Substitution at the indole 3 position yields highly potent indolecombretastatins against human tumor cells

Article abstract of DOI:10.1016/j.ejmech.2018.08.078

Resistance to combretastatin A-4 is mediated by metabolic modification of the phenolic hydroxyl and ether groups of the 3-hydroxy-4-methoxyphenyl (B ring). Replacement of the B ring of combretastatin A-4 by a N-methyl-5-indolyl reduces tubulin polymerization inhibition (TPI) and cytotoxicity against human cancer cell lines but cyano, methoxycarbonyl, formyl, and hydroxyiminomethyl substitutions at the indole 3-position restores potent TPI and cytotoxicity against sensitive human cancer cell lines. These highly potent substituted derivatives displayed low nanomolar cytotoxicity against several human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies, and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine site was suggested by molecular modeling studies. Substituted combretastatins displayed higher potencies than the isomeric isocombretastatins and the highest potencies were achieved for the hydroxyiminomethyl (21) and cyano (23) groups, with TPI values in the submicromolar range and cytotoxicities in the nanomolar and subnanomolar range. Dose-response and time-course studies showed that drug concentrations as low as 1 nM (23) or 10 nM (21) led to a complete G₂/M cell cycle arrest after 15 h treatment followed by a high apoptosis-like cell response after 48–72 h treatment. The P-glycoprotein antagonist verapamil increased 21 and 23 cytotoxicity to IC₅₀ values of 10^?10 M, and highly potentiated the cytotoxic activity in 100-fold of the CHO derivative (17), in A-549 human non-small cell lung cancer cells. The cyano substituted indolecombretastatin 23 is by itself highly potent against rather resistant HT-29 and A-549 cell lines. A 3,4,5-trimethoxyphenyl ring always afforded more potent derivatives than a 2,3,4-trimethoxyphenyl ring.

Full text of DOI:10.1016/j.ejmech.2018.08.078

Accepted Manuscript
Substitution at the indole 3 position yields highly potent indolecombretastatins against
human tumor cells
Raquel Álvarez, Consuelo Gajate, Pilar Puebla, Faustino Mollinedo, Manuel Medarde,
Rafael Peláez
PII:
S0223-5234(18)30750-5
10.1016/j.ejmech.2018.08.078
EJMECH 10689
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2018
Revised Date: 27 August 2018
Accepted Date: 27 August 2018
Please cite this article as: R. Álvarez, C. Gajate, P. Puebla, F. Mollinedo, M. Medarde, R. Peláez,
Substitution at the indole 3 position yields highly potent indolecombretastatins against human tumor
cells, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.078.
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ACCEPTED MANUSCRIPT
Substitution at the indole 3 position yields highly potent
indolecombretastatins against human tumor cells.
a,c,d,e
b,e
a,c,d
b
Raquel Álvarez,
Medarde
Consuelo Gajate, Pilar Puebla,
Faustino Mollinedo, Manuel
a,c,d
*,a,c,d
and Rafael Peláez
a
Laboratorio de Química Orgánica and Farmacéutica, Departamento de Ciencias
Farmacéuticas, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007
Salamanca, Spain.
b
Laboratory of Cell Death and Cancer Therapy, Department of Molecular Biomedicine, Centro
de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), E-
2
8040 Madrid, Spain.
c
Instituto de Investigación Biomédica de Salamanca (IBSAL), Facultad de Farmacia,
Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
d
Centro de Investigación de Enfermedades Tropicales de la Universidad de Salamanca
(CIETUS). Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno,
E-37007 Salamanca, Spain.
e
Both authors contribute equally
e-mail addresses:
Raquel Álvarez
Consuelo Gajate
Pilar Puebla
raquelalvarez@usal.es
cgajate@cib.csic.es
puebla@usal.es
Faustino Mollinedo
Manuel Medarde
Rafael Peláez
fmollin@cib.csic.es
medarde@usal.es
pelaez@usal.es
*
Corresponding author:
Phone: 00 34 923294528; 00 34 677554x890. FAX: 00 34 923294515.
e-mail: pelaez@usal.es.
Address: Laboratorio de Química Orgánica y Farmacéutica, Departamento de Ciencias
Farmacéuticas, Facultad de Farmacia, Campus Miguel de Unamuno, E-37007 Salamanca,
Spain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
N-methylindole combretastatins were evaluated for cytotoxicity and tubulin inhibition
3-substituted compounds displayed high tubulin polymerization inhibitory potency
Two of them disrupt microtubules and arrest cells in G /M phase at nM concentrations
2
Cell killing is mediated by caspase-3 activation
P-glycoprotein antagonist verapamil increases cytotoxicity to subnanomolar range

ACCEPTED MANUSCRIPT
Abstract
Resistance to combretastatin A-4 is mediated by metabolic modification of the phenolic
hydroxyl and ether groups of the 3-hydroxy-4-methoxyphenyl (B ring). Replacement of
the B ring of combretastatin A-4 by a N-methyl-5-indolyl reduces tubulin polymerization
inhibition (TPI) and cytotoxicity against human cancer cell lines but cyano,
methoxycarbonyl, formyl, and hydroxyiminomethyl substitutions at the indole 3-position
restores potent TPI and cytotoxicity against sensitive human cancer cell lines. These
highly potent substituted derivatives displayed low nanomolar cytotoxicity against
several human cancer cell lines due to tubulin inhibition, as shown by cell cycle
analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies,
and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine
site was suggested by molecular modeling studies. Substituted combretastatins
displayed higher potencies than the isomeric isocombretastatins and the highest
potencies were achieved for the hydroxyiminomethyl (21) and cyano (23) groups, with
TPI values in the submicromolar range and cytotoxicities in the nanomolar and
subnanomolar range. Dose-response and time-course studies showed that drug
concentrations as low as 1 nM (23) or 10 nM (21) led to a complete G2/M cell cycle
arrest after 15 h treatment followed by a high apoptosis-like cell response after 48-72 h
treatment. The P-glycoprotein antagonist verapamil increased 21 and 23 cytotoxicity to
-
10
IC values of 10 M, and highly potentiated the cytotoxic activity in 100-fold of the
50
CHO derivative (17), in A549 human non-small cell lung cancer cells. The cyano
substituted indolecombretastatin 23 is by itself highly potent against rather resistant
HT-29 and A549 cell lines. A 3,4,5-trimethoxyphenyl ring always afforded more potent
derivatives than a 2,3,4-trimethoxyphenyl ring.

