Synthesis and cytostatic properties of polyfunctionalized furanoallocolchicinoids

Article abstract of DOI:10.1016/j.ejmech.2016.11.020

A series of furan-based allocolchicinoids was prepared from commercially available colchicine via a nine-step reaction sequence. Cytostatic activity, cell cycle arrest, apoptosis, tubulin and F-actin expression were studied in vitro in 2D and 3D cultures of normal and tumor epithelial keratinocytes, endothelial and mesenchymal cells. Among the prepared furanoallocolchicine analogues, 14a and 7a displayed the most pronounced anti-cancer activity. These compounds induced two types of effects: (a) cell cycle arrest in the G2/M phase as a direct consequence of effective tubulin binding (metaphase effect), and (b) pronounced cell stress (as evidenced by the overexpression of tubulin and F-actin), which was caused by the hyperpolarization of mitochondrial and lysosomal membranes (interphase effect).

Full text of DOI:10.1016/j.ejmech.2016.11.020

Accepted Manuscript
Synthesis and cytostatic properties of polyfunctionalized furanoallocolchicinoids
Iuliia A. Gracheva, Iuliia V. Voitovich, Vladimir I. Faerman, Nikolay S. Sitnikov,
Ekaterina V. Myrsikova, Hans-Gunther Schmalz, Elena V. Svirshevskaya, Alexey Yu
Fedorov
PII:
S0223-5234(16)30958-8
10.1016/j.ejmech.2016.11.020
EJMECH 9053
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 August 2016
Revised Date: 8 November 2016
Accepted Date: 9 November 2016
Please cite this article as: I.A. Gracheva, I.V. Voitovich, V.I. Faerman, N.S. Sitnikov, E.V. Myrsikova,
H.-G. Schmalz, E.V. Svirshevskaya, A.Y. Fedorov, Synthesis and cytostatic properties of
polyfunctionalized furanoallocolchicinoids, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.11.020.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis and Cytostatic Properties of
Polyfunctionalized Furanoallocolchicinoids
Iuliia A. Grachevaa, Iuliia V. Voitovicha, Vladimir I. Faermanа, Nikolay S. Sitnikova, Ekaterina
V. Myrsikovab, Hans-Gunther Schmalzc, Elena V. Svirshevskayab,d*, Alexey Yu. Fedorova*
aDepartment of Organic Chemistry, Nizhni Novgorod State University, Gagarina av. 23, Nizhni
Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: afnn@rambler.ru
bShemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Tel/Fax (495) 330
40-11, E-mail: esvir@yandex.ru
cDepartment of Chemistry, University of Cologne, Greinstrasse 4, 50939 Köln, Germany
Fax: +49 221-470-3064, Tel: +49 221 470 3063, E-mail: schmalz@uni-koeln.de
dResearch Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of the Russian
Federation, Moscow
*Corresponding authors
Prof. Fedorov Alexey Yu.
aDepartment 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.
1

ACCEPTED MANUSCRIPT
dShemyakin-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
Keywords: colchicine, allocolchicinoids, cross-coupling, antitumor activity, tubulin binding
agents.
Abstract
A series of furan-based allocolchicinoids was prepared from commercially available colchicine
via a nine-step reaction sequence. Cytostatic activity, cell cycle arrest, apoptosis, tubulin and F-
actin expression were studied in vitro in 2D and 3D cultures of normal and tumor epithelial
keratinocytes, endothelial and mesenchymal cells. Among the prepared furanoallocolchicine
analogues, 14a and 7a displayed the most pronounced anti-cancer activity. These compounds
induced two types of effects: (a) cell cycle arrest in the G2/M phase as a direct consequence of
effective tubulin binding (metaphase effect), and (b) pronounced cell stress (as evidenced by the
overexpression of tubulin and F-actin), which was caused by the hyperpolarization of
mitochondrial and lysosomal membranes (interphase effect).
1. Introduction
The microtubule network is an essential component of the cytoskeleton of eukaryotic cells [1]. It
is formed via a dynamic polymerization-depolymerization process involving α- and β-tubulin
protein subunits. Inhibition of this process leads to the disruption of mitotic spindle formation,
cell cycle arrest and apoptosis. Compounds able to stabilize or destabilize the mitotic spindle are
2

ACCEPTED MANUSCRIPT
referred to as spindle poisons [1] or antimitotics, and are of common use in cancer therapy [2].
Colchicine (1), an alkaloid isolated from Colchicum autumnale, was the first discovered tubulin
destabilizing agent [3]. On the molecular level, it prevents self-assembly of tubulin by distorting
the interface between α/β-tubulin heterodimers (colchicine binding site of tubulin [4]). This
interaction results both in antimitotic activity and suppression of cell motility. In clinical
practice, colchicine is approved for the treatment of familial Mediterranean fever, Behcet’s
disease as well as acute gout, chondrocalcinosis and other types of microcrystalline arthritis [5-
8]. Potential indications include primary biliary cirrhosis, psoriasis, amyloidosis, various
dermatitis, relapsing polychondritis, necrotizing vasculitis, Sweet’s syndrome and
leukocytoclastic vasculitis [9-12]. Colchicine could also be useful for the treatment of
cardiovascular diseases [13, 14]. Importantly, colchicine can enter all tissues and is able to
accumulate in leukocytes [15-17]. Destabilisation of β-tubulin manifests both in antimitotic
activity and suppression of cell motility. However, high doses of colchicine induce general
toxicity mostly in the gastro-intestinal tract, which prevents its application in cancer
chemotherapy [18].
Although colchicine itself cannot be used for the treatment of cancer, structurally related
compounds (such as Z-stilbenes [19], 4-arylcoumarins [20], allocolchicine (2) [21] and their
analogs [22]) represent promising leads in the search for new anticancer agents. An advantage of
the antimitotics interacting with the colchicine binding site of tubulin is their ability to overcome
P-glycoprotein/β-III tubulin-mediated drug resistance (a common issue for Vinca alkaloids and
taxanes) as well as their pronounced antiangiogenic or antivascular activity [23]. While
colchicine is an old drug, it still represents an attractive profile for the treatment of many
diseases of different origin. Thus, the search for new colchicinoids can lead to the development
3

