Synthesis and biological evaluation of novel phosphatidylcholine analogues containing monoterpene acids as potent antiproliferative agents

Article abstract of DOI:10.1371/journal.pone.0157278

The synthesis of novel phosphatidylcholines with geranic and citronellic acids in sn-1 and sn-2 positions is described. The structured phospholipids were obtained in high yields (59-87%) and evaluated in vitro for their cytotoxic activity against several cancer cell lines of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards noncancer cell line BALB/3T3 (normal mice fibroblasts). The phosphatidylcholines modified with monoterpene acid showed a significantly higher antiproliferative activity than free monoterpene acids. The highest activity was observed for the terpene-phospholipids containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.

Full text of DOI:10.1371/journal.pone.0157278

RESEARCH ARTICLE
Synthesis and Biological Evaluation of Novel
Phosphatidylcholine Analogues Containing
Monoterpene Acids as Potent
Antiproliferative Agents
Anna Gliszczyńska1*, Natalia Niezgoda1, Witold Gładkowski1, Marta Czarnecka1,
Marta Świtalska2, Joanna Wietrzyk2
1 Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50–375
Wrocław, Poland, 2 Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of
Sciences, Department of Experimental Oncology, Weigla 12, 53–114 Wrocław, Poland
a11111
* anna.gliszczynska@wp.pl
Abstract
OPEN ACCESS
The synthesis of novel phosphatidylcholines with geranic and citronellic acids in sn-1 and
sn-2 positions is described. The structured phospholipids were obtained in high yields
(59–87%) and evaluated in vitro for their cytotoxic activity against several cancer cell lines
of different origin: MV4-11, A-549, MCF-7, LOVO, LOVO/DX, HepG2 and also towards non-
cancer cell line BALB/3T3 (normal mice fibroblasts). The phosphatidylcholines modified
with monoterpene acid showed a significantly higher antiproliferative activity than free
monoterpene acids. The highest activity was observed for the terpene-phospholipids con-
taining the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.
Citation: Gliszczyńska A, Niezgoda N, Gładkowski
W, Czarnecka M, Świtalska M, Wietrzyk J (2016)
Synthesis and Biological Evaluation of Novel
Phosphatidylcholine Analogues Containing
Monoterpene Acids as Potent Antiproliferative
Agents. PLoS ONE 11(6): e0157278. doi:10.1371/
journal.pone.0157278
Editor: Stephan Neil Witt, Louisiana State University
Health Sciences Center, UNITED STATES
Received: March 30, 2016
Accepted: May 26, 2016
Published: June 16, 2016
Introduction
Copyright: © 2016 Gliszczyńska et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Isoprenoids are one of the most widely occurring group of natural compounds. Many of them
exhibit wide range of biological activities. Among documented useful properties of isoprenoids
the following activities need to be mentioned: fungicidal, antibacterial, antiviral and anti-
inflammatory [1–4]. The results of many studies show that they inhibit proliferation of cancer
cell lines, causing inhibition of cell division and transition of cells into the phase of pro-
grammed cell death [5,6]. The administered doses, even in high concentrations do not cause
such an effect in the case of healthy cells [7].
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
In recent years there has been also a rapid progress in the field of bioengineering and phos-
pholipid technology [8,9]. Many new discoveries in this area completely changed the idea that
phospholipids are just the integral components of biomembrane ensuring their integrity and
functionality and energetic and supplementary material [10,11]. It is currently known that
phospholipids are also involved in many metabolic and neurological reactions [12,13]. They
regulate fundamental biological processes and play an important role in the transmission of
information as intracellular mediators [14,15]. For the description of phospholipids-protein
interactions, the mechanisms of their action in membrane as signaling molecules and relation
Funding: This project was financed by National
Science Center of Poland no. 2013/09/D/NZ9/02457.
Publication was supported by Wroclaw Centre of
Biotechnology, programme The Leading National
Research Centre (KNOW) for years 2014-2018
(http://know.wroc.pl).
Competing Interests: The authors have declared
that no competing interests exist.
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
between their structure and activity the key point is preparation of a great number of phospho-
lipid derivatives. Especially important in this area is development of new synthetic methods for
the preparation of biologically active phospholipids (PLs) with a defined structure and high
purity.
Keeping in view of the properties of isoprenoids and phospholipids the production of novel
hybrid molecules with the combination of these two biologically important products could
result in bioactive analogues—terpene-phospholipids, which could be characterized by
enhanced biological activities. It has been reported that phospholipid modifications of drugs is
the method of modulation their polarity, to prevent degradation and enhance the biological
activity of molecules attached to the PLs [16–20].
Materials and Methods
General Procedures
Geranic acid (mixture of cis/trans isomers; 23:67) (1a) and racemic citronellic acid (1b) were
purchased from Sigma–Aldrich Chemical Co. Phospholipase A2 (Lecitase 10L; 10,000 LEU/
mL) was a gift from Novozymes. The enantiomerically pure GPC was purchased from Bachem
and converted to the cadmium chloride complex (GPC × CdCl2) using the method described
elsewhere. All of the solvents used in liquid chromatography were of HPLC grade (LiChrosolv
Reagents) and were purchased from Merck.
Analytical TLC was performed on silica gel 60, F254 plates (Merck) with mixture of chloro-
form: methanol: water (65:25:4; v/v/v) as developing system. Products were detected by spraying
the plates with a solution of 10 g of Ce(SO4)2 and 20 g of phosphomolybdic acid in 1 L of 10%
H2SO4 followed by heating or 0.05% primuline solution (8: 2; acetone/H2O; v/v) followed by UV
(365 nm). Column chromatography was performed on silica gel (Kieselgel 60, 230–400 mesh
(Merck)). 1H, 13C, and 31P NMR spectra were recorded using a Bruker Advance II 600 MHz
spectrometer. Chemical shifts are given in ppm downfield from tetramethylsilane (TMS) as the
internal standard. In 31P NMR spectra, chemical shifts were referenced to 85% H3PO4 as a stan-
dard. Coupling constant (J) values are given in hertz. Elemental analyses were performed on
Waters ESI-Q-TOF Premier XE spectrometer using electron spray ionization (ESI) technique.
The purity of synthesized products were monitored by HPLC using an Ultimate 3000 DIO-
NEX chromatograph equipped with a DGP-3600A dual-pump fluid control module, a TCC-
3200 thermostatted column compartment, and a WPS-3000 autosampler. A Corona charged
aerosol detector (CAD) from ESA Biosciences was used. The acquisition range 100 pA, digital
filter set to none, the N2 pressure was 0.24 MPa. The system was controlled and data acquisi-
tion was carried out using the Chromeleon 6.80 software (Dionex Corporation). HPLC analysis
was carried out using a Betasil DIOL 5-μm column (Thermo, 150 × 2.1 mm).
The column temperature was maintained at 30°C. The gradient had a constant flow rate 1.5 mL
min-1 with solvent A = 1% HCOOH, 0.1% triethylamine (TEA) in water, B = hexane, and
C = propan-2-ol. Gradient time: 3/40/57 (%A/%B/%C), at 4 min 10/40/50, at 9 min 10/40/50, at
9.1 min 3/40/57 and at 19 min 43/40/57. The total time of analysis was 19 min. The injection vol-
ume was 15 μL in all of the experiments and the cooling temperature for the samples was 20°C.
Preparation of Cadmium Chloride Complex of GPC (GPC × CdCl2)
The solution of sn-glycero-3-phosphocholine (GPC) (10 g, 38.7 mmol) in methanol (65 mL)
was added to a solution of cadmium chloride (CdCl2 × 21/2H2O) (8.88 g, 38.7 mmol) in cold
water (30 mL). A heavy white precipitate appeared and was stored at 0°C for 4 h. The precipi-
tate (GPC × CdCl2) was next filtered, lyophilized and then dried in an Abderhalden’s drying
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
pistol over P2O5 at the boiling temperature of acetone to give 15.65 g of white powder in 91%
yield.
General procedure for the 1,2-diacylation of GPC × CdCl2 (3a, 3b)
The cadmium complex of GPC has been acetylated with isoprenoid acids in positions sn-1 and
sn-2. Geranic acid (1a) or citronellic acid (1b) (0.92 mmol) was dried by repeated evaporation
with an anhydrous CH2Cl2 and then dissolved in anhydrous CH2Cl2 (1 mL). To the 100 mg
(0.23 mmol) of GPC × CdCl2 complex added 56 mg (0.46 mmol) of DMAP and the solution of
200 mg of DCC (0.97 mmol) in 6 mL of the anhydrous CH2Cl2. The reaction flask was flushed
with dry N2 and next tightly sealed. The mixture was stirred at room temperature under pro-
tection from light for 72 h. After this time (TLC) the formed during the reaction course precipi-
tate was removed using a Schott funnel. The solution was mixed with ion-exchange resin
(DOWEX 50W X8, H+ form) for 30 min to dislodge the cadmium chloride and DMAP.
DOWEX was filtered off and the solvent was evaporated under vacuum. The crude PC was
purified by silica gel column chromatography (65: 25: 4, CHCl3/CH3OH/H2O, (v/v/v)). Frac-
tions containing products (Rf 0.3) were collected and evaporated under vacuum (45°C) to
afford 1,2-diisoprenoyl-sn-glycero-3-phosphocholine 3 as a mixture of two stereoisomers.
