Effects on tubulin polymerization and down-regulation of c-Myc, hTERT and VEGF genes by colchicine haloacetyl and haloaroyl derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.03.019

Several colchicine analogues in which the N-acetyl residue has been replaced by haloacetyl, cyclohexylacetyl, phenylacetyl and various aroyl moieties have been synthesized. The cytotoxic activities of the synthesized compounds have been measured on three tumor cell lines (HT-29, MCF-7 and A549) and on one non-tumor cell line (HEK-293). These compounds exhibit high antiproliferative activities at the nanomolar level, in many cases with a higher potency than colchicine itself. Some of the compounds, particularly the haloacetyl derivatives, inhibit the polymerization of tubulin in a similar manner as colchicine. As regards the cell cycle, the most active compounds are the chlorobenzoyl and bromobenzoyl derivatives, which cause cell cycle arrest at the G2/M phase when tested at 20 nM, and the bromoacetyl derivative, which arrests the cell cycle at 15 nM. In addition, these colchicine derivatives have shown fairly active downregulating the expression of the c-Myc, hTERT and VEGF genes, as well as VEGF protein secretion, at very low concentrations.

Full text of DOI:10.1016/j.ejmech.2018.03.019

Accepted Manuscript
Effects on tubulin polymerization and down-regulation of c-Myc, hTERT and VEGF
genes by colchicine haloacetyl and haloaroyl derivatives
Ana Marzo-Mas, Eva Falomir, Juan Murga, Miguel Carda, J. Alberto Marco
PII:
S0223-5234(18)30254-X
10.1016/j.ejmech.2018.03.019
EJMECH 10284
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 November 2017
Revised Date: 1 March 2018
Accepted Date: 5 March 2018
Please cite this article as: A. Marzo-Mas, E. Falomir, J. Murga, M. Carda, J.A. Marco, Effects
on tubulin polymerization and down-regulation of c-Myc, hTERT and VEGF genes by colchicine
haloacetyl and haloaroyl derivatives, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.019.
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ACCEPTED MANUSCRIPT
Effects on tubulin polymerization and down-regulation of c-Myc, hTERT and
VEGF genes by colchicine haloacetyl and haloaroyl derivatives
Ana Marzo-Mas, Eva Falomir, Juan Murga, Miguel Carda and J. Alberto Marco
Graphical Abstract
Several N-acyl colchicine analogues have been synthesized and their cytotoxicity, their effects on
inhibition of tubulin polymerization and cell cycle have been evaluated. Moreover their capacity to
downregulate some oncogenes at nontoxic dosis have been evaluated. The haloaroyl moiety enhances
oncogene down-regulation effect as regards colchicine itself. Outstanding results for m-
chlorobenzoylderivative 14 were obtained as this derivative is able to decrease the expression of
oncogenes involved in tumor aggressiveness at concentrations in which there is no antimitotic effect.
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Manuscript draft
Title: Effects on tubulin polymerization and down-regulation of c-Myc, hTERT and VEGF genes by
colchicine haloacetyl and haloaroyl derivatives
Article type: Original paper
Keywords: tubulin, colchicine, microtubules, polymerization, cell cycle, c-Myc, hTERT, VEGF,
oncogene.
Abstract:
Several colchicine analogues in which the N-acetyl residue has been replaced by haloacetyl,
cyclohexylacetyl, phenylacetyl and various aroyl moieties have been synthesized. The cytotoxic
activities of the synthesized compounds have been measured on three tumor cell lines (HT-29, MCF-7
and A549) and on one non-tumor cell line (HEK-293). These compounds exhibit high antiproliferative
activities at the nanomolar level, in many cases with a higher potency than colchicine itself. Some of
the compounds, particularly the haloacetyl derivatives, inhibit the polymerization of tubulin in a similar
manner as colchicine. As regards the cell cycle, the most active compounds are the chlorobenzoyl and
bromobenzoyl derivatives, which cause cell cycle arrest at the G2/M phase when tested at 20 nM, and
the bromoacetyl derivative, which arrests the cell cycle at 15 nM. In addition, these colchicine
derivatives have shown fairly active downregulating the expression of the c-Myc, hTERT and VEGF
genes, as well as VEGF protein secretion, at very low concentrations.
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EFFECTS ON TUBULIN POLYMERIZATION AND DOWN-REGULATION OF C-MYC, HTERT
AND VEGF GENES BY COLCHICINE HALOACETYL AND HALOAROYL DERIVATIVES
Ana Marzo-Mas,a Eva Falomir,* a Juan Murga,* a Miguel Carda a and J. Alberto Marcob
aDepart. de Q. Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
bDepart. de Q. Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
*Authors to whom correspondence should be addressed. E-Mail address: jmurga@uji.es,
efalomir@uji.es
ABSTRACT
Several colchicine analogues in which the N-acetyl residue has been replaced by haloacetyl,
cyclohexylacetyl, phenylacetyl and various aroyl moieties have been synthesized. The cytotoxic
activities of the synthesized compounds have been measured on three tumor cell lines (HT-29, MCF-7
and A-549) and on one non-tumor cell line (HEK-293). These compounds exhibit high antiproliferative
activities at the nanomolar level, in many cases with a higher potency than colchicine itself. Some of
the compounds, particularly the haloacetyl derivatives, inhibit the polymerization of tubulin in a similar
manner as colchicine. As regards the cell cycle, the most active compounds are the chlorobenzoyl and
bromobenzoyl derivatives, which cause cell cycle arrest at the G2/M phase when tested at 20 nM, and
the bromoacetyl derivative, which arrests the cell cycle at 15 nM. In addition, these colchicine
derivatives have shown fairly active downregulating the expression of the c-Myc, hTERT and VEGF
genes, as well as VEGF protein secretion, at very low concentrations.
KEYWORDS
tubulin, colchicine, microtubules, polymerization, cell cycle, c-Myc, hTERT, VEGF, oncogene.
1. Introduction
Colchicine is an alkaloid isolated from the poisonous plant meadow saffron Colchicum autumnale
L. This compound has been used in the treatment of several inflammatory diseases such as gout,
familial Mediterranean fever, pericarditis and Behçet's disease [1]. The primary mechanism of action of
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this natural product involves disruption of the tubulin microtubules. In fact, colchicine was the first
compound known to bind tubulin [2]. The microtubule disrupting action is responsible for the blockage
of migration of neutrophils to the damaged tissue thereby inhibiting the inflammatory process. Some
studies have revealed that the anti-inflammatory effect of colchicine may be mediated not only by
microtubule disruption but also by changes at transcriptional level. In this sense, it has been found that
colchicine was able to supress many genes related not only to the inflammation process but also to cell
cycle regulation. The handicap was that the concentration of natural product required to exert this
transcriptional regulation was higher than the therapeutic dose [3].
Colchicine is not clinically used in cancer treatments due to its relatively narrow therapeutic index.
Although a significant percentage of patients report adverse effects at daily doses when colchicine is
administered to treat the above mentioned diseases, colchicine may be administered to tolerant patients
for long periods of time without any side effect [4].
Many efforts have been made to find more effective and less toxic colchicine derivatives by means
of modifying its structure [5]. As a matter of fact, some derivatives of colchicine, such as
thiocolchicoside (Neoflax™, Muscoril™) are being used as anti-inflammatory and analgesic drugs.
In this sense, we have designed, synthesized and studied some hybrid molecules with a colchicine
moiety and a pironetin analogue fragment. We found that, in addition to binding to tubulin, these
compounds were able to downregulate the expression of the VEGF, hTERT and c-Myc genes. These
three genes are of paramount importance in the cancer generation process. Our results pointed to the
colchicine fragment being responsible for the observed biological activities, although high doses were
required (50 nM-15 µM). [6]
It has been reported that deregulation of c-Myc plays a major role in the carcinogenesis of human
malignancies and very often correlates with upregulation of VEGF and hTERT expression [7]. It has
also been determined that c-Myc upregulation is associated with low prognosis and that inactivation of
c-Myc or downstream targets of this one, might provide important therapeutic strategies [8].
In order to expand our research on colchicine derivatives with potential utility in cancer therapy not
only as anti-mitotic compounds but also as downregulators of some oncogenes, we decided to
investigate the influence of the N-acetyl group of the natural product in this latter activity. The purpose
was to get colchicine derivatives with enhanced anticancer properties and lower toxicity. Thus, we
designed a set of N-acyl derivatives (see Figure 1) that would let us observe the influence of the
electronic and steric nature of this residue in colchicine derivatives. In this family of colchicine
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derivatives we have included N-haloacetyl 1-4, N-cyclohexylacetyl 5, N-phenylacetyl 6, and N-aroyl
moieties 7-18.
