Synthesis and biological evaluation of α-tubulin-binding pironetin analogues with enhanced lipophilicity

Article abstract of DOI:10.1002/ejoc.201201283

Four new lipophilic analogues of the natural pyrone pironetin have been prepared. The C₉ side-chain of the latter has been replaced in one analogue by a 4-phenylbutyl chain and in the other three analogues by C ₁₃ or C₁₆ a

Full text of DOI:10.1002/ejoc.201201283

FULL PAPER
DOI: 10.1002/ejoc.201201283
Synthesis and Biological Evaluation of α-Tubulin-Binding Pironetin Analogues
with Enhanced Lipophilicity
Miguel Carda,*[a] Juan Murga,[a] Santiago Díaz-Oltra,[a] Jorge García-Pla,[a]
Julián Paños,[a] Eva Falomir,[a] Chiara Trigili,[b] J. Fernando Díaz,*[b] Isabel Barasoain,*[b]
and J. Alberto Marco*[c]
Keywords: Medicinal chemistry / Antitumor agents / Synthesis design / Proteins / Biological activity
Four new lipophilic analogues of the natural pyrone pirone-
tin have been prepared. The C9 side-chain of the latter has
been replaced in one analogue by a 4-phenylbutyl chain and
in the other three analogues by C13 or C16 aliphatic chains,
all of them bearing two stereogenic centres. Their cytotoxic
activities and interactions with tubulin have been investi-
gated. It was found that all four are cytotoxic towards two
either sensitive or resistant tumoral cell lines with similar
IC50 values in each case, which indicates that, like the parent
natural compound, they also display a covalent mechanism
of action. However, one of them operates in all likelihood
through a mechanism very similar to pironetin, whereas the
other three seem to operate through a different mechanism.
to influence the cell division process, not only of normal
cells, but also of tumoral cells. Because such an influence
may be exerted by molecules that bind to any of the tubulin
components, it is not surprising that tubulin-binding mole-
cules (TBMs) are a very important class of anticancer
agents.[4] TBMs are able to interfere with microtubule as-
sembly and functions, either by causing rupture of the
microtubules or through their stabilization. In both cases,
this results in mitotic arrest of eukaryotic cells and subse-
quent 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]
Introduction
Microtubules are dynamic polymers that play a central
role in a number of cellular processes, in particular, cell divi-
sion, as they are key constituents of the mitotic spindle.[1]
They can be described as hollow tubes with an external dia-
meter of about 25 nm made of a protein named tubulin.
The functional form of this protein is a heterodimer formed
by the non-covalent binding of two monomeric species,
namely two structurally related polypeptides of about 450
amino acid residues called α- and β-tubulin.[2] For cell divi-
sion to occur in a normal way, microtubules must be in a
constant state of assembly and disassembly, a process
named microtubule dynamics in which the hydrolysis of
GTP into GDP plays a key role.[3]
TBMs may be divided into two broad categories, those
that bind to α-tubulin and those that bind to β-tubulin. The
latter group is presently by far the most numerous and con-
tains compounds that cause either rupture or stabilization
of microtubules. Among the drugs that belong to this
It is easy to understand why any molecule that exerts
some type of action on microtubule dynamics will be able
[a] Departamento de Química Inorgánica y Orgánica, Universitat group, the well-known colchicine[6] exerts its effects by caus-
Jaume I,
ing rupture to the microtubules. In contrast, another re-
12071 Castellón, Spain
nowned representative of the same group, paclitaxel, was
the first-described tubulin-interacting drug with the ability
to stabilize microtubules.[7] Despite the fact that they exert
opposite effects on the mitotic spindle, both drugs are
known to bind to β-tubulin, although to different sites
within the protein subunit. The mechanisms of action[8] of
many of these TBMs and the molecular aspects[9] of their
interactions with tubulin have been studied by a broad
range of methods.[10]
Fax: +34-964-728214
E-mail: mcarda@qio.uji.es
Homepage: www.sinorg.uji.es
[b] Centro de Investigaciones Biológicas, Consejo Superior de
Investigaciones Científicas,
28040 Madrid, Spain
E-mail: i.barasoain@cib.csic.es
fer@cib.csic.es
Homepage: www.cib.csic.es
[c] Departamento de Química Orgánica, Universidad de Valencia,
c/Dr. Moliner, 50, 46100 Burjassot, Spain
Fax: +34-96-3544328
E-mail: alberto.marco@uv.es
Homepage: www.uv.es
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201283.
The number of compounds reported to bind to α-tubulin
is very small, the naturally occurring 5,6-dihydro-α-pyrone
pironetin (Figure 1) being the first example,[11] followed a
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Biological Evaluation of α-Tubulin-Binding Pironetin Analogues
short time later by the peptide-like hemiasterlin family.[12]
Pironetin proved a potent inhibitor of tubulin assembly and
was found to arrest cell cycle progression in the G2/M
phase.[13] This feature has motivated a number of groups to
undertake total syntheses of this natural compound[14] and
some synthetic and biological studies on modified variants
of pironetin have previously been published.[15]
Figure 1. Structures of two natural products reported to selectively
bind to α-tubulin.
