1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues of combretastatin A-4: Synthesis, molecular modeling and evaluation as cytotoxic agents and inhibitors of tubulin

Article abstract of DOI:10.1016/j.bmc.2008.03.049

A series of cis-restricted 1,5-disubstituted 1,2,3-triazole analogues of combretastatin A-4 (1) have been prepared. The triazole 12f, 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)aniline, displayed potent cytotoxic activity against severa

Full text of DOI:10.1016/j.bmc.2008.03.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4829–4838
1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues
of combretastatin A-4: Synthesis, molecular modeling
and evaluation as cytotoxic agents and inhibitors of tubulin
a
b
b
b,
´ ´
Kristin Odlo, Jean Hentzen, Jeremie Fournier dit Chabert, Sylvie Ducki,
Osman A. B. S. M. Gani,c Ingebrigt Sylte,c Martina Skrede,d
Vivi Ann Flørenesd and Trond Vidar Hansena,*
aSchool of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, PO Box 1068, N-0316 Oslo, Norway
bCentre for Molecular Design, Cockroft Building, University of Salford, Manchester M5 4WT, UK
cDepartment of Pharmacology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway
dDivision of Pathology, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
Received 20 December 2007; revised 12 March 2008; accepted 20 March 2008
Available online 23 March 2008
Dedicated to Professor Lars Skattebøl on the occasion of his 80th birthday.
Abstract—A series of cis-restricted 1,5-disubstituted 1,2,3-triazole analogues of combretastatin A-4 (1) have been prepared. The tri-
azole 12f, 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)aniline, displayed potent cytotoxic activity against several
cancer cell lines with IC50 values in the nanomolar range. The ability of triazoles to inhibit tubulin polymerization has been eval-
uated, and 12f inhibited tubulin polymerization with IC50 = 4.8 lM. Molecular modeling experiments involving 12f and the colchi-
cine binding site of a,b-tubulin showed that the triazole moiety interacts with b-tubulin via hydrogen bonding with several amino
acids.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Combretastatin A-4 (CA-4, 1) was isolated from the
bark of the South African tree Combretum caffrum in
1989 by Pettit et al.5 This cis-stilbene binds to the colchi-
cine binding site of tubulin and inhibits tubulin polymer-
ization. Combretastatin A-4 is also highly cytotoxic
against a variety of human cancer cell lines, including
multidrug resistant cell lines. Moreover, this natural
product disrupts the cell signalling pathways involved
in regulation and maintenance of the cytoskeleton of
endothelial cells in tumor vasculature, thereby leading
to selective shutdown of blood flow through tumors.
These biological effects and the structural simplicity of
1 make this natural product an attractive lead com-
pound in the development of new anticancer agents.6
Structure–activity relationship (SAR) studies showed
that a 3,4,5-trimethoxysubstituted A ring and a 4-meth-
oxysubstituted B ring separated by a double bond (two-
atom bridge) with cis configuration are important for
optimal cytotoxic activity.7 The major disadvantages
of combretastatin A-4 as a drug candidate are low water
solubility and isomerization to the less active trans-form.
A phosphate prodrug of combretastatin A-4 (CA-4P, 2)
Microtubules are cylindrical protein polymers composed
of a- and b-tubulin heterodimers. Microtubules are
essential for many cellular processes, such as mainte-
nance of cellular shape, intracellular transport and mito-
tic spindle assembly during cell division.1 Inhibition of
microtubule formation leads to mitotic arrest and pro-
motes vascular disruption in angiogenic vessels eventu-
ally leading to cell death by apoptosis.2 Hence, tubulin
is one of the most common and strategic targets for
development of new anticancer drugs.3,4
Keywords: Combretastatin A-4; Cytotoxic; 1,2,3-Triazoles; Molecular
modeling.
*
Corresponding author. Tel.: +47 22857450; fax: +47 22855947;
e-mail: t.v.hansen@farmasi.uio.no
´ ´
Present address: Laboratoire de Chimie des Heterocycles et Glucides,
´ ´
Clermont Universite, Ensemble Scientifique des Cezeaux, 63174
Aubie`re, France.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.049

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K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
Figure 1. Combretastatin A-4 1 and its prodrug 2.
has been prepared, and is currently in clinical trials8
(Fig. 1).
2. Results and discussion
2.1. Synthesis
A series of combretastatin analogues with a locked
cis-type bridge between the two phenyl rings have been
prepared to overcome the formation of the less active
trans-form of 1. This was achieved by the introduction
of a heterocyclic moiety between rings A and B.9 Hence,
we envisioned that a 1,5-disubstituted 1,2,3-triazole het-
erocyclic ring would serve as a suitable mimic for the cis
configuration present in 1. Moreover, some triazole ana-
logues of 1 have been reported.9a,10 Herein we report the
synthesis and in vitro cytotoxic activities of several 1,5-
disubstituted 1,2,3-triazole analogues of combretastatin
A-4, as well as the inhibition data of tubulin polymeriza-
tion for the most active compounds. Furthermore,
molecular modeling studies of the most active tubulin
inhibitor with the colchicine binding site of a,b-tubulin
revealed hydrogen-bonding interactions with several
amino acids in the colchicine binding site of b-tubulin.
1,2,3-Triazoles are available from the thermally induced
Huisgen cycloaddition reaction between azides and al-
kynes.11 This cycloaddition reaction usually affords mix-
tures of 1,4- and 1,5-disubstituted 1,2,3-triazoles.12
Recently, the groups of Sharpless and Meldal reported
that 1,4-disubstituted 1,2,3-triazoles are specifically pre-
pared from azides and terminal alkynes under copper-(I)
catalysis.13 The regioisomeric 1,5-disubstituted triazoles
are available from azides and terminal alkynes by the
use of either magnesium acetylides14 or ruthenium
catalysis.15
Our synthetic efforts toward the target triazoles 7a–7b
and 7d–7f depicted in Scheme 1, started with the synthe-
sis of the terminal alkyne 4 from the aldehyde 3 using the
Colvin rearrangement.16 The azides 6a–6d were obtained
Scheme 1. Synthesis of 1,5-disubstituted triazoles 7a–7f. Reagents and conditions: (i) LDA, TMSCHN2, THF, ꢀ78 ꢁC; (ii) HCl (aq), NaN3, NaNO2,
0 ꢁC; (iii) n-Bu4NF, THF; (iv) a—EtMgCl, THF, D; b—6a–6d; (v) n-Bu4NF, THF; (vi) H2, Pd/C, EtOH, H2SO4.

