Synthesis and biological evaluation of novel anticancer bivalent colchicine-tubulizine hybrids

Article abstract of DOI:10.1016/j.bmc.2012.05.072

A series of novel antimitotic hybrids were synthesized in good yields by linking of azide-containing colchicine congeners with acetylene-substituted tubulizine-type derivatives using copper-mediated 1,3-dipolar cycloaddition. Obtained compounds exhibit go

Full text of DOI:10.1016/j.bmc.2012.05.072

Bioorganic & Medicinal Chemistry 20 (2012) 4271–4278
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel anticancer bivalent
colchicine–tubulizine hybrids
Yulia B. Malysheva a, Sebastien Combes b, Diane Allegro c, Vincent Peyrot c, Paul Knochel d,
Andrei E. Gavryushin d, Alexey Yu. Fedorov a,
⇑
a Department of Organic Chemistry, Lobachevsky State University of Nizhni Novgorod, 23 Gagarin Avenue, 603950 Nizhni Novgorod, Russian Federation
b UMR-CNRS 6264, Université d’Aix-Marseille, Faculté des Sciences Saint-Jérôme, case 521, 13397 Marseille Cedex 20, France
c Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, CRO2 INSERM UMR 911, Universit_e d’Aix-Marseille, Facult_e de Pharmacie,
27 Boulevard Jean Moulin, Marseille 13005, France
d Department Chemie, Ludwig Maximilians Universität München, Butenandtstrasse 5-13, Haus F, 81377 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel antimitotic hybrids were synthesized in good yields by linking of azide-containing
colchicine congeners with acetylene-substituted tubulizine-type derivatives using copper-mediated
1,3-dipolar cycloaddition. Obtained compounds exhibit good cytotoxicity against HBL100 epithelial cell
Received 15 February 2012
Revised 15 May 2012
Accepted 25 May 2012
Available online 6 June 2012
lines (IC50 = 0.599–2.93 lM). Several newly synthesized compounds are the substoichiometric inhibitors
of microtubule assembly (R = 0.41–0.78). The results highlight the importance of the length of spacer
linking the tubulin binding ligands in heterodimeric molecules.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hybrid
Colchicine
Tubulizine
Click chemistry
Cytotoxicity
Tubulin
1. Introduction
individual ligands bind multiple receptors simultaneously to pro-
duce a greater biological effect.4 This can be exemplified by the
Microtubules, formed by tubulin molecules, are essential com-
ponents of the cytoskeleton in eukaryotic cells and are involved
in many important cellular processes including mitosis. As compo-
nents of the mitotic spindle, microtubules have emerged as a stra-
tegic target in anticancer therapy.1 Depending on the molecular
structure of tubulin poison (Fig. 1) it is able to act with different
binding sites on tubulin: with the vinca, taxane, colchicine and
tubulizine domains.2 Despite existence of the broad range of anti-
mitotic agents, only few examples reached so far clinical and com-
mercial success.2 The failure of the plurality of these molecules
could be attributed to poor therapeutic indexes—the balance be-
tween efficiency and toxicity, perhaps related to pharmacokinetics,
to the solubility problems, low affinity of therapeutic agents to
tubulin molecules in vivo trials and other unrecognized factors.1a
One way to improve some of these pharmacological parameters
is to construct therapeutic molecules in accordance with the con-
cept of multivalency.3
attachment of viruses and bacteria to cellular surfaces, by binding
of a cell to another cell, or by interactions of antibodies with patho-
gens.5 Several natural compounds possessing multiple binding
sites are represented by the homo- or heterodimers of bis-indole
alkaloid vincristine,6 bis-steroidal pyrazine cephalostatine,7 di-
meric sesquiterpene absinthine8 or bifunctional duocarmycin
derivatives.9 In fact, polyvalent interactions are normally much
stronger than the corresponding monovalent contacts. They can
provide the basis for the drug-receptor interactions, which are fun-
damentally different from those available in monovalent systems.3
The idea of combining different monovalent agents into biva-
lent hybrids was used for the construction of several types of novel
antimitotic agents like taxoid-colchicine4c (thiocolchicine10) or
vinca alkaloids-taxoid11 hybrids and podophyllotoxin-thiocolchi-
cine12 dynamic combinatorial library. Some of obtained dimeric
agents, however, were shown to be even less active in comparison
with their monomeric precursors. This can be explained by the use
of rather rigid and short linkers between the individual ligands,
making simultaneous binding of both ligands to tubulin
impossible.
This theory is based on the observation that in nature various
biological interactions are mediated multivalently, so that
We report herein the synthesis of new antimitotic bivalent li-
gands, connected by flexible spacers, prepared by linking of
⇑
Corresponding author. Tel.: +7 831 462 3232; fax: +7 831 465 8592.
E-mail address: afnn@rambler.ru (A.Yu. Fedorov).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.072

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Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
NHCOMe
NHR
N
N
O
N
H
N
N
H
MeO
OMe
OMe
MeO
O
OMe
MeO
i
Tubulizine A, R = -Pr
Tubulizine B, R = cyclo-C6H11
Colchicine
OH
Et
N
N
R2
O
OH
R1
NH
O
N
H
N
Ph
O
MeO2C
OH
O
MeO
H
Et
HO
OAc
H
OBz
OAc
CO2Me
Taxol, R1 = Ph, R2 = OAc
H
Me
OH
Taxotere, R1 = t-BuO, R2 = OH
Figure 1. Structure of some compounds with high affinity for different tubulin binding sites.
in the NH-alkyl fragment17 can be done without a significant loss of
antitumor activity. Therefore, a range of acylated deacetylcolchi-
cines 4a–c (Scheme 2) and tubulizines 9a–e (Scheme 3) were
proposed as building blocks for the heterodimers construction.
tubulizine site
ligand
colchicine site
ligand
N3
+
click-chemistry
2. Results and discussion
N
N
colchicine site
ligand
N
2.1. Synthesis of colchicine–tubulyzine heterodimers
tubulizine site
ligand
The colchicine site ligands 4a–c, bearing linkers of various
lengths, were synthesized from commercially available colchicine
1 in several steps (Scheme 2). The one-pot deacetylation of
colchicine18 afforded deacetylcolchicine 2 in 94% overall yield. It
Scheme 1. Synthesis of colchicine–tubulizine hybrids.
azide-containing deacetylcolchicine and acetylene-substituted
tubulizine entities using copper-mediated13 1,3-dipolar cycloaddi-
tion (Scheme 1). The colchicine binding site includes 239Cysß of
ß-tubulin,1b,14 while tubulizine interacts with 12Cysß of the same
protein molecule.2c 12Cysß is known to be a part of the exchange-
able GTP-binding site.2c Techniques for measuring fluorescence
resonance energy transfer (FRET) has been used to determine the
distance between colchicine and GTP-binding sites.15 However,
no FRET was observed between ligands bound to the colchicine
and GTP sites, indicating that these binding sites are at least 40 Å
apart.15b The exact distance between colchicine and GTP-binding
sites is unknown due to long range and allosteric effect.15a Tubuli-
zines’ binding to tubulin induces conformational changes of
protein.2c Therefore, we decided to synthesize several colchicine–
tubulizine hybrids with various length of the linker, in order to find
the optimal distance between the affine units and to realize their
simultaneous binding to colchicine and tubulizine domains of the
tubulin molecule.
was subjected to the Steglich amidation18a with a row of
x
-azido-
carboxylic acids N3(CH2)nCO2H (3a–c, n = 2, 5, 9), prepared from
the corresponding -azidoa-
-bromocarboxylic acids).19 Related
x
x
mides 4a–c were obtained in good to high yields (69–95%).
