Synthesis of isosteric phosphono analogs of biologically active alkylphosphocholines

Article abstract of DOI:10.1007/PL00010153

We describe a high yield reaction sequence for the conversion of long chain alcohols into 3-(trimethylammonio)propylphosphonates. The products are phospholipase D stable isosteric phosphono analogs of the respective alkylphosphocholines. The key step is t

Full text of DOI:10.1007/PL00010153

Monatshefte fuÈr Chemie 129, 173±185 (1998)
Synthesis of Isosteric Phosphono Analogs
of Biologically Active Alkylphosphocholines
Ã
È
Jorg T. Kley, Clemens Unger, and Ulrich Massing
Tumor Biology Center, Department of Medical Oncology, Division of Medical Research, D-79106
Freiburg, Germany
Summary. We describe a high yield reaction sequence for the conversion of long chain alcohols
into 3-(trimethylammonio)propylphosphonates. The products are phospholipase D stable isosteric
phosphono analogs of the respective alkylphosphocholines. The key step is the esteri®cation of 3-
chloropropylphosphonic acid with the respective long chain alcohols using DCC/DMAP.
Deprotection and various acylations of 2-hydroxy and 2-amino groups following the introduction
of the headgroup are described.
Keywords. Phosphonolipids, synthesis of; Alkylphosphocholines; Phospholipase D; Michaelis-
Becker reaction.
Synthese isosterer Phosphonoanaloga biologisch aktiver Alkylphosphocholine
Zusammenfassung. Wir beschreiben eine in sehr guten Ausbeuten verlaufende Reaktionsfolge zur
È
UberfuÈhrung langkettiger Alkohole in 3-(Trimethylammonium)propanphosphonate. Die Produkte
sind Phospholipase D-stabile isostere Phosphonoanaloga der entsprechenden Alkylphosphocholine.
È
SchluÈsselschritt ist die Veresterung von 3-Chlorpropanphosphonsaure mit den entsprechenden
langkettigen Alkoholen unter Verwendung von DCC/DMAP. EntschuÈtzung und verschiedene
Acylierungen von 2-Hydroxy- und 2-Amino-Gruppen nach EinfuÈhrung der Kopfgruppe werden
beschrieben.
Introduction
In most mammalian tissues, Phospholipase D (PLD) provides a major route for the
hydrolysis of phosphatidylcholine [1]. Furthermore, Ries has demonstrated that the
antineoplastic phospholipid hexadecylphosphocholine and the naturally occuring
dipalmitoyl phosphatidylcholine are hydrolyzed by PLD at a similar rate [2]. Thus,
it must be assumed that synthetic, pharmacologically interesting phospholipids
bearing a phosphocholine headgroup will be biodegraded by PLD and therefore
have a limited half-life, and toxic cleavage products may be formed. To avoid this
biodegradation by PLD and hence increase the half-life of the pharmacologically
interesting phospholipids in mammalian tissues, we prepared phosphono analogs of
antineoplastic n-alkylphosphocholines, 1-O-phosphocholine-2-N-acyl-octadecanes
(which are strong competitive inhibitors of 14 kDa phospholipases A2), and 1-O-

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J. T. Kley et al.
phosphocholine-2-O-acyl-octadecanes bearing the isosteric 3-(trimethylammonio)-
propylphosphonoyl headgroup without a cleavage point for PLD.
Results and Discussion
Synthesis of 3-chloropropylphosphonic acid (2)
The phosphonic acid 2 is usually prepared by acidic hydrolysis of diethyl-3-
chloropropylphosphonate which can be obtained by Michaelis-Arbuzov reaction of
1-bromo-3-chloropropane with triethylphosphite [3]. We investigated an alternative
route via dimethyl-3-chloropropylphosphonate (1) which was prepared by
Michaelis-Becker reaction1 from potassium dimethylphosphite and 1-bromo-3-
chloropropane in 67% yield (Scheme 1). Applying the Michaelis-Becker reaction,
neither high reaction temperatures nor distillation of the product were necessary.
We found that potassium dimethylphosphite can be conveniently generated in situ
by deprotonation of dimethylphosphite with the sterically hindered base potassium-
tert-butylate in THF. To our knowledge, the use of potassium-tert-butylate in the
Michaelis-Becker reaction has not yet been described previously. In the Michaelis-
Becker reaction, dialkylphosphites usually are deprotonated with metallic sodium,
sodium hydride, or sodium methoxide (or ethoxide), but it has been supposed that
primary alcoholates compete in the nucleophilic displacement of the halogen, thus
diminishing the yield [6]. 3-Chloropropylphosphonic acid (2) was obtained from 1
by acidic hydrolysis in fuming hydrochloric acid and subsequent crystallization
from chloroform in 94% yield.
Scheme 1. Preparation of 3-chloropropylphosphonic acid (2)
via Michaelis-Becker-reaction
ÐÐÐÐ
1
The Michaelis-Becker reaction, involving alkylation of (usually deprotonated) dialkyl phosphites,
generally has been less used than the Michaelis-Arbuzov reaction, although it is convenient and
dialkyl phosphites are commercially available [4, 5]. To our knowledge this is its first application to
phosphonolipid synthesis.