ACCEPTED MANUSCRIPT
of less toxic but more efficient compounds, offering new perspectives for the well-known
venerable drug.
Our group has recently synthesized a series of heterocyclic allocolchicine analogs, containing
indole (3-5) [24] or benzofurane (6) [25] fragments replacing the C-ring (Fig.1). These
derivatives represent the first examples of heteroarene-based allocolchicinoids [26]. Derivatives
3b, 4b, 5b, 6a and 6b, bearing a hydroxyl group in benzylic or pseudobenzylic position, possess
the highest antimitotic activity. Based on the accumulated SAR data, this work was aimed at the
preparation and investigation of the biological properties of furanoallocolchicinoids of type 7,
containing a hydroxyl group both in benzylic and pseudobenzylic position.
5
4
6
MeO
MeO
MeO
MeO
7
3
A
B
NHAc
NHAc
2
1
8
MeO
C
12
MeO
O
11
CO2Me
OMe
(-)-(aR, 7S) colchicine 1
(-)-allocolchicine 2
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
X
X
X
OH
OH
NHAc
MeO
MeO
MeO
MeO
MeO
MeO
MeO
N
N
O
Me
O
N
4
5
3
7
6
Me
Me
OH
3-5a X = O
R
R
6a R = H
6b R = Me
3-5b X = OH
3-5c X = NHAc
Fig. 1. Structures of colchicine (1), allocolchicine (2), pyrroloallocolchicinoids 3-5,
furanoallocolchicinoids 6, and the target molecules 7.
2. Results and discussion
Chemistry
A semi-synthetic approach to the target allocolchicinoids starting from colchicine (1) was
envisaged. We assumed that the furan ring in the tetracyclic scaffold 7 can be formed by a
transition metal-catalyzed Sonogashira coupling/cyclisation reaction sequence [27] (Scheme 1),
4

ACCEPTED MANUSCRIPT
starting from the tricyclic precursor 8. Ketone 8 is readily accessible from amine 9 through a
biomimetic transamination reaction [28]. The latter intermediate can be synthesized from
commercially available colchicine (1), following the previously reported procedures
(electrocyclic rearrangement/oxidation of colchiceine into N-acetyliodocolchinol [22g, 29]
followed by deacylation [30]).
Scheme 1. Strategy for the synthesis of furanoallocolchicinoids of type 7.
First, hydrolysis of the tropolone ether function of colchicine (1) was accomplished with HCl in
AcOH to afford colchiceine (10) in almost quantitative yield (Scheme 2). Oxidative ring
contraction in 10 according to the Windaus procedure (NaOH, I2, KI) yielded iodocolchinol (11)
[22g]. The hydroxy group and the NH of the acetamide in 11 were protected with
methoxymethylchloride and Boc2O, respectively, to afford 12 in 64% yield over two steps.
Deacylation of 12 with MeONa followed by the simultaneous MOM- and Boc-deprotection
under acidic conditions gave deacetyliodocolchinol (9) in a good yield. Amine 9 was readily
transformed into ketone 8 using Rapoport’s procedure [28]. The resulting product 8 smoothly
underwent catalytic tandem Sonogashira coupling – cyclization reactions with terminal alkynes
13a-d to give furanoallocolchicinoids 14a-d in 51-72% yield (Scheme 2, Table 1). The choice of
alkynes 13a-d was based on our previous SAR-studies [25], which indicated that the presence of
a CH2OH, CH(CH3)OH or CH2OAc group in the side chain of the furan ring leads to the
5

ACCEPTED MANUSCRIPT
improved in vitro antitumor activity of furanoallocolchicinoids 6. Finally, the ketones 14a-d
were reduced to the corresponding alcohols 7a-c. Reduction of the carbonyl group in 14d
resulted in the formation of alcohol 7a due to the hydrolysis of the ester functionality under basic
conditions.
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
Boc
Ac
NHAc
NHAc
O
NHAc
N
a
b
c, d
MeO
MeO
MeO
11
93%
53%
95%
MeO
O
OH
10
12
OMOM
83%
I
OMe
OH
I
O
(-)-(a , 7 ) colchicine 1
R
S
e, f
MeO
MeO
MeO
MeO
MeO
MeO
MeO
OH
O
R
N
NH2
TsO
O
MeO
13a-d
h
Me
MeO
MeO
g
DBU, DCM/DMF
MeO
MeO
O
O
R
rt
7a-d
14a-d
OH
8
OH
9
65%
I
I
R
Scheme 2. Synthesis of furanoallocolchicinoids 7. Reagents and conditions: (a) 0.1 M HCl,
AcOH, 100 °C, 3 h; (b) NaOH, I2, KI, H2O, 0-5 °C, 2 h; (c) MOMCl, DIPEA, DCM, 0-20 °C, 20
h; (d) Boc2O, DMAP, TEA, MeCN, reflux, 26 h; (e) NaOMe (20 mol%), MeOH, r.t., 1.5 h; (f)
HCl, EtOH, r.t., 20 h; (g) Pd(OAc)2 (0.05 mol%), CuI (0.1 mol%), AcOK (3 equiv.), Ph3P (0.15
mol%), MeCN, 70 °C 4-6 h; (h) NaBH4, THF/H2O/EtOH, r.t., 1-2 h.
Biology
In vitro cytostatic activity of the synthesized compounds against tumor and normal cell lines in
2D and 3D cultures.
The in vitro cytostatic activity of all synthesized compounds was first tested in 2-dimensional
(2D) cultures against a panel of tumor epithelial cell lines (BxPC-3, PANC-1, Colo-357,
MiaPaCa-2, and A549), nontransformed immortalized keratinocytes (HaCaT), the endothelial
cell line EA.hy926, and, finally, against completely normal bone marrow-derived mesenchymal
6

ACCEPTED MANUSCRIPT
stem cells (MSC), employing MTT staining after incubation of the cells with the compounds for
72 h. The calculated IC50 values are summarized in Table 1.
Table 1. Isolated Yields (from 8) of the Compounds and their In Vitro Cytotoxicity (IC50a , nM)
IC50 , nM
R
Yield,
%
CH2OH
70
16b
>800 >800
14a
16
16
64
16
16
c
CH(CH3)OH
(CH2)2OH
72
51
80
400
70
100 >100 40
-
-
-
14b
14c
400 >400 >400 400
-
-
-
CH2OAc
69
80
-
-
14d
16
16
16
16
CH2OH
CH(CH3)OH
66(83)d
>800 >800
7a
7b
16
30
16
30
16
64
16
64
16
80
16
-
66
-
-
(CH2)2OH
57
400
400 >400 >400 400
-
-
-
7c
80
80 160
160 400 >800 >800
80
1
aIC50 (nM) concentration inducing 50% inhibition of cell growth.
bThe most active compounds are shown in Italics Bold.
c not determined.
dThe yield of 7a from 14d.
Ketone 14a with a hydroxymethyl substituent at the furan ring, the corresponding
(biodegradable) acetate 14d, as well as diol 7a belonging to the same series, exhibited potent
antiproliferative activity against tumor cells with IC50 values in the nanomolar range (Table 1,
Fig. 2). Allocolchicinoids 14b and 7b with a 1-hydroxymethyl-substituted furan fragment proved
to be less active. Compounds 14c and 7c with a 2-hydroxyethyl side chain showed two orders of
magnitude lower activity in comparison to that of the derivatives 14a and 7a, containing a
7

