Exploring the effect of 2,3,4-trimethoxy-phenyl moiety as a component of indolephenstatins

Article abstract of DOI:10.1016/j.ejmech.2009.10.047

A new family of phenstatin analogues has been synthesized and assayed. This family simultaneously incorporates modifications of the A-ring (replacement of the 3,4,5-trimethoxyphenyl by the 2,3,4-trimethoxyphenyl arrangement), B-ring (N-alkyl-5-indolyl) and conversion of the Oxygen keto group into a substituted nitrogen (oximes, hydrazones, and their acetylderivatives). The conjunction of all this changes greatly diminishes the antimitotic and antiproliferative activities, but the maintenance of the keto bridge produces a potent analogue with the unusual 2,3,4-trimethoxyphenyl moiety.

Full text of DOI:10.1016/j.ejmech.2009.10.047

European Journal of Medicinal Chemistry 45 (2010) 588–597
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring the effect of 2,3,4-trimethoxy-phenyl moiety as a component
of indolephenstatins
a
a
b
a
´
´
´
´
´
´
Concepcion Alvarez , Raquel Alvarez , Purificacion Corchete , Concepcion Perez-Melero ,
a,
a,
*
*
´
Rafael Pelaez , Manuel Medarde
a Departamento de Qu´ımica Farmace´utica, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno s/n, Salamanca 37007, Spain
b Departamento de Fisiolog´ıa Vegetal, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno s/n, Salamanca 37007, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A new family of phenstatin analogues has been synthesized and assayed. This family simultaneously
incorporates modifications of the A-ring (replacement of the 3,4,5-trimethoxyphenyl by the 2,3,4-tri-
methoxyphenyl arrangement), B-ring (N-alkyl-5-indolyl) and conversion of the Oxygen keto group into
a substituted nitrogen (oximes, hydrazones, and their acetylderivatives). The conjunction of all this
changes greatly diminishes the antimitotic and antiproliferative activities, but the maintenance of the
keto bridge produces a potent analogue with the unusual 2,3,4-trimethoxyphenyl moiety.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 16 September 2009
Received in revised form
22 October 2009
Accepted 23 October 2009
Available online 3 November 2009
Keywords:
Phenstatin
Indole
Tubulin
Combretastatin
Cytotoxicity
Antitumour
Oxime
Hydrazone
Hydrazide
Acetylderivatives
2,3,4-Trimethoxyphenyl
1. Introduction
antiangiogenic agent [3]. These initial achievements were followed
by the testing of hydroxyphenstatin (VII), phenstatin analogue of
Phenstatin (I) was firstly prepared by Pettit during a research
directed at the study of the SAR of combretastatins, as an unex-
pected product of the oxidation of combretastatin A-4 (II, CA-4)
silyl derivative with Jacobsen’s complex [1]. Previously, the deox-
yderivative III had demonstrated a noticeable activity in the
inhibition of tubulin polymerization [2], but a diminished cyto-
toxicity in comparison with the highly potent reference compound
deoxycombretastatin (IV). Other compounds related to phenstatin,
which maintain the 3,4,5-trimethoxyphenyl moiety but with
different substitution patterns on B-ring (dimethoxy, methyl-
enedioxy, dichloro, difluoro), were less potent in both assays. On
the other hand, the phosphate derivative of phenstatin (V) is very
similar to combretastatin A-4 prodrug VI (CA-4P), a well known
combretastatin A-1 (VIII, CA-1), which also showed potent activity.
The behaviour of the phosphate derivative IX is very similar to the
CA-1 phosphate (X) [4].
Since then, several papers on phenstatins have been published
[5] and their application protected by patents [6]. Several groups
have introduced modifications on the basic benzophenone skeleton
of phenstatins, replacing either B-ring (guaiacol moiety) by other
aromatic systems or the carbonyl bridge by other bridges. The
B-ring has demonstrated to accept severe modifications without
losing cytotoxicity or antimitotic activity (Figs. 1 and 2). IC50 values
for cell growth inhibition are maintained in the low nanomolar
range for phenstatin (I), the 3-amino analogue XI or the 2-amino
analogue XII [5a,7], whereas the IC50s for the inhibition of tubulin
polymerization decrease to low micromolar values, being some of
them more potent than reference compound CA-4. The B-ring
(substituted phenyl) has also been successfully replaced by bicyclic
or/and heteroaromatic systems, such as thienyl [8], naphthyl [9],
benzo[b]thiophenyl [10], benzofuranyl [11], indolyl [12,13], carba-
zolyl [13] and others [14].
* Corresponding authors. Dr. R. Pela´ez and Prof. M. Medarde, Laboratorio de
´
´
´
Quımica Organica y Farmaceutica, Facultad de Farmacia, Universidad de Salamanca,
Campus Miguel de Unamuno, E-37007 Salamanca, Spain. Fax: þ34 923 294515.
E-mail addresses: pelaez@usal.es (R. Pela´ez), medarde@usal.es (M. Medarde).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.047

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C. Alvarez et al. / European Journal of Medicinal Chemistry 45 (2010) 588–597
589
Fig. 1. Structure of phenstatin (I), combretastatin A-4 (II), combretastatin A-1 (VIII) and related compounds, including their phosphates.
The replacement of the ketone bridge between both aromatic
systems by other one-atom length bridges has also led to potent
derivatives, which maintain the activity of the model compounds.
The carbonyl group (sp2 center) is important but no essential for
high potency [15], other bridges that retain the activity being the
methylene, methanol [16], oxygen [17], nitrogen [18] and sulfur
(also sulfone) atoms [19] or combinations of those with B-ring
modifications (Fig. 3A).
Finally, modification of the A-ring (3,4,5-trimethoxyphenyl
moiety) usually reduces the potency and it has been less thoroughly
explored. Removal of methoxy groups (Fig. 3B), such as in
4-methoxyphenyl, 3,5-dimethoxyphenyl and 2,6-dimethoxypyrid-
3-yl derivatives has been described as detrimental, as well as
changes in the methoxy groups distribution (2,3,4-trimethox-
yphenyl moiety) [10b,13,15,19c,20].
Continuing our work on antimitotic bridged diaryls, we have
assayed several compounds combining modifications on B-ring
(naphthyl, indolyl) with modifications on the bridge (1,1-bisaryl-
ethylenes, imines, oximes, hydrazones, etc. Fig. 3C). The unsub-
stituted naphthalene showed reduced potency, being preferable
the N-methyl-5-indolyl system [21]. The replacement of the
carbonyl oxygen by nitrogen produced varied results, depending on
the nature of the substituent on the nitrogen atom (oximes,
hydrazones), with members of this family maintaining the activity
of the parent compound [22]. The presence of an olefinic methylene
(1,1-diarylethylenes) at the bridge also maintains the activity [23],
thus indicating that the carbonyl group, proposed to establish an
additional hydrogen bond with the protein, is not mandatory and
that the shape conferred to the molecule by the bridge is more
important than its specific structure.
Recently, we have published the effect of the replacement of
B-ring by an indole nucleus (see compound XIII, Fig. 2), the effect of
modifications on the bridge and also on the A-ring (3,4,5- by
2,3,4-trimethoxyphenyl, previously unexplored in phenstatins
family. Fig. 3D) [9a,21,22]. In this paper we communicate the effect
of simultaneously introducing these three modifications. The
ability of tubulin to complex molecules of varied structure at the
colchicine (a 2,3,4-trimethoxybenzene derivative) site explains the
high potency for some of the derivatives obtained in this work.
