Design and synthesis of pironetin analogue/combretastatin A-4 hybrids and evaluation of their cytotoxic activity

Article abstract of DOI:10.1002/ejoc.201301725

We describe the preparation of a series of hybrid molecules containing a combretastatin A-4 moiety and a pironetin analogue fragment linked by an ester spacer of variable length. The cytotoxic activities of these compounds have been measured and the relationship between the structure and cytotoxicity is discussed. Some of the tested compounds showed cytotoxicity values of the same order of magnitude as those of the parental molecules, combretastatin A-4 and pironetin, and were less toxic than the latter for normal cells. The preparation of a series of hybrid molecules containing a combretastatin A-4 moiety and a pironetin analogue fragment linked by a spacer of variable length is described. The relationship between structure and cytotoxicity is discussed. Cytotoxicity values of some compounds were similar to those of the parent molecules, combretastatin A-4 and pironetin, and some were less toxic than the latter for normal cells. Copyright

Full text of DOI:10.1002/ejoc.201301725

FULL PAPER
DOI: 10.1002/ejoc.201301725
Design and Synthesis of Pironetin Analogue/Combretastatin A-4 Hybrids and
Evaluation of Their Cytotoxic Activity
Sandra Torijano-Gutiérrez,[a] Concepción Vilanova,[a] Santiago Díaz-Oltra,[a]
Juan Murga,*[a] Eva Falomir,[a] Miguel Carda,[a] and J. Alberto Marco*[b]
Keywords: Medicinal chemistry / Drug design / Cytotoxicity / Anticancer agents / Structure–activity relationships
We describe the preparation of a series of hybrid molecules
containing a combretastatin A-4 moiety and a pironetin ana-
logue fragment linked by an ester spacer of variable length.
The cytotoxic activities of these compounds have been mea-
sured and the relationship between the structure and cyto-
toxicity is discussed. Some of the tested compounds showed
cytotoxicity values of the same order of magnitude as those
of the parental molecules, combretastatin A-4 and pironetin,
and were less toxic than the latter for normal cells.
latter group is presently by far the most numerous and con-
tains products that cause either disruption or stabilization
of microtubules. Among the drugs that belong to this
group, the well-known colchicine[6] and the combretastat-
ins[7] (Figure 1) exert their effects by causing disruption of
microtubules. In contrast, another important representative
of the same group, paclitaxel, was the first described tubu-
lin-binding drug that was found to stabilize microtubules.[8]
Even though they display opposite effects, these drugs are
Introduction
It is widely known that cancer, one of the leading causes
of death in developed countries,[1] may be induced by a
plethora of both external and internal factors, including ge-
netic mutations. Accordingly, many types of therapeutic at-
tack have 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 act through their in-
teraction 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
composed of a protein named tubulin, the functional form
of which is a heterodimer formed through noncovalent
binding of two monomeric constituents, α- and β-tubulin.
For cell division to occur in a normal way, microtubules
must be in a constant state of formation and disruption, a
process named microtubule dynamic instability.[5] Any mo-
lecule that influences microtubule instability will also influ-
ence the cell division process, not only of normal cells but
also of tumoral cells. Therefore, it is not surprising that tub-
ulin-binding molecules (TBMs) constitute a very important
class of anticancer agents.
TBMs may be divided into two broad categories, those
that bind to α-tubulin and those that bind to β-tubulin. The
[a] Departamento de Química Inorgánica y Orgánica, Univ.
Jaume I,
12071 Castellón, Spain
E-mail: mcarda@qio.uji.es
http://www.sinorg.uji.es/index.htm
[b] Departamento de Química Orgánica, Univ. de Valencia,
50, c/D. Moliner, 46100 Burjassot, Spain
E-mail: alberto.marco@uv.es
http://www.uv.es/~marcoj/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301725.
Figure 1. Structures of some natural products reported to selec-
tively bind to β-tubulin.
2284
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296

Pironetin Analogue/Combretastatin A-4 Hybrids
known to bind to β-tubulin, albeit to different sites within fashion to the conjugated double bond of pironetin, thus
this protein subunit.[9–11]
forming a covalent bond with C-3 of the dihydropyrone
The number of products that bind to α-tubulin is very ring (Figure 3). It has also been suggested that the Asn258
small, with the naturally occurring 5,6-dihydro-α-pyrone pi- residue of α-tubulin holds the pironetin molecule through
ronetin being the first reported example,[12] followed, a two hydrogen bonds to the dihydropyrone carbonyl and the
short time later, by the peptide-like hemiasterlin family[13] methoxyl oxygen atoms.[14]
(Figure 2). Pironetin is a potent inhibitor of tubulin as-
The emergence of resistances to existing drugs has led to
sembly and has been found to arrest cell cycle progression a continuous need to develop new bioactive compounds
in the G2/M phase.[14]
that overcome such problems. Even though first observed
in the case of antibiotics,[15] resistances have been reported
to therapies with various types of anticancer agents.[16] The
investigation of new compounds with such biological prop-
erties therefore constitutes an important goal in chemistry
and pharmacology.
Concept and Design of Hybrid Tubulin-Binding Ligands
As a member of the small group of TBMs that bind to
α-tubulin, pironetin constitutes a pharmacologically inter-
esting target. Not surprisingly, appreciable numbers of total
syntheses of this natural compound have appeared in the
literature.[17] To develop SAR studies based upon the pi-
ronetin framework, two years ago[5a] we designed a simpli-
fied model structure in which all elements that had not yet
proven to be essential for biological activity were removed.
The target structures I/II are schematically shown in Fig-
ure 4. The elements that were maintained were the conju-
gated dihydropyrone ring and the side chain with the meth-
oxy group at C-9. The hydroxyl group at C-7 was removed
in some substrates (I) and retained in others (II), to assess
its influence on activity. All alkyl pendants (methyl groups
at C-8 and C-10, ethyl at C-4) and the isolated C12–C13
double bond were removed. The configurations of the two/
three remaining stereocenters were then varied in a system-
atic way. Thus, all four possible stereoisomers with general
constitution I, with no hydroxyl group at C-7, were pre-
pared. Likewise, all eight stereoisomers exhibiting general
structure II, with a hydroxyl group at C-7, were synthe-
sized.[5a]
Figure 2. Structures of two natural products reported to selectively
bind to α-tubulin.
Some structure-activity relationship (SAR) studies on pi-
ronetin have been reported. These studies have shown that
the presence of the conjugated C2–C3 double bond and of
the hydroxyl group at C-9, either free or methylated, are
essential for biological activity. The presence of a (7R)-hy-
droxyl group also seems to be relevant. It has been sug-
gested, although not yet conclusively demonstrated, that
the Lys352 residue of the α-tubulin chain adds in a Michael
Figure 4. General structures of simplified pironetin analogues of
the first generation.[5a]
The cytotoxic activity of these analogues and their inter-
actions 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 natural compound,
and compete for the same binding site in α-tubulin. As ob-
served for the parent compound, they also lead to disrup-
tion of the microtubule network.[5a]
Figure 3. Schematic model suggested for the covalent union of pi-
ronetin to its binding site at the α-tubulin surface.
Eur. J. Org. Chem. 2014, 2284–2296
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2285

J. Murga, J. A. Marco et al.
FULL PAPER
Figure 5. Structures of the pironetin analogue/combretastatin A-4 hybrids used in this study (1/8 and ent-1/ent-4) and of their synthetic
precursors (9–15), including combretastatin A-4 (13).
With the aim of extending the scope of our project, we
wanted to prepare cytotoxic TBMs with a dual ability to
bind to either α- or β-tubulin and exert a microtubule-de-
stabilizing effect. Since these properties are specifically ex-
hibited by pironetin (binds to α-tubulin) and combretasta-
tin A-4 (binds to β-tubulin), respectively, we decided to pre-
pare compounds with a hybrid structure such as 1–8 (Fig-
ure 5). These molecules contain a moiety of combretastatin
A-4 (itself numbered as 13 in Figure 5) and another of the
simplified pironetin type (14, ent-14 and 15), connected, in
turn, by a spacer of variable length. Some of these mo-
lecules (1–4 and ent-1 to ent-4) have been prepared in both
Scheme 1. Synthesis of combretastatin A-4 derivatives 9a–12a and
antipodal forms.[18]
9b–12b: Reagents and conditions: (a) K2CO3, DMF, room temp.,
24 h (9a: 92%; 10a: 92%; 11a: 91%; 12a: 90%); (b) aq. NaOH,
MeOH, room temp., 18 h (9b: 80%; 10b: 78%; 11b: 76%; 12b:
75%).
Results and Discussion
Chemical Results
Combretastatin A-4 (13) was prepared according to a presence of Grubbs first-generation catalyst Ru-I. This fur-
published procedure.[19] Its O-alkyl derivatives 9a–12a were nished dihydropyrone 19, deprotection of which provided
prepared by means of O-alkylation with four commercially 14 in good yield. Dihydropyrone ent-14 was prepared in the
available ω-bromo esters (Scheme 1). Saponification of es- same way, except that (+)-Ipc2BCl was used as the chiral
ters 9a–12a gave the corresponding acids 9b–12b.
boron reagent.
The synthesis of the pironetin fragments 14, ent-14 and
Dihydropyrone 15 was not connected as such with the
15 is depicted in Scheme 2 and was performed according combretastatin A-4 fragment, but as its O-silyl derivative
to the general concept used in previous papers.[5a,5b] Thus, 28 (Scheme 2), which was also prepared according to the
BrownЈs asymmetric allylation[20] of known aldehyde 16[21] previous methodology. BrownЈs asymmetric allylation of
afforded homoallyl alcohol 17. The required chiral allyl- known aldehyde 20[23] furnished alcohol 21, which was then
borane was prepared through reaction of allylmagnesium converted into methyl ether 22. The latter was then sequen-
bromide with commercially available (–)-diisopinocam- tially subjected to a two-step oxidative cleavage of the ole-
pheylboron chloride [(–)-Ipc2BCl]. Reaction of 17 with finic bond, followed by Brown’s asymmetric allylation to
acryloyl chloride at low temperature gave acrylate 18, which yield 23. Silylation of the hydroxyl group of 23 and repeti-
was subjected to ring-closing metathesis (RCM)[22] in the tion of the previous sequence gave alcohol 25, which was
2286
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296

Pironetin Analogue/Combretastatin A-4 Hybrids
Scheme 2. Synthesis of pironetin fragments 14, ent-14 and 15. Reagents and conditions: (a) (–)-Ipc2BCl, allylMgBr, Et2O, –78 °C, 1 h, then
addition of the aldehyde, 1 h, –78 °C (17: 83%, er 96:4; 21: 85%, er 94:6; 25, 78% overall yield from 24, dr 76:24); (b) CH2=CHCOCl,
CH2Cl2, iPr2NEt, –78 °C, 30 min (18: 94%; ent-18: 90%; 26: 91%); (c) 10% cat. Ru-I, CH2Cl2, Δ, 2 h (19: 81%; ent-19: 80%; 27: 83%);
(d) DDQ, wet CH2Cl2, 30 min (14: 69%; ent-14: 67%; 28: 74%); (e) (+)-Ipc2BCl, allylMgBr, Et2O, –78 °C, 1 h, then addition of the
aldehyde, 1 h, –78 °C (ent-17: 89%, er 96:4; 23: 90% overall yield from 22, dr 86:14); (f) NaH, THF, 0 °C, then MeI, room temp., overnight
(88%); (g) 1. OsO4, NMO, aq. tBuOH, THF, room temp., overnight. 2. Pb(OAc)4, CH2Cl2, 0 °C; (h) TBSOTf, 2,6-lut, 0 °C, 1 h (80%);
(i) PPTS (cat.), MeOH, Δ, overnight (85%). Acronyms and abbreviations: Ipc, isopinocampheyl; DDQ, 2,3-dichloro-5,6-dicyano-p-benzo-
quinone; TBS, tert-butyldimethylsilyl; Tf, trifluoromethanesulfonyl; NMO, N-methylmorpholine N-oxide; 2,6-lut, 2,6-lutidine; PPTS,
pyridinium p-toluenesulfonate.
esterified with acryloyl chloride. The resulting acrylate 26 then oxidatively deprotected[24] to 28. Desilylation of 28
was subjected to ring-closing metathesis to 27, which was gave 15, which was also used in the biological evaluations.
Dihydropyrones 1–8, including ent-1 to ent-4, were pre-
pared by esterification of acids 9b–12b with dihydropyrones
14, ent-14 and 28 by means of the Yamaguchi method[25]
(Scheme 3). In the case of compounds 5–8, an additional
desilylation step was required.
When the synthesis of all aforementioned hybrid mo-
lecules was complete, the compounds were investigated in
relation to their cytotoxic activity towards two types of tu-
moral lines and one normal cell line.
Biological Results
Cellular Effects of the Compounds; Cytotoxicity Values
We investigated the cytotoxicity of the hybrid molecules
1–4 and their respective enantiomers (ent-1 to ent-4), 5–8,
as well as combretastatin A-4 derivatives 9a–12a and 9b–
12b. Pironetin analogues 14, ent-14 and 15 were also tested.
Cytotoxicity assays were performed as described in the Ex-
perimental Section by using two types of tumoral cells, the
human colon adenocarcinoma HT-29 and the breast adeno-
carcinoma MCF-7 cell lines. In addition, one normal cell
line, the human embryonic kidney cell line (HEK-293),[26]
was employed in the assays for comparison. Cytotoxicity
Scheme 3. Synthesis of hybrid molecules 1–4, ent-1 to ent-4, and
5–8. Reagents and conditions: (a) 9b–12b, 2,4,6-trichlorobenzoyl
chloride, Et3N, room temp., then addition of the alcohol compo-
nent, room temp., 2 h (1: 38%; 2: 39%; 3: 37%; 4: 35%; ent-1: 42%;
ent-2: 43%; ent-3: 38%; ent-4: 40%; 29: 41%; 30: 40%; 31: 40%;
32: 36%); (b) PPTS (cat.), MeOH, Δ, overnight (5: 86%; 6: 85%;
7: 83%; 8: 81%).
Eur. J. Org. Chem. 2014, 2284–2296
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2287