Figure 1. Structure of the colchicine analogues investigated in this study
2. Results and discussion
2.1. Preliminary docking studies
Before starting the synthesis and biological evaluation of these colchicine derivatives, we carried out
docking studies in order to find out whether these derivatives preserved the ability to bind to tubulin
at/or near the colchicine binding site, in a similar conformation to the natural product. Thus, Autodock
4.2 [9] was used to perfom molecular docking calculations and the crystal structure of αβ tubulin (PDB
ID 1SA0) [10] was employed as a template. Figure 2 (Panel A) shows a superimposition of DAMA-
colchicine [N-deacetyl-N-(2- mercaptoacetyl)colchicine], obtained from the PDB ID 1SA0 over the
structures of the haloacetyl derivatives 1-4. These docking experiments suggest that derivatives 1-4
exhibit conformations very close to that of DAMA-colchicine. As an example, panel B shows the
location of floruoacetyl derivative 1 at the β-tubulin binding site. The docking score, based in free
energy of binding, is included in the Supplementary Information (Table SI-1). Structures 1-4 show the
same interactions that the crystallized structure of DAMA-colchicine (see 2D interactions in Figures
SI-1, SI-2 and SI-3 in Supplementary Information).
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Figure 2. Panel A corresponds to the superimposition of the structures of 1 (black), 2 (orange), 3 (red)
and 4 (purple) on the co-crystallized DAMA-colchicine (blue) at the colchicine binding site. Panel B
corresponds to the structure of fluoroacetyl derivative 1 at the colchicine binding site. The α- and β-
tubulin subunits are coloured in yellow and green, respectively.
As regards the aroyl derivatives, we selected the bromo derivatives 16-18 to perform docking
calculations. Figure 3 (Panel A) shows a superimposition of DAMA-colchicine over the structures of
these compounds.
Figure 3. Panel A corresponds to the superimposition of the structures of 16 (black), 17 (red) and 18
(purple) on the co-crystallized DAMA-colchicine (blue) at the colchicine binding site. Panel B, C and
D correspond, respectively, to the structures of 16, 17 and 18 at the colchicine binding site. The α- and
β-tubulin subunits are coloured in yellow and green, respectively.
As shown in Figure 3 (panels B, C and D), bromoaroyl derivatives 16-18 bind to the binding site of
the colchicine at the β-tubulin domain in a very close conformation to that of DAMA-colchicine, with
the bromoaroyl unit pointing towards a hydrophobic cavity of the adjacent alpha subunit of tubulin.
The bromoaroyl derivatives 16-18 showed lower binding energy when compared with compounds 1-4.
An extra hydrogen bond interaction between αAsn101 and the carbonyl group of acyl residue is formed
(see 2D interactions in Figures SI-4 and SI-5 in Supplementary Information). Also, the aromatic ring of
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bromoaryl unit shows an interaction with αAla180. These extra interacions could explain the lower
binding energy of compounds 16-18. According to the docking results, all these derivatives retain the
ability to bind to tubulin at the colchicine binding site.
2.2. Chemistry
The trifluoroacetate salt of N-deacetyl colchicine 19 [11] was used as the starting material to prepare
the N-acyl derivatives 1-18 as depicted in Scheme 1. Treatment of 19 with the corresponding
carboxylic acid in the presence of DCC and DMAP afforded compounds 1-18 with fair yields. Details
about the precise reaction conditions and yields are indicated in the Experimental Part together with
complete spectral data (graphical spectra are provided in the Supporting Information) [12].
Scheme 1. Synthesis of colchicine derivatives 1-18.
2.3. Biological evaluation
2.3.1. Effects on the inhibition of cell proliferation
The MTT assay was used to measure the ability of compounds 1-19 to inhibit cell proliferation,
expressed by means of their IC50 values towards the tumoral cell lines HT-29 (human colon
adenocarcinoma), MCF-7 (breast adenocarcinoma) and A549 (human alveolar lung epithelial
carcinoma cells), as well as towards the non-tumoral cell line HEK-293 (human embryonic kidney
cells). The results were compared with those of colchicine and are depicted in Table 1 along with the
calculated selectivity indexes SIA (for HT-29 cell line), SIB (for MCF-7 cell line) and SIc (for A549 cell
line), obtained by dividing the IC50 values of the non-tumoral cell line (HEK-293) by those of the
corresponding tumoral cell line. The SI value is a parameter that helps to estimate the possible
selectivity of compounds for cancer cells in relation to non-cancer cells. Thus, a higher SI index
indicates a higher therapeutic safety margin.
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Table 1. IC50 values (nM) of synthetic compounds 1-19 in cancer cell lines HT-29, MCF-7 and A549,
and one non-cancer cell line HEK-293.a
b
c
d
Compound
HT-29
MCF-7
12 ± 7
A549
HEK-293
5 ± 1
SIA
0.1
1.5
1.0
1.0
1.9
1.2
1.4
1.6
1.6
1.4
1.8
0.4
1.8
2.6
1.7
5.6
1.5
2.0
0.7
2.0
SIB
0.4
1.0
0.2
0.9
4.6
1.2
1.5
2.6
4.5
0.6
0.5
0.3
2.4
1.3
0.9
1.5
1.1
3.0
0.9
1.6
SIC
0.4
0.9
0.4
0.6
0.6
0.5
1.4
1.3
4.5
0.4
1.8
1.3
1.6
0.5
1.0
1.5
0.7
50 ± 3
12.2 ± 0.7
7.83 ± 0.15
12.3 ± 2.1
26 ± 5
Colchicine
4.7 ± 1.3
4.6 ± 0.4
13.7 ± 0.2
10 ± 1
7.3 ± 1.2
29 ± 1
7.1 ± 0.5
4.56 ± 0.08
14.36 ± 1.06
19 ± 1
1
2
16.4 ± 0.3
4.1 ± 0.8
9 ± 2
3
31 ± 3
4
8.7 ± 0.5
2.84 ± 0.04
8.1 ± 0.9
5.7 ± 0.5
2.23 ± 0.23
9.0 ± 2.2
4.2 ± 1.2
32.7 ± 2.1
2.3 ± 0.4
1.8 ± 0.3
1.79 ± 0.15
5.8 ± 0.3
0.56 ± 0.04
11.4 ± 1.2
12.4 ± 1.5
19.4 ± 3.1
2.8 ± 0.9
10 ± 3
10.5 ± 0.9
3.9 ± 0.7
13 ± 5
5
2.6 ± 0.9
5 ± 2
6
7
2.02 ± 0.04
5.6 ± 1.5
31 ± 8
2.0 ± 0.6
8.7 ± 1.3
8.8 ± 1.2
1.27 ± 0.4
36 ± 6
9.0 ± 1.2
3.1 ± 0.9
16.1 ± 0.6
1.6 ± 0.4
58 ± 11
8
9
10
11
12
13
14
15
16
17
18
19
5.8 ± 1.8
24 ± 4
4.5 ± 0.6
3.4 ± 0.6
6.7 ± 0.4
7.6 ± 1.9
13 ± 3
5.93 ± 0.06
3.0 ± 0.3
10.1 ± 2.4
8.5 ± 2.8
1.1 ± 0.3
7.8 ± 1.9
25.1 ± 0.3
3.1 ± 1.1
6.6 ± 1.0
13 ± 3
0.363 ± 0.013 14 ± 4
0.08
1.9
8.9 ± 0.7
4.2 ± 1.1
15.5 ± 1.5
16.8 ± 0.7
1.5
aIC50 values are expressed as the compound concentration (nM) that inhibits the cell growth by 50%. Data are the
b
c
average (±SD) of three experiments. SIA = IC50(HEK-293)/IC50(HT-29). SIB = IC50(HEK-293)/IC50(MCF-7).
dSIC = IC50(HEK-293)/IC50(A549).
The observed IC50 values are all in the low nanomolar range. In the case of the HT-29 cell line, all
compounds show a better antiproliferative activity than colchicine itself. It is worth noting that
derivative 17 exhibits in HT-29 and MCF-7 cell lines a very low IC50 value situated in the picomolar
range, therefore two orders of magnitude lower than the natural product. As for the A549 cell line, most
compounds exhibit IC50 values in the same range as the natural alkaloid. Interestingly, most compounds
exhibit SI values greater than colchicine. The haloacetyl derivatives 1-4 show lower SI values than
those having aroyl moieties, the most selective derivatives among the latter being 8 (2-MePh) and 15
(4-ClPh). Particularly worth mentioning is compound 17 (3-BrPh), which combines a picomolar anti-
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proliferative activity with high SI values in the HT-29 and MCF-7 cell lines. These synthetic
compounds therefore could offer the possibility of use in cancer therapy with lower dosages than
colchicine, this resulting in less acute toxicity problems than in the case of the natural alkaloid.
