Naphthylphenstatins as tubulin ligands: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2008.08.040

A new family of naphthalenic analogues of phenstatins with modifications on the ketone-bridge has been synthesised. The synthesised compounds have been assayed for tubulin polymerisation inhibitory activity as well as for cytotoxic activity against cancer

Full text of DOI:10.1016/j.bmc.2008.08.040

Bioorganic & Medicinal Chemistry 16 (2008) 8999–9008
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Naphthylphenstatins as tubulin ligands: Synthesis and biological evaluation
Concepción Álvarez a, Raquel Álvarez a, Purificación Corchete b, Concepción Pérez-Melero a,
a,
Rafael Peláez a, , Manuel Medarde
*
*
a Laboratorio de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
b Departamento de Fisiología Vegetal, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, 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 naphthalenic analogues of phenstatins with modifications on the ketone-bridge has been
synthesised. The synthesised compounds have been assayed for tubulin polymerisation inhibitory activ-
ity as well as for cytotoxic activity against cancer cell lines. The naphthalene has been confirmed as a
good surrogate for the isovanillin moiety (3-hydroxy-4-methoxyphenyl) of phenstatin, when combined
with the 3,4,5-trimethoxyphenyl ring, but not when combines with the 2,3,4-trimethoxyphenyl ring.
Binding models for the synthesised compounds have been generated and analysed in terms of a pharma-
cophore proposed for colchicine site ligands. The ketone is the optimal bridge substitution but E-acety-
loximes or acetylhydrazones are also tolerated. Significant differences with indole substituted
phenstatins are observed and discussed.
Received 12 May 2008
Accepted 20 August 2008
Available online 26 August 2008
Keywords:
Tubulin
Antimitotic
Cytotoxic
Combretastatins
Phenstatins
Naphthalene
Analogues
Docking
Ó 2008 Published by Elsevier Ltd.
1. Introduction
ing a strong geometrical preference of tubulin. We have also re-
placed several carbon atoms of the 2-naphthyl system of
Microtubules are essential cellular components involved in
intracellular transport, motility, shape maintenance, and division.1
The microtubules are mainly composed of a,b-tubulin dimers and
naphthylcombretastatins by nitrogen atoms, thus preparing quin-
oline and quinoxaline analogues of combretastatins, and found
that the position of the heteroatom is very influential on the activ-
ity but does not improve much the water solubility.6
drugs that interact with tubulin interfere with their dynamic equi-
librium, cause mitotic arrest and, often, trigger cell death.2 Ligands
binding at the colchicine site of tubulin have received much atten-
tion and some of them are undergoing clinical trials as antitumour
and/or vascular disrupting drugs, such as the combretastatin A-4
phosphate prodrug.3 Many of these ligands have two non-coplanar
polyoxygenated aromatic rings (most typically one trimethoxy-
phenyl and a 3-hydroxy-4-methoxyphenyl or related ring) linked
by a bridge of zero to more than four atoms (Fig. 1).4 Amongst
them, much effort has been devoted to combretastatins (Z stilb-
enes and derivatives) and more recently to phenstatins (bisarylke-
tones) and their structure–activity relationships.3,4
We have previously explored the possibility of replacing the
aryl rings of combretastatins by naphthyl systems (Fig. 2).5
Replacement of the 3,4,5-trimethoxyphenyl ring of combretastatin
A-4 by a 2-naphthyl system, and almost any other one, is highly
detrimental for the activity, but the 3-hydroxy-4-methoxyphenyl
ring can be replaced with no substantial loss of activity.5a,5b The
introduction of a 1-naphthyl system is highly detrimental, indicat-
* Corresponding authors. Tel.: +34 923 294528; fax: +34 923 294515 (R.P.).
Figure 1. Structure of colchicine, podophyllotoxin, combretastatin A-4 and its
E-mail address: pelaez@usal.es (R. Peláez).
phosphate prodrug and phenstatin.
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.08.040

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C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
Figure 2. Structure and TPI activity of previously described naphthylcombretastatin and naphthylphenstatins (X = O). Chemical structures of the new derivatives of
naphthylphenstatins modified on the bridge
We have also shown in a preliminary communication that a 2-
naphthophenone (naphthylphenstatin) bearing 2-naphthyl
were obtained by using n-BuLi for transmetallation, followed by
addition of the arylaldehyde. The by-products formed from the di-
rect addition of n-BuLi to the aldehyde were chromatographically
removed. Ketones 3 and 4 were obtained in high yields (higher
than 70%) by oxidation of the corresponding hydroxyderivatives
with KMnO4 in wet CH2Cl2 in the presence of a tetrabutylammo-
nium phase transfer catalyst, provided that the usual method for
oxidation used in phenstatin synthesis (PDC) proved unsuccessful.
Diarylmethanols were acetylated with acetic anhydride in pyridine
to yield the corresponding acetates 5 and 6. Diarylmethanol 1 was
also transformed into the imidazole derivative 9 by treatment with
imidazole in the presence of triphenylphosphine and iodine. Ke-
tone 3 was in turn the starting material to prepare the hydroxy
derivative 8, which bears a tetrasubstituted sp3 carbon-bridge, by
treatment with methylmagnesium bromide in THF.
a
system (in substitution for the 3-hydroxy-4-methoxyphenyl or re-
lated ring) combined with a 3,4,5-trimethoxyphenyl ring (3,4,
5-TM; such as the one present in combretastatins and podophyllo-
toxin) is a potent inhibitor of tubulin polymerisation and cytotoxic
compound.7 On the other hand, the combination of a 2-naphthyl
system with a 2,3,4-trimethoxyphenyl ring (2,3,4-TM, such as that
found in colchicine) is highly detrimental for the activity of phenst-
atin analogues (Fig. 2). In this paper, we disclose a full account of
the synthesis and biological activity of naphthylphenstatins and
of new derivatives modified on the ketone-bridge with nitrogen
containing substituents, in an attempt to further expand our
knowledge on the structure–activity relationships of the naphthyl-
phenstatins (Fig. 2).
Diarylketones 3 and 4 are key intermediates towards the syn-
thesis of analogues bearing a sp2 carbon-bridge. Thus, thioketone
7 was prepared from ketone 3 by treatment with Lawesson’s re-
agent under microwave irradiation. Nitrogenated derivatives were
synthesised from the corresponding ketones following standard
methodologies, through reaction with hydroxylamine, methoxy-
amine, hydrazine and methylhydrazine to yield oximes 10 and
11, methoxime 16, hydrazones 17 and 18 and methylhydrazone
21, respectively. These derivatives were mainly obtained as mix-
tures of Z and E isomers (see experimental section), which could
not be chromatographically resolved. For compounds of family I,
in which the methoxy groups are attached at positions 3,4,5 of
the phenyl ring, the Z/E ratio of isomers does not show a clear pref-
erence towards any of them. However, in the case of family II, with
a 2,3,4-trimethoxyphenyl ring, one of them is always preferred, as
it is observed for oxime 11, with a 2:1 ratio, and for hydrazone 18,
with only one isomer detected. Oximes and hydrazones were fur-
ther derivatised by formation of the acetylated analogues 12 and
13 (acetoximes) and 19 and 20 (acetylhydrazones). Oximes were
2. Results and discussion
2.1. Chemistry
The synthesis of the naphthylphenstatins is depicted in
Schemes 1–3. The compounds were grouped into two families
depending on the position at which the trimethoxyphenyl ring is
attached to the carbonyl group. Thus, family I comprises deriva-
tives bearing a 3,4,5-trimethoxyphenyl ring, whereas family II in-
cludes compounds with a 2,3,4-trimethoxyphenyl moiety. We
have recently described the preparation of the diarylmethanols,
diarylmethylacetates and diarylketones.7 Briefly, diarylmethanols
1 and 2 were prepared by reaction between naphthalen-2-yllithi-
um and 2,3,4- or 3,4,5-trimethoxybenzaldehyde. The alternative
approach, that is, the reaction between the corresponding 2,3,4-
or 3,4,5-trimethoxyphenyllithium and 2-naphthaldehyde, was dis-
carded due to the formation of dialkylated by-products which are
difficult to separate from the desired compound. The best results
Scheme 1. Synthesis of naphthylphenstatins 1–9. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C; (b) KMnO4, Bu4NþHSO4ꢀ, CH2Cl2; (c) Lawesson’s reagent, microwave
irradiation; (d) imidazole, PPh3, I2, CH2Cl2; (e) Ac2O, pyridine; (f) MeMgBr, THF.

C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
9001
Scheme 2. Synthesis of naphthylphenstatins 10–21. Reagents and conditions: (a) NH2OHꢁHCl, pyridine, MeOH; (b) Ac2O, pyridine; (c) (tBoc)2O, Na2CO3, dioxane/H2O (1:1);
(d) (tBoc)2O, NaH, THF; (e) MeONH2ꢁHCl, NaAcO, THF/MeOH (1:1); (f) NH2NH2, AcOH, MeOH; (g) MeNHNH2, AcOH, MeOH.