ACCEPTED MANUSCRIPT
Keywords
Combretastatins
-Substituted-N-methylindole derivatives
3
Tubulin polymerization inhibition
Low-nanomolar cytotoxicity
Caspase-3
Verapamil

ACCEPTED MANUSCRIPT
1. INTRODUCTION
Microtubules and their main constituents the αβ-tubulin heterodimers are the targets of many
anti-tumor and anti-parasitic drugs that disrupt their dynamical equilibrium. Microtubule-
targeting agents (MTAs) can affect cell shape maintenance, trafficking, cilia and flagella
functioning, signaling, and mitosis as some of the many functions of microtubules in the cells.
Several drug binding sites different from the nucleotide binding sites have been described,
with at least seven structurally characterized, including the taxanes, the vinca alkaloids, the
colchicine, the laulimalide/peloruside, the maytansine, the pironetin, and the hemiasterlin sites
located in both the α and β tubulins. Some MTA stabilize the microtubules, as for example
paclitaxel, while other destabilize them, as for instance colchicine, but at low concentrations all
of them affect the polymerization/depolymerization dynamics.[1]
The pivotal role of microtubules in mitosis, together with their role as scaffolds for signaling
molecules modulating apoptosis, makes them a major target in cancer chemotherapy, and
2
thus MTAs lead to cell cycle arrest at G /M phase and late apoptosis.[2] Inhibition of the
mitotic spindle is the most common signature of MTAs in cell treatment, and therefore they are
often referred to as anti-mitotic agents, although other mechanisms are probably important in
their in vivo anti-tumor effects.[3, 4] In fact, colchicine site binding drugs as for instance the
combretastatins (Figure 1) are often considered vascular disrupting agents (VDA), because in
vivo they cause a rapid collapse of the tumor associated vasculature and blood flow shutdown,
which is mainly responsible for tumor death.[5, 6] The emergence of VDAs holds great
potential in the treatment of malignant solid tumors, and the 3-O-phosphate prodrug of

ACCEPTED MANUSCRIPT
combretastatin A-4 (CA4P), the more effective CA1P (Oxi4503) and the amino analogue
AVE8062 are undergoing preclinical and clinical development.[7] However, despite a rapid
initial central tumor necrosis after VDAs treatment, a peripheral rim of resistant viable cancer
cells remains and eventually grow to the necrotic area causing recurrence and metastasis.[8,
9] Several strategies have been applied to increase the clinical efficiency of combretastatins,
including combinations with antiangiogenic drugs or with other antitumor agents or the
development of more potent derivatives.
Figure 1. Representative combretastatin type ligands
Innate or acquired resistance mechanisms employed by cancer cells against treatment with
combretastatin A-4 have been described, including adaptation to the hypoxic conditions and
reestablishment of the vasculature in response to the reduction in blood supply as well as
direct resistance to the drug. The B ring of combretastatin A-4 through its hydroxyl group
provides the attachment point for prodrug formation thus allowing pharmacokinetic
optimization but it is also its Achilles´ heel struck by phase I and II metabolic reactions
associated with its inactivation, as shown for resistant hepatocellular carcinoma or colon
adenocarcinoma-derived HT-29 cells.[10-12]
Several research groups have investigated the replacement of the B ring by heterocyclic
moieties, such as pyridine, pyrrole, furan, thiophene or their benzoanalogues.[13-21] In this
respect, we have previously shown that an indole ring is a suitable replacement for the B ring
of combretastatin A-4 in the context of a 1,1-diarylethene (isocombretastatin) skeleton (Fig.
1).[22] Substitutions of the indole 3 position did not result in improvements with respect to the

ACCEPTED MANUSCRIPT
parent unsubstituted indole. Surprisingly, the isomeric indole combretastatin (1,2-diarylethene)
showed much lower potency.[23] Here we show that introduction of substituents at the 3
position of indole combretastatin improves on the tubulin inhibitory activity. Furthermore, the
cytotoxicity of compounds with similar tubulin inhibitory potency against combretastatin A-4
resistant colon adenocarcinoma (HT-29) and lung adenocarcinoma (A-549) cell lines is
dependent on minor structural changes and can be further improved upon combination with
verapamil. Our results suggest that MDR efflux is an important factor in the observed
cytotoxicity differences and that substitution at the indole 3 position with appropriate
substituents such as the cyano group greatly reduces the compounds’ efflux and result in
apoptotic-like cell death after complete G /M cell cycle arrest after very low dose (1nM) drug
2
treatments.
2
2
2
. RESULTS AND DISCUSSION
.1. Chemistry
.1.1. Chemical synthesis
The synthesis of combretastatins was carried out by the usual Wittig procedure, followed by
derivatization at the 3 position of the indoles. The indole aldehydes used as starting materials
1-5, were obtained from commercial 1H-indole-5-carboxaldehyde (Scheme 1), whereas 1H-
benzo[d]imidazole-5-carboxylic acid (6) was used to prepare the corresponding 1-methyl-1H-
benzo[d]imidazole-5(6)-carbaldehydes (9 and 10, Scheme 2). Indolecombretastatins
unsubstituted at the 3 position 13, 15[23] and 16 were respectively synthesized by Wittig
olefination of 1 and 2 with 3,4,5-trimethoxybenzyl bromide (11) and 2 with 2,3,4-
trimethoxybenzyl bromide (12) (Scheme 1). 15 and 16 were readily formylated at the 3
position,[24] to yield 17 and 18 (Scheme 3), the two latter used to prepare derivatives at this
position 19-34.

ACCEPTED MANUSCRIPT
To complete the series of compounds to assay, the aldehyde 14, bromoderivatives 35 and 36
(Scheme 1), and the benzimidazolecombretastatins (Scheme 2) seemed adequate to fulfil the
structural modifications of indole related derivatives. The unresolved mixture of 1-methyl-1H-
benzimidazole-5(6)-carbaldehydes (9+10), obtained by methylation of 1H-benzimidazole-5(6)-
carbaldehyde (8), was used to produce the combretastatins 37+38/Z+E, which could only be
partially separated into 37+38/Z and 37+38/E.
Once separated from the E-aldehydes, several transformations aimed at increasing solubility
by means of additional nitrogen or oxygen atoms were made, Z-aldehydes 17 and 18 were
reduced to the alcohols 19 and 20, transformed into the oximes[25, 26] 21 (Z+E) and 22 (Z+E)
and oxidized to carboxylic acids[27] 25 and 26. In addition, the oximes by treatment with acetic
anhydride in pyridine, were converted into the nitriles 23 and 24 and the carboxylic acid 25
was transformed into its methyl ester 27.
Scheme 1: Synthesis of indolecombretastatin derivatives 13-16 and bromoderivatives 35 and
3
6.
Reagents and conditions: (a) (i) (3,4,5-(MeO)
0ºC, 1h; (ii) 1-ethyl-1H-indole-5-carbaldehyde (1), 1-methyl-1H-indole-5-carbaldehyde (2), 1-
3
2 3
PhCH )Ph PBr (11), THF, BuLi (1.6M), THF, -
4
ethyl-1H-indole-3,5-dicarbaldehyde (3), 1-methyl-3-bromo-1H-indole-5-carbaldehyde (4) or 1-
methyl-2,3-dibromo-1H-indole-5-carbaldehyde (5), THF, -40 ºC to rt, 24 h. (b) (i) (2,3,4-
(
MeO) PhCH )Ph PBr (12), THF, BuLi (1.6M), THF, -40ºC, 1h; (ii) 1-methyl-1H-indole-5-
3 2 3
carbaldehyde (2), THF, -40 ºC to rt, 24 h.

