Synthesis and biological evaluation of novel steroid-modified ether phospholipids

Article abstract of DOI:10.1016/j.chemphyslip.2005.07.007

Platelet activating factor is one of the most potent inflammatory ether phospholipid mediators known and structurally modified analogues are of considerable interest as potential therapeutic preparations. Inspired by the proposed structure for a novel end

Full text of DOI:10.1016/j.chemphyslip.2005.07.007

Chemistry and Physics of Lipids 138 (2005) 12–19
Synthesis and biological evaluation of novel steroid-modified
ether phospholipids
Haralabos C. Karantonisa, Emmanuel N. Pitsinosb, Smaragdi Antonopoulouc,
Elias A. Couladourosb,d, Constantinos A. Demopoulosa,∗
a
Laboratory of Biochemistry, Faculty of Chemistry, National and Kapodistrian University of Athens,
Panepistimioupolis, GR-157 71 Athens, Greece
Laboratory of Organic and Bio-Organic Chemistry, Institute of Physical Chemistry, NCSR “DEMOKRITOS”,
b
Aghia Paraskevi, P.O. Box 60228, GR-153 10 Athens, Greece
Department of Science of Dietetics-Nutrition, Harokopio University, 70 El. Venizelou Street, GR-176 71 Athens, Greece
c
d
Chemistry Laboratories, Agricultural University of Athens, Iera Odos 75, GR-118 55 Athens, Greece
Received 10 September 2004; received in revised form 17 December 2004; accepted 22 July 2005
Available online 19 September 2005
Abstract
Platelet activating factor is one of the most potent inflammatory ether phospholipid mediators known and structurally modified
analogues are of considerable interest as potential therapeutic preparations. Inspired by the proposed structure for a novel endogenous
hydroxy-PAF analogue isolated recently from gingival crevicular fluid, we designed and prepared two novel steroid-modified ether
phospholipids. These two novel compounds exhibit marked chemical and biological similarities to their endogenous prototype and
they antagonize it being less active in inducing washed platelet aggregation through PAF receptors.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Steroid ether phospolipids; Platelets; Platelet activating factor; PAF-acetylhydrolase; Periodontal disease
1. Introduction
number of physiological and pathological processes,
such as allergy, hypotension, anaphylaxis, thrombosis,
ischemia, acute infections in transplantation, nephritis,
gastric ulcer, etc. (Snyder, 1987). Activated inflamma-
tory cells challenged by bacterial lipopolysaccharides
produce and secrete inflammatory mediators like PAF
(Jakubowski et al., 2004), which is believed to be a key
regulator of various diseases like periodontal disease
(Antonopoulou et al., 2003). Moreover, PAF has been
shown to stimulate platelet degranulation and aggre-
gation (Snyder et al., 1989), to cause the contraction
of smooth muscles, bronchoconstriction, and coronary
vasoconstriction (Snyder et al., 1989), to increase
vascular permeability (Snyder et al., 1989; Shukla,
1991) and to stimulate immune response (Ruis et al.,
1991; Hayashi et al., 1991; Braquet et al., 1991). A large
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-
glycero-3-phosphocholine, PAF) occurs naturally in cell
membranes and is one of the most potent inflammatory
ether phospholipid mediators known (Blank et al.,
1981; Demopoulos et al., 1979; Montrucchio et al.,
2000). PAF (1, Fig. 1) plays an important role in a
Abbreviations: Ac, acetyl; Bn, benzyl; LPC, lysophosphatidyl-
choline; PAF, platelet-activating factor; Ph, phenyl; SM, sph-
ingomyelin; TBAF, tetrabutylammonium fluoride; TBDPS, tert-
butyldiphenylsilyl; THF, tetrahydrofuran
∗
Corresponding author. Present address: 39 Anafis Str., GR-113 64
Athens, Greece. Tel.: +30 210 7274265; fax: +30 210 7274265.
E-mail address: demopoulos@chem.uoa.gr (C.A. Demopoulos).
0009-3084/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2005.07.007

H.C. Karantonis et al. / Chemistry and Physics of Lipids 138 (2005) 12–19
13
Fig. 1. Naturally occurring ether phospholipids (1 and 2) and designed steroid-modified analogues (3a and 3b).
number of structurally modified analogues of PAF has
been synthesized and exhibit diverse biological activ-
ities ranging from specific PAF antagonism (Sablina
et al., 1995) and immunomodulation (O’Flaherty
and Wykle, 1983; Munder et al., 1976) to selective
tumor-cytotoxicity (Berdel et al., 1981; Hoffman et al.,
1984) and inhibition of HIV-1 replication (Kucera
et al., 1990). Because of the variety of their biological
effects, ether phospholipids are of considerable interest
as potential therapeutic preparations.
