Enzymatic Release of Antitumor Ether Lipids by Specific Phospholipase A2 Activation of Liposome-Forming Prodrugs

Article abstract of DOI:10.1021/jm031029r

An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing prop

Full text of DOI:10.1021/jm031029r

1694
J . Med. Chem. 2004, 47, 1694-1703
En zym a tic Relea se of An titu m or Eth er Lip id s by Sp ecific P h osp h olip a se A2
Activa tion of Lip osom e-F or m in g P r od r u gs
Thomas L. Andresen,*,†,‡ J esper Davidsen,‡ Mikael Begtrup,§ Ole G. Mouritsen,# and Kent J ørgensen‡
Department of Chemistry and LiPlasome Pharma A/ S, Technical University of Denmark, Building 207,
DK-2800 Lyngby, Denmark, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences,
Universitetsparken 2, DK-2100 Copenhagen, Denmark, and Physics Department, MEMPHYSsCenter for Biomembrane
Physics, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Received September 11, 2003
An enzymatically activated liposome-based drug-delivery concept involving masked antitumor
ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic
properties of AEL drugs as well as the membrane permeability enhancing properties of these
molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs
(proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were
investigated for enzymatic degradation by secretory phospholipase A2 (sPLA2), resulting in
the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-
glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phospho-
ethanolamine poly(ethylene glycol)350 (1-O-DPPE-PEG350), and 1-O-DPPE-PEG2000 of which 1-O-
DPPC was the main liposome component. All three phopholipids were synthesized from the
versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl
dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-
hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential
scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A
release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-
glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA2,
perturb membranes because of the detergent-like properties of the released hydrolysis products.
A hemolysis investigation was conducted on human red blood cells, and the results demonstrate
that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for
using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery
and prodrug concept in a single liposome system. Our investigation of the permeability-
enhancing properties of the AEL molecules imply that by encapsulating conventional
chemotherapeutic drugs, such as doxorubicin, in liposomes consisting of proAELs, an increased
effect of the encapsulated drug might be achievable due to an enhanced transmembrane drug
diffusion.
In tr od u ction
and tissues and in this way circumvent the general
problem with a very small difference between thera-
peutic and toxic doses.1 Of all drug-delivery systems,
liposomes are among the most extensively studied.
Being composed of naturally occurring substances, they
are biocompatible, biodegradable, and nontoxic. In
general, liposomes have a low degree of accumulation
in the heart, kidneys, and tissue of the nervous system,
and the toxicity of many drugs is therefore significantly
reduced when encapsulated into liposomal carriers.1 It
has been found that tumors in general have a vascula-
ture that is leaky toward small particles.2 This opens
up a possibility for small liposomes (<200 nm) to
extravasate and accumulate in tumors.3 To obtain a
passive diffusion of liposomes into the porous cancer
tissue, it is necessary to “coat” the liposome surface to
increase the vascular circulation time. Incorporation of
lipids with covalently attached poly(ethylene glycol)
(PEG) has been shown to increase the liposome circula-
tion time4 without causing considerable leakage of the
encapsulated material.5 After passive accumulation in
the porous cancer tissue, the encapsulated drugs will
eventually diffuse out of the liposomes, possibly by
Conventional cancer treatments, such as surgery,
radiotherapy, and chemotherapy, require early localiza-
tion of the tumor in order to be effective. Upon late
localization, surgery and radiotherapy in particular
become less effective and extensive chemotherapeutic
treatments are required. In recent years, an increased
understanding of human cancer has given rise to new
and improved chemotherapeutic agents. However, the
systemic use of these chemotherapeutics is severely
limited by the fact that anticancer agents in general are
extremely toxic to healthy as well as malign cells. The
search for tumor-targeting drugs and drug-delivery
systems to circumvent these complications has been
intense. The ultimate goal is to find a system that
targets pathological tissue and avoids healthy organs
* To whom correspondence should be addressed. Address: Depart-
ment of Chemistry, Technical University of Denmark, Building 207,
DK-2800 Lyngby, Denmark. E-mail: than@kemi.dtu.dk. Phone:
+45 45252139. Fax: +45 45883136.
† Department of Chemistry, Technical University of Denmark.
‡ LiPlasome Pharma A/S, Technical University of Denmark.
§ The Danish University of Pharmaceutical Sciences.
#
University of Southern Denmark.
10.1021/jm031029r CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/24/2004

Release of Antitumor Ether Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1695
F igu r e 1. Drug delivery principle. Secretory phospholipase
A2 (sPLA2) hydrolyzes the liposome membrane, thereby releas-
ing both activated antitumor ether lipids (AELs) and the
encapsulated drug. The AELs are cytotoxic to cancer cells.
Furthermore, synergistically with the generated fatty acids,
they function as permeability enhancers that promote drug
uptake by the cancer cells.
nonspecific degradation.6 However, it has also become
evident that today’s marketed PEG liposomes, contain-
ing high amounts of cholesterol, are very stable both in
the blood stream and in the cancer tissue, resulting in
a low and slow drug release in the tumor.7,8 It would,
therefore, be a substantial improvement if one could
trigger the release of drugs specifically at diseased sites.
It has been suggested that site-specific triggered release
can be achieved by the design of liposomes that are
sensitive to local hyperthermia,9,10 by light- and pH-
sensitive liposomes11-15 and by liposomes that are
destabilized by enzymes overexpressed in diseased
tissue.16
F igu r e 2. Chemical structures of the three different proan-
titumor ether lipids (proAELs) that have been synthesized and
investigated with respect to their physical properties and
ability to constitute a novel liposome-based drug-delivery
system: (1) (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phos-
phocholine (1-O-DPPC); (2) (R)-1-O-hexadecyl-2-palmitoyl-sn-
glycero-3-phosphoethanolamine poly(ethylene glycol)350 (1-O-
DPPE-PEG350); (3) 1-O-DPPE-PEG2000
.
Sch em e 1. Synthesis of 1-O-DPPCa
J ørgensen et al.17 have shown that polymer-covered
liposomes are susceptible to degradation by sPLA2,
which occurs at elevated levels in the evading zone of
tumor tissue,18 possibly as an important part of the
host’s defense mechanism.19-21 sPLA2 is furthermore
known to be up-regulated during infectious and inflam-
matory diseases.22-25 An interesting feature of the
enzyme is a much higher activity toward aggregated
lipid substrates, such as liposomes, compared to lipid
monomers. In addition, the enzyme activity is, to a large
extent, influenced by the physical properties of the
aggregated substrates, e.g., liposome size, charge, mi-
crostructure, and heterogeneity.26-28 This finding has
led us to develop a novel drug-delivery concept employ-
ing sPLA2 as a target-specific trigger.29 By formulating
liposomes that are sPLA2-degradable, it may be possible
to obtain a site-specific drug release and further profit
from the lysolipid’s detergent-like properties (Figure 1).
