Antiproliferative activity and molecular docking of novel double-modified colchicine derivatives

Article abstract of DOI:10.3390/cells7110192

Microtubules are tubulin polymer structures, which are indispensable for cell growth and division. Its constituent protein β-tubulin has been a common drug target for various diseases including cancer. Colchicine has been used to treat gout, but it has also been an investigational anticancer agent with a known antimitotic effect on cells. However, the use of colchicine as well as many of its derivatives in long-term treatment is hampered by their high toxicity. To create more potent anticancer agents, three novel double-modified colchicine derivatives have been obtained by structural modifications in C-4 and C-10 positions. The binding affinities of these derivatives of colchicine with respect to eight different isotypes of human β-tubulin have been calculated using docking methods. In vitro cytotoxicity has been evaluated against four human tumor cell lines (A549, MCF-7, LoVo and LoVo/DX). Computer simulations predicted the binding modes of these compounds and hence the key residues involved in the interactions between tubulin and the colchicine derivatives. Two of the obtained derivatives, 4-bromothiocolchicine and 4-iodothiocolchicine, were shown to be active against three of the investigated cancer cell lines (A549, MCF-7, LoVo) with potency at nanomolar concentrations and a higher relative affinity to tumor cells over normal cells.

Full text of DOI:10.3390/cells7110192

cells
Article
Antiproliferative Activity and Molecular Docking of
Novel Double-Modified Colchicine Derivatives
1
1
2
3
3
Urszula Majcher , Greta Klejborowska , Mahshad Moshari , Ewa Maj , Joanna Wietrzyk
,
4
5
1,
Franz Bartl , Jack A. Tuszynski and Adam Huczy n´ ski *
1
Department of Bioorganic Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b,
1-614 Poznan, Poland; urszula.majcher@amu.edu.pl (U.M.); greta.klejborowska@amu.edu.pl (G.K.)
Department of Chemistry, University of Alberta, Edmonton, AB T6G 1Z2, Canada; moshari@ualberta.ca
Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa
Weigla12, 53-114 Wrocław, Poland; ewa.maj@iitd.pan.wroc.pl (E.M.); wietrzyk@iitd.pan.wroc.pl (J.W.)
Institut für Biologie, AG Biophysikalische Chemie, Humboldt Universität zu Berlin, Invalidenstr, 42,
6
2
3
4
1
0099 Berlin, Germany; bartlfra@cms.hu-berlin.de
5
Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada; jack.tuszynski@gmail.com
Correspondence: adhucz@amu.edu.pl; Tel.: +48-61-829-1673
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 5 October 2018; Accepted: 31 October 2018; Published: 1 November 2018
Abstract: Microtubules are tubulin polymer structures, which are indispensable for cell growth
and division. Its constituent protein -tubulin has been a common drug target for various diseases
β
including cancer. Colchicine has been used to treat gout, but it has also been an investigational
anticancer agent with a known antimitotic effect on cells. However, the use of colchicine as well as
many of its derivatives in long-term treatment is hampered by their high toxicity. To create more
potent anticancer agents, three novel double-modified colchicine derivatives have been obtained
by structural modifications in C-4 and C-10 positions. The binding affinities of these derivatives of
colchicine with respect to eight different isotypes of human β-tubulin have been calculated using
docking methods. In vitro cytotoxicity has been evaluated against four human tumor cell lines (A549,
MCF-7, LoVo and LoVo/DX). Computer simulations predicted the binding modes of these compounds
and hence the key residues involved in the interactions between tubulin and the colchicine derivatives.
Two of the obtained derivatives, 4-bromothiocolchicine and 4-iodothiocolchicine, were shown to
be active against three of the investigated cancer cell lines (A549, MCF-7, LoVo) with potency at
nanomolar concentrations and a higher relative affinity to tumor cells over normal cells.
Keywords: colchicine binding site inhibitor; β-tubulin affinity; antimitotic agent; antiproliferative
activity; thiocolchicine
1
. Introduction
Microtubules, present in all eukaryotic cells, are cylindrical polymers composed of α/β-tubulin
heterodimers. They are involved in a wide range of key cellular processes, such as the maintenance
of cellular morphology and the active motor transport of cellular components throughout the
cytoplasm [1]. Another essential role microtubules play is the formation of mitotic spindles and
force generation during mitosis with the purpose of separating chromosomes [
2]. A failure within
this mitotic spindle apparatus leads to mitotic arrest and eventually apoptosis. This results in cell
death, which is a desirable outcome for cancer cells, but not for healthy tissues. With the objective of
promoting the former and avoiding the latter effect, microtubules have become the target for a large
number of antimitotic agents that act by either favoring or inhibiting microtubule polymerization by
binding at specific sites on the exposed surface of α/β-tubulin heterodimers [3–7]. Although there are
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Cells 2018, 7, 192
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multiple distinct binding sites on a tubulin heterodimer,
major microtubule-targeting drug families [8–10].
β-tubulin is the main binding partner for all
Among them colchicine (1), a well-known tropolone alkaloid isolated from Colchicum autumnale,
is of particular interest due to its powerful antimitotic properties. It has played an important role in
studies of mitosis and the therapeutic potential of using the colchicine binding site on
chemotherapy applications has generated much interest [ 11 16]. However, colchicine itself as well
as many of its derivatives, have not yet been used as successful drugs in long-term treatment because
of their detrimental side effects [ 11]. Up to now, many structure-activity relationship studies have
β-tubulin in
5
–7
,
–
6,7,
been performed to elucidate the structural features required for tubulin binding. These studies have
demonstrated great importance of the 9-keto function and the methoxy groups at C-1, C-2, and C-10 as
well as the importance of stereochemistry of 7-acetamido center, which is critical for antimitotic activity.
Ring B appears to be responsible for the irreversible nature of colchicine binding to tubulin, although
it may also contribute to its toxic effects [11,17]. Therefore, currently much interest has focused on
structural modification of 1 in the hope of improving its anticancer activity [18–33].
In 2011 Yasobu et al. published results of their studies on C-4 halogen substituted colchicine
derivatives [32]. On the evaluation of cell-growth inhibitory activity using mice transplanted with the
HCT116 human colorectal carcinoma cell line, some of the derivatives exhibited less toxicity in mice
and more potent cell-growth inhibitory activity than
thiomethyl group at C-10 called thiocolchicine, is also a potent inhibitor of tubulin polymerization
and cell growth, and binds to tubulin more rapidly than colchicine [34 36]. Thiocolchicine is not only
1. Moreover, another colchicine derivative with
–
easily available from colchicine after treatment with sodium methanethiolate, but also is more stable,
which allows for using harsher reaction conditions without formation of isomers.
Inspired by these reports, we decided to verify how double modification in C-4 and C-10 positions
influences the activity and selectivity of colchicine. Below, we report the synthesis and spectroscopic
analysis of a series of seven compounds, of which three are entirely novel compounds synthesized
for the first time. We also provide an evaluation of these derivatives as cytotoxic, tubulin-targeting
agents. The antiproliferative effect of seven colchicines derivatives (
four cancer cell lines and one normal murine embryonic fibroblast cell line. To better understand the
interactions between these colchicine derivatives and various isotypes of -tubulin, we investigated
potential binding modes of novel double-modified derivatives, 4-halocolchicines as well as colchicine
docked into the colchicine binding site (CBS) of eight different isotypes of -tubulin using AutoDock4
2–8) was tested in vitro using
β
β
software (version 2018.2.0, Tableau Research, Standford University, Seattle, WA, USA) under flexible
ligand and rigid receptor condition. A detailed discussion regarding differences between the structures
of the synthesized compounds and their ability to form complexes with CBS is provided below.
2
. Materials and Methods
2
.1. General
All precursors for the synthesis and solvents were obtained from Sigma-Aldrich (Merck KGaA,
Saint Louis, MO, USA) and were used as received without further purification. CDCl spectral grade
3
solvent was stored over 3 Å molecular sieves for several days. Thin layer chromatographywas carried
out on precoated plates (TLC silica gel 60 F254, Aluminum Plates Merck (Merck KGaA Saint Louis,
MO, USA)) and spots were detected by illumination with an ultra-violet (UV) lamp. All the solvents
used in flash chromatography were of HPLC grade (CHROMASOLV from Sigma-Aldrich, Merck
KGaA, Saint Louis, MO, USA) and were used as received. The elemental analysis of compounds was
carried out on Vario ELIII (Elementar, Langenselbold, Germany).
2
.2. Spectroscopic Measurements
1
13
The H, C spectra were recorded on a Varian VNMR-S 400 MHz spectrometer (Varian, Inc.,
1
−3
Palo Alto, CA, USA). H-NMR measurements of 2–8 (0.07 mol dm ) in CDCl were carried out at the
3