Further biological studies have been carried out in order to establish whether the observed
antiproliferative effect in cells is due to the interaction of these colchicine derivatives with tubulin.
These studies are detailed hereafter.
2.3.2. Effects on tubulin assembly
With the aim of studying the effects of our derivatives on tubulin self-assembly, the critical
concentration for compounds 1-19 was determined in glycerol-assembling buffer (GAB). The results
are compared with those achieved in the presence of colchicine and also of podophyllotoxin, a further
microtubule-destabilizing agent. A solution of 25 µM tubulin was incubated at 37ºC with 27.5 µM
concentrations of colchicine, podophyllotoxin and compounds 1-19. Table 2 summarizes the results
achieved.
Table 2. Critical concentration (CrC) for the assembly of purified tubulin in GAB in the presence of
colchicine, podophyllotoxin and the colchicine analogues 1-19.a
Compound
CrC (µM)
9± 2
Control
24.5 ± 0.3
23.5 ± 0.7
24.6 ± 0.3
24.7 ± 0.2
24.6 ± 0.6
24.2 ± 0.3
22.9 ± 1.5
24.1 ± 0.4
23.9 ± 0.7
19.9 ± 1.5
23.4 ± 1.1
21.1 ± 1.5
14.0 ± 0.9
22.9 ± 1.3
Colchicine
Podophyllotoxin
1
2
3
4
5
6
7
8
9
10
11
12
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24.1± 0.8
23.6 ± 0.5
24.9 ± 0.4
19.5 ± 0.6
23.5 ± 1.1
19.9 ± 1.4
20 ± 2
13
14
15
16
17
18
19
a Data are the average (±SD) of three experiments.
All colchicine derivatives inhibit microtubule formation with the exception of compound 11 (4-n-
PentPh), which shows very little activity. Indeed, the concentration of tubulin required to produce
assembly raises from 9 µM in the absence of the drugs (DMSO vehicle as control) to a maximum value
of 24.9 µM in the presence of compound 15 (4-ClPh), comparable with that showed by colchicine (24.5
µM) and podophyllotoxin (23.5 µM). The critical concentration values of analogues 1–19 (except for
11) therefore confirm that the observed antiproliferative effect in cells is due to their interaction with
tubulin.
Figure 4 depicts the effects of the ligands on the kinetics of tubulin self-assembly. As can be
observed, thirteen of the synthetic compounds showed a full inhibition of microtubule formation in the
same way as colchicine (CLC in the figure). Derivatives 8 (2-MePh), 10 (4-MePh), 16 (2-BrPh) and 18
(4-BrPh) and 19 caused partial inhibition of microtubule formation and also a delay on the
polymerization process.
Figure 4. Effects of colchicine and compounds 1-19 on the kinetics of tubulin assembly. The lines in
the figure show the turbidity time course of polymerization of tubulin alone (black) at 25 µM,
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colchicine (red), 8 (green), 10 (orange), 16 (gray) and 18 (purple) and 19 (pink). All compounds were
at 27.5 µM.
In order to quantify this delay of the nucleation and elongation process, we have calculated the It50
value, defined as the time needed to reach 50% of the polymerization equilibrium. The It50 values
obtained for the control (DMSO) and compounds 8 (2-MePh), 10 (4-MePh), 16 (2-BrPh) and 18 (4-
BrPh) and 19 are shown in Table 3.
Table 3. It50 of some compounds for tubulin self-assembly.a
Compound
It50 (min)
Control
8
10
16
18
19
27±5
16.5 ± 0.8
32.6 ± 0.3
28.8 ± 1.0
34.0 ± 0.5
27 ± 3
a Data are the average (±SD) of three experiments.
Compounds having a comparatively high value of critical concentration with respect to the control
(lower absorbance in the stationary phase), as well as a higher It50 value with respect to the control
(increased time required for nucleation and elongation) are considered as partial inhibitors of the
polymerization of tubulin. This is the case of compounds 8, 10, 16, 18 and 19.
2.3.3. Effects on the cell cycle
Having established that our colchicine derivatives inhibit in vitro microtubule assembly, it was
convenient to ascertain whether they also could inhibit microtubule polymerization inside cells causing
cell cycle arrest, and also to characterize their effects on microtubules, mitoses and DNA content. First
of all, we selected the most active compounds for a study of the effects on the morphology of A549
cells after 24 h treatment with different concentrations of our compounds. As a matter of fact, most of
the A549 cells became rounded mitotic cells in the presence of colchicine, deacetylcolchicine 19 and
compounds 1-4, 6-10, 13-15 and 16-18. However, the cell morphology was not significantly affected
after treatment with 5 (CH2C6H11), 11 (4-n-PentPh) and 12 (4-t-BuPh), as compared with the control
cells, with only some small, round and non-adherent cells being observed. For this reason, these three
derivatives were discarded for cell cycle studies. The main results are summarized in Table 4.
Table 4. Cell cycle distribution for colchicine and selected compounds.a
Compound
Control
Conc. (nM)
Sub G0
2 ± 1
G0/G1
73 ± 3
S
G2/M
11 ± 4
59 ± 17
15 ± 6
11 ± 2
Colchicine
50
3 ± 1
27 ± 14
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1
2
7.5
15
15
150
20
50
50
50
50
15
20
15
20
20
20
150
2 ± 1
2 ± 1
3 ± 1
7 ± 3
5 ± 1
4 ± 3
8 ± 2
16 ± 6
13 ± 5
3 ± 1
6 ± 1
2 ± 1
4 ± 3
4 ± 3
4 ± 3
5 ± 1
25 ± 6
22 ± 7
25 ± 3
17 ± 3
19 ± 2
29 ± 6
24 ± 5
29 ± 8
44 ± 6
11 ± 1
24 ± 5
27 ± 3
27 ± 1
29 ± 1
25 ± 3
47 ± 6
15 ± 9
20 ± 3
19 ± 3
13 ± 2
16 ± 2
28 ± 10
14 ± 2
10 ± 1
26 ± 2
26 ± 10
15 ± 1
14 ± 4
22 ± 4
16 ± 3
16 ± 1
15 ± 3
60 ± 13
55 ± 9
53 ± 4
62 ± 2
60 ± 2
40 ± 6
47 ± 5
45 ± 1
16 ± 1
55 ± 1
57 ± 8
48 ± 11
47 ± 6
51 ± 3
56 ± 1
33 ± 2
3
4
6
7
8
9
10
13
14
15
16
17
18
19
a
Concentration in italics corresponds approximately to the IC50 value. Data are the average (±SD) of two
experiments.
The observed results show that all compounds except 10 (4-MePh) and 19 (lacking any acyl group),
are able to arrest the cell cycle at the G2/M phase. Some of these derivatives have been studied at
different concentrations in order to see which are the most active compounds at the lowest
concentration. In all cases, a sub-G1 peak, presumably of cells undergoing apoptosis, was observed.
Ortho-, meta- and para-chloro and bromobenzoyl derivatives 13-18 cause interruption of the cell cycle
at the G2/M phase when tested at approximately 20 nM. However, at 3 nM concentration, derivatives
13-18 accumulate cells in G2/M phase in a higher percentage than in the control. Fluoroacetyl and
chloroacetyl derivatives 1 and 2, respectively, arrest the cell cycle at the G2/M phase at their IC50
concentration. Interestingly, the bromoacetyl derivative 3 is able to block the cell cycle at the G2/M
phase at half the value of its IC50. Iodoacetyl derivative 4 is the less active one of these series of
haloacetyl compounds.
2.3.4. Effect of selected derivatives on the expression of hTERT and c-MYC genes
In order to expand our research on colchicine derivatives with potential utility in cancer therapy,
we proceeded to investigate the effect they exert on the expression of c-Myc, hTERT and VEGF genes
as well as VEGF protein secretion on HT-29 cell line.
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First of all, a preliminary study was carried out by conventional PCR to qualitatively determine
which compounds were able to modify the expression of these three genes after 48 h of treatment in the
HT-29 cell line (data not shown). This preliminary test let us select twelve derivatives and their
minimal active concentration (see Figure 5) for a quantitative study of gene expression on the same cell
line.
Figure 5. Selected compounds and concentrations for a quantitative gene expression test
As regards the ability of our compounds for the inhibition of the c-Myc gene expression, we found
that colchicine was very active at a concentration of 40 nM but has no activity when tested at 10 nM
(see Figure 6, panel A). Special attention must be paid on chloroaroyl derivatives 13-15 and meta-
bromoaroyl compound 17, because they were able to inhibit more than 60% of c-MYC gene expression
at very low concentration (1.5 nM for all of them except for 17 that was 0.5 nM). Bromoacetyl
derivative 3 was also more active than colchicine itself as it was able to inhibit to 35% of gene
expression at a concentration 13 times lower than the natural product.
