Synthesis and biological evaluation of novel non-racemic indole-containing allocolchicinoids

Article abstract of DOI:10.1016/j.ejmech.2017.09.055

Two novel indole-containing allocolchicinoids were prepared from naturally occurring colchicine exploiting the Curtius rearrangement and tandem Sonogashira coupling/Pd-catalyzed cyclization as the key transformations. Their cytotoxic properties, apoptosis-inducing activity, tubulin assembly inhibition and short-time cytotoxic effects were investigated. Compound 7 demonstrated the most pronounced anti-cancer activity: IC₅₀ < 1 nM, cell cycle arrest in the G2/M phase, 25% apoptosis induction, as well as lower destructive short-time effects on HT-29 cell line in comparison with colchicine. Docking studies for prepared indole-derived allocolchicine analogues were carried out.

Full text of DOI:10.1016/j.ejmech.2017.09.055

Accepted Manuscript
Synthesis and biological evaluation of novel non-racemic indole-containing
allocolchicinoids
Ekaterina S. Shchegravina, Alexander A. Maleev, Stanislav K. Ignatov, Iuliia A.
Gracheva, Andreas Stein, Hans-Günther Schmalz, Andrey E. Gavryushin, Anastasiya
A. Zubareva, Elena V. Svirshchevskaya, Alexey Yu Fedorov
PII:
S0223-5234(17)30770-5
10.1016/j.ejmech.2017.09.055
EJMECH 9770
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 June 2017
Revised Date: 29 July 2017
Accepted Date: 25 September 2017
Please cite this article as: E.S. Shchegravina, A.A. Maleev, S.K. Ignatov, I.A. Gracheva, A. Stein, Hans.-
Gü. Schmalz, A.E. Gavryushin, A.A. Zubareva, E.V. Svirshchevskaya, A.Y. Fedorov, Synthesis and
biological evaluation of novel non-racemic indole-containing allocolchicinoids, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.055.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel non-racemic
indole-containing allocolchicinoids
a
a
a
a
Ekaterina S. Shchegravina , Alexander A. Maleev , Stanislav K. Ignatov , Iuliia A. Gracheva ,
b
b
c
d
Andreas Stein , Hans-Günther Schmalz , Andrey E. Gavryushin , Anastasiya A. Zubareva , Elena
e*
a*
V. Svirshchevskaya , Alexey Yu. Fedorov
a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, 603950
Nizhny Novgorod, Russian Federation, Fax: +7 (831) 462-30-85, *E-mail: afnn@rambler.ru
b
Department of Chemistry, University of Cologne, Greinstrasse 4, 50939 Cologne, Germany
c
Ludwig-Maximilians-Universität, Butenandtstrasse 5-13, 81377 Munich, Germany
d
Institute of Bioengineering, Research Center of Biotechnology RAS, Leninsky prospect 33,
119071 Moscow, Russian Federation
e
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya Street 16/10,
117997 Moscow, Russian Federation, *E-mail: esvir@yandex.ru
Abstract
Two novel indole-containing allocolchicinoids were prepared from naturally occurring
colchicine exploiting the Curtius rearrangement and tandem Sonogashira coupling/Pd-catalyzed
cyclization as the key transformations. Their cytotoxic properties, apoptosis-inducing activity,
tubulin assembly inhibition and short-time cytotoxic effects were investigated. Compound 7
demonstrated the most pronounced anti-cancer activity: IC < 1 nM, cell cycle arrest in the G2/M
5
0
phase, 25% apoptosis induction, as well as lower destructive short-time effects on HT-29 cell line in
comparison with colchicine. Docking studies for prepared indole-derived allocolchicine analogs
were carried out.

