1,2,3-Triazole analogs of combretastatin A-4 as potential microtubule-binding agents

Article abstract of DOI:10.1016/j.bmc.2010.07.032

A series of cis-restricted 1,4- and 1,5-disubstituted 1,2,3-triazole analogs of combretastatin A-4 (1) have been prepared. Cytotoxicity and tubulin inhibition studies showed that 2-methoxy-5-((5-(3,4,5-trimethoxyphenyl)-1H-1,2, 3-triazol-1-yl)methyl)aniline (5e) and 2-methoxy-5-(1-(3,4,5-trimethoxybenzyl)- 1H-1,2,3-triazol-5-yl)aniline (6e) were two of the most active compounds. Molecular modeling studies revealed that the N-2 and N-3 atoms in the triazole rings in 5e and 6e did not form hydrogen bonds with the amino acids in the anticipated pharmacophore.

Full text of DOI:10.1016/j.bmc.2010.07.032

Bioorganic & Medicinal Chemistry 18 (2010) 6874–6885
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,2,3-Triazole analogs of combretastatin A-4 as potential
microtubule-binding agents
Kristin Odlo a, Jérémie Fournier-Dit-Chabert b, Sylvie Ducki b, Osman A. B. S. M. Gani c, , Ingebrigt Sylte c,
Trond Vidar Hansen a,
*
a School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, PO Box 1068, N-0316 Oslo, Norway
b Clermont Université, ENSCCF, EA 987, LCHG, BP 10448, F-63000 Clermont-Ferrand, France
c Medical Pharmacology and Toxicology, Department of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cis-restricted 1,4- and 1,5-disubstituted 1,2,3-triazole analogs of combretastatin A-4 (1) have
been prepared. Cytotoxicity and tubulin inhibition studies showed that 2-methoxy-5-((5-(3,4,5-trime-
thoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)aniline (5e) and 2-methoxy-5-(1-(3,4,5-trimethoxybenzyl)-
1H-1,2,3-triazol-5-yl)aniline (6e) were two of the most active compounds. Molecular modeling studies
revealed that the N-2 and N-3 atoms in the triazole rings in 5e and 6e did not form hydrogen bonds with
the amino acids in the anticipated pharmacophore.
Received 4 June 2010
Revised 7 July 2010
Accepted 14 July 2010
Available online 17 July 2010
Keywords:
Combretastatin A-4
Cytotoxic
Ó 2010 Elsevier Ltd. All rights reserved.
12,3-Triazoles
Molecular Modeling
1. Introduction
phosphate prodrug of CA-4 (CA-4P, 2) has been prepared, and is
currently in several clinical trials.8 The synthesis and biological
Microtubule-binding agents (MBAs) are widely used in cancer
chemotherapy.1 There are two classes of MBAs: those that stabilize
microtubules and promote polymerization, and those that destabi-
lize the microtubules and promote depolymerization. Both types
interfere with the mitotic spindle assembly during cell division,
resulting in cell death. Studies have shown that most MBAs have
antivascular effects, antiangiogenic or vascular disrupting activi-
ties, or both.2
evaluation of several combretastatin analogs with a locked cis-type
bridge between the two phenyl rings have been reported.7a,9 How-
ever, very few analogs where a triazole moiety has replaced the cis-
olefin have been reported.10
Recently, we prepared a series of 1,5-disubstituted 1,2,3-triazole
analogs of CA-4 (type I).11 The analogs were evaluated for their cyto-
toxic activity and their ability to inhibit tubulin polymerization.12
One of the amino-substituted analogs (4e) exhibited potent cyto-
toxic activity and moderate activity as a tubulin inhibitor. The
molecular modeling studies of 4e revealed that the N-2 and N-3
atoms of the 1,2,3-triazole ring formed hydrogen bonds with the
main chain nitrogen atoms of bA250 and bD251 in loop T7 that con-
nects helices H7 and H8 of b-tubulin. To extend our knowledge of the
1,2,3-triazole as a suitable mimic for the cis-configuration present in
CA-4, we have prepared three new series of analogs of CA-4 (Fig. 2).
Several SAR studies have revealed that the two aryl rings of CA-4 can
be separated by more than a two-atom bridge.13 In order to investi-
gate this we replaced the cis-olefin in CA-4 with a 1,5-disubstituted
1,2,3-triazole ring coupled to a methylene group (type II), thereby
creating a three-atom bridge between the aryl rings. Furthermore,
basedonthehydrogenbondinginteractionsrevealedinourprevious
study,11 we wanted to investigate if a 1,4-disubstituted 1,2,3-triazol
ring would retain some of the biological activity of the 1,5-disubsti-
tuted triazole analogs, since trans-resveratrol is a stilbene that has
been found to be more cytotoxic than its cis-form.14 To further our
One MBA that has attracted much interest as a lead compound3
is combretastatin A-4 (CA-4, 1) isolated from the bark of the South
African tree Combretum caffrum.4 This natural product binds to the
colchicine binding site on tubulin and inhibits tubulin polymeriza-
tion.5 CA-4 is highly cytotoxic against a variety of human cancer
cell lines and is a vascular disrupting agent.6
Structure–activity relationship (SAR) studies have shown that a
3,4,5-trimethoxysubstituted A ring and a 4-methoxysubstituted B
ring separated by a double bond with cis-configuration are impor-
tant for optimal cytotoxic activity (Fig. 1).7
The major disadvantages of CA-4 as a drug candidate are low
water solubility and isomerization to the less active trans-form. A
* Corresponding author. Tel.: +47 22857450; fax: +47 22855947.
E-mail address: t.v.hansen@farmasi.uio.no (T.V. Hansen).
Present address: Department of Chemistry, Faculty of Science, University of
Tromsø, N-9037 Tromsø, Norway.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.032

K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
6875
Figure 1. Combretastatin A-4 (1) and its prodrug (2).
Figure 2. Structures of triazole analogs type I (1,5-disubstituted 1,2,3-triazole) 3a–3e and 4a–4e, type II (1,5-disubstituted 1,2,3-triazole with methylene group) 5a–5e and
6a–6e, type III (1,4-disubstituted 1,2,3-triazole) 7a–7e and 8a–8e, and type IV (1,4-disubstituted 1,2,3-triazole with methylene group) 9a–9e and 10a–10e.
knowledge of the possibility of mimicking the olefin with a four-
atom bridge, we prepared analogs where the aryl rings are separated
by a 1,4-disubstituted 1,2,3-triazole ring coupled to a methylene
group (type IV). Herein we report the synthesis and in vitro cytotoxic
activities of several 1,4- and 1,5-disubstituted 1,2,3-triazole analogs
of CA-4, as well as the inhibition data of tubulin polymerization for
the most active analogs. Furthermore, molecular modeling studies
of two of the prepared analogs (5e and 6e) with the colchicine bind-
verted into the benzyl azides (15a–15d and 15f).17 The phenyl
azides (18a–18d and 18f) were prepared either from the corre-
sponding anilines (16a–16c) using diazotization conditions10d or
from the corresponding boronic acids (17a and 17b) after reaction
with sodium azide.18 The phenolic groups were protected as
TBDMS-ethers when nedeed.19
The target triazoles (5a–5e, 6a–6e, 7a–7e, 8a–8e, 9a–9e, and
10a–10e) were prepared from the corresponding terminal alkynes
and benzyl or phenyl azides as depicted in Scheme 2 and Scheme 3.
The 1,4-disubstituted 1,2,3-triazoles (7a–7e, 8a–8e, 9a–9e, and
10a–10e) were prepared in a Cu(I)-catalyzed Huisgen cycloaddi-
tion reaction, either by the use of copper sulfate and sodium ascor-
bate, or copper turnings.20 The use of copper turnings proved to be
a convenient method for the synthesis of the 1,4-disubstituted
1,2,3-triazoles; however lower yields were obtained compared to
using the combination of copper sulfate and sodium ascorbate.
The 1,5-disubstituted 1,2,3-triazoles (5a–5e and 6a–6e) were
obtainedeitherbytheuseofmagnesiumacetylides21 orbyruthenium
catalysis.22 Removal of the TBDMS-group using TBAF afforded tria-
zoles 5c, 6c, and 8c, respectively.23 The amino-substituted triazoles
(5e, 6e, 7e, 8e, 9e, and 10e) were obtained by reduction of the nitro
group using NaBH4/CuSO4.24 All new compounds exhibited spectral
data in agreement with their assigned structures.
ing site of a,b-tubulin were performed together with a potential ana-
log 11.
