Drug–phospholipid conjugates as potential prodrugs: synthesis, characterization, and degradation...

Chemistry and Physics of Lipids

107 (2000) 143–157

Drug–phospholipid conjugates as potential prodrugs:synthesis, characterization, and degradation by pancreatic

phospholipase A2

Michael Kurz a, Gerhard K.E. Scriba b,*a Uni6ersity of Munster, Department of Pharmaceutical Chemistry, D-48149 Munster, Germany

b Department of Pharmaceutical Chemistry, Uni6ersity of Jena, School of Pharmacy, Philosophenweg 14, 07743 Jena, Germany

Received 10 April 2000; received in revised form 13 June 2000; accepted 14 June 2000

Abstract

The aim of the present study was the synthesis of phospholipids containing a drug molecule instead of a fatty acid.Valproic acid and ibuprofen served as model compounds. The target molecules were synthesized either starting fromsn-glycero-3-phosphocholine (1) or using (S)-2-O-benzyl-1-O-tritylglycerol (11) and (R)-2-O-benzyl-1-O-tert-butyldiphenylsilylglycerol (12), respectively, as key intermediates. With respect to the surface properties and theaggregation behavior, the drug–phospholipid conjugates resembled natural phosopholipids. Upon incubation withporcine pancreatic phospholipase A2, only compounds with a fatty acid in the sn-2 position of the glycerol backbonewere degraded. Derivatives with either ibuprofen in the sn-2 position or displaying the unnatural S-configurationwere resistant to enzymatic in vitro hydrolysis. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Drug-phospholipid conjugates; Prodrugs; Phospholipid; Phospholipase A2

www.elsevier.com/locate/chemphyslip

1. Introduction

Many promising pharmacological agents, dis-playing high in vitro efficacy, are less active oreven inactive after in vivo application, especiallyafter oral administration. One approach to over-come this problem is the synthesis of prodrugs.Among the prodrugs, triglyceride analogues, alsotermed pseudoglycerides, have been prepared by

substitution of one or more fatty acids of atriglyceride by a drug molecule in order to conferthe attached drugs to the metabolic pathways ofnatural lipids. Non-steroidal anti-inflammatorydrugs such as aspirin, indomethacin, ibuprofen, ornaproxen were covalently bound to diglycerides inorder to reduce the ulcerogenity of the com-pounds (Paris et al., 1979; Paris et al. 1980; Pariset al., 1982). The approach has also been utilizedfor an improved oral bioavailability of phenytoin(Scriba et al., 1995). Employing the absorption ofdietary fat, pseudoglycerides have been used fortargeting the lymphatic route in order to avoid

* Corresponding author. Tel.: +49-3641-949830; fax: +49-3641-949802.

E-mail address: [email protected] (G.K.E. Scriba).

1079-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0009 -3084 (00 )00167 -5

M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157144

the first-pass metabolism of L-DOPA (Garzon-Aburbeh et al., 1986) and/or target the lymphaticsystem for the treatment of lymphatic metastasis(Garzon-Aburbeh et al., 1983) or filiariasis (De-verre et al., 1992). Radioionated agents (Weichertet al., 1995) and compounds containing a nitroxylmoiety (Gallez et al., 1992) were coupled to glyce-rides as potential hepatographic agents in nuclearmedicine and nuclear magnetic resonance imaging.Improvement of the blood–brain barrier penetra-tion of GABA glycerides has been reported (Jacobet al., 1990). A related approach has been appliedto L-DOPA, glycine, valproic acid and theenkephalinase inhibitor thiorphan including amideisosters of glycerides (Mergen et al., 1991; Lambertet al., 1993).

The use of phospholipid analogues as drugcarriers has so far been utilized primarily forcytostatic nucleosides and nucleoside analoguessuch as AZT (van Wijk et al., 1992, 1994), acyclovir(Hostetler et al., 1990, 1993; van Wijk et al., 1992)or cytidine analogues (Turcotte et al., 1980; Mat-sushita et al., 1981; Ryu et al., 1982). In thesederivatives, nucleoside monophosphates, diphos-phates and triphosphates were bound to diacylglyc-erols, yielding derivatives with increased anti-neoplastic activity in vitro and in vivo. A phospho-lipid derivative obtained by coupling of a HIVprotease inhibitor to dipalmitoylphosphatidyl-ethanolamine has also been described (Hostetler et

al., 1994). ‘Pseudophospholipid’ prodrugs bearinga drug molecule instead of a fatty acid have not sofar been realized. The present study describes thesynthesis of such phospholipid derivatives usingthe anticonvulsant valproic acid and the non-steroidal anti-inflammatory drug ibuprofen asmodel compounds. The drugs were primarily at-tached to the sn-1 position, which is not hydrolyzedby pancreatic phospholipase A2. The surface activ-ity of the phospholipid analogues and the degrada-tion of the lipids by phospholipase A2 were alsoinvestigated.

2. Results and discussion

2.1. Synthesis

The synthetic sequence starting from sn-glyc-ero-3-phosphocholine (1), as depicted in Scheme1, essentially followed the procedure publishedby Hermetter et al. (1989) for the synthesis ofunsymmetrical phospholipids, except that theesterification of the hydroxy group of the tritylintermediate 2 with palmitic acid in the sn-2position was carried out in CH2Cl2 using dicyclo-hexylcarbodiimide (DCC) and 4-dimethy-laminopyridine (DMAP) instead of the acidimidazolide in dimethylsulphoxide (DMSO) in thepresence of sodium. Despite longer reaction

Scheme 1.

M. Kurz, G.K.E. Scriba / Chemistry and Physics of Lipids 107 (2000) 143–157 145

Scheme 2.

times, we found this procedure more suitable,especially due to the more facile work-up. Bothprocedures give comparable yields of compound 3.We found 77% compared with 74% of the publishedprocedure. The final product 1-O-valproyl-2-O-palmitoyl-sn-glycero-3-phosphocholine (4) was ob-tained in a one-pot reaction by removal of the tritylgroup with BF3 in the presence of valproic acidanhydride according to Hermetter et al. (1989). Theoverall yield of this sequence was 40%. However,attempts to synthesize the corresponding ibuprofenphospholipid by the same sequence failed.

The second synthetic sequence utilized the keyintermediates 11 and 12 (Peters et al., 1987), whichallowed the preparation of derivatives with thenatural R-configuration of phospholipids as well asderivatives with the unnatural S-configuration.Moreover, the corresponding lyso-phospholipidscan be obtained during this sequence. Compounds11 and 12 were synthesized as outlined in Scheme2.

D-Mannitol (5) was converted to the dibenzyli-dene acetal 6 by stirring in acidified dimethylfor-mamide (DMF) in the presence of an excess ofbenzaldehyde followed by reaction with KOH andbenzyl chloride in DMSO (Johnstone and Rose,1979) to give 2,5-di-O-benzyl-1,3:4,6-di-O-benzyli-dene-D-mannitol (7). Removal of the acetal pro-tecting groups upon refluxing with diluted HCl inethanol gave 2,5-di-O-benzyl-D-mannitol (8) witha yield of 93%. 2-O-Benzyl-1-O-trityl-D-mannitol(9) was obtained by reaction of compound 8 withtrityl chloride in the presence of DMAP as catalystand triethylamine as base. Malaprade oxidation ofcompound 9 using an excess of 1.5 equivalents ofsodium periodate in THF/water (10:1) resulted inaldehyde 10, which was reduced with sodiumborohydride to yield (S)-2-O-benzyl-1-O-trityl-glycerol (11) (Peters et al., 1987). The overall yieldof these three steps starting from compound 8 was90%. All steps can also be carried out withoutextensive isolation of the intermediates. (R)-2-O-


benzyl-1-O-tert-butyldiphenylsilylglycerol (12), thestarting material for phospholipids with the unnat-ural S-configuration, was obtained from com-pound 11 by protection of the free hydroxy groupas tert-butyldiphenylsilylether (Hanessian andLavallee 1975) followed by removal of the tritylgroup by refluxing with a 1% solution of pyri-dinium chloride in ethanol.