J. Murga, J. A. Marco et al.
FULL PAPER
values, expressed as the compound concentration [μmol/L] higher the therapeutic safety margin of the compound in
that causes 50% inhibition of cell growth (IC50), are shown the corresponding cell line. Thus, combretastatin A-4 itself
in Table 1. The observed values are, in most cases, in the shows good values of both coefficients, most particularly in
low to medium micromolar range. Compounds ent-1, 3, 7, the case of the MCF-7 cell line. Pironetin shows a similarly
10a, 11a, 12a and 12b showed the highest cytotoxicities for good value for the HT-29 cell line (α), albeit less so for
the HT-29 cell line, with IC50 values that were not very dif- the MCF-7 cell line. Among the compounds with a high
ferent (in some cases even lower) than those of combretas- cytotoxicity (low IC50 values), ent-1, 7, 11a, 12a and 12b
tatin A-4 and pironetin for this particular cell line. In the are particularly worth mentioning because they exhibit high
case of the MCF-7 cell line, the lowest IC50 values were values for both α and β coefficients. Compounds 3 showed
shown by compounds 7, 9b, 10a, 11a, 11b, 12a, 12b and 15. good selectivity only in the case of the HT-29 line (α Ͼ 30),
But a further aspect that is also worth mentioning is the whereas compounds 11b and 15 showed good selectivity in
fact that some of the synthetic compounds were found to the specific case of the MCF-7 line (βϾ60).
be much more toxic for tumoral cells than for normal cells,
The relationship between the structures and the observed
which is clearly a desirable feature. This can be better ap- cytotoxicity deserves comment. In the case of two subsets
preciated with the α and β coefficients, obtained by dividing of hybrid molecules[27] where there are differences in the
the IC50 values of the normal cell line (HEK-293) by those carbon chain length (e.g., 1–4 and 5–8), the cytotoxicity
of one of the tumoral cell lines (see footnote in Table 1). reaches a maximum for compounds 3 and 7, which have a
The higher the value of either the α or β coefficient, the ten-carbon chain in the combretastatin A-4 segment (the
cytotoxicities of the compounds of the enantiomeric series,
ent-1 to ent-4, are not very different to those of 1–4, al-
though the maximum cytotoxicity is found here for ent-1,
Table 1. Cytotoxicity of pironetin analogue/combretastatin A-4 hy-
brids 1–8 and ent-1 to ent-4, combretastatin A-4 derivatives 9a–12a
the compound with the three-carbon chain). It is difficult
to advance an explanation for these observations because
we do not yet know whether the compounds are interacting
with tubulin at the pironetin site (α-tubulin) or at the com-
bretastatin A-4 site (β-tubulin). Although the distance be-
tween the pironetin and the combretastatin A-4 binding
sites is not yet known with certainty, a cursory examination
of the available X-ray data of tubulin[10d] indicates that the
carbon chains of our compounds are not long enough to
permit simultaneous binding to both sites. Thus, the varia-
tion of the cytotoxicity with the length of the carbon chain
may be related to differential interactions with hydrophobic
zones on the tubulin surface. In any case, we have not tried
to prepare derivatives with longer chains because this would
give highly hydrophobic compounds with a too low solubil-
ity.
Although more research in this direction is still needed,
we compared the cytotoxicities of some of the hybrid com-
pounds with the cumulative effect of their separate “mono-
meric” precursors. In the case of the HT-29 cell line, an
equimolecular mixture of 9a and ent-14, the precursors of
ent-1, show higher IC50 values, thus less cytotoxicity, than
that of the latter compound alone. The same result is ob-
served with an equimolecular mixture of 11a and 15, the
precursors of 7. It is not known whether this indicates a
preferential interaction of the hybrid molecules at one or
the other binding site, or even the existence of some type
and 9b–12b, and pyrones 14, ent-14 and 15.[a]
HT-29
MCF-7
HEK-293 α[b],[d] β[c],[d]
CoA4
Pironetin
4.2Ϯ0.5 1Ϯ0.2
25Ϯ3
46Ϯ6
39Ϯ4
80Ϯ14
Ͼ 300
Ͼ 300
114Ϯ5
Ͼ 300
59Ϯ10
72Ϯ3
22Ϯ2
Ͼ 300
22Ϯ2
81Ϯ10
121Ϯ5
67Ϯ2
5.9
5.7
3.2
2.5
Ͼ 30
Ͼ 2.8
32
Ͼ 15
1.6
1.1
1.4
Ͼ 6.8
7.6
1.6
2
1.4
25
8Ϯ2
18Ϯ3
52Ϯ10
Ͼ 300
Ͼ 300
Ͼ 300
2.5
0.7
Ͻ 0.3
–
–
6.7
–
0.8
Ͻ 0.3
0.7
–
3.9
3.8
12
1
12Ϯ2
32Ϯ5
10Ϯ1
107Ϯ5
2
3
4
ent-1
ent-2
ent-3
ent-4
5
3.5Ϯ0.9 17Ϯ2
19Ϯ6
38Ϯ3
64Ϯ7
Ͼ 300
69Ϯ7
Ͼ 300
15.4Ϯ0.5 31Ϯ1
44Ϯ5 Ͼ 300
2.9Ϯ0.2 5.6Ϯ0.1
50.8Ϯ0.6 21Ϯ4
6
7
8
9a
60.1Ϯ0.5 10.4Ϯ0.5
9b
50Ϯ2
2.9Ϯ0.2
23
1
10a
10b
11a
11b
12a
12b
14
3.5Ϯ0.3 9.6Ϯ0.2
9.6Ϯ0.3 2.7
16Ϯ2
8Ϯ1
103Ϯ6
5Ϯ1
17Ϯ4
Ͼ 300
Ͼ 300
Ͼ 300
Ͼ 300
39Ϯ5
35Ϯ7
119Ϯ16
Ͼ 300
49Ϯ7
72Ϯ5
22Ϯ2
10Ϯ3
Ͼ 19 Ͼ 18
Ͼ 37 Ͼ 100
Ͼ 30 Ͼ 60
Ͼ 60 Ͼ 30
20
1.9
1.5
3Ϯ0.6
5Ϯ0.9
9.6Ϯ0.2
1.9Ϯ0.3 4.4Ϯ0.8
8.8
Ͻ 0.7
1.1
109Ϯ20
81Ϯ12
Ͼ 300
109Ϯ8
ent-14
15
9a+ent-14
11a+ent-14 33Ϯ3
9a+15
11a+15
25.4Ϯ0.2 2.5Ϯ0.4
Ͼ 12 Ͼ 120
41Ϯ3
23.9Ϯ0.9
52Ϯ5
1.2
2.2
0.9
0.2
2
1.4
0.7
1.4
25Ϯ2
56Ϯ4
32.2Ϯ0.5
7.1Ϯ0.1
[a] IC50 values are expressed as the compound concentration (μmol/ of synergy. In contrast, equimolecular mixtures of 11 and
L or μm) that causes 50% inhibition of cell growth and are the
ent-14 (the precursors of ent-3), and of 9a and 15 (the pre-
average (Ϯ s.d.) of three different measurements performed as de-
cursors of ent-5), show similar IC50 values to those of the
scribed in the Material and Methods section. The values refer to
hybrid molecules.
the hybrid molecules but also include their monomeric precursors,
As regards the MCF-7 cell line, the cytotoxicities of the
hybrid molecules are similar to those of their precursors
and to those of pironetin itself, but markedly lower than
those of combretastatin A-4. This may indicate that these
hybrid molecules preferentially interact with the tubulin of
such cells at the pironetin site. More definitive answers will
equimolecular mixtures of some of these and the parent natural
compounds combretastatin A-4 (CoA4) and pironetin. Com-
pounds with both high cytotoxicity towards one or both tumoral
cell lines and low cytotoxicity towards the normal cell line have
been highlighted. [b] α = IC50 (HEK-293)/IC50 (HT-29). [c] β = IC50
(HEK-293)/IC50 (MCF-7). [d] Values of α and β have been rounded
to one decimal place.
2288
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296

Pironetin Analogue/Combretastatin A-4 Hybrids
require an accurate knowledge of the geometry and location compounds pironetin and combretastatin A-4. More inter-
of the pironetin binding site.
esting was the fact that, for some of the compounds, the
We have also tested the cytotoxicity of the synthetic in- normal/tumoral cytotoxicity ratio was markedly higher
termediates used for the preparation of the aforementioned than in the case of the two aforementioned natural prod-
hybrid molecules. As regards combretastatin A-4 derivatives ucts, that is, they proved comparatively less cytotoxic
9–12a/b, a dependence of the cytotoxicity on the carbon towards normal cells (these compounds are highlighted in
chain length is also observed (Table 1). For the methyl es- Table 1). This may make these compounds of pharmacolog-
ters (9a–12a), a marked increase in the cytotoxicity is ob- ical interest.
served upon going from the compound with the three-car-
bon chain (9a) to those having longer chains (10a–12a),
with IC50 values of the latter three being very similar (with
both tumoral cell lines) and comparable to those of com-
bretastatin A-4 itself. As for the free acids (9b–12b), the
cytotoxicities vary in a rather erratic way and no systematic
correlation is perceived between the IC50 values and chain
the deuterated solvent (CDCl3) were taken as reference (the singlet
length. Furthermore, marked differences were also observed
Experimental Section
Chemical Procedures
General Experimental Procedures: 1H/13C NMR spectra were re-
corded at 500/125 MHz in CDCl3 solution at 25 °C. The signals of
at δ = 7.25 ppm for 1H NMR spectroscopic data and the triplet
in some cases between the IC50 values for the two tumoral
centered at δ = 77.00 ppm for 13C NMR spectroscopic data). Car-
cell lines.
bon atom types (C, CH, CH2, CH3) were determined with the
The cytotoxicities of the three pironetin analogues 14,
DEPT pulse sequence. High-resolution mass spectra were run by
ent-14 and 15 were also measured. Compound 14 and its
the electrospray technique (ESMS) in either the positive or the
enantiomer, which contain a single stereocenter, represent
the most simplified model possible for pironetin. From the
results presented in Table 1, it is evident that 14 and ent-14
show a very low cytotoxicity, much below of that observed
for the hybrid molecules (1–3, and ent-1 to ent-3) bearing
these fragments (exceptions to this are 4 and ent-4, which
show very high IC50 values, perhaps because of solubility
problems). Although theses conclusions are still provisory,
this seems to point to 1–3 and their enantiomers owing
their cytotoxicity to interactions with tubulin through the
combretastatin A-4 end. As regards compound 15, it has
three stereocenters and corresponds more closely to the pi-
ronetin models we have been investigating in recent years.[5]
The results listed in Table 1 show that its cytotoxicity for
the two tumoral cells is indeed much higher than those of
14/ent-14, particularly in the case of the MCF-7 cell line.
From the hybrid molecules 5–8 having this structural frag-
ment, the highest cytotoxicity is shown by 7, the molecule
that displays the ten-carbon chain, as commented above. It
is also worth noting that the IC50 value of 7 with the HT-
29 cell line is much lower than the IC50 values of both 11b
and 15, its two precursor components. This suggests that
hydrolysis of 7 into 11b and 15 is not taking place within
the cell.
negative ion mode. IR data are given only for compounds with
significant functions (OH, C=O). Optical rotations were measured
at 25 °C. Reactions that required an inert atmosphere were carried
out under N2 with flame-dried glassware. Et2O and THF were
freshly distilled from sodium/benzophenone ketyl and transferred
by using a syringe. Dichloromethane was freshly distilled from
CaH2. Tertiary amines were freshly distilled from KOH. Toluene
was freshly distilled from sodium wire. Commercially available rea-
gents were used as received. Unless detailed otherwise, “workup”
means pouring the reaction mixture into brine, followed by extrac-
tion with the solvent indicated in parenthesis. If the reaction me-
dium was acidic, an additional washing with 5% aq. NaHCO3 was
performed. If the reaction medium was basic, an additional wash-
ing with aq. NH4Cl was performed. Following washing with brine,
drying over anhydrous Na2SO4, and elimination of the solvent un-
der reduced pressure, purification was carried out by chromatog-
raphy on a silica gel column (60–200 μm) and elution with the indi-
cated solvent mixture. Where solutions were filtered through a Ce-
lite pad, the pad was additionally washed with the same solvent
used, and the washings were incorporated with the main organic
layer. All operations (including chromatographic separations) in-
volving combretastatin A-4 and its derivatives were performed with
minimum exposure to light.
Methyl (Z)-4-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]but-
anoate (9a): Combretastatin A-4 13 (3.16 g, 10 mmol) was dissolved
under N2 in anhydrous DMF (150 mL), treated with K2CO3 (3.3 g,
20 mmol) and stirred for 1 h at room temperature. After addition
of methyl 4-bromobutanoate (2.7 g, 15 mmol), the reaction mixture
was further stirred at room temperature for 24 h. Workup (extrac-
tion with Et2O) was followed by column chromatography of the
oily residue on silica gel (hexane/EtOAc, 9:1) to afford 9a (3.83 g,
92%) as an oil. 1H NMR (500 MHz): δ = 6.84 (dd, J = 8.3, 1.8 Hz,
1 H), 6.81 (d, J = 1.8 Hz, 1 H), 6.75 (d, J = 8.3 Hz, 1 H), 6.50 (s,
2 H), 6.47 (d, J = 12 Hz, 1 H), 6.42 (d, J = 12 Hz, 1 H), 3.84 (t, J
= 6.2 Hz, 2 H), 3.82 (s, 3 H), 3.81 (s, 3 H), 3.68 (s, 6 H), 3.65 (s, 3
H), 2.46 (t, J = 7.3 Hz, 2 H), 2.03 (br. quint, J ≈ 6.7 Hz, 2 H) ppm.
13C NMR (125 MHz): δ = 173.4, 152.8 (ϫ2), 148.7, 147.7, 137.1,
132.8, 129.8 (C), 129.6, 128.8, 122.1, 113.9, 111.4, 106.0 (ϫ2) (CH),
Conclusion
We have prepared a set of synthetic hybrid molecules
containing a combretastatin A-4 moiety and a fragment
that is structurally related to the natural product pironetin.
Some of these molecules have been synthesized in both en-
antiomeric forms. Their cytotoxic action (IC50 values)
towards a normal (HEK-293) and two tumoral (HT-29 and
MCF-7) cell lines has then been measured. Whereas most
of the synthetic derivatives proved to be cytotoxic towards
at least one of the two tumoral cell lines, some of them
67.7, 30.4, 24.4 (CH ), 60.7, 55.9 (ϫ3), 51.4 (CH ) ppm. IR: ν
˜
max
2
3
= 1737 (C=O) cm–1. HRMS (ESI): m/z calcd. for C23H28NaO7 [M
showed cytotoxic activities of the same order as the natural + Na+] 439.1728; found 439.1733.
Eur. J. Org. Chem. 2014, 2284–2296
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2289