K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
4831
from the anilines 5a–5d employing standard diazotiza-
tion conditions.17 The yield of the azide 6a was improved
to 93% yield using a recently published procedure.18 The
terminal alkyne 4 was treated with ethyl magnesium
chloride as previously described,9a followed by the addi-
tion of a THF-solution of the azides 6a–6d. The phenol
in azide 6c was protected in 56% yield as the correspond-
ing TBDMS-ether19 prior to the cycloaddition reaction.
After heating at reflux for 3 h, the 1,5-disubstituted
1,2,3-triazoles 7a–7d were obtained in 62–86% yields
after purification by column chromatography. Deprotec-
tion20 of the TBDMS-ether in 7c yielded triazole 7e in
79% yield after purification. Reduction of the nitro
group in triazole 7d by catalytic hydrogenation afforded
the amino triazole 7f in 80% yield.
difficult to obtain. Reduction of the nitro group in 12e–
12f was achieved by catalytic hydrogenation, while 13a
was reduced to 13b using NaBH4/CuSO4.22 Triazoles
12a–b, 12d–12f and 13a–13b were purified by chroma-
tography or recrystallization. All new compounds exhib-
ited spectral data in agreement with their assigned
structures.
2.2. Biological evaluation
All prepared triazoles have the four methoxy groups of
the A- and B-rings in the correct positions and a two-
atom bridge with a cis-similar arrangement between
the A- and B-rings required for optimal cytotoxicity.
Previous studies have shown that the hydroxyl group
on the B-ring of combretastatin A-4 is amenable for bio-
isosteric substitution9g,23 (Table 1).
Syntheses of the 1,5-disubstituted 1,2,3-triazoles 12a–
12b and 12d–12f followed the chemistry described above
(Scheme 2). Triazole 12e and its 1,4-disubstituted isomer
13a were obtained from the thermally induced Huisgen
cycloaddition reaction21 between azide 9 and alkyne
11d, since the magnesium acetylide of alkyne 11d proved
The target triazoles were submitted to the MTT assay24
for the evaluation of their cytotoxic effects against the
K562 leukemia cancer cell line. These results showed
that compounds 7a–7b and 7d–7f, in which the three
Scheme 2. Synthesis of triazoles 12a–12f and 13a–13b. Reagents and conditions: (i) LDA, TMSCHN2, THF, ꢀ78 ꢁC; (ii) HCl (aq), NaN3, NaNO2,
0 ꢁC; (iii) a—EtMgCl, THF, D; b—9; (iv) n-Bu4NF, THF; (v) 11d, H2O, EtOH, 9, D; (vi) H2, Pd/C, MeOH; (vii) NaBH4, CuSO4, EtOH, D.

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K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
Table 1. Cytotoxicity and inhibition of tubulin polymerization by
triazoles 7a–7b, 7d–7f, 12a–12b, 12d–12f and 13a–13b
antimitotic effects of these triazoles were not investi-
gated further, but triazole 12f and combretastatin A-4
(1) were submitted to several other cancer cell lines
(Table 2).
Compound
K562 cell assay,
a
IC50 (lM) in vitro
cytotoxicity
Inhibition of tubulin
polymerization
b
IC50 (lM)
These data showed that both 12f and combretastatin A-
4 (1) were cytotoxic in all six human cancer cell lines
with comparable activities with IC50 values ranging be-
tween 3.9–5.1 nM and 2.5–6.0, respectively.
7a
7b
4.00
3.60
>10
n.d.c
n.d.c
n.d.c
14.0
>20
7.0
7d
7e
0.41
0.57
0.027
0.057
0.035
0.46
0.011
>10
7f
12a
12b
12d
12e
12f
13a
13b
1
2.3. Molecular modeling
>20
12.6
>20
4.8
n.d.c
>20
0.6
Molecular modeling studies were performed to investi-
gate the binding ability of the triazoles to the colchicine
binding site of a,b-tubulin. Docking studies showed that
triazole 12f occupied the colchicine binding site of a,b-
tubulin (mostly buried in the b subunit) (Fig. 2a). The
binding energies of 12f for a- and b-tubulin were about
6 and ꢀ10 kcal/mol, respectively. The trimethoxyphenyl
moiety in ring A of triazole 12f was positioned in the
binding cavity surrounded by the strands S8 and S9,
and the helices H7 and H8 of b-tubulin. The thiol group
of bC241 in H7 formed a hydrogen bond with the oxy-
gen atom of one of the methoxy groups, and was close
1.30
0.010
a Results of three experiments.
b Results of two experiments.
c n.d., not determined.
N atoms in the triazole moiety were derived from the
B-ring azides 6a–6d, were less active than the triazoles
derived from the A-ring azide 9. Triazoles 7e and 7f
revealed the best cytotoxic activities with IC50 = 0.41
and 0.57 lM, respectively. For the regioisomeric tria-
zoles 12a–12b and 12d–12f, cytotoxic activities in the
nano- to micromolar range were observed for all
compounds; 12a and 12f showed best cytotoxic activi-
˚
(ꢁ4 A) to the oxygen of another methoxy group. Several
amino acids of b-tubulin formed hydrophobic interac-
tions with the trimethoxyphenyl moiety of 12f. bL248
and bL255 exhibited particular strong van der Waals
˚
interactions (distance <3.7 A) with the phenyl ring (ring
A) of the trimethoxyphenyl moiety, while bL242, bI378,
and bV318 interacted with the methyl group of the
methoxy moieties in ring A (Fig. 2b).
ties with IC50 = 27 and 11 nM, respectively (1, IC50
=
10 nM). In triazoles 12a and 12f the 30-hydroxyl group
in the lead compound was replaced by a hydrogen
atom and an amino group, respectively. Triazole 12d
with a hydroxyl group in the 30-position on ring B
was less active (IC50 = 35 nM) than both 12a and 12f.
The structure of 12f is more similar to the known 30-
amino analogue of 1, and this compound has been re-
ported to exhibit increased activity compared to 1 in
terms of in vitro cytotoxicity against some human can-
cer cell lines.25 Apparently, the position of the 1,2,3-tri-
azole moiety is important, since triazoles 12a and 12f
were more active than their regioisomers 7a and 7f.