Scheme 3. Products derived from oxime 10. Reagents: (a) LiAlH4, THF; (b) Ac2O, pyridine.
also transformed into the corresponding O-tert-butoxycarbonyl
derivatives 14 and 15. In that way, the size and the donor–acceptor
pattern of the bridge are also modified, so their influence on bio-
logical activity could be observed. The two geometrical isomers
have been separated in some cases, for example, acetoxime 13
and acetylhydrazone 19, and they are stable in solution for days
(data not shown). The stereochemistry has been assigned based
upon the 1H NMR chemical shifts of H3 of the 2-naphthyl system
and H2,6 of the 3,4,5-trimethoxyphenyl ring (H6 of 2,3,4-TM).
For a related indole substituted series, we have previously shown
that H6 of the 5-indolyl moiety is deshielded in the Z isomer re-
lated to the E one, whereas H2,6 of 3,4,5-TM show the opposite
trend.8 Hence, in compounds of family II the preferred isomer is
the Z isomer.
Attempts to obtain further nitrogenated derivatives by reacting
ketone 3 with several amines (ethylamine, benzylamine, 1,1-
dimethylhydrazine) in different conditions were unsuccessful. Thi-
oketone 7, more reactive, could not be used as starting material
due to its instability. Then, we decided to enlarge the group of
nitrogenated analogues by reducing the already obtained oximes.
Thus, the Z + E mixture of oximes 10 was treated with LiAlH4,
and the reaction crude was acetylated before further purification
for an easier separation of the products (Scheme 3). Three com-
pounds were isolated from this reaction mixture. The major one,
acetamide 23, produced by Beckmann rearrangement of the start-
ing Z oxime 10Z followed by reduction and acetylation. The minor
compounds are the expected acetamide 22 and acetoxime 12E,
produced by acetylation of unreacted E isomer 10E.
ble 1). In the case of one or both isomers being isolated, they were
assayed as individual compounds (indicated by E or Z following the
number). The compounds were first assayed at a single concentra-
tion (20–40 lM), and their degree of inhibition of tubulin polymer-
isation indicated as a percentage of the control (which represents
100% polymerisation). Phenstatin was used as reference. The re-
sults are shown in Table 1 and are in agreement with the struc-
ture–activity relationships for naphthylcombretastatins, which
indicate that the 2-naphthyl ring is a bioisostere of the 3-hydro-
xy-4-methoxyphenyl ring.5 Expressed in terms of a proposed phar-
macophore of the colchicine site ligands,4 the binding models
generated for naphthylphenstatins (Figs. 3 and 4) show that the
2-naphthylphenstatins occupy the R1 pharmacophoric planar
group with the naphthalene ring, the H2 hydrophobic centre and
the A2 hydrogen bond acceptor with the 3,4,5-trimethoxyphenyl
ring and, depending upon the bridge substitution (see below),
the A3 hydrogen bond acceptor with, for instance, the carbonyl
oxygen.
The most potent compound was ketone 3, for which an IC50 va-
lue of 1.1
tent than phenstatin (IC50 = 2.5
l
M was determined. This value makes it even more po-
M) and CA-4 (IC50 = 3–4 M). The
l
l
ten fold potency increase observed for naphthylphenstatin (3)
when compared with naphthylcombretastatin (Fig. 2) is consistent
with previous comparisons between equally substituted combre-
tastatins and phenstatins.9 An explanation for the greater TPI po-
tency of phenstatins relative to combretastatins has been
suggested.4 The presence of an additional hydrogen bond acceptor
(point A3) in phenstatins (the carbonyl oxygen) which could
hydrogen bond to the backbone N–H group of residues b248–
b249–b250 would explain their greater potency. The binding mod-
els that we have constructed for 3 are in good agreement with such
an explanation (Fig. 3).
2.2. Biological evaluation
The synthesised compounds were evaluated in vitro as inhibi-
tors of tubulin polymerisation as previously described.5b When
two geometric isomers were possible (Z and E), they were usually
tested as a mixture (indicated by the corresponding number in Ta-
Except for acetates 5 and 6, compounds of family I (bearing a
3,4,5-trimethoxyphenyl ring) are more potent inhibitors of tubulin
polymerisation than those of family II (bearing a 2,3,4-trimethoxy-

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C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
Table 1
Inhibition of tubulin polymerisation (TPI) and cytotoxicity results for the synthesised compounds
% TPI concentration
assayed (lM)
HeLa
A-549
HT-29
R
MeO
R
MeO
MeO
MeO
MeO
OMe
Family I
Family II
1 (R = H, OH)
3 (R = O)
0 (40)
0 (20)
100 (40)
4 (20)
5 (20)
26 (20)
3 (20)
0 (20)
0 (30)
0 (30)
10 (20)
38 (20)
0 (20)
15 (20)
0 (20)
2 (R = H, OH)
4 (R = O)
>10
0.043
1.7
3.8
2.7
>10
0.25
>10
>10
>10
>10
1.2
>10
>10
>10
1.8
5 (R = H, OAc)
6 (R = H, OAc)
8 (R = Me, OH)
1–10
9 (R = H, Im)
10 (R = N–OH)
1.5
0.4
>10
>10
11 (R = N–OH)
1–10
12 (R = N–OAc)
12E (R = N–OAc)
0.001–0.1
1.2
1–10
>10
>10
>10
>10
1–10
10
13Z (R = N–OAc)
>10
14 (R = N–OtBoc)
15 (R = N–OtBoc)
15Z (R = N–OtBoc)
0.3
16 (R = N–OMe)
17 (R = N–NH2)
4 (20)
0 (30)
0 (20)
35 (30)
0 (20)
0 (20)
31(30)
100 (20)
18Z (R = N–NH2)
20Z (R = N–NHAc)
19 (R = N–NHAc)
19Z (R = N–NHAc)
>10
2.7
>10
>10
21 (R = N–NHMe)
CA-4
0.003
0.032
TPI is expressed as the percentage of inhibition observed at the concentration assayed, related to the control; the values are the mean of at least three experiments.
Cytotoxicity is expressed as IC50 M); the values shown represent the mean of at least six experiments.
(
l
Figure 4. Superimposed binding models of naphthylphenstatin (3, carbons in blue),
N-methyl-5-indolyl analogue8 (carbons in orange) and podophyllotoxin (carbons in
cyan) in the colchicine site of tubulin. Tubulin is shown as ribbons and wireframe.23
The proposed hydrophobic site H1 of the pharmacophore is indicated by the dotted
sphere.4
Figure 3. Superimposed binding models of naphthylcombretastatin (carbons in
magenta), naphthylphenstatin (3, carbons in blue) and podophyllotoxin (carbons in
cyan) in the colchicine site of tubulin. Tubulin is shown as ribbons and wireframe.23
The carbonyl oxygen of naphthylphenstatin (3) and the backbone N of b250 are
highlighted as cpk. The proposed acceptor site A3 of the pharmacophore is
indicated by the light green dotted sphere.4
phenyl ring). The binding models of compounds of family II (not
shown) satisfy either pharmacophoric point H2 (trimethoxyphenyl
ring of podophyllotoxin) or R1 (plane of the methylenedioxyphenyl
ring of podophyllotoxin), thus suggesting that the molecular scaf-
fold precludes them from simultaneously fulfilling the overall geo-
metric and steric requirements of binding represented by
pharmacophoric points H2 and R1,4 and accounting for the lower
TPI potency.
With respect to bridge modifications, for compounds with a sp2
bridge carbon, only acetoxime 12E, acetylhydrazone (19) and

C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
9003
methylhydrazone (21), all of them belonging to family I, showed
moderate inhibitory activity. These results are in sharp contrast
with the results of TPI recently described by us for a related series
of compounds bearing a N-methyl-5-indolyl ring in place of the
naphthalene one.8 In the indolyl series, the oximes were even more
potent than the parent ketones, and their acetylation lead to a po-
tency decrease. A very different trend is seen here for the naphthyl-
phenstatins, whose oximes show almost no TPI activity and their
acetylation recovers some of it. A similar behaviour is observed
for the hydrazones and their acetates and for the sp3 bridged ana-
logues (alcohols and acetates). For them, the indole series show
higher potency for the hydrazones or alcohols compared to their
acetates, while the reverse is true for the naphthalene series. These
results suggest that, despite of the apparent high structural resem-
blance of the N-methylated-5-indole ring and the naphthalene one,
they behave in a very different way in these series and that com-
parisons of families with such a replacement should be cautious.
Their docking models show differences which might provide an
explanation: the N-methyl group of the N-methyl-5-indolyl ana-
logue occupies pharmacophoric point H1, which is not occupied
in the naphthylphenstatins (Fig. 4), thus allowing for a different
overall arrangement of the two fused ring systems. This in turn al-
lows for more bulky substituents on the bridge for the naphthalene
series. For the indole series, smaller substitutions are preferred,
due to the tighter fit of the bicyclic system in the H1 and R1 sites.
Whenever possible, a comparison of the tubulin polymerisation
inhibitory effect of mixtures of stereoisomers with isolated stereo-
isomers indicates that E isomers are more potent than the corre-
sponding Z isomers (as seen in Table 1 for acetyloxime 12 and
acetylhydrazone 19). This stereochemistry is the opposite to that
proposed for an oxime analogue of curacin10 on the basis of a gen-
erated binding model.4a However, this discrepancy only reflects
the very different scaffold characteristics of both families of com-
pounds. The preferred stereochemistry allows in each case for
the 3,4,5-trimethoxyphenyl ring to occupy the pharmacophoric
H2 hydrophobic centre and the A2 hydrogen bond acceptor site.
