Fluorescent phenylpolyene analogues of the ether phospholipid edelfosine for the selective labeling of cancer cells

Article abstract of DOI:10.1021/jm049808a

Edelfosine (ET-18-OCH₃), a synthetic antitumor ether lipid, is taken up by malignant but not by normal cells, triggering apoptosis in a large variety of human tumor cells. The synthesis of the first fluorescent edelfosine analogue (6), with apo

Full text of DOI:10.1021/jm049808a

© Copyright 2004 by the American Chemical Society
Volume 47, Number 22
October 21, 2004
Letters
to distinguish specific cytotoxic effects from nonspecific
interactions, such as the lytic disruption of cell mem-
branes. This limitation has now been overcome with the
finding3-5 of the selective proapoptotic effect on tumor
cells of edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-
3-phosphocholine, or ET-18-OCH3), a prototype for
synthetic antitumor ether lipids.
F lu or escen t P h en ylp olyen e An a logu es of
th e Eth er P h osp h olip id Ed elfosin e for
th e Selective La belin g of Ca n cer Cells
Ernesto Quesada,† J avier Delgado,† Consuelo Gajate,‡
Faustino Mollinedo,‡ A. Ulises Acun˜a,*,§ and
Francisco Amat-Guerri*,†
Instituto de Quı´mica Orga´nica, C.S.I.C.,
J uan de la Cierva 3, E-28006 Madrid, Spain, Centro de
Investigacio´n del Ca´ncer, Instituto de Biolog´ıa Molecular y
Celular del Ca´ncer, C.S.I.C.-Universidad de Salamanca,
Campus Miguel de Unamuno, E-37007 Salamanca, Spain,
and Instituto de Quı´mica Fı´sica Rocasolano, C.S.I.C.,
Serrano 119, E-28006 Madrid, Spain
Since apoptosis is a specific event that can be timed
and quantitated, the detection of an apoptotic response
provides a reliable signal and marker for the putative
antitumor activity of different ether lipids. Furthermore,
the apoptotic activity of edelfosine seems to account for
most of its antitumor activity.3 This remarkable selec-
tivity has been linked to the unidirectional uptake of
the ether lipid by the neoplastic cells, with an efficiency
of about an order of magnitude higher than that of
noncancer cells.3,4 However, the molecular mechanisms
involved in this process, as well as that of the antine-
oplastic and antiparasitic activities of edelfosine and
related EL, remain to be elucidated.
Fluorescent analogues of drugs are very useful tools
for unveiling their mechanism of action and therapeutic
targets,6 as well as for the development of new diag-
nostic assays. In the case of edelfosine, a fluorescent
analogue might also be of utility in the selective labeling
of tumor metastasis. Since the available information on
structure-apoptotic activity relationship on edelfosine
is rather scarce,3 we reasoned that replacement of the
C18 aliphatic chain by a lipophilic fluorescent group of
similar length might preserve drug activity and selec-
tivity. Previous studies have shown that the O-octadecyl
chain at C1 of edelfosine can hold some modifications,
including the presence of a double bond, preserving the
apoptotic activity.3 On these grounds, a conjugated
pentaene group may appear as a convenient candidate,
considering that this fluorophore has led to useful
Received March 8, 2004
Abst r a ct : Edelfosine (ET-18-OCH3), a synthetic antitumor
ether lipid, is taken up by malignant but not by normal cells,
triggering apoptosis in a large variety of human tumor cells.
The synthesis of the first fluorescent edelfosine analogue (6),
with apoptotic activity comparable to that of the parent drug,
is described. Fluorescence microscopy experiments show that
6 selectively labels human cancer cells, accumulating into
specific domains of the plasma membrane.
