Synthesis and biological evaluation of new disubstituted analogues of 6-methoxy-3-(3′,4′,5′-trimethoxybenzoyl)-1H-indole (BPR0L075), as potential antivascular agents

Article abstract of DOI:10.1016/j.bmc.2008.06.002

6-Methoxy-3-(3′,4′,5′-trimethoxybenzoyl)-1H-indole (BPR0L075) (1) is a potent inhibitor of tubulin polymerization which exhibits both in vitro and in vivo activities against a broad spectrum of solid tumors. This compound was designed as a heterocyclic an

Full text of DOI:10.1016/j.bmc.2008.06.002

Bioorganic & Medicinal Chemistry 16 (2008) 7494–7503
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new disubstituted analogues
of 6-methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-indole (BPR0L075),
as potential antivascular agents
Nancy Ty a, , Grégory Dupeyre a, , Guy G. Chabot b, Johanne Seguin b, François Tillequin a,
a,
Daniel Scherman b, Sylvie Michel a, , Xavier Cachet
*
*
a Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, UMR 8638 CNRS, Laboratoire de Pharmacognosie, 4 avenue de l’Observatoire, 75006 Paris, France
b Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, INSERM U 640—CNRS UMR 8151, Laboratoire de Pharmacologie Chimique et Génétique,
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
6-Methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-indole (BPR0L075) (1) is a potent inhibitor of tubulin poly-
merization which exhibits both in vitro and in vivo activities against a broad spectrum of solid tumors.
This compound was designed as a heterocyclic analogue of combretastatin A4 (CA-4), a natural stilbene
derivative that disrupts the tumor vasculature and causes tumor regression. In the present work, we
describe the design and synthesis of several new disubstituted analogues of 1, along with their biological
evaluation as potential antivascular agents. Compound 13, bearing a hydroxyl group at the 7-position of
the indole nucleus that mimics the hydroxyl group at the 3-position of the B-ring of CA-4, was identified
as a potent inhibitor of tubulin polymerization and also as a cytotoxic agent against B16 melanoma cells
at sub-micromolar concentration. In addition, compound 13 displayed marked morphological activity
(rounding up) at nanomolar concentrations on endothelial cells (EA.hy 926 cells), which is indicative
of potential antivascular activity. Interestingly, the corresponding 7-O-mesylate derivative 28 (an inter-
mediate in the synthesis of 13), was also found active in cellular assays, although it was moderately active
in the tubulin polymerization inhibition test. Finally, in order to better understand the SAR of disubsti-
tuted analogues of 1, two other position isomers (10 and 14), were synthesized and evaluated for their
biological activities. It was noted that the 7-hydroxysubstituted analogue 13 was more potent than the
5-hydroxysubstituted analogue 10. In conclusion, this work has allowed the identification of biologically
potent CA-4 analogues (13 and 28) and also contributes to a better understanding of the SAR of 1.
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 22 February 2008
Revised 29 May 2008
Accepted 4 June 2008
Available online 7 June 2008
Keywords:
Disubstituted 3-aroylindoles
BPR0L075
Combretastatin A4
Tubulin polymerization
Cytotoxicity
B16 melanoma cells
EA.hy 926 endothelial cells
Antivascular agents
1. Introduction
the colchicine domain, the vinca alkaloid site, and the paclitaxel
site.1
Microtubules are key components of the cytoskeleton and play
an essential role in all eukaryotic cells in the development and
maintenance of cell shape, in the transport of vesicles, mitochon-
dria and other components throughout cells, in cell signaling,
Tumour vasculature is an important and highly interesting tar-
get for anticancer therapy because of the requirement of the tumor
cells for a functional network of blood vessels and also because tu-
mor vasculature is profoundly different compared to normal blood
vessels.2 Two main strategies have been developed to target tumor
vasculature, the antiangiogenesis approach that prevents the for-
mation of new vessels in tumors, and the antivascular approach
that aims at disrupting the existing tumor vasculature. Compounds
that selectively target the pre-existing tumor vasculature and
cause damage to the endothelial cell layer are able to stop the tu-
mor blood supply and waste removal leading to tumor necrosis.
These compounds are called ‘vascular disrupting agents’ (VDA)
and several of them are currently undergoing phase I–III clinical
trials, most often in combination therapies with cytotoxic agents.3
In the vascular disrupting agents, combretastatin A-4 (CA-4),
first isolated from the bark of the South African bush willow tree
and in cell division and mitosis. Microtubules are composed of a-
tubulin and b-tubulin heterodimers that form highly dynamic
polymers. The central role of microtubules in the process of sepa-
rating duplicated chromosomes before cell division makes them a
particularly important target for anticancer drugs. Indeed, many
compounds which interfere with the dynamic of microtubules
are active anticancer drugs. Microtubule active drugs generally
bind to one of the three main classes of sites on tubulin, that is,
* Corresponding authors. Tel.: +33 1 53 73 98 12; fax: +33 1 40 46 96 58.
E-mail addresses: sylvie.michel@univ-paris5.fr (S. Michel), xavier.cachet@univ-
paris5.fr (X. Cachet).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.002

N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
7495
Combretum caffrum Kuntze (Combretaceae), emerged as lead com-
pound which displayed strong inhibitory activity against tubulin
polymerization.4,5 The corresponding water-soluble disodium
phosphate prodrug of CA-4 (CA-4P, brand name: ZybrestatTM) rep-
resents the lead compound in the group of tubulin binding small
molecules possessing tumor vascular disrupting activity. Devel-
oped by the Oxigene company, CA-4P recently entered phase II/
III studies in the US in anaplastic thyroid cancer.3e,6,7
Recently, the 6-methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-in-
dole 1 (BPR0L075), was designed as a heterocyclic analogue of
CA-4 (Fig. 1). Compound 1 was shown to be a potent inhibitor of
tubulin polymerization and binds tubulin at the colchicine site.8,9
It exhibits a strong cytotoxic activity (in the nanomolar range)
against a panel of human cancer cell lines, including multidrug-
resistant cells.10 This compound was also found to exert significant
antitumor activity in several human xenografted tumors and has
recently been selected for further preclinical development as an
antimitotic anticancer drug.10,11
We recently reported the in vitro activity of 1 on the morphol-
ogy of endothelial cells (rounding up at nanomolar concentra-
tions), suggesting its potential in vivo antivascular activity.9 Our
previous studies also disclosed a good correlation between the
rounding up effect of 1 on endothelial cells and its inhibition of
tubulin polymerization.9
Compound 1 and CA-4 share several common structural fea-
tures. Both possess two aryl rings linked by a two-carbon chain,
including an identical 3,4,5-trimethoxyphenyl ring, and a second
aromatic ring bearing a methoxy group (Fig. 1). Structure–activity
relationships (SAR) in the 3-aroylindole series have been exten-
sively studied, particularly modifications at the N1-position and
at the carbonyl bridge.8,9,12 An unsubstituted N1-position and a
sp2 center (carbonyl or thiocarbonyl9) are structural requirements
for potent cytotoxic and tubulin polymerization inhibitory activi-
ties,8,9 but some modifications are tolerated in these positions
without loss of potency (for example, replacement of the carbonyl
bridge by a methylene8,9 or a sulfur12,13; substitution at the N1-po-
sition by a phenyloxycarbonyl9,12 or a benzoyl12 group). Specific
examples include compounds 2–6 (Fig. 2).
Introduction of a methyl group at the C2-position, exemplified
by compound 7, led to a substantial improvement in cytotoxic
and antitubulin activities.8 In addition, in 2001, the Pinney14 and
the Flynn15 groups reported concomitantly the synthesis of 2-
phenylindole 8 that displayed strong tubulin polymerization inhib-
itory activities. However, this 3-aroylindole derivative was
designed (and is still considered) as a cis-restricted analogue of
CA-4, with an ethene bridge as part of a heterocycle (Fig. 2). Con-
cerning the substitution of the benzene ring of the indole nucleus,
monosubstituted indoles, mainly in positions 5 and 6 were inves-
tigated.8,9,12 These SAR studies underlined the critical role of the
methoxy group linked at the position 6 of the indole nucleus. Fur-
thermore, it mimics the p-methoxy group of CA-4, which is also
Figure 2. Selected analogues of BPR0L075.
known to play a crucial role in the activities of 1, particularly on
the ability to inhibit tubulin polymerization.8,9,12 SAR studies car-
ried out by Liou and coworkers also emphasize that the 6-methoxy
group can be replaced by another electron donating group (i.e., p-
dimethylamino and methyl groups) without unfavorable effects on
biological activities.12 In contrast, SAR of 3-aroylindoles containing
disubstituted indoles have not been studied in detail, probably due
to the difficulties encountered in the synthesis of the correspond-
ing indole precursors. The synthesis and biological evaluation of
a short series of 5,6-disubstituted-3-aroylindoles 9–11 (Fig. 3)
were reported by J.-P. Liou et al.8,12 The three compounds possess
a methoxy substituent in the 6-position and 10, particularly, an
additional hydroxyl group at the C5 position which could mimic
the 3-hydroxyl group on ring B of CA-4. Surprisingly, these ana-
logues were found to exhibit weak antimitotic and cytotoxic activ-
ities.10,14 Moreover, Duan et al. showed that substitution at the
7-position of indole with a bulky acetylene group (compound 12,
Fig. 3) led to a dramatic reduction in potency.16
Taking into account the above-mentioned SARs in the 3-aroylin-
dole series, we decided to explore the possibility of introducing a
hydroxyl group at the 7-position of the indole nucleus and to eval-
uate the influence of this modification on cytotoxic and antitubulin
Figure 1. BPR0L075, CA-4, CA-4P, and CA-1.