ACCEPTED MANUSCRIPT
Scheme 2: Preparation of benzimidazolecombretastatin derivatives 37+38.
Reagents and conditions: (a) (i) Ph P(3,4,5-MeO PhCH )I, THF, nBuLi (1.6M), THF, -40 ºC,
3
3
2
1h; (ii) 1-methyl-1H-benzimidazole-5(6)-carbaldehydes, THF, -40 ºC to rt, 24 h.
Scheme 3: Preparation of 3-substituted indolecombretastatin derivatives 17-34.
Reagents and conditions: (a) (i) POCl , DMF, 0 ºC, 1 h; (ii) 15 or 16, 60 ºC, 2 h. (b) NaBH ,
3
4
2
MeOH, rt, 30 min. (c) NH OH·HCl, MeOH, pyr (2 drops), rt, 24 h. (d) Ac
2
O, pyr, rt, 9 h. (e) 17
or 18 in tBuOH, 2-methyl-2-butene, THF; add NaClO , NaH PO , H O, rt, 12 h. (f) TMSCHN ,
2
2
4
2
2

ACCEPTED MANUSCRIPT
MeOH, 15 min. (g) 25, NH
2 2
CH COOEt·HCl; EDCI; 4-DMAP; rt, 48 h, inert atmosphere. (h) 25,
CH NHCH COOMe·HCl; EDCI; 4-DMAP; rt, 48 h, inert atmosphere. (i) 25,
3
2
2 2 2 2 2
NH CH CH COOEt; EDCI; 4-DMAP; rt, 48 h, inert atmosphere. (j) 25, (COOMe) CHNH ·HCl;
EDCI; 4-DMAP; rt, 48 h, inert atmosphere. (k) 28 or 29 or 30, KOH/MeOH, rt, 30 min.
Other transformations include the formation of several amides from the carboxylic acid 25,
using different aminoesters (Scheme 3).[28] Thus, the amides 28, 29, 30 and 31 were
obtained by treatment with glycine ethyl ester, sarcosine methyl ester, β-alanine ethyl ester
and dimethyl aminomalonate. By saponification with KOH/MeOH the carboxylic acids 32, 33
and 34 were obtained.
With these compounds, representative structures of the 3-substituted-N-methyl-1H-
indolecombretastatins were at hand in order to explore the effect of these substitutions on their
activities.
2.1.2. Aqueous solubility
Aqueous solubility in phosphate buffer at pH 7.0 was determined using a shake-flask
method, microfiltration and UV absorbance measurement of soluble compound. N-ethyl and N-
methyl indolecombretastatins with hydrogen at C-3 (13, 15 and 16) display aqueous solubility
similar to Combretastatin A-4 (Table 1). Cyano or bromine substituents do not improve
aqueous solubility but a carboxylic acid or amides of aminoacids increase the solubility
between 40 and 150 times. Low aqueous solubility is one of the major drawbacks for the
clinical use of combretastatin A-4, which is administered in the form of water soluble
phosphate prodrugs of the metabolically labile hydroxy group. The solubility of
indolecombretastatin analogues has been highly improved by means of carboxylic acid
derivatives but at the expense of a potency decrease, whereas for the highly potent derivatives
the hydroxyiminomethyl substituted indole 21 represents a modest solubility increase.

ACCEPTED MANUSCRIPT
Table 1. Aqueous solubility of representative compounds in aqueous buffer pH 7.0
Solubility
Solubility
(µg/mL)
80.0
1.0
60.0
380.0
352.0
2.0
Compound
CA-4
Compound
(µg/mL)
1.04[29]
2.0
2.6
2.0
6.0
3.0
8.0
1.0
25
27
29
33
34
35
36
13
15
16
17
19
21
23
<1.0
2.2. Biology.
2.2.1. Cell viability
The effect of the synthesized compounds on cell viability against four types of human cancer
cell lines: A-549 human lung carcinoma, HeLa human cervix epithelioid carcinoma, HL-60
human acute myeloid leukemia, and HT-29 human colon adenocarcinoma was assayed by the
XTT method.[30] The results are summarized in Table 2. The compounds are cytotoxic at
concentrations between micromolar and subnanomolar, thus indicating that the combination of
a trimethoxyphenyl and a 5-indolyl forming a Z stilbene skeleton is a very favorable scaffold for
cytotoxicity. The most potent compounds of the series inhibited proliferation of the indicated
human cancer cell lines at very low drug concentrations, with IC values in the nM and 0.1 nM
50
range. These IC50 values were up to 10−100 times lower than those shown by doxorubicin
IC at a range of 10 nM) and the IC values for the most potent derivative 23 were similar or
(
50
50
even lower than those shown by combretastatin A-4 (CA-4, Table 2). For most compounds the
IC50s are similar for HL60 and HeLa whereas they are higher for A-549 and HT-29, a
difference in sensitivity which has been previously observed for other colchicine site
ligands.[10, 22]
Table 2. Tubulin polymerization inhibitory activity, cytotoxic activity against human cancer
cell lines, and results of the docking studies.

