Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole analogs of combretastatin A-1

Article abstract of DOI:10.1016/j.bmc.2011.11.010

The synthesis, cytotoxicity, inhibition of tubulin polymerization data and anti-angiogenetic effects of seven 1,5-disubstituted 1,2,3-triazole analogs and two 1,4-disubstituted 1,2,3-triazole analogs of combretastatin A-1 (1) are reported herein. The biol

Full text of DOI:10.1016/j.bmc.2011.11.010

Bioorganic & Medicinal Chemistry 20 (2012) 234–242
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole
analogs of combretastatin A-1
Øyvind W. Akselsen a, Kristin Odlo a, Jing-Jy Cheng b, Giorgio Maccari c, Maurizio Botta c,
Trond Vidar Hansen a,
⇑
a School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, PO Box 1068, N-0316 Oslo, Norway
b National Research Institute of Chinese Medicine, 155-1 Li-Nung Street, Section 2, Shih-Pai, Taipei, Taiwan
c Dipartimento Farmaco Chimico Tecnologico, Università di Siena, Via Aldo Moro 2, I-53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, cytotoxicity, inhibition of tubulin polymerization data and anti-angiogenetic effects of
seven 1,5-disubstituted 1,2,3-triazole analogs and two 1,4-disubstituted 1,2,3-triazole analogs of
combretastatin A-1 (1) are reported herein. The biological studies revealed that the 1,5-disubstituted
1,2,3-triazoles 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diol (6),
3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diamine (8) and 5-(2,
3-difluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (9) were the three most
active compounds regarding inhibition of both tubulin polymerization and angiogenesis. Molecular mod-
eling studies revealed that combretastatins 1 and 2 and analogs 5–11 could be successfully docked into
Received 6 September 2011
Revised 1 November 2011
Accepted 5 November 2011
Available online 12 November 2011
Keywords:
Combretastatin A-1
Tubulin
Angiogenesis
1,2,3-Triazoles
Molecular modeling
the colchicine binding site of a,b-tubulin.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
the prodrug CA-4P (OXi2021, 4) derived from combretastatin A-4
(2).10 Prodrug 4 has entered several clinical trials and attracted
more interest as an anticancer agent than 3.7a,f Furthermore,
CA-1P (3) induces tumor growth delays and regressions resulting
in an enhanced in vivo activity compared to 4, most likely due to
a direct mode of action on the tumor mass.7a,11
Within the group microtubule-binding agents (MBAs), natural
products and their derivatives have been widely used in cancer che-
motherapy.1,2 There are two classes of MBAs; those that stabilize
microtubules and promote polymerization, and those that destabi-
lize the microtubules and promote depolymerization by inhibition
of tubulin.3 Both types interfere with the mitotic spindle assembly
during cell division, resulting in cell death. Studies have shown that
most MBAs have antivascular effects from anti-angiogenetic or
vascular disrupting activities, or both.4
The combretastatins, such as combretastatin A-1 (CA-1, 1) and
combretastatin A-4 (CA-4, 2) depicted in Figure 1, have attracted
much interest as anti-cancer agents.5 The combretastatins were
isolated from the bark of the South African bush willow tree
Combretum caffrum by Pettit et al.6 The phosphate prodrug of com-
bretastatin A-1 (CA-1P, 3, OXi4503) has entered clinical trials.7
Combretastatin A-1 (1) and its prodrug 3 are cytotoxic against a
variety of human cancer cell lines8 and is also a potent anti-vascular
agent (VDA).4j In addition, 1 has been reported to undergo oxidation
to its ortho-quinone derivative that also exhibits interesting biolog-
ical effects.9 Moreover, CA-1P (3) is an even more potent VDA than
Structure–activity relationship (SAR) studies have shown that a
3,4,5-trimethoxy substituted A ring (TMP) and a 4-methoxysubsti-
tuted B ring separated by a double bond with cis configuration are
important for optimal cytotoxic activity for the combretastatins.7a
The isomerization to the biologically less active trans-form may be
problematic for the further development of the combretastatins as
drug candidates.11 Hence, numerous combretastatin analogs with a
locked cis-type bridge between the two phenyl rings have been
prepared.12,13 Recently we prepared a series of 1,4- and 1,5-disub-
stituted 1,2,3-triazole analogs of combretastatin A-4 (2) that exhib-
ited potent cytotoxicity in the nanomolar range and tubulin
inhibitory activity in the low micromolar range.14a,b A few other
1,2,3-triazole analogs of combretastatin A-4 (2) have been repor-
ted.14c,d Furthermore, Welsh and co-workers reported that 3,
4-disubstituted-1,2,4-triazole analogs of 2 exhibited potent cyto-
toxic effects and inhibition of tubulin polymerization.14e Lee and
co-workers reported a few 4,5-disubstitued NH-1,2,3-triazoles
with potent cytotoxic effects.14f As of today, a growing number of
analogs of the naturally occurring stilbene 1 have been prepared
and biologically evaluated.9,15 Herein we present the synthesis,
⇑
Corresponding author. Tel.: +47 22857450; fax: +47 22855947.
E-mail address: t.v.hansen@farmasi.uio.no (T.V. Hansen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.010

Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
235
Figure 1. Structures of combretastatin A-1 (1) and A-4 (2), their prodrugs 3 and 4, respectively, and 1,2,3-triazole analogs 5–13.
