Synthesis of tocopheryl succinate phospholipid conjugates and monitoring of phospholipase A2 activity

Article abstract of DOI:10.1016/j.bmc.2012.05.024

Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells. In this study the enzyme activity of secretory phospholipase A₂ towards various succinate-phospholipid conjugates has been investigated. The synthesis of six novel phospholipids is described, including two TOS phospholipids conjugates. The studies revealed that the TOS conjugates are poor substrates for the enzyme whereas the phospholipids with alkyl and phenyl succinate moieties were hydrolyzed by the enzyme to a high extent.

Full text of DOI:10.1016/j.bmc.2012.05.024

Bioorganic & Medicinal Chemistry 20 (2012) 3972–3978
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of tocopheryl succinate phospholipid conjugates and monitoring
of phospholipase A2 activity
Palle J. Pedersen a, Hélène M.-F. Viart a, Fredrik Melander b,c, Thomas L. Andresen b,c, Robert Madsen a,
Mads H. Clausen a,c,
⇑
a Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 201, DK-2800 Kgs. Lyngby, Denmark
b Department of Micro- and Nanotechnology, Technical University of Denmark, Building 423, DK-2800 Kgs. Lyngby, Denmark
c Center for Nanomedicine and Theranostics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells.
In this study the enzyme activity of secretory phospholipase A2 towards various succinate-phospholipid
conjugates has been investigated. The synthesis of six novel phospholipids is described, including two
TOS phospholipids conjugates. The studies revealed that the TOS conjugates are poor substrates for the
enzyme whereas the phospholipids with alkyl and phenyl succinate moieties were hydrolyzed by the
enzyme to a high extent.
Received 11 September 2011
Revised 30 April 2012
Accepted 11 May 2012
Available online 17 May 2012
Keywords:
Drug delivery
Liposomes
Ó 2012 Elsevier Ltd. All rights reserved.
Vitamin E
Phospholipase
1. Introduction
studies in mice intravenous or intraperitoneal administration of a-
TOS in ethanol, DMSO or vegetable oil emulsions have been ap-
plied4,6 but these formulations are largely restricted to mouse tumor
models and have little clinical relevance. Non-covalent incorpora-
The lack of effective administration routes can be a bottleneck
in the development of pharmaceuticals from potent lead com-
pounds. Many promising lead molecules suffer from low water sol-
ubility and low stability during transport in the body due to labile
chemical functionalities or fast metabolic clearance, whereby the
use of intravenous or oral administration routes is precluded.
Therefore it is of great interest to develop drug delivery systems
that can enhance the drug stability through transport in the body
and hence increase bioavailability.
tion of a-TOS into liposomal drug delivery systems has so far pro-
vided the most promising administration route with regard to
clinical applicability.7 Nevertheless, the non-covalent incorporation
strategy suffers from low drug content in liposomes together with
an uncontrolled release mechanism and thereby also problems with
leakage from the carrier system. An improved drug delivery system
is therefore needed in order to make
humans.
a-TOS clinically applicable for
a-Tocopheryl succinate (a-TOS, Fig. 1) is a potent growth inhib-
itor towards various cancer cells while the toxicity towards healthy
cells is low.1–3 Structure–activity relationship (SAR) studies have
revealed that the succinate moiety is crucial for the activity and
In the present study incorporation of a-TOS into a recently
developed liposomal drug delivery system8,9 has been investigated.
The drug delivery system consists of secretory phospholipase A2
neither esters of
a
-TOS,
a
-tocopherol itself nor other members of
(sPLA2) IIA degradable liposomes, in which a-TOS is attached
the vitamin E family display cytotoxicity.1,3 In addition SAR studies
have demonstrated that succinates and malonates of TOS are more
active than the conjugates of longer dicarboxylic acids, like the glut-
arate analog.3 However the use of tocopheryl derivatives in cancer
treatment is restricted due to their low water solubility, which ham-
pers an intravenous administration route, and oral administration of
O
R
O
R
HO
O
O
a
-TOS is not effective due to hydrolysis of the succinate by esterases
during the transport through the gastrointestinal tract.4,5 For in vivo
α
-TOS: R = CH3
δ-TOS: R = H
⇑
Corresponding author. Tel.: +45 4525 2131; fax: +45 4593 3968.
E-mail address: mhc@kemi.dtu.dk (M.H. Clausen).
Figure 1. Structure of the potent antiproliferative compounds
nate ( -TOS) and d-tocopheryl succinate (d-TOS).
a-tocopheryl succi-
a
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.024

P. J. Pedersen et al. / Bioorg. Med. Chem. 20 (2012) 3972–3978
3973
O
P
O
Na+
2. Results and discussion
O
O
O
O
OH
2.1. Synthesis of alkyl and phenyl succinate phospholipid
conjugates and sPLA2 activity studies
O
OH
O
R
O
The synthesis of the phospholipids having a succinate moiety in
the sn-2 position was initiated from the known phospholipid
precursor3.23 Acylationof 3 withbenzyl succinate using dicyclohex-
ylcarbodiimide (DCC) and 4-(N,N-dimethylamino)pyridine (DMAP)
in CH2Cl2 gave the desired succinate. Hydrogenolysis of the benzyl
ester using Pd/C in a 1:1 mixture of MeOH and EtOAc afforded the
carboxylic acid 4 in 73% overall yield from 3 (Scheme 1). Precursor
4 was diversified into the desired phospholipids 5–8 by first a
carbodiimide mediated coupling with the corresponding alcohol
or phenol, followed by removal of the cyanoethyl and tert-butyl
dimethylsilyl (TBS) protection groups on the glycerol head group
using the reaction conditions previously described (Scheme 1).8,9
Phospholipid 8 was made applying a Yamaguchi coupling24 in the
esterification of 4 with 3,4,5-trimethylphenol.