Cells 2018, 7, 192
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operating frequency 402.64 MHz. The error of the chemical shift value was 0.01 ppm. The 13C-NMR
spectra were recorded at the operating frequency 101.25 MHz. The error of chemical shift value was
1
13
0
.1 ppm. All spectra were locked to deuterium resonance of CDCl . The H and C-NMR spectra are
3
shown in the Supplementary Materials.
The FT-IR spectra of in the mid infrared region were recorded in KBr. The spectra were taken
with an IFS 113v FT-IR spectrophotometer (Bruker, Karlsruhe, Germany) equipped with a deuterated
2–8
–1
triglycine sulfate detector (DTGS) detector; resolution 2 cm , NSS = 64. The Happ-Genzel apodization
function was used.
The ESI (Electrospray Ionization) mass spectra were recorded also on a Waters/Micromass (Waters
Corporation, Manchester, UK) ZQ mass spectrometer equipped with a Harvard Apparatus syringe
− −3
5
pump. The samples were prepared in dry acetonitrile (5
×
10 mol dm ). The sample was infused
−
1
into the ESI source using a Harvard pump at a flow rate of 20 mL min . The ESI source potentials
were: capillary 3 kV, lens 0.5 kV, extractor 4 V. The standard ESI mass spectra were recorded at the
◦
cone voltages: 10 and 30 V. The source temperature was 120 C and the desolvation temperature was
◦
3
−
1
3
00 C. Nitrogen was used as the nebulizing and desolvation gas at flow-rates of 100 dm h . Mass
spectra were acquired in the positive ion detection mode with unit mass resolution at a step of 1 m/z
unit. The mass range for ESI experiments was from m/z = 100 to m/z = 1000, as well as from m/z = 200
to m/z = 1500.
2
.3. Synthesis
2
.3.1. Synthesis of 2
To a mixture of
1 (500 mg, 1.25 mmol) in MeOH/water (1/1, v/v, 5 mL), the sodium
methanethiolate (solution 21% in H O, 0.83 mL, 2.5 mmol) was added. The mixture was stirred
2
in at RT for 72 h. Reaction time was determined by TLC. After that time, the reaction mixture was
quenched by the addition of water (150 mL). The whole mixture was extracted four times with CH Cl ,
2
2
and the combined organic layers were dried over MgSO , filtered, and evaporated under reduced
4
pressure. The residue was purified by CombiFlash® (hexane/EtOAc (1/1), then EtOAc/MeOH,
increasing concentration gradient) to give 2 with yield 78% [34].
The synthesis of compounds 4, 6 and 8 was carried out analogously to the above starting
respectively from the compounds 3, 5 and 7.
1
Compound
2
, H-NMR (403 MHz, CDCl )
δ
7.92 (s, 1H), 7.46 (s, 1H), 7.33 (d, J = 10.4 Hz, 1H), 7.10 (d,
3
J = 10.5 Hz, 1H), 6.55 (s, 1H), 4.72–4.64 (m, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 3.67 (s, 3H), 2.54 (dd, J = 13.0,
5
.8 Hz, 1H), 2.45 (s, J = 5.7 Hz, 3H), 2.43–2.26 (m, 2H), 1.99 (s, 3H), 1.94 (dd, J = 11.8, 5.5 Hz, 1H) ppm;
1
3
C-NMR (101 MHz, CDCl3)
δ 182.4, 170.0, 158.1, 153.6, 151.8, 151.1, 141.6, 138.6, 134.8, 134.4, 128.3,
1
1
26.7, 125.6, 107.3, 61.6, 61.4, 56.1, 52.3, 36.4, 29.9, 22.8, 15.1 ppm. FT-IR (KBr pellet): 3283, 2935, 1660,
−
1
605, 1541, 1485, 1461, 1425, 1404, 1349, 1321, 1286, 1236, 1195, 1155, 1138, 1095, 1023 cm . ESI-MS
+
+
+
(m/z): [M + H] calcd. 416, found 416, [M + Na] calcd. 438, found 438, [M + K] calcd. 454 found 454,
+
+
[2M + Na] calcd. 853, found 853, [3M + Na] calcd. 1268, found 1268.
1
Compound
4
, Amorphous yellow solid. H-NMR (403 MHz, CDCl )
δ
7.98 (d, J = 6,7 Hz, 1H), 7.44 (s,
3
1
H), 7.26 (d, J = 10.3 Hz, 1H), 7.08 (d, J = 10.8 Hz, 1H), 4.58 (dt, J = 13.1, 6.7 Hz, 1H), 3.98 (s, 3H), 3.96 (s,
3
(
1
2
H), 3.61 (s, 3H), 3.24 (dd, J = 13.5, 4.8 Hz, 1H), 2.44 (s, 3H), 2.27 (ddd, J = 18.0, 12.1, 6.0 Hz, 1H), 2.14
182.4, 170.1,
59.1, 151.3, 150.2, 149.7, 146.6, 137.3, 134.8, 131.7, 129.9, 128.1, 126.4, 122.1, 61.6, 61.5, 61.1, 52.2, 34.5,
5.9, 22.8, 15.1 ppm. FT-IR (KBr pellet): 3290, 2936, 1661, 1608, 1550, 1464, 1413, 1349, 1327, 1288, 1267,
td, J = 13.4, 6.2 Hz, 1H), 2.00 (s, 3H), 1.92–1.80 (m, 1H); 13C-NMR (101 MHz, CDCl3)
δ
−
1
+
+
1197, 1140, 1086, 1023 cm . ESI-MS (m/z): [M + H] calcd. 450, found 450, [M + Na] calcd. 472,
+
+
found 472, [2M + H] calcd. 889, found 889, [2M + Na] calcd. 921, found 921. Anal. Calcd. for C,
8.73; H, 5.38; Cl, 7.88; N, 3.11; O, 17.78; S, 7.13; found: C, 58.61; H 5.35; Cl, 7.93; N, 3.01; S, 7.25.
5

