Diarylmethyloxime and hydrazone derivatives with 5-indolyl moieties as potent inhibitors of tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2008.04.054

We describe the synthesis and biological evaluation of a series of diarylmethyloxime and diarylmethylhydrazone analogues that contain an indole ring and different modifications on the nitrogen of the bridge. Several compounds showed potent tubulin polymer

Full text of DOI:10.1016/j.bmc.2008.04.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5952–5961
Diarylmethyloxime and hydrazone derivatives with
5-indolyl moieties as potent inhibitors of tubulin polymerization
a
a
b
a
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´
´
´
´
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Concepcion Alvarez, Raquel Alvarez, Purificacion Corchete, Jose Luis Lopez,
a
a,
a,
*
*
´
´
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Concepcion Perez-Melero, Rafael Pelaez and Manuel Medarde
aDepartament de Quı´mica Farmace´utica, Facultad de Farmacia, Universidad de Salamanca,
Campus Miguel de Unamuno, 37007 Salamanca, Spain
bDepartamento de Fisiologı´a Vegetal, Facultad de Farmacia, Universidad de Salamanca,
Campus Miguel de Unamuno, 37007 Salamanca, Spain
Received 8 February 2008; revised 16 April 2008; accepted 23 April 2008
Available online 26 April 2008
Abstract—We describe the synthesis and biological evaluation of a series of diarylmethyloxime and diarylmethylhydrazone ana-
logues that contain an indole ring and different modifications on the nitrogen of the bridge. Several compounds showed potent tubu-
lin polymerization inhibitory action as well as cytotoxic activity against cancer cell lines. The N-methyl-5-indolyl substituted
analogues are more potent than ethyl substituted ones. The most potent inhibitors of tubulin polymerization are the diarylketones
and the diaryloximes. The cytotoxicity against several cancer cell lines is lower for the oximes than for the ketones. Other substi-
tutions on the imine nitrogen greatly reduce the tubulin inhibitory and/or cytotoxic potencies.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
rently in clinical development.2a,7 Many SAR studies
have established the key structural elements responsible
for high antitubulin and cytotoxic activities in combre-
tastatins and related compounds: (a) the trimethoxy-
phenyl ring (A) is seen as an essential element, (b) the
bridge may have zero to four atoms and must keep the
two aromatic rings in a cisoid disposition, such as that
forced by double bonds, small heterocycles or carbocy-
cles and (c) ring B allows more modifications than ring
A, while the 4-methoxy substituent is usually conserved
the 3-hydroxy is replaceable. More dramatic modifica-
tions—that is, the replacement of the B ring by bicyclic
systems such as a 2-naphthyl moiety or a 5-indolyl
unit—give rise to potent compounds (Fig. 1).8 These
SARs found for combretastatins have been extended
to many other structurally related compounds (e.g.,
phenstatins), with different degrees of fulfillment. In
the search for more potent analogues, these findings
are relevant to the establishment of broader SAR and
for the understanding of the rules that govern the bind-
ing to the colchicine site.
Microtubules are cylinder-shaped protein polymers
composed of a-tubulin and b-tubulin heterodimers ar-
ranged head to tail.1 Many useful anticancer drugs bind
to one of at least three distinct binding sites located on
the b subunit of tubulin: the colchicine, vinca and taxane
domains. Many natural and synthetic products of varied
structures bind to the colchicine site of tubulin,2 thus
suggesting a high plasticity of tubulin at the colchicine
site, and a common pharmacophore has been proposed
for colchicine site ligands.3,2d The X-ray crystal struc-
tures of tubulin complexes with DAMA-colchicine and
podophyllotoxin show very hydrophobic binding pock-
ets, which accommodate to the shape of the binding
ligands.4
Combretastatins are strong inhibitors of tubulin poly-
merization,5 which have attracted much attention owing
to their potent cytotoxic and vascular disrupting activi-
ties.6 The phosphate prodrugs of the natural products
combretastatin A-1 and combretastatin A-4 are cur-
A new family of tubulin polymerization inhibitors
termed phenstatins has recently attracted much atten-
tion, owing to their high potency and improved solubil-
ity characteristics.9,2d The phenstatins are bisarylketones
that share many of the SARs of combretastatins, such as
Keywords: Phenstatin; Indole; Tubulin; Combretastatin; Cytotoxicity;
Antitumour; Oxime; Hydrazone; Hydrazide.
*
Corresponding authors. Fax: +34 923 294515; e-mail addresses:
pelaez@usal.es; medarde@usal.es
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.054

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5953
Figure 1. Structure of colchicine, combretastatin A-4 and phenstatin.
the importance of the A ring and the possibility of hav-
ing a 2-naphthyl B ring10 or a 5-indolyl ring.11 However,
in some cases the SAR clearly deviates from that of
combretastatins thus opening the possibility of new tests
of structural modifications.8,9f,g There are no reports on
phenstatins carrying modifications on the central ke-
tone, other than the hydroxyl and acetoxy derivatives
produced as synthetic intermediates. Such modifications
may be of interest in the development of new antitu-
mour or vascular disrupting compounds with improved
characteristics, a largely explored possibility in combre-
tastatins (Fig. 2).7,12
sive as to the stereochemistry of each isomer. This issue
was finally solved by crystallization of one of the iso-
mers of compound 11, which was shown by X-ray dif-
fraction to have Z stereochemistry (Fig. 3). The
individual isomeric Z oximes (9Z and 9E) were prepared
by basic hydrolysis of the acetates, and no interconver-
sion occurred in time not even during column chroma-
tography. The stereochemistry of the remaining
compounds was established by spectroscopic compari-
son with 9 and 11 (Table 1). The main spectroscopic dif-
ferences between the isomers were observed for the
shielding of the two aromatic protons of the 3,4,5-trime-
thoxyphenyl ring (upfield-shifted in the Z isomers) and
for the proton at position 6 of the indole ring (opposite
trend).
2. Results and discussion
The diarylmethanols were synthesized by condensation
of the protected lithioindoles with 3,4,5-trimethoxybenz-
aldehyde (Scheme 1). The magnesium salts used to syn-
thesize the 2-naphthyl analogues of phenstatins proved
unsuccessful since at room temperature the reaction
does not occur. Upon heating, the addition occurs
mainly through C3 of the indole moiety, often with
the formation of double-addition products. Oxidations
of the methanols were carried out with KMnO4 and
phase transfer catalysis, since CCP or K2Cr2O7 did not
give the desired ketones in good yields.
The synthesized compounds were tested at a single con-
centration (20–40 lM) in the tubulin polymerization as-
say. The IC50 was determined for those compounds
exhibiting an inhibition over 50%. In most cases, the
N-methyl derivatives were more potent than the corre-
sponding ethyl derivatives. With respect to bridge sub-
stitution, the ketones (7 and 8) and the oximes (9 and
10) were the most potent analogues. Both the ketones
and the oximes were less potent than phenstatin itself,
although oxime 9, a mixture of the Z and E isomers,
was almost as potent as phenstatin. The low potency
shown by isomer 9Z suggests that isomer 9E must be
similar in potency to phenstatin. Replacement of the
3-hydroxy-4-methoxyphenyl ring by the N-methyl-5-
indolyl ring in combretastatins,8 combretastatin ana-
logues,13 and phenstatins11c,d causes slight changes in
potency, similar to those reported here. Neither the alco-
hols (3 and 4), the hydrazones (13, 14 and 16), or any of
the acylated derivatives (5, 6, 11, 12, 15 and 17) were
seen to be potent inhibitors of tubulin polymerization.
