search of novel tubulin polymerization inhibitors. This specific
trimethoxy motif is also critical for cytotoxicity, while some flexi-
bility was tolerated for the B-ring.
MeO
MeO
MeO
MeO
O
O
N
H
N
H
X
R2
R2
R3
3a-f,4a-f
9, 10
3a-f, 9: R1 = OMe, R2 = H
4a-f, 10: R1 = H , R2 = OMe
It is noteworthy that the A-ring modulation studied here, has
also proven beneficial in a series of derivatives with a methylene
bridge, the 1-benzyl-4,5,6-trimethoxyindoles, recently reported as
a novel class of potent antimitotic agents [23]. Therefore, we are
presently taking these data and our own results into account to
design novel and more potent CA4 analogues.
Compd
X
R3
H
IC50
(
m
M)
Compd
X
R3
IC50
B16a ITPb
>50
(mM)
B16a
ITPb
3a
3b
3c
3d
3e
3f
CH
CH
CeF
CH
CeNH2 Me
N
45
>20
9
>20
4
10
15
4a
4b
4c
4d
4e
4f
CH
CH
CeF
CH
CeNH2 Me
N
H
9
OMe >50
OMe
Me
OMe >50 12
OMe >50 20
Me
16
10
36
10
nt
>20
5. Experimental
c
nt c
OMe >50
16
0.005
OMe >50 18
33 18
5.1. Chemistry
9
CA4
e
e
>20
0.27
10
e
e
a
Dichloromethane was dried over P2O5, dioxane over Na/benzo-
phenone. Unless otherwise noted, all the materials were obtained
from commercial sources and were used without purification.
Aniline 5 and compound 3a were prepared as previously reported
[14]. 1H NMR spectra were recorded with a Bruker ACP 300 spec-
trometer at 300 MHz for 1H NMR and 75 MHz for 13C NMR spec-
Compound concentration required to reduce murine B16 melanoma cell
viability by 50% (48 h exposure time).
b
Compound concentration required to inhibit tubulin polymerization (ITP) by
50%. In vitro microtubule assembly was monitored using a fluorescent probe (DAPI)
[20].
c
nt, not tested.
troscopy. Chemical shifts are given as d values in ppm relative to the
residual solvent peak (CHCl3) as the internal reference, coupling
constants are given in Hertz. All 13C NMR spectra were recorded
with complete proton decoupling. Peak assignment was unambig-
uously performed using HMQC, HMBC and NOESY techniques. IR
spectra were obtained using an FT-IR PerkineElmer spectrometer.
Melting points were determined by the capillary method using an
Electrothermal 9200 apparatus and are uncorrected. Mass spectra
were recorded on a Waters ZQ 2000 system using electrospray
ionization (ESI). High-resolution mass spectra (ESI) were acquired
from the “imaGIF” service (CNRS-ICSN, 91198 Gif sur Yvette, France)
on a Waters LCT spectrometer. Reactions were followed with Merck
TLC silica gel 60 F254. Flash chromatographies were carried out on
Merck silica gel (320e400 mesh).
the CA4-refractory HT-29 [22] cells (Table 2). The 4,5,6-trime-
thoxyindole derivatives 3b, 3d and 3e showed similar activity
against HCT-116 and HT-29 cells (IC50: 1e4
mM), whereas CA4 was
ten-fold less potent on the HT-29 cell line (0.01 vs 0.001
mM).
Surprisingly, these compounds presented significant activity on the
HCT-15 cell line with IC50 in the submicromolar range. Neverthe-
less, the antiproliferative effects for these three compounds were
not in good agreement with their antitubulin activities, suggesting
that tubulin may not be their single target. In this series, compound
3e with an amino group displayed the most potent antiproliferative
activity (IC50: 0.3e2.0
mM). As previously observed, the 5,6,7-tri-
methoxyindole derivatives 4a and 4d were found inactive on the
three colon cancer cell lines (IC50 > 50 mM).