Figure 3. Structures of 5,6- and 6,7-disubstituted 3-aroylindoles.

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N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
activities. Thus, we now report our studies on the synthesis and the
biological evaluation of the 7-hydroxy-6-methoxy-3-(30,40,50-trim-
ethoxybenzoyl)-1H-indole (13).
led to a mixture of isomeric indoles 19a and 19b (a ratio of
55:45 was estimated by 1H NMR) which proved difficult to resolve.
Each of the two isomers was unambiguously identified on the basis
of HMBC and NOESY experiments.19 Particularly, the NOESY spec-
trum of 19a showed a strong correlation signal between the 5-
methoxy protons at d 3.88 ppm and the H-4 at d 6.91 ppm, whereas
for 19b, a NOESY coupling was observed between the 6-methoxy
signal at d 3.99 ppm and H-7 at d 7.54 ppm.
Compound 13 was found to inhibit tubulin polymerization at
low concentrations. In order to compare properly our results with
those already reported for 5,6-disubstituted analogues of 1, com-
pound 10, previously synthesized and reported by J.-P. Liou et
al.12 was resynthesized, using a new and efficient short-step meth-
od for the preparation of the indole intermediate precursor of 10.
Moreover, the new isomeric precursor 6-hydroxy-5-methoxydi-
substituted indole was concomitantly obtained in the same step.
Both precursors were further coupled with the 3,4,5-trim-
ethoxybenzoyl fragment and submitted to partial or total depro-
tection to afford five new 5,6-disubstituted 3-aroylindoles,
particularly the new analogue 14 and the desired compound 13
(Fig. 3).
Finally, seven new disubstituted 3-aroylindoles were synthe-
sized (five in the 5,6-disubstituted series and two in the 6,7-series)
and evaluated for their antiproliferative activities against murine
B16 melanoma cells, their capacity to inhibit tubulin polymeriza-
tion, and their ability to induce morphological changes on endo-
thelial cells (EA.hy 926).17 Our results provide new interesting
and important SAR in the series of disubstituted 3-aroylindoles,
and have allowed the identification of new biologically potent
CA-4 analogues.
In order to facilitate the chromatographic separation of both in-
doles, the latter mixture was directly treated with an excess of
methanesulfonylchloride in presence of Et3N and the correspond-
ing O-mesylates 20a and 20b were thus easily obtained pure in
47% and 38% isolated yield, respectively, from the indole 18.
Both indole mesylates were further selectively acylated at the
3-position under Friedel-Crafts conditions, using the 3,4,5-trim-
ethoxybenzoylchloride as electrophilic reactant (Scheme 2). The
corresponding coupling products were obtained in moderate to
good yields, depending on the substitution pattern of the starting
indole. Thus, the coupling step proceeded in 76% yield for the in-
dole 20a, which bears a methoxy group at the 5-position, and in
only 35% yield for the indole with a methoxy at the 6-position
(20b). The different protecting groups of the 3-aroylindoles 21a,b
were cleaved in two successive steps. First, the N-phenylsulfonyl
group was selectively removed by treatment with LiOH in metha-
nol at room temperature, to afford the free NH 3-aroylindoles 22a
or 22b in 73% and 76% yield, respectively. Thereafter, various con-
ditions of deprotection of the O-mesylate esters were tested. Under
classical basic conditions (KOH or K2CO3) only degradation prod-
ucts were formed. Finally, this step was cleanly achieved under
transesterification conditions using a large excess of magnesium
methoxide20 in refluxing methanol, to give the expected deprotec-
ted compounds 14 and 10 in 67% and 65% yield, respectively.
2. Chemistry
2.1. Synthesis of 5,6-disubstituted 3-aroylindoles
A previous publication of the synthesis of the 3-aroylindole 10
involved an acylation of 5-benzyloxy-6-methoxy-1H-indole with
3,4,5-trimethoxybenzoylchloride in the presence of EtMgBr, ZnCl2,
and AlCl3 and subsequent removal of the benzyl protecting group
under catalytic hydrogenation conditions.12 The aforementioned
indole could be prepared from isovanillin in four steps, including
a reductive cyclization of the corresponding 2-nitro-b-nitrosty-
rene, with a moderate overall yield of 28%. Moreover, the 6-benzyl-
oxy-5-methoxy-1H-indole isomer was similarly obtained from
vanillin in an overall yield of 27%.18 We have developed a new
efficient and simple method for the direct synthesis of the two
5,6-disubstituted indoles, as convenient precursors of the 3-aroy-
lindoles 10 and 14.
The starting material of this synthesis is the non-commercial
5,6-dimethoxy-N-benzenesulfonyl-1H-indole 18. This compound
was prepared on a gram-scale in a 73% overall yield from the anil-
line 15, according to a modified Bischler strategy previously devel-
oped in our laboratory (Scheme 1).9
3,4-Dimethoxyaniline 15 was quantitatively transformed into
its N-phenylsulfonyl derivative 16 by treatment with phenylsulfo-
nyl chloride, then alkylated by bromoacetaldehyde diacetal to give
17 (Scheme 1). Finally, the indole cyclization was performed in the
presence of boron trifluoride etherate and afforded 18 in 80% yield.
The mono-O-demethylation of 18 was achieved by treatment of
this compound with one equivalent of BBr3 in CH2Cl2 at ꢀ15 °C.
The reaction did not proceed with significant regioselectivity and
2.2. Synthesis of 6,7-disubstituted 3-aroylindoles
Synthesis of the 3-aroylindole 13 involves preparation of the 7-
hydroxy-6-methoxy-1H-indole with suitable protecting groups. In-
dole 27 was first synthesized according to a modified Fukuyama et
al.21 procedure. Particularly, O-mesylate ester instead of O-benzyl
ether was used to protect the phenol function, in order to allow
some comparison of the biological activities between this series
and the 5,6-disubstituted series (Scheme 3).
Thus, nitration of isovanillin 23 with fuming nitric acid22 in ace-
tone at ꢀ40 °C afforded the expected 2-nitro isomer 2421,22 as the
minor product (18% yield), while the 6-nitro isomer was identified
as the major product (31% yield). The use of NO2BF4 as nitrating
agent21 did not improve the yield, because both reactions suffered
from difficulties in purifying the reaction products. Mesylation of
24 with methanesulfonylchloride in the presence of triethylamine
in CH2Cl2 at 0 °C gave the 3-O-mesylate ester 25 in 83% yield. This
compound was treated under modified Henry’s conditions21
(CH3NO2, LHMDS in THF) to afford the 2-nitro-b-nitrostyrene 26
in 74% yield. The formation of the indolic ring was achieved in
54% yield, via a reductive cyclization under catalytic hydrogenation
conditions. Further acylation of the indole 27 with 3,4,5-trim-
ethoxybenzoylchloride in the presence of EtMgBr, ZnCl2, and
Scheme 1. Synthesis of N-phenylsulfonyl-5,6-dimethoxy-1H-indole (18). Reagents and conditions: (a) PhSO2Cl, pyridine, CH2Cl2, rt, 100%; (b) BrCH2CH(OEt)2, NaH, anhyd
DMF, reflux, 94%; (c) BF3ꢁEt2O, CH2Cl2, rt, 80%.

N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
7497
Scheme 2. Synthesis of 5,6-disubstituted 3-aroylindoles (10 and 14). Reagents and conditions: (a) BBr3, CH2Cl2, ꢀ15 °C; (b) MsCl, Et3N, rt, 15 min; (c) (OCH3)3PhCOCl, AlCl3,
CH2Cl2, rt, 2–6 h; (d) LiOH (2–3 equiv), MeOH, rt, 15 min–2 h; (e) Mg(OCH3)2 (1.4 M), MeOH, reflux, 12–24 h.
Scheme 3. Synthesis of 6,7-disubstituted 3-aroylindole (13). Reagents and conditions: (a) fuming HNO3, acetone, ꢀ40 °C, 1 h, 18%; (b) CH3SO2Cl, Et3N, CH2Cl2, 0 °C, 30 min,
83%; (c) i—CH3NO2, LHMDS in toluene, THF, ꢀ78 °C, 2.5 h; ii—AcONa, Ac2O, 60 °C, 3.5 h, 74%; (d) H2, 10% Pd/C, DMF/MeOH (1:3), rt, 16.5 h, 54%; (e) EtMgBr, ZnCl2,
(OCH3)3PhCOCl, AlCl3, CH2Cl2, rt, 7 h, 19%; (f) LDA, THF, ꢀ78 to 0 °C, 30 min, 100%.
AlCl323 afforded the 3-aroylindole 28 in low yield (19%). Finally, the
mesylate protecting group was cleanly removed using LDA at
ꢀ78 °C in THF to afford 13 in quantitative yield.
that induced a rounding-up of the cells was 0.047 lM for a 2 h
exposure time. For the mesylic ester 28, a slight decrease in tubulin
inhibition was observed (ITP = 2.7), in comparison with that of 13.
