Synthesis of a series of new racemic [2,3-bis(acyloxy)propyl]phosphonocholines

An efficient synthesis of [2,3-bis(acyloxy)propyl]phosphonocholines is described. The reaction pathway for the synthesis involves the Michaelis-Arbuzov reaction of allyl bromide with triethyl phosphite and epoxidation of the resulting diethyl (prop-2-en-1-yl)phosphonate ( 1 ) with m - CPBA to afford the phosphonated diol ( 3 ). The acylation of diol ( 3 ) with carboxylic acids gave a series of diacyloxypropylphosphonates ( 4 ). The introduction of choline to a pyridinium salt of [2,3-bis(acyloxy)propyl]phosphonic acid ( 5 ) was the most intensively studied reaction. The total yield of a six-step sequence synthesis of a series of phosphonolipids was in the range of 30-40%.


Introduction
Phosphonolipids constitute important class of organophosphorous compounds and a new class of cationic lipids. 1 The first naturally occurring phosphonolipids were isolated from the singlecelled microbes of Tetrahynema pyriformisare. 2 To date, phosphonolipids have been discovered in many species of marine animals, various bacteria, certain mammals and other sources. 3,46][7] Generally, phosphonolipids are considered to be components, which protect and stabilize cell-membrane, inhibit phospholipase and facilitate movement of DNA in cells. 8Additionally, many of these compounds have attracted attention for their biological effects, such as: anticancer, antiviral or enzyme inhibitor properties. 91][12] There is a distinct possibility to employ phosphonolipids as lipidic prodrugs. 13he first syntheses were focused on phosphonolipids with C-P bond on the polar side 14,15 and still, this kind of synthesis of these compounds is the most intensively studied.In the last few years researchers have demonstrated a synthetic route to lysophosphatidic acids (LPA) with C-P bond on the glycerol side. 16,17LPA reveal a wide variety of responses to cells and tissues.They were used as a factor that regulates cancer cell proliferation, invasion, angiogenesis, metastasis and also biochemical resistance to chemo-and radiotherapy induced apoptosis via interactions with G-protein coupled receptors. 18ne of the factors which determines the physical and biological properties of a lipid is the nature of fatty acids.][21][22] Here, an efficient synthesis of a series of seven racemic [2,3-bis(acyloxy)propyl] phosphonocholines with different fatty acids is presented.The obtained compounds could be considered as phosphono analogs of corresponding diacylophospholipids.In future, these compounds will be tested as substrates in enzymatic hydrolysis and transesterification reactions.Enzymatic reactions carried out on a series of phospho-and phosphonolipids with different aliphatic carbon chains as the hydrophobic part should provide some information concerning the influence of the C-P bond on the activity of phospholipases A1, A2 and D. The enzymatic hydrolysis of such molecules is very important in the future of applications of this type of compounds as prodrugs.
The phosphonate with CLA was synthesized because of many biological activities of CLA itself.It exhibited a considerable anti-proliferative effect on cancer cells in in vitro study and anti-tumor activity in in vivo tests. 23
The Michaelis-Arbuzov rearrangement was the first step of the synthesis.The reaction of allyl bromide with triethyl phosphite afforded diethyl (prop-2-en-1-yl)phosphonate (1) in a very high, practically quantitative, yield.The reaction was facilitated by an excess of allyl bromide (10%), which can be easily removed by distillation.The first attempts with of these reactions were carried out with the addition of iodide salt (NaI, KI).It is known that alkyl iodides are more reactive than bromides and these salts were very often used to accelerate the reaction. 24In our experiments with application of NaI or KI the yields were very high, but the product of the reaction had an undesirable dark color which caused the necessity of additional purification on a silica gel column.Therefore, we decided to perform this reaction without iodide salt.The yields of the trials were also very high and let us avoid purification of the product.The structure of diethyl allylphosphonate (1) was confirmed by NMR data.The presence of the allyl group confirms the signals of the olefin protons: multiplet of H-2 at 5.78 ppm and multiplet of CH2-3 in the range of 5.23-5.16ppm.Methylene protons of CH2-1 gave in the spectrum doublet (J = 21.9Hz) of doublets (J = 7.4 Hz) of triplets (J = 1.2 Hz).In the next step, the epoxidation of the diethyl (prop-2-en-1-yl)phosphonate (1), with 1.5 equiv of 3-chloroperbenzoic acid (m-CPBA) was carried out.The epoxyphosphonate (2) was obtained with 92% yield.The multiplets of H-2 (3.18 ppm) and CH2-3 (2.82 and 2.58 ppm) confirmed the presence of an oxirane ring in the product.