Some structure–activity (SAR) studies on pironetin have
also been reported.[13] These studies have shown that the
presence of the conjugated C2–C3 double bond and of the
hydroxy group at C-9, either free or methylated, are essen-
tial for biological activity. The presence of a (7R)-hydroxy
group also seems to be very important.[13c] Epoxidation of
the C12–C13 double bond has been shown to cause a de-
crease in activity,[13a,13b] but this may be a result of a delete-
rious effect of the oxirane ring, rather than a strict need for
this C=C bond. No data are available on the importance of
the remaining structural features.[15] It has been proposed
that the Lys352 residue of the α-tubulin chain undergoes a
Michael-type addition to the conjugated double bond of
the pyrone ring of pironetin to form a covalent bond with
C-3 (Figure 2). In addition, it has been suggested that the
Asn258 residue of α-tubulin fixes the pironetin molecule
through two hydrogen bonds with the pyrone carbonyl and
the methoxy oxygen atom.[13]
The occurrence of resistance to existing drugs has led to
a continuous need for the development of new bioactive
compounds that overcome such problems. Although first
observed for antibiotics, resistance has also been reported
for TBMs.[4c,4e,4h,16] The synthesis of new members of this
class of compounds therefore is an important goal in chem-
istry and pharmacology. As a member of the up-to-now
small group of compounds that bind to α-tubulin, pironetin
is a pharmacologically interesting target. Thus, the purpose
of our current research is the preparation of pironetin ana-
logues that retain a substantial proportion of the biological
activity of the natural metabolite but that have a more sim-
plified structure. Indeed, pironetin is not an extremely com-
plex molecule but, with six sp3 stereocentres, a total synthe-
Figure 2. Schematic model of the covalent union of pironetin with
its binding site at the α-tubulin surface.
at establishing which elements of the pironetin molecule are
essential for its activity and, desirably, at improving this ac-
tivity.
For SAR studies based upon the pironetin framework,
we started by considering a simplified model structure in
which all elements that have not yet proven to be essential
were removed. The elements that remained were the conju-
gated pyrone ring and the side-chain with the methoxy
group at C-9. The hydroxy group at C-7 was removed in
some substrates and retained in others to investigate its in-
fluence on the activity. All alkyl pendants (methyls at C-8
and C-10, ethyl at C-4) and the isolated C12–C13 double
bond were removed. The configurations of the three re-
maining stereocentres were varied systematically. Accord-
ingly, the selected target structures are schematically shown
in Figure 3. All four possible stereoisomers of 1, with no
hydroxy group at C-7, were prepared. In addition, all eight
stereoisomers of 2, with a hydroxy group at C-7, were syn-
thesized.[17]
Figure 3. General structures of the simplified pironetin analogues
1 and 2.[17]
The cytotoxic activities of these analogues and their in-
sis will be sufficiently lengthy that it is not very practical teractions with tubulin were subsequently investigated. To
for its preparation on a large scale. Our investigation aims measure the cytotoxic activities, ovarian carcinoma cells
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FULL PAPER
sensitive (A2780) and resistant (A2780AD) to chemother- structure 2.[17] Scheme 1 shows the details of the synthetic
apy by P-glycoprotein overexpression were used.[17] It was route to pyrone 3 in which n-decanal was the starting mate-
found on the one hand that the analogues are cytotoxic in rial.
the low micromolar range, that is, they are about three or-
ders of magnitude less active than the parent molecule.[17]
On the other hand, they behaved in the same way as pirone-
tin in that they killed both resistant and non-resistant cells
with a similar IC50, as expected for compounds with a cova-
lent mechanism of action.[13] The general conclusion was
that the synthetic pironetin analogues share the same
mechanism of action as the natural compound and compete
for the same binding site to α-tubulin, leading to rupture of
the microtubule network. It is worth mentioning that varia-
tions in the configurations of the three stereocentres (C-5,
C-7, C-9) does not translate into significant differences in
biological activity.[17]
In continuation of our efforts in this line of research, we
have now investigated the influence of the nature and size
of the lipophilic side-chain attached to the dihydropyrone
ring on the biological properties. It is our ongoing aim to
prepare tubulin-active molecules with a hybrid structure
and the ability to bind to two different points in the tubulin
network. Because one half of these hybrid molecules will be
pironetin-like, knowledge about the impact of the nature
and size of the required spacer fragment is of paramount
importance. In line with this reasoning, we have prepared
the four pironetin analogues 3–6 (Figure 4). As regards the
stereocentres, and in view of the aforementioned fact that
their configurations do not seem to have a significant effect
on their biological activity, compounds 3–6 were prepared
with the same configuration as natural pironetin at C-5, C-
7 and C-9. The difference between these compounds and
the previously reported analogues 1 and 2 resides in the
lipophilic end of the side-chain, which is much longer in 3
and 4, compound 5 is an O-methylated analogue of 4 and
compound 6 contains a phenyl ring instead of the aliphatic
chain.
