Design and synthesis of pironetin analogues with simplified structure and study of their interactions with microtubules

Article abstract of DOI:10.1016/j.ejmech.2011.02.011

The preparation of a series of pironetin analogues with simplified structure is described. Their cytotoxic activity and their interactions with tubulin have been investigated. It has been found that, while less active than the parent molecule, the pironetin analogues still share the mechanism of action of the latter and compete for the same binding site to α-tubulin. Variations in the configurations of their stereocenters do not translate into relevant differences between biological activities.

Full text of DOI:10.1016/j.ejmech.2011.02.011

European Journal of Medicinal Chemistry 46 (2011) 1630e1637
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of pironetin analogues with simplified structure and study
of their interactions with microtubules
a
,
b
b
b
b
c
*
J. Alberto Marco , Jorge García-Pla , Miguel Carda , Juan Murga , Eva Falomir , Chiara Trigili ,
c
c
,
c,
**
*
*
Sara Notararigo , J. Fernando Díaz
, Isabel Barasoain
a
Depart. de Q. Orgánica, Univ. de Valencia, c/ D. Moliner 50, E-46100 Burjassot, Valencia, Spain
Depart. de Q. Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
CIB, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 October 2010
Received in revised form
The preparation of a series of pironetin analogues with simplified structure is described. Their cytotoxic
activity and their interactions with tubulin have been investigated. It has been found that, while less
active than the parent molecule, the pironetin analogues still share the mechanism of action of the latter
29 January 2011
and compete for the same binding site to
centers do not translate into relevant differences between biological activities.
Ó 2011 Elsevier Masson SAS. All rights reserved.
a-tubulin. Variations in the configurations of their stereo-
Accepted 8 February 2011
Available online 22 February 2011
Keywords:
Tubulin
Tubulin-active compound
Microtubule
Microtubule destabilization
Pironetin analogue
Cytotoxicity
1
. Introduction
bind to any of the tubulin components, it is not surprising that
tubulin-binding molecules (TBM) constitute a most important class
of anticancer agents [4]. TBM are able to interfere with microtubule
assembly and functions, either by causing disruption of the
microtubules or else through their stabilization. In both cases, this
results in mitotic arrest of eukaryotic cells and subsequent cell
death. Most of the hitherto described active drugs are natural
products or derivatives thereof [5]. Major drugs can already be
found on the market and many other promising compounds are in
clinical trials [4,5].
Microtubules are dynamic polymers which play a central role in
a number of cellular processes, most particularly cell division, as
they are key constituents of the mitotic spindle [1]. Their shape can
be described as hollow tubes of about 25 nm external diameter
constituted of a protein named tubulin. The functional form of this
protein is a heterodimer formed in turn through non-covalent
binding of two monomeric constituents. These are two very similar
polypeptides of about 450 amino acid residues which are called
a-
and -tubulin [2]. For cell division to occur in a normal way,
b
TBM may be divided in two broad categories, those that bind to
microtubules must be in a constant state of formation and
disruption, a process named microtubule dynamics in which GTP
hydrolysis into GDP plays a key role [3].
It is easy to understand that any molecule which exerts some
type of action on microtubule dynamics will be able to influence
the cell division process, not only of normal cells but also of tumoral
ones. Since such an influence may be exerted by molecules that
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 venerable alkaloid colchicine
[6] exerts its effects by causing disruption of microtubules. In
contrast, another renowned representative of the same group,
paclitaxel, was the first-described tubulin-interacting drug that
was found to stabilize microtubules [7]. In spite of the fact that they
exert opposite effects on the mitotic spindle, both drugs are known
*
Corresponding author. Tel.: þ34 96 3544337.
to bind to
b-tubulin, even though to different sites within this
*
* Corresponding authors.
protein. The mechanisms of action [8] of many of these TBM and the
E-mail addresses: alberto.marco@uv.es (J.A. Marco), fer@cib.csic.es (J.F. Díaz),
i.barasoain@cib.csic.es (I. Barasoain).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.011

J.A. Marco et al. / European Journal of Medicinal Chemistry 46 (2011) 1630e1637
1631
molecular aspects [9] of their interactions with tubulin have been
studied using a broad palette of methods [10].
are essential for its activity and, desirably, at achieving an
improvement of the latter.