Cells 2018, 7, 192
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1
Compound
6
, Amorphous yellow solid. H-NMR (403 MHz, CDCl )
δ
7.68 (d, J = 6.6 Hz, 1H), 7.42 (s,
3
1
3
(
1
H), 7.26 (d, J = 9.6 Hz, 1H), 7.08 (d, J = 10.8 Hz, 1H), 4.61–4.52 (m, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.63 (s,
H), 3.27 (d, J = 8.0 Hz, 1H), 2.45 (s, 3H), 2.25 (dt, J = 13.4, 7.9 Hz, 2H), 2.01 (s, J = 1.6 Hz, 3H), 1.85
dd, J = 6.7, 4.1 Hz, 1H) ppm; 13C-NMR (101 MHz, CDCl3)
182.4, 170.0, 159.2, 151.2, 151.0, 150.4,
46.6, 137.4, 134.8, 133.4, 130.2, 128.1, 126.3, 113.5, 61.6, 61.5, 61.0, 52.2, 34.5, 29.0, 22.9, 15.2 ppm. FT-IR
δ
−
1
(
KBr pellet): 3267, 2930, 1659, 1603, 1559, 1462, 1410, 1347, 1138, 1074, 1053, 1014 cm . ESI-MS (m/z):
+
+
+
[M + H] calcd. 494, found 494, [M + 2 + H] 496, found 496, [M + Na] calcd. 516, found 516, [M + 2
+
+
+
+
Na] calcd. 518, found 518, [2M + H] calcd. 989, found 989, [2M + 2 + H] calcd. 991, found 991,
+
+
[
2M + Na] calcd. 1011, found 1011, [2M + 2 + Na] calcd. 1013, found 1013. Anal. Calcd. for C, 53.45;
H, 4.89; Br, 16.16; N, 2.83; O, 16.18; S, 6.49; found: C, 53.56; H 4.81; Br, 16.28; N, 2.89; S, 6.55.
1
Compound
8
, Amorphous yellow solid. H-NMR (403 MHz, CDCl )
δ
7.75 (d, J = 6.9 Hz, 1H), 7.42 (s,
3
1
(
(
H), 7.25 (d, J = 10.3 Hz, 1H), 7.09 (d, J = 10.8 Hz, 1H), 4.58–4.50 (m, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.63
s, 3H), 3.18 (dd, J = 13.7, 5.0 Hz, 1H), 2.46 (s, 3H), 2.40 (dd, J = 13.6, 6.2 Hz, 1H), 2.32–2.23 (m, 1H), 2.01
s, 3H), 1.85–1.79 (m, 1H); 13C-NMR (101 MHz, CDCl3)
182.4, 170.1, 159.1, 153.5, 151.4, 151.1, 145.6,
37.8, 136.8, 134.7, 129.7, 128.1, 126.3, 92.2, 61.6, 61.4, 60.8, 52.1, 34.5, 34.4, 22.9, 15.2 ppm. FT-IR (KBr
δ
1
−
1
pellet): 3288, 2936, 1660, 1607, 1547, 1461, 1406, 1346, 1318, 1288, 1262, 1197, 1138, 1081, 1019 cm
ESI-MS (m/z): [M + H] calcd. 542, found 542, [M + Na] calcd. 564, found 564, [M + K] calcd. 580,
.
+
+
+
found 580. Anal. Calcd. for C, 48.81; H, 4.47; I, 23.44; N, 2.59; O, 14.78; S, 5.92; found: C, 48.67; H 4.55; I,
2
3.59; N, 2.64; S, 5.98.
2
.3.2. Synthesis of 3
A mixture of N-chlorosuccinimide (175 mg, 1.31 mmol) and
1 (500 mg, 1.25 mmol) in acetonitrile
was stirred at RT under nitrogen atmosphere for the 72 h. Reaction time was determined by TLC.
The reaction was quenched with saturated aqueous Na S O . The whole mixture was extracted
2
2
3
four times with CH Cl , and the combined organic layers were dried over MgSO , filtered, and
2
2
4
®
evaporated under reduced pressure. The residue was purified by CombiFlash (EtOAc/MeOH,
increasing concentration gradient) to give 3 with yield 75% [32].
1H-NMR (403 MHz, CDCl3)
J = 11.2 Hz, 1H), 4.60–4.49 (m, 1H), 4.01 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 3.61 (s, 3H), 3.23 (dd, J = 13.7,
δ 8.29 (d, J = 6.2 Hz, 1H), 7.59 (s, 1H), 7.30 (d, J = 10.7 Hz, 1H), 6.87 (d,
5
.1 Hz, 1H), 2.31 (dq, J = 18.7, 6.2 Hz, 1H), 2.18–2.09 (m, 1H), 1.96 (s, 3H), 1.93–1.82 (m, 1H) ppm;
1
3
C-NMR (101 MHz, CDCl3)
δ 179.5, 170.2, 164.3, 152.0, 150.1, 149.7, 146.6, 135.8, 135.8, 131.7, 130.1,
1
1
29.8, 122.1, 112.5, 61.5, 61.5, 61.1, 56.5, 52.7, 34.5, 25.8, 22.7 ppm. FT-IR (KBr pellet): 3256, 2935, 1663,
−
1
618, 1591, 1556, 1456, 1412, 1397, 1351, 1290, 1272, 1243, 1171, 1136, 1080, 1021 cm . ESI-MS (m/z):
+
+
+
[M + H] calcd. 434, found 434, [M + Na] calcd. 456, found 456, [2M + Na] calcd. 889, found 889.
2
.3.3. Synthesis of 5
A mixture of N-bromosuccinimide (279 mg, 1.57 mmol) and
1 (500 mg, 1.25 mmol) in acetonitrile
was stirred at RT under nitrogen atmosphere for the 72 h. Reaction time was determined by TLC.
The reaction was quenched with saturated aqueous Na S O . The whole mixture was extracted
2
2
3
four times with CH Cl , and the combined organic layers were dried over MgSO , filtered, and
2
2
4
®
evaporated under reduced pressure. The residue was purified by CombiFlash (EtOAc/MeOH,
increasing concentration gradient) to give 5 with yield 95% [32].
1H-NMR (403 MHz, CDCl3)
δ
8.02 (s, 1H), 7.58 (s, 1H), 7.30 (d, J = 10.7 Hz, 1H), 6.88 (d, J = 11.1 Hz,
1
1
H), 4.59–4.49 (m, 1H), 4.03 (s, 3H), 3.99 (s, 3H), 3.96 (s, 3H), 3.63 (s, 3H), 3.27 (dd, J = 13.0, 4.3 Hz,
13
H), 2.26 (dd, J = 13.1, 5.2 Hz, 1H), 2.18 (d, J = 2.4 Hz, 1H), 1.99 (s, 3H), 1.78 (s, 1H) ppm; C-NMR
179.5, 170.2, 164.4, 151.8, 151.1, 150.4, 146.6, 135.8, 135.7, 133.4, 130.2, 130.0, 113.5,
12.4, 61.5, 61.5, 61.0, 56.5, 52.6, 34.5, 28.9, 22.8 ppm. FT-IR (KBr pellet): 3274, 2936, 1662, 1617, 1589,
(
101 MHz, CDCl3) δ
1
1
−
1
+
565, 1462, 1411, 1398, 1350, 1270, 1250, 1172, 1137, 1080, 1018 cm . ESI-MS (m/z): [M + Na] calcd.

Cells 2018, 7, 192
5 of 16
+
+
+
5
00, found 500, [M + 2 + Na] calcd. 502, found 502, [2M + 2 + Na] calcd. 979, found 979, [2M + Na]
+
calcd. 977, found 977, [2M + 4 + Na] calcd. 981, found 981.
2
.3.4. Synthesis of 7
A mixture of N-iodosuccinimide (560 mg, 2.49 mmol) and
stirred at 70 C under nitrogen atmosphere for the 20 h. Reaction time was determined by TLC.
1
(500 mg, 1.25 mmol) in AcOH was
◦
The reaction was quenched with saturated aqueous Na S O . The whole mixture was extracted
2
2
3
four times with CH Cl , and the combined organic layers were dried over MgSO , filtered, and
2
2
4
®
evaporated under reduced pressure. The residue was purified by CombiFlash (EtOAc/MeOH,
increasing concentration gradient) to give 7 with yield 95% [32].
1H-NMR (403 MHz, CDCl3)
J = 11.2 Hz, 1H), 4.55–4.47 (m, 1H), 4.04 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 3.63 (s, 3H), 3.21–3.15 (m,
δ 8.22 (d, J = 5.6 Hz, 1H), 7.61 (s, 1H), 7.30 (d, J = 10.7 Hz, 1H), 6.89 (d,
1
3
1
1
5
1
H), 2.40 (dd, J = 12.7, 5.0 Hz, 1H), 1.99 (s, 3H), 1.87–1.81 (m, 1H); C-NMR (101 MHz, CDCl3)
70.2, 164.4, 153.4, 152.0, 151.4, 145.6, 136.7, 136.2, 135.6, 130.1, 129.5, 112.5, 92.1, 61.5, 61.3, 60.7, 56.5,
2.6, 34.4, 34.4, 22.7 ppm; FT-IR (KBr pellet): 3274, 2934, 1662, 1617, 1588, 1563, 1461, 1406, 1393, 1346,
δ 179.5,
−
1
+
+
318, 1266, 1249, 1171, 1136, 1078, 1015 cm . ESI-MS (m/z): [M + H] calcd. 526, found 526 [M + Na]
calcd. 548, found 548.
2
.4. Antiproliferative Activity of Colchicine and Its Derivatives
Four human cancer cell lines and one murine normal cell line were used to evaluate
antiproliferative activity of colchicine and its derivatives: human lung adenocarcinoma (A549), human
breast adenocarcinoma (MCF-7), human colon adenocarcinoma cell lines sensitive and resistant to
doxorubicin (LoVo) and (LoVo/DX) respectively, and normal murine embryonic fibroblast cell line
(
(
BALB/3T3). The BALB/3T3 cell line was purchased from the American Type Culture Collection
ATCC, Manassas, VA, USA), A549 and MCF-7 cell lines—from European Collection of Authenticated
Cell Cultures (Salisbury, UK), LoVo cell line was purchased from the ATCC (ATCC, Manassas, VA,
USA), and LoVo/DX by courtesy of Prof. E. Borowski (Technical University of Gda n´ sk, Gda n´ sk,
Poland). All the cell lines are maintained in the Institute of Immunology and Experimental Therapy
(IIET), Wroclaw, Poland. Human lung adenocarcinoma cell line was cultured in mixture of OptiMEM
and RPMI 1640 (1:1) medium (IIET, Wroclaw, Poland), supplemented with 5% fetal bovine serum (GE
Healthcare, Logan, UT, USA) and 2 mM L-glutamine (Sigma-Aldrich, Merck KGaA, Saint Louis, MO,
USA). Human breast adenocarcinoma cell line was cultured in mixture of Eagle medium (IIET, Wroclaw,
Poland), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 8 µg/mL insulin and 1%
amino-acids (Sigma-Aldrich, Merck KGaA, Saint Louis, MO, USA). Human colon adenocarcinoma
cell lines were cultured in mixture of OptiMEM and RPMI 1640 (1:1) medium (IIET, Wroclaw, Poland),
supplemented with 5% fetal bovine serum (GE Healthcare, Logan UT, USA), 2 mM L-glutamine, 1 mM
sodium pyruvate (Sigma-Aldrich, Merck KGaA, Saint Louis, MO, USA) and 10 µg/100 mL doxorubicin
for LoVo/DX (Sigma-Aldrich, Merck KGaA, Saint Louis, MO, USA). Murine embryonic fibroblast cells
were cultured in Dulbecco medium (Life Technologies Limited, Paisley, UK), supplemented with 10%
fetal bovine serum (GE Healthcare, Logan, UT, USA) and 2 mM glutamine (Sigma-Aldrich, Merck
KGaA, Saint Louis, MO, USA). All culture media contained antibiotics: 100 U/mL penicillin and
1
00
µ
g/mL streptomycin (Polfa-Tarchomin, Warsaw, Poland). All cell lines were cultured during
◦
entire experiment in humid atmosphere at 37 C and 5% CO . Cells were tested for mycoplasma
2
contamination by mycoplasma detection kit for conventional PCR: Venor GeM Classic (Minerva
Biolabs GmbH, Berlin, Germany) and negative results was obtained. The procedure is repeated every
year or in the case of less frequently used lines: after thawing.