Ether glycerophospholipids may have one or two
hydrocarbon chains linked to the glycerol framework by
an ether bond.1 A number of synthetic ether lipids (EL),
collectively known as alkyl ether phospholipids or alkyl-
lysophospholipids, show antitumor activity as well as
other useful pharmacological properties,2 including a
potent antiparasitic effect. Interestingly, some EL have
been shown to inhibit in a selective way the proliferation
of cancer cells from a wide variety of solid tumors and
leukemias.3 However, because the therapeutic concen-
trations of these EL are usually close to their critical
micelle concentration (ca. 10-6 M), it has been difficult
* To whom correspondence should be addressed. For A.U.A.: phone,
+34915619400 (+1220); fax, +34915642431; e-mail, roculises@
iqfr.csic.es. For F.A.-G.: phone, +34915622900 (+394); fax,
+34915644853; e-mail, famat@iqog.csic.es.
† Instituto de Qu´ımica Orga´nica.
‡ Centro de Investigacio´n del Ca´ncer.
§ Instituto de Qu´ımica F´ısica Rocasolano.
10.1021/jm049808a CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/21/2004

5334 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Letters
Sch em e 1a
a
Reagents and conditions: (a) 2/1 mole ratio ) 1.3, Pd(PPh3)2Cl2 (7.5 mol %), CuI (25 mol %), Et2NH, THF, Ar, room temp, 2 h, 90%;
(b) Zn(Cu/Ag), MeOH/H2O 1:1, room temp, 24 h, 95%; (c) I2 (trace), hexane, Ar, 15 min reflux, 95%; (d) DIBAL-H, MePh, Ar, 0 °C, 93-
99%; (e) (i) 2-chloro-2-oxo-1,3,2-dioxaphospholane, Me3N, acetonitrile, pressure tube, -78 °C; (ii) room temp, 2 h, and 70 °C, 1 h, 23-35%.
fluorescent probes for lipid membranes.7,8 But this group
requires UV excitation at a wavelength (ca. 340 nm) not
readily available in a standard fluorescence microscope.
Here, we report the synthesis of the first fluorescent
analogue of edelfosine, with a conjugated ω-phenyltetra-
ene group instead of the n-octadecyl substituent in
position 1 (6, Scheme 1). Analogue 6 is characterized
by a strong absorption band at ca. 360 nm, a wavelength
widely used in fluorescence microscopy, and a bluish
fluorescence (λmax ≈ 442 nm) with a modest but still
adequate quantum yield (∼0.3 in a lipid bilayer). The
emitting ether lipid 6 showed both the selectivity for
F igu r e 1. Comparative apoptotic activity of edelfosine, the
cancer cells and the ensuing apoptotic activity of the
parent drug. The relatively simple synthetic method
allows for other fluorescent analogues with diverse
aromatic groups, chain length, number of conjugated
double bonds, or stereochemistry.
fluorescent analogue 6, and the inactive alcohol 5 in human
T-leukemic J urkat cells. Induction of apoptosis was determined
as the percentage of cells with a DNA content of less than G1
(hypodiploidy) in cell cycle analysis. Points were averaged
((SD) from three independent experiments. For further details
of the apoptotic assay, see refs 4 and 17.
Analogue 6 was obtained in five steps (Scheme 1), of
which the first three have been previously used in this
laboratory8 and in others9 for the synthesis of related
conjugated polyenes and represent an additional ex-
ample of the application of the acetylenic approach. The
first step was a Sonogashira-Hagihara cross-cou-
pling10,11 between the phenylbromotriene 1 (in the form
of 3:7 (1E)/(1Z) mixture of isomers) and the cis- or trans-
1,3-dioxane 2, with a terminal acetylenic group at the
chain in position 2. cis-2 and trans-2 were simulta-
neously obtained by acetalization of non-8-ynal with rac-
2-O-methylglycerol. The initial 3:7 (E)/(Z) ratio of 1 was
not changed by the cross-coupling reaction, and cis-3
and trans-3 were respectively isolated as the same (E)/
(Z) mixture. Pure isomers all-(E)-cis-3 and all-(E)-
trans-3 were easily separated from each (E)/(Z) mixture
by precipitation from a dichloromethane solution with
n-pentane. Partial reduction of the triple bond with
activated Zn (Boland method)12 in any of the isomeric
trienyne compounds 3 or mixtures thereof yielded the
corresponding (7′Z, ...)-tetraene, which after iodine
isomerization gave the respective all-(E)-cis-4 or all-(E)-
trans-4 isomers. The subsequent acetal cleavage/reduc-
tion of any cis/trans isomer 4 with DIBAL-H13 produced
the all-(E) alcohol 5. All the above reactions had >90%
yield. Eventually, one-pot reaction of 5 with 2-chloro-
2-oxo-1,3,2-dioxaphospholane and trimethylamine14 gave
the pure fluorescent analogue 6 with 23-35% yield.15
Compounds 3-6 are thermally stable for months if kept
frozen in a dark place.