Recently, a new endogenous phospholipid was iden-
tified in gingival crevicular fluid (Antonopoulou et al.,
1998). This molecule induced washed rabbit platelet
aggregation in a dose-dependent manner with PAF-like
aggregation tracing. Its EC50 value was in the order
of 0.1 ␮M, final concentration, being less active than
PAF, which induces aggregation with EC50 value in the
order of 0.1 nM, final concentration. Furthermore, it was
shown that this phospholipid acts through PAF receptors.
Enzymatic and chemical treatments along with ESMS
analysis demonstrated that the phospholipid has a glyc-
eryl ether backbone with an acetyl group at the sn-2
position and at least one free hydroxyl group at the sn-1
carbon chain, which can be acetylated under mild acety-
lation conditions. Unfortunately, the very small amount
ofmaterialisolatedhinderedfullstructuralelucidationof
this endogenous hydroxy-PAF analogue. Nevertheless,
the available structural information led to the proposed
structure 2 (Fig. 1) leaving ambiguous the exact nature
of the ether side chain.
The biological activity of this new ether phospholipid
and its proposed structure attracted our attention. In par-
ticular, the molecular formula C28H54O2 (Mr 422.7) pro-
posed for the hydroxylated ether unit intrigued us since
fatty acids or alcohols of such length are not common
in mammals. Thus, attempting to speculate on plausible
structures for this unit, based on the available chemical
and spectroscopic data, was haphazard.
Nevertheless, consideration of alternative molecular
formulae for the ether segment indicated the formula
C26H46O4 (Mr 422.6). This formula could accommo-
date the presence of four ring systems, an observation
that led us to consider derivatives of a steroid alcohol,
such as cholesterol or cholestanol, as alternative ether
units. The fact that, to the best of our knowledge, no
steroid-modified ether phospholipid has been previously
reported (Lal et al., 1994; Lebeau et al., 1991) along
with the important and diverse biological activity of ether
phospholipidspromptedustoinvestigatesuchnovelPAF
analogues despite the lack of any indication that they
might occur in nature.
Herein, we report the synthesis and some biological
properties of two steroid-modified ether phospholipid
analogues (3a and 3b). Apart from cholestanol derivative
3a, we opted to investigate the phospholipid derivative
of 24-nor-5␤-cholestane-3␣,7␣,12␣,25-tetrol (3b). This
C26-analogue of naturally occurring C27 bile alcohols
(Dayal et al., 1976) not only fits the available ESMS
spectroscopic data for 2 but also, we reasoned that due
to the steric demand of the neo-pentyl (C-12) and ter-

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H.C. Karantonis et al. / Chemistry and Physics of Lipids 138 (2005) 12–19
tiary (C-25) hydroxyls, only the C-7 hydroxyl would
be readily acetylated, thus accommodating the observed
chemical reactivity of the new hydroxy-PAF analogue.
chased from Rathburn (Walkerburn, Peebleshire, UK).
All standards were obtained from Sigma (St. Louis,
MO, USA). Semisynthetic PAF (80% C-16PAF and
20% C-18PAF) was synthesized in our laboratory as
previously described (Demopoulos et al., 1979). PAF-
acetylhydrolase from human serum has been purified
according to Stafforini (Stafforini et al., 1987). Bovine
serum albumin (BSA) and BN 52021 were obtained from
Sigma. Chrono-lume Reagent and Chrono-lume ATP
standard were purchased from Chrono-log Corporation
(Havertown, PA, USA).
2. Experimental
2.1. Materials and methods
All commercially available chemicals employed
(Merck, Darmstadt, Germany, or Aldrich, Steinheim,
Germany) were of reagent-grade and used without fur-
ther purification. All reactions were carried out under
a dry Ar atmosphere with anhydrous, freshly distilled
solvents under anhydrous conditions unless otherwise
noted. All reactions were magnetically stirred with
Teflon stir bars, and temperatures were measured exter-
nally. THF and toluene were freshly distilled over
sodium benzophenone ketyl. Reactions were monitored
by TLC carried out on 0.25 mm precoated glass plates
Merck Silica gel 60 F254. Visualisation was affected with
UV light (254 nm), iodine vapour chamber, 7% ethanolic
phosphomolybdenic acid-heat or p-anisaldehyde solu-
tion (2.5% p-anisaldehyde, 3.4% sulfuric acid, 1% acetic
acid in 95% ethanol) and heat as developing agent.
