Vol. 187, No. 2, 1992 September 16, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 603-608
SYNTHESIS AND ANTINEOPLASTIC PROPERTIES OF AN ETHER GLYCEROPHOSPHONOCHOLINE, AN ANALOG OF ET-18-OCHpGPC Hassan Salarit*, Sandra Howardt, and Robert Bittmans
TDepartment of Medicine, University of British Columbia, and Lipase Biotech, Inc., Vancouver, B.C., Canada, and 9 Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, NY 11367
Received
July
21,
1992
A glycerophosphonocholine analog of the ether-linked lipid, rat-1 -O-octadecyl2-0-methyl-glycero-3-phosphocholine (ET-l 8-OCHa-GPC), was synthesized in which the head group is nonhydrolyzable by phospholipase C. The phosphonate analog used in this study is rac-3-octadecyloxy-2-methoxy-propyl-phosphonocholine, CfsHs70CH2CH(OCHs)CHaP(O)(O)OCH&H2N+(CHs)s. The activity of the synthetic phosphonate was tested in the human leukemic cell line, HL-60, and the human undifferentiated cervical carcinoma, C-41. The glycerophosphonocholine inhibited [sH]thymidine uptake by HL-60 cells with an EC50 value of 5-7 PM. The glycerophosphate ET-18-OCHs-GPC had an EC58 value of approximately 2 f.rM against HL-60 cells. The ECacvalues estimated from cell viability experiments were similar to that for [sH]thymidine uptake. The EC50 value for C-41 cells was about 1O-l 5 PM. The data demonstrate that the glycerophosphonocholine is a promising anticancer drug for the treatment of both leukemia and solid tumors. Furthermore, the data demonstrate that phospholipase C-catalyzed hydrolysis of ET-l 8-OCHs-GPC does not play an important role in the cytotoxic action of the ether-linked glycerolipids. o 1992 Academic Press,Inc.
Ether lipids are non-DNA interacting
agents that have shown to possess
antineoplastic activity. The best known compound of this class of agents is l-0 octadecyl-2-0-methyl-racglycero-3-phosphocholine
(ET-18-OCHs-GPC),
which has
been shown to inhibit the growth of several different leukemic cell types (1). ET-18OCHs-GPC
is currently in phase II trial against non-small cell lung cancer
Although the mechanisms responsible for the cytotoxic
(2,3).
action of these ether lipids
have not been fully identified, it is clear that intracellular signalling is affected, probably *To whom correspondence should be addressed at 2660 Oak Street, B.C. Canada V6H 326. Fax: (604) 8754497. 0006-291X/92
603
$4.00
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No.
at several enzymes
sites, involved
(PLC) in Swiss was proposed gfycerol
by these
compounds.
in lipid turnover, which
(5). Moreover,
The cytotoxic
of these agents to serve
activity we synthesized
ether
COMMUNICATIONS
lipids
inhibit
that a metabolite, participates
various
phospholipase
C
1-O-alkyl-2-
in cytotoxicity
(5). It
for PLC have the ability to inhibit
both ET-l 8-OCHs-GPC
to inhibit protein kinase
(9) in various cell types; however,
effectiveness
RESEARCH
e.g. phosphatidylinositol-specific
is formed by PLC hydrolysis,
have been shown
OCHs-GPC
BIOPHYSICAL
that ether lipids that are substrate
cell growth
cytotoxicity
AND
3T3 cells (4). It has also been proposed
O-methyl-glycerol, neoplastic
BIOCHEMICAL
2, 1992
and 1 -O-alkyl-OCHs-
C (6-8) and diacyglycerol
kinase
the inhibition of this enzyme does not account for the
(10). In order to evaluate
as a substrate a phosphonate
whether
for PLC is involved
the ability of the ET-18-
in the cell growth
analog of ET-18-OCHs-GPC
inhibitory
and evaluated
its
against HL-60 and C-41 cell lines.