Scheme 1. Synthesis of pyrones 3–6. Reagents and conditions:
(a) (–)-Ipc2BCl, allylMgBr, Et2O, –78 °C, 1 h, then addition of n-
decanal, 2 h, –78 °C, 95% (er 96:4); (b) NaH, THF, 0 °C, then MeI,
r.t., overnight, 92%; (c) O3, CH2Cl2, –78 °C, then PPh3; (d) (+)-
Ipc2BCl, allylMgBr, Et2O, –78 °C, 1 h, followed by addition of the
aldehyde, 2 h, –78 °C, 70% overall from 8 (dr 88:12); (e) TBSOTf,
CH2Cl2, 2,6-lutidine, room temp., 1 h, 90%; (f) O3, CH2Cl2,
–78 °C, then PPh3; (g) (–)-Ipc2BCl, allylMgBr, Et2O, –78 °C, 1 h,
followed by addition of the aldehyde, 2 h, –78 °C, 47% overall yield
from 11 (dr Ͼ 95:5); (h) CH2=CHCOCl, CH2Cl2, iPr2NEt, –78 °C,
45 min, 77%; (i) 1. 10% cat. Ru-I, CH2Cl2, Δ, 4 h, 85%; 2. PPTS
(cat.), MeOH, Δ, overnight, 90%. TBS = tert-butyldimethylsilyl,
PPTS = pyridinium p-toluenesulfonate, Ipc = isopinocampheyl, Tf
= trifluoromethylsulfonyl.
Figure 4. Structures of new pironetin analogues 3–6.
Brown and co-workers’ asymmetric allylation of n-dec-
anal with a chiral allylborane afforded homoallyl alcohol 7.
The required borane was prepared by the reaction of allyl-
Results and Discussion
magnesium bromide with the commercially available (–)-di-
isopinocampheylboron chloride [(–)-Ipc2BCl].[18] Methyl-
Chemical Results
ation of the free hydroxy group of compound 7 yielded
Dihydropyrones 3–6 were prepared by using the method- methyl ether 8. Ozonolytic cleavage of the olefinic bond in
ology employed for the synthesis of compounds of general 8 gave the intermediate aldehyde 9, which was not isolated
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Biological Evaluation of α-Tubulin-Binding Pironetin Analogues
but directly subjected to asymmetric allylation, again by Table 1. Effect of pironetin analogues 3–6 on the growth of A2780
and A2780AD (MDR overexpressing P-glycoprotein) ovarian car-
using the chiral allylborane formed from allylmagnesium
bromide and (+)-Ipc2BCl. This gave rise to homoallyl
alcohol 10 as an inseparable mixture of diastereoisomers in
a ratio of 88:12. Silylation of this mixture gave 11, which
cinomas.[a]
Ligand
IC50 [μm][b]
R/S[c]
A2780
A2780AD
was subjected to ozonolytic cleavage of the C=C bond. Pironetin 0.0062Ϯ0.0012
0.0093Ϯ0.0014
38.6Ϯ1.1
12.5Ϯ0.353
10.9Ϯ0.05
48Ϯ0.7
1.5
0.97
0.90
1.2
3
4
5
6
39.7Ϯ0.5
14.1Ϯ0.424
9.2Ϯ0.3
Without purification, the intermediate aldehyde 12 was sub-
mitted to asymmetric allylation with the chiral allylborane
generated from (–)-Ipc2BCl and allylmagnesium bromide.
This provided homoallylic alcohol 13 as a single dia-
stereoisomer, the minor stereoisomer being removed during
the chromatographic separation. Reaction of 13 with acryl-
oyl chloride at low temperature gave acrylate 14, which was
then subjected to ring-closing metathesis[19] in the presence
of Grubbs first-generation catalyst Ru-I. This afforded the
corresponding dihydropyrone which, after acid-catalyzed
desilylation, yielded pironetin analogue 3.
An analogous reaction sequence starting from n-tri-
decanal served to prepare dihydropyrones 4 and 5, whereas
benzaldehyde was used for the synthesis of 6. The complete
details of these syntheses are given in the Supporting Infor-
mation.
54.7Ϯ5.4
0.9
[a] IC50 (50% inhibition of cell proliferation) values for pironetin
and compounds 3–6 in ovarian carcinomas. [b] IC50 values are
given as the meanϮstandard error of three independent experi-
ments. [c] Resistance index (the relative resistance of A2780AD cell
line, obtained by dividing the IC50 of the resistant cell line by that
of the parental A2780 cell line).