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Figure 6. Panel A: Expression percentage of the c-Myc gene after 48 h of incubation with DMSO
(control) and the selected compounds. Panel B: Expression percentage of the hTERT gene after 48 h of
incubation with DMSO (control), and the selected compounds. Panel C: Expression percentage of the
VEGF gene after 48 h of incubation with DMSO (control), and the selected compounds. Panel D:
Expression percentage of the VEGF protein secreted after 48 h of incubation with DMSO (control), and
the selected compounds. At least three measurements were performed in each case Gene expression
was normalized using β-ACTIN as endogenous gene. Gene expression was normalized using β-ACTIN
as endogenous gene. Percentages above 100% indicate that the corresponding compounds were less
active than the control. Error bars indicate standard errors of the mean. The statistical significance was
evaluated using one-sample t-tests (P <0.001).
As regards the ability of our compounds to inhibit hTERT gene expression, we found that
colchicine was able to inhibit to half the expression of this gene at a concentration of 40 nM but had no
activity when tested at 10 nM (see Figure 6, panel B). In this case, o- and m-chloroaroyl derivatives 13
and 14 were the most active ones as they caused more than 40% of hTERT gene inhibition at a
concentration of 1.5 nM. The iodoacetyl derivative 4 was also fairly active as it was able to
downregulate gene expression to 40% at a concentration of 3 nM.
13

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As regards inhibition of VEGF gene expression, we found that colchicine was active at a
concentration of 40 nM but had no activity when tested at 10 nM (see Figure 6, panel C). Again,
chloroaroyl derivatives 13-15 showed the best activities as they were able to inhibit more than 70% of
VEGF gene expression at a concentration of 1.5 nM. p-Bromoderivative 18 ahowed also a good
activity at 9 nM while o-bromoderivative 17 was able to inhibit about 40% of VEGF gene expression at
0.5 nM. In this case, the bromo and iodoacetyl derivatives 3 and 4 were also more active than
colchicine itself as they were able to inhibit more than 50% of gene expression at a concentration 13
times lower than the natural product.
In order to check how the downregulation of VEGF gene expression by our derivatives affected
VEGF protein secretion in HT-29 cell line, we evaluated the amount of this protein in the culture
medium after 48 h of treatment (see Figure 6, panel D).7 This evaluation was carried out by ELISA
measurements (see experimental section). The chloroaroyl derivatives 13-15 showed the best activities
as they were able to inhibit to the half the secretion of the VEGF protein at a concentration of 1.5 nM.
p-t-Butyl and p-bromoaroylderivatives 12 and 18 showed also activity similar to that of the natural
product but at lower concentrations (30 nM and 9 nM respectively). In this case, the haloacetyl
derivatives 1-4 were less active.
3. Conclusions
Colchicine analogues in which the N-acetyl residue has been replaced by N-haloacetyl, N-
cyclohexylacetyl, N-phenylacetyl and N-aroyl moieties have been synthetized and their
antiproliferative activities towards the tumor cell lines HT-29, MCF-7 and A549 and the non-tumor cell
line HEK-293 have been measured. All derivatives show IC50 values in the low nanomolar range, with
compound 17 showing a picomolar anti-proliferative action, two orders of magnitude more cytotoxic
than colchicine. As regards the Selectivity Index (SI) values, most of the synthetic derivatives exhibit
SI values greater than colchicine. Outstanding cases were those of derivatives 8 (2-MePh) and 15 (4-
ClPh), with high SI values in the three tumor cell lines assayed, and of compound 17 (3-BrPh) with
high SI values in the HT-29 and MCF-7 cell lines. These compounds therefore could offer the
possibility of use in cancer therapy with lower dosages than colchicine, this resulting in less acute
toxicity problems than in the case of the natural alkaloid.
As regards the tubulin polymerization process, compound 11 (4-n-PentPh) is the only one that
showed a low ability to prevent microtubule formation. Ortho- and para-methylbenzoyl derivatives (8
14

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and 10) and ortho- and para-bromobenzoyl derivatives (16 and 18) may be considered as partial
inhibitors of tubulin polymerization. The remaining derivatives behave like colchicine and can be
considered full tubulin polymerization inhibitors, compound 15 (4-ClPh) being the most active. The
critical concentration values of analogues 1–18 (except for 11) confirm that the observed
antiproliferative effect in cells is due to their interaction with tubulin.
It is worth mentioning that the tested derivatives cause extensive arrest of the cell cycle at the G2/M
phase with concentrations and incubation times identical to those of colchicine at 50 nM. Fluoro and
chloroacetyl derivatives 1 and 2, respectively, arrest the cell cycle at the G2/M phase when assayed at
their IC50 concentration. Interestingly, bromoacetyl derivative 3 is able to block the cell cycle at the
G2/M phase at half the value of its IC50 concentration. Ortho-, meta- and para-chloro and
bromobenzoyl derivatives 13-18 cause interruption of the cell cycle at the G2/M phase when tested at
approximately 20 nM.
These colchicine derivatives derivatives are also fairly active downregulating the expression of c-
MYC, hTERT and VEGF genes.
Figure 7. Percentage of the oncogene expressions and VEGF protein secretion in the presence of the
selected derivatives.
The most active derivatives, along with colchicine, were those with a chloroaroyl unit 13-15.
These were able to downregulate the expression of c-MYC, hTERT and VEGF genes at 1.5 nM, a much
lower concentration than the one needed to exert an anti-mitotic effect (see Table 4). The most active
was m-chloroaroylderivative 14, which reduced the expression of the c-Myc, hTERT and VEGF genes
to 20%, 59% and 30%, respectively. In this sense, it is also worth mentioning that the m-
15

ACCEPTED MANUSCRIPT
bromoaroylderivative 17 shows activity in downregulating the selected gene expressions at 0.5 nM, the
lowest concentration observed in all these derivatives.
In summary, we have found that haloaroyl moiety is the responsible for the enhanced oncogene
downregulation effect of these colchicine derivatives. We could conclude that the enhanced oncogene
downregulation effect of compounds 13-15, achieved at concentrations lower than their IC50 values,
might broaden the therapeutic window of these compounds. Indeed, they could be good anti-cancer
drug candidates because they decrease the expression of oncogenes involved in tumor aggressiveness at
concentrations in which there is no antimitotic effect.
4. Experimental section
4.1. General procedures
NMR spectra were measured at 25°C. The signals of the deuterated solvent (CDCl3) was taken as
the reference. Multiplicity assignments of 13C signals were made by means of the DEPT pulse
1
sequence. Complete signal assignments in H and 13C NMR spectra were made with the aid of 2D
homo- and heteronuclear pulse sequences (COSY, HSQC, HMBC). High resolution mass spectra were
run by the electrospray mode (ESMS). IR data were measured with oily films on NaCl plates (oils) and
are given only for relevant functional groups (C=O, NH). Optical rotations were measured at 25 °C.
Experiments which required an inert atmosphere were carried out under dry N2 in flame-dried
glassware. THF was freshly distilled from sodium/benzophenone ketyl and transferred via syringe.
Commercially available reagents were used as received. Where solutions were filtered through a Celite
pad, the pad was additionally washed with the same solvent used, and the washings incorporated to the
main organic layer.
4.2. General procedure to prepare colchicine analogues 1-18.
Compound 19 (283 mg, 0.6 mmol) and the appropriate acid (1.2 mmol) were dissolved under N2 in
dry CH2Cl2 (60 mL) and treated with N,N'-dicyclohexylcarbodiimide (743 mg, 3.6 mmol) and 4-(N,N-
dimethylamino)pyridine (147 mg, 1.2 mmol). The mixture was stirred for 3 h at room temperature and
then filtered through Celite. The filtrate was evaporated under reduced pressure, and the residue was
subjected to column chromatography on silica gel (EtOAc-acetone, 8:1) to afford the desired
compound 1-18. Yields ranked between 40 and 59%.
Yields: 1, 58%; 2, 56%; 3, 51%; 4, 43%; 5, 54%; 6, 52%; 7, 45%; 8, 59%; 9, 54%; 10, 54%; 11,
53%; 12, 46%; 13, 51%; 14, 55%; 15, 53%; 16, 49%; 17, 51%; 18, 58%.
16

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4.2.1.