For the naphthylphenstatin analogues here described, the E iso-
mers place the O-substituents toward the space occupied by ring
C and D of podophyllotoxin.
combined with the presence of naphthalene moiety, are detri-
mental for the inhibitory of tubulin polymerisation and inhibitory
tumour cell growth activities. For nitrogen derivatives at the
bridge having the possibility of Z,E-stereoisomerism, those with
the E-configuration (N-substituent directed towards the naphtha-
lene ring) showed to be more active than those with a Z-configu-
ration. The substitution pattern represented by 2,3,4-trimethoxy
in the ring A produced compounds with very low or no activity
in both types of assays, as it is usually observed for these classes
of antimitotics. These findings enlarge the knowledge of the
structure–activity relationships of the phenstatins, which are dif-
ferent than those displayed by the related combretastatins, and
show substantial departures in the structure–activity relation-
ships of 2-naphthyl substituted from N-methyl-5-indolyl phenst-
atin derivatives. These findings have important bearings on the
design of new families of colchicine site ligands, in particular of
phenstatin analogues.
4. Experimental
4.1. Chemistry
IR spectra were recorded on a Nicolet Impact 410 instrument, as
a thin film unless otherwise stated. 1H NMR and 13C NMR spectra
were recorded on Bruker AC 200 (200 MHz) or Bruker Advance
400 DRX (400 MHz). Chemical shift values (d) are given in ppm rel-
ative to TMS as internal standard, in CDCl3 as solvent unless other-
wise indicated. Mass spectra were recorder on a Hewlett-Packard
5890 Series II CG/MS and HRMS spectra were obtained on a VG
TS-250 spectrometer, using in both cases EI as ionization mode.
TLC analysis was performed on SDS precoated silica gel 60 F254
plates, 0.25 mm thick; spots were visualized with 254 and 336 nm
UV light and phosphomolybdic acid spray. Preparative TLC chroma-
tography was performed on Merck Si F254 precoated plates, 1 mm
thick. Column chromatography was performed with Merck 60
(0.063–0.200 mm or 0.040–0.063 mm) silica gel. Preparative HPLC
was performed on a Waters instrument, fitted with a Waters Delta
600 quaternary pump and a Waters 2996 photodiode array detec-
tor. A Waters X-TerraÒ Prep MS C18 column (5
was used, and the eluent was a gradient of acetonitrile/water. Com-
l
m, 10 ꢂ 150 mm)
The compounds were also evaluated as cytotoxic agents against
three human tumour cell lines: HeLa (cervix carcinoma), A-549
(lung carcinoma) and HT-29 (colon adenocarcinoma). The results
mercial THF was distilled over Na, under argon, prior to use.
4.1.1. General method A: synthesis of diarylmethanols
To a stirred suspension of the corresponding arylhalide (15.4–
45.2 mmol) in THF (35–60 mL), 1.6 M n-BuLi in hexanes (11.6–
34.0 mL, 1.2 mmol per mmol of halide) was added at ꢀ78 °C, under
argon. After 1.5 h, the arylaldehyde (1.2 mmol per mmol of halide)
was added, and the mixture was allowed to reach room tempera-
ture. After 12–24 h, the reaction mixture was poured onto ice
and extracted with dichloromethane. The combined organic layers
were dried (Na2SO4) and the solvent evaporated.
are shown in Table 1, expressed as IC50 (lM). All the compounds
were tested in a preliminary assay, and only the ones that showed
some cytotoxicity were taken further for determining their IC50 val-
ues. Combretastatin A-4 was used as reference. The most sensitive
cell line was HeLa, whereas A-549 and HT-29 were more resistant
to these agents, in contrast to the cytotoxicity shown by combre-
tastatin A-4 but in agreement with our previous reports on naph-
thylcombretastatins5 and indolephenstatins.8 The compounds
eliciting a more remarkable cytotoxicity belong to family I, bearing
the ketone (3) and the acetoxime group (12E) as the bridge between
the aromatic systems, in good agreement with the above discussed
TPI results. The IC50 values found for these analogues are compara-
ble to those of the reference compound CA-4, and follow previously
described trends for phenstatin analogues, which display lower
cytotoxicity than equally substituted combretastatins even though
they usually elicit higher TPI responses.8
4.1.2. General method B: synthesis of diarylketones
A 0.2 M solution of the corresponding diarylmethanol in CH2Cl2
(40–50 mL) was reacted with KMnO4 (1 mmol per mmol of alco-
hol) in the presence of n ꢀ Bu4NþHSOꢀ (about 1% w/w). The reac-
4
tion mixture was stirred for 8–12 h at room temperature and
then passed through silica gel, using dicloromethane and ethyl ace-
tate as eluents. The organic solvent was then evaporated.
3. Conclusions
4.1.3. General method C: acetylation
The naphthalene system is a good subrogate of the B ring of
combretastatins and phenstatins in the parent compounds (com-
A 10 mol excess of acetic anhydride was added to a 1 M solution
of the compound to be acetylated (0.3–1.0 mmol) in pyridine. After
2–4 h, the reaction mixture was diluted with ethyl acetate and
washed with 2 N HCl and saturated NaHCO3. The organic layer
was dried over Na2SO4 and the solvent evaporated.
bretastatin A-4 and phenstatin), with
a cis-olefin or ketone
bridges. As previously demonstrated for combretastatins and
now proven for phenstatins, further modifications at the bridge

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C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
4.1.4. General method D: synthesis of oximes
5.25 mmol) in the presence of tetra-n-butylammonium hydrogen-
sulfate (170 mg, 0.501 mmol), yielding ketone (1.42 g,
A mixture of hydroxylamine hydrochloride (10 mmol per mmol
of ketone), pyridine (2–3 drops) and a 0.05–0.06 M solution of ke-
tone in MeOH (15–25 mL) was refluxed for 12 h and then the sol-
vent was evaporated. The crude product was dissolved in
dichloromethane and washed with water. The organic layer was
dried (Na2SO4) and evaporated.
4
1
4.42 mmol, 85%). H NMR d 3.75 (3H, s, 20-OCH3), 3.92 (3H, s, 30-
OCH3 or 40-OCH3), 3.95 (3H, s, 40-OCH3 or 30-OCH3), 6.76 (1H, d,
J = 8,6, H50), 7.20 (1H, d, J = 8,6, H60), 7.51–7.59 (3H, m, H3, H6,
H7), 7.87–7.95 (3H, m, H4, H5, H8), 8.23 (1H, br s, H1). 13C NMR
d 56.2 (CH3), 61.0 (CH3), 61.8 (CH3), 106.8 (CH), 125.2 (2ꢂ) (CH),
125.9 (C), 126.7 (CH), 127.9 (CH), 128.1 (CH), 128.4 (CH), 129.6
(CH), 131.9 (CH), 132.4 (C), 135.6 (C), 135.8 (C), 142.2 (C), 152.8
(C), 156.3 (C), 195.5 (C). IR (cmꢀ1) 1590, 1652. HRMS m/z found
345.1097; calcd for C20H20O4 (+Na) 345.1091.
4.1.5. General method E: synthesis of hydrazones
Hydrazine hydrate (10 mmol per mmol of ketone) and acetic
acid (2–3 drops) were added to a 0.04–0.06 M solution of ketone
in MeOH (15–25 mL). The reaction mixture was refluxed for 12–
48 h. During this time, a large excess of hydrazine hydrate was
added to the reaction mixture in four portions. The solvent was
evaporated and the crude product was dissolved in dichlorometh-
ane and washed with water. The organic layer was dried (Na2SO4)
and evaporated.
4.1.10. [(2-Naphthyl)(3,4,5-trimethoxyphenyl)methyl]acetate (5)
Following general method C, compound 1 (100 mg, 0.31 mmol)
in pyridine (300
lL) was acetylated by treatment with acetic anhy-
dride (300 L, 3.1 mmol), yielding acetate 5 (80 mg, 0.22 mmol,
l
1
70%). H NMR d 2.22 (3H, s, COCH3), 3.82 (6H, s, 30-OCH3, 50-
OCH3), 3.83 (3H, s, 40-OCH3), 6.63 (2H, s, H20, H60), 7.00 (1H, s,
CH-OAc), 7.41–7.53(3H, m, H3, H6, H7), 7.81–7.88 (3H, m, H4,
H5, H8), 7.84 (1H, br s, H1). 13C NMR d 21.5 (CH3), 56.2 (2ꢂ)
(CH3), 60.9 (CH3), 77.2 (CH), 104.6 (2ꢂ) (CH), 124.9 (CH), 125.9
(CH), 126.4 (2ꢂ) (CH), 127.8 (CH), 128.2 (CH), 128.5 (CH), 133.0
(C), 133.1 (C), 135.7 (C), 137.4 (C), 137.8 (C), 153.4 (2ꢂ) (C),
170.2 (C). IR (cmꢀ1) 1592, 1739. HRMS m/z found 389.1358; calcd
for C22H22O5 (+Na) 389.1359.