2. Results and discussion
2.1. Synthesis
The syntheses of the starting materials are depicted in Scheme 1.
The terminal alkynes (13a–13d and 13f) were prepared from the
corresponding benzaldehydes (12a–12c and 12e–12f) using the
Colvin rearrangement.15 The benzyl azides were prepared from the
same starting materials, via the benzyl alcohols. The benzyl alcohols
(14b–14d) were obtained by a reduction of the aldehydes (12b, 12c,
and 12e) withsodium borohydride,16 whichwere subsequently con-

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K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
Scheme 1. Synthesis of starting materials. Reagents and conditions: (i) LDA, TMSCHN2, THF, ꢀ78 °C; (ii) NaBH4, EtOH, H2O, 0 °C; (iii) (PhO)2PON3, DBU, toluene, 0 °C; (iv) HCl
(aq), NaN3, NaNO2, 0 °C; (v) NaN3, CuSO4, MeOH; (vi) DIPEA, TBDMSCl, DMF; (vii) n-Bu4NF, THF.
Scheme 2. Synthesis of triazoles 5a–5e, 7a–7e, and 9a–9e. Reagents and conditions: (i) Na-ascorbate, CuSO4, t-BuOH/H2O (1:1); (ii) Cu-turnings, MeOH; (iii) EtMgCl, THF,
(iv) NaBH4, CuSO4, EtOH, ; (v) n-Bu4NF, THF.
D;
D
2.2. Biological evaluation
K562 leukemia cancer cells using the MTT assay (Table 1).25 These
results showed that the 1,5-disubstituted 1,2,3-triazoles in general
had greater cytotoxic activity than the 1,4-regioisomers, as
anticipated based on the work of Tron and Genazzani.10d The
The target triazoles (5a–5e, 6a–6e, 7a–7e, 8a–8e, 9a–9e, and
10a–10e) were evaluated for their ability to inhibit the growth of

K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
6877
Scheme 3. Synthesis of triazoles 6a–6e, 8a–8e, and 10a–10e. Reagents and conditions: (i) Na-ascorbate, CuSO4, t-BuOH/H2O (1:1); (ii) Cu-turnings, MeOH; (iii) EtMgCl, THF,
; (iv) CpꢂRuCl(COD), benzene, ; (v) n-Bu4NF, THF; (vi) NaBH4, CuSO4, EtOH,
D
D
D.
triazoles derived from the phenyl azides proved to be more cytotoxic
than the triazoles derived from the benzyl azides. Hence, the cyto-
toxic activity decreased with increasing bridge length; 2 > 3 > 4. As
inour previousstudy, theanalogswith theAringderivedfroma phe-
nylazide, resultedinaseriesofmoreactivetriazoleanalogs. Thesub-
stituent in the 30-position of the B-ring clearly affected the cytotoxic
activity, and the most potent compounds had an amino group (6e,
5e, and 8e), a hydrogen atom (6c, 5c, and 7c) or a phenolic group
(6a and 5a) in the 30-position, as shown in other studies.10c,26
Thetriazolesthatexhibitedcytotoxicactivitieswerefurthereval-
uated for their ability to inhibit the polymerization of tubulin.27
Unfortunately, none of the analogs proved to inhibit the polymeriza-
tion of tubulin in any significant degree. The 1,4-disubstituted 1,2,3-
triazoleringmightmimicthetrans-configurationofCA-4. Resultsre-
ported herein supplement the studies reported by Tron and Genaz-
zani.10d However, the 1,5-disubstituted 1,2,3-triazole ring with a
methylene group between the A and B rings might be used as a mi-
mic for the cis-configuration in CA-4 with regards to cytotoxicity.
The cytotoxic activity is retained in a larger degree than the tubulin
inhibitory activity for all triazole analogs reported herein.11
11, respectively. The corresponding values of (4e) in our previous
study were 6 and ꢀ10 kcal/mol.
Ring A of all three triazole analogs occupied a distinct binding
region at the interface of a- and b-tubulin. This moiety of 4e was
found to be buried completely inside of b-tubulin similar to what
is seen in the X-ray complex of DAMA-colchicine with tubulin28
(Fig. 4). The triazole ring in compounds 5e, 6e, and 11 did not form
hydrogen bonds with the surrounding residues, which is contrary
to 4e and the previously described pharmacophore model.11,29
Ring B of 5e occupied similar Cartesian space as corresponding
moieties in 4e and DAMA-colchicine, but the corresponding moiety
of 6e and 11 occupied a completely different space close to the GTP
binding region of a-tubulin (Fig. 4). The quite unexpected position
of this moiety may be responsible for the relatively low binding
affinities of 6e and 11 (ꢀ3.7 and ꢀ3.5 kcal/mol, respectively) com-
pared with 4e and 5e. Triazole analog 5e binds very similar to b-
tubulin as DAMA-colchicine in the X-ray complex with b-tubulin,
giving a more favorable binding energy of ꢀ8.5 kcal/mol. We also
assume that the low affinity of 6e and 11 for b-tubulin reflects
the weak binding affinity of 6e and 11 for a,b-tubulin. These results
suggest that the structure of 11 is not a suitable mimic for CA-4.
The binding energies also support our previous assumption that
b-tubulin is responsible for the strength of ligand binding.11 The
binding energies of the analogs is not in correlation with the ob-
served cytotoxic activities, indicating that other mechanisms most
likely are responsible for the cytotoxic effects.
2.3. Molecular modeling
Molecular modeling studies were performed to investigate the
difference in binding ability to the colchicine binding site of
a,b-tubulin of the less active triazole analogs (5e and 6e), and a po-
tential new triazole analog (11) (Fig. 3). The binding modes were
compared with that of the previously reported active amino-
substituted triazole analog (4e) that was docked in our previous
study.11 Upon docking all three compounds were located in the
The binding energies indicate that 4e showed >10-fold higher
affinity than 5e for b-tubulin (ꢁ1.4 kcal/mol difference in binding
energy causes about 10-fold difference in affinity). The higher
affinity of 4e (free energy of binding for b-tubulin ꢀ10 kcal/mol)
may be a result of favorable orientation of the trimethoxyphenyl
moiety inside the cavity of b-tubulin. This moiety of 4e overlapped
with the corresponding ring in the X-ray structure of DAMA-col-
chicine. However, the difference in orientation of the triazole ring
may also contribute to altered affinity for b-tubulin. The correct
position of the trimethoxy aryl moiety has been suggested to be
necessary, but not sufficient, to predict activity of potential
colchicine binding site of
a,b-tubulin (mostly into the b subunit).
The trimethoxy aryl moiety (ring A) and the triazole ring of the
analogs occupied similar regions of the binding cavity, but the ana-
logs had different orientation for the 3-amino-4-methoxy aryl moi-
ety (ring B) ( Fig. 4). For the three analogs the free energies of
binding were about 5 kcal/mol for
a-tubulin, but for b-tubulin
the energies were ꢀ8.5, ꢀ3.7, and ꢀ3.5 kcal/mol for 5e, 6e, and

6878
K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
Table 1
3. Conclusion
Cytotoxicity and inhibition of tubulin polymerization by triazoles 5a–5e, 6a–6e, 7a–
7e, 8a–8e, 9a–9e, and 10a–10e
The orientation of ring B of the triazole analogs of combretast-
atin A-4 proved to be very important for their binding energies,
as shown herein for compounds 5e and 6e. The present results
indicate that hydrogen bonding between the nitrogen atoms in
the triazole cis-olefin mimic may play a role for the affinity of
the analogs to tubulin, but based on other molecular modeling
studies it is probably less important for tubulin inhibition than
the orientation of the trimethoxy substituted A ring.
a
Compound
K562 cell assay, IC50
Inhibition of tubulin
polymerization IC50
b
(l
M) in vitro cytotoxicity
(
lM)
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
10a
10b
10c
10e
10e
4e
1
8.66
>10
1.83
>10
0.73
5.56
>10
0.53
>10
0.38
>10
>10
3.20
>10
>10
>10
>10
>10
>10
1.30
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.011
0.010
n.d.
n.d.
n.d.
n.d.
>20c
n.d.
n.d.
n.d.
n.d.
>20c
n.d.
n.d.
>20
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>20
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4.8
4. Experimental
4.1. General procedure
Unlessnoted otherwise, allreagents and solvents were used as pur-
chased without further purification. Melting points are uncorrected.