The fatty acid and lysolipid hydrolysis products, gener-
ated by sPLA2, have furthermore been shown to display
a synergistic effect as permeability enhancers.30
The aim of the present work has been to synthesize
and characterize phospholipids with a non-hydrolyzable
ether bond in the 1-position (1-O-phospholipids). These
lipids will not only be capable of forming liposomes and
carrying water-soluble drugs but will also function as
prodrugs using sPLA2 as the activating enzyme. When
sPLA2 hydrolyzes the 1-O-phospholipid-based liposomes,
highly cytotoxic lysolipids (antitumor ether lipids, AELs)
are formed. A large variety of AELs have been synthe-
sized and tested in a variety of cell and animal tumor
assays.31 Many are very potent, but a general problem
has been the toxicity toward red blood cells. The sPLA2
activation of proAELs reported herein circumvents the
a
(a) C16H33OH, NaH, THF, DMF, 16h, 80 °C; (b) palmitoyl
chloride, pyridine, petroleum ether, 16 h, room temp; (c) H2, Pd-
C, EtOAc, 1.5 h, room temp; (d) (i) POCl3, Et3N, CH2Cl2, 30 min,
room temp, (ii) pyridine, choline tosylate, 16 h, room temp.
hemolytic limitations of using AELs as drugs in cancer
treatment and furthermore offers a site-specific release
of a liposome-carried drug, such as doxorubicin.
We report the synthesis of (R)-1-O-hexadecyl-2-palmi-
toyl-sn-glycero-3-phosphocholine (1-O-DPPC) and two
analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-
3-phosphoethanolamine poly(ethylene glycol) (1-O-DPPE-
PEG) (Figure 2). We have investigated the physical
properties of liposomes composed of these lipids and
studied the sPLA2 activity toward different composi-
tions. In addition, the synergistic membrane perme-
ability enhancing properties of the hydrolysis products
have been studied. The results substantiate the idea of
using sPLA2 as a prodrug/drug-delivery trigger that can
specifically activate and release drugs in pathological
tissue. The work has been focused on clarifying funda-
mental questions involved in a rational development of
a novel anticancer liposome technology based on 1-O-
DPPC and various analogues as lipid prodrugs.
Resu lts a n d Discu ssion
Syn th esis of p r oAEL 1, 2, a n d 3 (F igu r e 2). The
synthetic approach to all three phospholipids involves
the synthesis of 7 (Scheme 1), from which either

1696 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Andresen et al.
a
Sch em e 2. Synthesis of 1-O-DPPE-PEG350 and 1-O-DPPE-PEG2000
a
(a) (i) MeOPOCl2, TMP, toluene, 16 h, room temp, (ii) N-BOC-ethanolamine, TMP, 24 h, room temp; (b) TFA, MeOH, CH2Cl2, 0.5 h,
0 °C; (c) 12 or 13, Et3N, CHCl3, 2 h, 40 °C; (d) NaI, 2-butanone, 2 h, 75 °C.
Sch em e 3. Synthesis of Activated PEGa
phosphatidylethanolamine or phosphatidylcholine is
formed. The ring opening of epoxide 4 with sodium cetyl
alcoholate was considerably improved compared to the
reported procedure by Hirth et al.32 by changing reaction
conditions. The original conditions, where DMF was
used as solvent, resulted in substantial dimerization,
which gave yields between 30% and 40%. We found that
THF/DMF 1:1 was a superior solvent system. 5 was
used without purification, and acylation using palmitoyl
chloride gave 6 in 70% yield over the two steps. We have
found that using palmitic acid with standard DCC
acylating conditions works equally well. Deprotection
of 6 using H2/Pd-C gave 7 in quantitative yield.
Compound 7 is not stable under acidic conditions
because it is prone to acyl migration. The rearrange-
ment is not detected under basic or neutral conditions
within 2 days and is therefore sufficiently stable to be
employed in the phosporylation reactions.
The coupling of the choline headgroup to form 1 was
performed by the use of phosphorus oxychloride and
choline tosylate, applying standard conditions.32;33 We
used a MB-3 ion-exchange column prior to column
chromatography to remove salts because washing is not
possible because of the amphiphilic properties of the
molecule. This reaction has suffered from inconsistent
yields,34 which we also experienced initially when using
CHCl3 as solvent (0-80% yields were obtained). Using
dichloromethane instead gave consistently high yields
around 77%. The overall yield in the synthesis of 1 from
4 was a very competitive 54%.
Preparation of the protected phosphatidylethanol-
amine 8 (Scheme 2) toward the synthesis of the polymer-
covered lipids was initially attempted with POCl3 as the
phosphorylation reagent.35-37 We encountered several
drawbacks with this reagent in the synthesis of 8. This
led us to develop a new phosporylation method using
commercially available methyl dichlorophosphate. The
first attempts were performed in chloroform and with
triethylamine as base. This resulted in complex mix-
tures, which were impossible to purify. A new attempt
was made using triethylamine and pyridine. The idea
was that pyridine would function as an activating agent
and triethylamine as a proton scavenger, keeping pH
high and preventing acyl migration; however, this also
a
(a) Et3N, ACN, 4 h, room temp.
resulted in complex mixtures. A more sterically hin-
dered base, tetramethyl piperidine (TMP), was tried,
and this resulted in the desired compound, easily
separated from the byproducts. Toluene was used
instead of chloroform, which resulted in an improved
yield. Furthermore, the use of toluene has the advan-
tage that salts precipitate and can be filtered off prior
to column chromatography. We have found this new
phosphorylation method superior when using cheap
headgroups that can be used in excess. The method gave
8 in 70% yield. The deprotection of 8 was performed
using TFA,38 giving 9 in quantitative yield, which
without purification was coupled to the activated poly-
(ethylene glycol) 12 and 13 (Scheme 3).39,40 10 was
purified by successively extracting toluene with water
to remove unreacted polymer, followed by column chro-
matography, resulting in a 74% yield. 11 could not be
purified the same way because of the water solubility
of the PEG2000. Unreacted PEG2000 could not be sepa-
rated from the desired product but was removed in the
final step. The deprotection of 10 and 11 was performed
with NaI,41,42 resulting in a 95% yield of 2 and a 70%
yield of 3 over two steps. Because of the size and polarity
of 3, it does not have simple properties in relation to
column chromatography. Even though TLC showed an
Rf of 0.3 in 12% methanol/dichloromethane, it ran
extremely slowly on a silica column. The polarity of the
solvent was raised to 50% MeOH/DCM before 3 came
through. This was ascribed to the size of the PEG2000
making 3 a macromolecule.

Release of Antitumor Ether Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1697
Ta ble 1. Differential Scanning Calorimetry Data Showing the Melting Enthalpy, ∆Hm, and Peak Position Tm of the Main Phase
Transition
95% 1-O-DPPC
90% 1-O-DPPC
95% 1-O-DPPC
1-O-DPPC
5% 1-O-DPPE-PEG350
10% 1-O-DPPE-PEG350
5% 1-O-DPPE-PEG2000
∆Hm (kcal/mol)
Tm (°C)
9.45
43.01
8.26
42.64
7.99
42.43
7.5
42.86
F igu r e 3. Differential scanning heat-capacity curves obtained
at a scan rate of 10 °C/h for 1-O-DPPC liposomes containing
0% 1-O-DPPE-PEG, 5% 1-O-DPPE-PEG350, 10% 1-O-DPPE-
PEG350, and 5% 1-O-DPPE-PEG2000
.
