Synthesis of diesteramide phospholipids of cationic type

Article abstract of DOI:10.1023/A:1022505916623

A promising approach to synthesis of diesteramide lipid structures of cationic type proceeding from menthol cycloamidophosphites was considered. The latter easily undergo an oxidative decyclization when treated with bromine. The bromoamidophosphates thus

Full text of DOI:10.1023/A:1022505916623

Russian Journal of Organic Chemistry, Vol. 38, No. 11, 2002, pp. 1615 1619. Translated from Zhurnal Organicheskoi Khimii, Vol. 38, No. 11, 2002,
pp. 1671 1675.
Original Russian Text Copyright
,
2002 by Predvoditelev, Kosarev, Suvorkin, Nifant ev.
Synthesis of Diesteramide Phospholipids of Cationic Type
D. A. Predvoditelev, G. V. Kosarev, S. V. Suvorkin, and E. E. Nifant,ev
Moscow State Pedagogical University, Moscow, 119021 Russia
Received January 8, 2002
Abstract A promising approach to synthesis of diesteramide lipid structures of cationic type proceeding
from menthol cycloamidophosphites was considered. The latter easily undergo an oxidative decyclization
when treated with bromine. The bromoamidophosphates thus obtained readily react with glycerol and
cholesterol derivatives providing models of lipo(P
N)cholines of cationic type which are among the most
important substances in the modern phospholipid chemistry.
We have demonstrated formerly that the use of
bromides instead of chlorides of the acids of penta-
valent phosphorus is promising for creating the
phosphorus site in the molecules of glycerophospho-
lipids and various phosphoramide analogs thereof.
Therewith we have established that the synthesis of
bromophosphates containing in their molecules hydro-
philic fragments of widespread phospholipids can be
accomplished by bromination of cyclic esters and
amides of alkylene phosphites and amidophosphites
[1]. Thus the common for the phospholipid chemistry
phosphorylation method based on the use of tradi-
tional chlorophosphates [2] was well supplemented
with the modern preparative achievements of the
organophosphorus chemistry [3, 4].
logically active substance, was brought into reaction
with cycloamidophosphite diethylamide (II) to obtain
previously unknown menthyl 2-hydroxyoxaaza-
phospholane (III). The menthol phosphorylation was
previously performed only with acyclic amidophos-
phites [5 7].
The homogeneity and structure of menthyl cyclo-
amidophosphate III were proved by a number of
1
physicochemical methods, among them H and 31P
NMR spectroscopy. The 31P NMR spectrum of com-
pound III consists of two singlets at
138.49 and
138.89 ppm due to the existence of the Pcompound as
1
a mixture of two diastereomers. In the H NMR
spectrum of cycloamidophosphate III appear the
proton signals from all groups of the assumed struc-
ture with the expected integral intensity: two doublet
Here we report on the synthesis of cationic
phospholipids based on glycerol and cholesterol
derivatives belonging to the class of diesteramido-
phosphates. We selected a menthyl bromoamido-
phosphate (IV) as a particular phosphorylating
agent. This bromide was prepared in two stages. In
the first stage l-menthol, an important natural bio-
3
signals ( 0.71 and 0.84 ppm, JHH 6.8 Hz) belonging
to the methyl protons of menthyl moiety, a multiplet
in the region 1.14 2.04 ppm (CH3C_H, OCHC_HCH2,
CH3C_HCH3 from menthyl), a doublet at 2.59 ppm
corresponding to the methyl protons from the PNCH3
group, multiplets at
2.94 and 4.18 ppm from
methylene protons of the phospholane ring, and a
multiplet at 3.62 ppm from the methine proton of
the POCH group. In the second stage menthol cyclo-
amidophosphate III was transformed into menthyl
bromoamidophosphate IV.
1070-4280/02/3811-1615$27.00 2002 MAIK Nauka/Interperiodica