J. Murga, J. A. Marco et al.
FULL PAPER
Methyl
1
(Z)-8-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]oct- conditions used for the synthesis of 9b. H NMR (500 MHz): δ =
anoate (10a): Obtained as an oil in 92% yield from 13 and methyl
6.87 (dd, J = 8.2, 1.8 Hz, 1 H), 6.86 (d, J = 1.8 Hz, 1 H), 6.79 (d,
J = 8.2 Hz, 1 H), 6.55 (s, 2 H), 6.52 (d, J = 12 Hz, 1 H), 6.47 (d,
8-bromooctanoate under the same conditions used for the synthesis
1
of 9a. H NMR (500 MHz): δ = 6.83 (dd, J = 8.2, 1.8 Hz, 1 H), J = 12 Hz, 1 H), 3.87 (s, 3 H), 3.86 (s, 3 H), 3.82 (t, J = 6.7 Hz, 2
6.82 (d, J = 1.8 Hz, 1 H), 6.75 (d, J = 8.2 Hz, 1 H), 6.51 (s, 2 H),
6.48 (d, J = 12 Hz, 1 H), 6.42 (d, J = 12 Hz, 1 H), 3.82 (s, 3 H), 7 Hz, 2 H), 1.66 (m, 2 H), 1.45–1.35 (br. m, 6 H) (OH signal not
3.81 (s, 3 H), 3.78 (t, J = 6.7 Hz, 2 H), 3.69 (s, 6 H), 3.65 (s, 3 H),
detected) ppm. 13C NMR (125 MHz): δ = 179.5, 152.9 (ϫ2), 148.7,
H), 3.73 (s, 6 H), 2.38 (t, J = 7.5 Hz, 2 H), 1.75 (br. quint, J ≈
2.29 (t, J = 7.5 Hz, 2 H), 1.70 (br. quint, J ≈ 7 Hz, 2 H), 1.62 (m, 148.0, 137.1, 132.9, 129.9 (C), 129.7, 128.7, 121.9, 113.5, 111.4,
2 H), 1.40–1.30 (br. m, 6 H) ppm. 13C NMR (125 MHz): δ = 174.1,
152.8 (ϫ2), 148.7, 148.0, 137.1, 132.9, 129.8 (C), 129.6, 128.7,
106.0 (ϫ2) (CH), 68.7, 34.0, 29.0, 28.9 (ϫ2), 25.7, 24.6 (CH2),
60.8, 55.9 (ϫ3) (CH ) ppm. IR: ν = 3500–2500 (br., OH), 1711
˜
3
max
121.8, 113.5, 111.3, 106.0 (ϫ2) (CH), 68.7, 34.0, 29.0 (ϫ2), 28.9, (C=O) cm–1. HRMS (ESI): m/z calcd. for C26H34O7-H [M – H]–
25.7, 24.8 (CH ), 60.7, 55.9 (ϫ3), 51.3 (CH ) ppm. IR: νmax = 1737 457.2226; found 457.2231.
˜
2
3
(C=O) cm–1. HRMS (ESI): m/z calcd. for C27H36NaO7 [M + Na+]
(Z)-11-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]undecanoic
Acid (11b): Obtained in 76% yield as an oil from 11a under the
same conditions used for the synthesis of 9b. 1H NMR (500 MHz):
δ = 6.85 (dd, J = 8.1, 1.8 Hz, 1 H), 6.84 (d, J = 1.8 Hz, 1 H), 6.77
(d, J = 8.1 Hz, 1 H), 6.53 (s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44
(d, J = 12 Hz, 1 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.80 (t, J = 6.8 Hz,
2 H), 3.70 (s, 6 H), 2.35 (t, J = 7.5 Hz, 2 H), 1.73 (br. quint, J ≈
7 Hz, 2 H), 1.64 (m, 2 H), 1.40–1.25 (br. m, 12 H) (OH signal not
detected) ppm. 13C NMR (125 MHz): δ = 179.5, 152.9 (ϫ2), 148.7,
148.0, 137.1, 132.9, 129.9 (C), 129.8, 128.7, 121.9, 113.5, 111.4,
106.0 (ϫ2) (CH), 68.8, 34.0, 29.4, 29.3 (ϫ2), 29.2, 29.0 (ϫ2), 25.8,
495.2359; found 495.2359.
Methyl (Z)-11-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]un-
decanoate (11a): Obtained in 91% yield from 13 and methyl 11-
bromoundecanoate under the same conditions used for the synthe-
sis of 9a. Solid; m.p. 39–40 °C. 1H NMR (500 MHz): δ = 6.85–6.80
(m, 2 H), 6.75 (d, J = 8.2 Hz, 1 H), 6.51 (s, 2 H), 6.48 (d, J =
12 Hz, 1 H), 6.42 (d, J = 12 Hz, 1 H), 3.82 (s, 3 H), 3.81 (s, 3 H),
3.78 (t, J = 6.7 Hz, 2 H), 3.69 (s, 6 H), 3.65 (s, 3 H), 2.29 (t, J =
7.5 Hz, 2 H), 1.71 (br. quint, J ≈ 7 Hz, 2 H), 1.60 (m, 2 H), 1.40–
1.25 (br. m, 12 H) ppm. 13C NMR (125 MHz): δ = 174.1, 152.8
(ϫ2), 148.7, 148.0, 137.1, 132.9, 129.8 (C), 129.7, 128.6, 121.8,
113.5, 111.3, 106.0 (ϫ2) (CH), 68.7, 34.0, 29.4, 29.3, 29.2, 29.1,
29.0, 28.9, 25.8, 24.8 (CH2), 60.7, 55.9 (ϫ3), 51.3 (CH3) ppm. IR:
24.7 (CH ), 60.8, 55.9 (ϫ3) (CH ) ppm. IR: νmax = 3500–2500 (br.,
˜
2
3
OH), 1709 (C=O) cm–1. HRMS (ESI): m/z calcd. for C29H40O7-H
[M – H]– 499.2696; found 499.2696.
ν
max = 1737 (C=O) cm–1. HRMS (ESI): m/z calcd. for C30H42NaO7
˜
[M + Na+] 537.2828; found 537.2826.
(Z)-16-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]hexadecanoic
Acid (12b): Obtained in 75% yield from 12a under the same condi-
Methyl (Z)-16-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]hexa-
decanoate (12a): Obtained in 90% yield from 13 and methyl 16-
bromohexadecanoate under the same conditions used for the syn-
1
tions used for the synthesis of 9b. Solid; m.p. 88–89 °C. H NMR
(500 MHz): δ = 6.85 (m, 2 H), 6.77 (d, J = 8.1 Hz, 1 H), 6.53 (s, 2
H), 6.50 (d, J = 12 Hz, 1 H), 6.45 (d, J = 12 Hz, 1 H), 3.85 (s, 3
H), 3.84 (s, 3 H), 3.81 (t, J = 6.8 Hz, 2 H), 3.71 (s, 6 H), 2.36 (t, J
= 7.5 Hz, 2 H), 1.74 (br. quint, J ≈ 7 Hz, 2 H), 1.64 (m, 2 H),
1.40–1.25 (br. m, 22 H) (OH signal not detected) ppm. 13C NMR
(125 MHz): δ = 179.2, 152.9 (ϫ2), 148.7, 148.1, 137.2, 132.9, 129.9
(C), 129.8, 128.7, 121.9, 113.5, 111.4, 106.0 (ϫ2) (CH), 68.9, 34.0,
29.6–29.0 (eleven partially overlapped peaks), 25.9, 24.7 (CH2),
1
thesis of 9a. Solid; m.p. 60–61 °C. H NMR (500 MHz): δ = 6.80–
6.75 (m, 2 H), 6.70 (d, J = 8.5 Hz, 1 H), 6.47 (s, 2 H), 6.43 (d, J =
12 Hz, 1 H), 6.37 (d, J = 12 Hz, 1 H), 3.77 (s, 3 H), 3.76 (s, 3 H),
3.73 (t, J = 6.8 Hz, 2 H), 3.64 (s, 6 H), 3.60 (s, 3 H), 2.24 (t, J =
7.5 Hz, 2 H), 1.68 (br. quint, J ≈ 7 Hz, 2 H), 1.56 (m, 2 H), 1.35–
1.20 (br. m, 22 H) ppm. 13C NMR (125 MHz): δ = 173.9, 152.7
(ϫ2), 148.5, 147.8, 137.0, 132.7, 129.6 (C), 129.5, 128.4, 121.6,
113.3, 111.1, 105.8 (ϫ2) (CH), 68.6, 33.8, 29.4–28.8 (eleven par-
tially overlapped peaks), 25.7, 24.7 (CH2), 60.5, 55.7 (ϫ3), 51.0
60.8, 55.9 (ϫ3) (CH ) ppm. IR: ν
= 3500–2500 (br., OH), 1710
˜
3
max
(C=O) cm–1. HRMS (ESI: m/z calcd. for C34H50O7-H [M – H]–
569.3478; found 569.3478.
(CH ) ppm. IR: νmax = 1739 (C=O) cm–1. HRMS (ESI): m/z calcd.
˜
3
for C35H52NaO7 [M + Na+] 607.3610; found 607.3625.
(R)-9-(4-Methoxybenzyloxy)non-1-en-4-ol (17): Allylmagnesium
bromide (1M in Et2O, 15 mL, 15 mmol) was added dropwise under
N2 by using a syringe to a cooled solution of (–)-Ipc2BCl (5.77 g,
ca. 18 mmol) in anhydrous Et2O (75 mL) (dry ice-acetone bath).
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 to
another flask by using a cannula. After cooling the flask to –78 °C,
a solution of aldehyde 16[21] (2.83 g, 12 mmol) in anhydrous Et2O
(35 mL) was added dropwise by using a syringe. The resulting solu-
tion was further stirred at –78 °C for 2 h, then the reaction mixture
was quenched through addition of phosphate pH 7 buffer solution
(15 mL), MeOH (15 mL) and 30% H2O2 (7 mL). After stirring for
30 min, the mixture was poured onto satd. aq. NaHCO3 and
worked up (extraction with Et2O). The residue was subjected to
careful column chromatography on silica gel (hexane, then hexane/
EtOAc, 7:3) to afford 17 (2.87 g, 86%) as an oil. The enantiomeric
ratio was found to be 96:4 by means of NMR analysis of the corre-
(Z)-4-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]butanoic Acid
(9b): Methyl ester 9a (3.33 g, 8 mmol) was dissolved in MeOH
(400 mL) at room temperature. After addition of a 4 m aqueous
solution of NaOH (25 mL, 100 mmol), the reaction mixture was
stirred for 24 h at room temperature. Workup (extraction with
Et2O) was followed by column chromatography of the oily residue
on silica gel (hexane/EtOAc, 9:1) to afford acid 9b (2.57 g, 80%) as
1
an oil. H NMR (500 MHz): δ = 6.87 (dd, J = 8.3, 1.8 Hz, 1 H),
6.82 (d, J = 1.8 Hz, 1 H), 6.77 (d, J = 8.3 Hz, 1 H), 6.52 (s, 2 H),
6.49 (d, J = 12 Hz, 1 H), 6.45 (d, J = 12 Hz, 1 H), 3.86 (t, J =
6.2 Hz, 2 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.70 (s, 6 H), 2.54 (t, J
= 7.3 Hz, 2 H), 2.06 (br. quint, J ≈ 6.7 Hz, 2 H) (OH signal not
detected) ppm. 13C NMR (125 MHz): δ = 178.0, 153.0 (ϫ2), 148.8,
147.7, 137.1, 133.0, 129.9 (C), 129.7, 128.8, 122.4, 114.0, 111.4,
106.0 (ϫ2) (CH), 67.7, 30.4, 24.3 (CH2), 60.9, 55.9 (ϫ3)
(CH ) ppm. IR: ν
= 3500–2500 (br., OH), 1710 (C=O) cm–1.
max
˜
3
HRMS (ESI): m/z calcd. for C22H25O7 [M – H]– 401.1606; found
401.1600.
1
sponding Mosher ester. [α]D = +3.1 (c = 1.15, CHCl3). H NMR
(Z)-8-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]octanoic Acid (500 MHz): δ = 7.26 (br. d, J ≈ 8 Hz, 2 H), 6.88 (d, J ≈ 8 Hz, 2 H),
(10b): Obtained in 78% yield as an oil from 10a under the same 5.83 (m, 1 H), 5.15–5.05 (m, 2 H), 4.44 (s, 2 H), 3.80 (s, 3 H), 3.63
2290
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296