Reaction of the ketones with hydroxylamine, hydrazine,
and N-methylhydrazine led to the corresponding oximes
and hydrazones as roughly 1:1 mixtures of the E and Z
isomers, which could not be separated chromatographi-
cally. In order to attempt a chromatographic separation
and to modify the hydrogen bond donor–acceptor nat-
ure of the bridge substitutions, the oximes and hydra-
zones were acetylated. Thus, E and Z acetyloximes
were separated, but nOe experiments were not conclu-
The synthesized compounds were assayed following a
previously described procedure8 against several cancer
cell lines, including HeLa human cervix epitheloid carci-
noma, A-549 human lung carcinoma, HL-60 human leu-
kemic, and HT-29 human colon adenocarcinoma. The
results were compared with those of CA4 (Table 2).
The most sensitive cell line was HeLa whereas the other
cell lines were more resistant to the compounds. Often,
but not always, phenstatin analogues displayed lower
cytotoxic potencies than their combretastatin counter-
parts, even though they were more potent inhibitors of
tubulin polymerization. The same reduction in cytotox-
icity with respect to tubulin inhibitory activity has also
been reported for the replacement of the 3-hydroxy-4-
Figure 2. Representative structures of potent combretastatins modified
on the bridge (left) and suggested analogous modifications on related
phenstatins (right).

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5954
Scheme 1. Synthesis of compounds 1–18. Reagents and conditions: (a) NaOH, MeI, CH2Cl2, HSO4 ꢀ Bu4N+; (b) NaOH, EtBr, CH2Cl2, HSO4
ꢀ
ꢀ
Bu4N+; (c) n-BuLi, THF, ꢀ40 ꢁC, 3,4,5-trimethoxybenzaldehyde; (d) Ac2O, pyr; (e) KMnO4, HSO Bu4N+, CH2Cl2; (f) NH2OHÆHCl, MeOH
4
reflux, AcOH; (g) NH2NH2ÆHCl, MeOH reflux, AcOH; (h) NH2NHMeÆHCl, MeOH reflux, AcOH; (i) (tBOC)2O, dioxane/water, Na2CO3; (j)
NaBH4, MeOH; (k) NaOH, MeOH.
Table 1. Chemical shifts of the relevant protons for the establishment
of the stereochemistry of compounds 9–17 by comparison with those
of 9 and 11 (italics)
C6–H
d (ppm)
Compound
C20, C60–H
d (ppm)
E
Z
E
Z
7.27
7.31
7.22
7.20
7.10
7.04
7.02
7.08
7.25
7.56
7.56
7.65
7.63
7.68
7.63
7.75
7.70
7.64
9
6.74
6.76
6.81
6.81
6.78
6.76
6.85
6.78
6.83
6.65
6.68
6.55
6.55
6.52
6.51
6.43
6.51
6.59
10
11
12
13
14
15
16
17
new (double) prodrugs of phenstatin analogues with im-
proved solubility, pharmacokinetics, or tumor-targeting
properties.
Figure 3. Ortep drawing of the X-ray crystal structure of compound
11Z.
methoxyphenyl ring by the N-methyl-5-indolyl
ring.8,11c,d,13 In our case the ketones were unaffected
by any of these unfavorable aspects and showed cyto-
toxic activities comparable to those of CA4, despite their
being slightly less potent tubulin polymerization inhibi-
tors. On the other hand, the oximes underwent a signif-
icant decrease in cytotoxic potency. The lower
cytotoxicity of the oximes as compared with their parent
ketones indicates that they are not hydrolyzed under the
assay conditions, in conformity with the hydrolysis
kinetics of ketoprophen oximes, designed as prodrugs.14
The lack of activity of the acylated oxime analogues
makes them suitable starting points for the design of
The synthesized compounds were cross-docked15 in a
combined podophyllotoxin–colchicine site using the Sur-
flex docking program.16 The preferred poses fit well into
both sites (that of the podophyllotoxin complex and that
of the colchicine complex), many ligands binding in both
sites with minor energy differences between the poses.
However, visual inspection of the docked poses at the
colchicine site revealed that the binding site for the trime-
thoxyphenyl ring is often different from that of colchi-
cine, in sharp contrast with its essential role in many
diverse colchicine-site binding ligands.7 We therefore
decided to give preference to the poses docked at the
podophyllotoxin site. Most of these poses placed the

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5955
Table 2. Activity results for the synthesized compounds
Compound
%TPI
IC50 TPI
HeLa
HL-60
A-549
HT-29
CA48
Phenstatin
99 (20)
97 (20)
86 (20)
42 (20)
41 (20)
0 (20)
3 (1–4)7,10
2.5
18
0.003
0.03
>1.0
>1.0
0.0064
>1.0
0.034
0.016
0.36
—
0.002
0.03
>1.0
>1.0
0.26
>1.0
—
0.003
0.3
0.032
1.8
3
>10
>10
>10
>10
0.12
0.14
>10
—
>10
>10
0.24
>10
—
4
—
—
5
6
—
7
98 (40)
95 (20)
82 (30)
23 (20)
87 (20)
41 (20)
38 (20)
54 (20)
26 (20)
35 (30)
7 (30)
7.9
5.7
1.8
—
8
9a
—
0.034
0.11
—
0.33
—
9Z
10
11Z
11E
12Z
12E
13
14
15
16
17Z
17E
5.9
—
>1.0
—
>1.0
—
>10
—
>1.0
>10
>10
>1.0
>1.0
>10
>1.0
>1.0
>1.0
>1.0
>1.0
—
—
—
—
—
>10
>1.0
—
>10
>1.0
—
>10
>10
—
—
—
—
—
>1.0
2.3
>1.0
—
>10
>10
>1.0
>1.0
>10
14 (30)
20 (20)
17 (20)
10 (20)
—
—
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
—
Tubulin polymerization inhibitory activity expressed as the percentage of polymerization of the sample with respect to the control (%TPI) at a single
micromolar concentration (under parenthesis) or as the IC50 (lM) for the most potent derivatives (IC50 TPI). Cytotoxicity against 4 cell lines
expressed as the IC50 (lM). Literature IC50(TPI) values for CA4 are given under parenthesis for comparison.
a 50% mixture of E/Z isomers.
as the acyl derivatives). In the prediction of a pharmaco-
phore for colchicine site inhibitors,3 the carbonyl oxygen
of phenstatins has been proposed as a hydrogen bond
acceptor pharmacophoric point additional to those
found in combretastatins. These predictions agree with
the results described here. The N-methyl-5-indolyl unit
superimposes onto the methylenedioxyphenyl ring of
podophyllotoxin (Fig. 4). The ethyl group of N-ethyl-5-
indolyl analogues slightly protudes over the methylenedi-
oxyphenyl ring and displaces the indole moiety. This
suggests that the moiety is too large and it explains the
observed modest potency decrease.