drug edelfosine (Figure 1), whereas the control alcohol
5, lacking the essential phosphocholine headgroup, was
inactive. The apoptotic response of 6 was further
evidenced by the typical internucleosomal DNA frag-
mentation in multiples of 180-200 bp (data not shown).
In addition, no previous effects were observed in any
cell cycle phase in the concentration range assayed here.
On the other hand, analogue 6 spared normal peripheral
blood lymphocytes, inducing less than 2% apoptosis.
The selective uptake of the fluorescent analogue 6 was
demonstrated by comparing in vitro its incorporation
into cancer versus normal nontransformed cells. Human
T-leukemic J urkat cells have been reported to incorpo-
rate high levels of edelfosine, whereas normal human
peripheral blood lymphocytes failed to take up signifi-
cant amounts of the lipid.4,16 Thus, both cell types were
incubated with the fluorescent analogue 6, and after
exhaustive washing to remove the loose cell-surface-
bonded ether lipid, the cells were analyzed by fluores-
cence microscopy.17 Whereas a faint labeling could be
hardly observed in normal cells (Figure 2a), 6 was taken
up by the plasma membrane of leukemic cells in large
quantities (Figure 2b) from which it could be reversibly
displaced by adding edelfosine. Interestingly, the fluo-
rescent signal from leukemic cells was not homoge-
neously distributed over the membrane but accumulated
into well-defined domains of the cell plasma membrane.
The lateral segregation of the ether lipid, which can
easily be detected with fluorescent analogue 6, may be
related to its ability to cluster membrane lipid rafts18
and suggests its putative enrichment in these lipid
Analogue 6 induced apoptosis in human T-leukemia
J urkat cells, although to a lower extent than the parent

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5335
(7) (a) Souto, A. A.; Acun˜a, A. U.; Amat-Guerri, F. A general and
practical synthesis of linear conjugated pentaenoic acids. Tet-
rahedron Lett. 1994, 35, 5907-5910. (b) Reyes Mateo, C.; Souto,
A. A.; Amat-Guerri, F.; Acun˜a, A. U. New fluorescent octadeca-
pentaenoic acids as probes of lipid membranes and protein-lipid
interactions. Biophys. J . 1996, 71, 2177-2191.
(8) (a) Quesada, E.; Acun˜a, A. U.; Amat-Guerri, F. New transmem-
brane polyene bolaamphiphiles as fluorescent probes in lipid
bilayers. Angew. Chem., Int. Ed. 2001, 40, 2095-2097. (b)
Quesada, E.; Acun˜a, A. U.; Amat-Guerri, F. Synthesis of car-
boxyl-tethered symmetric conjugated polyenes as fluorescent
transmembrane probes of lipid bilayers. Eur. J . Org. Chem.
2003, 1308-1318.
(9) Crousse, B.; Alami, M.; Linstrumelle, G. Stereocontrolled syn-
thesis of (E,E,E)-chlorotrienes: Efficient intermediates for the
construction of all E conjugated polyenes. Tetrahedron Lett.
1997, 38, 5297-5300.
(10) (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. A convenient
synthesis of acetylenes: catalytic substitutions of acetylenic
hydrogen with bromoalkenes, iodoarenes and bromopyridines.
Tetrahedron Lett. 1975, 16, 4467-4470. (b) Takahashi, S.;
Kuroyama, Y.; Sonogashira, K.; Hagihara, N. A convenient
synthesis of ethynylarenes and diethynylarenes. Synthesis 1980,
627-630.