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Key words: colchicine, heterocyclic allocolchicinoids, tubulin, apoptosis, cytotoxicity, short-time
effects, antimitotics, colchicine site of tubulin.
1. Introduction
Tubulin proteins play an essential role in many cellular functions of eukaryotic cells such as
cell proliferation, the development of cell shape, cell motility, and cell signaling [1]. Colchicine (1),
isolated from Colchicinum autumnale, was the first tubulin-binding agent ever discovered [2]. For a
long time, colchicine was considered as a perspective antitumor agent. However, its significant
general toxicity even in therapeutic doses prevented its use in cancer therapy [3]. On the other hand,
the tubulin-destabilizing properties of colchicine allow its use in clinical practice for the treatment
of familial Mediterranean fever, Behcet’s disease, as well as several types of microcrystalline
arthritis [4]. Recently, colchicine was suggested as a drug for the treatment of cardiovascular
diseases, such as acute pericarditis, atrial fibrillation caused by inflammation, and ischemic diseases
[
5]. These properties, obviously, are associated with the ability of colchicine to accumulate in the
immune system cells, leading to the suppression of inflammatory reactions. Potentially, colchicine
and its analogs may be useful in the treatment of autoimmune, allergic and neurodegenerative
diseases, and in therapy of chronic infections [6, 7]. Therefore, the search for less toxic colchicine-
binding site inhibitors [2c] with improved bioavailability and tissue distribution might lead to the
elaboration of novel therapeutic agents, useful in the treatment of inflammatory, cardiovascular,
autoimmune and oncological diseases.
Our group has recently synthesized a series of heterocyclic allocolchicine analogs of
colchicine, containing indole (2-4) [8] or benzofurane (5 and 6) [9] fragments replacing the C-ring
in the parent compound (Figure 1).

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Figure 1. Colchicine and its heterocyclic analogues.
These molecules have demonstrated higher activity as tubulin polymerization and cell
proliferation inhibitors compared to colchicine, while their general cytotoxicity was relatively low.
Thus, furano-allocolchicinoids 5 inhibit tumor growth in mice without displaying symptoms of
acute toxicity such as weight loss or neurological disorders, typical for colchicine poisoning.
2
2
. Results and discussion
.1. Chemistry
Racemic pyrrolo-allocolchicinoids 2-4 were prepared previously in 11 steps starting from
,4,5-trimethoxyphenylpropionic acid [8]. Herein, we report a conceptionally different ten-steps
3
semi-synthetic approach (Scheme 1) towards non-racemic pyrrolo-allocolchicine 7 (Figure 1)
starting from naturally occurring (-)-(aR, 7S)-colchicine 1. Previously, we observed that the
presence of a hydroxymethyl substituent either at C-7 of ring B (compounds 2-4) [8a] or on the
heterocyclic fragment (compounds 5 and 6) [9] significantly increases the anti-proliferative activity.
Thus, a similar 2’’-hydroxymethyl substitution pattern in the pyrrole fragment was chosen for these
new synthetic targets.
The bromination of colchicine (1) using NBS in TFA/AcOH mixture afforded 4-
bromocolchicine in almost quantitative yield. It was transformed into 4-bromoallocolchicinic acid 8
by applying the previously described base-induced ring contraction [10] (85% yield over two

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stages). Allocolchicinic acid 8 was subjected to the Curtius degradation yielding the corresponding
aniline 9 in 45% yield. The followed α-iodination reaction (NIS/AcOH) and subsequent acylation
gave allocolchicinoid 10 in 64% yield. Finally, the desired N-methylated pyrrolo-allocolchine 7
was prepared in 3 steps from the amide 10. The TBDMS-protected indolyl-2-methanol 11 was
obtained through tandem Sonogashira reaction/cyclization sequence in 51% yield [11], using O-
protected propargyl alcohol. After the N-methylation of the indole nitrogen (NaH/MeI) and
selective cleavage of the silyl group, compound 7 was isolated in 55% yield over two steps.
Scheme 1. The synthetic route to pyrrolo-allocolchicinoids 7 and 12. Reagents and conditions: (a)
HC≡C-CH -OTBDMS (1.2 equiv.), Pd(dppf)Cl (0.05 equiv.), CuI (0.1 equiv.), DIPEA (3.0 equiv.), MeCN,
2
2
8
0 °C, 8 h; (b) HC≡C-CH OH (1.2 equiv.), Pd(OAc) (0.05 equiv.), CuI (0.1 equiv.), PPh (0.15 equiv.),
2 2 3
AcOK (3.0 equiv.), MeCN, 80 °C, 8 h. See Experimental Section.
In order to determine the influence of substituents at the nitrogen atom on the biological
activity, N-H pyrrolo-allocolchine 12 was also prepared, starting from amide 10 via two-step one-
pot Sonogashira/Larock-type cyclization [11], using unprotected propargyl alcohol for the coupling
stage. It has been demonstrated, that the presence of a halogen atom in the A-ring of colchicinoids
slightly enhances their cell inhibitory activity [12], thus the presence of bromine in the final
products was considered as positive.
2.2. Biology
2.2.1. Cytotoxicity

