A Glucose-Containing Ether Lipid (Glc-PAF) as an Antiproliferative Analogue of the Platelet-Activating Factor

Full text of DOI:10.1002/(SICI)1521-3773(19980216)37:3<351::AID-ANIE351>3.0.CO;2-P

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an AA'MM'XX' pattern, 2H; Rh H); 31P{1H} NMR (C6D6, 85%
H3PO4): d ˆ 57.4 (AA' part of an AA'XX' pattern); 29Si{1H} NMR
(C6D6, TMS): d ˆ 157.8 (t, 2J(P,Si) ˆ 50 Hz), 28.7 (tt, 2J(P,Si) ˆ 50,
Experimental Section
3: A solution of 1 (264 mg, 0.24 mmol) in toluene (5 mL) was stirred for 2 h
at 708C. During the reaction the color of the solution changed from yellow
to orange. Gas-chromatographic analysis of the solution showed quanti-
tative formation of benzene. The 31P{1H} NMR spectrum of the mixture
after the reaction showed only the signal for 3. Addition of pentane caused
separation of 3 as yellow-orange crystals, which were collected by filtration
and recrystallized from toluene/pentane (71 mg, 29% yield). The similar
reaction of 2 gave 4 in 51% yield.
1
1J(Rh,Si) ˆ 8 Hz). 4: H NMR (C6D6, TMS): d ˆ 6.76 ± 7.88 (m, 20H;
aromatic H), 1.12 (m, 6H; P CH), 0.86 (m, 36H; CH3), 17.60 (AA'
part of an AA'MM'XX' pattern, 2H; Rh H); 31P{1H} NMR (C6D6,
85% H3PO4): d ˆ 58.2 (AA' part of an AA'XX' pattern).
[11] a) M. Auburn, M. Ciriano, J. A. K. Howard, M. Murray, N. J. Pugh,
J. L. Spencer, F. G. A. Stone, P. Woodward, J. Chem. Soc. Dalton
Trans. 1980, 659; b) D. E. Hendriksen, A. A. Oswald, G. B. Ansell, S.
Leta, R. V. Kastrup, Organometallics 1989, 8, 1153; c) S. K. Thomson,
G. B. Young, ibid. 1989, 8, 2068; d) P. Burger, R. G. Bergman, J. Am.
Chem. Soc. 1993, 115, 10462; e) T. Rappert, O. Nürnberg, H. Werner,
Organometallics 1993, 12, 1359; f) M. Baum, B. Windmüller, H.
Werner, Z. Naturforsch. B 1994, 49, 859; g) D. Huang, R. H. Heyn,
J. C. Bollinger, K. G. Caulton, Organometallics 1997, 16, 292.
The kinetic measurements of the conversion were carried out in a
thermostatted (708C) NMR probe by following the changes in the
intensities of the 1H NMR signal for the hydrido ligands of 1. Dioxane
was used as an internal standard.
Received: August 19, 1997 [Z10834IE]
German version: Angew. Chem. 1998, 110, 364 ± 366
Keywords: bridging ligands ´ rhodium ´ Si ligands
A Glucose-Containing Ether Lipid (Glc-PAF)
as an Antiproliferative Analogue of the
Platelet-Activating Factor**
[1] a) W. A. G. Graham, J. Organomet. Chem. 1986, 300, 81, and
references therein; b) U. Schubert, Adv. Organomet. Chem. 1990,
Ä
30, 151, and references therein; c) R. Carreno, V. Riera, M. A. Ruiz, Y.
Jeannin, M. Philoche-Levisalles, J. Chem. Soc. Chem. Commun. 1990,
15; d) M. D. Fryzuk, L. Rosenberg, S. J. Rettig, Organometallics 1991,
10, 2537; ibid. 1996, 15, 2871; e) H. Suzuki, T. Takao, M. Tanaka, Y.
Moro-oka, J. Chem. Soc. Chem. Commun. 1992, 476; f) T. Takao, S.
Yoshida, H. Suzuki, M. Tanaka, Organometallics 1995, 14, 3855; g)
B. K. Campion, R. H. Heyn, T. D. Tilley, ibid. 1992, 11, 3918; h) R. S.
Simons, C. A. Tessier, ibid. 1996, 15, 2604.
Michael Mickeleit, Thomas Wieder, Michael Arnold,
Christoph C. Geilen, Johann Mulzer, and
Werner Reutter*
It has long been known that the platelet-activating factor
(PAF) is a biologically highly active phosphoglyceride,[1] and
various PAF analogues have been reported as inhibitors of
proliferation.[2] However, since synthetic phospholipids are
strongly cytotoxic, their therapeutic use has hitherto been
restricted to topical applications.[3] We reported earlier the
synthesis of a new type of glyceroglucolecithin (Glc-PC)[4]
which displayed antiproliferative activity without cytotoxicity
[2] a) A. Heine, D. Stalke, Angew. Chem. 1993, 105, 90; Angew. Chem. Int.