2. Results and discussion
The diarylmethanols (3 and 4) were synthesized by condensa-
tion of the lithiated N-alkylindoles with 2,3,4-trimethox-
ybenzaldehyde (Scheme 1). The magnesium salts used to
synthesize the 2-naphthyl analogues of phenstatins proved
unsuccessful since at room temperature the reaction does not occur
and upon heating complex mixtures were produced. The use of two
equivalents of butyllithium (2 mol BuLi/mol N-alkylindole) ensures
reaction through metalated position C-5 as, otherwise, trans-
metalation between position 5 and position 2 proceeds with pref-
erential reaction at position 2, whereas if this position is
deprotonated by the excess of butyllithium, the reaction takes place
through the more reactive position 5. Oxidations of the methanols
were carried out with KMnO4 and phase transfer catalysis, since
PCC or K2Cr2O7 did not give the desired ketones in good yields.
Following this procedure, the N-methyl- and N-ethyl-2,3,4-tri-
methoxy-5-indolophenones (5 and 6 respectively) were produced
in high yields starting from 5-bromoindoles 1 and 2, followed by
Fig. 2. Aminophenstatins XI, and XII, and other types of phenstatin analogues carrying naphthalene, benzothiophene and indolyl moieties, that display high antitubulin activity and
cytotoxicity.

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Fig. 3. A) Bridge modifications replacing the keto group by thioethers, amines or derivatives, which produce active analogues. B) Modifications of the A-ring, yielding analogues of
reduced potency. C) Modifications on the bridge and D) modification of the A-ring described from our group.
oxidation of the methanols 3 and 4, which are readily obtained by
the afore cited reaction.
indolyl moieties, Z and E stereoisomers were produced, as depicted
in Fig. for derivatives of (3,4,5-trimethoxyphenyl)(2-naph-
4
Once the ketones were obtained, representative derivatives
produced by modifications of the bridge replacing the keto-oxygen
by substituted nitrogen were synthesized by standard procedures.
During the preparation of related derivatives with naphthyl and
thyl)ketone (Fig. 4A), (3,4,5-trimethoxyphenyl)(5-indolyl)ketone
(Fig. 4B) and (2,3,4-trimethoxyphenyl)(2-naphthyl)ketone (Fig. 4C).
For the (2,3,4-trimethoxyphenyl)(5-indolyl)ketone derivatives
obtained in the present work, only one stereoisomer was produced
Scheme 1. Synthesis of compounds 1–16 and structure of compound 17. Reagents and conditions: a) NaOH, CH2Cl2, HSOꢀ4 Bu4Nþ, r.t., 1 h; then MeI, reflux. b) NaOH, CH2Cl2, HSOꢀ4
Bu4Nþ, r.t., 1 h; then EtBr, reflux. c) nBuLi, THF, ꢀ78 ꢁC, 1–2 h; then 2,3,4-trimethoxybenzaldehyde, ꢀ78 ꢁC to r.t., 12–24 h. d) KMnO4, HSO4ꢀ Bu4Nþ, CH2Cl2, 8–12 h. e) NH2OH$HCl,
MeOH, pyridine (2–4 drops), reflux, 12–48 h. f) Ac2O, pyridine, 2–4 h. g) NH2NH2$HCl, MeOH, AcOH (2–4 drops), reflux, 12–48 h. h) NH2NHMe$HCl, MeOH, AcOH (2–4 drops), reflux,
12–48 h.

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Fig. 4. 1H NMR shifts of significant protons for Z and E isomers of nitrogen derivatives of: A) (3,4,5-trimethoxyphenyl)(2-naphthyl)ketone, B) (3,4,5-trimethoxyphenyl)(5-indo-
lyl)ketone, and C) (2,3,4-trimethoxyphenyl)(2-naphthyl)ketone. Framed: intervals observed for (2,3,4-trimethoxyphenyl)(5-indolyl)ketone derivatives obtained in the present work.
in all the cases. Because none of the compounds 7–16 yielded
crystals for X-ray diffraction studies, the trend of the 1H -NMR shifts
of related analogues was taken into consideration for assigning
their stereochemistries.
Accordingly, as shown in Fig. 4, there are recognized intervals
for representative protons in the Z and E stereoisomers of related
compounds previously synthesized [21,22]. These trends allowed
us to assign the Z-stereochemistry to compounds 7–16, taking into
consideration the shifts shown in Table 1, which clearly fit within
the intervals of indole H-6 (Fig. 4B, deshielded), phenyl H-60
(Fig. 4C, shielded) and phenyl 20-methoxy group (Fig. 4C, deshiel-
ded) for Z- when compared to those for E-stereoisomers.
The synthesized compounds were tested at a single concentra-
IC50s were determined for those compounds exhibiting higher
potency in the preliminary assay for the inhibition of tubulin
polymerization. As it was observed for the (3,4,5-trimethox-
yphenyl) (N-alkyl-5-indolyl)ketone derivatives, the N-methyl
compounds are more potent than the N-ethyl compounds (IC50
values of TPI: 5 < 6 and 7 < 8, see Table 2). Neither the alcohols (3
and 4), nor the hydrazones (11, 12, 15 and 16), or any of the acylated
derivatives (9, 10, 13 and 14) are potent inhibitors of tubulin poly-
merization. Ketone 6 and oximes 7 and 8 were less potent than
phenstatin itself, but ketone 5, surprisingly, is almost one order of
magnitude more potent than phenstatin as inhibitor of tubulin
polymerization. Taking into account that replacement of the
3-hydroxy-4-methoxyphenyl ring by the N-methyl-5-indolyl ring
in combretastatins [24], combretastatin analogues [25], and
phenstatins [12,13,21] only causes slight changes in potency, the
effect produced by the combination of such replacement with the
modificaton of the trimethoxyphenyl substitution pattern is
noteworthy.
tion (between 20 and 40 mM) in the tubulin polymerization assay
(Table 2). Only ketones 5 and 6 and oximes 7 and 8 showed more
than 40% inhibition at the tested concentrations, in full agreement
with the results for the 3,4,5-trimethoxyphenyl analogues [21]. The
The synthesized compounds were assayed against several
cancer cell lines, including HeLa human cervix epitheloid carci-
noma, A-549 human lung carcinoma, HL-60 human leukemic, and
Table 1
Chemical shifts of the relevant protons for the establishment of the Z-stereochem-
istry for compounds 9–16 by comparison with those of related compounds (see
intervals in Fig. 4).
HT-29 human colon adenocarcinoma following
a previously
described procedure [21]. The results were compared with those of
CA-4 and phenstatin (Table 2).