Analytical TLC was performed using silica gel 60 F254 plates (Merck)
or RP-18 F254s plates (Merck). Flash column chromatography was per-
formed on silica gel 60 (40–63 lm, Fluka) or Versapak C18 cartridge
(Supelco). NMR (1H, 13C) spectra were recorded on a Bruker DPX-
300 MHz spectrometer. Coupling 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). Compounds 8d, 8e, 12e,
13a–13d, 13f, and 18a–18f have been described in our previous
work.11,31 Compounds 7a and 7c,10d 8a and 8c,10d 13e,13b 14b,32
14c,33 14d,34 15a,35 15c,36 and 15d,37 are known compounds. Combre-
tastatin A-4 (1) was prepared according to a literature procedure.38
0.6
4.2. Syntheses of benzyl alcohols: general procedure
n.d., not determined.
a
The corresponding benzaldehyde (10.00 mmol) was dissolved
in ethanol (10 mL) and stirred at 0 °C. A solution of sodium borohy-
dride (0.19 g, 5.02 mmol) in ethanol/H2O 1:1 (12 mL) was added
dropwise. The resulting mixture was stirred at room temperature
for 2 h. Ice was added, and the mixture was acidified using concen-
trated HCl. The mixture was extracted with ethyl acetate (4ꢃ
20 mL). The combined organic layers were washed with brine
(25 mL), dried over anhydrous magnesium sulfate and the solvent
was removed in vacuo.
Results of three experiments.
Results of two experiments.
Determined using the fluorescence based tubulin polymerization assay kit (cat.
b
c
# BK011) from cytoskeleton.
compounds binding to the colchicine binding site on tubulin. Most
of the reported molecular modeling studies of tubulin inhibitory
analogs of CA-4 describe hydrogen bonding interactions between
the methoxy group(s) of ring A and bC241.10a,29,30 For 5e and 6e,
the large difference in affinity for b-tubulin may be due to different
orientations of ring B. From our modeling studies the orientation of
this moiety seems to be very important for tubulin binding of all
1,2,3-triazole analogs of CA-4. On the other hand, the triazole ana-
logs might be too rigid to enter the colchicine binding site, thus not
exhibiting tubulin inhibitory activity despite their binding affinity
to the binding site in the molecular modeling studies.
4.2.1. (3-Bromo-4-methoxyphenyl)methanol (14b)
Pale yellow oil (>99%). Rf = 0.88 (hexane/EtOAc 1:1). 1H NMR
(300 MHz, CDCl3): d = 1.68 (br s, 1H), 3.89 (s, 3H), 4.60 (s, 2H),
6.88 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 8.4, 2.1 Hz, 1H), 7.56 (d,
J = 2.1 Hz, 1H). 13C NMR (75 MHz, CDCl3): d = 56.30, 64.29,
111.69, 111.87, 127.30, 132.25, 134.51, 155.39. HRMS calcd. for
C8H9BrO2 (M+): 215.9786. Found 215.9791.
We are currently evaluating the antivascular effects of triazole
analogs such as 4e. Moreover, based on the results reported herein,
other triazole mimics of the combretastatins will be prepared.
These efforts will be reported in due course.
4.2.2. (3,4,5-Trimethoxyphenyl)methanol (14c)
Colorless oil (>99%). Rf = 0.82 (dichloromethane/methanol 4:1).
1H NMR (300 MHz, CDCl3): d = 1.97 (br s, 1H), 3.83 (s, 3H), 3.86
Figure 3. Structures of triazole analogs used in molecular modeling (4e, 5e, 6e, and 11).

K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
6879
Figure 4. Binding mode of triazole analogs 4e, 5e, 6e, and 11 after docking into the colchicine binding site of tubulin (PDB entry 1SA0). The figure shows the superposition of
receptor–ligand complexes where the X-ray structure of tubulin is shown as ribbon representation ( -tubulin in red, and b-tubulin in blue) and stick models are used for
a
ligands (with atom type color: carbon-white, nitrogen-blue, oxygen-red, hydrogen-gray). The orientations of the trimethoxy aryl moiety (ring A) and the 3-amino-4-methoxy
aryl moiety (ring B) in the ligands are indicated for 4e, 5e, 6e, and 11. Side-views (ꢀ90° rotation relative to a and b) of superimposed ligands are also shown, indicating their
relative positions of ring A and ring B.
(s, 6H), 4.62 (s, 2H), 6.59 (s, 2H). 13C NMR (75 MHz, CDCl3):
d = 56.04, 60.81, 65.45, 103.76, 136.63, 137.27, 153.32. HRMS calcd.
for C10H14O4 (M+): 198.0892. Found 198.0887.
2H), 7.24 (d, J = 8.7 Hz, 2H). 13C NMR (75 MHz, CDCl3): d = 54.39,
55.28, 114.18, 127.38, 129.72, 159.61. HRMS calcd. for C8H9N3O
(M+): 163.0746. Found 163.0745.
4.3.2. 4-(Azidomethyl)-2-bromo-1-methoxybenzene (15b)
The crude product was purified by chromatography (hexane/
EtOAc 8:3, Rf = 0.71) affording a colorless oil (86%). 1H NMR
(300 MHz, CDCl3): d = 3.90 (s, 3H), 4.25 (s, 2H), 6.89 (d, J = 8.4 Hz,
1H), 7.23 (dd, J = 8.4, 2.2 Hz, 1H), 7.51 (d, J = 2.2 Hz, 1H). 13C NMR
(75 MHz, CDCl3): d = 53.64, 56.25, 111.90, 128.45, 128.86, 133.21,
155.84. HRMS calcd. for C8H8BrN3O (M+): 240.9851. Found
240.9861.
4.2.3. (3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl)-
methanol (14d)
Colorless oil (98%). Rf = 0.51 (petroleum ether/EtOAc 3:1). 1H
NMR (300 MHz, CDCl3): d = 0.16 (s, 6H), 1.00 (s, 9H), 1.69 (br s,
1H), 3.80 (s, 3H), 4.56 (s, 2H), 6.82 (d, J = 8.1 Hz, 1H), 6.86–6.93
(m, 2H). 13C NMR (75 MHz, CDCl3): d = ꢀ4.64, 18.42, 25.70, 55.52,
65.05, 111.99, 120.07, 120.45, 133.66, 145.06, 150.55. HRMS calcd.
for C14H24O3Si (M+): 268.1495. Found 268.1490.
4.3.3. 5-(Azidomethyl)-1,2,3-trimethoxybenzene (15c)
4.3. Syntheses of benzyl azides: general procedure
The crude product was purified by chromatography (hexane/
EtOAc 8:3, Rf = 0.39) affording a colorless oil (99%). 1H NMR
(300 MHz, CDCl3): d = 3.85 (s, 3H), 3.88 (s, 6H), 4.28 (s, 2H), 6.53
(s, 2H). 13C NMR (75 MHz, CDCl3): d = 55.12, 56.13, 60.83, 105.12,
130.97, 137.90, 153.46. HRMS calcd. for
223.0957. Found 223.0954.
A mixture of the corresponding benzyl alcohol (7.67 mmol) and
diphenylphosphorazidate (1.9 mL, 8.82 mmol) was dissolved in dry
toluene (15 mL) under argon. The mixture was cooled to 0 °C, and
neat 1,8-diazabicyclo[5.4.0]undec-7-ene (1.4 mL, 9.37 mmol) was
added dropwise. The reaction was stirred for 2 h at 0 °C and then
at room temperature for 19 h. The mixture was washed with water
(2ꢃ 20 mL) and aqueous 5% HCl (2ꢃ 20 mL). The organic layer was
dried over anhydrous magnesium sulfate and the solvent was re-
moved in vacuo.
C
10H13N3O3 (M+):
4.3.4. (5-(Azidomethyl)-2-methoxyphenoxy)(tert-butyl)di-
methylsilane (15d)
The crude product was purified by chromatography (hexane/
EtOAc 9:1, Rf = 0.56) affording a colorless oil (67%). 1H NMR
(300 MHz, CDCl3): d = 0.16 (s, 6H), 1.00 (s, 9H), 3.81 (s, 3H), 4.21 (s,
2H), 6.81–6.89 (m, 3H). 13C NMR (75 MHz, CDCl3): d = ꢀ4.64, 18.44,
25.70, 54.41, 55.48, 112.02, 121.15, 121.84, 127.75, 145.20, 151.11.
HRMS calcd. for C14H23N3O2Si ([M+Na]+): 316.1451. Found 316.1447.
