Synthesis and biological evaluation of furanoallocolchicinoids

Article abstract of DOI:10.1021/jm501678w

A series of conformationally flexible furan-derived allocolchicinoids was prepared from commercially available colchicine in good to excellent yields using a three-step reaction sequence. Cytotoxicity studies indicated the potent activity of two compounds against human epithelial and lymphoid cell lines (AsPC-1, HEK293, and Jurkat) as well as against Wnt-1 related murine epithelial cell line W1308. The results of in vitro experiments demonstrated that the major effect of these compounds was the induction of cell cycle arrest in the G2/M phase as a direct consequence of effective tubulin binding. In vivo testing of the most potent furanoallocolchicinoid 10c using C57BL/6 mice inoculated with Wnt-1 tumor cells indicated significant inhibition of the tumor growth.

Full text of DOI:10.1021/jm501678w

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Article
Synthesis and Biological Evaluation of Furanoallocolchicinoids
Yuliya V. Voitovich, Ekaterina S. Schegravina, Nikolay S. Sitnikov, Vladimir I. Faerman,
Valery V. Fokin, Hans-Guenther Schmalz, Sebastien Combes, Diane Allegro, Pascale
Barbier, Irina P. Beletskaya, Elena V. Svirshchevskaya, and Aleksey Yurievich Fedorov
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2014
Downloaded from http://pubs.acs.org on December 12, 2014
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The Synthesis and Biological Evaluation of
Furanoallocolchicinoids
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a
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Yuliya V. Voitovich , Ekaterina S. Shegravina , Nikolay S. Sitnikov , Vladimir I. Faerman ,
a
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Valery V. Fokin , Hans-Gunther Schmalz , Sebastien Combes , Diane Allegro , Pascal Barbier ,
e
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Irina P. Beletskaya , Elena V. Svirshchevskaya *, Alexey Yu. Fedorov *
a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny
Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: afnn@rambler.ru
b
Department of Chemistry, University of Cologne, Greinstrasse 4, 50939 Koln (Germany)
Fax: +49 221-470-3064, Tel: +49 221 470 3063, E-mail: schmalz@uni-koeln.de
c
CRCM, CNRS UMR7258, Laboratory of Integrative Structural and Chemical Biology
(
ISCB), INSERM, U1068, Institut Paoli-Calmettes, Aix-Marseille Universit´e, UM105,
F-13009, Marseille, France, Fax: +33 (0)491 164 540; Tel: +33 (0)86 97 73 31, E-mail:
sebastien.combes@univ.provence.fr,
d
Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, CRO2 INSERM
UMR 911, Universite d’Aix-Marseille, Faculte de Pharmacie, 27 Boulevard Jean Moulin,
Marseille 13005, France Tel : +33 (0)491 835 616, E-mail : pascale.barbier@univ-amu.fr
e
Department of Chemistry, M.V. Lomonosov Moscow State University, Vorobyevy Gory,
119992 Moscow, Russia, Tel: +7 (495) 938 36 18, E-mail: beletska@org.chem.msu.ru
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Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Laboratory of Cell
Interactions, GSP-7, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation,
Tel/Fax +7 (495) 330 40-11, E-mail: esvir@yandex.ru
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Corresponding authors
Prof. Fedorov Alexey Yu.
a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny
Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: afnn@rambler.ru
Dr. Svirshchevskaya Elena V.
e
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Laboratory of Cell
Interactions, GSP-7, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation,
Tel/Fax (495) 330 40-11, E-mail: esvir@yandex.ru
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Keywords: colchicine, allocolchicinoids, cross-coupling, antitumor activity, tubulin binding
agents.
Abstract
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A series of conformationally flexible furan-derived allocolchicinoids was prepared from
commercially available colchicine in good to excellent yields using a three-step reaction
sequence. Cytotoxicity studies indicated potent activity of two compounds against human
epithelial and lymphoid cell lines (AsPC-1, HEK293 and Jurkat) as well as against Wnt-1 related
murine epithelial cell line W1308. The results of in vitro experiments demonstrated that the
major effect of these compounds was the induction of cell cycle arrest in the G2/M phase as a
direct consequence of effective tubulin binding. In vivo testing of the most potent
furanoallocolchicinoid 10c using C57BL/6 mice inoculated with Wnt-1 tumor cells indicated
significant inhibition of the tumor growth.
1
. Introduction
Due to the essential role of microtubules in cell growth and division, inhibition of microtubule
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formation is considered promising in the treatment of cancer. Microtubules are formed via a
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polymerization process involving α- and β-tubulin protein subunits. Interference with the
dynamic equilibrium between the assembly of tubulin into microtubules or, inversely, the
depolymerization of microtubules leads to the arrest of cell division and eventually to apoptosis.
Compounds inhibiting either of these important processes are referred to as spindle poisons or
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antimitotics.
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The search for selective inhibitors of tubulin assembly or disassembly has led to the development
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of some of the most useful antitumor drugs currently in clinical use. These are the naturally
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occurring Vinca alkaloids as well as the taxoids, which are widely used for the treatment of
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breast, ovarian, and non-small cell lung carcinomas. However, these structurally rather complex
molecules share several drawbacks: they are difficult to synthesize (thus expensive) and prone to
1,5
the development of resistance.
Therefore, the development of novel small-molecule
antimitotics with low-toxicity remains a clinically relevant challenge.
Colchicine (1) (Figure 1), isolated from Colchicum autumnale, was the first tubulin-destabilizing
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agent ever discovered. Although it has been employed since ancient times in the treatment of
acute gout and familial Mediterranean fever, high cardio- and neurotoxic adverse effects prevent
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its use in cancer chemotherapy. However, structural analogs of colchicine, such as
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allocolchicine (2), combretastatin A-4 (3), 4-arylcoumarins (4), (Figure 1) and their
derivatives, represent promising leads in the search for new anticancer agents addressing the
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colchicine binding site of tubulin. The ability of colchicine and its analogs to block mitosis by
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affecting the microtubule dynamics is well studied,
and several colchicine analogs such as
AbT-751 and indibulin are currently in clinical trials for patients suffering from advanced solid
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tumors.
Clinical trials of other colchicine analogues have been discontinued due to poorly
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controlled drug absorption, neurotoxicity or low efficacy. Nevertheless, the search for novel
colchicinoids remains an interesting challenge because, besides acting as mitosis inhibitors,
recent studies have shown that microtubule-targeting agents may also exhibit effects in the areas
of (i) mitosis-independent cell death and metastasis, (ii) tumor angiogenesis, and (iii) vascular-
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disrupting activity. Moreover, the development of drugs overcoming Pgp/β-III-tubulin-
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mediated drug resistance is a key task in cancer research.