Preparative column chromatography: silica gel (Merck
Si 60; 40–63 ␮m). Chromatographic material used for
TLC lipid isolation was silica gel H-60 (Merck, Darm-
stadt, Germany). The solvents used for HPLC were pur-
2.2. Preparation of steroid-modified ether
phospholipids
For the preparation of the targeted modified
ether phospholipids, Bittmans’s synthetic strategy was
employed (Guivisdalsky and Bittman, 1989a,b). Thus,
highly regioselective opening of the epoxide ring of
racemic, tert-butyldiphenylsilyl-protected glycidol 5 by
cholestanol (4a) was catalyzed by BF3-etherate to pro-
vide alcohol 6a in 53% isolated yield – based on the
starting material used – (Scheme 1) (Erukulla et al.,
1995). In preparation for the introduction of the phos-
phocholine moiety, benzylation followed by cleavage of
the silyl-ether with TBAF furnished alcohol 8a (72%
yieldovertwosteps). Phosphorylation, using2-chloro-2-
oxo-1,3,2-dioxaphospholane in benzene in the presence
Scheme 1. Synthesis of steroid-modified ether phospholipids 3a and 3b. a series: R3 = H, R4 = CH2CH(CH3)2; b series: R3 = OH, R4 = C(OH)(CH3)2.
Reagents and conditions: (i) 5, BF3·Et2O, CH2Cl2; (ii) PhCH2Br, NaH, THF, 0–25 ◦C; (iii) TBAF, THF; (iv) 2-chloro-2-oxo-1,3,2-dioxaphospholane,
Et3N, toluene, 0 ◦C; (v) Me3N, CH3CN, 65–70 ◦C; (vi) H2, Pd(OH)2/C, MeOH/H2O (9:1, v/v); (vii) Ac2O, CHCl3, 25 ◦C.

H.C. Karantonis et al. / Chemistry and Physics of Lipids 138 (2005) 12–19
15
of 1 equivalent triethylamine, followed by treatment
of the phosphate thus obtained with excess anhydrous
trimethylamine in dry acetonitrile furnished phospho-
choline 10a (75% yield over two steps) (Thuong and
Chabrier, 1974). Finally, the lyso-PAF analogue 11a,
obtained by hydrogenolysis of the benzyl ether moiety,
was acetylated (Ac2O, CHCl3, rt, 12 h) to provide the tar-
geted cholestanyl-ether phospholipid 3a in 35% yield.
The reported selective glucuronidation of 24-nor-
5␤-cholestane-3␣,7␣,12␣,25-tetrol (4b) at C-3 (Dayal
et al., 1993) fuelled our hopes that this tetrahydroxy-
lated steroid could be used in the BF3-etherate catalyzed
epoxide ring opening step without prior protection of
the hydroxyls at C-7, -12 and -25. Indeed, when gly-
cidol 5 was reacted with the above tetrol, alcohol 6b
was obtained in 45% yield. Further transformation of
this alcohol to the targeted ether phospholipid 3b was
accomplishedinanalogytothepreparationofetherphos-
pholipid 3a from alcohol 6a described above.
26-CH3 + 27-CH3); 0.99 (d, J = 6.33 Hz, 3 H, steroid-
21-CH3); 0.88 (s, 3 H, steroid-19-CH3); 0.67 (s, 3 H,
steroid-18-CH3).
Compound 3a: 1H NMR (CDCl3, 250 MHz): δ
5.14–5.02 (broad m, 1 H, CHOAc); 4.28 (broad
s, 2 H, choline-CH2); 3.98–3.87 (broad m, 2 H,
CH2OPO3); 3.78 (broad s, 2 H, choline-CH2); 3.62–3.48
(broad m, 2 H, CH2OCH); 3.33 (s, 9 H, N+(CH3)3);
3.24–3.12 (m, 1 H, CH2OCH); 2.06 (s, 3 H, CH3COO);
1.99–0.54 (m, 46 H, steroid). MS (ESI+) m/z for
C37H68NNaO7P(M + Na+), calc.: 692.5; found: 692.7
Compound 3b: 1H NMR (CDCl3, 250 MHz): δ
5.11–4.90 (broad m, 1 H, CHOAc); 4.30 (broad s, 2 H,
choline-CH2); 3.93–2.94 (broad m, 9 H, steroid-H12␤
,
H3␤, H7␤ + CH2OPO3 + choline-CH2 + CH2OCH); 3.36
(broad s, 9 H, N(CH3)3); 2.31–0.86 (m, 25 H, steroid);
2.06 (s, 3 H, CH3COO); 1.15 (s, 6 H, steroid-26-
CH3 + 27-CH3); 0.99 (d, J = 5.58 Hz, 3 H, steroid-21-
CH3);0.86(s, 3H, steroid-19-CH3);0.65(s, 3H, steroid-
18-CH3). MS (ESI+) m/z for C36H67NO10P (M + H+),
calc.: 704.4; found 704.2.