3. Conclusions
Several bridge modifications have been introduced for the
first time in a new family of phenstatin analogues, and the
oximes have been shown to maintain tubulin inhibitory
Figure 4. Superimposition of the lowest-energy docked pose of
compound 8 (in wireframe, carbons colored orange) over podophyl-
activity. These results indicate that the oximes of phenst-
atins and their derivatives are potentially good starting
points for the design of new cytotoxic compounds with
improved characteristics. This functionalization of the
bridge of phenstatins also opens up the possibility to pre-
pare new derivatives able to act as prodrugs. The N-
methyl-5-indolyl has been confirmed as a good surrogate
for the 3-hydroxy-4-methoxyphenyl ring of phenstatin.
lotoxin at its binding site at tubulin (protein is represented as a
wireframe and podophyllotoxin as thicker lines).
trimethoxyphenyl ring in close proximity to the one in
podophyllotoxin, and the resulting ranking of the poses
agreed more closely with the experimental TPI results.
These results are in good concordance with the fact that
the phenstatins resemble the structure of podophyllo-
toxin (a bisarylmethane) more closely than that of colchi-
cine (a phenyltropolone). The ketones and the oximes
hydrogen bond with the backbone NH group of residue
b248 and place the carbonyl oxygen atom (or iminic
nitrogen) close to the position occupied by the carbonyl
oxygen atom of podophyllotoxin, explaining hence the
difficulty of replacing it by more bulky substituents (such
4. Experimental
4.1. Chemistry
4.1.1. Materials and methods. Reagents were used as
purchased without further purification. Solvents

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5956
(THF, DMF, CH2Cl2, benzene) were dried and freshly
distilled before use according to procedures reported
in the literature. 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). Melting points were determined
4.1.5. Oxidation of diarylmethanols to diarylketones. One
millimole of KMnO4 per mmol of alcohol and a 1% w/w
of n-Bu4N+ 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 eluants, and the organic layers evaporated un-
der vacuum to yield the diarylketones.
on a Buchi 510 apparatus and are uncorrected. 1H
¨
4.1.6.
Oxime
preparation.
1.2–15.5 mmol
of
NMR and 13C NMR spectra were recorded on a Bru-
ker AC200 spectrometer at 200/50 MHz or on a Bru-
ker SY spectrometer at 400/100 MHz. Chemical shifts
(d) are given in ppm downfield from tetramethylsilane
as internal standard, and coupling constants (J values)
are in Hertz. GC–MS analyses were carried out on a
Hewlett-Packard 5890 Series II apparatus (70 eV). For
FABHRMS analyses, a VG-TS250 apparatus (70 eV)
was used. A Helios-R UV-320 from Thermo-Spectron-
ic was used for UV experiments and absorption spec-
tra. HPLC analysis were run on an HP-1100 device
from Agilent Technologies or a Delta 600 device from
Waters instruments, using X-Terraꢂ MS C18 5 lm
(4.6 · 150 mm), X-Terraꢂ MS C8 5 lm (4.6 ·
150 mm), and X-Terraꢂ MS Phenilic 5 lm (4.6 · 150
mm) columns with water–acetonitrile or water–metha-
nol gradients. Combretastatin A4 and phenstatin were
synthesized following literature protocols.8,9
NH2OHÆHCl (10 mmol/mmol of ketone) and 2–4 drop-
lets 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 res-
idue was re-dissolved in CH2Cl2, and washed with brine,
and the organic layers were dried, filtered, and
evaporated.
4.1.7. Hydrazone synthesis. To a 0.02–0.06 M solution of
the ketone in methanol were added 10 mmol of the cor-
responding hydrazine per mmol of ketone and 2–4 drop-
lets 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
in vacuum, re-dissolved in CH2Cl2 and washed with
brine, and the organic layers were dried over anhydrous
Na2SO4, filtered and evaporated in vacuum.
4.1.2. Protection of the indole nitrogen. Two millimole of
finely ground NaOH per mmol of indole derivative and
a 10% w/w (with respect to indole derivative) of tetrabu-
tylammonium 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.
4.1.8. Synthesis of key intermediates and analogs.
4.1.8.1. 1-Methyl-5-bromo-1H-indole (1). Following
the general method, 2.5 g of 5-bromo-1H-indole and
1
2.3 mL of MeI yielded 2.3 g (86%) of 1. H NMR dH
(ppm) (400 MHz): 3.77 (s, 3H), 6.42 (d, J = 3.1 Hz, 1H),
7.05 (d, J = 3.1 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 7.30
(dd, J1 = 8.8 Hz, J2 = 1.7 Hz, 1H), 7.76 (d, J = 1.7 Hz,
1H).
4.1.8.2. 1-Ethyl-5-bromo-1H-indole (2). Following the
general method, 2.5 g of 5-bromo-1H-indole and 1.9 mL
1
of EtBr yielded 1.9 g (55%) of 2. H NMR dH (ppm)
(400 MHz): 1.46 (t, J = 7.5 Hz, 3H), 4.15 (q,
J = 7.5 Hz, 2H), 6.44 (d, J = 3.1 Hz, 1H), 7.12 (d,
J = 3.1 Hz, 1H), 7,21 (d, J = 8.8 Hz, 1H), 7.29 (dd,
J1 = 8.8 Hz, J2 = 1.8, 1H); 7.76 (d, J = 1.8 Hz, 1H).
4.1.3. Synthesis of diaryl methanols. Three to 35 mL
(2.2 mmol per mmol of N-alkyl-2-bromoindole) of
1.6 M n-BuLi in hexanes was added to a stirred sus-
pension of 2.4–45.2 mmol of the N-alkylated-5-bro-
mo-1H-indol in dry THF (20–60 mL) at ꢀ78 ꢁC
under an Argon atmosphere. After 1–2 h, 1.2 mmol
of the aldehyde per mmol of the bromoindole was
slowly added to the resulting yellowish solution, allow-
ing 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 un-
der vacuum.
4.1.8.3.
(1-Methyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methanol (3). 34.7 mL (55.2 mmol) of 1.6 M
n-BuLi in hexanes was reacted with 5.3 g (25.2 mmol)
of 1 in 40 mL of dry THF and with 6.5 g (33.5 mmol)
of 3,4,5-trimethoxybenzaldehyde dissolved in 20 mL of
dry THF to yield, after treatment according to the gen-
eral procedure, 6.5 g of a white solid, which after column
chromatography gave 4 g (50%) of 3. Mp: 125–127 ꢁC
(CH2Cl2/Hex). IR (KBr): 3386, 1591, 1125, 734 cmꢀ1
.
1H NMR dH (ppm) (400 MHz): 3.77 (s, 3H), 3.81 (s,
6H), 3.83 (s, 3H), 5.88 (br s, 1H), 6.47 (d, J = 3.3 Hz,
1H), 6.67 (s, 2H), 7.06 (d, J = 3.3 Hz, 1H), 7.22 (d,
J = 8.6 Hz, 1H), 7.29 (br d, J = 8.6 Hz, 1H), 7.62 (br s,
1H). 13C NMR dC (ppm) (100 MHz): 33.0 (CH3), 56.1
(CH3) (2), 60.9 (CH3), 76.9 (CH), 101.2 (CH), 103.5
(CH) (2), 109.5 (CH), 119.2 (CH), 120.7 (CH), 128.4
(C), 129.5 (CH), 135.1 (C), 136.4 (C), 140.4 (C) (2),
4.1.4. Acetylation reactions. A 10 mol excess of acetic
anhydride was added to a solution of the hydroxyl deriv-
ative 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.