4.1.6. (2-Naphthyl)(3,4,5-trimethoxyphenyl)methanol (1)
Following general method A, 2-bromonaphthalene (3.2 g,
16.3 mmol) in dry THF (35 mL), 1.6 M n-BuLi in hexanes (11.6 mL,
18.6 mmol) and 3,4,5-trimethoxybenzaldehyde (3.6 g, 18.4 mmol)
were reacted to obtain alcohol 1 (4.7 g, 14.5 mmol, 89%), which
was purified by crystallisation from dichloromethane/hexane. 1H
NMR d 3.82 (6H, s, 30-OCH3, 50-OCH3), 3.83 (3H, s, 40-OCH3), 5.94
(1H, br s, CH–OH), 6.65 (2H, s, H20, H60), 7.43–7.51 (3H, m, H3, H6,
H7), 7.80–7.88 (3H, m, H4, H5, H8), 7.89 (1H, br s, H1). 13C NMR d
56.2 (2ꢂ) (CH3), 60.9 (CH3), 76.4 (CH), 104.1 (2ꢂ) (CH), 124.8 (CH),
125.1 (CH), 126.1 (CH), 126.2 (CH), 127.7 (CH), 128.1 (CH), 128.4
(CH), 133.0 (C), 133.3 (C), 137.3 (C), 139.5 (C), 141.1 (C), 153.4 (2ꢂ)
(C). IR (cmꢀ1) 1130, 1600, 3425. HRMS m/z found 347.1235; calcd
for C20H20O4 (+Na) 347.1254. Mp 134–136 °C (CH2Cl2/hexane).
4.1.11. [(2-Naphthyl)(2,3,4-trimethoxyphenyl)methyl]acetate (6)
Following general method C, compound 2 (200 mg, 0.62 mmol)
in pyridine (600
lL) was acetylated by treatment with acetic anhy-
dride (600 L, 6.2 mmol), obtaining acetate 6 (180 mg, 0.49 mmol,
l
79%). 1H NMR d 2.19 (3H, s, COCH3), 3.80 (3H, s, 20-OCH3), 3.85 (3H,
s, 30-OCH3 or 40-OCH3), 3.87 (3H, s, 40-OCH3 or 30-OCH3), 6.66 (1H,
d, J = 8,8, H50), 7.06 (1H, d, J = 8,8, H60), 7.31 (1H, s, CH-OAc), 7.44–
7.49 (3H, m, H3, H6, H7), 7.75–7.90 (4H, m, H1, H4, H5, H8). 13C
NMR d 21.3 (CH3), 56.0 (CH3), 60.7 (2ꢂ) (CH3), 72.1 (CH), 107.4
(CH), 122.1 (CH), 125.1 (CH), 125.8 (CH), 126.1 (2ꢂ) (CH), 127.7
(CH), 128.1 (2ꢂ) (CH), 133.0 (C), 133.1 (C), 137.9 (C), 142.4 (C),
145.9 (C), 153.8 (2ꢂ) (C), 169.8 (C). IR (cmꢀ1) 1601, 1741. HRMS
m/z found 389.1359; calcd for C22H22O5 (+Na) 389.1359.
4.1.7. (2-Naphthyl)(2,3,4-trimethoxyphenyl)methanol (2)
Following general method A, 2-bromonaphthalene (9.4 g,
45.2 mmol) in dry THF (60 mL), 1,6 M n-BuLi in hexane (34.0 mL,
54.2 mmol) and 2,3,4-trimethoxybenzaldehyde (10.6 g, 54.2 mmol)
were reacted to obtain alcohol 2 (13.5 g, 41.6 mmol, 92%). 1H NMR d
3.65 (3H, s, 20-OCH3), 3.83 (3H, s, 30-OCH3 or 40-OCH3), 3.85 (3H, s, 40-
OCH3 or 30-OCH3), 6.11 (1H, br s, CH-OH), 6.62 (1H, d, J = 8,6, H50),
6.99 (1H, d, J = 8,6, H60), 7.43–7.48 (3H, m, H3, H6, H7), 7.72–7.88
(3H, m, H4, H5, H8), 7.91 (1H, br s, H1). 13C NMR d 56.0 (CH3), 60.8
(2ꢂ) (CH3), 72.4 (CH), 107.1 (CH), 122.6 (CH), 124.8 (CH), 125.0
(CH), 125.8 (CH), 126.1 (CH), 127.7 (CH), 128.0 (CH), 128.1 (CH),
129.9 (C), 132.8 (C), 133.3 (C), 141.5 (C), 142.2 (C), 151.5 (C), 153.5
(C). IR (cmꢀ1) 1600, 3425. HRMS m/z found 347.1248; calcd for
4.1.12. (2-Naphthyl)(3,4,5-trimethoxyphenyl)methanothione (7)
Lawesson0s reagent (32 mg, 0.08 mmol) was added to ketone 3
(50 mg, 0.16 mmol) in a test tube, that was placed into a vessel filled
with alumina and irradiated at 750 W for 10 min (5 periods of
2 min). The reaction progress was checked by TLC until completion.
The reaction mixture was then passed through silica gel, using ethyl
acetate as eluent, yielding thioketone 7 (30 mg, 0.089 mmol, 55%).
1H NMR d 3.85 (6H, s, 30-OCH3, 50-OCH3), 3.98 (3H, s, 40-OCH3), 7.08
(2H, s, H20, H60), 7.54–7.62 (3H, m, H3, H6, H7), 7.87–7.95 (3H, m,
H4, H5, H8), 8.16 (1H, br s, H1). MS m/z (rel. int.) 338 (M+, 100).
C20H20O4 (+Na) 347.1254.
4.1.8. (2-Naphthyl)(3,4,5-trimethoxyphenyl)methanone (3)
FollowinggeneralmethodB, alcohol1 (3.0 g, 9.3 mmol)indichlo-
romethane (40 mL) was oxidised with KMnO4 (1.5 g, 9.3 mmol) in
the presence of tetra-n-butylammonium hydrogensulfate (30 mg,
0.088 mmol), yielding ketone 3 (2.2 g, 6.8 mmol, 73%), which is puri-
fied by crystallisation from dichloromethane/hexane. 1H NMR d 3.88
(6H, s, 30-OCH3, 50-OCH3), 3.97 (3H, s, 40-OCH3), 7.13 (2H, s, H20, H60),
7.58–7.64 (3H, m, H3, H6, H7), 7.85–8.05 (3H, m, H4, H5, H8), 8.29
(1H, br s, H1). 13C NMR d 56.4 (2ꢂ) (CH3), 61.0 (CH3), 108.0 (2ꢂ)
(CH), 125.9 (CH), 126.9 (CH), 127.9 (CH), 128.3 (2ꢂ) (CH), 129.4
(CH), 131.4 (CH), 132.3 (C), 132.9 (C), 135.1 (C), 135.2 (C), 142.0
(C), 153.0 (2ꢂ) (C), 195.7 (C). IR (cmꢀ1) 1600. HRMS m/z found
345.1080; calcd for C20H18O4 (+Na) 345.1097. Mp 108–110 °C
(CH2Cl2/hexane).
4.1.13. 1-(2-Naphthyl)-1-(2,3,4-trimethoxyphenyl)ethanol (8)
A 3 M solution of methylmagnesium chloride in THF (3.1 mL,
9.33 mmol) was added to a solution of ketone 4 (1.0 g, 3.1 mmol)
in THF (40 mL), at -40 °C. The crude product was percolated
through silica gel using dichloromethane as eluent, obtaining com-
pound 8 (550 mg, 1.63 mmol, 52%). 1H NMR d 1.90 (3H, s, CCH3),
3.07 (3H, s, 20-OCH3), 3.81 (3H, s, 30-OCH3 or 40-OCH3), 3.90 (3H,
s, 40-OCH3 or 30-OCH3), 4.75 (1H, br s, OH), 6.64 (1H, d, J = 8.8,
H50),7.27 (1H, d, J = 8.8, H60), 7.40–7.47 (3H, m, H3, H6, H7),
7.73–7.84 (3H, m, H4, H5, H8), 7.89 (1H, s, H1). 13C NMR d 30.2
(CH3), 56.0 (CH3), 60.1 (CH3), 60.6 (CH3), 76.2 (C), 106.4 (CH),
121.0 (CH), 123.0 (CH), 124.1 (CH), 125.6 (CH), 126.0 (CH), 127.6
(2ꢂ) (CH), 128.1 (CH), 132.3 (C), 133.2 (C), 133.4 (C), 142.9 (C),
148.2 (C), 151.8 (C), 153.6 (C). IR (cmꢀ1) 1599, 3513. HRMS m/z
found 361.1436; calcd for C21H22O4 (+Na) 361.1410.
4.1.9. (2-Naphthyl)(2,3,4-trimethoxyphenyl)methanone (4)
Following general method B, alcohol 2 (1.7 g, 5.2 mmol) in
dichloromethane (50 mL) was oxidised with KMnO4 (829 mg,

C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
9005
4.1.14. 1-[(2-Naphthyl)(3,4,5-trimethoxyphenyl)methyl]-1H-
imidazole (9)
mixture 1:1 of Z and E isomers. 1H NMR d 2.05 (3H, s, CH3CO, Z
or E isomer), 2.17 (3H, s, CH3CO, E or Z isomer), 3.75 (6H, s, 30-
OCH3, 50-OCH3, Z or E isomer), 3.81 (6H, s, 30-OCH3, 50-OCH3, E or
Z isomer), 3.88 (3H, s, 40-OCH3, Z or E isomer), 3.96 (3H, s, 40-
OCH3, E or Z isomer), 6.59 (2H, s, H20, H60, Z isomer), 6.84 (2H, s,
H20, H60, E isomer), 7.42–7.58 (6H, m, H3, H6, H7), 7.75–7.95 (8H,
m, H1, H4, H5, H8). 13C NMR d 19.7 (CH3), 19.9 (CH3), 56.3 (4ꢂ)
(CH3), 61.0 (2ꢂ) (CH3), 106.4 (2ꢂ) (CH), 106.8 (2ꢂ) (CH), 125.0
(CH), 126.0 (CH), 126.6 (CH), 126.8 (CH), 127.5 (CH), 127.8 (CH),
127.9 (4ꢂ) (CH), 128.3 (CH), 128.6 (CH), 128.8 (CH), 130.0 (2ꢂ)
(C), 130.2 (C), 130.4 (CH), 132.1 (C), 132.6 (C), 132.8 (C), 133.7
(C), 134.5 (C), 139.0 (C), 140.8 (C), 153.1 (4ꢂ) (C), 164.5 (C),
164.9 (C), 168.7 (C), 168.9 (C). IR (cmꢀ1) 1577, 1653, 1741. HRMS
m/z found 402.1326; calcd for C22H21NO5 (+Na) 402.1312.