4.3.1. 1-(Azidomethyl)-4-methoxybenzene (15a)
The crude product was purified by chromatography (hexane/
EtOAc 8:3, Rf = 0.66) affording a colorless oil (89%). 1H NMR
(300 MHz, CDCl3): d = 3.81 (s, 3H), 4.27 (s, 2H), 6.91 (d, J = 8.7 Hz,

6880
K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
4.3.5. 4-(Azidomethyl)-1-methoxy-2-nitrobenzene (15f)
4.5.3. 4-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)
-1H-1,2,3-triazole (8d)
The crude product was purified by chromatography (hexane/
EtOAc 8:3, Rf = 0.37) affording
a
yellow oil (51%). 1H NMR
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.37) affording a yellow solid (91%). Mp 229–
230 °C (hexane/ether 9:1). 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,
(300 MHz, CDCl3): d = 3.98 (s, 3H), 4.36 (s, 2H), 7.11 (d, J = 8.6 Hz,
1H), 7.51 (dd, J = 8.6, 2.3 Hz, 1H), 7.82 (d, J = 2.2 Hz, 1H). 13C NMR
(75 MHz, CDCl3): d = 53.30, 56.65, 113.94, 125.39, 127.89, 133.74,
139.54, 152.82. HRMS calcd. for C8H8N4O3 (M+): 208.0596. Found
208.0599.
4.4. Syntheses of phenyl azides: general procedure
144.99, 151.73, 153.46. HRMS calcd. for
386.1226. Found 386.1234.
C
18H18N4O6 (M+):
Sodium azide (78 mg, 1.2 mmol) and copper sulfate (25 mg,
0.1 mmol) were placed in a flask. Methanol (3 mL) and the corre-
sponding boronic acid (1.0 mmol) were added subsequently. The
reaction mixture was stirred vigorously at room temperature for
6 h. Water (10 mL) was added, followed by an extraction with hex-
ane (3ꢃ 15 mL). The combined organic layers were washed with
aqueous 5% NH3 (2ꢃ 10 mL), dried over anhydrous magnesium sul-
fate and the solvent was removed in vacuo.
4.5.4. 1-(4-Methoxybenzyl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,
3-triazole (9a)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.28) affording a blank semisolid (92%). 1H NMR
(300 MHz, DMSO-d6): d = 3.67 (s, 3H), 3.74 (s, 3H), 3.83 (s, 6H),
5.55 (s, 2H), 6.95 (d, J = 8.7 Hz, 2H), 7.14 (s, 2H), 7.32 (d,
J = 8.7 Hz, 2H), 8.57 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d = 52.51, 55.05, 55.81, 59.96, 102.38, 114.08, 121.06, 126.21,
127.78, 129.42, 137.08, 146.63, 153.17, 159.07. HRMS calcd. for
4.4.1. 1-Azido-4-methoxybenzene (18a)
Yellow oil (93%). Rf = 0.76 (hexane/EtOAc 2:1). 1H NMR
(300 MHz, DMSO-d6): d = 3.74 (s, 3H), 6.95–6.99 (m, 2H), 7.03–
7.07 (m, 2H); 13C NMR (75 MHz, DMSO-d6): d = 55.32, 115.25,
120.09, 131.33, 156.70; MS (EI): 150.0 (M+1).
C
19H21N3O4 (M+): 355.1532. Found 355.1529.
4.5.5. 1-(3-Bromo-4-methoxybenzyl)-4-(3,4,5-trimethoxyphen
yl)-1H-1,2,3-triazole (9b)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.20) affording a pale yellow solid (89%). Mp 113–
114 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.68
(s, 3H), 3.83 (s, 6H), 3.84 (s, 3H), 5.58 (s, 2H), 7.12–7.15 (m, 3H),
7.37 (dd, J = 8.5, 2.2 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H), 8.60 (s, 1H).
13C NMR (75 MHz, DMSO-d6): d = 51.73, 55.81, 56.22, 59.97,
102.42, 110.53, 112.80, 121.19, 126.12, 128.90, 129.42, 132.54,
137.13, 146.68, 153.18, 155.22. HRMS calcd. for C19H20BrN3O4
(M+): 433.0637. Found 433.0642.
4.4.2. 5-Azido-1,2,3-trimethoxybenzene (18c)
Pale orange solid (93%). Mp 42–45 °C. Rf = 0.68 (hexane/EtOAc
2:1). 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, 134.77, 134.87, 153.66. HRMS calcd. for C9H11N3O3 (M+):
209.0800. Found 209.0795.
4.5. Syntheses of 1,4-disubstituted 1,2,3-triazoles using copper
sulfate: general procedure
4.5.6. 2-Methoxy-5-((4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-tria
zol-1-yl)methyl)phenol (9c)
A mixture of the corresponding azide (4.21 mmol) and the cor-
responding alkyne (4.21 mmol) was dissolved in t-BuOH/H2O 1:1
(20 mL). Sodium ascorbate (167.0 mg, 20 mol %) and copper sulfate
(52.6 mg, 5 mol %) were added. After stirring overnight water/ice
(40 mL) was added. The product was either worked up by filtration,
followed by rinsing with aqueous 5% NH3 (ꢃ3) and cold ether (ꢃ2),
or by extraction with dichloromethane (4ꢃ 100 mL). The combined
organic layers were washed with aqueous 5% NH3 (3ꢃ 100 mL) and
brine (100 mL), and dried over anhydrous magnesium sulfate. The
solvent was removed in vacuo.
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.15) affording a white solid (76%). Mp 142–
143 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.67
(s, 3H), 3.75 (s, 3H), 3.83 (s, 6H), 5.47 (s, 2H), 6.74–6.79 (m, 2H),
6.92 (d, J = 8.1 Hz, 1H), 7.15 (s, 2H), 8.55 (s, 1H), 9.09 (br s, 1H).
13C NMR (75 MHz, DMSO-d6): d = 52.72, 55.55, 55.81, 59.97,
102.37, 112.20, 115.03, 118.82, 121.12, 126.23, 128.28, 137.06,
146.60, 147.56, 153.18. HRMS calcd. for
371.1481. Found 371.1477.
C
19H21N3O5 (M+):
4.5.1. 1-(3-Bromo-4-methoxyphenyl)-4-(3,4,5-trimethoxyphe
nyl)-1H-1,2,3-triazole (7b)
4.5.7. 1-(4-Methoxy-3-nitrobenzyl)-4-(3,4,5-trimethoxyphenyl)-
1H-1,2,3-triazole (9d)
Pale brown solid (97%). Mp 154–156 °C (hexane/ether 9:1). 1H
NMR (300 MHz, DMSO-d6): d = 3.71 (s, 3H), 3.87 (s, 6H), 3.94 (s,
3H), 7.24 (s, 2H), 7.36 (d, J = 9.0 Hz, 1H), 7.95 (dd, J = 8.9, 2.6 Hz,
1H), 8.17 (d, J = 2.6 Hz, 1H), 9.26 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 55.84, 56.63, 60.00, 102.52, 111.10, 113.21, 119.48,
120.48, 124.22, 125.65, 130.36, 137.39, 147.20, 153.28, 155.39.
HRMS calcd. for C18H18BrN3O4 (M+): 419.0481. Found 419.0475.
Pale yellow solid (96%). Mp 159–160 °C (hexane/ether 9:1). 1H
NMR (300 MHz, DMSO-d6): d = 3.68 (s, 3H), 3.83 (s, 6H), 3.92 (s,
3H), 5.67 (s, 2H), 7.15 (s, 2H), 7.40 (d, J = 8.8 Hz, 1H), 7.66 (dd,
J = 8.7, 2.3 Hz, 1H), 7.97 (d, J = 2.2 Hz, 1H), 8.63 (s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 51.44, 55.82, 56.73, 59.97, 102.44,
114.81, 121.32, 124.73, 126.07, 128.11, 134.27, 137.17, 138.84,
146.73, 151.84, 153.19. HRMS calcd. for
400.1383. Found 400.1377.
C
19H20N4O6 (M+):
4.5.2. 1-(4-Methoxy-3-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)
-1H-1,2,3-triazole (7d)
Yellow solid (92%). Mp 257–258 °C (hexane/ether 9:1). 1H NMR
(300 MHz, DMSO-d6): d = 3.71 (s, 3H), 3.88 (s, 6H), 4.03 (s, 3H), 7.24
(s, 2H), 7.65 (d, J = 9.2 Hz, 1H), 8.25 (dd, J = 9.1, 2.7 Hz, 1H), 8.48 (d,
J = 2.7 Hz, 1H), 9.34 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 55.87,
57.18, 60.03, 102.58, 115.78, 116.58, 119.69, 125.58, 129.06,
137.49, 139.03, 143.51, 147.40, 151.67, 153.31. HRMS calcd. for
4.5.8. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxybenzyl)-1H-
1,2,3-triazole (10a)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.27) affording a white solid (53%). Mp 157–
158 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.64
(s, 3H), 3.76 (s, 6H), 3.78 (s, 3H), 5.51 (s, 2H), 6.74 (s, 2H), 7.00
(d, J = 8.9 Hz, 2H), 7.77 (d, J = 8.9 Hz, 2H), 8.50 (s, 1H). 13C NMR
C
18H18N4O6 (M+): 386.1226. Found 386.1232.