Compound ¼N-G
R
d
(ppm)
d (ppm) d (ppm) d (ppm) d (ppm)
H-4
H-6
H-5’
H-6’
MeO-2’
Only those compounds showing higher potency in the inhibition
of tubulin polymerization assay showed cytotoxicity in the sub-
micromolar range, with the exception of N-methylhydrazone 16,
which is moderately potent as cytotoxic and inactive as tubulin
polymerization inhibitor. In accordance with the general trend of
phenstatins in relation to combretastatins, a reduction in cytotox-
icity with respect to tubulin inhibitory activity is observed, as well as
the reduction that has also been reported for the replacement of the
3-hydroxy-4-methoxyphenyl ring by the N-methyl-5-indolyl ring
7
8
9
10
11
12
13
14
15
16
]N–OH
Me 7.59
Et 7.62
Me 7.72
Et 7.73
Me 7.52
Et 7.52
7.59
7.58
7.71
7.70
7.70
7.70
7.74
7.73
7.70
7.67
6.81
6.80
6.83
6.73
6.79
6.79
6.81
6.78
6.78
6.75
6.96
6.93
6.72
6.82
6.86
6.85
6.81
6.78
6.84
6.80
3.76
3.76
3.79
3.72
3.75
3.75
3.80
3.67
3.74
3.70
]N–OH
]N–OAc
]N–OAc
]N–NH2
]N–NH2
]N–NHAc Me 7.60
]N–NHAc Et 7.57
]N–NHMe Me 7.51
]N–NHMe Et 7.47

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592
Table 2
Activity results for the synthesized compounds. Tubulin polymerization inhibitory activity, expressed as the percentage of polymerization with respect to the control %TPI at
a single micromolar concentration (under parenthesis) or as the IC50 M) for the most potent derivatives (IC50 TPI). Cytotoxicity against 4 cell lines expressed as the IC50 M).
Literature IC50(TPI) values for CA-4 are given under parenthesis for comparison.
(m
(m
Compound
%TPI (
m
M)
IC50 TPI
m
M
HeLa
m
M
HL-60
mM
A-549
m
M
HT-29 mM
X, Y
R
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
CA-4
OH, H
OH, H
]O
]O
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
9 (20)
–
–
2.4
–
>10
–
5.0
–
2.0
0.58
–
2.0
2.0
2.0
–
1.0
–
>10
>10
0.51
>10
1.0
>10
>10
–
>10
>10
>10
–
>10
1.9
–
0 (20)
98 (20)
47 (20)
82 (30)
52 (30)
20 (30)
29 (20)
25 (20)
12 (20)
22 (20)
5 (20)
<1.0
0.017
0.42
0.076
2.0
1.0
–
2.0
–
1.0
–
1.0
0.29
–
0.003
0.03
0.034
>10
0.021
2.0
0.27
2.0
1.5
–
2.0
>10
2.0
–
2.0
1.2
–
0.032
1.8
–
0.4
5.6
9.0
43.1
–
–
–
–
–
–
–
–
–
]N–OH
]N–OH
]N–OAc
]N–OAc
]N–NH2
]N–NH2
]N–NHAc
]N–NHAc
]N–NHMe
]N–NHMe
0 (20)
17 (30)
19 (20)
99 (20)
97 (20)
98 (40)
–
–
3.0 (1–4)
2.5
6.9
0.002
0.03
–
0.003
0.3
0.12
Phenstatin
XIII
]O
Me
[12,13,23c]. The ketones (especially 5) have noticeable activity in
both assays and, among the derivatives produced by replacement of
the keto-oxygen by an imine nitrogen, only the oxime (7) showed
some activity, with a 5–20-fold decrease with respect tothe ketones.
After these results, we centred our attention on ketones I (phen-
statin), XIII [21] and 5, and we also synthesized compound 17 (the
2,3,4-trimethoxyphenyl isomer of phenstatin. Scheme 1), by depro-
tection of its TBDMS derivative, described in a previous paper [23d].
There are remarkable differences in potency observed between
the pairs with 2,3,4-trimethoxy- and 3,4,5-trimethoxy-phenyl
moieties for 3-hydroxy-4-methoxysubstituted (I and 17) and
N-methylindole substituted (XIII and 5) phenstatins: in the second
Fig. 5. Upper: Structure and TPI activity of ketones I, XIII, 5 and 17. Bottom left: Superposition of the preferred conformations for compounds 17, I (phenstatin), 5 and XIII with their
dipole moments. Bottom right: MEP for 17, I (Phenstatin), 5 and XIII in their docked conformations.

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593
Fig. 6. Docked models of Phenstatin (I) (A, carbons in magenta), 17 (A, carbons in cyan), XIII (B, carbons in magenta), and 5 (B, carbons in cyan). Podophyllotoxin is shown in thinner
lines in red and the protein backbone as a ribbon, with protein sidechains close to the ligands as wireframe. The figure shows the higher steric bulkiness of the 2,3,4-trime-
thoxyphenylrings (cyan) when compared to the 3,4,5-trimethoxyphenylrings (magenta), closer to the trimethoxyphenyl ring of podophyllotoxin. When 5 (right) and 17 (left) are
compared, the trimethoxyphenyl ring of the latter is placed further from that of podophyllotoxin than that of the former, thus suggesting an explanation for the differences in
potency observed.
pair both compounds are potent TPI inhibitors, whereas in the first
one, phenstatin (I) is a much stronger inhibitor than 17 (see Fig. 5,
upper part). In an attempt to justify such a difference, we have
analyzed the preferred conformations for the two pairs and their
associated dipole moments, and we have found very similar
behaviours (Fig. 5, bottom left). As shown, compounds with
a 3,4,5-trimethoxyphenyl ring (I and XIII) are quite similar to those
with a 2,3,4-trimethoxyphenyl ring moiety (5 and 17), except for the
fact that the 2,3,4-trimethoxyphenyl ring can adopt two dispositions.
Furthermore, there are almost no difference between the two pairs
methylenedioxy moiety of podophyllotoxin. Compound 17, the
2,3,4-trimethoxyphenyl isomer of phenstatin, also suffers from the
higher size of the 3-hydroxy-4-methoxyphenyl ring when
compared with the N-methyl-5-indolyl system of compound 5,
thus accounting for the higher potency of the latter in TPI assays. A
similar argument seems to operate on the bridge, were only the
smallest substituents are allowed.
Thus, the unique combination of 2,3,4-trimethoxyphenyl ring
(higher steric demand for binding) with the N-methylindol-5-yl
residue (less demanding) and the ketone on the bridge represents
a singular case of potent tubulin polymerization inhibitor having
the 2,3,4-trimethoxy substitution pattern on the A-ring.
(molecules with
a N-methylindole ring and molecules with
a 3-hydroxy-4-methoxyphenylring) to explain the different observed
behaviour. Also, the dipole moments are similar for the molecules
within a pair. We have also calculated the molecular electrostatic
potential of the molecules in the calculated docked conformation (see
below), and they look very similar to each other (Fig. 5, bottom right).
Therefore, we decided to further study the possible interaction
of these compounds with tubulin by means of docking studies
(Fig. 6). The synthesized ketones 5 and 6 were cross-docked [26] in
a combined podophyllotoxin-colchicine site using the Surflex
docking program [27], as previously described [21,22]. Similar
results were obtained with AutoDock. Regardless of the program
used, molecules with a 3,4,5-trimethoxyphenyl ring docked more
often in conformations with the phenyl rings close to those of
reference ligands such as podophyllotoxin or colchicine than
compounds with a 2,3,4-trimethoxyphenyl ring. This observation is
in good agreement with the fact that the former are in general more
potent than the latter, and consistent with the SAR for these
families of compounds which give a paramount importance to the
binding of the trimethoxyphenyl, thus suggesting a common
binding site for this moiety across the different families (as seen for
instance for podophyllotoxin and DAMA-colchicine in the X-ray
structures) [28].