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The cytotoxicity of the synthesized compounds towards human pancreatic adenocarcinomas
COLO-357 and MiaPaCa-2, and human embryonic kidney cell line HEK293 was investigated in
vitro. A tetrazolium-based assay was used to determine the drug concentration required to inhibit
cell growth by 50% (IC ) after incubation in the culture medium for 72 h. The calculated IC
5
0
50
values are summarized in Table 1. Compound 12 was less active than colchicine. In contrast, N-
methylated analog 7 inhibited cell proliferation already at subnanomolar concentrations, lower than
of colchicine. We presume that the presence of alkyl on the N-1 of the pyrrolo-allocolchicine
skeleton is essential for the antiproliferative activity of the prepared compounds.
a
Table 1. Cytotoxic properties of synthesized compounds, (IC , nM) .
5
0
Compound
7
12
Colchicine 1
HEK293
COLO-357
MiaPaCa-2
6.4
0.3
160
3.5
10
32
1.3
1.3
<0.1
a
Each drug concentration was tested in triplicate, and the SE of each point is <10%.
.2.2. Cell cycle analysis
2
The main mechanism of the colchicine action is the inhibition of tubulin assembly, which
leads to the block of cell proliferative cycle and subsequent apoptosis. We found that both new
compounds demonstrate colchicine-specific tubulin interaction (Figures 2 and 3). When compared
in the equal concentrations, the only difference found was the higher activity of compound 7 in cell
cycle arrest (Figure 2, Table 2) expressed by the increased number of cells in G2/M phase, and
higher percentage of cells in the apoptotic fraction.

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B
A
D
C
Figure 2. Cell cycle analysis of colchicine analogues. COLO-357 were incubated without (A) or
with 5 µM colchicine 1 (B), 12 (C), or 7 (D) compounds for 72 h, permeabilized, stained with
propidium iodide and analyzed by flow cytometry. Regions H2 and H4 correspond to apoptotic
fraction, H3 and H5 – to G2/M cells. Quantitative results are shown in Table 2.
Table 2. Cell cycle inhibition induced by colchicine analogues in COLO-357 cells.
Apoptosis
G
G2/M
G1/G2
Control
12.4
12.4
23
56.6
12.6
16
31
75
61
63
1.8
0.2
0.3
0.2
Colchicine
1
2
7
25
12

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Similarly, a higher activity in tubulin assembly inhibition was found in cultures stained with
anti-β
-tubulin antibodies (Figure 3a-d).
Figure 3. Tubulin organization in COLO-357 cells. COLO-357 were incubated without (a, e) or
with 5 µM colchicine 1 (b), 12 (c), or 7 (d, f) compounds for 24 h (a-d) or 3 h (e-f), permeabilized,
stained with anti-β-tubulin antibody (red), and analyzed by confocal microscopy. Mitotic spindles
are shown with white arrows; rearranged chromosomes or apoptotic bodies - with green arrows.
Nuclei are stained with Hoechst 33342 (blue). Scale bar 12-20 µm.

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Tubulin assembly inhibition, resulting in the mitotic spindle block and G2/M cell cycle arrest,
requires at least 20-24 h to proceed completely (see Figure 2A-D). However, colchicine and its
analogs bind to tubulin much faster. Incubation of cells with compound 7 for 3 h already re-
localized β-tubulin (Figure 3e-f). The cytotoxic effects of colchicine and its analogues in vitro are
rather mild, and require 72 h to reach maximum 60-80 % inhibition. Short-time cell incubation with
colchicine derivatives demonstrated that cytotoxic effect depended both on time and the cell type
(Figure 4).
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
0
.5
.4
A
B
0
0.3
0.2
0
.1
.0
Colch
7
0
0.1
1
10
100 1000 10000 100000
nM
0.1
1
10
100 1000 10000 100000
nM
-
0.1
-
0.1
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
D
C
0.1
10
1000
100000
0.1
10
1000
100000
-0.1
-0.2
nM
nM
Figure 4. Effect of colchicine (Colch) and compound 7 short time incubation on cell proliferation.
Human colon HT-29 (A), pancreatic COLO-357 (B), PANC-1 (C) carcinoma cells, and embryonic
kidney HEK293 (D) cells were incubated with colchicine 1 or colchicinoid 7 for 24 h and their