The aqueous or THF (95%) solutions of HCl, H2SO4, HClO4 and acetic acid were tried as reagents for opening the oxirane ring in diethyl (2,3-epoxypropyl)phosphonate (2).The best results were obtained for acetic acid solution (pH<2) in water.The diethyl (2,3duhydroxypropyl)phosphonate (3) was obtained in a 79% yield.Other experiments gave lower yields.The structure of diethyl (2,3-dihydroxypropyl)phosphonate (3) was confirmed by its spectral data ( 1 H and 13 C NMR, MS).The multiplet of H-2 proton was observed in the 1 H-NMR spectra together with multiplets of -OCH2 protons in the range of 4.23 -4.01 ppm and the doublets of doublets of CH2-3 are located at 3.70 and 3.54 ppm.The attempts of obtaining (2,3dihydroxypropyl)phosphonate (3) by direct oxidation of diethyl allylphosphonate (1) with potassium permanganate in water as well as acetone solution failed.A non-separable complex mixture of products was obtained.
The acylation of diethyl (2,3-dihydroxypropyl)phosphonate (3) with carboxylic acids was carried out in the presence of coupling agent (DCC).The molar excess of carboxylic acid per one mole of diol (3) was established in the experiments on the synthesis of diethyl [2,3-bis(palmitoyloxy)propyl]phosphonate (4c).The trials were carried out with 2.4, 3.0 and 4.0 equiv. of acid pere one mole of phosphonate (3).The results showed that the highest yield (92%) of product was obtained in the reaction with 3.0 molar equiv. of acid per one mole of diethyl (dihydroxypropyl)phosphonate (3).
The purity of the phosphonates (4a-g) was confirmed by HPLC (Figure 1, Chromatogram A).In the course of these analyses the influence of the carbon chain length of carboxylic acid on the retention time (Rt) of phosphonates (4a-g) was observed.Increase in the length of the carbon chain of fatty acid leads to higher Rt (HPLC: ODS2 Sperisorb column).In contrast to double bond in the rest of oleic acid (4e), linoleic acid (4f) and conjugated linoleic acid (4g) decrease their R t in comparison to the R t of diethyl [2,3-bis(stearoyloxy)propyl]phosphonate (4d) (Figure 1, Chromatogram B).The structures of diacyl phosphonates (4a-g) were confirmed by their spectral data ( 1 H and 13 C NMR, 31 P NMR).Comparing the 1 H NMR spectra of both substrate and products it is noticed that the signals from the proton H-2 were shifted downfield from 4.23 -4.10 ppm in the spectrum of diol (3) to above 5 ppm for [2,3-bis(acyloxy)propyl]phosphonates (4a-g).Similarly, the signals belonging to CH2-3 protons were shifted from 3.70 and 3.54 ppm to above 4 ppm.Structures of the fatty acids residue were also confirmed by spectral data.The signals of the olefin protons were in the same region of the spectrum as signals of H-2. 1 H NMR and 13 C NMR spectra of (4g) were analyzed according to earlier studies. 25sterification of (2,3-epoxypropyl)phosphonate ( 2) with palmitic acid anhydride was also tested.The reaction was carried out according to the procedure of Ali and Bittman. 26The best yield (57.6%) of (4c) was obtained.So we decided to apply a two-step synthesis of phosphonates (4a-g) via diol (3).