To a solution of compound 1 (100 mg, 0.30 mmol) in CH2Cl2
(2.0 mL), imidazole (84 mg, 1.2 mmol), triphenylphosphine
(79 mg, 0.30 mmol) and iodine (312 mg, 1.23 mmol) were added.
The reaction mixture was stirred at room temperature, under ar-
gon, for 8 h. It was diluted with CH2Cl2 and washed with 10% so-
dium thiosulfate and water. The organic layer was dried over
Na2SO4 and the solvent evaporated. The crude product was chro-
matographed on silica using CH2Cl2/MeOH (9:1) as eluent, obtain-
ing imidazole derivative 9 (30 mg, 0.080 mmol, 27%). 1H NMR d
3.73 (6H, s, 30-OCH3, 50-OCH3), 3.86 (3H, s, 40-OCH3), 6.35 (2H, s,
H20, H60), 6.62 (1H, br s, (Ar)2-CH-im), 6.92 (1H, br s, H3 im or
H4 im), 7.14 (1H, br s, H4 im or H3 im), 7.25 (1H, dd, J1 = 8.6,
J2 = 1.8, H3), 7.46 (1H, m, H6 or H7), 7.48 (1H, s, H1), 7.52 (1H, s,
H2 im), 7.52 (1H, m, H7 or H6), 7.63 (1H, m, H5 or H8), 7.78 (1H,
m, H8 or H5), 7.84 (1H, d, J = 8.6, H4). 13C NMR d 56.2 (2ꢂ) (CH3),
60.8 (CH3), 65.3 (CH), 105.4 (2ꢂ) (CH), 119.4 (CH), 125.5 (CH),
126.7 (CH), 127.7 (CH), 128.1 (CH), 128.4 (CH), 128.7 (CH), 129.0
(CH), 131.9 (CH), 132.1 (CH), 133.0 (C), 133.1 (C), 134.3 (C), 136.3
(C), 137.4 (C), 153.6 (2ꢂ) (C). IR (cmꢀ1) 1592, 1713. HRMS m/z
found 397.1504; calcd for C23H22N2O3 (+Na) 397.1523.
4.1.18. (Z)-(2-Naphthyl)(2,3,4-trimethoxyphenyl)methanone
acetoxime (13Z)
Following general method C, oxime 11 (200 mg, 0.59 mmol) in
pyridine (600 lL) was acetylated with acetic anhydride (600 lL,
6.35 mmol), yielding acetoxime 13 (180 mg, 85%) as a 2:1 mixture
of Z and E isomers. This mixture was subjected to chromatography
on silica using CH2Cl2 as eluent, only allowing the isolation of iso-
mer Z (40 mg, 0.11 mmol, 19%). 1H NMR d 2.13 (3H, s CH3CO), 3.69
(3H, s, 20-OCH3), 3.91 (3H, s, 30-OCH3 or 40-OCH3), 3.94 (3H, s, 40-
OCH3 or 30-OCH3), 6.76 (1H, d, J = 8.6, H50), 6.86 (1H, d, J = 8.6,
H60), 7.45–7.51 (2H, m, H6, H7), 7.75–7.92 (4H, m, H1, H4, H5,
H8), 7.89 (1H, dd, J1 = 8.6, J2 = 1.8, H3). 13C NMR d 20.0 (CH3),
56.2 (CH3), 61.1 (2ꢂ) (CH3), 107.1 (CH), 123.8 (CH), 124.6 (CH),
126.5 (CH), 127.5 (CH), 127.9 (CH), 128.3 (CH), 128.9 (CH), 129.6
(CH), 132.3 (C), 132.9 (C), 134.6 (C), 142.2 (C), 147.7 (C), 151.4
(C), 155.0 (C), 162.4 (C), 169.0 (C). IR (cmꢀ1) 1652, 1761. HRMS
m/z found 402.1312; calcd for C22H21NO5 (+Na) 402.1311.
4.1.15. (Z + E)-(2-Naphthyl)(3,4,5-trimethoxyphenyl)
methanone oxime (10)
According to general method D, to a solution of ketone 3
(260 mg, 0.81 mmol) in methanol (15 mL) were added hydroxyl-
amine (535 mg, 8.1 mmol) and pyridine (2 drops), yielding oxime
10 (250 mg, 0.74 mmol, 91%) as a 1:1 mixture of Z and E isomers.
1H NMR d 3.74 (6H, s, 30-OCH3, 50-OCH3, Z or E isomer), 3.83 (6H,
s, 30-OCH3, 50-OCH3, E or Z isomer), 3.89 (3H, s, 40-OCH3, Z or E iso-
mer), 3.96 (3H, s, 40-OCH3, E or Z isomer), 6.70 (2H, s, H20, H60, Z iso-
mer), 6.76 (2H, s, H20, H60, E isomer), 7.46–7.58 (6H, m, H3, H6, H7),
7.78–7.95 (8H, m, H1, H4, H5, H8). 13C NMR d 56.2 (4ꢂ) (CH3), 61.0
(2ꢂ) (CH3), 105.5 (2ꢂ) (CH), 106.6 (2ꢂ) (CH), 124.4 (CH), 126.5
(2ꢂ) (CH), 126.8 (CH), 127.0 (2ꢂ) (CH), 127.9 (4ꢂ) (CH), 128.2
(CH), 128.6 (3ꢂ) (CH), 129.2 (C), 130.0 (C), 133.5 (C), 133.6 (C),
4.1.19. (Z + E)-(2-Naphthyl)(3,4,5-trimethoxyphenyl)
methanone tert-butoxycarbonyloxime (14)
Di-tert-butyl dicarbonate (196 mg, 0.90 mmol) and Na2CO3
(300 mg, 2.83 mmol) were added to a solution of oxime 10
(300 mg, 0.90 mmol) in 1:1 dioxane/water (14 mL). The reaction
mixture was stirred at room temperature for 48 h. After extraction
with dichloromethane (3ꢂ), the combined organic layers were
dried (Na2SO4) and the solvent evaporated to yield tert-but-
oxycarbonyloxime 14 (290 mg, 0.66 mmol, 74%) as a 1:1 mixture
of Z and E isomers. 1H NMR d 1.53 (18H, s, (CH3)3C), 3.77 (6H, s,
30-OCH3, 50-OCH3, Z or E isomer), 3.82 (6H, s, 30-OCH3, 50-OCH3, E
or Z isomer), 3.89 (3H, s, 40-OCH3, Z or E isomer), 3.97 (3H, s, 40-
OCH3, E or Z isomer), 6.62 (2H, s, H20, H60, Z isomer), 6.83 (2H, s,
H20, H60, E isomer), 7.44–7.60 (4H, m, H6, H7), 7.46 (1H, dd,
J1 = 8.4, J2 = 1.8, H3, Z or E isomer), 7.80 (1H, d, J = 8.4, H4, E or Z iso-
mer), 7.80–7.95 (4H, m, H5, H8), 7.88 (1H, dd, J1 = 8.4, J2 = 1.8, H3, E
or Z isomer), 7.92 (1H, d, J = 8.4, H4, Z or E isomer), 7.92 (2H, br s,
H1). 13C NMR d 27.4 (3ꢂ) (CH3), 27.8 (3ꢂ) (CH3), 56.3 (4ꢂ) (CH3),
61.0 (2ꢂ) (CH3), 83.7 (C). 85.2 (C), 106.7 (4ꢂ) (CH), 125.1 (CH),
126.3 (CH), 126.6 (2ꢂ) (CH), 127.3 (CH), 127.5 (CH), 127.7 (3ꢂ)
(CH), 127.9 (CH), 128.1 (CH), 128.7 (CH), 129.0 (CH), 130.1 (2ꢂ)
(CH) and (2ꢂ) (C), 132.3 (2ꢂ) (C), 132.8 (2ꢂ) (C), 133.7 (2ꢂ) (C),
134.5 (2ꢂ) (C), 146.6 (2ꢂ) (C), 153.1 (4ꢂ) (C), 163.5 (2ꢂ) (C). IR
143.5 (C), 144.0 (C), 153.1 (4ꢂ) (C), 158.0 (2ꢂ) (C). IR (cmꢀ1
)
1852, 3419. HRMS m/z found 360.1212; calcd for C20H19NO4
(+Na) 360.1206.