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5957
153.2 (C) (2). HRMS (C19H21 NO4 + Na): calcd
350.1363, found 350.1321.
125.5 ꢁC (CH2Cl2/Hex). IR (KBr): 1649, 1582, 820,
761 cmꢀ1 1H NMR dH (ppm) (400 MHz): 3.75 (s,
.
3H), 3.81 (s, 6H), 3.90 (s, 3H), 6.53 (d, J = 3.3 Hz,
1H), 7.04 (s, 2H), 7.08 (d, J = 3.3 Hz, 1H), 7.32 (d,
J = 8.8 Hz, 1H), 7.74 (br d, J = 8.8 Hz, 1H), 8.09 (br s,
1H). 13C NMR dC (ppm) (100 MHz): 33.0 (CH3), 56.3
(CH3) (2), 60.9 (CH3), 102.9 (CH), 107.6 (CH) (2),
109.1 (CH), 123.7 (CH), 125.0 (CH), 127.7 (C), 129.3
(C), 130.6 (CH), 134.3 (C), 138.9 (C), 141.3 (C), 152.8
(C) (2), 196.3 (C). HRMS (C19H19NO4 + Na): calcd
348.1206, found 348.1185.
4.1.8.4.
(1-Ethyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methanol (4). 32 mL (51 mmol) of 1.6 M n-BuLi
in hexanes was reacted with 5.2 g (23.2 mmol) of 2 in
40 mL of dry THF and with 5.5 g (27.8 mmol) of 3,4,5-tri-
methoxybenzaldehyde dissolved in 20 mL of dry THF to
yield, after treatment according to the general procedure,
6.2 g of a white solid, which after column chromatogra-
phy gave 3.5 g (44%) of 4. Mp: 114–116 ꢁC (Hex/CH2Cl2).
IR (KBr): 3457, 1592, 1505 cmꢀ1. H NMR dH (ppm)
1
(400 MHz): 1.46 (t, J = 7.1 Hz, 3H), 3.83 (s, 9H), 4.17
(q, J = 7.1 Hz, 2H), 5.89 (br d, J = 3.3 Hz, 1H), 6.48 (d,
J = 3.0 Hz, 1H), 6.68 (s, 2H), 7.13 (d, J = 3.0 Hz, 1H),
7.21 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 7.32 (d,
J = 8.4 Hz, 1H), 7.62 (br s, 1H). 13C NMR dC (ppm)
(100 MHz): 15.5 (CH3), 41.1 (CH2), 56.2 (CH3) (2), 60.8
(CH3), 77.1 (CH), 101.4 (CH), 103.8 (CH) (2), 109.5
(CH), 119.3 (CH), 120.6 (CH), 127.6 (CH), 128.6 (C),
135.1 (C), 135.4 (C), 140.4 (C) (2), 153.2 (C) (2). HRMS
(C20H23NO4 + Na): calcd 364.1519, found 364.1511.
HPLC: 9.72 (C18), 9.34 (C8), 9.14 (Phen).
4.1.8.8.
(1-Ethyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methanone (8)11d. 2.0 g (5.9 mmol) of 4 was oxi-
dized according to the general procedure to yield, after
8 h, 1.4 g of 8 (70%) as a white solid. Mp: 101–103 ꢁC
(Hex/CH2Cl2). IR (KBr): 1645, 1582 cmꢀ1. 1H NMR dH
(ppm) (400 MHz): 1.51 (t, J = 7.3 Hz, 3H), 3.88 (s, 6H),
3.94 (s, 3H), 4.23 (q, J = 7.3 Hz, 2H), 6.61 (d,
J = 3.1 Hz, 1H), 7.08 (s, 2H), 7.21 (d, J = 3.1 Hz, 1H),
7.42 (d, J = 8.8 Hz, 1H), 7.79 (dd, J1 = 8.8 Hz,
J2 = 1.5 Hz, 1H), 8,14 (d, J = 1.5 Hz, 1H). 13C NMR dC
(ppm) (100 MHz): 15.4 (CH3), 41.2 (CH2), 56.3 (CH3)
(2), 60.9 (CH3), 103.0 (CH), 107.8 (CH) (2), 109.1 (CH),
123.6 (CH), 125.1 (CH), 127.9 (C), 128.7 (CH), 129.3
(C), 134.3 (C), 138.0 (C), 141.5 (C), 152.8 (C) (2), 196.1
(C). HRMS (C20H21NO4 + Na): calcd 362.1363, found
362.1351. HPLC: 11.63 (C18), 10.90 (C8), 10.59 (Phen).
4.1.8.5.
(1-Methyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methylacetate (5). 600 lL of acetic anhydride
was added to a solution of 200 mg of 3 in 600 lL of pyr-
idine. After 2 h, 160 mg of 5 (70%), was obtained as a
1
colourless oil. IR (film): 1739, 1592 cmꢀ1. H NMR dH
(ppm) (400 MHz): 2.18 (s, 3H), 3.76 (s, 3H), 3.83 (s,
6H), 3.85 (s, 3H), 6.48 (d, J = 3.2 Hz, 1H), 6.64 (s,
2H), 7.00 (s, 1H), 7.06 (d, J = 3.2 Hz, 1H), 7.22 (br d,
J = 8.6 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.62 (br s,
1H). 13C NMR dC (ppm) (100 MHz): 21.5 (CH3), 32.9
(CH3), 56.2 (CH3) (2), 60.8 (CH3), 77.9 (CH), 101.4
(CH), 104.3 (CH) (2), 109.4 (CH), 120.0 (CH), 121.2
(CH), 128.4 (C), 129.6 (CH), 131.1 (C), 136.4 (C),
136.7 (C), 137.5 (C), 153.3 (C) (2), 170.2 (C). HRMS
(C21H23NO5 + Na): calcd 392.1468, found 392.1493.
HPLC: 11.13 (C18), 10.68 (C8), 10.37 (Phen).
4.1.8.9. (E/Z)-(1-Methyl-1H-indol-5-yl)(3,4,5-trime-
thoxyphenyl)methanone oxime (9). Following the general
procedure, 90 mg (0.28 mmol) of 7 yielded 86 mg (90%)
of 9 as a 45(Z):55(E) mixture. IR (film): 3256,
1
1582 cmꢀ1. H NMR dH (ppm) (400 MHz): 3.74 (s, 3H,
E), 3.76 (s, 6H, E), 3.81 (s, 3H, Z), 3.85 (s, 6H, Z), 3.86
(s, 3H, E), 3.93 (s, 3H, Z), 6.41 (d, J = 2.9 Hz, 1H, E),
6.48 (d, J = 3.1 Hz, 1H, Z), 6.65 (s, 2H, Z), 6.74 (s, 2H,
E), 7.00 (d, J = 2.9 Hz, 1H, E), 7.06 (d, J = 3.1 Hz, 1H,
Z), 7.27 (d, J = 8.8 Hz, 1H, E), 7.30 (d, J = 8.8 Hz, 1H,
Z), 7.31 (m, 1H, E), 7.55 (br d, J = 8.8 Hz, 1H, Z), 7.60
(br s, 1H, Z), 7.72 (br s, 1H, E). 13C NMR dC (ppm)
(100 MHz): 33.0 (CH3, E), 33.1 (CH3, Z), 56.2 (CH3,
Z + E) (2), 60.9 (CH3, Z + E), 101.8 (CH, E), 101.9
(CH, Z), 105.9 (CH, E) (2), 106.7 (CH, Z) (2), 108.9
(CH, E), 109.2 (CH, Z), 121.2 (CH, Z), 121.6 (CH,
Z + E), 122.7 (CH, E), 123.2 (CH, Z), 123.5 (C, Z + E),
124.0 (CH, E), 128.0 (C, E), 128.1 (C, Z), 129.7 (CH, Z),
133.2 (C, Z + E), 136.9 (C, E), 137.4 (C, Z), 138.2 (C,
Z), 139.1 (C, E), 153.0 (C, E) (2), 153.1 (C, Z + E) (2),
158.4 (C, Z + E). HRMS (C19H20N2O4 + Na): calcd
363.1315, found 363.1365. HPLC: 9.41 and 9.59 (C18),
9.05 and 9.20 (C8), 9.00 and 9.12 (Phen).