1',2'-di(E)-geranoyl)-sn-glycero-3'-phosphocholine + 1',2'-di(Z)-geranoyl)-sn-glycero-3'-
phosphocholine (3a). Colourless greasy solid (82% yield, 99% purity (according to HPLC));
1H NMR (600 MHz, CDCl3/CD3OD 2:1 (v/v)), δ: 1.29 (two s, 12H, CH3-9sn-1 (A), CH3-9sn-1
(B), CH3-9sn-2 (A), CH3-9sn-2 (B)), 1.36 (s, 12H, CH3-8sn-1 (A), CH3-8sn-1 (B), CH3-10sn-2 (A),
CH3-10sn-2 (B)), 1.81–1.85 (m, 8H, CH2-4sn-1 (A), CH2-4sn-1 (B), CH2-5sn-2 (A), CH2-5sn-2
(B)), 2.91 (s, 18H, -N(CH3)3 (A), N(CH3)3 (B)), 3.32–3.35 (m, 4H, CH2-β (A), CH2-β (B)),
3.70–3.77 (m, 4H, CH2-3' (A), CH2-3' (B)), 3.91 (dd, J = 12.0, 6.6 Hz, 2H, one of CH2-1' (A),
one of CH2-1' (B)), 3.96–4.01 (m, 4H, CH2-α (A), CH2-α (B), 4.06 (m, 2H, one of CH2-1' (A),
one of CH2-1' (B), 4.80 (m, 2H, H-6sn-1 (B), H-6sn-2 (B)), 4.84 (m, 2H, H-6sn-1 (A), H-6sn-2
(A)), 4.94 (m, 2H, H-2' (A), H-2' (B)), 5.35 (s, 4H, H-2sn-1 (A), H-2sn-1 (B), H-2sn-2 (A), H-2sn-2
(B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 15.39, 15.44, 15.46, 15.49, 24.70, 24.74,
24.88 (C-8sn-1 (A), C-8sn-1 (B), C-8sn-2 (A), C-8sn-2 (B), C-10sn-1 (A), C-10sn-1 (B), C-10sn-2 (A),
C-10sn-2 (B)), 16.88, 16.89, 16.93, 16.95 (C-9sn-1 (A), C-9sn-1 (B), C-9sn-2 (A), C-9sn-2 (B)),
25.58, 25.59, 26.26, 26.28 (C-5sn-1 (A), C-5sn-1 (B), C-5sn-2 (A), C-5sn-2 (B)), 40.52, 40.54, 40.55,
40.57 (C-4sn-1 (A), C-4sn-1 (B), C-4sn-2 (A), C-4sn-2 (B)), 53.65 (t, J = 3.7 Hz, -N(CH3)3 (A), -N
(CH3)3 (B)), 58.59 (d, J = 4.8 Hz, C-α (A), C-α (B)), 61.73 (C-1' (A), C-1' (B)), 63.24 (d, J = 4.8
Hz, C-3' (A), C-3' (B)), 66.10 (m, C-β (A), C-β (B)), 70.28 (d, J = 8.4 Hz, C-2' (A), C-2' (B)),
114.49, 114.60, 115.00, 115.10 (C-2sn-1 (A), C-2sn-1 (B), C-2sn-2 (A), C-2sn-2 (B)), 121.85,
121.88, 122.35, 122.37 (C-6sn-1 (A), C-6sn-1 (B), C-6sn-2 (A), C-6sn-2 (B)), 128.11, 128.15
(C-7sn-1 (A), C-7sn-1 (B), C-7sn-2 (A), C-7sn-2 (B)), 131.44, 132.13 (C-3sn-1 (A), C-3sn-1 (B),
C-3sn-2 (A), C-3sn-2 (B)), 161.05, 161.19, 165.69, 165.78 (C-1sn-1 (A), C-1sn-1 (B), C-1sn-2 (A),
C-1sn-2 (B)); 31P NMR (243 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: -0.63; HRMS (ESI): m/z calcd.
for C28H48NO8P [M + H]+ 558.3196; found 558.3217
(2'R)-1',2'-di[(3R)-3,7-dimethylocta-6-enyl]-sn-glycero-3'-phosphocholine + (2'R)-1',2'-
di[(3S)-3,7-dimethylocta-6-enyl]-sn-glycero-3'-phosphocholine (3b). Colourless greasy
solid (86% yield, 99% purity (according to HPLC)); 1H NMR (600 MHz, CDCl3/CD3OD 2:1
(v/v)), δ: 0.70, 0.71 (two d, J = 7.8 Hz, 12H, CH3-9sn-1 (A), CH3-9sn-1 (B), CH3-9sn-2 (A), CH3-
9sn-2 (B)), 0.99 (m, 4H, one of CH2-4sn-1 (A), one of CH2-4sn-1 (B), one of CH2-4sn-2 (A), one of
CH2-4sn-2 (B)), 1.12 (m, 4H, one of CH2-4sn-1 (A), one of CH2-4sn-1 (B), one of CH2-4sn-2 (A),
one of CH2-4sn-2 (B)), 1.36, 1.44 (two s, 24H, CH3-8sn-1 (A), CH3-8sn-1 (B), CH3-10sn-1 (A),
CH3-10sn-1 (B), CH3-8sn-2 (A), CH3-8sn-2 (B), CH3-10sn-2 (A), CH3-10sn-2 (B)), 1.66–1.81 (m,
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
12H, CH2-5sn-1 (A), CH2-5sn-1 (B), CH2-5sn-2 (A), CH2-5sn-2 (B), H-3sn-1 (A), H-3sn-1 (B), H-
3sn-2 (A), H-3sn-2 (B)), 1.86–1.95 (m, 4H, one of CH2-2sn-1 (A), one of CH2-2sn-1 (B), one of
CH2-2sn-2 (A), one of CH2-2sn-2 (B)), 2.07–2.14 (m, 4H, one of CH2-2sn-1 (A), one of CH2-2sn-1
(B), one of CH2-2sn-2 (A), one of CH2-2sn-2 (B)), 2.99 (s, 18H, -N(CH3)3 (A), N(CH3)3 (B)),
3.38–3.39 (m, 4H, CH2-β (A), CH2-β (B)), 3.77 (m, 4H, CH2-3' (A), CH2-3' (B)), 3.92 (m, 2H,
one of CH2-1' (A), one of CH2-1' (B)), 4.03 (m, 4H, CH2-α (A), CH2-α (B)), 4.19 (m, 2H, one
of CH2-1' (A), one of CH2-1' (B)), 4.84 (t, J = 7.2 Hz, 4H, H-6sn-1 (A), H-6sn-1 (B), H-6sn-2 (A),
H-6sn-2 (B)), 5.00 (m, 2H, H-2' (A), H-2' (B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1 (v/v))
δ: 16.91, 24.96 (C-8sn-1 (A), C-8sn-1 (B), C-8sn-2 (A), C-8sn-2 (B), C-10sn-1 (A), C-10sn-1 (B), C-
10sn-2 (A), C-10sn-2 (B)), 18.77, 18.78. 18.85 (C-9sn-1 (A), C-9sn-1 (B), C-9sn-2 (A), C-9sn-2 (B)),
24.88 (C-5sn-1 (A), C-5sn-1 (B), C-5sn-2 (A), C-5sn-2 (B)), 29.49, 29.53, 29.56 (C-3sn-1 (A), C-3sn-1
(B), C-3sn-2 (A), C-3sn-2 (B)), 36.20, 36.23 (C-4sn-1 (A), C-4sn-1 (B), C-4sn-2 (A), C-4sn-2 (B)),
40.99, 41.15, 41.16 (C-2sn-1 (A), C-2sn-1 (B), C-2sn-2 (A), C-2sn-2 (B)), 53.58 (t, J = 3.5 Hz, -N
(CH3)3 (A), -N(CH3)3 (A)), 58.75 (d, J = 4.8 Hz, C-α (A), C-α (B)), 62.21 (C-1' (A), C-1' (B)),
63.27 (d, J = 4.7 Hz, C-3' (A), C-3' (B)), 65.93 (m, C-β (A), C-β (B)), 69.89, (d, J = 7.7 Hz, C-2'
(A)), 69.91 (d, J = 8.3 Hz, C-2' (B)), 123.64 (C-6sn-1 (A), C-6sn-1 (B), C-6sn-2 (A), C-6sn-2 (B)),
131.09 (C-7sn-1 (A), C-7sn-1 (B), C-7sn-2 (A), C-7sn-2 (B)), 172.55, 172.56, 172.97 (C-1sn-1 (A),
C-1sn-1 (B), C-1sn-2 (A), C-1sn-2 (B)); 31P NMR (243 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: -0.79;
HRMS (ESI): m/z calcd. for C28H52NO8P [M + H]+ 562.3509; found 562.3530
General procedure for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
enzymatic hydrolysis
Hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (5) was carried out according to
the method described by Morgado et al. [21]. The detailed data were described earlier [22].
1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (6) was obtained in 99% yield and its
physical and spectroscopic data were consistent with those found in literature [23].
General procedure for 1-palmitoyl-2-isoprenoyl-sn-glycero-
3-phosphocholine (7a, 7b) synthesis
Terpene acids (1a or 1b, 0.604 mmol), solution of DMAP (74 mg, 0.6 mmol) in 3 mL of
CH2Cl2 and finally a solution of DCC (267 mg, 1.3 mmol) in 5 mL of CH2Cl2 were added to
the solution of 1-palmitoyl-2-hydroxy-sn-3-glycero-phosphocholine (6) (150 mg, 0.302 mmol)
dissolved in 4 mL of anhydrous CH2Cl2. The reaction was carried out for 72 h under a nitrogen
atmosphere in the dark at 40°C. The product was extracted according to the method described
for compound 3a and 3b. 1-Palmitoyl-2-isoprenoyl-sn-glycero-3-phosphocholine (7) was
obtained as the mixture of two stereoisomers.