Compounds with 2,3.4-trimethoxyphenyl rings docked with the
center of this ring slightly displaced from the center of the trime-
thoxyphenyl ring of podophyllotoxin, in order to alleviate the
higher steric bulkiness of the three methoxy groups on one side of
the ring. Thus, depending on the bulkiness of the other aromatic
ring, the compounds reproduced more or less easily the disposition
of the aromatic rings of podophyllotoxin and colchicine. Therefore,
compound 6 (lower potency), with the larger N-ethyl substituent,
showed a similar accommodation as 5, but having destabilizing
interactions with the protein in the region occupied by the
3. Conclusions
The usually unexplored modification of phenstatins, replacing
the 3,4,5-trimethoxyphenyl (A-ring) by the 2,3,4-trimethox-
yphenyl arrangement, has been carried out in a series of N-alkyl-
5-indolyl derivatives. Surprisingly, this modification proved to be
very efficient when the keto group is maintained on the bridge
and N-methyl-5-indolyl is the B-ring equivalent. The IC50 for the
inhibition of tubulin polymerization in the submicromolar range
displayed by compound 5 is one of the lower ever described for
combretastatin and phenstatin analogues or the reference
compounds.
When this modification of the A-ring is accompanied by
modifications on the bridge, these compounds lack inhibitory
activity of tubulin polymerization and cytotoxicity, only maintained
for oxime 7. This lack of activity is in agreement with a similar effect
observed in previously described analogues maintaining the
3,4,5-trimethoxyphenyl A-ring.
As a general trend in this type of compounds, although they
display higher inhibition of tubulin polymerization, their cytotoxic
effect is lower than the observed for similar combretastatins with
lower effect on the polymerization of tubulin.
4. Experimental section
4.1. Chemistry
4.1.1. Materials and methods
Reagents were used as purchased without further purification.
Solvents (THF, DMF, CH2Cl2, benzene) were dried and freshly

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594
distilled before use according to procedures reported in the liter-
ature. Chromatographic separations were performed on silica gel
columns by flash (Kieselgel 40, 0.040–0.063; Merck) or gravity
column (Kieselgel 60, 0.063–0.200 mm; Merck) chromatography.
TLC was performed on precoated silica gel polyester plates
(0.25 mm thickness, with UV 254 fluorescent indicator. Polychrom
SI F254) and stained with sulphuric acid (10% in EtOH) or phos-
phomolibdic acid (10% in EtOH). Melting points were determined
on a Bu¨chi 510 apparatus and are uncorrected. 1H NMR and 13C
NMR spectra were recorded on a Bruker AC200 spectrometer at
200/50 MHz or on a Bruker SY spectrometer at 400/100 MHz, using
residue was re-dissolved in CH2Cl2, and washed with brine, and the
organic layers were dried, filtered, and evaporated.
4.1.1.5. Hydrazone synthesis. To a 0.02–0.06 M solution of the
ketone in methanol were added 10 mmol of the corresponding
hydrazine per mmol of ketone and 2–4 droplets of acetic acid. The
reaction was refluxed for 48 h, with the addition of 10 mmol of
hydrazone per mmol of ketone every 12 h. The crude product was
evaporated under vacuum, redissolved in CH2Cl2 and washed with
brine, and the organic layers were dried over anhydrous Na2SO4,
filtered and evaporated under vacuum.
CDCl3 as solvent (unless otherwise stated). Chemical shifts (d) are
given in ppm downfield from tetramethylsilane as internal stan-
dard, and coupling constants (J values) are in Hertz. GC–MS anal-
yses were carried out on a Hewlett–Packard 5890 Series II
4.1.1.6. Acetylation reactions. A 10 mol excess of acetic anhydride
was added to a solution of the oxime or hydrazone derivative in
pyridine. After 2–4 h, the reaction was poured onto EtOAc, washed
with 2 N HCl, 5% NaHCO3 and brine, and the organic layers were
dried over anhydrous Na2SO4, filtered, and evaporated under
vacuum.
4.1.1.6.1. 1-Methyl-5-bromo-1H-indole (1) and 1-ethyl-5-bromo-
1H-indole (2). Following the general method, both compounds
were prepared as described previously [21].
apparatus (70 eV), equipped with
a silicon SPB-1 column
(12 m ꢂ 0.2 mm, 0.33
mm thickness) and using a temperature
program (5 min 100 ꢁC – heating 5 ꢁC/min – 15 min at 300 ꢁC). For
FABHRMS analyses, a VG-TS250 apparatus (70 eV) was used. A
Helios-a UV-320 from Thermo-Spectronic was used for UV spectra
and turbidimetric measurements. IR spectra were acquired in a FT-
IR Nicolet Impact 410 equipment (film, unless otherwise stated)
4.1.1.6.2. (1-Methyl-1H-indol-5-yl)(2,3,4-trimethox-
and
y
values are expressed in cmꢀ1. HPLC analysis were run on a HP-
yphenyl)methanol (3). 3.3 mL (5.2 mmol) of 1.6 M nBuLi in hexanes
was reacted with 500 mg (2.4 mmol) of 1 in 20 mL of dry THF and
with 564 mg (2.9 mmol) of 2,3,4-trimethoxybenzaldehyde dis-
solved in 5 mL of dry THF to yield, after treatment according to the
general procedure and column chromatography, 550 mg (71%) of 3.
IR (KBr): 3445, 1600, 1513 cmꢀ1 1H NMR dH (ppm) (400 MHz): 3.65
(s; 3H), 3.76 (s; 3H), 3.85 (s; 3H), 3.87 (s; 3H), 6.11 (br s; 1H), 6.46 (d,
J ¼ 3.1 Hz, 1H), 6.65 (d; J ¼ 8.4 Hz, 1H), 7.04 (d, J ¼ 3.1 Hz, 1H), 7.07
(d; J ¼ 8.4 Hz, 1H), 7.27 (br s; 2H), 7.64 (br s, 1H). 13C NMR dC (ppm)
(100 MHz): 32.9 (CH3), 56.1 (CH3), 60.9 (CH3) (2), 72.5 (CH), 101.2
(CH), 107.1 (CH), 109.1 (CH), 118.9 (CH), 120.9 (CH), 122.2 (CH), 128.4
(C), 129.3 (CH), 131.0 (C), 135.3 (C), 136.2 (C), 142.2 (C), 151.3 (C),
153.1 (C). HRMS (C19H21NO4 þ Na): calcd 350.1368, found 350.1332.
HPLC: 9.71 (C18), 9.29 (C8), 9.05 (Phen).
1100 device from Agilent Technologies or a Delta 600 device from
Waters instruments, using X-TerraÒ MS C18 5
m
m (4.6 ꢂ 150 mm),
X-TerraÒ MS C8 5
m
m (4.6 ꢂ 150 mm), and X-TerraÒ MS Phenylic
5
m
m (4.6 ꢂ 150 mm) columns with water–acetonitrile or water–
methanol gradients. Combretastatin A4 [24] and phenstatin [1]
were synthesized following literature protocols.