Ed. Engl. 1993, 32, 121; b) A. Heine, R. Herbest-Irmer, D. Stalke, J.
Chem. Soc. Chem. Commun. 1993, 1729.
[3] a) P. Braunstein, M. Knorr, B. Hirle, G. Reinhard, U. Schubert, Angew.
Chem. 1992, 104, 1641; Angew. Chem. Int. Ed. Engl. 1992, 31, 1583; b)
M. Knorr, P. Braunstein, A. Tiripicchio, F. Ugozzoli, Organometallics
1995, 14, 4910; c) M. Knorr, E. Hallauer, V. Huch, M. Veith, P.
Braunstein, ibid. 1996, 15, 3868.
[4] W. Lin, S. R. Wilson, G. S. Girolami, Organometallics 1994, 13, 2309.
[5] R. Bender, P. Braunstein, A. Dedieu, Y. Dusausoy, Angew. Chem.
1989, 101, 931, Angew. Chem. Int. Ed. Engl. 1989, 28, 923.
1
at concentrations below 10 mmolL . We now present the
glycoside of the ether analogue [1-O-octadecyl-2-O-a-d-
glucopyranosyl-sn-glycero(3)]phosphorylcholine (Glc-PAF,
1), which is formally derived from the PAF by exchanging
the 2-acyl group for a glucose molecule.
The glycerol skeleton is provided by the starting material
(S)-isopropylidene glycerol (2),[5] whose hydroxyl group is
protected as the allyl ether functionality in 3 for introduction
of the end groups (Scheme 1). The primary hydroxyl group of
the diol 4 released upon acid hydrolysis cannot react directly
to form an ether, and it is therefore first converted into a
terminal benzoate ester group.[6] Compound 5 undergoes
[6] K. Osakada, T. Koizumi, T. Yamamoto, Organometallics 1997, 16, 2063.
[7] 2: 1H NMR (C6D6, TMS): d ˆ 7.59 (dd, 3J(H,H) ˆ 9, 3J(H,F) ˆ 2 Hz,
12H; aromatic H), 6.90 (t, 3J(H,H) ˆ 3J(H,F) ˆ 9 Hz, 12H; aromatic
H), 1.54 (m, 3J(P,H) ˆ 7 Hz, 6H; P CH), 0.82 (br, 36H; CH3), 12.55
(tt, 1J(Rh,H) ˆ 58, 2J(P,H) ˆ 25 Hz, 1H; Rh-H-Rh), 16.07 (AA' part
of an AA'MM'XX' pattern, 2H; Rh H); 31P{1H} NMR (C6D6, 85%
H3PO4): d ˆ 54.2 (AA' part of an AA'XX' pattern).
[8] Crystallographic data for 4: C48H64ClF5P2Rh2Si2, Mr ˆ 1095.41, mono-
clinic, space group P21/c (no. 14), a ˆ 18.146(10), b ˆ 12.806(8), c ˆ
1
21.512(9) Š, b ˆ 90.39(3)8,
V
ˆ 4998 Š3, Z ˆ 4, m ˆ 8.74 cm
,
F(000) ˆ 2248, 1calcd ˆ 1.455 g cm 3. The final R factor was 0.075
(Rw ˆ 0.059) for 7999 reflections with I > 3s(I). Data collection on a
RIGAKU-AFC5R diffractometer at 298 K with graphite-monochro-
mated MoKa radiation (l ˆ 0.71073 Š). The structure was solved with
the program teXsan. Crystallographic data (excluding structure
factors) for the structure reported in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC-100766. Copies of the data can be obtained
free of charge on application to CCDC, 12 Union Road, Cambridge
CB21EZ, UK (fax: int. code ‡ (49)1223-336033; e-mail: depos-
it@ccdc.cam.ac.uk).
[*] Prof. Dr. W. Reutter, Dr. T. Wieder
Institut für Molekularbiologie und Biochemie der Freien Universität
Arnimallee 22, D-14195 Berlin (Germany)
Fax: Int. code ‡ (49)30838-2141
Prof. Dr. J. Mulzer
Institut für Organische Chemie der Universität Wien (Austria)
Dr. M. Mickeleit
Institut für Organische Chemie der Freien Universität Berlin
[9] a) K. W. Muir, J. A. Ibers, Inorg. Chem. 1970, 9, 440; b) M.-J.