1'-palmitoyl-2'-(E)-geranoyl-sn-glycero-3'-phosphocholine + 1'-palmitoyl-2'-(Z)-gera-
noyl-sn-glycero-3'-phosphocholine (7a). Colourless greasy solid (59% yield, 98% purity
(according to HPLC)); 1H NMR (600 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 0.60 (t, J = 6.6 Hz, 6H,
CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 1.98–1.02 (m, 48H, CH3(CH2)12CH2CH2C(O)
(A), CH3(CH2)12CH2CH2C(O) (B)), 1.32 (m, 4H, CH3(CH2)13CH2CH2C(O) (A),
CH3(CH2)13CH2CH2C(O) (B)), 1.35 (s, 6H, CH3-9 (A), CH3-9 (B)), 1.41 (s, 12H, CH3-8 (A),
CH3-8 (B), CH3-10 (A), CH3-10 (B)), 1.85–1.91 (m, 8H, CH2-4 (A), CH2-4 (B), CH2-5 (A),
CH2-5 (B)), 2.03 (m, 4H, CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 2.94 (s, 18H,
-N(CH3)3 (A), -N(CH3)3 (B)), 3.31–3.35 (m, 4H, CH2-β (A), CH2-β (B)), 3.72 (m, 4H, CH2-3'
(A), CH2-3' (B)), 3.92 (m, 2H, one of CH2-1' (A), one of CH2-1' (B), 3.96–4.00 (m, 4H, CH2-α
(A), CH2-α (B)), 4.14 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)), 4.80, 4.85 (two t, J = 6.6
Hz, 2H, H-6 (A), H-6 (B)), 4.96–5.05 (two m, 2H, H-2' (A), H-2' (B)), 5.39 (s, 2H, H-2 (A), H-2
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
(B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 13.37 (CH3CH2(CH2)11CH2CH2C(O)
(A), CH3CH2(CH2)11CH2CH2C(O) (B)), 16.94, 16.95 (C-9 (A), C-9 (B)), 22.15
(CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 24.33, 24.34
(CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 24.90 (C-8 (A), C-
10 (A), C-8 (B), C-10 (B)), 25.53, 25.55 (C-5(A), C-5 (B)), 28.63, 28.77, 28.84, 28.98, 29.11,
29.13, 29.16 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B), 33.54
(CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 40.53, 40.56 (C-4
(A), C-4 (B)), 53.56 (m, -N(CH3)3 (A), -N(CH3)3 (B)), 58.59 (d, J = 4.7 Hz, C-α (A), C-α (B)),
62.20 (C-1' (A), C-1' (B)), 63.06, 63.11 (two d, J = 5.2 Hz, C-3' (A), C-3' (B)), 65.95–65.99 (two
m, C-β (A), C-β (B)), 70.24, 70.31 (two d, J = 7.8 Hz, C-2' (A), C-2' (B)), 114.93, 114.41 (C-2
(A), C-2 (B)), 121.78, 121.80 (C-6 (A), C-6 (B)), 126.89, 127.14 (C-7 (A), C-7 (B)), 127.70,
128.12 (C-3 (A), C-3 (B)), 161.45, 165.68 (C-1 (A), C-1 (B)), 173.57, 173.68
(CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)); 31P NMR (243
MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 0.64; HRMS (ESI): m/z calcd. for C34H64NO8P [M + H]+
646.4448; found 646.4451
(2'R)-1'-palmitoyl-2'-[(3R)-3,7-dimethylocta-6-enyl]-sn-glycero-3'-phosphocholine + (2'R)-
1'-palmitoyl-2'-[(3S)-3,7-dimethylocta-6-enyl]-sn-glycero-3'-phosphocholine (7b). Colourless
greasy solid (62% yield, 99% purity (according to HPLC)); 1H NMR (600 MHz, CDCl3/CD3OD
2:1 (v/v)), δ: 0.64 (t, J = 6.6 Hz, 6H, CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 0.72 (d,
J = 7.2 Hz, 6H, CH3-9 (A), CH3-9 (B)), 0.96–1.05 (m, 50H, CH3(CH2)12CH2CH2C(O) (A),
CH3(CH2)12CH2CH2C(O) (B), one of CH2-4 (A), one of CH2-4 (B)), 1.12 (m, 2H, one of CH2-4
(A), one of CH2-4 (B)), 1.31–1.39 (m, 4H, CH3(CH2)13CH2CH2C(O) (A), CH3(CH2)13CH2CH2C
(O) (B)), 1.36, 1.44 (two s, 12H, CH3-8 (A), CH3-8 (B), CH3-10 (A), CH3-10 (B)), 1.65–1.81
(m, 6H, CH2-5 (A), CH2-5 (B), H-3 (A), H-3 (B)), 1.87–1.93 (two dd, J = 15.0, 8.4 Hz, 2H,
one of CH2-2 (A), one of CH2-2 (B)), 2.07 (t, J = 7.8 Hz, 4H, CH3(CH2)13CH2C(O) (A),
CH3(CH2)13CH2C(O) (B)), 2.09–2.15 (two dd, J = 15.0, 6.0 Hz, 1H, one of CH2-2 (A), one of
CH2-2 (B)), 2.99 (s, 18H, -N(CH3)3 (A), -N(CH3)3 (B)), 3.39 (m, 4H, CH2-β (A), CH2-β (B)), 3.77
(m, 4H, CH2-3' (A), CH2-3’ (B), 3.92 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)), 4.03 (m, 4H,
CH2-α (A), CH2-α (B)), 4.17 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)), 4.84 (t, J = 6.6 Hz,
2H, H-6 (A), H-6 (B)), 5.00 (m, 2H, H-2' (A), H-2' (B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1
(v/v)) δ: 13.44 (CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 16.97, 25.02 (C-8 (A), C-8 (B), C-10
(A), C-10 (B)), 18.93 (C-9 (A), C-9 (B)), 22.20 (CH3CH2(CH2)13C(O) (A), CH3CH2(CH2)13C(O)
(B)), 24.45 (CH3(CH2)12CH2CH2C(O) (A), (CH3(CH2)12CH2CH2C(O) (B)), 24.94 (C-5 (A), C-5
(B)), 28.67, 28.87, 28.89, 29.06, 29.19, 29.21, 29.23, 31.47 (CH3CH2(CH2)11CH2CH2C(O) (A),
CH3CH2(CH2)11CH2CH2C(O) (B)), 29.55, 29.56 (C-3 (A), C-3 (B)), 33.78 (CH3(CH2)13CH2C
(O) (A), CH3(CH2)13CH2C(O) (B)), 36.28 (C-4 (A), C-4 (B)), 41.06 (C-2 (A), C-2 (B)), 53.66 (t,
J = 3.6 Hz, -N(CH3)3 (A), -N(CH3)3 (B)), 58.79 (d, J = 4.9 Hz, C-α (A), C-α (B)), 62.12, 62.14 (C-
1' (A), C-1' (B)), 63.39 (d, J = 5.1 Hz, C-3' (A), C-3' (B)), 65.00 (m, C-β (A), C-β (B)), 69.95 (d,
J = 7.8 Hz, C-2' (A), C-2' (B)), 123.68 (C-6 (A), C-6 (B)), 131.13 (C-7 (A), C-7 (B)), 172.95 (C-1
(A), C-1 (B)), 173.19 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B); 31P NMR (243
MHz, CDCl3/CD3OD 2:1 (v/v)) δ: -0.83; HRMS (ESI): m/z calcd. for C34H66NO8P [M + H]+
648.4604; found 648.4615
General procedure the synthesis of 1-isoprenoyl-2-hydroxy-sn-glycero-
3-phosphocholine (9a, 9b)
The synthesis of 1-isoprenoyl-2-hydroxy-sn-glycero-3-phosphocholine (9a, 9b) was carried
out according to the procedure described by Niezgoda et al. [24]. GPC (300 mg, 1.16 mmol)
and dibutyltin oxide (DBTO) (290 mg, 1.17 mmol) were suspended in 10 mL of anhydrous
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
propan-2-ol and refluxed for 1 h. The reaction was cooled to an ambient temperature and TEA
(2.4 mmol) was added followed by terpene chloride (281 mg, 2.4 mmol). After 1 h of the reac-
tion, the mixture was filtreted using diatomaceous earth (Celite1 545) and washed CH2Cl2.
The organic solvent was evaporated under vacuum (45°C), after which the crude residue was
purified on a silica gel column (65: 25: 4 CHCl3/CH3OH/H2O, (v/v/v)). Product-containing
fractions of Rf 0.1 were collected and solvent evaporated under vacuum (45°C) to gave 1-iso-
prenoyl-2-hydroxy-sn-glycero-3-phosphocholine (9a, 9b). The chemical structure was con-
firmed by 1H and 13C NMR spectra.