4.1.16. (Z + E)-(2-Naphthyl)(2,3,4-trimethoxyphenyl)
methanone oxime (11)
According to general method D, to a solution of ketone 4
(500 mg, 1.55 mmol) in methanol (25 mL) were added hydroxyl-
amine (1.02 g, 15.5 mmol) and pyridine (3 drops), yielding oxime
11 (448 mg, 1.33 mmol, 86%) as a 2:1 mixture of Z and E isomers.
1H NMR (DMSO) d 3.17 (3H, s, 20-OCH3, E isomer), 3.57 (3H, s, 20-
OCH3, Z isomer), 3.67 (3H, s, 30-OCH3 or 40-OCH3, E isomer), 3.77
(3H, s, 30-OCH3 or 40-OCH3, Z isomer), 3.82 (3H, s, 40-OCH3 or 30-
OCH3, E isomer), 3.85 (3H, s, 40-OCH3 or 30-OCH3, Z isomer), 6.83
(1H, d, J = 8.4, H50, Z isomer), 6.87 (1H, d, J = 8.4, H50, E isomer),
6.91 (1H, d, J = 8.4, H60, Z isomer), 7.13 (1H, d, J = 8.4, H60, E isomer),
7.44–7.56 (6H, m, H3, H6, H7), 7.80–7.90 (8H, m, H1, H4, H5, H8).
13C NMR d 55.9 (2ꢂ) (CH3), 60.4 (4ꢂ) (CH3), 107.6 (CH), 107.9 (CH),
123.4 (2ꢂ) (CH), 124.0 (2ꢂ) (CH), 124.9 (CH), 126–128 (11ꢂ) (CH)
and (2ꢂ) (C), 132.4 (C), 132.6 (2ꢂ) (C), 133.0 (C), 134.4 (2ꢂ) (C),
141.9 (2ꢂ) (C), 150.7 (C), 151.8 (C), 153.1 (C), 153.5 (C), 153.7
(C), 154.3 (C). HRMS m/z found 360.1239; calcd for C20H19NO4
(+Na) 360.1206.
(cmꢀ1
25H27NO6 (+Na) 460.1731.
) 1582, 1773. HRMS m/z found 460.1736; calcd for
C
4.1.20. (Z + E)-(2-Naphthyl)(2,3,4-trimethoxyphenyl)
methanone tert-butoxycarbonyloxime (15)
4.1.17. (Z + E)-(2-Naphthyl)(3,4,5-trimethoxyphenyl)
methanone acetoxime (12)
To a mixture of NaH (29 mg, 1.2 mmol) in THF (15 mL), oxime
11 (200 mg, 0.56 mmol) and di-tert-butyl dicarbonate (118 mg,
0.54 mmol) were added. The reaction mixture was stirred at room
temperature, under argon, for 8 h. The mixture was then perco-
lated through silica, using CH2Cl2 as eluent. The organic layer
Following general method C, oxime 10 (350 mg, 1.04 mmol) in
pyridine (900
lL) was acetylated with acetic anhydride (900 lL,
9.45 mmol), yielding acetoxime 12 (340 mg, 0.90 mmol, 87%) as a

9006
C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
was washed with water and dried over Na2SO4, yielding tert-but-
oxycarbonyloxime 15 (200 mg, 0.46 mmol, 82%) as a 2:1 mixture
of Z and E isomers. This mixture was chromatographed on silica,
with hexane/ethyl acetate (95:5) as eluent, allowing the separation
of the Z isomer 15Z (20 mg, 8%). 15Z 1H NMR d 1.51 (9H, s,
(CH3)3C), 3.73 (3H, s, 20-OCH3), 3.92 (3H, s, 30-OCH3 or 40-OCH3),
3.94 (3H, s, 40-OCH3 or 30-OCH3), 6.77 (1H, d, J = 8.6, H50), 6.89
(1H, d, J = 8.6, H60), 7.44–7.51 (2H, m, H6, H7), 7.75–7.86 (3H, m,
H1, H5, H8), 7.81 (1H, d, J = 8.4, H4), 7.95 (1H, dd, J1 = 8.4,
J2 = 1.8, H3). 15E (from Z + E mixture) 1H NMR d 1.51 (9H, s,
(CH3)3C), 3.27 (3H, s, 20-OCH3), 3.77 (3H, s, 30-OCH3 or 40-OCH3),
3.91 (3H, s, 40-OCH3 or 30-OCH3), 6.73 (1H, s, H50), 7.34 (1H, d,
J = 8.6, H60), 7.47–8.00 (6H, m, H1, H4, H5, H6, H7, H8), 7.62 (1H,
dd, J1 = 8.7, J2 = 1.4, H3). 13C NMR (Z + E mixture) d 27.9 (6ꢂ)
(CH3), 56.1 (2ꢂ) (CH3), 61.0 (4ꢂ) (CH3), 83.5 (2ꢂ) (C), 107.0 (2ꢂ)
(CH). 119.3 (2ꢂ) (C), 123.9 (2ꢂ) (CH), 124.6 (2ꢂ) (CH), 126.4
(2ꢂ) (CH), 127.3 (2ꢂ) (CH), 127.7 (2ꢂ) (CH), 128.1 (2ꢂ) (CH),
128.8 (2ꢂ) (CH), 129.3. (2ꢂ) (CH), 132.5 (2ꢂ) (C), 132.9 (2ꢂ) (C),
134.5 (2ꢂ) (C), 142.2 (2ꢂ) (C), 151.5 (2ꢂ) (C) , 152.4 (2ꢂ) (C),
154.9 (2ꢂ) (C), 161.3 (2ꢂ) (C). IR (cmꢀ1) 1596, 1772. HRMS m/z
found 460.1735; calcd for C25H27NO6 (+Na) 460.1731.
H5, H8), 7.79 (1H, d, J = 8.8, H4), 8.02 (1H, dd, J1 = 8.8, J2 = 1.8,
H3). 13C NMR d 56.2 (CH3), 61.1 (2ꢂ) (CH3), 108.3 (CH), 118.9 (C),
123.7 (CH), 124.6 (CH), 126.1 (3ꢂ) (CH), 127.6 (CH), 127.9 (CH),
128.4 (CH), 133.3 (2ꢂ) (C), 136.5 (C), 142.9 (C), 146.7 (C), 151.8
(C), 154.6 (C). IR (cmꢀ1) 1597, 3405. HRMS m/z found 359.1362;
calcd for C20H20N2O3 (+Na) 359.1366.
4.1.24. (Z + E)-(2-naphthyl)(3,4,5-trimethoxyphenyl)
methanone acetylhydrazone (19)
Following general method C, hydrazone 17 (200 mg,
0.60 mmol) in pyridine (600 lL) was treated with acetic anhydride
to obtain acetylhydrazone 19 (170 mg, 0.45 mmol, 75%) as a 6:4
mixture of Z and E isomers. This mixture was chromatographed
on silica, allowing the isolation of
Z isomer 19Z (20 mg,
0.053 mmol, 9%). 19Z 1H NMR d 2.50 (3H, s, CH3CO), 3.85 (6H, s,
30-OCH3, 50-OCH3), 3.97 (3H, s, 40-OCH3), 6.47 (2H, s, H20, H60),
7.45–7.52 (2H, m, H6, H7), 7.63 (1H, br s, H1), 7.70–7.80 (2H, m,
H5, H8), 7.84 (1H, d, J = 8.4, H4), 8.10 (1H, dd, J1 = 8.4, J2 = 1.8,
H3). 19E (from Z + E mixture): 2.46 (3H, s, CH3CO), 3.76 (6H, s,
30-OCH3, 50-OCH3), 3.87 (3H, s, 40-OCH3), 6.83 (2H, s, H20, H60),
7.28 (1H, dd, J1 = 8.4, J2 = 1.8, H3), 7.60 (2H, m, H6, H7), 7.70–
7.90 (2H, m, H5, H8), 7.78 (1H, br s, H1), 8.02 (1H, d, J = 8.4, H4).