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2
.4.1. The Antiproliferative Assays In Vitro
Twenty-four hours before adding the tested compounds, all cell lines were seeded in 96-well
4
plates (Sarstedt, Nümbrecht, Germany) in appropriate media with 10 cells per well. All cell lines
were exposed to each tested agent at four different concentrations in the range 100–0.01
µg/mL for
7
2 h. Cells were also exposed to the reference drug cisplatin (Teva Pharmaceuticals Polska, Warsaw,
Poland) and doxorubicin (Accord Healthcare Limited, Middlesex, UK). Additionally, all cell lines were
exposed to DMSO (solvent used for tested compounds) (POCh, Gliwice, Poland) at concentrations
corresponding to those present in tested agents’ dilutions. After 72 h sulforhodamine B assay (SRB)
was performed [37].
2
.4.2. SRB
After 72 h of incubation with the tested compounds, cells were fixed in situ by gently adding of
L per well of cold 50% trichloroacetic acid TCA (POCh, Gliwice, Poland) and were incubated at
5
4
0 µ
◦
C for one hour. Following, wells were washed four times with water and air dried. Next, 50 µL
of 0.1% solution of sulforhodamine B (Sigma-Aldrich, Merck KGaA, Saint Louis, MO, USA) in 1%
acetic acid (POCh, Gliwice, Poland) were added to each well and plates were incubated at room
temperature for 0.5 h. After incubation time, unbound dye was removed by washing plates four times
with 1% acetic acid whereas stain bound to cells was solubilized with 10 mM Tris base (Sigma-Aldrich,
Steinheim, Germany). Absorbance of each solution was read at Synergy H4 Hybrid Multi-Mode
Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) at the 540 nm wavelength.
Results are presented as mean IC50 (concentration of the tested compound, that inhibits cell
proliferation by 50%)
±
standard deviation. IC50 values were calculated in Cheburator 0.4, Dmitry
Nevozhay software (version 1.2.0 software by Dmitry Nevozhay, 2004–2014, http://www.cheburator.
nevozhay.com, freely available) for each experiment [38]. Compounds at each concentration were
tested in triplicates in single experiment and each experiment was repeated at least three times
independently. Results are summarized in Table 1. The Resistance Index (RI) was defined as the ratio
of IC50 for a given compound calculated for resistant cell line to that measured for its parental drug
sensitive cell line (Table 1).
2
.5. Molecular Docking Simulations
A combination of different theoretical methods was used to explore ligand-tubulin interactions.
The ligand structures were first minimized and then fully optimized based on the RHF/cc-pVDZ level
of theory in GAMESS-US version 2010-10-01. Since there is no crystal structure available for human
tubulin (TBB5_HUMAN), we obtained its sequence from UniProt (ID: Q13509). We used the tubulin
structure 1SA0.pdb as a template to construct the homology model for I tubulin using MOE2015.
βI
β
We then docked the small library of colchicine derivatives to the protein using the AutoDock4 program
under flexible ligand and rigid receptor conditions (Table 2). AutoDock4 software (version 2018.2.0,
Tableau Research, Standford University, Seattle, WA, USA) is designed to predict how drug candidates
bind to a receptor of a known 3D structure and consists of two main programs: AutoDock performs
the docking of the ligand to a set of grids describing the target protein; AutoGrid pre-calculates these
grids. The estimated Moriguchi octanol-water partition coefficient, MlogP, of the compounds were
calculated by ADMET Predictor 8.0 (ADMET Predictor, Simulations Plus, Lancaster, CA, USA).
3
. Results
3
.1. Chemistry
The synthetic routes to colchicines derivatives
was treated with sodium methanethiolate to give thiocolchicine (
previously described method [34]. 4-chlorocolchicine ( ), 4-bromocolchicine (
) were synthesized from by treatment with NCS, NBS, and NIS with yields from 75% up to
2
–
8
are outlined in Scheme 1. Colchicine (
) with yield 78% according to the
), and 4-iodocolchicine
1)
2
3
5
(7
1

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9
4
5%, respectively, based on the methods developed earlier [32]. For 4-chlorocolchicine (
3
) and
-bromocolchicine (
5
), the application of milder conditions, i.e., the replacement of acetic acid (the
solvent) by acetonitrile followed by reacting at room temperature, also allowed to obtain the same final
yields. Compounds were then treated with sodium methanethiolate to give double-modified
derivatives (4, 6, 8) with yields from 71% to 75%.
3, 5, 7
Scheme 1. Synthesis of colchicine derivatives (
RT; (b) for 5, NBS, acetonitrile, RT; (c) for 7, NIS, AcOH, 70 C; (d) MeOH/H O, CH SNa, RT.
2
–
8
). Reagents and conditions: (a) for
3
, NCS, acetonitrile,
◦
2
3
The structures of all products
2
–
8
were determined using the elemental analysis, ESI-MS, FT-IR,
1H- and 13C-NMR methods and are shown in Supplementary Material. In the C-NMR spectra of
13
the 4-halo derivatives a resonance for the C-4 carbon atom of the A aromatic ring was observed at
1
22.1 ppm for
After the introduction of thiomethyl group in C-10 positions shifts of the signal for the C-20 carbon
atom in compounds and were observed in the range 15.1–15.2 ppm, while in unmodified as
) shifts of the signal for the C-20 carbon atom were observed in the
3, at 113.5 ppm for 5 and at 92.1 ppm for 7, while in 1 it was observed at 107.3 ppm.
2,
4,
6
8
1
well as 4-halo derivatives (
3, 5, 7
range 56.1–56.5 ppm.
3
.2. In Vitro Determination of Drug-Induced Inhibition of Human Cancer Cell Line Growth
The synthesized colchicine derivatives ( ) and starting material ( ) were evaluated for their
2–
8
1
in vitro antiproliferative effect on normal and cancer cells. Each compound was tested on four human
cancer cell lines, including one cell line displaying various level of drug resistance, namely human lung
adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human colon adenocarcinoma cell
line (LoVo) and doxorubicin-resistant subline (LoVo/DX). The antiproliferative effect was also studied
on normal murine embryonic fibroblast cell line (BALB/3T3) for better description of cytotoxic activity
of the compounds studied. The mean values of IC50
±
SD of the tested compounds are collected in
Table 1. To evaluate the agents’ activity against the cells with MDR (multidrug resistance) phenotype,
one drug resistant cancer cell line, i.e., LoVo/DX, was tested and the indexes of resistance (RI) were
calculated (see Table 1). The RI values indicate how many times more resistant is the subline in
comparison to its parental cell line.
All obtained derivatives with single modification at either the C-4 or C-10 position as well as
double-modified compounds showed better antiproliferative activity against all tested cancer cell lines
than unmodified
1 and some common chemotherapeutics such as doxorubicin and cisplatin. The IC50
values of novel 4-halothiocolchines are better than for single-modified colchicines in C-4 positions and
remain at a level similar to the cytotoxicity of 2 for the A549 and MCF-7 cell lines.