The number of products that bind to
a-tubulin is very small, the
naturally occurring 5,6-dihydro- -pyrone pironetin (Fig. 1) being
a
2. Concept and design of analogues
the first-reported example [11]. The compound is a potent inhibitor
of tubulin assembly and has been found to arrest cell cycle
progression in the G2/M phase [12]. This feature has motivated
a number of groups to undertake total syntheses of this natural
compound [13]. Some synthetic and biological studies on modified
variants of pironetin have recently been published [14].
Some structure-activity (SAR) studies on pironetin have been
reported [12]. These studies have shown that the presence of the
conjugated C2ꢀC3 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 also seems to be very rele-
vant [12c]. The epoxidation of the C12eC13 double bond has been
shown to cause a decrease in the activity [12a,b] but this may
perhaps involve a deleterious effect of the oxirane ring, rather than
the necessity of this C]C bond. No data are available about the
importance of the remaining structural features [14]. It has been
In order to develop SAR studies based on the pironetin frame-
work, we have considered a very simplified, starting structure
where all elements that have not yet proven to be essential have
been removed. In the initial phase of our research, the elements
that have been maintained are, as commented above, the conju-
gated pyrone ring and the methoxy group at C-9. The hydroxyl
group at C-7 has been removed in some substrates and retained in
others, in order to see the degree of its influence on the activity. The
configurations of the stereocenters have also been varied. All alkyl
pendants (methyl, ethyl) and the isolated C12ꢀC13 double bond
have been removed. Accordingly, the selected target structures are
shown in Chart 1. From these, compounds 1e4, which lack the
hydroxyl group at C-7, are all four possible stereoisomers with this
constitution (two racemates, 1/3 and 2/4). Likewise, structures
5e12, where the C-7 hydroxyl is present, represent all eight
possible stereoisomers with such a constitution (four racemates,
5/11, 6/12, 7/10 and 8/9).
proposed that the Lys352 residue of the a-tubulin chain adds in
a Michael fashion to the conjugated double bond of pironetin,
therefore forming a covalent bond with C-3 of the pyrone ring
(
a
Fig.1). In addition, it has been suggested that the Asn258 residue of
-tubulin holds the pironetin molecule through two hydrogen
3. Synthetic work
bonds to the pyrone carbonyl and the methoxyl oxygen atoms [12].
The appearance 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 [15], resistances have also been reported to TBM
The synthesis of pironetin analogues 1e4 (Chart 1) was per-
formed according to the general concept depicted with detail in
Scheme 1 for the case of compound 1. Brown’s asymmetric allyla-
tion [17] of n-hexanal afforded homoallyl alcohol 13 [17c,d]. The
required chiral allylborane was prepared through reaction of
allylmagnesium bromide with the commercially available (þ)-dii-
[4c,e,h,16]. The investigation of new members of this compound
class therefore constitutes an important goal in chemistry and
pharmacology. As a member of the up to now small group of
sopinocampheylboron chloride, (þ)-Ipc BCl. Methylation of the
2
free hydroxyl group of compound 13 yielded methyl ether 14 [18].
products that bind to
a-tubulin, pironetin constitutes a pharmaco-
Ozonolytic cleavage of the olefinic bond in 14 was followed by
Wadsworth-Emmons olefination of the intermediate aldehyde 15
under mild conditions [19] to yield the conjugate ester 16. Hydro-
genation of the olefinic bond and reduction of the ester function
gave primary alcohol 18, which was then oxidized to the corre-
sponding aldehyde. The latter was then subjected to the same
asymmetric allylation conditions as the starting n-hexanal to yield
homoallyl 19 as a 86:14 mixture of diastereomers. Reaction of 19
with acryloyl chloride at low temperature gave acrylate 20, which
logically interesting target. Thus, the purpose of our present
research is the preparation of pironetin analogues that retain
a substantial proportion of the biological activity of the natural
metabolite while displaying a more simplified structure. Indeed,
3
pironetin is not an extremely complex molecule but, with six sp
stereocenters, a total synthesis will be anyway lengthy enough as to
make not too practical a preparation at large scale. Our investiga-
tion aims at establishing which elements of the pironetin molecule
Fig. 1. (a) Structure of pironetin. (b) Schematic model of the covalent union of pironetin to its binding site at the a-tubulin surface.