2.3. Analytical data for selected new compounds
Compound 6a: 1H NMR (CDCl3, 250 MHz): δ
7.75–7.65 (m, 4 H, C6H5SiO); 7.46–7.31 (m, 6 H,
C6H5SiO); 3.89–3.80 (m, 1 H, CHOH); 3.71 (d,
J = 5.21 Hz, 2 H, CH2OTBDPS); 3.68–3.47 (m, 2 H,
CH2OCH); 3.28–3.15 (m, 1 H,CH2OCH); 2.52 (broad
d, J = 5.21 Hz, 1 H, CHOH); 2.04–0.55 (m, 55 H,
steroid+(CH3)3CSiO).
2.4. Purification of synthetic phospholipids
Botth diastereomers of compound 3a (1-O-choles-
tanol-2-acetyl-sn-glycero-3-phosphocholine and 1-
phosphocholine-2-acetyl-sn-glycero-3-O-cholestanol)
and compound 3b (1-O-24-nor-5␤-cholestane-3␣,7␣,
12␣,25-tetrol-2-acetyl-sn-glycero-3-phosphocholine
and 1-phosphocholine-2-acetyl-sn-glycero-3-O-24-nor-
5␤-cholestane-3␣,7␣,12␣,25-tetrol) referred as (d,l)-
3a and (d,l)-3b, respectively, based on the absolute
configuration of the glycerol moiety, were further
purified.
After synthesis the lipid compounds were extracted
according to Bligh–Dyer (Bligh and Dyer, 1959). The
extracts were evaporated to dryness under a stream of
nitrogen and re-dissolved in a small volume of chloro-
form/methanol (1:1, v/v) and were further purified by
TLC and HPLC.
Compound 6b: 1H NMR (CDCl3, 250 MHz): δ
7.60–7.56 (m, 4 H, C6H5SiO); 7.36–7.26 (m, 6 H,
C6H5SiO); 3.87 (bs, 1 H, steroid-H12␤); 3.78–3.72
(broad m, 2 H, CHOH + steroid-H7␤); 3.63–3.58 (m, 2 H,
CH2OTBDPS); 3.55–3.37 (m, 3 H, CH2OCH+steroid-
H3␤); 3.09–2.95 (broad m, 1 H, OH); 2.85–2.69 (broad
m, 1 H, OH); 2.32–0.76 (m, 25 H, steroid); 1.10 (s, 6 H,
steroid-26-CH3 + 27-CH3); 0.98 (s, 9 H, (CH3)3CSiO);
0.91 (d, J = 6.33 Hz, 3 H, steroid-21-CH3); 0.79 (s, 3 H,
steroid-19-CH3); 0.58 (s, 3 H, steroid-18-CH3).
Compound 8a: 1H NMR (CDCl3, 250 MHz):
δ 7.36–7.30 (m, 5 H, C6H5CH2O); 4.67 (ABq,
ꢀv = 21.02 Hz, J = 11.71 Hz,
2
H, C6H5CH2O);
2.4.1. TLC purification
3.81–3.48 (m, 5 H, CH2CHCH2); 3.29–3.17 (m, 1 H,
CH2OCH); 2.40 (broad t, J = 7.44 Hz, 1 H, CH2OH);
1.99–0.56 (m, 46 H, steroid).
TLC plates were 20 cm × 20 cm glass plates coated
with silica gel H and activated by heating at
120 ◦C for 1 h. Their thickness was 0.5 mm (analyt-
ical). The chromatogram was developed in chloro-
form/methanol/glacial acetic acid/water (100:65:20:15,
v/v) (Kates, 1975). Detection of compounds was per-
formed with iodine vapour. Both (d,l)-3a (RF = 0.42)
and (d,l)-3b (RF = 0.32) migrated between sphin-
gomyelin (SM; RF = 0.51) and lysophosphatidylcholine
(LPC; RF = 0.30).