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proliferation was analyzed by MTT assay. Vertical lines in a and b show differences in the
sensitivity of cells to the preparations.
Among all, colon cell line HT-29 was the most sensitive to colchicinoid 7, followed by
COLO-357 cells. At the same time, PANC-1 and HEK293 cells were not affected at that time
(
Figure 4). The sensitivity of cell lines to colchicinoids at early time points of incubation correlates
with the cells division rates. These early effects may translate into systemic toxicity in vivo.
Multiple studies demonstrated that colchicine and its analogues affect
lysosomal/endosomal/autophagial pathways, induce endoplasmic reticulum (EPR) and
mitochondrial stress, disrupt Golgi apparatus, and stimulate heart beating [13]. Other multiple
effects on cell signaling have also been demonstrated [4e]. So far, it is not quite clear which
pathway of colchicine activity is the most important for the systemic effects. Clinical studies have
demonstrated that the most often adverse effects in humans are diarrhea and vomiting, causing loss
of liquid [14].
Earlier, we have shown a hyperpolarization of mitochondrial and lysosomal membranes
induced by allocolchicinoids in 3D cultures of pancreatic cancer cells [9b]. To understand the role
of different cell compartment disturbances induced by colchicinoids, we compared short time effect
on several organelles in colon cells HT-29. Minor differences between control and cultures treated
with colchicine and compound 7 was found in Golgi apparatus, EPR, endosomes, membrane
integrity (data not shown). The cell mitochondria responded to colchicine by significant increase in
the membrane potential, their number, and relocation from perinuclear position to cytoplasm
(Figure 5). The compound 7 also stimulated mitochondria movement, although the effect was less
significant (p<0.05). Possibly, a higher specific activity of the compound 7 is responsible for this
difference. Analysis of the lysosomal membrane potential demonstrated that the colon cells
responded to colchicine decreasing it, while the compound 7 did not affect lysosomes at all (Figure
5g). A stronger effect of 7 was found against pancreatic tumor cells COLO-357 mitochondria
(Figure 5h and 5i).

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Figure 5. Short-time effect of colchicinoids on mitochondria and lysosomes. HT-29 (a-g) or
COLO-357 (h, i) were incubated for 3 h without (a, d, h), with 5 µM colchicine (1) (b, e) or
compound 7 (c, f, i), and stained with red mitochondrial (a-c, h, i) or lysosomal tracker (d-f). Nuclei
in (h, i) are stained with Hoechst 33342 (blue). Scale bar 8-15 µm. g. Flow cytometry analysis of
lysosomal and mitochondrial tracker staining in HT-29 cells.
Generally, it might be assumed that a lesser effect against colon cells of the new compound 7
will result in a lesser toxicity to gastrointestinal tract while preserving its antitumor potential.
2.3. Docking studies
In order to assess the ability of newly synthesized compounds for inhibition of intracellular
tubulin functions, we carried out the docking study of complexes between compounds 7 and 12 and

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tubulin dimer and estimated their binding energy in comparison with the colchicine-tubulin
complex, studied earlier [15]. The calculated binding energy (∆Gbind) of the most favorable
complexes of 7 and 12 with tubulin are close to the values of the colchicine-tubulin complex (Table
3).
Table 3. Binding energies of the most favorable conformations of complexes between tubulin
dimer and colchicinoids 1, 7, and 12.
Compound
Binding energy, kcal/mol
–8.66
Colchicine (1)
a
7
–7.96 (–7.82 )
b
1
2
–8.26 (–7.47 )
a
–
binding energy of 12 in the conformation similar to the most favorable structure of 7-tubuline
complex;
b
–
binding energy of 7 in the conformation similar to the most favorable structure of 12-tubuline
complex.
The obtained geometry of colchicine-tubulin complex with the colchicine site of tubulin
coincides with the one previously reported [15]. The geometries of most favorable conformations
for compounds 7, 12 in the colchicine site of tubulin are remarkably different from the colchicine
structure (Figure 6A). On Figure 6B, a superposition of the most energetically favored
conformation of 7 (∆G = -7.96 kcal/mol), and the conformation of 12, corresponding to a local
minimum (∆G = -7.82 kcal/mol) is presented.

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A A
B
C
D
Figure 6. Accommodation of colchicinoids 1, 7 and 12 in the colchicine site of tubulin. (А) red -
colchicine 1, blue - 7, yellow - 12. (B) colchicine 1 (red) and completely superposed pyrrolo-
allocolchicinoids 7 and 12 (blue) in colchicine site of tubulin. Amino acids environment of 7 (C)
and 12 (D).
In the case of the most favorable complex of pyrrolo-allocolchicinoid 12 with tubulin, the 2-
methoxy (colchicine numeration) group in A ring contacts with N-H peptide group of Asn101α
residue (hydrogen bond length is 1.95 Å) (Figure 6A and 6C). The acetamide group of aliphatic B
ring is located in the region of the week polar contacts with Alaβ354 and Leuβ248 residues. The C
ring (indole moiety) is placed in zone of contacts with Lysβ352, Asnβ258, Asnβ350, Alaβ316,
Valβ315, Thrβ314, Metβ259 and Valα181, wherein hydroxymethyl fragment forms the hydrogen
bonds with peptide C=O group of Valβ315 (1.90Å).