The drug–phospholipid conjugates with the nat-ural R-configuration were synthesized from com-

pound 11 as outlined in Scheme 3. Upon reactionwith 1.2 equivalents methyldichlorophosphite and2.5 equivalents of diisopropylethylamine in te-trahydrofuran (THF) at −78°C, followed by theaddition of 1.5 equivalents of 2-bromoethanol andoxidation with hydrogen peroxide (Martin et al.,1994), ((R)-(2-O-benzyl-3-O-tritylglyceryl)-2-bro-moethyl-methyl phosphate (13) was produced as aviscous oil that was converted to ((R)-2-O-benzyl-glyceryl)-2-bromoethyl-methyl phosphate (15) by

Scheme 3.


Fig. 1. Surface pressure-apparent molecular area isotherms ofcompounds 30 and 32 at the air–water interface at 20°C.

that removal of the silyl ether of compound 14to give ((S)-2-O-benzylglyceryl)-2-bromoethyl-methyl phosphate (16) was carried out with tetra-butylammonium fluoride in THF. The yields ofthis sequence were comparable with the R-configurated series.

2.2. Physicochemical characterization

With respect to the physicochemical properties,the surface activity and the particle size of aggre-gates formed in aqueous solutions of compounds30 and 32 were determined. The surface activityof the compounds was determined by the Wil-helmy method. The surface pressure apparentmolecular area curves of compounds 30 and 32are shown in Fig. 1. Lift off areas of 90 and 110A, 2/molecule were found for compounds 30 and32, respectively. Transition to the solid-condensedphase occurred continuously. The collapse pointswere 45 and 55 A, 2/molecule, the collapse pressurevalues were 45 and 43 mN/m. Values of 40 A, 2/mol and 60 and 40 mN/m have been reported forthe standard phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, respectively (Bredlowet al., 1992). These differences compared with theibuprofen-containing pseudophospholipids maybe explained by the smaller size of the lipophilicpart of the molecule due to the substitution of apalmitic acid residue with ibuprofen.

The aggregation behavior of the ibuprofenphospholipids 30 and 32 in aqueous solution wascharacterized by dynamic laser light scattering.An average particle size of about 160–180 nmwas determined for a 0.1% aqueous dispersion ofcompound 30. This suggests the formation ofspherical particles by compound 30 (Eibl, 1984).The value was not significantly altered upon in-creasing the temperature from 20 to 37°C. Incontrast, compound 32 containing ibuprofen inthe sn-2 position displayed a different behavior.While two maxima at approximately 500 and1000 nm could be observed when no particle sizewas preselected (Fig. 2A), a single maximum atabout 550 nm was recorded when a particle sizeof 500 nm (Fig. 2B) or 1000 nm (data not shown)was preselected. This behavior did not change

refluxing with pyridinium chloride in ethanol. De-pending on the target molecule the free hydroxygroup was esterified with either ibuprofen,palmitic acid, or valproic acid to give compounds17, 19 and 20, respectively. Hydrogenolytic depro-tection and esterification with the appropriate car-boxylic acid gave compounds 22, 23 and 25–27,which were converted to the pseudophospholipids29, 30, and 32–34 by treatment with trimethy-lamine. The lyso-phospholipid 28 was obtained bytreatment of compound 21 with triethylamine. Allreactions of the sequence did not affect the stereo-chemistry of the chiral sn-2 carbon atom. Thechiral purity of all compounds was estimated bymeasuring the optical rotation.

The pseudophospholipid 31 with the unnaturalS-configuration was obtained from compound 12in the same synthetic sequence as described forthe R-configurated compounds (Scheme 3) except


upon dilution of the solution to 0.01% or uponincreasing the temperature from 20 to 37°C. The

fact that the maximum at 1000 nm is twice thesize of the maximum at 500 nm suggested theexistence of ‘monomers’ and ‘dimers’. The‘dimers’ should disappear upon dilution. How-ever, no significant change was observed upondilution to 0.01%. As the solution could not bediluted further due to a strong concomitant de-crease of the light scattering intensity, the pres-ence of ‘dimers’ cannot be unequivocally ruledout. An alternative explanation of the observedphenomenon might be the existence of hexagonalstructures as the fitting software cannot distin-guish between light scattering at spherical orcylindric shape particles.

2.3. Enzymatic degradation

The ibuprofen-containing phospholipids 29–32were incubated in the presence of porcine pan-creatic phospholipase A2 in order to estimate theactivity of the enzyme towards the drug–phos-pholipid conjugates. The incubations were ana-lyzed by high-performance liquid chromatography(HPLC). Under the conditions applied, only con-jugate 30, which contained ibuprofen in the sn-1position and palmitic acid in the sn-2 positionand possesses the natural R-configuration, washydrolyzed. No degradation of the other com-pounds was observed for 96 h. This is in agree-ment with the fact that pancreatic phospholipaseA2 only cleaves R-configurated phospholipidsand not derivatives with the S-configuration, i.e.compound 31. Apparently, compounds containingibuprofen at the sn-2 position are not substratesfor the enzyme under the conditions applied.This may be explained by steric hindrance ofthe branched 2-methyl-phenylacetic acid moietyand/or by a different spatial arrangement ofthe acid moieties compared with natural phospho-lipids with linear fatty acids.

The time course of the degradation of com-pound 30 by pancreatic phospholipase A2 ata starting concentration of 500 mM is shown inFig. 3. Between 0 and 3 h, the hydrolysis followsapparent zero-order kinetics with a half-life of2.0 h, while the kinetic becomes apparent firstorder after 5–6 h with a half-life of 13.1 h.

Fig. 2. Distribution of particle size of compound 32 deter-mined by dynamic laser light scattering. (A) No preselectedparticle size; (B) preselected particle size, 500 nm.

Fig. 3. Time course of the in vitro degradation of compound30 by porcine pancreatic phospholipase A2. Each time pointrepresents the mean of three determinations9S.D.


3. Conclusions

The target ‘pseudophospholipids’ containing ei-ther valproic acid or ibuprofen instead of a fattyacid were synthesized. The surface properties andaggregation behavior resembled the properties ofnatural phospholipids. Upon incubation withpancreatic phospholipase A2, only the com-pounds containing palmitic acid in the sn-2 posi-tion of the glycerol backbone were degraded,while the compounds containing either ibuprofenin the sn-2 position or displaying the unnaturalS-configuration were resistant to enzymatic hy-drolysis. The pharmacological activity of thecompounds is currently under investigation.

4. Experimental

4.1. Synthesis

sn-Glycero-3-phosphocholine was a gift fromNattermann Phospholipid GmbH (Cologne, Ger-many). All other chemicals were obtained fromcommercial suppliers and were used without fur-ther purification unless noted otherwise. THFand diethyl ether were distilled from sodium andbenzophenone ketyl, chloroform was distilledfrom phosphorous pentoxide, and the amines andDMF were distilled from calcium hydride. Reac-tions involving air- or moisture-sensitive reagentswere carried out under nitrogen. Column chro-matography was performed using Merck silica gel60 (70–230 mesh). Melting points are uncor-rected. Nuclear magnetic resonance (NMR) spec-tra were obtained on either a Varian Gemini 200or a Varian Unityplus 600. Mass spectra wereobtained on a Varian MAT 44S. The opticalrotation, [a ]D20, was recorded using a Perkin-Elmer341 instrument. Elemental analyses were within90.4% of the theoretical values.

4.1.1. 1-O-Trityl-sn-glycero-3-phosphocholine (2)Compound 2 was synthesized from 10.3 g sn-

glycero-3-phosphocholine (1) (40 mmol), 5.5 gZnCl2 (40 mmol) and 12.2 g trityl chloride (44mmol) according to Hermetter et al. (1989).Yield: 14.3 g (72%); melting point (m.p.), 228–

230°C. [a ]D20= −10.0° (c=4.0 in MeOH). 1H-NMR (MeOD, 200 MHz): d (ppm)=3.14 (s,11H), 3.55 (m, 2H), 3.95 (m, 2H), 4.04 (m, 1H),4.22 (m, 2H), 7.17–7.48 (m, 15H). 13C-NMR(MeOD, 50 MHz): d (ppm)=54.58, 60.31, 66.07,67.29, 68.82, 71.28, 87.85, 128.13, 128.81, 129.85,145.32. MS (chemical ionization (CI), ammonia):m/z=500 ([M+H]+).