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A molecular modeling study suggested that a key structural feature of colchicine binding site
inhibitors is the presence of two closely neighbored non-coplanar hydrophobic aromatic
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fragments A and B, bearing hydrogen bond donor or acceptor functionalities (Figure 1).
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Indeed, the structural similarity of compounds 1 to 4 arises from the presence of two adjacent
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polyoxygenated aromatic rings A and B, which are non-coplanar.
Figure 1
For a long time, the unique substitution pattern of ring B in molecules such as 1-4 was
considered essential for the high affinity towards the colchicine binding site of tubulin. However,
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recent investigations by the groups of Wang, Simonini, and in our laboratories demonstrated
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that replacement of the aromatic B-ring by a hydrophobic benzoheterocyclic moiety leads to
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highly active compounds (Figure 2). For instance, the indole derivatives 5 and 7 were found
to inhibit in vitro microtubule assembly in a substoichiometric mode of action. The phosphate
prodrugs 6 demonstrated high antitumor activity both in vitro and in vivo, notwithstanding a
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minimal effect on microtubule organization with respect to 3 (CA-4). The indole-derived
allocolchicine analogs 8 and 9 caused proliferation inhibition and apoptosis induction at
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nanomolar or even in sub-nanomolar concentrations. These derivatives actually represent the
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first examples of heteroarene-based allocolchicinoids.
Figure 2
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As structural analogs of pyrrole-fused colchicinoids 8 and 9 we considered the corresponding
furan-derived allocolchicinoids of type 10 to be promising compounds for biological testing. We
herein report a short and convenient semi-synthetic access to such compounds (of type 10) and
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demonstrate their promising antitumor activity (including in vivo mice experiments) in
comparison with colchicine (1) and combretastatin A-4 (3).
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. Results and Discussion
Chemistry
Compounds of type 10 were prepared in only four steps starting from commercially available
colchicine (1) by making use of the previously described colchiceine - N-acetyliodocolchinol
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rearrangement
and transition metal-catalyzed methods for the construction of the furan ring
(Scheme 1). First, the tropolone ether function of colchicine (1) was hydrolyzed by heating with
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HCl in AcOH to afford colchiceine (11) in 97% yield. The oxidative degradation of 11 to
iodocolchinol (12) was also achieved in high yield using the Windaus procedure (NaOH, I , KI,
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H O).
The Sonogashira coupling of iodocolchinol (12) with terminal alkynes (13) then
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afforded the alkyne intermediates of type 14, which under the reaction conditions (Pd/Cu
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catalysis, base) directly underwent cyclisation to afford the desired benzofurans of type 10.
Scheme 1
Table 1
The Sonogashira coupling of iodocolchinol (12) with phenylacetylene (13a) or 2-
pyridylacetylene (13b) was best performed at 65 °C using Pd(OAc) /CuI/PPh (5:10:15 mol%) as
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the catalytic system in MeCN in the presence of K PO as a base to afford the cyclization
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products 10a and 10b, respectively, in 86 and 83% isolated yield (Table 1). In contrast, when
iodocolchinol 12 was reacted with non-(hetero)aryl-substituted terminal alkynes, such as
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propargyl alcohol (13c), under the same conditions, the cyclization products were obtained in
much lower yield (e.g. 30% of 10c). However, as the yield of the furanoallocolchicinoids 10
proved to be strongly base-dependent, different bases (NEt , DIPEA, K CO , Cs CO , t-BuOK,
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or AcOK) were tested in the reaction between 12 and 13c using the same catalytic system as
before. Under the optimized conditions (Pd(OAc) /CuI/PPh (5:10:15 mol%), AcOK (3 equiv.),
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o
MeCN, 70 C) the reaction of iodocolchinol (12) with different functionalized terminal alkynes
13c-k) afforded the products 10a-i in good to high yields (Table 1). Thus, the desired
(
benzofurans of type 10 were efficiently obtained in only three preparative steps starting from
commercially available colchicine (1).
An attempt to prepare benzofuran 10j with an aminomethyl group in the side chain
revealed that this compound was rather unstable. Noteworthy, we did not succeed at all in
synthesizing the TMS-substituted furanoallocolchicine 10k, because the reaction of 12 with
trimethylsilylacetylene (13k) stopped at the stage of intermediate 14k, which was isolated in
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2% yield. This correlates well with the literature reports that the Pd/Cu-catalyzed cyclization of
ortho-trimethylsilylethynyl- and ortho-ethynyl-substituted phenols, anilines or thiols require
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rather harsh conditions (thermolysis or photolysis). The pyrrolidinomethyl-substituted
benzofuran 10l was obtained in 80% yield by reacting 12 with propargylbromide 13l in the
presence of catalytic amounts of Pd(PPh ) Cl and CuI in pyrrolidine as a solvent (Scheme 2).
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This type of reaction might also be useful for the preparation of related aminomethyl-substituted
furanoallocolchicinoids employing different primary and secondary amines.
Scheme 2
One should note that furan-derived allocolchicinoids 10 were prepared using semi-synthetic
pathway in three steps from commercially available colchicine with the retention of the absolute
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configuration at C7. Proposed methodology seems to be advantageous in comparison with the
eight-to-twelve-step synthesis used for the preparation of allocolchicinoids 8 and 9, affording the
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target compounds as a racemic mixture.
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Biology
In vitro cytotoxicity of new compounds against cancer cell lines
The in vitro cytotoxicity of all synthesized compounds against human cancer cell lines was
investigated by MTT assay after the incubation of the cells with the compounds for 72 h. The
representative inhibitory curves for HEK293, Jurkat, and AsPC-1 cells are shown in Figure 3.
For comparison, effects of all 10 compounds synthesized are shown only for HEK293 cells in
two sets (Figure 3 A and B), where A demonstrates the effects of the compounds with high
activity, and B – with low activity. The results for Jurkat and AsPC-1 cells are presented only for
the compounds with high activity (Figures 3C and 3D).
Figure 3
All compounds, including 10c and 10d as the most toxic ones, induced ca. 60% inhibition
even at very high concentrations (Figure 3, black dotted lines) showing the absence of direct
necrotic or pro-apoptotic toxicity. The same is true for many previously published colchicine
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analogues. Activity of compounds inducing 50% of the maximal cell growth inhibition (IC ,
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Figure 3, grey dotted lines) are summarized in Table 2. The sensitivity of different cell lines to
these compounds slightly varied due to variations in cell concentrations, different rates of
division, and experimental errors. For the comparison we have also included IC50 for
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indoloallocolchicinoids 8a and 9a synthesized by us earlier, the most active compounds from 8
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and 9 series.