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In the case of most favorable complex of colchicinoid 7 with tubulin (Figure 6B and 6D), 2-
methoxy group in A ring contacts with OH-group of Ser-α178 residue (the hydrogen bond length is
1.79 Å). The acetamide fragment of aliphatic B ring, like in the case of compound 12, is located in
the field of week polar interactions (Lysβ352, Valα181, Asnβ258 and Metβ259 residues). The
hydroxymethyl moiety of indole side chain forms H-bond with the peptide C=O group of Valβ238
(1.81 Å), while the N-Me substituent is arranged in the hydrophobic pocket constituted by residues
of Leu255, Ile378 and Val318.
Although colchicinoid 7 is by two orders of magnitude more active than compound 12, the
difference of the calculated binding energies for these two molecules are close. To explain the
significant difference in cytotoxicity of two compounds, we can speculate that allocolchicinoid 12,
in contrast to its analog 7, is able to shape the intramolecular H-bond between N-H in pyrrole core
and oxygen in hydroxymethyl group of the side chain. This intramolecular hydrogen bonding can
be competitive with the “target” 12/tubulin interactions, leading to serious decrease in activity. At
the same time, due to the intermolecular hydrogen bonds, the molecules of 12 can form relatively
stable dimeric associates having significantly lower membranotropicity in comparison with
monomeric units of homolog 7.
To explain very potent antimitotic activity of colchicinoids 2-7 (Figure 1, Scheme 1),
containing hydroxyl groups in benzylic or pseudobenzylic positions, we hypothesized that these
compounds are even under physiologic conditions capable to form a pseudobenzylic cation like 13
that immediately attacks the free cysteine fragments of tubulin, forming a covalent bond (Scheme
2).

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Br
Br
Br
MeO
MeO
MeO
MeO
MeO
NHAc
NHAc
NHAc
N Me
MeO
HS-Tubulin
OMe
OMe
OMe
N Me
OH
N
Me
7
13
14
S-Tubulin
N Me
N Me
N H
N H
cation, obtained
cation, obtained
More stable than
from 7
from
12
Scheme 2. An illustration of hypothesis of possible covalent bounding of colchicinoid 7 to tubulin.
This idea is indirectly confirmed by the ability of furano-allocolchicinoids 5 to react readily
with various thiols, leading to the corresponding sulfides in quantitative yields [16]. Our docking
studies revealed the proximal location of the hydroxymethyl moiety of colchicinoid 7 to the Cys241
of tubulin. The N-methylindole fragment of 7 should form a more stable cation than the N-
unsubstituted indole (compound 12, Scheme 2). This might explain the differences in their
biological activity. Experimental work directed at the proof of this concept is currently in progress.
3
. Conclusions
Starting from naturally occurring colchicine, highly cytotoxic pyrrolo-allocolchicinoids 7 and
2 were synthesized in ten and eight steps consequently as non-racemic compounds.
1
Allocolchicinoid 7 exhibits cytotoxic properties at sub-nanomolar concentration, and efficiently
arrests the cell cycle in G2/M phase, thus increasing the number of cells undergoing apoptosis in
comparison with colchicine. Short-time incubation of cells with 7 demonstrated that the cytotoxic
effect strongly depends both on time and the cell type. It can be assumed that a lesser effect of 7
against some types of cells in comparison with colchicine (for example, against colon cells) could
possibly result in a lesser toxicity to gastrointestinal tract while preserving its antitumor potential.
4. Experimental