However, 28 was slightly more cytotoxic than 13, with an IC50 va-
lue of 0.17
observation that 28 could cause the rounding up of endothelial
cells in the nanomolar concentration range (0.013 M).
Figure 4 depicts representative changes in the morphology of
endothelial cells (EA.hy 926) after a brief 2 h exposure time to
reference compounds and to newly synthesized combretastatin
analogues 13 and 28. Control cells exposed to the solvent alone
(1% DMSO) did not cause a rounding up of the endothelial cells.
As expected, for the reference compounds CA-4 and compound
1 (BPR0L075), a rounding up of the cells was clearly observed
at concentrations as low as 0.008 and 0.007 lM, respectively.
Compounds 13 and 28 presented clear rounding up activity
(Fig. 4).
Other compounds 14, 21a,b, and 22a,b were either weak inhib-
itors of tubulin polymerization (14 and 21a,b) or inactive (22a,b).
However, compounds 14 and 21a,b exhibited inhibitory effects
on the proliferation of B16 melanoma cells, but these effects were
lM. The most surprising and interesting result was the
3. Biological evaluation
l
The biological activities of the compounds are summarized in
Table 1 and were evaluated using three complementary assays,
that is, the inhibition of tubulin polymerization, cytotoxicity
against murine B16 melanoma cells, and the induction of a rapid
morphological change of EA.hy 926 endothelial cells (rounding
up), which is considered a useful model of potential in vivo anti-
vascular effects.24 BPR0L075 (1) was used as the reference standard
in the present study, thus allowing a direct comparison with its
disubstituted analogues.
Within the series of newly synthesized compounds, only com-
pounds 13 and 28 presented significant biological activities. Com-
pound 13 was the best inhibitor of tubulin polymerization
reported to date within the series of disubstituted 3-aroylindoles
and was nearly as potent as the reference 1 with an ITP value of
1.3.25 In terms of cytotoxicity against B16 melanoma cells, the
IC50 value was about 18-fold higher than that of the reference com-
observed only in the micromolar range (5.07, 3.14, and 3.29
respectively). Furthermore, a morphological rounding up effect
was noted for 21a at a relatively low concentration (1.30 M), as
depicted in Figure 4. It is interesting to note that the good morpho-
lM,
pound 1, but was still in the submicromolar range (IC50 = 0.24 lM).
Both activities were well correlated with a potent morphological
effect on EA.hy 926 endothelial cells, and the lowest concentration
l

7498
N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
Table 1
Finally, the 5-hydroxy-6-methoxy-1H-indole 10 showed very
similar biological activities to those previously described in the liter-
ature.12 This molecule also exhibited low micromolar cytotoxicity
Inhibition of tubulin polymerization, cytotoxicity on B16 melanoma cells and
morphological effects on EA.hy 926 endothelial cells of disubstituted 3-aroylindoles
Compound
ITPa,b (IC50 compound Cytotoxicity
Morphological activity on
against B16 melanoma cells (IC50 = 12 lM vs 0.013 lM for 1), as well
endothelial cellsd
(lM)
c
IC50 colchicine)
IC50
(l
M)
as no significant effects on the morphology of endothelial cells.
This study provides new SAR information, particularly regarding
the tolerance for substitution at the 7-position. As mentioned in Sec-
tion 1, the previously reported analogues of BPR0L075, substituted
at either position 5 or 7 of the indole nucleus, were found to loose
the antitubulin and cytotoxic activities of the parent compound.
Particularly, substitution at the 7-position with a bulky acetylene
group was not tolerated. Results obtained with compound 13 indi-
cate that position 7 of 1 can accommodate a hydroxyl without a dra-
matic loss in biological activities. However, it should be mentioned
that compound 13 was slightly less potent than the parent com-
pound 1. Apart from quite obvious structural differences between
both the 3-aroylindole and the stilbene skeletons, we hypothesize
that the indolic nitrogen atom may also play a role in the SAR. In-
deed, we found that 13 was sensitive to air oxidation and quickly
turned brown on standing without precautions. This finding may
suggests that a O-quinoneimine type derivative was formed upon
oxidation of the 2-aminophenol structure. Similar observations
were noted in the case of combretastatin A1 (CA-1), a 2-hydroxyl-
ated analogue of CA-4 (Fig. 1). Although CA-1 was approximately
200- to 300-fold less cytotoxic than CA-4,4b this compound (pre-
cisely its disodium phosphate diester prodrug, designated as
OXi4503), demonstrated a greater in vivo antitumor activity than
CA-4P26 and is currently undergoing phase II clinical trials.7
BPR0L075 (1) 0.4e
0.013 0.004
0.031 0.003
0.003 0.001
12.18 5.06
0.24 0.14
5.07 0.91
3.14 2.17
3.29 1.15
32.42 2.24
13.67 1.40
0.17 0.05
0.007
0.003
0.008
17.49
0.047
8.74
Colchicine
CA-4
1012
13
1.0f
0.7g
31e
1.3
14
12
21a
18
1.30
21b
22a
22b
28
15
10.86
28.71
14.35
0.013
Inactiveb
Inactiveb
2.7
a
Inhibition of tubulin polymerization (ITP) is the ratio of the IC50 value of a
synthesized compound over the IC50 value of colchicine, used as the reference.
b
Compounds were designated as ‘inactive’ when the measured ratio was greater
than 40.
c
IC50 SEM of 3–7 independent triplicate experiments (B16 melanoma cells line).
Morphological activity (rounding up) on modified HUVEC cells (EA.hy 926) is
d
expressed as the lowest concentration (
lM) at which cell rounding up was observed
after a 2 h incubation period with the test compound. Experiments done in triplicate.
e
Experimental data. Values of IC50 from Ref. 12: 2.8 0.3
M for compound 10.
IC50 (colchicine) varies from 1.8 to 6.7 M for the different experiments
lM for BPR0L075 and
30
4 l
f
l
according to the tubulin concentration.
g
Value of IC50 from Ref. 32: 2.2 0.2 lM.
logical activity of compound 21a, is not correlated with a strong
antitubulin activity.
Figure 4. Morphological effects of combretastatin A4 (CA-4) analogues. Endothelial cells (EAhy 926 cells) were exposed to the test compounds and incubated for 2 h at the
indicated concentrations. Representative photographs were taken at a magnification of 360ꢂ.

N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
7499
The relationship between the chemical structure and the in-
creased morphological activity of the 7-O-mesylate 28 deserves
further investigation. Indeed, 28 displayed a noteworthy effect on
endothelial cell shape, at a similar concentration to that of the ref-
erence compounds CA-4 and 1. Moreover, among all the new
disubstituted 3-aroylindoles synthesized in this study, 28 was
the most cytotoxic against B16 melanoma cells. However, these ef-
fects were not well correlated with inhibition of tubulin polymer-
ization, that was somewhat weaker compared to 1 and 13.
Contrary to 13, compound 28 could be isolated as a stable crystal-
line form and it would be therefore interesting to determine
whether 28 could act as a prodrug of 13.
spectra were performed at the Laboratoire de Spectrométrie de
Masse (I.C.S.N./C.N.R.S., Gif sur Yvette, France). Elemental analyses
were performed at the Laboratoire de microanalyses (Université
Paris 7 - Denis Diderot, Paris, France). Flash column chromatogra-
phy was done using silica gel (SDS 60 ACC 35–70 lM). The reac-
tions were monitored by thin-layer chromatography (TLC) using
Merck Kieselgel 60 F254 silica gel; zones were detected visually
under ultraviolet irradiation (254 and 366 nm) and read after
spraying with sulfuric vanillin followed by heating. All solvents
were dried according to standard procedures. All reagents were
used as purchased without further treatment unless otherwise
stated.
Introduction of a hydroxyl or a mesylic ester group at the 5-po-
sition of 1 (compounds 10 and 22b, respectively) is deleterious for
both antitubulin and cytotoxic activities, which confirms previous
observations.8,12 Shifting the methoxy group from position 6 to 5 of
the indole ring (compounds 14 and 22a), also affect biological
activities, but, surprisingly, 14 was about twofold more active in
all the three tests than its isomer 10.
Finally, for analogues containing a N-phenylsulfonyl protecting
group (i.e., 21a,b), the SARs remains somewhat unclear, particularly
since the biological activities were not obviously correlated (e.g.,
poor antitubulin activity and good morphological effect for 21a or
cytotoxicity not correlated with antitubulin activity for both com-
pounds). However, these findings suggest that other mechanisms
of action are involved, particularly with regard to the morphological
activity on endothelial cells (e.g., actin polymerization, signaling
pathways, or interaction with microtubule associated proteins).