cytotoxicity, inhibition of tubulin polymerization data and anti-
angiogenetic effects of several 1,5-disubstituted 1,2,3-triazole ana-
logs of combretastatin A-1 (1) as well as two 1,4-disubstituted
1,2,3-triazoles (Fig. 1). Furthermore, molecular modeling studies
Reacting azide 15 with the magnesium acetylide of alkyne 30 as
previously reported14,26 produced the 1,5-disubstituted 1,2,3-tria-
zole 31 in 14% yield (Scheme 2). Deprotection of the bis-methoxy-
methyl functionality in 31 according to a literature procedure
generated triazole 5 in 86% yield.27 Using the previously men-
tioned cycloaddition conditions for reacting azide 32 with the
magnesium acetylide of alkyne 19, produced triazole 33 in 59%
yield. In the same manner, triazole 9 was obtained in excellent
89% yield. Deprotection27 of the bis-methoxymethyl functionality
in 33 in aqueous HCl produced triazole 6 in 77% total yield (Scheme
2). Triazoles 7 and 12 were obtained in a thermally induced cyclo-
addition reaction, as described by Huisgen et al.,28 between alkyne
27 and azide 32. The two triazoles 7 and 12 were isolated in a 1:1
ratio. Separation of 7 and 12 was achieved by column chromatog-
raphy, affording the desired 1,5-disubstituted product 7 in 43%
yield. The amino-substituted triazole 8 was obtained in 28% yield
by reduction of the nitro groups in 7 using NaBH4 and CuSO4 in
EtOH (Scheme 2).29
Reacting the magnesium acetylides of alkynes 23a and 23b with
azide 32 followed by deprotection as described above, afforded the
desired triazoles 10 and 11 in 48% and 59% total yield, respectively,
over two steps (Scheme 3). The 1,4-disubstituted 1,2,3-triazole 12
was obtained after reacting alkyne 27 and azide 32 using Sharpless
and co-workers copper catalysed azide-alkyne cycloaddition
(CuAAC) conditions.30 However, we observed a sluggish reaction
that required molar equivalents of CuSO4 for decent conversion
of the starting materials. Reduction of the nitro groups in 12 affor-
ded triazole 13 in 13% overall yield from alkyne 27 (Scheme 3).29
with the colchicine binding site of
a,b-tubulin were performed
with all 1,2,3-triazoles 5–13. No molecular docking studies of the
natural product 1 or any of its analogs in the colchicine binding si-
te4a,16 on tubulin have been reported.
2. Results and discussion
2.1. Synthesis
The syntheses of the starting materials are depicted in Scheme 1.
The known bromide 1417 was converted into the azide 15 after
reacting first with n-BuLi and then with tosylazide.18 Unfortunately,
purification of this product proved difficult, therefore the crude
phenyl azide 15 was used in the cycloaddition reaction. Catechol
17 was prepared from commercially available 16 using a literature
procedure.19 Then 17 was protected as its bis-methoxymethyl ether
according to a standard protocol20 affording benzaldehyde 18.
Benzaldehyde 18 was converted to alkyne 19 using a Colvin rear-
rangement reaction21 (Scheme 1). The known phenol 20a22 reacted
in an ortho-formylation reaction23 to the corresponding salicylalde-
hyde 21a that was protected using MOMCl and DIPEA,19 affording
the aldehyde 22a. The Colvin rearrangement reaction was also
applied for the synthesis of alkyne 23a. Using the same three step
protocol starting from phenol 20b, alkyne 23b was obtained. Al-
kyne 27 was prepared by nitration of commercially available benz-
aldehyde 24 affording compounds 25 and 26 in approximately 1:1
ratio.15e Compounds 25 and 26 were separated by column chroma-
tography affording the desired benzaldehyde 26 in 41% yield.
Neither the Colvin rearrangement nor the Corey–Fuchs reaction24
converted benzaldehyde 26 into alkyne 27. However, the Ohira–
Bestmann reaction25 produced the desired alkyne 27 in 75% yield
from 26. The same conditions were applied to aldehyde 28 afford-
ing alkyne 29.
2.2. Biological evaluation
The prepared 1,2,3-triazoles 5–13, as well as both lead com-
pounds 1 and 2, were evaluated for their ability to inhibit the
growth of four human cancer cell lines (MCF-7; breast cancer cell
line, NCIH460; human non-small lung cancer cell line, HT-29; colo-
rectal cancer cell line and CEM; leukemia cancer cell line) and a
non-cancerous mammalian fibroblast cell line using the alamar

236
Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
Scheme 1. Synthesis of starting materials. Reagents and conditions: (i) (a) n-BuLi, THF, À78 °C; (b) TsN3, THF, À78 °C; (ii) BCl3, CH2Cl2; (iii) DIPEA, MOMCl, CH2Cl2, 0 °C; (iv)
LDA, TMSCHN2, THF, À78 °C; (v) MgCl2, Et3N, (CH2O)n, THF, ; (vi) HNO3, H2SO4, 0 °C; (vii) (a) separation by chromatography; (b) aldehyde 26, CH3COCH(N2)P(O)(OMe)2,
K2CO3, MeOH; (viii) CH3COCH(N2)P(O)(OMe)2, K2CO3, MeOH.
D
BlueÒ-assay.31,32 Triazoles 8, 9 and 11 inhibited the growth of the
MCF-7 cancer cell line with IC50-values in the micromolar range
comparable to CA-4 (2), and were all more potent than CA-1 (1)
(Table 1). Noteworthy, compound 7 was more potent than both
combretastatins. Triazoles 5, 6, 10, 12 and 13 were all less potent
than both CA-1 (1) and CA-4 (2). Triazole 7 was also the most po-
than CA-1 (1) (IC50 = 3.2
potent than 8 and 9 with IC50 = 0.01
derived from the alkyne 30, was inactive as an inhibitor in this
angiogenesis assay (IC50 > 10 M). Triazole 11, with the dimethyl
acetal moiety in the B-ring, was also inactive in this assay. None
of the 1,4-disubstituted 1,2,3-triazoles 12 and 13 exhibited any
l
M). However, CA-4 (2) was 10-fold more
l
M. Triazole 5, with the A-ring
l
tent analog (IC50 = 0.7
l
M) in the H460 lung cancer cell line com-
activity (IC50 > 10 lM).
pared to all compounds tested. In this cell line the 1,4-
disubstituted analogs 12 and 13 exhibited IC50-values in the micro-
molar range, but were less active than the combretastatins and the
1,5-disubstituted 1,2,3-triazoles evaluated. In the HT-29 colorectal
cancer cell assay all triazoles 5–13 exhibited lower potency than
both CA-1 (1) and CA-4 (2). The most potent triazoles in this cell
Next we investigated the inhibition of tubulin assembly.34 All of
the tested 1,5-disubstituted 1,2,3-triazoles exhibited lower activi-
ties than CA-1 (1). This was also observed for 1,2,3-triazole analogs
of CA-4 (2) in our previous study.14a,b Furthermore, all of the pre-
pared triazoles exhibited significant lower inhibition of tubulin
assembly compared to CA-4 (2) (Table 1). The most potent triazole
in the tubulin inhibition assay was the diamino triazole 8 with
assay were 6 and 9 with IC50 = 1.9
lM for both compounds, com-
pared to IC50 = 0.9 M for CA-1 (1). Noteworthy, both combretast-
l
IC50 = 5.2
CA-4 (2), respectively. Triazoles 6, 9 and 10 exhibited weak tubulin
inhibition activities with IC50-values of 10.1, 15.6 and 17.2 M,
respectively. The 1,2,3-triazoles 5, 7 and 11 were all inactive as
lM compared to IC50 = 3.5 and 0.6 lM for CA-1 (1) and
atin A-1 (1) and A-4 (2) as well as all the prepared triazoles 5, 6 and
8–13 were inactive in the leukemia cancer cell line (Table 1).