2-Fluoro-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}acetamide: yellowish solid, mp 103-105 °C, lit. [11a] mp 137-142 °C; [α] −140 (c, 1; CHCl3); 1H
D
NMR (CDCl3) δ 7.65 (1H, br d, J ~ 6.5 Hz, NH), 7.40 (1H, s), 7.25 (1H, d, J = 10.7 Hz), 6.80 (1H, d, J
= 10.7 Hz), 6.50 (1H, s), 4.80-4.60 (3H, m), 3.95 (3H, s), 3.90 (3H, s), 3.87 (3H, s), 3.61 (3H, s), 2.51
13
(1H, dd, J = 13.7, 6.5 Hz), 2.39 (1H, apparent td, J = 13.2, 6.8 Hz), 2.26 (1H, m), 1.97 (1H, m); C
NMR (CDCl3) δ 179.2, 167.1 (doublet with 2JC F ~ 18.5 Hz), 163.9, 153.4, 151.1, 150.3, 141.6, 136.0,
−
133.9, 125.4 (C), 135.1, 130.6, 112.1, 107.3, 51.6 (CH), 80.0 (center point of doublet with 1JC F ~ 185
−
Hz), 36.4, 29.7 (CH2), 61.2, 61.1, 56.2, 56.0 (CH3); IR νmax 3270 br (NH), 1680 (C=O) cm−1; HR
ESMS m/z 440.1485 (M+Na+). Calcd. for C22H24FNNaO6, 440.1485.
4.2.2.
2-Chloro-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl}
1
acetamide: yellowish solid, mp 116-118 °C, lit. [11a] mp 204-206 °C; [α]D −82 (c, 1; CHCl3); H
NMR (CDCl3) δ 8.15 (1H, br d, J ~ 6.5 Hz, NH), 7.45 (1H, s), 7.30 (1H, d, J = 10.7 Hz), 6.84 (1H, d, J
= 10.7 Hz), 6.51 (1H, s), 4.63 (1H, apparent dt, J = 11.7, 6.5 Hz), 4.04-4.00 (2H, AB system, J = 14.5
Hz). 3.97 (3H, s), 3.91 (3H, s), 3.87 (3H, s), 3.62 (3H, s), 2.51 (1H, dd, J = 13.7, 6 Hz), 2.38 (1H,
apparent td, J = 13.2, 6.7 Hz), 2.28 (1H, m), 1.94 (1H, m); 13C NMR (CDCl3) δ 179.3, 166.0, 164.1,
153.6, 151.2, 151.0, 141.7, 136.4, 134.0, 125.5 (C), 135.4, 130.6, 112.6, 107.4, 52.8 (CH), 42.5, 36.5,
29.8 (CH2), 61.5, 61.4, 56.4, 56.1 (CH3); IR νmax 3270 br (NH), 1681 (C=O) cm−1; HR ESMS m/z
456.1194 (M+Na+). Calcd. for C22H2435ClNNaO6, 456.1190.
4.2.3.
2-Bromo-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}acetamide: brown-yellowish solid, mp 140-142 °C, lit. [11a] mp 203-206 °C; [α]D −81 (c, 1;
CHCl3); 1H NMR (CDCl3) δ 8.40 (1H, br d, J ~ 6.5 Hz, NH), 7.52 (1H, s), 7.34 (1H, d, J = 10.7 Hz),
6.87 (1H, d, J = 10.7 Hz), 6.52 (1H, s), 4.63 (1H, apparent dt, J = 11.7, 6.5 Hz), 3.99 (3H, s), 3.93 (3H,
s), 3.89 (3H, s), 3.95-3.85 (2H, m overlapped by the OMe signals), 3.64 (3H, s), 2.51 (1H, dd, J = 13.5,
13
6 Hz), 2.38 (1H, apparent td, J = 13, 6.5 Hz), 2.28 (1H, m), 1.90 (1H, m); C NMR (CDCl3) δ 179.3,
166.0, 164.0, 153.6, 151.4, 151.1, 141.7, 136.5, 134.0, 125.4 (C), 135.4, 130.4, 112.7, 107.4, 52.9
(CH), 36.4, 29.7, 28.7 (CH2), 61.4, 61.3, 56.4, 56.1 (CH3); IR νmax 3260 br (NH), 1681 (C=O) cm−1;
HR ESMS m/z 500.0684 (M+Na+). Calcd. for C22H2479BrNNaO6, 500.0685.
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4.2.4.
2-Iodo-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}acetamide: brown-yellowish solid, mp 161-163 °C, lit. [11a] mp 195 °C; [α] −85 (c, 1; CHCl3); 1H
D
NMR (CDCl3) δ 8.70 (1H, br d, J ~ 7 Hz, NH), 7.64 (1H, s), 7.36 (1H, d, J = 10.7 Hz), 6.89 (1H, d, J =
10.7 Hz), 6.51 (1H, s), 4.63 (1H, apparent dt, J = 11.7, 6.5 Hz), 4.00 (3H, s), 3.93 (3H, s), 3.89 (3H, s),
3.82 (1H, d, J = 10.5 Hz), 3.77 (1H, d, J = 10.5 Hz), 3.65 (3H, s), 2.48 (1H, dd, J = 13, 6 Hz), 2.40-2.25
13
(2H, br m), 1.86 (1H, m); C NMR (CDCl3) δ 179.5, 167.6, 164.0, 153.5, 152.2, 151.1, 141.7, 136.8,
134.0, 125.4 (C), 135.5, 130.5, 112.9, 107.4, 52.9 (CH), 36.4, 29.8, −0.1 (CH2), 61.5, 61.3, 56.4, 56.1
(CH3); IR νmax 3260 br (NH), 1675 (C=O) cm−1; HR ESMS m/z 548.0537 (M+Na+). Calcd. for
C22H24INNaO6, 548.0546.
4.2.5.
2-Cyclohexyl-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl}acetamide: brown-yellowish solid, mp 226-229 °C; [α]D −138 (c, 1; CHCl3); H NMR (CDCl3)
δ 7.49 (1H, s), 7.35 (1H, br d, J ~ 7 Hz, NH), 7.28 (1H, d, J = 10.7 Hz), 6.82 (1H, d, J = 10.7 Hz), 6.49
(1H, s), 4.64 (1H, apparent dt, J = 11.7, 6.5 Hz), 3.96 (3H, s), 3.91 (3H, s), 3.86 (3H, s), 3.63 (3H, s),
2.46 (1H, dd, J = 13, 6 Hz), 2.34 (1H, apparent td, J = 13.2, 6.8 Hz), 2.22 (1H, m), 2.05 (2H, apparent
d, J = 7 Hz), 1.83 (1H, m), 1.75-1.50 (6H, br m), 1.20-1.00 (3H, br m), 0.87 (2H, m); 13C NMR
(CDCl3) δ 179.3, 172.1, 163.9, 153.3, 152.0, 151.1, 141.5, 136.5, 134.1, 125.6 (C), 135.0, 130.7, 112.4,
107.3, 52.0, 34.9 (CH), 44.1, 36.7, 33.1, 33.0, 29.8, 26.1, 25.9, 25.8 (CH2), 61.5, 61.2, 56.2, 56.0
(CH3); IR νmax 3280 br (NH), 1656 (C=O) cm−1; HR ESMS m/z 504.2364 (M+Na+). Calcd. for
C28H35NNaO6, 504.2362.
4.2.6.
2-Phenyl-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl}acetamide: yellowish solid, mp 122-125 °C; [α]D −99 (c, 1; CHCl3); H NMR (CDCl3) δ 7.40-7.25
(7H, br m), 6.80 (1H, d, J = 10.7 Hz), 6.50 (1H, s), 5.90 (1H, br d, J ~ 7 Hz, NH), 4.62 (1H, apparent
dt, J = 11.7, 6.5 Hz), 4.00 (3H, s), 3.94 (3H, s), 3.89 (3H, s), 3.66 (3H, s), 3.57 (2H, br s), 2.48 (1H, dd,
J = 13.5, 6.5 Hz), 2.38 (1H, apparent td, J = 13.2, 6.5 Hz), 2.13 (1H, m), 1.65 (1H, apparent td, J = 12,
7 Hz); 13C NMR (CDCl3) δ 179.3, 170.6, 163.9, 153.4, 151.6, 151.1, 141.6, 136.5, 134.8, 134.1, 125.6
(C), 135.1, 130.6, 129.2 (x 2), 128.7 (x 2), 127.0, 112.4, 107.3, 52.2 (CH), 43.1, 36.6, 29.8 (CH2), 61.5,
61.2, 56.2, 56.0 (CH3); IR νmax 3280 br (NH), 1653 (C=O) cm−1; HR ESMS m/z 476.2083 (M+H+).
Calcd. for C28H30NO6, 476.2073.
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4.2.7.