Alkyne-substituted tubulizine analogs 6 and 9a–f were pre-
pared from commercially available 2,4,6-trichloro-1,3,5-triazine
(cyanuric chloride, Scheme 3). Its reaction with p-methoxybenzyl-
amine in aqueous acetone led to 2-chloro-4,6-di-(p-methoxyben-
zylamino)-1,3,5-triazine
5
in 92% yield.2c Its treatment with
propargylamine in dioxane at 100 °C gave alkynyl analog of tubu-
lizine A (6) in a good yield. Triazine 5 reacted with 2-aminoethanol
to give hydroxyl-substituted tubulizine analog 7 in 71% yield.
Esterification of 7 with alkynoic acids (8a–e, m = 2, 3, 4, 8, 15)
afforded acetylenic esters 9a–e in excellent yields (Scheme 3).
The tubuline ligands 4a–c, 6 and 9a–e, bearing correspondingly
terminal azide- and alkyne-fragments, were linked using ‘click’
[3+2] cycloaddition reaction,13 applying copper sulfate/magnesium
ascorbate catalytic system in THF-water solution (Scheme 4). This
methodology allows to synthesize exclusively 1,4-regioisomeric
triazoles 10a–g in 47–82% yields (Table 1).
Recently, it has been shown that the functionalization of the
colchicine core in the position 716 as well as the tubulizine molecule
O
MeO
MeO
H
MeO
MeO
MeO
MeO
N3
N
NH2
O
N
H
n
(d)
Ac (a), (b), (c)
MeO
MeO
MeO
O
O
OMe
1
2
4a-c
OMe
OMe
4a: n = 2 69%
4b: n = 5 70%
4c: n = 9 95%
Scheme 2. Synthesis of colchicine derivatives containing an azide group. Reagents and conditions: (a) (Boc)2O, DMAP, Et3N, CH3CN, 100 °C, 4 h; (b) MeONa, MeOH, rt, 1.5 h;
(c) TFA, CH2Cl2, rt, 2 h, 94% (yield of 3 steps); (d) N3(CH2)nCOOH (3a–c, n = 2, 5, 9), EDC, DMAP, CH2Cl2, 0 °C—1 h, rt—4 h, 69–95%.

Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
4273
OMe
OH
OMe
OMe
H2N
Cl
N
HN
N
Cl
N
N
N
(a)
(c)
N
N
N
H
+
N
N
Cl
Cl
HN
N
H
N
H
OMe
OMe
5
7
(b)
(d)
OMe
9a:
9b:
OMe
m = 2 96%
m = 3 99%
HN
HN
N
N
N
N
O
m
9c: m = 4 92%
9d: m = 8 87%
9e:
N
NH
N
H
N
O
HN
HN
m = 15 94%
OMe
OMe
9a-e
6
Scheme 3. Synthesis of tubulizine derivatives containing a terminal alkyne group. Reagents and conditions: (a) acetone, H2O, 0 °C—20 min, rt—20 h, 92%; (b) NH2CH2CBCH,
dioxane, 100 °C, 60 h, 61%; (c) NH2(CH2)2OH, dioxane, 100 °C, 60 h, 71%; (d) HCBC(CH2)mCOOH (8a–e, m = 2, 3, 4, 8, 15), DCC, DMAP, THF, rt, 24 h, 87–99%.
O
MeO
MeO
MeO
N3
NH
N
H
n
N
N
+
H
N
4a-c
N
6,9a-e
Z
MeO
NH
O
OMe
(a)
MeO
n = 2, 5, 9
Z = CH2 or (CH2)mC(O)O(CH2)2
m = 2, 3, 4, 8, 15
N
MeO
Z
N
N
O
NH
MeO
MeO
N
H
n
N
N
N
H
N
N
H
O
OMe
OMe
MeO
10a-g
Scheme 4. Synthesis of triazole-joined colchicine–tubulizine hybrids using click chemistry. Reagents and conditions: CuSO4ꢀ5H2O, 0.1 equiv, MgAsc, 0.2 equiv, THF–H2O
(1:1), rt, 24 h, 57–82%.
2.2. Evaluation of in vitro biological activity
Table 1
The yields of colchicines-tubulizine hybrids
In vitro cytotoxicity of the synthesized colchicine–tubulizine
hybrids 10a–h as well as the modified tubuline ligands 2, and 7
was investigated toward HBL100 human mammary cell line. A
tetrazolium-based assay was used for determination of the drug
concentration required to inhibit cell growth by 50% after the incu-
bation in the culture medium for 72 h. The obtained values are sum-
marized in Table 2. Furthermore, to confirm that the potential
activity of these compounds are due to interaction with tubulin
binding site, studies on their effect on microtubules assembly were
performed. As an example, Figure 2 shows the effects of 10e on the
40,6-diamidino-2-phenylindole (DAPI) fluorimetric time course of
in vitro microtubule assembly from pure tubulin. A clear inhibition
was observed, and the rate of assembly as well as the final amount of
microtubules was lower in the presence of 10e than in the control
experiment. The inset of Figure 2 show that the extent of inhibition
increased monotonically with the mole ratio of the total ligand to
total tubulin in the solution (R). The activity of microtubule assem-
bly inhibition for hybrids 10b–h decreases along with the linker
length. Very importantly, that hybrids with l = 14–20 are the substo-
ichiometric inhibitors of microtubule assembly (R = 0.41–0.71,
Table 2) with the same order of activity as diacetylcolchicine
Compound
n
m
la
Yields (%)
10a
10b
10c
10d
10e
10f
9
2
2
5
2
9
5
—
2
4
3
8
16
14
16
18
20
22
30
47
57
82
73
63
69
64
3
15
10g
a
Linker length (l) was calculated as the number of atoms between two NH-
groups inclusively.
Additionally to 10a–g triazole-linked compounds, polyethyl-
eneglycol-bridged colchicine–tubulizine heterodimer 10h with
the longest spacer, containing 24 carbon and 11 oxygen atoms,
was prepared using two-step synthetic procedure (Scheme 5). At
the first stage, tubulizine analog 7 was subjected to the Steglich
esterification with poly(ethylene glycol)-600 bis-dicarboxylic acid,
affording intermediate 11 in 55% yield. The subsequent reaction of
11 with deacetylcolchicine 2 led to PEG-bridged colchicine–tubuli-
zine dimer 10h in 37% overall yield (Scheme 5).