Only triazole analogue 7 exhibited activity in the non-cancerous
cell line. Interestingly, none of the other 10 triazoles and neither
lead compound 1, nor 2 displayed any growth inhibition activity
in the non-cancerous cell line, thus indicating greater selectivity
for cancer cells. The biological results for the 1,2,3-triazole analogs
of CA-1 (1) reported herein, support the previous findings that 1,5-
disubstituted 1,2,3-triazoles with the A ring derived from a phenyl
azide, are the most cytotoxic analogs.14a,b
l
tubulin inhibitors (IC50 > 20 lM).
2.3. Molecular modeling
Molecular modeling studies were performed to investigate the
binding ability of the 1,2,3-triazoles to the colchicine binding site
of
disubstituted 1,2,3-triazoles 5–11 as well as CA-1 (1) and CA-4
(2) occupied the colchicine binding site of ,b-tubulin mostly bur-
a
,b-tubulin (pdb: 1SA0).35 Docking studies showed that 1,5-
The anti-angiogenetic activity of the most cytotoxic triazole
analogs 5–11 was also evaluated (Table 1).33 The 1,5-disubstitued
1,2,3-triazoles 6–11 with the A-ring derived from the azide 32
exhibited anti-angiogenetic activity in the low micromolar range
a
ied in the b subunit (Fig. 2). The docking scores varied from À8.53
to À7.50 kcal/mol. The colchicine-binding pocket could not accom-
modate compounds 12 and 13, and the docking score values were
2 kcal/mol lower than the average 1,5-disubstituted 1,2,3-triazole
score values.
with IC50-values between 0.3 and 4.4
triazoles were 8 and 9 with IC50 = 0.3
Interestingly, these two triazoles were about 10-fold more potent
lM; the two most potent
l
M for both compounds.

Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
237
Scheme 2. Synthesis of 1,2,3-triazoles 5–9. Reagents and conditions: (i) Alkyne 30, then EtMgCl, THF,
D; (ii) HCl, H2O, THF; (iii) alkyne 19 or alkyne 29, then EtMgCl, THF, D;
(iv) alkyne 27, toluene, H2O,
D
; (v) (a) separation by chromatography; (b) triazole 7, then NaBH4, CuSO4Á5H2O, EtOH.
Scheme 3. Synthesis of 1,2,3-triazoles 10–13, 34a and 34b. Reagents and conditions: (i) (a) EtMgCl, THF,
CuSO4Á5H2O, t-BuOH/H2O (1:1); (iv) NaBH4, CuSO4Á5H2O, EtOH.
D; (b) azide 32; (ii) HCl, H2O, THF; (iii) azide 32, Na-ascorbate,
There was no correlation between the docking scores and the
IC50-values for the compounds that exhibited tubulin inhibition
properties. Since the compounds are structural similar, this obser-
vation was not unexpected as scoring of such structural similar
compounds still remains a challenge.36 In general, after energy
minimization, the TMP moiety and B-ring of the 1,5-disubstituted
1,2,3-triazole analogs were superimposed over DAMA-colchicine
in the 1SA0 tubulin structure. The 4-methoxy group is a possible

238
Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
Table 1
Cytotoxicity, inhibition of angiogenesis and inhibition of tubulin
Compound MCF-7 cell assay H460 cell assay HT-29 cell assay
CEM cell assay
Fibroblast cell assay
Inhibition of
angiogenesisa
Tubulin
inhibitionb
(lM)
a
a
a
a
a
IC50
(l
M)
IC50
(lM)
IC50
(lM)
IC50
(
lM)
IC50
(lM)
(
lM)
1
2
5
6
7
8
9
10
11
12
13
75.9
48.9
>100
>100
6.1
38.8
26.7
>100
46.3
>100
>100
1.8
4.4
26.6
6.4
0.7
17.3
4.9
7.4
16.8
18.2
29.8
0.9
0.4
>100
>100
>100
>100
26.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
31.5
>100
>100
>100
>100
>100
>100
3.2
3.5
0.6
0.01
>10
3.2
4.4
0.3
0.3
3.4
>10
>10
>10
64.3
1.9
12.0
17.2
1.9
>20
10.1
>20
5.2
15.6
17.1
>20
n.d.
n.d.
8.8
>100
>100
>100
cn.d. = not determined.
a
Results of three experiments performed as triplicates.
Results of two experiments performed as triplicates.
b
4. Experimental
4.1. General procedure
Unless noted otherwise, all reagents and solvents were used as
purchased without further purification. Melting points are uncor-
rected. Analytical TLC was performed using silica gel 60 F254 plates
(Merck) or RP-18 F254s plates (Merck). Flash column chromatogra-
phy was performed on silica gel 60 (40–63 l
m, Fluka). NMR (1H,
13C) spectra were recorded on a Bruker DPX-300 MHz or DPX-
200 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), DMSO-d6 (2.50 ppm
for 1H and 39.43 ppm for 13C), acetonitrile-d3 (1.94 ppm for 1H and
1.32 ppm for 13C), methanol-d4 (4.87 ppm for 1H and 49.15 for 13C),
acetone-d6 (2.05 ppm for 1H and 29.84 ppm for 13C). Starting mate-
rials 14,17 20a38 and 20b39 as well as intermediates 17,6a 18,15h
21a,21 21b,40 22a,41 25,15e 2615e and 3214b are known compounds.
Aldehyde 28 was purchased from Alfa Aesar. Combretastatins A-1
(1)17 and A-4 (2)42 were synthesized as previously reported and
used as positive controls for all biological evaluations.
Figure 2. Compounds 5–11, CA-1 (1) and CA-4 (2) occupy the same Cartesian space
as colchicine (teal); -tubulin (dark gray); b-tubulin (light gray).
a
4.2. Syntheses of aryl azides
4.2.1. 1-Azido-4-methoxy-2,3-bis(methoxymethoxy)benzene (15)
n-BuLi (1.6 M, 3.3 mL, 5.12 mmol) was added dropwise to a solu-
tion of 1-bromo-4-methoxy-2,3-bis(methoxymethoxy)-benzene
(14) (0.86 g, 2.80 mmol) in dry THF (35 mL) at À78 °C under argon,
and the mixture was stirred at À78 °C for 0.5 h. A solution of tosy-
lazide (1.174 g, 5.95 mmol) in dry THF (20 mL) was added dropwise
at À78 °C, and the mixture was stirred at À78 °C for 1 h, followed by
stirring at room temperature for 14 h. Water (50 mL) was added,
followed by extraction with dichloromethane (3 Â 50 mL). The
combined organic layers were dried over anhydrous magnesium
sulfate and the solvent removed in vacuo affording a brown oil.