1'-(E)-geranoyl-2'-hydroxy-sn-glycero-3'-phosphocholine + 1'-(Z)-geranoyl-2'-hydroxy-
sn-glycero-3'-phosphocholine) (9a). Colourless greasy solid (75% yield, 99% purity (accord-
ing to HPLC)); 1H NMR (600 MHz, CDCl3/CD3OD 2:1 (v/v)), δ: 1.38 (s, 6H, CH3-9 (A),
CH3-9 (B)), 1.45 (s, 24H, CH3-8 (A), CH3-8 (B), CH3-10 (A), CH3-10 (B)), 1.89–1.84 (m, 8H,
CH2-4 (A), CH2-4 (B), CH2-5 (A), CH2-5 (B)), 2.99 (s, 18H, -N(CH3)3 (A), -N(CH3)3 (B)),
3.37–3.39 (m, 4H, CH2-β (A), CH2-β (B)), 3.65 (m, 4H, one of CH2-3' (A), one of CH2-3' (B)),
3.71–3.78 (two m, 4H, H-2' (A), H-2' (B), one of CH2-3' (A), one of CH2-3' (B)), 3.87–3.96
(two m, 4H, CH2-1' (A), CH2-1' (B)), 4.02–4.05 (m, 4H, CH2-α (A), CH2-α (B)), 4.86 (m, 2H,
H-6 (A), H-6 (B)), 5.48 (s, 2H, H-2 (A), H-2 (B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1
(v/v)) δ: 17.00, 18.24 (C-9 (A), C-9 (B)), 24.97 (C-8 (A), C-8 (B), C-10 (A), C-10 (B)), 25.58
(C-5 (A), C-5 (B)), 40.56, 41.19 (C-4 (A), C-4 (B)), 53.63 (t, J = 3.8 Hz, -N(CH3)3 (A), -N
(CH3)3 (B)), 58.63 (d, J = 4.9 Hz, C-α (A), C-α (B)), 64.88 (C-1' (A), C-1' (B)), 66.03 (m, C-β
(A), C-β (B)), 66.41 (d, J = 5.6 Hz, C-3' (A)), 66.58 (d, J = 5.7 Hz, C-3' (B)), 68.24 (d, J = 6.6 Hz,
C-2' (A)), 68.41 (d, J = 6.8 Hz, C-2' (B)), 113.22, 114.58 (C-2 (A), C-2 (B)), 122.41, 123.12 (C-6
(A), C-6 (B)), 131.51, 132.13 (C-7 (A), C-7 (B)), 141.68 (C-3 (A), C-3 (B)), 160.88, 166.63 (C-1
(A), C-1 (B)); 31P NMR (243 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 0.02; HRMS (ESI): m/z calcd.
for C18H34NO7P [M + H]+ 408.2151; found 408.2175
(2'R)-1'-[(3R)-3,7-dimethylocta-6-enyl]-2'-hydroxy-sn-glycero-3'-phosphocholine +
(2'R)-1'-[(3S)-3,7-dimethylocta-6-enyl]-2'-hydroxy-sn-glycero-3'-phosphocholine (9b).
Colourless greasy solid (87% yield, 98% purity (according to HPLC)); 1H NMR (600 MHz,
CDCl3/CD3OD 2:1 (v/v)), δ: 0.72 (d, J = 6.6 Hz, 6H, CH3-9 (A), CH3-9 (B)), 0.96–1.15 (two m,
4H, CH2-4 (A), CH2-4 (B)), 1.37, 1.45 (two s, 12H, CH3-8 (A), CH3-8 (B), CH3-10 (A), CH3-
10 (B)), 1.70–1.82 (m, 6H, CH2-5 (A), CH2-5 (B), H-3 (A), H-3 (B)), 1.93 (dd, J = 15.0, 8.4 Hz,
2H, one of CH2-2 (A), one of CH2-2 (B)), 2.14 (dd, J = 15.0, 5.4 Hz, 2H, one of CH2-2 (A), one
of CH2-2 (B)), 2.99 (s, 18H, -N(CH3)3 (A), -N(CH3)3 (B)), 3.37–3.39 (m, 4H, CH2-β (A), CH2-
β (B)), 3.62–3.73 (two m, 4H, CH2-3' (A), CH2-3' (B)), 3.74–3.77 (m, 2H, H-2' (A), H-2' (B)),
3.87–3.95 (two m, 4H, CH2-1' (A), CH2-1' (B)), 4.02–4.04 (m, 4H, CH2-α (A), CH2-α (B)), 4.85
(t, J = 7.2 Hz, 2H, H-6 (A), H-6 (B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 16.92,
24.98 (C-8 (A), C-8 (B) C-10 (A), C-10 (B)), 18.86 (C-9 (A), C-9 (B)), 24.91 (C-5 (A), C-5 (B)),
29.53 (C-3 (A), C-3 (B)), 36.30 (C-4 (A), C-4 (B)), 41.09 (C-2 (A), C-2 (B)), 53.61 (t, J = 3.8 Hz,
-N(CH3)3 (A), -N(CH3)3 (B), 58.62 (d, J = 5.1 Hz, C-α (A), C-α (B)), 64.49 (C-1' (A), C-1' (B)),
66.01 (m, C-β (A), C-β (B)), 66.47 (d, J = 5.6 Hz, C-3' (A), C-3' (B)), 68.30 (d, J = 6.5 Hz, C-2'
(A), C-2' (B)), 123.72 (C-6 (A), C-6 (B)), 131.08 (C-7 (A), C-7 (B)), 173.39 (C-1 (A), C-1 (B));
31P NMR (243 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: -0.03; HRMS (ESI): m/z calcd. for
C18H36NO7P [M + H]+ 410.23075; found 410.2311
General procedure for the synthesis of 1-isoprenoyl-2-palmitoyl-sn-
glycero-3-phosphocholine (10a, 10b)
Compounds 10a and 10b were prepared from 1-isoprenoyl-2-hydroxy-sn-glycero-3-phospho-
cholines (9a, 9b) (0.14 mmol) by adding palmitic acid (0.56 mmol) dissolved in 2 mL of
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
anhydrous CH2Cl2, DMAP (0.28 mmol) and finally a solution of DCC (0.58 mmol) in 3 mL of
CH2Cl2. The reaction was carried out for 72 h under a nitrogen atmosphere in the dark at
40°C. The product was extracted according to the method described for compounds 3a, 3b and
pure 1-isoprenoyl-2-palmitoyl-sn-glycero-3-phosphocholine (10) was obtained as the mixture
of two stereoisomers.
1'-(E)-geranoyl-2'-palmitoyl-sn-glycero-3'-phosphocholine + 1'-(Z)-geranoyl-2'-palmi-
toyl-sn-glycero-3'-phosphocholine) (10a). Colourless greasy solid (67% yield, 99% purity
(according to HPLC)); 1H NMR (600 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 0.63 (t, J = 6.6 Hz, 6H,
CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 1.03–1.04 (m, 48H, CH3(CH2)12CH2CH2C(O)),
1.35 (m, 4H, CH3(CH2)13CH2CH2C(O) (A), CH3(CH2)13CH2CH2C(O) (B)), 1.36 (s, 6H, CH3-
9 (A), CH3-9 (B)), 1.43 (s, 12H, CH3-8, CH3-10), 1.86–1.92 (two m, 8H, CH2-4 (A), CH2-4 (B),
CH2-5 (A), CH2-5 (B)), 2.07 (t, J = 6.6 Hz, 4H, CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C
(O) (B)), 2.98 (s, 18H, -N(CH3)3 (A), -N(CH3)3 (B),), 3.36–3.39 (m, 4H, CH2-β (A), CH2-β
(B)), 3.77 (m, 4H, CH2-3' (A), CH2-3' (B)), 3.94 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)),
4.00–4.05 (m, 4H, CH2-α (A), CH2-α (B)), 4.18 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)),
4.83 (m, 2H, H-6 (A). H-6 (B)), 4.96–5.03 (m, 2H, H-2' (A), H-2' (B)), 5.41 (s, 2H, H-2 (A), H-
2 (B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 13.38 (CH3CH2(CH2)11CH2CH2C
(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 16.95, 16.97 (C-9 (A), C-9 (B)), 22.16
(CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 24.38, 24.43
(CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 24.92 (C-8 (A), C-8
(B), C-10 (A), C-10 (B)), 25.55 (C-5 (A), C-5 (B)), 28.59, 28.63, 28.82, 28.85, 29.01, 29.14,
29.16, 29.18 (CH3CH2(CH2)11CH2CH2C(O) (A), CH3CH2(CH2)11CH2CH2C(O) (B)), 33.70,
33.76 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 40.54, 41.13 (C-4 (A), C-4 (B)),
53.57 (t, J = 3.2 Hz, -N(CH3)3 (A), -N(CH3)3 (A)), 58.69 (d, J = 4.7 Hz, C-α (A), C-α (B)), 62.50
(C-1' (A), C-1' (B)), 63.17 (d, J = 5.1 Hz, C-3' (A)), 63.37 (d, J = 4.8 Hz, C-3' (B)), 65.94 (m, C-β
(A), C-β (B)), 69.85 (d, J = 7.8 Hz, C-2' (A)), 70.08 (d, J = 8.0 Hz, C-2' (B)), 113.31, 114.32 (C-2
(A), C-2 (B)), 122.32, 123.03 (C-6 (A), C-6 (B)), 131.48, 132.09 (C-7 (A), C-7 (B)), 141.46 (C-3
(A), C-3 (B)), 161.13, 166.16 (C-1 (A), C-1 (B)), 173.13, 173.23 (CH3CH2(CH2)11CH2CH2C(O)
(A), CH3CH2(CH2)11CH2CH2C(O) (B)); 31P NMR (243 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 0.83;
HRMS (ESI): m/z calcd. for C34H64NO8P [M + H]+ 646.4448; found 646.4474
(2'R)-2'-palmitoyl-1'-[(3R)-3,7-dimethylocta-6-enyl]-sn-glycero-3'-phosphocholine +
(2'R)-2'-palmitoyl-1'-[(3S)-3,7-dimethylocta-6-enyl]-sn-glycero-3'-phosphocholine (10b).