Fernandez, P. M. Bailey, P. O. Bentz, J. S. Ricci, T. F. Koetzle, P. M.
Maitlis, J. Am. Chem. Soc. 1984, 106, 5458; c) F. L. Joslin, S. R. Stobart,
J. Chem. Soc. Chem. Commun. 1989, 504; d) D. L. Thorn, R. L.
Harlow, Inorg. Chem. 1990, 29, 2017; e) K. Osakada, K. Hataya, Y.
Nakamura, M. Tanaka, T. Yamamoto, J. Chem. Soc. Chem. Commun.
1993, 576; ibid. 1995, 2315.
Priv.-Doz. Dr. Dr. C. C. Geilen, M. Arnold
Haut- und Poliklinik des Universitätsklinikums Benjamin Franklin
Freie Universität Berlin
[**] These investigations were supported by the Bundesministerium für
Bildung, Wissenschaft, Forschung und Technologie (W.R.), zentrale
Mittel (FNK) der Freien Universität Berlin, the Fonds der Chem-
ischen Industrie, and the Deutsche Forschungsgemeinschaft (Ge 641/
3-3).
[10] 3: 1H NMR (C6D6, TMS): d ˆ 6.9 ± 8.2 (m, 25H; aromatic H), 1.18 (m,
3J(P,H) ˆ 7 Hz, 6H; P CH), 0.95 (m, 36H; CH3), 17.40 (AA' part of
Angew. Chem. Int. Ed. 1998, 37, No. 3
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Scheme 1. Synthesis of 1. a) NaH, allyl bromide, THF, 08C (98%);
b) AcOH/H2O (60/40) (73%); c) BzCl, DMAP, py, 08C (76%);
d) 2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl fluoride, AgClO4, SnCl2,
4-Š molecular sieves, Et2O, 158C (81%); e) KOH/MeOH (30%), MeOH,
(91%); f) NaH, RBr, THF, 808C (83 ± 98%); g) Pd/C (10%), p-toluene-
sulfonic acid, MeOH/iPrOH/H2O (4/4/1), 808C (70 ± 94%); h) 1. POCl3,
Et3N, CHCl3, 08C; 2. choline tosylate, py, 08C (47 ± 74%); i) H2 (2 atm),
Pd/C (5%), MeOH (71 ± 97%); Bn ˆ benzyl, Bz ˆ benzoyl, DMAP ˆ 4-
(dimethylamino)pyridine, py ˆ pyridine, R ˆ CnH2n‡1, n ˆ 18, 16, 14, 12.
reaction with 2,3,4,6-tetra-O-benzyl-b-d-glucopyranosyl fluo-
ride[8] at the secondary alcohol functionality to provide
glycoside 6[9] according to Mukaiyama et al.[7] After saponi-
fication of the benzoate, the resulting compound 7 is
converted into the ether with the appropriate alkyl bromide.
Since the activity of choline-containing phospholipid ana-
logues strongly depends on the length of the alkyl chain,[10] the
chain length of the ether introduced at this stage (C18, C16, C14,
and C12) corresponds to that of a biologically relevant
derivative. Formation of the ether is highly temperature-
dependent, and complete conversion into 8 does not occur
until 808C.
Figure 1. Influence of 1 (n ˆ 18) on the viability (a) and proliferation (b) of
HaCaT cells. Both are expressed as a percentage A of the value measured
for the control experiment; c ˆ concentration. Four experiments were
carried out per data point. See the Experimental Section for details.
Before introducing the final glycerol substituent (i.e. the
phosphate), the original allyl ether must be cleaved by
hydrogenolysis.[11] Alcohol 9 is then esterified with phosphoryl
chloride, and the resulting dichloroester is immediately
converted into the desired phosphocholine derivative 10 with
choline tosylate.[12,13] Cleavage of the benzyl protecting groups
presented considerable problems in the synthesis of the ester
analogue Glc-PC. The ethers are, however, much more
reactive, and the products are obtained in good yields after
only short periods of hydrogenation. Starting from 2, gram
quantities of 1 were synthesized in nine steps with a total yield
of 25%.[14]
shown in Figure 1b, nontoxic concentrations of 1 (n ˆ 18) as
1
low as 4 mmol L inhibit proliferation. The inhibition curve
shows a half-maximal inhibitor concentration (IC50) of
1
4.8 mmol L , which is well below the LD50 value. In view of
the detergentlike structure of 1, cytotoxicity may be attributed
to cell lysis and/or the initiation of programmed cell death
(apoptosis),[15] which is characterized biochemically by the
appearance of ordered arrangements of internucleosomal
DNA fragments.[16] To further characterize the cell death
caused by 1, we determined the proportion of apoptotic
HaCaT cells by measuring the number of cytosolic nucleo-
somes after treatment of the cells for 20 h with the synthetic
ether lipid. At low but antiproliferative concentrations of 1
The biological activity of 1 was determined in a serum-free
cell-culture system of human keratinocytes (HaCaT cells) by
measuring three typical cell parameters. First, it was shown
1
(n ˆ 18; 1.5, 4.5 and 7.4 mmol L ), the number of apoptotic
1
cells showed
a
concentration-dependent increase; the
that 1 (n ˆ 18) is nontoxic at concentrations below 4 mmol L
1
greatest (tenfold) increase was observed for 7.4 mmol L
(Figure 2). At concentrations of 14.9 and 29.8 mmol L 1 there
was a marked decrease in the number of apoptotic cells, as
(Figure 1a). Higher concentrations cause marked cell dam-
age; the median lethal dose (LD50) was 9 mmolL . The
1
growth-inhibitory properties of 1 were then investigated. As
352
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centrifuged for 10 min at 13000 g to remove nonbound antibodies.