4.1.2. 1-O-Trityl-2-O-palmitoyl-sn-glycero-3-phosphocholine (3)

Compound 3 was prepared from 8.0 g com-pound 2 (16 mmol), 5.0 g palmitic acid (20mmol), 5.0 g DCC (24 mmol) and 1 g DMAP(8.2 mmol) in 80 ml CH2Cl2. Filtration, evapora-tion of the solvent and recrystallization frommethanol:water (4:1, v/v) followed by recrystal-lization from acetone, yielded 5.7 g (77%). [a ]D20=−7.4° (c=4.0 in MeOH). 1H-NMR (CDCl3, 200MHz): d (ppm)=0.88 (t, 3H, 3J=5.8 Hz), 1.25(s, 24H), 1.60 (m, 2H), 2.34 (t, 2H, 3J=7.5 Hz),3.14 (s, 11H), 3.65 (m, 2H), 3.95 (m, 2H), 4.13(m, 2H), 5.24 (m, 1H), 7.16–7.42 (m, 15H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=14.15, 22.71,25.05, 29.30, 29.73, 31.94, 34.61, 54.23, 59.14,63.05, 64.20, 66.21, 72.44, 86.46, 127.09, 127.84,128.90, 143.70, 173.42. MS (CI, ammonia): m/z=738 (M+).

4.1.3. 1-O-Valproyl-2-O-palmitoyl-sn-glycero-3-phosphocholine (4)

Compound 4 was prepared from compound 3(3.0 g, 4 mmol), valproic acid anhydride (2.2 g,8 mmol) and 16 mmol BF3·Et2O according to theprocedure by Hermetter et al. (1989). Yield: 1.85g (77%). [a ]D20= +6.4° (c=4.0 in CHCl3). 1H-NMR (CDCl3, 200 MHz): d (ppm)=0.88 (t, 9H,3J=7.6 Hz), 1.26 (s, 28H),1.50 (m, 6H), 2.29(t, 2H, 3J=7.6 Hz), 2.34 (m, 1H), 3.38 (s, 9H),3.80 (m, 2H), 3.89 (m, 2H), 4.30 (m, 2H), 4.41(m, 2H), 5.18 (m, 1H). 13C-NMR (CDCl3, 50MHz):d (ppm)=14.12, 20.57, 22.71, 24.99, 29.27, 29.75,31.97, 34.39, 34.49, 45.14, 54.44, 59.45, 62.92,63.55, 66.55, 70.76, 173.11, 176.21. MS (CI,ammonia): m/z=623 ([M+H]+). C32H64NO8P·1.2H2O (643.45): Calc.: C, 59.73; H, 10.40; N,2.18; found: C, 59.44; H, 10.33; N, 2.26.


4.1.4. 2,5-Di-O-benzyl-1,3:4,6-di-O-benzylidene-D-mannitol (7)

Compound 7 was synthesized according toJohnstone and Rose (1979) from 45 g 1,3:4,6-di-O-benzylidene-D-mannitol (6) (Baggett and Strib-blehill, 1977), 60 g KOH (1 mol) and 60 ml benzylchloride (0.5 mol). Yield: 61 g (90%); m.p., 108–110°C. [a ]D20= −37.5° (c=1.3 in CHCl3). 1H-NMR (CDCl3, 200 MHz): d (ppm)=3.76 (t,2J=3J=9.8 Hz, 2H), 3.90–4.11 (m, 4H), 4.42(dd,2J=10.0 Hz, 3J=4.4 Hz, 2H), 4.64 (s, 4H),5.46 (s, 2H), 7.16–7.60 (m, 20H). 13C-NMR(CDCl3, 50 MHz): d (ppm)=67.00, 69.70, 72.72,77.58, 101.13, 126.30, 127.97, 128.02, 128.51,128.86, 137.85, 138.08. MS (EI): m/z=538 (M+).

4.1.5. 2,5-Di-O-benzyl-D-mannitol (8)Compound 7 (54 g, 0.1 mol) was refluxed for 4

h in 400 ml ethanol containing 40 ml of 0.5 MHCl. After neutralization with NaHCO3, the sol-vents were removed in vacuo. The residue wasdissolved in hot ethyl acetate and washed oncewith warm water. Upon removal of the ethylacetate, the residue was suspended in a mixture ofdiethyl ether (200 ml) and xylene (200 ml), andstirred for 0.5 h. The white solid was filtered,washed with petrol ether and dried. Yield: 33.7 g(93%); m.p., 123–125°C. [a ]D20= −8.1° (c=1.0EtOH). 1H-NMR (MeOD, 200 MHz): d (ppm)=3.61 (m, 2H), 3.75 (dd, 2J=11.8 Hz, 3J=4.5 Hz,2H), 3.93 (dd, 2J=11.8 Hz, 3J=3.6 Hz, 2H), 3.98(d, 3J=7.9 Hz, 2H), 4.58 (d, 2J=11.6 Hz, 1H),4.73 (d, 2J=11.6 Hz, 1H), 7.20–7.50 (m, 10H).13C-NMR (MeOD, 50 MHz): d (ppm)=62.28,70.41, 73.54, 81.42, 128.55, 128.97, 129.25, 140.01.MS (CI, ammonia): m/z=379 ([M+NH3]+).

4.1.6. 2,5-Di-O-benzyl-1,6-di-O-trityl-D-mannitol(9)

Compound 8 (36.2 g, 0.1 mol), 1 g DMAP and35 ml triethylamine (250 mmol) were suspended in250 ml CH2Cl2, 61.3 g tritylchloride (220 mmol)were added and the mixture was stirred for 6 h atroom temperature. The organic phase was washedwith water and evaporated. The residue was re-crystallized from 2-propanol. Yield: 83 g (98%);m.p., 64–66 °C. [a ]D20= −22.1°. 1H-NMR(CDCl3, 200 MHz): d (ppm)=2.84 (d, 3J=5.7

Hz, 2H), 3.27 (dd, 2J=10.0 Hz, 3J=5.2 Hz, 2H),3.37 (dd, 2J=10.0 Hz, 3J=4.2 Hz, 2H), 3.74 (m,2H), 3.97 (dd, 3J=5.7, 6.5 Hz, 2H), 4.53 (d,2J=11.3 Hz, 2H), 4.70 (d,2J=11.3 Hz, 2H),7.18–7.46 (m, 40H). 13C-NMR (CDCl3, 50 MHz):d (ppm)=63.83, 69.90, 73.31, 79.89, 87.13,127.08, 127.73, 127.84, 127.91, 128.01, 128.06,128.43, 128.46, 128.79, 138.34, 144.02. MS (CI,ammonia): m/z=863 ([M+NH3]+).

4.1.7. (R)-2-O-Benzyl-3-O-tritylglycerol aldehyde(10)

To 83 g of compound 9 (100 mmol) dissolved in400 ml THF, 32 g sodium periodate (150 mmol)in 100 ml water were added and stirred for 2 h.Then, 400 ml ethyl acetate and 300 ml water areadded, and the aqueous phase was extracted withethyl acetate. The combined organic phases werewashed with brine and evaporated. Compound 10was obtained as a colourless oil. Yield: 78.7 g(95%). [a ]D20= +2.5° (c=1.1 in CHCl3). 1H-NMR (CDCl3, 200 MHz): (ppm)=3.41 (dd, 2J=10.1 Hz, 3J=5.3 Hz, 1H), 3.46 (dd, 2J=10.1 Hz,3J=4.4 Hz, 1H), 3.93 (dd, 3J=4.4, 5.3 Hz, 1H),4.68 (s, 2H), 7.19–7.46 (m, 20H), 9.72 (s, 1H).13C-NMR (CDCl3, 50 MHz): d (ppm)=63.15,72.57, 82.85, 87.10, 127.17, 127.26, 127.84, 127.89,127.96, 128.02, 128.53, 128.71, 128.76, 138.42,143.53, 201.74. MS (CI, ammonia): m/z=440([M+NH3]+).