ACCEPTED MANUSCRIPT
hydroxyl group in the pseudobenzylic position. These observations correlate well with the results
obtained by us earlier [25], indicating that the presence of a hydroxy function in the
pseudobenzylic position of the furan ring in the type compounds of type 6 is a crucial structural
feature, which determines the cytostatic activity.
The major activity of colchicine and the two most active analogues 14a and 7a is a cytostatic
effect, as even at the highest doses the inhibition never reaches 100% (Fig. 2, a-b). This seems to
be specific for all colchicinoids, as previously shown also for furanoallocolchicinoids and
combretastatin A4 [24, 25].
In addition to pancreatic epithelial tumor cells, we also studied the effect of allocolchicinoids
14a and 7a on normal MSC and immortalized keratinocytes HaCaT, and endothelial cells EA.hy
926. Among these cell lines, HaCaT cells were as sensitive as tumor cells (Table 1, Fig. 2, b)
while both MSC and endothelial cells EA.hy926 appeared to be resistant to these compounds
(Table 1, Fig. 2, c, d). EA.hy926 and MSC differ from all other cells used (including HaCaT) by
a lower proliferation rate, which may explain their resistance. Multicellular tumor spheroids, also
called 3-dimensional (3D) cultures, are used as a model for avascular tumors [31, 32]. Many
authors show that the cells in 3D cultures are more resistant to chemotherapeutic drugs [33, 34].
As far as we have found, the effect of tubulin-destabilizing drugs has never been studied in 3D
cultures. Thus, we tried to estimate the effect of colchicine and its analogues 14a and 7a on the
growth of cells in 3D cultures using one sensitive cell line (Colo-357) and one resistant cell line
(EA.hy926). We found no differences between 2D and 3D cultures both with respect to their
sensitivity towards colchicinoids and the level of activity (Fig. 2, e, f). Endothelial cells
EA.hy926 were resistant to the drugs both in 2D and 3D cultures, while Colo-357 responded by a
decreased proliferation and IC50, coincided in 2D and 3D cultures.
8

ACCEPTED MANUSCRIPT
Fig. 2. Cytostatic effects of colchicine and allocolchicinoids 14a and 7a.
Pancreatic ductal adenocarcinoma Colo-357 (a, e), keratinocytes HaCaT (b), mesenchymal stem
cells (c), and endothelial EA.hy 926 (d, f) cells were incubated without or with colchicine
(Colch), 14a, or 7a for 72 h in 2D (a-d) and 3D (e-f) cultures. The results are shown as inhibitory
indexes (II) calculated as II=1-ODexp/ODcontrol, where OD – optical density in control and
9

ACCEPTED MANUSCRIPT
experimental (exp) cultures. Dashed lines in (a) and (b) show 50% cutoff. Intersections of these
lines with abscissa determine IC50 for the drugs.
Cell cycle inhibition by colchicine and its synthetic analogues
Cell cycle inhibition by colchicine 1 and allocolchicinoids 14a and 7a was studied in 2D and 3D
cultures. In the 2D cultures, all the compounds effectively induced accumulation of the cells in
G2/M phase, which steadily resulted in apoptosis (Fig. 3, a-f). Samples 14a and 7a were slightly
more effective (Fig. 3, d), possibly due to lower IC50. Analysis of cell cycle in 3D cultures was
fulfilled using Colo-357 and MiaPaCa-2 cell lines. We demonstrated that both cell lines
responded to tubulin-destabilizing agents with the accumulation of cells in G2/M phase (Fig. 3,
g-n) as it was found in 2D cultures.
10

ACCEPTED MANUSCRIPT
Fig. 3. Induction of cell cycle arrest by colchicine and compounds 14a and 7a.
Colo-357 (a-j) or MiaPaCa-2 (k-n) cells were incubated in 2D (a-f) or 3D (g-n) cultures for 72 h
without (c, g, k) or with 1 µM of the drugs, trypsinized, permeabilized, stained with propidium
iodide and analyzed by flow cytometry. Gating of cells is shown in dot plots (a-b) with grey
lines. a-b. Dot plots of control (a) and 7a treated (b) cultures clearly show cell accumulation in
G2/M phase (blue arrows) and apoptosis induction (red arrows) in 7a treated cells. c. Cell cycle
11

ACCEPTED MANUSCRIPT
analysis of control (blue histogram) and colchicine treated (red histogram) cells. d. Percentage of
cells in different phases. e-f. Cell cycle distribution in 14a (e) and 7a (f) treated cultures. Arrows
interlink different phases in dot plots and histograms. g-n. Histograms correspond to control
(blue), colchicine 1 (red), 14a (green), and 7a (purple) cultures. Apoptotic cells were not
included.
Effect of allocolchicinoids on
β
-tubulin expression and the mitotic spindle formation
-tubulin assembly
The effect of colchicine 1 and the two most active derivatives 14a and 7a on
β
and mitotic spindle formation was studied using the Colo-357 cell line which is characterized by
a high percentage of cells staying in the G2/M phase in control cultures. We found that all the
compounds studied effectively disrupted the mitotic spindle, which led to the chromosome
scattering and the inability of the cells to divide (Fig. 4, inserts). To estimate the effect of cell
treatment with colchicine 1 and its analogues on total
fields were analyzed by ImageJ soft and red pixels were counted. We found that treatment with 1
induced -tubulin overexpression in Colo-357 cells.
β-tubulin expression, several microscopic
β
12

ACCEPTED MANUSCRIPT
Fig. 4. Effect of colchicine and compounds 14a and 7a on mitotic spindle formation and β-
tubulin expression in pancreatic epithelial cells Colo-357.
Colo-357 cells were incubated for 24 h without (a) or with 1 µM of colchicine 1 (b), 14a (c), or
7a (d), fixed, stained with anti-β-tubulin antibody (red) and nuclei stain Hoechst 33342 (blue).
Mean fluorescence intensity (MFI) was counted on several fields by red pixel numbers using
ImageJ soft. Statistical difference (p<0.05) is pointed by asterisk. Grey inserts show DNA
condition. Scale bars correspond to 10-20 µm.
13