4.1.8.6.
(1-Ethyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methylacetate (6). 600 lL of acetic anhydride
was added to a solution of 200 mg of 4 in 600 lL of pyr-
idine. After 2 h, 195 mg of 5 (86%) was obtained as a
colourless oil. IR (film): 1739, 1592, 1507 cmꢀ1 1H
.
NMR dH (ppm) (400 MHz): 1.45 (t, J = 7.4 Hz, 3H),
2.20 (s, 3H), 3.83 (s, 9H), 4.15 (q, J = 7.4 Hz, 2H),
6.50 (d, J = 3.3 Hz, 1H), 6.67 (s, 2H), 7.01 (s, 1H),
7.13 (d, J = 3.3 Hz, 1H), 7.22 (dd, J1 = 8.4 Hz,
J2 = 1.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.63 (br s,
1H). 13C NMR dC (ppm) (100 MHz): 15.5 (CH3), 21.4
(CH3), 41.1 (CH2), 56.2 (CH3) (2), 60.8 (CH3), 77.8
(CH), 101.5 (CH), 104.6 (CH) (2), 109.4 (CH), 120.1
(CH), 121.1 (CH), 127.8 (CH), 128.6 (C), 131.1 (C),
135.5 (C), 136.7 (C), 153.3 (C) (2), 170.1 (C). HRMS
(C22H25NO5 + Na): calcd 406.1625, found 406.1623.
HPLC: 11.85 (C18), 11.30 (C8), 10.89 (Phen).
4.1.8.10.
(E/Z)-(1-Ethyl-1H-indol-5-yl)(3,4,5-trime-
thoxyphenyl)methanone oxime (10). Following the general
procedure, 60 mg (0.2 mmol) of 8 yielded 54 mg (85%) of
10 as a 1:1 mixture of the E and Z isomers. IR (film): 3421,
1582 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.46 (t,
.
J = 7.3 Hz, 1H, E), 1.50 (t, J = 7.3 Hz, 1H, Z), 4.17 (q,
J = 7.3 Hz, 1H, E), 4.21 (q, J = 7.3 Hz, 1H, Z), 3.75 (s,
6H, Z), 3.82 (s, 6H, E), 3.88 (s, 3H, Z), 3.95 (s, 6H, E),
6.47 (d, J = 3.3 Hz, 1H, Z), 6.53 (d, J = 3.3 Hz, 1H, E),
6.68 (s, 2H, Z), 6.76 (s, 2H, E), 7.12 (d, J = 3.3 Hz, 1H,
4.1.8.7.
(1-Methyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methanone (7)11d. 3.1 g (9.5 mmol) of 3 was oxi-
dized according to the general procedure to yield, after
8 h, 2.5 g of 7 (77%) as a white solid. Mp: 124.5–

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5958
Z), 7.17 (d, J = 3.3 Hz, 1H, E), 7.30 (d, J = 8.9 Hz, 1H, Z),
7.31 (dd, J1 = 8.6 Hz, J2 = 1.4 Hz, 1H, E), 7.41 (d,
J = 8.6 Hz, 1H, E), 7.51 (dd, J1 = 8.9 Hz, J2 = 1.8 Hz,
1H, Z), 7.62 (d, J = 1.4 Hz, 1H, Z), 7.76 (d, J = 1.8 Hz,
1H, E). 13C NMR dC (ppm) (100 MHz): 15.6 (CH3) (2),
41.2 (CH2) (2), 56.3 (CH3) (4), 61.0 (CH3) (2), 102.0
(CH), 102.1 (CH), 105.8 (CH) (2), 106.6 (CH) (2), 109.0
(CH), 109.4 (CH), 121.1 (CH), 121.9 (CH), 123.0 (CH)
(2), 127.8 (C), 128.1 (CH), 128.3 (CH), 128.3 (C), 129.0
(C), 133.0 (C), 136.0 (C), 136.5 (C), 138.2 (C), 139.2 (C),
142.4 (C), 142.8 (C), 153.0 (C) (2), 153.1 (C) (2), 158.9
(C) (2). HRMS (C20 H22N2O4 + Na): calcd 377.1472,
found 377.1474. HPLC: 10.25 and 10.40 (C18), 9.85 and
9.97 (C8), 9.67 and 9.80 (Phen).
(CH3) (2), 61.0 (CH3), 102.4 (CH), 106.3 (CH) (2), 109.3
(CH), 122.0 (CH), 123.6 (CH), 125.7 (C), 128.1 (CH),
128.8 (C) (2), 137.2 (C), 142.8 (C), 153.0 (C) (2), 165.9
(C), 169.2 (C). HRMS (C22H24N2O5 + Na): calcd
419.1577, found 419.1567.
4.1.8.12.2. (E)-(1-Ethyl-1H-indol-5-yl)(3,4,5-trime-
thoxyphenyl)methanone O-acetyloxime (12E).IR (film):
1
1762, 1574 cmꢀ1. H NMR dH (ppm) (400 MHz): 1.52
(t, J = 7.3 Hz, 3H), 2.11 (s, 3H), 3.78 (s, 6H), 3.88 (s,
3H), 4.23 (q, J = 7.3 Hz, 2H), 6.54 (d, J = 3.3 Hz, 1H),
6.81 (s, 2H), 7.20 (d, J = 3.3 Hz, 1H), 7.20 (dd,
J1 = 8.4 Hz, J2 = 1.5 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H),
7.64 (d, J = 1.5 Hz, 1H). 13C NMR dC (ppm)
(100 MHz): 15.5 (CH3), 19.9 (CH3), 41.2 (CH2), 56.4
(CH3) (2), 61.0 (CH3), 102.1 (CH), 107.1 (CH) (2),
108.8 (CH), 123.0 (CH) (2), 123.0 (C), 128.0 (C), 128.1
(CH), 131.5 (C), 136.3 (C), 141.1 (C), 153.0 (C) (2),
166.1 (C), 169.2 (C). HRMS (C22H24N2O5 + Na): calcd
419.1577, found 419.1564.