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1
13
4
.1. Materials. H NMR and C NMR spectra were recorded on Agilent DDR2 400 spectrometer at
2
5 °C. Chemical shifts (δ) are reported in ppm for the solution of compound in DMSO-d with
6
internal 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. Optical
rotation [α] was measured on JASCO P-2000 polarimeter at 19 °C and λ=589 nm (cuvette length:
D
1.0 dm, volume: 1.0 mL). Concentration is given in g/100 mL. 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 prior to use under reduced pressure (b.p. 75 °C/80 mm Hg). Solvents
were purified according to standard procedures. The petroleum ether refers to the fraction with
distillation range 40 – 70 °C.
4.2. Synthesis of pyrrolo-allocolchicinoids
4.2.1. Synthesis of 1ʹ-bromo-2ʹ, 3ʹ, 4ʹ-trimethoxybenzo[5ʹ, 6ʹ:4, 5]1H-(aR, 1S)1-acetamido-6,7-
dihydro-cyclohepta[3, 4-f]1H-2-hydroxymethylindole (12)
Into a Schlenk flask with a stirring bar, were subsequently added compound 10 (60.0 mg,
0.090 mmol), palladium acetate (1.1 mg, 0.004 mmol), copper (I) iodide (1.7 mg, 0,009 mmol),
triphenylphosphine (3.5 mg, 0.013 mmol) and potassium acetate (26.5 mg, 0.270 mmol). The flask
was filled with argon. Acetonitrile (3.0 mL) was added under inert atmosphere. Then, propargylic
alcohol (6.2 mg, 0.110 mmol) was added, the temperature was raised to 80 °C, and the mixture
stirred for 8 h. The volatiles were removed under reduced pressure, and the residue was purified by
column chromatography (petroleum ether – ethyl acetate – ethanol 15:1:1). The product was
1
isolated as white solid, 40% yield; mp 144-145 °C. H NMR: (400 MHz, DMSO-d ) δ 11.27 (s, 1H,
6
NH-indole), 8.53 (d, J = 8.4 Hz, 1H, NHAc), 7.47 (s, 1H, C5”-H), 7.33 (s, 1H, C4”-H), 6.47 (s, 1H,
C3”-H), 5.18 (d, J = 12.8 Hz, 2H, CH OH), 4.52 – 4.47 (m, 1H, C1-H), 3.90 (s, 3H, OMe), 3.86 (s,
2
3
1
H, OMe), 3.04 – 3.00 (m, 1H, C6-H), 2.06 – 1.93 (m, 2H, C7-H), 1.90 (s, 3H, C(O)CH ), 1.86 –
3
1
3
.79 (m, 1H, C6-H). C NMR: (101 MHz, DMSO-d ) δ 168.4, 150.1, 149.2, 145.9, 136.3, 134.6,
6

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1
2
33.7, 133.6, 130.8, 125.9, 124.5, 121.3, 112.5, 105.7, 102.2, 61.2, 60.7, 60.3, 59.0, 48.4, 31.3,
2.7, 20.7. MS: m/z (%) = 412.94 (100%), 410.90 (57%), 472.90 (74%), 470.94 (53%), 473.94
(61%), 471.91 (73%), 428.90 (45%), 430.96 (34%), 488.02 (27%), 488.96 (10%), 490.81 (7%).
1
9
Anal. Calcd for C H BrN O : C, 56.45; H, 5.15. Found: C, 56.39; H, 5.19. [α] = -28.2 (c =
2
3
25
2
5
D
0.59, Chloroform).
4.2.2 Synthesis of 1ʹ-bromo-2ʹ, 3ʹ, 4ʹ-trimethoxybenzo[5ʹ, 6ʹ:4, 5]1H-(aR, 1S)1-acetamido-6, 7-
dihydrocyclohepta[3, 4-I]1H-2-tert-butyldimethylsilyloxymethylindole (11)
Into a Schlenk flask with a stirring bar were placed compound 10 (80.0 mg, 0.120 mmol),
palladium(diphenylphosphinoferrocene) dichloride (4.4 mg, 0.006 mmol), and copper (I) iodide
(2.3 mg, 0.012 mmol). The flask was filled with argon. Acetonitrile (3.0 mL) was added under inert
atmosphere. N,N-diisopropylethylamine (0.095 mL, 0.366 mmol) and tert-butyldimethyl(2-
propynyloxy)silane (0.030 mL, 0.146 mmol) were added to the reaction mixture, the temperature
was raised to 90 °C and kept for 8 h. The volatiles were removed under reduced pressure and the
residue was purified by column chromatography (petroleum ether-ethyl acetate-ethanol 5:1:1). The
1
product was obtained as pale-beige oil in 51% yield. H NMR: (400 MHz, DMSO-d ) δ 11.06 (s,
6
1H, NH-indole), 8.55 (d, J = 8.5 Hz, 1H, NHAc), 7.42 (s, 1H, C5”-H), 7.31 (s, 1H, C4”-H), 6.31 (s,
1
H, C3”-H), 4.78 (s, 2H, CH OTBDMS), 4.48 – 4.41 (m, 1H, C1-H), 3.89 (s, 3H, OMe), 3.84 (s,
2
3
H, OMe), 3.36 (s, 3H, OMe), 2.07 – 1.96 (m, 4H, C6-H, C7-H), 1.90 (s, 3H, C(O)CH ), 0.89 (s,
3
t
13
9
H, Si-Bu ), 0.09 (s, 6H, SiMe ). C NMR: (101 MHz, DMSO-d ) δ 168.7, 150.3, 149.2, 146.0,
2
6
139.0, 136.3, 133.9, 133.9, 131.1, 126.2, 124.3, 121.1, 112.6, 105.7, 99.5, 61.3, 60.9, 60.5, 58.8,
4
8.6, 36.8, 29.9, 26.0, 22.8, 18.2, -5.0. Anal. Calcd for C H BrN O Si: C, 57.70; H, 6.51. Found:
29
39
2
5
C, 58.00; H, 6.55.
4.2.3 Synthesis of 1ʹ-bromo-2ʹ, 3ʹ, 4ʹ-trimethoxybenzo[5ʹ, 6ʹ:4, 5]1H-(aR, 1S)1-acetamido-6, 7-
dihydro-cyclohepta[3, 4-f]1H-1-methyl-2-hydroxymethylindole (7)
Pyrrolo-allocolchicinoid 11 (48.0 mg, 0.079 mmol) was treated with sodium hydride (60% in
mineral oil, 0.190 mmol, 7.6 mg) and methyl iodide (17.7 mg, 0.125 mmol) in THF at 0 °C. The