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Table 1. Antiproliferative activity (IC ) of colchicine (1) and its derivatives (2–8) compared with antiproliferative activity of standard anticancer drugs doxorubicin
50
and cisplatin and the calculated values of the resistance index (RI) and selectivity index (SI) of tested compounds [19,39].
A549
MCF-7
LoVo
LoVo/DX
IC50 (µM)
BALB/3T3
IC50 (µM)
Compound
IC50 (µM)
SI
1.4
IC50 (µM)
SI
1.6
IC50 (µM)
SI
SI
RI
1
2
0.149 ± 0.009
0.011 ± 0.001
0.128 ± 0.135
0.010 ± 0.002
0.108 ± 0.025
0.021 ± 0.006
1.9
5.5
2.65 ± 0.96
0.398 ± 0.075
0.1
0.3
24.5
19.0
0.208 ± 0.042
0.114 ± 0.072
10.1
11.9
3
4
5
6
7
8
0.046 ± 0.035
0.022 ± 0.002
0.105 ± 0.008
0.010 ± 0.0001
0.094 ± 0.006
0.011 ± 0.002
0.258 ± 0.044
6.367 ± 1.413
3.0
1.0
1.4
10.3
1.4
10.5
0.023 ± 0.005
0.022 ± 0.002
0.027 ± 0.008
0.015 ± 0.002
0.098 ± 0.029
0.017 ± 0.006
0.386 ± 0.118
10.70 ± 0.753
6.0
1.0
5.3
6.9
1.4
6.8
0.4
0.4
0.069 ± 0.012
0.022 ± 0.002
0.084 ± 0.021
0.014 ± 0.004
0.010 ± 0.002
0.007 ± 0.002
0.092 ± 0.018
4.37 ± 0.73
2.0
1.0
1.7
0.784 ± 0.28
0.111 ± 0.044
1.55 ± 0.17
0.135 ± 0.012
2.78 ± 0.45
0.642 ± 0.084
4.75 ± 0.99
5.70 ± 0.63
0.2
0.2
0.1
0.8
0.1
0.2
<0.1
0.7
11.4
5.1
18.5
9.6
278.0
91.7
51.6
1.3
0.138 ± 0.069
0.022 ± 0.002
0.142 ± 0.073
0.103 ± 0.089
0.135 ± 0.056
0.115 ± 0.044
0.166 ± 0.074
3.90 ± 1.50
7.4
13.5
16.4
1.8
Doxorubicin
Cisplatin
0.6
0.6
0.9
The IC50 value is defined as the concentration of a compound at which 50% growth inhibition is observed. Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7),
human colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX), normal murine embryonic fibroblast cell line (BALB/3T3). The SI (Selectivity Index) was
calculated for each compound using the formula: SI = IC50 for normal cell line BALB/3T3/IC50 for respective cancerous cell line. A beneficial SI > 1.0 indicates a drug with efficacy against
tumor cells greater than the toxicity against normal cells. The RI (Resistance Index) indicates how many times a resistant subline is chemoresistant relative to its parental cell line. The RI
was calculated for each compound using the formula: RI = IC50 for LoVo/DX/IC50 for LoVo cell line. When RI is 0–2, the cells are sensitive to the compound tested, RI in the range 2–10
means that the cell shows moderate sensitivity to the drug tested, RI above 10 indicates strong drug resistance.

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As many as three of the compounds tested on the LoVo cell line (
6–
8), including two novel
double-modified derivatives (
even better than the activity of
6
2
,
8
), exhibited extremely high activity (IC50 = 0.007–0.014
µ
M), which is
(IC50 = 0.021
µM). During the tests on the doxorubicin-resistant subline
(LoVo/DX), compounds 4 and 6 showed the best activity among all tested compounds. However,
the RI values of the tested compounds indicated that colchicines did not break the drug resistance
of LoVo/DX (RI = 9.64–278). Comparison between the cancer cell lines and the normal cell line
(
BALB/3T3) was made to define the Selectivity Index (SI) as a measure of therapeutic potential.
This parameter seems to be especially important for drug-like molecules based on a scaffold of a toxic
compound. The SI values showed that compounds and mostly targeted cancer cells, and fewer
targeted normal cells (SI = 10.08–10.45, SI = 6.76–11.85, SI = 5.45–16.43 for A549, MCF-7, LoVo cancer
cell lines, respectively). Also compounds and indicated good SI values for MCF-7 cell line (SI = 6.00,
SI = 5.26, respectively), as well as compound 7 indicated good SI value for LoVo cell line (SI = 13.5).
2,
6
8
3
5
3
.3. Molecular Docking: in Silico Determination of Drug-Induced Inhibition of βI Tubulin
To further investigate the ability to inhibit tubulin aggregation by the new colchicine derivatives
in cancer cell growth assays, binding energies between the new compounds and βI tubulin, one of
the subunits of microtubules in the cytoskeleton structure of every eukaryotic cell, were calculated
using docking methodology. The eight structures of colchicine and its derivatives described above
were docked into the βI tubulin CBS and ranked according to their binding affinity (Table 2).
Since the binding energy shows how strong the interaction between the distributed drug in the cell
and β-tubulin protein can be, the partition coefficient (MlogP) values were calculated and considered for
the ability of the drugs to diffuse into the cells (Table 2). The MlogP values can be a beneficial factors in
estimating and comparing the distribution of the novel drugs within the cells, organs and the body [40].
Based on our computational predictions, compounds
energies of 8.6, 8.6, 8.4 and 8.3 kcal/mol, respectively. In the experimental part of the study,
the lowest IC50 values showed compounds having higher activity than (Table 1).
To investigate in more detail why the computational results did not show better binding energies of
compound than and , the interactions between double-modified colchicine derivatives and the
6, 4, 5 and 3 show the lowest binding
−
−
−
−
4
,
6
,
8
3, 5, 7
8
3
5
CBS of βI tubulin were studied.
As shown in the diagrams representing schematic interactions of the
4 and 6 compounds with the
CBS of βI tubulin Lys 352, Met 259 and Asn 258 residues interact with oxygen of the carbonyl of the C
ring (sidechain acceptor), with hydrogen of thiomethyl group on ring C and hydrogen of C-11 on ring
C (sidechain donor) and with ring C (arene-H), respectively.
However, Lys 352, Met 259 and Val 315 residues of the CBS of βI tubulin interact with oxygen of the
carbonyl of the C ring (sidechain acceptor), with hydrogen of C-11 on ring C (sidechain donor) and with
hydrogens of methoxyl groups on ring C (backbone donors) of the 3 and 5 compounds, respectively.
Table 2. Binding energies interactions between eight different colchicine derivatives and βI tubulin
and the estimated Moriguchi octanol-water partition coefficient, MlogP for each colchicine derivative.
Binding Energy
Structure
1
Interactions
MlogP
Active Residues
(
Kcal/mol)
−
8.09
1.37
Asn258

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Table 2. Cont.
Binding Energy
Kcal/mol)
Structure
Interactions
MlogP
Active Residues
(
2
Met259 Asn258
Lys352
−
−
−
−
−
8.13
8.33
8.57
8.40
8.60
1.56
3
Met259 Lys352
Val315
1.58
2.34
1.93
2.69
4
Met259 Asn258
Lys352
5
Met259 Val315
Lys352
6
Met259 Asn258
Lys352