Synthesis of Analogs of Alkylphosphocholines
175
Synthesis of 1-hydroxy-2-O-benzyl-octadecane (5), 1-hydroxy-2-N-
phthalimido-octadecane (6) and 1-hydroxy-2-N-formyl-octadecane (8)
The alcohols 5, 6, and 8 needed for the esteri®cation with 3-chloropropylphos-
phonic acid (2) to obtain the respective phosphonates were synthesized from racemic
1,2-octadecanediol (see Scheme 2). 5 and 6 were prepared as described elsewhere
[7]. Brie¯y, the primary hydroxy group of 1,2-octadecanediol was selectively
protected as a trityl ether. The secondary hydroxy group of the trityl ether was then
either protected as benzyl ether or substituted with phthalimide under complete
inversion of con®guration by the Mitsunobu reaction. Acidic cleavage of the trityl
ethers lead to 5 and 6. Starting from enantiomerically pure 1,2-octadecanediol, this
reaction sequence also allows the preparation of pure enantiomers of 5 and 6 [7].
Compound 8 was prepared from 6 via 1-hydroxy-2-amino-octadecene (7).
Dephthalimidation of 6 was achieved in 72% yield according to Osby's method by
reduction with sodium borohydride and subsequent acidic cleavage of the resulting
hydroxymethylbenzoylamide [8]. 7 was selectively N-formylated using formic acid
and DCC as condensing agent [9]. Complete removal of the dicyclohexyl urea
formed was not necessary for the following phosphonylation step.
Scheme 2. Preparation of the alcohols 5, 6, and 8 from 1,2-octadecanediol
Formation of the 3-chloropropylphosphonoyl headgroup. Synthesis
of the phosphonates 9, 10, 11, 12, and 13
Phosphonolipids bearing the 2-(trimethylammonio)ethylphosphonoyl headgroup have been prepared
since the sixties, whereas the synthesis of phosphonolipids with a 3-(trimethylammonio)propyl-
phosphonoyl headgroup has not been described before 1991 (Turcotte et al. and Ries et al. [3, 10,
11]). The methods described by these authors include the esteri®cation of the corresponding long
chain alcohols with 3-chloro(bromo)-propylphosphonic acid monochloride which ®rst has to be
generated from the corresponding phosphonic acid. None of the methods described was satisfactory

176
J. T. Kley et al.
for the phosphonylation of our alcohols, because the overall yield of these strategies was described to
be lower than (partly far below) 50%.
During our search for a simple and generally applicable method to introduce the 3-
(trimethylammonio)propylphosphonoyl headgroup in higher yields, we tested the
widely used condensing agent DCC to carry out the esteri®cation in one step from
the alcohol (5, 6, 8 or the commercially available hexadecanol or erucanol) and 3-
chloro-propylphosphonic acid (2) (Scheme 3). Using DCC as condensing agent and
THF as solvent, we observed quantitative turnover of the alcohols. Interestingly, in
the case of the simple alcohols hexadecanol and erucanol (without side chain in the
2-position) the overall yields are the lowest (57% or 61%, respectively). Higher
yields were achieved with alcohols 5, 6, or 8 bearing a formamido, phthalimido, or
benzyloxy group in the 2-position. The crude phosphonic acid esters were treated
with lithium bromide in butanone (Finkelstein reaction) to exchange the terminal
chloride against bromide and then reacted with trimethylamine (without previous
isolation of the intermediates) affording the respective 3-(trimethylammonio)-
propylphosphonates. The complete reaction sequence (outlined in Scheme 3) gave
overall yields ranging from 57% to 90%.
Scheme 3. Preparation of the 3-(trimethylammonio)propylphosphonates 9±13

Synthesis of Analogs of Alkylphosphocholines
177
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-hydroxy-octadecane (11)
and 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-amino-octadecane (12)
The O-benzyl-protected hydroxyl group in 11 was deprotected by hydrogenolysis
in THF/1N HCl with palladium on activated charcoal as catalyst in quantitative
yield. Deprotection of the N-phthalimide-protected amino group in 12 was
achieved in high yields according to the method of Osby by reduction of the
phthalimide with sodium borohydride in 2-propanol/water with the addition of
toluene followed by acidic cleavage of the arising 2-hydroxymethylbenzoylamide
which in this case was achieved by simply adding 6 N HCl to the solution and
heating to 60ꢀC [7, 8].
Scheme 4. Formation and acylation of 1-O-(3-(trimethylammonio)propylphosphonoyl)-
2-hydroxy-octadecane (14)
Acylation of 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-hydroxy-
octadecane (11) and 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-amino-
octadecane (12) with various acid chlorides, imidazolides, and methyl
chloroformate
To minimize the total number of reaction steps, acylation of the 2-hydroxy or 2-
amino group in 11 and 12 with different acyl groups was performed as the last
reaction step (Schemes 4 and 5). Only in the case of formamide 13 the acylation
was carried out before the headgroup was introduced. This procedure was
necessary because the separation of the acylation product 1-O-(3-(trimethylam-
monio)propylphosphonoyl)-2-N-formyl-octadecane (13) from the respective