ACCEPTED MANUSCRIPT
Antiproliferative activity
Binding
energy
b
c
TPI
IC s (µM)
50
(
Cluster
number
// % of
a
IC50
N
R
N
X
Y
Z
R
2
HL-60 A-549 HeLa HT-29
(
µM)
d
conf)
-
(
-
(
10.31
1//93)
10.40
1//65)
1
2
3
13
14
15
Et
Et
OMe
OMe
OMe
H
H
H
CH
C-CHO
CH
H
H
H
2.0
1.2
2.0
0.172
0.03
0.919
>10
0.27
0.029
n.d.
0.434
3.10
0.36
e
-9.80
1//96)
9.24
2//41)
Me
n.d.
0.47
(
-
4
5
6
16
(E)-16
17
Me
Me
Me
H
H
OMe
OMe
H
CH
CH
H
H
H
>5
4-5
0.5
0.0059
0.303
1.7
0.062
0.07
0.086
1.262
(
f
0.462
d.n.b.
-
(
10.34
1//85)
OMe
C-CHO
0.00271
1.36 0.00397 0.942
-
9.74
7
8
9
18
19
20
Me
Me
Me
H
OMe
H
OMe
H
C-CHO
H
H
H
>5
1.2
≈20
0.0087
0.028
8.5
7.9
0.045
3.9
0.043
0.032
1.2
0.6
0.033
1.0
(
-
(
2//23)
10.47
1//79)
C-CH
C-CH
2
OH
OH
-
9.62
2//20)
10.93
E)
OMe
2
(
-
(
(
-
1//20)
11.10
1
0
21
Me
Me
OMe
H
H
C-CH=NOH
C-CH=NOH
H
H
0.5
0.00298 0.0140 0.00323 0.01605
(Z)
(
-
1//13)
10.47
(
-
E) (1//5)
9.84 (Z)
3//9)
1
1
22
OMe
≈20
5.6
0.76
0.37
1.0
(
-
(
-
10.78
1//48)
10.10
1
1
1
1
1
1
1
1
2
2
3
4
5
6
7
8
9
0
23
24
25
26
27
29
30
31
33
Me
Me
Me
Me
Me
Me
Me
Me
Me
OMe
H
H
OMe
H
C-CN
C-CN
H
H
H
H
H
H
H
H
H
0.3 0.000347 0.0027 0.00030 0.00177
>5
1.4
0.21
0.0622
3.1
0.7
0.471 0.0561
3.0 3.9
0.13
0.41
1.142
2.9
(
2//6)
9.73
1//53)
9.17
2//24)
-
(
-
(
-
(
OMe
H
C-COOH
C-COOH
OMe
H
>20
0.7
10.16
1//19)
OMe
OMe
OMe
OMe
OMe
C-COOMe
0.0192 0.2092 0.0210 0.2034
C-CON(Me)
H
>20
3.0
0.305
0.0452
0.239
0.266
0.159
1.6
0.235
0.395
0.306
0.21
d.n.b.
d.n.b.
d.n.b.
(
CH
C-CONH
CH COOEt
C-CON(Me)
2 2
) COOMe
H
0.316 0.0588
(
2 2
)
H
n.d.
>20
0.156
0.112
1.17
CH2(COOMe)
C-CON(Me)
2
-6.23
(11//3)
H
0.185
CH
C-CONH
CH COOH
2
COOH
2
2
2
1
2
3
34
35
36
Me
Me
Me
OMe
OMe
OMe
H
H
H
H
H
13
2.5
1.1
0.404
0.0368
0.165
>1
1.42
0.309
0.695
1.47
0.346
0.191
d.n.b.
(
2 2
)
C-Br
0.442
0.194
d.n.b.
-
(
10.07
2//47)
C-Br
Br
-
9.40
1//100)
9.13
1//96)
8.35
1//96)
2
2
2
4
5
6
37/38
37/38
CA-4
Me
OMe
OMe
-
H
H
-
N
NMe
-
H
H
-
2.2
≈20
3
0.032
0.03
0.034
0.22
0.031
0.028
0.003
0.032
0.029
0.032
(
-
(
-
(
-
-
0.013
0.003

ACCEPTED MANUSCRIPT
a
b
Compound number. All the compounds are the Z isomers unless otherwise stated.
c
Tubulin polymerization inhibitory (TPI) IC . Values are derived from concentration-response
50
curves using the XTT assay as described in the Experimental Section. Data are shown as
means from at least three independent experiments performed in triplicate. Standard
d
deviations were less than 15% of the mean. AutoDock binding energies in kcal/mol for the
best pose of the cluster corresponding to the same ring dispositions as combretastatin A-4.
The numbers in parenthesis are the ranking number of the cluster and the number of poses in
e
f
the cluster out of a total of 100. n.d.: not determined. d.n.b.: did not bind.
For all the pairs differing only in the disposition of the methoxy groups of ring A (e.g.,
compare the matched couples 15/16Z, 17/18, 19/20, 21/22, 23/24 and 25/26), the compounds
with a 2,3,4-trimethoxyphenyl (methoxy groups in a similar arrangement to those of colchicine)
were less potent than those with a 3,4,5-trimethoxyphenyl ring (like those of podophyllotoxin
and combretastatin A-4), similar to what has been reported previously.[31] Comparison of 13
and 14 with 15 and 17 respectively, shows that a methyl is slightly preferred over an ethyl on
the indole nitrogen.
Substitutions at the indole 3 position (e.g. 17, 19, 21, 23 and 25) result in an overall potency
increase with respect to the unsubstituted 15 for the 3,4,5-trimethoxyphenyl substituted
compounds whereas an opposite trend is seen for compounds with a 2,3,4-trimethoxyphenyl
(e.g., compare 18, 20, 22, 24 and 26 with 16Z). This different behavior probably arises from
steric hindrance, as larger groups result in more reduced potencies, in agreement with
previous results on the isocombretastatins where indole substitution causes a reduction in
potency.[22] We have previously shown that substituents protruding out of the plane of ring B
in isocombretastatins result in less potent compounds.[32]
The optimal substitutions at the indole 3 position are the hydroxyiminomethyl (21) and the
cyano (23) groups, which are nanomolar or subnanomolar in the four cell lines assayed. The
formyl substituent (17) provides also high potency against HeLa and HL60, but there is a
significant potency drop down to the micromolar range for A549 and HT-29. For the second
potency level (two digit nanomolar), carboxy (25), methoxycarbonyl (27) and hydroxymethyl