To study the effect of compounds 3–6 on the microtubule
cytoskeleton, we incubated cells in the presence of these
compounds for 24 hours (Figure 5). Pironetin at a concen-
tration of 50 nm completely depleted the cytoplasmic micro-
tubules (Figure 5, C,D and inset): cells are arrested in the
prometaphase[13a,13b] and type III mitotic spindles can be
observed[21] with the chromosomes being arranged in a ball
of condensed DNA enclosing one or more star-shaped ag-
gregates of microtubules.
Biological Results
With 100 μm 3, 25 μm 4 and 15 μm 5, disorganization and
some depolymerization of the microtubule cytoskeleton
were observed: the cells become rounded and detach from
Cellular Effects of the Compounds
We determined the IC50 values for the action of ana- the plastic substrate on which they are growing, with
logues 3–6 on A2780 and A2780AD human ovary carci- shrinking of the cell nucleus occurring in some cases (Fig-
nomas and compared them with those of pironetin ure 5, E–J). No mitotic cells were observed in these cell
(Table 1). Pironetin proved active in both the parental and preparations. However, compound 6 at a concentration of
resistant cell lines, as expected for a compound with a cova- 200 μm was found to cause microtubule depolymerization
lent mechanism of action.[20] Although about three orders and cell arrest in the prometaphase, as in the case of pirone-
of magnitude less active than pironetin, analogues 3–6 were tin (see Figure 5, K,L and inset).
also found to be cytotoxic towards the A2780 and
A2780AD cells and were able to kill both resistant and non- of blocking cells in the G2/M phase of the cell cycle of
resistant cells with similar IC50 values. A549, as other microtubule modulating agents do. We incu-
We next studied whether compounds 3–6 were capable
Figure 5. Effect of compounds 3–6, in comparison with the parent molecule pironetin, on the microtubule network and nucleus mor-
phology of A549 cells. Cells were incubated for 24 h with drug vehicle DMSO (A,B), 50 nM pironetin (C,D), 100 μm 3 (E,F), 25 μm 4
(G,H), 15 μm 5 (I,J) and 200 μm 6 (K,L). Microtubules were stained with α-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,K,L) are mitotic spindles of the same preparation. The scale bar in L represents 10 μm.
All panels and insets have the same magnification.
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M. Carda, J. F. Díaz, I. Barasoain, J. A. Marco et al.
FULL PAPER
bated these cells for 20 hours in the presence of the different Conclusions
compounds 3–6 or the drug vehicle (Figure 6). Pironetin at
Four new pironetin derivatives 3–6 with an extended li-
pophilic side-chain have been synthesized with the aim of
exploring the influence of the side-chain on their biological
activities. All the compounds are cytotoxic in the micromo-
lar range against both non-resistant and resistant P-glyco-
protein overexpressing, multidrug ovarian carcinoma cell
lines, similar IC50 values being found in both cell lines.
However, although all the compounds are able to inhibit
microtubule assembly, both in vitro and in cell cultures,
thus sharing the general mechanism for the inhibition of
action of tubulin assembly, compounds 3–5, which contain
a long aliphatic side-chain, differ from pironetin and com-
pound 6 in that they do not accumulate cells in the G2/M
phase of the cell cycle. This indicates that, in contrast to
pironetin and 6, compounds 3–5 trigger an alternative
mechanism for cytotoxicity that leads to cell death.
a concentration of 50 nM almost completely arrested the
cells in the G2/M phase and, interestingly, so did 6 at a
concentration of 200 μm. In contrast, compounds
3
(100 μm), 4 (50 μm) and 5 (10 μm) only caused a decrease in
the number of G2/M cells with the appearance of subG1
cells, presumably dying cells. These results indicate that, al-
though markedly less active, compound 6 is the only com-
pound that behaves like pironetin. In addition, the results
indicate that compounds 3–5, although being active against
the tubulin cytoskeleton, may exert their cytotoxicity
through an alternative pathway.
Experimental Section
Chemical Procedures
General: 1H and 13C NMR spectra were recorded at 500 and
125 MHz in CDCl3 solution at 25 °C. The signals of the deuteriated
solvent (CDCl3) were used as references (the singlet at δ = 7.25 ppm
for 1H NMR and the triplet centred at δ = 77.00 ppm for 13C
NMR). Carbon atom types (C, CH, CH2, CH3) were determined
by using the DEPT pulse sequence. HRMS were recorded in elec-
trospray mode (ESMS). IR data are given only for compounds with
significant functions (OH, C=O) and were recorded as oily films
on NaCl plates (oils) or as KBr pellets (solids). Optical rotations
were measured at 25 °C. Reactions that required an inert atmo-
sphere were carried out under N2 in flame-dried glassware. Et2O
and THF were freshly distilled from sodium/benzophenone ketyl
and transferred through a syringe. Dichloromethane was freshly
distilled from CaH2, tertiary amines were freshly distilled from
KOH, and toluene was freshly distilled from sodium wire. Com-
mercially available reagents were used as received. Unless detailed
otherwise, “work-up” means pouring the reaction mixture into
brine followed by extraction with the solvent indicated in parenthe-
ses. If the reaction medium was acidic, the mixture was also washed
with 5% aq. NaHCO3. If the reaction medium was basic, the mix-
ture was also washed with aq. NH4Cl. Further washing with brine,
drying over anhydrous Na2SO4 and elimination of the solvent un-
der reduced pressure were followed by chromatography on a silica
gel column (60–200 μm) and elution with the solvent mixture indi-
cated. When solutions were filtered through a Celite pad, the pad
was additionally washed with the same solvent and the washings
incorporated into the main organic layer.