Table 2
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Compounds 10a and 10b bearing an aromatic (or heteroaromatic) moiety in the side chain of the
benzofuran fragment were not active. In contrast, compounds 10c and 10d, with a
hydroxymethyl or a 1-hydroxyethyl substituent at the furan ring, exhibited potent anti-
proliferative activity with IC values in the nanomolar range. While allocolchicinoid 10e also
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contains a hydroxyl group in the pseudo-benzylic position (like 10c and 10d) it exhibited lower
cytotoxicity, possibly because the hydrophobic cyclopentane ring shields the OH group.
Interestingly, compound 10f with a 2-hydroxyethyl side chain showed a two-order of magnitude
lower activity in comparison with the hydroxylated compounds 10c and 10d. Compound 10g,
which can be regarded as an ester prodrug of 10c, exhibits a significantly lower activity in
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comparison with the “parent” compound 10c. A low cytotoxicity was also observed for the
hydrophobic ester 10h.
22b
The most cytotoxic among the 8-series compounds
was 8a, an indolyl analog of
furanoallocolchicinoid 10. It was less active than 10c. On the other hand, the isomeric
colchicinoid 9a manifested the same order of activity as 10c (Table 2).
These results demonstrate that a hydroxymethyl fragment in the α-position of the furan ring of
type 10 compounds significantly improves the pharmacophore. Therefore it was interesting to
also investigate the cytotoxic activity of nitrogen-containing analogs of 10c and 10d. While the
aminomethyl-substituted allocolchicinoid 10j turned out to be unstable, the N,N-
dialkylaminomethyl derivatives 10i and 10l were successfully synthesized and tested. However,
both compounds were found to be inactive up to a concentration of 1000 µM.
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Among all compounds tested, 10c and 10d were identified as the most active ones and were
therefore used for further in vitro and in vivo studies.
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Effects of compounds 10c and 10d on tubulin polymerization
As structural analogues of colchicine, compounds of type 10 were expected to interact with the
colchicine binding site of tubulin and, consequently, to inhibit the formation of microtubules. To
prove this, compounds 10c and 10d were examined in a tubulin polymerization assay. Figures
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A and 4B show the effects on the time-resolved turbidity change reflecting the microtubule
assembly from pure tubulin. For both compounds a clear inhibition of polymerization was noted,
and the rate of assembly as well as the final amount of microtubules was lower in the presence of
the compounds than in the control experiment. Figures 4C and 4D show that the extent of
inhibition by 10c and 10d, respectively, increases monotonically with the increase in the molar
ratio (R) of the total ligand to total tubulin in the solution. From this it becomes evident that 50%
inhibition occurs at a molar ratio of ca. 0.08 and 0.3 for 10c and 10d, respectively. A similar
experiment performed with combretastatin A-4 (3) revealed an R value of 0.09 mol (data not
shown). It should be mentioned that the “substoichiometric” poisoning of microtubule assembly
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by colchicine (R = 0.38) is well established.
Figure 4
Although less potent in tubulin assembly inhibition, 10d also exhibited a “substoichiometric”
mode of action, like 10c which in turn showed a slightly higher activity than combretastatin A-4
(3) and was almost one order of magnitude more effective than colchicine (1). Interestingly,
compound 10c, which was identified as the most potent cytotoxic agent in this series, also
showed the strongest tubulin depolymerization activity. This suggests that the cytotoxic effect of
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compounds of type 10 indeed is a consequence of their tubulin-binding properties.
Effects of compound 10c on apoptosis induction
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As the cell proliferation inhibition assay has revealed (Figure 3), compound 10c does not show
an acute (non-specific) toxicity against both epithelial and lymphoid cells. To support this
conclusion, we also studied the induction of apoptosis in W1308 cells incubated with compounds
1
0c, 10d, and 9a at 200 nM for 48 hrs. For this purpose, cells were stained with DiOC
6
and PI
is a mitochondria membrane potential-sensitive dye, while PI is able to penetrate
the membranes of dead and apoptotic cells. Double staining permits the identification of a
population of cells with a decreased DiOC and increased PI fluorescence (Figure 5) specific to
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dyes. DiOC
6
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apoptotic cells. After 48 h of incubation the rate of apoptosis in 10c culture was comparable with
the control one (Figure 5 A and B), while both 10d and 9a induced significant apoptosis. These
results demonstrate that compounds of type 10, in particular 10c and 10d, partially differ in the
mechanisms of action. Similar to 10c effects were described for colchicine and its analogues,
which were able to induce early apoptosis only in the presence of tumor necrosis factor alpha
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(TNF-alpha), a direct apoptosis inducer. However, the inhibition of microtubule assembly
inevitably results in late stage apoptosis.
Figure 5
Late apoptosis was studied simultaneously with cell cycle arrest after incubating cells with
compound 10c for 72 hours. Representative histograms for AsPC-1 and W1308 are shown in
Figure 6 A-D and the data for the different cell lines tested are summarized in Table 3. Treatment
of HEK293, Jurkat, AsPC-1, and W1308 cells with 10c for 72 hours led to profound changes in
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the cell cycle state (Figure 6). The flow cytometric analysis using PI-stained cells indicates that
10c induces a massive accumulation of cells in the G2/M phase (Table 3). At the same time,
even after 72 h of incubation with 10c, the apoptotic rate was not very high (up to 6% depending
on cell line) (Table 3). The absence of apoptotic cells in 72 cultures could be a result of dead cell
elimination during sample preparation. To visualize the full population of cells after 10c
treatment monolayer cultures were analyzed by confocal microscopy.
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Different stages of apoptosis were observed in cell cultures after 72 h of incubation with 10c:
double nucleated cells (Figure 6, C-D), cell fractionation and apoptotic body formation (Figure 6,
E-F), mitochondria enlargement and depolarization (Figure 6, C-E). These results clearly
indicate that the cell cycle arrest caused by 10c, as evidenced by the inability of the incubated
cells to pass through the G2/M phase (see G1/G2 ratio, Table 3), leads to a consequent apoptosis
(Figure 6).
Table 3
In vivo antitumor activity of the compound 10c
The results of the in vitro experiments revealed tubulin as a target of furanoallocolchicine 10c,
which induces late apoptosis as a result of cell cycle arrest. As compound 10c exhibited higher
antitubulin polymerization activity than the natural products colchicine (1) and combretastatin A-
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(3) (Figures 4, 5C) it may have pharmacological advantages. In addition, compound 10c is
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configurationally stable, in contrast to 3, which tends to undergo Z/E isomerization. Therefore,
10c was chosen for the preliminary in vivo acute toxicity and antitumor activity investigation
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to define the therapeutic dose of 10c, we first determined the LD100 and LD acute toxicity doses
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by injecting single doses of 10c either intraperitoneally (i.p.) or intravenously (i.v.) into C57BL/6
mice. On average injection of 10c i.p. was more toxic than i.v. and resulted in 0.8 and 2 mg/kg
LD , respectively. Corresponding LD for i.p. and i.v. injections were 0.3 and 0.8 mg/kg.