4.1.8. (S)-2-O-Benzyl-1-O-tritylglycerol (11)Compound 10 (78.5 g, 186 mmol) was dissolved

in 300 ml MeOH, 8 g sodium borohydride (200mmol) added and the mixture was stirred for 4 hat room temperature. The solvent was removed invacuo, the residue was dissolved in diethyl etherand washed with water. The residue obtainedupon evaporation of the ether was purified bycolumn chromatography (cyclohexane/ethyl ace-tate 10:1). Yield: 76.7 g (92%); m.p., 80–83°C.[a ]D20= −26.3° (1.2% in CHCl3). 1H-NMR(CDCl3, 200 MHz): d (ppm)=2.01 (s, 1H), 3.24(dd, 2J=11.0 Hz, 3J=5.8 Hz, 1H), 3.31 (dd,2J=11.0 Hz, 3J=4.8 Hz, 1H), 3.50–3.80 (m,3H), 4.53 (d, 2J=11.7 Hz, 1H), 4.68 (d, 2J=11.7Hz, 1H), 7.15–7.55 (m, 20H). 13C-NMR (CDCl3,


50 MHz): d (ppm)=63.24, 63.65, 72.31, 78.84,87.09, 127.16, 127.32, 127.84, 127.87, 127.94,128.00, 128.03, 128.54, 128.78, 138.43, 143.97.

4.1.9. (R)-2-O-Benzyl-1-O-tert-butyldiphenylsilylglycerol (12)

Twenty-eight grams of tert-butyldiphenylsilylchloride (100 mmol) were added to 40 g com-pound 11 (94 mmol) and 15 g imidazole (220mmol) in 125 ml DMF. The mixture was stirredat room temperature overnight, poured into 400ml water and extracted with CH2Cl2. The productobtained upon evaporation of the solvent wasrefluxed without further purification in 150 mlethanol containing 1.25% of pyridinium chloridefor 2 h. Upon addition of 1.1 equivalents ofNaHCO3 in water, the solvents were removed invacuo. The residue was dissolved in CH2Cl2 andwashed with brine. The organic phase was evapo-rated in vacuo. Column chromatography (cyclo-hexane/ethyl acetate, 10:1) yielded 33.4 g (84.5%)of a colourless oil. [a ]D20= +23.7° (c=4.7 inCHCl3). 1H-NMR (CDCl3, 200 MHz): d (ppm)=1.06 (s, 9H), 2.07 (brs, 1H), 3.55–3.90 (m, 5H),4.51 (d, 2J=11.7 Hz, 1H), 4.63 (d, 2J=11.7 Hz,1H), 7.10–7.75 (m, 15H). 13C-NMR (CDCl3, 50MHz): d (ppm)=19.31, 26.97, 62.96, 63.74, 72.26,79.79, 127.82, 127.85, 128.11, 128.18, 128.53,128.82, 129.89, 130.27, 133.33, 133.44, 135.70,135.73, 138.50. MS (CI, ammonia): m/z=437([M+NH3]+).

4.1.10. ((R)-2-O-Benzyl-3-O-tritylglyceryl)-2-bromoethyl-methyl phosphate (13)

Ten grams of methyldichlorophosphite (75mmol) and 30 ml diisopropylethylamine (175mmol) were dissolved in 200 ml THF under nitro-gen and cooled to −78°C. Then, 26.5 g com-pound 11 (62.5 mmol) dissolved in 50 ml THFwere added slowly over a period of 1 h. Afterstirring for an additional hour, 7.1 ml 2-bro-moethanol (100 mmol) were added dropwise. Thereaction mixture was allowed to warm to roomtemperature and stirred for 2 h prior to evapora-tion of the solvents. The residue was dissolved inCH2Cl2 and treated for 2 h with 20 ml hydrogenperoxide solution (30%, 200 mmol) The organic

phase was washed with water. Column chro-matography (cyclohexane/ethyl acetate, 3:1)yielded a colourless oil. Yield: 30.5 g (78%).[a ]D20= −5.5° (c=0.8 in CHCl3). 1H-NMR(CDCl3, 200 MHz): d (ppm)=3.26 (d, 3J=5.1Hz, 2H), 3.42 (dt, 3J=6.2 Hz, 4JH,P=0.8 Hz,2H), 3.64–3.78 (m, 2JH,P=11.3 Hz, 4H), 4.15–4.30 (m, 4H), 4.62 (s, 2H), 7.19–7.47 (m, 20H).13C-NMR (CDCl3, 50 MHz): d (ppm)=29.86,54.88, 63.14, 67.17, 68.14, 72.77, 77.38, 86.96,126.27, 126.87, 127.12, 127.58, 127.74, 127.80,127.87, 128.18, 128.29, 128.40, 128.73, 138.17,143.83.

4.1.11. ((S)-2-O-Benzyl-3-O-tert-butyldiphenyl-silylglyceryl)-2-bromoethyl-methylphosphate (14)

Compound 12 (26.3 g, 62.5 mmol) was reactedas described for compound 13. Yield: 28.0 g(72%). [a ]D20= +8.4° (c=1.3 in CHCl3). 1H-NMR (CDCl3, 200 MHz): d (ppm)=1.06 (s, 9H),3.44 (dt, 3J=6.2 Hz, 4JH,P=0.7 Hz, 2H), 3.71–3.77 (m, 3JH,P=11.2 Hz, 6H), 4.18–4.30 (m, 4H),4.58 (s, 2H), 7.24–7.69 (m, 15H). 13C-NMR(CDCl3, 50 MHz): d (ppm)=19.26, 26.89, 29.42,54.49, 62.66, 66.73, 67.25, 72.24, 78.16, 127.73,127.80, 128.38, 129.84, 133.21, 133.31, 135.62,135.66, 138.17. MS (EI): m/z=623 (M+).

4.1.12. ((R)-2-O-Benzyl-glyceryl)-2-bromoethyl-methyl phosphate (15)

Compound 12 (27.1 g, 43.3 mmol) was refluxedwith 1.2 g pyridinium chloride (10 mmol) in 200ml EtOH for 2 h. Upon addition of 1.1 equiva-lents of NaHCO3 in water, the solvents wereremoved in vacuo. The residue was dissolved inCH2Cl2 and washed with brine. Evaporation ofthe organic solvent yielded 14.5 g (87%) of acolourless oil. [a ]D20= +7.0° (c=0.9 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=2.72 (s,1H), 3.50 (2× dt, 3J=6.1 Hz, 4JH,P=0.8 Hz,2H), 3.64–3.74 (m, 3H), 3.79 (2× d, 3JH,P=11.3Hz, 3H), 4.16–4.36 (m, 4H), 4.62 (d, 2J=11.7 Hz,1H), 4.71 (d, 2J=11.7 Hz, 1H), 7.25–7.40 (m,5H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=29.46, 54.70, 61.09, 66.40, 66.95, 72.22, 77.73,127.86, 127.98, 128.54, 137.88. MS (EI): m/z=383 (M+).


4.1.13. ((S)-2-O-Benzyl-glyceryl)-2-bromoethyl-methyl phosphate (16)

Compound 14 (9.9 g, 19 mmol) was dissolvedin 32 ml 1 M tetrabutylammonium fluoride inTHF and stirred for 1 h. Then, 200 ml ethylacetate were added and the solution was washedwith brine. Column chromatography (diethylether/acetone, 5:1) gave compound 18 as a colour-less oil. Yield: 5.5 g (90%). [a ]D20= −6.9° (c=1.0in CHCl3). The other spectroscopic data wereidentical to compound 15.

4.1.14. Esterification of the sn-1-position —general procedure

Compound 15 or 16 (3.8 g, 10 mmol) wasdissolved in 50 ml CH2Cl2 and 12 mmol car-boxylic acid, 3.1 g DCC (15 mmol) and 250 mgDMAP (2 mmol) were added and stirred for 2 h.After filtration, 50 ml CH2Cl2 were added and theorganic phase was washed with 0.1 M HCl, satu-rated NaHCO3 and brine. The solvent was evapo-rated and the residue purified by columnchromatography (cyclohexane/ethyl acetate 5:1–2:1). Compound 18 was not extensively character-ized but used directly for the next synthetic step.