4.1.8.11. (E/Z)-(1-Methyl-1H-indol-5-yl)(3,4,5-trime-
thoxyphenyl)methanone O-acetyloxime (11). 400 mg of
oxime 9 was acetylated to yield 400 mg of a mixture of
acetyloximes, which were separated by column chroma-
tography, giving 172 mg (38%) of 11Z and 125 mg (28%)
of 11E.
4.1.8.13.
5-[(E/Z)-hydrazono(3,4,5-trimethoxy-
4.1.8.11.1. (Z)-(1-Methyl-1H-indol-5-yl)(3,4,5-tri-
methoxyphenyl)methanone O-acetyloxime (11Z). IR
(film): 1762, 1583 cmꢀ1. 1H NMR dH (ppm) (400 MHz):
2.14 (s, 3H), 3.82 (s, 9H), 3.95 (s, 3H), 6.49 (d,
J = 3.2 Hz, 1H), 6.55 (s, 2H), 7.07 (d, J = 3.2 Hz, 1H),
7.31 (d, J = 8.8 Hz, 1H), 7.65 (dd, J1 = 8.8 Hz,
J2 = 1.4 Hz, 1H), 7.73 (d, J = 1.4 Hz, 1H). 13C NMR dC
(ppm) (100 MHz): 19.9 (CH3), 33.1 (CH3), 56.3 (CH3)
(2), 61.0 (CH3), 102.3 (CH), 106.3 (CH) (2), 109.3 (CH),
122.2 (CH), 123.4 (CH), 125.8 (C), 128.1 (C), 128.8 (C),
130.0 (CH), 130.0 (C), 138.2 (C), 153.0 (C) (2), 165.9
(C), 169.2 (C). HRMS (C21H22N2 O5 + Na): calcd
405.1421, found 405.1402.
phenyl)methyl]-1-methyl-1H-indole (13). Following the
general procedure, 120 mg (0.4 mmol) of 7 yielded
110 mg (88%) of 13 as a 40(Z):60(E) mixture. IR (film):
1
3396, 1733, 1584 cmꢀ1. H NMR dH (ppm) (400 MHz):
3.74 (s, 9H, Z), 3.82 (s, 3H, Z), 3.84 (s, 9H, E), 3.93 (s,
3H, E), 6.42 (d, J = 2.9 Hz, 1H, Z), 6.52 (s, 2H, Z), 6.53
(d, J = 2.9 Hz, 1H, E), 6.78 (s, 2H, E), 7.01 (d,
J = 2.9 Hz, 1H, Z), 7.10 (d, J = 2.9 Hz, 1H, E), 7.10 (br
d, J = 8.4 Hz, 1H, E), 7.27 (d, J = 8.4 Hz, 1H, E), 7.46
(d, J = 8.8 Hz, 1H, Z), 7.66 (br s, 1H, Z), 7.68 (br d,
J = 8.8 Hz, 1H, Z), 7.70 (br s, 1H, E). 13C NMR dC
(ppm) (100 MHz): 33.0 (CH3, Z + E), 56.2 (CH3, Z + E)
(2), 60.9 (CH3, Z + E), 101.5 (CH, Z), 101.8 (CH, E),
104.2 (CH, Z) (2), 105.6 (CH, E) (2), 109.2 (CH, E),
110.3 (CH, Z), 120.2 (CH, Z), 120.3 (CH, E), 121.5
(CH, E), 122.0 (CH, Z), 123.2 (C, Z + E), 128.0 (C, Z),
128.8 (C, E), 129.4 (CH, Z), 129.8 (CH, E), 129.0 (C, E),
130.1 (C, Z), 135.1 (C, Z + E), 136.7 (C, Z + E), 138.3
(C, Z), 140.7 (C, E), 150.4 (C, Z), 151.0 (C, E), 152.9 (C,
Z) (2), 154.2 (C, Z) (2). HRMS (C19H21N3O3 + Na): calcd
362.1475, found 362.1461. HPLC: 9.12 and 9.39 (C18),
9.73 and 9.93 (C8), 8.76 and 8.95 (Phen).
4.1.8.11.2. (E)-(1-Methyl-1H-indol-5-yl)(3,4,5-trime-
thoxyphenyl)methanone O-acetyloxime (11E). IR (film):
1
1762, 1583 cmꢀ1. H NMR dH (ppm) (400 MHz): 2.10
(s, 3H), 3.78 (s, 6H), 3.86 (s, 3H), 3.88 (s, 3H), 6.54 (d,
J = 3.3 Hz, 1H), 6.81 (s, 2H), 7.14 (d, J = 3.3 Hz, 1H),
7.22 (dd, J1 = 8.4 Hz, J2 = 1.4 Hz, 1H), 7.37 (d,
J = 8.4 Hz, 1H), 7.64 (d, J = 1.4 Hz, 1H). 13C NMR dC
(ppm) (100 MHz): 19.8 (CH3), 33.0 (CH3), 56.3 (CH3)
(2), 61.0 (CH3), 102.0 (CH), 107.0 (CH) (2), 108.8 (CH),
122.9 (CH), 123.0 (CH), 123.1 (C), 127.8 (C), 130.0
(CH), 130.8 (C), 131.4 (C), 137.2 (C), 153.0 (C) (2),
166.2 (C), 169.1 (C). HRMS (C21H22N2O5 + Na): calcd
405.1421, found 405.1409.
4.1.8.14. 5-[(E/Z)-hydrazono(3,4,5-trimethoxyphenyl)
methyl]-1-ethyl-1H-indole (14). Following the general
procedure, 80 mg (0.2 mmol) of 8 yielded 52 mg (61%)
of 14 as a 1(Z):2(E) mixture. IR (film): 3285, 1669,
4.1.8.12.
(E/Z)-(1-Ethyl-1H-indol-5-yl)(3,4,5-trime-
1583 cmꢀ1 1H NMR dH (ppm) (400 MHz): 1.43 (t,
.
thoxyphenyl)methanone O-acetyloxime (12). 300 mg
(0.9 mmol) of oxime 10 was acetylated to yield 310 mg
of a 45(Z):55(E) mixture of acetyloximes, which were
separated by column chromatography, giving 130 mg
(39%) of 12Z and 115 mg (35%) of 12E.