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Table 2. Cont.
Binding Energy
Kcal/mol)
Structure
7
Interactions
MlogP
Active Residues
(
−
−
7.53
1.50
Ala317 Cys241
8
Ala316 Ala317
Cys241
7.33
2.00
The study was continued for compound
understanding over the effect of interaction of I tubulin’s residues with novel derivatives on better
activities of the derivatives. Compound interacts with Ala317 residue of I tubulin, iodine on ring A
backbone donor) and Cys241 residue of I tubulin, hydrogen of methoxyl group on ring A (sidechain
donor) and Ala 316 residue of βI tubulin, the oxygen of carbonyl on ring B (sidechain acceptor).
Since the IC50 is a cell-based assay and the I tubulin isotype is not the only isotype of tubulin
8 with the highest binding energy for better
β
8
β
(
β
β
expressed in the cell, we decided to check interactions of the synthesized compounds with the other
tubulin isotypes present in the referred cell lines. Tubulin isotypes are highly conserved in all mammals
as discussed by Luduena [41]. As is commonly the case, both normal and cancer cells in humans
contain the same tubulin isotypes. However, their expression levels differ and specifically
tubulin is very narrowly distributed in normal cells while it is almost always found in cancer cells
and it is often correlated with drug resistance [42 44]. Furthermore, I isotype (TUBB) is the most
abundant isotype in most tumors, followed by, IVb (TUBB4B), IIa (TUBB2A), V (TUBB6), and III
TUBB3) with 47%, 38%, 8.9%, 3.1%, and 2.2% respectively and with IVa (TUBB4), IIb (TUBB2B) and
VI (TUBB1) levels below 0.5% of the total tubulin [42]. Interestingly, the binding sites for common
βIII (TUBB3)
–
β
β
β
β
β
(
β
β
β
β
tubulin-binding agents do not vary significantly between tubulin isotypes except for the CBS [8].
Tubulin isotype classes βIVa and βIVb comprised more than 50% of the total β tubulin in breast
cancer (MCF-7) and colon cancer cells. The expression of βI, βIII and βIV (a and b) in the MCF-7
cell line has been reported as 39.1%, 2.5% and 58.4% respectively [45]. In another study, the ratio
for MCF-7 cell line was reported as 55% to 6% to 39% [46]. In colon cancer cells, the percentage
expression of the referred tubulin isotypes is 61.8%, 0.2% and 38%, respectively. The ratio of the isotype
expression in lung cancer cells (A549) is given as 71.9% to 1.6% to 26.5% [45]. Consequently, each
of the cell lines investigated has a distinct distribution of tubulin isotypes, which affects the overall
response to a cytotoxic agent whose affinity for each of these isotypes is different making the problem
of computational prediction complex.
To quantify the assumption that these compounds have different binding free energies for each of
the tubulin isotypes, docking simulations between the novel colchicine derivatives and
ID: P07437), IIa (UniProt ID: Q13885), IIb (UniProt ID: Q9BVA1), III (UniProt ID: Q13509),
UniProt ID: P04350), IVb (UniProt ID: P68371), V (UniProt ID: Q9BUF5), and VI (UniProt ID:
βI (UniProt
β
β
β
βIVa
(
β
β
β

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Q9H4B7), isotypes were performed. Tubulin structure 1SA0.pdb was used as the homology model
template for all tubulin isotypes using MOE2015. To visualize the results, a heat map was prepared
to better illustrate the comparison of binding energies between the investigated compounds and
the different tubulin isotypes using AutoDock Tableau Desktop (version 2018.2.0, Tableau Research,
Standford University, Seattle, WA, USA) (see Table 3).
Table 3. Heat map of binding energies interactions between the eight different colchicine derivatives
and βI, βIII βIIa, βIIb, βIVa, βIVb, βV and βVI tubulin isotypes.
1
2
3
4
5
6
7
8
βI
−8.090
−7.420
−7.050
−7.490
−7.610
−7.260
−7.480
−7.730
−8.130
−7.190
−6.680
−7.150
−7.300
−7.210
−7.180
−7.050
−8.330
−7.890
−6.800
−7.470
−7.100
−7.420
−7.320
−7.270
−8.570
−7.640
−6.900
−7.620
−7.300
−7.610
−7.260
−7.710
−8.400
−8.000
−6.890
−7.850
−7.210
−7.370
−7.250
−7.590
−8.600
−7.460
−7.050
−7.780
−7.430
−7.620
−7.340
−7.870
−7.530
−8.300
−6.430
−8.200
−6.970
−7.180
−6.500
−8.320
−7.330
−8.200
−7.040
−7.710
−7.010
−6.340
−7.190
−8.300
βIIa
βIIb
βIII
βIVa
βIVb
βV
βVI
−
8.600
−6.340
As can be seen in the heat map above, the binding energy between compound
isotype IIa as well as
their interaction with
8
and tubulin
β
β
VI are good examples of high binding energies while for compounds
I tubulin and IIa dominates. For compound 7 βVI, IIa and III are the
3 and
5
β
β
β
β
strongest binding tubulin isotypes. These differences might be the reasons of a discrepancy between
experimental and computational data. However, even data concerning the level of tubulin isotypes
expression reported for the same cell line in the literature, differ from each other. Precise levels of
isotype expression are not only hard to determine but they fluctuate in the same cell line as a result
of exposure to various drugs [47]. Therefore, more detailed experimental studies on the isotype
expression among different cell lines are still needed to give a better insight in this issue in the future.
In terms of a mechanistic explanation, specific residues of
interactions with compound are Asn 256 that interacts with C-20 on ring C (backbone donor) and
with ring C (arene-H) and Cys 239 that interacts with hydrogen of methoxyl group on ring A (sidechain
donor). The residues of VI tubulin that are involved in interactions with compound are Asn256,
βIIa tubulin that are involved in
8
β
8
which interacts with carbon atoms of two methoxyl groups on ring A (sidechain donors), Ala248,
interacts with the oxygen of carbonyl on ring C (sidechain acceptor) and Asn247 that interacts with
C-20 on ring C (sidechain donor) (see Table 4).
The analysis of interactions between compounds
VI shows that an arene-H interaction between ring C Asn256 or Asn258 and a sidechain acceptor
interaction between the oxygen of carbonyl on ring C and either Lys352 or Ala248 can result in a
strong binding effect. As mentioned before, the probability of the expression of IIa in most tumors is
approximately 9% versus less than 0.5% for each isotype in the group of βIVa, βVI and βIIb [42].
To the best of the authors’ knowledge of the literature, ligands binding to alpha and tubulin
4, 6 and βI and compound 8 and βIIa and
β
β
β
are exclusive, except of course of ATP, and therefore, we do not expect any cross-interactions of our
compounds with alpha tubulin [48]. Concerning interactions with ABC transporters, it is quite possible
that our compounds are substrates for these multidrug resistance enzymes, but this is common
to many chemotherapy drugs, including taxanes. Therefore, in order to inhibit this interaction,
it would most advantageous to use our compounds in combination with some of their modulators,
e.g., verapamil [49].

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Table 4. Binding energies interactions between compound
8 and βIIa and βVI tubulin as well as the
estimated Moriguchi octanol-water partition coefficient, MlogP for compound 8.
Tubulin
Isotype
Binding Energy
(Kcal/mol)
Interactions
MlogP
Active Residues
βIIa
−8.20
1.99
Asn256 Cys239
Asn256 Asn247
Ala248
βVI
−8.30
1.99
4
. Discussion
We synthesized three novel double-modified 4-halothiocolchicines (
biological activity according to the in vitro antiproliferative tests as well as the molecular docking.
For a better comparison, also the activity of single-modified colchicine derivatives ( ) as well
as colchicine itself ( ) was evaluated on four human cancer cell lines and normal murine embryonic
fibroblast cell line. The results of our study clearly showed that the antiproliferative activity of novel
-halothiocolchines ( ) is better than the activity of 4-halocolchicines ( ) and remain at a level
similar to the cytotoxicity of 2 for the A549, MCF-7 and LoVo cell lines. Furthermore, the cytotoxicity
of compounds and is higher than cytotoxicity of unmodified colchicine ( ) and commonly used
chemotherapeutics such as doxorubicin and cisplatin.
The introduction of thiomethyl group in C-10 position significantly increased the cytotoxicity in
comparison to single-modified 4-halo derivatives ( ) as well as allowed to reduce the toxicity for
-bromo and 4-iodo derivatives. Compounds 4-bromothiocolchine ( ) and 4-iodothiocolchicine (
4, 6, 8) and evaluated their
2, 3, 5, 7
1
4
4
,
6
,
8
3, 5, 7
4,
6
8
1
3, 5, 7
4
6
8)
proved to be less toxic to normal murine fibroblast cells than the currently used anticancer drugs, such
as cisplatin and doxorubicin, which is confirmed by their high SI values. The appropriate modification
of colchicine molecule and synthesis of its analogs might overcome the toxicity, which is a major
challenge in designing a potential colchicine-based drug candidate. However, it is still challenging to
draw clear conclusions from the molecular-level calculations. Compounds
lowest binding energies of 8.6, 8.6, 8.4 and 8.3 kcal/mol, respectively. The results only partially
correlate with in vitro determined IC values. This may be explained by several additional effects
6, 4, 5 and 3 showed the
−
−
−
−
5
0
taking place in living cells compared to the computational simulations that focus only on the binding
mode of the compounds to the target. Specifically, off-target interactions involving efflux pumps with
different affinities for the individual compounds may explain the observed partial correlation between
IC50 values and binding free energies. Additionally, differences in the solubility values and membrane
permeability may have to be accounted for when ranking the various compounds in biological assays
and comparing them to computational predictions based on binding affinity for the target alone.
We have partially addressed this issue by performing docking simulations for the remaining tubulin
isotypes, several of them may be expressed in cancer cells in a manner different than in normal cells.

