Design and synthesis of pironetin analogue/combretastatin A-4 hybrids containing a 1,2,3-triazole ring and evaluation of their cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2014.09.053

We here describe the preparation of a series of hybrid molecules containing a combretastatin A-4 moiety and a pironetin analogue fragment connected through a spacer of variable length which includes a 1,2,3-triazole ring. The cytotoxic activities of these compounds have been measured. Relations between structure and cytotoxicity are discussed. Some of the tested compounds showed cytotoxicity values of the same order of magnitude as combretastatin A-4 and were less toxic than the latter compound for normal cells.

Full text of DOI:10.1016/j.ejmech.2014.09.053

European Journal of Medicinal Chemistry 87 (2014) 125e130
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of pironetin analogue/combretastatin A-4
hybrids containing a 1,2,3-triazole ring and evaluation of their
cytotoxic activity
a
a
a
a, *
Concepci oꢀ n Vilanova , Sandra Torijano-Guti eꢀ rrez , Santiago Díaz-Oltra , Juan Murga
,
a
a
b, *
Eva Falomir , Miguel Carda , J. Alberto Marco
Depart. de Q. Inorg aꢀ nica y Org aꢀ nica, Univ. Jaume I, E-12071 Castell oꢀ n, Spain
a
b
Depart. de Q. Org aꢀ nica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2014
Received in revised form
We here describe the preparation of a series of hybrid molecules containing a combretastatin A-4 moiety
and a pironetin analogue fragment connected through a spacer of variable length which includes a 1,2,3-
triazole ring. The cytotoxic activities of these compounds have been measured. Relations between
structure and cytotoxicity are discussed. Some of the tested compounds showed cytotoxicity values of
the same order of magnitude as combretastatin A-4 and were less toxic than the latter compound for
normal cells.
12 September 2014
Accepted 14 September 2014
Available online 16 September 2014
©
2014 Published by Elsevier Masson SAS.
Keywords:
Combretastatin A-4
Pironetin analogues
Hybrid molecules
Click reaction
Tubulin-active compounds
Cytotoxicity
1
. Introduction
named microtubule dynamic instability [5]. Any molecule which
influences microtubule instability will also influence the cell divi-
sion process, not only of normal cells but also of tumoral cells.
Therefore, it is not surprising that tubulin-binding molecules
(TBMs) constitute a very important class of anticancer agents.
TBMs may be divided in two broad categories, those that bind to
It is widely known that cancer, one leading cause of death in
developed countries [1], may be induced by a plethora of both
external and internal factors, including genetic mutations.
Accordingly, a number of types of therapeutic attack has been
investigated [2,3]. One of these involves the use of cytotoxic drugs,
which exert their effect in many cases by means of inducing various
mechanisms of cell death [4]. As a matter of fact, many such drugs
owe this property to interaction with the microtubule network.
Microtubules are dynamic polymers that play a central role in a
number of cellular processes, most particularly cell division, as they
are key constituents of the mitotic spindle. Microtubules are
constituted of a protein named tubulin, the functional form of
which, and the main component, is a heterodimer formed through
a-tubulin and those that bind to b-tubulin. The latter group is
presently by far the most numerous and contains products which
cause either disruption or stabilization of microtubules. Among the
drugs that belong to this group, the well-known colchicine [6] and
the combretastatins [7] (Fig. 1) exert their effects by causing
disruption of microtubules. In contrast, another important repre-
sentative of the same group, paclitaxel, was the first-described
tubulin-binding drug that was found to stabilize microtubules [8].
Even though they display opposite effects, these drugs are known
non-covalent binding of two monomeric constituents, called
-tubulin. For cell division to occur in a normal way, microtubules
must be in a constant state of formation and disruption, a process
a
- and
to bind to b-tubulin, whenever to different sites within this protein
subunit [9e11].
b
The number of products that bind to a-tubulin is very small, the
naturally occurring 5,6-dihydro-a-pyrone pironetin being the first-
reported example [12], followed a short time later by the peptide-
like hemiasterlin family [13] (Fig. 2). Pironetin is a potent inhibitor
*
Corresponding authors.
E-mail addresses: jmurga@qio.uji.es (J. Murga), alberto.marco@uv.es (J. Alberto
Marco).
http://dx.doi.org/10.1016/j.ejmech.2014.09.053
0223-5234/© 2014 Published by Elsevier Masson SAS.