K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
6881
(75 MHz, DMSO-d6): d = 53.17, 55.04, 55.83, 59.87, 105.67, 114.17,
120.26, 123.20, 126.42, 131.20, 137.26, 146.44, 152.94, 158.89.
HRMS calcd. for C19H21N3O4 (M+): 355.1532. Found 355.1531.
60.01, 102.55, 107.56, 110.39, 112.42, 119.28, 125.85, 130.06,
137.31, 147.01, 147.25, 147.88, 153.26. HRMS calcd. for
C
18H19N3O5 (M+): 357.1325. Found 357.1323.
4.5.9. 4-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-trimethoxyben
zyl)-1H-1,2,3-triazole (10b)
4.6.3. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole (8a)
White solid (91%). Mp 132–133 °C (hexane/ether 9:1). 1H NMR
(300 MHz, DMSO-d6): d = 3.64 (s, 3H), 3.77 (s, 6H), 3.87 (s, 3H), 5.52
(s, 2H), 6.74 (s, 2H), 7.18 (d, J = 8.7 Hz, 1H), 7.84 (dd, J = 8.6, 2.1 Hz,
1H), 8.05 (d, J = 2.1 Hz, 1H), 8.60 (s, 1H). 13C NMR (75 MHz, DMSO-
d6): d = 53.28, 55.84, 56.21, 59.87, 105.74, 110.95, 112.93, 120.88,
124.67, 125.63, 129.37, 131.00, 137.29, 145.11, 152.95, 154.90.
HRMS calcd. for C19H20BrN3O4 (M+): 433.0637. Found 433.0638.
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.60) affording a pale yellow solid (51%). Mp 181–
182 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.73
(s, 3H), 3.81 (s, 3H), 3.90 (s, 6H), 7.07 (d, J = 8.8 Hz, 2H), 7.25 (s,
2H), 7.86 (d, J = 8.8 Hz, 2H), 9.17 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 55.11, 56.20, 60.14, 97.79, 114.33, 118.74, 122.72,
126.57, 132.47, 137.21, 147.00, 153.44, 159.19. HRMS calcd. for
C
18H19N3O4 (M+): 341.1376. Found 341.1366.
4.5.10. 2-Methoxy-5-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazol-4-yl)phenol (10c)
4.6.4. 4-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphen
yl)-1H-1,2,3-triazole (8b)
The crude product was purified by chromatography (chloro-
form/EtOAc 1:1, Rf = 0.15) affording a white solid (86%). Mp 156–
157 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.64
(s, 3H), 3.76 (s, 6H), 3.78 (s, 3H), 5.50 (s, 2H), 6.74 (s, 2H), 6.96
(d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.3, 2.1 Hz, 1H), 7.29 (d, J = 2.1 Hz,
1H), 8.45 (s, 1H), 9.09 (br s, 1H). 13C NMR (75 MHz, DMSO-d6):
d = 53.15, 55.53, 55.83, 59.88, 105.68, 112.37, 112.45, 116.25,
120.27, 123.57, 131.25, 137.25, 146.63, 147.45, 152.93. HRMS
calcd. for C19H21N3O5 (M+): 371.1481. Found 371.1476.
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.56) affording a pale yellow solid (86%). Mp 164–
165 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.72
(s, 3H), 3.89 (s, 6H), 3.90 (s, 3H), 7.24–7.27 (m, 3H), 7.92 (dd,
J = 8.6, 2.0 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 9.25 (s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 56.21, 56.30, 60.16, 97.73, 111.11, 113.09,
119.33, 124.20, 125.74, 129.50, 132.37, 137.29, 145.66, 153.48,
155.22. HRMS calcd. for C18H18BrN3O4 ([M+H]+): 420.0553. Found
420.0562.
4.5.11. 4-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyben
zyl)-1H-1,2,3-triazole (10d)
4.6.5. 4-(3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (8c-protected)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.55) affording a pale yellow solid (68%). Mp 117–
118 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 0.17
(s, 6H), 0.99 (s, 9H), 3.73 (s, 3H), 3.82 (s, 3H), 3.90 (s, 6H), 7.11 (d,
J = 8.5 Hz, 1H), 7.25 (s, 2H), 7.39 (d, J = 2.1 Hz, 1H), 7.50 (dd, J = 8.4,
2.1 Hz, 1H), 9.15 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = ꢀ4.74,
18.10, 25.50, 55.32, 56.23, 60.12, 97.91, 112.67, 117.38, 118.88,
119.06, 123.08, 132.48, 137.26, 144.45, 146.81, 150.48, 153.42.
HRMS calcd. for C24H33N3O5Si (M+): 471.2189. Found 471.2185.
The crude product was purified by chromatography (chloro-
form/EtOAc 1:1, Rf = 0.23) affording a yellow solid (89%). Mp
154–155 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6):
d = 3.64 (s, 3H), 3.77 (s, 6H), 3.96 (s, 3H), 5.55 (s, 2H), 6.75 (s,
2H), 7.45 (d, J = 8.9 Hz, 1H), 8.14 (dd, J = 8.8, 2.2 Hz, 1H), 8.32 (d,
J = 2.2 Hz, 1H), 8.68 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d = 53.35, 55.85, 56.72, 59.87, 105.78, 114.88, 121.22, 121.44,
123.31, 130.70, 130.89, 137.33, 139.41, 144.50, 151.33, 152.97.
HRMS calcd. for C19H20N4O6 (M+): 400.1383. Found 400.1388.
4.6. Syntheses of 1,4-disubstituted 1,2,3-triazoles using copper
turnings: general procedure
4.7. Syntheses of 1,5-disubstituted 1,2,3-triazoles using magne
sium acetylides: general procedure
A mixture of the corresponding azide (2 mmol) and the corre-
sponding alkyne (2 mmol) was dissolved in methanol (5 mL). A
copper turning was added and the mixture was stirred violently
for 48 h. The solvent was removed in vacuo.
The corresponding terminal alkyne (3.3 mmol) dissolved in dry
THF (3 mL) was added dropwise to an oven dried flask containing a
solution of EtMgCl in dry THF (1.5 mL, 2.0 M, 3.0 mmol) under ar-
gon at room temperature. After the alkyne was added, the solution
was heated to 50 °C for 15 min and cooled to room temperature.
The corresponding azide (3.0 mmol) dissolved in dry THF (3 mL)
was added dropwise. The reaction mixture was heated to 50 °C
for 1.5 h. After quenching with aqueous NH4Cl (6 mL), the product
was extracted with dichloromethane (3ꢃ 40 mL). The combined
organic layers were washed with aqueous NH4Cl (2ꢃ 60 mL), dried
over anhydrous magnesium sulfate and the solvent was removed
in vacuo.
4.6.1. 1-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole (7a)
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.56) affording a pale pink solid (53%). Mp 169–
170 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.71
(s, 3H), 3.85 (s, 3H), 3.87 (s, 6H), 7.18 (d, J = 9.1 Hz, 2H), 7.25 (s,
2H), 7.84 (d, J = 9.0 Hz, 2H), 9.20 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 55.49, 55.86, 60.02, 102.56, 114.83, 119.41, 121.43,
125.84, 129.96, 137.34, 147.09, 153.27, 159.19. HRMS calcd. for
4.7.1. 1-(4-Methoxybenzyl)-5-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole (5a)
C
18H19N3O4 (M+): 341.1376. Found 341.1377.