1632
J.A. Marco et al. / European Journal of Medicinal Chemistry 46 (2011) 1630e1637
reaction. Reaction of 23 with acryloyl chloride at low temperature
gave acrylate 24, which was subjected to RCM in the presence of
Grubbs first-generation catalyst Ru-I to furnish dihydropyrone 25
in good yield. Acid-catalyzed cleavage of the silyl group afforded
pironetin analogue 5.
Pyrones 6e12 were prepared along the same sequence of
reactions by using in each case the appropriate chiral allylborane,
either (þ)- or (ꢀ)-Ipc
2
BCl. Yields and stereoisomeric ratios were
very similar to those observed in 5 (for experimental details, see the
Supporting information).
After the synthesis of pironetin analogues 1e12 was completed,
they were investigated in relation to their cytotoxic activity
towards two types of tumoral lines and to their interactions as
ligands with tubulin, always in comparison with the parental
molecule, pironetin.
4. Results and discussion
4.1. Cellular effects of the compounds
In order to check whether the newly synthesized compounds
were cytotoxic through a mechanism similar to that of pironetin,
we determined the IC50 values for all simplified analogs 1e12 and
compared them with that of pironetin on A2780 and A2780AD
human ovary carcinomas (Table 1). Pironetin was found active
(IC50 3 nM) in both the parental and the resistant cell lines.
Furthermore, it has been found more cytotoxic than pironetin
3
analogues 1e12 by 5e18 ꢁ 10 times in the case of A2780 cells and
3
by 3e16 ꢁ 10 times in A2780AD cells. Indeed, and according to
IC50 values, pironetin is active at concentrations in the nanomolar
range whereas the pironetin analogues are active in the micromolar
range. The latter killed both resistant and non resistant cells with
a similar IC50 in the same way as pironetin, as expected for
compounds with a covalent mechanism of action [21].
In order to establish whether compounds 1e12 act as microtu-
bule depolymerizers in the same way as pironetin [12a,b], we
studied the effect of these ligands on the microtubule cytoskeleton.
We incubated cells in the presence of these ligands for 4 h (Fig. 2) or
for 24 h (Figure A in the Supporting information). Pironetin at
5
0 nM concentration completely depleted cellular microtubules (C,
D). In comparison, compounds 8 (E, F), 1 (G, H), 4 (I, J) and 3 (K, L)
Fig. 2) and 2 (Figure A in the Supporting information) were also
active in microtubule depolymerization but at 200 M concentra-
(
Chart 1. Structures of simplified pironetin analogues.
m
3
tion, i.e. 4 ꢁ 10 times less active than pironetin. In the prepara-
tions, we observed metaphase mitotic cells with type III mitotic
spindles [22], with the DNA forming a ring surrounding a mono-
aster of microtubules, as with pironetin. With 24 h incubation and
was subjected to ring-closing metathesis (RCM) [20] in the pres-
ence of Grubbs first-generation catalyst Ru-I. This provided dihy-
dropyrone 1 in high yield.
200 mM concentration of 5, 6, 7, 9, 10, 11 and 12 (Figure A in the
Pyrones 2e4 were prepared along the same sequence of reac-
tions by using in each case the appropriate chiral allylborane, either
Supporting information), the microtubule cytoskeleton was also
disorganized and depolymerized as with compounds 1, 2, 3, 4 and
8. Aberrant mitosis was also present in all preparations.
(
þ)- or (ꢀ)-Ipc
2
BCl. Yields and stereoisomeric ratios were very
similar to those observed in 1 (for experimental details, see the
Supporting information).
We next studied whether ligands 1e12 were capable of blocking
cells in the G2/M phase of the cell cycle of A549 (Fig. 3), as other
microtubule modulating agents do. We incubated these cells for
20 h in the presence of the different ligands (1e12) or the drug
vehicle. Pironetin at 50 nM concentration almost completely
arrested cells in the G2/M phase. In the case of compounds 1e12, an
increase in the number of cells in this phase with respect to the
control cells was observed. This effect was more pronounced with
compounds 5e12, the ligands where the C-7 hydroxyl is present
(Fig. 3). At higher concentrations of the ligands, however, we did
not see an increase in the number of cells arrested in the G2/M
phase but a great cytotoxic effect.
