Prostaglandin phospholipid conjugates with unusual biophysical and cytotoxic properties

Article abstract of DOI:10.1016/j.bmcl.2010.06.054

The synthesis of two secretory phospholipase A₂ IIA sensitive 15-deoxy-Δ^12,14-prostaglandin J₂ phospholipid conjugates is described and their biophysical and biological properties are reported. The conjugates spontaneously

Full text of DOI:10.1016/j.bmcl.2010.06.054

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4456–4458
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Prostaglandin phospholipid conjugates with unusual biophysical and
cytotoxic properties
Palle J. Pedersen a, Sidsel K. Adolph b, Thomas L. Andresen c, Mogens W. Madsen b, Robert Madsen a,
Mads H. Clausen a,
*
a Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 201, DK-2800 Kgs. Lyngby, Denmark
b LiPlasome Pharma A/S, Technical University of Denmark, Diplomvej 378, DK-2800 Kgs. Lyngby, Denmark
c Department of Micro- and Nanotechnology, Technical University of Denmark, DK-4000 Roskilde, Denmark
a r t i c l e i n f o
a b s t r a c t
The synthesis of two secretory phospholipase A2 IIA sensitive 15-deoxy-D
12,14-prostaglandin J2 phospho-
Article history:
Received 3 May 2010
Revised 8 June 2010
Accepted 8 June 2010
Available online 12 June 2010
lipid conjugates is described and their biophysical and biological properties are reported. The conjugates
spontaneously form particles in the liposome size region upon dispersion in an aqueous buffer and both
phospholipids are hydrolyzed by phospholipase A2, but with different conversion rates and extent of
hydrolysis. The cytotoxicity was evaluated in HT-29 and Colo205 cells and the conjugates induced cell
death in the presence of phospholipase A2 and surprisingly also in the absence of the enzyme.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Phospholipds
Prostaglandins
Drug delivery
Liposomes
Since von Euler1 and Goldblatt2 independently isolated and
studied prostaglandins for the first time, these fatty acids have at-
tracted attention due to their involvement in many important bio-
logical functions.3–5 The biosynthetic precursor for prostaglandins
is arachidonic acid, which by a number of enzyme catalyzed reac-
tions is converted into the diverse selections of prostaglandins
known today.3–5 Our interest in prostaglandins arose when we
started to develop a new generation of liposomal drug delivery sys-
tem consisting of secretory phospholipase A2 (sPLA2) IIA degrad-
able liposomes with potential in cancer treatment.6,7
chains in the sn-1-position linked through either an ether or an es-
ter functionality (see Fig. 2).
The sn-1-ether conjugate 1 was synthesized by a tetrazole med-
iated coupling of the known primary alcohol 3 and the phospho-
amidite 56 (Scheme 1) followed by oxidation to the phosphate
with tBuOOH. The p-methoxybenzyl group was removed with
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in moist CH2Cl2
affording the secondary alcohol that was coupled to 15-deoxy-
D
12,14-PGJ2 applying a dicyclohexylcarbodiimide (DCC) mediated
ester coupling. The conjugate 1 was procured after removal of
The anticancer agents are covalently attached to the sn-2-posi-
tion of the lipid backbone and thereby the drug is an integral part
of the lipophilic liposome membrane. The design uses bioactive
compounds with a carboxylic acid moiety, and therefore, we
decided to investigate prostaglandins. Many prostaglandins have
shown antiproliferative activity in tumor cells, but among the most
studied and active prostaglandins are the dienone prostaglandins
the cyanoethyl group with 1,8-diazabicyclo[5.4.0]undec-7-ene
O
O
OH
OH
O
O
OH
OH
like
D D
12-PGJ2 and 7-PGA1 (see Fig. 1).8 Likewise, 15-deoxy-
Δ
12-PGJ2
Δ7-PGA1
D
12,14-PGJ2, a metabolic derivative of
D
12-PGJ2, has demonstrated
high antitumor activity against L1210 leukemia cells9 and for the
investigation of the liposomal drug delivery system that compound
is more suitable than the former prostaglandins since 15-deoxy-
OH
O
D
12,14-PGJ2 has a higher lipophilicity. The phospholipid conjugates
O
are designed to have phosphatidylglycerol headgroups and C18
15-deoxy-Δ12,14-PGJ2
Figure 1. Potent antiproliferative prostaglandins.
* Corresponding author. Tel.: +45 45252131; fax: +45 45933968.
E-mail address: mhc@kemi.dtu.dk (M.H. Clausen).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.054