N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl}benzamide:
yellowish solid, mp 171-173 °C, lit. [11e] mp 150-152 °C; [α] −40 (c, 1.1; CHCl3), lit. [15]1 [α]
D
D
−107; 1H NMR (CDCl3) δ 7.90 (1H, br d, J ~ 7 Hz, NH), 7.77 (2H, d, J = 7.8 Hz), 7.63 (1H, s), 7.35-
7.25 (2H, m), 7.19 (2H, t, J = 7.8 Hz), 6.85 (1H, d, J = 10.7 Hz), 6.53 (1H, s), 4.86 (1H, apparent dt, J
= 11.5, 7 Hz), 3.97 (3H, s), 3.95 (3H, s), 3.89 (3H, s), 3.73 (3H, s), 2.49 (1H, dd, J = 13, 6.5 Hz), 2.43
(1H, apparent td, J = 13.5, 7 Hz), 2.40 (1H, m), 2.00 (1H, m); 13C NMR (CDCl3) δ 179.2, 166.9, 163.9,
153.4, 152.1, 151.1, 141.6, 136.5, 134.2, 133.3, 125.6 (C), 135.2, 131.3, 130.7, 128.1 (x 2), 127.0 (x 2),
112.4, 107.3, 52.7 (CH), 36.3, 29.9 (CH2), 61.5, 61.2, 56.2, 56.0 (CH3); IR νmax 330 br (NH), 1654
(C=O) cm−1; HR ESMS m/z 462.1915 (M+H+). Calcd. for C27H28NO6, 462.1916.
4.2.8.
2-Methyl-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl}benzamide: yellowish solid, mp 202-205 °C; [α]D −160 (c, 1.1; CHCl3); H NMR (CDCl3) δ 7.46
(1H, s), 7.40 (1H, d, J = 7.5 Hz), 7.35-7.30 (2H, m), 7.20 (2H, m), 6.83 (1H, d, J = 10.7 Hz), 6.58 (1H,
s), 6.20 (1H, br d, J ~ 7 Hz, NH), 4.85 (1H, apparent dt, J = 11.5, 7 Hz), 4.00 (3H, s), 3.97 (3H, s), 3.93
(3H, s), 3.72 (3H, s), 2.58 (1H, dd, J = 13, 6.5 Hz), 2.50 (1H, apparent td, J = 13.5, 7.2 Hz), 2.38 (3H,
s), 2.35 (1H, m), 1.85 (1H, apparent td, J = 11.7, 6.8 Hz); 13C NMR (CDCl3) δ 179.2, 169.3, 163.9,
153.4, 151.2, 151.0, 141.6, 136.2, 136.0, 135.6, 134.1, 125.6 (C), 135.0, 130.8, 130.7, 129.7, 126.8,
125.5, 112.1, 107.3, 52.2 (CH), 36.9, 29.8 (CH2), 61.4, 61.2, 56.1, 56.0, 19.7 (CH3); IR νmax 3260 br
(NH), 1653 (C=O) cm−1; HR ESMS m/z 476.2067 (M+H+). Calcd. for C28H30NO6, 476.2073.
4.2.9.
3-Methyl-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 145-147 °C; [α] −48 (c, 1; CHCl3); 1H NMR (CDCl3) δ 7.57 (1H,
D
s), 7.54 (1H, br d, J ~ 7 Hz), 7.46 (1H, s), 7.33 (1H, d, J = 10.7 Hz), 7.30-7.25 (2H, m) 6.83 (1H, d, J =
10.7 Hz), 6.70 (1H, br d, J ~ 7 Hz, NH), 6.57 (1H, s), 4.86 (1H, apparent dt, J = 11.7, 7 Hz), 4.00 (3H,
s), 3.97 (3H, s), 3.92 (3H, s), 3.73 (3H, s), 2.59 (1H, dd, J = 13, 6.5 Hz), 2.50 (1H, apparent td, J = 13,
13
7 Hz), 2.37 (3H, s), 2.40-2.35 (1H, m), 1.95 (1H, apparent td, J = 11.7, 6.8 Hz); C NMR (CDCl3) δ
179.2, 166.9, 163.9, 153.4, 152.1, 151.1, 141.6, 137.8, 136.6, 134.2, 133.2, 125.6 (C), 135.1, 132.0,
130.8, 128.0, 127.5, 124.1, 112.5, 107.3, 52.7 (CH), 36.4, 29.9 (CH2), 61.5, 61.3, 56.2, 56.0, 20.9
(CH3); IR νmax 3290 br (NH), 1653 (C=O) cm−1; HR ESMS m/z 476.2079 (M+H+). Calcd. for
C28H30NO6, 476.2073.
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4.2.10.
4-Methyl-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 179-181 °C; [α] −28 (c, 1; CHCl3); 1H NMR (CDCl3) δ 7.67 (2H,
D
d, J = 7.8 Hz), 7.49 (1H, s), 7.33 (1H, d, J = 10.7 Hz), 7.16 (2H, d, J = 7.8 Hz) 6.90 (1H, br d, J ~ 7
Hz, NH), 6.83 (1H, d, J = 10.7 Hz), 6.56 (1H, s), 4.86 (1H, apparent dt, J = 11.7, 7 Hz), 3.99 (3H, s),
3.96 (3H, s), 3.91 (3H, s), 3.73 (3H, s), 2.57 (1H, dd, J = 13, 6.5 Hz), 2.48 (1H, apparent td, J = 13, 7
Hz), 2.40-2.30 (1H, m), 2.35 (3H, s), 1.95 (1H, apparent td, J = 11.7, 6.8 Hz); 13C NMR (CDCl3) δ
179.2, 166.8, 163.9, 153.4, 152.1, 151.2, 141.6 (x 2), 136.6, 134.2, 130.4, 125.6 (C), 135.1, 130.7,
128.8 (x 2), 127.0 (x 2), 112.4, 107.3, 52.7 (CH), 36.4, 29.9 (CH2), 61.5, 61.3, 56.2, 56.0, 21.2 (CH3);
IR νmax 3300 br (NH), 1652 (C=O) cm−1; HR ESMS m/z 476.2070 (M+H+). Calcd. for C28H30NO6,
476.2073.
4.2.11.
4-(n-Pentyl)-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl}benzamide: yellowish solid, mp 136-138 °C; [α]D −20 (c, 0.96; CHCl3); H NMR (CDCl3) δ 7.67
(2H, d, J = 7 Hz), 7.47 (1H, s), 7.33 (1H, d, J = 10.7 Hz), 7.18 (2H, d, J = 7 Hz), 6.82 (2H, m), 6.56
(1H, s), 4.86 (1H, apparent dt, J = 11.7, 7 Hz), 3.98 (3H, s), 3.96 (3H, s), 3.91 (3H, s), 3.73 (3H, s),
2.65-2.55 (3H, br m), 2.48 (1H, apparent td, J = 13, 7 Hz), 2.40-2.30 (1H, m), 1.94 (1H, apparent td, J
= 11.5, 7 Hz), 1.65-1.55 (2H, m), 1.35-1.20 (4H, m), 0.89 (3H, t, J = 6.5 Hz); 13C NMR (CDCl3) δ
179.3, 166.9, 164.0, 153.4, 151.7, 151.2, 146.8, 141.7, 136.4, 134.2, 130.8, 125.7 (C), 135.1, 130.7,
128.3 (x 2), 127.0 (x 2), 112.4, 107.4, 52.6 (CH), 36.7, 35.7, 31.3, 30.7, 29.9, 22.4 (CH2), 61.6, 61.3,
56.2, 56.0, 13.9 (CH3); IR νmax 3290 br (NH), 1653 (C=O) cm−1; HR ESMS m/z 532.2705 (M+H+).
Calcd. for C32H38NO6, 532.2699.
4.2.12.
4-(tert-Butyl)-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 163-165 °C; [α] −28 (c, 1; CHCl3); 1H NMR (CDCl3) δ 7.70 (2H,
D
d, J = 7 Hz), 7.44 (1H, s), 7.43 (2H, d, J = 7 Hz), 7.33 (1H, d, J = 10.7 Hz), 6.82 (1H, d, J = 10.7 Hz),
6.60 (1H, br d, J ~ 7 Hz, NH), 6.57 (1H, s), 4.87 (1H, apparent dt, J = 11.7, 7 Hz), 3.99 (3H, s), 3.97
(3H, s), 3.92 (3H, s), 3.74 (3H, s), 2.57 (1H, dd, J = 13, 6.5 Hz), 2.50 (1H, apparent td, J = 13, 7 Hz),
2.40-2.30 (1H, m), 1.92 (1H, apparent td, J = 11.5, 7 Hz), 1.33 (9H, s); 13C NMR (CDCl3) δ 179.3,
166.9, 163.9, 154.8, 153.4, 151.8, 151.2, 141.6, 136.4, 134.2, 130.6, 125.7, 34.7 (C), 135.1, 130.7,
126.9 (x 2), 125.2 (x 2), 112.4, 107.4, 52.6 (CH), 36.6, 29.9 (CH2), 61.6, 61.3, 56.2, 56.0, 31.0 (x 3)
(CH3); IR νmax 3300 br (NH), 1652 (C=O) cm−1; HR ESMS m/z 518.2549 (M+H+). Calcd. for
C31H36NO6, 518.2543.