Figure 6. Cell cycle histograms of A549 lung carcinoma cells un-
treated and treated with pironetin and the pironetin analogues 3–
6. The lowest concentration that induces maximal effect on the cell
cycle is depicted.
Tubulin Assembly
The critical concentration of tubulin required for as-
sembly was determined in glycerol assembling buffer (GAB)
in the presence of a large excess (100 μm) of compounds 3–
6. As shown in Table 2, the concentration of tubulin re-
quired to produce assembly (critical concentration[22]) oscil-
lates between 3.3 μm in the absence of pironetin analogue
and 4.9 μm in the presence of 5, the most active of the com-
pounds in this respect. The observed increase in the critical
concentration required indicates that, as expected for a pi-
ronetin analogue, compounds 3–6 also inhibit the assembly
of tubulin.
Table 2. Critical concentrationd (Cr) of tubulin required for micro-
(R)-Tridec-1-en-4-ol (7): Allylmagnesium bromide (commercial 1 m
solution in Et2O, 15 mL, 15 mmol) was added dropwise under N2
through a syringe to a cooled solution (dry ice–acetone bath) of
(–)-Ipc2BCl (5.77 g, ca. 18 mmol) in dry Et2O (75 mL). After the
addition, the dry ice–acetone bath was replaced by an ice bath and
the mixture was stirred for 1 h. The solution was allowed to stand,
whereby precipitation of magnesium chloride took place. The su-
pernatant solution was carefully transferred to another flask
through a cannula. After cooling this flask to –78 °C, a solution of
n-decanal (2.25 mL, 1.87 g, 12 mmol) in dry Et2O (35 mL) was
added dropwise through a syringe. The resulting solution was fur-
ther stirred at –78 °C for 2 h. The reaction mixture was quenched
tubule assembly induced by pironetin analogues 3–6.[a]
Ligand
Cr [μm][b]
Control
Docetaxel
3.3Ϯ0.3
1.3Ϯ0.4
3.8Ϯ0.9
4.3Ϯ1.3
4.9Ϯ0.8
3.5Ϯ0.8
3
4
5
6
[a] Concentrations are 25 μm for docetaxel and 100 μm for com-
pounds 3–6. [b] Cr values are the mean Ϯstandard error of three
independent experiments.
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Biological Evaluation of α-Tubulin-Binding Pironetin Analogues
by the addition of a phosphate pH 7 buffer solution (15 mL),
MeOH (15 mL) and 30% H2O2 (7 mL). After stirring for 30 min,
the mixture was poured into satd. aq. NaHCO3 and worked up
(extraction with Et2O). The residue was subjected to careful col-
umn chromatography on silica gel (hexanes, then hexanes/EtOAc,
9:1) to afford 7 (2.26 g, 95%) as an oil. [α]D = +3.3 (c = 1.1,
CHCl3). The physical and spectroscopic data are as reported.[23]
The enantiomeric ratio was found to be 96:4 by means of chiral
HPLC using a Kromasil 5-AmyCoat column (4.6ϫ20 mm). Elu-
tion was performed with hexane/2-propanol (99:1) and a flux of
0.4 mL/min. Elution times of both enantiomers: 21.21 (S) and
22.87 min (R).
H), 5.15–5.05 (br. m, 2 H), 3.95 (m, 1 H), 3.48 (m, 1 H), 3.37 (s, 3
H), 2.90 (br. s, 1 H, OH), 2.25 (m, 2 H), 1.70–1.40 (br. m, 4 H),
1.35–1.25 (br. m, 14 H), 0.89 (t, J = 7 Hz, 3 H) ppm. 13C NMR
(125 MHz): δ = 135.0, 79.3, 68.0 (CH), 117.5, 42.3, 39.0, 33.1, 31.9,
29.8, 29.6, 29.5, 29.3, 25.4, 22.7 (CH2), 56.7, 14.1 (CH3) ppm.
HRMS (ES): calcd. for C16H32NaO2 [M + Na]+ 279.2300; found
279.2302.