P. J. Pedersen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4456–4458
4457
R
O
P
O
O
O
O
OH
O
OH
Na+
O
O
15-deoxy-Δ12,14-PGJ2 phospholipid conjugates
1: R = H2
2: R = O
Figure 2. Targeted 15-deoxy-
D
12,14-PGJ2 phospholipid conjugates.
CN
R
O
P
a-f
1: R = H2 (17%)
2: R = O (13%)
C17H35
O
OH
OPMB
N
O
OTBDMS
OTBDMS
3: R = H2
4: R = O
5
Scheme 1. Synthesis of the 15-deoxy-
Reagents: (a) tetrazole, CH2Cl2, MeCN; (b) tBuOOH; (c) DDQ, H2O, CH2Cl2; (d) 15-
deoxy-
12,14-PGJ2, DCC, DMAP, CH2Cl2; (e) DBU, CH2Cl2; (f) HF, H2O, CH2Cl2, MeCN.
D 1 and 2.
12,14-PGJ2 lipid conjugates
D
(DBU) and the TBDMS-groups with aqueous HF in MeCN and
CH2Cl2. The corresponding sn-1-ester conjugate 2 (Scheme 1) was
accessed in a similar way starting from the acylated glycerol 4
(see Supplementary data).
Figure 3. MALDI-TOF MS monitoring of snake (Naja mossambica mossambica)
venom sPLA2 activity on the 15-deoxy-
(bottom).
D 1 (top) and 2
12,14-PGJ2 conjugates
The prostaglandin conjugates 1 and 2 were hydrated in 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
(pH 7.5) at 20 °C yielding clear solutions. Dynamic light scattering
(DLS) analysis of the lipid suspension of 2 revealed that particles
with an average diameter of around 100 nm were formed, indicat-
ing that the prostaglandin conjugates spontaneously form small
unilamellar vesicles (SUVs) at 20 °C. Self-aggregation into SUVs
upon dispersion in a buffer is very rare for phospholipids and has
only been reported in the literature once before.10 Extrusion
through a 100 nm filter narrowed the average diameter and the
polydispersity of the vesicles (Table 1 and Supplementary data),
presumably because filtration removed the minor population of
flocculated particles. Twelve days after formulation, the particle
distribution of the lipid suspensions was investigated with DLS
again. As evident from Table 1, neither the diameter nor the poly-
dispersity changed significantly, showing that the vesicles formed
maintain their size and does not aggregate into larger particles.
The formulated lipid suspensions of 1 and 2 were investigated
for their susceptibility to undergo sPLA2-mediated hydrolysis. Puri-
fied snake venom sPLA2 from Agkistrodon piscivorus piscivorus and
Naja mossambica mossambica were used. Previously, these enzymes
have shown activity towards a broad range of phospholipids and
the substrate specificity is comparable to human sPLA2 IIA.11 The
enzyme activity was investigated with matrix-assisted laser
desorption–ionization time of flight (MALDI-TOF) MS, which we
and others have demonstrated to be a reliable and valid tool for
detection of phospholipids.6,7,12,13 For the measurements, 2,5-dihy-
droxybenzoic acid (DHB) and CF3COONa in methanol containing
1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) as an inter-
nal standard was used as the matrix, which did not interfere with
the regions of interest in the mass spectrometry spectra for the 15-
deoxy-D
12,14-PGJ2 conjugates.
The formulated lipids 1 and 2 were subjected to sPLA2 and stir-
red at 37 °C and samples for MALDI-TOF analysis were taken after
2, 24 and 48 h. As evident from the MS spectra in Figure 3 the sn-1-
ester 15-deoxy-
sumed by sPLA2 after 24 h. However, the sn-1-ether 15-deoxy-
12,14-PGJ2 conjugate (1) was not completely degraded after 48 h,
D
12,14-PGJ2 conjugate (2) was completely con-
D
the conversion was estimated to be 70%. This difference in hydro-
lysis rate and extent for the sn-1-ester versus the sn-1-ether phos-
pholipids is remarkable and to our knowledge has not been
observed before, but the observation verifies that sn-1-ester phos-
pholipids are better substrates for sPLA2 than sn-1-ether phospho-
lipids.14 For both conjugates 15-deoxy- 12,14-PGJ2 and the
D
lysophospholipids were detected as the released products (data
not shown). Also evident from Figure 3 is that sPLA2 is required
for hydrolysis of both 1 and 2.
Table 2
IC50 values for 15-deoxy-D
12,14-PGJ2, lysolipids and the conjugates 1 and 2 in HT-29
and Colo205 colon cancer cell linesa
Compound
IC50 (lM)
Table 1
DLS analysis of the 15-deoxy-
12,14-PGJ2 conjugates formulated by extrusion
0 days
12 daysb
Diameter
b
D
HT-29
1.6 0.3
HT-29 + sPLA2
Colo205
15-Deoxy-
D
12,14-PGJ2
nd
nd
nd
2.2 0.2
6.4 0.4
4
25
22
9
2
2
3
2
5
Compound
sn-1-Ether lysolipid
sn-1-Ester lysolipid
1
2
11
7
6
1
1
2
Diameter
PdIc
PdIc
1
51
112
81
0.26
0.45
0.35
61
98
76
0.26
0.39
0.41
6
2a
2
32
17
a
Cytotoxicity was measured using the MTT assay as cell viability 48 h after
Data was obtained immediately after the formulation and 12 days later
incubation with the indicated substances for 24 h and shown by mean SD (n P3);
nd = not determined; Snake (Agkistrodon piscivorus piscivorus) venom sPLA2 caused
no change in cell viability within 24 h.
a
Not extruded.
The lipid suspensions were stored at 4 °C for 12 days.
b
c
b
PdI = polydispersity index.
sPLA2 was added to the cell media to a final concentration of 5 nm.