5.1.1. 2-(2,2-Dibromovinyl)-4,5,6-trimethoxyaniline (6)
Aniline 6, prepared from 2-nitro-3,4,5-trimethoxybenzaldehyde
[24] (12 g, 50 mmol) following the procedure reported by Fang and
Lautens [15], was obtained after purification by flash chromatog-
raphy (cyclohexane/ethyl acetate 9/1) as a beige solid (70% over the
2 steps): mp 87e88 ꢀC, nmax(CH2Cl2)/cmꢁ1 3452, 3376, 3052, 2939,
1588, 1490, 1464, 1355, 1198, 1128, 1077; dH (300 MHz; CDCl3) 7.33
(1H, s), 6.76 (1H, s), 3.90 (3H, s), 3.89 (3H, s), 3.80 (3H, s), 3.75 (2H,
br s); dC (75 MHz; CDCl3) 145.4, 143.1, 133.3, 132.4, 116.2, 107.8, 91.5,
60.9, 60.5, 56.6; m/z (ESI) 388 (50%), 389 (100), 392 (40): [M þ Na]þ;
HRMS (ESI) m/z 365.9358 [M þ H]þ, C11H14NO739Br2 requires
365.9340.
Table 2
Antiproliferative effects of selected trimethoxyaroylindoles against human colon
carcinoma cell lines.
Compd
IC50 (m
M)a
HCT-116
HCT-15
HT-29
3b
3d
3e
4a
4d
CA4
4.2
2.8
2.0
>50
>50
0.001
0.45
0.61
0.32
>50
>50
1.24
1.09
0.99
>50
>50
0.001
0.013
a
Compound concentration required to inhibit human tumoral cell proliferation
by 50% after 72 h incubation.
5.1.2. General procedure for the domino reactions
The autoclave and the magnetic stirring bar were dried in an
oven and then cool to room temperature under an argon atmo-
sphere. Boronic acid (1.1 mmol), K2CO3 (5 mmol, flame dried prior
to use) and Pd(PPh3)4 (0.05 mmol) were introduced then the
autoclave was flushed with argon for 5 min. A degassed solution
(argon bubbling for 10 min) of aniline 5 or 6 (1 mmol) in dry
dioxane (10 mL) was added and the autoclave was flushed three
times with CO and pressurized to 12 bar.
4. Conclusions
The present study was undertaken to explore the possibility to
replace the usual trimethoxyphenyl ring present in a large majority
of CA4 analogues with a more bulky moiety, a trimethoxyindole
ring. Thus, several 2-aroyltrimethoxyindoles have been prepared

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M. Arthuis et al. / European Journal of Medicinal Chemistry 46 (2011) 95e100
After heating at 85 ꢀC in an oil bath for the appropriate time
(75 MHz; CDCl3) 186.1, 154.9, 147.1, 144.7, 137.1, 136.3, 135.0, 133.3,
130.4, 127.0, 119.8, 116,1, 115.0, 110.7, 89.0, 61.5, 61.0, 56.1, 17.6; m/z
(ESI) 341 [M þ H]þ; HRMS (ESI) m/z 363.1329 [M þ Na]þ,
C19H20N2O4Na requires 363.1321.
(24 h for adducts 3ae3e, 60 h for adducts 3f, 4ae4f, 9 and 10), the
autoclave was cooled to room temperature and then cautionary
discharged of the gas excess. Reaction mixture was diluted in ethyl
acetate (10 mL) and washed with water (10 mL), saturated aqueous
NH4Cl (10 mL) and brine (10 mL). The aqueous layers were
combined, saturated with NaCl, acidified (by adding HCl 1 M until
pH ¼ 2) and extracted with ethyl acetate (2 ꢂ 20 mL). Organic layers
were combined, dried over MgSO4, filtered and concentrated under
reduce pressure. The crude residue was purified by flash chroma-
tography and crystallized in the indicated solvents to give the
attempted compounds.
5.1.2.5. (4,5,6-Trimethoxy-1H-indol-2-yl)(6-methoxypyridin-3-yl)
methanone (3f). Compound 3f was isolated after chromatography
(cyclohexane/ethyl acetate 7/3) and recrystallization (dichloro-
methane/hexane) as yellow crystals in 48% yield: mp 161e162 ꢀC;
nmax(CH2Cl2)/cmꢁ1 3442, 3307, 3058, 2939, 2834, 2571, 2359, 1617,
1601, 1519, 1492, 1466, 1372, 1272, 1257, 1147, 1108, 1016; dH
(300 MHz; CDCl3) 9.35 (1H, br s), 8.87 (1H, dd, J 2.4 and 0.6), 8.17
(1H, dd, J 8.6 and 2.4), 7.22 (1H, dd, J 2.2 and 0.8), 6.86 (1H, dd, J 8.6
and 0.6), 6.62 (1H, d, J 0.8), 4.11 (3H, s), 4.05 (3H, s), 3.92 (3H, s), 3.87
(3H, s); dC (75 MHz; CDCl3) 183.2, 166.3, 155.4, 149.0, 147.1, 139.3,
136.4, 135.2, 132.9, 127.6, 116.3, 111.1, 110.8, 88.8, 61.5, 61.0, 56.2,
54.1; m/z (ESI) 343 [M þ H]þ; HRMS (ESI) m/z 343.1300 [M þ H]þ,
C18H19N2O5 requires 343.1294.