4.1.1.1. Protection of the indole nitrogen. 2 mmol of finely ground
NaOH per mmol of indole derivative and a 10% w/w (with respect to
indole derivative) of tetrabutylammonium hydrogenosulfate were
added to a 0.25 M solution of the 5-bromo-1H-indole in 50 mL of
dry CH2Cl2. After stirring for 1 h at room temperature under an
Argon atmosphere, 2–3 mmol of the alkylating agent (MeI or EtBr)
was added, and the reaction was refluxed until completion (as
determined by TLC) for 48–144 h. The reaction mixture was then
washed with brine and the organic layers were dried, filtered, and
rotary-evaporated.
4.1.1.6.3. (1-Ethyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methanol (4). 11 mL (17.6 mmol) of 1.6 M nBuLi in hexanes
was reacted with 1.8 g (8.0 mmol) of 2 in 25 mL of dry THF and with
1.88 g (9.6 mmol) of 2,3,4-trimethoxybenzaldehyde dissolved in
5 mL of dry THF to yield, after treatment according to the general
procedure, 6.5 g of crude reaction product. By column chromatog-
raphy (hexanes/EtOAc 6:4), 1.4 g (51%) of 4 were isolated. IR (KBr):
3458, 1559 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.46 (t; J ¼ 7.0 Hz,
3H), 3.68 (s; 3H), 3.86 (s; 3H), 3.87 (s; 3H), 4.12 (q; J ¼ 7.0 Hz, 2H),
6.11 (br s,1H), 6.11 (d, J ¼ 3.3 Hz,1H), 6.66 (d, J ¼ 8.5 Hz,1H), 7.08 (d;
J ¼ 8.5 Hz, 1H), 7.12 (d, J ¼ 3.3 Hz, 1H), 7.23 (dd; J1 ¼8.3 Hz,
J2 ¼1.3 Hz, 1H), 7.32 (d; J ¼ 8.3, 1H), 7.62 (br s, 1H). 13C NMR dC
(ppm) (100 MHz): 15.5 (CH3), 41.1 (CH2), 56.0 (CH3), 60.8 (CH3) (2),
72.6 (CH), 101.3 (CH), 107.1 (CH), 109.2 (CH), 119.0 (CH), 120.7 (CH),
122.2 (CH), 127.4 (CH), 128.6 (C), 130.9 (C), 135.1 (C) (2), 142.2 (C),
151.3 (C), 153.1 (C). HRMS (C20H23NO4 þ Na): calcd 364.1525, found
364.1515. HPLC: 10.56 (C18), 10.10 (C8), 9.77 (Phen).
4.1.1.2. Synthesis of diarylmethanols. 3–35 mL (2.2 mmol per mmol
of N-alkyl-5-bromoindole) of 1.6 M nBuLi in hexanes was added to
a stirred suspension of 2.4–45.2 mmol of the N-alkylated-5-bromo-
1H-indol in dry THF (20–60 mL) at ꢀ78 ꢁC under an Argon atmo-
sphere. After 1–2 h, 1.2 mmol of the aldehyde per mmol of the
bromoindole was slowly added to the resulting yellowish solution,
allowing the reaction to slowly reach room temperature. After 12–
24 h, the mixture was poured over ice and extracted with CH2Cl2.
The organic layers were dried over anhydrous Na2SO4, filtered, and
evaporated under vacuum.
4.1.1.3. Oxidation of diarylmethanols to diarylketones. 1 mmol of
KMnO4 per mmol of alcohol and a 1% w/w of n-Bu4Nþ HSO4ꢀ were
added to a 0.15–0.25 M solution of the alcohol in CH2Cl2, and the
reaction was allowed to proceed until completion (TLC) for 8–12 h.
The reaction crude was filtered through silica, using CH2Cl2 and
ethyl acetate as eluents, and the organic layers evaporated under
vacuum to yield the diarylketones.
4.1.1.6.4. (1-Methyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methanone (5). 2.0 g (6.2 mmol) of 3 was oxidized accord-
ing to the general procedure to yield, after 8 h, 1.8 g of 5 (89%). Mp:
104–106 ꢁC (Hex/CH2Cl2). IR (KBr): 1651 cmꢀ1 1H NMR dH (ppm)
(400 MHz): 3.77 (s; 3H), 3.83 (s; 3H), 3.92 (s; 3H), 3.93 (s; 3H), 6.55
(d, J ¼ 3.2 Hz,1H), 6.73 (d, J ¼ 8.6 Hz,1H), 7.10 (d; J ¼ 3.2 Hz,1H), 7.11
(d, J ¼ 8.6 Hz, 1H), 7.35 (dd; J ¼ 8.6 Hz, 1H), 7.85 (d; J1 ¼8.6 Hz,
J2 ¼1.6 Hz,1H), 8.10 (d; J ¼ 1.6 Hz,1H).13C NMR dC (ppm) (100 MHz):
33.0 (CH3), 56.1 (CH3), 61.0 (CH3), 61.8 (CH3), 103.1 (CH), 106.8 (CH),
108.9 (CH), 123.4 (CH), 124.4 (CH), 125.4 (CH), 127.8 (C) (2), 130.4
(CH), 139.3 (C), 139.9 (C), 142.2 (C), 152.4 (C), 155.5 (C), 195.7 (C).
HRMS (C19H19NO4 þ Na): calcd 348.1212, found 348.1205.
4.1.1.4. Oxime
preparation. 1.2–15.5 mmol
of
NH2OH$HCl
(10 mmol/mmol of ketone) and 2–4 droplets of pyridine were
added to a 0.02–0.06 M solution of the corresponding ketone in
10–25 mL of MeOH, and the reaction was refluxed for 12–48 h. The
reaction was cooled, the solvent was evaporated off, and the

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595
4.1.1.6.5. (1-Ethyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methanone (6). 2.3 g (6.8 mmol) of
1597 cmꢀ1 1H NMR dH (ppm) (400 MHz): 3.73 (s; 3H), 3.75 (s; 3H),
3.92 (s; 3H), 3.94 (s; 3H), 6.39 (d, J ¼ 3.2 Hz, 1H), 6.79 (d; J ¼ 8.4 Hz,
1H), 6.86 (d; J ¼ 8.4 Hz, 1H), 6.98 (d; J ¼ 3.2 Hz, 1H), 7.25 (d;
J ¼ 8.8 Hz, 1H), 7.52 (d; J ¼ 1.6 Hz, 1H), 7.70 (dd; J1 ¼8.8 Hz,
J2 ¼1.6 Hz, 1H). 13C NMR dC (ppm) (100 MHz): 32.9 (CH3), 56.2
(CH3), 61.1 (CH3) (2), 101.7 (CH), 108.1 (CH), 109.1 (CH), 119.9 (CH),
120.2 (CH), 124.6 (CH), 124.6 (C), 128.8 (C), 129.2 (CH), 130.7 (C),
136.8 (C), 142.7 (C), 148.8 (C), 151.7 (C), 154.3 (C). HRMS
(C19H21N3O3 þ Na): calcd 362.1481, found 362.1469. HPLC: 9.50
(C18), 9.09 (C8), 8.99 (Phen).