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P. J. Pedersen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4456–4458
20 lM (Table 2) and complete cell death was observed when high-
er concentrations were used (see Supplementary data). However,
since Colo205 cells secrete sPLA2 it is not possible to conclude
whether the hydrolysis of the conjugates was brought about
through the action of sPLA2, but it is promising that the 15-
deoxy-
as well.
In conclusion, we have synthesized a novel class of phospho-
lipid conjugates with 15-deoxy-
12,14-PGJ2 attached to the sn-2-
D
12,14-PGJ2 conjugates show activity in this cancer cell line
D
position. The conjugates spontaneously form SUVs upon dispersion
in HEPES buffer, which is rare for phospholipids. Furthermore, we
observed a significant difference in rate and extend of sPLA2 hydro-
lysis between the sn-1-ester and the sn-1-ether conjugates. In the
cytotoxicity studies we observed that the 1 and 2 induced cell
death in Colo205 cells and in HT-29 cells both in the presence
and absence of sPLA2.
Acknowledgment
deoxy-
D
12,14-PGJ2 conjugates; 1 (j), 1 + sPLA2 (d), 2 (
Figure 4. Dose–response curves for the treatment oNf HT-29 cells with the 15-
) and 2 + sPLA2 (.).
We thank the Danish Research Council for Strategic Research
(NABIIT Program) for financial support.
The cytotoxicity of the conjugates was evaluated in two colon
cancer cell lines, HT-29 and Colo205. HT-29 cells do not secrete
sPLA2 whereas Colo205 cells do. Therefore, the activity of the con-
jugates towards HT-29 cells was investigated in the presence and
in the absence of sPLA2, which enabled us to evaluate the impor-
tance of enzymatic activity for cytotoxicity. Surprisingly, we
observed that the conjugates induced significant cell death (see
Table 2 and Fig. 4) also in the absence of the enzyme, albeit to a
lesser degree than when sPLA2 was present. Despite having studied
a number of other phospholipid prodrugs,6,7 this is the first time
we have observed cytotoxicity of the phospholipid conjugates in
the absence of sPLA2. We speculate that this behavior can arise
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.054.
References and notes
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through spontaneous cellular uptake of the 15-deoxy-D
12,14-PGJ2
conjugates followed by metabolic breakdown in the cytosol. This
facile uptake is likely a consequence of the dynamic behavior of
15-deoxy-D
12,14-PGJ2 conjugates, which also manifests itself dur-
8. Sasaki, H.; Fukushima, M. Anti-Cancer Drugs 1994, 5, 131.
9. Kato, T.; Fukushima, M.; Kurozumi, S.; Noyori, R. Cancer Res. 1986, 46, 3538.
10. Linderoth, L.; Peters, G. H.; Madsen, R.; Andresen, T. L. Angew. Chem. Int. Ed.
2009, 48, 1823.
11. Leidy, C.; Linderoth, L.; Andresen, T. L.; Mouritsen, O. G.; Jørgensen, K.; Peters,
G. H. Biophys. J. 2006, 90, 3165.
12. Harvey, D. J. J. Mass Spectrom. 1995, 30, 1333.
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ing formulation, where self-aggregation into SUVs were observed
(vide supra).
The cytotoxicity of the release compounds 15-deoxy-D12,14
-
PGJ2 (see Table 2) and the lysophospholipids7 was also obtained.
As evident, the IC50 values for the released compounds are in the
same range as for the conjugates 1 and 2 indicating that the activ-
ity originate from 15-deoxy-D
12,14-PGJ2 and the lysophospholipids,
released either extra- or intracellularly. The conjugates were also
able to induce cell death in Colo205 cells, with IC50 values below