5.1. N-Benzenesulfonamide-3,4-dimethoxyphenylaniline (16)
To a solution of 4-aminoveratrole 15 (8.14 g, 53.2 mmol) in a 2/
8 mixture of pyridine and anhydrous CH2Cl2 (120 mL), benzenesul-
fonyl chloride (8.18 mL, 63.8 mmol) was slowly added at 0 °C. After
5 min of stirring at room temperature, the reaction mixture was
evaporated to dryness in vacuo and the residue was taken up with
CH2Cl2 (50 mL). The organic solution was washed with water, 10%
aqueous HCl, water and brine, dried over MgSO4, and concentrated
in vacuo to give 15.6 g of analytically pure 16 as transparent crys-
tals (quantitative yield). Recrystallization from a 9:1 mixture of
CH2Cl2 and MeOH gave prismatic transparent crystals. Mp 127–
130 °C. 1H NMR (CDCl3) d 3.73 (s, 3H, 3OCH3); 3.79 (s, 3H,
4OCH3); 6.56 (dd, J = 8.5 Hz, J = 2.4 Hz, 1H, H6); 6.66 (s, J = 8.5 Hz,
1H, H5); 6.68 (s, J = 2.4 Hz, 1H, H2); 7.16 (br s, D2O exch., 1H,
0
0
0
0
0
NH); 7.41 (m, 2H, H;3 -5 ); 7.52 (m, 1H, H4 ); 7.74 (m, 2H, H2 -6 );
13C NMR (CDCl3) d 55.9 (3OCH3); 56.0 (4OCH3); 108.0 (C2); 111.3
4. Conclusion
0
0
0
0
(C5); 115.8 (C6); 127.3 ðC2 -6 Þ; 128.9 ðC3 -5 Þ; 129.2 (C1); 132.9
0
0
ðC4 Þ; 138.8 ðC1 Þ; 147.4 (C4); 149.2 (C3); MS (ZQ2000/ES+) m/z
316 [M+Na]+; HRMS (ES+) (C14H16NO4S, [M+H]+): found:
294.0802; calcd: 294.0800.
In the present study, 7 new disubstituted 3-aroylindoles were
synthesized and evaluated in terms of inhibition of tubulin polymer-
ization, cytotoxicity against the B16 melanoma cancer cell line, and
morphologicaleffectson endothelial cells. Thisworkcontributesto a
better understanding of the SARs of 6-methoxy-3-(30,40,50-trim-
ethoxybenzoyl)-1H-indole (BPR0L075, 1). Particularly, we demon-
strated that, contrary to the 5-position, the 7-position of the indole
nucleus can accommodate a hydroxyl group without dramatic loss
ofcytotoxicand inhibitionofpolymerizationactivities. The7-hydro-
xy-substituted analogue of 1 (compound 13) was shown to be more
potent than the 5-hydroxy-substituted analogue 10. Furthermore,
the corresponding 6-O-mesylate 28 displayed a strong activity in
the endothelial cell morphology assay. Although 28 was not a strong
inhibitor of tubulin polymerization, morphological effect was ob-
served in the low nanomolar range, similar to those of BPR0L075
and CA-4. Compound28 thereforedeserves furtherin vivo investiga-
tions to fully assess its potential antitumor antivascular activity and
also to elucidate its molecular mechanism of action.
5.2. N-(2,2-Diethoxyethyl)-N-(3,4-dimethoxyphenyl)
benzenesulfonamide (17)
To a solution of 16 (1.1 g, 3.75 mmol) in anhydrous DMF
(10 mL), NaH (216 mg, 4.50 mmol) was added at room tempera-
ture. The suspension was stirred for a few minutes, and then,
bromoacetaldehyde diethyl acetal (846 lL, 5.60 mmol) was added
and the mixture was warmed at 110 °C. After 24 h, another
1.87 mmol of NaH (90 mg) and bromoacetaldehyde diethyl acetal
(282 lL) were added and heating was continued for an additional
24 h. The reaction mixture was quenched with MeOH and DMF
was removed under reduced pressure. The residue was taken up
with CH2Cl2 and the organic solution was washed with water
and brine, dried over MgSO4, and concentrated in vacuo to yield
1.4 g of analytically pure 17 (91%) as an amber oil. 1H NMR (CDCl3)
d 1.11–1.14 (m, 6H, 2ꢂ CH3); 3.48 (m, 2H, CH2CH3); 3.61 (d,
J = 5.5 Hz, 2H, –CH2–); 3.62 (m, 2H, CH2CH3); 3.69 (s, 3H, 3OCH3);
3.84 (s, 3H, 4OCH3); 4.60 (t, J = 5.5 Hz, 1H, CH); 6.51 (d, J = 2.0 Hz,
1H, H2); 6.55 (dd, J = 8.5 Hz, J = 2.0 Hz, 1H, H6); 6.72 (d, J = 8.5 Hz,
5. Experimental
Melting points were determined on a LEICA VM microscope
equipped with a heating stage and are uncorrected. Infrared spec-
tra were obtained with a Nicolet 510 FT-IR apparatus. NMR spec-
tra were recorded at 300 or 400 MHz (1H NMR) and at 75 or
100 MHz (13C NMR) with a AC 300 and a Avance 400 BRUKER
spectrometers; chemical shifts are given in parts per million
(ppm, d) relative to solvent peaks as internal standards (d: CDCl3:
7.27 ppm (1H), 77 ppm. (13C); DMSO-d6: 2.50 ppm (1H), 40.6 ppm
0
0
0
1H, H5); 7.43 (m, 2H, H3 -5 ); 7.55 (m, 1H, H4 ); 7.60 (m, 2H,
0
0
H
2 -6 ); 13C NMR (CDCl3) d 15.2 (2ꢂ CH3); 53.5 (–CH2–); 55.8
(4OCH3); 55.9 (3OCH3); 62.1 (2ꢂ CH2CH3); 100.8 (CH); 110.7 (C5);
0
0
0
0
0
112.5 (C2); 121.2 (C6); 127.8 ðC2 -6 Þ; 128.7 ðC3 -5 Þ; 132.6 ðC4 Þ;
0
132.8 (C1); 138.3 ðC1 Þ; 148.7 (C3+C4); MS (ZQ2000/ES+) m/z 448
[M+K]+, 432 [M+Na]+. Anal. (C20H27NO6S). Found: C, 58.90%; H,
6.93%; N, 3.53%; calcd: C, 58.66%; H, 6.65%; N, 3.42%.
(
13C); acetone-d6: 2.05 ppm (1H), 29.8 and 206.0 ppm (13C)); 1H
and 13C signals were unambiguously attributed by 2D NMR exper-
iments; coupling constants are given in Hertz (Hz, J). Mass spectra
(MS) were measured with a Nermag R 10-10C mass spectrometer
(CI/NH3) or with a ZQ2000 Waters mass spectrometer (ESI); HRMS
5.3. N-Phenylsulfonyl-5,6-dimethoxy-1H-indole (18)
To a magnetically stirred solution of 17 (1.14 g, 2.78 mmol) in
anhydrous CH2Cl2 (10 mL), BF3–Et20 (526 lL, 4.17 mmol) was

7500
N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
added dropwise under argon at 0 °C. The reaction mixture was
quenched after 15 min with a saturated aqueous solution of NaH-
CO3. The aqueous layer was separated and extracted with CH2Cl2.
The combined organic extracts were washed with brine, dried over
MgSO4 and concentrated in vacuo. Flash chromatography using
cyclohexane/EtOAc (7:3) gave 705 mg of 18 (80%) as a white solid.
Recrystallization from a 9:1 mixture of CHCl3 and n-hexane gave
transparent crystals. Mp 130–133 °C. 1H NMR (CDCl3) d 3.86 (s,
3H, 6OCH3); 3.96 (s, 3H, 5OCH3); 6.55 (d, J = 3.6 Hz, 1H, H3); 6.94
pounds were recrystallized as colorless crystals from a 9:1 mixture
of CH2Cl2 and MeOH.
5.8. N-Phenylsulfonyl-6-methanesulfonyloxy-5-methoxy-1H-
indole (20a)
Mp 174–177 °C; IR (NaCl film) m0 (cmꢀ1) 3385, 1474, 1423, 1365,
1221, 1182, 1139, 1108, 1085, 1019, 964, 871, 785, 723; 1H NMR
(CDCl3)
d
3.19 (s, 3H, CH3); 3.88 (s, 3H, 5OCH3); 6.60 (d,
0
0
0
0
0
(s, 1H, H4); 7.39–7.43 (m, 3H, H2 þ H3 -5 ); 7.51 (m, 1H, H4 ); 7.56
J = 3.4 Hz, 1H, H3); 7.07 (s, 1H, H4); 7.45 (m, 2H, H3 -5 ); 7.50–7.58
(s, 1H, H7); 7.82 (m, 2H, H2 -6 ); 13C NMR (CDCl3) d 56.1 (6OCH3);
(m, 3H, H2 þ H4 ); 7.90 (m, 2H, H2 -6 ); 8.01 (s, 1H, H7); 13C NMR
0
0
0
0
0
56.3 (5OCH3); 97.3 (C7); 102.7 (C4); 109.6 (C3); 123.6 (C3a); 125.0
(CDCl3) d 38.2 (CH3); 56.3 (5OCH3); 104.1 (C4); 109.4 (C3); 110.3
0
0
0
0
0
0
0
(C2); 126.5 ðC2 -6 Þ; 129.2 ðC3 ꢀ5 Þ; 129.3 (C7a); 133.7 ðC4 Þ; 138.2
(C7); 126.8 ðC2 -6 Þ; (C2); 148.7 (C5); MS (ZQ2000/ES+) m/z 404
[M+Na]+; HRMS (ES+) (C16H16NO6S2, [M+H]+): found: 382.0421;
calcd: 382.0419.