13C NMR d 20.8 (2ꢂ) (CH3), 56.3 (4ꢂ) (CH3), 61.0 (2ꢂ) (CH3),
105.2 (4ꢂ) (CH), 123.3 (CH), 125.2 (CH), 126.6 (CH), 126.8 (2ꢂ)
(C), 127.2 (CH), 127.3 (CH), 127.7 (2ꢂ) (CH), 128.1 (CH), 128.3
(3ꢂ) (CH), 128.7 (2ꢂ) (CH), 130.0 (CH), 132.6 (C), 133.0 (C), 133.4
(C), 133.7 (C), 134.0 (C), 134.3 (C), 138.8 (C), 139.5 (C), 150.1 (C),
150.3 (C), 153.2 (2ꢂ) (C), 154.6 (2ꢂ) (C), 172.8 (C), 173.0 (C). IR
(cmꢀ1) 1583, 1683, 3317. HRMS m/z found 401.1464; calcd for
4.1.21. (Z + E)-(2-naphthyl)(3,4,5-trimethoxyphenyl)
methanone methoxime (16)
Methoxyamine hydrochloride (84 mg, 6.4 mmol) and NaAc-
Oꢁ3H2O (850 mg, 6.25 mmol) were added to a solution of ketone
3 (100 mg, 0.31 mmol) in 1:1 THF/MeOH (10 mL). The reaction
mixture was stirred at room temperature for 10 days, checking
by TLC until completion. The solvent was evaporated, the residue
dissolved in CH2Cl2 and the resulting solution washed with water,
dried (Na2SO4) and evaporated to yield methoxime 16 (75 mg,
0.21 mmol, 69%) as a 35:65 mixture of Z and E isomers. 1H NMR
d 4.02 (3H, s, CH3O–N, E isomer), 4.07 (3H, s, CH3O–N, Z isomer),
3.79 (6H, s, 30-OCH3, 50-OCH3, E isomer), 3.84 (6H, s, 30-OCH3, 50-
OCH3, Z isomer), 3.89 (3H, s, 40-OCH3, E isomer), 3.96 (3H, s, 40-
OCH3, Z isomer), 6.62 (2H, s, H20, H60, Z isomer), 6.77 (2H, s, H20,
H60, E isomer), 7.48–7.55 (6H, m, H3, H6, H7), 7.76–7.85 (8H, m,
H1, H4, H5, H8). 13C NMR d 56.2 (4ꢂ) (CH3), 61.0 (2ꢂ) (CH3),
62.6 (2ꢂ) (CH3), 105.5 (2ꢂ) (CH), 106.5 (2ꢂ) (CH), 124.6 (CH),
126.0–129.0 (13ꢂ) (CH), 130.7 (2ꢂ) (C), 132.1 (2ꢂ) (C), 132.9
(2ꢂ) (C), 133.0 (C), 133.8 (C), 139.4 (2ꢂ) (C), 153.1 (4ꢂ) (C),
156.6 (2ꢂ) (C). IR (cmꢀ1) 1585, 1653. HRMS m/z found 374.1364;
calcd for C21H21NO4 (+Na) 374.1363.
C22H22N2O4 (+Na) 401.1472.
4.1.25. (Z)-(2-naphthyl)(2,3,4-trimethoxyphenyl)methanone
acetylhydrazone (20Z)
Following general method C, hydrazone 18Z (200 mg,
0.60 mmol) in pyridine (600 lL) was treated with acetic anhydride
to obtain acetylhydrazone 20Z (180 mg, 0.48 mmol, 80%). 1H NMR
d 2.50 (3H, s, CH3CO), 3.69 (3H, s, 20-OCH3), 3.93 (3H, s, 30-OCH3 or
40-OCH3), 3.95 (3H, s, 40-OCH3 or 30-OCH3), 6.83 (2H, s, 50H, 60H),
7.42–7.48 (2H, m, H6, H7), 7.62 (1H, br s, H1), 7.65–7.90 (2H, m,
H5, H8), 7.83 (1H, d, J = 8.8, H4), 8.09 (1H, dd, J1 = 8.8, J2 = 1.8,
H3), 8.45 (1H, br s, NH). 13C NMR d 20.8 (CH3), 56.3 (CH3), 61.1
(2ꢂ) (CH3), 108.4 (CH), 117.4 (C), 123.4 (CH), 124.1 (CH), 126.4
(CH), 126.9 (CH), 127.7 (CH), 128.0 (CH), 128.2 (CH), 128.6 (CH),
133.0 (C), 134.0 (C), 134.9 (C), 143.0 (C), 148.1 (C), 151.5 (C),
155.6 (C), 173.0 (C). IR (cmꢀ1) 1595, 1670. HRMS m/z found
401.1477; calcd for C22H22N2O4 (+Na) 401.1472.
4.1.22. (Z + E)-(2-naphthyl)(3,4,5-trimethoxyphenyl)
methanone hydrazone (17)
Following general method E, ketone 3 (200 mg, 0.62 mmol) in
methanol (15 mL) reacted with excess hydrazine in the presence
of acetic acid (2 drops) to give hydrazone 17 (200 mg, 0.59 mmol,
95%) as a 6:4 mixture of Z and E isomers. 1H NMR d 3.74 (6H, s, 30-
OCH3, 50-OCH3, E isomer), 3.86 (6H, s, 30-OCH3, 50-OCH3, Z isomer),
3.86 (3H, s, 40-OCH3, E isomer), 3.96 (3H, s, 40-OCH3, Z isomer), 6.52
(2H, s, H20, H60, Z isomer), 6.76 (2H, s, H20, H60, Z isomer), 7.30–7.50
(6H, m, H3, H6, H7), 7.53 (1H, br s, H1, E isomer), 7.70–8.00 (7H, m,
H1, Z isomer, H4, H5; H8). IR (cmꢀ1) 1588, 3403. HRMS m/z found
359.1361; calcd for C20H20N2O3 (+Na) 359.1366.
4.1.26. (E + Z)-(2-naphthyl)(3,4,5-trimethoxyphenyl)
methanone methylhydrazone (21)
Methylhydrazine (286 mg, 6.2 mmol) and acetic acid (2 drops)
were added to a solution of ketone 3 (200 mg, 0.62 mmol) in MeOH
(20 mL). The reaction mixture was refluxed for 48 h, and in this
time a large excess of methylhydrazine was added in four portions.
The solvent was evaporated, the residue dissolved in CH2Cl2 and
the resulting solution washed with water, dried (Na2SO4) and
evaporated to yield methylhydrazone 21 (200 mg, 0.57 mmol,
92%) as a 40:60 mixture of Z and E isomers. 1H NMR d 3.03 (3H,
s, CH3NH, E isomer), 3.10 (3H, s, CH3NH, Z isomer), 3.75 (6H, s,
30-OCH3, 50-OCH3, E isomer), 3.85 (9H, s, 40-OCH3, E isomer, 30-
OCH3, 50-OCH3, Z isomer), 3.97 (3H, s, 40-OCH3, Z isomer), 5.47
(1H, br s, NH, Z or E isomer), 5.60 (1H, br s, NH, E or Z isomer),
6.52 (2H, s, 20H, 60H, Z isomer), 6.77 (2H, s, 20H, 60H, E isomer),
7.33–7.46 (4H, m, H5, H8), 7.35 (1H, dd, J1 = 8.6, J2 = 1.8, H3, E iso-
mer), 7.53 (1H, br s, H1, E isomer), 7.75 (1H, br s, H1, Z isomer),
7.77–8.12 (4H, m, H6, H7), 7.78 (1H, d, J = 8.6, H4, Z isomer), 7.99
(1H, d, J = 8.6, H4, E isomer), 8.08 (1H, dd, J1 = 8.6, J2 = 1.8, H3, Z iso-
4.1.23. (Z)-(2-naphthyl)(2,3,4-trimethoxyphenyl)methanone
hydrazone (18Z)
Following general method E, ketone 4 (500 mg, 1.55 mmol) in
methanol (25 mL) reacted with excess hydrazine in the presence
of acetic acid (3 drops) to give the Z hydrazone 18Z (480 mg,
1
1.43 mmol, 92%). H NMR d 3.74 (3H, s, 20-OCH3), 3.94 (3H, s, 30-
OCH3 or 40-OCH3,), 3.95 (3H, s, 40-OCH3 or 30-OCH3,), 5.60 (2H, br
s, NH2), 6.82 (1H, d, J = 8.4, H50), 6.88 (1H, d, J = 8.4, H60), 7.35-
7.50 (2H, m, H6, H7), 7.58 (1H, d, J = 1.8, H1), 7.68-7.81 (2H, m,

C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
9007
mer). 13C NMR d 38.1 (2ꢂ) (CH3), 56.2 (4ꢂ) (CH3), 61.0 (2ꢂ) (CH3),
103.8 (2ꢂ) (CH), 105.8 (2ꢂ) (CH), 123.7 (CH), 126.0 (CH), 126.1
(CH), 126.2 (2ꢂ) (CH), 126.5 (CH), 126.7 (CH), 127.0 (CH), 127.7
(CH), 128.0 (CH), 128.3 (2ꢂ) (CH), 128.8 (CH), 129.0 (C), 129.4
(CH), 130.9 (C), 133.1 (C), 133.3 (2ꢂ) (C), 133.6 (C), 134.5 (C),
136.1 (C), 138.1 (C), 139.0 (C), 144.8 (2ꢂ) (C), 153.1 (2ꢂ) (C),
154.4 (2ꢂ) (C). HRMS m/z found 373.1517; calcd for C21H22N2O3
(+Na) 373.1523.
ufacturer’s instructions. Briefly, a freshly prepared mixture solu-
tion of XTT labelling reagent and PMS (N-methylphenazinium
methylsulfate) electron coupling reagent was added to each well
at an amount of 50 lL. The resulting mixtures were further incu-
bated 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 ref-
erence wavelength of 655 nm. The IC50 was then calculated as the
drug concentration causing a 50% inhibition of cell proliferation. At
least six experiments were carried out for each compound assayed.
4.1.27. (E)-(2-naphthyl)(2,3,4-trimethoxyphenyl)methanone
oxime (10E), N-[(2-naphthyl)(3,4,5-trimethoxyphenyl)
methyl]acetamide (22) and N-(2-naphthyl)-N-(3,4,
4.2.2. Isolation of microtubular protein
5-trimethoxybenzyl)acetamide (23)
Calf brain microtubule protein (MTP) was purified by two cycles
of temperature-dependent assembly/disassembly, according to the
method of Shelanski,11 modified as described in the literature.12
Protein concentrations were determined by the method of Brad-
ford,13 using BSA as standard. Six different MTP preparations were
used in the tubulin assembly assays.