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Lethality was observed 24-48 hrs after injections.
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To analyze the therapeutic activity of 10c, C57BL/6 mice inoculated with breast tumors Wnt-1
were injected once a week for 4 weeks with 0.4 mg/kg (i.v.) of 10c dissolved in saline. At the
initiation of 10c treatment both control and experimental groups had palpable tumors with a
3
comparable average volume (42±12 and 32±11 mm accordingly). The selected dose
significantly (p<0.01) inhibited the tumor growth (Figure 7) as was estimated by parametric
statistics (Student’s t-test). No weight loss, neurological symptoms or change in the behavior of
mice were registered. Taken collectively, these experiments demonstrate the existence of the
therapeutic window for the compound 10c.
In vivo interphase microtubule destabilization activity of colchicine and its analogues
Combretastatin A4 (CA-4, 3) is considered to be unique among cytotoxic agents as it appears to
cause rapid shutdown in the vascular function of the tumor, leading to blood and oxygen
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deprivation and consequently to ischemic or hemorrhagic necrosis of the tumor. We studied the
effect of a short exposure to colchicine (1), CA-4 (3) and 10c on normal vasculature in mice.
Mice were treated with 1 mg/kg (i.v.) of the compounds mixed with the fluorescent dye
rhodamine. Four hours later, the mice were sacrificed, organs collected, and rhodamine was
extracted from each organ. Note that 4 hours of exposure is not sufficient to affect mitosis and
thus this study reflects the effect of compounds on the cells in interphase. The results
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demonstrated a catastrophic leakage of rhodamine through the gut, and significant influxes of the
dye into the heart and spleen (Figure 7B), corresponding to the most common side effects of
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solid tumor therapy with CA-4 analogues: hypertension, anemia, lymphopenia. Confocal
analysis of tissue sections supported the results of rhodamine extraction (Figure 7, C-F). In
contrast, the effect of the compounds on blood-brain barrier (BBB) endothelial cells was quite
opposite. There was ca. 40% decrease in the permeability of BBB. This difference can be
explained by the differences in the adhesion between endothelial cells forming BBB and normal
endothelium. BBB endothelial cells express tight junction proteins additionally to a set of
integrin ligands expressed by normal endothelial cells. Evidently, BBB endothelial cells lost
integrin type of adhesion but not the tight junction one. Thus, leakage of blood proteins and
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oxygen deprivation of the brain cells can explain the neurotoxicity of colchicine analogues.
Figure 7
The higher toxicity of 10c after i.p. administration in comparison to the i.v. route also supports
the conclusion that the gut endothelium is highly sensitive to tubulin destabilization. Our results
demonstrated that colchicine was significantly more toxic to the gut than CA-4 and 10c (Figure
7
, B).
Structural aspects
Another interesting feature of compound 10c is its relatively high barrier of rotation around the
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22b
aryl-aryl axis.
As in the case of indoloallocolchinoids of type 8 and 9, two distinct atrop-
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diastereomers are observed in the H and C NMR spectra under ambient conditions (Figure
41,42
8
).
The ratio of the atrop-diastereomers varies strongly with the polarity of the solvent (CDCl3
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or DMSO), possibly as a consequence of subtle solvation effects (see supporting information,
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page S37).
Figure 8
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A question that has never been addressed so far is the axial configuration of the biologically
active conformers of allocolchicine and its analogues. Also, differences in the conformational
flexibility may contribute to the different biological activities of structurally related
allocolchicinoids. Thus, synthesis of compounds with adjustable conformational mobility and
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examination of their conformational dynamics,
followed by correlation of these properties
with biological activity, would be worth purusing.
3
. Conclusion
We have devised and synthesized a series of furan-derived allocolchicinoids of type 10 as a
novel class of analogs of the natural product colchicine (1). These compounds were efficiently
obtained in only three preparative steps starting from commercially available colchicine under
conservation of the absolute configuration at C(7). Compounds 10c and 10d, containing a
hydroxyl group in the pseudo-benzylic position of the furan side chain, exhibited high
cytotoxicity toward epithelial and lymphoid cell lines in the nanomolar concentration range. In
comparison with colchicine (1) and combretastatin A-4 (3) the allocolchicinoid 10c proved to be
a more effective tubulin assembly inhibitor, acting in a substoichiometric mode. Biochemical
assays and in vitro experiments clearly demonstrated 10c and 10d to effectively inhibit both
tubulin polymerization and proliferation of endothelial cells as a consequence of two major
effects: (1) disruption of the interphase and (2) induction of cell cycle arrest, both as a direct
consequence of tubulin binding. The in vivo experiments also demonstrated that endothelial cells
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treated with 10c quickly lose adhesion and become “leaky”. In contrast, BBB endothelial cells
become more tightly packed, what possibly leads to an insufficient supply of the brain with
nutrients and oxygen. Still, treatment of mice with 10c resulted in the inhibition of tumor growth.
Evidently, metaphase and interphase effects of colchicine analogues cannot be easily segregated.
The synthetic accessibility and significant antitumor activity exhibited by the new colchicinoids
described here makes them promising objects for a more detailed biological investigations. We
consider compound 10c as a promising lead in the search for novel anticancer agents with
improved properties.
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4. Experimental section
Materials
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H NMR and C NMR spectra were recorded on Agilent DD2 400 spectrometer. Chemical shifts
(
δ) are reported in ppm for the solution of compound in CDCl or DMSO-d with internal
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reference TMS and J values in hertz. EI mass spectra (70 eV) were obtained on a DSQ II mass-
spectrometer (Thermo Electron Corporation) with quadrupole mass-analyzer. Combustion
analysis was performed using Elementar (Vario Micro Cube) apparatus, and the final compounds
have a purity of > 95%. Separation by column chromatography was performed using Merck
Kieselgel 60 (70 – 230 mesh). All reactions were performed with commercially available
reagents («Aldrich», «Alfa Aesar», «Acros», «Serva»). Phenylacetylene was distilled under low
pressure (b.p. 75 °C/80 mm Hg). Solvents were purified according to standard procedures. The
petroleum spirit refers to the fraction with distillation range 40 – 70 °C.
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.2. Preparation of compounds 10-12.