178
J. T. Kley et al.
Scheme 5. Formation and acylation of 1-O-(3-(trimethylammonio)propylphosphonoyl)-
2-amino-octadecane (15)
hydroxy compound 12 is dif®cult due to very similar Rf values. All acylations were
carried out in a 0.5 mmol scale by stirring the educts in chloroform at room
temperature. The reaction times ranged from 1 h to 3 days depending on the type of
acylation. According to earlier results, puri®cation of phospholipids on silica gel
causes a loss of substance which is not neglectible when small quantities are to be
puri®ed. In our case (0.5 mmol ˆ about 300 mg phospholipid, 10 g silica gel) the
recovery is about 75±80%.
Acylation of 11 and 12 with acid chlorides in the presence of triethylamine or
DMAP gave good yields only in the case of chloroacetamide 212 and carbamate 23.
In the case of the esters 16 and 17, best yields were obtained with acid chlorides
and without addition of any base. For the amides 18, 19, 20, and 22, the
corresponding acylimidazoles proved to be the most suitable acylating agents.
ÐÐÐÐ
2
Although 21 was formed quantitatively (TLC control), it could be isolated in only 29% yield
after purification on silica due to rapid decomposition, probably nucleophilic displacement of the
chloride. The isolated product is not stable at room temperature over a longer period of time and
soon becomes reddish.

Synthesis of Analogs of Alkylphosphocholines
179
To prepare the ¯uoroacetamide 22 we developed a new method that allows the use
of the non-volatile sodium ¯uoroacetate as acylating agent. N-Fluoro-
acetylimidazole was formed in situ from N,N0-carbonyldiimidazole and ¯uoro-
acetic acid which was liberated from the sodium salt by perchloric acid (70%).
Quantitative acylation was achieved when the surplus of carbonyldiimidazole
added was at least equimolar to the amount of water contained in the perchloric
acid.
Experimental
All chemicals were of reagent grade and used without further puri®cation. THF and chloroform
(ethanol free, stabilized with 60 ppm 2-methyl-2-butene) used for the acylations were dried over
molecular sieve (0.4 nm). Removal of solvents was carried out in a rotatory evaporator under reduced
pressure. Residual water, if not otherwise speci®ed, was removed by azeotropic distillation with
ethyl acetate. Solvent mixtures for extractions and chromatography are given in volume ratios,
`solvent X' thereby stands for chloroform/methanol/ammonia (25% aqueous solution) 60:40:X. All
chromatographic separations were performed in glass columns (diameter 2 cm, fraction size 10±
20 ml). Silica gel (Kieselgel 60, 0.063±0.100 mm) and TLC plates were obtained from Merck
1
(Darmstadt, FRG). H NMR spectra (ꢀ in ppm) were recorded with a Bruker AC 250 spectrometer
(250 MHz, TMS ˆ 0 ppm) or a Bruker AM 400 spectrometer (400 MHz, TMS ˆ 0 ppm). Infrared
spectra were measured using a Perkin Elmer 16 PC FT-IR spectrometer. Peaks are given in cmꢁ1 and
labelled `ss' (very strong) `s' (strong), and `m' (medium). N-Acetylimidazole, N-propionylimida-
zole, and N-palmitoylimidazole were prepared according to the method described by Staab
from imidazole and the acid chlorides and used without puri®cation [12]. 1-Hydroxy-2-O-
benzyl-octadecane (5) and 1-hydroxy-2-N-phthalimido-octadecane (6) were prepared from racemic
È
1,2-octadecanediol as described elsewhere [7]. Erucanol (4) was a gift from H. Eibl, Gottingen,
FRG.
Dimethyl-3-chloropropylphosphonate (1)
Dimethylphosphite (50.5 ml, 60.6 g, 550 mmol) was added dropwise to a stirred solution of
potassium-tert-butylate (56.1 g, 500 mmol) in 200 ml THF within 15 minutes. After cooling to room
temperature, the resulting suspension was added dropwise to a stirred solution of 1-bromo-3-
chloropropane (64.0 ml, 102 g, 650 mmol) in 100 ml THF within 40 minutes. The mixture was heated
under re¯ux for 15 minutes. After cooling to room temperature, the precipitate (potassium bromide)
was ®ltered off with suction and washed twice with 200 ml diisopropyl ether. Solvents, educts, and
tert-butanol were evaporated (20 mbar, up to 170ꢀC) to yield 62.4 g (334 mmol, 67%) pure 1.
Rf ˆ 0.60 (acetone); IR (Film): 2955 (s), 2850 (m), 1240 (s) P=O, 1055 (ss) P-O-C, 1030 (ss) P-
1
O-C, 825 (s) C-Cl; H NMR (400 MHz, CDCl3): 1.86±1.98 (m, 2H, P-CH2 CH2), 2.01±2.13 (m, P-
3
CH2-), 3.61 (dt, JP;H ˆ 1.0 Hz, J ˆ 6.2 Hz, 2H, -CH2Cl), 3.76 (d, JP;H ˆ 10.7 Hz, 6H, O-CH3).
3-Chloropropylphosphonic acid (2)
Compound 1 (15.1 g, 80.9 mmol) was dissolved in 150 ml (181 mmol) hydrochloric acid (37%) and
re¯uxed for 9 h. The solvent was removed and the residue dried by azeotropic distillation with
toluene. The jelly residue was crystallized from 200 ml chloroform to yield 12.1 g (76.3 mmol, 94%)
2 as colorless crystals.
Rf ˆ 0.63 (methanol); IR (KBr): 3300±2400 (sb) OH, 2965 (m), 2310 (s), 1285 (m), 1235 (m)
P=O, 1020 (s), 1000 (ss), 955 (s); 1H NMR (250 MHz, D2O): 1.41±1.52 (m, 2H, P-CH2-), 1.52±1.70