Nucleosomes were then determined in the lysate and in the control with
a commercially available enzyme-linked immunosorbent assay (ELISA)
from Boehringer Mannheim according to the instructions provided by the
manufacturer. An aliquot of each sample (20 mL) was pipetted into a well
of a streptavidin-coated microtiter plate followed by immune reagent
(80 mL; made up of 1 part peroxidase-conjugated anti-DNA antibody, 1
part biotinylated anti-histone antibody, and 18 parts incubation buffer) and
incubated for 2 h at room temperature. After threefold washing incubation
buffer, and the quantity of bound peroxidase-conjugated anti-DNA
antibody was quantified by a peroxidase-catalyzed color reaction with
measurment at 405 nm in the ELISA reader. The increase in the number of
apoptotic cells was calculated by comparing the extinction of the treated
sample with that of the corresponding control. The specificity of the
method was tested by applying it to cells treated with suitable apoptosis-
inducing substances (cell membrane-penetrating ceramides). In addition,
electron microscopy of the treated cells revealed typical apoptotic changes,
such as membrane evaginations and chromatin condensations, which were
correlated with the results of the ELISA test.
Received: July 2, 1997 [Z10624IE]
German version: Angew. Chem. 1997, 110, 371 ± 373
Keywords: apoptosis ´ glycosides ´ phospholipids ´ platelet-
activating factor ´ proliferation inhibitors
Figure 2. Influence of 1 (n ˆ 18) on the apoptosis of HaCaT cells, which is
expressed as the percentual enrichment A of apoptotic cells in comparison
to the control experiment; c ˆ concentration. Four experiments were
carried out per data point. See the Experimental Section for details.
[1] D. J. Hanahan, Annu. Rev. Biochem. 1986, 55, 483 ± 509.
[2] M. Modollel, R. Andreesen, W. Pahlke, U. Brugger, P. G. Munder,
Cancer Res. 1979, 39, 4681 ± 4686; E. M. Scholar, Cancer Lett. 1986, 33,
199 ± 204; T. Wieder, A. Haase, C. C. Geilen, C. E. Orfanos, Lipids
1995, 30, 389 ± 393.
[3] C. Unger, W. Damenz, E. A. M. Fleer, D. J. Kim, A. Breiser, P.
Hilgard, J. Engel, G. Nagel, H. Eibl, Acta Oncol. 1989, 28, 213 ± 217; S.
Clive, R. C. F. Leonard, Lancet 1997, 349, 621 ± 622.
determined by the presence of cytosolic nucleosomes. This
indicates that the cells were damaged by lysis at these higher
concentrations. According to these data, 1 is a proliferation
inhibitor which initiates programmed cell death in keratino-
1
cytes at concentrations below 7.5 mmol L . Compared with
[4] M. Mickeleit, T. Wieder, K. Buchner, C. Geilen, J. Mulzer, W. Reutter,
Angew. Chem. 1995, 107, 2879 ± 2881; Angew. Chem. Int. Ed. Engl.
1995, 34, 2667 ± 2669.
the above-mentioned Glc-PC (LD50 ˆ 17 mmol L 1 and IC50 ˆ
1
9 mmol L
under the experimental conditions described
[5] R. M. Hanson, Chem. Rev. 1991, 91, 437 ± 475.
above), 1 (n ˆ 18) is not only biologically more active, but
also more toxic. The synthesis of other derivatives with 2-
glycosidic substituents and different alkyl side chains will be
necessary to determine the structure ± activity relationships of
this new type of compound.