(4R,6R)-4-(tert-Butyldimethylsilyloxy)-6-methoxypentadec-1-ene
(11): Alcohol 10 (1.025 g, 4 mmol) was dissolved under N2 in dry
CH2Cl2 (20 mL) and treated sequentially with 2,6-lutidine (700 μL,
6 mmol) and TBSOTf (1.15 mL, 5 mmol). The reaction mixture
was then stirred for 1 h at room temp. and worked up (extraction
with CH2Cl2). Column chromatography on silica gel (hexanes/
EtOAc, 19:1) afforded 11 (1.33 g, 90%) as an oil containing 88:12
mixture of diastereoisomers which were very difficult to separate
and thus the mixture was used as such in the next step. For analyti-
cal purposes, an aliquot could be concentrated to about 95% purity
by means of careful column chromatography. [α]D = –8.2 (c = 0.1,
(R)-4-Methoxytridec-1-ene (8): Sodium hydride (60% slurry in min-
eral oil, amount equivalent to 20 mmol) under N2 was washed
twice with dry hexane and once with dry THF. THF (75 mL) was
added and the suspension was cooled in an ice bath. Alcohol 7
(1.98 g, 10 mmol) was then dissolved in dry THF (25 mL) and
added dropwise to the sodium hydride suspension. The mixture
was then allowed to warm to room temperature. Subsequently,
methyl iodide (1.87 mL, ca. 30 mmol) was added in one portion
and the mixture was stirred overnight at room temp. Work-up
(Et2O) was followed by column chromatography on silica gel (hex-
anes/EtOAc, 19:1) to afford 8 (1.95 g, 92%) as an oil. [α]D = +2.9
(c = 0.1, CHCl3). IR and 1H NMR spectroscopic data are as re-
ported.[24] 13C NMR (125 MHz): δ = 135.1, 80.6 (CH), 116.7, 37.8,
33.4, 31.9, 29.8, 29.7, 29.6, 29.4, 25.3, 22.7 (CH2), 56.5, 14.1
(CH3) ppm.
1
CHCl3). H NMR (500 MHz): δ = 5.82 (ddt, J = 17, 10, 7 Hz, 1
H), 5.10–5.00 (br. m, 2 H), 3.93 (m, 1 H), 3.34 (m, 1 H), 3.30 (s, 3
H), 2.24 (m, 2 H), 1.60–1.40 (br. m, 4 H), 1.35–1.25 (br. m, 14 H),
0.90 (12 H, strong singlet of 9 H overlapping a methyl triplet at δ
= 0.89 ppm), 0.08 (s, 6 H) ppm. 13C NMR (125 MHz): δ = 18.1
(C), 134.9, 77.1, 68.6 (CH), 117.0, 42.8, 41.7, 33.0, 31.9, 29.9, 29.7,
29.6, 29.3, 24.8, 22.7 (CH2), 55.7, 26.0 (ϫ3), 14.1, –4.1, –4.7
(CH3) ppm. HRMS (ES): calcd. for C22H46NaO2Si [M + Na]+
393.3165; found 393.3162.
(4S,6R,8R)-6-(tert-Butyldimethylsilyloxy)-8-methoxyheptadec-
1-en-4-ol (13): Prepared in two steps from 11 (via the non-isolated
aldehyde 12) in 47% overall yield by using the same experimental
conditions as in the synthesis of 7. Careful chromatography on sil-
ica gel (hexane/Et2O, 9:1, then 8:2) permitted the isolation of dia-
(4R,6R)-6-Methoxypentadec-1-en-4-ol (10): Olefin 8 (1.275 g, ca.
6 mmol) was dissolved in CH2Cl2 (100 mL) and cooled to –78 °C.
A stream of ozone-containing air was then bubbled through the
solution until complete consumption of the starting material (TLC
monitoring). Ozone residues were then eliminated by bubbling a
stream of N2 through the mixture, which was then allowed to warm
to room temperature, treated with PPh3 (3.15 g, ca. 12 mmol) and
stirred for 2 h. After removal of the solvent under reduced pressure,
the crude residue was stirred for 10 min in cold pentane (40 mL)
and filtered. The solution was then concentrated under reduced
pressure and the crude residue containing 9 was used directly in
the next step.
stereomerically pure (by NMR) 13 as an oil. [α]D = –4.1 (c = 0.1,
1
CHCl ). IR: ν
= 3400 (br., OH) cm–1. H NMR (500 MHz): δ
˜
3
max
= 5.82 (ddt, J = 17, 10, 7 Hz, 1 H), 5.15–5.05 (br. m, 2 H), 4.18
(m, 1 H), 4.05 (m, 1 H), 3.50 (br. s, 1 H, OH), 3.29 (s, 3 H), 3.26
(m, 1 H), 2.30–2.10 (br. m, 2 H), 1.70–1.50 (br. m, 4 H), 1.35–1.25
(br. m, 16 H), 0.90 (12 H, strong singlet of 9 H overlapping a methyl
triplet at δ = 0.89 ppm), 0.12 (s, 3 H), 0.10 (s, 3 H) ppm. 13C NMR
(125 MHz): δ = 17.9 (C), 135.0, 77.3, 69.3, 68.1 (CH), 117.0, 42.4,
42.1, 41.0, 32.8, 31.9, 29.9, 29.7, 29.6, 29.3, 24.7, 22.7 (CH2), 55.6,
25.9 (ϫ3), 14.1, –4.4, –4.9 (CH3) ppm.