The crude product was partially purified by chromatography (hex-
ane/EtOAc 1:2, Rf = 0.64) affording a red oil of 15 (64%). 1H NMR
(300 MHz, CDCl3): d = 7.04 (d, J = 8.0 Hz, 1H), 6.72 (d, J = 8.0 Hz,
1H), 5.19 (s, 2H), 5.14 (s, 2H), 3.86 (s, 3H), 3.57 (s, 3H), 3.55 (s,
3H). 13C NMR (75 MHz, CDCl3): d = 155.08, 149.82, 138.72, 125.03,
122.23, 107.89, 99.98, 98.74, 57.88, 57.54, 56.11.
hydrogen bond acceptor of the thiol proton of Cys b241. The amide
side chain of Asn b258 can be seen as coplanar to the aromatic B-
ring that could indicate aliphatic interaction between the two. The
triazole moiety was observed in a hydrophobic pocket consisting of
the following residues; Leu b248, Ala b250, Lys b254, Leu b55 and
Asn b258 as reported earlier.14a,37
3. Conclusion
The 1,5-disubstiuted 1,2,3-triazole analogs 6, 8 and 9 of com-
bretastatin A-1 (1) exhibited cytotoxic effects against several can-
cer cell lines in the low micromolar range. In addition, these
triazoles exhibited modest inhibition of tubulin assembly and
anti-angiogenetic effects with IC50-values in the low micromolar
range. Based on the data from our biological evaluations, triazoles
6, 8 and 9, most likely effect their mode of actions, at least in part,
due to binding to the colchicine binding site of a,b-tubulin. This is
the same mode of action as the lead compounds 1 and 2. The initial
molecular modeling studies also support these observations. The
ability to induce vascular disrupting effects is one of the most
interesting biological features of the combretastatins A-1 (1) and
A-4 (2) as potential new remedies against cancer. The triazoles re-
ported herein are currently undergoing biological evaluations as
VDAs. These studies will be reported in due time.
4.3. 4-(Methoxymethoxy)-2,2-dimethylbenzo[d][1,3]-dioxole-5-
carbaldehyde (22b)
The benzaldehyde 21b (550 mg, 2.84 mmol) was dissolved in dry
dichloromethane under argon and cooled to 0 °C. Diisopropylethyl-
amine (1.50 ml, 8.50 mmol) was added dropwise. Chloromethyl

Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
239
methyl ether (0.43 ml, 5.68 mmol) was added dropwise. The reac-
tion was stirred at 0 °C for 2 h, and then warmed to room tempera-
ture over night. Brine (30 mL) was added and the aqueous layer was
extracted with dichloromethane (30 mL). The combined organic lay-
ers were washed with acetic acid (2 Â 15 mL, 10%), satd NaHCO3
(15 ml) and brine (30 mL), dried over anhydrous magnesium sulfate
and the solvent was removed in vacuo. The compound was used
without further purification. Yellow solid (88%); mp 45–47 °C. 1H
NMR (200 MHz, CDCl3) d = 10.25 (d, J = 0.7 Hz, 1H), 7.44 (d,
J = 8.3 Hz, 1H), 6.58 (dd, J = 8.3, 0.7 Hz, 1H), 5.34 (s, 2H), 3.53 (s,
3H), 1.72 (s, 7H). 13C NMR (75 MHz, CDCl3) d = 188.11, 154.22,
142.31, 137.05, 124.18, 123.44, 120.09, 104.28, 97.20, 57.33, 25.82.
HRMS calcd. for C12H14O5 (MÅ+): 238.0841. Found 238.0844.
dried over anhydrous magnesium sulfate. After filtration and evap-
oration of the solvent in vacuo the desired alkyne was obtained.
4.5.1. 1-Ethynyl-2,3-dinitro-4-methoxybenzene (27)
The product was purified by chromatography (heptane/EtOAc
3:7, Rf = 0.42) affording a yellow solid (75%); mp 162–163 °C. 1H
NMR (300 MHz, d6-acetone): d = 7.96 (d, J = 9.0 Hz, 1H), 7.71 (d,
J = 9.0 Hz, 1H), 4.13 (s, 4H). 13C NMR (75 MHz, d6-acetone):
d = 206.23, 153.16, 138.45, 118.55, 109.22, 86.55, 76.07, 58.50.
HRMS calcd. for C9H6N2O5 (MÅ+): 222.0277. Found 222.0280.
4.5.2. 1-Ethynyl-2,3-difluoro-4-methoxybenzene (29)
Colorless crystals that sublimated under reduced pressure in
>85% yield; (heptane/EtOAc 1:1, Rf = 0.54); mp. 71–72 °C. 1H
NMR (300 MHz, CDCl3): d = 7.17 (ddd, J = 9.4, 7.3, 2.4 Hz, 1H),
6.73–6.64 (m, 1H), 3.91 (s, 3H), 3.25 (s, 1H). 13C NMR (75 MHz,
CDCl3): d = 152.17 (dd, J = 252.5, 11.2 Hz), 149.95 (dd, J = 8.0,
3.3 Hz), 140.95 (dd, J = 248.3, 13.8 Hz), 127.79 (dd, J = 4.4, 1.8 Hz),
107.94 (dd, J = 3.4, 1.3 Hz), 104.34 (d, J = 13.0 Hz), 81.78 (d,
J = 3.4 Hz), 75.99 (dd, J = 3.9, 0.8 Hz), 56.56 (s). HRMS calcd. for
C9H6F2O (MÅ+): 168.0387. Found 168.0387.
4.4. Syntheses of alkynes using the Colvin rearrangement:
general procedure
Trimethylsilyldiazomethane (3.2 mL, 6.40 mmol, 2.0 M hexane
solution) was added dropwise to a solution of lithium diisopropyl-
amide (3.9 mL, 7.02 mmol, 1.8 M in heptane/THF/ethylbenzene) at
À78 °C under argon, and the mixture was stirred at À78 °C for 1 h.