180
J. T. Kley et al.
3
1
(m, 2H, -CH2CH2-P), 3.26 (dt, J ˆ 6.0 Hz, JP;H ˆ 1.0 Hz, 2H, -CH2Cl), 4.40 (s, OH); H NMR
(250 MHz, CD3CN): 1.81±2.15 (m, 4H, P-CH2CH2-), 3.68 (dt, 3J ˆ 6.3 Hz, JP;H ˆ 0.7 Hz, 2H,
-CH2Cl), 9.46 (s, 2H, P-OH).
1-Hydroxy-2-amino-octadecane (7)
1-Hydroxy-2-N-phthalimido-octadecane (6, 4.16 g, 10.0 mmol) was dissolved in 90 ml 2-propanol
and 15 ml water. Sodium borohydride (1.9 g, 50 mmol) was added stepwise within 2 h. The reaction
mixture was stirred for further 12 h. Then, 30 ml glacial acetic acid were added, and the mixture was
heated to 80ꢀC for 4 h. After removal of the solvent the residue was dissolved in 90 ml
dichloromethane/methanol 2:1 and extracted with 60 ml ammonia (10% acqueous solution). The
aqueous phase was reextracted with 60 ml dichloromethane/methanol 5:1, and the solvents of the
combined organic layers were removed. The residue was puri®ed by recrystallization from 40 ml
dichloromethan to yield 2.05 g (7.18 mmol, 72%) pure 7 as shimmering crystals.
Rf ˆ 0.05 (ethyl acetate/methanol 2:1); IR (KBr): 3345 (s) NH2, 3285 (s) NH2, 2925 (ss) -CH2-,
2850 (ss) -CH2-, 1605 (s), NH, 1470 (s), 1070 (s); 1H NMR (250 MHz, CDCl3): 0.88 (t, 3J ˆ 6.6 Hz,
3H, -CH3), 1.62 (s, 30H, (3±17)-CH2-), 2.01 (sb, 3H, OH and NH2, H/D-exchangeable), 2.76±2.87
3
2
3
2
(m, 1H, 2-CH), 3.26 (dd, J ˆ 7.9 Hz, J ˆ 10.6 Hz, 1H, 1-CH), 3.58 (dd, J ˆ 3.8 Hz, J ˆ 10.6 Hz,
1H, 1-CH0).
1-Hydroxy-2-N-formyl-octadecane (8)
A 2 M solution of formic acid in THF (12.6 ml, 25.2 mmol) was added slowly to an ice-cold solution
of DCC (2.60 g, 12.6 mmol) in 20 ml THF. After stirring the mixture for 20 min, a solution of 1-
hydroxy-2-amino-octadecane (7, 1.80 g, 6.3 mmol) in 70 ml THF/pyridine 3:1 was added. After 1 day,
10 ml of water were added. The precipitate was ®ltered off with suction after another day, and the
solvents were removed. Recrystallization from 35 ml cyclohexane afforded 2.25 g crude 8 which was
phosphonylated according to the general procedure described below without further puri®cation. For
analytical purposes, a small amount of 8 was puri®ed on silica gel (eluent: chloroform/methanol 95:5).
Rf ˆ 0.63 (ethyl acetate/methanol 2:1); IR (KBr): 2915 (ss) -CH2-, 2850 (s) -CH2-, 1650 (ss)
C=O, 1560 (m), 1465 (m), 1385 (m), 1070 (m), 725 (s); 1H NMR (250 MHz, CDCl3): 0.88 (t,
3J ˆ 6.4 Hz, 3H, -CH3), 1.25 (s, 28H, (4±17)-CH2-), 1.41±1.64 (m, 2H, 3-CH2-), 2.58 (t, 3J ˆ 4.9 Hz,
1H, -OH, H/D-exchangeable), 3.60 (ddd, after H/D-exchange dd, 2J ˆ 11.1 Hz, 3J ˆ 5.5 Hz, JH;OH
ˆ
4.9 Hz, 1H, 1-CH), 3.71 (ddd, after H/D-exchange dd, 2J ˆ 11.1 Hz, 3J ˆ 3.5 Hz, JH;OH ˆ 4.9 Hz, 1H,
1-CH0), 3.94±4.09 (m, 1H, 2-CH), 5.75 (d, J ˆ 7.0 Hz, 1H, NH, H/D-exchangeable), 8.21 (s, 1H,
formyl-H).
General procedure for the formation of the 3-(trimethylammonio)propylphosphonoyl headgroup
The alcohols 3, 4, 5, 6, or 8 (6.30 mmol), 3-chloropropylphosphonic acid (2) (1.10 g, 6.94 mmol),
DCC (2.9 g, 14 mmol), and DMAP (70 mg, 0.6 mmol) were stirred in 60 ml THF for 24 h. To
hydrolyze the surplus of DCC, 10 ml of water were added. After stirring the mixture for further 24 h,
the precipitate was ®ltered off with suction, the solvent was removed, and lithium bromide (5.47 g,
63 mmol) was added. Butanone (50 ml) was added, and the mixture was re¯uxed for 4 h. After
removal of the solvent, 30 ml methanol and 15 ml trimethylamine in ethanol (4.2 mol/l, 63 mmol)
were added, and the sealed ¯ask was kept at 50ꢀC for 72 h with stirring. The volatile components
were evaporated, and the residue was dissolved in 180 ml chloroform/methanol 1:1.2 and extracted
with 80 ml water. The aqueous phase was extracted twice with 50 ml chloroform/methanol 4:1. The
solvents of the combined organic layers were removed, and the crude phosphonates (9, 10, 11, 12, or
13) were puri®ed or further reacted as described.