The phospholipids 5–8 were formulated as liposomes in 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer by
extrusion25 and dynamic light scattering (DLS) analysis revealed
that particles with diameters close to 100 nm were formed, indi-
cating formation of liposomes. The formulated solutions of 5–8
were subjected to snake (Naja mossambica mossambica) venom
sPLA2 and incubated at 37 °C for 48 h. Samples were taken after
2, 24 and 48 h and the enzyme activity was monitored by ma-
trix-assisted laser desorption/ionization time of flight mass spec-
troscopy (MALDI-TOF MS) using a 2,5-dihydroxy benzoic acid
(DHB) matrix.8,26 As evident from Figure 3 the octyl succinate
phospholipid 5 was completely degraded by the enzyme within
24 h, demonstrating that the succinate moiety itself is tolerated
by sPLA2. The enzyme activity on the phenyl succinate phospholip-
ids 6, 7 and 8 was almost as good as on 5, although small peaks for
the starting phospholipids remain in the MS spectra (Fig. 3), but the
degree of hydrolysis is still >70% for 6–8. The MS spectra also con-
firmed that the desired lysolipid was released in the enzyme cata-
lyzed hydrolysis (see Supplementary data). In the absence of sPLA2,
no degradation of the phospholipids was detected (Fig. 3). From the
activity studies on 5–8 it can therefore be concluded that the incor-
poration of a succinate moiety into the sn-2 position does not ob-
struct the ability of sPLA2 to liberate molecules from the sn-2
position. However, alkyl succinates are slightly better substrates
for the enzyme than phenyl succinates, but as the extent of
degradation for the phenyl succinate phospholipids were >70%
O
R
1
: R = CH3
2: R = H
Figure 2. Structure of the targeted TOS phospholipid conjugates.
covalently to the sn-2 position of the phospholipid backbone, see
Figure 2. sPLA2 IIA is part of the PLA2 superfamily, which consists
of a broad range of enzymes defined by their ability to catalyze
the hydrolysis of the ester bond at the sn-2 position in phospholip-
ids yielding the carboxylic acid and lysolipids.10,11 Overexpression
of sPLA2 IIA in human tumors12,13 have, together with the enhanced
permeability and retention (EPR) effect,14,15 made selective delivery
of anticancer agents to cancer tissue possible by the use of sPLA2
sensitive liposomes.16,17 Advantageously, this liposomal formula-
tion will scavenge the biologically important carboxylate moiety
in a-TOS by formation of an ester bond whereby only tissue with
an over-expression of sPLA2 will be affected by the circulating
liposomes.
In addition to
a
-TOS, the less potent d-tocopheryl succinate.3
(d-TOS, Fig. 1) was included in the investigation in order to provide
additional information concerning enzyme activity and cytotoxo-
city. The d-TOS phospholipid conjugate (2) is shown in Figure 2.
Previous studies have shown that sPLA2 is less reactive to-
wards substrates with substituents in close proximity of the sn-
2 position,18–21 and therefore we decided to investigate the sPLA2
activity towards phospholipids with a succinate moiety in the sn-
2 position prior to studying the TOS phospholipid conjugates. In
addition to the alkyl succinate phospholipid conjugate
5
(Scheme 1) we also opted to study the enzyme activity on the
phenyl succinate phospholipid conjugates 6–8 (Scheme 1). This
was done in order to investigate the importance of the methyl-
substitution on the aromatic ring for the enzymatic activity. The
extensively substituted phenyl-moiety in
a-TOS provides a bulky
environment close to the active site, which may lead to an imper-
fect positioning of the phospholipid in the active site of sPLA2
and/or block the entrance of water via the lipophilic channel in
the enzyme.20,22
O
P
O
O
P
O
C18H37
O
O
O
OTBS
OTBS
C18H37
O
O
O
O
OTBS
OTBS
a
OH
O
O
3
4
73%
CN
CN
HO
O
P
O
Na+
C18H37
O
O
O
O
OH
b or c
O
O
OH
RO
5: R = C8H17 (31%)
6
: R = Ph (25%)
: R = 2,6-(CH3)2Ph (14%)
8: R = 3,4,5-(CH3)3Ph (71%)
7
Scheme 1. Synthesis of the succinate phospholipids 5, 6, 7 and 8. Reagents: (a) (i) Benzyl succinate, DCC, DMAP, CH2Cl2; (ii) H2, Pd/C, EtOAc, MeOH; (b) (i) C8H17OH, PhOH, or
2,6-dimethylphenol, DCC, DMAP, CH2Cl2; (ii) DBU, CH2Cl2; (iii) HF, H2O, CH2Cl2, MeCN; (c) (i) 3,4,5-trimethylphenol, 2,4,6-Cl3C6H2COCl, DMAP, THF; (ii) DBU, CH2Cl2; (iii) HF,
H2O, CH2Cl2, MeCN.