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(19) indoles and the N-methylbenzimidazoles (37, 38) show similar potencies in the four cell
lines. Larger substitutions at the 3 position (29 - 34) result in a significant potency reduction,
although most of them are still in the submicromolar range. Of these extended amides, that of
the beta-alanine ethylester (30) is the more potent representative.
2.2.2. Tubulin polymerization inhibition
The effect of the compounds on in vitro bovine brain tubulin assembly was studied at
concentrations between 5 and 20 µM. For those compounds inhibiting tubulin polymerization
more than 50% at 20 µM we determined the IC50 values of tubulin polymerization inhibition
(TPI) (Table 2). These values allowed us to compare the potencies of different compounds
and to analyze the relationship between structure and TPI. Most of the compounds showed
potent TPI inhibitory activity with values below 20 µM, many better than combretastatin A-4
(
IC = 3 µM). As seen for the cytotoxicity assays, the 3,4,5-trimethoxyphenyl and 3 substituted
50
N-methylindole combination was optimal, with several compounds (e.g. 17, 21, 23 and 27)
showing submicromolar values. The compounds with a 2,3,4-trimethoxyphenyl ring showed
reduced potencies, typically a ten - fold reduction as for example from 19 to 20. Substitution of
the indole N-methyl by an N-ethyl modestly reduces the TPI potency. The TPI results show
that aldehyde (17), hydroxyiminomethyl (21), nitrile (23), and methoxycarbonyl (27)
substituents at the 3-position of the indole are optimal for tubulin inhibition. These groups
2
share a sp carbonyl or equivalent conjugated with the indole ring and both hydrogen-bond
donors and acceptors are allowed. Comparison of the TPI and cytotoxicity IC50 values shows
that there is a good overall agreement between the TPI and the cytotoxic potencies against
HeLa and HL60 cells, while the effects against A-549 and HT-29 show in some cases
significant deviations. These results suggest either a difference in the target (different tubulin
isotype expression) or the involvement of an additional effect (drug efflux, drug metabolism, or
other) to account for the observed potency reduction in these cell lines.

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Figure 2. Effects of compounds 17, 21 and 23 on the microtubule network of HeLa cells. Cells
were incubated in the absence (Control) or in the presence of 10 or 100 nM of compounds 17,
21 and 23 for 15 h and then fixed and processed to analyze microtubules (red fluorescence)
and nuclei (blue fluorescence) as described in the Experimental Section. Bar: 25 µm. The
photomicrographs shown are representative of at least three independent experiments
performed.
The three compounds that showed a lower IC , namely 17, 21 and 23, led to a drastic and
50
severe disruption of the microtubule network of HeLa cells at nanomolar concentrations
Figure 2), further confirming that the observed effects were due to interaction with tubulin.
Dose-response and time-course analyses of the most active compounds synthesized in this
(

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2
work (16E, 17, 21, 23, 25, 27, 30) showed a major cell cycle arrest at G /M phase (Figure 3,
and Supplementary Table S1), followed by the induction of apoptotic cell death, as assessed
by an increase in the percentage of cells at the sub-G /G region, representing cells
0
1
undergoing chromosomal DNA degradation (Supplementary Table S1).
2.2.3. Effects on the cell cycle
Compound 23 was the most active one in arresting most of the cells (74.2%) at the G2/M
phase at only 1 nM concentration and after 15 h incubation, followed by compound 21 (79.9%)
and 17 (59.3%), which required 10 nM concentration (Supplementary Table S1, and Figure 3).
Figure 4 shows a time-course of the effects of the most active analogs 23 (active at 1 nM) and
2
1 (active at 10 nM) on the cell cycle profile of HeLa cells. G /M cell cycle arrest was evident
2
even at only 9 h treatment, and the onset of apoptosis took place after 24 h incubation, and
the cell death response was almost complete after 72 h treatment (Figure 4). Taken together,
these data indicate that the actions of 23 and 21 on microtubules lead to a total disruption of
microtubules that triggers a potent apoptotic response, rendering a complete cell demise in the
drug-treated population. To further support this induction of apoptosis, we found that
compound 23 induced caspase-3 activation in HeLa cells, as assessed by the cleavage of the
typical caspase-3 substrate PARP, using a monoclonal antibody that detects both the 116-kDa
intact form and the 89-kDa cleaved form of PARP, as well as the processing of procaspase-3
into the p17/p19 active form (Supplementary Figure S1). These results are in agreement with
the previously reported involvement of a caspase-3-induced apoptotic response to
microtubule-disrupting agents that has been previously reported.[33] On the other hand, we
found that preincubation with the P-glycoprotein 1/multidrug resistance protein 1 (MDR1)
inhibitor verapamil increased the cytotoxic activity of compounds 21 and 23 (Figure 5). Thus
21 or 23 combined with 10 µM verapamil, a concentration that had been previously reported
not to decrease cell viability,[34, 35] highly potentiated the cytotoxic activity of both 21 and 23

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to IC50s of 0.3 - 0.5 nM in A549 cells (Figure 5). It is interesting to note that verapamil
pretreatment induced an increase in compound 21 efficiency of 100-fold, while this increase
was only 10-fold for compound 23 (Figure 5). On these grounds, it might be envisaged that
compound 23 displays a reduced drug resistance. Compound 17 also became very active
against A549 cells (IC50 = 59.8 nM) (about 100-fold increase) when combined with 10 µM
verapamil. These data suggest that the P-glycoprotein antagonist verapamil highly potentiates
the cytotoxicity rendered by these newly synthetized combretastatin A-4 derivatives. In this
regard, verapamil has been previously reported to increase cytotoxicity of microtubule-
targeting agents against cancer cells.[34, 36] Nevertheless, as mentioned above, the highly
active compound 23 shows a lower increase in cytotoxic activity following verapamil treatment
as compared to other derivatives, thus suggesting a very potent activity (nM range) that is not
so dependent on drug-resistance mechanisms.

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Figure 3. Effect of different compounds on the distinct cell cycle phases in HeLa cells.
Compounds 16E, 17, 21, 23, 25, 27 and 30 were incubated for 15 h and then their DNA
content was analyzed by flow cytometry as described in the Experimental Section. The
different cell cycle phases were quantified and represented in pie charts to easily visualize the
changes in G /M arrest. Untreated control cells were run in parallel. Data shown are
2
representative of at least three independent experiments performed.

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Figure 4. Time-course of the effects of compounds 21 and 23 on cell cycle in HeLa cells. Cells
were incubated with 1 or 10 nM of 21 and 23 for the indicated times, and their DNA content
was analyzed by fluorescence flow cytometry. The positions of the G /G1 and G /M peaks are
0
2
indicated by arrows, and the proportion of cells in each phase of the cell cycle was quantified
by flow cytometry. The cell population in the sub-G /G1 region represents cells with
0
hypodiploid DNA content, an indicator of apoptosis, and the percentages of cells at this region
are indicated in each histogram. Untreated control cells were run in parallel. The experiments
shown are representative of three performed.