A solution of the corresponding benzaldehyde (5.11 mmol) in dry
THF (10.2 mL) was added dropwise at À78 °C. The mixture was
stirred at À78 °C for 2 h, then at room temperature for 4 h. The
reaction was quenched with brine (5 mL), and the mixture ex-
tracted with EtOAc (3 Â 15 mL). The combined organic layers were
washed with brine (2 Â 15 mL), dried over anhydrous magnesium
sulfate and the solvent was removed in vacuo.
4.6. Syntheses of 1,5-disubstituted 1,2,3-triazoles using
magnesium acetylides: general procedure
The corresponding terminal alkyne (1.1 mmol) dissolved in dry
THF (1.5 mL) was added dropwise to an oven dried flask containing
a solution of EtMgCl in dry THF (0.5 mL, 2.0 M, 1.0 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 (1.0 mmol) dissolved in dry THF
(1.5 mL) was added dropwise. The reaction mixture was heated
to 50 °C for 3 h. After quenching with aqueous NH4Cl (6 mL), the
product was extracted with dichloromethane (3 Â 50 mL). The
combined organic layers were washed with aqueous NH4Cl
(2 Â 50 mL), dried over anhydrous magnesium sulfate and the sol-
vent was removed in vacuo.
4.4.1. 1-Ethynyl-4-methoxy-2,3-bis(methoxymethoxy)-benzene
(19)
The product was purified by chromatography (hexane/EtOAc
2:1, Rf = 0.56) affording a yellow oil (68%). 1H NMR (300 MHz,
CDCl3): d = 7.20 (d, J = 8.7 Hz, 1H), 6.64 (d, J = 8.7 Hz, 1H), 5.27 (s,
2H), 5.11 (s, 2H), 3.85 (s, 3H), 3.63 (s, 3H), 3.59 (s, 3H), 3.18 (s,
1H). 13C NMR (75 MHz, CDCl3): d = 154.81. 152.58, 138.99,
129.41, 109.96, 107.88, 99.35, 98.80, 80.26, 80.00, 57.84, 57.54,
56.14. HRMS calcd. for C13H16O5 (MÅ+): 252.0998. Found 252.0998.
4.6.1. 1-(4-Methoxy-2,3-bis(methoxymethoxy)phenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,3-triazole (31)
4.4.2. 5-Ethynyl-4-(methoxymethoxy)benzo[d][1,3]dioxole (23a)
The product was purified by chromatography (hexane/EtOAc
6:4, Rf = 0.62) affording a pale yellow solid (57%); mp 35–37 °C.
1H NMR (200 MHz, CDCl3) d = 7.01 (d, J = 8.1 Hz, 1H), 6.54 (d,
J = 8.1 Hz, 1H), 5.98 (s, 2H), 5.33 (s, 2H), 3.58 (s, 3H), 3.17 (s,
1H).13C NMR (50 MHz, CDCl3) d = 149.86, 141.14, 137.66, 127.90,
109.61, 103.93, 101.61, 96.82, 79.83, 79.51, 57.01. HRMS calcd.
for C11H10O4 (MÅ+): 206.0579. Found 206.0588.
The product was purified by chromatography (hexane/EtOAc
1:2, Rf = 0.30) affording an orange semisolid (14%). 1H NMR
(300 MHz, CDCl3): d = 7.83 (s, 1H), 6.96 (d, J = 8.9 Hz, 1H), 6.73 (d,
J = 9.0 Hz, 1H), 6.48 (s, 2H), 5.07 (s, 2H), 4.96 (s, 2H), 3.87 (s, 3H),
3.80 (s, 3H), 3.65 (s, 6H), 3.44 (s, 3H), 2.99 (s, 3H). 13C NMR
(75 MHz, CDCl3): d = 155.30, 153.47, 146.96, 139.73, 139.61,
138.73, 131.88, 124.77, 123.39, 122.29, 107.63, 105.38, 99.62,
98.74, 61.03, 57.55, 56.90, 56.44, 56.17. HRMS calcd. for
4.4.3. 5-Ethynyl-4-(methoxymethoxy)-2,2-dimethylbenzo-[d]-
[1,3]dioxole (23b)
C
22H27N3O8 (MÅ+): 461.1798. Found 461.1782.
The crude product was purified by chromatography (hexane/
EtOAc 8:2, Rf = 0.43) affording a pale yellow solid (59%); mp 67–
68 °C. 1H NMR (300 MHz, CDCl3) d = 6.96 (d, J = 8.1 Hz, 1H), 6.46
(d, J = 8.1 Hz, 1H), 5.29 (s, 2H), 3.58 (s, 3H), 3.15 (s, 1H), 1.68 (s,
7H). 13C NMR (75 MHz, CDCl3) d = 149.63, 141.03, 138.00, 127.21,
119.21, 109.10, 104.00, 96.94, 80.17, 79.19, 57.01, 25.74. HRMS
calcd. for C13H14O4 (MÅ+): 234.0892. Found 234.0892.
4.6.2. 5-(4-Methoxy-2,3-bis(methoxymethoxy)phenyl)-1-(3,4,5-
trimethoxyphenyl)-1H-1,2,3-triazole (33)
The product was purified by chromatography (hexane/EtOAc
1:2, Rf = 0.42) affording an orange semisolid (59%). 1H NMR
(300 MHz, DMSO-d6): d = 7.94 (s, 1H), 6.97 (d, J = 8.7 Hz, 1H),
6.93 (d, J = 8.8 Hz, 1H) 6.67 (s, 2H), 4.98 (s, 2H), 4.89 (s, 2H), 3.81
(s, 3H), 3.67 (s, 3H), 3.62 (s, 6H), 3.35 (s, 3H), 2.91 (s, 3H). 13C
NMR (75 MHz, DMSO-d6): d = 154.57, 152.81, 148.59, 138.43,
137.36, 134.27, 134.24, 132.23, 125.85, 114.49, 108.67, 101.96,
98.79, 97.74, 60.08, 56.52, 56.11, 55.85. HRMS calcd. for
4.5. Syntheses of alkynes using the Ohira–Bestmann reaction:
general procedure
C
22H27N3O8 (MÅ+): 461.1798. Found 461.1800.