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P. J. Pedersen et al. / Bioorg. Med. Chem. 20 (2012) 3972–3978
Figure 3. MALDI-TOF MS monitoring of snake (Naja mossambica mossambica) venom sPLA2 activity on the succinate phospholipid 5 (top left), 6 (top right), 7 (bottom left) and
8 (bottom right).
we found it relevant to continue with the incorporation of
and d-TOS.
a-TOS
achieved from succinate 4 in 48% yield over the three steps
(Scheme 2).
The TOS phospholipid conjugates 1 and 2 were hydrated in
HEPES buffer yielding milky solutions and differential scanning
calorimetry (DSC) scans (20–65 °C) were obtained. However, no
main phase transition temperature was observed in the tested
range, suggesting that 1 and 2 could be in a fluid state at 20 °C.
These findings were further supported during the formulation
where it was possible to extrude the solutions of 1 and 2 through
a 100 nm filter at 20 °C, yielding clear and transparent solutions.
DLS analysis of the formulated phospholipids revealed that
particles with an average diameter of 115 nm were formed indicat-
ing formation of unilamellar vesicles.
2.2. Synthesis, enzyme activity and cytotoxicity of tocopheryl
succinate phospholipid conjugates
The
synthetic steps from the secondary alcohol 3 (Scheme 2). Acylation
of 3 with -TOS gave the desired ester bond and the following
a-TOS phospholipid conjugate 1 was accessible in three
a
deprotections of the cyanoethyl and the TBS groups were achieved
using the same conditions as in synthesis of 5–8 (Scheme 1), giving
the phospholipid conjugate 1 in 61% overall yield. In a similar fash-
ion the synthesis of the d-TOS phospholipid conjugate 2 was
O
P
O
O
P
O
Na+
C18H37
O
O
O
OTBS
OTBS
C18H37
O
O
O
O
OH
a, c
OH
O
OH
O
61%
CN
RO
3
1
: ROH =
α
-Tocopherol
O
P
O
O
P
O
Na+
O
O
OTBS
OTBS
O
O
O
OH
OH
C18H37
O
C18H37
O
b, c
O
O
O
O
O
48%
CN
RO
HO
2: ROH = δ-Tocopherol
4
Scheme 2. Synthesis of the phospholipid conjugate 1 and 2. Reagents: (a) (i)
a-TOS, EDCI, DMAP, EtOAc, CH2Cl2; (b) (i) d-Tocopherol, 2,4,6-Cl3C6H2COCl, DMAP, THF; (c) (i)
DBU, CH2Cl2; (ii) HF, H2O, CH2Cl2, MeCN.

P. J. Pedersen et al. / Bioorg. Med. Chem. 20 (2012) 3972–3978
3975
Figure 4. MALDI-TOF MS monitoring of snake (Naja mossambica mossambica) venom sPLA2 activity on the TOS conjugates 1 (left) and 2 (right).
The sPLA2 activity was investigated with snake (Naja mossamb-
ica mossambica) venom sPLA2 and human sPLA2. Upon addition of
the enzymes the phospholipid solutions were incubated at 37 °C
for 48 h and the activity was monitored by MALDI-TOF MS. How-
ever, as evident from the MS spectra in Figure 4 (see also Supple-
further degradation, we determined the IC50 of 1 to 40
icantly higher than an all-trans retinoic acid derivative (IC50
l
M, signif-
:
8
lM), which has previously been successfully formulated and re-
leased by sPLA2 (see Supplementary data). 9 To further ensure that
the lack of hydrolysis observed was not due to inhibition of the en-
zyme, we co-formulated (1:1) 1 and 2 with 1,2-distearoyl-sn-gly-
cero-3-phosphoglycerol (DSPG), a known substrate for sPLA2. The
mixed liposomes were subjected to snake and human sPLA2 (see
Supplementary data) and the experiments showed that DSPG
was degraded efficiently by the enzymes, whereas neither 1 nor
2 were hydrolyzed, similar to the monocomponent liposomes de-
scribed above. Taken together, these experiments demonstrate
that the inertness to sPLA2 degradation displayed by 1 and 2 is
inherent to their structure and not an effect stemming from the
properties of the formulated liposomes or inhibition of the
enzyme.
mentary data), the
a-TOS conjugate 1 is a very poor substrate for
the enzyme and based on the MS spectra for 1 no significant deg-
radation occurred within 48 h, neither by using the snake venom
nor the human sPLA2. Based on the MS spectra the d-TOS conjugate
2 is a slightly better substrate for the enzyme and significant
hydrolysis is observed after 24 and 48 h (see Fig. 4), which is con-
firmed by detection of the lysolipid and d-TOS in the MS spectra
(see Supplementary data).