Pironetin Analogue/Combretastatin A-4 Hybrids
(m, 1 H), 3.45 (m, 2 H), 2.30 (m, 1 H), 2.15 (m, 1 H), 1.70–1.60 [α]D = –87.1 (c = 0.89, CHCl3). 1H NMR (500 MHz): δ = 6.88 (m,
(br. m, 3 H), 1.50–1.30 (br. m, 6 H) ppm. 13C NMR (125 MHz): δ
= 158.7, 130.7 (C), 134.8, 129.2 (ϫ2), 113.7 (ϫ2), 70.6 (CH), 118.0,
1 H), 6.02 (br. d, J ≈ 9.8 Hz, 1 H), 4.43 (m, 1 H), 3.65 (t, J =
6.6 Hz, 2 H), 2.40–2.30 (m, 2 H), 1.68 (m, 1 H), 1.70–1.35 (br. m,
72.5, 70.0, 41.9, 36.8, 29.7, 26.2, 25.5 (CH2), 55.2 (CH3) ppm. IR: 8 H) ppm. 13C NMR (125 MHz): δ = 164.6 (C), 145.0, 121.4, 77.8
ν
= 3430 (br., OH) cm–1. HRMS (ESI): m/z calcd. for (CH), 62.7, 34.8, 32.5, 29.4, 25.5, 24.6 (CH ) ppm. IR: νmax = 1712
˜
2
˜
max
C17H26NaO3 [M + Na]+ 301.1780; found 301.1777.
(C=O) cm–1. HRMS (ESI): m/z calcd. for C10H17O3 [M + H]+
185.1178; found 185.1176.
(S)-9-(4-Methoxybenzyloxy)non-1-en-4-ol (ent-17): Obtained with
similar yield and er through the same procedure as above except
that the chiral reagent (+)-Ipc2BCl was used. [α]D = –2.9 (c = 1.65,
CHCl3). Spectral data as for 17.
(6S)-6-(5-Hydroxypentyl)-5,6-dihydro-2H-pyran-2-one (ent-14): Ob-
tained from ent-19 in 67% yield as an oil by using the same pro-
cedure as for the preparation of 14. [α]D = +84.1 (c = 1.3, CHCl3).
Spectral data as for 14.
(R)-9-(4-Methoxybenzyloxy)non-1-en-4-yl Acrylate (18): Alcohol 17
(1.4 g, 5 mmol) was dissolved under N2 in anhydrous CH2Cl2
(100 mL), then cooled to –78 °C and treated sequentially with ethyl
diisopropylamine (2.6 mL, 15 mmol) and acryloyl chloride (800 μL,
10 mmol). The reaction mixture was stirred for 1 h at –78 °C and
then worked up (extraction with CH2Cl2). Column chromatog-
raphy on silica gel (hexane/EtOAc, 7:3) afforded 18 (1.56 g, 94%)
(S)-1-(4-Methoxybenzyloxy)hex-5-en-3-ol (21): Obtained with 85%
yield and 94:6 er as an oil by using the same procedure used for
the preparation of 17. [α]D = –3.6 (c = 1.6, CHCl3). 1H NMR
(500 MHz): δ = 7.27 (br. d, J = 8.3 Hz, 2 H), 6.89 (d, J = 8.3 Hz,
2 H), 5.83 (ddt, J = 17, 10.3, 7.3 Hz, 1 H), 5.15–5.05 (m, 2 H), 4.47
(s, 2 H), 3.85 (m, 1 H), 3.81 (s, 3 H), 3.68 (m, 1 H), 3.61 (m, 1 H),
2.90 (br. s, 1 H, OH), 2.24 (t, J = 6.6 Hz, 2 H), 1.80–1.70 (br. m, 2
H) ppm. 13C NMR (125 MHz): δ = 159.2, 130.1 (C), 134.8, 129.2
(ϫ2), 113.8 (ϫ2), 70.3 (CH), 117.4, 72.8, 68.5, 41.9, 35.8 (CH2),
1
as an oil. [α]D = +12.5 (c = 0.22, CHCl3). H NMR (500 MHz): δ
= 7.25 (br. d, J ≈ 8 Hz, 2 H), 6.87 (d, J ≈ 8 Hz, 2 H), 6.38 (dd, J
= 17.5, 1.5 Hz, 1 H), 6.10 (dd, J = 17.5, 10 Hz, 1 H), 5.80–5.70 (m,
2 H), 5.10–4.95 (m, 3 H), 4.41 (s, 2 H), 3.79 (s, 3 H), 3.42 (t, J =
6.8 Hz, 2 H), 2.40–2.30 (m, 2 H), 1.65–1.50 (br. m, 4 H), 1.40–1.25
(br. m, 4 H) ppm. 13C NMR (125 MHz): δ = 165.8, 159.0, 130.7
(C), 133.6, 130.3, 129.2 (ϫ2), 113.7 (ϫ2), 73.4 (CH), 128.8, 117.6,
72.5, 69.9, 38.5, 33.5, 29.5, 26.0, 25.1 (CH2), 55.2 (CH3) ppm. IR:
55.2 (CH ) ppm. IR: ν
= 3460 (br., OH) cm–1. HRMS (ESI):
max
˜
3
m/z calcd. for C14H20NaO3 [M + Na]+ 259.1310; found 259.1313.
(S)-1-Methoxy-4-[(3-methoxyhex-5-en-1-yloxy)methyl]benzene (22):
Sodium hydride (60% slurry in mineral oil, amount equivalent to
20 mmol) was washed two times under N2 with anhydrous hexane
and once with anhydrous THF. Then, THF (75 mL) was added
and the suspension was cooled in an ice bath. Alcohol 21 (2.36 g,
10 mmol) was then dissolved in anhydrous THF (25 mL) and
added dropwise to the sodium hydride suspension. The mixture
was then allowed to reach room temperature and methyl iodide
(1.87 mL, ca. 30 mmol) was added in one portion and the mixture
was stirred overnight at room temperature. Workup (Et2O) was fol-
lowed by column chromatography on silica gel (hexanes/EtOAc,
9:1) to afford 22 (2.2 g, 88%) as an oil. [α]D = +20.4 (c = 1.4,
CHCl3). 1H NMR (500 MHz): δ = 7.30 (br. d, J = 8.3 Hz, 2 H),
6.92 (d, J = 8.3 Hz, 2 H), 5.86 (ddt, J = 17, 10.3, 7.2 Hz, 1 H),
5.15–5.10 (m, 2 H), 4.48 (s, 2 H), 3.83 (s, 3 H), 3.65–3.55 (m, 2 H),
3.45 (m, 1 H), 3.38 (s, 3 H), 2.30 (t, J = 6.6 Hz, 2 H), 1.85–1.75
(br. m, 2 H) ppm. 13C NMR (125 MHz): δ = 159.1, 130.6 (C),
134.5, 129.1 (ϫ2), 113.6 (ϫ2), 77.4 (CH), 116.9, 72.5, 66.5, 37.8,
33.8 (CH2), 56.6, 55.1 (CH3) ppm. HRMS (ESI): m/z calcd. for
C15H22NaO3 [M + Na]+ 273.1467; found 273.1459.
ν
max = 1718 (C=O) cm–1. HRMS (ESI): m/z calcd. for C20H28NaO4
˜
[M + Na]+ 355.1885; found 355.1880.
(S)-9-(4-Methoxybenzyloxy)non-1-en-4-yl Acrylate (ent-18): Ob-
tained from ent-17 as an oil in 90% yield by using the same pro-
cedure used for the preparation of 18. [α]D = –10.8 (c = 0.2,
CHCl3). Spectral data as for 18.
(6R)-6-[5-(4-Methoxybenzyloxy)pentyl]-5,6-dihydro-2H-pyran-2-one
(19): Diolefin 18 (1.33 g, 4 mmol) was dissolved under N2 in anhy-
drous, degassed CH2Cl2 (500 mL) and treated with Grubbs ruth-
enium catalyst Ru-I (320 mg, ca. 0.4 mmol). The mixture was
stirred and heated at reflux until consumption of the starting mate-
rial (ca. 2–3 h; reaction monitored by TLC). DMSO was then
added (1 mL) and the mixture was further stirred overnight at room
temperature. Solvent removal under reduced pressure and column
chromatography of the residue on silica gel (hexanes/EtOAc, 7:3)
furnished dihydropyrone 19 (987 mg, 81%) as an oil. [α]D = –49.7
1
(c = 1.1, CHCl3). H NMR (500 MHz): δ = 7.25 (br. d, J ≈ 8 Hz,
(4R,6R)-6-Methoxy-8-(4-methoxybenzyloxy)oct-1-en-4-ol (23): Ole-
fin 22 (1.5 g, ca. 6 mmol) was dissolved in a THF/tBuOH/H2O mix-
ture (4:10:1, 120 mL), and the solution was treated with NMO
(820 mg, 7 mmol) and 4% aq. OsO4 (1 mL, ca. 0.15 mmol). The
reaction mixture was then stirred overnight at room temperature
and quenched by the addition of solid Na2SO3 (7.5 g), followed by
additional stirring for 30 min. Extraction with EtOAc was followed
by desiccation of the organic layer on anhydrous MgSO4 and sol-
vent removal under reduced pressure. The crude 1,2-diol was then
dissolved in anhydrous CH2Cl2 (75 mL), cooled in an ice bath and
treated with Pb(OAc)4 (3.1 g, 7 mmol). The reaction mixture was
then stirred at 0 °C until consumption of the starting material (ca.
2–3 h; reaction monitored by TLC). The reaction was then
quenched by addition of solid Na2CO3 (6 g), and the mixture was
stirred for 1 h. Filtration through Celite and solvent removal under
reduced pressure gave a crude aldehyde, which was used without
purification in the next step.
2 H), 6.87 (m, 3 H), 6.00 (br. d, J ≈ 9.8 Hz, 1 H), 4.41 (s, 2 H),
4.40 (m, 1 H), 3.80 (s, 3 H), 3.44 (t, J = 6.5 Hz, 2 H), 2.35–2.25
(m, 2 H), 1.80 (m, 1 H), 1.70–1.35 (br. m, 7 H) ppm. 13C NMR
(125 MHz): δ = 164.5, 159.1, 130.7 (C), 145.0, 129.2 (ϫ2), 121.4,
113.7 (ϫ2), 77.8 (CH), 72.5, 69.8, 34.8, 29.6, 29.3, 26.0, 24.6 (CH2),
55.2 (CH ) ppm. IR: ν
= 1719 (C=O) cm–1. HRMS (ESI): m/z
max
˜
3
calcd. for C18H24NaO4 [M + Na]+ 327.1572; found 327.1576.
(6S)-6-[5-(4-Methoxybenzyloxy)pentyl]-5,6-dihydro-2H-pyran-2-one
(ent-19): Obtained from ent-18 as an oil in 80% yield by using the
same procedure used for the preparation of 19. [α]D = +21.8 (c =
0.15, CHCl3). Spectral data as for 19.
(6R)-6-(5-Hydroxypentyl)-5,6-dihydro-2H-pyran-2-one (14): PMB
derivative 19 (152 mg, 0.5 mmol) was dissolved in a CH2Cl2/H2O
mixture (20:1, 10 mL) and treated with DDQ (160 mg, ca.
0.7 mmol). The mixture was then stirred at room temperature until
consumption of the starting material (45–60 min; reaction moni-
tored by TLC). Workup (extraction with CH2Cl2) was followed by
column chromatography of the oily residue on silica gel (hexanes/
Allylmagnesium bromide (1M in Et2O, 8 mL, 8 mmol) was added
dropwise under N2 by using a syringe to a cooled solution of (+)-
EtOAc, from 1:1 to 1:3) to provided 19 (64 mg, 69%) as an oil. Ipc2BCl (3.2 g, ca. 10 mmol) in anhydrous Et2O (50 mL) (dry ice-
Eur. J. Org. Chem. 2014, 2284–2296
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2291