THODS Octadecyl ally1 ether was prepared from 1-octadecanol, ally1 bromide, and sodium hydride in dimethyl sulfoxide-toluene (4:1, v/v) under nitrogen by modification of a previous method (11). To octadecyl ally1 ether dissolved in freshly distilled 1,2dimethoxyethane were added 2.5 equivalents of each of the following: iodine, zinc oxide, and methanol. The mixture was refluxed for 24 h, giving rao3-octadecyloxy-2methoxyiodopropane (12). The solvent was removed by evaporation under reduced pressure, the residue was extracted with hexane, and zinc oxide was removed by filtration. The filtrate was washed with 10% aqueous sodium thiosulfate until colorless, then with water, and finally with 5% aqueous sodium sulfate. The organic layer was dried over MgSO4. The solvent was removed and the iodo diether was purified by silica gel chromatography (elution with hexane-ethyl acetate mixtures). Reaction with an excess of tris(trimethylsilyl) phosphite at 120 “C under nitrogen as previously described (13, 14), followed by hydrolysis, gave rat-3-octadecyloxy-2methoxypropylphosphonic acid. The phosphonic acid was converted into the corresponding phosphonocholine as follows. To 18.6 mg (0.044 mmol) of 3-octadecyl2-rat-methoxypropyl-1-phosphonic acid in 3 ml of dry pyridine was added 98 mg (0.36 mmol) of dry choline tosylate and 275 PI of trichloroacetonitrile (Scheme 1). The reaction mixture was heated with stirring at 50 “C for 48 h. The solvent was removed under reduced pressure; toluene and 2-propanol were added and the solvents were reevaporated. The residue was dissolved in tetrahydrofuran and passed through an Amberlite MB-3 ion-exchange column (elution with THF-water 9:l). The eluate was evaporated, and water was removed by azeotropic distillation with 2-propanol. The residue (11.4 mg, 49% yield) was dissolved in chloroform-methanol 3:l and purified by chromatography on silica gel (elution with chloroform-methanol-water, 65:25:5). The fractions that contained the product were combined, the solvents were evaporated, and the residue was dissolved in chloroform and passed through a Metricel filter (Gelman Sciences) three times to remove suspended silica gel. The product was further purified by lyophilization from benzene. Yield, 7.9 mg (34%). Rf 0.15 (chloroform-methanol-water, 65:25:5). The product was characterized by its 1H NMR and FAB-MS spectra. HL-60 cell and human peripheral blood lymphocytes were maintained in culture at 37 “C in Corning plastic dishes (Corning Glass Laboratories, NY) in RPMI 1640 604
Vol.
187,
No.
2, 1992
%1%70H
BIOCHEMICAL
AND
BIOPHYSICAL
II
I
I
r
HOCH2CH2NfMe3 OTs) Cl,CCN, Py, 50’
COMMUNICATIONS
I. (MqSiO),P 2. H,O
CHOCH3
ZnO
CHZoC18H37
CHOCH, 0 I CH,f! -OH
I
CH,OH, I,, -
NaH
RESEARCH
oC18H37
~-0cH3
Li
POCH,CH,p(CH,), I 0.
bH Scheme I. Synthesis
of rac-3-octadecyloxy-2-methoxypropyIphosphonocholine.
medium supplemented with 10% fetal bovine serum (GIBCO Laboratories, Grand Island, NY). The human cervical carcinoma cell line C-41 (15), and normal mouce fibrobtasts were maintained in DMEM medium (GIBCO) in the presence of 10% FBS. All media contained antibiotics (50 ug/ml streptomycin and 50 units/ml of penicillin). Normal peripheral blood lymphocytes were isolated using Ficoll-Hypaque as reported elsewhere (16). Cells were grown in 96-well plates at 2 x 104/ml for 24, 48, or 72 h in the presence or absence of the glycerophosphonocholine. [sH]Thymidine (specific activity 70-85 Ci/mmoI) was purchased from Amersham Radiochemical Co. (Arlington Heights, IL). A stock solution of the phosphonate (0.1 M) in ethanol was diluted in the medium. [sH]Thymidine (0.1 uCi in 5 ul) was added to the wells, and after various days the non-adherent cells (HL-60, and blood lymphocytes) were harvested in a Brandel Model M-12 cell harvester. The adherent cells (mouse fibroblasts, and C-41 cells) were solubilized with 0.1% sodium dodecyl sulfate, and the solubilized cells were counted in a liquid scintillation counter. Cells were diluted 1 :lO in a solution of 2.5% trypan blue (Sigma), and 1 mm3 was counted under a light microscope. The adherent cells were first treated with trypsin before staining with trypan blue. Cells that took up the dye were considered dead. Cytotoxicity was determined as the percentage of dead cells in drug-treated cells compared with control (nontreated) cells.
RESULTS The antiproliferative cells is shown fashion,
DISCUSSION
of the glycerophosphonocholine
in Fig. 1. The drug inhibited
thymidine
uptake
against
HL-60
in a dose-dependent
with EC50 values for 24 h, 48 h, and 72 h of about 3-5 PM. To investigate
cells,
activity
and
cell viability
whether was
inhibition
examined
of thymidine
with trypan 605
uptake
was due to the death of
blue. Dead cells incorporate
dye and
Vol.
187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fiaure 1. Effect of various concentrations of the glycerophosphonocholine on [aH]thymidine uptake by HL-60 cells after 24 ( q ), 48 (a), and 72 h (0). Data are mean of 5 experiments f S.D.
appear blue under light microscopic examination. In this study two cancerous cell lines (HL-60 and C-41) and two normal cell types (human peripheral lymphocytes and mouse fibroblasts) were tested for their sensitivity to the cytotoxic action of the glycerophosphonocholine. Cells were incubated with three different concentrations of drug for 24 h, and the number of viable cells was determined after 48 h. We also compared the efficacy of the synthetic glycerophosphonocholine with ET-1 6-OCHsGPC.
Table 1 shows that the EC50 values for glycerophosphonocholine
60 and C-41 cells are