+
0
ðC1 Þ; 147.2 (C6); 148.2 (C5); MS (CI/NH3) m/z 318 [M+H] , 178.
Anal. (C16H15NO4S, 0.6H20). Found: C, 58.41%; H, 4.90%; N, 4.32%;
calcd: C, 58.55%; H, 4. 99%; N, 4.27%.
5.9. N-Phenylsulfonyl-5-methanesulfonyloxy-6-methoxy-1H-
5.4. N-Phenylsulfonyl-6-hydroxy-5-methoxy-1H-indole (19a)
and N-phenylsulfonyl-5-hydroxy-6-methoxy-1H-indole (19b)
indole (20b)
Mp 143–145 °C; IR (NaCl film) m0 (cmꢀ1) 3144, 3120, 2941, 2836,
1618, 1583, 1529, 1482, 1447, 1365, 1322, 1217, 1178, 1128, 1085,
1015, 972, 867, 816, 754, 727, 684; 1H NMR (CDCl3) d 3.17 (s, 3H,
CH3); 3.97 (s, 3H, 6OCH3); 6.60 (d, J = 3.0 Hz, 1H, H3); 7.45–7.51 (m,
To a solution of 18 (88 mg, 0.28 mmol) in anhydrous CH2Cl2
(5 mL), a molar solution of BBr3 in CH2Cl2 (332 lL, 0.33 mmol)
was added dropwise at ꢀ15 °C. The reaction mixture was immedi-
ately quenched by addition of chilled MeOH (1 mL) and then, a sat-
urated solution of aqueous NaHCO3 was added. The aqueous layer
was separated and extracted with CH2Cl2. The combined organic
extracts were dried over MgSO4, and concentrated in vacuo to af-
ford 19a and 19b in a hardly separable mixture (79 mg, 94%). A ra-
tio of 55:45 was estimated by 1H NMR. Pure analytical samples of
each isomer were obtained by a slow chromatography of the mix-
0
0
0
4H, H4 þ H3 -5 þ H2); 7.58 (m, 1H, H4 ); 7.64 (s, 1H, H7); 7.86 (m,
2H, H2 -6 ); 13C NMR (CDCl3) d 38.0 (CH3); 56.3 (6OCH3); 97.8 (C7);
0
0
0
0
109.0 (C3); 116.6 (C4); 123.5 (C3a); 126.1 (C2); 126.5 ðC2 -6 Þ;
0
0
0
0
129.3 ðC3 -5 Þ; 133.5 (C7a); 134.1 ðC4 Þ; 135.8 (C5); 137.8 ðC1 );
149.5 (C6); MS (ZQ2000/ES+) m/z 404 [M+Na]+. Anal.
(C16H15NO6S2). Found: C, 50.66%; H, 4.21%; N, 3.77%; calcd: C,
50.38%; H, 3.96%; N, 3.67%.
ture on 20–45 lm silica gel (eluent: cyclohexane/EtOAc (8:2)).
5.10. 6-Methanesulfonyloxy-5-methoxy-N -phenylsulfonyl-3-
(30,40,50-trimethoxybenzoyl)-1H-indole (21a)
5.5. N-Phenylsulfonyl-6-hydroxy-5-methoxy-1H-indole (19a)
Mp 154–155 °C (CH2Cl2/n-hexane (9:1), colorless crystals); 1H
NMR (CDCl3) d 3.88 (s, 3H, OCH3); 5.83 (br s, D2O exch., 1H, OH);
6.54 (d, J = 3.5 Hz, 1H, H3); 6.91 (s, 1H, H4); 7.39–7.44 (m, 3H,
To a magnetically stirred suspension of AlCl3 (171 mg,
1.28 mmol) in anhydrous CH2Cl2, 3,4,5-trimethoxybenzoyl chlo-
ride (222 mg, 0.96 mmol) was added at 25 °C and the mixture
was stirred for 15 min to afford a red suspension. A solution of in-
dole 20a (245 mg, 0.64 mmol) in anhydrous CH2Cl2 was added
dropwise and the mixture was stirred at 25 °C for 2 h and poured
onto crushed ice. The aqueous layer was separated and extracted
with CH2Cl2, and the combined organic extracts were washed with
saturated aqueous NaHCO3 and brine, dried over MgSO4, and con-
0
0
0
H3 -5 þ H2); 7.52 (m, 1H, H4 ); 7.60 (s, 1H, H7); 7.85 (m, 2H,
H
2 -6 ); 13C NMR (CDCl3) d 56.2 (OCH3); 99.8 (C7); 102.0 (C4);
0
0
0
0
109.2 (C3); 123.3 (C3a); 124.9 (C2); 126.7 ðC2 -6 Þ; 129.2
0
0
0
0
ðC7a þ C3 -5 Þ; 133.6 ðC4 Þ; 138.3 ðC1 Þ; 144.5 (C6); 144.7 (C5); MS
(ZQ2000/ES+) m/z 342 [M+K]+, 326 [M+Na]+; HRMS (ES+)
(C15H14NO4S, [M+H]+): found: 304.0640; calcd: 304.0644.
centrated in vacuo. Chromatography on 20–45 lm silica gel, using
5.6. N-Phenylsulfonyl-5-hydroxy-6-methoxy-1H-indole (19b)
cyclohexane/EtOAc (6:4) gave 276 mg of 21a (76%) as a yellowish
powder. Recrystallization from a 9:1 mixture of CH2Cl2 and MeOH
gave orange crystals. Mp 216–220 °C; IR (NaCl film) m0 (cmꢀ1) 3113,
3000, 2934, 2832, 1638, 1583, 1532, 1501, 1470, 1408, 1365, 1326,
1233, 1217, 1186, 1155, 1124, 1015, 898, 867, 762, 723; 1H NMR
Colorless oil: 1H NMR (CDCl3) d 3.99 (s, 3H, OCH3); 5.60 (br s,
D2O exch., 1H, OH); 6.53 (d, J = 3.6 Hz, 1H, H3); 7.01 (s, 1H, H4);
0
0
7.41 (d, J = 3.6 Hz, 1H, H2); 7.43 (m, 2H, H3 -5 ); 7.54 (m, 2H,
0
0
H4 þ H7); 7.82 (m, 2H, H2 -6 ); 13C NMR (CDCl3) d 56.4 (OCH3);
(CDCl3) d 3.20 (s, 3H, SO2CH3); 3.94 (s, 6H, 3 -5 OCH3); 3.96 (s, 3H,
0
0
0
0
5OCH3); 3.98 (s, 3H, 4 OCH3); 7.20 (s, 2H, H2 -6 ); 7.50 (m, 2H,
0
0
96.6 (C7); 105.3 (C4); 109.6 (C3); 124.3 (C3a); 125.3 (C2); 126.5
0
0
0
0
0
0
00
00-500
00-600
ðC2 -6 Þ; 129.2 ðC3 -5 þ C7aÞ; 133.7 ðC4 Þ; 138.4 ðC1 Þ; 143.4 (C5);
H3 ); 7.62 (m, 1H, H4 ); 7.89 (s, 1H, H7); 7.96 (m, 2H, H2 );
145.6 (C6); MS (ZQ2000/ES+) m/z 326 [M+Na]+.
8.06 (s, 2H, H4 + H2); 13C NMR (CDCl3) d 38.4 (SO2CH3); 56.3
0
0
40
5
0
0
ð
3 -5 OCH3Þ; 56.4 ( OCH3); 61.1 ð OCH3Þ; 105.3 (C4); 106.6 ðC2 -6 );
00-600
00-500
5.7. N-Phenylsulfonyl-6-methanesulfonyloxy-5-methoxy-1H-
indole (20a) and N-phenylsulfonyl-5-methanesulfonyloxy-6-
methoxy-1H-indole (20b)
110.3 (C7); 119.8 (C3); 127.2 ðC2 Þ; 128.8 (C7a); 129.8 ðC3 Þ;
0
0
0
0
0
133.7 (C2); 133.8 ðC1 Þ; 134.9 ðC4 Þ; 137.0 ðC1 Þ; 137.3 (C6); 142.3
0
0
0
ðC4 Þ; 149.9 (C5); 153.2 ðC3 ꢀ5 Þ; 189.6 (CO); MS (ZQ2000/ES+) m/z
614 [M+K]+, 598 [M+Na]+. Anal. (C26H25NO10S2, 0.5H20). Found:
C, 53.32%; H, 4.53%; N, 2.43%; calcd: C, 53.25%, H, 4.51%; N, 2.39%.