J. Murga, J. A. Marco et al.
FULL PAPER
acetone bath). 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 mag-
nesium chloride took place. The supernatant solution was carefully
transferred to another flask by using a cannula. After cooling this
flask at –78 °C, a solution of the crude aldehyde from above dis-
solved in anhydrous Et2O (15 mL) was added dropwise by using a
syringe. The resulting solution was further stirred at –78 °C for 2 h.
The reaction mixture was quenched through addition of phosphate
pH 7 buffer solution (40 mL), MeOH (40 mL) and 30 % H2O2
(20 mL). After stirring for 30 min, the mixture was poured onto
satd. aq. NaHCO3 and worked up (extraction with Et2O). The resi-
due was subjected to careful column chromatography on silica gel
(hexane/EtOAc, 4:1) to afford 23 (1.58 g, 90% overall from 22) as
an 86:14 mixture of diastereoisomers, which was used as such in
the next step. An aliquot was purified for analytical purposes: oil.
[α]D = –2.2 (c = 1.3, CHCl3). 1H NMR (500 MHz): δ = 7.28 (br. d,
J = 8.3 Hz, 2 H), 6.90 (d, J = 8.3 Hz, 2 H), 5.84 (ddt, J = 17, 10.3,
7.2 Hz, 1 H), 5.15–5.10 (m, 2 H), 4.45 (s, 2 H), 3.94 (m, 1 H), 3.81
(s, 3 H), 3.67 (br. qd, J ≈ 6.5, 4 Hz, 1 H), 3.60–3.50 (m, 2 H), 3.38
(s, 3 H), 3.00 (br. s, 1 H, OH), 2.30–2.20 (m, 2 H), 1.95 (dq, J =
14, 6.3 Hz, 1 H), 1.80 (m, 1 H), 1.71 (ddd, J = 14, 9.5, 4 Hz, 1 H),
1.60 (ddd, J = 14, 7, 3 Hz, 1 H) ppm. 13C NMR (125 MHz): δ =
159.1, 130.4 (C), 134.8, 129.1 (ϫ2), 113.7 (ϫ2), 76.6, 67.9 (CH),
117.4, 72.6, 66.4, 42.2, 39.2, 33.5 (CH2), 57.0, 55.1 (CH3) ppm. IR:
from 25 under the same conditions used for the preparation of 18.
[α]D = +16.9 (c = 1.75, CHCl3). 1H NMR (500 MHz): δ = 7.24
(br. d, J = 8.3 Hz, 2 H), 6.85 (d, J = 8.3 Hz, 2 H), 6.34 (dd, J =
17.5, 1.5 Hz, 1 H), 6.07 (dd, J = 17.5, 10.5 Hz, 1 H), 5.80–5.70 (m,
2 H), 5.10–5.05 (m, 3 H), 4.41 (br. s, 2 H), 3.85 (m, 1 H), 3.80 (s,
3 H), 3.55–3.40 (br. m, 3 H), 3.30 (s, 3 H), 2.40–2.30 (br. m, 2 H),
1.85–1.65 (br. m, 5 H), 1.57 (ddd, J = 14, 6.5, 4.5 Hz, 1 H), 0.87
(s, 9 H), 0.04 (s, 3 H), 0.03 (s, 3 H) ppm. 13C NMR (125 MHz): δ
= 165.6, 159.1, 130.6, 18.0 (C), 133.3, 130.3, 129.2 (ϫ 2), 113.7
(ϫ2), 75.2, 71.1, 66.8 (CH), 128.9, 117.1, 72.7, 66.5, 43.0, 41.8,
39.0, 33.9 (CH2), 56.1, 55.2, 25.9 (ϫ3), –4.3, –4.4 (CH3) ppm. IR:
ν
˜
= 1723 (C=O) cm– 1 . HRMS (ESI): m/z calcd. for
m a x
C28H46NaO6Si [M + Na]+ 529.2961; found 529.2963.
(6S)-6-{(2S,4R)-[2-(tert-Butyldimethylsilyloxy)-4-methoxy-6-(4-
methoxybenzyloxy)hexyl]}-5,6-dihydro-2H-pyran-2-one (27): Ob-
tained from 26 in 83% yield by using the same procedure used for
the preparation of 19: oil. [α]D = –14 (c = 1, CHCl3). 1H NMR
(500 MHz): δ = 7.25 (br. d, J = 8.3 Hz, 2 H), 6.88 (m, 3 H), 6.00
(br. d, J ≈ 9.8 Hz, 1 H), 4.58 (m, 1 H), 4.42 (s, 2 H), 4.09 (m, 1 H),
3.78 (s, 3 H), 3.55–3.45 (m, 2 H), 3.40 (m, 1 H), 3.30 (s, 3 H), 2.30
(m, 2 H), 2.00 (ddd, J = 14, 9.3, 3.5 Hz, 1 H), 1.85–1.60 (br. m, 5
H), 0.88 (s, 9 H), 0.07 (s, 3 H), 0.06 (s, 3 H) ppm. 13C NMR
(125 MHz): δ = 164.1, 159.1, 130.5, 18.0 (C), 145.1, 129.2 (ϫ2),
121.4, 113.7 (ϫ2), 75.2, 74.4, 65.9 (CH), 72.6, 66.3, 43.3, 43.2, 33.9,
29.9 (CH ), 56.1, 55.2, 25.9 (ϫ3), –4.4, –4.5 (CH ) ppm. IR: ν
˜
max
2
3
ν
˜
= 3450 (br., OH) cm–1. HRMS (ESI): m/z calcd. for
max
= 1728 (C=O) cm–1. HRMS (ESI): m/z calcd. for C26H42NaO6Si
C17H26NaO4 [M + Na]+ 317.1729; found 317.1723.
[M + Na]+ 501.2648; found 501.2653.
(4R,6R)-4-(tert-Butyldimethylsilyloxy)-6-methoxy-8-(4-methoxy-
benzyloxy)oct-1-ene (24): Alcohol 23 (1.18 g, 4 mmol) was dissolved
under N2 in anhydrous CH2Cl2 (30 mL), cooled in an ice bath, 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 2 h at 0 °C and worked up (extraction with CH2Cl2). Column
chromatography on silica gel (hexane/EtOAc, 9:1) afforded 24
(1.3 g, 80%) as an oil. [α]D = –25.6 (c = 1.5, CHCl3). 1H NMR
(500 MHz): δ = 7.28 (br. d, J = 8.3 Hz, 2 H), 6.90 (d, J = 8.3 Hz,
2 H), 5.84 (ddt, J = 17, 10.3, 7.2 Hz, 1 H), 5.10–5.05 (m, 2 H), 4.45
(s, 2 H), 3.95 (m, 1 H), 3.82 (s, 3 H), 3.60–3.50 (m, 3 H), 3.36 (s, 3
H), 2.30–2.20 (br. m, 2 H), 1.86 (dq, J = 14, 6.3 Hz, 1 H), 1.77 (m,
1 H), 1.64 (ddd, J = 14, 8.8, 3.5 Hz, 1 H), 1.52 (ddd, J = 14, 8.8,
3.5 Hz, 1 H), 0.92 (s, 9 H), 0.10 (s, 3 H), 0.09 (s, 3 H) ppm. 13C
NMR (125 MHz): δ = 159.1, 130.6, 18.1 (C), 134.8, 129.2 (ϫ2),
113.7 (ϫ 2), 74.9, 68.5 (CH), 117.0, 72.7, 66.5, 42.6, 42.0, 33.6
(CH2), 56.0, 55.2, 25.9 (ϫ3), –4.1, –4.7 (CH3) ppm. HRMS (ESI):
m/z calcd. for C23H40NaO4Si [M + Na]+ 431.2594; found 431.2591.
(6S)-6-{(2S,4R)-[2-(tert-Butyldimethylsilyloxy)-6-hydroxy-4-
methoxyhexyl]}-5,6-dihydro-2H-pyran-2-one (28): Obtained from 27
in 74% yield by using the same procedure as for the preparation
of 14: amorphous solid; [α]D = –2.2 (c = 1, CHCl3). 1H NMR
(500 MHz): δ = 6.89 (dt, J = 9.5, 4.5 Hz, 1 H), 6.01 (dt, J = 9.5,
1.8 Hz, 1 H), 4.59 (m, 1 H), 4.08 (m, 1 H), 3.80–3.70 (m, 2 H), 3.47
(m, 1 H), 3.34 (s, 3 H), 2.40 (br. s, 1 H, OH), 2.33 (m, 2 H), 1.98
(ddd, J = 14, 9.6, 3.3 Hz, 1 H), 1.90–1.75 (br. m, 2 H), 1.70–1.60
(br. m, 3 H), 0.88 (s, 9 H), 0.09 (s, 3 H), 0.07 (s, 3 H) ppm. 13C
NMR (125 MHz): δ = 164.1, 18.0 (C), 145.2, 121.4, 77.2, 74.4, 65.7
(CH), 60.1, 43.0, 42.6, 35.7, 29.9 (CH2), 56.2, 25.8 (ϫ3), –4.4, –4.6
(CH ) ppm. IR: νmax = 1720 (C=O) cm–1. HRMS (ESI): m/z calcd.
˜
3
for C18H34NaO5Si [M + Na]+ 381.2073; found 381.2064.
(6S)-6-[(2S,4R)-2,6-Dihydroxy-4-methoxyhexyl]-5,6-dihydro-2H-
pyran-2-one (15): Silylated compound 28 (72 mg, 0.2 mmol) was
dissolved in MeOH (8 mL) and treated with PPTS (10 mg,
0.04 mmol) and water (0.1 mL). The mixture was then heated at
reflux for 18 h, cooled, and neutralized by addition of solid
NaHCO3 (10 mg). After filtering, the solution was evaporated un-
der reduced pressure, and the oily residue was subjected to column
chromatography on silica gel (hexane/EtOAc, 1:1) to provide 15
(4S,6R,8R)-6-(tert-Butyldimethylsilyloxy)-8-methoxy-10-(4-meth-
oxybenzyloxy)dec-1-en-4-ol (25): Obtained (75% of a 76:24 mixture
of diastereoisomers) from olefin 24 under the same conditions used
for the preparation of 23: oil. [α]D = –25.7 (c = 2.5, CHCl3). 1H
NMR (500 MHz): δ = 7.26 (br. d, J = 8.3 Hz, 2 H), 6.88 (d, J =
8.3 Hz, 2 H), 5.84 (ddt, J = 17, 10.3, 7.2 Hz, 1 H), 5.15–5.05 (m, 2
H), 4.43 (s, 2 H), 4.17 (m, 1 H), 4.03 (m, 1 H), 3.80 (s, 3 H), 3.55–
3.45 (m, 3 H), 3.42 (m, 1 H), 3.30 (s, 3 H), 2.30–2.15 (br. m, 2 H),
1.86 (dq, J = 14, 6.5 Hz, 1 H), 1.80–1.65 (br. m, 4 H), 1.57 (ddd, J
= 14, 4, 2.5 Hz, 1 H), 0.90 (s, 9 H), 0.11 (s, 3 H), 0.09 (s, 3 H) ppm.
13C NMR (125 MHz): δ = 159.1, 130.4, 17.8 (C), 134.8, 129.1 (ϫ2),
113.7 (ϫ2), 75.0, 69.0, 67.8 (CH), 117.1, 72.7, 66.3, 42.3, 41.8, 41.1,
1
(42 mg, 85%) as an oil; [α]D = –7.1 (c = 0.71, CHCl3). H NMR
(500 MHz): δ = 6.89 (ddd, J = 9.8, 5.5, 3.5 Hz, 1 H), 6.01 (dt, J =
9.8, 1.5 Hz, 1 H), 4.72 (m, 1 H), 4.23 (tt, J = 9.5, 2.5 Hz, 1 H),
3.80–3.70 (br. m, 3 H), 3.45 (br. s, 1 H, OH), 3.39 (s, 3 H), 2.60
(br. s, 1 H, OH), 2.40–2.35 (m, 2 H), 1.95–1.60 (br. m, 6 H) ppm.
13C NMR (125 MHz): δ = 164.5 (C), 145.4, 121.3, 77.9, 75.0, 64.7
(CH), 59.8, 42.8, 39.8, 35.5, 30.0 (CH ), 56.8 (CH ) ppm. IR: ν
˜
max
2
3
= 3400 (br., OH), 1710 (C=O) cm–1. HRMS (ESI): m/z calcd. for
C12H20NaO5Si [M + Na]+ 267.1208; found 267.1209.
33.4 (CH ), 55.9, 55.1, 25.8 (ϫ3), –4.5, –4.9 (CH ) ppm. IR: ν
˜
max
2
3
5-[(2R)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-4-[2-Methoxy-
5-(3,4,5-trimethoxystyryl)phenoxy]butanoate (1): Acid 9b (104 mg,
0.25 mmol) was dissolved under N2 in anhydrous THF (5 mL) and
= 3460 (br., OH) cm–1. HRMS (ESI): m/z calcd. for C25H44NaO5Si
[M + Na]+ 475.2856; found 475.2851.
(4S,6S,8R)-6-(tert-Butyldimethylsilyloxy)-8-methoxy-10-(4-meth- treated sequentially with Et3N (105 μL, 0.75 mmol) and 2,4,6-tri-
oxybenzyloxy)dec-1-en-4-yl Acrylate (26): Obtained in 91% yield chlorobenzoyl chloride (80 μL, ca. 0.5 mmol). The mixture was
2292
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296