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Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
OMe
OH
OMe
HN
N
HN
N
O
N
N
N
N
(a)
N
H
NH(CH2)2O
O
O
OH
z
O
HN
HN
OMe
OMe
(b)
7
11
O
OMe
OMe
O
O
HN
O
N
H
z
O
N
N
OMe
O
N
H
N
N
H
MeO
OMe
z = 10
MeO
10h
Scheme 5. Synthesis of PEG-joined colchicine–tubulizine hybrid. Reagents and conditions: (a) PEG-diacid, DCC, DMAP, THF, rt, 24 h, 55%; (b) deacetylcolchicine, EDC, DMAP,
CH2Cl2, 0 °C—1 h, rt—4 h, 68%.
cytotoxic activity of the synthesized compounds depends on hydro-
phobicity (LogD) of heterodimers. The cell growth inhibiting activ-
ity increases with the rise of hydrophobicity of hybrids what can be
explained by augment of membranotropicity for these compounds.
While heterodimers 10a–h manifested relatively high cytotoxic
Table 2
Cytotoxicity and inhibitory effects on microtubule assembly of colchicine–tubulizine
hybrids
Compound
l
IC50
(
l
M)
Ra
LogD
2
7
—
—
0.045 0.002
3.14 0.24
0.699 0.021
2.59 0.03
1.62 0.09
1.86 0.07
0.687 0.013
1.01 0.06
0.599 0.06
2.93 0.01
0.17
0.34
0.99
0.48
0.41
0.48
0.71
3.40
2.75
3.44
—
—
activity (IC50 =0.599–2.93 lM), all of them appeared to be less active
10a
10b
10c
10d
10e
10f
10g
10h
16
14
16
18
20
22
30
40
8.465
4.815
5.591
6.367
7.612
8.738
9.814
2.568
than deacetylcolchicine 2, but more active compare to tubulizine 7
(Table 2). Such decrease of the activity in comparison with the
parent compound can be attributed to such factors as cell availabil-
ity and fast biological transformations of the newly prepared
compounds. Compound 10e with l = 20, contain, probably, the opti-
mal spacer between the tubulin-binding units, as it manifest the
best combination of antiproliferative and tubulin binding activity
a
R—half inhibitory molar ratio (ligand/tubulin) of microtubule formation
(IC50 = 0.687 0.013
group between triazole fragment and tubulizine core showed
relatively good cytotoxic and tubulin binding activity (IC50
0.699 0.021 M, R = 0.99), like 10e compound containing such
lM, R = 0.71, Table 2). Hybrid 10a without ester
in vitro.
=
l
ester linkage. The lowest cytotoxicity and inhibition of microtubule
formation were found for the long chain PEG-bridged colchicine–
tubulizine dimer 10h which characterized with low hydrophobicity
2 (R = 0.17, Table 2). The decrease of tubulin inhibition by heterodi-
mers with l = 22–40 can be argued by increase of the steric encum-
brance adversely influencing the effective binding to the catalytic
sites of protein in the case of 10f–h agents. On the contrary, the
(IC50 = 2.93 0.01 lM, R = 3.44, Table 2).
4.5
4
100
90
80
70
60
50
40
30
20
10
0
3.5
3
2.5
2
1.5
1
0.5
0
0
0.2
0.4
0.6
Ratio (R)
0.8
1
1.2
1.4
0
10
20
30
40
50
60
70
80
Time (minutes)
Figure 2. Effect of 10e on the 40,6-diamidino-2-phenylindole (DAPI) fluorimetric time course of in vitro microtubule assembly (excitation at 355 nm). The reaction was
started by warming the solution to 37 °C. Panel shows tubulin polymerization at 15 M alone (line 1) and with 1, 5, 10, 20, 30 M of 10e (line 2–6). Colchicine derivative 2
M, respectively. Inset shows the percentage of fluorescence inhibition as a function of
l
l
(line 7) and tubulizine derivative 7 (line 8) were used as positive controls at 5 and 10
l
the molar ratio of ligand to tubulin in the solution (R).

Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
4275
147.29, 141.71, 137.17, 137.07, 133.78, 131.65, 124.37, 114.48,
107.81, 61.28, 61.15, 56.78, 56.15, 53.93, 35.78, 29.50. MS (ESI),
m/z (%): 356 (13) [MꢂH]+, 249 (95). HRMS (ESI), m/z: Calcd for
3. Conclusions
Several new hybrid tubulin-interacting agents were synthesized
by linking the modified small tubuline ligands, using modular ap-
proach and the ‘click’ chemistry. Exclusively 1,4-triazole regioisom-
ers of colchicine–tubulizine hybrids 10a–g were obtained in 47–
82% yields. The cytotoxic activity of the synthesized heterodimers
with several different linker lengths was investigated toward
HBL100 human mammary cell line. All of the tested heterodimers
C
20H24NO5 358.1654. Found 358.1649 [M+H]+.
4.1.3. General procedure for the preparation of colchicine deriv
atives (4a–c)
A solution of 3-azidopropionic acid (173 mg, 1.5 mmol) in
CH2Cl2 (2 ml) was added to 2 (268 mg, 0.75 mmol), EDCꢀHCl
(288 mg, 1.5 mmol) and DMAP (46 mg, 0.375 mmol) at 0 °C under
nitrogen atmosphere. The obtained solution was stirred for 1 h at
0 °C, then for 4 h at room temperature. The solvent was removed
and the residue was purified by flash chromatography on silica
gel (EtOAc/acetone, 2:1, then EtOAc/acetone/EtOH, 2:1:1) to give
N-(3-azidopropionyl)deacetylcolchicine 4a (163 mg, 0.359 mmol,
48%) as a yellow solid, mp 150.0–152.1 °C.
presented substantial cytotoxic activity (IC50 = 0.599–2.93 lM), all
of them appeared to be less active than deacetylcolchicine 2, but
more active compare to tubulizine 7. Analysis of the structure–
activity relationship for synthesized hybrids showed that their
activity of microtubule formation inhibition decreases along with
the linker length and cytotoxic activity depends on hydrophobicity
of the molecule. Several newly synthesized compounds 10b–e
are the substoichiometric inhibitors of microtubule assembly (R =
0.41–0.71). The highest activity among the heterodimers was
1H NMR (599 MHz, CDCl3) d 8.08 (s, 1H), 7.66 (s, 1H), 7.39 (d,
J = 10.8 Hz, 1H), 6.93 (d, J = 10.8 Hz, 1H), 6.52 (s, 1H), 4.74–4.64
(m, 1H), 3.99 (s, 3H), 3.92 (s, 3H), 3.88 (s, 3H), 3.64 (s, 3H), 3.57–
3.42 (m, 2H), 2.61–2.43 (m, 3H), 2.39–2.20 (m, 2H), 1.92–1.82
(m, 1H). 13C NMR (151 MHz, CDCl3) d 179.68, 169.93, 163.79,
153.77, 153.30, 151.20, 141.70, 137.78, 135.96, 134.39, 131.04,
125.41, 113.70, 107.52, 61.68, 61.46, 56.66, 56.20, 52.42, 47.24,
36.72, 35.06, 29.97. MS (ESI), m/z (%): 453 (16) [MꢂH]+, 249 (14).