F igu r e 2. Fluorescence images of (a) normal human periph-
eral blood lymphocytes and (b) human T-leukemic J urkat cells,
both treated for 7 h with 20 µM fluorescent edelfosine analogue
6.17 Practical detection limit was ∼5 µM. The bar represents
10 µm.
domains. Further studies on the biochemical basis of
this selectivity are in progress.
Ack n ow led gm en t. This work was supported by
Grants BQU2000-1500, BQU2003-4413, FIS-02/1199,
and 1FD97-0622 from the Ministerio de Ciencia y
Tecnolog´ıa, Fondo de Investigacio´n Sanitaria (Spain),
and the European Commission. E.Q. and J .D. acknowl-
edge predoctoral grants from the same sources.
(11) Reviews: (a) Campbell, I. B. The Sonogashira Cu-Pd-catalyzed
alkyne coupling reaction. In Organocopper Reagents; Taylor, R.
J . K., Ed.; I.R.L. Press: Oxford, 1994; pp 217-235. (b) Sono-
gashira, K. Cross-coupling reactions to sp carbon atoms. In Metal
Catalyzed Cross-Coupling Reactions; Diederich, F., Stang, P. J .,
Eds.; Wiley-VCH: Weinheim, Germany, 1998; pp 203-229. (c)
Sonogashira, K. Development of Pd-Cu catalyzed cross-coupling
of terminal acetylenes with sp2-carbon halides. J . Organomet.
Chem. 2002, 653, 46-49. (d) Tykwinski, R. R. Evolution in the
palladium-catalyzed cross-coupling of sp- and sp2-hybridized
carbon atoms. Angew. Chem., Int. Ed. 2003, 42, 1566-1568.
(12) Boland, W.; Pantke S. (Z)-Selective reduction of conjugated
alkynes with Zn (Cu/Ag). J . Prakt. Chem. 1994, 336, 714-715.
(13) Takano, S.; Akiyama, M.; Sato, S.; Ogasawara, K. A facile
cleavage of benzylidene acetals with diisobutylaluminum hy-
dride. Chem. Lett. 1983, 1593-1596.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and analytical and spectral data for 1-6 and ad-
ditional fluorescence data of 6. This material is available free
of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Paltauf, F. Ether lipids in biomembranes. Chem. Phys. Lipids
1994, 74, 101-139.
(14) J ia, A.; Haines, H. Diamide analogs of phosphatidyl choline as
potential anti-AIDS agents. J . Chem. Soc., Perkin Trans. 1 1993,
2521-2523.
(2) (a) Munder, P. G.; Westphal, O. Antitumoral and other biomedi-
cal activities of synthetic ether lysophospholipids. Chem. Im-
munol. 1990, 49, 206-235. (b) Houlihan, W. J .; Lohmeyer, M.;
Workman, P.; Cheon, S. H. Phospholipid antitumor agents. Med.
Res. Rev. 1995, 15, 157-223. (c) Arthur, G.; Bittman, R. The
inhibition of cell signaling pathways by antitumor ether lipids.
Biochim. Biophys. Acta 1998, 1390, 85-102. (d) Bittman, R.;
Perkins, W. R.; Swenson, C. E. TLC ELL-12. Drugs Future 2001,
26, 1052-1058. (e) J endrossek, V.; Handrick, R. Membrane
targeted anticancer drugs: potent inducers of apoptosis and
putative radiosensitisers. Curr. Med. Chem. 2003, 3, 343-353.
(f) Croft, S. L.; Seifert, K.; Duchene, M. Antiprotozoal activities
of phospholipid analogues. Mol. Biochem. Parasitol. 2003, 126,
165-172.
(15) Lower yields were obtained with the two-step phosphorylation
frequently used in the synthesis of related phospholipids: (a)
Berchtold, R. Preparation of 1-O-octadecyl-2-O-methyl-sn-glyc-
ero-3-phosphocholine. Chem. Phys. Lipids 1982, 30, 389-392.
(b) Hirth, G.; Barner, R. Synthesis of glyceryletherphosphatides.