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Synthesis of colchiceine (11). A mixture of colchicine (1) (300.0 mg, 0.75 mmol), glacial acetic
acid (3 mL) and hydrochloric acid (0.1 N 18 mL) was stirred for 3 h at 100 °C, then solid
Na CO was added until pH become neutral. Resulting solution was extracted with CHCl
10
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(3×250 mL), the combine organic layer was washed with brine and dried over Na SO . The
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solvent was removed under reduced pressure. Colchiceine (11) was obtained as yellow-green
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1
crystals (275.0 mg, 95%) and directly used at the next step. mp 150 ºС. mp lit 150 ºС. Н NMR:
(
400 MHz, CDCl ) δ 7.49 (s, 1H), 6.87 (s, 1H), 6.54 (s, 1H), 5.91 (s, 1H), 5.75 (d, J = 8.1 Hz,
3
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H), 4.81 – 4.72 (m, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.53 (s, 3H), 2.54 – 2.26 (m, 4H), 2.04 (s,
1
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3H). C NMR (101 MHz, CDCl ): δ = 29.64, 37.26, 52.61, 55.94, 61.22, 61.27, 107.22, 119.95,
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22.53, 125.71, 134.31, 136.37, 141.32, 150.69, 151.57, 153.47, 170.36, 175.59.
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Synthesis of iodocolchinol (12) Colchiceine (11) (275.0 mg, 0.71 mmol) was dissolved in
water (37 mL), solid NaOH (300.0 mg, 7.50 mmol) was added and the solution was cooled to 0
°
C. The solution of NaI×2H O (2,439.0 mg, 16.00 mmol) and iodine (543.0 mg, 2.14 mmol) in
2
water (190 mL) was added dropwise to the reaction mixture and the resulting solution was stirred
for 1 h at 0 °C. The temperature was allowed to rise until 20 °C. Corresponding quantity of
Na SO was added to the solution to remove the excess of iodine. Then the mixture was acidified
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with conc. HCl to pH=2. The obtained yellow-green precipitate was isolated by filtration and
dried under reduced pressure at 50 – 60 °C. The residual solution was extracted with ethyl
acetate (3×250 ml), the combined organic layer was washed with brine, dried over Na SO . The
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crude product obtained after the solvent removing together with the precipitate were purified by
column chromatography, eluent petroleum spirit - ethyl acetate - ethanol (4:1:1), to afford 338.0
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mg, 93% of iodocolchinol (12). mp 238 ºС. mp lit 238 ºС. H NMR (400 MHz, DMSO-d ) δ
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.95 (s, 1H), 8.37 (d, J = 8.1 Hz, 1H), 7.17 (s, 1H), 6.86 (s, 1H), 6.76 (s, 1H), 4.42 (dt, J = 12.3,
1
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6.2 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.42 (s, 3H), 2.08 (s, 4H), 1.87 (s, 3H). С NMR (101
MHz, DMSO-d ): 22.51, 29.87, 37.78, 48.13, 55.72, 60.41, 81.29, 107.98, 109.96, 123.14,
26.49, 134.65, 139.06, 140.41, 142.30, 150.06, 152.10, 155.52, 168.20.
General procedure for the synthesis of compounds 10a-i. Iodocolchinol (12) (100.0 mg, 0.21
mmol), Pd(OAc) (2.3 mg, 0.01 mmol), CuI (3.9 mg, 0.02 mmol), PPh (8.1 mg, 0.03 mmol) and
AcOK (60.6 mg, 0.62 mmol) were placed into the two-neck flask under an inert atmosphere. In
the case of 10a and 10b compounds synthesis 131.1 mg (0.62 mmol) of K PO was used as a
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base instead of AcOK. Then anhydrous acetonitrile (4 mL) and the corresponding alkyne (0.25
mmol) were added. The mixture was stirred at 60 °C for 1 – 1.5 h, and then the temperature was
raised up to 80 °C until the completeness of the reaction (TLC control). The solvent was
removed under reduce pressure, a solid residue was purified by column chromatography to
afford the target products.
N-((1S)-2"-phenyl-1� ,2� ,3� -trimethoxy-6,7-dihydro-1H-benzo[5',6':5,4]cyclohepta[3,2-
f]benzofuran-1-yl)acetamide (10a). Purified by column chromatography, eluent petroleum spirit-
1
EtOAc-EtOH (5:1:1), as brownish-yellow powder, 86%, mp 140 ºС. H NMR (400 MHz, DMSO-
d6) δ 8.50 (d, J = 8.5 Hz, 1H), 7.93 (s, 1H), 7.91 (s, 1H), 7.57 (s, 2H), 7.52 (t, J = 7.7 Hz, 2H),
7.44 (s, 1H), 7.41 (t, J = 7.3 Hz, 1H), 6.81 (s, 1H), 4.66 – 4.59 (m, 1H), 3.85 (s, 3H), 3.80 (s,
13
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H), 3.46 (s, 3H), 2.29 – 1.97 (m, 4H), 1.93 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 168.53,
6
68.50, 155.13, 153.73, 152.36, 150.41, 138.26, 134.80, 132.42, 129.91, 129.30, 129.09, 128.96,
26.98, 124.57, 121.98, 108.08, 105.75, 101.99, 60.62, 60.56, 55.85, 48.54, 30.05, 22.75, 22.72.
MS: m/z (%) = 354 (19), 367 (40), 383 (45), 398 (100), 399 (30), 458 (81). Found С, 73.45; Н,
5.97. С Н NO requires: C, 73.51; H, 5.95%.
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N-((1S)-2"-(pyridin-2-yl)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide (10b). Purified by column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (4:1:1), as brown polycrystalline
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powder, 83%, mp 140 ºC. H NMR (400 MHz, DMSO-d
6
) δ 8.69 (d, J = 4.7 Hz, 1H), 8.51 (d, J
= 8.4 Hz, 1H), 7.96 (d, J = 3.6 Hz, 2H), 7.65 (s, 1H), 7.60 (d, J = 4.9 Hz, 2H), 7.41 (dd, J = 8.9,
4
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.5 Hz, 1H), 6.81 (s, 1H), 4.66 – 4.60 (m, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.46 (s, 3H), 2.26 –
1
3
6
.95 (m, 4H), 1.93 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 168.47, 154.76, 154.10, 152.38,
150.40, 150.00, 148.32, 140.61, 139.20, 137.35, 134.76, 129.53, 126.56, 124.43, 123.43, 122.64,
1
3
19.40, 108.06, 105.97, 104.87, 60.60, 60.56, 55.83, 48.57, 30.95, 30.01, 22.70. MS: m/z (%) =
68 (35), 384 (39), 399 (100), 400 (27), 415 (12), 458 (62). Found С, 70.81; Н, 5.67.