Caution: CO is a highly toxic odorless and colorless gas. Reactions
involving Carbon Monoxide must be performed in a well-ventilated
hood with a Carbon Monoxide detector nearby.
5.1.2.1. (4,5,6-Trimethoxy-1H-indol-2-yl)(4-methoxyphenyl)metha-
none (3b). Compound 3b was isolated after chromatography
(toluene/ethyl acetate 9/1) and recrystallization (dichloromethane/
hexane) as
a
yellow crystalline powder in 55% yield: mp
5.1.2.6. (5,6,7-Trimethoxy-1H-indol-2-yl)(phenyl)methanone
(4a). Compound 4a was isolated after chromatography (toluene/
ethyl acetate 9/1) and recrystallization (dichloromethane/hexane)
as yellow crystals in 61% yield: mp 179e180 ꢀC; nmax(CH2Cl2)/cmꢁ1
3436, 2940, 2839, 1626, 1530, 1493, 1302, 1253, 1230, 1124, 1107; dH
(300 MHz; CDCl3) 9.30 (1H, br s), 7.95 (2H, dt, J 7.1 and 1.4), 7.59 (1H,
tt, J 7.5 and 1.4), 7.51 (2H, ddt, J 7.5, 7.1 and 1.4), 7.03 (1H, d, J 2.3),
6.82 (1H, s), 4.08 (3H, s), 3.95 (3H, s), 3.89 (3H, s); dC (75 MHz;
CDCl3) 186.5, 150.4, 141.6, 139.0, 138.2, 134.3, 132.2, 129.1, 128.4,
127.6, 123.4, 112.8, 98.0, 61.5, 61.2, 56.2; m/z (ESI) 312 [M þ H]þ;
HRMS (ESI) m/z 334.1067 [M þ Na]þ, C18H17NO4Na requires
334.1055.
162e163 ꢀC; nmax(CH2Cl2)/cmꢁ1 3443, 3056, 2935, 1619, 1570, 1500,
1466, 1375, 1272, 1264, 1256, 1170, 1143, 1108, 1032; dH (300 MHz;
CDCl3) 9.40 (1H, br s), 8.02 (2H, d, J 6.8), 7.18 (1H, dd, J 2.2 and 0.8, 3-
H), 7.02 (2H, d, J 6.8), 6.64 (1H, d, J3,7 0.8, 7-H), 4.11 (3H, s), 3.91 (6H,
s), 3.87 (3H, s); dC (75 MHz; CDCl3) 184.9 (CO), 163.0, 154.9, 147.0,
136.2, 134.9, 133.2, 131.3, 130.8, 116.1, 113.7, 110.2 (CH-3), 88.9 (CH-
7), 61.5, 61.0, 56.2, 55.5; m/z (ESI) 342 [M þ H]þ; HRMS (ESI) m/z
342.1352 [M þ H]þ, C19H20NO5 requires 342.1341.
5.1.2.2. (3-Fluoro-4-methoxyphenyl)(4,5,6-trimethoxy-1H-indol-2-
yl)methanone (3c). Compound 3c was isolated after chromatog-
raphy (toluene/ethyl acetate 8/2 to 7/3) and recrystallization
(ethanol/heptane) as yellow crystals in 73% yield: mp 211e212 ꢀC;
nmax(CH2Cl2)/cmꢁ1 3443, 3054, 2986, 1621, 1574, 1513, 1501, 1276,
1260, 1121; dH (300 MHz; CDCl3) 9.26 (1H, br s), 7.82 (1H, ddd, J 8.4,
2.0 and 1.2), 7.75 (1H, dd, J 11.6 and 2.0), 7.19 (1H, dd, J 2.2 and 0.8),
7.07 (1H, t, J 8.4), 6.62 (1H, d, J 0.8), 4.11 (3H, s), 3.99 (3H, s), 3.93
(3H, s), 3.87 (3H, s); dC (75 MHz; CDCl3) 183.7, 155.3, 151.9 (J 246),
151.3 (J 10),147.1,136.0,135.1,132.8,131.0 (J 5.3),126.3 (J 3.6),116.9 (J
19.3), 116.3, 112,5, 110.5, 88.9, 61.5, 61.0, 56.3, 56.2; m/z (ESI) 360
[M þ H]þ; HRMS (ESI) m/z 360.1256 [M þ H]þ, C19H19FNO5 requires
360.1247.