Pironetin Analogue/Combretastatin A-4 Hybrids
then stirred at room temperature for 3 h. Subsequently, a solution
of alcohol 14 (41 mg, 0.22 mmol) and DMAP (61 mg, 0.5 mmol)
in anhydrous THF (2 mL) was added dropwise. The mixture was
then further stirred at room temperature for 2 h. Workup (extrac-
tion with Et2O) was followed by column chromatography of the
oily residue on silica gel (hexane/EtOAc, 3:2) to provide ester 1
(54 mg, 38% based on 14) as an amorphous solid. [α]D = +30.9 (c
= 1.88, CHCl3). 1H NMR (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.76
(d, J = 8.3 Hz, 1 H), 6.52 (s, 2 H), 6.49 (d, J = 12 Hz, 1 H), 6.44
(d, J = 12 Hz, 1 H), 6.01 (br. d, J ≈ 9.8 Hz, 1 H), 4.40 (m, 1 H),
4.07 (t, J = 6.6 Hz, 2 H), 3.86 (t, J = 6.4 Hz, 2 H), 3.83 (s, 6 H),
3.70 (s, 6 H), 2.48 (t, J = 7.3 Hz, 2 H), 2.32 (m, 2 H), 2.05 (br. quint,
J ≈ 7 Hz, 2 H), 1.80 (m, 1 H), 1.70–1.35 (br. m, 7 H) ppm. 13C
NMR (125 MHz): δ = 173.1, 164.4, 152.9 (ϫ2), 148.7, 147.8, 137.2,
132.9, 129.9 (C), 144.9, 129.6, 128.9, 122.2, 121.5, 114.0, 111.5,
106.0 (ϫ2), 77.8 (CH), 67.8, 64.3, 34.7, 30.7, 29.4, 28.5, 25.7, 24.5
ν
max = 1721 (C=O) cm–1. HRMS (ESI): m/z calcd. for C39H54NaO9
˜
[M + Na+] 689.3666; found 689.3665.
5-[(2S)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-11-[2-Meth-
oxy-5-(3,4,5-trimethoxystyryl)phenoxy]undecanoate (ent-3): Ob-
tained in 38% yield from 11b and ent-14 under the same conditions
used for the preparation of 1: amorphous solid. [α]D = –20.3 (c =
1.86, CHCl ). IR: ν
= 1718 (C=O) cm–1. HRMS (ESI): m/z
˜
3
max
calcd. for C39H54NaO9 [M + Na+] 689.3666; found 689.3671.
NMR spectroscopic data as for 3.
5-[(2R)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-16-[2-Meth-
oxy-5-(3,4,5-trimethoxystyryl)phenoxy]hexadecanoate (4): Obtained
in 35% yield from 12b and 14 under the same conditions used for
the preparation of 1: amorphous solid. [α]D = +17.4 (c = 2, CHCl3).
1H NMR (500 MHz): δ = 6.85–6.80 (m, 3 H), 6.74 (d, J = 8.3 Hz,
1 H), 6.50 (s, 2 H), 6.47 (d, J = 12 Hz, 1 H), 6.41 (d, J = 12 Hz, 1
H), 6.00 (br. d, J ≈ 9.8 Hz, 1 H), 4.39 (m, 1 H), 4.04 (t, J = 6.6 Hz,
2 H), 3.81 (s, 3 H), 3.80 (s, 3 H), 3.77 (t, J = 6.8 Hz, 2 H), 3.68 (s,
6 H), 2.35–2.25 (m, 4 H), 1.80–1.50 (br. m, 10 H), 1.45–1.20 (br. m,
24 H) ppm. 13C NMR (125 MHz): δ = 173.9, 164.4, 152.9 (ϫ2),
148.7, 148.0, 137.1, 132.9, 129.9 (C), 144.9, 129.7, 128.7, 121.8,
121.5, 113.5, 111.3, 106.0 (ϫ2), 77.7 (CH), 68.8, 64.0, 34.7, 34.3,
29.6–29.0 (twelve partially overlapped signals), 28.5, 25.9, 25.7,
(ϫ 2) (CH ), 60.8, 55.9 (ϫ 3) (CH ) ppm. IR: ν = 1731
˜
max
2
3
(C=O) cm–1. HRMS (ESI): m/z calcd. for C32H40NaO9 [M + Na+]
591.2570; found 591.2570.
5-[(2S)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-4-[2-Methoxy-
5-(3,4,5-trimethoxystyryl)phenoxy]butanoate (ent-1): Obtained in
42% yield from 9b and ent-14 under the same conditions used for
the preparation of 1: amorphous solid. [α]D = –25 (c = 1.74,
24.9, 24.5 (CH ), 60.8, 55.9 (ϫ 3) (CH ) ppm. IR: ν = 1726
˜
max
2
3
CHCl ). IR: νmax = 1726 (C=O) cm–1. HRMS (ESI): m/z calcd. for
(C=O) cm–1. HRMS (ESI): m/z calcd. for C44H64NaO9 [M + Na+]
˜
3
C32H40NaO9 [M + Na+] 591.2570; found 591.2567. NMR spectro-
759.4448; found 759.4456.
scopic data as for 1.
5-[(2S)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-16-[2-Meth-
oxy-5-(3,4,5-trimethoxystyryl)phen-oxy]hexadecanoate (ent-4): Ob-
tained in 40% yield from 12b and ent-14 under the same conditions
used for the preparation of 1: amorphous solid. [α]D = –21.2 (c =
5-[(2R)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-8-[2-Methoxy-
5-(3,4,5-trimethoxystyryl)phenoxy]octanoate (2): Obtained in 39%
yield from 10b and 14 under the same conditions used for the prep-
aration of 1: amorphous solid. [α]D = +24.9 (c = 2, CHCl3). 1H
NMR (500 MHz): δ = 6.85–6.80 (m, 3 H), 6.75 (d, J = 8.3 Hz, 1
H), 6.51 (s, 2 H), 6.48 (d, J = 12 Hz, 1 H), 6.42 (d, J = 12 Hz, 1
H), 6.00 (br. d, J ≈ 9.8 Hz, 1 H), 4.40 (m, 1 H), 4.05 (t, J = 6.6 Hz,
2 H), 3.82 (s, 3 H), 3.81 (s, 3 H), 3.78 (t, J = 6.8 Hz, 2 H), 3.69 (s,
6 H), 2.35–2.25 (m, 4 H), 1.80 (m, 1 H), 1.75–1.30 (br. m, 17
H) ppm. 13C NMR (125 MHz): δ = 173.7, 164.3, 152.9 (ϫ2), 148.6,
147.9, 137.2, 132.9, 129.9 (C), 144.9, 129.7, 128.7, 121.8, 121.4,
113.5, 111.3, 106.0 (ϫ2), 77.7 (CH), 68.7, 64.0, 34.7, 34.2, 29.3
(ϫ2), 29.0 (ϫ2), 28.5, 25.7, 25.6, 24.8, 24.4 (CH2), 60.8, 55.9 (ϫ3)
1.68, CHCl ). IR: ν
= 1726 (C=O) cm–1. HRMS (ESI): m/z
˜
3
max
calcd. for C44H64NaO9 [M + Na+] 759.4448; found 759.4456.
NMR spectroscopic data as for 4.
(3R,5S)-5-(tert-Butyldimethylsilyloxy)-3-methoxy-6-[(S)-6-oxo-
3,6-dihydro-2H-pyran-2-yl]hexyl 4-[2-Methoxy-5-(3,4,5-trimethoxy-
styryl)phenoxy]butanoate (29): Obtained in 41% yield from 9b and
28 under the same conditions used for the preparation of 1:
amorphous solid. [α]D = –24.5 (c = 0.2, CHCl3). 1H NMR
(500 MHz): δ = 6.90–6.80 (m, 3 H), 6.76 (d, J = 8.2 Hz, 1 H), 6.50
(s, 2 H), 6.48 (d, J = 12 Hz, 1 H), 6.43 (d, J = 12 Hz, 1 H), 6.00
(br. d, J ≈ 9.8 Hz, 1 H), 4.58 (m, 1 H), 4.20–4.10 (m, 3 H), 3.87 (t,
J = 6 Hz, 2 H), 3.83 (s, 6 H), 3.70 (s, 6 H), 3.39 (m, 1 H), 3.30 (s,
3 H), 2.48 (t, J = 7.3 Hz, 2 H), 2.32 (m, 2 H), 2.10–1.95 (br. m, 3
H), 1.82 (m, 2 H), 1.75–1.55 (br. m, 3 H), 0.88 (s, 9 H), 0.10 (s, 3
H), 0.08 (s, 3 H) ppm. 13C NMR (125 MHz): δ = 173.0, 164.2,
152.9 (ϫ2), 148.8, 148.0, 137.2, 132.9, 130.0, 18.0 (C), 145.1, 129.6,
128.9, 122.2, 121.5, 114.1, 111.5, 106.0 (ϫ2), 74.8, 74.4, 65.9 (CH),
67.8, 61.1, 43.3, 43.2, 32.7, 30.7, 30.0, 24.5 (CH2), 60.8, 56.1, 55.9
(CH ) ppm. IR: νmax = 1726 (C=O) cm–1. HRMS (ESI): m/z calcd.
˜
3
for C36H48NaO9 [M + Na+] 647.3196; found 647.3196.
5-[(2S)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-8-[2-Methoxy-
5-(3,4,5-trimethoxystyryl)phenoxy]octanoate (ent-2): Obtained in
43% yield from 10b and ent-14 under the same conditions used for
the preparation of 1: amorphous solid. [α]D = –25.4 (c = 1.81,
CHCl ). IR: νmax = 1726 (C=O) cm–1. HRMS (ESI): m/z calcd. for
˜
3
C36H48NaO9 [M + Na+] 647.3196; found 647.3193. NMR spectro-
scopic data as for 2.
(ϫ 3), 25.9 (ϫ 3), –4.4, –4.5 (CH ) ppm. IR: ν
= 1729
˜
3
max
(C=O) cm–1. HRMS (ESI): m/z calcd. for C40H58NaO11Si [M +
5-[(2R)-6-Oxo-3,6-dihydro-2H-pyran-2-yl]pentyl (Z)-11-[2-Meth-
oxy-5-(3,4,5-trimethoxystyryl)phenoxy]undecanoate (3): Obtained in
37% yield from 11b and 14 under the same conditions used for the
preparation of 1: amorphous solid. [α]D = +21.1 (c = 1.3, CHCl3).
1H NMR (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.74 (d, J = 8.3 Hz,
Na+] 765.3646; found 765.3639.
(3R,5S)-5-(tert-Butyldimethylsilyloxy)-3-methoxy-6-[(S)-6-oxo-
3,6-dihydro-2H-pyran-2-yl]hexyl 8-[2-Methoxy-5-(3,4,5-trimethoxy-
styryl)phenoxy]octanoate (30): Obtained in 40% yield from 10b and
1 H), 6.52 (s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44 (d, J = 12 Hz, 1 28 under the same conditions used for the preparation of 1:
H), 6.02 (br. d, J ≈ 9.8 Hz, 1 H), 4.41 (m, 1 H), 4.07 (t, J = 6.6 Hz, amorphous solid. [α]D = –7.2 (c = 0.8, CHCl3). 1H NMR
2 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.80 (t, J = 6.8 Hz, 2 H), 3.70 (s, (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.76 (d, J = 8 Hz, 1 H), 6.52
6 H), 2.35–2.25 (m, 4 H), 1.80 (m, 1 H), 1.75–1.55 (br. m, 9 H), (s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44 (d, J = 12 Hz, 1 H), 6.02
1.50–1.25 (br. m, 14 H) ppm. 13C NMR (125 MHz): δ = 173.9, (br. d, J ≈ 9.8 Hz, 1 H), 4.58 (m, 1 H), 4.20–4.10 (m, 3 H), 3.84 (s,
164.4, 152.9 (ϫ 2), 148.7, 148.0, 137.2, 132.9, 129.9 (C), 144.9,
129.7, 128.7, 121.8, 121.5, 113.5, 111.4, 106.0 (ϫ2), 77.7 (CH),
68.8, 64.0, 34.7, 34.3, 29.4–29.0 (seven partially overlapped signals),
28.5, 25.9, 25.7, 24.9, 24.5 (CH2), 60.8, 55.9 (ϫ3) (CH3) ppm. IR:
3 H), 3.83 (s, 3 H), 3.78 (t, J = 6.6 Hz, 2 H), 3.70 (s, 6 H), 3.40 (m,
1 H), 3.31 (s, 3 H), 2.35–2.25 (br. m, 4 H), 2.00 (ddd, J = 14, 9.3,
3.5 Hz, 1 H), 1.85–1.55 (br. m, 9 H), 1.45–1.30 (br. m, 6 H), 0.89
(s, 9 H), 0.10 (s, 3 H), 0.08 (s, 3 H) ppm. 13C NMR (125 MHz): δ
Eur. J. Org. Chem. 2014, 2284–2296
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2293

