New Colchicine-Derived Triazoles and Their Influence on Cytotoxicity and Microtubule Morphology

Article abstract of DOI:10.1021/acsmedchemlett.5b00418

A series of new colchicinoids with a variable triazole unit at C-7 was synthesized through Cu(I)-catalyzed 1,3-dipolar cycloaddition (click-chemistry) of a colchicine-derived azide with various alkynes and the cytotoxicity against THP-1 and Jurkat cancer cell lines was used for structural optimization. Three particularly active compounds (IC₅₀ ≤ 5 nM) were additionally investigated with respect to their efficacy against relevant solid tumor cell lines (HeLa, A549, and SK MES 1). Besides distorting the microtubule morphology by tubulin depolymerization, one compound also exhibited a pronounced centrosome declustering effect in triple negative breast cancer cells (MDA-MB-231) and nonsmall cell lung cancer cells (H1975).

Full text of DOI:10.1021/acsmedchemlett.5b00418

Letter
pubs.acs.org/acsmedchemlett
New Colchicine-Derived Triazoles and Their Influence on Cytotoxicity
and Microtubule Morphology
Persefoni Thomopoulou,† Julia Sachs,‡ Nicole Teusch,‡ Aruljothi Mariappan,§ Jay Gopalakrishnan,§
and Hans-Gunther Schmalz*,†
̈
†Department of Chemistry, University of Cologne, Greinstr. 4, 50939 Cologne, Germany
‡Technische Hochschule Koeln, Kaiser-Wilhelm-Allee, Building E39, 51373 Leverkusen, Germany
§Center for Molecular Medicine and Institute of Biochemistry II of the University of Cologne, Robert-Koch-Str. 21, 50931 Cologne,
Germany
S
* Supporting Information
ABSTRACT: A series of new colchicinoids with a variable triazole
unit at C-7 was synthesized through Cu(I)-catalyzed 1,3-dipolar
cycloaddition (click-chemistry) of a colchicine-derived azide with
various alkynes and the cytotoxicity against THP-1 and Jurkat
cancer cell lines was used for structural optimization. Three
particularly active compounds (IC50 ≤ 5 nM) were additionally
investigated with respect to their efficacy against relevant solid
tumor cell lines (HeLa, A549, and SK MES 1). Besides distorting
the microtubule morphology by tubulin depolymerization, one
compound also exhibited a pronounced centrosome declustering
effect in triple negative breast cancer cells (MDA-MB-231) and
nonsmall cell lung cancer cells (H1975).
KEYWORDS: Colchicine, click chemistry, tubulin, antitumoral compounds, resistance
icrotubules are highly dynamic polymers of α,β-tubulin
heterodimers that play a key role in essential cellular
processes such as accurate cell division, intracellular transport,
and cell motility.1 Therefore, the tubulin polymerization/
depolymerization equilibrium represents an attractive target for
the development of anticancer drugs.2−5 Colchicine (1, Figure
1) is a long-known and powerful antimitotic agent extracted
chain at the colchicine core (Scheme 1).15−17 The method is
based on the microwave-assisted Cu-catalyzed 1,3-dipolar
M
Scheme 1. Synthesis of C7-Modified Colchicine Derivatives
a
through Click-Chemistry
a
Reagents and conditions: CuSO4·5H2O, (5 mol %), sodium ascorbate
(10 mol %), H2O/tert-BuOH (1:1), μW (300 W), 85 °C, 20−45 min.
See Chart 1 for structures and isolated yields.
Figure 1. Natural product colchicine (1).
cycloaddition18,19 of various alkynes to azide 2, which is readily
prepared from colchicine (1) in only four steps. Furthermore, it
was shown that some of the resulting triazoles of type 3 exhibit
promising levels of activity. We here report the continuation of
this study, which has led to the identification of some new and
highly active compounds.
from Colchicum autumnale and clinically used mainly in the
treatment of gout.6 It acts by destabilizing microtubules via
depolymerization leading to cell cycle arrest in the metaphase
and, as a consequence, apoptotic cell death. While the use of
colchicine (1) in cancer chemotherapy is hampered by its
toxicity,7 its remarkable biological activity motivates the search
for new analogues with improved pharmacological proper-
ties.8−14
Received: October 28, 2015
Accepted: December 29, 2015
In this context, we recently developed a “click conjugation”
approach allowing a fast and efficient variation of the C7-side
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00418
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ACS Medicinal Chemistry Letters
Letter
Following an improved protocol (see Supporting Informa-
tion) the azide 2 was obtained from N-deacetyl-colchi-
cin15−17,20 through diazo transfer in 86% yield. The triazoles
of type 3 were then prepared through “click chemistry” by
microwave-induced heating of 2 and a terminal alkyne to 85 °C
in the presence of 5 mol % of CuSO4·5H2O and 10 mol % of
sodium ascorbate in a H2O/tert-BuOH (1:1) solvent mixture.
