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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].