Cells 2018, 7, 192
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We have demonstrated that a higher affinity for
βVI tubulin of the compounds investigated may explain
the differences in their biological activities. Our studies clearly show the potential of the obtained
double-modified compounds. In particular, 4-halothiocolchicines are worthwhile for a continuation of
the search for strong and broad-spectrum anticancer agents. Inspired by these preliminary results we
plan subsequent modifications in C-7 position to obtain a series of triple-modified derivatives. Further
evaluation should help to find more detailed structure-activity relationships of microtubule-targeting
drugs and CBS inhibitors, which can help in rational drug design in the future.
Supplementary Materials: The following are available online, Figure S1: The 13C-NMR spectrum of
2 in CDCl3,
1
in CDCl , Figure S3: The 13C-NMR spectrum of
Figure S2: The H-NMR spectrum of
2
3
in CDCl , Figure S4:
3
3
1
13
1
The H-NMR spectrum of
3
in CDCl , Figure S5: The C-NMR spectrum of
4
in CDCl , Figure S6: The H-NMR
in CDCl , Figure S8: The H-NMR spectrum of
in CDCl , Figure S10: The H-NMR spectrum of
3
3
1
3
1
spectrum of
4
in CDCl , Figure S7: The C-NMR spectrum of
5
5
3
3
13
1
in CDCl , Figure S9: The C-NMR spectrum of
6
6
in CDCl3,
3
3
Figure S11: The 13C-NMR spectrum of
7
in CDCl , Figure S12: The H-NMR spectrum of
1
7
in CDCl , Figure S13:
3
3
1
3
1
The C-NMR spectrum of 8 in CDCl , Figure S14: The H-NMR spectrum of 8 in CDCl .
3
3
Author Contributions: Conceptualization, A.H., U.M. and G.K.; Methodology, A.H., J.W., J.A.T.; Software, M.M.;
Investigation, A.H., U.M., G.K., M.M., E.M.; Resources, U.M., G.K.; Data Curation, U.M., G.K.; Writing-Original
Draft Preparation, G.K., U.M., M.M.; Writing-Review and Editing, G.K., A.H., J.A.T., F.B.; Visualization, G.K.,
M.M.; Supervision, A.H.; Project Administration, A.H.; Funding Acquisition, A.H.
Funding: Financial support by grant of the Polish National Science Centre (NCN)—No. 2016/21/B/ST5/00111 is
gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Hyams, J.; Stebbings, H. The mechanism of microtubule associated cytoplasmic transport. Cell Tissue Res.
979, 196, 103–116. [CrossRef] [PubMed]
1
2
3
.
.
Dustin, P. Microtubules; Springer: Berlin, Germany, 1984; ISBN 978-3-642-69652-7.
Jordan, M.A.; Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 2004, 4, 253–265.
[CrossRef] [PubMed]
4
5
.
.
Dutcher, S.K. The tubulin fraternity: alpha to eta. Curr. Opin. Cell Biol. 2001, 13, 49–54. [CrossRef]
Vindya, N.G.; Sharma, N.; Yadav, M.; Ethiraj, K.R. Tubulins—The target for anticancer therapy. Curr. Top.
Med. Chem. 2015, 15, 73–82. [CrossRef] [PubMed]
6.
7.
8.
9.
Seligmann, J.; Twelves, C. Tubulin: An example of targeted chemotherapy. Future Med. Chem. 2013, 5,
3
39–352. [CrossRef] [PubMed]
Katsetos, C.D.; Dráber, P. Tubulins as therapeutic targets in cancer: from bench to bedside. Curr. Pharm. Des.
012, 18, 2778–2792. [CrossRef] [PubMed]
Huzil, J.T.; Ludueña, R.F.; Tuszynski, J. Comparative modelling of human
for drug binding. Nanotechnology 2006, 17, S90–S100. [CrossRef] [PubMed]
2
β
tubulin isotypes and implications
Ravanbakhsh, S.; Gajewski, M.; Greiner, R.; Tuszynski, J.A. Determination of the optimal tubulin isotype
target as a method for the development of individualized cancer chemotherapy. Theor. Biol. Med. Model.
2
013, 10, 1–18. [CrossRef] [PubMed]
1
0. Kumar, B.; Kumar, R.; Skvortsova, I.; Kumar, V. Mechanisms of tubulin binding ligands to target cancer
cells: Updates on their therapeutic potential and clinical Trials. Curr. Cancer Drug Targets 2017, 17, 357–375.
[
CrossRef] [PubMed]
1. Avendaño, C.; Menéndez, J.C. Medicinal Chemistry of Anticancer Drugs; Elsevier: Amsterdam, The Netherlands,
008; ISBN 9780444528247.
2. Slobodnick, A.; Shah, B.; Pillinger, M.H.; Krasnokutsky, S. Colchicine: Old and new. Am. J. Med. 2015, 128,
61–470. [CrossRef] [PubMed]
3. Nerlekar, N.; Beale, A.; Harper, R.W. Colchicine—A short history of an ancient drug. Med. J. Aust. 2014, 201,
87–688. [CrossRef] [PubMed]
4. Grattagliano, I.; Bonfrate, L.; Ruggiero, V.; Scaccianoce, G.; Palasciano, G.; Portincasa, P. Novel therapeutics
for the treatment of familial mediterranean fever: From colchicine to biologics. Clin. Pharmacol. Ther. 2013
5, 89–97. [CrossRef] [PubMed]
1
1
1
1
2
4
6
,
9