Synthesis of Analogs of Alkylphosphocholines
181
Hexadecyl-3-(trimethylammonio)propylphosphonate (9)
Hexadecanol (3, 1.53 g, 6.30 mmol) was converted into the corresponding phosphonate 9 according
to the general procedure. Workup as described there and puri®cation on silica gel (20 g) using ®rst
solvent 2 and then solvent 7 as eluents afforded 1.46 g (3.60 mmol, 57%) 9 as colorless solid.
Rf ˆ 0.21 (solvent 7); IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2, 1495 (m), 1470 (m), 1200 (s)
P=O, 1075 (ss) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.89 (t, 3J ˆ 6:7 Hz, 3H, -CH3),
1.27 (s, 26H, (3±15)-CH2-), 1.56 (dt, JPH ˆ 17.0 Hz, 3J ˆ 7.1 Hz, 2H, P-CH2), 1.62 (quint,
3J ˆ 6.7 Hz, 2H, 2-CH2-), 1.92±2.05 (m, 2H, P-CH2-CH2-), 3.12 (s, 9H, N(CH3)3), 3.41±3.46 (m,
3
2H, -CH2-N), 3.84 (quart, J ˆ 6.7 Hz, JPH ˆ 6.7 Hz, 2H, P-O-CH2-).
Erucyl-3-(trimethylammonio)propylphosphonate (10)
13-cis-Docosenol (erucanol, 4; 2.04 g, 6.30 mmol) was reacted according to the general procedure.
Workup and puri®cation as described for compound 9 afforded 1.87 g (3.83 mmol, 61%) 10 as
colorless solid.
Rf ˆ 0.25 (solvent 7); IR (KBr): 2925 (ss) -CH2-, 2855 (s) -CH2-, 1465 (m), 1200 (s) P=O,
1075 (ss) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD(2:1)): 0.89 (t, 3J ˆ 6.7 Hz, 3H, -CH3), 1.27 (s,
30H, (3±11, 16±21)-CH2-), 1.58 (dt, JPH ˆ 17.1 Hz, 3J ˆ 7.2 Hz, 2H, P-CH2), 1.62 (quint, 3J
ˆ 6.7 Hz, 2H, 2-CH2-), 1.93±2.05 (m, 6H, P-CH2-CH2- and allylic -CH2-), 3.13 (s, 9H, N(CH3)3),
3.41±3.47 (m, 2H, -CH2-N), 3.84 (quart, 3J ˆ 6.7 Hz, JPH ˆ 6.7 Hz, 2H, P-O-CH2-), 5.33±5.37
(m, 2H, vinylic CH).
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-hydroxy-octadecane (14)
1-Hydroxy-2-O-benzyl-octadecane (5, 2.37 g, 6.30 mmol) was reacted according to the general
procedure. The crude phosphonate 11 was dissolved in 15 ml THF and stirred with activated charcoal
(100 mg) for 2 h. Palladium on activated charcoal (10% Pd, 75 mg) in 6 ml hydrochloric acid (1 mol/
l) was added. Hydrogenolysis was peformed under intensive stirring at room temperature and
atmospheric pressure until hydrogen uptake was completed (24 h). Charcoal and the catalyst were
®ltered off, and the solvents were removed. The crude product was puri®ed on silica gel (30 g)
using ®rst solvent 2 and then solvent 7 as eluents to yield 2.55 g (5.67 mmol, 90%) 14 as colorless
solid.
Rf(11) ˆ 0.39, Rf(14) ˆ 0.14 (solvent 7); IR (KBr): 3420 (s) OH, 2915 (ss) -CH2-,2850 (s) -CH2-,
1470 (s), 1200 (s) P=O, 1055 (s) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.88
(t, 3J ˆ 6.8 Hz, 3H, -CH3), 1.27 (s, 28H, (4±17)-CH2-), 1.45 (s, 2H, 3-CH2-), 1.66 (dt, JP;H ˆ 17.2 Hz,
3J ˆ 7:2 Hz, 2H, P-CH2-) 1.96±2.08 (m, 2H, P-CH2CH2-), 3.15 (s, 9H, N(CH3)3), 3.43±3.50 (m, 2H,
-CH2-N), 3.68±3.76 (m, 2H, 1-CH2), 3.85±3.93 (m, 1H, 2-CH).
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-amino-ctadecane (15)
1-Hydroxy-2-N-phthalimido-octadecane (6, 2.62 g, 6.30 mmol) was reacted according to the general
procedure. The crude phosphonate 12 was dissolved in 30 ml 2-propanol/water/toluene (6:1:1.5).
The solution was cooled to 0ꢀC and sodium borohydride (1.13 g, 30 mmol) was added stepwise
within 30 minutes. The mixture was stirred for 12 h; then, hydrochloric acid (10 ml, 6 mol/l) was
added dropwise. The solution was stirred for 3.5 h at 60ꢀC and cooled to room temperature. Then,
50 ml 2 N sodium hydroxide, 60 ml methanol, and 50 ml chloroform were added. The aqueous phase
was reextracted with 50 ml chloroform/methanol 4:1, and the solvents of the combined organic
layers were removed. Puri®cation on silica gel (30 g) using ®rst solvent 5 and then solvent 10 as
eluents afforded 2.02 g (4.50 mmol, 71%) 15 as colorless solid.