Interestingly, triazole 13b, which is the 1,4-regioisomer
of 12f, also exhibited cytotoxic activity (IC50 = 1.3 lM)
in this assay.
The N-2 and N-3 atoms of the 1,2,3-triazole ring of 12f
formed hydrogen bonds with the main chain nitrogen
atoms of bA250 and bD251 in loop T7 that connects
H7 and H8 of b-tubulin. The amino group of the 3-ami-
no-4-methoxy phenyl moiety formed hydrogen bonds
with the main chain carbonyl oxygen atom of aT179
of loop T5 (the loop connecting strand 5 and helix 5
of a-tubulin). The methoxy oxygen atom in ring B of
12f formed hydrogen bonds with the main chain nitro-
gen atom of aV181. The side chain of bK352 was close
both to the amino group and the methoxy oxygen atom
˚
˚
(3.8 A and 4.2 A, respectively), indicating the possibility
of hydrogen bonds with both groups. The side chains of
aV181 and bM259 and bK352 (Cc atom) formed strong
˚
˚
The triazoles that exhibited significant cytotoxic activi-
ties in the K562 cell assay were further evaluated for
their ability to inhibit tubulin assembly.26 Triazoles
12a and 12f showed inhibition of tubulin assembly with
IC50 = 7.0 and 4.8 lM, respectively, while the other
compounds tested showed IC50 values >10 lM. How-
ever, the lead compound combretastatin A-4 (1) inhib-
ited tubulin assembly with IC50 = 0.6 lM. Hence, the
van der Waals contacts (distances of 3.5 A, 3.8 A, and
˚
3.6 A, respectively) with the methoxy methyl group.
The binding energies of 12f with different tubulin sub-
units indicate that b-tubulin is responsible for the
strength of the binding. The suggested binding mode
of triazole 12f is in agreement with the X-ray structure
complex of DAMA-colchicine–tubulin27 (Fig. 2a), a
Table 2. IC50 values (nM)a for in vitro cytotoxicity data of CA-4 (1) and 12f
Compound MDA-MB231 cell assay SK-BR 3 cell assay SKOV cell assay OVCAR cell assay WM35 cell assay WM239 cell assay
1
12f
6.00
>10
5.10
4.00
3.30
4.90
3.30
4.70
2.50
3.90
2.80
5.10
a Data are medians of three experiments.

K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
4833
We are currently preparing other triazole mimics of the
combretastatins, and these efforts, together with their
biological evaluation and docking studies, will hopefully
further elucidate the importance of the 1,2,3-triazole
moiety with regard to tubulin inhibition and cytotoxic-
ity. These efforts will be reported in due course.
3. Conclusion
Compound 12f exhibited potent cytotoxic activity in
several cancer cell lines as well as moderate inhibition
of tubulin polymerization. These activities are most
likely, at least in part, due to binding to the colchicine
binding site of a,b-tubulin in the b subunit, as supported
by our molecular modeling studies.
4. Experimental
4.1. General procedure
Unless noted otherwise, all reagents and solvents were
used as purchased without further purification. Melting
points are uncorrected. Analytical TLC was performed
using silica gel 60 F254 glass plates (Merck). Flash col-
umn chromatography was performed on silica gel 60
(40–63 lm, Fluka). NMR (1H, 13C) spectra were re-
corded on a Bruker DPX-300 MHz spectrometer. Cou-
pling constants (J) are reported in hertz, and chemical
shifts are reported in parts per million (d) relative to
CDCl3 (7.26 ppm for 1H and 77.00 ppm for 13C) or
DMSO-d6 (2.50 ppm for 1H and 39.43 ppm for 13C).
Intermediates 4,29 6a,30 6d,31 9,32 10c,33 11a,34 and
11c35 are known compounds. Combretastatin A-4 (1)
was prepared according to a literature procedure.19b
Figure 2. The structure of 12f docked into the colchicine binding site
of tubulin (PDB entry 1SA0). (a) Superposition of the docked
conformation of 12f (stick model with atom type color: carbon-white,
nitrogen-blue, oxygen-red, hydrogen-gray) on top of the X-ray
structure of DAMA-colchicine (black wire model). The backbone of
tubulin is shown as ribbon representation (a-tubulin: red, and b-
tubulin: blue). Secondary structures important for ligand binding are
labeled. GTP is shown as stick model within a-tubulin. (b) A closer
view of 12f at the binding site. Triazole 12f and its hydrogen bonding
4.2. Syntheses of azides: general procedure
The corresponding aniline (10.00 mmol) was suspended
in HCl/H2O (1:1, 34 mL) and cooled to 0 ꢁC. An aque-
ous solution of sodium nitrite (811 mg, 11.75 mmol)
was added dropwise, and the reaction mixture stirred
at 0 ꢁC for 1 h. An aqueous solution of sodium azide
(780 mg, 11.96 mmol) was added dropwise (caution!).
The reaction mixture was stirred at room temperature
for 3 h, and extracted with ethyl acetate (2· 150 mL).
The combined organic layers were washed with brine
(2· 100 mL), dried over anhydrous magnesium sulfate
and the solvent was removed in vacuo. The azides were
used as such without purification.
˚
partners are shown as stick models. Hydrogen bonds (distance 63.5 A)
are shown as dotted lines. The amino acids of tubulin within 4.2 A
from 12f are shown as wire models.
˚
study of 1,2,4-triazole-based tubulin inhibitors,9a and
with a suggested structure-based pharmacophore model
of tubulin.28 In agreement with the first two studies, dif-
ferent moieties (trimethoxyphenyl, triazole, and 3-ami-
no-4-methoxyphenyl) of 12f occupied a Cartesian
space corresponding to that of colchicine. The binding
mode is also in agreement with the pharmacophore
model which identified specific receptor–ligand hydro-
gen bonds and hydrophobic interactions.27 In addition,
we observed the following interactions which have not
been reported previously: (a) main chain carbonyl oxy-
gen of aT179 participated in hydrogen bonds to 12f;
(b) strong hydrophobic interactions of several b-tubulin
amino acids (bL248, bL255, bL242, bI378, and bV318)
to the trimethoxyphenyl moiety; and (c) strong polar
and hydrophobic interactions of bK352 to 12f.
4.2.1. 1-Azido-4-methoxybenzene (6a). Brown oil (13%).