Pironetin analogues 5e12 (Chart 1) were prepared using the
same synthetic concept. Scheme 2 illustrates this with detail in the
case of pyrone 5. Aldehyde 15 (Scheme 1) was subjected to asym-
metric allylation with the chiral allylborane derived from
2
(þ)-Ipc BCl to yield homoallyl alcohol 21, obtained as a 92:8
mixture of diastereoisomers. Silylation to 22 was followed by
ozonolytic cleavage of the C]C bond. Without purification, the
intermediate aldehyde was submitted to asymmetric allylation
using again the same chiral allylborane as before. This provided
homoallyl alcohol 23 as a single diastereoisomer. This indicates that
the minor stereoisomer was removed after this step, either during
the chromatographic separation or via a kinetic resolution in the
We now conclude that, even if these pironetin analogues are less
cytotoxic than pironetin itself, they also perturb the microtubular

J.A. Marco et al. / European Journal of Medicinal Chemistry 46 (2011) 1630e1637
1633
ꢃ
ꢃ
Scheme 1. Synthesis of pironetin analogues 1e4. (a) (þ)-Ipc
2
BCl, allylMgBr, Et
NEt, (EtO) P(O)CH
2
O, ꢀ78 C, 1 h, then addition of n-hexanal, 1 h, ꢀ78 ꢃC, 95% (e.r. 91:9); (b) NaH, THF, 0 C, then MeI,
ꢃ
RT, overnight; (c) O
3
, CH
2
Cl
2
, ꢀ78 C, then PPh
3
; (d) LiCl, iPr
2
2
2
CO
2
Et, MeCN, RT, overnight (60% overall from 13); (e) H
2
, Pd/C, EtOH, RT (quant.); (f) DIBAL, hexane,
ꢃ
ꢃ
O, ꢀ78 ꢃC, 1 h, followed by addition of the aldehyde, 1 h, ꢀ78 C (55% overall from 18, d.r. 86:14); (h) CH
ꢃ
ꢀ
78 Ce0 C (98%); (g) Swern oxidation, then (þ)-Ipc
2
BCl, allylMgBr, Et
NEt, ꢀ78 C, 45 min (90%, d.r. 86:14); (i) 10% cat. Ru-I, CH
2
2
]
ꢃ
CHCOCl, CH
2
Cl
2
, iPr
2
2 2
Cl , D, 2 h (90%, d.r. 86:14). Acronyms and abbreviations: Ipc, isopinocampheyl; DIBAL,
diisobutylaluminum hydride.
network. Analogues 1, 2, 5, 6, 7, 8, 9, 10, 11 and 12 accumulate cells
in the G2/M phase but no apoptotic cells were observed, even with
pironetin itself. Furthermore, they induce aberrant mitosis and give
rise to a depolymerized microtubule cytoskeleton. Analogues 3 and
shown by compounds 1e4 lacking the hydroxyl group at C-7 (Cr for
1, 3.73 ꢂ 0.35 M; Cr for 2, 3.75 ꢂ 0.11 M; Cr for 3, 3.79 ꢂ 0.10 M;
Cr for 4, 3.85 ꢂ 0.16 M), whereas the weakest compounds were 9
(Cr, 3.39 ꢂ 0.07 M). For the sake of
m
m
m
m
m
M) and 12 (Cr, 3.44 ꢂ 0.03
m
4
also induce aberrant mitosis and cause microtubule cytoskeleton
comparison, values for docetaxel (a microtubule-stabilizing agent)
and pironetin under these conditions are, respectively,
depolymerization but are less effective in accumulating cells in the
G2/M phase of the cell cycle.
0.40 ꢂ 0.12
mM and >15 mM. The values measured for analogues
1e12 confirm that the observed effect in cells is due to their
interaction with tubulin. Moreover, the observed in vitro effect
correlates well with the measured cytotoxicities.