J = 7.3 Hz, 3H, Z), 1.50 (t, J = 7.3 Hz, 3H, E), 3.73 (s,
6H, E), 3.81 (s, 3H, E), 3.82 (s, 6H, Z), 3.93 (s, 3H, Z),
4.14 (q, J = 7.3 Hz, 2H, Z), 4.18 (q, J = 7.3 Hz, 2H, E),
6.40 (d, J = 3.3 Hz, 1H, Z), 6.52 (d, J = 3.3 Hz, 1H, E),
6.51 (s, 2H, Z), 6.76 (s, 2H, E), 7.04 (dd, J1 = 8.8 Hz,
J2 = 1.8 Hz, 1H, E), 7.06 (d, J = 3.3 Hz, 1H, Z), 7.19 (d,
J = 3.3 Hz, 1H, E), 7.63 (dd, J1 = 8.8 Hz, J2 = 1.8 Hz,
1H, Z), 7.28 (d, J = 8.8 Hz, 1H, Z), 7.46 (d, J = 8.8 Hz,
1H, E), 7.48 (d, J = 1.8 Hz, 1H, Z), 7.54 (d, J = 1.8 Hz,
1H, E). 13C NMR dC (ppm) (100 MHz): 15.5 (CH3,
Z + E), 20.9 (CH3, Z + E), 41.2 (CH2, Z + E), 56.2
(CH3, Z + E) (2), 60.9 (CH3, Z + E), 101.6 (CH, Z),
101.9 (CH, E), 104.3 (CH, Z) (2), 105.5 (CH, E) (2),
109.3 (CH, E), 110.4 (CH, Z), 120.0 (CH, E), 120.4
4.1.8.12.1. (Z)-(1-Ethyl-1H-indol-5-yl)(3,4,5-trime-
thoxyphenyl)methanone O-acetyloxime (12Z).IR (film):
1
1762, 1574 cmꢀ1. H NMR dH (ppm) (400 MHz): 1.47
(t, J = 7.3 Hz, 3H), 2.13 (s, 3H), 3.81 (s, 6H), 3.94 (s,
3H), 4.18 (q, J = 7.3 Hz, 2H), 6.49 (d, J = 3.2 Hz, 1H),
6.55 (s, 2H), 7.14 (d, J = 3.2 Hz, 1H), 7.32 (d,
J = 8.7 Hz, 1H), 7.63 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1H),
7.72 (d, J = 1.8 Hz, 1H). 13C NMR dC (ppm)
(100 MHz): 15.5 (CH3), 19.9 (CH3), 41.2 (CH2), 56.3

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C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5959
(CH, E), 121.9 (CH, Z), 122.1 (CH, Z), 127.6 (C, Z), 128.0
(CH, E), 128.0 (C, E), 128.3 (CH, Z), 128.3 (C, E) (2),
129.3 (C, Z), 129.9 (C, Z), 135.1 (C, E), 135.8 (C, Z),
144.2 (C, E), 150.6 (C, Z), 151.3 (C, E) (2), 152.9 (C, Z)
(2), 154.1 (C, Z + E). HRMS (C20H23N3O3 + Na): calcd
376.1632, found 376.1642. HPLC: 10.28 (C18), 9.76 (C8),
9.68 (Phen).
subsequently added and the mixture was stirred at room
temperature for 48 h. The crude was diluted in CH2Cl2,
was washed with brine, and the organic layers were dried,
evaporated and chromatographied to yield 63 mg (28%)
of 17Z and 57 mg of 17E (20%).
4.1.8.17.1. O-tert-Butoxycarbonyl-(2Z)-2-[(1-methyl-
1H-indol-5-yl)(3,4,5-trimethoxyphenyl)methanone]oxime
1
(17Z).IR (film): 1769, 1575 cmꢀ1. H NMR dH (ppm)
4.1.8.15. N0-[(1E/Z)-(1-Methyl-1H-indol-5-yl)(3,4,5-
trimethoxyphenyl)methylene]acetohydrazide (15). 100 mg
(0.3 mmol) of 13 gave after acetylation, 75 mg of a 40:60
mixture of the E and Z isomers (82%). IR (film): 3317,
(400 MHz): 1.52 (s, 9H), 3.81 (s, 9H), 3.94 (s, 3H), 6.48
(d, J = 3.2 Hz, 1H), 6.59 (s, 2H), 7.06 (d, J = 3.2 Hz, 1H),
7.30 (d, J = 8.8 Hz, 1H), 7.64 (br d, J = 8.8 Hz, 1H), 7.73
(br s, 1H). 13C NMR dC (ppm) (100 MHz): 27.9 (CH3)
(3), 30.0 (CH3), 56.2 (CH3) (2), 61.0 (CH3), 83.3 (C),
102.2 (CH), 106.6 (CH) (2), 109.2 (CH), 122.2 (CH),
123.3 (CH), 125.9 (C), 128.1 (C), 128.5 (C), 129.8 (CH),
138.1 (C), 140.2 (C), 153.0 (C) (2), 153.1 (C), 165.0 (C).
HRMS (C24H28N2O6 + Na): calcd 463.1840, found
463.1846.
1
1674, 1585 cmꢀ1. H NMR dH (ppm) (400 MHz): 2.44
(s, 3H, Z), 2.47 (s, 3H, E), 3.76 (s, 3H, E + Z), 3.81 (s,
3H, Z), 3.83 (s, 6H, Z), 3.87 (s, 6H, E), 3.94 (s, 3H, E),
6.43 (s, 2H, Z), 6.46 (d, J = 3.0 Hz, 1H, Z), 6.53 (d,
J = 3.0 Hz, 1H, E), 6.85 (s, 2H, E), 7.02 (br d,
J = 8.4 Hz, 1H, E), 7.05 (d, J = 3.0 Hz, 1H, Z), 7.17 (d,
J = 3.0 Hz, 1H, E), 7.31 (d, J = 8.4 Hz, 1H, Z), 7.46 (d,
J = 8.4 Hz, 1H, E), 7.50 (br s, 1H, E), 7.59 (br s, 1H, Z),
7.75 (br d, J = 8.4 Hz, 1H, Z), 8.40 (br s, 1H, Z), 8.50
(br s, 1H, E). 13C NMR dC (ppm) (100 MHz): 20.7
(CH3, Z + E), 33.1 (CH3, Z + E), 56.2 (CH3, Z + E) (2),
61.0 (CH3, Z + E), 101.7 (CH, Z), 102.1 (CH, E), 105.2
(CH, Z + E) (2), 109.3 (CH, E), 110.5 (CH, Z), 120.4
(CH, Z), 121.3 (CH, Z + E), 121.8 (CH, E), 127.8 (C,
E), 128.2 (C, Z), 128.4 (C, E), 129.0 (C, Z), 129.8 (CH,
E), 130.4 (CH, Z), 133.5 (C, Z), 137.1 (C, E), 137.5 (C,
E), 139.8 (C, Z), 151.7 (C, Z) (2), 153.0 (C, E) (2), 154.4
(C, Z + E), 172.6 (C, Z), 172.9 (C, E). HRMS
(C21H23N3O4 + Na): calcd 404.1581, found 404.1590.
HPLC: 9.53 (C18), 9.04 and 9.22 (C8), 9.02 and 9.19
(Phen).
4.1.8.17.2. O-tert-Butoxycarbonyl-(2E)-2-[(1-methyl-
1H-indol-5-yl)(3,4,5-trimethoxyphenyl)
methanone]-
oxime (17E).IR (film): 2923, 1769, 1575 cmꢀ1. 1H NMR
dH (ppm) (400 MHz): 1.50 (s, 9H), 3.77 (s, 6H), 3.81 (s,
3H), 3.88 (s, 3H), 6.53 (d, J = 3.3 Hz, 1H), 6.83 (s, 2H),
7.11 (d, J = 3.3 Hz, 1H), 7.25 (br d, J = 8.8 Hz, 1H),
7.36 (d, J = 8.8 Hz, 1H), 7.67 (br s, 1H). 13C NMR dC
(ppm) (100 MHz): 27.9 (CH3) (3), 33.0 (CH3), 56.3
(CH3) (2), 61.0 (CH3), 83.3 (C), 101.9 (CH), 106.9 (CH)
(2), 108.9 (CH), 122.9 (CH), 123.2 (CH), 127.8 (C),
129.8 (C), 131.6 (C), 129.8 (CH), 137.2 (C), 140.2 (C),
152.7 (C), 152.9 (C) (2), 164.8 (C). HRMS (C24H28N2
O6 + Na): calcd 463.1840, found 463.1844.