5.1.2.7. (5,6,7-Trimethoxy-1H-indol-2-yl)(4-methoxyphenyl)meth-
anone (4b). Procedure on 5 mmol scale. Compound 4b was isolated
after flash chromatography (cyclohexane/ethyl acetate 8/2) and
recrystallization (dichloromethane/heptane) as yellow crystals
(1.0 g) in 59% yield: mp 154e155 ꢀC; nmax(CH2Cl2)/cmꢁ1 3684, 3436,
2939, 2840, 1623, 1602, 1529, 1491, 1464, 1302, 1254, 1170, 1124,
1108; dH (300 MHz; CDCl3) 9.28 (1H, br s), 7.99 (2H, dt, J 8.8 and 2.7),
7.02 (1H, s, 3-H), 7.00 (2H, dt, J 8.9 and 2.1), 6.84 (1H, s, 4-H), 4.08
(3H, s), 3,94 (3H, s), 3.91 (3H, s), 3.90 (3H, s); dC (75 MHz; CDCl3)
185.1 (CO), 163.1, 150.3, 141.3, 139.0, 134.4, 131.4, 130.8, 127.2, 123.4,
113.7, 111.9 (CH-3), 98.0 (CH-4), 61.5, 61.2, 56.3, 55.5; m/z (ESI) 342
[M þ H]þ; HRMS (ESI) m/z 342.1353 [M þ H]þ, C19H20NO5 requires
342.1341.
5.1.2.3. (4,5,6-Trimethoxy-1H-indol-2-yl)(p-tolyl)methanone
(3d). Compound 3d was isolated after chromatography (toluene/
ethyl acetate 95/5) and recrystallization (dichloromethane/hexane)
as bright yellow needles in 55% yield: mp 190e191 ꢀC;
nmax(CH2Cl2)/cmꢁ1 3443, 3063, 2961, 2931, 2860, 2359, 2341, 1722,
1619, 1519, 1499, 1466, 1376, 1272, 1141, 1108; dH (300 MHz; CDCl3)
9.41 (1H, br s), 7.89 (2H, d, J 8.1), 7.32 (2H, d, J 8.1), 7.19 (1H, dd, J 2.2
and 0.9), 6.64 (1H, d, J 0.8), 4.10 (3H, s), 3.91 (3H, s), 3.86 (3H, s), 2.46
(3H, s); dC (75 MHz; CDCl3) 185.1, 155.1, 147.1, 142.8, 136.3, 135.5,
135.1, 133.2, 129.2, 129.1, 116.2, 110.8, 88.9, 61.5, 61.0, 56.1, 21.6; m/z
(ESI) 326 [M þ H]þ; HRMS (ESI) m/z 326.1403 [M þ H]þ, C19H20NO4
requires 326.1392.
5.1.2.8. (3-Fluoro-4-methoxyphenyl)-(5,6,7-trimethoxy-1H-indol-2-
yl)-methanone (4c). Compound 4c was isolated after chromatog-
raphy (toluene/ethyl acetate 9/1) and recrystallization (chloroform/
heptane) as
a yellow crystalline powder in 46% yield: mp
192e193 ꢀC; nmax(CH2Cl2)/cmꢁ1 3540, 3006, 2939, 2842, 1624, 1610,
1578,1530,1517,1530,1517,1490,1464,1428,1302,1279,1229,1132,
1103; dH (300 MHz; CDCl3) 9.26 (1H, br s), 7.80 (1H, ddd, J 8.4, 2.0
and 0.9), 7.76 (1H, dd, J 11.6 and 2.0), 7.07 (1H, t, J 8.2), 7.04 (1H, d, J
2.1), 6.84 (1H, s), 4.08 (3H, s), 3.99 (3H, s), 3.95 (3H, s), 3.90 (3H, s);
dC (75 MHz; CDCl3) 183.9 (J 2), 151.9 (J 246), 151.3 (J 10.3), 150.4,
141.5, 139.0, 133.9, 131.0 (J 5.4), 127.5, 126.3 (J 3.3), 123.4, 117.1 (J
19.9), 112.5 (J 3.3), 112.1, 97.9, 61.5, 61.2, 56.4, 56.3; m/z (ESI) 360
[M þ H]þ; HRMS (ESI) m/z 360.1258 [M þ H]þ, C19H19FNO5 requires
360.1247.