4.2. Inhibition of microtubule assembly by pironetin analogues 1e12
The observation of a weak microtubule assembly inhibition
activity in cells by compounds 1e12 led us to study their ability to
inhibit purified tubulin microtubule assembly. This aims at ascer-
taining whether the observed activity is exerted by means of
tubulin-binding in a pironetin-like way. With this purpose in mind,
the critical concentration [23] required for tubulin assembly was
4.3. Competition between pironetin and analogues as to their
binding ability to tubulin
When a ligand does not show a visible change in a measurable
property (e.g. absorbance or fluorescence) upon binding to
a protein, the accurate characterization of such an interaction may
become a complex task. In those cases it is possible to observe the
existence of the aforementioned interaction by means of compe-
tition of the ligand under study with a reference ligand for the same
binding site. When such a competition actually takes place, the
determined in GAB in the presence of a large excess (100
pironetin analogues 1e12. Indeed, the concentration of tubulin
required to produce assembly raises from 3.30 ꢂ 0.10 M in the
absence of the pironetin analogues (DMSO vehicle) to a maximum
value of 3.85 ꢂ 0.16 M in their presence. The highest activity was
mM) of
m
m

1634
J.A. Marco et al. / European Journal of Medicinal Chemistry 46 (2011) 1630e1637
a-tubulin ligand [12d], is the reference ligand. In order to confirm
that compounds 1e12 share with the parent molecule the same
binding site in tubulin, competition experiments between pir-
onetin and some of its analogues were performed. As commented
above, pironetin and, expectedly, also each one of ligands 1e12
become covalently attached to tubulin (Fig. 1). Assuming that
a percentage of the pironetin binding sites is blocked by the ligand,
it may be expected that a proportion of pironetin remains
unreacted (i.e. not covalently bound) and can thus be measured by
means of extraction with an organic solvent (for details, see
Experimental, Section 6.2.4).
Fig. 4 shows that, in the absence of tubulin (Control), all pir-
onetin (30
presence of the vehicle (DMSO), 23.4
bound to tubulin since only 6.6 M can be extracted by the organic
m
M) remains unreacted in the liquid phase. In the
m
M of pironetin is covalently
m
solvent. When the microtubules were previously incubated with
the analogues, a significant percentage of the pironetin binding
sites get blocked. As for the tubulin assembly inhibition, the
compounds lacking the hydroxyl at C7 are the most powerful. In the
presence of compounds 2, 3 and 4, nearly 10
binding sites are protected and approximately 15
remains unreacted. In the presence of compounds 6, 9 and 10
approximately 4 M of the pironetin binding sites remain protected
and approximately 10 M pironetin remains unreacted. Thus,
mM of the pironetin
mM pironetin
m
m
concentrations of pironetin analogues 2, 3, 4, 6, 9 and 10 similar to
those required to kill tumoral cells result in an increase of the
critical concentration of purified tubulin required for the assembly,
thus indicating that tubulin is actually their cellular target. The high
concentrations of the analogues required to block the pironetin
Scheme 2. Synthesis of pironetin analogues 5e12. (a) (þ)-Ipc
2
BCl, allylMgBr, Et
Cl , 2,6-
BCl,
2
O,
ꢃ
ꢃ
ꢀ
78 C, 1 h, then addition of aldehyde, 1 h, ꢀ78 C (d.r. 92:8); (b) TBSOTf, CH
2
2
ꢃ
ꢃ
lutidine, 0 C, 1 h (75% from 15); (c) O
3
, CH
2
Cl
2
, ꢀ78 C, then PPh
3
; (d) (þ)-Ipc
2
ꢃ
ꢃ
allylMgBr, Et
2
O, ꢀ78 C, 1 h, followed by addition of the aldehyde, 1 h, ꢀ78 C (90%,
ꢃ
d.r. > 95:5); (e) CH
CH Cl
2
]CHCOCl, CH
2
Cl
2
, iPr
2
NEt, ꢀ78 C, 45 min (90%); (f) 10% cat. Ru-I,
2
2
,
D
, 2 h (86%); (g) PPTS (cat.), MeOH,
D
, overnight (69%). Acronyms and abbre-
binding site in
a-tubulin indicate that they compete with pironetin
viations: TBS, tert-butyldimethylsilyl; Tf, trifluoromethanesulfonyl; PPTS, pyridinium
p-toluenesulfonate.
for the same binding site, even though with a lower affinity.