4.1.8.18. (1-Methyl-1H-indol-5-yl)(3,4,5-trimethoxy-
phenyl)methylamine (18). 30 mg (0.1 mmol) of 11 in
10 mL of MeOH was stirred for 2 h under H2 (1 atm)
in the presence of 0.1 mmol of Pd/C. The reaction crude
was then filtered through silica and the solvent was
evaporated to yield 15 mg (52%) of 18, which readily
4.1.8.16. 5-[(E/Z)-Methylhydrazono(3,4,5-trimethoxy-
phenyl)methyl]-1-methyl-1H-indole (16). Following the
general procedure, 190 mg (0.6 mmol) of 7 yielded
170 mg (90%) of 16 as a 1(Z):2(E) mixture. IR (film):
3100, 1579, 1504 cmꢀ1
.
1H NMR dH (ppm)
decomposes. H NMR dH (ppm) (400 MHz): 3.75 (s,
1
(400 MHz): 3.00 (s, 3H, E), 3.04 (s, 3H, Z), 3.76 (s,
6H, E), 3.78 (s, 3H, Z), 3.84 (s, 6H, E), 3.85 (s, 6H,
Z), 3.94 (s, 3H, Z), 6.41 (d, J = 3.3 Hz, 1H, Z), 6.51
(s, 2H, Z), 6.53 (d, J = 3.3 Hz, 1H, E), 6.78 (s, 2H, E),
7.00 (d, J = 3.3 Hz, 1H, Z), 7.08 (dd, J1 = 8.4 Hz,
J2 = 1.5 Hz, 1H, E), 7.14 (d, J = 3.3 Hz, 1H, E), 7.27
(d, J = 8.4 Hz, 1H, Z), 7.45 (d, J = 8.4 Hz, 1H, E),
7.49 (d, J = 1.5 Hz, 1H, Z), 7.54 (d, J = 1.5 Hz, 1H,
E), 7.70 (dd, J1 = 8.4 Hz, J2 = 1.5 Hz, 1H, Z). 13C
NMR dC (ppm) (100 MHz): 33.0 (CH3, Z + E), 38.0
(CH3, Z + E), 56.2 (CH3, Z + E) (2), 61.0 (CH3,
Z + E), 101.5 (CH, Z), 101.7 (CH, E), 103.9 (CH, Z)
(2), 105.7 (CH, E) (2), 109.1 (CH, E), 110.3 (CH, Z),
119.9 (CH, E), 121.5 (CH, Z), 120.1 (CH, E), 122.3
(CH, Z), 124.0 (C, Z + E), 128.1 (C, E), 128.8 (C, Z)
129.3 (CH, E), 129.8 (CH, Z), 130.0 (C, Z), 130.3 (C,
E), 135.3 (C, Z + E), 136.6 (C, Z + E), 146.7 (C, Z),
147.5 (C, E), 152.9 (C, Z) (2), 154.1 (C, E) (2).
3H), 3.79 (s, 6H), 3.80 (s, 3H), 6.44 (d, J = 3.2 Hz,
1H), 6.66 (s, 2H), 6.78 (br s, 1H), 7.04 (d, J = 3.2 Hz,
1H), 7.07 (br d, J = 8.2 Hz, 1H), 7.26 (d, J = 8.2 Hz,
1H), 7.60 (br s, 1H).
4.2. Tubulin isolation
Calf brain microtubule protein (MTP) was purified by
two cycles of temperature-dependent assembly/disas-
sembly, according to the method of Shelanski,17 modi-
fied as described in the literature.18 The MTP solution
was stored at ꢀ80 ꢁC. Protein concentrations were
determined by the method of Bradford,19 using BSA
as standard. Six different MTP preparations were used
in the tubulin assembly assays.
4.3. Tubulin assembly
In vitro tubulin self-assembly was monitored turbidi-
metrically at 450 nm, using a thermostated Thermospec-
tronic Helios a spectrophotometer fitted with a Peltier
temperature controller 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%,
4.1.8.17. O-tert-Butoxycarbonyl-(2E/Z)-2-[(1-methyl-
1H-indol-5-yl)(3,4,5-trimethoxyphenyl) methanone]oxime
(17). 200 mg (0.6 mmol) of 9 was dissolved in a 1:1 mix-
ture of dioxane:water, and 127 mg of ditertbutyldicar-
bonate (0.6 mmol) and 300 mg of Na2CO3 were

´
C. Alvarez et al. / Bioorg. Med. Chem. 16 (2008) 5952–5961
5960
which has been reported not to interfere with the assem-
bly process.20 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 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.
a single file.24 The combined tubulin sites and the podo-
phyllotoxin and colchicine ligands were used to generate
a combined protomol with the Surflex docking pro-
gram.16 The synthesized compounds together with
roughly 300 combretastatins and phenstatin analogues
were manually constructed in silico25 and docked into
the combined sites (cross-docking), in an attempt to bet-
ter reproduce the receptor flexibility by using different
configurations of the protein.15 Additionally, the test
set of ACD compounds used in the Surflex validation
were equally docked.16 These compounds were consid-
ered a negative control group: that is, lacking biological
activity. The combined results were analyzed by receiver
operating characteristics (ROCs)26; the ACD com-
pounds and the analogues of the colchicine site with
published TPI worse than 20 lM were considered inac-
tive. The enrichment factors achieved were similar to
those described for similar systems. The structures of
the best scored complexes in the colchicine and podo-
phyllotoxin sites were inspected visually and compared
with the TPI results.27
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 incuba-
tion and was determined graphically. At least two-inde-
pendent experiments (or more when required for the
most potent inhibitors) with different MTP preparations
were carried out for each compound tested.
4.4. XTT procedure
100 lL of exponentially growing HeLa (1.5 · 103 cells/
well), HT-29 (3 · 103 cells/well), or A-549 (5 · 103 cells/
well) cells were seeded in 96-well flat-bottomed microti-
ter 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 plates. HL-60 cells were seeded at 3 · 103
(100 lL) cells per well. Then, cells were incubated with
different concentrations of the assayed compound at
37 ꢁC under the 5% CO2/95% air atmosphere for 72 h.
Cell proliferation was quantified using the XTT (3,30-
[4-(phenylaminocarbonyl)-2,3-tetrazolium]-bis-(4-meth-
oxy-6-nitro)benzene sulfonic 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-methylphenazini-
um methylsulfate) electron coupling reagent was added
to each well at an amount of 50 lL. The resulting mix-
tures were further incubated for 4 h in a humidified
atmosphere (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.
Acknowledgments
´
We thank the ‘Junta de Castilla y Leon’ (Refs. SA
090A06 and SA 009A06), Spanish MEC (Ref.
CTQ2004-00369/BQU) 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.
Supplementary data
Elemental analysis and NMR data for compounds 3–18.
CCDC 675444 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic
Data Centre via http://www.ccdc.cam.ac.uk/data_re-
quest/cif. Supplementary data associated with this arti-
cle can be found, in the online version, at doi:10.1016/
j.bmc.2008.04.054.
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