F igu r e 4. sPLA2 hydrolysis of 1-O-DPPC liposomes. Lag-time
measurements (solid line) show a minimum just above the
main phase transition. The degree of hydrolysis 1000 s after
the burst measured by HPLC is also illustrated (dashed line).
Th er m a l An a lysis. Figure 3 shows differential scan-
ning calorimetry results obtained for four different
multilamellar liposome systems. We have studied pure
1-O-DPPC liposomes together with mixtures of 1-O-
DPPC and 1-O-DPPE-PEG. The thermograms reveal
that all formulations form liposomes. The DSC scan of
1-O-DPPC shows the main phase transition Tm at 43
°C and a pretransition where the lipid membrane goes
from the gel to the rippled phase. This is the usual phase
behavior of diacylphosphatidylcholines,43 thereby show-
ing that the C-1 ether linkage does not change the phase
behavior drastically. The main phase-transition tem-
perature is situated slightly above Tm of DPPC, indicat-
ing that there are more hydrophobic interactions be-
tween the aliphatic chains. However, the width of the
transition, T1/2, of multilamellar vesicles (MLVs) is
larger for 1-O-DPPC (T1/2 ) 0.24 °C) than for DPPC (T1/2
) 0.17 °C), indicating a less abrupt change in the
membrane structure. The increase in the enthalpy of
melting, compared to the enthalpy of melting of DPPC
(∆Hm ) 8.39 kcal/mol 44), suggests that 1-O-DPPC (∆Hm
) 9.45 kcal/mol) forms more stable liposomal mem-
branes because of a tighter packing of the lipids. Tm and
∆Hm values for the multilamellar liposomes of 1-O-
DPPC with 5% and 10% 1-O-DPPE-PEG350 and 5% 1-O-
DPPE-PEG2000 are given in Table 1. It can be seen that
incorporation of polymer-coated lipids lowers ∆Hm,
indicating weaker interactions in the bilayer. In par-
ticular, the decrease in ∆Hm for 1-O-DPPE-PEG2000
suggests that a large headgroup has an impact on the
packing properties of the aliphatic chains. ∆Hm remains
nearly the same for both concentrations of PE-PEG350
in accordance with results presented by Kenworthy et
al.45
sP LA2 La g-Tim e Mea su r em en ts on 1-O-DP P C
Lip osom es. Figure 4 shows the observed sPLA2 lag
times in the temperature region of the main phase
transition (38.5-46.5 °C), together with the degree of
hydrolysis measured by HPLC 1000 s after the burst.
The lag time, τ, reflects the elapsed time prior to
accelerated hydrolysis (burst), which can be monitored
by intrinsic fluorescence from the enzyme (see Figure
5a). Simultaneously with the sudden increase in the
measured fluorescence, a decrease in the 90° static light
scattering is observed. This is due to a change in the
lipid morphology as nonbilayer-forming lysophospho-
lipids and fatty acids are formed. Comparison of the
DSC scan and the lag-time measurements shows that
the lag-time minimum correlates with the main phase
transition. This is in accordance with results found by
Hønger et al.26 Interestingly, the minimum in the lag
time furthermore corresponds to the maximum in the
degree of hydrolysis (Figure 4).
Ca lcein Relea se by Lysop h osp h olip id P er tu r ba -
tion . Calcein release from D-O-SPC liposomes can be
used as a model system for analyzing and substantiating
the idea of using a liposomal carrier system as a
combined prodrug and drug-delivery system that en-
hances the membrane permeability, resulting in an
increased diffusion of the released drugs over the cell
membrane.46 The drug carriers are unilamellar lipo-
somes of 1-O-DPPC lipids with different concentrations
of 1-O-DPPE-PEG lipids. These liposomes are stable and
long-circulating in the bloodstream because of the steric
repulsion of the polymers but are succeptible to sPLA2
attack, resulting in liposome destabilization followed by

1698 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Andresen et al.
F igu r e 5. (a) Charateristic reaction time profile at 40.5 °C
for sPLA2 (A. piscivorus piscivorus) acting on 1-O-DPPC
liposomes, measured by intrinsic fluorescence from the enzyme
(black line) and at 90° static light scattering (gray line) from
the lipid suspension. The scale is arbitrary. (b) Released calcein
from D-O-SPC target liposomes at 40.5 °C. The measurements
show that calcein release succeeds hydrolysis of the drug
carrier liposomes, as should be expected.
F igu r e 6. Release of the fluorescent calcein from the target
D-O-SPC liposomes as a function of time. sPLA2 was added
after 20 min to a liposome suspension of D-O-SPC and 1-O-
DPPC incorporated with 1-O-DPPE-PEG lipids: (a) temper-
ature was 40.5 °C; (b) temperature was 38.5 °C. The experi-
ments were stopped, after the burst had occurred, by addition
of Triton X-100, which leads to complete release (100%) of
calcein.
lysolipid (AEL) and fatty acid release. The D-O-SPC
liposomes with encapsulated calcein are inert to sPLA2
degradation and serve as a model of the membrane of
the tumor cells. This makes it possible to measure the
permeability-enhancing properties of the released AEL
molecules from sPLA2 hydrolysis of the carrier lipo-
somes simply by measuring the amount of calcein
released from the target liposomes.
the enzyme activity is lower. This does not reveal
anything about the perturbing ability of the two lipids
but reflects a lower enzyme affinity and/or accessibility
to the membrane surface at this temperature.47 1-O-
DPPE-PEG350 is expected to be less perturbing than
1-O-DPPE-PEG2000 based on molecular-shape consid-
erations. The large headgroup of 1-O-DPPE-PEG2000 is
expected to induce a more pronounced curvature stress
in the membrane. However, to answer the question of
which hydrolyzed 1-O-DPPE-PEG (1-O-2-lyso-PPE-
PEG) possesses the highest ability to perturb the D-O-
SPC membrane and thereby enhances drug uptake, one
needs to account for the effective concentration of the
1-O-2-lyso-PPE-PEG in the target membrane, which is
a result of the water-solubility difference of the mono-
mers. It is notable, however, that 1-O-DPPC with 10%
1-O-DPPE-PEG350 shows nearly complete calcein re-
lease. From HPLC measurements (data not shown), we
know that this formulation gives a very high degree of
hydrolysis at 38.5 °C, which could be the reason for the
more complete calcein release. Another explanation
could be that 1-O-2-lyso-PPE-PEG350 perturbs the mem-
brane more than 1-O-2-lyso-PPC does because of the
difference in the overall molecular shape.