Cells 2018, 7, 192
15 of 16
1
5. Cocco, G.; Chu, D.C.C.; Pandolfi, S. Colchicine in clinical medicine. A guide for internists. Eur. J. Intern. Med.
2
010, 21, 503–508. [CrossRef] [PubMed]
®
1
6. Yang, L.P.H. Oral colchicine (Colcrys ). Drugs 2010, 70, 1603–1613. [CrossRef] [PubMed]
1
7. Marangon, J.; Christodoulou, M.S.; Casagrande, F.V.M.; Tiana, G.; Dalla Via, L.; Aliverti, A.; Passarella, D.;
Cappelletti, G.; Ricagno, S. Tools for the rational design of bivalent microtubule-targeting drugs.
Biochem. Biophys. Res. Commun. 2016, 479, 48–53. [CrossRef] [PubMed]
1
1
2
2
2
2
2
2
8. Huczy n´ ski, A.; Rutkowski, J.; Popiel, K.; Maj, E.; Wietrzyk, J.; Stefa n´ ska, J.; Majcher, U.; Bartl, F. Synthesis,
antiproliferative and antibacterial evaluation of C-ring modified colchicine analogues. Eur. J. Med. Chem.
2
014, 90, 296–301. [CrossRef] [PubMed]
9. Huczy n´ ski, A.; Majcher, U.; Maj, E.; Wietrzyk, J.; Janczak, J.; Moshari, M.; Tuszynski, J.A.; Bartl, F. Synthesis,
antiproliferative activity and molecular docking of Colchicine derivatives. Bioorg. Chem. 2016, 64, 103–112.
[CrossRef] [PubMed]
0. Zhang, X.; Kong, Y.; Zhang, J.; Su, M.; Zhou, Y.; Zang, Y.; Li, J.; Chen, Y.; Fang, Y.; Zhang, X.; et al. Design,
synthesis and biological evaluation of colchicine derivatives as novel tubulin and histone deacetylase dual
inhibitors. Eur. J. Med. Chem. 2015, 95, 127–135. [CrossRef] [PubMed]
1. Nicolaou, K.C.; Valiulin, R.A.; Pokorski, J.K.; Chang, V.; Chen, J.S. Bio-inspired synthesis and biological
evaluation of a colchicine-related compound library. Bioorg. Med. Chem. Lett. 2012, 22, 3776–3780. [CrossRef]
[
PubMed]
2. Chang, D.-J.; Yoon, E.-Y.; Lee, G.-B.; Kim, S.-O.; Kim, W.-J.; Kim, Y.-M.; Jung, J.-W.; An, H.; Suh, Y.-G. Design,
synthesis and identification of novel colchicine-derived immunosuppressant. Bioorg. Med. Chem. Lett. 2009
9, 4416–4420. [CrossRef] [PubMed]
,
1
3. Marzo-Mas, A.; Barbier, P.; Breuzard, G.; Allegro, D.; Falomir, E.; Murga, J.; Carda, M.; Peyrot, V.; Marco, J.A.
Interactions of long-chain homologues of colchicine with tubulin. Eur. J. Med. Chem. 2017, 126, 526–535.
[CrossRef] [PubMed]
4. Johnson, L.; Goping, I.S.; Rieger, A.; Mane, J.Y.; Huzil, T.; Banerjee, A.; Luduena, R.; Hassani, B.; Winter, P.;
Tuszynski, J.A. Novel Colchicine derivatives and their anti-cancer activity. Curr. Top. Med. Chem. 2017, 17.
[CrossRef] [PubMed]
5. Kumar, B.; Sharma, P.; Gupta, V.P.; Khullar, M.; Singh, S.; Dogra, N.; Kumar, V. Synthesis and biological
evaluation of pyrimidine bridged combretastatin derivatives as potential anticancer agents and mechanistic
studies. Bioorg. Chem. 2018, 78, 130–140. [CrossRef] [PubMed]
2
2
6. Kumar, A.; Sharma, P.R.; Mondhe, D.M. Potential anticancer role of colchicine-based derivatives. Anticancer
Drugs 2017, 28, 250–262. [CrossRef] [PubMed]
7. Shchegravina, E.S.; Maleev, A.A.; Ignatov, S.K.; Gracheva, I.A.; Stein, A.; Schmalz, H.G.; Gavryushin, A.E.;
Zubareva, A.A.; Svirshchevskaya, E.V.; Fedorov, A.Y. Synthesis and biological evaluation of novel
non-racemic indole-containing allocolchicinoids. Eur. J. Med. Chem. 2017, 141, 51–60. [CrossRef] [PubMed]
8. Bartusik, D.; Tomanek, B.; Lattová, E.; Perreault, H.; Tuszynski, J.; Fallone, G. Derivatives of thiocolchicine
and its applications to CEM cells treatment using 19F Magnetic Resonance ex vivo. Bioorg. Chem. 2010, 38,
2
1
–6. [CrossRef] [PubMed]
2
9. Raspaglio, G.; Ferlini, C.; Mozzetti, S.; Prislei, S.; Gallo, D.; Das, N.; Scambia, G. Thiocolchicine dimers:
A novel class of topoisomerase-I inhibitors. Biochem. Pharmacol. 2005, 69, 113–121. [CrossRef] [PubMed]
0. Kozaka, T.; Nakagawa-Goto, K.; Shi, Q.; Lai, C.Y.; Hamel, E.; Bastow, K.F.; Brossi, A.; Lee, K.H. Antitumor
agents 273. Design and synthesis of N-alkyl-thiocolchicinoids as potential antitumor agents. Bioorg. Med.
Chem. Lett. 2010, 20, 4091–4094. [CrossRef] [PubMed]
3
3
3
3
1. Nakagawa-Goto, K.; Chen, C.X.; Hamel, E.; Wu, C.-C.; Bastow, K.F.; Brossi, A.; Lee, K.-H. Antitumor agents.
Part 236: Synthesis of water-soluble colchicine derivatives. Bioorg. Med. Chem. Lett. 2005, 15, 235–238.
[CrossRef] [PubMed]
2. Yasobu, N.; Kitajima, M.; Kogure, N.; Shishido, Y.; Matsuzaki, T.; Nagaoka, M.; Takayama, H. Design,
synthesis, and antitumor activity of 4-halocolchicines and their pro-drugs activated by cathepsin B. ACS Med.
Chem. Lett. 2011, 2, 348–352. [CrossRef] [PubMed]
3. Shchegravina, E.S.; Knyazev, D.I.; Beletskaya, I.P.; Svirshchevskaya, E.V.; Schmalz, H.G.; Fedorov, A.Y.
Synthesis of nonracemic pyrrolo-allocolchicinoids exhibiting potent cytotoxic activity. Eur. J. Org. Chem.
2
016, 2016, 5620–5623. [CrossRef]

Cells 2018, 7, 192
16 of 16
3
3
4. Shi, Q.; Verdier-Pinard, P.; Brossi, A.; Hamel, E.; Lee, K.H. Antitumor agents-CLXXV. Anti-tubulin action of
+)-thiocolchicine prepared by partial synthesis. Bioorg. Med. Chem. 1997, 5, 2277–2282. [CrossRef]
5. Banerjee, A.; Kasmala, L.T.; Hamel, E.; Sun, L.; Lee, K.-H. Interaction of novel thiocolchicine analogs with
(
the tubulin isoforms from bovine brain. Biochem. Biophys. Res. Commun. 1999, 337, 334–337. [CrossRef]
[PubMed]
3
3
6. Prajapati, P.B.; Bodiwala, K.B.; Marolia, B.P. Oxidative degradation kinetic study of thiocolchicoside using
stability indicating high performance thin layer chromatographic method. Pharm. Methods 2014, 5, 1–10.
[CrossRef]
7. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.;
Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82,
1
107–1112. [CrossRef] [PubMed]
3
3
8. Nevozhay, D. Cheburator software for automatically calculating drug inhibitory concentrations from in vitro
screening assays. PLoS ONE 2014, 9, e106186. [CrossRef] [PubMed]
9. Majcher, U.; Urbaniak, A.; Maj, E.; Moshari, M.; Delgado, M.; Wietrzyk, J.; Bartl, F.; Chambers, T.C.;
Tuszynski, J.A.; Huczy n´ ski, A. Synthesis, antiproliferative activity and molecular docking of thiocolchicine
urethanes. Bioorg. Chem. 2018, 81, 553–566. [CrossRef] [PubMed]
4
4
4
0. Devalapally, H.; Chakilam, A.; Amiji, M.M. Role of nanotechnology in pharmaceutical product development.
J. Pharm. Sci. 2007, 96, 2547–2565. [CrossRef] [PubMed]
1. Ludueña, R.F. Multiple forms of tubulin: Different gene products and covalent modifications. Int. Rev. Cytol.
1
997, 178, 207–275.
2. Leandro-García, L.J.; Leskelä, S.; Landa, I.; Montero-Conde, C.; López-Jiménez, E.; Letón, R.; Cascón, A.;
Robledo, M.; Rodríguez-Antona, C. Tumoral and tissue-specific expression of the major human
isotypes. Cytoskeleton 2010, 67, 214–223. [CrossRef] [PubMed]
β-tubulin
4
3. Mozzetti, S.; Ferlini, C.; Concolino, P.; Filippetti, F.; Raspaglio, G.; Prislei, S.; Gallo, D.; Martinelli, E.;
Ranelletti, F.O.; Ferrandina, G.; et al. Class III -tubulin overexpression is a prominent mechanism of
paclitaxel resistance in ovarian cancer patients. Clin. Cancer Res. 2005, 11, 298–305. [PubMed]
4. Sève, P.; Dumontet, C. Is class III -tubulin a predictive factor in patients receiving tubulin-binding agents?
Lancet Oncol. 2008, 9, 168–175. [CrossRef]
5. Hiser, L.; Aggarwal, A.; Young, R.; Frankfurter, A.; Spano, A.; Correia, J.J.; Lobert, S. Comparison of β-tubulin
β
4
4
β
mRNA and protein levels in 12 human cancer cell lines. Cell Motil. Cytoskeleton 2006, 63, 41–52. [CrossRef]
PubMed]
[
4
4
6. Davis, A.; Martinez, S.; Nelson, D.; Middleton, K. A tubulin polymerization microassay used to compare
ligand efficacy. In Methods in Cell Biology; Elsevier: Amsterdam, The Netherlands, 2010; Volume 95(C),
pp. 331–351. ISBN 9780123748157.
7. Tseng, C.Y.; Mane, J.Y.; Winter, P.; Johnson, L.; Huzil, T.; Izbicka, E.; Luduena, R.F.; Tuszynski, J.A.
Quantitative analysis of the effect of tubulin isotype expression on sensitivity of cancer cell lines to a
set of novel colchicine derivatives. Mol. Cancer 2010, 9, 1–19. [CrossRef] [PubMed]
8. Dumontet, C.; Jordan, M.A. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat. Rev.
Drug Discov. 2010, 9, 790–803. [CrossRef] [PubMed]
4
4
9. Choi, Y.H.; Yu, A.-M. ABC transporters in multidrug resistance and pharmacokinetics, and strategies for
drug development. Curr. Pharm. Des. 2014, 20, 793–807. [CrossRef] [PubMed]
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