Colourless greasy solid (70% yield, 99% purity (according to HPLC)); 1H NMR (600 MHz,
CDCl3/CD3OD 2:1 (v/v)) δ: 0.64 (t, J = 6.6 Hz, 6H, CH3(CH2)14C(O) (A), CH3(CH2)14C(O)
(B)), 0.70 (d, J = 6.6 Hz, 6H, CH3-9 (A), CH3-9 (B)), 0.90–1.34 (m, 50H, CH3(CH2)12CH2CH2C
(O) (A), CH3(CH2)12CH2CH2C(O) (B), one of CH2-4 (A), one of CH2-4 (B)), 1.13 (m,
2H, one of CH2-4 (A), one of CH2-4 (B)), 1.35–1.40 (m, 4H, CH3(CH2)13CH2CH2C(O) (A),
CH3(CH2)13CH2CH2C(O) (B)), 1.36, 1.44 (two s, 12H, CH3-8 (A), CH3-8 (B), CH3-10 (A), CH3-
10 (B)), 1.68–1.78 (m, 6H, CH2-5 (A), CH2-5 (B), H-3 (A), H-3 (B)), 1.87–1.92 (m, 2H, one of
CH2-2 (A), one of CH2-2 (B)), 2.06–2.12 (m, 2H, one of CH2-2 (A), one of CH2-2 (B)), 2.09
(t, J = 6.6 Hz, 4H, CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O) (B)), 2.99 (s, 18H, -N(CH3)3
(A), -N(CH3)3 (B)), 3.39–3.40 (m, 4H, CH2-β (A), CH2-β (B)), 3.78 (m, 4H, CH2-3' (A), CH2-3’
(B), 3.93 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)), 4.01–4.04 (m, 4H, CH2-α (A), CH2-α
(B)), 4.20 (m, 2H, one of CH2-1' (A), one of CH2-1' (B)), 4.84 (t, J = 6.0 Hz, 2H, H-6 (A), H-6
(B)), 4.99 (m, 2H, H-2' (A), H-2' (B)); 13C NMR (151 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: 13.61
(CH3(CH2)14C(O) (A), CH3(CH2)14C(O) (B)), 17.14, 25.19 (C-8 (A), C-8 (B), C-10 (A), C-10
(B)), 18.94, 18.97 (C-9 (A), C-9 (B)), 22.31 (CH3CH2(CH2)13C(O) (A), CH3CH2(CH2)13C(O)
(B)), 24.49 (CH3(CH2)12CH2CH2C(O) (A), (CH3(CH2)12CH2CH2C(O) (B)), 25.03 (C-5 (A), C-5
(B)), 28.78, 28.94, 28.99, 29.14, 29.27, 29.29, 29.30, 29.32, 31.57 (CH3CH2(CH2)11CH2CH2C(O)
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
(A), CH3CH2(CH2)11CH2CH2C(O) (B)), 29.69, 29.71 (C-3 (A), C-3 (B)), 33.72 (CH3(CH2)13CH2C
(O) (A), CH3(CH2)13CH2C(O) (B)), 36.34, 36.36 (C-4 (A), C-4 (B)), 41.30 (C-2 (A), C-2 (B)), 53.81
(t, J = 3.5 Hz, -N(CH3)3 (A), -N(CH3)3 (B)), 58.86 (d, J = 4.8 Hz, C-α (A), C-α (B)), 62.41, 62.43
(C-1' (A), C-1' (B)), 63.42 (d, J = 4.8 Hz, C-3' (A), C-3' (B)), 66.09 (m, C-β (A), C-β (B)), 69.95 (d,
J = 7.8 Hz, C-2' (A)), 69.96 (d, J = 7.7 Hz, C-2' (B)), 123.76 (C-6 (A), C-6 (B)), 131.27 (C-7 (A), C-7
(B)), 172.66, 172.68 (C-1 (A), C-1 (B)), 173.71 (CH3(CH2)13CH2C(O) (A), CH3(CH2)13CH2C(O)
(B)); 31P NMR (243 MHz, CDCl3/CD3OD 2:1 (v/v)) δ: -0.88; HRMS (ESI): m/z calcd. for
C34H66NO8P [M + H]+ 648.4604; found 648.4630
Biological studies
Cell cultures. Established in vitro, human cancer cell lines: MV4-11 (human biphenotypic
B myelomonocytic leukaemia), A-549 (non-small cell lung cancer), MCF-7 (breast cancer),
LoVo (colon cancer), LoVo/DX (colon cancer drug resistant), HepG2 (liver cancer) and BALB/
3T3 (normal mice fibroblasts) were used.
All lines were obtained from American Type Culture Collection (Rockville, Maryland, U.S.
A.). The cell line is being maintained in the Institute of Immunology and Experimental Ther-
apy, Wroclaw, Poland.
The leukaemia cell line was cultured in RPMI 1640 medium (IIET, Wroclaw, Poland) with
supplemented with 2 mM L-glutamine adjusted to contain 1.0 mM sodium pyruvate, 10% fetal
bovine serum (all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany). A549, LoVo and
LoVo/DX cells were cultured in the mixture of RPMI 1640+Opti-MEM (1:1) (both from Gibco,
Scotland, UK), MCF-7 cells in Eagle medium (IIET, Wroclaw, Poland), HepG2 in DMEM
medium (Gibco, Scotland, UK), BALB/3T3 in DMEM medium (IIET, Wroclaw, Poland) supple-
mented with 2 mM L-glutamine and 1.0 mM sodium pyruvate, 10% fetal bovine serum (all from
Sigma-Aldrich, Germany). The MCF-7 cell culture was supplemented with 0.8 mg/L of insulin
(Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and the LoVo/DX cell culture was supple-
mented with 0.1 μg/mL of doxorubicin (Accord). All culture media were supplemented with 100
units/mL penicillin, and 100 μg/mL streptomycin (both from Polfa Tarchomin S.A., Warsaw,
Poland). All cell lines were grown at 37°C with 5% CO2 humidified atmosphere.
Compounds. Prior to usage, the compounds were dissolved in DMSO or in mixture of
99.8% ethanol and DMSO (1:1) to the concentration of 25 or 50 mM, and subsequently diluted
in culture medium to reach the required concentrations (ranging from 5 to 625 μM).
Antiproliferative assay in vitro. 24 hours before addition of the tested compounds, the
cells were plated in 96-well plates at density of 1×104 cells per well. An assay was performed
after 72 hours exposure to varying concentrations of the tested agents.
Cytotoxic test MTT was applied for the cytotoxicity screening against leukaemia cells grow-
ing in suspension culture and were carried out according to the method described before [25].
Cytotoxic test SRB was applied against A549, MCF-7, LoVo, LoVo/DX, BALB/3T3 and HepG2
cells and were carried out according to the method described before [25].
The results were calculated as an IC50 (inhibitory concentration 50)–the concentration of
tested agent, which inhibits proliferation of 50% of the cell population. IC50 values were calcu-
lated for each experiment separately and mean values SD is presented in the Table 1. Each
compound in each concentration was tested in triplicate in a single experiment, which was
repeated 3–5 times.
Using the obtained IC50 values, the resistance indexes (RI) were calculated by dividing the
IC50 values of the compounds tested against the cells of drug resistant cell LOVO/DX line by
respective values obtained against the cells of drug sensitive LoVo line. According to Harker
et al. [26] three categories of the cells could be distinguished: (a) the cells are drug-sensitive—if
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
Table 1. Antiproliferative activity of terpene-phospholipids against selected cell lines.
Compounds
Acyl
Cell line /IC50 SD (μM)
residue
sn-1 sn-2 MV4-11
A-549
MCF-7
LOVO
263.1 32
LOVO/DX
HepG2
BALB/3T3
1a geranic acid
-
-
310.77 96.41 n.a.
254.49 67.95 n.a
447.09 72.02
452.9 96.0
492.3 44.5
n.a.
n.a.
n.a.
1b citronellic acid
-
-
249.3 26.4 533.2 36.7
n.a
3a
GA
CA
PA
PA
GA
CA
GA
CA
-
GA
CA
GA
CA
-
96.66 33.94* 274.29 9.42 236.33 21.33* 248.5 9.3
278.8 10.8* 285.3
251.4 23.4^ 280.7 1.8
54.96 2.12* 199.6 6.1
5
273.16 6.76
283.7 6.3
185.76 15.2
3b
130.08 37.8^ 203.1 47.7
242.8 45.1^
104.66 6.51*
155.0 88.3^
522.96 97.24
n.a
193.6 22^
7a
60.89 10.86*
72.1 14.4
61.2 6.0
51.05 5.1*
7b
45.66 7.63^
53.9 2.97^ 57.1 2.5^
246.2 9.9 526 89.6
286.7 35.1 551.9 18.5
103.4 11.1 111.9 29.3
9a
323.5 115.62 n.a.
381.58 24.49 n.a
n.a.
n.a.
9b
-
n.a.
n.a
10a
PA
PA
-
38.21 9.3*
57.06 6.37^
1.3 0.47
-
49.54 1.88
52.45 5.9*
208.7 25.7^
8.1 0.03
-
34.6 1.6*
52.4 3.6^
2.56 0.35
51.9 6.7*
57 1.8^
3.17 0.2
70.3 8.6
65.8 3.34
10b
60.2 6.4
8.6 0.7
-
232.9 14.7 92.9 15.3
Cisplatin
Doxorubicin
2.38 0.64
-
4.2 1.1
n.a.