1616
PREDVODITELEV et al.
The oxidative bromination was carried out in
(CH2CH2, CHC_H2CH, CH3C_H, OCHC_HCH2,
CH3C_HCH3 of menthyl group), singlets at 1.27 and
1.35 from the methyl protons of isopropylidene group,
a doublet at 2.65 ppm (3JPH 9.59 Hz) belonging to
PNCH3 group, a multiplet at 3.30 3.36 ppm from
methylene protons in the PNCH2CH2Br group, a
complex multiplet in the 3.62 4.08 ppm region
from methylene and methine protons of the
(C_H2CHCH_ 2OPOC_H) group, and also a multiplet at
4.24 ppm of the methine proton attached to the
-carbon of the glycerol skeleton. The proton spectra
of phosphoglycerol derivative IX and cholesterol
phospholipid* X were fully consistent with the
assumed structures (see EXPERIMENTAL).
chloroform under mild conditions (at 20 C). As
shown by TLC and 1H and 31P NMR data bromide IV
formed in quantitative yield. The freshly prepared
bromophosphate IV was not subjected to further
purification and was directly used after evaporation
of solvent as a phosphorylating agent in reaction with
1,2-O-isopropylideneglycerol (V), 1,2-distearoyl
glycerol (VI), and natural cholesterol VII.
The reaction of 1,2-O-isopropylideneglycerol (V)
with bromoamidophosphate IV was carried out in
benzene in the presence of pyridine at 60 C for 12 h.
The reaction with 1,2-distearoyl glycerol (VI) and
cholesterol (VII) required heating of the reaction mix-
ture to 80 C for 30 h (here dioxane was used as
solvent). After the purification by column chromato-
graphy on silica gel the yields of amidophosphate
phospholipids VIII X amounted respectively to
63, 58, and 49%.
In the final stage of the study the -bromoethylene-
amidophosphates VIII X were converted into
phospholipids with cationic groups by treatment with
trimethylamine.
The reaction with trimethylamine was accomplish-
ed at 80 C within 16 h and furnished the target
products XI XIII in respective yields 73, 75, and
69%. The 31P NMR spectra of the cationic amido-
phosphate lipid analogs XI XIII obtained were
virtually identic to those of menthyl amidophosphates
The homogeneity and structure of menthyl amido-
phosphates VIII X were confirmed by physico-
chemical methods. In the 31P NMR spectrum of
phospholipids VIII X a broad singlet at 9 10 ppm
P
indicated that the obtained menthyl amidophosphates
contained mixtures of diastereomers. We failed to
register separate signals of the diastereomers in the
31P NMR spectra due to insufficient resolution ability
of our spectrometer.
In the 1H NMR spectrum of menthyl amido-
phosphates VIII X appear the proton signals from
all groups of the assumed structures with the cor-
responding integral intensities. In the 1H NMR
spectrum of the glycerol derivative VIII are observed
doublet signals at 0.75 and 0.84 ppm from methyl
groups in menthyl moiety of the molecule, a multi-
plet signal in the 0.91 2.24 ppm region
1
VIII X. Unlike the H NMR spectra of the latter in
the spectra of cationic phospholipids XI XIII occur-
red the downfield shift of methylene protons multiplet
belonging to the group PN(CH3)CH2C_H2, and the
appearance of a new singlet at 3.5 ppm corresponding
to the methyl protons of the cationic center.
*
1
In the analysis of H NMR spectra of the cholesterol-contain-
ing phospholipids X, XIII we took into account the detailed
description and assignment of the spectral signals of protons
in cholesterol published in [8].
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 38 No. 11 2002