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.21) affording a pale yellow solid (81%). Mp 97–
98 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.69
(s, 3H), 3.70 (s, 3H), 3.71 (s, 6H), 5.61 (s, 2H), 6.66 (s, 2H), 6.88
(d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 7.92 (s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 50.73, 55.02, 55.88, 59.99, 106.01,
113.97, 121.79, 128.03, 128.20, 132.97, 137.37, 138.05, 153.01,
4.6.2. 2-Methoxy-5-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)phenol (7c)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.17) affording a pale yellow solid (35%). Mp 184–
185 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.70
(s, 3H), 3.85 (s, 3H), 3.87 (s, 6H), 7.13 (d, J = 8.8 Hz, 1H), 7.25 (s,
2H), 7.29 (dd, J = 8.7, 2.6 Hz, 1H), 7.37 (d, J = 2.6 Hz, 1H), 9.17 (s,
1H), 9.64 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 55.83, 55.87,
158.71. HRMS calcd. for
355.1528.
C
19H21N3O4 (M+): 355.1532. Found

6882
K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
4.7.2. 1-(3-Bromo-4-methoxybenzyl)-5-(3,4,5-trimethoxyphe
nyl)-1H-1,2,3-triazole (5b)
4.8.1. 5-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-trimethoxyben
zyl)-1H-1,2,3-triazole (6b)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.20) affording a pale yellow solid (87%). Mp 165–
166 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.70
(s, 3H), 3.74 (s, 6H), 3.80 (s, 3H), 5.63 (s, 2H), 6.66 (s, 2H), 7.05
(s, 1H), 7.06 (s, 1H), 7.29 (s, 1H), 7.92 (s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 50.04, 55.91, 56.18, 60.01, 106.08, 110.41, 112.69,
121.67, 127.87, 129.54, 131.74, 133.05, 137.41, 138.13, 153.05,
154.89. HRMS calcd. for C19H20BrN3O4 (M+): 433.0637. Found
433.0646.
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.17) affording a white solid (94%). Mp 105–
106 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.60
(s, 3H), 3.64 (s, 6H), 3.89 (s, 3H), 5.58 (s, 2H), 6.32 (s, 2H), 7.22
(d, J = 8.6 Hz, 1H), 7.48 (dd, J = 8.5, 2.2 Hz, 1H), 7.66 (d, J = 2.2 Hz,
1H), 7.91 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 51.36, 55.62,
56.40, 59.88, 104.44, 110.89, 112.83, 120.12, 129.48, 131.22,
132.82, 133.11, 135.96, 136.87, 152.84, 156.01. HRMS calcd. for
C
19H20BrN3O4 (M+): 433.0637. Found 433.0638.
4.7.3. 1-(3-(tert-Butyldimethylsilyloxy)-4-methoxybenzyl)-5-
(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (5c-protected)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.35) affording a pale yellow solid (84%). Mp 98–
100 °C (hexane/ether 9:1). 1H NMR (300 MHz, CDCl3): d = 0.09 (s,
6H), 0.94 (s, 9H), 3.73 (s, 6H), 3.76 (s, 3H), 3.87 (s, 3H), 5.45 (s,
2H), 6.42 (s, 2H), 6.62–6.67 (m, 2H), 6.76 (d, J = 8.2 Hz, 1H), 7.72
(s, 1H). 13C NMR (75 MHz, CDCl3): d = ꢀ4.67, 18.39, 25.62, 51.44,
55.51, 56.13, 60.93, 106.23, 112.09, 119.63, 120.29, 121.97,
128.28, 132.80, 138.99, 145.38, 150.87, 153.47. HRMS calcd. for
4.8.2. 5-(4-Methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxybenzyl)-
1H-1,2,3-triazole (6d)
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.14) affording a pale orange solid (94%). Mp 151–
152 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.59
(s, 3H), 3.63 (s, 6H), 3.96 (s, 3H), 5.63 (s, 2H), 6.32 (s, 2H), 7.47
(d, J = 8.8 Hz, 1H), 7.75 (dd, J = 8.8, 2.3 Hz, 1H), 7.98–7.99 (m, 2H).
13C NMR (75 MHz, DMSO-d6): d = 51.47, 55.59, 56.93, 59.85,
104.49, 114.88, 118.76, 124.85, 130.98, 133.50, 134.44, 135.27,
136.90, 139.23, 152.19, 152.86. HRMS calcd. for C19H20N4O6 (M+):
400.1383. Found 400.1384.
C
25H35N3O5Si ([M+H]+): 486.2418. Found 486.2433.
4.7.4. 1-(4-Methoxy-3-nitrobenzyl)-5-(3,4,5-trimethoxyphenyl)-
1H-1,2,3-triazole (5d)
4.9. Deprotection of TBDMS-ethers: general procedure
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.17) affording a pale orange solid (30%). Mp 184–
185 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.68
(s, 3H), 3.73 (s, 6H), 3.87 (s, 3H), 5.70 (s, 2H), 6.67 (s, 2H), 7.31
(d, J = 8.8 Hz, 1H), 7.37 (dd, J = 8.7, 2.1 Hz, 1H), 7.58 (d, J = 2.0 Hz,
1H), 7.91 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 49.86, 55.91,
56.69, 59.96, 106.15, 114.67, 121.58, 123.90, 128.20, 133.10,
133.38, 137.55, 138.16, 138.67, 151.51, 153.07. HRMS calcd. for
Tetra-n-butyl ammonium fluoride in THF (1 M, 7.5 mL,
7.5 mmol) was added dropwise to a solution of the corresponding
TBDMS-ether (5.72 mmol) in dry THF (11.5 mL) under argon. The
reaction mixture was stirred at room temperature for 1.5 h and
then treated with water (11.5 mL). The mixture was extracted with
chloroform (3ꢃ 40 mL). The combined organic layers were washed
with water (40 mL) and brine (40 mL), and dried over anhydrous
magnesium sulfate and the solvent was removed in vacuo.
C
19H20N4O6 (M+): 400.1383. Found 400.1380.
4.9.1. 2-Methoxy-5-((5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)methyl)phenol (5c)
4.7.5. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxybenzyl)-1H-
1,2,3-triazole (6a)
The crude product was purified by chromatography (hexane/
EtOAc 1:5, Rf = 0.47) affording a white solid (75%). Mp 171–
172 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6):
d = 3.69 (s, 3H), 3.70 (s, 9H), 5.53 (s, 2H), 6.47 (dd, J = 8.2,
2.1 Hz, 1H), 6.52 (d, J = 2.1 Hz, 1H), 6.66 (s, 2H), 6.84 (d,
J = 8.3 Hz, 1H), 7.93 (s, 1H), 9.07 (br s, 1H). 13C NMR (75 MHz,
DMSO-d6): d = 50.85, 55.56, 55.85, 59.99, 105.95, 112.23, 113.97,
117.48, 121.76, 128.67, 132.95, 137.37, 138.04, 146.62, 147.16,
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.16) affording a pale yellow solid (72%). Mp 115–
116 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.60
(s, 3H), 3.62 (s, 6H), 3.80 (s, 3H), 5.56 (s, 2H), 6.28 (s, 2H), 7.06 (d,
J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.85 (s, 1H). 13C NMR
(75 MHz, DMSO-d6): d = 51.14, 55.23, 55.61, 59.86, 104.41, 114.42,
118.70, 130.01, 131.33, 132.67, 136.81, 137.31, 152.78, 159.91.
HRMS calcd. for C19H21N3O4 (M+): 355.1532. Found 355.1531.
152.99. HRMS calcd. for
371.1490.
C
19H21N3O5 (M+): 371.1481. Found
4.7.6. 5-(3-(tert-Butyldimethylsilyloxy)-4-methoxyphenyl)-1-
(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazole (6c-protected)
The crude product was purified by chromatography (hexane/
EtOAc 1:1, Rf = 0.38) affording a yellow solid (58%). Mp 108–110 °C
(hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 0.05 (s, 6H),
0.90 (s, 9H), 3.61 (s, 3H), 3.63 (s, 6H), 3.80 (s, 3H), 5.56 (s, 2H), 6.26
(s, 2H), 6.84 (d, J = 1.6 Hz, 1H), 7.10–7.11 (m, 2H), 7.89 (s, 1H). 13C
NMR (75 MHz, DMSO-d6): d = ꢀ4.97, 17.95, 25.34, 51.00, 55.43,
55.60, 59.83, 103.87, 112.73, 118.82, 120.24, 122.65, 131.58,
132.64, 136.82, 137.21, 144.28, 151.38, 152.91. HRMS calcd. for
4.9.2. 2-Methoxy-5-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazol-5-yl)phenol (6c)
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.19) affording a pale yellow solid (83%). Mp 194–
195 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.60
(s, 3H), 3.63 (s, 6H), 3.81 (s, 3H), 5.54 (s, 2H), 6.30 (s, 2H), 6.87–
6.90 (m, 2H), 7.04 (d, J = 8.9 Hz, 1H), 7.81 (s, 1H), 9.32 (br s, 1H).