The poor activity of sPLA2 on 1 and 2 were confirmed by evalu-
ation of the cytotoxicity in U2OS human osteosarcoma cells. U2OS
cells are free of sPLA2, which allowed us to explore the cytotoxicity
both in the presence and absence of the enzyme. As evident from
Figure 5 (and Supplementary data) no significant cell death was
observed when 1 or 2 were subjected to the cells neither with
nor without sPLA2. In contrast, the TOS’s and the lysolipid alone in-
duced significantly higher cell death than 1 and 2 verifying the low
extent of hydrolysis.
Based on the enzyme activity data for the succinate conjugates
5–8 and the TOS’s conjugates 1 and 2 the difference in enzyme
activity cannot directly be explained by the substituted phenyl
moiety in tocopherols. Rather, it is apparent that the long lipophilic
tail in tocopherols either blocks the access of water or prevents the
phospholipids 1 and 2 from obtaining a productive positioning in
the active site. That structural motifs so far away from the active
site can influence the enzyme activity in such a strong manner
In order to rule out the possibility that small amounts of liber-
ated TOS is acting as an inhibitor of the enzyme and preventing
Figure 5. Dose-response curves for the treatment of U2OS cells with 1 (ꢀ), 1 + sPLA2 (j), lysolipid (d) and a-TOS (N). The cytotoxicity was measured using the MTT assay as
cell viability 48 h after incubation with the indicated substances for 24 h and shown by mean + SD (n = 3). Snake (Agkistrodon piscivorus piscivorus) venom sPLA2 was added to
a final concentration of 5 nM in the studies.

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P. J. Pedersen et al. / Bioorg. Med. Chem. 20 (2012) 3972–3978
has never been observed for sPLA2 and the current findings are
valuable in the efforts to better understand the ability of sPLA2 to
bind and hydrolyze a broad range of phospholipid substrates.
0.059 mmol) was added and the mixture was concentrated in va-
cuo and purified by column chromatography (CH2Cl2/MeOH 20:1
then CH2Cl2/MeOH 4:1) to give 30 mg (61%) of 1 as a colorless
amorphous solid. Rf = 0.09 (CH2Cl2/MeOH 9:1). 1H NMR
(300 MHz, CDCl3/CD3OD 4:1): d 5.22–5.14 (m, 1H), 4.05–3.98 (m,
2H), 3.98–3.90 (m, 2H), 3.86–3.79 (m, 1H), 3.67–3.56 (m, 4H),
3.47–3.40 (m, 2H), 2.98–2.90 (m, 2H), 2.83–2.76 (m, 2H), 2.60 (t,
J = 6.4 Hz, 2H), 2.09 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H), 1.84–1.74
(m, 2H), 1.59–1.48 (m, 8H), 1.47–1.01 (m, 48H), 0.91–0.83 (m,
15H). 13C NMR (75 MHz, CDCl3/CD3OD 4:1): d 172.4, 171.8,
149.7, 140.7, 126.9, 125.2, 123.3, 117.8, 75.4, 72.6, 72.1, 71.1,
69.1, 66.7, 64.4, 62.4, 39.7 (2C), 37.7 (3C), 37.6, 33.1, 33.0, 32.2,
3. Conclusion
In conclusion we have shown that sn-2 succinate phospholipid
conjugates can form liposomes that are good substrates for sPLA2
when they are either alkyl or phenyl substituted. However, low en-
zyme activity was observed for the a-TOS and d-TOS conjugates 1
and 2, and taking the results from the enzyme studies on phospho-
lipid 5–8 into account, the slow hydrolysis of 1 and 2 cannot directly
be associated with neither the phenyl ring nor methyl substituents
in tocopherols, and therefore we hypothesize that the long lipo-
philic tail in combination with the ring systems in TOS’s influences
the enzyme activity. This means that liposomal formulation of
tocopherol succinates for sPLA2-triggered release is not viable and
future efforts would have to consider other approaches, such as
non-covalent incorporation in liposomes7,27 or the use of alternative
enzymes for triggered release, for example matrix metalloprotein-
ases.28 Nevertheless, the overall conclusion from the present work
is that succinate conjugation provides a promising opportunity for
liposomal delivery of lipophilic alcohols, which may otherwise lack
administration routes due to low water solubility.
31.4, 30.0 (13C), 29.3, 29.0, 28.2, 26.3, 25.1, 24.7, 24.2/23.731
,
23.0, 22.9, 22.8, 21.3, 20.9, 20.0, 19.9, 14.3, 13.1, 12.2, 12.0. 31P
NMR (202 MHz, CDCl3/CD3OD 4:1): d 3.43. IR (neat) 3423, 2923,
2853, 1722, 1696, 1660, 1397, 1264, 1062, 804 cmÀ1. HRMS
(ESI+) calcd for C57H103O12PNa [M+H]+ 1033.7085, found
1033.7094.