ACCEPTED MANUSCRIPT
Confocal microscopy is a semi-quantitative technique. To verify confocal data, Colo-357 cells
were incubated in 3D cultures with allocolchicinoids for 48 h, trypsinized, and β-tubulin
expression was analyzed by flow cytometry, which permits the simultaneous analysis of many
cells. The results of flow cytometry demonstrated that both colchicine 1 and its analogues 14a
and 7a significantly increased total tubulin expression (Fig. 5, a, c).
Interphase effect induced by allocolchicinoids
In addition to the overexpression of β-tubulin, the allocolchicinoids also induced overexpression
of F-actin (Fig. 5, b). Actin is another protein involved in cell division. It forms microfilaments
(F-actin from filamentous) which are essential for the cellular mobility and cell contraction
during the division. The effect of allocolchicinoids on F-actin expression depended on the type
of cells. A significant portion of Colo-357 cells treated with colchicine and 14a lost F-actin,
while other cells overexpressed it (Fig. 5, b). At the same time, all allocolchicinoids induced only
F-actin overexpression in MiaPaCa-2 cells (Fig. 5, d). Colo-357 cells form dense spheroids in 3D
cultures (Fig. 5, e), while MiaPaCa-2 under the same condition grows as a film. The difference in
growth conditions can possibly explain the different effects of the allocolchicinoids on these
cells.
Figure 5c shows an overview of PANC-1, Colo-357, and MiaPaCa-2 3D cultures before and
after the treatment. Colchicine and its derivatives 14a and 7a disrupted preformed spheroids and
changed their internal architecture due to interphase effects.
14

ACCEPTED MANUSCRIPT
Fig. 5. Effects of colchicine and compounds 14a and 7a on tubulin and F-actin expression in 3D
cultures.
15

ACCEPTED MANUSCRIPT
Cells were incubated in 3D cultures for 48 h without or with 1 µM of the drugs, trypsinized,
permeabilized, stained with anti-tubulin antibodies and phalloidin-AlexaFluor488 (F-actin), and
analyzed by flow cytometry. a-d. Effect of colchicine 1 (red), 14a (blue) or 7a (green) on β-
tubulin (a, c) and F-actin (b, d) expression by Colo-357 (a-b) and MiaPaCa-2 (c-d). Control is
shown in grey. e. Confocal images of 3D cultures formed by PANC-1, Colo-357, MiaPaCa-2
after the treatment. Spheroids were fixed, permeabilized, stained with anti-β-tubulin antibody
(red), and PhalloidinAlexaFluor488 (green). Nuclei are stained with Hoechst 33342 (blue). Scale
bar corresponds to 30-50 µm.
Interphase effect of allocolchicinoids is not restricted to the overexpression of β-tubulin and F-
actin. Other protein systems also react to mitotic poisons. We have analyzed the effect of
allocolchicinoids on mitochondrial and lysosomal membrane potential using pH sensitive
molecular probes DiOC6 (a mitochondrial dye) and LysoTrackRed (a lysosomal dye). We found
out that both mitochondrial and lysosomal membranes were hyperpolarized (Fig. 6, a-c). Cell
membrane permeability was analyzed by propidium iodide staining. As was expected,
allocolchicinoids also increased cell membrane permeability (Fig. 6, d).
16

ACCEPTED MANUSCRIPT
Fig. 6. Interphase effects of colchicine 1, the compounds 14a and 7a in 3D cultures.
Cells were incubated in 3D cultures for 48 h without or with 1 µM of the drugs, trypsinized, and
stained with mitochondrial dye DiOC6 (a, b), lysosomal dye LysoTrackerRed (c) or propidium
iodide (d). DiOC6 staining is shown for Colo-357 (a) and MiaPaCa-2 (b).
3. Conclusions
We have synthesized a series of furan-derived allocolchicinoids of type 7, containing a hydroxyl
group both in benzylic and pseudobenzylic position, as a novel class of analogs of the natural
product colchicine (1). These compounds were efficiently obtained in nine preparative steps
17

ACCEPTED MANUSCRIPT
starting from commercially available colchicine. Compounds 14a and 7a exhibited cytotoxicity
towards epithelial cell lines in the nanomolar concentration range. Low proliferating cells such as
endothelial cells EA.hy 926, as well as mesenchymal stem cells showed a decreased
susceptibility to the drugs. Colchicinoids 14a and 7a effectively inhibited the cell cycle and
induced cell accumulation in the G2/M phase both in 2D and 3D cultures. Beside their
metaphase effect, colchicine, 14a and 7a induced a panel of interphase effects such as
overexpression of tubulin and F-actin, as well as hyperpolarization of mitochondrial and
lysosomal membranes.
4. Experimental
1
13
4.1. Materials. H NMR and C NMR spectra were recorded on an Agilent DD2 400 MHz
spectrometer. Chemical shifts ( ) are reported in ppm for the solution of compound in CDCl3 or
DMSO-d6, with internal reference TMS and values in Hertz. EI mass spectra (70 eV) were
δ
J
obtained on a DSQ II mass-spectrometer (Thermo Electron Corporation) with a quadrupole
mass-analyzer. Combustion analysis was performed using an Elementar (Vario Micro Cube)
apparatus. Column chromatography separation was performed using Merck Kieselgel 60 (70-230
mesh). All reactions were performed with commercially available reagents (Aldrich, Alfa Aesar,
Acros, and Serva). 3-Butyn-2-ol was distilled previously under low pressure (b.p. 66-67 °C/150
mm Hg). Solvents were purified according to the standard procedures. Petroleum ether used was
of bp 40-70 °C.
4.2. Preparation of Compounds.
Synthesis of Colchiceine (10) [22g]. 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
Na2CO3 was added until the pH became neutral. The resulting solution was extracted with CHCl3
18