182
J. T. Kley et al.
Rf (12) ˆ 0.37, Rf (15) ˆ 0.11 (solvent 7); IR (KBr): 2915 (ss) -CH2-, 2850 (s) -CH2-, 1465 (s),
1
1200 (s) P=O, 1075 (s) P-O-C, 1050 (s) P-O-C; H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.88
(t, 3J ˆ 6.8 Hz, 3H, -CH3), 1.27 (s, 28H, (4±17)-CH2-), 1.45±1.56 (m, 2H, 3-CH2-), 1.61 (dt,
JP;H ˆ 17.2 Hz, 3J ˆ 7.2 Hz, 2H, P-CH2-), 1.93±2.07 (m, 2H, P-CH2CH2-), 3.05±3.13 (m, 1H,
2-CH), 3.14 (s, 9H, N(CH3)3), 3.41±3.47 (m, 2H, -CH2-N), 3.67±3.75 (m, 1H, 1-CH), 3.93±4.00
(m, 1H, 1-CH0).
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-formyl-octadecane (13)
Crude 1-hydroxy-2-N-formyl-octadecane (8, 2.25 g, prepared from 6.3 mmol 7 as described above)
was reacted according to the general procedure (3.5). Workup as described there and puri®cation on
silica gel (30 g) using solvent 7 as eluent afforded 1.79 g (3.76 mmol, 60% with respect to 7) 13 as
slightly yellowish powder.
Rf ˆ 0.14 (solvent 7); IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2-, 1670 (ss) C=O, 1465 (m), 1195
1
3
(ss) P=O, 1065 (ss) P-O-C; H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.89 (t, J ˆ 6.7 Hz, 3H,
-CH3), 1.27 (s, 28H, alkyl-(4±17)-CH2-), 1.43±1.62 (m, 4H, 3-CH2- and P-CH2-), 1.90±2.05 (m, 2H,
P-CH2-CH2-), 3.12 (s, 9H, N(CH3)3), 3.38±3.44 (m, 2H, -CH2N), 3.81 (ddd, 2J ˆ 10.5 Hz,
3J ˆ 6.4 Hz, JPH ˆ 6.4 Hz, 1H, 1-CH), 3.89 (ddd, 2J ˆ 10.5 Hz, 3J ˆ 3.8 Hz, JPH ˆ 6.1 Hz, 1H,
1-CH0), 4.02±4.10 (m, 1H, 2-CH), 8.11 (s, 1H, formyl-H).
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-O-acetyl-octadecane (16)
and 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-O-palmitoyl-octadecane (17)
A solution of 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-hydroxy-octadecan (14, 225 mg,
0.500 mmol) and 5.00 mmol of the corresponding acid chloride (acetyl chloride: 0.355 ml, 393 mg;
palmitoyl chloride: 3.00 ml, 2.73 g) in 10 ml chloroform was stirred in a stoppered ¯ask at room
temperature for 24 h. 12 ml methanol were added, and after 2 h of stirring the solution was extracted
with 10 ml ammonia (10% aqueous solution). The aqueous phase was reextracted twice with 5 ml
chloroform/methanol 4:1; then the solvents of the combined organic layers were removed. The crude
products were puri®ed on silica gel (10 g) using ®rst solvent 2 and then solvent 7 as eluents to yield
the respective esters as colorless solids.
a) 1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-O-acetyl-octadecane (16)
Yield: 117 mg (0.238 mmol, 48%); Rf ˆ 0.26 (solvent 7); IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2-,
1735 (s) C=O, 1245 (s), 1075 (s) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.88
(t, 3J ˆ 6.8 Hz, 3H, alkyl-CH3), 1.27 (s, 28H, alkyl-(4±17)-CH2-), 1.52±1.63 (m, 4H, P-CH2
and 3-CH2-), 1.92±2.05 (m, 2H, P-CH2CH2-), 2.07 (s, 3H, acetyl-CH3), 3.13 (s, 9H, N(CH3)3),
3.39±3.46 (m, 2H, -CH2-N) 3.82±3.89 (m, 1H, 1-CH), 3.91±3.97 (m, 1H, 1-CH0), 3.97±4.05 (m,
1H, 2-CH).
b) 1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-O-palmitoyl-octadecane (17)
Yield: 152 mg (0.221 mmol, 44%); Rf ˆ 0.53 (solvent 7); IR (KBr): 2915 (ss) -CH2-, 2850 (s) -CH2-,
1725 (s) C=O, 1465 (m), 1200 (s) P=O, 1075 (s) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD
(2:1)): 0.89 (t, 3J ˆ 6.8 Hz, 6H, -CH3), 1.27 (s, 52H, alkyl-(4±17)-CH2- and acyl-(4±15)-CH2-, 1.52±
1.68 (m, 6H, P-CH2-, alkyl-3-CH2- and acyl-3-CH2-), 1.92±2.04 (m, 2H, P-CH2CH2-), 2.25±2.38
(m, 2H, acyl-2-CH2-), 3.12 (s, 9H, N(CH3)3), 3.40±3.46 (m, 2H, -CH2-N), 3.82±3.96 (m, 2H, 1-
CH2), 3.99±4.06 (m, 1H, 2-CH).