5
. Conclusion
attachment of the reference ligand to the binding site becomes
perturbed by the presence of other ligands, whereby it may be
presumed that they are competing for the same binding site [24].
This is a widely used technique in many situations, including the
case of tubulin, which exhibits multiple binding sites, both acti-
vating and deactivating ones [25e28].
As a general conclusion, it is worth highlighting that the
synthesized pironetin analogues still retain a measurable propor-
tion of the cytotoxicity of the natural product in spite of the deep-
seated simplifying modifications performed on the structure of the
natural product. Most importantly, they share the mechanism of
action of the latter and compete with it for same binding site. From
the various biochemical and biological data acquired, it seems that
the configurations of the stereocenters do not seem to exert an
outstanding influence on the cytotoxicity and on the ability of the
In the present investigation, the ligands under study are pir-
onetin analogues 1e12, whereas pironetin, a well-characterized
Table 1
compounds to bind to a-tubulin. Indeed, in the different assays
Cytotoxicity of pironetin analogues 1e12 in ovarian carcinoma cells sensitive
performed the stereoisomeric analogues exhibit figures of the same
order of magnitude. In contrast with that observed with pironetin
itself, where the presence of a hydroxyl group at C-7 was found to
be important [12], the removal of this hydroxyl group (compounds
1e4) does not cause a suppression of the cytotoxicity or of the
ability to depolymerize microtubules. However, these compounds
are less effective in accumulating cells in the G2/M phase when
compared with those having the C-7 hydroxy group. All these
aspects will be taken into account when planning and designing the
structures of the next generation of pironetin analogues. Reports in
this sense will be published in due course.
(
A2780) and resistant (A2780AD) to chemotherapy by P-glycoprotein
a
overexpression .
Drug
A2780 (
m
M)b
A2780AD (
m
M)
R/Sc
Taxol
Pironetin
0.00082 ꢂ 0.0002
0.0029 ꢂ 0.001
20.6 ꢂ 2.4
14.9 ꢂ 1.3
14.4 ꢂ 3.2
17.1 ꢂ 5
0.949 ꢂ 0.38
0.003 ꢂ 0.0002
9 ꢂ 3.2
1157
1
1
2
3
4
5
6
7
8
9
0.45
11.5 ꢂ 1.3
20.49 ꢂ 5
12.2 ꢂ 1.9
48 ꢂ 0.01
26.3 ꢂ 4
0.77
1.42
0.7
0.89
0.74
0.74
0.93
1.3
53.7 ꢂ 3
35.4 ꢂ 6.3
33.7 ꢂ 9
25.2 ꢂ 0.86
46.3 ꢂ 6.8
57.5 ꢂ 0.35
30 ꢂ 1.3
49.4 ꢂ 3.4
44 ꢂ 2.8
6. Experimental
10
11
12
22.9 ꢂ 4.4
54.5 ꢂ 6
1.3
1.67
0.62
91.5 ꢂ 23.6
18.1 ꢂ 3.2
6.1. Chemistry. General procedures
28.9 ꢂ 7
a
IC50 (50% inhibition of cell proliferation) of the ligands determined in ovarian
The general reaction conditions and the physical and spectral
carcinomas.
data of all synthetic intermediates and final compounds are
described in detail in the Supporting Information. The samples of
compounds 1e12 used for the biological studies were purified to
>95% by means of preparative HPLC.
b
IC50 values (
m
M) are the mean ꢂ standard error of two independent experiments
done in duplicate.
c
The relative resistance of A2780AD cell line, obtained dividing the IC50 of the
resistant cell line by the IC50 of the parental A2780 cell line.

J.A. Marco et al. / European Journal of Medicinal Chemistry 46 (2011) 1630e1637
1635
Fig. 2. Effect of pironetin analogues 1, 3, 4 and 8 as compared to the parent molecule pironetin on the microtubule network and on nucleus morphology. A549 cells were incubated
for 4 h with either drug vehicle DMSO (A, B), 50 nM pironetin (C, D), 200 M 8 (E, F), 200 M 1 (G, H), 200 M 4 (I, J) and 200 M 3 (K, L). Microtubules are stained with -tubulin
antibodies (A, C, E, G, I, K) whereas DNA (B, D, F, H, J, L) was stained with Hoechst 33342. Insets (A, B, C, D, E, F, I, J) are mitotic spindles from the same preparation. The scale bar (L)
represents 10 m. All panels and insets have the same magnification.
m
m
m
m
a
m
6
6
.2. Biological studies. Materials and methods
cultured as above with the addition of 0.25 units/mL of bovine
insulin.