ACCEPTED MANUSCRIPT
(3 × 250 mL), and the combined organic layers were washed with brine and dried over Na2SO4.
The solvent was removed under reduced pressure. Colchiceine (10) was obtained as yellow-
green crystals (275.0 mg, 95%) and was directly used at the next step; mp 150 °C; mp lit. [22g]
1
150 °C. H NMR: (400 MHz, CDCl3)
δ
7.49 (s, 1H, NH), 6.87 (s, 1H, CH), 6.54 (s, 1H, CH),
5.91 (s, 1H, CH), 5.75 (d,
J = 8.1 Hz, 1H, CH), 4.80−4.75 (m, 1H, CHNH), 3.92 (s, 3H, CH3O),
3.88 (s, 3H, CH3O), 3.53 (s, 3H, CH3O), 2.46−2.27 (m, 4H, CH2-CH2), 2.04 (s, 3H, CH3C(O)).
13C NMR (101 MHz, CDCl3):
δ
= 175.59, 170.36, 153.47, 151.57, 150.69, 141.32, 136.37,
134.31, 125.71, 122.53, 119.95, 107.22, 61.27, 61.22, 55.94, 52.61, 37.26, 29.64.
Synthesis of Iodocolchinol (11) [22g] Colchiceine (10) (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 × 2H2O (2.976 g, 16.00 mmol) and iodine (543.0 mg, 2.14 mmol) in
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 to 20 °C. A corresponding quantity of
Na2SO3 was added to the solution to remove the excess of iodine. Then, the mixture was
acidified 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 layers were washed with brine and dried over
Na2SO4. The crude product obtained after solvent removal together with the precipitate was
purified by column chromatography, eluent: petroleum ether - ethyl acetate - ethanol (4:1:1), to
afford 338.0 mg (93%) of iodocolchinol (11) [22g]; mp 238 °C; mp lit. [22g] 238 °C. 1H NMR:
(400 MHz, DMSO-d6)
δ 10.28 (s, 1H, OH), 8.38 (d, J = 8.0 Hz, 1H, NH), 7.56 (s, 1H, CH), 6.86
(s, 1H), 6.76 (s, 1H, CH), 4.38 – 4.33 (m, 1H, CHNH), 3.82 (s, 3H, CH3O), 3.77 (s, 3H, CH3O),
13
3.48 (s, 3H, CH3O), 2.16 – 1.90 (m, 4H, CH2-CH2), 1.87 (s, 3H, CH3C(O)). C NMR: (101
19

ACCEPTED MANUSCRIPT
MHz, DMSO-d6): 168.20, 155.52, 152.10, 150.06, 142.30, 140.41, 139.06, 134.65, 126.49,
123.14, 109.96, 107.98, 81.29, 60.41, 55.72, 48.13, 37.78, 29.87, 22.51.
Synthesis
dibenzo[a,c]cycloheptene-5-yl)-acetamide. A solution of DIPEA (72.0
was cooled to 0 °C and MOMCl (26.6 L) was added. The resulting mixture was added
of
N-((5S)-2-iodo-9,10,11-trimethoxy-3-(methoxymethoxy)-6,7-dihydro-5Н-
µ
L) in CH2Cl2 (1 mL)
µ
dropwise with stirring to a solution of iodocolchinol (11) (100.0 mg, 0.21 mmol) in 1 mL of
CH2Cl2 at 0 °C under an inert atmosphere. The temperature was allowed to rise to 20 °C and the
mixture was stirred until the reaction was complete (TLC control). After removing the solvent
under reduced pressure, the compound was purified by column chromatography, eluent:
petroleum ether - ethyl acetate - ethanol (7:1:1), to afford 93.0 mg, 88% of desired product as
white crystals; mp 187 º
С
. 1H NMR: (400 MHz, DMSO-d6)
δ 8.41 (d, J = 8.4 Hz, 1H, NH), 7.68
(s, 1H, CH), 7.08 (s, 1H, CH), 6.78 (s, 1H, CH), 5.28 (q,
J
= 6.7 Hz, 2H, CH2OCH3), 4.46 (dt,
J
= 12.2, 7.7 Hz, 1H, CHNH), 3.83 (s, 3H, CH3O), 3.78 (s, 3H, CH3O), 3.51 (s, 3H, CH3O), 3.47
(s, 3H, CH2OCH3), 2.21 – 1.90 (m, 4H, CH2-CH2), 1.87 (s, 3H, CH3C(O)). 13C NMR: (101 MHz,
DMSO-d6)
δ 168.49, 154.69, 152.54, 150.15, 142.53, 140.51, 139.50, 134.83, 129.38, 122.74,
109.86, 108.20, 94.72, 84.23, 60.66, 60.55, 56.17, 55.83, 48.21, 38.17, 29.97, 22.58 MS: m/z (%)
= 527 (100), 468 (24), 440 (43), 423 (34), 401 (12), 358 (39), 341 (10), 326 (15), 311 (12), 283
(20), 281 (14), 268 (12), 207 (12). Found C, 50.17; H, 4.95. C22H26INO6 requires C, 50.11; H,
4.97%.
Synthesis of N-tert-butoxycarbonyl,N-((5S)-2-iodo-9,10,11-trimethoxy-3-(methoxymethoxy)-6,7-
dihydro-5Н-dibenzo[a,c]cycloheptene-5-yl)-acetamide (12). A half of the solution of Boc2O
(119.4 mg, 0.55 mmol) in MeCN (2 mL) and 33.2
µL of TEA were added to a mixture of N-
((5S)-2-iodo-9,10,11-trimethoxy-3-(methoxymethoxy)-6,7-dihydro-5
Н-
20

ACCEPTED MANUSCRIPT
dibenzo[a,c]cycloheptene-5-yl) –acetamide, prepared in the previous step (63.0 mg, 0.12 mmol)
and DMAP (14.5 mg, 0.12 mmol) in MeCN (3 mL) under an inert atmosphere. The resulting
solution was stirred at 100 ºC for 1 h. Then, the second half of the solution of Boc2O was added,
the mixture was stirred for 2 h at 90 ºC, and overnight at room temperature. After the reaction
completion (TLC control), citric acid was added to the solution, and the mixture was extracted
with CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and the crude
product obtained after solvent removal was purified by column chromatography, eluent:
petroleum ether - ethyl acetate - ethanol (10:1:1), to afford 44.0 mg (78%) of compound 12 as
1
white solid; mp 105 º
С
. H NMR: (400 MHz, DMSO-d6)
δ
7.70 (s, 1H, CH), 7.12 (s, 1H, CH),
6.80 (s, 1H, CH), 5.30 (d,
J = 6.8 Hz, 1H, CHNH), 5.19 (d, J = 6.8 Hz, 2H, CH2OCH3), 3.83 (s,
3H, CH3O), 3.78 (s, 3H, CH3O), 3.44 (s, 6H, CH3O and CH2OCH3), 2.69 – 2.60 (m, 2H, CH2),
13
2.28 (s, 3H, C(O)CH3), 2.18 – 2.06 (m, 2H, CH2), 1.51 (s, 9H, (CH3)3C). C NMR: (101 MHz,
DMSO-d6)
δ 154.69, 153.22, 152.64, 150.37, 140.64, 140.11, 139.47, 134.70, 129.57, 122.75,
111.52, 108.17, 94.88, 84.61, 83.79, 60.63, 60.61, 56.00, 55.80, 34.73, 30.13, 27.98, 27.73,
27.36. MS: m/z (%) = 627 (100), 527 (34), 468 (23), 423 (28), 358 (21), 326 (15), 281 (14), 268
(10). Found C, 51.64; H, 5.42. C27H34INO8 requires C, 51.68; H, 5.46%.
Synthesis of tert-butyl((5S)-2-iodo-9,10,11-trimethoxy-3-(methoxymethoxy)-6,7-dihydro-5H-
dibenzo[a,c]cycloheptene-5-yl)carbamate. MeOH (5 mL) and 2M solution of freshly prepared
sodium methoxide in methanol (60.0 µL) were added into the flask containing compound 12
(360.0 mg, 0.59 mmol) under an inert atmosphere. The resulting solution was stirred for 1.5 h at
room temperature. After evaporating the solvent, the product was separated between CH2Cl2 and
H2O, the organic layer was washed with brine and dried over Na2SO4. After removal of all
volatiles,
tert-butyl((5S)-2-iodo-9,10,11-trimethoxy-3-(methoxymethoxy)-6,7-dihydro-5H-
21