SYNTHESIS OF DIESTERAMIDE PHOSPHOLIPIDS OF CATIONIC TYPE
1617
It should be noted in conclusion that the applica-
tion of bromophosphates instead of chlorophosphates
as phosphorylating agents in lipid synthesis is a
convenient preparative method for creating the
phosphorus site and provides a possibility to obtain
under relatively mild conditions complex phosphorus
derivatives of lipids, in particular those containing in
the structure fragments of important biologically
active natural compounds.
sphere of dry argon. The solvents applied were dried
by standard procedures.
2-Diethylamino-3-methyl-1,3,2-oxaazaphospholane
(II) was prepared by method [9] and possessed con-
stants identical to those published.
3-Methyl-2-(3-menthyloxy)-1,3,2-oxaazaphos-
pholane (III). A solution of a mixture of 1.55 g of
l-menthol (I) and 1.52 g of cycloamidophosphite (II)
in 3 ml of benzene were kept for 4 h at 80 C distilling
off the forming diethylamine. Menthyl cycloamido-
phosphite (III) was isolated as individual compound
EXPERIMENTAL
1H NMR spectra of compounds III, IV, VIII XIII
were recorded on spectrometer Bruker WM-250
(250 MHz) from solutions in CDCl3, internal refer-
ence TMS; the proton signals were assigned with the
help of double resonance spectra. NMR spectra
31P {1H} of compounds III, IV, VIII XIII were
registered on spectrometer Bruker WP-80 SY from
solutions in CHCl3 at 32.4 MHz with an external
standard (85% phosphoric acid).
by vacuum distillation at 1 10 mm Hg and bath
4
temperature 82 C. Yield 2.25 g (87%), n2D0 1.4822,
Rf (elution system): 0.42 (A), 0.38 (B), 0.83 (C).
1H NMR spectrum, , ppm: 0.71 d (3H), 0.84 d [6H,
3
CH3 (menthyl), JHH 6.8 Hz], 1.14 2.04 m [9H,
C_H2, (CH3)2C_H (menthyl)], 2.59 d (3H, PNCH3,
3JPH 10.4 Hz), 2.94 m (2H, NC_H2CH2O), 3.62 m
[1H, POCH (menthyl)], 4.18 m (2H, NCH2C_H2O).
31P NMR spectrum, , ppm: 138.49 s, 138.89 s.
Found, %: C 59.98; H 10.12; P 11.87. C13H26NO2P.
Calculated, %: C 60.21; H 10.11; P 11.94.
For column chromatography was used a column
of 15 mm diameter packed with silica gel L 100
250 ; the Rf values were measured by TLC on
Silufol UV-254 plates using as eluents benzene di-
oxane, 3: 1 (A), hexane dioxane, 3: 1 (B), chloro-
form methanol, 3: 1 (C), and methanol water, 4: 1
(D). Vizualizing of spots was done by iodine vapor
and calcination at 250 300 C. The melting points
were determined in sealed capillaries heating at a rate
O-(3-Menthyl)-[N-(2-bromoethyl-N-methyl]-
amidobromophosphate (IV). To a solution of
2-menthylphospholane III in 10 ml of chloroform at
30 C while vigorous stirring was added dropwise a
solution of 1.23 g of bromine in 5 ml of chloroform.
In 10 min the solvent was removed in a vacuum, and
the compound IV obtained was kept at 1 mm Hg and
40 C for 4 h. Yield 3.23 g (100%), n2D0 1.4924, Rf
(elution system): 0.84 (A), 0.67 (B), 0.94 (C).
1H NMR spectrum, , ppm: 0.74 d (3H), 0.85 d [6H,
1
1 deg min .
All the syntheses with the use of trivalent
phosphorus compounds were carried out under atmo-
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 38 No. 11 2002