Compound 8b: 1H NMR (CDCl3, 250 MHz):
δ 7.36–7.28 (m, 5 H, C6H5CH2O); 4.66 (ABq,
ꢀv = 20.84 Hz, J = 11.91 Hz, 2 H, C6H5CH2O); 3.95
(broad s, 1 H, steroid-H12␤); 3.81–3.46 (m, 7
H, CH2CHCH2 + steroid-H3␤ + steroid-H7␤); 3.17–3.07
(broad m, 1 H, OH); 2.98–2.36 (broad m, 2 H, 2 × OH);
2.31–0.84 (m, 25 H, steroid); 1.18 (s, 6 H, steroid-

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H.C. Karantonis et al. / Chemistry and Physics of Lipids 138 (2005) 12–19
Fig. 2. HPLC purification of compounds 3a, 3b, (l)-11b and (d)-3b. (a) HPLC purification of (d,l)-3a after TLC purification. (b) HPLC purification
of (d,l)-3b after TLC purification. (c) HPLC separation of (l)-11b and (d)-3b after PAF-acetylhydrolase treatment.
2.4.2. HPLC purification
et al., 1988) with PAF and experiments with its spe-
cific inhibitor BN 52021 (0.1 ␮M) were also performed.
ATP secretion was measured by a sensitive luminescent
(firefly luciferin–luciferase) assay for extracellular ATP.
Luminescence is created by chrono-lume reagent (fire-
fly luciferin–luciferase) reacting with ATP secreted by
dense granules in washed platelets. The luminescence
technique is calibrated during the testing procedure by
use of chrono-lume ATP standard (2 nmol). Chrono-
lume reagent was added (0.09 ␮M, final concentration)
2 min before the addition of the aggregating agent or the
chrono-lume ATP standard and luminescence was mea-
sured simultaneously with aggregation.
HPLC was performed at room temperature on a HP
Series 1100 liquid chromatography model (Hewlett-
Packard, Waldbronn, Germany) equipped with a 100 ␮L
loop Rheodyne (7725 i) loop valve injector, a degaser
G1322A, a quat gradient pump G1311A and a HP UV
spectrophotometer G1314A as a detection system. The
spectrophotometer was connected to a Hewlett-Packard
model HP-3395 integrator-plotter. A Sphereclone 5u
NH2 normal phase column (250 mm × 4.6 mm i.d.) from
Phenomenex, (Hurdsfield, Cheshire, UK) with a NH2
silica (20 mm × 4.0 mm i.d.) precolumn cartridge was
used. This step of purification of the synthetic com-
pounds was performed with an isocratic elution sys-
tem consisted of acetonitrile/methanol (75:25, v/v) at
210 nm. Both synthetic lipids eluted between SM and
LPC (Fig. 2).
2.6. PAF-acetylhydrolase assay
A volume of 40 ␮L of Tris buffer solution (50 mM,
pH 7.5) and 37 ␮L of the examined compound in BSA
(2.5 mg/mL saline), corresponding to a quantity as much
as five times the quantity capable of inducing platelet
aggregation, were added and mixed vigorously to a pre-
warmed at 37 ◦C test tube. From this mixture the aggre-
gatory activity of the one-fifth is tested and defined as
the activity at zero time. Afterwards, human serum PAF-
acetylhydrolase was added and the whole mixture is
incubated at 37 ◦C. At different time intervals, aliquots
were taken to test the ability, of the possibly partially
catabolizedlipidsample, toinducewashedrabbitplatelet
aggregation. As control the same procedure is carried
out with PAF (80% C-16PAF and 20% C-18PAF) – in
an amount that induces platelet aggregation equivalent
to that of the examined compound – instead of the lipid
compound.
2.5. Biological evaluation
PAF-induced aggregation was measured in a Chrono-
Log (Havertown, PA, USA) aggregometer coupled to
a Chrono-Log recorder (Havertown, PA, USA). The
synthetic compounds were tested for their biological
activity toward washed rabbit platelets according to
the method of Demopoulos et al. (1979). The purified
synthetic compounds were dissolved in 2.5 mg bovine
serum albumin per mL saline. The platelet aggregation
induced by PAF (5 × 10−10 M, final concentration) was
measured as PAF-induced aggregation, in washed rab-
bit platelets before (considered as 0% inhibition) and
after the addition of various concentrations of the exam-
ined compounds. Desensitization experiments (Lazanas

H.C. Karantonis et al. / Chemistry and Physics of Lipids 138 (2005) 12–19
17
2.7. PAF-acetylhydrolase treatment and acetylation
treatment with PAF-acetylhydrolase and subsequent
TLC analysis indicated two products. Based on the
known substrate selectivity of the enzyme (Demopoulos
and Antonopoulou, 1996), the first one (RF = 0.18)
was attributed to (l)-11a, while the second one
(RF = 0.42), which resisted further treatment with PAF-
acetylhydrolase, was attributed to compound (d)-3a.