HRMS (ESI), m/z: Calcd for C23H25N4O6 453.1774. Found 453.1760
[MꢂH]+.
achieved for ligand 10e (IC50 = 0.687 0.013 lM, R = 0.71). The pro-
posed modular synthesis and simple ‘click’ chemistry methodology
makes it easy to expand the bivalent hybrid library with a variety of
new members.
4. Experimental section
4.1. Chemistry
4.1.4. N-(6-Azidocaproyl)deacetylcolchicine (4b)
4.1.1. General information
Yellow solid (70%), mp 85.5–89.5 °C, 1H NMR (599 MHz, CDCl3)
d 7.52 (s, 1H), 7.38 (s, 1H), 7.32 (d, J = 10.8 Hz, 1H), 6.85 (d,
J = 10.8 Hz, 1H), 6.50 (s, 1H), 4.73–4.54 (m, 1H), 3.98 (s, 3H), 3.91
(s, 3H), 3.87 (s, 3H), 3.63 (s, 3H), 3.36–3.10 (m, 2H), 2.63–2.08
(m, 5H), 1.95–1.24 (m, 7H). 13C NMR (151 MHz, CDCl3) d 179.56,
172.59, 164.11, 153.62, 152.25, 151.30, 141.76, 136.83, 135.58,
134.31, 130.68, 125.68, 112.88, 107.45, 61.71, 61.49, 56.51, 56.21,
52.36, 51.30, 36.89, 35.95, 30.03, 28.64, 26.47, 24.96. MS (ESI),
m/z (%): 495 (51) [MꢂH]+, 340 (6), 156 (17).HRMS (ESI), m/z: Calcd
for C26H33N4O6 497.2400. Found 497.2392 [M+H]+.
1H and 13C NMR spectra were recorded on 300 and 600 MHz
spectrometers Varian NMR-System INOVA 300, INOVA 600. Chem-
ical shifts are given as ppm relative to the residual solvent peak
(chloroform-d1: 7.26/77.0 ppm). Column chromatography purifi-
cation was performed on Merck silica gel 60 (0.040–0.063 mm,
230–400 mesh ASTM). Melting points are uncorrected and were
measured on a Büchi B.540 apparatus. Mass spectra were recorded
on a Finnigan MAT 90 and Finnigan MAT 95Q instrument. Com-
mercial reagents were used without prior purification.
4.1.2. Synthesis of N-deacetylcolchicine (2)
Di(tert-butyl) pyrocarbonate (6 g, 28 mmol) was added portion-
wise to a mixture of colchicine 1 (2.4 g, 6 mmol), Et3N (1.7 mL,
12 mmol), and DMAP (0.73 g, 6 mmol) in CH3CN (50 mL), and the
mixture was stirred for 4 h at 100 °C. The reaction was quenched
by the addition of 150 mL of CH2Cl2 and was washed with
3 ꢁ 100 mL of saturated aqueous citric acid. The combined aque-
ous layers were back extracted with CH2Cl2, and the organic layers
combined. The organic layer was washed with saturated brine and
then concentrated to a brown solid. 0.65 ml of 2 M solution of
NaOMe in MeOH was added to a solution of N-boccolchicine in
3 ml of MeOH. The reaction mixture was stirr0d for 1.5 h at room
temperature. It was then neutralized by the addition of NH4Cl
and and extracted with CH2Cl2. The organic layer was washed with
saturated brine and then concentrated to a brown solid. The resi-
due was purified by flash chromatography on silica gel (AcOEt/ace-
tone, 4:1) to give of N-[(tert-butoxy)carbonyl]deacetylcolchicine
(2.67 g, 5.8 mmol) as a pale yellow solid. A solution of N-[(tert-But-
oxy)carbonyl]deacetylcolchicine (2.67 g, 5.8 mmol) in CH2Cl2
(50 mL) containing TFA (5 mL) was stirred for 3 h at room temper-
ature. Toluene was then added, the mixture was concentrated in
vacuo, and the residue was purified by flash chromatography on
silica gel (CH2Cl2/EtOH, 9:1) to give deacetylcolchicine 2 (2.03 g,
5.7 mmol, 95%) as a yellow solid, mp 172.4–178.8 °C. 1H NMR
4.1.5. N-(10-Azidodecanoyl)deacetylcolchicine (4c)
Yellow oil (95%), 1H NMR (300 MHz, CDCl3) d 7.68 (s, 1H), 7.40
(d, J = 10.8 Hz, 1H), 7.18 (br s, 1H), 6.92 (d, J = 10.8 Hz, 1H), 6.53 (s,
1H), 4.75–4.60 (m, 1H), 4.01 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H), 3.64
(s, 3H), 3.29–3.13 (m, 2H), 2.61–2.15 (m, 5H), 2.03–1.83 (m, 1H),
1.68–1.45 (m, 4H), 1.44–1.14 (m, 10H). 13C NMR (75 MHz, CDCl3)
d 177.18, 173.09, 164.11, 153.78, 153.25, 151.29, 141.80, 137.68,
136.31, 134.42, 130.72, 125.58, 113.68, 107.56, 61.77, 61.49,
56.62, 56.24, 52.37, 51.57, 37.09, 36.35, 30.08, 29.38, 29.33,
29.31, 29.16, 28.91, 26.76, 25.49. MS (ESI), m/z (%): 553 (100)
[M+H]+, 523 (4). HRMS (ESI), m/z: Calcd for C30H41N4O6
553.3026. Found 553.3023 [M+H]+.
4.1.6. General procedure for the preparation of (6,7)
A suspension of cyanuric chloride (17.8 g, 97 mmol) in acetone
(100 mL) was cooled on ice and treated dropwise with 4-methoxy-
benzylamine (25 mL, 193 mmol). After the mixture was stirred at
that temperature for 20 min, 200 mL of a 1 N sodium hydroxide
aqueous solution was added dropwise at rt, followed by stirring
for 20 h at room temperature. The precipitate was collected by
filtration and washed with EtOH to obtain 2-chloro- 4,6-di
(4-methoxybenzylamino)-1,3,5-triazine 5 (34 g, 89 mmol, 92%) as
a white solid. To a suspension of 5 (0.75 g, 2 mmol) and monop-
ropargyliamine (0.35 ml, 5 mmol) in dioxane (5 mL) was added
DIPEA (1 ml, 3 mmol). The mixture was heated for 60 h at 100 °C
and the solvent was removed on vacue. The compound was iso-
lated by flash chromatography on silica gel (EtOAc/EtOH 10:1) to
(300 MHz, CDCl3)
d 9.15 (br s, 2H), 7.69 (s, 1H), 7.37 (d,
J = 11.3 Hz, 1H), 6.95 (d, J = 11.3 Hz, 1H), 6.54 (s, 1H), 4.05 (s, 1H),
3.98–3.78 (m, 9H), 3.55 (s, 3H), 2.69–2.47 (m, 2H), 2.46–2.11 (m,
2H). 13C NMR (75 MHz, CDCl3) d 179.44, 163.93, 154.28, 151.00,

4276
Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
give
2,4-di(40-methoxybenzylamino)-6-(propargylamino)-1,3,5-
4H), 4.17 (t, J = 5.2 Hz, 2H), 3.78 (s, 6H), 3.62 (br s, 2H), 2.30 (t,
J = 7.6 Hz, 2H), 2.17 (td, J = 7.0, 2.6 Hz, 2H), 1.93 (t, J = 2.6 Hz, 1H),
1.73–1.13 (m, 12H). 13C NMR (75 MHz, CDCl3) d 173.86, 159.07,
130.73, 129.02, 114.12, 84.85, 68.25, 63.10, 55.41, 44.33, 39.90,
34.26, 29.22, 29.19, 29.01, 28.78, 28.55, 24.96, 18.50, 1.14. MS
(ESI), m/z (%): 573 (100) [MꢂH]+, 285 (16), 283 (21), 269 (25).