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temperature was raised to 65 °C. The mixture was stirred for 6 h, then the solvent was removed and
the residue was filtered through a short pad of silica. The crude product was dissolved in 4.0 mL of
THF, 0.045 mL of 1N TBAF in THF was added, and the mixture was stirred for 3 h. The volatiles
were removed, and the product was isolated using column chromatography (petroleum ether – ethyl
acetate – ethanol (15:1:1)). The product 7 was obtained as white solid in 55% yield (for 2 steps), mp
1
1
75-177 °С. H NMR: (400 MHz, DMSO-d ) δ 8.51 (d, J = 8.3 Hz, 1H, NHAc), 7.46 (s, 1H, C5”-
6
H), 7.37 (s, 1H, C4”-H), 6.38 (s, 1H, C3”-H), 5.22 (t, J = 5.0 Hz, 1H, OH), 4.64 (d, J = 5.1 Hz, 2H,
CH OH), 4.60 – 4.56 (m, 1H, C1-H), 3.91 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.77 (s, 3H, NMe),
2
3.34 (s, 3H, OMe), 3.04 – 2.99 (m, 1H, C6-H), 2.10 – 2.05 (m, 1H, C6-H), 2.02 – 1.97 (m, 1H, C7-
1
3
H), 1.93 (s, 3H, C(O)CH ), 1.84 – 1.79 (m, 1H, C7-H). C NMR: (101 MHz, DMSO-d ) δ 168.5,
3
6
150.1, 149.1, 145.8, 140.7, 137.2, 133.8, 133.7, 130.2, 125.2, 124.3, 121.2, 112.5, 103.6, 100.1,
61.2, 60.7, 60.2, 55.5, 48.3, 37.0, 29.8, 29.7, 22.80. MS: m/z (%) = 502.08 (100%), 504.09 (91%),
443.09 (79%), 445.08 (78%), 431.10 (50%), 429.08 (39%) 414.06 (61%), 416.08 (60%), 401.07
(29%), 399.09 (28%). Anal. Calcd for C H BrN O : C, 57.26; H, 5.41. Found: C, 57.33; H, 5.44.
24 27 2 5
1
9
[
α] = -37.6 (c = 0.59, Chloroform).
D
4.3. Cell cultures. Pancreatic human cell lines COLO-357, MiaPaCa-2, PANC-1; nontransformed
human embryonic kidney cell line HEK293, human colon cell line HT-29 were grown in DMEM
medium supplemented with 10% fetal calf serum (FCS), pen-strep-glut (all from PanEco, Moscow,
Russian Federation). Adherent cells were detached using 0.05% trypsin-EDTA (PanEco, Moscow),
counted and sub-cultured. Twenty-four hours before the assays, cells were seeded in the appropriate
5
plates (96- or 24-well plates), adjusted to 3×10 cells/mL, and incubated overnight to achieve
standardized growth conditions.
4.4. MTT-assay. Cytotoxic effect of colchicine and the compounds 7 and 12 was estimated by a
standard 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test as
described earlier [9]. All the compounds were dissolved in dimethyl sulfoxide to 20 mM
o
concentration and stored at -20 C until the assay. Different dilutions of the new compounds from