J. Murga, J. A. Marco et al.
FULL PAPER
= 173.7, 164.2, 152.9 (ϫ2), 148.6, 148.0, 137.0, 132.9, 129.8, 18.0
ate (6): Obtained in 85% yield from 30 under the same conditions
(C), 145.1, 129.7, 128.7, 121.8, 121.4, 113.4, 111.2, 105.9 (ϫ2), used for the preparation of 15: oil. [α]D = –6.3 (c = 0.54, CHCl3).
74.8, 74.4, 65.8 (CH), 68.7, 60.7, 43.3, 43.2, 34.3, 32.7, 30.0, 29.0
1H NMR (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.77 (d, J = 8.4 Hz,
1 H), 6.52 (s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44 (d, J = 12 Hz, 1
H), 6.02 (br. d, J ≈ 9.8 Hz, 1 H), 4.74 (m, 1 H), 4.28 (br. t, J ≈
9 Hz, 1 H), 4.17 (t, J = 6.5 Hz, 2 H), 3.84 (s, 3 H), 3.83 (s, 3 H),
3.80 (t, J = 6.8 Hz, 2 H), 3.71 (s, 6 H), 3.60 (m, 1 H), 3.37 (s, 3 H),
3.20 (br. s, 1 H, OH), 2.38 (m, 2 H), 2.30 (t, J = 7.5 Hz, 2 H), 2.00
(m, 1 H), 1.90–1.50 (br. m, 7 H), 1.45–1.20 (br. m, 8 H) ppm. 13C
NMR (125 MHz): δ = 173.7, 164.3, 152.9 (ϫ2), 148.7, 148.0, 137.2,
132.9, 129.9 (C), 145.1, 129.7, 128.7, 121.9, 121.4, 113.6, 111.4,
106.0 (ϫ2), 76.5, 74.9, 64.5 (CH), 68.8, 61.0, 43.0, 39.7, 34.3, 32.4,
30.0, 29.7, 29.1, 29.0, 25.8, 24.8 (CH2), 60.9, 57.1, 55.9 (ϫ 3)
(ϫ3), 25.8, 24.8 (CH2), 60.8, 56.1, 55.9 (ϫ3), 25.9 (ϫ3), –4.4, –4.5
(CH ) ppm. IR: νmax = 1728 (C=O) cm–1. HRMS (ESI): m/z calcd.
˜
3
for C44H66NaO11Si [M + Na+] 821.4272; found 821.4277.
(3R,5S)-5-(tert-Butyldimethylsilyloxy)-3-methoxy-6-[(S)-6-oxo-
3,6-dihydro-2H-pyran-2-yl]hexyl 11-[2-Methoxy-5-(3,4,5-trimeth-
oxystyryl)phenoxy]undecanoate (31): Obtained in 40% yield from
11b and 28 under the same conditions used for the preparation
of 1: amorphous solid. [α]D = –8.3 (c = 1.23, CHCl3). 1H NMR
(500 MHz): δ = 6.90–6.80 (m, 3 H), 6.76 (d, J = 8.1 Hz, 1 H), 6.52
(s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44 (d, J = 12 Hz, 1 H), 6.02
(dt, J = 9.8, 1.5 Hz, 1 H), 4.58 (m, 1 H), 4.20–4.10 (m, 3 H), 3.84
(s, 3 H), 3.83 (s, 3 H), 3.79 (t, J = 6.7 Hz, 2 H), 3.70 (s, 6 H), 3.40
(m, 1 H), 3.31 (s, 3 H), 2.35–2.25 (br. m, 4 H), 2.00 (ddd, J = 14,
9.3, 3.5 Hz, 1 H), 1.82 (m, 2 H), 1.75–1.60 (br. m, 7 H), 1.40–1.25
(br. m, 12 H), 0.88 (s, 9 H), 0.10 (s, 3 H), 0.08 (s, 3 H) ppm. 13C
NMR (125 MHz): δ = 173.7, 164.2, 152.9 (ϫ2), 148.7, 148.0, 137.0,
132.9, 129.8, 18.0 (C), 145.1, 129.7, 128.7, 121.8, 121.4, 113.5,
111.4, 105.9 (ϫ2), 74.9, 74.4, 65.9 (CH), 68.8, 60.8, 43.3, 43.2, 34.3,
32.7, 30.0, 29.5–29.0 (six partially overlapped signals), 25.9, 24.8
(CH2), 60.7, 56.1, 56.0 (ϫ3), 25.8 (ϫ3), –4.4, –4.5 (CH3) ppm. IR:
(CH ) ppm. IR: ν
= 3500 (br., OH), 1726 (C=O) cm–1. HRMS
max
˜
3
(ESI): m/z calcd. for C38H52NaO11 [M + Na+] 707.3407; found
707.3408.
(3R,5S)-5-Hydroxy-3-methoxy-6-[(S)-6-oxo-3,6-dihydro-2H-pyran-
2-yl]hexyl 11-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]undec-
anoate (7): Obtained in 83% yield from 31 under the same condi-
tions used for the preparation of 15: oil. [α]D = –4.2 (c = 1.1,
1
CHCl3). H NMR (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.77 (d, J
= 8 Hz, 1 H), 6.52 (s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44 (d, J =
12 Hz, 1 H), 6.02 (br. d, J ≈ 9.8 Hz, 1 H), 4.75 (m, 1 H), 4.28 (br. t,
J = 9.5 Hz, 1 H), 4.17 (t, J = 6.6 Hz, 2 H), 3.85 (s, 3 H), 3.83 (s, 3
H), 3.80 (t, J = 6.8 Hz, 2 H), 3.71 (s, 6 H), 3.60 (m, 1 H), 3.38 (s,
3 H), 3.10 (br. s, 1 H, OH), 2.38 (m, 2 H), 2.30 (t, J = 7.5 Hz, 2
H), 2.00 (m, 1 H), 1.90–1.55 (br. m, 9 H), 1.40–1.20 (br. m, 12
H) ppm. 13C NMR (125 MHz): δ = 173.8, 164.3, 152.9 (ϫ2), 148.7,
148.0, 137.2, 132.9, 129.9 (C), 145.1, 129.7, 128.7, 121.9, 121.4,
113.6, 111.4, 106.0 (ϫ2), 76.6, 74.9, 64.5 (CH), 68.8, 61.0, 43.0,
39.6, 34.3, 32.4, 30.0, 29.5–29.0 (six partially overlapped signals),
ν
= 1732 (C=O) cm– 1 . HRMS (ESI): m/z calcd. for
˜
m a x
C47H72NaO11Si [M + Na+] 863.4742; found 863.4744.
(3R,5S)-5-(tert-Butyldimethylsilyloxy)-3-methoxy-6-[(S)-6-oxo-
3,6-dihydro-2H-pyran-2-yl]hexyl 16-[2-Methoxy-5-(3,4,5-trimeth-
oxystyryl)phenoxy]hexadecanoate (32): Obtained in 36% yield from
12b and 28 under the same conditions used for the preparation
of 1: amorphous solid. [α]D = –2.2 (c = 0.62, CHCl3). 1H NMR
(500 MHz): δ = 6.90–6.80 (m, 3 H), 6.77 (d, J = 8.1 Hz, 1 H), 6.52
(s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.44 (d, J = 12 Hz, 1 H), 6.02
(br. d, J ≈ 9.8 Hz, 1 H), 4.58 (m, 1 H), 4.20–4.10 (m, 3 H), 3.84 (s,
3 H), 3.83 (s, 3 H), 3.80 (t, J = 6.6 Hz, 2 H), 3.70 (s, 6 H), 3.40 (m,
1 H), 3.32 (s, 3 H), 2.35–2.25 (br. m, 4 H), 2.00 (ddd, J = 14, 9.5,
3.7 Hz, 1 H), 1.85 (m, 2 H), 1.75–1.55 (br. m, 7 H), 1.40–1.25
25.9, 25.0 (CH ), 60.9, 57.1, 56.0 (ϫ3) (CH ) ppm. IR: νmax = 1726
˜
2
3
(C=O) cm–1. HRMS (ESI): m/z calcd. for C41H58NaO11 [M + Na+]
749.3877; found 749.3871.
(3R,5S)-5-Hydroxy-3-methoxy-6-[(S)-6-oxo-3,6-dihydro-2H-pyran-
(br. m, 22 H), 0.89 (s, 9 H), 0.10 (s, 3 H), 0.08 (s, 3 H) ppm. 13C 2-yl]hexyl 16-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]hexa-
NMR (125 MHz): δ = 173.7, 164.2, 152.9 (ϫ2), 148.7, 148.1, 137.1, decanoate (8): Obtained in 81% yield from 32 under the same con-
132.9, 129.9, 18.0 (C), 145.1, 129.8, 128.7, 121.8, 121.5, 113.5, ditions used for the preparation of 15: oil. [α]D = –2 (c = 0.88,
1
111.4, 106.0 (ϫ2), 74.9, 74.5, 65.9 (CH), 68.8, 60.7, 43.3, 43.2, 34.3, CHCl3). H NMR (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.77 (d, J
32.7, 30.0, 29.7–29.0 (eleven partially overlapped signals), 25.9, 24.9
= 8 Hz, 1 H), 6.52 (s, 2 H), 6.50 (d, J = 12 Hz, 1 H), 6.45 (d, J =
(CH2), 60.8, 56.1, 56.0 (ϫ3), 25.8 (ϫ3), –4.4, –4.5 (CH3) ppm. IR: 12 Hz, 1 H), 6.02 (br. d, J ≈ 9.8 Hz, 1 H), 4.75 (m, 1 H), 4.28 (br. t,
ν
= 1733 (C=O) cm– 1 . HRMS (ESI): m/z calcd. for
J ≈ 9 Hz, 1 H), 4.17 (t, J = 6.6 Hz, 2 H), 3.85 (s, 3 H), 3.84 (s, 3
H), 3.81 (t, J = 6.8 Hz, 2 H), 3.71 (s, 6 H), 3.60 (m, 1 H), 3.38 (s,
3 H), 3.10 (br. s, 1 H, OH), 2.38 (m, 2 H), 2.30 (t, J = 7.5 Hz, 2
H), 2.00 (m, 1 H), 1.90–1.50 (br. m, 11 H), 1.40–1.20 (br. m, 20
H) ppm. 13C NMR (125 MHz): δ = 173.9, 164.3, 152.9 (ϫ2), 148.7,
148.1, 137.2, 133.0, 129.9 (C), 145.1, 129.8, 128.8, 121.9, 121.4,
113.6, 111.4, 106.0 (ϫ2), 76.6, 74.9, 64.5 (CH), 68.9, 61.0, 43.0,
39.6, 34.3, 32.4, 30.0, 29.7–29.0 (eleven partially overlapped sig-
˜
m a x
C52H82NaO11Si [M + Na+] 933.5524; found 933.5533.
(3R,5S)-5-Hydroxy-3-methoxy-6-[(S)-6-oxo-3,6-dihydro-2H-pyran-
2-yl]hexyl 4-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]butano-
ate (5): Obtained in 86% yield from 29 under the same conditions
used for the preparation of 15: oil. [α]D = –0.9 (c = 0.79, CHCl3).
1H NMR (500 MHz): δ = 6.90–6.80 (m, 3 H), 6.77 (d, J = 8.2 Hz,
1 H), 6.52 (s, 2 H), 6.49 (d, J = 12 Hz, 1 H), 6.45 (d, J = 12 Hz, 1
H), 6.02 (br. d, J ≈ 9.8 Hz, 1 H), 4.74 (m, 1 H), 4.26 (m, 1 H), 4.18
(m, 2 H), 3.88 (t, J = 6 Hz, 2 H), 3.83 (s, 6 H), 3.71 (s, 6 H), 3.60
(m, 1 H), 3.37 (s, 3 H), 3.10 (br. s, 1 H, OH), 2.50 (t, J = 7.5 Hz,
2 H), 2.37 (m, 2 H), 2.10–1.95 (br. m, 2 H), 1.90–1.65 (br. m, 3 H),
1.65–1.45 (br. m, 3 H) ppm. 13C NMR (125 MHz): δ = 173.1,
164.3, 152.9 (ϫ 2), 148.8, 147.8, 137.2, 132.9, 130.0 (C), 145.1,
129.6, 128.9, 122.2, 121.5, 114.1, 111.5, 106.0 (ϫ2), 76.5, 74.9, 64.5
(CH), 67.8, 61.2, 43.0, 39.6, 32.3, 30.7, 30.1, 24.5 (CH2), 60.8, 57.1,
nals), 25.9, 25.0 (CH ), 60.9, 57.1, 55.9 (ϫ3) (CH ) ppm. IR: ν
˜
max
2
3
= 3470 (br., OH), 1726 (C=O) cm–1. HRMS (ESI): m/z calcd. for
C46H68NaO11 [M + Na+] 819.4659; found 819.4679.
Biological Procedures
Cell Culture: Cell culture media were purchased from Gibco
(Grand Island, NY, USA). Fetal bovine serum (FBS) was a product
of Harlan–Seralab (Belton, U.K.). Supplements and other chemi-
cals 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 μg/mL and stored at –20 °C
until use.
55.9 (ϫ 3) (CH ) ppm. IR: ν
= 3470 (br., OH), 1726
˜
3
m a x
(C=O) cm–1. HRMS (ESI): m/z calcd. for C34H44NaO11 [M + Na+]
651.2781; found 651.2777.
(3R,5S)-5-Hydroxy-3-methoxy-6-[(S)-6-oxo-3,6-dihydro-2H-pyran-
2-yl]hexyl 8-[2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy]octano-
2294
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296