4.1.2. 1-O-Octadecyl-2-(4-(d-tocopheroxy)-4-oxobutanoyl)-sn-
glycero-3-phospho-(S)-glycerol (2)
Carboxylic acid 4 (70 mg, 0.084 mmol) and d-tocopherol (50 mg,
0.12 mmol) were dissolved in THF (1.1 mL). 2,4,6-Trichlorobenzoyl
chloride (17 lL, 0.11 mmol) and then DMAP (15 mg, 0.12 mmol)
were added and the reaction mixture was stirred at 20 °C for 17 h,
after which the mixture was concentrated in vacuo and then puri-
fied by column chromatography (heptane/EtOAc 4:1 then hep-
tane/EtOAc 2:1) to give 65 mg of the desired ester that was
4. Experimental procedures
4.1. Organic synthesis
dissolved in CH2Cl2 (4.8 mL) along with DBU (8 lL, 0.053 mmol).
Starting materials, reagents, solvents and sPLA2 from Naja mos-
sambica mossambica were purchased from Sigma–Aldrich Chemical
Co. and used without further purification. CH2Cl2 was dried over
4 Å molecular sieves and THF was dried over sodium/benzophe-
none and distilled before use. Evaporation of solvents was done un-
der reduced pressure (in vacuo). Thin layer chromatography (TLC)
was performed on Merck aluminum sheets precoated with silica
gel 60 F254. Column chromatography was performed using Matrex
60 Å silica gel. The purity of all tested compounds was found to be
>95% by HPLC. HPLC was performed on a Waters Alliance HPLC
equipped with a diode array detector, using a LiChrospher Si 60
column and eluting with water/isopropanol/hexane mixtures.29,30
NMR spectra were recorded using a Bruker AC 200 MHz spectrom-
eter, a Varian Mercury 300 MHz spectrometer or a Varian Unity
Inova 500 MHz spectrometer. Chemical shifts were measured in
ppm and coupling constants in Hz, and the field is indicated in each
case. IR analysis was carried out on a Bruker Alpha FT-IR spectrom-
eter. HRMS was recorded on an Ionspec Ultima Fourier transform
mass spectrometer.
The resulting mixture was stirred for 1 h and then purified directly
by column chromatography (heptane/EtOAc 1:1 then CH2Cl2/MeOH
9:1) to give 59 mg of a colorless oil, which was dissolved in a mix-
ture of MeCN (4.3 mL) and CH2Cl2 (1.5 mL) and cooled to 0 °C. Aque-
ous HF (40%, 255 lL) was added dropwise and the reaction mixture
was allowed to reach 20 °C. After 3.5 h, excess reagent was
quenched by dropwise addition of MeOSiMe3 (1.0 mL) and the mix-
ture was stirred for 30 min after which solid NaHCO3 (13 mg) was
added and the mixture was concentrated in vacuo and purified by
column chromatography (CH2Cl2/MeOH 10:1 then CH2Cl2/MeOH
4:1) to give 40 mg (48%) of 2 as a colorless amorphous solid. 1H
NMR (300 MHz, CDCl3/CD3OD 4:1): d 6.66 (d, J = 2.7 Hz, 1H), 6.62
(d, J = 2.7 Hz, 1H), 5.24–5.15 (m, 1H), 4.07–3.91 (m, 4H), 3.89–3.81
(m, 1H), 3.67–3.57 (m, 4H), 3.47–3.38 (m, 2H), 2.88–2.71 (m, 6H),
2.14 (s, 3H), 1.86–1.72 (m, 2H), 1.62–1.02 (m, 56H), 0.91–0.84 (m,
15H). 13C NMR (50 MHz, CDCl3/CD3OD 4:1): d 170.6, 170.3, 148.2,
140.7, 125.6, 119.4 (2C), 117.3, 75.0, 70.6, 70.2, 69.2, 67.1, 64.9,
62.7, 60.5, 38.5, 37.7, 35.7 (3C), 31.1 (2C), 30.3, 30.0, 29.3 (19C),
28.0, 26.3, 24.3, 23.1, 22.8, 22.3, 20.9, 19.3, 18.0, 14.3, 12.3. 31P
NMR (202 MHz, CDCl3/CD3OD 4:1): d –1.93. IR (neat) 3368, 2922,
2852, 1738, 1466, 1222, 1148, 1046 cmÀ1. HRMS (ESI+) calcd for
4.1.1. 1-O-Octadecyl-2-(4-(
glycero-3-phospho-(S)-glycerol (1)
Alcohol (48 mg, 0.062 mmol),
a-tocopheroxy)-4-oxobutanoyl)-sn-
C
55H99O12PNa [M+H]+ 1005.6766, found 1005.6736.