28 26 2 5
С Н N O requieres C, 70.73; H, 5.72%.
N-((1S)-2"-(hydroxymethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide (10c). Purified by column
chromatography, eluent petroleum spirit-EtOAc-EtOH (3:1:1), as yellow polycrystalline powder,
1
8
4%, mp 110 ºС. H NMR (400 MHz, DMSO-d
6
) δ 8.46 (d, J = 8.5 Hz, 1H), 7.51 (s, 1H), 7.46
(s, 1H), 6.79 (s, 1H), 6.75 (s, 1H), 5.43 (t, J = 5.8 Hz, 1H), 4.64 – 4.54 (m, 3H), 3.84 (s, 3H),
13
3
1
1
3
6
.79 (s, 3H), 3.41 (s, 3H), 2.22 – 1.70 (m, 7H). C NMR (101 MHz, DMSO-d ) δ 168.39,
58.42, 153.73, 152.23, 150.37, 140.59, 137.42, 134.75, 128.69, 126.18, 124.73, 121.81, 108.01,
05.52, 103.21, 60.59, 60.45, 56.22, 55.82, 48.40, 30.95, 30.05, 22.67. MS: m/z (%) = 294 (17),
6
21 (57), 337 (43), 352 (100), 353 (23), 411 (55). Found С, 67.07; Н, 6.24. С23H25NO requires
C, 67.14; H, 6.12%.
N-((1S)-2"-(1-hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
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10d). Purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (4:1:1),
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as yellow polycrystalline powder, 93%, mp 80 ºC. H NMR (400 MHz, DMSO-d
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8
.4 Hz, 1H), 7.50 (s, 1H), 7.45 (s, 1H), 6.79 (s, 1H), 6.70 (s, 1H), 5.51 (s, 1H), 4.84 (q, J = 6.5
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Hz, 1H), 4.63 – 4.55 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.29 – 1.93 (m, 4H), 1.90
1
3
(s, 3H), 1.47 (d, J = 6.5 Hz, 3H). C NMR (101 MHz, DMSO-d
6
) δ 168.43, 162.01, 161.98,
53.48, 152.24, 150.39, 140.61, 137.24, 134.77, 128.65, 126.17, 124.79, 121.79, 108.03, 105.55,
01.22, 62.30, 60.60, 60.46, 56.03, 55.83, 48.43, 30.06, 26.45, 22.68, 18.56. MS: m/z (%) = 321
1
1
(24), 335 (42), 351 (42), 366 (100), 367 (26), 425 (62). Found С, 67.76; Н, 6.42. С24
requires C, 67.75; H, 6.40%.
H27NO
6
N-((1S)-2''-(1-hydroxyсусlopentyl)-1',2',3'-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide (10e). Purified by column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (6:2:1), as dark-yellow oil, 86%.
1
H NMR (400 MHz, DMSO-d
H), 6.70 (s, 1H), 5.25 (s, 1H), 4.62 – 4.55 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.16
td, J = 12.1, 5.9 Hz, 2H), 2.05 (dd, J = 12.4, 6.5 Hz, 2H), 1.99 (s, 1H), 1.90 (s, 3H), 1.74 (dd, J
6
) δ 8.42 (d, J = 8.5 Hz, 1H), 7.48 (s, 1H), 7.44 (s, 1H), 6.79 (s,
1
(
1
3
= 12.2, 8.0 Hz, 3H), 1.68 – 1.55 (m, 1H), 1.54 – 1.43 (m, 1H), 1.20 – 0.99 (m, 2H). C NMR
101 MHz, DMSO-d ) δ 168.40, 163.40, 153.53, 152.21, 150.37, 140.61, 136.98, 134.76, 128.56,
26.32, 124.83, 121.61, 108.02, 105.49, 100.87, 78.09, 60.60, 60.45, 55.82, 48.42, 34.19, 30.05,
(
6
1
2
6
6.34, 24.78, 23.48, 22.68, 13.96. Found C, 69.85; H, 6.64. C27
.71%.
6
H31NO requieres C, 69.66; H,
N-((1S)-2"-(2-hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide (10f). Purified by column
chromatography, eluent petroleum spirit-EtOAc-EtOH (4:1:1), as yellow polycrystalline powder,
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4%, mp 125 ºС. H NMR (400 MHz, DMSO-d
(s, 1H), 6.78 (s, 1H), 6.62 (s, 1H), 4.81 (t, J = 5.4 Hz, 1H), 4.63 – 4.56 (m, 1H), 3.84 (s, 3H),
.79 (s, 3H), 3.78 – 3.73 (m, 2H), 3.41 (s, 3H), 2.91 (t, J = 6.6 Hz, 2H), 2.31 – 1.91 (m, 4H), 1.90
6
) δ 8.43 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.42
3
10
11
12
13
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15
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17
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30
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38
39
40
41
42
43
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45
46
47
48
49
50
51
52
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55
56
57
58
59
60
1
3
(
s, 3H). C NMR (101 MHz, DMSO-d
6
) δ 168.37, 156.91, 153.46, 152.18, 150.36, 140.59,
136.65, 134.75, 128.49, 126.69, 124.85, 121.21, 107.99, 105.25, 102.91, 62.78, 60.60, 60.48,
5
3
9.03, 55.80, 48.38, 31.93, 30.06, 22.67. MS: m/z (%) = 321 (15), 335 (57), 351 (36), 366 (100),
67 (26), 425 (57). Found С, 67.82; Н, 6.36. С24 requires C, 67.75; H, 6.40%.
H
27NO
6
N-((1S)-2"-(acetoxymethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide (10g). Purified by column
chromatography, eluent petroleum spirit-EtOAc-EtOH (6:1:1), as light-beige polycrystalline
1
powder, 41%. mp 82 ºC. H NMR (400 MHz, DMSO-d
6
) δ 8.47 (d, J = 8.3 Hz, 1H), 7.56 (s,
1
H), 7.49 (s, 1H), 6.99 (s, 1H), 6.79 (s, 1H), 5.22 (s, 2H), 4.58 (dd, J = 11.8, 7.5 Hz, 1H), 3.84 (s,
3H), 3.79 (s, 3H), 3.42 (s, 3H), 2.23 – 2.11 (m, 2H), 2.08 (s, 3H), 2.03 (dd, J = 12.7, 5.6 Hz, 2H),
1
3
1
1
.91 (s, 3H). C NMR (101 MHz, DMSO-d
6
) δ 169.95, 168.45, 153.95, 152.33, 152.12, 150.37,
40.60, 138.54, 134.74, 129.11, 125.70, 124.51, 122.29, 108.05, 107.03, 105.75, 60.59, 60.49,
57.95, 55.83, 48.50, 30.61, 29.98, 22.66, 20.56. MS: m/z (%) = 334 (30), 351 (45), 363 (33), 394
100), 395 (26), 453 (77). Found С, 66.11; Н, 6.12. С25 requires C, 66.21; H, 6.00%.