triazine 6 (0.49 g, 61%) as a yellow oil. 1H NMR (300 MHz, CDCl3)
d 7.20 (d, J = 8.5 Hz, 4H), 6.82 (d, J = 8.5 Hz, 4H), 5.68 (br s, 3H),
4.47 (s, 4H), 4.15 (br s, 2H), 3.77 (s, 6H), 2.16 (s, 1H). 13C NMR
(75 MHz, CDCl3) d 165.51, 164.47, 158.93, 131.23, 129.00, 114.03,
71.14, 67.20, 55.38, 44.22, 30.47. MS (EI), m/z (%): 404 (100)
[M]+, 403 (43), 283 (24), 136 (20), 121 (62). HRMS, m/z: Calcd for
HRMS (ESI), m/z: Calcd for
C32H43N6O4 575.3346. Found
C
22H24N6O2 404.1961. Found 404.1966 [M]+.
575.3342 [M+H]+.
4.1.7. 2-(20-Hydroxyethylamino)-4,6-di(40-methoxybenzyl-
amino)-1,3,5-triazine (7)
4.1.12. 2,4-Di(40-methoxybenzylamino)-6-[20-(1700-
octadecynoyloxy)ethylamino]-1,3,5-triazine (9e)
White solid (71%), mp 106.4–108.0 °C, 1H NMR (300 MHz,
CDCl3) d 7.17 (d, J = 7.9 Hz, 4H), 6.80 (d, J = 7.9 Hz, 4H), 6.52 (br s,
1H), 5.98 (br s, 1H), 4.95 (br s, 2H), 4.44 (s, 4H), 3.76 (s, 6H), 3.69
(s, 2H), 3.46 (br s, 2H). 13C NMR (75 MHz, CDCl3) d 164.44,
163.82, 158.85, 130.71, 128.84, 113.94, 62.87, 55.26, 44.11, 43.75.
MS (EI), m/z (%): 410 (90) [M]+, 380 (12), 289 (19), 245 (14), 136
(40), 121 (100). HRMS, m/z: Calcd for C21H26N6O3 410.2066. Found
410.2056 [M]+.
Yellow solid (94%), mp 63.5–64.3 °C, 1H NMR (300 MHz, CDCl3)
d 7.20 (d, J = 8.5 Hz, 4H), 6.82 (d, J = 8.5 Hz, 4H), 5.70–5.45 (m, 3H),
4.47 (s, 4H), 4.16 (t, J = 5.2 Hz, 2H), 3.78 (s, 6H), 3.60 (br s, 3H), 2.28
(t, J = 7.6 Hz, 2H), 2.17 (td, J = 7.0, 2.6 Hz, 2H), 1.93 (t, J = 2.6 Hz,
1H), 1.70–0.70 (m, 26H). 13C NMR (75 MHz, CDCl3) d 173.89,
165.64, 164.02, 158.97, 131.11, 128.95, 114.05, 84.93, 68.16,
63.24, 55.37, 44.28, 39.86, 34.29, 29.77, 29.76, 29.72, 29.62,
29.58, 29.47, 29.40, 29.28, 29.23, 28.88, 28.62, 25.00, 18.51,
14.23. MS (ESI), m/z (%): 671 (63) [MꢂH]+, 390 (100). HRMS (ESI),
m/z: Calcd for C39H57N6O4 673.4441. Found 673.4439 [M+H]+.
4.1.8. General procedure for the preparation of tubulyzine
derivatives (9a–e)
A
solution of
7
(1 g, 2.4 mmol), 4-pentinoic acid (0.27 g,
4.1.13. General procedure for the preparation of triazole-joined
hybrids (10a–g)
2.6 mmol), DCC (0.7 g, 3.1 mmol) and DMAP (0.37 g, 3 mmol) in
THF (4 ml) was stirred for 24 h at room temperature under nitro-
gen atmosphere. The solvent was removed and the residue was
purified by flash chromatography on silica gel (EtOAc/pentane,
1:1, then EtOAc) to give 2,4-di(40-methoxybenzylamino)-6-[20-
(400-pentynoyloxy)ethylamino]-1,3,5-triazine 9a (1.12 g, 2.3 mmol,
96%) as a white oil. 1H NMR (300 MHz, CDCl3) d 7.19 (d, J = 8.5 Hz,
4H), 6.82 (d, J = 8.5 Hz, 4H), 5.88–5.20 (m, 3H), 4.47 (s, 4H), 4.19 (t,
J = 5.1 Hz, 2H), 3.78 (s, 6H), 3.61 (br s, 2H), 2.64–2.35 (m, 4H), 1.99
(t, J = 2.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) d 171.77, 165.37,
165.18, 158.93, 131.25, 128.91, 114.04, 82.60, 69.34, 63.83, 55.38,
44.23, 39.69, 33.34, 14.47. MS (ESI), m/z (%): 489 (100) [MꢂH]+,
391 (16), 269 (53), 223 (29). HRMS (ESI), m/z: Calcd for
Compound 4a (55.2 mg, 0.1 mmol) and 6 (44.5 mg, 0.11 mmol)
were dissolved in THF mixture (1 mL). 0.04 M aqueous solution of
magnesium ascorbate (0.5 ml, 0.02 mmol) was added to a virgious-
ly stirred mixture, followed by addition of 0.02 M aqueous solution
of CuSO4 (0.5 ml, 0.01 mmol). The resulting mixture was stirred for
24 at room temperature. After distillation of the solvent, the corre-
sponding product was isolated by flash chromatography on silica
gel (CH2Cl2/EtOH, 10:1) to give 10a (45 mg, 0.047 mmol, 47%), as
a yellow solid, mp 128.0–130.8 °C. 1H NMR (599 MHz, CDCl3) d
7.48 (s, 1H), 7.29 (d, J = 10.7 Hz, 1H), 7.25–6.98 (m, 5H), 6.92–
6.70 (m, 5H), 6.51 (s, 1H), 5.89 (br s, 3H), 4.73–4.34 (m, 7H),
4.31–4.02 (m, 3H), 3.96 (s, 3H), 3.93 (s, 3H), 3.88 (s, 3H), 3.75 (s,
6H), 3.64 (s, 3H), 2.49 (dd, J = 13.6, 6.5 Hz, 1H), 2.37 (td, J = 13.2,
6.8 Hz, 1H), 2.31–2.17 (m, 3H), 1.90–1.67 (m, 3H), 1.61–1.50 (m,
2H), 1.36–1.15 (m, 9H). 13C NMR (151 MHz, CDCl3) d 179.55,
172.93, 165.34, 164.74, 164.10, 158.77, 158.00, 153.56, 151.85,
151.33, 141.76, 136.62, 135.29, 134.33, 131.42, 130.88, 128.86,
128.47, 125.79, 122.16, 113.95, 112.55, 107.45, 77.37, 77.16,
76.95, 61.72, 61.50, 56.44, 56.22, 55.36, 52.23, 51.59, 50.35,
44.09, 43.97, 36.90, 36.28, 30.21, 30.05, 29.24, 29.08, 28.84,
26.37, 25.40. MS (ESI), m/z (%): 955 (31) [MꢂH]+, 786 (40), 616
(28). HRMS (ESI), m/z: Calcd for C52H65N10O8 957.4987. Found
957.4983 [M+H]+.