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Figure 5. Effect of verapamil on the sensitivities of A549 cancer cells to 21 and 23. Cells were
co-incubated with 10 µM verapamil and different concentrations of 21 and 23. The dose–
response curves were drawn using the GraphPad Prism 5 software. Results are mean values
±
S.D. of a representative experiment in triplicate, out of three performed.
2.3. Computational studies
Combretastatins (1,2-diarylethenes) and their isomeric isocombretastatins (1,1-
diarylethenes) are structurally quite similar but it has been previously noticed that significant
differences in SAR are common.[14, 22, 23, 37] Here, a cyano group at the indole 3 position of
a combretastatin yields the most potent compound of the series with a 100 fold improvement
with respect to the unsubstituted analogue, whereas in isocombretastatins (Fig. 1) the same

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substitution has almost no effect.[22] We therefore set to investigate the structural basis of
these different SARs in an attempt to facilitate future drug design. The different effect of the 3-
substituents on the indole ring of isomeric combretastatins and isocombretastatins on their
potency could be explained by their topological differences. Therefore, the preferred
conformations of the compounds, including the two pairs of isomers (15 and 23 and their
corresponding isocombretastatins depicted in Fig. 1), were calculated, DFT-optimized and
those conformations placing the aromatic rings closer to those of the X-ray crystal structure of
combretastatin A-4 when in complex with tubulin (pdb ID 1LYJ)[38] were selected as the
putative conformations binding to the colchicine site of tubulin. Figure 6a-f on the left-hand
side shows the superimposition of the unsubstituted (a, 15) and 3-cyano indole
combretastatins (b, 23 with a 3,4,5-trimethoxyphenyl ring and c, 24 with a 2,3,4-
trimethoxyphenyl ring) and the isocombretastatins (in d the isomer of 15 and in e the isomer of
23) onto combretastatin A-4 (carbon atoms colored in cyan). All of them place the phenyl rings
close to those of combretastatin A-4 to comply with the essential role assigned by
pharmacophoric models for ligands of the colchicine site to their spatial arrangement. The
2
single atom sp hybridized connection between the phenyl rings of isocombretastatins results
in a wider opening angle between the ring centroids and the bridge than in a stilbene (Fig 6d
and 6e). When in complex with tubulin this disposition of the rings either projects the 3
substituents at positions further away probably colliding with the site walls or forces the
displacement of the trimethoxyphenyl ring in the opposite direction towards the site walls thus
resulting in the observed potency reduction. Similarly, a 2,3,4-trimethoxyphenyl ring is more
sterically demanding (Fig 6c) in the plane of the B – ring and trying to accommodate it in the
colchicine site pushes the indole ring in the opposite sense. Substituents at the indole 3-
position further worsen the steric problem and explains the potency decrease proportional to
substituent size.

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The favorable effect on the TPI activity here observed for electron withdrawing groups at the
indole 3 position of combretastatins can be explained by a combination of steric hindrance,
changes in the electronic density and favorable interactions with the target. The favorable
substituents are always conjugate systems placed in the same plane as the B ring. We have
previously shown that substituents protruding out of the plane are detrimental for the activity.
[
32] Furthermore, hydrogen bond donors (such as hydroxy or amino groups, as in
combretastatin A-4,[38] AVE-8062 or rigosertib[39]) and acceptors (carbonyl groups of
MTC[40] or colchicine[41]) are well tolerated and make hydrogen bonds to both the backbone
and the sidechains of the B5-H5 loop of α-tubulin and of the H10-B9 loop and B9 of the β-
subunit, mostly within the B ring plane and adapting to different geometries and lengths. We
have previously described, by means of molecular dynamics simulations, similar effects in
isocombretastatins.[22] Furthermore, changes in electron density of the indole ring and the
nitrogen substituents caused by the electron withdrawing substituents can favor interactions
with tubulin, as for instance with Asn258 which stacks on top of B ring. In order to better
characterize the interaction mode of the indolecombretastatins here described with tubulin we
have performed docking calculations.
N
N
O
O
2
3
O
a)
c)
b)
d)

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e)
f)
Figure 6. Best superimpositions of the X-ray structure of combretastatin A-4 in cyan (a-f) in
the complex with tubulin and the conformations of representative compounds showing the
effects of the structural modifications and the corresponding docking results (the protein is
shown in grey): a) with 15, b) with 23, c) with 24, d) with the 1,1-diarylethene isomer
(isocombretastatin) of 15, e) with the 1,1-diarylethene isomer (isocombretastatin) of 24, and f)
a combination of b,c and e.
Docking experiments using AutoDock 4.2 on the X-ray structure of tubulin in complex with
combretastatin A-4 (pdb ID: 5LYJ) yielded results in excellent agreement with the experimental
TPI and cytotoxicity results (Table 2) and with the results of the conformational study (Figure
6). Other X-ray structures of tubulin (15 different structures obtained from X-ray crystal
structures of tubulin in complex with less structurally related ligands of the colchicine site) or
structures derived from molecular dynamics simulations, as previously described[22] yielded
worse results. The docking binding energy differences between compounds with a 3,4,5-
trimethoxyphenyl (15, 17, 19, 21, 23, and 25) and their matched pairs with a 2,3,4-
trimethoxyphenyl ring (16, 18, 20, 22, 24, and 26, respectively) is ca 0.6 kcal/mol, in good
agreement with the differences observed for TPI IC50 values. Also, for the former group the
lowest energy clusters correspond to the same ring dispositions as seen for combretastatin A-
4, whereas for the second group the clusters with this ring disposition are usually second or
worse by energy, being usually surpassed by dispositions with the two phenyl rings switched.
For the most potent analogues (15, 17, 19, 21, 23, 25 and 27) the rank order of binding
energies (21, 23, 19, 17, 27, 15, 25, CA-4) roughly matches the TPI values (23, 21, 17, 27, 19,
25, 15, CA-4), in spite of the fact that the energy differences between them are probably too

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small to be safely trusted. For the extended amides (29-34) the results are similar to those
described for the 2,3,4-trimethoxyphenyl rings.
3. CONCLUSIONS
The replacement of the B ring of combretastatin A-4 by 3 substituted indoles results in highly
potent tubulin inhibition and cytotoxicity against several human cancer cell lines. This effect of
the indole 3 substituents is highly context dependent, as it has been shown to be detrimental
in the isomeric isocombretastatins or in compounds with a 2,3,4-trimethoxyphenyl ring. The
optimal substituent is the cyano group which displays a high potency in all the studied cell
lines, whereas compounds with other substituents show different potencies in different cell
lines. Co-treatment with verapamil restores high potency in these latter compounds, thus
suggesting a resistance based on MDR efflux proteins. The lower effect observed of the co-
treatment for the cyano substituted indolecombretastatin 23 suggests a lower sensitivity to
MDR efflux. The more potent compounds displayed low nanomolar cytotoxicity against several
2
human cancer cell lines due to tubulin inhibition, complete G /M cell cycle arrest after 15 h
followed by a high apoptosis-like cell response after 48-72 h mediated by caspase-3
activation. Molecular modeling studies support binding at the colchicine site and provide an
explanation for the aforementioned SAR. These compounds are highly potent against cell lines
intrinsically resistant to combretastatin A-4 and therefore the structural modifications here
described, in particular the 3-cyanoindole moiety, could be applied to prevent resistance.
4. EXPERIMENTAL SECTION
4.1. Chemistry