126
C. Vilanova et al. / European Journal of Medicinal Chemistry 87 (2014) 125e130
conjugated dihydropyrone ring and the side chain with the
methoxy group at C-9. The hydroxyl group at C-7 was removed in
some substrates (I) and retained in others (II), in order to see its
influence on the activity. All alkyl pendants (methyl groups at C-8
and C-10, ethyl at C-4) and the isolated C12eC13 double bond were
removed. The configurations of the two/three remaining stereo-
centres were then varied in a systematic way. Thus, all four possible
stereoisomers with general constitution I, with no hydroxyl group
at C-7, were prepared. Likewise, all eight stereoisomers exhibiting
general structure II, with a hydroxyl group at C-7, were synthesized
[5a].
The cytotoxic activity of these analogues and their interactions
with tubulin were subsequently investigated. It was found, on one
hand, that analogues I/II were cytotoxic in the low micromolar
range, about three orders of magnitude less active than the parent
molecule. On the other hand, we also found that they behave in the
same way as pironetin, share the mechanism of action of the nat-
ural compound and compete for the same binding site in a-tubulin.
As observed for the parent compound, they also lead to disruption
of the microtubule network [5a].
With the aim at extending our project, our idea was to prepare
cytotoxic TBMs with a dual ability to bind to either the a- or the b-
tubulin subunits [19] and exert a microtubule-destabilizing effect.
Since these properties are specifically exhibited by pironetin (binds
to
tively, we decided to prepare compounds with a hybrid structure
20] such as 1e8 (Fig. 4). These molecules display a moiety of
a-tubulin) and combretastatin A-4 (binds to b-tubulin), respec-
[
combretastatin A-4 (itself numbered as 13 in Fig. 4) and another of
the simplified pironetin type (14, ent-14 and 15), connected in turn
by a spacer of variable length containing a 1,2,3-triazole ring
generated by means of a “click-type reaction” [21]. Such hetero-
cyclic rings are known to exhibit pharmacologically interesting
Fig. 1. Structures of some natural products reported to selectively bind to b-tubulin.
of tubulin assembly and has been found to arrest cell cycle pro-
gression in the G2/M phase [14].
Some structureeactivity relationship (SAR) studies on pironetin
have been reported. These studies have shown that the presence of
the conjugated C2eC3 double bond and of the hydroxyl group at C-
9
, either free or methylated, are essential for the biological activity.
The presence of a (7R)-hydroxyl group might also be relevant. It has
been suggested that a nucleophilic residue, yet to be conclusively
identified, of the a-tubulin chain adds in a Michael fashion to the
conjugated double bond of pironetin [14], therefore forming a co-
valent bond with C-3 of the dihydropyrone ring [15].
The emergence of resistances to existing drugs has led to a
continuous need of developing new bioactive compounds that
overcome such problems. Even though first observed in the case of
antibiotics [16], resistances have been reported to therapies with
various types of anticancer agents [17]. The investigation of new
compounds with such biological properties therefore constitutes
an important goal in chemistry and pharmacology.
Fig. 2. Structure of two natural products reported to selectively bind to a-tubulin.
features that are beyond a mere connecting role [22] and, for
example, may act as peptide isosters [21]. Some of the hybrid
2
. Concept and design of hybrid tubulin-binding ligands
As a member of the up to now small group of TBMs that bind to
-tubulin, pironetin constitutes a pharmacologically interesting
a
target. Not surprisingly, an appreciable number of total syntheses of
this natural compound has appeared in the literature [18]. In order
to develop SAR studies based upon the pironetin framework, we
designed two years ago [5a] a simplified model structure where all
elements that had not yet proven to be essential for the biological
activity were removed. The target structures I/II are schematically
shown in Fig. 3. The elements that were maintained are the
Fig. 3. General structures of simplified pironetin analogues of the first generation
([5a]).

C. Vilanova et al. / European Journal of Medicinal Chemistry 87 (2014) 125e130
127
Fig. 4. Structures of the pironetin analogue/combretastatin A-4 hybrids used in this study (1e8) and of their synthetic precursors (9e15), including combretastatin A-4 (13).
molecules (1 to 4 and ent-1 to ent-4) have been prepared in both
antipodal forms [23].
azide in DMF furnished dihydropyrones 14, ent-14 and 24. Desily-
lation of the latter gave 15 [23].