C
26H31N6O4 491. 2407. Found 491.2399 [M+H]+.
4.1.9. 2-[20-(500-Hexynoyloxy)ethylamino]-4,6-di(40-
methoxybenzylamino)-1,3,5-triazine (9b)
Colorless oil (99%), 1H NMR (300 MHz, CDCl3 6.82 (d, J = 8.4 Hz,
4H), 5.97–5.16 (m, 3H), 4.47 (s, 4H), 4.17 (t, J = 5.2 Hz, 2H), 3.78 (s,
6H), 3.60 (br s, 2H), 2.44 (t, J = 7.3 Hz, 2H), 2.24 (td, J = 6.9, 2.6 Hz,
2H), 1.97 (t, J = 2.6 Hz, 1H), 1.91–1.74 (k, J = 7.1 Hz, 2H). 13C NMR
(75 MHz, CDCl3) d 173.10, 165.31, 164.36, 158.90, 131.16, 128.93,
114.05, 83.34, 69.37, 63.46, 55.38, 44.22, 39.79, 32.84, 23.61,
17.97. MS (ESI), m/z (%): 503 (100) [MꢂH]+, 450 (30), 391 (14).
HRMS (ESI), m/z: Calcd for C27H33N6O4 505.2563. Found 505.2555
[M+H]+.
4.1.14. 4-{30-[300,500-Di(4000-methoxybenzylamino)-200,400,600-
triazinylaminoethyloxy]-30-oxopropyl}-1-{30-[(100,200,300,1000-
tetramethoxy-900-oxo-500,600,700,900-tetrahydrobenzo[a]heptalen-
700-yl)amino]-30-oxopropyl}-1,2,3,-triazole (10b)
4.1.10. 2-[20-(600-Heptynoyloxy)ethylamino]-4,6-di(40-
methoxybenzylamino)-1,3,5-triazine (9c)
Colorless oil (92%), 1H NMR (300 MHz, CDCl3) d 7.19 (d,
J = 8.3 Hz, 4H), 6.82 (d, J = 8.3 Hz, 4H), 5.80–5.26 (m, 3H), 4.47 (s,
4H), 4.16 (t, J = 5.2 Hz, 2H), 3.77 (s, 6H), 3.60 (br s, 2H), 2.31 (t,
J = 7.3 Hz, 2H), 2.19 (td, J = 7.0, 2.6 Hz, 2H), 1.95 (t, J = 2.6 Hz, 1H),
1.82–1.63 (m, 2H), 1.62–1.45 (m, 2H). 13C NMR (75 MHz, CDCl3)
d 173.39, 165.63, 164.71, 158.93, 131.23, 128.92, 114.03, 84.05,
68.80, 63.40, 55.37, 44.22, 39.79, 33.68, 27.91, 24.00, 18.22. MS
(ESI), m/z (%): 517 (100) [MꢂH]+, 391 (14). HRMS (ESI), m/z: Calcd
for C28H35N6O4 519.2720. Found 519.2716 [M+H]+.
Yellow solid (57%), mp 138.8–140.6 °C, 1H NMR (599 MHz,
CDCl3) d 8.01 (br s, 1H), 7.41 (br s, 2H), 7.28 (d, J = 10.7 Hz, 1H),
7.17 (br s, 4H), 6.88–6.70 (m, 5H), 6.52 (s, 1H), 5.94–5.09 (m,
3H), 4.64–4.34 (m, 7H), 4.15 (br s, 2H), 3.94 (s, 3H), 3.93 (s, 3H),
3.88 (s, 3H), 3.76 (s, 6H), 3.69–3.47 (m, 5H), 2.99 (br s, 2H), 2.88
(br s, 2H), 2.65 (br s, 2H), 2.53–2.43 (m, 1H), 2.41–2.30 (m, 1H),
2.21–2.09 (m, 1H), 1.95–1.79 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 179.42, 172.77, 169.31, 164.20, 164.00, 158.85, 158.80, 157.65,
153.59, 151.22, 141.67, 138.57, 136.57, 135.34, 134.38, 131.34,
131.11, 128.97, 125.71, 122.49, 113.98, 112.57, 107.48, 63.66,
61.56, 61.49, 56.41, 56.21, 55.38, 52.53, 46.18, 44.22, 39.76,
36.55, 36.38, 33.83, 30.01, 21.14. MS (ESI), m/z (%): 943 (42)
[MꢂH]+, 604 (11), 532 (9). HRMS (ESI), m/z: Calcd for
4.1.11. 2,4-Di(40-methoxybenzylamino)-6-[20-(1000-
undecynoyloxy)ethylamino]-1,3,5-triazine (9d)
Colorless oil (87%), 1H NMR (300 MHz, CDCl3) d 7.20 (d,
J = 8.3 Hz, 4H), 6.83 (d, J = 8.3 Hz, 4H), 6.13–5.52 (m, 3H), 4.49 (s,
C
49H57N10O10 945.4259. Found 945.4257 [M+H]+.

Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
4277
4.1.15. 4-{50-[300,500-Di(4000-methoxybenzylamino)-200,400,600-
triazinylaminoethyloxy]-50-oxopentyl}-1-{30-[(100,200,300,1000-
tetramethoxy-900-oxo-500,600,700,900-tetrahydrobenzo[a]heptalen-
700-yl)amino]-30-oxopropyl}-1,2,3,-triazole (10c)
6.82 (s, 5H), 6.52 (s, 1H), 5.49 (br s, 3H), 4.68–4.61 (m, 1H), 4.50
(s, 4H), 4.27 (s, 2H), 4.18 (s, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.89
(s, 3H), 3.77 (s, 6H), 3.71–3.52 (m, 5H), 2.75 (s, 2H), 2.56–2.45
(m, 1H), 2.43–2.31 (m, 3H), 2.31–2.12 (m, 3H), 2.09–1.93 (m,
2H), 1.93–1.73 (m, 3H), 1.63–1.47 (m, 2H), 1.41–1.01 (m, 10H).