(
H27NO
7
N-((1S)-2"-(8-(metoxycarbonyl)octyl)-1′,2′,3′-trimetoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10h). Purified by column
1
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (5:1:1), as brownish oil, 49%. H
NMR (400 MHz, DMSO-d
6
) δ 8.42 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 6.78 (s, 1H),
6
.57 (s, 1H), 4.63 – 4.54 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.57 (s, 3H), 3.42 (s, 3H), 2.75 (t, J
=
7.4 Hz, 2H), 2.22 – 1.76 (m, 7H), 1.68 (dd, J = 14.3, 7.1 Hz, 2H), 1.54 – 1.47 (m, 2H), 1.41 –
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1
.15 (m, 10H). C NMR (101 MHz, DMSO-d
6
) δ 168.41, 156.02, 153.73, 152.22, 150.35,
140.57, 137.19, 134.76, 128.63, 126.17, 124.75, 121.55, 120.59, 108.02, 105.52, 104.90, 62.78,
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0.57, 60.51, 55.83, 48.90, 48.38, 46.47, 30.05, 22.70, 12.07. MS: m/z (%) = 447 (10), 461 (19),
78 (19), 492 (100), 493 (30), 551 (38). Found С, 69.73; Н, 7.47. С32 requires C, 69.67;
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H41NO
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H, 7.49%.
N-((1S)-2"-((diethylamino)methyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10i). Purified by column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (2:1:1), as light-brown
1
polycrystalline powder, 72%, mp 148 ºC. H NMR (400 MHz, DMSO-d ) δ 8.47 (d, J = 8.6 Hz,
6
1
3
H), 7.47 (d, J = 6.8 Hz, 2H), 6.79 (s, 1H), 6.75 (s, 1H), 4.59 (dd, J = 6.9, 3.9 Hz, 1H), 3.84 (s,
H), 3.79 (s, 3H), 3.75 (s, 2H), 3.43 (s, 3H), 2.56 – 2.51 (m, 4H), 2.21 – 2.10 (m, 2H), 2.04 (dd,
1
3
J = 12.5, 7.1 Hz, 2H), 1.90 (s, 3H), 1.03 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, DMSO-d ) δ
6
168.41, 156.02, 153.73, 152.22, 150.35, 140.57, 137.19, 134.76, 128.63, 126.17, 124.75, 121.55,
1
08.02, 105.52, 104.90, 62.78, 60.57, 60.51, 55.83, 48.90, 48.38, 46.47, 30.05, 22.70, 12.07. MS:
m/z (%) = 335 (44), 336 (18), 368 (16), 394 (100), 395 (34), 466 (16). Found С, 69.46; Н, 7.38.
С H N O requires C, 69.50; H, 7.35%.
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5
Synthesis
of
N-((1S)-2"-(pyrollidino)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10l). Iodocolchinol (12) (100.0
mg, 0.21 mmol), Pd(PPh ) Cl (7.2 mg, 0.01 mmol), CuI (3.9 mg, 0.02 mmol) were added into
3
2
2
the two-neck flask, and the flask was filled with argon. Pyrrolidine (4 ml) was appended to the
mixture and the solution was cooled to 0°С, then the solution of propargylbromide (29.4 mg,
0
.25 mmol) in toluene was added dropwise. The mixture was stirred at 50 °С. After the
completeness of the reaction the resulting solution was poured into 15 mL of water and the
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resulting mixture was extracted with ethyl acetate (3×10 ml). The combined organic layer was
washed with brine, dried over Na SO . The solvent was removed under reduced pressure. A solid
2
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residue was purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol
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56
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58
59
60
1
(
3:1:1) to afford product 10l as brownish polycrystalline powder, 80%, mp 123 °С. H NMR
(400 MHz, DMSO-d ) δ 8.60 (d, J = 8.5 Hz, 1H), 7.48 (s, 2H), 6.78 (s, 1H), 6.74 (s, 1H), 4.61 –
6
4
–
.56 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.76 (d, J = 3.7 Hz, 2H), 3.42 (s, 3H), 2.56 (s, 4H), 2.18
1
3
2.09 (m, 2H), 2.04 (dd, J = 12.4, 6.6 Hz, 2H), 1.90 (s, 3H), 1.71 (s, 4H). C NMR (101 MHz,
DMSO-d ) δ 168.43, 156.10, 153.66, 152.23, 150.36, 140.58, 137.33, 134.77, 128.67, 126.19,
6
1
24.75, 121.63, 108.02, 105.55, 104.28, 60.58, 60.50, 55.82, 53.29, 51.73, 51.59, 48.43, 42.53,
30.06, 23.33, 23.21, 22.68. Found C, 69.88; H, 6.89. C H N O requires C, 69.81; H, 6.94%.
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Preparation of lamb brain tubulin. Tubulin was purified from lamb brain by ammonium
sulfate fractionation and ion-exchange chromatography. The pure protein was stored in liquid
44
nitrogen and prepared as described.
Protein concentrations were determined
spectrophotometrically with a Perkin Elmer spectrophotometer Lambda 800 and an extinction
-
1
-1
-1
-1
coefficient at 275 nm of 1.07 L.g .cm in 0.5% SDS in neutral aqueous buffer or 1.09 L.g .cm
in 6 M guanidine hydrochloride.
Tubulin polymerization. Microtubule assembly was performed in 20 mM sodium phosphate
buffer, 1 mM EGTA, 10 mM MgCl , and 3.4 M glycerol, 1 mM GTP pH 6.5. The reaction was
2
started by warming the samples at 37 °C and the mass of polymer formed was monitored by
turbidimetry at 350 nm with a POLARstar BMG Labtech spectrophotometer using 96-well plate.
Samples containing the compound and controls had less than 1% residual Me SO.
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Cell cultures. HEK-293, human embryonic kidney cell line (ATCC, CRL-1573), Jurkat human
T-lymphoma (ATCC, TIB-152), AsPC-1 pancreatic human adenocarcinoma cell line (ATCC,
35
CRL-1682), and Wnt-1 related murine epithelial cell line W1308, generated by us earlier, were
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used in the study. HEK-293 and AsPC-2 were grown in DMEM supplemented with 10% fetal
–
5
calf serum (FCS), pen-strep-glut, and 2-ME 5 x 10 M (all from PanEco, Moscow, Russian
Federation). Jurkat and W1308 cells were grown in RPMI-1640 with the same supplements.