20

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4.2.13.
2-Chloro-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 153-155 °C; [α] −134 (c, 1; CHCl3); 1H NMR (CDCl3) δ 7.64 (1H,
D
d, J = 7.5 Hz), 7.48 (1H, s), 7.40-7.30 (4H, br m), 6.85-6.80 (2H, m), 6.57 (1H, s), 4.87 (1H, apparent
dt, J = 11.5, 7 Hz), 3.99 (3H, s), 3.96 (3H, s), 3.92 (3H, s), 3.72 (3H, s), 2.58 (1H, dd, J = 13, 6.5 Hz),
2.49 (1H, apparent td, J = 13.2, 7 Hz), 2.40 (1H, m), 1.92 (1H, apparent td, J = 11.5, 6.5 Hz); 13C NMR
(CDCl3) δ 179.2, 165.8, 163.9, 153.4, 151.1, 150.5, 141.6, 136.0, 134.4, 134.0, 130.5, 125.6 (C), 134.9,
131.1, 131.0, 130.0, 129.8, 126.8, 112.1, 107.3, 52.6 (CH), 36.8, 29.8 (CH2), 61.4, 61.2, 56.1, 56.0
(CH3); IR νmax 3250 br (NH), 1666 (C=O) cm−1; HR ESMS m/z 496.1523 (M+H+). Calcd. for
C27H2735ClNO6, 496.1527.
4.2.14.
3-Chloro-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl}benzamide: yellowish solid, mp 105-107 °C; [α]D −15 (c, 0.92; CHCl3); H NMR (CDCl3) δ 7.85
(1H, br s, NH), 7.74 (1H, s), 7.62 (2H, m), 7.47 (1H, d, J = 10.7 Hz), 7.33 (1H, d, J = 7.8 Hz), 7.17
(1H, t, J = 7.8 Hz), 6.89 (1H, d, J = 10.7 Hz), 6.56 (1H, s), 4.87 (1H, apparent dt, J = 11.5, 7 Hz), 4.00
(3H, s), 3.96 (3H, s), 3.91 (3H, s), 3.73 (3H, s), 2.57 (1H, dd, J = 13, 6.5 Hz), 2.50-2.35 (2H, br m),
2.07 (1H, apparent td, J = 11.5, 6.5 Hz); 13C NMR (CDCl3) δ 179.2, 165.2, 164.1, 153.5, 152.4, 151.2,
141.6, 136.9, 134.8, 134.3, 130.5, 125.6 (C), 135.5, 131.3, 130.7, 129.5, 127.5, 125.0, 112.9, 107.4,
53.0 (CH), 36.2, 29.9 (CH2), 61.6, 61.3, 56.4, 56.1 (CH3); IR
ESMS m/z 496.1527 (M+H+). Calcd. for C27H2735ClNO6, 496.1527.
ν
max 3290 br (NH), 1662 (C=O) cm−1; HR
4.2.15.
4-Chloro-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 122-124 °C; [α] +56.5 (c, 0.92; CHCl3); 1H NMR (CDCl3) δ 7.73
D
(2H, d, J = 7.8 Hz), 7.62 (1H, s), 7.39 (1H, d, J = 10.7 Hz), 7.20-7.15 (3H, m), 6.90 (1H, d, J = 10.7
Hz), 6.55 (1H, s), 4.85 (1H, apparent dt, J = 11.5, 7 Hz), 4.00 (3H, s), 3.96 (3H, s), 3.91 (3H, s), 3.75
(3H, s), 2.60-2.55 (1H, m), 2.50-2.35 (2H, br m), 2.10-2.00 (1H, m); 13C NMR (CDCl3) δ 179.1, 165.7,
164.0, 153.5, 152.8, 151.2, 141.6, 137.4, 137.0, 134.2, 131.3, 125.5 (C), 135.6, 130.4, 128.5 (x 2),
128.3 (x 2), 112.9, 107.4, 53.2 (CH), 36.0, 30.0 (CH2), 61.6, 61.3, 56.3, 56.1 (CH3); IR νmax 3290 br
(NH), 1662 (C=O) cm−1; HR ESMS m/z 496.1527 (M+H+). Calcd. for C27H2735ClNO6, 496.1527.
4.2.16.
2-Bromo-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 174-176 °C; [α] −152 (c, 1; CHCl3); 1H NMR (CDCl3) δ 7.59 (1H,
D
d, J = 7.8 Hz), 7.55 (1H, d, J = 7.8 Hz), 7.50 (1H, s), 7.40-7.30 (3H, br m), 6.82 (1H, d, J = 10.8 Hz),
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6.57 (1H, s), 6.50 (1H, br d, J ~ 7 Hz, NH), 4.88 (1H, apparent dt, J = 11.3, 7 Hz), 4.00 (3H, s), 3.97
(3H, s), 3.93 (3H, s), 3.72 (3H, s), 2.58 (1H, dd, J = 13, 6.5 Hz), 2.50 (1H, apparent td, J = 13.2, 6.5
13
Hz), 2.44 (1H, m), 1.90 (1H, apparent td, J = 11.5, 6.5 Hz); C NMR (CDCl3) δ 179.2, 166.8, 163.9,
153.4, 151.1, 150.5, 141.6, 137.1, 136.1, 134.0, 125.6, 119.1 (C), 135.0, 133.0, 131.2, 131.1, 129.6,
127.4, 112.1, 107.4, 52.6 (CH), 36.9, 29.8 (CH2), 61.4, 61.2, 56.1, 56.0 (CH3); IR νmax 3250 br (NH),
1665 (C=O) cm−1; HR ESMS m/z 540.1019 (M+H+). Calcd. for C27H2779BrNO6, 540.1022.
4.2.17.
3-Bromo-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl}benzamide: yellowish solid, mp 137-139 °C; [α]D −11 (c, 0.96; CHCl3); H NMR (CDCl3) δ 8.50
(1H, br d, J ~ 7 Hz, NH), 7.90 (1H, br s), 7.68 (1H, s), 7.64 (1H, d, J = 7.8 Hz), 7.40 (1H, br d, J ~ 8
Hz), 7.38 (1H, d, J = 10.7 Hz), 6.99 (1H, t, J = 8 Hz), 6.90 (1H, d, J = 10.7 Hz), 6.52 (1H, s), 4.85 (1H,
apparent dt, J = 11.5, 7 Hz), 3.99 (3H, s), 3.95 (3H, s), 3.89 (3H, s), 3.72 (3H, s), 2.49 (1H, dd, J = 13,
6 Hz), 2.45-2.30 (2H, br m), 2.13 (1H, m); 13C NMR (CDCl3) δ 179.2, 165.2, 164.1, 153.5, 152.4,
151.2, 141.6, 136.9, 134.8, 134.3, 130.5, 125.6 (C), 135.5, 131.3, 130.7, 129.5, 127.5, 125.0, 112.9,
107.4, 53.0 (CH), 36.2, 29.9 (CH2), 61.6, 61.3, 56.4, 56.1 (CH3); IR νmax 3290 br (NH), 1654 (C=O)
cm−1; HR ESMS m/z 540.1025 (M+H+). Calcd. for C27H2779BrNO6, 540.1022.
4.2.18.
4-Bromo-N-{(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl}benzamide: yellowish solid, mp 154-156 °C; [α] +61.6 (c, 0.92; CHCl3); 1H NMR (CDCl3) δ 7.80
D
(1H, br s, NH), 7.66 (2H, d, J = 8 Hz), 7.57 (1H, s), 7.40-7.35 (3H, m), 6.88 (1H, d, J = 10.8 Hz), 6.55
(1H, s), 4.84 (1H, apparent dt, J = 11.5, 7 Hz), 4.01 (3H, s), 3.97 (3H, s), 3.92 (3H, s), 3.74 (3H, s),
2.57 (1H, dd, J = 13, 6.5 Hz), 2.50-2.35 (2H, br m), 2.05-2.00 (1H, m); 13C NMR (CDCl3) δ 179.0,
165.7, 164.0, 153.5, 152.8, 151.1, 141.6, 137.0, 134.2, 131.7, 126.0, 125.5 (C), 135.6, 131.2 (x 2),
130.3, 128.7 (x 2), 113.0, 107.3, 53.2 (CH), 35.8, 29.9 (CH2), 61.6, 61.2, 56.3, 56.0 (CH3); IR νmax
3290 br (NH), 1654 (C=O) cm−1; HR ESMS m/z 540.1022 (M+H+). Calcd. for C27H2779BrNO6,
540.1022.