13C NMR (151 MHz, CDCl3) d 179.53, 173.36, 172.84, 164.08,
159.00, 153.56, 151.67, 151.34, 141.77, 138.75, 138.56, 136.62,
135.19, 134.35, 131.06, 130.79, 129.05, 128.74, 125.82, 114.06,
112.50, 112.44, 107.48, 63.19, 61.71, 61.51, 56.43, 56.23, 55.40,
52.14, 50.38, 44.31, 44.08, 39.92, 37.04, 36.42, 33.46, 30.23,
30.08, 29.22, 29.06, 28.82, 26.39, 25.44, 24.95, 24.66. MS (ESI), m/
z (%): 1055 (21) [MꢂH]+, 717 (32), 716 (100), 668 (12). HRMS
(ESI), m/z: Calcd for C57H73N10O10 1057.5511. Found 1057.5511
[M+H]+.
Yellow solid (82%), mp 143.6–147.8 °C, 1H NMR (599 MHz,
CDCl3) d 7.86 (br s, 1H), 7.41 (s, 1H), 7.31 (s, 1H), 7.28 (d,
J = 10.7 Hz, 1H), 7.17 (s, 4H), 6.91–6.67 (m, 5H), 6.52 (s, 1H), 5.43
(br s, 3H), 4.64–4.31 (m, 7H), 4.15 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H),
3.89 (s, 3H), 3.76 (s, 6H), 3.64 (s, 3H), 3.65–3.46 (m, 2H), 2.98–
2.79 (m, 2H), 2.67 (s, 2H), 2.53–2.44 (m, 1H), 2.42–2.23 (m, 3H),
2.23–2.10 (m, 1H), 1.95–1.81 (m, 1H), 1.62 (s, 4H). 13C NMR
(151 MHz, CDCl3)
d 179.40, 173.57, 169.29, 165.57, 164.08,
158.83, 153.58, 151.37, 151.23, 147.78, 141.67, 138.53, 136.51,
135.34, 134.37, 131.42, 131.10, 128.96, 125.72, 122.07, 113.96,
112.53, 107.46, 63.45, 61.55, 61.49, 56.41, 56.20, 55.38, 52.52,
46.05, 44.20, 39.87, 36.59, 36.20, 33.95, 29.99, 28.80, 25.26, 24.42.
MS (ESI), m/z (%): 971 (37) [MꢂH]+, 669 (5), 560 (12). HRMS (ESI),
m/z: Calcd for C51H61N10O10 973.4572. Found 973.4565 [M+H]+.
4.1.19. 4-{160-[300,500-Di(4000-methoxybenzylamino)-200,400,600-
triazinylaminoethyloxy]-160-oxohexadecanyl}-1-{60-
[(100,200,300,1000-tetramethoxy-900-oxo-500,600,700,900-
tetrahydrobenzo[a]heptalen-700-yl)amino]-60-oxohexyl}-1,2,3,-
triazole (10g)
4.1.16. 4-{40-[300,500-Di(4000-methoxybenzylamino)-200,400,600-
triazinylaminoethyloxy]-40-oxobutyl}-1-{60-[(100,200,300,1000-
tetramethoxy-900-oxo-500,600,700,900-tetrahydrobenzo[a]heptalen-
700-yl)amino]-6’-oxohexyl}-1,2,3,-triazole (10d)
Yellow solid (64%), mp 88.0–91.7 °C, 1H NMR (599 MHz, CDCl3)
d 7.42 (s, 1H), 7.34 (s, 1H), 7.30 (d, J = 10.7 Hz, 1H), 7.19 (s, 4H), 7.01
(br s, 1H), 6.92–6.69 (m, 5H), 6.52 (s, 1H), 5.46 (br s, 3H), 4.67–4.56
(m, 1H), 4.48 (s, 4H), 4.34–4.21 (m, 2H), 4.15 (s, 2H), 3.97 (s, 3H),
3.93 (s, 3H), 3.89 (s, 3H), 3.77 (s, 6H), 3.70–3.46 (m, 5H), 2.73–
2.62 (m, 2H), 2.51 (dd, J = 13.5, 6.3 Hz, 1H), 2.39 (td, J = 13.2,
6.8 Hz, 1H), 2.34–2.13 (m, 5H), 1.94–1.76 (m, 4H), 1.72–1.48 (m,
6H), 1.43–1.06 (m, 23H). 13C NMR (151 MHz, CDCl3) d 179.47,
173.93, 172.38, 164.13, 158.87, 153.59, 151.61, 151.33, 148.39,
141.78, 136.54, 135.36, 134.26, 131.52, 131.38, 130.88, 128.93,
125.76, 121.02, 120.85, 113.99, 112.52, 107.45, 63.37, 61.68,
61.50, 56.46, 56.22, 55.38, 52.39, 51.67, 51.47, 50.05, 44.25,
39.83, 39.74, 38.97, 36.85, 35.85, 34.31, 30.12, 30.02, 29.81,
29.77, 29.75, 29.71, 29.69, 29.64, 29.57, 29.52, 29.45, 29.38,
29.27, 26.17, 25.85, 25.01, 24.89. MS (ESI), m/z (%): 1167 (23)
[MꢂH]+, 943 (7), 829 (51). HRMS (ESI), m/z: Calcd for
Yellow solid (73%), mp 102.8–105.7 °C, 1H NMR (599 MHz,
CDCl3) d 7.61–7.35 (m, 3H), 7.29 (d, J = 10.7 Hz, 1H), 7.18 (s, 4H),
6.89–6.70 (m, 5H), 6.52 (s, 1H), 5.42 (br s, 3H), 4.65–4.56 (m, 1H),
4.56–4.38 (m, 4H), 4.28 (s, 2H), 4.16 (s, 2H), 3.96 (s, 3H), 3.93 (s,
3H), 3.89 (s, 3H), 3.76 (s, 6H), 3.64 (s, 3H), 3.63–3.48 (m, 2H), 2.75
(s, 2H), 2.54–2.45 (m, 1H), 2.44–2.27 (m, 3H), 2.26–2.12 (m, 3H),
1.99 (s, 2H), 1.93–1.74 (m, 3H), 1.69–1.57 (m, 1H), 1.57–1.45 (m,
1H), 1.33–1.17 (m, 2H). 13C NMR (151 MHz, CDCl3) d 179.46,
173.50, 172.44, 164.13, 158.87, 153.59, 151.61, 151.33, 146.76,
141.78, 138.62, 138.48, 136.56, 135.32, 134.30, 131.42, 131.01,
128.98, 125.79, 114.05, 113.99, 112.52, 107.47, 63.49, 61.70, 61.51,
56.45, 56.23, 55.40, 52.41, 50.18, 44.24, 39.80, 39.62, 36.79, 35.95,
33.52, 30.03, 29.82, 26.11, 24.90, 24.63. MS (ESI), m/z (%): 999 (15)
[MꢂH]+, 774 (10), 696 (23), 661 (29), 660 (100). HRMS (ESI), m/z:
Calcd for C53H65N10O10 1001.4885. Found 1001.4877 [M+H]+.
C
65H89N10O10 1169.6763. Found 1169.6753 [M+H]+.