Cells were passed by trypsinization using Trypsin /EDTA solution (PanEco, Moscow, Russian
Federation) twice a week. Twenty four hours before assays, cells were seeded in the appropriate
5
plates (96- or 24-well plates) adjusted to 3x10 cells/mL and incubated overnight to achieve
standardized growth conditions.
Confocal analysis. For confocal analysis AsPC-1 cells were grown overnight on sterile cover
slips in 200 µl of complete culture medium in 6-well plates (Costar). Compound 10c in different
concentrations was dissolved in 4 ml of complete medium and added to the wells. Cells were
cultivated for 72 hrs. Nuclei dye Hoecst 33342 (Sigma) and mitochondrial tracker MitoTreckRed
(Invitrogen) were added for 1 hr. Finally cells were fixed with 1% paraformaldehyde, washed,
and polymerized with Mowiol 4.88 medium (Calbiochem, Germany). Slides were analyzed using
Eclipse TE2000 confocal microscope (Nikon, Japan).
MTT-assay. Cytotoxic effect of the prodrugs was estimated by a standard 3-(4, 5-dimethyl-2-
45
thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test as was described earlier.
In short, different dilutions of the new compounds using series of 10 times dilutions from 10 µM
to 1 nM were prepared on a separate plate and then transferred in 100 ul to the plates with the
cells. Non-treated cells served as controls. Plates were incubated for 72 hrs. For the last 6 hours
250 µg/ml of MTT was added in 10 ul to each well. After the incubation culture medium was
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removed and 100 µl dimethylsulfoxide was added to each well. Plates were incubated at shaking
for 15 min to dissolve formazan. Optical density was read on spectrophotometer Titertek (UK) at
5
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40 nm. Results were analyzed by Excel package (Microsoft). Cytotoxic concentration giving
0% of dead cells (IC50) was calculated from the titration curves. The inhibition of proliferation
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(
inhibition index, II) was calculated as [1 – (ODexperiment / ODcontrol)], where OD was MTT optical
density.
Analysis of apoptosis by flow cytometry. Percentage of apoptotic cell was analyzed by 3,3'-
dihexyloxacarbocyanine iodide (DiOC
6
) and propidium iodide (PI) (Sigma Chemical Co) double
(Invitrogen) was used to evaluate
staining. The cationic lypophilic fluorochrome DiOC
6
4
6
transmembrane potential in mitochondria. Cells were harvested, washed in pre-warmed PBS
6
supplemented with 2% of FCS, resuspended at 10 cells/ml in PBS/2%FCS, 40 µM DiO C
6
and
o
5
ug/ml PI and incubated for 30 minutes at 37 C. Cells were washed with PBS/2% FCS and
analyzed by flow cytometry using FACScan device (BD, USA). Total 2000 events were
collected. The results were analyzed using CellQuest software (BD Biosciences).
Cell cycle analysis. Cell cycle was analyzed using PI-stained DNA. Two million cells were
harvested at indicated time, washed in ice-cold PBS, fixed by the addition of 70% ethanol and
o
left for 2 hours at -20 C. Thereafter, the cells were washed twice in PBS, stained with 50 μg/ml
of PI (Sigma Chemical Co) in PBS and analyzed by flow cytometry.
Animals. C57BL/6 mice were purchased from Pushchino Affiliation of Shemyakin-Ovchinnikov
Institute of Bioorganic Chemistry RAS, Moscow. All mice were 6–8 wk old and maintained in
minimal pathogen animal facility at the Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry RAS, Moscow. All studies were conducted in an AAALAC accredited facility in
compliance with the PHS Guidelines for the Care and Use of Animals in Research.
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Wnt-1 tumor growth and treatment in vivo. Wnt-1 tumor cells (10 /mouse) were obtained as
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7
described, and inoculated subcutaneously into the left inguinal mouse fat pad (MFP #4). The
injection of cells in 50 µl of PBS was performed through the skin of anesthetized mice. Tumor
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size was measured with vernier calipers twice a week and calculated using the formula
2
(
W xL)/2, where W and L corresponded to width and length of tumors. This protocol was
efficient in the induction of tumors in 100% of mice approximately on day 30 after the cell
inoculation. Experiment was started only in mice (n=7 per group) with palpable tumors at day
3
2
7. The volume of tumors was comparable (100 mm ) in both groups before 10c treatment.
Compound 10c was injected in 200µl of saline intravenously into the orbital sinus of mice once a
week at days 27, 34, 41, and 48. Control group was injected with saline only. The experiment
was terminated at day 51 when two control mice died.
Short term effect of colchicine analogues on endothelial cells (interphase effect). Colchicine,
combretastatin A4, compound 10c were dissolved in dimethyl sulfoxide at 20 mM concentration.
Four µl of each were transferred to 200 µl of saline (1 mg/kg of weight), mixed with 50 µl of
rhodamine B (100 µl/ml of water) and injected i.v. into C57BL/6 intact mice (3 per group).
Control animals were injected with dimethyl sulfoxide and rhodamine B only. Four hours later
mice were sacrificed by cervical dislocation. Major organs were collected, cut in pieces, and
o
extracted with 0.3N acidic acid in ethanol for 2 hours at 37 C. Supernatants were collected,
centrifuged 10 min at 10000 rpm, and cleared fraction was transferred into 96-well black plates
(
SPL). Fluorescence was measured using GlomaxMulti reader (Promega) with 540 nm filter. For
confocal analysis small pieces (1x1 mm) of nonfixed tissue were transferred on the microscopic
glass, layed with Mowiol 4.88 medium (Calbiochem, Germany), covered with the cover glass
and squeezed. Slides were analyzed using Eclipse TE2000 confocal microscope (Nikon, Japan).
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Statistical analysis
Statistical analysis was performed using Excel Student's t-test. Comparison values of p <0.05
were considered statistically significant.
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Acknowledgments
We thank the Russian Foundation for Basic Research (projects 14-03-91342 and 12-03-00214-a),
The Ministry of Education and Science of The Russian Federation (project 4.619.2014/K). The
research is partly supported by the grant № 02.В.49.21.0003 of The Ministry of Education and
Science of the Russian Federation to N.I. Lobachevsky State University of Nizhni Novgorod.
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References
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microtubule-targeting agents. Breast Cancer Res. and Treat. 2012, 133, 821-830. (c)
Katsetos, C.D.; Dráber, P. Tubulins as therapeutic targets in cancer: from bench to bedside.
Curr. Pharm. Des. 2012, 18, 2778-2792. (d) Stanton, R.A.; Gernert, K.M.; Nettles, J.H.;
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