-
-
0.117 0.012 6.53 0.93
n.a.—no activity in concentration of 5, 25, 125, 625 μM. IC50 –compound concentration leading to 50% inhibition of cell proliferation. Data are presented
as mean SD of 3–5 independent experiments.
*—results within column which are significantly different in comparison to geranic acid, p < 0.05.
^—results within column which are significantly different in comparison to citronellic acid, p < 0.05.
doi:10.1371/journal.pone.0157278.t001
the ratio approaches 0–2; (b) the cells are moderately drug-resistant—if the ratio ranges from 2
to 10; (c) the cells are markedly drug-resistant—if the ratio is higher than 10.
Statistical analysis
Statistical analysis was performed using STATISTICA version 10 (StatSoft Inc., USA). Mann-
Whitney U Test was used in the analysis and the results in Table 1 are given with statistical sig-
nificance in comparison to geranic acid (Ã) or citronellic acid (^), p < 0.05.
Results and Discussion
One of the most widely studied phospholipids is phosphatidylcholine (PC). Yamamoto pro-
posed the transphosphatidylation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with
terpene alcohols (geraniol, nerol, perillyl alcohol, myrtenol, farnesol, geranylgeraniol, phytol
[19,20] as the method for joining two biologically active groups of molecules. In this way the
novel carrier-linked prodrugs—phospholipids containing terpenes in the polar part of PC,
which showed antiproliferative effects on human leukaemia cells (HL-60) and prostate cancer
(PC-3) cells were prepared [19,20]. Promising results obtained by Yamamoto inspired us to
create a novel strategy for the synthesis of phosphatidylcholine enriched in biologically active
terpenes. In our aproach we decided to leave polar part of phosphatidylcholine (PC)
untouched, because choline is biologically active compound, which is a substrate for the syn-
thesis of the neurotransmitter acetylcholine and directly affects many vital functions such as
breathing, heart rate, memory processes. It also protects the liver cells, prevents accumulation
of fat in hepative cells, lowers cholesterol levels, reduces the risk of atherosclerosis and heart
diseases by the reduction of homocysteine level [27,28]. Instead, we used two monoterpene car-
boxylic acids (geranic and citronellic) as acyl donors and introduced them into apolar part of
PC in order to extend their application field and ensure their efficient transport in the human
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
body. Geranic acid is known as a strong inhibitor of the melanine synthesis and the most
potent isoprenoid with antifungal activity towards Fusarium graminearum and Colletotrichum
graminicola [29,30]. Its structural 2,3-dihydroanalogue named citronellic acid occurs in many
essential oils and as the carrier of specific odor exhibiting antimicrobial activity is often used in
the cosmetic industry as valuable ingredient of various preparations [31]. The biological activi-
ties of phospholipids depend on the composition of fatty acids in the sn-1 and sn-2 positions.
From that reason three groups of terpene-modified phosphatidylcholines were synthesized.
First group contained the same residue of geranic acid (GA) (1a) or cytronellic acid (CA) (1b)
in the sn-1 and sn-2 positions. In second variant, GA or CA was attached to the sn-2 position
whereas in sn-1 position saturated fatty acid (palmitic acid) was introduced as it is observed in
natural phospholipids. Third group of phospholipids possessed geranic or cytronellic acid resi-
due in sn-1 position and palmitic acid in sn-2 position.
The first group of PC containing the same residue of geranic acid (1a) or citronellic acid
(1b) in the sn-1 and sn-2 positions (3a, 3b) was obtained by known Steglich esterification
method [32–34]. The cadmium complex of sn-glycero-3-phosphocholine (GPC×CdCl2) (2)
was acylated with terpene acid (TA) 1a and 1b in the presence of 4-(N,N-dimethylamino)pyri-
dine (DMAP) and a coupling agent N,N’-dicyclohexylcarbodiimide (DCC). All the reagents
and solvent (dichloromethane) were freshly dried just before reaction to avoid the hydrolysis
of DCC to dicyclohexylurea. In described condition using the molar ratio 1: 4: 2: 4.2 of GPC/
TA/DMAP/DCC, 1,2-digeranoyl-sn-glycero-3-phosphocholine (3a) and 1,2-dicitroneloyl-sn-
glycero-3-phosphocholine (3b) were obtained in 82% and 86% yields respectively after 72
hours of reaction (Fig 1).
The structures of products 3a and 3b were characterized based on comprehensive spectro-
scopic data interpretation. In the 1H NMR spectrum one can see signals from the incorporated
isoprenoids. Signals from olefinic protons of terpene acids in the range δ 4.84–5.43 and protons
from three methyl groups in δ 1.38–1.45 were observed. The carbon atoms of double bond sys-
tems present in terpenes acid residues gave signals in 13C NMR spectrum in the range 114.49–
132.13 ppm for 3a and in the range 123.64–131.09 ppm for 3b. Signals in the 31P NMR spec-
trum at -0.63 ppm for 3a and -0.79 ppm for 3b proved that the phosphatidyl parts of molecules
have been retained. Detailed assignments are given in the supplementary material (S1–S40
Figs).
The synthesis of asymmetrically substituted phospholipids containing terpene acids (TA) in
the sn-2 position and palmitic acid in sn-1 position was started by acetylation of cadmium
complex of GPC with palmitic acid (PA) (Fig 2). Then 1,2-dipalmitoyl-sn-3-glycero-phospho-
choline was converted to the 2-lysophospholipid (6) by treatment with phospholipase A2.
Fig 1. Synthesis of 1,2-diacyl-sn-glycero-3-phosphocholine containing the GA and CA residues (3a, 3b).
doi:10.1371/journal.pone.0157278.g001
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
Fig 2. Synthesis of 1-palmitoyl-2-isoprenoyl-sn-glycero-3-phosphocholine (7a, 7b).
doi:10.1371/journal.pone.0157278.g002
Subsequent reacetylation of 2-OH group of known 1-palmitoyl LPC by isoprenoid acids
yielded the mixed diacyl phosphatidylcholines (7a, 7b) [35].
The time-course of esterification of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine
by isoprenoid acids proceeded very slowly (Fig 3). We prolonged the time of reaction but still
the isolated yields of asymmetrically substitued phosphatidylocholines were lower than in the
case of symmetrically substituted products. The progress of the reaction was monitored by
HPLC and showed that even after 120 hours the content of structured PC (7a, 7b) in the reac-
tion mixture was on the level of 58–84%. We decided to use then the higher molar excess of
Fig 3. The time-course of esterification of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine by
isoprenoid acids.
doi:10.1371/journal.pone.0157278.g003
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
Fig 4. Synthesis of 1-isoprenoyl-2-palmitoyl-sn-glycero-3-phosphocholine (10a, 10b).
doi:10.1371/journal.pone.0157278.g004
DCC and DMAP in relation of 1-palmitoyl-2-lysophospholipids and isoprenoid acids and
increase temperature of reaction to 40°C which resulted the full conversion of substrate after
72 h of the reaction.
The structures of obtained products 7a and 7b were established based on their spectral data.
The 1-palmitoyl-2-geranoyl-sn-glycero-3-phosphocholine (7a) was synthesized earlier by Bio-
drowska in 27% yield but in our investigations using higher ratio of DCC and DMAP resulted
in significantly higher yield (59%) [36]. The 1-pamitoyl-2-citroneloyl-sn-glycero-3-phospho-
choline (7b) has not been reported in the literature and was obtained in 62% yield.
The synthesis of 1-isoprenoyl-2-palmitoyl-sn-glycero-3-phosphocholines is a task requiring
regioselective incorporation of different acyl donors in two positions of the GPC backbone.
The literature presents several useful methods for this type of mixed-chain phosphatidylcho-
lines synthesis [29,24,37]. In all procedures the key step is production of 1-acyl-2-lyso-sn-gly-
cero-3-phosphocholine. Initially, we applied chemoenzymatic strategy similar to that applied
in the synthesis of compounds 7a and 7b. It was based on the hydrolysis of 1,2-diacyl-sn-gly-
cero-3-phosphocholines (3a, 3b) obtained previously (Scheme 1) and subsequent reesterifica-
tion of 1-acyl LPC with palmitic acid (PA) using DCC and DMAP. Unfortunately the yields of
hydrolysis of terpene-PLs carried out with the phospholipase A2 (PLA2) were very low and we
decided to use tin-mediated mono-functionalization of GPC described by D’Arrigo [35]. As it
is presented in the Fig 4, in this process, GPC was first transformed into cyclic stannylene ketal
by treatment with DBTO and then selectively acylated with isoprenoyl chlorides. Geranoyl and
citroneloyl chlorides were obtained in situ using the procedure described by Mattson et al. [38].
We used the quantities of reagents suggested by D’Arrigo et al. [35], for the synthesis of 1-acyl-
LPCs. Crude products were purified by a silica gel column chromatography to afford two new
1-isoprenoyl-2-hydroxy-sn-glycero-3-phosphocholines (9a, 9b) with 75% and 87% yields
respectively. The last step was acylation of 9a and 9b with palmitic acid (PA) in the presence of
DCC and DMAP, affording two new products 10a and 10b in 67 and 70% yields respectively.
The structures of the final products were confirmed by their spectroscopic data.