To a solution of the mixture of 19a and 19b (65 mg, 0.99 mmol)
in CH2Cl2 (5 mL), Et3N (280
lL, 2.01 mmol), and methanesulfonyl
chloride (160 L, 2.03 mmol) were successively added at room
temperature. The reaction mixture was quenched after 15 min by
l
5.11. 5-Methanesulfonyloxy-6-methoxy-N -phenylsulfonyl-3-
(30,40,50-trimethoxybenzoyl)-1H-indole (21b)
addition of few drops of aq 10% HCl, and then, was evaporated in
vacuo and purified by chromatography on 20–45
using cyclohexane/EtOAc (6.5:3.5) in order to obtain 168 mg of
20b (38% from 18) and 165 mg of 20a (47% from 18). Both com-
l
M silica gel,
The same procedure was performed with 20b (226 mg,
0.59 mmol), 237 mg (1.78 mmol) of AlCl3 and 205 mg (0.89 mmol)
of 3,4,5-trimethoxybenzoyl chloride. The mixture was in this case,

N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
7501
stirred for 6 h. Chromatography on 20–45
l
m silica gel, using
0.5H20). Found: C 54.11%; H, 4.94%; N, 3.13%; calcd: C, 54.04%; H,
5.00%; N, 3.15%.
cyclohexane/EtOAc (6:4) gave 122 mg of 21b (35%) as a yellowish
powder. Recrystallization from a 9:1 mixture of CH2Cl2 and MeOH
gave yellow transparent crystals. Mp 234–236 °C; IR (NaCl film) m0
(cmꢀ1) 3132, 3004, 2934, 2844, 1630, 1587, 1536, 1505, 1482,
1447, 1412, 1369, 1330, 1241, 1182, 1128, 1089, 1019, 999, 968,
906, 867, 805, 762, 731, 684; 1H NMR (CDCl3) d 3.24 (s, 3H,
5.14. 6-Hydroxy-5-methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-
indole (14)
To a freshly prepared 1.4 M solution of Mg(OCH3)2 in dry MeOH
(3 mL), 22a (40 mg, 0,09 mmol) was added at room temperature
and the reaction mixture was refluxed for 12 h. Solvent was evap-
orated under reduced pressure and the residue was taken up with
EtOAc. The organic solution was washed with saturated aqueous
NH4Cl, brine and dried over MgSO4, and then, concentrated in va-
0
0
0
SO2CH3); 3.92 (s, 6H, 3 -5 OCH3); 3.97 (s, 3H, 4 OCH3); 4.01 (s, 3H,
6OCH3); 7.10 (s, 2H, H2 -6 ); 7.53 (m, 2H, H3 ); 7.64–7.68 (m, 2H,
0
0
00-500
00
000-600
H7 þ H4 ); 7.92 (m, 2H, H2 ); 7.99 (s, 1H, H2); 8.16 (s, 1H, H4);
40
0
13
6
C NMR (CDCl3) d 56.3 ð3 -5 OCH3Þ; 56.5 ( OCH3); 61.0 ð OCH3Þ;
0
0
97.4 (C7); 106.6 ðC2 -6 Þ; 117.9 (C4); 120.5 (C3); 121.5 (C3a); 126.9
00-600
00-500
0
ðC2 Þ; 129.8 ðC3 Þ; 132.1 (C2); 133.7 ðC1 Þ; 133.9 (C7a); 134.9
cuo. Chromatography on 20–45 lm silica gel, using EtOAc gave
ðC4 Þ; 137.1 (C5); 137.4 ðC1 Þ; 142.2 ðC4 Þ; 151.0 (C6); 153.1
22 mg of 14 as a colorless amorphous powder (67%). 1H NMR
0
0 0
00
00
0
+
ðC3 -5 Þ; 188.9 (CO); MS (ZQ2000/ES+) m/z 598 [M+Na] . Anal.
(C26H25NO10S2). Found: C, 54.69%; H, 4.87%; N, 2.63%; calcd: C,
54.25%; H, 4.38%; N, 2.43%.
(CDCl3) d 3.91 (s, 6H, 3 ꢀ5 OCH3); 3.94 (s, 3H, 4 OCH3); 4.02 (s, 3H,
0
0
5OCH3); 5.88 (br s, D2O exch., 1H, OH); 7.00 (s, 1H, H7); 7.12 (s,
0
0
2H, H2 -6 ); 7.59 (d, J = 2.7 Hz, 1H, H2); 7.93 (s, 1H, H4); 8.55 (br s,
0
0
13
D2O exch., 1H, NH); C NMR (CDCl3) d 56.3 ð3 -5 OCH3Þ; 56.4
40
5
5.12. 6-Methanesulfonyloxy-5-methoxy-3-(30,40,50-trimethoxy-
benzoyl)-1H-indole (22a)
( OCH3); 61.0 ð OCH3Þ; 96.5 (C7); 103.0 (C4); 106.3 ðC2 -6 Þ; 116.9
0
0
0
0
(C3); 119.3 (C3a); 130.9 (C7a); 131.8 (C2); 136.0 ðC1 Þ; 140.9 ðC4 Þ;
0
0
144.5 (C5); 144.8 (C6); 153.0 ðC3 -5 Þ; 190.5 (CO); MS (ZQ2000/
ES+) m/z 380 [M+Na]+. Anal. (C19H19NO6). Found: C, 64.45%; H,
5.40%; N, 3.88%; calcd: C, 63.86%; H, 5.36%; N, 3.92%.
To a magnetically stirred solution of 21a (190 mg, 0.35 mmol)
in a 3:4 mixture of MeOH and THF (35 mL), LiOH (17 mg,
0.69 mmol) was added at room temperature. The reaction mixture
was quenched after 15 min by addition of few drops of 10% aque-
ous HCl. The reaction mixture was evaporated in vacuo and the
residue was taken up with CH2Cl2. The organic solution was
washed with brine, dried over MgSO4, and concentrated under re-
duced pressure. Flash chromatography using EtOAc/cyclohexane
(7:3) gave 110 mg of 22a (73%). Recrystallization from a 9:1 mix-
ture of CH2Cl2/MeOH gave yellow crystals. Mp 148–151 °C; IR
(NaCl film) m0 (cmꢀ1) 3327, 3000, 2934, 2832, 1614, 1575, 1497,
1478, 1412, 1361, 1326, 1237, 1178, 1124, 1104, 1073, 999, 968,
871, 832, 766, 735, 672; 1H NMR (CDCl3) d 3.22 (s, 3H, SO2CH3);
5.15. 5-Hydroxy-6-methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-
indole (10)12
The same procedure was performed with 22b (66 mg,
0.15 mmol), though the reaction mixture was refluxed for 24 h.
Flash chromatography using EtOAc gave 35 mg of 10 as a colorless
amorphous powder (65%). 1H NMR (DMSO-d6) d 3.73 (s, 3H,
0
0
0
4 OCH3); 3.79 (s, 3H, 6OCH3); 3.82 (s, 6H, 3 ꢀ5 OCH3); 6.95 (s, 1H,
0
0
H7); 7.02 (s, 2H, H2 -6 ); 7.63 (s, 1H, H4); 7.80 (d, J = 2.8 Hz, 1H,
H2); 8.68 (br s, D2O exch., 1H, OH); 11.62 (br s, D2O exch., 1H,
0
0
0
0
0
3.92 (s, 6H, 3 -5 OCH3); 3.95 (s, 3H, 4 OCH3); 4.00 (s, 3H, 5OCH3);
NH); C NMR (DMSO-d6) d 56.1 (6OCH3); 56.4 ð3 -5 OCH3Þ; 60.5
13
40
0
0
0
0
7.11 (s, 2H, H2 -6 ); 7.47 (s, 1H, H7); 7.76 (s, 1H, H2); 8.08 (s, 1H,
ð OCH3Þ; 95.7 (C7); 106.4 ðC2 ꢀ6 Þ; 106.8 (C4); 115.0 (C3); 120.2
H4); 8.90 (s, D2O exch., 1H, NH); 13C NMR (CDCl3) d 38.1 (SO2CH3);
(C3a); 130.9 (C7a); 134.1 (C2); 136.5 ðC1 Þ; 140.2 ðC4 Þ; 144.1 (C5);
0
0
0
0
40
5
56.3 ð3 -5 OCH3); 56.4 ( OCH3); 61.0 ð OCH3Þ; 104.9 (C4); 106.3
146.8 (C6); 153.0 ðC3 ꢀ5 Þ; 189.2 (CO); MS (ZQ2000/ES+) m/z 380
0
0
ðC2 -6 Þ; 108.0 (C7); 116.5 (C3); 125.7 (C3a); 129.7 (C7a); 134.2 (C2);
[M+Na]+.
0
0
0
0
0
0
135.6 ðC1 Þ; 136.4 (C6); 141.1 ðC4 Þ; 148.3 (C5); 153.1 ðC3 -5 Þ;
190.3 (CO); MS (ZQ2000/ES+) m/z 458 [M+Na]+; HRMS (ESꢀ)
(C10H20NO8S, [MꢀH]+): found: 434.0906; calcd: 434.0910.
5.16. 3-Hydroxy-4-methoxy-2-nitrobenzaldehyde (24)21,22
Compound 24 was synthesized according a previously de-
scribed procedure.22 Isovanilline 23 (3 g, 19.7 mmol) was nitrated
in presence of 1.2 equiv of fuming nitric acid (2 mL) to afford
1.26 g of 3-hydroxy-4-methoxy-6-nitrobenzaldehyde (31%) and
700 mg of 24 (18%), after purification by flash chromatography
using cyclohexane/acetone (7:3). Mp 142–144 °C (EtOH) (lit.