[6] The dibenzoate forms in 9% and the regioisomeric monobenzoate in
12% yield. The regioisomers were separated by preparative HPLC.
[7] T. Mukaiyama, Y. Murai, S. Shoda, Chem. Lett. 1981, 431 ± 432.
[8] G. H. Posner, S. H. Haines, Tetrahedron Lett. 1985, 26, 5 ± 8.
[9] The anomers are formed in the ratio a:b ˆ 9:1.
[10] C. C. Geilen, A. Haase, T. Wieder, D. Arndt, R. Zeisig, W. Reutter, J.
Lipid Res. 1994, 35, 625 ± 632.
[11] R. Boss, R. Scheffold, Angew. Chem. 1976, 88, 578 ± 579; Angew.
Chem. Int. Ed. Engl. 1976, 15, 558 ± 559.
Experimental Section
Cell culture: The spontaneously immortalized human keratinocyte cell line
HaCaT[17] was incubated in serum-free keratinocyte growth medium.[18]
Compound 1 was dissolved directly in the keratinocyte growth medium.
[12] G. Hirth, H. Saroka, W. Bannwarth, R. Barner, Helv. Chim. Acta 1983,
66, 1210 ± 1240.
[13] At this stage the anomers were separated by HPLC (MeOH/CH2Cl2
4/1). The final product 1 is the pure a-anomer
Cytotoxicity test: Confluent HaCaT cells were incubated for 6 h with
different concentrations of 1. The cytotoxicity of 1 for HaCaT cells was
determined in comparison with the control experiments (which did not
contain 1) by measuring the activity of alkaline phosphatase as de-
scribed.[19]
[14] Selected physical properties and spectroscopic data of 1 (n ˆ 18):
colorless, amorphous solid; m.p. 2258C, [a]2D0 ˆ‡ 50.9 (c ˆ 0.7 in
CH3OH); 1H NMR (500 MHz, CD3OD): d ˆ 0.90 (t, J ˆ 6.25 Hz, 3 H),
1.18 ± 1.38 (m, 30 H), 1.56 (mc, 2 H), 3.20 (s, 9 H), 3.31 ± 3.49 (m, 4 H),
3.58 ± 3.80 (m, 8 H), 3.95 ± 4.08 (m, 3 H), 4.30 (mc, 2 H), 5.06 (d, J ˆ
Measurement of proliferation: HaCaT cells were sown at a cell density of
about 20000 cells per cm2. After adhesion the cells were incubated for 48 h
with different concentrations of 1. The cell count was determined as
described[20] after staining with 0.1% crystal violet in PBS, and compared
with the control.
‡
3.75 Hz, 1 H); MS (FAB ): m/z (%): 672 (4.0), 510 (5.6), 224 (9.4), 184
(39.4), 166 (26.4), 86 (100), 58 (65.7).
[15] J. Kerr, A. Wyllie, A. Currie, Br. J. Cancer 1972, 26, 239 ± 257.
[16] A. Wyllie, Nature 1980, 284, 555 ± 556.
[17] P. Boukamp, R. T. Petrussevska, D. Breitkreutz, J. Hornung, A.
Markham, N. E. Fusenig, J. Cell Biol. 1988, 106, 761 ± 771. The cells
were provided by Dr. N. E. Fusenig (DKFZ, Heidelberg) und
maintained in a liquid-culture medium.
[18] C. C. Geilen, M. Bektas, T. Wieder, C. E. Orfanos, FEBS Lett. 1996,
378, 88 ± 92.
[19] J. G. Culvenor, A. W. Harris, T. E. Mandel, A. Whitelaw, E. Ferber, J.
Immunol. 1981, 126, 1974 ± 1977.
Measurement of apoptosis: Confluent HaCaT cells adapted to keratinocyte
growth medium were treated for 20 h with different concentrations of 1.
The medium was removed, and the adherent cells released from the dish
with a solution containing 0.1% trypsin and 0.02% ethylenediaminetetra-
acetic acid (EDTA). The cells were then washed off with serum-containing
medium (10% fetal-calf serum in Rosewell Park Memorial Institute
(RPMI) medium) and combined with the previously removed medium. The
cells were pelleted by centrifugation for 5 min at 300 g, washed once with
serum-containing RPMI medium, and pelleted again. The cell pellet was
incubated for 30 min in 1 mL of lysis buffer, and the resulting lysate
[20] R. J. Gillies, N. Didier, M. Denton, Anal. Biochem. 1986, 159, 109 ±
113; C. C. Geilen, R. Haase, K. Buchner, T. Wieder, F. Hucho, W.
Reutter, Eur. J. Cancer 1991, 27, 1650 ± 1653.
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353