Dimethyl-1-diazo-2-oxopropylphosphonate
(280 mg,
1.44 mmol) was added to a suspension of the aldehyde (1.2 mmol)
and K2CO3 (336 mg, 2.4 mmol) in MeOH (15 mL). Stirring was con-
tinued for 1.5 h. Then the reaction mixture was diluted with Et2O
(25 mL), washed with an aq solution of NaHCO3 (10 mL, 5%) and
4.6.3. 5-(2,3-Difluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (9)
The crude product was purified by chromatography (hexane/
EtOAc 1:2, Rf = 0.31) affording a pale yellow solid (89%); mp

240
Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
107–108 °C. 1H NMR (300 MHz, DMSO-d6): d = 8.10 (d, J = 1.2 Hz,
1H), 7.14 (d, J = 8.6 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 6.75 (s, 2H),
3.90 (s, 3H), 3.71 (s, 3H), 3.68 (s, 6H). 13C NMR (75 MHz, DMSO-
d6): d = 153.05, 149.64 (dd, J = 7.5, 3.2 Hz), 147.72 (dd, J = 248.6,
11.2 Hz), 140.07 (dd, J = 246.2, 14.3 Hz), 138.00, 134.29 (d,
J = 2.4 Hz), 131.61, 130.80 (d, J = 2.6 Hz), 125.47 (dd, J = 4.0,
2.6 Hz), 109.35 (d, J = 2.3 Hz), 107.94 (d, J = 11.9 Hz), 102.94,
4.8. Reduction of nitro groups: general procedure
The triazole (0.08 mmol) was suspended in MeOH/H2O (10 mL,
2:1) and saturated CuSO4Á5H2O solution (200
lL) was added and
stirred at ambient temperature. NaBH4 was added (15 mg,
0.40 mmol) and an exothermic reaction occurred. The reaction
mixture turned black. Additional portions of NaBH4 were added
until the color of the solution changed from brown to black/green.
RP-18 TLC (CH3CN/H2O, 6:4) and normal phase TLC analysis using a
(EtOAc) was used to monitor the reaction. When complete conver-
sion of the starting material was observed, the reaction was diluted
with HCl (10 mL, 0.5 M) and EtOAc (5 mL). The organic phase was
extracted a second time with HCl (5 mL, 0.5 M). The combined
aqueous extracts was made basic with NaOH (1 M) and extracted
with EtOAc (4 Â 10 mL). The combined EtOAc fractions were
washed with aqueous ammonia (2 Â 5 mL, 5%) before dried over
anhydrous magnesium sulfate and then concentrated in vacuo.
60.18, 56.78, 56.18. HRMS calcd. for
377.1187. Found 377.1188.
C
18H17F2N3O4 (MÅ+):
4.6.4. 5-(4-(Methoxymethoxy)benzo[d][1,3]dioxol-5-yl)-1-
(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (34a)
The product was purified by chromatography (hexane/EtOAc
1:2, Rf = 0.30) affording an orange solid (88%); mp 86–88 °C. 1H
NMR (200 MHz, CDCl3) d = 7.80 (s, 1H), 6.66 (s, 2H), 6.61 (d,
J = 2.8 Hz, 2H), 6.01 (s, 2H), 5.04 (s, 2H), 3.85 (s, 3H), 3.72 (s, 6H),
3.13 (s, 3H). 13C NMR (75 MHz, CDCl3) d = 153.25, 150.52, 138.22,
138.05, 137.59, 134.81, 133.81, 132.47, 124.53, 114.50, 103.98,
101.73, 101.69, 96.91, 60.92, 56.77, 56.11. HRMS calcd. for
4.8.1. 3-Methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-5-yl)benzene-1,2-diamine (8)
C
20H21N3O7 (MÅ+): 415.1380. Found 415.1367.
The product was purified by chromatography (CH2Cl2/EtOAc,
2:8–1:9) affording a brown solid (28%); mp 69–70. Rf = 0.58
(CH3CN/H2O 6:4 °C). 1H NMR (300 MHz, acetonitrile-d3): d = 7.74
(s, 1H), 6.75 (s, 2H), 6.44 (d, J = 8.5 Hz, 1H), 6.38 (d, J = 8.5 Hz,
1H), 3.80 (s, 3H), 3.73 (br s, 4 H, 2 Â NH2), 3.71 (s, 3H), 3.66 (s,
6H). 13C NMR (75 MHz, acetonitrile-d3): d = 154.25, 149.71,
138.96, 136.57, 135.57, 135.10, 133.48, 124.34, 121.42, 107.15,
103.09, 102.54, 60.94, 56.80, 56.44. HRMS calcd. for C18H21N5O4
(MÅ+): 371.1594. Found 371.1586.
4.6.5. 5-(4-(Methoxymethoxy)-2,2-dimethylbenzo[d][1,3]-
dioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (34b)
The product was purified by chromatography (hexane/EtOAc
6:4, Rf = 0.23) affording an orange solid (90%); mp 122–124 °C. 1H
NMR (300 MHz, CDCl3) d = 7.79 (s, 1H), 6.67 (s, 2H), 6.54 (d,
J = 8.1 Hz, 1H), 6.48 (d, J = 8.1 Hz, 1H), 5.00 (s, 2H), 3.83 (s, 3H),
3.70 (s, 6H), 3.08 (s, 3H), 1.68 (s, 6H). 13C NMR (75 MHz, CDCl3)
d = 153.19, 150.20, 138.12, 137.94, 137.92, 134.86, 134.00,
132.52, 123.78, 119.46, 114.19, 104.13, 101.60, 97.02, 60.91,
56.67, 56.02, 25.67. HRMS calcd. for C22H25N3O7 (MÅ+): 443.1693.
Found 443.1683.
4.8.2. 3-Methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-4-yl)benzene-1,2-diamine (13)
The product was purified by chromatography (Rf = 0.50, EtOAc)
affording a brown oil (19%). 1H NMR (300 MHz, acetonitrile-d3):
d = 8.50 (s, 1H), 7.16 (s, 2H), 7.03 (d, J = 8.6 Hz, 1H), 6.48 (d,
J = 8.6 Hz, 1H), 5.31 (s, 2H, NH2), 3.92 (s, 6H), 3.85 (s, 3H), 3.79 (s,
3H), 3.67 (s, 2H, NH2). 13C NMR (75 MHz, acetonitrile-d3)
d = 155.02, 149.95, 148.69, 139.07, 135.58, 133.98, 124.54,
119.98, 118.68, 109.15, 102.52, 100.99, 99.41, 61.05, 57.13, 56.45.
HRMS calcd. for C18H21N5O4 (MÅ+): 371.1594. Found 371.1590.