As a triazole of type 3 with an amino acid-derived side chain
had shown significant activity in our previous study,15−17 we
focused on compounds derived from (N-protected) amino acid
propargyl esters. The synthesized library of new colchicine
conjugates (Chart 1), however, not only comprises compounds
Table 1. Cytotoxic Activity of Colchicine-Derived
Compounds against THP-1 and Jurkat Cells
a
compd
IC50 [nM] THP-1
IC50 [nM] Jurkat
1
20.4 2.7
20.9 0.7
8.1 1.8
13.5 4.7
2
3a
3b
3c
3d
3e
3f
6.6 5.5
17.3 6.6
10.6 9.3
7.3 2.3
4.1 1.7
15.3 3.5
84.2 5.9
21.4 3.8
25.1 2.9
3g
3h
3i
25.5 0.9
23.3 4.8
24.0 6.1
139.8 34.6
31.0 0.8
35.2 12.8
12.7 2.4
9.3 0.2
Chart 1. New Colchicine-Derived Triazoles of Type 3
a
Synthesized According to Scheme 1
3j
3k
3l
16.7 7.1
9.7 3.1
4.9 1.4
2.9 1.6
5.0 1.5
9.4 4.0
3m
3n
3o
3p
3q
3r
5.1 1.1
5.7 2.2
5.4 0.3
23.1 10.1
a
Values represent cytotoxic activity (IC50) after 24 h (THP-1) or 48 h
(Jurkat) compound incubation. Data shown are mean values SD
from at least 3 independent experiments with biological replicates ≥3,
respectively.
the colchicine core (i.e., the main pharmacophore), as does also
the benzylester 3k. Shortening or lengthening the linking unit
to the lipophilic group resulted in a decrease of activity13 as
illustrated by compounds 3i−3l or 3p−3r (Table 1). While
phenylalanine derivatives 3g−3i differing from 3f only in the N-
protecting group did not exhibit improved activities as
compared to 3f, the introduction of an additional fluorine
substituent in the aryl moiety of 3k led to an increase of the
cytotoxic activity (compounds 3m−3o). These results indicate
a favorable hydrophobic interaction between the side chain and
a lipophilic pocket of the target protein.
The three most active compounds, that is the phenylalanine
derivative 3f, the 4-fluorobenzyl ester 3o, and the β-alanine
ester 3p, were further evaluated with respect to their cytotoxic
activity against solid tumor cell lines, that is HeLa cervix
carcinoma cells as well as A549 and chemotherapy resistant SK
MES 1 lung cancer cells (Table 2). As a reference, paclitaxel, a
marketed chemotherapeutic drug, was used as a strongly
mictrotubule-stabilizing compound. Interestingly, compound
3o proved to exhibit remarkable cytotoxic efficacy by inhibiting
a
Yields of purified products are given in brackets. *3i was prepared
from 3f by treatment with TFA.
Table 2. Cytotoxicity and Tubulin Polymerization Inhibition
a
Activity of Compounds 3f, 3o, and 3p
derived from amino acids (3a−3i) but also compounds with an
O-benzylated carboxylic acid substituent (3j−3o) or a N-Boc-
protected aminoalkyl side chain (3p−3r), respectively.
IC50 [nM]
HeLa
IC50 [nM]
A549
IC50 [nM] SK
MES1
IC50 [μM]
tubulin
compd
The cytotoxicity (IC50) of all compounds was determined by
means of a bioluminescence cell viability assay21,22 using THP-
1 acute monocytic leukemia cells as well as Jurkat T cell
lymphocytes. While all compounds were found to exhibit
cytotoxic activity (Table 1), the phenylalanine derivative 3f and
the β-alanine ester 3p stood out in the initial screening by
inhibiting cell growth even at low nanomolar concentrations
(IC50 < 10 nM). Interestingly, these compounds carry a
lipophilic moiety (phenyl or tert-butyl) in a certain distance to
paclitaxel
18.9 1.7
21.8 1.1
70.7 3.1
4.9 2.7
13.2 3.1
23.9 4.6
73.7 20.5
5.5 1.7
13.5 3.2
18.6 1.8
39.2 14.2
3.5 0.9
1
6.7 0.05
3.2 0.3
2.1 0.6
2.44 0.4
3f
3o
3p
20.0 2.2
25.4 4.1
9.6 1.6
a
Cytotoxic activity (IC50) after 48 h (n = 3). Inhibition of tubulin
polymerization (n = 2). Data shown are mean values SD from n
independent experiments.