Allylmagnesium bromide (commercial 1 m solution in Et2O, 8 mL,
8 mmol) was added dropwise under N2 through a syringe to a
cooled solution (dry ice–acetone bath) of (+)-Ipc2BCl (3.2 g,
≈10 mmol) in dry Et2O (50 mL). After finishing the addition, the
dry ice–acetone bath was replaced by an ice bath and the mixture
was stirred for 1 h. The solution was allowed to stand, whereby
precipitation of magnesium chloride took place. The supernatant
solution was carefully transferred into another flask through a can-
nula. After cooling this flask to –78 °C, a solution of the crude
aldehyde 9 from above in dry Et2O (15 mL) was added dropwise
through a syringe. The resulting solution was stirred at –78 °C for
a further 2 h. The reaction mixture was quenched by the addition
of a phosphate pH 7 buffer solution (40 mL), MeOH (40 mL) and
30% H2O2 (20 mL). After stirring for 30 min, the mixture was
poured into satd. aq. NaHCO3 and worked up (extraction with
Et2O). The residue was subjected to careful column chromatog-
raphy on silica gel (hexanes, then hexanes/EtOAc, 19:1) to afford
10 (1.077 g, 70% overall from 8) as an oil containing an 88:12 mix-
ture of diastereoisomers which were very difficult to separate and
thus the mixture was used as such in the next step. For analytical
purposes, an aliquot could be concentrated to about 95% purity
by means of careful column chromatography. [α]D = –22.8 (c = 0.8,
(4S,6S,8R)-6-(tert-Butyldimethylsilyloxy)-8-methoxyheptadec-
1-en-4-yl acrylate (14): Compound 13 (415 mg, ca. 1 mmol) was
dissolved under N2 in dry CH2Cl2 (30 mL) cooled to –78 °C and
treated sequentially with N,N-diisopropylethylamine (2.1 mL,
12 mmol) and acryloyl chloride (815 μL, ca. 10 mmol). The reac-
tion mixture was stirred at –78 °C until consumption of the starting
material was complete (about 45 min, TLC monitoring). Work-up
(extraction with CH2Cl2) and column chromatography on silica gel
(hexane/Et2O, 9:1) provided 14 (361 mg, 77%) as an oil. [α]D = +2.2
(c = 0.5, CHCl ). IR: ν
= 1727 (C=O) cm–1 1H NMR
.
˜
3
max
(500 MHz): δ = 6.38 (dd, J = 17.3, 1.5 Hz, 1 H), 6.10 (dd, J = 17.3,
10.5 Hz, 1 H), 5.80 (dd, J = 10.5, 1.5 Hz, 1 H), 5.78 (ddt, J = 17,
10.2, 7 Hz, 1 H,), 5.10–5.00 (br. m, 3 H,), 3.86 (m, 1 H), 3.29 (s, 3
H), 3.28 (m, 1 H), 2.40 (m, 2 H), 1.85–1.40 (br. M, 6 H), 1.35–1.25
(14 H, br. m), 0.89 (12 H, strong singlet of 9 H overlapping a methyl
triplet at δ = 0.89 ppm), 0.05 (s, 3 H), 0.04 (s, 3 H) ppm. 13C NMR
(125 MHz): δ = 165.7, 18.0 (C), 133.4, 129.0, 77.5, 71.2, 67.0 (CH),
130.2, 118.0, 42.7, 42.0, 39.0, 33.2, 31.9, 29.9, 29.7, 29.6, 29.3, 24.7,
22.7 (CH2), 55.8, 26.0 (ϫ3), 14.1, –4.3, –4.4 (CH3) ppm. HRMS
(ES): calcd. for C27H52NaO4Si [M + Na]+ 491.3533; found
491.3529.
1
CHCl3). H NMR (500 MHz): δ = 5.85 (ddt, J = 17, 10, 7 Hz, 1
Eur. J. Org. Chem. 2013, 1116–1123
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1121

M. Carda, J. F. Díaz, I. Barasoain, J. A. Marco et al.
FULL PAPER
(6S)-6-[(2S,4R)-2-(tert-Butyldimethylsilyloxy)-4-methoxytridecyl]-
5,6-dihydro-2H-pyran-2-one (15): Diolefin 14 (234 mg, ca.
