Synthesis and Biological Evaluation of Novel Phosphatidylcholine Analogues Containing Monoterpene Acids as Potent Antiproliferative Agents

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 containing the isoprenoid acids in sn-1 position of phosphatidylcholine and palmitic acid in sn-2.


Introduction
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 antiinflammatory [1][2][3][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 programmed cell death [5,6]. The administered doses, even in high concentrations do not cause such an effect in the case of healthy cells [7].
In recent years there has been also a rapid progress in the field of bioengineering and phospholipid 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 between their structure and activity the key point is preparation of a great number of phospholipid 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][17][18][19][20].

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 A 2 (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 × CdCl 2 ) 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, F 254 plates (Merck) with mixture of chloroform: 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(SO 4 ) 2 and 20 g of phosphomolybdic acid in 1 L of 10% H 2 SO 4 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)). 1 H, 13 C, and 31 P 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 31 P NMR spectra, chemical shifts were referenced to 85% H 3 PO 4 as a standard. 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 N 2 pressure was 0.24 MPa. The system was controlled and data acquisition 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).
pistol over P 2 O 5 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 × CdCl 2 (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 CH 2 Cl 2 and then dissolved in anhydrous CH 2 Cl 2 (1 mL). To the 100 mg (0.23 mmol) of GPC × CdCl 2 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 CH 2 Cl 2 . The reaction flask was flushed with dry N 2 and next tightly sealed. The mixture was stirred at room temperature under protection from light for 72 h. After this time (TLC) the formed during the reaction course precipitate 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, CHCl 3 /CH 3 OH/H 2 O, (v/v/v)). Fractions containing products (R f 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.
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 1×10 4 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 growing 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 IC 50 (inhibitory concentration 50)-the concentration of tested agent, which inhibits proliferation of 50% of the cell population. IC 50 values were calculated 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

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 significance 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 proposed 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 synthesis 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 carboxylic 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 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 activities 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 residue 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][33][34]. The cadmium complex of sn-glycero-3-phosphocholine (GPC×CdCl 2 ) (2) was acylated with terpene acid (TA) 1a and 1b in the presence of 4-(N,N-dimethylamino)pyridine (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-snglycero-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 spectroscopic data interpretation. In the 1 H 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 systems present in terpenes acid residues gave signals in 13 C 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 31 P NMR spectrum 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 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 reaction mixture was on the level of 58-84%. We decided to use then the higher molar excess of  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 Biodrowska 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-phosphocholine (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 phosphatidylcholines synthesis [29,24,37]. In all procedures the key step is production of 1-acyl-2-lyso-sn-glycero-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-glycero-3-phosphocholines (3a, 3b) obtained previously (Scheme 1) and subsequent reesterification 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 A 2 (PLA 2 ) 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.

Biological studies
All terpene-modified PCs (1a, 1b, 3a, 3b, 7a, 7b, 9a, 9b, 10a, 10b) were tested for their antiproliferative 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 IC 50 -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 activity 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 phospholipids (3a, 3b) containing terpene acids (TA) in the sn-1 and sn-2 position possessed the antiproliferative 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 IC 50 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 demonstrated against leukaemia and colon cancer LoVo cells (IC 50 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 IC 50 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 Pgp-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 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 terpene 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 further work is also to elucidate the mechanism of the cytotoxicity terpene-modified PCs.