Synthesis of Analogs of Alkylphosphocholines
183
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-acetyl-octadecane (18),
1-O-(3-(trimethylammonio)propylphosphonoyl)-2-N-propionyl-octadecane (19),
and 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-N-palmitoyl-octadecane (20)
A solution of 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-amino-octadecane (15, 225 mg,
0.500 mmol) and 5.00 mmol of the corresponding N-acylimidazole (N-acetylimidazole: 551 mg;
N-propionylimidazole: 621 mg; N-palmitoylimidazole: 1.53 g) in 15 ml chloroform was stirred in a
stoppered ¯ask at room temperature for 3 days. Methanol (18 ml) was added, and the solution was
extracted with 15 ml water. The aqueous phase was extracted twice with 10 ml chloroform/methanol
4:1, and the solvents of the combined organic layers were removed. The crude amides were puri®ed
on silica gel (10 g) using ®rst solvent 2 and then solvent 7 as eluents to yield the respective amides as
colorless solids.
a) 1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-acetyl-octadecane (18)
Yield: 162 mg (0.330 mmol, 66%); Rf ˆ 0.17 (solvent 7); IR (KBr): 2915 (ss) -CH2-, 2850 (s) -CH2-,
1
1650 (s) C=O, 1200 (s) P=O, 1065 (s) P-O-C; H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.89
(t, 3J ˆ 6.8 Hz, 3H, alkyl-CH3), 1.27 (s, 28H, alkyl-(4±17)-CH2-), 1.42±1.49 (m, 2H, 3-CH2-), 1.51±
1.63 (m, 2H, P-CH2-), 1.92±2.06 (m, 2H, P-CH2CH2-), 1.98 (s, 3H, acetyl-CH3), 3.12 (s, 9H,
N(CH3)3), 3.39±3.45 (m, 2H, -CH2-N), 3.76±3.83 (m, 1H, 1-CH), 3.83±3.89 (m, 1H, 1-CH0), 3.92±
3.99 (m, 1H, 2-CH).
b) 1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-propionyl-octadecane (19)
Yield: 187 mg (0.370 mmol, 74%); Rf ˆ 0.22 (solvent 7); IR (KBr): 2915 (ss) -CH2-, 2850 (s) -CH2-,
1
1635 (ss) C=O, 1540 (s), 1465 (m), 1195 (ss) P=O, 1070 (ss) P-O-C; H NMR (400 MHz, CDCl3/
CD3OD (2:1)): 0.89 (t, 3J ˆ 6.8 Hz, 3H, Alkyl-CH3), 1.15 (t, 3J ˆ 7.6 Hz, 3H, propionyl-CH3), 1.27
(s, 28H, alkyl-(4±17)-CH2-), 1.41±1.61 (m, 4H, 3-CH2- and P-CH2-), 1.91±2.05 (m, 2H, P-CH2CH2-
2
), 2.21 (dquart, 3J ˆ 7.6 Hz, JAB ˆ 14.5 Hz, 1H, C(O)CHAHBCH3), 2.24 (dquart, 3J ˆ 7.6 Hz,
2JAB ˆ 14.5 Hz, 1H, C(O)CHAHBCH3), 3.12 (s, 9H, N(CH3)3), 3.39±3.45 (m, 2H, -CH2-N), 3.79
(ddd, 2J ˆ 10.5 Hz, 3J ˆ 7.0 Hz, JPH ˆ 5.8 Hz, 1H, 1-CH), 3.86 (ddd, 2J ˆ 10.5 Hz, 3J ˆ 4.0 Hz,
JPH ˆ 6.2 Hz, 1H, 1-CH0), 3.93±4.00 (m, 1H, 2-CH).
c) 1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-palmitoyl-octadecane (20)
Yield: 178 mg (0.259 mmol, 52%); Rf ˆ 0.50 (solvent 7); IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2-,
1
1640 (s) C=O, 1200 (s) P=O, 1070 (s) P-O-C; H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.89 (t,
3J ˆ 6.8 Hz, 6H, -CH3), 1.27 (s, 52H, alkyl-(4±17)-CH2- and acyl-(4±15)-CH2-), 1.41±1.49 (m, 2H,
alkyl-3-CH2-), 1.51±1.67 (m, 4H, P-CH2- and acyl-3-CH2-), 1.91±2.04 (m, 2H, P-CH2CH2), 2.12±
2.26 (m, 2H, acyl-2-CH2-), 3.12 (s, 9H, N(CH3)3), 3.38±3.44 (m, 2H, -CH2-N), 3.75±3.87 (m, 2H,
1-CH2), 3.93±4.01 (m, 1H, 2-CH).
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-chloroacetyl-octadecane (21)
A solution of 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-amino-octadecane (15, 225 mg,
0.500 mmol), DMAP (305 mg, 2.50 mmol), and chloroacetylchloride (0.159 ml, 226 mg, 2.00 mmol)
in 15 ml chloroform was stirred in a stoppered ¯ask at room temperature for 1 h. 0.4 ml
Triethylamine and 20 ml methanol were added, and the solution was extracted with 15 ml water.
After reextracting the aqueous phase twice with 15 ml chloroform/methanol 4:1, the solvents were
removed. Puri®cation on silica gel (10 g) using ®rst solvent 2 and then solvent 7 as eluents afforded
75 mg (143 mmol, 29%) 21 as reddish solid.