The transformation of diethyl [2,3-bis(acyloxy)propyl]phosphonates (4) to phosphonic acid required a careful choice of reagent.Bromotrimethylsilane (TMSBr) is commonly used for this purpose. 27,28After several experiments carried out in different solvents (CH3CN or CH2Cl2), at several temperatures (20, 35 o C and under reflux) and with various molar excesses of TMSBr we decided to carry out this reaction in CH2Cl2 with a 10 molar excess of TMSBr at room temperature.The [2,3-bis(acyloxy)propyl]phosphonic acids were very unstable so they were transformed to more stable and reactive form of monopyridinium salts (5a-g) 24,29,30 The 1 H and 13 C NMR spectral data of the pyridinium salts (5a-g) confirmed their structures.The signals of aromatic protons are present in the 1 H NMR spectrum at approximately 8.6, 8.0, 7.5 ppm and the signals of pyridine carbon atoms at 148.5, 137.7 and 124.5 ppm.Reaction conditions: 1eq of phosphonic acid (A) or monopyridinium salt of phosphonic acid (5c) (B), choline tosylate (2 molar eq), DCC (3 molar eq), 48 h, room temp., N2, solvent (TEAtrietylamine) The introduction of choline moiety into [2,3-bis(acyloxy)propyl]phosphonic acid was achieved in the reaction of pyridinium salt of phosphonic acid (5a-g) with choline tosylate (Scheme 1).The reaction of phosphonic acid with an active molecule, such as choline, is usually the crucial step in the synthesis of modified phospho-and phosphonolipids, including prodrugs. 13,31The first experiments on this reaction were carried out for [2,3bis(palmitoyloxy)propyl]phosphonic acid and its pyridinium form (5c) in the presence of DCC (3 molar excess) as coupling agent in dry pyridine or in dry CH2Cl2.Results presented in Table 1 showed that the protonic form of phosphonic acid was not a good substrate for synthesis of 6c.The yield of the reaction was below 2%.The pyridinium salt form (5c) was a much more reactive substrate. 29The yields of the reaction were in the range of 37-51% (Table 1).So in the next experiments pyridinium salt of phosphonic acid (5c) was used.
The results of the esterification carried out in various solvents (CH 2 Cl 2 , pyridine, CCl 3 CN, toluene, trietylamine) led to the conclusion that the best results were obtained for pyridine and CCl3CN.We decided to use pyridine as a solvent for the next reactions, because the pyridine dissolves the substrates and the products more effectively.The molar excess of choline tosylate was also established in the experiments on synthesis of (6c).The common condition used for esterification of phosphatidic acid with choline needs the use of 10 molar excess of choline. 26In our method the 2 molar excess of choline tosylate was sufficient.It is a result of the application of the more reactive form (pyridinium salt) of phosphonic acid.Three coupling agents were tested in the reaction: N,N'-dicyclohexylcarbodiimide (DCC), p-toluenesulfonyl chloride (TsCl) and mesitylene-sulfonyl chloride (MeCl).The DCC was the most effective with a yield of 50,8%.Conditions elaborated for the reaction of the introduction of choline to the phosphonic acid molecule let us obtain the phosphonates (6a-g) in good yields (60 -72%).Structures of [2,3-bis(acyloxy)propyl]phosphonocholine (6a-g) were confirmed by their NMR spectral data.Signals from protons of the choline group were present in the 1 H NMR spectra as a multiplets cumulated between 4.5 -3 ppm.A singlet of -N(CH3)3 group protons was observed at about 3.17 ppm.

Conclusions
An efficient method of synthesis of phosphonolipids with choline as a polar head was elaborated.The introduction of the choline to phosphonic acid in known methods requires a high excess of choline, even 10 molar equiv. 26Lower excess of choline moiety could be applied for the synthesis with a more reactive formpyridinium salt of phosphonic acid (5).Our method allows to use only 2 molar equiv. of choline tosylate.The presented method could be applied for synthesis of different phosphonolipids as potential prodrugs.
We hope that studies on enzymatic hydrolysis of a series of phosphonolipids with different aliphatic carbon chains as the hydrophobic part can afford interesting information on influence of fatty acid carbon chain length on the efficiency of enzymatic hydrolysis of such compounds.
Comparable studies on hydrolysis of phospholipids and their phosphono analogues by phospholipase A1, A2, D could afford valuable information on the influence of C-P bond on these enzyme activity.
ESI-MS were measured on a Bruker micrOTOF-Q and GC-MS analyses were measured on a mass spectrometer (MS), equipped with an ion-trap analyzer, set at 1508 for all analyses with an electron multiplier voltage of 1350 V. Scanning (1 scan s -1 ) was performed in the range of 39-400 m/z and using electron impact ionization at 70eV.NMR spectra were measured on a Bruker Avance II 600 MHz.Chemical shifts ( 1 H and 13 C) () are given in ppm with TMS as the internal standard, in 31 P NMR chemical shifts were referenced to 85% H3PO4 as an external standard.Coupling constant (J) values are in Hz.Assignments of signals to corresponding protons and carbons were made on the basis of homoand heteronuclear correlation ( 1 H-1 H COSY, 1 H-13 C HMQC).
Gas chromatography analysis was carried out on a Varian CP-3380 instrument (FID, carrier gas H2) using TR-5 (30 m x 0.32 mm x 1.0 μm) with the following temp.program: 110 -210 o C at 30 o C/min, then with rate 30 o C/min to 300 o C and hold for 1 min.The total run time was 9.6 min.