Heating acetylenic compounds 9e12 with the aforementioned
dihydropyrones under copper(I) catalysis gave rise to a 1,3-dipolar
cycloaddition [21] with regioselective formation of the desired
3
. Synthetic work
1
,2,3-triazoles 1 to 4, ent-1 to ent-4 and 5 to 8 (Scheme 3).
Combretastatin A-4 (13) was prepared according to a literature
After the synthesis of the hybrid molecules was completed, the
compounds were investigated in relation to their cytotoxic activity
towards two types of tumoral cell lines and one normal cell line.
procedure [24]. Its O-alkyl derivatives 9e12 were prepared as
depicted in Scheme 1 by means of alkylation with -haloalkynes
6e19 (for experimental details, see the Supporting Information).
The synthesis of the pironetin fragments 14, ent-14 and 15 was
u
1
performed as depicted in Scheme 2. Thus, the known pyrones 20,
ent-20 and 22, prepared according to the general concept used in
our previous papers [5a,b], were first tosylated to, respectively, 21,
ent-21 and 23. Treatment of the latter compounds with sodium
4
4
4
. Biological work
.1. Cellular effects of the compounds
.1.1. Cytoxicity values
We carried out a measurement of the cytotoxicity of the syn-
thetic combretastatin A-4 derivatives 1e4 and their respective
enantiomers (ent-1 to ent-4), and 5e8. Cytotoxicity assays were
performed as described in the Experimental Section using two
types of tumoral cells, the human colon adenocarcinoma HT-29 and
the breast adenocarcinoma MCF-7 cell lines. In addition, one
normal cell line, the human embryonic kidney cell line (HEK-293)
[25] was employed in the assays for comparison. Cytotoxicity
values, expressed as the compound concentration ( mol/L) that
m
causes 50% inhibition of cell growth (IC50), are shown in Table 1.
The observed values are in the medium to low micromolar
range. Compounds 2, 3, ent-1, ent-4, 6 and most particularly 8
showed the highest cytotoxicities for the HT-29 cell line, with IC50
values which were not very different of (in the case of 8 visibly
lower than) those of combretastatin A-4 for this particular cell line.
In the case of the MCF-7 cell line, the lowest IC50 value, not very
different of that of combretastatin A-4, was shown by compound 6,
followed by ent-3. A further aspect also worth mentioning is the
2 3
Scheme 1. Synthesis of O-alkylated combretastatin A-4 derivatives 9e12: (a) K CO ,
DMF, 24 h, r.t. (yields: 9, 83%; 10, 82%; 11, 83%; 12, 88%). Acronyms and abbreviations:
DMF, N,N-dimethylformamide.

128
C. Vilanova et al. / European Journal of Medicinal Chemistry 87 (2014) 125e130
explanation for this fact, as we do not yet know whether these
compounds are interacting with tubulin at the pironetin site (
tubulin) or else at the combretastatin A-4 site ( -tubulin). The fact
a
-
b
that the IC50 values of the hybrid molecules are much more similar
to those of combretastatin A-4 than to those of pironetin suggests
that these compounds are possibly interacting with tubulin
through the combretastatin A-4 site at
would conclude that the reversible non-covalent interaction at the
-tubulin site is kinetically faster than the covalent irreversible
union at the -tubulin site. More research in this direction will be
b-tubulin. If this is true, one
b
a
necessary in order to definitively clarify this and other issues, such
as the possible influence of the triazole fragment [26].
5. Summary and conclusions
We have prepared a set of synthetic hybrid molecules contain-
ing a combretastatin A-4 moiety and a fragment structurally related
to the natural product pironetin. The two structural moieties have
been connected through a spacer of variable length containing a
1,2,3-triazole ring, generated in turn by means of a 1,3-dipolar
cycloaddition (“click reaction”). Some of these molecules have
been synthesized in both enantiomeric forms. Their cytotoxic ac-
tion (IC50 values) towards a normal (HEK-293) and two tumoral
(HT-29 and MCF-7) cell lines has then been measured. Several of
the synthetic derivatives proved cytotoxic towards at least one of
the two tumoral cell lines, with some of them showing cytotoxic
activities of the same order as combretastatin A-4. For some com-
pound/cell line combinations, most particularly 8 and the HT-29
cell line, the normal vs. tumoral cytotoxicity ratio was higher
than in the case of the aforementioned natural product, that is, they
proved comparatively much less cytotoxic towards normal cells
(these compounds are highlighted in Table 1). This feature may
potentially show pharmacological interest.