Rf = 0.89 (hexane/EtOAc 1:1). 1H NMR (300 MHz,
DMSO-d6): d = 3.74 (s, 3H), 6.96–7.00 (m, 2H), 7.02–
7.06 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
d = 55.30, 115.23, 120.06, 131.32, 156.69. MS (EI):
150.0 (M+1).
4.2.2. 4-Azido-2-bromo-1-methoxybenzene (6b). Dark
brown oil (87%). Rf = 0.82 (hexane/EtOAc 1:1). 1H
NMR (300 MHz, CDCl3): d = 3.87 (s, 3H), 6.86 (d,
J = 8.8 Hz, 1H), 6.94 (dd, J = 8.8, 2.6 Hz, 1H), 7.23 (d,

4834
K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
J = 2.6 Hz, 1H). 13C NMR (75 MHz, CDCl3): d = 56.54,
112.51, 112.67, 118.82, 123.86, 133.27, 153.35. HRMS
calcd for C7H6BrN3O (M+): 226.9694. Found 226.9691.
114.48, 121.47, 127.37, 129.17, 132.50, 137.61, 137.85,
152.74, 159.82. HRMS calcd for C18H19N3O4 (M+):
341.1376. Found 341.1379.
4.2.3. 5-Azido-2-methoxyphenol (6c). Brown oil (30%).
Rf = 0.88 (hexane/EtOAc 1:5). 1H NMR (300 MHz,
DMSO-d6): d = 3.74 (s, 3H), 6.48–6.52 (m, 2H), 6.94
(dd, J = 7.4, 1.5 Hz, 1H), 9.40 (br s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 55.86, 106.34, 109.23,
113.31, 131.57, 145.40, 147.70. HRMS calcd for
C7H7N3O2 (M+): 165.0538. Found 165.0539.
4.3.2. 1-(3-Bromo-4-methoxyphenyl)-5-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (7b). The crude product was
purified by chromatography (hexane/EtOAc 1:1,
Rf = 0.26) affording a pale yellow solid (86%). Mp
1
186–187 ꢁC. H NMR (300 MHz, DMSO-d6): d = 3.64
(s, 6H), 3.66 (s, 3H), 3.91 (s, 3H), 6.62 (s, 2H), 7.26 (d,
J = 8.9 Hz, 1H), 7.45 (dd, J = 8.8, 2.6 Hz, 1H), 7.82 (d,
J = 2.5 Hz, 1H), 8.13 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 55.79, 56.65, 60.01, 106.17, 110.35,
112.59, 121.25, 126.80, 129.65, 130.42, 132.60, 137.85,
138.01, 152.77, 156.17. HRMS calcd for C18H18BrN3O4
(M+): 419.0481. Found 419.0480.
4.2.4. (5-Azido-2-methoxyphenoxy)(tert-butyl)dimethyl-
silane (6c-protected). Brown semisolid (56%). Rf = 0.83
(hexane/EtOAc 2:1). H NMR (300 MHz, DMSO-d6):
1
d = 0.13 (s, 6H), 0.95 (s, 9H), 3.75 (s, 3H), 6.52 (d,
J = 2.7 Hz, 1H), 6.71 (dd, J = 8.7, 2.7 Hz, 1H), 7.02 (d,
J = 8.7 Hz, 1H). 13C NMR (75 MHz, DMSO-d6):
d = ꢀ4.85, 18.04, 25.41, 55.59, 111.42, 112.11, 113.40,
131.49, 145.17, 148.18. HRMS calcd for C13H21N3O2Si
(M+): 279.1403. Found 279.1404.
4.3.3. 1-(3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl)-
5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (7c). The
crude product was purified by chromatography (hex-
ane/EtOAc 1:1, Rf = 0.42) affording a pale orange solid
1
(62%). Mp 129–130 ꢁC. H NMR (300 MHz, DMSO-
4.2.5. 4-Azido-1-methoxy-2-nitrobenzene (6d). Brown so-
d6): d = 0.01 (s, 6H), 0.86 (s, 9H), 3.62 (s, 6H), 3.65 (s,
3H), 3.82 (s, 3H), 6.57 (s, 2H), 6.74 (d, J = 2.1 Hz,
1H), 7.12 (dd, J = 8.7, 2.2 Hz, 1H), 7.16 (d, J = 8.7 Hz,
1H), 8.06 (s, 1H). 13C NMR (75 MHz,, DMSO-d6):
d = ꢀ5.17, 17.96, 25.27, 55.68, 59.86, 106.06, 112.36,
117.90, 119.39, 121.64, 128.94, 132.58, 137.54, 137.89,
144.21, 151.39, 152.80. HRMS calcd for C24H33N3O5Si
(M+): 471.2189. Found 471.2192.
1
lid (89%). Mp 80 ꢁC. Rf = 0.69 (hexane/EtOAc 1:1). H
NMR (300 MHz, DMSO-d6): d = 3.91 (s, 3H), 7.36–
7.44 (m, 2H), 7.64 (d, J = 2.4 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 56.89, 115.43, 115.73,
124.75, 131.89, 139.52, 148.98. HRMS calcd for
C7H6N4O3 (M+): 194.0440. Found 194.0439.
4.2.6. 5-Azido-1,2,3-trimethoxybenzene (9). Dark red so-
lid (81%). Mp 42–45 ꢁC. Rf = 0.81 (hexane/EtOAc 1:1).
1H NMR (300 MHz, DMSO-d6): d = 3.62 (s, 3H), 3.78
(s, 6H), 6.40 (s, 2H). 13C NMR (75 MHz, DMSO-d6):
d = 55.94, 60.02, 96.68, 134.78, 134.87, 153.66. HRMS
calcd for C9H11N3O3 (M+): 209.0800. Found 209.0795.
4.3.4. 1-(4-Methoxy-3-nitrophenyl)-5-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (7d). The crude product was
purified by chromatography (hexane/EtOAc 1:5,
Rf = 0.45) affording a yellow solid (67%). Mp 192–
193 ꢁC. 1H NMR (300 MHz, DMSO-d6): d = 3.65 (s,
6H), 3.67 (s, 3H), 3.99 (s, 3H), 6.64 (s, 2H), 7.52 (d,
J = 9.1 Hz, 1H), 7.74 (dd, J = 9.0, 2.7 Hz, 1H), 8.15–
8.16 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
d = 55.84, 57.18, 60.02, 106.32, 115.09, 121.02, 122.62,
128.32, 131.64, 132.86, 138.07, 138.14, 138.82, 152.26,
152.87. HRMS calcd for C18H18N4O6 (M+): 386.1226.
Found 386.1226.