ACCEPTED MANUSCRIPT
dibenzo[a,c]cycloheptene-5-yl)carbamate (339.0 mg, 98%) was obtained as white crystals and
1
was used directly at the next step; mp 111 º
CH), 7.52 (d,
CH2OCH3), 5.22 (d,
С
. H NMR: (400 MHz, DMSO-d6)
δ
7.66 (s, 1H,
= 6.8 Hz, 1H,
= 6.8 Hz, 1H, CH2OCH3), 4.16 – 4.09 (m, 1H, CHNH), 3.83 (s, 3H,
J
= 8.3 Hz, 1H, NH), 7.12 (s, 1H, CH), 6.78 (s, 1H, CH), 5.31 (d, J
J
CH3O), 3.77 (s, 3H, CH3O), 3.56 (s, 3H, CH3O), 3.45 (s, 3H, CH2OCH3), 2.08 (m,
12.6, 6.7 Hz, 4H, CH2-CH2), 1.34 (s, 9H, (CH3)3C). 13C NMR: (101 MHz, DMSO-d6)
J
= 19.8,
154.84,
δ
154.72, 152.55, 150.07, 142.94, 140.47, 139.48, 134.87, 129.24, 122.64, 109.79, 108.10, 94.73,
84.05, 77.90, 60.74, 60.50, 59.74, 56.07, 55.83, 54.91, 50.17, 29.93, 28.17. MS: m/z (%) = 585
(100), 530 (20), 529 (100), 484 (15), 440 (40), 423 (45), 350 (24), 341 (34), 327 (45), 326 (64),
314 (52), 281 (64), 268 (58), 252 (35), 237 (25), 181 (30). Found C, 51.35; H, 5.53. C25H32INO7
requires C, 51.29; H, 5.51%.
Synthesis
of
(5S)-6,7-dihydro-3-hydroxy-2-iodo-9,10,11-trimethoxy-5H-dibenzo[a,c]-
cycloheptene-5-yl-amine (9). tert-Butyl((5S)-2-iodo-9,10,11-trimethoxy-3-(methoxymethoxy)-
6,7-dihydro-5H-dibenzo[a,c]cycloheptene-5-yl)carbamate, prepared in the previous step (200.0
mg, 0.35 mmol) was placed into the flask and dissolved in a minimum amount of EtOH. Conc.
HCl (10 mL)) was added to the solution, and the reaction was stirred at room temperature for 1 h.
Then, 5 mL more of conc. HCl were added, and the temperature was raised to 40 °C. After the
reaction was completed (TLC control), the solution was neutralized with NaOH. After
evaporating the solvent, the dark-yellow oily residue was extracted with CH2Cl2, washed with
brine and dried over Na2SO4. After removal of all volatiles, the pure compound 9 was obtained
1
as white crystals (132.0 mg, 85%); mp 155 º
С
. H NMR (400 MHz, DMSO-d6)
δ
7.57 (s, 1H,
= 11.3,
= 12.9, 7.4 Hz,
CH), 7.16 (s, 1H, CH), 6.75 (s, 1H, CH), 3.82 (s, 3H, CH3), 3.75 (s, 3H, CH3), 3.57 (dd,
7.0 Hz, 1H, CHNH2), 3.51 (s, 3H, CH3), 2.30 – 2.25 (m, 2H, CH2), 2.06 (td,
J
J
22

ACCEPTED MANUSCRIPT
13
1H, CH2), 1.69 (td,
J
= 11.8, 7.8 Hz, 1H, CH2). C NMR: (101 MHz, DMSO-d6)
δ
155.62,
152.15, 150.06, 140.42, 139.15, 135.10, 126.62, 123.09, 110.51, 107.96, 81.38, 60.52, 60.49,
55.82, 50.06, 40.45, 30.22. MS: m/z (%) = 441 (100), 424 (100), 409 (100), 393 (74), 366 (42),
314 (52), 297 (41), 282 (40), 267 (34), 254 (25), 212 (12), 207 (25), 181 (22). Found C, 50.04;
H, 4.59. C18H20INO4 requires C, 48.99; H, 4.57%.
Synthesis of 3-hydroxy-2-iodo-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-5-
yl-1-one (8). N-methyl-4-formylpyridinium benzolsulfonate (64.0 mg, 0.23 mmol) was added to
a stirred solution of compound 9 (78.0 mg, 0.18 mmol) in 4 mL CH2Cl2 / DMF (1:1) under an
inert atmosphere. The resulting mixture was stirred for 10 h at room temperature, and 59.4 µL of
DBU were added. The mixture was stirred for 30 min, 3 mL of saturated aqueous solution of
oxalic acid were added, and stirring was continued for 12 h. The resulting solution was separated
between CH2Cl2 and H2O. The organic layer was concentrated under reduced pressure. The pure
product (8) was isolated by column chromatography as yellow crystals (50.4 mg, 65%), eluent:
1
petroleum ether - ethyl acetate - ethanol (3:1:1); mp 185 º
С
. H NMR: (400 MHz, DMSO-d6)
δ
7.77 (s, 1H, CH), 6.90 (s, 1H, CH), 6.82 (s, 1H, CH), 3.82 (s, 3H, CH3), 3.74 (s, 3H, CH3), 3.49
(s, 3H, CH3), 2.99 (d,
J
= 18.7 Hz, 1H, CH2), 2.89 – 2.83 (m, 1H, CH2), 2.65 (dd,
J
= 28.5, 14.1
Hz, 2H, CH2). 13C NMR: (101 MHz, DMSO-d6)
δ
205.49, 155.84, 152.58, 151.14, 141.04,
140.78, 140.46, 135.73, 125.64, 122.22, 112.41, 107.77, 88.38, 60.62, 60.46, 55.82, 47.42, 29.10.
MS: m/z (%) = 440 (100), 425 (100), 412 (68), 383 (80), 366 (28), 339 (33), 311 (30), 298 (40),
283 (53), 255 (45), 220 (28), 184 (28). Found C, 49.14; H, 3.94. C18H17IO5 requires C, 49.11; H,
3.89%.
Synthesis of compound 14a. Compound 8 (162.0 mg, 0.37 mmol), Pd(OAc)2 (4.1 mg, 0.02
mmol), CuI (7.0 mg, 0.04 mmol), PPh3 (15.0 mg, 0.05 mmol), and AcOK (61.0 mg, 1.10 mmol)
23