4.1.15. (3-O-[2-(4-Isobutylphenyl propanoyl)]-(R)-2-O-benzylglyceryl)-2-bromoethyl-methylphosphate (17)

Yield: 91%. [a ]D20= +1.1° (c=1.0 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.88 (d,3J=6.7 Hz, 6H), 1.49 (2× d, 3J=7.2 Hz, 3H),1.82 (m, 1H), 2.43 (d, 3J=7.1 Hz, 2H), 3.45 (m,2H), 3.64–3.84 (m, 2H), 3.74 (2× d, 3JH,P=11.3Hz, 3H), 3.96–4.36 (m, 6H), 4.54 (2× s, 2H),7.05–7.40 (m, 9H). 13C-NMR (CDCl3, 50 MHz):d (ppm)=18.41, 18.45, 22.40, 29.40, 30.15, 45.09,45.13, 54.55, 62.90, 63.02, 66.54, 66.83, 72.25,72.35, 75.21, 75.41, 127.18, 127.23, 127.83, 128.42,129.40, 137.50+137.53, 137.75, 140.61, 174.33.MS (EI): m/z=571 (M+).

4.1.16. (3-O-Palmitoyl-(R)-2-O-benzylglyceryl)-2-romoethyl-methyl phosphate (19)

Yield: 88%. [a ]D20= +0.9° (c=1.3 inCHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.87 (t, 3J=6.5 Hz, 3H), 1.29 (s, 24H), 1.60 (m,2H), 2.31 (t, 3J=7.6 Hz), 3.49 (m, 2H), 3.74 (2×

d, 3JH,P=11.3 Hz, 3H), 3.83 (m, 1H), 4.06–4.35(m, 6H), 4.64 (s, 2H), 7.30–7.40 (m, 5H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=14.14, 22.72,24.93, 29.18, 29.31, 29.44, 29.64, 29.72, 31.95,34.18, 54.68, 62.33, 66.60, 66.87, 72.27, 75.18,127.89, 128.17, 128.48, 137.66, 173.47. MS (EI):m/z=621 (M+).

4.1.17. (3-O-[2-Propyl pentanoyl]-(R)-2-O-benzylglyceryl)-2-bromoethyl-methyl phosphate(20)

Yield: 92%. [a ]D20= +0.6° (c=1.3 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.87 (t,3J=7.3 Hz, 6H), 1.16–1.68 (m, 8H), 2.39 (m,1H), 3.47 (m, 2H), 3.77 (2× d, 3JH,P=11.3 Hz,3H), 3.83 (m, 1H), 4.05–4.38 (m, 6H), 4.65 (s,2H), 7.25–7.45 (m, 5H). 13C-NMR (CDCl3, 50MHz): d (ppm)=13.96, 20.63, 29.39, 34.57, 45.24,54.60, 62.14, 66.70, 66.86, 72.24, 75.44, 127.87,128.25, 128.75, 137.72, 176.01. MS (EI): m/z=509 (M+).

4.1.18. Deprotection and esterification of thesn-2-position — general procedure

Compounds 17–20 (6 mmol) were dissolved in50 ml CH2Cl2 and 750 mg Pd/C (10% m/m) wereadded. The suspension was stirred for 2 h underan atmosphere of hydrogen (1.05 bar). The reac-tion mixture was filtered and removed under re-duced pressure to isolate compound 21. In allother cases, 7.5 mmol respective carboxylic acid,1.85 g DCC (9 mmol) and 250 mg DMAP (2mmol) were directly added and the solution wasstirred for 2 h. Work-up as described for com-pounds 17–20 yielded compounds 22–27 ascolourless, viscous oils. Compound 24 was notextensively characterized but used directly for thenext synthetic step.

4.1.19. (3-O-[2-(4-Isobutylphenyl propanoyl)]-(R)-2-glyceryl)-2-bromoethyl-methyl phosphate (21)

Yield: 90%. 1H-NMR (CDCl3, 200 MHz): d

(ppm)=0.89 (d, 3J=6.6 Hz, 6H), 1.50 (d, 3J=7.2 Hz, 3H), 1.84 (m, 1H), 2.45 (d, 3J=7.0 Hz,2H), 2.62 (s, 1H), 3.52 (t, 3J=6.1 Hz, 2H), 3.73(q, 3J=7.2 Hz, 2H), 3.79 (d, 3JH,P=11.3 Hz, 3H),3.94–4.08 (m, 4H), 4.16 (d, 3J=4.5 Hz, 2H),4.31 (dd, 3J=6.1 Hz, 3J=14.1 Hz, 2H), 7.09 (d,


3J=8.1 Hz, 2H), 7.19 (d, 3J=8.1 Hz, 2H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=18.41, 22.44,29.42 (d, 3JC,P=8.7 Hz), 30.21, 45.01, 45.09, 54.85(d, 2JC,P=7.3 Hz), 64.62+64.73, 67.12+67.16,68.68+68.73 (d, 3JC,P=5.5 Hz), 68.89 (d, 2JC,P=5.5 Hz), 127.18, 129.49, 137.53, 140.82, 174.69.

4.1.20. ((R)-2,3-Di-O-[2-(4-isobutylphenylpropanoyl)]-glyceryl)-2-bromoethyl-methylphosphate (22)

Yield: 77%. [a ]D20= +0.5° (c=1.3 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.89 (d,3J=6.5 Hz, 12H), 1.36–1.53 (m, 6H), 1.84 (m,2H), 2.44 (d, 3J=7.1 Hz, 4H), 3.38–3.55 (m, 2H),3.57–3.67 (4× q, 3J=4.4 Hz, 2H), 3.70–3.78(4× d, 3JH,P=11.3 Hz, 3H), 3.98–4.42 (m, 6H),5.17 (m, 1H), 7.05–7.25 (m, 8H). 13C-NMR(CDCl3, 50 MHz): d (ppm)=18.16, 18.25, 18.34,18.45, 22.22, 22.36, 29.29, 30.12, 44.84, 44.95,45.06, 54.46, 54.57, 61.65, 61.80, 65.22, 65.47,66.80, 66.87, 69.68, 69.73, 127.12, 129.33, 137.22,137.30, 140.59, 173.56, 173.62, 173.89, 173.98. MS(CI, ammonia): m/z=689 ([M+NH3]+).

4.1.21. (3-O-[2-(4-Isobutylphenyl propanoyl)]-(R)-2-O-palmitoylglyceryl)-2-bromoethyl-methylphosphate (23)

Yield: 75%. [a ]D20= +0.3° (c=1.4 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.85(d+ t, 3J=6.4 Hz, 9H), 1.26 (s, 24H), 1.49 (2×d, 3J=7.2 Hz, 3H), 1.57 (m, 2H), 1.82 (m, 1H),2.23 (t, 3J=7.4 Hz, 2H), 2.44 (d, 3J=7.1 Hz,2H), 3.52 (t, 3J=6.2 Hz, 2H), 3.58–3.66 (m, 1H),3.77 (d, 3JH,P=11.3 Hz, 3H), 3.98–4.40 (m, 6H),5.20 (m, 1H), 7.09 (d, 3J=8.1 Hz, 2H), 7.19 (d,3J=8.1 Hz, 2H). 13C-NMR (CDCl3, 50 MHz): d

(ppm)=14.12, 18.33, 22.42, 22.72, 24.88, 29.16,29.32, 29.46, 29.73, 30.19, 31.97, 34.09, 45.05,45.11, 54.44, 61.80, 61.92, 65.61, 66.96, 69.26,69.40, 127.20, 129.40, 137.38, 140.73, 172.72,174.11. MS (CI, ammonia): m/z=739 ([M+NH3]+).