5.1.2.4. (3-Amino-4-methylphenyl)(4,5,6-trimethoxy-1H-indol-2-yl)
methanone (3e). Compound 3e was isolated after chromatography
(toluene/ethyl acetate 8/2 to 1/1) and recrystallization (dichloro-
methane/hexane) as a yellow crystalline powder in 45% yield: mp
204e205 ꢀC; nmax(CH2Cl2)/cmꢁ1 3687, 3444, 3059, 2936, 2834,1618,
1568, 1519, 1500, 1466, 1374, 1285, 1253, 1187, 1136, 1107, 1040; dH
(300 MHz; CDCl3) 9.30 (1H, br s), 7.33 (1H, dd, J 7.7 and 1.7), 7.26 (1H,
d, J 1.7), 7.21 (1H, dd, J 2.2 and 0.8), 7.18 (1H, d, J 7.7), 6.61 (1H, s), 4.09
(3H, s), 3.91 (3H, s), 3.86 (3H, s), 3.81 (2H, br s), 2.26 (3H, s); dC
5.1.2.9. (5,6,7-Trimethoxy-1H-indol-2-yl)(p-tolyl)methanone
(4d). Compound 4d was isolated after chromatography (cyclo-
hexane/ethyl acetate 85/15) and recrystallization (chloroform/
heptane) as pale yellow crystals in 46% yield: mp 232e233 ꢀC;

M. Arthuis et al. / European Journal of Medicinal Chemistry 46 (2011) 95e100
99
nmax(CH2Cl2)/cmꢁ1 3452, 2939, 2838, 1622, 1606, 1529, 1490, 1464,
1404, 1301, 1259, 1180, 1124, 1108; dH (300 MHz; CDCl3) 9.24 (1H, br
s), 7.88 (2H, d, J 8.1), 7.32 (2H, d, J 8.1), 7.04 (1H, d, J 2.3), 6.83 (1H, s),
4.09 (3H, s), 3.95 (3H, s), 3.90 (3H, s), 2.46 (3H, s); dC (75 MHz; CDCl3)
186.2,150.3,142.9,141.4,139.0,135.5,134.4,129.3,129.1,127.4,123.4,
112.3, 98.0, 61.5, 61.2, 56.3, 21.7; m/z (ESI) 348 [M þ Na]þ; HRMS
(ESI) m/z 326.1400 [M þ H]þ, C19H20NO4 requires 326.1392.
compounds were initially dissolved in DMSO at a stock concentration
of 2.5 mg/mL and were further diluted in cell culture medium. For
comparative purposes, CA4 was routinely included in the experi-
ments as reference compound. Exponentially growing B16 cells were
plated onto 96-well plates at 5000 cells per well in 100
medium. Twenty-four h after plating, 100 l of medium containing
the compound of interest at final concentrations ranging from 0.01 to
30 M were added to the wells (in triplicate) containing the cells, and
ml of culture
m
m
5.1.2.10. (3-Amino-4-methylphenyl)(5,6,7-trimethoxy-1H-indol-2-yl)
methanone (4e). Compound 4e was isolated after chromatography
(cyclohexane/ethyl acetate 7/3) and recrystallization (chloroform/
heptane) as an orange amorphous solid in 54% yield: nmax(CH2Cl2)/
cmꢁ1 3690, 3451, 2939, 2838, 1619, 1571, 1530, 1491, 1464, 1302,
1227, 1122, 1107; dH (300 MHz; CDCl3) 9.23 (1H, br s), 7.33 (1H, dd, J
7.7 and 1.5), 7.18 (1H, d, J 7.7), 7.07 (1H, d, J 2.2), 6.83 (1H, s), 4.08
(3H, s), 3.96 (3H, s), 3.89 (3H, s), 3.83 (2H, br s), 2.27 (3H, s); dC
(75 MHz; CDCl3) 186.4, 150.3, 144.7, 141.4, 139.0, 137.1, 134.5, 130.3,
127.3, 127.2, 123.4, 119.9, 98.0, 61.5, 61.2, 56.3, 17.6; m/z (ESI) 341
[M þ H]þ; HRMS (ESI) m/z 341.1496 [M þ H]þ, C19H21N2O4 requires
341.1501.