.2.1. Cell culture
Human A549 non small lung carcinoma cells were cultured in
6.2.2. Cytotoxicity assays, indirect immunofluorescence and cell
cycle
Cytotoxic evaluation was performed with A2780 and A2780AD
cells with the MTT assay modified as previously described in Ref.
RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotics
as previously described in Ref. [21]. Human ovarian carcinomas
A2780 and A2780AD (MDR overexpressing P-glycoprotein) were
Fig. 3. Cell cycle histograms of A549 lung carcinoma cells untreated or treated with pironetin analogues 1e12. The lowest ligand concentration that induces maximal arrest in the
G2/M phase is depicted.

1636
J.A. Marco et al. / European Journal of Medicinal Chemistry 46 (2011) 1630e1637
reacted pironetin was then determined by means of HPLC in an
Agilent 1100 series instrument. Samples containing pironetin and
compounds 6, 9 and 10 were analyzed using a Zorbax Eclipse XDB-
C18 column (4.6 ꢁ 150 mm, 5 micron). Elution was performed with
a gradient of 50e80% acetonitrile in water at a flow rate of 1 mL/min
for 30 min, with detection being followed through the UV absor-
bance at
l
¼ 230 nm. Samples containing pironetin and compounds
2, 3 and 4 were analyzed using two sequentially connected columns,
a Zorbax Eclipse XDB-C18 (4.6 ꢁ 150 mm, 5 micron) column and
a Supercosil LC18DB (4.6 ꢁ 250 mm, 5 micron) column. Elution was
performed with a gradient of 60e80% acetonitrile in water at a flow
rate of 1 mL/min for 30 min. As above, detection was followed
through the UV absorbance at
l
¼ 230 nm.
Acknowledgements
Financial support has been granted by the Spanish Ministry of
Education and Science (CTQ2008-02800), by the Consellería
d’Empresa, Universitat i Ciencia de la Generalitat Valenciana
Fig. 4. Protection of the pironetin binding site by 200
analogues 2, 3, 4, 6, 9 and 10. Concentration of pironetin extracted with CH
the samples after 15 min of incubation of 30 M pironetin with tubulin at 37
control means the absence of tubulin in the buffer). Data represent average values of
three measurements, errors are the standard errors of the average.
m
M concentrations of pironetin
(ACOMP09/113) and by the BANCAJA-UJI Foundation (P1-1B2002-
2
Cl from
2
ꢃ
06 and P1-1B-2008-14). The biological work has been supported in
part by grants from the Spanish Ministry of Education and Science
m
C
(
(BIO2007-61336 and BIO2010-16351) and from the Comunidad de
Madrid (BIPPED-CM) (both to J.F.D.). We further thank the Mata-
dero Municipal Vicente de Lucas in Segovia for providing the calf
brains which were the source of tubulin.
[
29]. Indirect immunofluorescence was performed in A549 cells
that had been cultured overnight in 12 mm round coverslips and
incubated for further 24 h in the absence (drug vehicle DMSO) or in
the presence of different ligand concentrations. Attached cells were
permeabilized with Triton X100 and fixed with 3.7% formaldehyde.
Appendix. Supplementary information
Microtubules were specifically stained with DM1A
a
-tubulin
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.011.
monoclonal antibodies and DNA with Hoechst 33342 as previously
described in Ref. [30]. The preparations were examined using
a Zeiss axioplan epifluorescence microscope and the images were
recorded in a Hamamatsu 4742-95 cooled CCD camera. Progression
through the cell cycle analysis was assessed by flow cytometry DNA
determination with propidium iodide. Cells were fixed, treated
with RNase and stained with propidium iodide as previously
described in Ref. [31]. Analysis was performed with a Coulter Epics
XL flow cytometer.
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