All synthesized products (3a, 3b, 7a, 7b, 9a, 9b, 10a, 10b) were obtained as the mixture of
diastereoisomers. In the case of phospholipids containing geranic acid (3a, 7a, 9a, 10a) it was
the results of composition of cis/trans (23:67) isomers of starting isoprenoid. In the case of
phosphatidylcholines modified with citronellic acid (3b, 7b, 9b, 10b) the formation of two
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
diastereoisomers (50:50) was caused by introduction of racemic citronellic acid into optically
pure sn-glycero-3-phosphocholine (GPC).
Biological studies
All terpene–modified PCs (1a, 1b, 3a, 3b, 7a, 7b, 9a, 9b, 10a, 10b) were tested for their antipro-
liferative activity towards human cancer cell lines, such as MV4-11 (human leukaemia), A549
(lung cancer), MCF-7 (breast cancer), HepG2 (liver cancer), LoVo (colon cancer) and also
against doxorubicin-resistant colon cancer LoVo/DX (P-gp-dependent, MRP-, LRP-dependent
multidrug resistance). The experiments of cytotoxicity were also performed towards BALB/
3T3 (normal mice fibroblasts). The data for the in vitro anticancer activity (Table 1) were
expressed as the IC50 –concentration of the compound (in μM) that inhibits proliferation of
cells by 50% compared to the untreated control cells. The results were compared with the activ-
ity of free terpene acids (TA). Cisplatin and doxorubicin hydrochloride (for LoVo and LoVo/
Dx cells) were used as a positive control.
The aim of this study was to determine whether a joining the terpenes with PC has impact on
their biological activity. Increasing the lipophilicity of active compounds was expected to raise
their cytotoxicity. We observed that almost all synthesized compounds were cytotoxic against
tested cancer cell lines. The exception is compound 9a that is not active towards cells of A549 and
HepG2 lines and 9b, which inhibits only the proliferation of leukaemia and colon cancer LoVo
cells but in high concentration on the level 381.58 μM and 286.7 μM, respectively. Upon careful
evaluation of data it can be concluded that cytotoxic activity of terpene-modified PCs is depended
on the position of terpene moiety in the backbone of PC. Symmetrically substituted phospholip-
ids (3a, 3b) containing terpene acids (TA) in the sn-1 and sn-2 position possessed the antiproli-
ferative activity 2-3-fold higher than free acids. These compounds stronger than corresponding
acids inhibit the proliferation of cancer cells. Free terpene acids (1a, 1b) did not showed any
reduction of cells growth of A549, HepG2 even at 625 μM. The highest activity was observed for
mixed-chain PC (7a, 7b, 10a, 10b). Their IC50 values were more than 9-fold lower than those
determined for free terpene acids 1a, 1b towards leukaemia, LoVo, LOVO/DX and breast cell
lines. For cells of lung cancer and HepG2 lines the activity was exhibited only after introduction
of terpene acids into PC. Among eight tested terpene-PCs compound 10a showed the strongest
antiproliferative effect towards all tested cell lines. Very high antiproliferative activity was demon-
strated against leukaemia and colon cancer LoVo cells (IC50 38.21 and 34.6 μM, respectively).
During the experiments we observed also that tested compounds exhibit activity against normal
mice fibroblasts (BALB/3T3). However it is worth to notice that cytotoxic activity of compounds
7a, 7b, 10a and 10b against BALB/3T3 cells was in most cases lower in comparison to cytotoxicity
exhibited against cancer cell lines, which is very promising in contest of potential application of
tested compounds as the anticancer drugs. Generally, activity of terpene-modified PC increased
in the following sequence: 1-isoprenoyl-LPC < diisoprenoyl-PC < monoisoprenoyl-PC.
We calculated also the resistance indexes (RI) by dividing the IC50 values of the compounds
tested against the cells of drug resistant cell LoVo/DX by respective values obtained against the
drug sensitive LoVo cell line (Table 2). All compounds were able to overcome the barrier of P-
gp-dependent resistance with exception of two compounds: free terpene acids 1b and 9a,
which revealed moderated ability to overcome drug resistance. Compounds 7a, 7b and 10b
have the highest ability to overcome the barrier of resistance (RI = 1.08, 1.06 and 1.09).
In conclusion, all novel phospholipids containing the isoprenoid residue of geranic and
citronellic acid in the non-polar part of phosphatidylcholine were synthesized in good yields
(59–87%). It is the first report on the evaluation of anticancer activity of geranic and citronellic
acid in the free form and after incorporation into PC. The results of cytotoxic studies confirmed
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
Table 2. Resistance index (RI) values of terpene-phospholipids.
Compounds
Acyl residue
sn-1
sn-2
RI
1a geranic acid
-
-
1.87
2.14
1.12
1.3
1b citronellic acid
-
-
3a
GA
CA
PA
PA
GA
CA
GA
CA
-
GA
CA
GA
CA
-
3b
7a
1.08
1.06
2.14
1.93
1.5
7b
9a
9b
-
10a
10b
DOX
PA
PA
-
1.09
55.81
DOX–doxorubicin
RI was calculated according to the formula RI = (IC50 estimated against resistant cell line)/(IC50 estimated
against non-resistant cell line); values range: 0<RI<2-indicate that the tested compound is able to
overcome drug resistance; 2<RI<10 –defines the moderate ability of the compound to overcome drug
resistance; RI>10 –defines no influence on the drug resistance phenomenon.
doi:10.1371/journal.pone.0157278.t002
that phospholipid modifications with geranic and citronellic acids enhanced their biological
activity. It was indicated by the lower active doses of terpene-PCs comparison to doses of free ter-
pene acids. The low activity observed for lysoderivatives may be a result of significantly increased
polarity of those compounds comparing to 1,2-diacylphosphatidylcholines. We found that there
is structure-activity relationship among the tested terpene-modified PCs but it is also important
to determine how the structure of isoprenoid residue (the chain length, number of double bond
etc.) influences on the activity of whole molecule. These studies are in progress. The aim of fur-
ther work is also to elucidate the mechanism of the cytotoxicity terpene-modified PCs.
Supporting Information
S1 Fig. 1H NMR spectrum of 3a.
(DOCX)
S2 Fig. 13C NMR spectrum of 3a.
(DOCX)
S3 Fig. 31P NMR spectrum of 3a.
(DOCX)
S4 Fig. 1H – 1H COSY spectrum of 3a.
(DOCX)
S5 Fig. HSQC spectrum of 3a.
(DOCX)
S6 Fig. 1H NMR spectrum of 3b.
(DOCX)
S7 Fig. 13C NMR spectrum of 3b.
(DOCX)
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
S8 Fig. 31P NMR spectrum of 3b.
(DOCX)
S9 Fig. 1H – 1H COSY spectrum of 3b.
(DOCX)
S10 Fig. HSQC spectrum of 3b.
(DOCX)
S11 Fig. 1H NMR spectrum of 7a.
(DOCX)
S12 Fig. 13C NMR spectrum of 7a.
(DOCX)
S13 Fig. 31P NMR spectrum of 7a.
(DOCX)
S14 Fig. 1H – 1H COSY spectrum of 7a.
(DOCX)
S15 Fig. HSQC spectrum of 7a.
(DOCX)
S16 Fig. 1H NMR spectrum of 7b.
(DOCX)
S17 Fig. 13C NMR spectrum of 7b.
(DOCX)
S18 Fig. 31P NMR spectrum of 7b.
(DOCX)
S19 Fig. 1H – 1H COSY spectrum of 7b.
(DOCX)
S20 Fig. HSQC spectrum of 7b.
(DOCX)
S21 Fig. 1H NMR spectrum of 9a
(DOCX)
S22 Fig. 13C NMR spectrum of 9a.
(DOCX)
S23 Fig. 31P NMR spectrum of 9a.
(DOCX)
S24 Fig. 1H – 1H COSY spectrum of 9a.
(DOCX)
S25 Fig. HSQC spectrum of 9a.
(DOCX)
S26 Fig. 1H NMR spectrum of 9b.
(DOCX)
S27 Fig. 13C NMR spectrum of 9b.
(DOCX)
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Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
S28 Fig. 31P NMR spectrum of 9b.
(DOCX)
S29 Fig. 1H – 1H COSY spectrum of 9b.
(DOCX)
S30 Fig. HSQC spectrum of 9b.
(DOCX)
S31 Fig. 1H NMR spectrum of 10a.
(DOCX)
S32 Fig. 13C NMR spectrum of 10a.
(DOCX)
S33 Fig. 31P NMR spectrum of 10a.
(DOCX)
S34 Fig. 1H – 1H COSY spectrum of 10a.
(DOCX)
S35 Fig. HSQC spectrum of 10a.
(DOCX)
S36 Fig. 1H NMR spectrum of 10b.
(DOCX)
S37 Fig. 13C NMR spectrum of 10b.
(DOCX)
S38 Fig. 31P NMR spectrum of 10b.
(DOCX)
S39 Fig. 1H – 1H COSY spectrum of 10b.
(DOCX)
S40 Fig. HSQC spectrum of 10b.
(DOCX)
Acknowledgments
This project was financed by National Science Center of Poland no. 2013/09/D/NZ9/02457.
Publication was supported by Wroclaw Centre of Biotechnology, programme The Leading
National Research Centre (KNOW) for years 2014–2018 (http://know.wroc.pl).
Special thanks for Mr Paweł Dąbrowski (Wroclaw University of Technology) for NMR
spectrum.