3
a-tocopheryl succinate
(0.032 M in EtOAc, 1.5 mL, 0.048 mmol) and DMAP (18 mg,
0.14 mmol) were dissolved in CH2Cl2 (4 mL). EDCI (28 mg,
0.14 mmol) was added and the reaction mixture was stirred for
1 h at 20 °C, then concentrated in vacuo and purified by column
chromatography (heptane/EtOAc 2:1 then heptane/EtOAc 1:2) to
give 61 mg of the desired ester, which was dissolved in CH2Cl2
4.1.3. 1-O-Octadecyl-2-(4-hydroxy-4-oxobutanoyl)-sn-glycero-
3-(2-cyanoethylphospho)-(S)-2,3-di-O-tert-butyldimethylsilyl-
glycerol (4)
Alcohol 3 (96 mg, 0.12 mmol) and benzyl succinate (51 mg,
0.25 mmol) were dissolved in CH2Cl2 (2.5 mL). DCC (76 mg,
0.37 mmol) and DMAP (15 mg, 0.12 mmol) were added and the
reaction mixture was stirred at 20 °C for 1 h, then concentrated in
vacuo and purified by column chromatography (heptane then hep-
tane/EtOAc 2:1) to afford 81 mg of the desired ester, which was dis-
solved in a mixture of EtOAc (0.7 mL) and MeOH (0.7 mL). Pd/C
(10 wt%, 12 mg, 0.012 mmol) was added and the mixture was stir-
red under an atmosphere of H2 for 2 h at 20 °C, after which filtration
through celite gave 74 mg (73%) of 4 (two diastereoisomers 1:1) as a
colorless oil. 1H NMR (300 MHz, CDCl3, two diastereoisomers): d
(1.3 mL) along with DBU (8 lL, 0.050 mmol). The resulting mixture
was stirred at 20 °C for 40 min and then purified directly by col-
umn chromatography (CH2Cl2/MeOH 10:1) to afford 41 mg that
was dissolved in MeCN (2.3 mL) and CH2Cl2 (0.8 mL) and cooled
to 0 °C. Aqueous HF (40%, 0.14 mL) was added dropwise and the
reaction mixture was allowed to reach 20 °C. After 3 h the reaction
was quenched by dropwise addition of MeOSiMe3 (0.6 mL) and the
mixture was stirred for 30 min, after which solid NaHCO3 (5 mg,

P. J. Pedersen et al. / Bioorg. Med. Chem. 20 (2012) 3972–3978
3977
5.23–5.16 (m, 1H), 4.32–4.13 (m, 5H), 4.06–3.97 (m, 1H), 3.92–3.84
(m, 1H), 3.62–3.53 (m, 4H), 3.48–3.39 (m, 2H), 2.77 (t, J = 6.3 Hz,
2H), 2.70–2.64 (m, 4H), 1.59–1.49 (m, 2H), 1.32–1.23 (m, 30H),
0.92–0.86 (m, 21H), 0.10 (s, 3H), 0.09 (s, 3H), 0.07 (s, 6H). 13C NMR
(50 MHz, CDCl3, two diastereoisomers): d 175.1, 171.5, 116.5, 72.1
(2C), 71.2, 69.7, 68.2, 66.7, 64.1, 62.2, 32.1, 29.8 (13C), 29.0 (2C),
26.0 (7C), 22.8, 19.6, 18.4 (2C), 14.2, À4.6 (2C), À5.3 (2C). 31P NMR
(202 MHz, CDCl3, two diastereoisomers): d À0.94, À1.13. IR (neat):
2924, 2854, 1737, 1463, 1252, 1152, 1102. HRMS (ESI+) calcd for
4:1): d 7.06–7.05 (m, 3H), 5.20–5.15 (m, 1H), 4.05–3.97 (m, 2H),
3.95–3.89 (m, 2H), 3.82–3.77 (m, 1H), 3.63–3.56 (m, 4H), 3.47–
3.38 (m, 2H), 2.95 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.7 Hz, 2H), 2.14
(s, 6H), 1.55–1.49 (m, 2H), 1.30–1.22 (m, 30H), 0.88 (t, J = 6.9 Hz,
3H). 13C NMR (50 MHz, CDCl3/CD3OD 4:1): d 172.4, 171.1, 148.3,
130.4 (2C), 129.0 (2C), 126.3, 72.6, 72.2, 71.2, 69.2, 66.8, 64.5,
62.5, 32.2, 30.0, 29.3 (13C), 28.9, 26.3, 23.0, 16.4 (2C), 14.3. 31P
NMR (202 MHz, CDCl3/CD3OD 4:1): d À2.62. IR (neat): 3331,
2922, 2853, 1753, 1738, 1233, 1139, 1058. HRMS (ESIÀ) calcd for
C
43H86NO11PSi2 [M+H]+ 880.5793, found 880.5822.
C
36H62O11P [MÀNa]À 701.4035, found 701.4017.