4.7. Thermal [3+2] Huisgen cycloaddition reaction
Alkyne 27 (100 mg, 0.42 mmol) and azide 32 (92 mg,
0.42 mmol) was dissolved in toluene (2.5 mL) and H2O (2.5 mL)
was added. The reaction mixture was heated to reflux for 3 days.
Additional amounts of toluene (2.5 mL each time, total of four
times) was added when it had evaporated from the reaction mix-
ture. The solvent was removed in vacuo and the products were dis-
solved in THF and absorbed on celite for purification by flash
chromatography (CH2Cl2/EtOAc, 9:1–1:1).
4.9. Deprotection of methoxymethyl groups: general procedure
The corresponding MOM-protected triazole (0.24 mmol) was
dissolved in 3 M HCl/THF (1:1) (10 mL), and the mixture was stir-
red at room temperature or heated to reflux (compounds 10 and
11) for 5 h. Water (20 mL) was added, and the reaction mixture
was extracted with dichloromethane (3 Â 50 mL). The combined
organic layers were dried over anhydrous sodium sulfate and the
solvent removed in vacuo.
4.7.1. 5-(4-Methoxy-2,3-dinitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (7)
Chromatography (CH2Cl2/EtOAc 8:2, Rf = 0.34) affording a or-
ange solid (43%); mp 162–163 °C. 1H NMR (300 MHz, CDCl3):
d = 7.75 (s, 1H), 7.35 (d, J = 8.9 Hz, 1H), 7.25 (d, J = 8.9 Hz, 1H),
6.55 (s, 2H), 3.99 (s, 3H), 3.81 (s, 3H), 3.72 (s, 6H). 13C NMR
(75 MHz, CDCl3): d = 153.59, 152.71, 143.08, 138.88, 134.66,
134.27, 134.08, 130.60, 130.54, 116.18, 113.46, 102.46, 77.00,
60.85, 57.52, 56.21. HRMS calcd. for C18H17N5O8 (MÅ+): 431.1077.
Found 431.1064.
4.9.1. 3-Methoxy-6-(5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-1-yl)benzene-1,2-diol (5)
The product was purified by recrystallization in hexane/diethyl
ether (1:1) affording a yellow solid (86%); mp 127–129 °C. 1H NMR
(300 MHz, DMSO-d6): d = 9.32 (s, OH), 9.08 (s, OH), 8.12 (s, 1H),
6.79 (d, J = 8.8 Hz, 1H), 6.65 (s, 2H), 6.63 (d, J = 8.9 Hz, 1H), 3.84
(s, 3H), 3.64 (s, 3H), 3.61 (s, 6H). 13C NMR (75 MHz, DMSO-d6):
d = 152.75, 149.71, 142.55, 138.52, 137.72, 134.69, 131.52,
122.10, 118.79, 118.27, 104.80, 102.69, 60.01, 56.07, 55.65. HRMS
calcd. for C18H19N3O6 (MÅ+): 373.1274. Found 373.1276.
4.7.2. 4-(4-Methoxy-2,3-dinitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)-1H-1,2,3-triazole (12)
Chromatography (CH2Cl2/EtOAc 8:2, Rf = 0.54) affording a white
solid (45%); mp 221–222 °C. 1H NMR (300 MHz, DMSO-d6):
d = 9.31 (s, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 9.2 Hz, 1H),
7.25 (s, 2H), 4.08 (s, 3H), 3.90 (s, 6H), 3.73 (s, 3H). 13C NMR
(75 MHz, DMSO-d6): d = 153.56, 151.32, 140.92, 140.43, 137.74,
133.76, 133.33, 132.04, 122.49, 118.22, 115.46, 98.41, 80.02,
80.00, 60.23, 58.11, 56.33. HRMS calcd. for C18H17N5O8 (MÅ+):
431.1077. Found 431.1066.
4.9.2. 3-Methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-5-yl)benzene-1,2-diol (6)
The product was purified by recrystallization in hexane/diethyl
ether (1:1) affording a pale beige solid (77%); mp 214–216 °C. 1H

Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
241
NMR (300 MHz, DMSO-d6): d = 8.97 (s, OH), 8.82 (s, OH), 7.83 (s,
1H), 6.72 (s, 2H), 6.57 (d, J = 8.6 Hz, 1H), 6.52 (d, J = 8.6 Hz, 1H),
3.78 (s, 3H), 3.68 (s, 3H), 3.65 (s, 6H). 13C NMR (75 MHz, DMSO-
d6): d = 152.74, 149.49, 144.64, 137.33, 135.02, 134.01, 133.92,
132.52, 120.53, 107.74, 103.21, 101.91, 60.11, 55.92, 55.84. HRMS
calcd. for C18H19N3O6 (MÅ+): 373.1274. Found 373.1269.
wells were incubated for 30 min with ECs (30,000 cells/well). After
adhesion of the cells, the medium was removed and replaced by
fresh medium supplemented with triazole analogs with five differ-
ent concentrations ranging from 10 lM to 0.001 lM and incubated
at 37 °C for 18 h. The tubes of growth were visualized with an in-
verted ZEISS microscope at a magnification of 10. The length of
the capillary network was quantified with a map scale calculator
(KURABO Angiogenesis Image Analysis Software).
4.9.3. 5-(1-(3,4,5-Trimethoxyphenyl)-1H-1,2,3-triazol-5-
yl)benzo[d][1,3]dioxol-4-ol (10)
The product was purified by chromatography (hexane/EtOAc
1:2, Rf = 0.33) affording a white solid (54%); mp 216–217 °C. 1H
NMR (300 MHz, methanol-d4) d = 7.91 (s, 1H), 7.82 (s, 2H), 6.74
(s, 3H), 6.71 (d, J = 8.1 Hz, 2H), 6.48 (d, J = 8.1 Hz, 2H), 5.97 (s,
3H), 3.79 (s, 5H), 3.73 (s, 10H). 13C NMR (75 MHz, methanol-d4)
d = 154.81, 151.79, 140.86, 139.68, 136.79, 136.54, 135.26,
134.29, 125.70, 111.52, 103.40, 103.14, 102.01, 61.33, 56.85. HRMS
calcd. for C18H17N3O6 (MÅ+): 371.1117. Found 371.1123.