incubated for 48 h at 37 ꢀC and 5% CO2. After the 48 h exposure period
to the test compounds, cell viability was assayed using the MTT test
[25] and absorbance was read at 562 nm in a microplate reader
(BioKinetics Reader, EL340). Appropriate controls with DMEM only
and MTT were run to subtract background absorbance. The concen-
tration of compound that inhibited cell viability by 50% (inhibitory
concentration for 50% of cells, or IC50) was determined using the
GraphPad Prism software. Results are presented as the mean of three
independent experiments each run in triplicate.
5.2.2. Antiproliferative assays on human colon carcinoma cell lines
These assays were monitored at the laboratory “Ciblothèque
Cellulaire”, ICSN-CNRS, Gif sur Yvette, France. Human colon carci-
noma cells (HCT-116, HCT-15 and HT-29) were grown in RPMI 1640
medium supplemented with 10% fetal calf serum, streptomycin and
fungizone, and maintained at 37 ꢀC in a humidified atmosphere
containing 5% CO2. Cells (500e800/well) were seeded in 96-well
5.1.2.11. (5,6,7-Trimethoxy-1H-indol-2-yl)(6-methoxypyridin-3-yl)
methanone (4f). Compound 4f was isolated after chromatography
(cyclohexane/ethyl acetate 7/3) and recrystallization (chloroform/
heptane) as beige crystals in 63% yield: mp172-173 ꢀC;
nmax(CH2Cl2)/cmꢁ1 3450, 2940, 2839, 1620, 1601, 1530, 1487, 1464,
1372, 1301, 1292, 1258, 1126, 1108; dH (300 MHz; CDCl3) 9.24 (1H, br
s), 8.86 (1H, dd, J 2.2 and 0.5), 8.17 (1H, dd, J 8.7 and 2.3), 7.06 (1H, d,
J 2.2), 6.87 (1H, dd, J 8.7 and 0.5), 6.83 (1H, s), 4.09 (3H, s), 4.05 (3H,
s), 3.95 (3H, s); 3.91 (3H, s); dC (75 MHz; CDCl3) 183.6, 166.4, 150.5,
149.2, 141.6, 139.3, 138.9, 134.1, 127.7, 127.6, 123.4, 112.2, 111.1, 98.0,
61.5, 61.2, 56.7, 54.1; m/z (ESI) 343 [M þ H]þ; HRMS (ESI) m/z
343.1309 [M þ H]þ, C18H19N2O5 requires 343.1294.
microplates containing 200
the cells were exposed to varying concentrations (0.5 nMe10
the tested compound dissolved in DMSO (less than 1% in each
preparation). After 72 h of incubation, 40 L of the MTS reagent
mL growth medium. After 24 h of culture,
mM) of
m
(Promega) was added for 2 h before the absorbance at 490 nM was
recorded. The IC50 corresponds to the concentration of the tested
compound eliciting a 50% inhibition of cell growth.
5.2.3. Inhibition of tubulin polymerization
5.1.2.12. (E)-1-(4,5,6-Trimethoxy-1H-indol-2-yl)-3-phenylprop-2-en-
1-one (9). Compound 9 was isolated after chromatography (toluene/
ethyl acetate 95/5) and recrystallization (dichloromethane/hexane)
as yellow crystals in 61% yield: mp 219e220 ꢀC; nmax(CH2Cl2)/cmꢁ1
3452, 2961, 2932, 1720, 1645, 1629, 1519, 1281, 1170; dH (300 MHz;
CDCl3) 9.25 (1H, br s), 7.88 (1H, d, J15.7), 7.69e7.66(2H, m), 7.50(1H, d,
J 15.7), 7.46e7.41 (4H, m), 6.62 (1H, s), 4.16 (3H, s), 3.93 (3H, s), 3.88
(3H, s); dC (75 MHz;CDCl3) 179.9,155.3,147.1,142.6,136.4,135.3,134.9,
130.4, 129.0, 128.4, 121.5, 116.2, 108.0, 88.9, 61.5, 61.0, 56.2; m/z (ESI)
338[Mþ H]þ;HRMS(ESI)m/z338.1403 [Mþ H]þ, C20H20NO4 requires
338.1392.