B
DOI: 10.1021/acsmedchemlett.5b00418
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ACS Medicinal Chemistry Letters
Letter
cell growth already at concentrations below 6 nM clearly
surpassing paclitaxel and colchicine (1). In addition, com-
pounds 3f, 3o, and 3p inhibited tubulin polymerization by 50%
at concentrations of 2−3 μM in an in vitro assay. This
demonstrates the strong cytotoxic activity of these compounds
resulting from their microtubule-destabilizing activity, as
expected.
In an additional set of experiments we explored the effect of
colchicinoids 3f, 3o, and 3p on the microtubule cytoskeleton
morphology of MDA-MB-231 breast cancer cells by means of
immune fluorescence microscopy. For this purpose, the cells
were incubated with 3f, 3o, or 3p at concentrations of 100 nM
and microtubules, centrosomes, and DNA were visualized using
fluorescence stains (Figure 2). Centrosomes are the major
which contributes to cancer cell invasion.24,25 The amplified
centrosomes in cancer cells are clustered together so that
cancer cells are able to avoid multipolar mitosis. Thus,
preventing centrosome clustering has been recognized to be
an attractive cancer target.26,27 Interestingly, the phenylalanine
derivative 3f (as the only one of the investigated compounds)
was found to exhibit a strong centrosome declustering effect on
MDA-MB-231 breast cancer cells (G) and also on H1975 small
cell lung cancer cells (H). Notably, H1975 cells have been
identified to be a therapy resistant cancer against currently
available tyrosine receptor kinase inhibitors.28
In conclusion, we have demonstrated that readily available
colchicine-derived triazoles of type 3 may exhibit remarkable
biological effects in dependence of the side chain structure.
Besides pronounced cytotoxic activities the remarkable
centrosome-declustering effect exhibited by compound 3f
may justify further investigation. Since tubulin heterodimers
are core structural components also of centrosomes,29 targeting
their regulatory mechanisms by small molecules may offer
interesting new options for therapeutic intervention in cancer
chemotherapy.30
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00418.
Experimental procedures, characterization of all com-
pounds, and protocols for biological assays (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: schmalz@uni-koeln.de. Tel: +49 (0)221 470 3063.
Fax: +49 (0)221 470 3064.
Author Contributions
P.T. performed the synthesis, purification, and characterization
of new colchicinoids. J.S. carried out the antiproliferation and
tubulin polymerization assays. A.M. performed the immune
fluorescence microscopic studies. N.T., J.G., and H.G.S.
initiated and supervised the project. The manuscript was
written, based on a first draft by P.T., through contributions of
all authors. All authors have given approval to the final version
of the manuscript.
Figure 2. Microtubule morphology of MDA-MB-231 breast cancer
cells after 24 h incubation with different compounds: (A) control
(untreated cells); (B) paclitaxel (100 nM); (C) 1 (100 nM); (D) 3f
(100 nM); (E) 3o (100 nM); (F) 3p (100 nM). Centrosome
declustering: (G) 3f (100 nM) in MDA-MB-231 breast cancer cells;
(H) 3f (500 nM) in H1975 lung cancer cells. Microtubules (red) and
centrosomes (green) were stained with antibodies, while DAPI (4,6-
diamidino-2-phenylindole) was used to visualize DNA (blue). The
white scale bars correspond to a distance of 10 μm.
Funding
This work was supported by the Jurgen Manchot foundation
̈
(Ph.D. fellowship to P.T.), the German Science Foundation
(Projects SCHM 857/18-1 and GO 2301/2-1), and the
German Federal Ministry of Economy and Energy (Ph.D.
fellowship to J.S.).
microtubule organizing centers of animal cells.1,23 While
untreated cells (A) showed a typical microtubule network,
incubation with 100 nM of paclitaxel as a microtubule-
stabilizing agent resulted in the expected segregation of the
cells (B). As a second control, colchicine completely collapsed
the microtubule network (C). As Figure 2 clearly shows,
compounds 3f, 3o, and 3p also suppressed the formation of the
microtubule network (at 100 nM); however, short microtubule
fragments (tending to localize around the cell nucleus) were
still visible (D−F).13,14
Notes
The authors declare no competing financial interest.
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