0.5 mmol) was dissolved under N2 in dry, degassed CH2Cl2 (50 mL)
and treated with Grubbs first-generation catalyst [PhCH=RuCl2-
Tubulin Assembly Inhibition Assay: The effect of compounds 3–6
on the assembly of purified tubulin was determined by incubating
20 μm purified tubulin at 37 °C for 30 min in GAB (glycerol as-
sembling buffer, 3.4 m glycerol, 10 mm sodium phosphate, 1 mm
(PCy3)2] (41 mg, ca. 0.05 mmol). The mixture was heated at reflux EGTA, 1 mm GTP and 6 mm MgCl2 at pH 6.5) in the presence of
until consumption of the starting material (ca. 4 h, TLC monitor-
ing). Removal of the solvent under reduced pressure and column
chromatography on silica gel (hexane/EtOAc, 19:1) yielded py-
ranone 15 (187 mg, 85%) as an oil. [α]D = –6.5 (c = 0.2, CHCl3).
25 μm docetaxel, 100 μm of one of the analogues 3–6 or 2 μL
DMSO (drug vehicle). The samples were processed and the critical
concentrations for tubulin assembly[22] in the presence of the com-
pound were calculated as described previously.[27]
IR: νmax = 1732 (C=O) cm–1. 1H NMR (500 MHz): δ = 6.86 (dt, J
˜
Supporting Information (see footnote on the first page of this arti-
= 9.7, 4 Hz, 1 H), 6.00 (br. d, J = 9.7 Hz, 1 H), 4.58 (m, 1 H), 4.06
(m, 1 H), 3.28 (s, 3 H), 3.26 (quint., J ≈ 6 Hz, 1 H), 2.30 (m, 2 H),
2.00 (m, 1 H), 1.65–1.55 (br. m, 3 H), 1.55–1.40 (br. m, 2 H), 1.35–
1.20 (br. m, 14 H), 0.87 (12 H, strong singlet of 9 H overlapping a
methyl triplet at δ = 0.89 ppm), 0.07 (s, 3 H), 0.06 (s, 3 H) ppm.
13C NMR (125 MHz): δ = 164.2, 18.0 (C), 145.1, 121.5, 77.7, 74.5,
66.3 (CH), 43.3, 43.1, 33.2, 31.9, 29.9, 29.7, 29.6, 29.5, 29.3, 24.7,
22.6 (CH2), 55.8, 25.9 (ϫ3), 14.1, –4.4, –4.5 (CH3) ppm. HRMS
(ES): calcd. for C25H48NaO4Si [M + Na]+ 463.3219; found
463.3218.
cle): Experimental procedures and details of the preparation of py-
1
rones 4–6 and of all required synthetic intermediates, and H and
13C NMR spectra of all new compounds.
Acknowledgments
Financial support has been granted to M. C. by the Spanish Minis-
try of Education and Science (project numbers CTQ2008-02800
and CTQ2011-27560), by the Consellería d’Empresa, Universitat i
Ciencia de la Generalitat Valenciana (ACOMP09/113) and by the
BANCAJA-UJI Foundation (P1-1B2002-06, P1-1B-2008-14 and
PI-1B2011-37). The biological work has been supported in part by
grants from the Spanish Ministry of Education and Science (grant
number BIO2010-16351) and from the Comunidad de Madrid
(grant number S2010/BMD-2457 BIPEDD2-CM), both to J. F. D.
We further thank the Matadero Municipal Vicente de Lucas in
Segovia for providing the calf brains, which were the source of tub-
ulin.
(6S)-6-[(2S,4R)-2-Hydroxy-4-methoxytridecyl]-5,6-dihydro-2H-
pyran-2-one (3): Compound 15 (132 mg, 0.3 mmol) was dissolved
in MeOH (15 mL) and treated with PPTS (15 mg, 0.06 mmol) and
water (0.15 mL). The mixture was then heated at reflux overnight,
cooled and neutralized by addition of solid NaHCO3. After filter-
ing, the solution was evaporated under reduced pressure and the
oily residue was subjected to column chromatography on silica gel
(hexanes/EtOAc, 1:4) to yield 3 (88 mg, 90%) as an oil. [α]D = –16
(c = 1, CHCl ). IR: νmax = 1712 (C=O) cm–1. 1H NMR (500 MHz):
˜
3
δ = 6.86 (m, 1 H), 6.00 (d, J = 9.5 Hz, 1 H), 4.72 (m, 1 H), 4.22
(m, 1 H), 3.45 (m, 1 H), 3.35 (br. s, 4 H, OMe + OH), 2.45–2.30
(br. m, 2 H), 1.90–1.40 (br. m, 6 H), 1.35–1.20 (br. m, 14 H), 0.87
(t, J = 6.8 Hz, 3 H) ppm. 13C NMR (125 MHz): δ = 164.4 (C),
145.2, 121.4, 79.6, 75.1, 64.7 (CH), 42.9, 39.4, 32.8, 31.9, 30.0, 29.7,
29.6, 29.5, 29.3, 25.5, 22.6 (CH2), 56.6, 14.1 (CH3) ppm. HRMS
(ES): calcd. for C19H34NaO4 [M + Na]+ 349.2354; found 349.2358.
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Received: September 27, 2012
Published Online: January 7, 2013
Eur. J. Org. Chem. 2013, 1116–1123
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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