4.3. Syntheses of 1,5-disubstituted 1,2,3-triazoles: general
procedure
The corresponding terminal alkyne (0.79 mmol) dis-
solved in dry THF (1 mL) was added dropwise to an
oven dried flask containing a solution of EtMgCl in
dry THF (0.36 mL, 2.0 M) under argon at room temper-
ature. After the alkyne was added, the solution was
heated to 50 ꢁC for 15 min and cooled to room temper-
ature. The corresponding azide (0.72 mmol) dissolved in
dry THF (1 mL) was added dropwise. The reaction mix-
ture was heated to 50 ꢁC for 3 h. After quenching with
aqueous NH4Cl (4 mL), the product was extracted with
dichloromethane (3· 40 mL). The combined organic
layers were washed with aqueous NH4Cl (2· 60 mL),
dried over anhydrous magnesium sulfate and the solvent
removed in vacuo.
4.3.5. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
1H-1,2,3-triazole (12a). The crude product was purified
by chromatography (hexane/EtOAc 1:1, Rf = 0.28)
1
affording an orange solid (64%). Mp 126 ꢁC. H NMR
(300 MHz, DMSO-d6): d = 3.68 (s, 6H), 3.72 (s, 3H),
3.76 (s, 3H), 6.74 (s, 2H), 6.98 (d, J = 8.9 Hz, 2H),
7.27 (d, J = 8.9 Hz, 2H), 8.04 (s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 55.18, 56.10, 60.13, 103.70,
114.14, 118.41, 129.74, 131.91, 132.26, 137.50, 137.87,
152.95, 159.72. HRMS calcd for C18H19N3O4 (M+):
341.1376. Found 341.1382.
4.3.1. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
1H-1,2,3-triazole (7a). The crude product was purified
by chromatography (hexane/EtOAc 1:1, Rf = 0.27)
affording a pale orange solid (83%). Mp 98–99 ꢁC. H
NMR (300 MHz, DMSO-d6): d = 3.61 (s, 6H), 3.66 (s,
3H), 3.82 (s, 3H), 6.57 (s, 2H), 7.10 (d, J = 9.0 Hz,
2H), 7.39 (d, J = 9.0 Hz, 2H), 8.13 (s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 55.69, 55.71, 59.98, 105.91,
4.3.6. 5-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-tri-methoxy-
phenyl)-1H-1,2,3-triazole (12b). The crude product was
purified by chromatography (hexane/EtOAc 1:1,
Rf = 0.22) affording a pale yellow solid (67%). Mp 76–
78 ꢁC. 1H NMR (300 MHz, DMSO-d6): d = 3.71 (s,
6H), 3.72 (s, 3H), 3.85 (s, 3H), 6.80 (s, 2H), 7.14 (d,
J = 8.7 Hz, 1H), 7.27 (dd, J = 8.6, 2.2 Hz, 1H), 7.63 (d,
1

K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
4835
J = 2.2 Hz, 1H), 8.13 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 56.19, 56.32, 60.16, 104.00, 110.54,
112.58, 119.84, 129.04, 131.69, 132.60, 132.67, 136.23,
138.09, 152.99, 155.82. HRMS calcd for C18H18BrN3O4
(M+): 419.0481. Found 419.0467.
3 M H2SO4 9:1 (25 mL). The mixture was stirred under
hydrogen at room temperature for 64 h. The mixture
was filtered through Celite. Aqueous NaOH (4 M) was
added to pH 10, followed by extraction with ethyl ace-
tate (4· 30 mL). The combined organic layers were dried
over anhydrous magnesium sulfate and the solvent re-
moved in vacuo affording a pale brown solid (80%).
4.3.7. 5-(3-(tert-Butyldimethylsilyloxy)-4-methoxy-phe-
nyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (12c).
The crude product was purified by chromatography
(hexane/EtOAc 1:1, Rf = 0.41) affording a brown semi-
solid (83%). 1H NMR (300 MHz, DMSO-d6):
d = ꢀ0.05 (s, 6H), 0.84 (s, 9H), 3.68 (s, 6H), 3.71 (s,
3H), 3.77 (s, 3H), 6.60 (d, J = 2.0 Hz, 1H), 6.74 (s,
2H), 7.06 (d, J = 8.5 Hz, 1H), 7.11 (dd, J = 8.4, 2.0 Hz,
1H), 8.04 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d = ꢀ5.14, 17.85, 25.27, 55.40, 56.02, 59.93, 103.80,
112.51, 118.57, 119.89, 122.48, 131.99, 132.05, 137.32,
137.96, 143.95, 151.28, 153.09. HRMS calcd for
C24H33N3O5Si (M+): 471.2189. Found 471.2183.
1
Mp 73–74 ꢁC. Rf = 0.21 (hexane/EtOAc 1:2). H NMR
(300 MHz, DMSO-d6): d = 3.62 (s, 6H), 3.66 (s, 3H),
3.82 (s, 3H), 5.13 (br s, 2H), 6.55 (dd, J = 8.4, 2.6 Hz,
1H), 6.61 (s, 2H), 6.66 (d, J = 2.5 Hz, 1H), 6.91 (d,
J = 8.5 Hz, 1H), 8.11 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 55.61, 55.70, 60.00, 105.61, 110.28,
110.56, 113.27, 121.63, 129.67, 132.27, 137.21, 137.82,
138.62, 146.79, 152.73. HRMS calcd for C18H20N4O4
(M+): 356.1485. Found 356.1478.
4.6. Syntheses of alkynes: general procedure
Trimethylsilyldiazomethane (9.4 mL, 18.8 mmol, 2.0 M
hexane solution) was added dropwise to a solution of
lithium diisopropylamide (11.5 mL, 20.7 mmol, 1.8 M
in heptane/THF/ethylbenzene) at ꢀ78 ꢁC under argon,
and the mixture was stirred at ꢀ78 ꢁC for 1 h. A solu-
tion of the corresponding benzaldehyde (15.0 mmol) in
dry THF (33 mL) was then added dropwise at ꢀ78 ꢁC.
The mixture was stirred at ꢀ78 ꢁC for 1 h, then at room
temperature for 2 h. The reaction was quenched with
brine (15 mL), and the mixture extracted with ethyl ace-
tate (4· 30 mL). The combined organic layers were
washed with brine (30 mL), dried over anhydrous mag-
nesium sulfate and the solvent removed in vacuo.