I. Preparation of 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phos-
phorylcholine (platelet activating factor) of its enantiomer and
of some analogous compounds. Helv. Chim. Acta 1982, 65, 1059-
1084. (c) Stekar, J .; No¨ssner, G.; Kutscher, B.; Engel, J .; Hilgard,
P. Synthesis, antitumor activity, and tolerability of phospholipids
containing nitrogen homologs. Angew, Chem. Int. Ed. Engl. 1995,
34, 238-240.
(16) Cabaner, C.; Gajate, C.; Macho, A.; Mun˜oz, E.; Modolell, M.;
Mollinedo, F. Induction of apoptosis in human mitogen-activated
peripheral blood T-lymphocytes by the ether phospholipid ET-
18-OCH3: involvement of the Fas receptor/ligand system. Br.
J . Pharmacol. 1999, 127, 813-825.
(3) Mollinedo, F.; Ferna´ndez-Luna, J . L.; Gajate, C.; Martı´n-Mart´ın,
B.; Benito, A.; Mart´ınez Dalmau, R.; Modolell, M. Selective
induction of apoptosis in cancer cells by the ether lipid ET-18-
OCH3 (edelfosine): molecular structure requirements, cellular
uptake, and protection by Bcl-2 and Bcl-XL. Cancer Res. 1997,
57, 1320-1328.
(4) Gajate, C.; Fonteriz, R. I.; Cabaner, C.; AÄ lvarez-Noves, G.;
AÄ lvarez-Rodr´ıguez, Y.; Modolell, M.; Mollinedo, F. Intracellular
triggering of Fas, independently of FasL, as a new mechanism
of antitumor ether lipid-induced apoptosis. Int. J . Cancer 2000,
85, 674-682.
(17) Human normal peripheral blood lymphocytes and human T-
leukemia J urkat cells (2.5 × 105 cells/mL) were treated with 20
µM 6 for 7 h in RPMI-1640 culture medium containing 10% fetal
calf serum and washed three times with phosphate buffered
saline containing 1% bovine serum albumin to eliminate loosely
bound 6 to the cells surface. Then 2 × 105 6-treated cells were
settled onto poly-L-lysine-coated slides, fixed in 4% formaldehyde
for 25 min at room temperature, and analyzed using a Zeiss
Axioplan 2 fluorescence microscope (Oberkochen, Germany) and
a Hamamatsu ORCA-ER-1394 digital camera. The extent of
apoptosis was assessed (flow cytometry) as the percentage of cells
with a DNA content of less than G1 (hypodiploidy) in cell cycle
analysis, using propidium iodide staining in permeabilized cells,
after 24 h of incubation with edelfosine or with 5 and 6 in the
presence of 10% fetal calf serum. Untreated control cells run in
parallel showed 2.3 ( 1.0% apoptosis.
(5) Gajate, C.; Mollinedo, F. Biological activities, mechanisms of
action and biomedical prospect of the antitumor ether phospho-
lipid ET-18-OCH3 (edelfosine), a proapoptotic agent in tumor
cells. Curr. Drug Metab. 2002, 3, 491-525.
(6) For the case of the antitumor drug Taxol, see the following. (a)
Souto, A. A.; Acun˜a, A. U.; Andreu, J . M.; Barasoain, I.; Abal,
M.; Amat-Guerri, F. New fluorescent water-soluble taxol deriva-
tives. Angew. Chem., Int. Ed. Engl. 1995, 34, 2710-2712. (b)
Abal, M.; Souto, A. A.; Amat-Guerri, F.; Acun˜a, A. U.; Andreu,
J . M.; Barasoain, I. Centrosome and spindle pole microtubules
are main targets of a fluorescent Taxoid inducing cell death. Cell
Motil. Cytoskeleton 2001, 49, 1-15. (c) Lillo, M. P.; Can˜adas,
O.; Dale, R. E.; Acun˜a, A. U. Location and properties of the taxol
binding center in microtubules: a picosecond laser study with
fluorescent taxoids. Biochemistry 2002, 41, 12436-12449.
(18) Gajate, C.; Mollinedo, F. The antitumor ether lipid ET-18-OCH3
induces apoptosis through translocation and capping of Fas/
CD95 into membrane rafts in human leukemic cells. Blood 2001,
98, 3860-3863.
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