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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187,

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

Synthesis and antineoplastic properties of an ether glycerophosphonocholine, and analog of ET-18-OCH3-GPC.

A glycerophosphonocholine analog of the ether-linked lipid, rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3-GPC), was synthesized in...
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