143–145 °C)22; IR (NaCl film) m0 (cmꢀ1) 3216, 2848, 1673, 1285;
1H NMR (CDCl3) d 4.14 (s, 3H, OCH3); 7.14 (d, J = 8.4 Hz, 1H, H6);
7.50 (d, J = 8.4 Hz, 1H, H5); 10.10 (s, 1H, CHO); 10.50 (s, 1H, OH);
MS (ZQ2000/ES+) m/z 236 [M+K]+, 220 [M+Na]+.
5.13. 5-Methanesulfonyloxy-6-methoxy-3-(30,40,50-trimethoxy-
benzoyl)-1H-indole (22b)
To a magnetically stirred solution of 21b (93 mg, 0.16 mmol) in
a 1:2 mixture of MeOH and THF (25 mL), LiOH (8 mg, 0.32 mmol)
was added at room temperature. The reaction mixture was
quenched after 2 h by addition of few drops of 10% aqueous HCl.
The reaction mixture was evaporated in vacuo and the residue
was taken up with CH2Cl2. The organic solution was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
Chromatography on 20–45
l
m silica gel using EtOAc gave 53 mg of
5.17. 4-Methoxy-3-methanesulfonyloxy-2-nitrobenzaldehyde
(25)
22b (76%). Recrystallization from a 9:1 mixture of CH2Cl2 and
MeOH gave yellowish needles. Mp 189–193 °C; IR (NaCl film) m0
(cmꢀ1) 3350, 3124, 3004, 2937, 2836, 1571, 1525, 1501, 1459,
1412, 1357, 1322, 1272, 1233, 1159, 1124, 1065, 999, 968, 875,
832, 809, 770, 731; 1H NMR (CDCl3) d 3.21 (s, 3H, SO2CH3); 3.79
To a solution of 24 (256 mg, 1.29 mmol) in anhydrous CH2Cl2
(7 mL), Et3N (260
lL, 2.62 mmol) was added at 0 °C, and then,
methanesulfonyl chloride (205
lL, 2.64 mmol) was added 10 min
0
0
0
(s, 3H, 6OCH3); 3.85 (s, 6H, 3 -5 OCH3); 3.90 (s, 3H, 4 OCH3); 6.91
later. The reaction was followed by TLC (cyclohexane/acetone
(1:1)) and quenched after 30 min by adding 30 mL of distillated
water. The aqueous layer was separated and extracted with CH2Cl2.
The combined organic extracts were washed with brine, dried over
MgSO4, filtered and concentrated under reduced pressure. The res-
idue was purified by flash chromatography using cyclohexane/
EtOAc (3:2) to give 295 mg of 25 (83%) which was recrystallized
0
0
(s, 1H, H7); 7.05 (s, 2H, H2 -6 ); 7.64 (s, 1H, H2); 8.22 (s, 1H, H4);
9.74 (br s, D2O exch., 1H, NH); 13C NMR (CDCl3) d 38.2 (SO2CH3);
0
0
40
6
56.1 ( OCH3); 56.2 ð3 -5 OCH3Þ; 60.9 ð OCH3Þ; 95.6 (C7); 106.3
0
0
0
ðC2 -6 Þ; 116.5 (C3); 116.9 (C4); 119.3 (C3a); 133.8 (C2); 135.2 ðC1 Þ;
0
0
0
135.3 (C7a); 135.5 (C5); 141.0 ðC4 Þ; 149.3 (C6); 152.9 ðC3 -5 Þ;
190.2 (CO); MS (ZQ2000/ES+) m/z 458 [M+Na]+. Anal. (C20H21NO8S,

7502
N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
0
0
40
from EtOH. Mp 133–135 °C; IR (NaCl film) m0 (cmꢀ1) 2848, 1697,
1371, 1290; 1H NMR (acetone-d6) d 3.55 (s, 3H, CH3), 4.19 (s, 3H,
OCH3); 7.68 (d, J = 8.8 Hz, 1H, H6); 8.14 (d, J = 8.8 Hz, 1H, H5);
9.92 (s, 1H, CHO); MS (ZQ2000/ES+) m/z 298 [M+Na]+.
6H, 3 -5 OCH3); 3.96 (s, 3H, 6OCH3); 4.01 ð OCH3Þ; 7.10 (d,
0
0
J = 8.8 Hz, 1H, H5); 7.13 (s, 2H, H2 -6 ); 7.71 (d, J = 2.9 Hz, 1H, H2);
8.30 (d, J = 8.8 Hz, J = 0.4 Hz, 1H, H4); 9.18 (br s, D2O exch., 1H,
0
0
NH); 13C NMR (CDCl3)
d
37,6 (CH3); 56.3
ð
3 -5 OCH3Þ; 57.1
40
6
0
0
( OCH3); 61.0 ð OCH3Þ; 106.3 ðC2 -6 Þ; 109.3 (C5); 117.2 (C3);
5.18. 6-Methoxy-2-nitro-3-(2-nitrovinyl)phenyl
methanesulfonate (26)
122.1 (C4); 123.2 (C7); 123.4 (C3a); 131.2 (C7a); 133.6 (C2); 135.5
0
0
0
0
ðC1 Þ; 141.2 ðC4 Þ; 148.3 (C6); 153.1 ðC3 -5 Þ; 190.1 (CO); MS
(ZQ2000/ES+) m/z 476 [M+K]+, 458 [M+Na]+; HRMS (ESꢀ)
(C10H20NO8S, [MꢀH]+): found: 434.0906; calcd: 434.0910.
To a stirred solution of dry CH3NO2 (720 lL, 13.3 mmol) in dry
THF (10 mL) cooled at ꢀ78 °C were successively added a 1 M solu-
tion of LHMDS in toluene (6.65 mL) and, 10 min later, a solution of
25 (915 mg, 3.33 mmol) in dry THF (25 mL). After stirring for 2.5 h
at the same temperature, the reaction mixture was quenched with
saturated aqueous NH4Cl (100 mL) and extracted with EtOAc. The
combined organic layers were successively washed with water,
brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was dissolved in Ac2O (6 mL), and then, AcO-
Na (90 mg, 1.10 mmol) was added. The reaction mixture was
heated at 60 °C for 3.5 h, then diluted with toluene and concen-
trated in vacuo. The crude product was taken up with CH2Cl2 and
the organic solution was washed with water, brine, dried over
MgSO4, filtered, and evaporated under reduced pressure to afford
787 mg of 26 as an amorphous solid used without further purifica-
tion (74%). 1H NMR (CDCl3) d 3.38 (s, 3H, CH3), 4.07 (s, 3H, OCH3),
7.24 (d, J = 8.9 Hz, 1H, H5), 7.50 (d, J = 13.5 Hz, 1H, Ha), 7.60 (dd,
J = 8.9 Hz,J = 0.4 Hz, 1H, H6), 7.86 (d, J = 13.5 Hz, 1H, Hb).
5.21. 7-Hydroxy-6-methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-
indole (13)
To a solution of 28 (20 mg, 0.046 mmol) in anhydrous THF
(3 mL) cooled at ꢀ78 °C, a 2 M solution of LDA in THF (115
lL,
0.23 mmol) was slowly added and the reaction mixture was stirred
for 5 min at the same temperature, then further 30 min at 0 °C. The
reaction mixture was quenched with saturated aqueous NH4Cl and
extracted with EtOAc. The combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated under re-
duced pressure. The residue was purified by flash chromatography
using cyclohexane/EtOAc (3:7) to give pure 13 as a pink oil
(16.4 mg, 100%). IR (NaCl film) m0 (cmꢀ1) 3332, 2939, 2836, 1575,
1515, 1416, 1360, 1125, 1026; 1H NMR (CDCl3) d 3.90 (s, 6H,
0
0
0
3 -5 OCH3); 3.94 (s, 3H, 6OCH3); 3.98 (s, 3H, 4 OCH3); 5.97 (br s,
D2O exch., 1H, OH); 7.02 (d, J = 8.7 Hz, 1H, H5); 7.12 (s, 2H,
0
0
H2 -6 ); 7.68 (br s, 1H, H2); 7.87 (d, J = 8.6 Hz, 1H, H4); 9.09 (br s,
0
0
13
5.19. 6-Methoxy-7-methanesulfonyloxy-1H-indole (27)
D2O exch., 1H, NH); C NMR (CDCl3) d 56.3 ð3 -5 OCH3Þ; 57.4
40
6
0
0
( OCH3); 61.0 ð OCH3Þ; 106.3 ðC2 -6 Þ; 109.0 (C5); 113.4 (C4);
To a solution of 26 (787 mg, 2.47 mmol) in a 4:1 mixture of
MeOH/DMF (28 mL), 10% Pd/C (263 mg, 0.25 mmol) was added
and the reaction mixture was stirred at room temperature under
hydrogen for 16.5 h. The suspension was filtered on a Celite layer
and concentrated under pressure. The residue was purified by flash
chromatography using cyclohexane/EtOAc (4:1) to afford 27 as a
pale pink solid, recrystallized from a mixture of CH2Cl2 and n-hex-
ane. Mp 130–131 °C; IR (NaCl film) m0 (cmꢀ1) 3408, 2938, 2834,
1636, 1452, 1353, 1325, 1254, 1173, 1087; 1H NMR (CDCl3) d
3.25 (s, 3H, CH3); 3.93 (s, 3H, OCH3); 6.49 (dd, J = 3.1 Hz, J = 2.1
Hz, 1H, H3); 6.87 (d, J = 8.7 Hz, 1H, H5); 7.16 (dd, J = 3.1 Hz,
J = 2.4 Hz, 1H, H2); 7.48 (d, J = 8.7 Hz, 1H, H4); 8.64 (br s, D2O exch.,
1H, NH); 13C NMR (CDCl3) d 37.4 (CH3), 57.2 (OCH3), 102.7 (C3),
107.0 (C5), 119.8 (C4), 123.9 (C7), 125.2 (C2), 125.4 (C3a), 130.5
(C7a), 146.9 (C6); MS (ZQ2000/ES+) m/z 280 [M+K]+, 264 [M+Na]+,
242 [M+H]+; HRMS (ES+) (C10H11NO4NaS, [M+Na]+): found:
264.0308; calcd: 264.0306.