4.1.22. ((R)-2-O-[2-(4-Isobutylphenyl propanoyl)]-3-O-palmitoylglyceryl)-2-bromoethyl-methylphosphate (25)

Yield: 73%. [a ]D20= +2.7° (c=1.1 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.87

(d+ t, 3J=6.6 Hz, 9H), 1.25 (s, 24H), 1.49 (2×d, 3J=7.1 Hz, 3H), 1.54 (m, 2H), 1.84 (m, 1H),2.15+2.28 (2× t, 3J=7.8 Hz, 2H), 2.43 (d,3J=7.1 Hz, 2H), 3.44+3.51 (2× dt, 3J=7.1 Hz,4JH,P=0.8 Hz, 2H), 3.66 (m, 1H), 3.77 (d, 3JH,P=11.3 Hz, 3H), 4.02–4.40 (m, 6H), 5.21 (m, 1H),7.10 (d, 3J=7.6 Hz, 2H), 7.20 (d, 3J=7.6 Hz,2H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=14.16, 18.43, 22.42, 22.73, 24.77, 24.87, 29.17,29.31, 29.46, 29.73, 30.21, 31.95, 33.88, 34.05,44.84, 44.98, 45.08, 54.70, 61.52, 61.43, 65.45,65.83, 66.90, 67.10, 69.71, 69.78, 127.18, 128.18,137.21, 137.26, 140.70, 173.16, 173.82. MS (CI,ammonia): m/z=739 ([M+NH3]+).

4.1.23. (3-O-Palmitoyl-(R)-2-O-[2-propylpentanoyl])-2-bromoethyl-methyl phosphate (26)

Yield: 76%. [a ]D20= +2.3° (c=1.3 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.85 (m,9H), 1.10–.32 (m, 28H), 1.38 (m, 2H), 1.44–.63(m, 4H), 2.25 (t, 3J=7.5 Hz, 2H), 2.35 (m, 1H),3.48 (t, 3J=6.1 Hz, 2H), 3.75 (2× d, 3JH,P=11.2Hz, 3H), 4.05–.34 (m, 6H), 5.22 (m, 1H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=13.88, 14.01,20.45, 20.49, 22.62, 24.78, 29.08, 29.13, 29.19,29.34, 29.53, 29.61, 31.86, 33.95, 34.45, 34.50,45.06, 45.16, 54.58, 61.69, 65.65, 65.71, 66.91,69.17, 173.00, 175.33. MS (CI, ammonia): m/z=676 ([M+NH3]+).

4.1.24. ((R)-2,3-Di-O-[2-propyl pentanoyl]-glyceryl)-2-bromoethyl-methyl phosphate (27)

Yield: 79%. [a ]D20= +1.1° (c=1.3 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.80 (t,3J=7.1 Hz, 12H), 1.08–.62 (m, 16H), 2.21–.38(m, 2H), 3.45 (t, 3J=6.1 Hz, 2H), 3.71 (d, 3JH,P=11.3 Hz, 3H), 3.99–.35 (m, 6H), 5.17 (m, 1H).13C-NMR (CDCl3, 50 MHz): d (ppm)=13.81,20.42, 29.16, 34.32, 34.36, 44.96, 45.06, 54.43,61.47, 65.54, 65.60, 66.84, 69.25, 175.17, 175.57.MS (CI, ammonia): m/z=564 ([M+NH3]+).

4.1.25. Synthesis of the choline moiety — generalprocedure

Compound 21 (4 mmol) or compounds 22–27in 10 ml toluene and 20 ml freshly condensed(−50°C) trimethylamine were heated for 2 daysat 50°C in an autoclave. After cooling to −20°C,


the solution was poured into a mixture of 200 mlmethanol, 100 ml of 1 M HCl and 100 ml water,and extracted with CH2Cl2. The organic phasewas evaporated. Column chromatography(methanol) gave the products as waxy solids.

4.1.26. 1-O-[2-(4-Isobutylphenyl propanoyl)]-2-lyso-sn-glycero-3-phosphocholine (28)

Yield: 71%. [a ]D20= +3.3° (c=1.4 in CHCl3).1H-NMR (CDCl3, 600 MHz): d (ppm)=0.87 (d,3J=6.6 Hz, 3H), 1.43 (d, 3J=7.0 Hz, 3H), 1.81(sept., 3J=6.7 Hz, 1H), 2.41 (s, 3J=7.1 Hz, 2H),3.22 (s, 9H), 3.54 (m, 0.5 H), 3.65–3.72 (m, 3H),3.78 (m, 0.5H), 3.89 (m, 2H), 3.97+4.15 (2× m,2H), 4.25 (m, 2H), 7.06 (d, 3J=7.5 Hz, 2H), 7.16(d, 3J=7.5 Hz, 2H). 13C-NMR (CDCl3, 150MHz): d (ppm)=18.80, 18.97, 22.34, 30.12, 44.83,44.93, 53.97, 59.31, 65.51, 65.68, 65.88, 66.94,68.66, 127.20, 129.27, 137.55, 137.70, 140.45,174.75. MS (CI, ammonia): m/z=416([M�C2H6]+). C21H36NO7P·1.2 H2O (467.11):calc.: C, 54.00; H, 8.29; N, 3.05; found: C, 53.77;H, 8.49; N, 3.05.

4.1.27. 1,2-Di-O-[2-(4-Isobutylphenyl propanoyl)]-sn-glycero-3-phosphocholine (29)

Yield: 78%. [a ]D20= +8.0° (c=1.0 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.86 (d,3J=6.7 Hz, 12H), 1.27–1.43 (m, 6H), 1.80 (sept.,3J=6.7 Hz, 2H), 2.40 (d, 3J=7.1 Hz, 4H), 3.19(s, 9H), 3.53–3.65 (2× q, 3J=7.0 Hz, 2H), 3.63(m, 2H), 3.74–3.95 (m, 2H), 3.95–4.30 (m, 3H),4.30–4.50 (m, 1H), 5.04–5.24 (m, 1H), 6.98 (m,8H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=18.27, 18.38, 18.53, 18.71, 22.40, 30.13, 44.78,44.87, 44.95, 54.24, 59.34, 63.12, 63.36, 66.13,70.94, 127.21, 127.31, 129.29, 137.46, 137.57,140.52, 174.01, 174.35. MS (CI, ammonia): m/z=634 (M+). C34H52NO8P·1.5 H2O (660.77): calc.:C, 61.63; H, 8.40; N, 2.11; found: C, 61.39; H,8.49; N, 1.92.

4.1.28. 1-O-[2-(4-Isobutylphenyl propanoyl)]-2-O-palmitoyl-sn-glycero-3-phosphocholine (30)

Yield: 77%. [a ]D20= +8.3° (c=1.0 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.85 (m,9H), 1.24 (s, 24H), 1.46 (d, 3J=6.8 Hz, 3H), 1.50(m, 2H), 1.81 (m, 1H), 2.13–2.23 (m, 2H), 2.41 (d,

3J=7.1 Hz, 2H), 3.27 (s, 9H), 3.60–3.95 (m, 5H),3.99–4.47 (m, 4H), 5.28 (m, 1H), 7.04 (d, 3J=7.9Hz, 2H), 7.14 (d, 3J=7.9 Hz, 2H). 13C-NMR(CDCl3, 50 MHz): d (ppm)=14.10, 18.56, 22.42,22.71, 24.95, 29.26, 29.39, 29.62, 29.75, 30.19,31.95, 34.28, 45.04, 45.09, 54.35, 59.33, 63.28,63.49, 66.39, 70.54, 127.21, 129.33, 137.50, 137.57,140.55, 173.04, 174.47. MS (CI, ammonia): m/z=684 (M+). C37H66NO8P·H2O (701.91): calc.; C,63.31; H, 9.76; N, 2.00; found: C, 63.15; H, 10.15;N, 1.99.