ACCEPTED MANUSCRIPT
were placed into the two-neck flask under an inert atmosphere. Anhydrous acetonitrile (6 mL)
and propargyl alcohol (21.0 mg, 0.37 mmol) were added. The mixture was stirred at 100 °C for 1
h, then the temperature was reduced to 90 °C and stirring was continued until the reaction
completed (TLC control). The solvent was removed under reduced pressure, the solid residue
was purified by column chromatography, eluent: petroleum ether - ethyl acetate - ethanol (7:1:1),
to afford 95.0 mg (70%) of compound 14a; mp 100 º
С δ 7.65
. 1H NMR: (400 MHz, DMSO-d6)
(s, 1H, CH), 7.56 (s, 1H, CH), 6.87 (s, 1H, CH), 6.86 (s, 1H, CH), 5.58 (s, 1H, OH), 4.61 (s, 2H,
CH2OH), 3.84 (s, 3H, CH3O), 3.76 (s, 3H, CH3O), 3.40 (s, 3H, CH3O), 3.03 (d,
J = 17.5 Hz, 1H,
CH2), 2.89 – 2.82 (m, 1H, CH2), 2.76 – 2.71 (m, 2H, CH2). 13C NMR: (100 MHz, DMSO-d6)
δ
206.77, 162.13, 153.47, 153.27, 152.16, 141.48, 136.65, 136.33, 131.01, 128.56, 124.15, 123.73,
109.83, 108.01, 103.72, 63.38, 60.62, 55.93, 49.38, 47.79, 29.18. MS: m/z (%) = 368 (100), 352
(10), 340 (18). Found C, 68.44; H, 5.45. C21H20O6 requires C, 68.47; H, 5.47%.
Synthesis of compound 7a. NaBH4 (3.0 mg, 0.09 mmol) was added to a mixture of compound
(14a) (63.0 mg, 0.17 mmol), 2 mL of THF and 2.4 mL of 40% aqueous methanol. The resulting
solution was stirred for 30 min at room temperature. As the reaction completed (TLC control),
5.5 mL of water were added. After evaporating the solvent, the resulting solution was extracted
with MTBE. The organic layer was concentrated under reduced pressure. Pure product (7a) as
white crystals (41.0 mg, 66%) was isolated by column chromatography, eluent: petroleum ether -
ethyl acetate - ethanol (10:1:1); mp 128 º
7.50 (s, 1H, CH), 6.77 (s, 1H, CH), 6.75 (s, 1H, CH), 5.46 (s, 1H, HOCH2), 5.32 (s, 1H, HOCH),
4.57 (s, 2H, CH2OH), 4.36 (dd, = 7.4, 3.4 Hz, 1H, CHOH), 3.83 (s, 3H, CH3O), 3.77 (s, 3H,
С δ 7.67 (s, 1H, CH),
. 1H NMR (400 MHz, DMSO-d6)
J
CH3O), 3.45 (s, 3H, CH3O), 2.47 – 2.34 (m, 2H, CH2), 2.08 – 2.02 (m, 1H, CH2), 1.72 (dd,
J =
13
18.6, 10.9 Hz, 1H, CH2). C NMR (100 MHz, DMSO-d6)
δ 158.94, 154.42, 152.74, 150.84,
24

ACCEPTED MANUSCRIPT
141.03, 140.75, 135.76, 127.97, 126.50, 124.92, 122.02, 108.23, 105.89, 103.66, 68.42, 60.63,
60.52, 56.34, 55.89, 41.23, 29.82. MS: m/z (%) = 370 (100), 352 (10), 339 (11), 311 (12), 181
(30). Found C, 68.06; H, 5.97. C21H22O6 requires C, 68.10; H, 5.99%.
4.3. Cell cultures. Pancreatic cell lines BxPC-3, PANC-1, Colo-357, MiaPaCa-2, lung
adenocarcinoma A549; nontransformed immortalized keratinocytes HaCaT were grown in
DMEM medium supplemented with 10% fetal calf serum (FCS), pen-strep-glut (all from
PanEco, Moscow, Russian Federation). Endothelilal cell line EA.hy926 was a gift from Freindlin
I.S. Mesenchymal stem cells (MSC), used in this study, were harvested with the informed
consent of the donor as approved by an Institutional Review Board of the Research Center for
Obstetrics, Gynecology and Perinatology, Ministry of Healthcare of the Russian Federation,
Moscow. Bone marrow derived MSC were obtained by iliac crest puncture of a 25 years old
patient. MSC were grown under sterile conditions and used at early passages (P) P2 to P6. All
cell lines used were routinely tested for mycoplasma. Adherent cells were detached using 0.05%
trypsin-EDTA (PanEco, Moscow), counted and sub-cultured. Twenty-four hours before assays,
cells were seeded in the appropriate plates (96- or 24-well plates), adjusted to 3x105 cells/mL,
and incubated overnight to achieve standardized growth conditions.
To prepare multicellular spheroids (3D cultures), plates (96- or 12-well, Costar, USA) were
coated with poly(2-hydroxyethyl methacrylate) (PolyHEMA, Sigma), dried and used at request.
4.4. MTT-assay. Cytotoxic effect of the allocolchicinoids was estimated by a standard 3-(4, 5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test as was described
earlier.45 All the compounds were dissolved in dimethylsulfoxide to 20 mM concentration and
stored at -20oC until the assay. Different dilutions of the new compounds from 20 µM to 0.1 nM
were prepared separately and transferred in 100 µl to the plates with the cells. Non-treated cells
25