4.4. Deprotection of TBDMS-ethers: general procedure
Tetra-n-butyl ammonium fluoride in THF (1 M, 1.9 mL,
1.9 mmol) was added dropwise to a solution of the cor-
responding TBDMS-ether (1.44 mmol) in dry THF
(2.9 mL). The reaction mixture was stirred at room tem-
perature for 3 h and then treated with water. The mix-
ture was extracted with dichloromethane (3· 30 mL).
The combined organic layers were washed with water
(60 mL) and brine (2· 60 mL), and dried over anhy-
drous magnesium sulfate and the solvent removed in
vacuo.
4.4.1. 2-Methoxy-5-(5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)phenol (7e). The crude product was purified
by chromatography (hexane/EtOAc 1:2, Rf = 0.28)
affording a white solid (79%). Mp 202–203 ꢁC. 1H
NMR (300 MHz, DMSO-d6): d = 3.62 (s, 6H), 3.66 (s,
3H), 3.82 (s, 3H), 6.59 (s, 2H), 6.82–6.86 (m, 2H), 7.06
(d, J = 8.9 Hz, 1H), 8.11 (s, 1H), 9.61 (br s, 1H). 13C
NMR (75 MHz, DMSO-d6): d = 55.72, 55.80, 59.99,
105.82, 112.04, 113.06, 116.77, 121.53, 129.19, 132.44,
137.37, 137.87, 146.88, 148.57, 152.74. HRMS calcd
for C18H19N3O5 (M+): 357.1325. Found 357.1323.
4.6.1. 5-Ethynyl-1,2,3-trimethoxybenzene (4). The crude
product was purified by chromatography (hexane/
EtOAc 2:1, Rf = 0.65) affording a yellow solid (91%).
Mp 69–70 ꢁC. 1H NMR (300 MHz, CDCl3): d = 3.03
(s, 1H), 3.85 (s, 9H), 6.73 (s, 2H). 13C NMR (75 MHz,
CDCl3): d = 56.13, 60.93, 76.19, 83.68, 109.34, 117.00,
139.28, 153.03. HRMS calcd for C11H12O3 (M+):
192.0786. Found 192.0783.
4.6.2. 1-Ethynyl-4-methoxybenzene (11a). The crude
product was purified by chromatography (hexane/
1
EtOAc 8:3, Rf = 0.72) affording a yellow oil (68%). H
4.4.2. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-5-yl)phenol (12d). The crude product was purified
by chromatography (hexane/EtOAc 1:2, Rf = 0.39)
affording a pale yellow solid (77%). Mp 171–172 ꢁC.
1H NMR (300 MHz, DMSO-d6): d = 3.69 (s, 6H), 3.72
(s, 3H), 3.77 (s, 3H), 6.74–6.76 (m, 4H), 6.95 (d,
J = 9.0 Hz, 1H), 7.98 (s, 1H), 9.25 (br s, 1H). 13C
NMR (75 MHz, DMSO-d6): d = 56.08, 56.10, 60.11,
103.62, 112.07, 115.34, 118.62, 119.63, 131.98, 132.19,
137.64, 137.87, 146.36, 148.39, 152.93. HRMS calcd
for C18H19N3O5 (M+): 357.1325. Found 357.1330.
NMR (300 MHz, CDCl3): d = 3.00 (s, 1H), 3.81 (s,
3H), 6.85 (d, J = 8.9 Hz, 2H), 7.44 (d, J = 8.9 Hz, 2H).
13C NMR (75 MHz, CDCl3): d = 55.25, 75.76, 83.62,
113.89, 114.10, 133.55, 159.88. MS (EI): 133.0 (M+1).
4.6.3. 2-Bromo-4-ethynyl-1-methoxybenzene (11b). The
crude product was purified by chromatography (hex-
ane/EtOAc 8:3, Rf = 0.62) affording a white solid
(68%). Mp 90–91 ꢁC. 1H NMR (300 MHz, CDCl3):
d = 3.02 (s, 1H), 3.91 (s, 3H), 6.83 (d, J = 8.5 Hz, 1H),
7.41 (dd, J = 8.5, 2.0 Hz, 1H), 7.68 (d, J = 2.0 Hz, 1H).
13C NMR (75 MHz, CDCl3): d = 56.27, 76.87, 82.06,
111.28, 111.42, 115.56, 132.58, 136.77, 156.37. HRMS
calcd for C9H7BrO (M+): 209.9680. Found 209.9686.
4.5. Synthesis of aminosubstituted triazole 7f
4.5.1. 2-Methoxy-5-(5-(3,4,5-trimethoxyphenyl)-1H-1,2,
3-triazol-1-yl)aniline (7f). 1-(4-Methoxy-3-nitrophenyl)-
5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (7d) (386 mg,
1 mmol) and Pd/C 10% (107 mg) were added to EtOH/
4.6.4. tert-Butyl(5-ethynyl-2-methoxyphenoxy)dimethyl-
silane (11c). 3-(tert-Butyldimethylsilyloxy)-4-methoxy-
benzaldehyde (10c) was prepared as previously describe-

4836
K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
d.19a The crude product was purified by chromatogra-
phy (petroleum ether/EtOAc 4:1, Rf = 0.54) affording a
pale yellow oil (96%). 1H NMR (300 MHz, CDCl3):
d = 0.17 (s, 6H), 1.00 (s, 9H), 3.89 (s, 3H), 6.95 (d,
J = 8.3 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.47 (dd,
J = 8.3, 2.0 Hz, 1H), 9.82 (s, 1H). 13C NMR (75 MHz,
CDCl3): d = ꢀ4.65, 18.41, 25.63, 55.56, 111.16, 120.07,
126.23, 130.20, 145.55, 156.58, 190.87. MS (EI): 267.2
(M+1). 10c was used for preparing 11c according to
the general procedure. The crude product was purified
by chromatography (petroleum ether/EtOAc 8:1,
Rf = 0.76) affording a yellow oil (81%). 1H NMR
(300 MHz, CDCl3): d = 0.16 (s, 6H), 1.00 (s, 9H), 2.97
(s, 1H), 3.81 (s, 3H), 6.77 (d, J = 8.4 Hz, 1H), 6.98 (d,
J = 2.0 Hz, 1H), 7.09 (dd, J = 8.3, 2.0 Hz, 1H). 13C
NMR (75 MHz, CDCl3): d = ꢀ4.68, 18.42, 25.67,
55.38, 75.43, 83.69, 111.65, 114.19, 124.46, 126.31,
144.66, 152.04. HRMS calcd for C15H22O2Si (M+):
262.1389. Found 262.1393.
4.8. Syntheses of amino substituted triazoles 12f and 13b
4.8.1. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,
3-triazol-5-yl)aniline (12f). 5-(4-Methoxy-3-nitrophenyl)-
1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (12e) (200 mg,
0.5 mmol) and Pd/C 10% (56 mg) were dissolved in meth-
anol (15 mL). The mixture was stirred under hydrogen
(balloon) at room temperature overnight. The mixture
was filtered through Celite and the solvent removed in va-
cuo affording a white solid (98%). Mp 165–166 ꢁC.