A solution of oxime 10 (200 mg, 0.59 mmol) in THF was treated
with LiAlH4 (236 mg, 5.90 mmol) at room temperature for 2 h. The
reaction mixture was filtered through silica and the solvent evap-
orated. The crude product was acetylated according to general
method C, by treatment with acetic anhydride (600
lL, 6.35 mmol)
in pyridine (600 L). The reaction product was then chromato-
l
graphed on silica, using hexane/ethyl acetate as eluent, yielding
60 mg (0.16 mmol, 28%) of acetamide 23, 20 mg (0.053 mmol,
9%) of acetoxime 12E and 10 mg (0.027 mmol, 5%) of acetamide 22.
4.2.3. Tubulin assembly
In vitro tubulin self-assembly was monitored turbidimetrically
at 450 nm, using a thermostated Thermospectronic Helios k spec-
trophotometer fitted with a Peltier temperature controller and a
circulating water bath. The ligands were dissolved in DMSO and
stored at ꢀ20 °C. The amount of DMSO in the assays was 4%. The
increase in turbidity was followed simultaneously in a batch of
six or seven cuvettes (containing 1.0 mg/mL MTP in 0.1 M MES buf-
fer, 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. The samples were preincubated
for 30 min at 20 °C in order to allow binding of the ligand, and then
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 polymerisation
after 20 min of incubation and was determined graphically. At
least three independent experiments with different MTP prepara-
tions were carried out for each compound tested.
1
Compound 12E. H NMR d 2.06 (3H, s, CH3CO), 3.77 (6H, s, 30-
OCH3, 50-OCH3), 3.89 (3H, s, 40-OCH3), 6.84 (2H, s, H20, H60), 7.42-
7.65 (4H, m, H6, H7), 7.44 (1H, br d, J = 8.4, H3), 7.81-7.91 (3H,
m, H4, H5, H8), 7.95 (1H, br s, H1). 13C NMR d 20.0 (CH3), 56.3
(2ꢂ) (CH3), 61.0 (CH3), 106.8 (2ꢂ) (CH), 126.0 (CH), 126.8 (CH),
127.4 (CH), 127.9 (2ꢂ) (CH), 128.6 (CH), 128.8 (CH), 130.0 (C),
130.1 (C), 132.6 (C), 133.7 (C), 140.8 (C), 153.1 (2ꢂ) (C), 164.9
(C), 168.7 (C). IR (cmꢀ1) 1573, 1653, 1769. HRMS m/z found
402.1302; calcd for C22H21NO5 (+Na) 402.1312.
1
Compound 22. H NMR d 2.10 (3H, s, CH3CO), 3.76 (6H, s, 30-
OCH3, 50-OCH3), 3.83 (3H, s, 40-OCH3), 6.23 (1H, br d, J = 8.0, NH),
6.34 (1H, d, J = 8.0, AcNH-CH), 6.48 (2H, s, H20, H60), 7.32 (1H, dd,
J1 = 8.4, J2 = 1.8, H3), 7.45-7.55 (2H, m, H6, H7), 7.68 (1H, br s,
H1), 7.75–7.90 (3H, m, H4, H5, H8). 13C NMR d 23.5 (CH3), 56.2
(2ꢂ) (CH3), 60.9 (CH3), 57.3 (CH), 104.9 (2ꢂ) (CH), 125.6 (CH),
125.9 (CH), 126.2 (CH), 126.4 (CH), 127.7 (CH), 128.0 (CH), 128.6
(CH), 132.8 (C), 133.3 (C), 137.2 (C), 137.3 (C), 138.6 (C), 153.5
(2ꢂ) (C), 169.2 (C). IR (cmꢀ1) 1592, 1651, 3287. HRMS m/z found
388.1504; calcd for C22H23NO4 (+Na) 388.1519.
4.2.3.1. Molecular modelling.
The compounds were docked
into the colchicine site of tubulin following a described proto-
col.14 The X-ray structures of the tubulin complexes with podo-
phyllotoxin and DAMA-colchicine were retrieved from the
protein data bank15, while chains C, D and E and the correspond-
ing hetero-groups were removed by hand. The pdb files were en-
ergy-minimized and subjected to molecular dynamics
simulations at 300 K.16 We initially restrained the backbone,
and then it was set free. The relaxed structures were superim-
posed and a combined site was generated by shifting the tubu-
1
Compound 23. H NMR d 1.91 (3H, s, CH3CO), 3.71 (6H, s, 30-
OCH3, 50-OCH3), 3.81 (3H, s, 40-OCH3), 4.87 (1H, s, CH2–N), 6.42
(2H, s, H20, H60), 7.09–7.53 (2H, m, H6, H7), 7.11 (1H, br d, J = 8.0,
H3), 7.73–7.85 (4H, m, H1,H4, H5, H8). 13C NMR d 22.9 (CH3),
56.1 (2ꢂ) (CH3), 60.9 (CH3), 106.2 (2ꢂ) (CH), 126.2 (CH), 126.9
(3ꢂ) (CH), 127.8 (2ꢂ) (CH), 129.7 (CH), 132.5 (C), 133.1 (C),
133.6 (C), 137.4 (C), 140.2 (C), 153.1 (2ꢂ) (C), 170.6 (C). IR
(cmꢀ1
) 1660, 1714. HRMS m/z found 388.1515; calcd for
C22H23NO4 (+Na) 388.1519.
lin–podophyllotoxin complex 30 Å along the X
axes.17 The
combined tubulin sites and the podophyllotoxin and DAMA-col-
chicine ligands were used to generate a combined protomol with
the Surflex docking program.18 The individual sites were also
used in separate docking experiments with AutoDock 3.19 The
synthesised compounds together with roughly 300 combretasta-
tins and phenstatin analogues were manually constructed in sil-
ico20 and docked into the combined sites (cross-docking), in an
attempt to better reproduce the receptor flexibility by using dif-
ferent configurations of the protein.21 Additionally, the test set
of ACD compounds used in the Surflex validation were equally
docked.18 These compounds were considered a negative control
group: that is, lacking biological activity. The combined results
were analysed by receiver operating characteristics (ROCs);22
the ACD compounds and the analogues of the colchicine site
4.2. Biological evaluation
4.2.1. Cytotoxicity. XTT procedure
One hundred micro litres of exponentially growing HeLa
(1.5 ꢂ 103 cells/well), HT-29 (3 ꢂ 103 cells/well), or A-549
(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
plates. HL-60 cells were seeded at 3 ꢂ 103 (100
lL) cells per well.
Then, cells were incubated with different concentrations of the as-
sayed compound at 37 °C under the 5% CO2/95% air atmosphere for
72 h. Cell proliferation was quantified using the XTT (3,3-[4-(phe-
nylaminocarbonyl)-2,3-tetrazolium]-bis-(4-methoxy-6-nitro)ben-
zene sulfonic acid sodium salt hydrate) cell proliferation kit (Roche
Molecular Biochemicals, Mannheim, Germany) following the man-
´
with published TPI worse than 20
lM were considered inactive.
The enrichment factors achieved were similar to those described

9008
C. Álvarez et al. / Bioorg. Med. Chem. 16 (2008) 8999–9008
6. Pérez-Melero, C.; Maya, A. B.; del Rey, B.; Pelaez, R.; Caballero, E.; Medarde, M.
Bioorg. Med. Chem. Lett. 2004, 14, 3771–3774.
7. Alvarez, C.; Alvarez, R.; Corchete, P.; Pérez-Melero, C.; Pelaez, R.; Medarde, M.
Bioorg. Med. Chem. Lett. 2007, 17, 3417–3420.
for similar systems. The structures of the best scored complexes
in the colchicine and podophyllotoxin sites were inspected visu-
ally and compared with the TPI results.23
8. Alvarez, C.; Alvarez, R.; Corchete, P.; Pérez-Melero, C.; Pelaez, R.; Medarde, M.
Bioorg. Med. Chem. 2008, 18. DOI: 10.1016/j.bmc.2008.04.054.
9. Liou, J. P.; Chang, J. Y.; Chang, C. W.; Chang, C. Y.; Mahindroo, N.; Kuo, F. M.;
Hsieh, H. P. J. Med. Chem. 2004, 47, 2897–2905.
Acknowledgments
10. Wipf, P.; Reeves, J. T.; Balachandran, R.; Day, B. W. J. Med. Chem. 2002, 45,
1901–1917.
11. Shelanski, M. L.; Gaskin, F.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A. 1973, 70,
765–768.
12. Dumortier, C.; Gorbunoff, M.; Andreu, J. M.; Engelborghs, Y. Biochemistry 1996,
35, 4387–4395.
13. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
14. Pelaez, R.; López, J. L.; Medarde, M. Application of chemoinformatic tools for
the analysis of virtual screening studies of tubulin inhibitors. In Advances in Soft
Computing; Corchado, E., Corchado, J. M., Abraham, A., Eds.; Springer Verlag:
Berlin, 2007; Vol. 44, pp 411–417.
Financial support from Ministerio de Educación y Ciencia (Ref.
CTQ2004-00369/BQU), Junta de Castilla y León (Ref. SA090A06
and SA 009A06), Universidad de Salamanca (Ref. USAL2005-02)
and EU (structural funds). R.A. thanks Fundación Ramón Areces
and R.A. and C.A. thank Ministerio de Educación y Ciencia for predoc-
toral fellowships.
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