ACCEPTED MANUSCRIPT
served as controls. Plates were incubated for 72 hrs. For the last 6 hours, 5 mg/ml of MTT was
added in the amount of 10 µl to each well. After the incubation, culture medium was removed
and 100 µl dimethylsulfoxide was added to each well. Plates were incubated at shaking for 15
min to dissolve the formed formazan product. Optical density was read on spectrophotometer
Titertek (UK) at 540 nm. Results were analyzed by Excel package (Microsoft). Cytotoxic
concentration giving 50% of the maximal toxic effect (IC50) was calculated from the titration
curves. The inhibition of proliferation (inhibition index, II) was calculated as [1 – (ODexperiment
/
ODcontrol)], where OD was MTT optical density. 3D cultures were run in the same way using
polyHEMA precoated plates. Both 2D and 3D cultures were generated until adhesion to plastic
or spheroid formation before the addition of the drugs.
4.5. Confocal analysis. For confocal analysis, 2D cells were grown overnight on sterile cover
slips in 200 µl of complete culture medium in 6-well plates (Costar). For 3D culture analysis
spheroids were formed in bulk cultures in 12-well plates precoated with polyHEMA. Colchicine
or new compounds 14a and 7a in different concentrations was dissolved in 4 ml of complete
medium and added to the wells. Cells were cultivated for 72 hrs. After incubation, 2D or 3D cells
were fixed with 1% paraformaldehyde, permeabilized by 0.1% Triton X100 in PBS, washed, and
treated with Mowiol 4.88 medium (Calbiochem, Germany). Spheroids were treated in the same
way and transferred onto the slides, treated by Mowiol 4.88 medium, covered with cover slips.
Tubulin was identified by anti-tubulin antibody (SantaCruz, USA) followed by anti-mouse IgG-
AlexaFluor555 (Molecular Probes, Invitrogen, USA). PhalloidinAlexaFluor488 (Molecular
Probes, Invitrogen, USA) was used to identify F-actin expression. Hoechst 33342 (Sigma) was
used to visualize nuclei. Slides were analyzed using Eclipse TE2000 confocal microscope
(Nikon, Japan).
26

ACCEPTED MANUSCRIPT
4.6. Cell cycle analysis by flow cytometry
.
Cell cycle was analyzed using PI-stained DNA.
Cells from 2D or 3D cultures were collected at indicated time, trypsinized, washed in ice-cold
o
PBS, fixed by the addition of 70% ethanol and left for 2 hours at -20 C. Thereafter, the cells
were washed twice in PBS, stained with 50 µg/ml of propidium iodide (Sigma Chemical Co) in
PBS, 10 µg/ml of DNAse and analyzed by flow cytometry using FACScan device (BD, USA).
Total 2000 events were collected. The results were analyzed using Flowing 2.5.1 software
(Finland). Gating strategy is shown in Figure 3(a, b).
4.7. Interphase effects of allocolchicinoids. Mitochondrial tracker 3,3′-dihexyloxacarbocyanine
iodide (DiOC6), lysotracker LysoTrackRed (Molecular Probes, USA), propidium iodide (Sigma
Chemical Co) were added for 1 h to live cells. The cationic lypophilic fluorochrome DiOC6 was
used to evaluate transmembrane potential in mitochondria46, while LysoTrackRed was used to
estimate the potential of lysosomal membranes. Cells were harvested, trypsinized, washed in pre-
warmed PBS supplemented with 2% of FCS, resuspended at 106 cells/ml in PBS/2%FCS, and
analyzed by flow cytometry using FACScan device (BD, USA).
ACKNOWLEDGEMENTS
We thank the Russian Science Foundation (project16-13-10248).
Appendix A. Supplementary data
The following is the supplementary data related to this article:
General information, syntheses and characterization of compounds, 1H and 13C NMR spectra.
Corresponding Authors
*(E.V.S.) Tel/Fax: (495) 330 40-11, E-mail: esvir@yandex.ru.
*(A.Yu.F.) Fax: +7 831-462-30-85. E-mail: afnn@rambler.ru.
27

ACCEPTED MANUSCRIPT
Notes
The authors declare no competing financial interest.
REFERENCES
[1]
(a) K.H. Downing, E. Nogales, Tubulin and microtubule structure, Curr. Opin. Cell Biol.
10 (1998) 16−22. (b) E. Stec-Martyna, M. Ponassi, M. Miele, S. Parodi, L. Felli, C. Rosano,
Structural comparison of the interaction of tubulin with various ligands affecting microtubule
dynamics, Curr. Cancer Drug Targets 12 (2012) 658−666.
[2]
(a) J. Seligmann, C. Twelves, Tubulin: an example of targeted chemotherapy, Future
Med. Chem. 5 (2013) 339−352. (b) J. Cortes, M. Vidal, Beyond taxanes: the next generation of
microtubule targeting agents, Breast Cancer Res. Treat. 133 (2012) 821−830. (c) C.D. Katsetos,
P. Dráber, Tubulins as therapeutic targets in cancer: from bench to bedside, Curr. Pharm. Des. 18
(2012) 2778−2792. (d) R.A. Stanton, K.M. Gernert, J.H. Nettles, R. Aneja, Drugs that target
dynamic microtubules: a new molecular perspective, Med. Res. Rev. 31 (2011) 443−481. (e) C.
Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer therapeutics,
Nat. Rev. Drug Discovery 9 (2010) 790−803. (f) M. Aeluri, S. Chamakuri, B. Dasari, S.K.R.
Guduru, R. Jimmidi, S. Jogula, P. Arya, Small molecule modulators of protein − protein
interactions: selected case studies, J. Med. Chem. 114 (2014) 4640−4694. (g) D.G.I. Kingston,
Tubulin-interactive natural products as anticancer agents, J. Nat. Prod. 72 (2009) 507−515.
[3]
Brossi, Ed., Academic Press: New York (1984) Vol. 23, p. 1. (b) C. Le Hello, In The Alkaloids:
Chemistry and Biology (Ed.: G.A. Cordell), Academic Press: San Diego, CA (1999) Vol. 53 p.
287. (c) T. Graening, H.-G. Schmalz, Total syntheses of colchicine in comparison: a journey
(a) H.-G. Capraro, A. Brossi, Tropolonic Colchicum Alkaloids, In The Alkaloids, A.
,
28