Pironetin Analogue/Combretastatin A-4 Hybrids
[7]
a) A. Cirla, J. Mann, Nat. Prod. Rep. 2003, 20, 558–564; b)
V. Srivastava, A. S. Negi, J. K. Kumar, M. M. Gupta, S. P. S.
Khanuja, Bioorg. Med. Chem. 2005, 13, 5892–5908; c) G. U.
Dachs, A. J. Steele, C. Coralli, C. Kanthou, A. C. Brooks, S. P.
Gunningham, M. J. Currie, A. I. Watson, B. A. Robinson,
G. M. Tozer, BMC Cancer 2006, 6, 280–290; d) G. C. Tron, T.
Pirali, G. Sorba, F. Pagliai, S. Busacca, A. A. Genazzani, J.
Med. Chem. 2006, 49, 3033–3044; e) Y. Shan, J. Zhang, Z. Liu,
M. Wang, Y. Dong, Curr. Med. Chem. 2011, 18, 523–538.
Cell lines were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing glucose (1 g/L), glutamine (2 mm), penicillin
(50 IU/mL), streptomycin (50 μg/mL) and amphoterycin (1.25 μg/
mL), supplemented with 10% FBS.
Cytotoxicity Assays: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO)
dye reduction assay in 96-well microplates was used, as described
previously.[28] Approximately 5ϫ103 cells of HT-29, MCF-7 or
HEK-293 cells in a total volume of 100 μL of their respective
growth media were incubated with serial dilutions of the tested
compounds. After 3 d incubation (37 °C, 5% CO2 in a humid at-
mosphere), 10 μL MTT (5 mg/mL in PBS) was added to each well
and the plate was incubated for a further 4 h (37 °C). The resulting
formazan was dissolved in 0.04 n HCl/2-propanol (150 μL) and
read at 550 nm. All determinations were carried out in triplicate.
[8]
[9]
Y. Fu, S. Li, Y. Zu, G. Yang, Z. Yang, M. Luo, S. Jiang, M.
Wink, T. Efferth, Curr. Med. Chem. 2009, 16, 3966–3985.
a) M. A. Jordan, Curr. Med. Chem. Anticancer Drugs 2002, 2,
1–17; b) M. Abal, J. M. Andreu, I. Barasoain, Curr. Cancer
Drug Targets 2003, 3, 193–203.
a) J. J. Correia, S. Lobert, Curr. Pharm. Des. 2001, 7, 1213–
1228; b) J. Jiménez-Barbero, F. Amat-Guerri, J. P. Snyder, Curr.
Med. Chem. Anticancer Drugs 2002, 2, 91–122; c) J. F. Díaz,
J. M. Andreu, J. Jiménez-Barbero, Top. Curr. Chem. 2009, 286,
121–149; d) B. Gigant, A. Cormier, A. Dorléans, R. B. G. Rav-
elli, M. Knossow, Top. Curr. Chem. 2009, 286, 259–278; e)
E. M. Daly, R. E. Taylor, Curr. Chem. Biol. 2009, 3, 367–379.
[10]
Supporting Information (see footnote on the first page of this arti-
1
cle): Copies of H and 13C NMR spectra of all new compounds.
[11]
a) V. M. Sánchez-Pedregal, C. Griesinger, Top. Curr. Chem.
2009, 286, 151–208; b) J. H. Nettles, K. H. Downing, Top. Curr.
Chem. 2009, 286, 209–257; c) M. Botta, S. Forli, M. Magnani,
F. Manetti, Top. Curr. Chem. 2009, 286, 279–328.
F. Sarabia, M. García-Castro, A. Sánchez-Ruiz, Curr. Bioact.
Compd. 2006, 2, 269–299.
Acknowledgments
Financial support was granted by the Spanish Government (Minis-
terio de Economía y Competitividad (MEC), grant numbers
CTQ2008-02800 and CTQ2011-27560), by the Consellería d’Em-
presa, Universitat i Ciencia de la Generalitat Valenciana (grant
numbers PROMETEO/2013/027 and ACOMP09/113) and by the
Universitat Jaume I, Castellón, Spain (grant numbers P1-1B-2008-
14 and PI-1B-2011-37). S. T.-G. thanks the Generalitat Valenciana
for a predoctoral fellowship of the Santiago Grisolía program. C. V.
thanks the Ministerio de Economía y Competitividad for a predoc-
toral fellowship of the FPI program. J. A. M. thanks the COST
Action CM0804 for aid in establishing cooperations with other re-
search groups.
[12]
[13]
[14]
H. J. Anderson, J. E. Coleman, R. J. Andersen, M. Roberge,
Cancer Chemother. Pharmacol. 1997, 39, 223–226.
a) M. Kondoh, T. Usui, S. Kobayashi, K. Tsuchiya, K. Nishi-
kawa, T. Nishikiori, T. Mayumi, H. Osada, Cancer Lett. 1998,
126, 29–32; b) M. Kondoh, T. Usui, T. Nishikiori, T. Mayumi,
H. Osada, Biochem. J. 1999, 340, 411–416; c) H. Watanabe,
H. Watanabe, T. Usui, M. Kondoh, H. Osada, T. Kitahara,
J. Antibiot. 2000, 53, 540–545; d) T. Usui, H. Watanabe, H.
Nakayama, Y. Tada, N. Kanoh, M. Kondoh, T. Asao, K.
Takio, H. Watanabe, K. Nishikawa, T. Kitahara, H. Osada,
Chem. Biol. 2004, 11, 799–806.
a) L. M. Rossi, P. Rangasamy, J. Zhang, X.-Q. Qiu, G. Y. Wu,
J. Pharm. Sci. 2007, 97, 1060–1070; b) P. Courvalin, J. Int.
Med. Res. 2008, 264, 4–16; c) M. Gualtieri, F. Baneres-Roquet,
P. Villain-Guillot, M. Pugniere, J.-P. Leonetti, Curr. Med.
Chem. 2009, 16, 390–393.
[1] M. Garcia, A. Jemal, E. M. Ward, M. M. Center, Y. Hao, R. L.
Siegel, M. J. Thun, Global Cancer Facts & Figures 2007, Ameri-
can Cancer Society, Atlanta, GA, 2007.
[2] a) D. Hanahan, R. A. Weinberg, Cell 2000, 100, 57–70; b)
M. R. Stratton, P. J. Campbell, P. A. Futreal, Nature 2009, 458,
719–724; c) D. Hanahan, R. A. Weinberg, Cell 2011, 144, 646–
674.
[15]
[16]
[17]
a) M. Kavallaris, Nat. Rev. Cancer 2010, 10, 194–204; b) C.
Holohan, S. Van Schaeybroeck, D. B. Longley, P. G. Johnston,
Nat. Rev. Cancer 2013, 13, 714–726.
[3] a) F. T. Boyle, G. F. Costello, Chem. Soc. Rev. 1998, 27, 251–
261; b) J. B. Gibbs, Science 2000, 287, 1969–1973.
a) K. Yasui, Y. Tamura, K. Nakatani, K. Kawada, M. Ohtani,
J. Org. Chem. 1995, 60, 7567–7574; b) M. K. Gurjar, J. T.
Henri Jr., D. S. Bose, A. V. R. Rao, Tetrahedron Lett. 1996, 37,
6615–6618; c) N. Chida, M. Yoshinaga, T. Tobe, S. Ogawa,
Chem. Commun. 1997, 1043–1044; d) H. Watanabe, H. Watan-
abe, M. Bando, M. Kido, T. Kitahara, Tetrahedron 1999, 55,
9755–9776; e) G. E. Keck, C. E. Knutson, S. A. Wiles, Org.
Lett. 2001, 3, 707–710; f) L. C. Dias, L. G. de Oliveira, M. A.
de Sousa, Org. Lett. 2003, 5, 265–268; g) X. Shen, A. S. Was-
muth, J. Zhao, C. Zhu, S. G. Nelson, J. Am. Chem. Soc. 2006,
128, 7438–7439; h) D. Enders, S. Dhulut, D. Steinbusch, A.
Herrbach, Chem. Eur. J. 2007, 13, 3942–3949; i) C. Bressy, J.-
P. Vors, S. Hillebrand, S. Arseniyadis, J. Cossy, Angew. Chem.
2008, 120, 10291; Angew. Chem. Int. Ed. 2008, 47, 10137–
10140; j) M. T. Crimmins, A.-M. R. Dechert, Org. Lett. 2009,
11, 1635–1638.
S. Torijano-Gutiérrez, Ph. D. Thesis, University Jaume I, 2013.
K. Gaukroger, J. A. Hadfield, L. A. Hepworth, N. J. Lawrence,
A. T. McGown, J. Org. Chem. 2001, 66, 8135–8138.
a) P. V. Ramachandran, G.-M. Chen, H. C. Brown, Tetrahedron
Lett. 1997, 38, 2417–2420; b) for pinane-based versatile “allyl”
boranes, see:P. V. Ramachandran, Aldrichim. Acta 2002, 35,
23–35.
[4] a) L. Z. Penn, Curr. Opin. Invest. Drugs 2001, 2, 684–692; b)
B. Zhou, Z.-L. Liu, Pure Appl. Chem. 2005, 77, 1887–1903; c)
H.-J. Park, H.-J. Jung, K.-T. Lee, J. Choi, Nat. Prod. Sci. 2006,
12, 175–192; d) L. Portt, G. Norman, C. Clapp, M. Green-
wood, M. T. Greenwood, Biochim. Biophys. Acta 2011, 1813,
238–259; e) F. Torres-Andón, B. Fadeel, Acc. Chem. Res. 2013,
46, 733–742.
[5] For additional comments on tubulin structure and function,
see our previous papers in this area, see: a) J. A. Marco, J.
García-Pla, M. Carda, J. Murga, E. Falomir, C. Trigili, S. Not-
ararigo, J. F. Díaz, I. Barasoain, Eur. J. Med. Chem. 2011, 46,
1630–1637; b) M. Carda, J. Murga, S. Díaz-Oltra, J. García-
Pla, J. Paños, E. Falomir, C. Trigili, J. F. Díaz, I. Barasoain,
J. A. Marco, Eur. J. Org. Chem. 2013, 1116–1123; c) M. Carda,
J. Murga, J. Paños, C. A. Angulo-Pachón, J. García-Pla, S.
Díaz-Oltra, J. A. Marco, C. Trigili, M. Redondo-Horcajo, J. F.
Díaz, I. Barasoain, Curr. Med. Chem. 2013, 20, 1173–1182; d)
J. Paños, S. Díaz-Oltra, M. Sánchez-Peris, J. García-Pla, J.
Murga, E. Falomir, M. Carda, M. Redondo-Horcajo, J. F.
Díaz, I. Barasoain, J. A. Marco, Org. Biomol. Chem. 2013, 11,
5809–5826.
[18]
[19]
[20]
[6] J. Chen, T. Liu, X. Dong, Y. Hu, Mini-Rev. Med. Chem. 2009,
9, 1174–1190.
Eur. J. Org. Chem. 2014, 2284–2296
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2295

J. Murga, J. A. Marco et al.
FULL PAPER
[21] D. Tripathi, S. K. Pandey, P. Kumar, Tetrahedron 2009, 65,
2226–2231.
[24] K. Horita, T. Yoshioka, T. Tanaka, Y. Oikawa, O. Yonemitsu,
Tetrahedron 1986, 42, 3021–3028.
[22] For general reviews on metathesis, some with particular em-
phasis in RCM, see: a) A. Fürstner, Angew. Chem. 2000, 112,
3140; Angew. Chem. Int. Ed. 2000, 39, 3012–3043; b) L. Jafarp-
our, S. P. Nolan, Adv. Organomet. Chem. 2000, 46, 181–222; c)
T. M. Trnka, R. H. Grubbs, Acc. Chem. Res. 2001, 34, 18–29;
d) Handbook of Metathesis (Ed.: R. H. Grubbs), Wiley-VCH,
Weinheim, Germany, 2003; e) R. H. Grubbs, Tetrahedron 2004,
60, 7117–7140; f) D. Astruc, New J. Chem. 2005, 29, 42–56; g)
A. H. Hoveyda, A. R. Zhugralin, Nature 2007, 450, 243–251;
[25] J. Inanaga, K. Hirata, H. Saeki, T. Katsuki, M. Yamaguchi,
Bull. Chem. Soc. Jpn. 1979, 52, 1989–1993.
[26] N. Arden, M. J. Betenbaugh, Trends Biotechnol. 2004, 22, 174–
180.
[27] For some selected reviews on the interest and utility of hybrid
molecules in medicine and pharmacology, see: a) S. Hanessian,
ChemMedChem 2006, 1, 1300–1330; b) B. Meunier, Acc. Chem.
Res. 2008, 41, 69–77; c) M. Decker, Curr. Med. Chem. 2011,
18, 1464–1475.
h) Metathesis in Natural Product Synthesis (Eds.: J. Cossy, S. [28] S. Rodríguez-Nieto, M. A. Medina, A. R. Quesada, Anticancer
Arseniyadis, C. Meyer), Wiley-VCH, Weinheim, Germany,
2010.
[23] R. D. Cink, C. J. Forsyth, J. Org. Chem. 1997, 62, 5672–5673.
Res. 2001, 21, 3457–3460.
Received: November 16, 2013
Published Online: February 6, 2014
2296
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2284–2296