4.1.4. 1-O-Octadecyl-2-(4-octanyloxy-4-oxobutanoyl)-sn-
glycero-3-phospho-(S)-glycerol (5)
4.1.7. 1-O-Octadecyl-2-(4-(3,4,5-trimethylphenyloxy)-4-
oxobutanoyl)-sn-glycero-3-phospho-(S)-glycerol (8)
Carboxylic acid 4 (26 mg, 0.031 mmol) and octanol (7.2
l
L,
Carboxylic acid 4 (70 mg, 0.084 mmol) and 3,4,5-trimethylphe-
nol (16 mg, 0.12 mmol) were dissolved in THF (1.1 mL). 2,4,6-Tri-
chlorobenzoyl chloride (17 lL, 0.11 mmol) and then DMAP
(15 mg, 0.12 mmol) were added and the reaction mixture was stir-
red at 20 °C for 17 h, after which the mixture was concentrated in
vacuo and purified by column chromatography (heptane/EtOAc
4:1 then heptane/EtOAc 2:1) to give 65 mg of the desired ester, that
0.045 mmol) were dissolved in CH2Cl2 (0.5 mL). DCC (8 mg,
0.04 mmol) and DMAP (2 mg, 0.016 mmol) were added and the
reaction mixture was stirred at 20 °C for 1 h, after which the mix-
ture was purified directly by column chromatography (heptane/
EtOAc 4:1 then heptane/EtOAc 2:1) to give 12 mg of the desired es-
ter, that was dissolved in CH2Cl2 (1.1 mL) along with DBU (1.9 lL,
0.013 mmol). The resulting mixture was stirred for 1 h and then
purified directly by column chromatography (heptane/EtOAc 1:1
then CH2Cl2/MeOH 9:1) to give 11 mg of a colorless oil, that was
dissolved in a mixture of MeCN (1.0 mL) and CH2Cl2 (0.33 mL)
was dissolved in CH2Cl2 (6.0 mL) along with DBU (10 lL,
0.068 mmol). The reaction mixture was stirred for 1 h and then
purified directly by column chromatography (heptane/EtOAc 1:1
then CH2Cl2/MeOH 9:1) to give 54 mg of a colorless oil that was dis-
solved in a mixture of MeCN (5.5 mL) and CH2Cl2 (1.9 mL) and
and cooled to 0 °C. Aqueous HF (40%, 60 lL) was added dropwise
and the reaction mixture was allowed to reach 20 °C. After 3.5 h
cooled to 0 °C. Aqueous HF (40%, 325 lL) was added dropwise and
excess reagent was quenched by dropwise addition of MeOSiMe3
(225 lL) and the mixture was stirred for 30 min, after which solid
the reaction mixture was allowed to reach 20 °C. After 3.5 h excess
reagent was quenched by dropwise addition of MeOSiMe3 (1.2 mL)
and the mixture was stirred for 30 min, after which solid NaHCO3
(16 mg) was added, the mixture was concentrated in vacuo and
purified by column chromatography (CH2Cl2/MeOH 10:1 then
CH2Cl2/MeOH 4:1) to give 44 mg (71%) of 8 as a colorless amorphous
solid. 1H NMR (500 MHz, CDCl3/CD3OD 4:1): d 6.71 (s, 2H), 5.22–
5.15 (m, 1H), 4.06–3.90 (m, 4H), 3.88–3.80 (m, 1H), 3.67–3.56 (m,
4H), 3.47–3.37 (m, 2H), 2.89–2.84 (m, 2H), 2.79–2.73 (m, 2H), 2.27
(s, 6H), 2.14 (s, 3H), 1.58–1.48 (m, 2H), 1.35–1.20 (m, 30H), 0.88
(t, J = 6.7 Hz, 3H). 13C NMR (50 MHz, CDCl3/CD3OD 4:1): d 171.6,
171.2, 147.3, 137.2 (2C), 132.3, 119.6 (2C), 71.4 (2C), 70.2, 68.0,
66.8, 64.5, 61.7, 31.4, 29.2 (14C), 28.6, 25.5, 22.2, 19.9 (2C), 14.3,
13.5. 31P NMR (202 MHz, CDCl3/CD3OD 4:1): d À1.56. IR (neat):
3362, 2922, 2852, 1738, 1465, 1147, 1045 cmÀ1. HRMS (ESI+) calcd
for C37H65NaO11P [M+H]+ 739.4157, found 739.4144.
NaHCO3 (3 mg, 0.035 mmol) was added and the mixture was con-
centrated in vacuo and purified by column chromatography
(CH2Cl2/MeOH 10:1 then CH2Cl2/MeOH 4:1) to give 7 mg (31%)
of 5 as a colorless amorphous solid. Rf = 0.38 (CH2Cl2/MeOH 4:1).
1H NMR (300 MHz, CDCl3/CD3OD 4:1): d 5.18–5.11 (m, 1H), 4.08
(t, J = 6.8 Hz, 2H), 3.97–3.90 (m, 4H), 3.85–3.79 (m, 1H), 3.66–
3.62 (m, 2H), 3.60–3.55 (m, 2H), 3.47–3.40 (m, 2H), 2.68–2.61
(m, 4H), 1.67–1.58 (m, 2H), 1.58–1.50 (m, 2H), 1.35–1.23 (m,
40H), 0.89 (t, J = 6.0 Hz, 3H), 0.88 (t, J = 5.8 Hz, 3H). 13C NMR
(50 MHz, CDCl3/CD3OD 4:1): d 172.3, 172.0, 71.8 (2C), 70.4, 68.1,
67.3, 65.1 (2C), 62.2, 31.7 (2C), 29.6 (18C), 25.8, 24.0, 22.5 (2C),
13.8 (2C). 31P NMR (202 MHz, CDCl3/CD3OD 4:1): d À1.61. IR
(neat): 3352, 2922, 2853, 1736, 1220, 1162, 1118, 1053. HRMS
(ESIÀ) calcd for C36H70O11P [MÀNa]À 709.4660, found 709.4687.