The results obtained by the lag-time measurements
show a strong time-dependent correlation with the
calcein release. Noticeably, the burst indicated by
intrinsic fluorescence from sPLA2 happens prior to
calcein release. Figure 5 shows that we can use the
calcein release measurements as a consistent and direct
indication of the sPLA2 lag-time (burst) behavior.47
Intrinsic fluorescence and HPLC measurements of the
mixed liposome systems are very similar to the pure
1-O-DPPC system (data not shown). The lag time is,
however, shorter because of the negative charge intro-
duced by the 1-O-DPPE-PEG lipids. Figure 6a shows
the degree of calcein release from D-O-SPC liposomes
at 40.5 °C as a function of time after the addition of
sPLA2 to the different liposome suspensions. A small
release of calcein is seen shortly after addition of sPLA2.
This release is difficult to explain; however, early sPLA2
studies have shown that a short initial burst occurs
followed by a lag phase in sPLA2 hydrolysis of zwitte-
rionic phospholipid vesicles.48 The time-release profiles
for the four liposome systems investigated show that
liposomes containing 1-O-DPPE-PEG lead to a fast
calcein release in accordance with lag-time experiments
on polymer-covered liposomes.28 It is seen that there is
very little difference in the calcein release for 1-O-
DPPE-PEG2000 and 1-O-DPPE-PEG350 when incorpo-
rated in the same concentration, which indicates a
similar susceptibility of these membrane systems for
sPLA2 hydrolysis. However, at 38.5 °C (Figure 6b) the
difference is more substantial because the steric barrier
of the PEG2000 polymer becomes more dominating when
There is evidence that the negative charge on 1-O-
DPPE-PEG lipids is the main contributor to the en-
hanced sPLA2 activity. The length of the PEG2000 should
give rise to a much higher steric repulsion of the enzyme
than PE-PEG350 does. However, the apparent steric
repulsion observed at 40.5 °C is overshadowed by the
electrostatic attraction, resulting in a short lag time and
fast hydrolysis of the liposomes. At lower temperatures
(38.5 °C), the steric repulsion of PE-PEG2000 compared
to PE-PEG350 becomes more pronounced and results in
an increased lag time. These results substantiate the
results presented in early articles,49-52 describing re-
duced enzyme interaction, and the finding that sPLA2

Release of Antitumor Ether Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1699
products of sPLA2 possess a membrane-perturbing effect
and cause a substantial calcein leakage from the target
liposomal membranes. This indicates, in relation to
medical applications in cancer treatment, that there will
be an increased diffusion of the released drugs into the
cancer cells. This could potentially lead to an enhanced
efficacy of the carried antitumor drugs. Hemolytic
experiments on red blood cells have demonstrated that
proAEL liposomes composed of pure 1-O-DPPC and 1-O-
DPPC doped with 5% 1-O-DPPE-PEG2000 have a very
low hemolytic toxicity.
The results presented are promising in relation to the
development of a new liposome-based drug and drug-
delivery system that target cancer tissue with elevated
levels of sPLA2. The proAEL liposomes offer a cancer-
targeted release of a carried drug, such as doxorubicin,
through their susceptibility to sPLA2 degradation. In
addition, the idea combines the use of liposomes as drug
carriers with a unique and novel lipid-based prodrug
that is activated by sPLA2 specifically in tumor tissue.
The resulting antitumor ether lipids (AELs) are known
to display promising anticancer activity but have so far
been limited in use because of their hemolytic proper-
ties. The proAEL concept is designed to circumvent
these limitations and might even enhance the efficacy
of the carried drug through the increased permeability
of the target cell membrane. The results presented
herein constitute a model study that is necessary for
clarifying the suitability and potential of using sPLA2
as a site-specific trigger for the release of antitumor
ether lipids specifically in tumor tissue. We have
provided the fundamental building blocks of the concept,
which are required in order to take the next rational
steps into biological studies.
F igu r e 7. Percent hemolysis of human red blood cells as a
function of lipid concentration. The liposomes were composed
of 100% 1-O-DPPC (squares) and 95% 1-O-DPPC with 5% 1-O-
DPPE-PEG2000 (triangles) and compared with ET-18-OCH3
(diamonds) (forms micelles).
has increased activity toward PE-PEG doped lipo-
somes47 because of the introduction of a net negative
charge.28
In Vitr o Stu d y of Red Blood Cell Hem olysis by
p r oAEL Lip osom es. The thoroughly investigated AEL
compound ET-18-OCH3 (Edelfosine) is known to be very
cytotoxic toward tumor cells. However, the therapeutic
use of ET-18-OCH3 as an anticancer drug has been
restricted by its strong hemolytic properties.53 The
disruptive nature of ET-18-OCH3 on the membrane of
red blood cells is closely associated with its detergent-
like properties. It has been shown that the association
of ET-18-OCH3 with stable liposomes greatly reduces
the hemolytic effects in vitro and in vivo.54,55 However,
the toxic dose is still very low. Two of the liposome
systems studied in the present paper have been com-
pared with ET-18-OCH3. The synthesized proAELs are
close proanalogues of ET-18-O-CH3 and become cyto-
toxic once activated by sPLA2. These proAEL analogues
are consequently very interesting as chemotherapeutic
agents if the severe side effects, such as hemolysis, can
be removed by localized activation by sPLA2 specifically
in tumor tissue. Figure 7 shows the comparison of the
hemolytic properties of two liposome proAEL systems
with ET-18-O-CH3. It is clear that the masked proAELs
are significantly less hemolytic and nontoxic against
human red blood cells. These results suggest that the
proAEL liposome systems may be worthy candidates for
the design of novel tumor-targeting drug-delivery for-
mulations that can be administered intravenously.
Exp er im en ta l Section
Ma ter ia ls a n d Lip osom e P r ep a r a tion . Edelfosine (ET-
18-OCH3), which was used as reference in the blood hemolysis
study, was purchased from Avanti Polar Lipids, Birmingham,
AL. All other lipids were synthesized in our laboratories as
described in Results and Discussion. 1-O-DPPC multilamellar
liposomes were prepared by hydrating the lipids in a Hepes
buffer solution for 1 h at 55 °C. The lipid suspension was
vortexed every 15 min. Mixed liposomes of 1-O-DPPC and 1-O-
DPPE-PEG were prepared by dissolving the weighed amounts
in chloroform. The solvent was removed using a gentle stream
of nitrogen, after which the lipid film was dried overnight
under reduced pressure. The lipid film was hydrated in Hepes
buffer using the same conditions as for 1-O-DPPC. Unilamellar
liposomes of narrow size distribution were made by extrusion
of the multilamellar liposome suspension 10 times through two
stacked 100 nm polycarbonate filters.57 The Hepes buffer used
was prepared from 0.15 M KCl, 1 mM NaN3, 0.03 mM CaCl2,
0.01 mM EDTA, 0.01 M Hepes (pH 7.5). The employed sPLA2
(Agkistrodon piscivoros piscivoros) was isolated and purified
from snake venom.58 This enzyme is structurally similar to
mammalian sPLA2, indicating a similar catalytic hydrolysis
mechanism for phospholipid substrates.59
Differ en tia l Sca n n in g Ca lor im etr y. Differential scan-
ning calorimetry (DSC) of 5 mM multilamellar liposomes was
performed using a Microcal MC-2 (Northhampton, MA) ultra-
sensitive power-compensating scanning calorimeter equipped
with a nanovoltmeter. The scans were performed in the upscan
mode at a scan rate of 10 °C/h. An appropriate baseline has
been subtracted from the calorimetric curves.