13C NMR (75 MHz, DMSO-d6): d = 51.07, 55.60, 59.87, 104.42,
112.38, 115.69, 118.94, 119.75, 131.41, 132.46, 136.81, 137.56,
C
25H35N3O5Si ([M+H]+): 486.2418. Found 486.2425.
146.70, 148.55, 152.78. HRMS calcd. for
371.1481. Found 371.1478.
C
19H21N3O5 (M+):
4.8. Syntheses of 1,5-disubstituted 1,2,3-triazoles using
ruthenium catalysis: general procedure
4.9.3. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-4-yl)phenol (8c)
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.38) affording a white solid (94%). Mp 211–
212 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 3.72
A mixture of the corresponding azide (1 mmol), the correspond-
ing alkyne (1.1 mmol) and CpꢂRuCl(COD) (3.8 mg, 10 mol %) was
refluxed in dry benzene (10 mL) in a Schlenk flask under argon
for 3 h. The solvent was removed in vacuo.

K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
6883
(s, 3H), 3.82 (s, 3H), 3.90 (s, 6H), 7.03 (d, J = 8.4 Hz, 1H), 7.26 (s, 2H),
7.32 (dd, J = 8.3, 2.0 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 9.12 (s, 1H),
9.17 (br s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 55.57, 56.20,
60.11, 97.73, 112.42, 112.59, 116.41, 118.69, 123.08, 132.48,
137.16, 146.76, 147.23, 147.75, 153.43. HRMS calcd. for
132.19, 136.79, 138.12, 138.17, 146.99, 152.75. HRMS calcd. for
19H22N4O4 (M+): 370.1641. Found 370.1638.
C
4.10.3. 2-Methoxy-5-(4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)benzenamine (7e)
C
18H19N3O5 ([M+H]+): 358.1397. Found 358.1413.
Recrystallization in methanol and ether afforded a pale pink so-
lid (86%). Mp 166–167 °C (methanol/ether 3:1). 1H NMR (300 MHz,
DMSO-d6): d = 3.70 (s, 3H), 3.84 (s, 3H), 3.87 (s, 6H), 5.20 (br s, 2H),
6.97–6.98 (m, 2H), 7.21 (d, J = 1.6 Hz, 1H), 7.25 (s, 2H), 9.10 (s, 1H).
13C NMR (75 MHz, DMSO-d6): d = 55.56, 55.88, 60.02, 102.54,
105.01, 106.96, 110.54, 119.22, 125.97, 130.44, 137.26, 138.83,
4.9.4. 5-Ethynyl-2-methoxyphenol (13e)
The crude product was purified by chromatography (hexane/
EtOAc 9:1, Rf = 0.15) affording a pale yellow solid (90%). Mp 61–
62 °C (hexane/ether 9:1). 1H NMR (300 MHz, DMSO-d6): d = 2.97
(s, 1H), 3.89 (s, 3H), 5.61 (s, 1H), 6.78 (d, J = 8.1 Hz, 1H), 7.01–
7.06 (m, 2H). 13C NMR (75 MHz, DMSO-d6): d = 55.91, 75.60,
83.54, 110.36, 114.86, 118.01, 124.80, 145.23, 147.30. HRMS calcd.
for C9H8O2 (M+): 148.0524. Found 148.0526.
146.24, 146.88, 153.26. HRMS calcd. for
356.1485. Found 356.1477.
C
18H20N4O4 (M+):
4.10.4. 2-Methoxy-5-((4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)methyl)aniline (9e)
The crude product was purified by reversed phase chromatogra-
phy (MeOH/H2O 2:1 (1% TFA), Rf = 0.38) affording a pale yellow so-
lid (62%). Mp 114–115 °C (methanol/ether 3:1). 1H NMR (300 MHz,
DMSO-d6): d = 3.67 (s, 3H), 3.74 (s, 3H), 3.83 (s, 6H), 4.81 (br s, 2H),
5.41 (s, 2H), 6.53–6.56 (m, 2H), 6.78 (d, J = 8.2 Hz, 1H), 7.15 (s, 2H),
8.51 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 53.11, 55.25, 55.81,
59.96, 102.37, 110.34, 112.79, 115.64, 121.04, 126.28, 128.10,
137.04, 137.87, 146.15, 146.56, 153.17. HRMS calcd. for
4.9.5. 5-(Azidomethyl)-2-methoxyphenol (15e)
The crude product was purified by chromatography (hexane/
EtOAc 8:3, Rf = 0.40) affording a pale yellow oil (85%). 1H NMR
(300 MHz, CDCl3): d = 3.90 (s, 3H), 4.23 (s, 2H), 5.68 (br s, 1H),
6.81 (dd, J = 8.2, 1.9 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.90 (d,
J = 1.8 Hz, 1H). 13C NMR (75 MHz, CDCl3): d = 54.50, 55.96,
110.62, 114.60, 120.13, 128.48, 145.82, 146.58. HRMS calcd. for
C8H9N3O2 (M+): 179.0695. Found 179.0693.
C
19H22N4O4 (M+): 370.1641. Found 370.1638.
4.10. Syntheses of amino-substituted triazoles: general
procedure
4.10.5. 2-Methoxy-5-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazol-4-yl)aniline (10e)
Pale brown solid (70%). Mp 126–127 °C (methanol/ether 3:1).
1H NMR (300 MHz, DMSO-d6): d = 3.64 (s, 3H), 3.76 (s, 6H), 3.78
(s, 3H), 4.80 (br s, 2H), 5.49 (s, 2H), 6.73 (s, 2H), 6.82 (d,
J = 8.3 Hz, 1H), 6.97 (dd, J = 8.2, 2.1 Hz, 1H), 7.16 (d, J = 2.1 Hz,
1H), 8.35 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 53.08, 55.23,
55.83, 59.87, 105.67, 110.55, 113.37, 119.94, 123.38, 131.31,
137.23, 137.73, 146.16, 147.17, 152.91. HRMS calcd. for
Saturated copper sulfate solution (0.8 mL) was added to the
corresponding nitro-substituted triazole (2.01 mmol) dissolved in
ethanol (10 mL). The mixture was cooled to 0 °C, and a solution of
sodium borohydride (380 mg, 10.05 mmol) in ethanol/H2O 1:1
(6 mL) was added dropwise to the reaction mixture. The mixture
was refluxed for 4 h. After cooling, saturated copper sulfate solution
(0.8 mL) and a solution of sodium borohydride (380 mg, 10.05
mmol) were added. The mixture was refluxed overnight. After cool-
ing, ethyl acetate was added and the mixture extracted with 1 M HCl
(3ꢃ 45 mL). The pH of the combined aqueous layers was adjusted to
pH 10 using 4 M NaOH, and extracted with ethyl acetate (3ꢃ 50 mL).
The combined organic layers were dried over anhydrous magnesium
sulfate and the solvent was removed in vacuo.
C
19H22N4O4 (M+): 370.1641. Found 370.1642.
4.11. Biological assays
4.11.1. Cancer cell growth inhibition on K562 human leukemia
cell line
The method applied was previously described by Edmondson
and coworkers.25a K562 human chronic myelogeneous leukemia
cells were cultivated in RMPI medium, free of antibiotics and
4.10.1. 2-Methoxy-5-((5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)methyl)aniline (5e)
containing 2-mercaptoethanol (2 lM) and L-glutamine (2 mM),
Recrystallization in methanol and ether afforded a pale yellow
solid (36%). Mp 69–71 °C (methanol/ether 3:1). 1H NMR
(300 MHz, DMSO-d6): d = 3.69 (s, 3H), 3.70 (s, 6H), 3.71 (s, 3H),
4.83 (br s, 2H), 5.48 (s, 2H), 6.23 (dd, J = 8.2, 2.0 Hz, 1H), 6.40 (d,
J = 2.0 Hz, 1H), 6.68 (s, 2H), 6.71 (d, J = 8.2 Hz, 1H), 7.93 (s, 1H).