Compound (d)-3a did not cause platelet aggregation.
Compound (d,l)-3b was less active than control PAF.
Thus, a final concentration of 1 × 10−4 M, based on
phosphorus determination, was required to achieve bio-
logical activity equal to that of 2.5 × 10−11 M PAF (final
concentration), causing reversible platelet aggregation
and 81% desensitization of control PAF (b, Fig. 3). Fur-
thermore, compound (d,l)-3b (1 × 10−4 M, final con-
centration) acts through PAF receptors since BN 52021
inhibits its activity at 96%. It also causes ATP secretion
(0.31 nmol) as PAF (2.5 × 10−11 M, final concentration)
does (0.21 nmol).
The examined compound was dissolved in BSA
(0.25 mg/mL saline) and 800 ␮L of Tris buffer 50 mM,
pH 7.4 were added as well as 100 ␮L of human serum
PAF-acetylhydrolase. The whole mixture was main-
tained at 37 ◦C for 45 min. Afterwards, lipid compounds
were extracted according to Bligh–Dyer, evaporated to
dryness under a stream of nitrogen, redissolved in a
small volume of chloroform/methanol (1:1, v/v) and sub-
jected to TLC separation. Acetylation of the products
was performed by the addition of 1 mL acetic anhy-
dride and incubation at 60 ◦C for 45 min. After that, the
reaction mixture was evaporated and extracted accord-
ing to Bligh–Dyer (Bligh and Dyer, 1959) and tested
for its ability to induce washed rabbit platelet aggrega-
tion. Acetylation of the initial compound, without human
serum PAF-acetylhydrolase was also performed using
the same procedure.
In order to gain additional information, an amount of
(d,l)-3b was subjected to acetylation giving (d,l)-3bAc,
while a quantity as much as two times the above was
subjected to PAF-acetylhydrolase treatment giving (l)-
11b and unaffected (d)-3b. The products were extracted
according to the Bligh–Dyer method and isolated by
HPLC (c, Fig. 2). Acetylation of (l)-11b gave (l)-
11bAc. All the above compounds in a final concentrat-
3. Results and discussion
Compound (d,l)-3a at a final concentration of
2.5 × 10−8 M, based on phosphorus determination
(Bartlett, 1959), exhibited biological activity smaller
than control PAF (5 × 10−10 M, final concentration)
causing reversible platelet aggregation (a, Fig. 3). Pre-
Fig. 3. Biological activity of compounds 3a and 3b. (a) Biological activity of compound (d,l)-3a toward washed platelets: 1, PAF (5 × 10−10 M, final
concentration); 2, compound (d,l)-3a (2.5 × 10−8 M, final concentration). (b) Biological activity of compound (d,l)-3b toward washed platelets:
1, PAF (2.5 × 10−11 M, final concentration); 2, compound (d,l)-3b (1 × 10−4 M, final concentration); 3, addition of PAF (2.5 × 10−11 M, final
concentration) for desensitization.

18
H.C. Karantonis et al. / Chemistry and Physics of Lipids 138 (2005) 12–19
These compounds may be useful in inflammatory
conditions treatment, since PAF antagonists are potential
therapeutic agents. Rupatadine (Izquierdo et al., 2003),
for example, is a new agent, which exhibits strong antag-
onist activity toward, both, histamine H1 and PAF recep-
tors. This agent is used for the management of diseases
with allergic inflammatory conditions and it is clinically
effective in relieving symptoms in patients with seasonal
and perennial allergic rhinitis in which PAF plays a piv-
otal role (Labrakis-Lazanas et al., 1988). Moreover, it
is more effective than agents that exhibit only antihis-
taminic effect (Izquierdo et al., 2003).
Fig. 4. Biological activity of 3b and related compounds. PAF-
acetylhydrolase assay for (d,l)-3b (0.6 × 10−6 M, final concen-
tration), (d,l)-3bAc (0.6 × 10−6 M, final concentration) and PAF
(5 × 10−10 M, final concentration).
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ing, based on phosphorus determination (Bartlett, 1959),
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Dayal, B., Shefer, S., Tint, G.S., Salen, G., Mosbach, E.H., 1976. C26
-
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