ACCEPTED MANUSCRIPT
2
0 µM to 0.1 nM were prepared separately and transferred in 100 µL to the plates with the cells.
Non-treated cells served as controls. Plates were incubated for 72 h. For the last 6 h, 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 dimethyl sulfoxide was added to each well. Plates were incubated in a shaker
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 (IC ) was calculated from the titration
5
0
curves. The inhibition of proliferation (inhibition index, II) was calculated as [1 – (ODexperiment
ODcontrol)], where OD was MTT optical density.
/
4.5. Confocal analysis. For confocal analysis, cells were grown overnight on sterile cover slips
immersed into 6-well plates (Costar) in 200 µL of complete culture medium. Colchicine or new
compounds 12 and 7 were dissolved in 4 mL of complete medium and added to the wells. After the
incubation, cells were stained with different trackers. Wheat germ agglutinin (WGA)-
AlexaFluor555, LysoTrackerRed, MitoTrackerRed, and Transferrin-AlexaFluot555 (all from
Molecular Probes, Invitrogen, USA) were used to stain membranes and Golgi apparatus, lysosomes,
mitochondria and endosomes accordingly. After the incubation with the trackers, cells were fixed
with 1% paraformaldehyde, washed, and treated with Mowiol 4.88 medium (Calbiochem,
Germany). Tubulin was identified by anti-tubulin antibody (SantaCruz, USA) followed by anti-
mouse IgG-AlexaFluor555 (Molecular Probes, Invitrogen, USA) in cells permeabilized by 0.1%
Triton X100 in PBS. Hoechst 33342 (Sigma) was used to visualize nuclei. Slides were analyzed
using Eclipse TE2000 confocal microscope (Nikon, Japan).
4.6. Flow cytometry. Cell cycle was analyzed using PI-stained DNA. Cells from colchicinoid-
treated cultures were collected at indicated time, trypsinized, washed in ice-cold PBS, fixed by the
o
addition of 70% ethanol and left for 2 h at -20 C. Thereafter, the cells were washed twice in PBS,
stained with 50 µg/mL of propidium iodide (Sigma Chemical Co) in PBS, treated with 10 µg/mL of
RNAse and analyzed by flow cytometry using FACScan device (BD, USA). Total 2000 events were

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collected. The results were analyzed using Flowing 2.5.1 software (Finland). To analyze lysosomal
and mitochondrial membrane potentials, cells were incubated with colchicinoids for 3 h, stained
with the trackers for the last 30 min, detached by trypsin-EDTA and analyzed by flow cytometry.
Total 20000 events were collected.
4.7. Docking study. Docking calculations were carried out using the AutoDock 4.2 program [17].
The default settings were used with the MM potential of MMFF94 type. The necessary hydrogen
atoms, Kollman united atom charges, and solvation parameters were added using the
3
AutoDockTools [17]. Grid maps of 30×30×30 Å with 3Å mesh size were generated with AutoGrid
[
17]. Lamarckian genetic algorithm combined with the Solis-Wets [18] local optimization algorithm
were used for the structure optimization. Each docking experiment was resultant from 200 that were
to come to an end after a maximum of 2500000 energy evaluations. The geometry of the ligands
(colchicine 1, and of compounds 7 and 12) was obtained on the basis of full geometry optimization
at the DFT level (B3LYP/6-31G(d,p)) using the Gaussian 03 program [19].
Notes
The authors declare no competing financial interest.
Acknowledgements
We thank the Russian Science Foundation (Project 16-13-10248).
Appendix A. Supplementary data
The following is the supplementary data related to this article:
1
13
General information, syntheses and characterization of compounds, H and C NMR spectra,
biological assays, docking study.
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Highlights:
•
•
•
•
Two pyrrole-based allocolchicinoids were prepared from commercial colchicine
Compounds exhibit excellent cytotoxicity towards epithelial cell lines
7 and 12 induced cell accumulation in the G2/M phase in 2D cultures
7 has lower destructive short-time effects on HT-29 cell line in comparison with colchicine