Rf = 0.13 (hexane/EtOAc 1:2). 1H NMR (300 MHz,
DMSO-d6): d = 3.69 (s, 6H), 3.72 (s, 3H), 3.76 (s, 3H),
4.88 (br s, 2H), 6.46 (dd, J = 8.2, 2.1 Hz, 1H), 6.63 (d,
J = 2.1 Hz, 1H), 6.72 (s, 2H), 6.81 (d, J = 8.3 Hz, 1H),
7.90 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 55.26,
56.05, 60.11, 103.33, 110.30, 113.19, 116.45, 118.64,
132.06, 132.10, 137.75, 137.80, 138.22, 146.87, 152.85.
HRMS calcd for C18H20N4O4 (M+): 356.1485. Found
356.1485.
4.6.5. 4-Ethynyl-1-methoxy-2-nitrobenzene (11d). The
crude product was purified by chromatography (hex-
ane/EtOAc 8:3, Rf = 0.42) affording a yellow solid
(56%). Mp 101–102 ꢁC. H NMR (300 MHz, CDCl3):
d = 3.08 (s, 1H), 3.98 (s, 3H), 7.04 (d, J = 8.7 Hz, 1H),
7.64 (dd, J = 8.7, 2.1 Hz, 1H), 7.96 (d, J = 2.1 Hz, 1H).
13C NMR (75 MHz, CDCl3): d = 56.66, 78.03, 80.95,
113.52, 114.58, 129.28, 137.56, 139.38, 153.03. HRMS
calcd for C9H7NO3 (M+): 177.0426. Found 177.0423.
4.8.2. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-4-yl)aniline (13b). Saturated copper sulfate solu-
tion (0.9 mL) was added to 4-(4-methoxy-3-nitro-
phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (13a)
(750 mg, 1.94 mmol) dissolved in ethanol (11.5 mL).
The mixture was cooled to 0 ꢁC, and a solution of so-
dium borohydride (435 mg, 11.5 mmol) in ethanol/H2O
1:1 (11.5 mL) was added dropwise to the reaction mix-
ture. The mixture was refluxed for 4 h. After cooling,
saturated copper sulfate solution (0.9 mL) and a solu-
tion of sodium borohydride (435 mg, 11.5 mmol) were
added. The mixture was refluxed overnight. After cool-
ing, ethyl acetate was added and the mixture extracted
with 1 M HCl (3· 45 mL). The pH of the combined
aqueous layers was adjusted to pH 10 using 4 M NaOH,
and extracted with ethyl acetate (3· 50 mL). The com-
bined organic layers were dried over anhydrous magne-
sium sulfate and the solvent removed in vacuo.
Recrystallization in methanol and ether afforded a pale
brown solid (49%). Mp 166–168 ꢁC. Rf = 0.25 (hexane/
1
4.7. Huisgen cycloaddition reaction
A mixture of azide 9 (628 mg, 3.0 mmol) and alkyne 11d
(532 mg, 3.0 mmol) was dissolved in ethanol (15 mL).
The mixture was refluxed for 24 h. Water (5 mL) was
added and the mixture was refluxed for another 24 h.
The precipitate was washed with ether. The filtrate was
concentrated in vacuo. The products were purified by
chromatography (hexane/EtOAc 1:1).
1
4.7.1. 5-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (12e). Pale yellow solid (34%).
EtOAc 1:2). H NMR (300 MHz, DMSO-d6): d = 3.72
(s, 3H), 3.81 (s, 3H), 3.90 (s, 6H), 4.88 (br s, 2H), 6.90
(d, J = 8.4 Hz, 1H), 7.08 (dd, J = 8.2, 2.1 Hz, 1H), 7.26
(s, 2H), 7.28 (d, J = 2.1 Hz, 1H), 9.05 (s, 1H). 13C
NMR (75 MHz, DMSO-d6): d = 55.27, 56.20, 60.11,
97.70, 110.61, 113.58, 118.37, 122.91, 132.53, 137.11,
137.88, 146.43, 147.81, 153.42. HRMS calcd for
C18H20N4O4 (M+): 356.1485. Found 356.1477.
1
Mp 79–80 ꢁC. Rf = 0.13 (hexane/EtOAc 1:1). H NMR
(300 MHz, DMSO-d6): d = 3.71 (s, 6H), 3.72 (s, 3H),
3.93 (s, 3H), 6.83 (s, 2H), 7.42 (d, J = 8.9 Hz, 1H),
7.56 (dd, J = 8.8, 2.3 Hz, 1H), 7.93 (d, J = 2.2 Hz, 1H),
8.21 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d = 56.20, 56.86, 60.13, 104.07, 114.68, 118.50, 124.83,
131.44, 132.92, 133.99, 135.67, 138.21, 138.92, 152.15,
153.08. HRMS calcd for C18H18N4O6 (M+): 386.1226.
Found 386.1221.
4.9. Biological assays
4.9.1. Cancer cell growth inhibition on K562 human
leukemia cell line. The method applied was that de-
scribed by Edmondson et al.24 K562 human chronic
myelogeneous leukemia cells were cultivated in RMPI
medium, free of antibiotics and containing 2-mercap-
toethanol (2 lM) and L-glutamine (2 mM), supple-
mented with fetal calf serum (FCS) (10% v/v). The
cells were adjusted to a concentration depending on
their observed doubling time, (ca 40,000 cells/mL), in
RPMI medium supplemented with FCS (10% v/v). The
candidate drug was dissolved in DMSO. A drug solu-
tion of 100 ll in medium was added to 100 ll of cell
4.7.2. 4-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (13a). Yellow solid (22%).