Scheme 2. Synthesis of azides 14, ent-14 and 15: (a) TsCl, Et
r.t. (21: 83%; ent-21: 84%; 23: 80%); (b) NaN , DMF, 3e4 h, r.t. (14: 85%; ent-14: 85%; 24,
2%); (c) 48% aq HF, MeCN, 4 h, r.t. (95%). Acronyms and abbreviations: Ts, p-tolue-
3 2 2
N, DMAP, CH Cl , 3e4 h,
3
8
nesulfonyl; DMAP, 4-(N,N-dimethylamino)pyridine; TBS, t-butyldimethylsilyl.
6
6
6
. Experimental
fact that some of the synthetic compounds are more toxic for one or
the other tumoral cell type than for normal cells, a desirable
.1. Chemistry
feature. This can be better appreciated with the
obtained by dividing the IC50 values of the normal cell line (HEK-
93) by those of one or the other tumoral cell line (see footnote in
the Table). The higher value of either the or the coefficient, the
a and b coefficients,
.1.1. General procedures
The general reaction conditions and the physical and spectral
2
data of all synthetic intermediates and final compounds are
described in detail in the Supporting Information. The samples of
compounds used for the biological studies were purified to >95% by
means of preparative HPLC.
a
b
higher the safety margin of the compound in the corresponding cell
line. Thus, combretastatin A-4 itself showed good values of both
coefficients, most particularly in the case of the MCF-7 cell line.
Pironetin, with IC50 values in the low nanomolar range, showed
similar values for the HT-29 and the MCF-7 cell lines. Among the
compounds with an appreciable cytotoxicity (low IC50 values), ent-
6.2. Biological studies
6
6
.2.1. Materials and methods
.2.1.1. Reagents and cell culture. Cell culture media were pur-
1, 6 and 8 are worth mentioning as they exhibit good a values, with
that of 8 being particularly high. Compound 6 also show a good
b
chased from Gibco (Grand Island, NY, USA). Fetal bovine serum
FBS) was a product of Harlan-Seralab (Belton, U.K.). Supplements
value.
(
Some aspects related to the relation between the structures and
the observed cytotoxicity deserve comment. In the case of two
subsets of hybrid molecules [20] where there are differences be-
tween in the carbon chain length (e.g. 1e4 and 5e8), differences in
the degree of cytotoxicity are also observed but no obvious relation
between both features are perceived. Thus, for compounds 1e4,
which display a very simplified pironetin moiety, the lowest IC50
values are found for the two compounds that have the intermediate
values of the spacer carbon chain length (2, n ¼ 7 and 3, n ¼ 10). As
for the compounds of the enantiomeric series (ent-1 to ent-4), their
cytotoxicities are not very different from those of 1e4. However, for
compounds 5e8, which display a more complex pironetin part, the
lowest IC50 value is clearly that of the compound with the longest
spacer carbon chain (8, n ¼ 15). It is difficult to propose now an
and other chemicals not listed in this section were obtained from
Sigma Chemicals Co. (St. Louis, Mo., USA). Plastics for cell culture
were supplied by Thermo Scientific™ BioLite. All tested compounds
were dissolved in DMSO at a concentration of 10
m
g/mL and stored
ꢁ
at ꢀ20 C until use.
Cell lines were maintained in Dulbecco's modified Eagle's me-
dium (DMEM) containing glucose (1 g/L), glutamine (2 mM),
penicillin (50 IU/mL), streptomycin (50
mg/mL) and amphoterycin
(
1.25 g/mL), supplemented with 10% FBS.
m
6.2.1.2. Cytotoxicity assays
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT; Sigma Chemical Co., St. Louis, MO) dye reduction assay
in 96-well microplates was used, as previously described [27].

C. Vilanova et al. / European Journal of Medicinal Chemistry 87 (2014) 125e130
129
Scheme 3. Synthesis of hybrid molecules 1 to 4, ent-1 to ent-4, and 5 to 8: (a) THF/MeOH, r.t, then addition of Cu powder and CuSO
ent-1: 50%; ent-2: 55%; ent-3: 58%; ent-4: 52%; 5: 55; 6: 58%; 7: 53%; 8: 55%).