P h osp h olip a se A2 La g-Tim e Mea su r em en ts. Assay con-
ditions for the sPLA2 lag-time measurements were 0.15 mM
unilamellar liposomes, 150 nM sPLA2, 0.15 M KCl, 1 mM
NaN3, 0.03 mM CaCl2, 0.01 mM EDTA, and 0.01 M Hepes (pH
Con clu sion
Results have been presented that support and elabo-
rate earlier results by Vermehren et al.47,56 and J ør-
gensen et al.28 It is shown that there is an increased
sPLA2 activity toward liposomes composed of zwitter-
ionic phospholipids, such as 1-O-DPPC, when incorpo-
rated with negatively charged 1-O-DPPE-PEG. Through
the synthesis of prodrug lipids of 1-O-DPPC and 1-O-
DPPE-PEG, it has been possible to substantiate that
sPLA2 does not express a significant difference in
activity toward liposomes composed of 1-O-phospholip-
ids compared to liposomes composed of diacylphospho-
lipids.47 By employment of D-O-SPC liposomes with
encapsulated calcein as a simplified model of a target
membrane, it has furthermore been possible to show
that the generated AELs and fatty acid hydrolysis

1700 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Andresen et al.
7.5). The catalytic reaction was initiated by adding 8.9 µL of
a 42 µM sPLA2 stock solution to 2.5 mL of a thermostated
liposome suspension equilibrated 20 min prior to addition of
the enzyme. The time elapsed between addition of sPLA2 and
a sudden increase in the intrinsic fluorescence emission from
sPLA2 (tryptophan), due to a burst in the sPLA2 activity, is
defined as the characteristic lag time of the enzyme.26 The
emission takes place at 340 nm after excitation at 285 nm.
The total amount of hydrolyzed lipid 1000 s after the sPLA2
burst was determined by HPLC using a 5 µL diol column, a
mobile phase composed of CHCl3/MeOH/H2O (730:230:25, v/v),
and an evaporative light scattering detector.
P h osp h olip a se A2 Ca lcein R elea se Mea su r em en t s.
Assay conditions for the sPLA2 calcein release measurements
performed in order to investigate the permeability-enhancing
effect of the generated AELs and fatty acid hydrolysis products
were 25 µM unilamellar proAEL liposomes, 25 µM 1,2-bis-O-
octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes
with encapsulated calcein, 25 nM sPLA2, 0.15 M KCL, 1 mM
NaN3, 0.03 mM CaCl2, 0.01 mM EDTA, and 0.01 M Hepes (pH
7.5). The catalytic reaction was initiated by adding 1.5 µL of
a 42 µM sPLA2 stock solution to 2.5 mL of a thermostated
liposome suspension equilibrated 20 min prior to addition of
the enzyme. The calcein release from the D-O-SPC liposomes
was measured by the fluorescent intensity at 520 nm after a
492 nm excitation. The D-O-SPC liposomes were prepared by
hydrating a D-O-SPC lipid film with a Hepes buffer (pH 7.5)
containing 20 mM calcein for 1 h at 65 °C. The lipid suspension
was vortexed every 15 min. The multilamellar liposomes were
extruded as described above, and the resulting unilamellar
liposomes were separated from free calcein using a chromato-
graphic column packed with sephadex G-50. The measure-
ments were performed at temperatures where both the carrier
and the target liposomes are in the gel state. The amount of
calcein released is calculated as
1-O-Hexa d ecyl-3-O-ben zyl-sn -glycer ol (5). To a flame-
dried flask (100 mL) containing washed NaH (0.153 g, 6.40
mmol) under N2 at 0 °C was added cetyl alcohol (1.48 g, 6.10
mmol) in dry THF (25 mL). The NaH was washed by a
continuous extraction of a NaH dispersion (60% mineral oil)
with dry petroleum ether. The reaction mixture was heated
to 80 °C for 1 h. (R)-O-Benzyl glycidol (4) (0.5 g, 3.05 mmol)
was added and then DMF (25 mL) dropwise over 5 min. The
reaction mixture was stirred overnight at 80 °C. The reaction
mixture was cooled to room temperature and stirred for 20
min upon addition of water (2.5 mL). Removal of solvent in
vacuo gave a brown residue, which was redissolved in ether
(40 mL), washed with brine (3 × 30 mL), and dried over Na2-
SO4. The resulting yellow solid was purified on a short column
(5 cm) using 20% ether/CH2Cl2 to give 1-O-hexadecyl-3-O-
benzyl-sn-glycerol (5), crude, Rf ) 0.44 (ether/CH2Cl2 1:4),
which was used without further purification.
1-O-Hexa d ecyl-2-p a lm itoyl-3-O-ben zyl-sn -glycer ol (6).
Crude 5 was dissolved in petroleum ether (60 mL). Pyridine
(1.46 mL, 18.3 mmol) and palmitoyl chloride (4.5 mL, 14,7
mmol) dissolved in petroleum ether (80 mL) were added, and
the mixture was stirred for 16 h at 80 °C, after which TLC
(petroleum ether/CH2Cl2/ether 20:12:3) indicated that the
reaction was complete. The reaction was quenched with water
(15 mL) and the mixture was stirred 30 min, after which
petroleum ether (100 mL) was added. The solution was washed
with 0.8 mL of HCl (2 × 50 mL), H2O (2 × 50 mL), NaHCO3
(4 × 25 mL), and H2O (2 × 50 mL), dried over MgSO4, and
concentrated. Purification by flash chromatography (petroleum
ether/CH2Cl2/ether 60:12:3) gave 2,39 g (70%) of 6. Rf ) 0.51
(petroleum ether/CH2Cl2/ether 20:12:3). Anal. Calcd: C, 78.21;
H, 11.78. Found: C, 78,24; H, 11.59. 1H NMR (300 MHz,
CDCl3): δ 7.32 (m, 5H, Ph), 5.18 (quintet, 1H, J ) 5.1 Hz,
CH), 4.48-4.60 (AB, 2H, J ) 12.2 Hz, CH2Ph), 3.62 (d, 2H, J
) 5.4 Hz, CH2CHCH2), 3.57 (d, 2H, J ) 5.2 Hz, CH2CHCH2),
3.42 (m, 2H, C15H31CH2O), 2.33 (t, 2H, J ) 7.4 Hz, CH2COO),
1.62 (quintet, 2H, â-CH2), 1.52 (quintet, 2H, â-CH2), 1.26 (br
s, 50H, 25 × CH2), 0.88 (t, 6H, 2 × CH3). 13C NMR (75 MHz,
CDCl3): δ 173.5 (COO), 138.1, 128.4, 127.7 127.6 (Ph), 73.2,
71.5, 69.1, 68.7 (4 × CH2O), 71.2 (OCH), 34.4 (CH2COO), 31.8,
29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.0, 25.9, 24.9, 22.6 (CH2
aliphatic), 14.0 (2 × CH3).