4.3. Cell culture studies
Cell culture media were purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was a
product of Harlan-Seralab (Belton, U.K.). Supplements and other chemicals not listed in this section
were obtained from Sigma Chemical Co. (St. Louis, MO). Plastics for cell culture were supplied by
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Thermo Scientific BioLite. All tested compounds were dissolved in DMSO at a concentration of 10
mM and stored at −20°C until use.
HT-29, MCF-7, A-549 and HEK-293 cell lines were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing glucose (1g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin
(50 µg/mL), and amphotericin B (1.25 µg/mL), supplemented with 10% FBS.
4.4. MTT assays
In order to study the antiproliferative capacity of our derivatives, we used MTT assay on HT-29,
MCF-7, A-549 or HEK-293. 5,000 cells/well were placed in 96-well microtiter plate in a total volume
of 100 µL of growth media. After 24 h, they were incubated with serial dilutions of the tested
compounds. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma
Chemical Co.) dye reduction assay in 96-well microplates was used, as previously described [26]. After
2 days of incubation (37 °C, 5% CO2 in a humid atmosphere), 10 µL of MTT (5 mg/mL in phosphate-
buffered saline, PBS) was added to each well, and the plate was incubated for a further 3 h (37 °C). The
supernatant was discarded and replaced by 100 µL of DMSO to dissolve formazan crystals. The
absorbance was then read at 540 nm by spectrophotometry. For all concentrations of compound, cell
viability was expressed as the percentage of the ratio between the mean absorbance of treated cells and
the mean absorbance of untreated cells. Three independent experiments were performed, and the IC50
values (i.e., concentration half inhibiting cell proliferation) were graphically determined.
4.5. Tubulin self-assembly measurements
Purified tubulin was used for these measurements. Tubulin polymerization was carried out in a 96
well plate. In each well 50 µL of a solution of 25 µM of tubulin in GAB buffer was added to 50 µL of
27.5 µM solution of the corresponding compounds in GAB buffer (20 mM sodium phosphate, 10 mM
MgCl2, 1 mM EGTA, 30% glycerol) and 0.1 mM GTP at pH = 6.5. Then, the plate was incubated at 37
ºC in Multiskan (R) and absorbance at 340 nM was registered every 30 seconds during 2 hours.
4.6. Cell cycle studies
Progression of the cell cycle was analysed by DNA determination by means of flow cytometry with
propidium iodide. A549 cells were fixed, treated with RNase and stained with propidium iodide
following instructions of BD CycletestTM DNA Kit. Analysis was performed with a BD AccuriTM C6
flow cytometer.
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4.7. RT-qPCR Assay
HT-29 cells at 70–80% confluence were collected and 1.5 x 105 cells were placed in a six well plate
in 1.5 mL of medium. After 24h, cells were incubated with the corresponding compounds for 48 h.
Cells were collected and the total cellular RNA from HT-29 cells was isolated using Ambion RNA
extraction Kit according to the manufacturer’s instructions. The cDNA was synthesized by MMLV-RT
with 1–21 µg of extracted RNA and oligo(dT)15 according to the manufacturer’s instructions. Genes
were amplified by use of a thermal cycler and StepOnePlus ™ Taqman ® probes. TaqMan ® Gene
Expression Master Mix Fast containing the appropriate buffer for the amplification conditions, dNTPs,
thermostable DNA polymerase enzyme and a passive reference probe was used. To amplify each of the
genes the predesigned primers were used and sold by Life Technologies TaqMan ® Gene Expression
Assays, Hs99999903-m1 (β-actin), Hs00972646-m1 (hTERT), Hs00153408-m1 (c-MYC) and
Hs00900055-m1 (VEGF-A).
4.8. ELISA analysis
HT-29 cells at 70–80% confluence were collected and 1.5 x 105 cells were placed in a six well plate
in 1.5 mL of medium. After 24h, cells were incubated with the corresponding compounds for 48 h.
Culture supernatants were collected and VEGF secreted by HT-29 cells was determined using
Invitrogen Human Vascular Endothelial Growth Factor ELISA Kit according to the manufacturer’s
instructions.
4.9. Molecular docking studies
Molecular docking was performed using Autodock 4.2 [9]. The crystal structure of αβ tubulin (PDB
ID 1SA0) was used as a template. Nevertheless, the C and D subunits, stathmin, water molecules and
bound ligands were previously removed from the protein structure in order to perform docking
simulation. The GaussView 5.0 program [13] was used to build the structures of the colchicine
derivatives 1-4 and 16-18. Local docking was made in such a way that the grid box covered the entire
αβ-tubulin interface. The grid map was used with 126 points equally in each x, y and z direction and
with grid spacing 0.247 Å. The cluster was compared on the basis of the free energy of binding. The
Lamarckian genetic algorithm (LGA) was employed with the default parameters; g_eval was set to
2500000 (medium) and 100 LGA runs were conducted. Molecular graphics were done with the
program Visual Molecular Dynamics (VMD) [14]. Discovery Studio Visualizer program [15] has been
used to show the interaction in 2-D between the compounds and the tubulin.
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Acknowledgments
This research has been funded by the Ministerio de Economía y Competitividad (project CTQ2014-
52949-P), by the Universitat Jaume I (project PI-1B2015-75) and by the Conselleria d’Educaciò,
Investigaciò, Cultura i Sport de la Generalitat Valenciana (project PROMETEO 2013/027). A. M.-M.
thanks the Conselleria d’Educaciò, Investigaciò, Cultura i Esport de la Generalitat Valenciana for a Ph.
D. grant. The authors are also grateful to the SCIC of the Universitat Jaume I for providing NMR and
mass spectrometry facilities.
Supplementary Information
Additional NMR data of all new synthetic compounds are provided in the Supplementary
Information.
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List of captions
Figure 1. Structure of the colchicine analogues investigated in this study.
Figure 2. Panel A corresponds to the superimposition of the structures of 1 (black), 2 (orange), 3 (red)
and 4 (purple) on the co-crystallized DAMA-colchicine (blue) at the colchicine binding site. Panel B
corresponds to the structure of fluoroacetyl derivative 1 at the colchicine binding site. The α- and β-
tubulin subunits are coloured in yellow and green, respectively.
Figure 3. Panel A corresponds to the superimposition of the structures of 16 (black), 17 (red) and 18
(purple) on the co-crystallized DAMA-colchicine (blue) at the colchicine binding site. Panel B, C and
D correspond, respectively, to the structures of 16, 17 and 18 at the colchicine binding site. The α- and
β-tubulin subunits are coloured in yellow and green, respectively.
Scheme 1. Synthesis of colchicine derivatives 1-18.
Table 1. IC50 values (nM) of synthetic compounds 1-19 in cancer cell lines HT-29, MCF-7 and A549,
and one non-cancer cell line HEK-293.a
Table 2. Critical concentration (CrC) for the assembly of purified tubulin in GAB in the presence of
colchicine, podophyllotoxin and the colchicine analogues 1-19.a
Figure 4. Effects of colchicine and compounds 1-19 on the kinetics of tubulin assembly. The lines in
the figure show the turbidity time course of polymerization of tubulin alone (black) at 25 µM,
colchicine (red), 8 (green), 10 (orange), 16 (gray) and 18 (purple) and 19 (pink). All compounds were
at 27.5 µM.
Table 3. It50 of some compounds for tubulin self-assembly.a
Table 4. Inhibitory Effects on Microtubule Assembly at 37ºC and 15 µM of tubulin.a
Figure 5. Selected compounds and concentrations for a quantitative gene expression test
Figure 6. Panel A: Expression percentage of the c-Myc gene after 48 h of incubation with DMSO
(control) and selected compounds. Panel B: Expression percentage of the hTERT gene after 48 h of
incubation with DMSO (control), and selected compounds. Panel C: Expression percentage of the
VEGF gene after 48 h of incubation with DMSO (control), and selected compounds. Panel D:
Expression percentage of the VEGF protein secreted after 48 h of incubation with DMSO (control), and
selected compounds. At least three measurements were performed in each case Gene expression was
normalized using β-ACTIN as endogenous gene. Gene expression was normalized using β-ACTIN as
endogenous gene. Percentages above 100% indicate that the corresponding compounds were less active
than the control. Error bars indicate standard errors of the mean. The statistical significance was
evaluated using one-sample t-tests (P <0.001).
28