4
·5H
2
O, r.t., 3e4 h (1: 46%; 2: 51%; 3: 51%; 4: 52%;
3
Some 5 ꢂ 10 cells of HT-29, MCF-7 or HEK-293 cells in a total
volume of 100 mL of their respective growth media were incubated
with serial dilutions of the tested compounds. After 3 days of in-
ꢁ
cubation (37 C, 5% CO
2
in a humid atmosphere), 10
ml of MTT
(
5 mg/ml in PBS) were added to each well and the plate was
Table 1
ꢁ
Cytotoxicity of pironetin analogue/combretastatin A-4 hybrids 1e8 and ent-1 to ent-
incubated for further 4 h (37 C). The resulting formazan was dis-
solved in 150 L of 0.04 N HCl/2-propanol and read at 550 nm. All
a
4
.
m
ab
bc
determinations were carried out in triplicate.
Compound HT-29
MCF-7
1 ± 0.2
HEK-293
CoA4
4.2 ± 0.5
25 ± 3
5.9
2.6
0.04 <0.1
25
2.8
Pironetin
0.0064 ± 0.0007 0.006 ± 0.002 0.017 ± 0.001
1
2
40 ± 2
8 ± 1
40 ± 0.3
39 ± 3
29 ± 6
66 ± 4
20 ± 5
32 ± 4
13 ± 3
37 ± 3
23 ± 6
7 ± 2
1.7 ± 0.3
9.3 ± 0.2
4.5 ± 0.3
16 ± 1
34 ± 10
40 ± 1
13 ± 3
2.6 ± 1.3
30 ± 5
1.2
0.5
0.7
3.7
3.6
0.7
0.3
1.2
2.6
2.3
<0.1
0.2
0.2
1.7
1.2
1
<0.1
1.3
3
3
9 ± 1
4
22 ± 3
Acknowledgments
ent-1
ent-2
ent-3
ent-4
5
9.3 ± 0.8
11 ± 1
17.5 ± 0.1
8 ± 3
24.0 ± 0.2
8 ± 1
Financial support has been granted by the Spanish Government
(MINECO, Ministerio de Economía y Competitividad, projects
CTQ2008-02800 and CTQ2011-27560), by the Consellería
d'Empresa, Universitat i Ciencia de la Generalitat Valenciana (pro-
jects PROMETEO/2013/027, ACOMP09/113 and ACOMP/2014/272)
and by the Universitat Jaume I (projects P1-1B-2008-14 and PI-1B-
6
21 ± 4
40 ± 5
32 ± 10
7
8
17.3 ± 0.4
0.40 ± 0.05
20 ± 3
63 ± 2
2
0.5
80
a
IC50 values, which include those of the parent compounds combretastatin A-4
mol/L or M)
2011-37). S.T.-G. thanks the Generalitat Valenciana for a predoc-
(
CoA4) and pironetin, are expressed as the compound concentration (
m
m
toral fellowship of the Santiago Grisolía program. C.V. thanks the
MINECO for a predoctoral fellowship of the FPI program. J.A.M.
thanks the COST Action CM0804 for aid in establishing collabora-
tions with other research groups. We further thank Prof. C.M. Cerda
García-Rojas, from the Departamento de Química, Centro de
Investigaci oꢀ n y de Estudios Avanzados del Instituto Polit eꢀ cnico
that causes 50% inhibition of cell growth. The values are the average (±s.d.) of three
different measurements performed as described in the Material and Methods
section.
b
a
¼ IC50 (HEK-293)/IC50 (HT-29).
c
b
¼ IC50 (HEK-293)/IC50 (MCF-7). Values of
a and b have been rounded off to a
decimal figure. Compounds with both high cytotoxicity towards one tumoral cell
line and comparatively low cytotoxicity towards the normal cell line have been
highlighted in boldface italics.
Nacional, Mexico, for the kind sending of pironetin samples.

130
C. Vilanova et al. / European Journal of Medicinal Chemistry 87 (2014) 125e130
Appendix A. Supplementary data
K. Takio, H. Watanabe, K. Nishikawa, T. Kitahara, H. Osada, Chem. Biol. 11
2004) 799e806.
(
[
15] For two recent reviews on chemical and pharmacological aspects of 5,6-
dihydropyran-2-ones, see: (a) J.A. Marco, M. Carda, J. Murga, E. Falomir, Tet-
rahedron 63 (2007) 2929e2958;
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.053.
(b) J.A. Marco, M. Carda, in: T. Janecki (Ed.), Natural Lactones and Lactams.
Synthesis, Occurrence and Biological Activity, Wiley-VCH, 2014, pp. 51e100.
16] (a) L.M. Rossi, P. Rangasamy, J. Zhang, X.-Q. Qiu, G.Y. Wu, J. Pharm. Sci. 97
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