4.1.29. 3-O-[2-(4-Isobutylphenyl propanoyl)]-2-O-palmitoyl-sn-glycero-1-phosphocholine (31)

Yield: 73%. [a ]D20= −7.9° (c=1.2 in CHCl3).1H-NMR (CDCl3, 600 MHz): d (ppm)=0.88(d+ t, 3J=8.5 Hz, 9H), 1.26 (s, 24H), 1.45 (d,3J=6.9 Hz, 3H), 1.52 (m, 2H), 1.83 (sept., 3J=6.6 Hz, 1H), 2.20 (m, 2H), 2.42 (s, 3J=6.9 Hz,2H), 3.29 (s, 9H), 3.63–3.71 (m, 1H), 3.74 (m,2H), 3.84–3.95 (m, 2H), 4.13 (m, 1H), 4.26 (m,2H), 4.36+4.42 (2× d, 2J=11.2 Hz, 1H), 5.17(m, 1H), 7.06 (d, 3J=7.5 Hz, 2H), 7.15 (d, 3J=7.5 Hz, 2H). 13C-NMR (CDCl3, 150 MHz): d

(ppm)=14.02, 18.32, 18.42, 22.29, 22.59, 24.78,25.19, 29.11, 29.27, 29.30, 29.39, 29.50, 29.53,29.57, 29.63, 30.08, 31.83, 34.09, 34.11, 44.85,44.93, 54.20, 59.19, 59. 23, 63.21, 63.25, 63.25,63.35, 66.14, 66.18, 70.22, 70.28, 70.33, 127.10,127.11, 129.18, 129.24, 137.31, 137.38, 140.43,172.92, 172.99, 174.36. MS (CI, ammonia): m/z=684 (M+). C37H66NO8P·H2O (701.91): calc.: C,63.31; H, 9.76; N, 2.00; found: C, 62.99; H, 10.27;N, 2.40.

4.1.30. 1-O-Palmitoyl-2-O-[2-(4-isobutylphenylpropanoyl)]-sn-glycero-3-phosphocholine (32)

Yield: 79%. [a ]D20= +9.1° (c=1.0 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.88(d+ t, 3J=6.6 Hz, 9H), 1.20 (s, 24H), 1.34–1.60(m, 5H), 1.80 (m, 1H), 2.12 (m, 2H), 2.40 (d,3J=7.2 Hz, 2H), 3.25+3.29 (2× s, 9H),3.60–3.98 (m, 5H), 4.01–4.44 (m, 4H), 5.27 (m,1H), 7.04 (d, 3J=8.0 Hz, 2H), 7.14 (d, 3J=8.0Hz, 2H). 13C-NMR (CDCl3, 50 MHz): d

(ppm)=14.08, 18.49, 18.83, 22.40, 22.73, 24.78,24.89, 29.21, 29.35, 29.57, 29.72, 30.17, 31.94),33.94, 34.14, 44.98, 45.24, 45.07, 54.34, 59.31,


62.94, 63.40, 66.30, 71.05,127.18, 127.32, 129.25,129.31, 137.50, 137.62, 140.44, 140.55, 173.35,173.44, 174.09, 174.16. MS (CI, ammonia): m/z=684 (M+). C37H66NO8P·2.5 H2O (728.94): calc.: C,60.97; H, 9.82; N, 1.92; found: C, 60.99; H, 9.99;N, 1.85.

4.1.31. 1-O-Palmitoyl-2-O-[2-propylpentanoyl]-sn-glycero-3-phosphocholine (33)

Yield: 74%. [a ]D20= +0.7° (c=1.0 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.85 (t,3J=7.1 Hz, 9H), 1.22 (m, 28H), 1.28–1.64 (m,6H), 2.18–2.40 (m, 3H), 3.33 (s, 9H), 3.68–3.84(m, 2H), 3.90–4.10 (m, 3H), 4.20–4.35 (m, 3H),5.17 (m, 1H). 13C-NMR (CDCl3, 50 MHz): d

(ppm)=14.03, 20.54, 22.65, 24.87, 24.97, 29.15,29.22, 29.28, 29.31, 29.43, 29.51, 29.57, 29.66,31.90, 34.12, 34.16, 34.45, 45.06, 45.20, 54.26,59.40, 63.23, 64.96, 66.20, 69.00, 175.75, 176.42.MS (CI, ammonia): m/z=621 (M+). C32H64NO8-P·2.5 H2O (666.87): calc.: C, 57.33; H, 10.43; N,2.09; found: C, 56.99; H, 10.41; N, 2.26.

4.1.32. 1,2-Di-O-[2-propylpentanoyl]-sn-glycero-3-phosphocholine (34)

Yield: 75%. [a ]D20= +6.2° (c=1.0 in CHCl3).1H-NMR (CDCl3, 200 MHz): d (ppm)=0.83 (t,3J=7.0 Hz, 12H), 1.10–1.62 (m, 16H), 2.29 (m,2H), 3.36 (s, 9H), 3.80 (m, 2H), 3.85–4.15 (m, 2H),4.28 (m, 3H), 4.46 (m, 1H), 5.15 (m, 1H). 13C-NMR (CDCl3, 50 MHz): d (ppm)=14.00, 20.53,34.33, 34.44, 45.09, 45.19, 54.41, 59.32, 63.16,66.46, 70.65, 175.69, 176.20. MS (CI, ammonia):m/z=510 (M+). C24H48NO8P·0.5 H2O (518.61):calc.: C, 55.20; H, 9.53; N, 2.68; found: C, 55.01;H, 9.91; N, 2.60.4.2. Physicochemical measurements

4.2.1. Surface tensionThe surface tension of the compounds was de-

termined using a computer-assisted Nima Type622 Wilhelmy film balance. The Teflon trough(200×600 mm2) was rinsed with ethanol, distilledwater and bidistilled water before use. The mono-layers were formed by spreading 50 ml chloroformsolution containing 100 nmol phospholipids ontothe surface of the water. After a period of 10 min,in order for the organic solvent to evaporate, the

monolayers were continuously compressed by themotor-driven Teflon barriers at 20 A, 2/molecule/min. Every experiment was performed three times.

4.2.2. Laser light scatteringThe size of the aggregates was determined by

dynamic laser light scattering (scattering angle,90°) using a ZetaMasterS Autosizer 4700 (Mal-vern Instruments, operating with a He–Ne laser at633 nm) and fitting software. The size of theaggregates is expressed as the mean diameter ofthree determinations. The solutions were preparedby dissolving 100 mg phospholipid in 100 mlbidistilled water and sonicating for 2 h at 40°C.

4.3. Enzymatic studies

4.3.1. HPLC analysisThe HPLC system consisted of a Shimadzu LC

6A solvent delivery module, a Shimadzu SPD 6AUV detector operated at 220 nm, and a ShimadzuC-R6A integrator. The chromatographic separa-tion was obtained on a LiChrospher Si 60 column(250×4.6 mm2, 5 mm) (Merck, Germany). Themobile phase consisted of acetronitrile/methanol/phosphoric acid (70:30:0.5, v/v/v), and the flowrate was 1.5 ml/min. Phospholipid and lyso-phos-pholipid concentrations were calculated from cali-bration curves obtained by analysis of the purecompounds under identical chromatographicconditions.

4.3.2. KineticsEnzymatic hydrolysis was studied at 3790.5°C

in 100 mM tris(hydroxymethyl)aminomethane(TRIS) buffer, pH 8.0, containing 10 mM CaCl2and 100 U porcine pancreatic phospholipase A2

(assayed by Sigma using soybean L-a-phos-phatidylcholine as substrate). Two micromoles ofthe compounds were incubated in 4 ml buffer andthe mixtures were vigorously stirred during thekinetic run. Aliquots were sampled at selected timeintervals and extracted with methanol/chloroform/2 N HCl (2:2:2, v/v/v) according to Bligh andDyer (1959). The organic phase was dried under astream of nitrogen. The residue was dissolved inn-hexane/2-propanol (3:1, v/v) and analyzed byHPLC.


Acknowledgements

The gift of sn-glycero-3-phosphocholine byNattermann Phospholipid GmbH and the finan-cial support by the Fonds der Chemischen Indus-trie are gratefully acknowledged. We also thankDr. Claus Rudiger for assistance with the filmbalance measurements and Dr. Jorg Breitkreutzfor assistance with laser light scatteringexperiments.

References

Baggett, N., Stribblehill, P., 1977. Asymmetric reduction ofketones by using complexes of lithium tetrahydridoalumi-nate(III) with 1,4:3,6-dianhydro-D-mannitol and 1,3:4,6-di-O-benzylidene-D-mannitol. J. Chem. Soc. Perkin Trans. 1,1123–1126.

Bligh, E., Dyer, W., 1959. A rapid method of total lipidextraction and purification. Can. J. Biochem. Physiol. 37,911–917.

Bredlow, A., Galla, H., Bergelson, L., 1992. Influence offluorescent lipid probes on the packing of their environ-ment. A monolayer study. Chem. Phys. Lipids 62, 293–301.