IF(t) - IB
% release ) 100 ×
(1)
IT - IB
IF(t) is the measured fluorescence at time t, IB is the
background fluorescence, and IT is the fluorescence measured
after complete calcein release upon addition of Triton X-100.
Calcein is entrapped in a self-quenching concentration at 20
mM, which is the reason for increased fluorescence upon
release from D-O-SPC liposomes. A linear relation between
the fluorescence measured and calcein released exists with the
D-O-SPC liposome/calcein concentration used in the experi-
ment.
Hem olysis Assa y. The hemolysis assays were performed
as described by Perkins et al.60 0.5 mL of the lipid suspensions
was mixed with 0.5 mL of a washed human red blood cell
(RBC) suspension [4% in PBS (v/v)] and incubated with
constant agitation at 37 °C for 20 h. A negative reference (0%
hemolysis) was prepared by mixing 0.5 mL of the RBC
suspension with 0.5 mL of PBS. A positive reference (100%
hemolysis) was prepared by adding 0.5 mL of 10% Triton X-100
to 0.5 mL of RBC suspension. The incubated samples were
centrifuged (2000 rpm) for 10 min at 20 °C. An amount of 400
µL of the supernatant was diluted with 2 mL of buffer solution
(PBS). Percent hemolysis was measured as absorbance at 550
nm using a Perkin-Elmer 320 spectrophotometer.
Syn th esis. Reactions involving air-sensitive reagents were
carried out under N2 using the syringe-septum technique.
THF was freshly distilled over sodium/benzophenone ketyl.
DMF, CH2Cl2, CHCl3, toluene, pyridine, TMP, and Et3N were
dried over 3 Å molecular sieves. Ether refers to diethyl ether.
Reagents were purchased from Sigma-Aldrich Chemical Co.
and used without further purification. Column chromatogra-
phy was performed using silica gel (35-70 µm, 230-400 mesh).
HPLC analysis was performed using a 5 µL diol column, a
mobile phase consisting of CHCl3/MeOH/H2O (730:230:25, v/v),
and an evaporative light scattering detector. 1H NMR and 13C
NMR spectra were recorded at 300 and 75 MHz, respectively.
1-O-Hexa d ecyl-2-p a lm itoyl-sn -glycer ol (7). To a flame-
dried flask (50 mL) with Pd-C 10% (22 mg) under N2 was
added ethyl acetate (5 mL), and then 6 (195 mg, 0.30 mmol)
was dissolved in ethyl acetate (10 mL). The flask was fitted
with a balloon containing H2, and the mixture was stirred for
90 min. The solution was filtered through a glass filter, which
was washed successively with CH2Cl2. Concentration in vacuo
gave 166 mg (99%) of 7 as a white solid, which was dried at
0.1 mmHg for 1 h and used in the next step. 1H NMR (300
MHz, CDCl3): δ 4.99 (quintet, 1H, J ) 5.1 Hz, CH), 3.82 (d,
2H, J ) 4.8 Hz, CH2CHCH2), 3.62 (ABX, 2H, J ) 5.0 Hz, CH2-
CHCH2), 3.45 (m, 2H, C15H31CH2O), 2.29 (t, 2H, J ) 7.2 Hz,
CH2COO), 1.58 (quintet, 4H, 2 × â-CH2), 1.26 (br s, 50H. 25
× CH2), 0.88 (t, 6H, 2 × CH3).
1-O-Hexa d ecyl-2-p a lm itoyl-sn -glycer o-3-p h osp h och o-
lin e (1). To a solution of POCl3 (35.1 µL, 0.388 mmol) in dry
CH2Cl2 (1.5 mL) at 0 °C was added a solution of 7 (160 mg,
0.290 mmol) and Et3N (54 µL, 0.388 mmol) in CH2Cl2 (3.5 mL)
dropwise over 20 min. The reaction mixture was stirred for
30 min under N2 at room temperature, after which pyridine
(200 µL, 2.49 mmol) and choline tosylate (128 mg, 0.465 mmol)
were added. The reaction was stirred overnight at room
temperature, resulting in a yellow solution. Water (0.2 mL)
was added, and the reaction mixture was stirred for 40 min
and concentrated to a white foam by azeotropic distillation
with ethanol. The residue was dissolved in THF/H2O 9:1 and
slowly passed through an MB-3 column (5 cm), and the solvent
was removed by azeotropic distillation with ethanol in vacuo.
The crude product was purified by column chromatography
(CH2Cl2/MeOH/H2O 65:25:4), giving 1 in 159 mg (77%) as a
white solid. Rf ) 0.37 (CH2Cl2/MeOH/H2O 65:25:4), >99% pure
by HPLC, [R]25 -3.37° (c 0.5, CHCl3/MeOH 1:1) (lit.,61 [R]25
D
D

Release of Antitumor Ether Lipids
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1701
-3.38°, c 0.53 CHCl3/MeOH 1:1). Phospholipase A2 showed
>95% conversion. Anal. Calcd for C40H82NO7P‚H2O: C, 65.09;
H, 11.47; N, 1.90. Found: C, 64.94; H, 11.42; N, 1.90. 1H NMR
(300 MHz, CDCl3): δ 5.13 (quintet, 1H, CH), 4.28 (m, 2H,
POCH2CH2N+), 3.92 (m, 2H, CH2CHCH2), 3.79 (m, 2H,
concentrated to give the crude product as a white solid. The
crude product was purified by flash chromatography (16%
MeOH/Toluene), which gave 1.0 g (82%) of the title compound
as a white solid together with an impurity of N-hydroxysuc-
cinimide. Rf ) 0.15 (eluent, 16% MeOH/toluene). 1H NMR (300
MHz, CDCl3): δ 4.47 (ABX2, 2H, J ) 4.6 Hz, OCH2CH2OCOO),
3.81 (ABX2, 2H, J ) 4.6 Hz, OCH2CH2OCOO), 3.71-3.61 (m,
180H, (CH2CH2O)n), 3.59-3.53 (m, 2H, CH3OCH2), 3.38 (s, 3H,
CH3O), 2.85 (s, 4H. 2 × CH2CO).
POCH2CH2N+), 3.55 (m, 2H, CH2CHCH2), 3.40 (m, 2H, C15H31
-
CH2O), 3.33 (s, 9H, (CH3)3N+), 2.30 (t, 2H, J ) 7.3 Hz, CH2-
COO), 1.59 (quintet, 2H, â-CH2), 1.51 (quintet, 2H, â-CH2), 1.26
(br s, 50H. 25 × CH2), 0.88 (t, 6H, 2 × CH3). 13C NMR (75
MHz, CDCl3): δ 173.6 (COO), 72.0 (CH2CHCH2, J ) 7.9 Hz),
71.6 (C15H31CH2O), 69.3 (CH2CHCH2), 66.1 (POCH2CH2N+, J
) 5.7 Hz), 63.8 (CH2CHCH2, J ) 5.4 Hz), 59.3 (POCH2CH2N+,
J ) 4.8 Hz), 54.2 ((CH3)3N+), 34.4 (CH2COO), 31.8, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 26.0, 24.9, 22.5 (CH2 aliphatic), 13.9 (2
× CH3).