4
was oxidized
according to the general procedure to yield, after 8 h, 2.1 g of 6
(91%). IR (KBr): 1651, 1597 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.45
(t, J ¼ 7.3 Hz, 3H), 3.77 (s; 3H), 3.90 (s; 6H), 4.17 (q, J ¼ 7.3 Hz, 2H),
6.53 (d; J ¼ 3.2 Hz, 1H), 6.70 (d; J ¼ 8.8 Hz, 1H), 7.08 (d; J ¼ 8.8 Hz,
1H), 7.14 (d, J ¼ 3.2 Hz, 1H), 7.35 (d, J ¼ 8.8 Hz, 1H), 7.83 (dd;
J1 ¼8.8 Hz, J2 ¼ 1.8 Hz,1H), 8.06 (d, J ¼ 1.8 Hz,1H). 13C NMR dC (ppm)
(100 MHz): 15.4 (CH3), 41.3 (CH2), 56.2 (CH3), 61.0 (CH3), 61.8 (CH3),
103.3 (CH), 106.8 (CH), 109.0 (CH), 123.3 (CH), 124.4 (CH), 125.6
(CH), 128.0 (C), 128.6 (CH), 130.1 (C), 138.4 (C) (2), 142.2 (C), 152.4
(C), 155.4 (C), 195.2 (C). HRMS (C20H21NO4 þ Na): calcd 362.1368,
found 362.1351. HPLC: 11.59 (C18), 10.95 (C8), 10.59 (Phen).
4.1.1.6.6. Z-(1-methyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methanone oxime (7). Following the general procedure,
75 mg (0.20 mmol) of 5 yielded 72 mg (92%) of 7. IR (film): 3289,
1596, 1494 cmꢀ1 1H NMR dH (ppm) (400 MHz): 3.76 (s; 6H), 3.94 (s;
3H), 3.96 (s; 3H), 6.43 (d, J ¼ 3.2 Hz,1H), 6.81 (d, J ¼ 8.6 Hz,1H), 6.96
(d, J ¼ 8.6 Hz, 1H), 7.02 (d; J ¼ 3.2 Hz, 1H), 7.25 (d; J ¼ 8.6 Hz, 1H),
7.59 (br d; J ¼ 8.6 Hz, 1H), 7.59 (br s; 1H). 13C NMR dC (ppm)
(100 MHz): 33.0 (CH3), 56.1 (CH3), 61.0 (CH3) (2), 101.9 (CH), 107.3
(CH), 109.3 (CH), 120.4 (CH), 120.9 (CH), 123.3 (CH), 125.4 (C), 127.6
(C), 128.2 (C), 129.5 (CH), 137.4 (C), 142.4 (C), 151.4 (C), 154.2 (C),
156.7 (C). HRMS (C19H20N2O4 þ Na): calcd 363.1321, found
363.1320. HPLC: 9.72 (C18), 9.37 (C8), 9.18 (Phen).
4.1.1.6.11. Z-1-Ethyl-5-[hydrazono(2,3,4-trimethox-
yphenyl)methyl]-1H-indole (12). Following the general procedure,
93 mg (0.3 mmol) of 6 yielded 82 mg (80%) of 12. IR (film): 3392,
1596 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.42 (t, J ¼ 7.3 Hz, 3H),
3.75 (s; 3H), 3.90 (s; 3H), 3.94 (s; 3H), 4.12 (q, J ¼ 7.3 Hz, 2H), 6.40
(d; J ¼ 3.3 Hz, 1H), 6.79 (d; J ¼ 8.8 Hz, 1H), 6.85 (d; J ¼ 8.8 Hz, 1H),
7.05 (d; J ¼ 3.3 Hz, 1H), 7.30 (d; J ¼ 8.8 Hz, 1H), 7.52 (br s; 1H), 7.70
(dd; J1 ¼8.8 Hz, J2 ¼ 1.8 Hz, 1H). 13C NMR dC (ppm) (100 MHz): 15.5
(CH3), 41.1 (CH2), 56.2 (CH3), 61.1 (CH3) (2), 101.8 (CH), 108.1 (CH),
109.2 (CH),120.0 (CH) (2),124.6 (CH),127.4 (CH),128.3 (C),130.6 (C),
135.2 (C), 135.8 (C), 142.7 (C), 148.8 (C), 151.7 (C), 154.3 (C). HRMS
(C20H23N3O3 þ H): calcd 354.1818, found 354.1794. HPLC: 10.39
(C18), 9.91 (C8), 9.71 (Phen).
4.1.1.6.12. Z-N0-[(1-methyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methylene] acetohydrazide (13). 100 mg. (0.3 mmol) of 11
gave after acetylation, 88 mg of acetyl derivative 13 (80%). IR (film):
3318, 1680, 1597 cmꢀ1 1H NMR dH (ppm) (400 MHz): 2.47 (s; 3H),
3.68 (s; 3H), 3.80 (s; 3H), 3.92 (s; 3H), 3.95 (s; 3H), 6.44 (d;
J ¼ 3.0 Hz, 1H), 6.81 (br s, 2H), 7.03 (d; J ¼ 3.0 Hz, 1H), 7.30 (d,
J ¼ 8.9 Hz, 1H), 7.60 (br s; 1H), 7.74 (br d; J ¼ 8.9 Hz, 1H), 8.33 (br s;
1H). 13C NMR dC (ppm) (100 MHz): 20.8 (CH3), 33.0 (CH3), 56.3
(CH3), 61.0 (CH3) (2), 102.0 (CH), 108.2 (CH), 109.3 (CH), 118.4 (C),
120.4 (CH), 121.1 (CH), 124.2 (CH), 128.2 (C), 129.1 (C), 129.6 (CH),
137.5 (C), 143.0 (C), 149.5 (C), 151.5 (C), 155.2 (C), 172.8 (C). HRMS
(C21H23N3O4 þ Na): calcd 404.1586, found 404.1592. HPLC: 9.84
(C18), 9.42 (C8), 9.33 (Phen).
4.1.1.6.13. Z-N0-[(1-ethyl-1H-indol-5-yl)(2,3,4-trimethylphenyl)-
methylene] acetohydrazide (14). 300 mg. (0.9 mmol) of 12 gave
after acetylation, 244 mg of acetyl derivative 14 (73%). 1H NMR dH
(ppm) (400 MHz): 1.42 (t, J ¼ 7.3 Hz, 3H), 2.45 (s; 3H), 3.67 (s; 3H),
3.90 (s; 6H), 4.13 (q; J ¼ 7.3 Hz, 2H), 6.41 (d; J ¼ 2.9 Hz, 1H), 6.78 (br
s; 2H), 7.07 (d; J ¼ 2.9 Hz, 1H), 7.30 (d, J ¼ 8.8 Hz, 1H), 7.57 (d,
J ¼ 1.8 Hz, 1H), 7.73 (dd; J1 ¼8.8 Hz, J2 ¼ 1.8 Hz, 1H), 8.36 (br s; 1H).
13C NMR dC (ppm) (100 MHz): 15.5 (CH3), 20.8 (CH3), 41.2 (CH2),
56.3 (CH3), 61.1 (CH3) (2), 102.1 (CH), 108.3 (CH), 109.4 (CH), 118.4
(C), 120.2 (CH), 121.3 (CH), 124.2 (CH), 127.9 (CH), 128.4 (C), 129.0
(C), 136.5 (C), 142.9 (C), 149.6 (C), 151.5 (C), 155.2 (C), 172.9 (C).
HRMS (C22H25N3O4 þ Na): calcd 418.1743, found 418.1726.