Deverre, J.R., Loiseau, P., Puisieux, F., Gayral, P., Letourneux,Y., Couvreur, P., Benoit, J.P., 1992. Synthesis of the orallymacrofilaricidal and stable glycerolipidic prodrug ofmelphalan, 1,3-dipalmitoyl-2-(4%(bis(2%-chlorethyl)amino)-phenylalaninoyl)glycerol. Arzneim.-Forsch./Drug Res. 42,1153–1156.

Eibl, H., 1984. Phospholipide als funktionelle Bausteine biolo-gischer Membranen. Angew. Chem. 96, 247–314.

Gallez, B., Demeure, R., Debuyst, R., Leonard, D., Dejehet, F.,Dumont, P., 1992. Evaluation of nonionic nitroxyl lipids aspotential organ-specific contrast agents for magnetic reso-nance imaging. Magn. Reson. Imaging 10, 445–455.

Garzon-Aburbeh, A., Poupaert, J.H., Claesen, M., Dumont, P.,Atassi, G., 1983. 1,3-Dipalmitoylglycerol ester of chloram-bucil as a lymphotropic, orally administrable antineoplasticagent. J. Med. Chem. 26, 1200–1203.

Garzon-Aburbeh, A., Poupaert, J.H., Claesen, M., Dumont, P.,1986. A lymphotropic prodrug of L-DOPA: synthesis, phar-macological properties and pharmacokinetic behaviorof 1,3-dihexadecanoyl-2-[(S)-2-amino-3-(3,4-dihydroxy-phenyl)-propanoyl]propane1,2,3-triol. J. Med. Chem. 29,687–691.

Hanessian, S., Lavallee, P., 1975. The preparation and syntheticutility of tert-butyldiphenylsilyl ethers. Can. J. Chem. 53,2975–2977.

Hermetter, A., Stutz, H., Franzmair, R., Paltauf, F., 1989.1-O-Trityl-sn-glycerophosphocholine: a new intermediatefor the facile preparation of mixed-acid 1,2-diacylglyc-erophosphocholines. Chem. Phys. Lipids 50, 57–62.

Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van denBosch, H., Richman, D.D., 1990. Synthesis and antiretrovi-ral activity of phospholipid analogs of azidothymidineand other antiviral nucleosides. J. Biol. Chem. 265, 6112–6117.

Hostetler, K.Y., Parker, S., Sridhar, C.N., Martin, M.J., Li,J.-L., Stuhmiller, L.M., van Wijk, G.M.T., van den Bosch,H., Gardner, M.F., Aldern, K.A., Richman, D.D., 1993.Acyclovir diphosphate dimyristoylglycerol: a phospholipidprodrug with activity against acyclovir-resistant herpes sim-plex virus. Proc. Natl. Acad. Sci. USA 90, 11835–11839.

Hostetler, K.Y., Richman, D.D., Forssen, E.A., Selk, L.,Basava, R., Gardner, M.F., Parker, S., Basava, C., 1994.Phospholipid prodrug inhibitors of the HIV protease. An-tiviral activity and pharmacokinetics in rats. Biochem.Pharmacol. 48, 1399–1404.

Jacob, J.N., Hesse, G.W., Shashoua, V.E., 1990. Synthesis,brain uptake, and pharmacological properties of a glyceryllipid containing GABA and the GABA-T inhibitor g-vinyl-GABA. J. Med. Chem. 33, 733–736.

Johnstone, R., Rose, M., 1979. A rapid, simple, and mildprocedure for alkylation of phenols, alcohols, amines andacids. Tetrahedron 35, 2169–2173.

Lambert, D.M., Neuvens, L., Mergen, F., Gallez, B., Ghysel-Burton, J., Maloteaux, J.-M., Dumont, P., 1993. 1,3-Diacy-laminopropan-2-ols and corresponding 2-acyl derivatives asdrug carriers: unexpected pharmacological properties. J.Pharm. Pharmacol. 45, 186–191.

Martin, S., Josey, J., Wong, Y., Dean, W., 1994. Generalmethod for the synthesis of phospholipid derivatives of1,2-O-diacyl-sn-glycerols. J. Org. Chem. 59, 4805–4820.

Matsushita, T., Ryu, E.K., Hong, C.I., MacCoss, M., 1981.Phospholipid derivatives of nucleoside analogs as prodrugswith enhanced catabolic stability. Cancer Res. 41, 2707–2713.

Mergen, F., Lambert, D.M., Goncalves Saraiva, J.C.,Poupaert, J.H., Dumont, P., 1991. Antiepileptic acitivity of1,3-dihexadecanoylamino-2-valproyl-propan-2-ol, a pro-drug of valproic acid endowed with a tropism for the centralnervous system. J. Pharm. Pharmacol. 43, 815–816.

Paris, G.Y., Garmaise, D.L., Cimon, D.G., Swett, L., Carter,G.W., Young, P., 1979. Glycerides as Prodrugs. 1. Synthesisand antiinflammatory activity of 1,3-bis(alkanoyl)-2-(O-acetylsalicyloyl)glycerides (aspirin glycerides). J. Med.Chem. 22, 683–687.

Paris, G.Y., Garmaise, D.L., Cimon, D.G., Swett, L., Carter,G.W., Young, P., 1980. Glycerides as Prodrugs. 3. Synthesisand antiinflammatory activity of [1-(p-chlorbenzoyl)-5-methoxy-2-methylindole-3-acetyl]glycerides (indomethacinglycerides). J. Med. Chem. 23, 9–12.

Paris, G.Y., Cimon, D.G., Garmaise, D.L., Swett, L., Carter,G.W., Young, P.R., 1982. Glycerides as Prodrugs. 4. Syn-thesis and antiinflammatory activity of 1,3-dialkanoyl-2-arylalkanoylglycerides. Eur. J. Med. Chem. 17, 193–195.

Peters, U., Bankowa, W., Wetzel, P., 1987. Platelet activatingfactor synthetic studies. Tetrahedron 43, 3803–3816.


Ryu, E.K., Ross, R.J., Matusushita, T., MacCoss, M., Hong,C.I., West, C.R., 1982. Phospholipid-nucleoside conjugates.3. Synthesis and preliminary biological evaluation of 1-b-D-arabinofuranosylcytosine 5%-monophosphate-L-1,2-dipalmitin and selected of 1-b-D-arabinofuranosylcytosine5%-diphosphate-L-1,2-diacylglycerols. J. Med. Chem. 25,1322–1329.

Scriba, G.K.E., Lambert, D.M., Poupaert, J.H., 1995.Bioavailability of phenytoin following oral administration ofphenytoin-lipid conjugates to rats. J. Pharm. Pharmacol. 47,945–948.

Turcotte, J.G., Srivastava, S.P., Meresak, W.A., Rizkalla, B.A.,Louzon, F., Wunz, T.P., 1980. Cytotoxic liponucleotideanalogs. I. Chemical synthesis of CDP-diacylglycerol analogscontaining the cytosine arabinoside moiety. Biochim. Bio-phys. Acta 619, 604–618.

van Wijk, G.M.T., Hostetler, K.Y., van den Bosch, H., 1992.Antiviral nucleoside diphosphate diglycerides: improvedsynthesis and faciliated purification. J. Lipid Res. 33,1211–1219.

van Wijk, G.M.T., Hostetler, K.Y., Kroneman, E., Richman,D.D., Sridhar, C.N., Kumar, R., van den Bosch, H., 1994.Synthesis and antiviral activity of 3%-azido-3%-de-oxythymidine triphosphate distearoylglycerol: a novelphospholipid conjugate of the anti-HIV agent AZT. Chem.Phys. Lipids 70, 213–222.

Weichert, J.P., Longino, M.A., Bakan, D.A., Spigarelli, M.G.,Chou, T., Schwendner, S.W., Counsell, R.E., 1995. Polyio-dinated triglyceride analogs as potential computed tomog-raphy imaging agents for the liver. J. Med. Chem. 38,636–646.

.

Drug–phospholipid conjugates as potential prodrugs: synthesis, characterization, and degradation...

Documents

Transcript of Drug–phospholipid conjugates as potential prodrugs: synthesis, characterization, and degradation...