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4
.1.1. General chemical techniques
General techniques. Reagents were used as purchased without further purification. Solvents
THF, DMF, CH Cl , toluene) were dried and freshly distilled before use according to
(
2
2
procedures described in the literature. TLC was performed on precoated silica gel polyester
plates (0.25 mm thickness) with a UV fluorescence indicator 254 (Polychrom SI F254).
Chromatographic separations were performed on silica gel columns by flash (Kieselgel 40,
0.040-0.063; Merck) or gravity (Kieselgel 60, 0.063-0.200 mm; Merck) chromatography.
1
Melting points were determined on a Buchi 510 apparatus and are uncorrected. H NMR and
1
3
C NMR spectra were recorded in CDCl
3
on a Bruker WP 200-SY spectrometer at 200/50
) are given in ppm
MHz or on a Bruker SY spectrometer at 400/100 MHz. Chemical shifts (
δ
downfield from tetramethylsilane and coupling constants (J values) are in Hertz. IR spectra
were run on a Nicolet Impact 410 Spectrophotometer. For FABHRMS analyses, a VG-TS250
apparatus (70 eV) was used. HPLCs were run on Waters X-Terra® MS C18 (5 mm, 4.6x150
mm) or Agilent HP series 1100 equipment with acetonitrile/water solvent gradients. Elemental
analyses were run on a Perkin Elmer 2400 CHN apparatus. All the compounds described here
were obtained with at least 95% of purity by quantitative HPLC and/or elemental analysis,
unless otherwise stated.
4.1.2. Chemical synthesis
1
-Ethyl-1 -indole-5-carbaldehyde (1) and 1-Methyl-1H-indole-5-carbaldehyde (2)
H
+
-
4
1
.04 g (26 mmol) of NaOH and 20 mg of n-Bu
4
N HSO
were added to a stirred solution of 1
H-
indole-5-carbaldehyde (2.0 g, 13.8 mmol) in 40 mL of dry CH
2
Cl . After 1 hour at atmosphere
2
temperature 3 mL (40.2 mmol) of ethyl bromide or methyl iodide were added and the reaction
was heated at 50ºC. After 3 days the reaction mixture was concentrated, re-dissolved in

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2 2 2 4
CH Cl , washed with brine, dried over anhydrous Na SO , filtered and concentrated in
vacuum to obtain 1.48 g (62%) of 1 and 1.10 g (50.1%) of 2.
1-Ethyl-1
H-indole-5-carbaldehyde (1): M.p.: 64-66 ºC (CH Cl /Hex). IR (KBr): 1703, 1605,
2 2
-
1 1
1
307, 769 cm . H NMR (200 MHz, CDCl ): δ 1.49 (3H, t, J = 7.2), 4.22 (2H, m), 6.66 (1H, d,
J
J
3
=
=
3.3); 7.22 (1H, d,
J
= 3.3), 7.42 (1H, d,
J
= 8.4), 7.78 (1H, dd,
J = 8.4 and 1.4), 8.15 (1H, d,
13
1.4), 10.0 (1H, s). C NMR (50 MHz, CDCl
3
): 15.4 (CH ), 41.2 (CH
δ
3
2
), 103.4 (CH), 109.9
(CH), 121.6(CH), 126.5 (CH), 128.4 (C), 128.5 (CH), 129.3 (C), 139.0 (C), 192.5 (CH).
1
1
-Methyl-1
CDCl3): δ 3.76 (3H, s), 6.55 (1H, d,
dd, = 8.8 and 1.9), 8.05 (1H, d,
H
-indole-5-carbaldehyde (2): M.p.: 85-86ºC (Ether). H NMR (200 MHz,
J
J
= 3.3), 7.10 (1H, d
J
= 3.3), 7.41(1H, d,
J
= 8.8), 7.80 (1H,
): 32.6
13
J
= 1.9), 9.92 (1H, s). C NMR (50 MHz, CDCl
3
δ
(CH3), 103.1 (CH), 109.8 (CH),121.4 (CH), 126.1 (CH), 128.2(C), 129.1 (C), 130.9 (CH), 139.8
(C), 192.3 (CH).
1
-
1
1
-Ethyl-1
MHz, CDCl3): δ 1.54 (3H, t, J = 7.2), 4.25 (2H, c, J = 7.2), 7.44 (1H, d,
.85 (1H, dd, = 8.6 and 1.6), 8.73 (1H, d, = 1.6), 9.99 (1H, s), 10.04 (1H, s). C NMR (50
MHz, CDCl ): 15.0 (CH ), 42.1 (CH ), 110.8 (CH), 118.8 (C), 123.4 (CH),125.0 (C), 126.8
CH), 131.7 (C), 139.7 (CH), 140.1 (C), 184.5 (CH), 192.1 (CH).
H
-indole-3,5-dicarbaldehyde (3): IR (film): 1687, 1662, 1191 cm . H NMR (200
J
= 8.6), 7.84 (1H, s),
13
7
J
J
3
δ
3
2
(
3-Bromo-1-methyl-1H-indole-5-carbaldehyde (4) and 2,3-Dibromo-1-methyl-1H-indole-5-
carbaldehyde (5)
To a solution of 1-methyl-1
H
-indole-5-carbaldehyde (2, 2.0 g, 12.58 mmol) in 4 mL of
-bromosuccinimide were slowly added. After 24 h stirring
chloroform, 2.9 g (16.35 mmol) of
N
at 0ºC, the reaction mixture was evaporated to obtain 5.38 g of crude. After column
chromatography on silica gel (Hex/EtOAc 7/3) 540 mg (2.27 mmol, 18.03%) of compound 4
and 611 mg (1.93 mmol, 15.33%) of compound 5 were obtained.
1
3
-Bromo-1-methyl-1
H
-indole-5-carbaldehyde (4): H NMR (200 MHz, CDCl
3
): δ 3.84 (3H,
s), 7.26 (1H, s), 7.40 (1H, d,
J
= 8.8), 7.84 (1H, dd,
J
= 8.8 and 1.9), 8.09 (1H, d, = 1.9),
J