1-O-H exa d ecyl-2-p a lm it oyl-sn -glycer o-3-p h osp h o-O-
m eth yleth a n ola m in e P oly(eth ylen e glycol)350 (10). Acti-
vated polymer 12 (126 mg, 0.256 mmol) was added to a
solution of 9 (87 mg, 0.128 mmol) and Et3N (75µL, 0.54 mmol)
in dry CHCl3 (5 mL), and the reaction mixture was stirred for
2 h at 40 °C. The reaction mixture was concentrated to give a
yellow solid, which was redissolved in toluene (50 mL) and
washed with water (10 × 10 mL) to remove uncoupled polymer.
Toluene was removed under reduced pressure and the residue
was purified by column chromatography (16% MeOH/toluene),
resulting in 104 mg (74% over two steps) of a white solid. Rf
1-O-H exa d ecyl-2-p a lm it oyl-sn -glycer o-3-p h osp h o-O-
m eth yl-N-BOC-eth a n ola m in e (8). To a solution of MeO-
POCl2 (136 µL, 1.36 mmol) and TMP (240 µL, 1.42 mmol) in
dry toluene (2 mL) under N2 at -25 °C was added 7 (343 mg,
0.62 mmol) in toluene (10 mL) by dropwise addition. The
reaction mixture was stirred overnight at room temperature,
after which only traces of starting material were indicated by
TLC. TMP (366 µL, 2.17 mmol) and N-BOC-ethanolamine (575
µL, 3.71 mmol) were added, and the reaction mixture was
stirred overnight at room temperature. TLC indicated traces
of monochlorophosphate, Rf ) 0.81 (CH2Cl2/EtOAc 7:3). The
formed TMP-Cl salt was removed by filtration, and the solvent
was removed under reduced pressure. The product was puri-
fied by column chromatography (CH2Cl2/EtOAc 7:3) to give 343
mg (70%) of 8 as a white solid. Rf ) 0.32 (CH2Cl2/EtOAc 7:3).
Anal. Calcd for C43H86NO9P: C, 65.20; H, 10.94; N, 1.77.
Found: C, 65.32; H, 11.01; N, 1.83. 1H NMR (300 MHz,
CDCl3): δ 5.17 (m, 1H, J ) 5.2 Hz, CH), 4.21, 4.10 (m, 4H,
POCH2CH2N CH2CHCH2), 3.78 (dd, 3H, J ) 11.2 Hz, 3.6 Hz,
CH3OP), 3.56 (d, 2H, J ) 5.3 Hz, CH2CHCH2), 3.43 (m, 4H,
POCH2CH2N C15H31CH2O), 2.34 (t, 2H, J ) 7.4 Hz, CH2COO),
1.62 (quintet, 2H, â-CH2), 1.54 (quintet, 2H, â-CH2), 1.45 (s,
9H, (CH3)3), 1.26 (br s, 50H. 25 × CH2), 0.88 (t, 6H, 2 × CH3).
13C NMR (75 MHz, CDCl3): δ 173.3 (d, CH2COO), 155.9 (NCO),
79.6 (C(CH3)3), 71.8 (C15H31CH2O), 70.5 (CH2CHCH2, J ) 7.4
Hz), 68.2 (CH2CHCH2), 67.1 (POCH2CH2N, J ) 5.9 Hz), 66.1
(dd, CH2CHCH2, J ) 4.9 Hz), 54.4 (CH3OP, J ) 4.8), 40.8
(POCH2CH2N, J ) 5.1 Hz), 34.1 (CH2COO), 31.8, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 26.0, 24.9, 22.5, (CH2 aliphatic), 28.2
((CH3)3), 14.0 (2 × CH3).
1
) 0.35 (16% MeOH/toluene). >99% pure by HPLC. H NMR
(300 MHz, CDCl3): δ 5.15 (m, 1H, CH), 4.28-4.04 (m, 6H,
POCH2CH2N NCOOCH2 CH2CHCH2), 3.76 (dd, 3H, J ) 11.2
Hz 3.7 Hz, CH3OP), 3.70-3.59 (m, 20H, (CH2CH2O)n), 3.57-
3.50 (m, 4H, NCOOCH2CH2O CH3OCH2), 3.49-3.37 (m, 4H,
CH2CHCH2 C15H31CH2O), 3.36 (s, 3H, CH3O), 2.32 (t, 2H, J )
7.4 Hz, CH2COO), 1.60 (m, 2H, â-CH2), 1.52 (m, 2H, â-CH2),
1.26 (br s, 50H. 25 × CH2), 0.88 (t, 6H, 2 × CH3). 13C NMR
(75 MHz, CDCl3): δ 173.2 (COO), 156.5 (NCO), 71.9 (CH2-
OCH3), 71.8 (C15H31CH2O), 70.9-70.1 ((CH2CH2O)n CH2CHCH2),
69.4 (NCOOCH2CH2O), 68.2 (CH2CHCH2), 66.8 (POCH2CH2N,
J ) 5.7 Hz), 66.1 (t, CH2CHCH2, J ) 5.9 Hz), 64.1 (NCOOCH2-
CH2O), 59.0 (CH3O), 54.4 (CH3OP, J ) 6.0 Hz), 41.2 (CH2NCO,
J ) 6.3 Hz), 34.1 (CH2COO), 31.8, 29.6, 29.5, 29.4, 29.3, 29.2,
29.1, 26.0, 24.9, 22.5 (CH2 aliphatic), 13.9 (2 × CH3).
1-O-Hexa d ecyl-2-p a lm itoyl-sn -glycer o-3-p h osp h oeth a -
n ola m in e P oly(eth ylen e glycol)350 (2). Phospholipid (10) (88
mg, 0.081 mmol) was dissolved in 2-butanone (5 mL), and
sodium iodide (122 mg, 0.81 mmol) was added. The reaction
mixture was stirred for 2 h at 75 °C with the flask immersed
to its neck in an oil bath, after which it was concentrated to
give a yellow residue. The residue was redissolved in dichlo-
romethane (50 mL) and washed with 1 M Na2SO3 (5 × 10 mL)
and then with water (5 × 10 mL). The crude product was
purified by column chromatography (12% MeOH/CH2Cl2) to
give 85 mg (95%) of 2 as a white solid. Rf ) 0.31 (12% MeOH/
CH2Cl2). >99% pure by HPLC; phospholipase A2 shows >95%.
1H NMR (300 MHz, CDCl3): δ 5.13 (m, 1H, CH), 4.21 (m, 2H,
NCOOCH2), 3.94 (m, 4H, POCH2CH2N CH2CHCH2), 3.80-3.61
(m, 22H, (CH2CH2O)n), 3.60-3.50 (m, 4H, NCOOCH2CH2O
CH3OCH2), 3.48-3.27 (m, 4H, CH2CHCH2 C15H31CH2O), 3.37
(s, 3H, CH3O), 2.30 (t, 2H, J ) 7.9 Hz, CH2COO), 1.59 (m, 2H,
â-CH2), 1.51 (m, 2H, â-CH2), 1.26 (br s, 50H. 25 × CH2), 0.88
(t, 6H, 2 × CH3). 13C NMR (75 MHz, CDCl3): δ 173.7 (COO),
156.9 (NCO), 72.0 (CH2OCH3), 71.7 (C15H31CH2O), 70.9-69.1
((CH2CH2O)n CH2CHCH2 NCOOCH2CH2O CH2CHCH2), 64.4
(POCH2CH2N), 63.9 (CH2CHCH2), 63.1 (NCOOCH2CH2O),
59.2 (CH3O), 41.4 (CH2NCO), 34.4 (CH2COO), 32.0, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 26.0, 24.9, 22.5 (CH2 aliphatic), 14.0 (2
× CH3).