Author Contributions
Conceived and designed the experiments: AG MŚ. Performed the experiments: AG NN MC
MŚ. Analyzed the data: AG WG MŚ JW. Contributed reagents/materials/analysis tools: AG
MŚ JW. Wrote the paper: AG WG MŚ.
References
1. Fujita K, Kubo I. Multifunctional action of antifungal polygodial against Saccharomyces cerevisiae:
involvement of pyrrole formation on cell surface in antifungal action. Bioorg Med Chem. 2005; 13(24):
6742–6747. PMID: 16122929
PLOS ONE | DOI:10.1371/journal.pone.0157278 June 16, 2016
16 / 18

Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
2. Peng J, Franzblau SG, Zhang F, Hamann MT. Novel sesquiterpenes and a lactone from the Jamaican
sponge Myrmekioderma styx. Tetrahedron Lett. 2002; 43: 9699–9702.
3. Singh SB, Felock P, Hazuda DJ. Chemical and enzymatic modifications of integric acid and HIV-1 inte-
grase inhibitory activity. Bioorg Med Chem Lett. 2000; 10(3): 235–238. PMID: 10698443
4. Hernandez V, Manez S, Recio MC, Giner RM, Rios JL. Anti-inflammatory profile of dehydrocostic acid,
a novel sesquiterpene acid with a pharmacophoric conjugated diene. Eur J Pharma Sci. 2005; 26:
162–169.
5. Burke YD, Stark MJ, Roach SL, Sen SE, Crowell PL. Inhibition of pancreatic cancer growth by the die-
tary isoprenoids farnesol and geraniol. Lipids. 1997; 32(2): 151–156. PMID: 9075204
6. Adany I, Yazlovitskaya EM, Haug JS, Voziyan PA, Melnykovych G. Differences in sensitivity to farnesol
toxicity between neoplastically- and non-neoplastically-derived cells in culture. Cancer Lett. 1994; 79:
175–179. PMID: 8019976
7. Rioja A, Pizzey AR, Marson CM, Thomas NSB. Preferential induction of apoptosis of leukaemic cells
by farnesol. FEBS Lett. 2000; 467: 291–295. PMID: 10675556
8. Vance DE, Vance JE. Biochemistry of Lipids, Lipoproteins and Membranes, Elsevier; 2002.
9. Szuhaj BF, Nieuwenhuyzen WV, Nutrition and Biochemistry of Phospholipids, Illinois: AOCS Press;
2003.
10. Bezrukov SM. Functional consequences of lipid packing stress. Curr Opin Colloid In Sci. 2000; 5: 237–
243.
11. White SH, Ladokhin AS, Jayasinghe S, Hirstova K. How membranes shape protein structure. J Biol
Chem. 2001; 276: 32395–32398. PMID: 11432876
12. Schlegel RA, Halleck MS, Williamson P. Nutrition and Biochemistry of Phospholipids Illinois: AOCS
Press; 2003.
13. Lee TC. Biosynthesis and possible biological functions of plasmalogens. Biochim Biophys Acta. 1998;
1394: 129–145. PMID: 9795186
14. Irvine RF, Schell MJ. Back in the water: the return of the inositol phosphates. Nat Rev Mol Cell Biol.
2001; 2(5): 327–338. PMID: 11331907
15. Chrencik JE, Roth CB, Terakado M, Kurata H, Omi R, Kihara Y, I in. Crystal structure of antagonist
bound human lysophosphatidic acid receptor 1. Cell. 2015; 161: 1633–1643. doi: 10.1016/j.cell.2015.
06.002 PMID: 26091040
16. Lambert DM. Rationale and applications of lipids as prodrugs carriers. Eur J Pharm Sci. 2000; 11:
S15–27. PMID: 11033424
17. Hidaka N, Takami M, Suzuki YJ. Enzymatic phosphatidylation of thiamin, pantothenic acid and their
derivatives. Nutr Sci Vitaminol. 2008; 54: 255–261.
18. Takami M, Hidaka N, Miki S, Suzuki Y. Synthesis of novel phosphatidyldihydroxyacetone by phospholi-
pase D. Biosci Biotechnol Biochem. 1994; 58: 1716–1717.
19. Yamamoto Y, Hokosawa M, Kurihara H, Miyashita K. Preparation of phosphatidylated terpenes via
phospholipase D-mediated transphosphatidylation. J Am Oil Chem Soc. 2008; 85: 313–320.
20. Yamamoto Y, Masashi H, Kurihara H, Maoka T, Miyashita K. Synthesis of phosphatidylated-monoter-
pene alcohols catalyzed by phospholipase D and their antiproliferative effects on human cancer cells.
Bioorg Med Chem Lett. 2008; 18: 4044–4046. doi: 10.1016/j.bmcl.2008.05.113 PMID: 18556205
21. Morgado MAP, Cabral JMS, Prazeres DMF. Hydrolysis of lecithin by phospholipase A2 in mixed
reversed micelles of lecithin and sodium dioctyl sulphosuccinate. J Chem Technol Biot. 1995; 63: 181–
189.
22. Niezgoda N, Gliszczyńska A, Gładkowski W, Kempińska K, Wietrzyk J, Wawrzeńczyk C. Phosphatidyl-
choline with cis-9,trans-11 and trans-10,cis-12 conjugated lin oleic acid isomers: synthesis and cyto-
toxic studiem. Aust J Chem. 2015; 68: 1065–1075.
23. Fasoli E, Arnone A, Caligiuri A, D’Arrigo P, de Ferra L, Servi S. Tin-mediated Synthesis of Lysopho-
spholipids. Org Biomol Chem. 2006; 4: 2974–2978. PMID: 16855747
24. Niezgoda N, Mituła P, Kempińska K, Wietrzyk J, Wawrzeńczyk C. Synthesis of Phosphatidylcholine
with conjugated linoleic acid and studies on its cytotoxic activity. Aust J Chem. 2013, 66:354–361.
25. Gliszczyńska A, Gładkowski W, Świtalska M, Wietrzyk J, Szumny A, Gębarowska E, Wawrzeńczyk C.
Dehalogenation activity of selected fungi towards δ-jodo-γ-lactone derived from trans, trans-farnesol.
Chem Biodivers. 2016; 13: 477–482. doi: 10.1002/cbdv.201500127 PMID: 26923572
26. Harker WG, Slade DL, Drake FH, Parr RL. Mitoxantrone resistance in HL-60 leukemia cells: reduced
nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced
expression of the topoisomerase II beta isoform. Biochem J. 1991; 30: 9953–9961.
PLOS ONE | DOI:10.1371/journal.pone.0157278 June 16, 2016
17 / 18

Synthesis and Biological Evaluation of Novel Terpene-Phospholipids
27. Innis SM, Davidson A, Geoge F, Melynk S, James SJ. Choline-related supplements improve abnormal
plasma methioninehomocysteine metabolites and glutathione status in children with cystic fibrosis. Am
J Clin Nutr. 2007; 85: 702–708. PMID: 17344490
28. Wang T. Phospholipid Technology and Applications. The Oily Press Lipid Library Series. 2008.
29. Masuda T, Odaka Y, Ogawa N, Nakamoto K, Kuninaga H. Identification of Geranic Acid, a Tyrosinase
Inhibitor in Lemongrass (Cymbopogon citratus). J Agric Food Chem. 2008; 56: 597–601. PMID:
18081247
30. Yang T, Stoopen G, Yalpani N, Vervoort J, de Vos R, Voster A, Verstappen FWA, Bouwmeester HJ,
Jongsma MA. Metabolic engineering of geranic acid in maize to achieve fungal resistance is compro-
mised by novel glycosylation patterns. Metab Eng. 2011; 13: 414–425. doi: 10.1016/j.ymben.2011.01.
011 PMID: 21296182
31. Wuryatmo E, Klieber A, Scott ES. Inhibition of Citrus Postharvest Pathogens by Vapor of Citral and
Related Compounds in Culture. J Agric Food Chem. 2003; 51: 2637–2640. PMID: 12696950
32. Gupta CM, Radhakrishnan R, Khorana HG. Glycerophospholipid synthesis: improved general method
and new analogs containing photoactivable groups. Proc Natl Acad Sci. USA 1977; 74: 4315–4319.
PMID: 270675
33. Singh A. An efficient synthesis of phosphatidylcholines. J Lipid Res. 1990; 31: 1522–1525. PMID:
2280193
34. Ichihara K, Iwasaki H, Ueda K, Takizawa R, Naito H, Tomosugi M. Synthesis of phosphatidylcholine:
an improved method without using the cadmium chloride complex of sn-glycero-3-phosphocholine.
Chem Phys Lipids. 2005; 137: 94–99. PMID: 16054615
35. D’Arrigo P, Fasoli E, Pedrocchi-Fantoni G, Rossi C, Saraceno C, Tessaro D, Servi S. A practical selec-
tive synthesis of mixed short/long chains glycerophosphocholines. Chem Phys Lipids. 2007; 147: 113–
118. PMID: 17499652
36. Biodrowska K, Draus A, Gliszczyńska A, Gładkowski W, Leśniak A, Wawrzeńczyk C. Synthesis of iso-
prenoid phospholipids. Przem Chem. 2012; 91: 692–696.
37. D’Arrigo P, Servi S. Synthesis of Lysophospholipids. Molecules. 2010; 15: 1354–1377. doi: 10.3390/
molecules15031354 PMID: 20335986
38. Mattson FH, Volpenhein RA. Synthesis and properties of glycerides. J Lipid Res. 1962; 3: 281–296.
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