4.11.3. Inhibition of Tubulin Assembly
The method applied was that described by Lawrence et al.34
Tubulin was isolated from porcine brain and stored at À78 °C. Sam-
ples 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
tilled water, pH 6.6)), GTP (20
(50 l, 11 mg/ml in Mes buffer) and the candidate drug (10
l
l (0.1 M Mes, 1 mM EGTA, 0.5 mM MgCl2, dis-
l, 5 mM in Mes buffer), tubulin
l,
l
l
l
Csample in DMSO). The tubulin/drug samples were immediately
placed in a 96-well plate reader, alongside blank samples contain-
ing Mes buffer (198 ll) and the candidate drug (10 ll, same con-
4.9.4. 2,2-Dimethyl-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-5-yl)benzo[d][1,3]dioxol-4-ol (11)
The product was purified by chromatography (hexane/EtOAc
1:2, Rf = 0.40) affording a white solid (65%); mp 204–206 °C. 1H
NMR (300 MHz, CDCl3) d = 7.69 (s, 1H), 6.65 (s, 2H), 6.53 (d,
J = 8.1 Hz, 1H), 6.37 (d, J = 8.1 Hz, 1H), 3.81 (s, 3H), 3.68 (s, 6H),
1.68 (s, 6H).13C NMR (75 MHz, CDCl3) d = 153.15, 149.75, 138.61,
138.00, 135.35, 134.13, 134.05, 132.40, 123.48, 119.68, 109.05,
101.79, 101.76, 60.97, 56.06, 25.82. HRMS calcd. for C20H21N3O6
(MÅ+): 399.1430. Found 399.1431.
centration). The absorbance (k 350 nm) was recorded at ambient
temperature for a period of 60 min, and the results were compared
to untreated controls to evaluate the relative degree of change in
optical 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.
4.12. Molecular modeling
4.10. Synthesis of 4-(4-methoxy-2,3-dinitrophenyl)-1-(3,4,5-
trimethoxyphenyl)-1H-1,2,3-triazole (12) using CuSO4 and
sodium ascorbate
4.12.1. General
The compounds were drawn in Maestro (v. 9.8) and with the
Macromodel package the structures were energy minimized using
the OPLS2005 force field. All molecular modeling calculations were
performed using the software Glide (v. 5.6) running on Linux
x86_64 workstation. The conformation of colchicine was taken
from the tubulin-ligand complex filed in the Brookhaven Protein
Data Bank (pdb entry code 1SA0). The protein complex was pre-
pared for docking with Maestro/Macromodel (v. 9.1.207) Protein
Preparation Wizard where bond orders were assigned, hydrogens
added, water molecules removed, hydrogen bond optimized and fi-
nally the protein energy minimized (converge threshold 0.30 kcal/
mol). The native ligand was redocked into the processed protein to
verify that the program in use could identify the correct binding
mode of the ligand. Figure 2 was prepared with Pymol (v. 1.3) in
Windows 7.
To a suspension of 1-ethynyl-4-methoxy-2,3-dinitrobenzene
(27) (22 mg, 0.1 mmol) and 5-azido-1,2,3-trimethoxybenzene
(32) (21 mg, 0.1 mmol) in t-BuOH (1 mL), a solution of CuSO4Á5H2O
(25 mg, 0.1 mmol in 0.5 mL H2O) and sodium ascorbate (10 mg,
0.05 mmol) were added. The reaction mixture was stirred over-
night at 45 °C where after the reaction was complete as observed
by TLC and H2O (10 mL) was added. The aqueous phase was ex-
tracted with EtOAc (3 Â 5 mL) and the combined organic phases
was washed with aqueous ammonia (2 Â 5 mL, 5%) and brine be-
fore dried with MgSO4 and concentrated in vacuo. Purified by flash
chromatography (CH2Cl2/EtOAc 8:2, Rf = 0.54) affording a white so-
lid (67%). Physical and spectral data (1H- and 13C-NMR, HRMS)
were in accordance for structure of 12 with those data obtained
by the thermal Huisgen cycloaddition.
4.12.2. Docking with Glide
4.11. Biological Assays
Standard parameters were used for the with Glide grid genera-
tion. A mesh of 0.375 Å and 56 Â 60 Â 50 number of points were
used for the grid size. The grid was centered on the mass center
of the DAMA-colchicine coordinates. Docking in Glide was per-
formed with standard parameters in standard precision mode.
Ten poses per ligand were generated. As a standard of comparison
both CA-1 (1) and CA-4 (2) were docked together with the set of
analogues 5–13.
4.11.1. Cancer Cell Growth Inhibition
To assess cell viability, the alamarBlueÒ (AB) assay (dye pur-
chased from Biosource International, Nivelles, Belgium) was used
as previously described.31,32 This involved aspirating medium at
the end of each treatment period and adding 100 ll of fresh med-
ium containing 10% v/v AB to control and treated wells. Plates were
incubated at 37 °C for six hours prior to measuring the absorbance
at 540 nm and at 595 nm wavelengths using a spectrophotometric
plate reader (DYNEX Technologies, USA). Experimental data were
normalized to control values.
4.12.3. Energy minimization
The result complexes from the Glide docking were energy min-
imized with Macromodel after the reinsertion of GDP and GTP that
were removed with the protein preparation wizard. Minimization
was performed with Maestro/Macromodel using OPLS2005 as force
field in implicit water phase. Minimization method was PRCG with
maximum iterations 30000, converge on gradient and convergence
threshold 0.01.
4.11.2. Inhibition of Angiogenesis
Endothelial cell tube formation assay was modified from a
method previously described.33 Matrigel (12.5 mg/ml) was thawed
at 4 °C, and 50 Al were quickly added to each well of a 96-well
plate and allowed to solidify for 10 min at 37 °C. Once solid, the

242
Ø. W. Akselsen et al. / Bioorg. Med. Chem. 20 (2012) 234–242
Sun, C.-M.; Lin, L.-G.; Yu, H.-J.; Cheng, C.-Y.; Tsai, Y.-C.; Chu, C.-W.; Din, Y.-H.;
Acknowledgments
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. Russ. Chem.
Bull. 2007, 56, 2460; (f) Xue, N.; Yang, X.; Wu, R.; Chen, J.; He, Q.; Yang, B.; Lu,
X.; Hu, Y. Bioorg. Med. Chem. 2008, 16, 2550.
Financial support to Ø.W.A. from the School of Pharmacy,
University of Oslo; Norsk Farmasøytisk Selskap; MolecularLife-
Sciences@UiO; Ella og Robert Wenzins legat and COST CM0602-
Angiokem are all gratefully appreciated.
14. (a) Odlo, K.; Hentzen, J.; Fournier dit Chabert, J.; Ducki, S.; Gani, O. A. B. S. M.;
Sylte, I.; Skrede, M.; Flørenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16,
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