Tubulin microtubule assembly in microtubules was carried out
using the fluorescent dye DAPI (40,6-diamidino-2-phenylindole) [26]
in a 96-well plate format as described by Barron et al. [20b] and Bane
et al. [27] The standard assay was performed as follows: wells were
charged with tubulin (Cytoskeleton, 97% pure, final concentration
1 mg/ml) in PME buffer (100 mM PIPES (1,4-piperazinebis(ethane-
sulfonic acid)); 1 mM MgSO4; 2 mM EGTA) with 10 mM DAPI and
varying concentrations of the test compounds using colchicine as an
internal control. After a preincubation of 45 min at room tempera-
ture, 5 ml of 1 mM GTP was added to each well to initiate tubulin
polymerization, and the plate was then transferred to a thermostated
Victor plate reader at 37 ꢀC for an additional 2 h. Fluorescence was
then read at the excitation wavelength of 360 nm and emission of
5.1.2.13. (E)-1-(5,6,7-Trimethoxy-1H-indol-2-yl)-3-phenylprop-2-en-
1-one (10). Compound 10 was isolated after chromatography
(cyclohexane/ethyl acetate 85/15) and recrystallization (chloro-
form/heptane) as yellow crystals in 49% yield: mp 154e155 ꢀC;
nmax(CH2Cl2)/cmꢁ1 3451, 3004, 2939, 2838, 1648, 1594, 1530, 1493,
1311,1220,1195,1170,1111; dH (300 MHz; CDCl3) 9.28 (1H, br s,), 7.90
(1H, d, J 15.6), 7.69e7.66 (2H, m), 7.47 (1H, d, J 15.6), 7.45e7.42 (3H,
m), 7.25 (1H, d, J 2.3), 6.86 (1H, s), 4.08 (3H, s), 3.95 (3H, s), 3.92 (3H,
s); dC (75 MHz; CDCl3) 180.4, 150.4, 143.0, 141.6, 139.0, 136.4, 134.9,
130.5,129.0,128.5,127.7,123.4,121.6,109.5, 98.0, 61.5, 61.2, 56.3; m/z
(ESI) 338 [M þ H]þ; HRMS (ESI) m/z 338.1405 [M þ H]þ, C20H20NO4
requires 338.1392.
450 nm. The percent inhibition was determined as follows:1 ꢁ (
(sample/ F control ¼ F(no inhibition) ꢁ F
F(control))) ꢂ 100, where
(complete inhibition), and
F sample ¼ F(sample) ꢁ F(complete
DF
D
D
D
inhibition with colchicine). The IC50 for compound-induced inhibi-
tion of tubulin polymerization is the concentration of compound at
which the extent of inhibition of polymerization is 50% of the
maximum value as determined from the semi-logarithmic plot of
percent inhibition as a function of the drug concentration.
Acknowledgments
This work was financially supported by the Centre National de la
Recherche Scientifique (CNRS), the Institut Curie, the Institut
National de la Santé et de la Recherche Médicale (Inserm), and by
a grant from the Institut National du Cancer (INCa, Boulogne Bill-
ancourt, France). We acknowledge Dr Thierry Cresteil (ICSN,
CNRS-UPR 2301, Gif sur Yvette, France) for accurate in vitro cyto-
toxicity evaluation on human cells. M.A. is grateful to the Ministère
5.2. Biological evaluation procedures
5.2.1. Evaluation of cytotoxicity in murine B16 melanoma cells
Murine B16 melanoma cells were grown in DMEM medium
containing 2 mM
penicillin and 100
L
-glutamine, 10% fetal bovine serum, 100 U/mL
m
g/mL streptomycin (37 ꢀC, 5% CO2). All

100
M. Arthuis et al. / European Journal of Medicinal Chemistry 46 (2011) 95e100
(b) D. Simoni, R. Romagnoli, R. Baruchello, R. Rondanin, G. Grisolia, M. Eleopra,
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C. Pisano, J. Med. Chem. 51 (2008) 6211e6215;
de l’Enseignement Supérieur et de la Recherche for a Ph.D. Fellow-
ship Grant. We also thank Thomas Perseille for expert technical
assistance.
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