Mp 229–230 ꢁC. Rf = 0.35 (hexane/EtOAc 1:1). 1H
NMR (300 MHz, DMSO-d6): d = 3.73 (s, 3H), 3.90 (s,
6H), 3.99 (s, 3H), 7.25 (s, 2H), 7.53 (d, J = 8.9 Hz,
1H), 8.20 (dd, J = 8.8, 2.2 Hz, 1H), 8.37 (d, J = 2.2 Hz,
1H), 9.34 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d = 56.20, 56.80, 60.13, 97.81, 115.13, 119.93, 121.40,
122.81, 130.84, 132.27, 137.38, 139.35, 144.99, 151.73,
153.46. HRMS calcd for C18H18N4O6 (M+): 386.1226.
Found 386.1234.

K. Odlo et al. / Bioorg. Med. Chem. 16 (2008) 4829–4838
4837
solution (40,000 cells/mL) in a 96-well microtiter test-
plate (4 ll of the drug solution diluted in medium in or-
der to reach decreasing concentrations). This series of
dilutions was continued to afford samples at different
concentrations leaving one cell solution free of drug act-
ing as a control. The plates were incubated at 37 ꢁC (5%
CO2 in air) for 5 days. The plate was then removed from
the incubator and 50 ll of a solution of MTT (3 mg/mL
in PBS) was added to each well. After incubation (37 ꢁC,
5% CO2 in air, 3 h) the medium was carefully removed
from each well by suction and the resulting formazan
precipitate redissolved in 200 ll DMSO. The optical
density of each well was read at two wavelengths (k
540 and 690 nm) using a Titretek Multiscan MCC/340
platereader. After processing and analysis through the
application of an ‘in-house’ software package, the re-
sults enabled the calculation of the drug dose required
to inhibit cell growth by 50% (IC50 value), determined
by graphical means as percentage of the control growth.
last 24 h before harvesting using a Filtermate har-
vester (Packard Instrument Company, Meriden, CT).
[3H]Thymidine incorporation was assessed in a Pack-
ard Microplate Scintillation Counter (Packard Instru-
ment Company). Controls were incubated with
medium containing solvent only.
4.10. Molecular modeling
The X-ray crystallographic structures of a- and b-tubu-
lin (PDB entry 1SA027; chain A and chain B, respec-
tively) in complex with guanosine-50-triphosphate
(GTP) were loaded into the Internal Coordinate
Mechanics36,37 (ICMv.3.5) program and hydrogen
atoms were subsequently added. ECEPP/3 atom charges
were assigned,38 and followed by energy minimization to
relieve atomic clashes. A grid map including the
DAMA-colchicine binding site of 1SA0 was calculated.
Triazole 12f was modeled, optimized, and assigned
MMFF9439 partial charges, and docked as a flexible li-
gand into the rigid a,b-tubulin conformation. The li-
gand–tubulin complex showing the most favorable
docking energy was further refined using the flexible-li-
gand, flexible-target method of ICM.40 The free energy
of binding of the refined target-ligand complex was cal-
culated in order to predict the contributions of different
subunits for binding the triazole 12f.
4.9.2. Inhibition of tubulin assembly. The method applied
was that described by Lawrence et al.26 Tubulin was iso-
lated from porcine brain and stored at ꢀ78 ꢁC. Samples
were prepared directly in a 96-well microtiter testplate
that was preincubated at 4 ꢁC in the fridge for 30 min
and contained Mes buffer [128 ll (0.1 M Mes, 1 mM
EGTA, 0.5 mM MgCl2, distilled water, pH 6.6)], GTP
(20 ll, 5 mM in Mes buffer), tubulin (50 ll, 11 mg/mL
in Mes buffer) and the candidate drug (20 ll, Csample in
DMSO). The tubulin/drug samples were immediately
placed in a 96-well plate reader, alongside blank samples
containing Mes buffer (198 ll) and the candidate drug
(10 ll, same concentration). The absorbance (k
350 nm) was recorded at 25 ꢁC temperature for a period
of 60 min, and the results were compared to untreated
controls to evaluate the relative degree of change in opti-
cal density. The results enabled the calculation of the
drug dose required to inhibit the assembly of tubulin
by 50% (IC50 value), determined by graphical means
as percentage of the control assembly.
Acknowledgment
Financial support to K.O. from the School of Pharmacy,
University of Oslo, is gratefully appreciated.
References and notes
1. (a) Desai, A.; Mitchison, T. J. Annu. Rev. Cell Dev. Biol.
1997, 13, 83; (b) Jordan, A.; Hadfield, J. A.; Lawrence, N.
J.; McGown, A. T. Med. Res. Rev. 1998, 18, 259; (c)
Hadfield, J. A.; Ducki, S.; Hirst, N.; McGown, A. T. In
´ ´
Progress in Cell Cycle Research; Meijer, L., Jezequel, A.,
4.9.3. Cancer cell growth inhibition of human cancer cell
lines. The human melanoma (WM35 and WM239),
ovarian (SKOV, OVCAR) and breast carcinoma cell
lines (MDA-MB231, SK-BR 3) were cultivated in
RPMI 1640 medium (BioWhittaker Europe, Verviers,
Belgium) containing 5% (melanoma) and 10% FCS
(PAA laboratories, Pasching, Austria) and 2 mM L-
glutamine (BioWhittaker Europe, Verviers, Belgium).
The SK-BR 3 cell line medium was in addition supple-
mented with 12,5 lM insulin (Sigma–Aldrich, St.
Louis, MO, USA). The WM35 and WM239 cell lines
were kindly provided by Dr. Meenhard Herlyn (Wis-
tar Institute, Philadelphia, PA, USA) whereas the
ovarian and breast carcinoma cell lines were obtained
from American Type Culture Collection (ATCC)
(Manassas, VA, USA). Five thousand cells in 100 ll
regular growth medium were plated per well in 96-well
plates and left to attach overnight. Thereafter, a dilu-
tion series of candidate drug (in 100 ll) was added.
Proliferation was measured after 72 h following label-
ing of the cells with 1 lCi [3H]thymidine (American
Radiolabeled Chemicals, Inc., St. Louis, MO) for the
Roberge, M., Eds.; Life in Progress Editions: Roscoff,
2003; Vol. 5, pp 309–325.
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