184
J. T. Kley et al.
Rf ˆ 0.25 (solvent 7); IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2-, 1650 (s) C=O, 1560 (m), 1470
(m), 1190 (s) P=O, 1070 (ss) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.89 (t, 3J ˆ 6.8 Hz,
3H, -CH3), 1.27 (s, 28H, alkyl-(4-17)-CH2-), 1.47±1.63 (m, 4H, 3-CH2- and P-CH-), 1.91±2.05 (m,
2H, P-CH2-CH2-), 3.12 (s, 9H, N(CH3)3), 3.39±3.44 (m, 2H, -CH2N), 3,82 (ddd, 2J ˆ 10.7 Hz,
3J ˆ 6.7 Hz, JPH ˆ 6.2 Hz, 1H, 1-CH), 3.90 (ddd, 2J ˆ 10.7 Hz, 3J ˆ 3.9 Hz, JPH ˆ 6.2 Hz, 1H, 1-
2
CH0), 3.97±4.05 (m, 1H, 2-CH), 4.02 and 4.04 (2d, JAB ˆ 13.5 Hz, 2H, -CHAHBCl).
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-¯uoroacetyl-octadecane (22)
Perchloric acid (70%, 0.171 ml, 2.00 mmol) was added to a suspension of sodium ¯uoracetate
(0.20 g, 2.00 mmol) and carbonyldiimidazole (1.17 g, 7.20 mmol) in 8 ml THF. The mixture was
stirred for 30 min, the precipitate was ®ltered off, and a solution of 1-O-(3-(trimethylammonio)-
propylphosphonoyl)-2-amino-octadecane (15, 225 mg, 0.50 mmol) in 8 ml chloroform was added.
The mixture was stirred for 1 day, then the solvents were removed. The residue was dissolved in 12
ml chloroform and 15 ml methanol, and the solution was extracted with 12 ml water. The aqueous
phase was reextracted twice with 10 ml chloroform/methanol 5:1, and the solvents of the combined
organic layers were removed. Puri®cation on silica gel (10 g) using ®rst solvent 2 and then solvent 7
as eluent afforded 184 mg (0.362 mmol, 72%) 22 as slightly yellowish solid.
Rf ˆ 0.19 (solvent 7); IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2-, 1660 (ss) C=O, 1560 (m),
1470 (m), 1190 (s) P=O, 1050 (ss) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.89 (t,
3J ˆ 6.8 Hz, 3H, -CH3), 1.27 (s, 28H, alkyl-(4±17)-CH2-), 1.48±1.66 (m, 4H, 3-CH2- and P-CH2-),
1.92±2.05 (m, 2H, P-CH2-CH2-), 3.13 (s, 9H, N(CH3)3), 3.39±3.45 (m, 2H, -CH2N), 3.84 (ddd,
3
2J ˆ 10.6 Hz, 3J ˆ 6.4 Hz, JPH ˆ 6.4 Hz,1H, 1-CH), 3.91 (ddd, 2J ˆ 10.6 Hz, 3J ˆ 3.9 Hz,
JPH ˆ 6.0 Hz, 1H, 1-CH0), 4.04-4.12 (m, 1H, 2-CH), 4.74 and 4.86 (2s, 2H, -CHAHBF)
1-O-(3-(Trimethylammonio)propylphosphonoyl)-2-N-methoxycarbonyl-octadecane (23)
A solution of 1-O-(3-(trimethylammonio)propylphosphonoyl)-2-amino-octadecane (15, 225 mg,
0.500 mmol), DMAP (100 mg, 0.82 mmol), triethylamine (0.28 ml, 202 mg, 2.00 mmol), and methyl
chloroformate (0.154 ml, 189 mg, 2.00 mmol) in 15 ml chloroform was stirred in a stoppered ¯ask at
room temperature for 24 h. Methanol (18 ml) was added, and the solution was extracted with 15 ml
ammonia (5% aqueous solution). The aqueous phase was extracted with 10 ml chloroform/methanol
4:1, and the solvents of the combined organic layers were removed. Puri®cation of the crude
carbamate on silica gel (10 g) using ®rst solvent 2 and then solvent 7 as eluents yielded 173 mg
(341 mmol, 68%) 23 as slightly yellowish solid.
Rf ˆ 0.23 (solvent 7): IR (KBr): 2920 (ss) -CH2-, 2850 (s) -CH2-, 1710 (ss) C=O, 1545 (m), 1470
(m), 1195 (s) P=O, 1075 (ss) P-O-C; 1H NMR (400 MHz, CDCl3/CD3OD (2:1)): 0.88 (t, 3J ˆ 6.8 Hz,
3H, alkyl-CH3), 1.27 (s, 28H, alkyl-(4±17)-CH2-), 1.42±1.61 (m, 2H, 3-CH2-), 1.57 (dt,
3
JPH ˆ 17.1 Hz, J ˆ 7:2 Hz, 2H, P-CH2-), 1.91±2.04 (m, 2H, P-CH2CH2-), 3.12 (s, 9H, N(CH3)3),
3), 3.38±3.45 (m, 2H, -CH2-N), 3.63 (s, 3H, -O-CH3), 3.64±3.72 (m, 1H, 2-CH), 3.76±3.88 (m,
2H, 1-CH2).
Acknowledgements
This work was supported by the Hermann-Schlosser-Stiftung, Frankfurt, FRG.
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Synthesis of Analogs of Alkylphosphocholines
185
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Received July 17, 1997. Accepted July 28, 1997