117.0 (C3); 122.7 (C3a); 126.1 (C7a); 131.2 (C7); 133.5 (C2); 136.0
0
0
0
0
ðC1 ); 140.9 ðC4 ); 142.5 (C6); 152.9 ðC3 -5 Þ; 190.5 (CO); MS
(ZQ2000/ES+) m/z 396 [M+K]+, 380 [M+Na]+, 358 [M+H]+; HRMS
(ES+) (C19H20NO6, [M+H]+): found: 358.1287; calcd: 358.1291.
5.22. Tubulin-binding assay
Calf brain tubulin was purified according to the method of She-
lanski et al.27 by three cycles of assembly–disassembly and then
dissolved at a final concentration of 2–3 mg/mL in the assembly
buffer (pH 6.6) containing 0.1 M MES, 0.5 mM MgCl2, 2 mM EGTA,
and 1 mM GTP. Tubulin assembly was monitored and recorded
continuously by turbidimetry at 400 nm in a UV spectrophotome-
ter, equipped with a thermostated cell at 37 °C.28 The IC50 value,
defined as the concentration of inhibitor which decreased by 50%
the maximum assembly rate of tubulin in control cell without test
compound, was determined for each newly synthesized com-
pound. The IC50 of all compounds were compared to the IC50 of col-
chicine (Sigma–Aldrich), measured the same day under the same
conditions. The results are presented as inhibition of tubulin poly-
merization (or ITP), which is the ratio of the IC50 value of a given
synthesized compound to the IC50 value of colchicine (ITP = IC50
test compound/IC50 colchicine).
5.20. 7-Methanesulfonyloxy-6-methoxy-3-(30,40,50-trimethoxy-
benzoyl)-1H-indole (28)
To a suspension of 27 (120 mg, 0.50 mmol) and anhydrous
ZnCl2 (135.6 mg, 1.0 mmol) in dry CH2Cl2 (1.5 mL), a 3 M solution
of EtMgBr in Et2O (215 lL, 0.65 mmol) was added dropwise. The
suspension was stirred for 1 h, then a solution of 3,4,5-trim-
ethoxybenzoyl chloride (230 mg, 1.0 mmol) in dry CH2Cl2
(2.0 mL) was added dropwise. The reaction mixture was stirred
for another 1 h followed by the addition of AlCl3 (67 mg,
0.5 mmol). The resultant suspension was vigorously stirred for
7 h and was quenched with water and extracted with CH2Cl2.
The combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The res-
idue was purified by flash chromatography using cyclohexane/
EtOAc (1:1) and 41.4 mg of pure 28 (19%) was obtained as brown
crystals after crystallization in a mixture of CH2Cl2/MeOH. Mp
209–210 °C; IR (NaCl film) m0 (cmꢀ1) 3372, 2935, 2844, 1580,
1415, 1365, 1125; 1H NMR (CDCl3) d 3.32 (s, 3H, CH3); 3.93 (s,
5.23. Evaluation of cytotoxicity in murine B16 melanoma cells
Murine B16 melanoma cells were grown in DMEM containing
2 mM
L-glutamine, 10% fetal bovine serum, 100 U/mL penicillin,
and 100
lg/mL streptomycin (37 °C, 5% CO2). All 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, CA-4 (synthesized according to Ref. 29) and colchicine
(Sigma–Aldrich) were routinely included in the experiments as ref-
erence control compounds. Exponentially growing B16 cells were
plated onto 96-well plates at 5000 cells per well in 100
ture medium. Twenty-four hours after plating, 100 L of medium
containing the compound of interest (final concentrations ranging
lL of cul-
l

N. Ty et al. / Bioorg. Med. Chem. 16 (2008) 7494–7503
7503
Dark, G. G.; Dicker, A. P.; Eskens, F. A.; Horsman, M. R.; Marmé, D.; LoRusso, P.
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E.; Chaplin, D. J.; Blakey, D. C. Cancer Res. 2003, 63, 1144; (k) Griggs, J.; Metcalfe,
J. C.; Hesketh, R. Lancet Oncol. 2001, 2, 82.
from 0.25 ng/mL to 25 lg/mL, in 10-fold dilutions) was added to
the wells (in triplicate) containing the cells, and 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 test30 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. Results are presented
as percent of controls containing 1% DMSO, which was not cyto-
toxic at this concentration. The concentration of compound that
inhibited cell viability by 50% (inhibitory concentration for 50%
of cells, or IC50) was determined using the GraphPad Prism soft-
ware. Results are presented as means SEM of 3–7 independent
experiments each run in triplicate.
4. (a) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall,
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5.24. Effect on the morphology of transformed HUVEC cells
(EA.hy 926 cells)
7. Oxigene company Web site. Phase II/III Study Assessing ZybrestatTM
(Combretastatin), A Potential New Treatment for Anaplastic Thyroid Cancer.
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8. Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C. C.; Yang,
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To assess the effects of the compounds on the morphology of
endothelial cells, we used the EA.hy 926 cell line which is derived
from the fusion of human umbilical vein endothelial cells (HUVEC)
with the permanent human cell line A549.17 The EA.hy 926 cell line
is considered as one of the best immortalized HUVEC cell lines be-
cause these cells express most of the biochemical markers of
parental HUVEC.31 EA.hy 926 cells, originally obtained from Dr.
Cora-Jean S. Edgell (Pathology Department, University of North
Carolina, Chapel Hill, NC 27599-7525, USA) were used with her
13. Silvestri and coworkers have recently published SAR studies of 3-
arylthioindoles. SAR findings for this series, were found to be markedly
different from those of 3-aroylindoles (a) De Martino, G.; La Regina, G.;
Coluccia, A.; Edler, M. C.; Barbera, M. C.; Brancale, A.; Wilcox, E.; Hamel, E.;
Artico, M.; Silvestri, M. J. Med. Chem. 2004, 47, 6120; (b) La Regina, G.; Edler, M.
C.; Brancale, A.; Kandil, S.; Coluccia, A.; Piscitelli, F.; Hamel, E.; De Martino, G.;
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permission, and were grown in DMEM containing 2 mM
mine, 10% fetal bovine serum, 100 U/mL penicillin, and 100
mL streptomycin (37 °C, 5% CO2). Exponentially growing EA.hy
926 cells were plated onto 96-well plates at 5000 cells/100 L/
well. Twenty-four h after plating, the medium was aspirated, and
100 L of medium containing the test compound was added to
L
-gluta-
lg/
l
l
14. (a) Pinney, K.; Wang, F.; Hadimani, M.; Del Pilar Mejia, M. PCT Int. Appl. WO
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50, 1001.
the well containing the cells (in triplicate) in 10-fold dilutions,
and incubated for 2 h at 37 °C and 5% CO2. After the 2 h-incubation
period, digital photographs were taken of srepresentative center
areas of each well at a magnification of 360ꢂ. Combretastatin A4
(CA-4) and colchicine were routinely included in the experiments
as internal standards.
Acknowledgment
17. Edgell, C. J.; McDonald, C. C.; Graham, J. B. Proc. Natl. Acad. Sci. U.S.A. 1983, 80,
3734.
18. Benigni, J.; Mimis, D. J. Heterocycl. Chem. 1965, 2, 387.
19. See Supplementary data.
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35, 4584.
G.D. and N.T. were supported by studentships from the Ministè-
re de l’Education et de la Recherche.
Supplementary data
23. Yang, C.; Patel, H.; Ku, Y.-Y.; Shah, R.; Sawick, D. Synth. Commun. 1997, 27,
2125.
24. (a) Galbraith, S. M.; Chaplin, D. J.; Lee, F.; Stratford, M. R.; Locke, R. J.; Vojnovic,
B.; Tozer, G. M. Anticancer Res. 2001, 21, 93; (b) Kanthou, C.; Tozer, G. M. Blood
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NMR data. 1H and 13C spectra for compounds 10, 13, 14, 19a,b,
21a,b, 22a,b, and 28 are available, as well as HMQC, HMBC, and
NOESY spectra for compounds 19a and 19b. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2008.06.002.
25. Note: In a report on preclinical studies of 1, published during the course of our
studies, 13 was identified as an in vitro metabolite of 1.11
26. (a) Holwell, S. E.; Cooper, P. A.; Thompson, M. J.; Pettit, G. R.; Lippert, L. W., III;
Martin, S. W.; Bibby, M. C. Anticancer Res. 2002, 22, 3933; (b) Holwell, S. E.;
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C. Anticancer Res. 2002, 22, 707.
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