4.1.1.6.14. 5-[2-(methylhydrazono)(2,3,4-trimethox-
4.1.1.6.7. Z-(1-ethyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methanone oxime (8). Following the general procedure,
100 mg (0.3 mmol) of 6 yielded 83 mg (81%) of 8. IR (film): 3347,
1597 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.45 (t, J ¼ 7.3 Hz, 3H), 3.76
(s; 3H), 3.93 (s; 6H), 4.16 (q, J ¼ 7.3 Hz, 2H), 6.43 (d; J ¼ 3.1 Hz, 1H),
6.80 (d; J ¼ 8.6 Hz, 1H), 6.93 (d; J ¼ 8.6 Hz, 1H), 7.09 (d, J ¼ 3.1 Hz,
1H), 7.29 (d, J ¼ 8.8 Hz, 1H), 7.58 (br d; J ¼ 8.8 Hz, 1H), 7.62 (br s, 1H).
13C NMR dC (ppm) (100 MHz): 15.5 (CH3), 41.2 (CH2), 56.1 (CH3),
61.0 (CH3) (2), 102.1 (CH), 107.3 (CH), 109.3 (CH), 120.2 (CH), 121.1
(CH), 124.0 (CH), 127.5 (CH), 127.7 (C) (2), 128.4 (C), 134.5 (C), 136.4
(C), 142.5 (C), 151.2 (C), 154.2 (C). HRMS (C20H22N2O4 þ Na): calcd
377.1477, found 377.1481. HPLC: 10.56 (C18), 10.14 (C8), 9.85 (Phen).
4.1.1.6.8. Z-(1-methyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methanone O-acetyloxime (9). 200 mg (0.6 mmol) of
oxime 7 was acetylated to yield 140 mg of acetyloxime 9 (62%). 1H
NMR dH (ppm) (400 MHz): 2.23 (s; 3H), 3.70 (s; 3H), 3.79 (s; 3H),
3.90 (s; 3H), 3.93 (s; 3H), 6.45 (d, J ¼ 3.2 Hz, 1H), 6.72 (d, J ¼ 8.6 Hz,
1H), 6.83 (d, J ¼ 8.6 Hz, 1H), 7.04 (d; J ¼ 3.2 Hz, 1H), 7.30 (d,
J ¼ 8.0 Hz, 1H), 7.71 (br d; J ¼ 8.0 Hz, 1H), 7.72 (br s; 1H). 13C NMR dC
(ppm) (100 MHz): 19.9 (CH3), 33.0 (CH3), 56.1 (CH3), 61.2 (CH3) (2),
102.2 (CH),106.9 (CH),109.3 (CH),120.6 (CH),121.7 (CH),122.4 (CH),
126.0 (C), 128.2 (C), 129.8 (CH), 130.3 (C), 142.1 (C), 144.8 (C), 154.6
(C), 154.7 (C), 167.8 (C), 169.3 (C). HRMS (C21H22N2O5 þ Na): calcd
405.1426, found 405.1420.
4.1.1.6.9. Z-(1-ethyl-1H-indol-5-yl)(2,3,4-trimethox-
yphenyl)methyl]-1-methyl-1H-indole (15). Following the general
procedure, 150 mg (0.4 mmol) of 5 yielded 140 mg (90%) of 15. IR
(film): 3312, 1651, 1596 cmꢀ1 1H NMR dH (ppm) (400 MHz): 3.05 (s;
3H), 3.71 (s; 3H), 3.74 (s; 3H), 3.91 (s; 3H), 3.93 (s; 3H), 6.38 (d,
J ¼ 3.2 Hz, 1H), 6.78 (d, J ¼ 8.8 Hz, 1H), 6.84 (d, J ¼ 8.8 Hz, 1H), 6.97
(d; J ¼ 3.2 Hz, 1H), 7.25 (d, J ¼ 8.8 Hz, 1H), 7.51 (br s; 1H), 7.70 (br d;
J ¼ 8.8 Hz,1H). 13C NMR dC (ppm) (100 MHz): 32.9 (CH3), 38.2 (CH3),
56.1 (CH3), 61.1 (CH3) (2), 101.6 (CH), 108.2 (CH), 109.1 (CH), 119.5
(CH), 120.2 (CH), 120.5 (C), 124.8 (CH), 128.2 (C), 129.1 (CH), 130.9
(C), 136.7 (C), 142.7 (C), 145.0 (C), 151.7 (C), 154.2 (C). HRMS
(C20H23N3O3 þ Na): calcd 376.1637, found 376.1633.
yphenyl)methanone O-acetyloxime (10). 266 mg (0.8 mmol) of
oxime 8 was acetylated to yield 280 mg of acetyloxime 10 (94%). 1H
NMR dH (ppm) (400 MHz): 1.44 (t, J ¼ 7.3 Hz, 3H), 2.10 (s; 3H), 3.72
(s; 3H), 3.91 (s; 3H), 3.92 (s; 3H), 4.16 (q, J ¼ 7.3 Hz, 2H), 6.45 (d;
J ¼ 3.3 Hz, 1H), 6.73 (d; J ¼ 8.6 Hz, 1H), 6.82 (d; J ¼ 8.6 Hz, 1H), 7.11
(d; J ¼ 3.3 Hz, 1H), 7.31 (d, J ¼ 8.8 Hz, 1H), 7.70 (dd; J1 ¼8.8 Hz,
J2 ¼ 1.8 Hz, 1H), 7.73 (br s; 1H). 13C NMR dC (ppm) (100 MHz): 15.5
(CH3), 19.9 (CH3), 41.2 (CH2), 56.1 (CH3), 61.0 (CH3) (2), 102.3 (CH),
106.9 (CH), 109.4 (CH), 121.5 (CH), 122.9 (CH), 123.7 (CH), 125.8 (C),
128.0 (CH), 128.3 (C), 137.2 (C) (2), 142.1 (C), 151.4 (C), 154.6 (C),
163.5 (C), 169.3 (C). HRMS (C22H24N2O5 þ Na): calcd 419.1583,
found 419.1559.
4.1.1.6.15. 5-[(2-methylhydrazono)(2,3,4-trimethox-
yphenyl)methyl]-1-ethyl-1H-indole (16). Following the general
procedure, 123 mg (0.4 mmol) of 6 yielded 117 mg (84%) of 16. IR
(film): 3289, 1650, 1597 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.43 (t,
J ¼ 7.3 Hz, 3H), 3.02 (s; 3H), 3.70 (s; 3H), 3.88 (s; 3H), 3.90 (s; 3H),
4.1.1.6.10. Z-5-[hydrazono(2,3,4-trimethoxyphenyl)methyl]-1-
methyl-1H-indole (11). Following the general procedure, 200 mg
(0.6 mmol) of 5 yielded 190 mg (90%) of 11. IR (film): 3318, 1680,

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C. Alvarez et al. / European Journal of Medicinal Chemistry 45 (2010) 588–597
596
4.13 (q, J ¼ 7.3 Hz, 2H), 6.35 (d; J ¼ 3.1 Hz, 1H), 6.75 (d; J ¼ 8.4 Hz,
1H), 6.80 (d; J ¼ 8.4 Hz, 1H), 7.01 (d; J ¼ 3.1 Hz, 1H), 7.24 (d;
J ¼ 8.4 Hz, 1H), 7.47 (br s; 1H), 7.67 (dd; J1 ¼8.4 Hz, J2 ¼1.5 Hz, 1H).