4.1.20. Synthesis of poly(ethylene glycol) 2-[30,50-di(400-
methoxybenzylamino)-20,40,60-triazinylaminoethyloxy]-2-
oxoethyl 2-[(10,20,30,100-tetramethoxy-90-oxo-50,60,70,90-
4.1.17. 4-{90-[300,500-Di(4000-methoxybenzylamino)-200,400,600-
triazinylaminoethyloxy]-90-oxononyl}-1-{30-[(100,200,300,1000-
tetramethoxy-900-oxo-500,600,700,900-tetrahydrobenzo[a]heptalen-
700-yl)amino]-30-oxopropyl}-1,2,3,-triazole (10e)
tetrahydrobenzo[a]heptalen-70-yl)amino]-2-oxoethyl ether
(10h)
Yellow solid (63%), mp 115.0–117.7 °C, 1H NMR (599 MHz,
CDCl3) d 7.78 (br s, 1H), 7.43 (s, 1H), 7.31 (s, 1H), 7.28 (d,
J = 10.7 Hz, 1H), 7.19 (s, 4H), 6.88–6.71 (m, 5H), 6.52 (s, 1H), 5.33
(br s, 3H), 4.68–4.35 (m, 7H), 4.16 (s, 2H), 3.95 (s, 3H), 3.93 (s,
3H), 3.89 (s, 3H), 3.76 (s, 6H), 3.65 (s, 3H), 3.63–3.51 (m, 2H),
3.01–2.90 (m, 1H), 2.90–2.80 (m, 1H), 2.64 (s, 2H), 2.49 (dd,
J = 13.3, 6.5 Hz, 1H), 2.38 (td, J = 13.3, 6.5 Hz, 1H), 2.31–2.22 (m,
2H), 2.23–2.13 (m, 1H), 1.94–1.81 (m, 1H), 1.64–1.51 (m, 4H),
1.37–1.16 (m, 8H). 13C NMR (151 MHz, CDCl3) d 179.44, 173.92,
169.27, 164.25, 158.86, 153.59, 151.33, 151.25, 141.69, 138.58,
136.49, 135.35, 134.35, 131.47, 131.36, 131.19, 128.96, 125.74,
121.80, 113.98, 112.53, 110.13, 107.46, 63.37, 61.55, 61.49, 56.42,
56.21, 55.38, 52.49, 46.07, 44.24, 39.85, 36.67, 36.18, 34.31,
32.04, 30.01, 29.81, 29.42, 29.18, 29.12, 25.64, 24.96, 22.81. MS
(ESI), m/z (%): 1027 (38) [MꢂH]+, 761 (4). HRMS (ESI), m/z: Calcd
for C55H69N10O10 1029. 5198. Found 1029.5198 [M+H]+.
A solution of 7 (0.409 g, 1 mmol), poly(ethylene glycol) bis(car-
boxymethyl) ether (average Mn = 600) (1.2 g, 2 mmol), DCC
(0.206 g, 1 mmol) and DMAP (0.061 g, 0.5 mmol) in THF (5 ml)
was stirred for 3 h at room temperature under nitrogen atmo-
sphere. The solvent was evaporated, followed by addition of
CH2Cl2. The precipitate was removed and the solution was washed
with water, dried over Na2SO4 and concentrated to colorless oil. It
was purified by flash chromatography on silica gel (CH2Cl2/MeOH,
1:1) to give 11 (0.544 g, 0.55 mmol, 55%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) d 7.20 (d, J = 8.2 Hz, 4H), 6.81 (d, J = 8.2 Hz, 4H),
4.55–4.38 (m, 4H), 4.23 (t, J = 5.4 Hz, 2H), 4.13 (br s, 2H), 3.90 (br
s, 2H), 3.77 (s, 6H), 3.73–3.49 (m, 42H). 13C NMR (75 MHz, CDCl3)
d 175.39, 175.20, 170.60, 170.56, 158.88, 131.39, 128.95, 113.99,
71.02–68.65, 63.87, 55.38, 44.18, 39.63. MS (ESI), m/z (%): 965
(98) [MꢂH]+, 921 (89), 877 (70), 833 (46), 789 (26), 735 (18),
691 (11). HRMS (ESI), m/z: Calcd for C45H71N6O17 967.4876. Found
967.4876 [M+H]+. A solution of 11 (238 mg, 0.24 mmol) in CH2Cl2
(2 ml) was added to 2 (86 mg, 0.24 mmol), EDCꢀHCl (50 mg,
0.26 mmol) and DMAP (15 mg, 0.12 mmol) at 0 °C under nitrogen
atmosphere. The obtained solution was stirred for 1 h at 0 °C, then
for 48 h at room temperature. The solvent was removed and the
residue was purified by flash chromatography on silica gel
(CH2Cl2/EtOH, 4:1) to give 10h (213 mg, 0.16 mmol, 68%) as a
4.1.18. 4-{40-[300,500-Di(4000-methoxybenzylamino)-200,400,600-
triazinylaminoethyloxy]-40-oxobutyl}-1-{100-[(100,200,300,1000-
tetramethoxy-900-oxo-500,600,700,900-tetrahydrobenzo[a]heptalen-
700-yl)amino]-100-oxodecanyl}-1,2,3,-triazole (10f)
Yellow solid (69%), mp 110.2–112.3 °C, 1H NMR (599 MHz,
CDCl3) d 7.50 (s, 1H), 7.29 (d, J = 10.6 Hz, 1H), 7.21 (br s, 5H),

4278
Y. B. Malysheva et al. / Bioorg. Med. Chem. 20 (2012) 4271–4278
yellow solid, mp 58.5–60.8 °C. 1H NMR (300 MHz, CDCl3) d 7.98 (br
s, 1H), 7.48 (s, 1H), 7.39–7.13 (m, 6H), 6.96–6.70 (m, 5H), 6.55 (s,
1H), 4.76–4.59 (m, 2H), 4.62–4.37 (m, 4H), 4.37–3.29 (m, 63H),
2.55 (dd, J = 10.4, 6.2 Hz, 1H), 2.51–2.33 (m, 1H), 2.33–2.07 (m,
2H), 2.11–1.85 (m, 2H). 13C NMR (75 MHz, CDCl3) d 179.56,
159.10, 153.82, 153.65, 151.26, 141.73, 136.12, 134.46, 134.44,
132.99, 129.11, 114.12, 114.12, 70.15–68.11, 63.43, 61.54, 61.48,
56.25, 56.25, 55.41, 44.37, 36.58, 29.85. MS (ESI), m/z (%): 1307
(40) [M+H]+, 1263 (33), 1175 (18), 1131 (9), 775 (29), 731 (56),
709 (99), 687 (98), 643 (67), 621 (39) 610 (32). HRMS (ESI), m/z:
Calcd for C65H92N7O211306.6346. Found 1306.6353 [M+H]+.
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4.2.3. Microtubules assembly monitored by fluorescence
Microtubules assembly was performed on a Fluoroscan Ascent
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was set at 460 nm. Experiments were carried out at 37 °C and per-
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were done in triplicate.
Acknowledgements
This work was supported by Russian Foundation for Basic Re-
search (09-03-00647-a and 09-03-97038-r_povolzh’e_a), German
Academic Research Service (A/08/81119) and the Council on Grants
at the President of the Russian Federation (MD-5606.2010.3).
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