4.1.5. 1-O-Octadecyl-2-(4-phenyloxy-4-oxobutanoyl)-sn-
glycero-3-phospho-(S)-glycerol (6)
4.1.8. d-Tocopheryl succinate (d-TOS)
d-Tocopherol (30 mg, 0.075 mmol), succinic anhydride (19 mg,
0.19 mmol) and DMAP (9 mg, 0.075 mmol) were dissolved in
CH2Cl2 (0.3 mL). The reaction mixture was stirred at 20 °C for
14 h, concentrated in vacuo and purified by column chromatogra-
phy (heptane/EtOAc 3:1 then CH2Cl2/MeOH 9:1) to give 26 mg
The synthesis was performed as for 5, starting from carboxylic
acid 4 (30 mg, 0.034 mmol) and phenol (10 mg, 0.10 mmol) and
gave 6 mg (25%) of 6 as a colorless amorphous solid. Rf = 0.14
(CH2Cl2/MeOH 4:1). 1H NMR (500 MHz, CDCl3/CD3OD 4:1): d 7.38
(t, J = 7.8 Hz, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 7.8 Hz, 2H),
5.22–5.17 (m, 1H), 4.05–3.96 (m, 2H), 3.93–3.89 (m, 2H), 3.79–
3.76 (m, 1H), 3.63–3.57 (m, 4H), 3.47–3.38 (m, 2H), 2.90 (t,
J = 6.7 Hz, 2H), 2.79 (t, J = 6.7 Hz, 2H), 1.55–1.49 (m, 2H), 1.31–
1.22 (m, 30H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (50 MHz, CDCl3/
CD3OD 4:1): d 172.5, 171.7, 150.9, 129.8 (2C), 126.3, 121.8 (2C),
72.6, 72.2, 71.2, 69.2, 66.8, 64.5, 62.5, 32.2, 30.0 (15C), 26.3, 23.0,
14.3. 31P NMR (202 MHz, CDCl3/CD3OD 4:1): d À0.64. IR (neat):
3333, 2922, 2852, 1763, 1737, 1251, 1198, 1131, 1068. HRMS
(ESI+) calcd for C34H58O11P [MÀNa]À 673.3721, found 673.3702.
(68%) of d-TOS as
a
colorless amorphous solid. 1H NMR
(300 MHz, CDCl3): d 6.68 (d, J = 2.6 Hz, 1H), 6.63 (d, J = 2.6 Hz,
1H), 2.88–2.68 (m, 6H), 2.14 (s, 3H), 1.85–1.68 (m, 2H), 1.61–
1.02 (m, 24H), 0.87 (d, J = 6.8 Hz, 6H), 0.86 (d, J = 6.5 Hz, 3H), 0.85
(d, J = 6.8 Hz, 3H).3
4.2. Procedures for biophysical and biological characterization
4.2.1. Liposome preparation and particle size determination
The phospholipid prodrugs were dissolved in CHCl3 in a test
tube and dried under vacuum for 15 h to form a thin film. The
phospholipid prodrugs (2 mM) were solubilized by addition of
4.1.6. 1-O-Octadecyl-2-(4-(2,6-dimethylphenyloxy)-4-
oxobutanoyl)-sn-glycero-3-phospho-(S)-glycerol (7)
The synthesis was performed as for 5, starting from carboxylic
aqueous buffer (0.15 M KCl, 30 lM CaCl2, 10 lM EDTA, 10 mM
HEPES, pH 7.5) and vortexed periodically over 1 h at 20 °C. Subse-
quently, the solutions were extruded through a 100 nm polycar-
bonate cutoff membrane (20–30 repetitions) using a Hamilton
syringe extruder (Avanti Polar Lipids, Birmingham, AL). The
acid
4 (71 mg, 0.086 mmol) and 2,6-dimethylphenol (32 mg,
0.26 mmol) and gave 9 mg (14%) of 7 as a colorless amorphous so-
lid. Rf = 0.09 (CH2Cl2/MeOH 4:1). 1H NMR (500 MHz, CDCl3/CD3OD

3978
P. J. Pedersen et al. / Bioorg. Med. Chem. 20 (2012) 3972–3978
particle size distribution of the formulated lipids was measured by
DLS. The DLS measurements were obtained using a Zetasizer Nano
Particle Analyzer (ZS ZEN3600, Malvern Instrument, Westborough,
MA). DSC was performed using a MicroCal MC-2 (Northhampton,
MA) on samples at a scan rate of 10 °C/h. A DSC scan was also per-
formed on the HEPES buffer solution and was used to subtract a
baseline from the thermograms of 1 and 2.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.024.
References and notes
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l
l
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l
l
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Acknowledgments
We thank the Danish Council for Strategic Research (NABIIT
Program) and the Karen Krieger Foundation for financial support.
Dr. Ahmad Arouri and Professor Ole G. Mouritsen, MEMPHYS-
Center for Biomembrane Physics, University of Southern Denmark,
are acknowledged for DSC experiments and IC50 measurements.