ACCEPTED MANUSCRIPT
13
1
0.07 (1H, s). C NMR (50 MHz, CDCl3): δ 33.3 (CH ), 91.7 (C), 110.2 (CH), 122.4 (CH),
3
124.9 (CH), 127.3 (C), 129.5 (CH), 129.8 (C), 139.4 (C), 192.0 (CH).
1
2
,3-Dibromo-1-methyl-1
= 8.4), 7,85 (1H, dd,
s). C NMR (50 MHz, CDCl ): 32.7 (CH ), 94.9 (C), 100.2 (C), 110.3 (CH), 117.0 (C), 118.4
C), 122.8 (CH), 126.9 (CH), 130.3 (C), 191.8 (CHO).
H
-indole-5-carbaldehyde (5): H NMR (200 MHz, CDCl
3
)
: δ 3.84
(3H, s), 7,40 (1H, d,
J
J
= 8.4 and 1.0), 8.05 (1H, d,
J
= 1.0), 10.06 (1H,
13
3
δ
3
(
Preparation of compounds 7-10
To a suspension of 2.0 g (12.3 mmol) of 1
dry THF, in inert atmosphere was added 983 mg (24,7 mmol) of LiAlH
5ºC. The reaction mixture was poured into EtOAc/EtOH and was filtered, by evaporation to
dryness 1.4 g (77%) of compound 7 was obtained.
H-benzo[
d]imidazole-5-carboxylic acid in 50 mL of
4
and stirred 48 h at
6
4
.94 g (56.7 mmol) of MnO were added to a solution of 1.40 g (9.45 mmol) of 7 in 50 mL of
2
MeOH. After 48 h at rt, the mixture was filtered over sílica and by evaporation 1.24 g (90%) of
compound 8 was isolated.
To a solution of 1.24 g (8.49 mmol) of 8 in 50 mL of CH Cl :H O (3:2) was added 700 mg (17.5
2
2
2
+
mmol) of NaOH and and 40 mg of n-Bu N HSO . After 1 h stirring 1.33 mL (21.2 mmol) of MeI
4
4
were added. After 3 days the reaction mixture was concentrated, re-dissolved in CH Cl . By
2
2
chromatography on silica gel (CH Cl /EtOAc 85/15), 600 mg (44%) of a mixture 9 and 10 were
2
2
obtained
1
1
H
-Benzo[
1H, dd;
CDCl ):
46.0 (CH).
d
]imidazol-5-yl)methanol (7): H NMR (200 MHz, CDCl ): δ 4.60 (2H, s), 7.08
3
13
(
J
= 8.4 and 1.6); 7,45 (1H, d, J = 8.4), 7.49 (1H, bs), 7.94 (1H, s). C NMR (50 MHz,
3
δ
65.9, (CH ), 115.2 (CH), 116.3 (CH), 121.3 (CH), 136.1 (C), 140.6 (C), 141.0 (C),
2
1

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1
1H-Benzo[
d
]imidazole-5-carbaldehyde (8): H NMR (200 MHz, CDCl
3
): δ 7.55 (1H, d,
J
=
13
8
.4), 7.65 (1H, dd,
116.3 (CH), 120.5 (CH), 124.5 (CH), 133.1 (C), 139.7 (C), 142.8 (C), 145.9
CH), 194.0 (CH).
J = 8,4 and 1.4), 8.01 (1H, bs), 8.24 (1H, s), 9.85 (1H, s). C NMR (50
MHz, CDCl3):
δ
(
1
6
8
-Methyl-1
H
-benzo[
d
]imidazole-5-carbaldehyde (9) and 1-Methyl-1H-benzo[d]imidazole-
1
-carbaldehyde (10): H NMR (200 MHz, CDCl
3
)
: δ 3.60 (3H, s); 3.63 (3H, s), 7.17 (1H, d,
= 8.4 and 1.6), 7.59 (1H, d, = 8.4), 7.61
= 1.6), 7.98 (1H, d, = 1.6), 9.78 (1H, s), 9.79 (1H, s). C
), 110.1 (CH), 111.8 (CH), 120.2 (CH), 123.2 (CH),
J =
.4), 7,56 (1H, dd,
J
= 8.4 and 1.6), 7.56 (1H, dd,
J
J
13
(1H, s), 7.77 (1H, s), 7.82 (1H, d,
J
J
NMR (50 MHz, CDCl3):
δ
31.2 (2) (CH
3
1
23.5 (CH), 123.8 (CH), 131.3 (C), 131.5 (C), 134.5 (C), 138.7 (C); 143.4 (C), 145.9 (CH),
47.0 (CH), 148.1 (C), 191.7 (CH), 191.9 (CH).
1
(Z)-1-Ethyl-5-(3,4,5-trimethoxystyryl)-1H-indole (13)
To a suspension of triphenyl(3,4,5-trimethoxybenzyl)phosphonium bromide (11, 1.67 g, 3.19
mmol) in dry THF (40 mL), at -40 ºC in argon atmosphere, BuLi (1.6 M in hexane, 2.0 mL, 3.2
mmol) was added and stirred for 1 h. A solution of 1-ethyl-1 -indole-5-carbaldehyde (1, 500
n
H
mg, 2.89 mmol) in dry THF (10 mL) was slowly added at -40 ºC and progressively warmed to
room temperature. After 24 h, the reaction mixture was poured into ammonium chloride
solution and extracted with CH Cl . The organic layers were washed to neutrality with brine,
2
2
dried over anhydrous Na
silica gel (Hex/EtOAc 85/15), 130 mg (0.40 mmol, 14%) of 13
of a mixture 13Z/ 3E were obtained.
2
SO
4
, filtered and evaporated to dryness. After chromatography on
Z
and 161 mg (0.51 mmol, 18%)
1
(
Z
)-1-Ethyl-5-(3,4,5-trimethoxystyryl)-1
H
-indole (13): IR (film): 1685, 1579, 1127, 1007, 796,
22 cm . H NMR (200 MHz, CDCl ): δ 1.51 (3H, t, = 7.3), 3.62 (6H, s), 3.85 (3H, s), 4.18
= 12.3), 6.57 (2H, s), 6.72 (1H, d,
-
1 1
7
J
3
(2H, c, J = 7.3), 6.41 (1H, d,
J
= 3.1); 6.43 (1H, d,
J
J =
13
12.3), 7.08 (1H, d,
J
= 3.1), 7.20 (2H, bs), 7.60 (1H, bs). C NMR (50 MHz, CDCl3): δ 15.5