13C NMR dC (ppm) (100 MHz): 15.5 (CH3), 38.2 (CH3), 41.3 (CH2),
56.2 (CH3), 61.9 (CH3) (2), 101.8 (CH), 108.0 (CH), 109.0 (CH), 119.6
(CH), 120.2 (CH), 124.5 (C), 125.7 (CH), 128.3 (C), 128.6 (CH), 129.8
(C), 138.4 (C), 142.1 (C), 145.1 (C), 152.4 (C), 154.2 (C). HRMS
(C21H25N3O3 þ Na): calcd 390.1794, found 390.1803.
[4-(phenylaminocarbonyl)-2,3-tetrazolium]-bis-(4-methoxy-6-
nitro)benzenesulfonic acid sodium salt hydrate) cell proliferation
kit (Roche Molecular Biochemicals, Mannheim, Germany)
following the manufacturer’s instructions. Briefly,
a freshly
prepared mixture solution of XTT labelling reagent and PMS
(N-methylphenazinium methylsulfate) electron coupling reagent
was added to each well at an amount of 50 mL. The resulting
mixtures were further incubated for 4 h in a humidified atmo-
sphere (37 ꢁC, 5% CO2), and the absorbance of the formazan product
generated was measured with a microtiter plate reader at a test
wavelength of 490 nm and a reference wavelength of 655 nm. The
IC50 (50% inhibitory concentration) was then calculated as the drug
concentration causing a 50% inhibition of cell proliferation.
4.1.1.6.16. (3-Hydroxy-4-methoxyphenyl)(2,3,4-trimethox-
yphenyl)methanone (17). To a stirred solution 75 mg of the pro-
tected phenol [23d] in 5 mL of dry THF, 450 mg of TBAF in 2 mL of
dry THF was aded and stirred for 1 h under Ar. The reaction mixture
was poured into water and extracted with ether, washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated in
vacuum. The residues were purified by flash chromatography and
crystallized (CH2Cl2/hexanes) to yield 45 mg (82%) of compound 17.
1H NMR dH (ppm) (200 MHz): 3.74 (s; 3H), 3.86 (s; 3H), 3.87 (s; 3H),
3.88 (s; 3H), 6.67 (d; J ¼ 8.6 Hz, 1H), 6.82 (d; J ¼ 8.6 Hz, 1H), 7.03 (d;
J ¼ 8.6 Hz, 1H), 7.32 (dd; J1 ¼8.6 Hz, J2 ¼ 2.3 Hz, 1H), 7.39 (d;
J ¼ 2.3 Hz, 1H). 13C NMR dC (ppm) (100 MHz): 56.2 (CH3) (2), 61.1
(CH3), 61.9 (CH3), 106.7 (CH), 109.7 (CH), 115.9 (CH), 123.8 (CH),
124.6 (CH), 126.8 (C), 131.8 (C), 142.1 (C), 145.2 (C), 150.6 (C), 152.4
(C), 155.7 (C), 194.3 (C). HRMS (C17H18O6 þ Na): calcd 341.1001,
found 341.1020.
4.1.1.10. Molecular modeling. The compounds were docked into the
colchicine site of tubulin following a described protocol [33]. The X-
ray structures of the tubulin complexes with podophyllotoxin and
DAMA-colchicine were retrieved from the protein data bank [34],
while chains C, D and E and the corresponding hetero-groups were
removed manually. The pdb files were energy-minimized and
subjected to molecular dynamics simulations at 300 K [35]. Initially
the backbone was restrained, and then it was set free. The relaxed
structures were superimposed and the tubulin–podophyllotoxin
complex was shifted 30ꢁ Å along the X axes, prior to combining the
two protein complexes in a single file [36]. The combined tubulin
sites and the podophyllotoxin and colchicine ligands were used to
generate a combined protomol with the Surflex docking program
[27] The synthesized compounds together with roughly 300 com-
bretastatins and phenstatin analogues were manually constructed
in silico [37] and docked into the combined sites (cross-docking), in
an attempt to better reproduce the receptor flexibility by using
different configurations of the protein [26]. Additionally, the test set
of ACD compounds used in the Surflex validation was equally
docked [27]. These compounds were considered a negative control
group: i.e., lacking biological activity. The combined results were
analyzed by receiver operating characteristics (ROCs) [38]; the ACD
compounds and the analogues of the colchicine site with published
4.1.1.7. Tubulin isolation. Calf brain microtubule protein (MTP) was
purified by two cycles of temperature-dependent assembly/disas-
sembly, according to the method of Shelanski [29], modified as
described in the literature [30]. The MTP solution was stored at
ꢀ80 ꢁC. Protein concentrations were determined by the method of
Bradford [31], using BSA as standard.
4.1.1.8. Tubulin assembly. In vitro tubulin self-assembly was moni-
tored turbidimetrically at 450 nm, using a thermostated Thermo-
spectronic
Helios
a
spectrophotometer
connected
to
a thermostated bath and a circulating water carrousel system. The
ligands were dissolved in DMSO and stored at ꢀ20 ꢁC. The amount
of DMSO in the assays was 4%, which has been reported not to
interfere with the assembly process in related assays [32]. The
increase in turbidity was followed simultaneously in a batch of six
cuvettes (containing 1.0 mg/mL MTP in 0.1 M MES buffer, 1 mM
TPI higher than 20 mM were considered inactive. The enrichment
factors achieved were similar to those described for similar
systems. The structures of the best scored complexes in the
colchicine and podophyllotoxin sites were inspected visually and
compared with the TPI results [39]. A similar procedure was
employed to dock the synthesized ligands, independently at each
site, with AutoDock [40]. The results were analyzed in the same
way as those obtained with Surflex
EGTA, 1 mM MgCl2, 1 mM
b-ME, 1.5 mM GTP, pH 6.7, and the
measured ligand concentration), with a control (i.e., with no ligand)
always being included.
The samples were preincubated for 30 min at 20 ꢁC in order to
allow binding of the ligand, and were cooled on ice for 10 min. The
cuvettes were then placed in the spectrophotometer at 4 ꢁC. The
assembly process was initiated by a shift in the temperature to
37 ꢁC. The IC50 was calculated as the concentration of drug causing
50% inhibition of polymerization after 20 min of incubation and
was determined analytically by fitting the curves of TPI% versus
drug concentration to exponential curves. At least two independent
experiments (or more when required for the most potent inhibi-
tors) with different MTP preparations were carried out for each
compound tested.
Acknowledgments
´
We thank the ‘‘Junta de Castilla y Leon’’ (Refs SA 090A06, SA
067A09, SA 063A09), Spanish MEC (Ref CTQ2004-00369/BQU) and
MICINN (Ref SAF2008-04242; BFU2-02876) and the EU (Structural
Funds) for financial support. C.A. and R.A. thank the Spanish MEC
´
´
for FPI predoctoral grants and R.A. thanks the Fundacion Ramon
Areces for a predoctoral grant.
4.1.1.9. XTT procedure. 100
m
L
of exponentially growing HeLa
103 cells/well), or A-549
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ꢂ
103 cells/well), HT-29 (3
ꢂ
(5 ꢂ 103 cells/well) cells were seeded in 96-well flat-bottomed
microtiter plates, and incubated at 37 ꢁC in a humidified atmo-
sphere of 5% CO2/95% air for 24 h to allow the cells attach to the
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