13C NMR (75 MHz, DMSO-d6): d = 51.17, 55.26, 55.85, 59.98,
105.94, 110.35, 111.69, 114.24, 121.84, 128.58, 132.89, 137.37,
137.81, 138.00, 145.78, 152.98. HRMS calcd. for C19H22N4O4 (M+):
370.1641. Found 370.1650.
supplemented 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 supple-
mented with FCS (10% v/v). The candidate drug was dissolved
in DMSO. A drug solution of 100
100 l of cell solution (40,000 cells/mL) in a 96-well microtitre
testplate (4 l of the drug solution diluted in medium in order
ll in medium was added to
l
l
to reach decreasing concentrations). This series of dilutions was
continued to afford samples at different concentrations leaving
one cell solution free of drug acting as a control. The plates were
incubated at 37 °C (5% CO2 in air) for 5 days. The plate was then
4.10.2. 2-Methoxy-5-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazol-5-yl)aniline (6e)
removed from the incubator and 50 ll of a solution of MTT
The crude product was purified by reversed phase chromatogra-
phy (MeOH/H2O 2:1, Rf = 0.43) and recrystallized in methanol and
ether affording a yellow solid (53%). Mp 148–150 °C (methanol/
ether 3:1). 1H NMR (300 MHz, DMSO-d6): d = 3.60 (s, 3H), 3.63 (s,
6H), 3.80 (s, 3H), 4.95 (br s, 2H), 5.54 (s, 2H), 6.31 (s, 2H), 6.64
(dd, J = 8.2, 2.0 Hz, 1H), 6.74 (d, J = 2.0 Hz, 1H), 6.91 (d, J = 8.2 Hz,
1H), 7.76 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d = 50.98, 55.33,
55.60, 59.86, 104.49, 110.63, 113.42, 116.35, 118.95, 131.46,
(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

6884
K. Odlo et al. / Bioorg. Med. Chem. 18 (2010) 6874–6885
8. (a) Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma, R.; Simpson, M. J.; Boyd,
M. R.; Rener, G. A.; Bansal, N. Anti-Cancer Drug Des. 1995, 10, 299; (b) Siemann,
D. W.; Chaplin, D. J.; Walicke, P. A. Expert Opin. Invest. Drugs 2009, 18, 189.
9. (a) Peifer, C.; Stoiber, T.; Unger, E.; Totzke, F.; Schächtele, C.; Marmé, D.; Brenk,
R.; Klebe, G.; Schollmeyer, D.; Dannhardt, G. J. Med. Chem. 2006, 49, 1271; (b)
Pirali, T.; Busacca, S.; Beltrami, L.; Imovilli, D.; Pagliai, F.; Miglio, G.; Massarotti,
A.; Verotta, L.; Tron, G. C.; Sorba, G.; Genazzani, A. A. J. Med. Chem. 2006, 49,
5372; (c) Quintin, J.; Roullier, C.; Thoret, S.; Lewin, G. Tetrahedron 2006, 62,
4038; (d) Sun, C.-M.; Lin, L.-G.; Yu, H.-J.; Cheng, C.-Y.; Tsai, Y.-C.; Chu, C.-W.;
Din, Y.-H.; Chau, Y.-P.; Don, M.-J. Bioorg. Med. Chem. Lett. 2007, 17, 1078; (e)
Tsyganov, D. V.; Yakubov, A. P.; Konyushkin, L. D.; Firgang, S. I.; Semenov, V. V.
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Yang, B.; Lu, X.; Hu, Y. Bioorg. Med. Chem. 2008, 16, 2550.
10. (a) Cafici, L.; Pirali, T.; Condorelli, F.; Del Grosso, E.; Massarotti, A.; Sorba, G.;
Canonico, P. L.; Tron, G. C.; Genazzani, A. A. J. Comb. Chem. 2008, 10, 732; (b)
Demko, Z.; Borella, C.; Chen, S.; Sun, L. U.S. Patent 2007238699 A1, 2007.; (c)
Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga,
Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T. Bioorg. Med. Chem. Lett. 1998, 8, 3153; (d)
Pagliai, F.; Pirali, T.; Del Grosso, E.; Di Brisco, R.; Tron, G. C.; Sorba, G.;
Genazzani, A. A. J. Med. Chem. 2006, 49, 467; (e) Pati, H. N.; Wicks, M.; Holt, H. L.
J.; LeBlanc, R.; Weisbruch, P.; Forrest, L.; Lee, M. Heterocycl. Commun. 2005, 11,
117; (f) Zhang, Q.; Peng, Y.; Wang, X. I.; Keenan, S. M.; Arora, S.; Welsh, W. J. J.
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I.; Skrede, M.; Flørenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16, 4829.
12. The MTT assay does not distinguish between growth inhibition and cell
death.
13. (a) Getahun, Z.; Jurd, L.; Chu, P. S.; Lin, C. M.; Hamel, E. J. Med. Chem.
1992, 35, 1058; (b) Kaffy, J.; Pontikis, R.; Florent, J.-C.; Monneret, C. Org.
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Med. Chem. Lett. 2009, 19, 1318.
cell growth by 50% (IC50 value), determined by graphical means
as percentage of the control growth.
4.11.2. Inhibition of tubulin assembly
The method applied was that described by Lawrence and
coworkers.27 Tubulin was isolated from porcine brain and stored
at ꢀ78 °C. Samples were prepared directly in a 96-well microtitre
testplate that was preincubated at 4 °C in the fridge for 30 min
and contained Mes buffer [128
0.5 mM MgCl2, and distilled water, pH 6.6)], GTP (20
Mes buffer), tubulin (50 l, 11 mg/mL in Mes buffer), and the can-
didate drug (20 l, Csample in DMSO). The tubulin/drug samples
were immediately placed in a 96-well plate reader, alongside blank
samples containing Mes buffer (198 l) and the candidate drug
(10 l, same concentration). The absorbance (k 350 nm) was re-
l
l
(0.1 M Mes, 1 mM EGTA,
ll, 5 mM in
l
l
l
l
corded at ambient temperature for a period of 60 min, and the re-
sults were compared to untreated controls to evaluate the relative
degree of change in optical density. The results enabled the calcu-
lation 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.
4.12. Molecular modeling
Three triazoles, 5e, 6e, and 11, were modeled by the Internal
Coordinate Mechanics (ICM39,40) v.3.5-ln program package. The
ligands were geometry-optimized and assigned partial atomic
charges according to the MMFF94 force field.41 The X-ray crystallo-
14. Pettit, G. R.; Grealish, M. P.; Jung, M. K.; Hamel, E.; Pettit, R. K.; Chapuis, J.-C.;
Schmidt, J. M. J. Med. Chem. 2002, 45, 2534.
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Koketsu, M.; Yamamoto, H.; Kobayashi, M.; Maruyama, T.; Ohuchida, S.; Nakai,
H.; Kondo, K.; Toda, M. Bioorg. Med. Chem. 2006, 14, 5562.
17. Thompson, A. S.; Humphrey, G. R.; DeMarco, A. M.; Mathre, D. J.; Grabowski, E.
J. J. J. Org. Chem. 1993, 58, 5886.
graphic structures of a
- and b-tubulin (PDB entry 1SA0;28 chain A
and chain B, respectively) in complex with guanosine-50-triphos-
phate (GTP) were loaded into ICM, and hydrogen atoms were
added. ECEPP/342 atom charges were assigned, followed by energy
minimization to relieve atomic clashes. A grid map was calculated
that included the ligand binding site of 1SA0. The grid size was
kept similar to our previous docking of triazole analog 4e.11 The tri-
18. Tao, C.-Z.; Cui, X.; Li, J.; Liu, A.-X.; Liu, L.; Guo, Q.-X. Tetrahedron Lett. 2007, 48,
3525.
19. Lombardo, L. Tetrahedron Lett. 1984, 25, 227.
azole analogs were docked as flexible-ligands on the rigid
a,b-
20. (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2002, 41, 2596; (b) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
2002, 67, 3057.
21. (a) Akimova, G. S.; Chistokletov, V. N.; Petrov, A. A. Zh. Org. Khim. 1967, 3, 968;
(b) Krasin´ ski, A.; Fokin, V. V.; Sharpless, K. B. Org. Lett. 2004, 6, 1237.
22. Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.; Sharpless, K. B.; Fokin,
V. V.; Jia, G. J. Am. Chem. Soc. 2005, 127, 15998.
tubulin conformation. The docked ligand conformation that exhib-
ited the most favorable docking energy was further refined using a
flexible-ligand, flexible-receptor method in ICM.43 The free binding
energy of the refined tubulin-ligand complex was calculated to
predict the contributions of different subunits in binding the differ-
ent triazole analogs.
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