1-O-H exa d ecyl-2-p a lm it oyl-sn -glycer o-3-p h osp h o-O-
m eth yleth a n ola m in e (9). To 8 (100 mg, 0.128 mmol) in
dichloromethane (3 mL) at 0 °C was added CF3COOH (3 mL),
and the reaction mixture was stirred 35 min at 0 °C. TLC
showed 100% conversion, and the solvent was removed under
reduced pressure. Toluene was added to remove CF3COOH by
azeotropic distillation. The resulting viscous oil was used
without purification in the next reaction. Rf ) 0.05 (CH2Cl2/
EtOAc 7:3).
N-Su ccin im ide P oly(eth ylen e glycol)350 Car bon ate (12).
Poly(ethylene glycol)350 (100 mg, 0.286 mmol) was dissolved
in acetonitrile (1.5 mL) in a flame-dried flask under N2. To
this solution was added triethylamine (120 µL, 0.86 mmol) and
disuccinimide carbonate (146 mg, 0.571 mmol). The clear
solution was stirred for 4 h at room temperature and then
concentrated to give the crude product as a white solid. The
crude product was purified by flash chromatography (8%
MeOH/CH2Cl2), which gave 226 mg (80%) of the title compound
as a white solid. Rf ) 0.54 (eluent, 10% MeOH/toluene). 1H
NMR (300 MHz, CDCl3): δ 4.47 (ABX2, 2H, J ) 4.6 Hz,
OCH2CH2OCOO), 3.81 (ABX2, 2H, J ) 4.6 Hz, OCH2CH2-
OCOO), 3.72-3.61 (m, 26H, (CH2CH2O)n), 3.59-3.51 (m, 2H,
CH3OCH2), 3.39 (s, 3H, CH3O), 2.85 (s, 4H, 2 × CH2CO).
1-O-H exa d ecyl-2-p a lm it oyl-sn -glycer o-3-p h osp h o-O-
m eth yleth a n ola m in e P oly(eth ylen e glycol)2000 (11). Ac-
tivated polymer 13 containing N-hydroxysuccinimide (1 g,
0.467 mmol) was added to a solution of 9 (87 mg, 0.128 mmol)
and Et3N (150µL, 1.076 mmol) in dry CHCl3 (10 mL), and the
reaction mixture was stirred for 2 h at 40 °C. The reaction
mixture was concentrated to give a yellow solid, which was
redissolved in dichloromethane (50 mL) and was washed with
water (10 × 10 mL). Dichloromethane was removed under
reduced pressure and the residue was purified by column
chromatography (8% MeOH/CH2Cl2), resulting in a white solid,
which was used without further purification in the next
reaction. Rf ) 0.45 (10% MeOH/CH2Cl2).
N-Su ccin im id e P oly(eth ylen e glycol)2000 Ca r bon a te
(13). Poly(ethylene glycol)2000 (1.15 g, 0.572 mmol) was dis-
solved in acetonitrile (4 mL) in a flame-dried flask under N2.
To this solution was added triethylamine (240 µL, 1.72 mmol)
and disuccinimide carbonate (292 mg, 1.14 mmol). The clear
solution was stirred for 4 h at room temperature and then

1702 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Andresen et al.
(13) Shin, J .; Shum, P.; Thompson, D. H. Acid-triggered release via
dePEGylation of DOPE liposomes containing acid-labile vinyl
ether PEG-lipids. J . Controlled Release 2003, 91, 187-200.
(14) Guo, X.; Szoka, F. C., J r. Steric stabilization of fusogenic
1-O-Hexa d ecyl-2-p a lm itoyl-sn -glycer o-3-p h osp h oeth a -
n ola m in e P oly(eth ylen e glycol)2000 (3). Phospholipid 11
(0.128 mmol) was dissolved in 2-butanone (50 mL), and sodium
iodide (180 mg, 1.28 mmol) was added. The reaction mixture
was stirred 2 h at 75 °C with the flask immersed to its neck
in an oil bath, after which the yellow solution was concentrated
to give a yellow residue. The residue was redissolved in
dichloromethane (40 mL) and washed with 1 M Na2SO3 (5 ×
10 mL) and then with water (5 × 10 mL). The crude product
was purified by column chromatography (12% f 50% MeOH/
CH2Cl2) to give 245 mg (70% over three steps) of 3 as a white
solid. Rf ) 0.25 (10% MeOH/CH2Cl2). >99% pure by HPLC;
phospholipase A2 shows >95% conversion. 1H NMR (300 MHz,
CDCl3): δ 5.13 (m, 1H, CH), 4.21 (m, 2H, NCOOCH2), 4.00-
3.86 (m, 4H, POCH2CH2N CH2CHCH2), 3.78-3.60 (m, 168H,
(CH2CH2O)n), 3.60-3.50 (m, 4H, NCOOCH2CH2O CH3OCH2),
3.48-3.28 (m, 4H, CH2CHCH2 C15H31CH2O), 3.38 (s, 3H,
CH3O), 2.29 (t, 2H, J ) 6.9 Hz, CH2COO), 1.59 (m, 2H, â-CH2),
1.51 (m, 2H, â-CH2), 1.26 (br s, 50H, 25 × CH2), 0.88 (t, 6H, 2
× CH3). 13C NMR (75 MHz, CDCl3): δ 173.4 (COO), 156.7
(NCO), 71.9 (CH2OCH3), 71.5 (C15H31CH2O), 70.9-69.0 ((CH2-
CH2O)n CH2CHCH2 NCOOCH2CH2O CH2CHCH2), 64.0
(POCH2CH2N), 63.7 (CH2CHCH2), 63.1 (NCOOCH2CH2O),
59.2 (CH3O), 42.3 (CH2NCO), 34.3 (CH2COO), 31.8, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 26.0, 24.9, 22.5 (CH2 aliphatic), 14.0 (2
× CH3).
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Ack n ow led gm en t. This work was supported by the
Danish Technical Research Academy, the Danish Na-
tional Research foundation via a grant to the MEM-
PHYS Center of Biomembrane Physics, The Hasselblad
Foundation, LiPlasome Pharma A/S, and The Medical
Research Council via the Center for Drug Design and
Transport. We furthermore thank J ette Klausen and
Rene O. Holte for laboratory assistance. The sPLA2
(Agkistrodon piscivoros piscivoros) was kindly provided
by Prof. Rodney L. Biltonen, University of Virginia
Health System, as a gift.
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