1618
PREDVODITELEV et al.
3
CH3 (menthyl), JHH 6.4 Hz], 1.25 2.04 m [9H, CH2
C_H2CH2C(O)], 2.02 m [4H, CH2C_H2C(O)], 2.75 d
(3H, PNCH3, JPH 9.89 Hz), 3.37 3.58 m (4H,
3
3
and CH (menthyl)], 2.62 d (3H, PNCH3, JPH
9.39 Hz), 3.35 m (4H, NCH2CH2Br), 3.96 m [1H,
POCH (menthyl)]. 31P NMR spectrum, , ppm:
2.99 s, 3.67 s. Found, %: C 37.31; H 6.20; P 7.44.
C13H26Br2NO2P. Calculated, %: C 37.25; H 6.25;
P 7.39.
NCH2CH2Br), 4.10m [1H, POCH, (menthyl)], 4.18m
(2H, C_H2CHCH2OP), 4.37 m (2H, CH2CHCH_ 2OP),
5.30 m (1H, CH2C_HCH2OP). 31P NMR spectrum,
, ppm: 10.28 br.s. Found, %: C 64.40; H 10.71;
P 3.25. C52H101BrNO7P. Calculated, % : C 64.84;
H 10.57; P 3.22.
O-(1, 2-O-Isopropylidene-3-glycerol)-O-(3-
menthyl)-[N-(2-bromoethyl-N-methyl]amidophos-
phate (VIII). To a solution of 0.84 g of bromide IV
in 4 ml of benzene was added dropwise at stirring a
solution of a mixture of 0.26 g of 1,2-)-isopropyl-
ideneglycerol (V) and 0.16 g of freshly distilled
pyridine in 1 ml of benzene. The reaction mixture
was heated to 60 C for 12 h. The separated preci-
pitate of pyridine hydrobromide was filtered off, the
solvent was removed in a vacuum. Glycerophosphate
VIII was isolated as an individual compound by
chromatography on a column charged with silica gel
(10 g) and filled with benzene. A mixture of benzene
dioxane, 7: 1 (250 ml) was used as eluent. The
solvents were removed in a vacuum, and the com-
pound obtained was kept at 1 mm Hg and 40 C for
3 h. Yield 0.59 g (63%), n2D0 1.4782, Rf (elution
system): 0.47 (A), 0.32 (B), 0.87 (C). 1H NMR
spectrum, , ppm: 0.75d (3H), 0.84d [6H, CH3
O-(3-Menthyl)-O-(3-cholesteryl)-[N-(2-bromo-
ethyl-N-methyl]amidophosphate (X). Likewise from
0.54 g of bromide IV, ).5 g of cholesterol (VII), and
0.1 g of pyridine in 10 ml of anhydrous dioxane by
heating to 80 C for 26 h was obtained compound X.
Cholesteryl phosphate X was isolated as an individual
compound by chromatography on a column charged
with silica gel (10 g) and filled with benzene. A mix-
ture of benzene dioxane, 8: 1 (250 ml) was used as
eluent. The solvents were removed in a vacuum, and
the oily compound obtained was kept at 1 mm Hg and
40 C for 3 h. Yield 0.46 g (48%), Rf (elution system):
1
0.69 (A), 0.46 (B), 0.84 (C). H NMR spectrum, ,
ppm: 0.68 2.47 (H, cholesterol)*, 0.79 2.50 m [9H,
3
CH2 and CH (menthyl)], 2.68 d (3H, PNCH3, JPH
9.52 Hz), 3.40 3.47 m (4H, NC_H2C_H2Br), 4.11
4.32 m [2H, POCH (menthyl) and C3H (cholesterol)],
5.38 m [1H, HC6 (cholesterol)]. 31P NMR spectrum,
, ppm: 9.81 br.s. Found, %: C 66.12; H 9.82; P
4.21. C40H71BrNO3P. Calculated, %: C 66.28;
H 9.87; P 4.27.
3
(menthyl), JHH 6.7 Hz], 0.91 2.24 m [9H, CH2
and CH (menthyl)], 1.27 s (3H), 1.35 s [3H,
3
C(CH3)2], 2.65d (3H, PNCH3, JPH 9.59 Hz), 3.30
3.36m (4H, NCH2CH2Br), 3.62 4.08 m (5H,
C_H2CHCH_ 2OPOC_H), 4.24 m (1H, CH2C_HCH2OP,
3JHH 5.6 Hz). 31P NMR spectrum, , ppm: 9.15 br.s.
Found, %: C 48.33; H 7.87; P 6.52. C19H37BrNO5P.
Calculated, %: C 48.51; H 7.93; P 6.58.
O-(1, 2-O-Isopropylidene-3-glycerol)-O-(3-
menthyl)-[N-methyl-N-(2-trimethylammonioethyl)]-
amidophosphate bromide (XI). A sealed ampule
with solution of 0.5 g of amidophosphate VIII and
0.63 g of trimethylamine in 2 ml of anhydrous benz-
ene was heated to 80 C for 6 h. The solvent was
evaporated in a vacuum, and the residue was dis-
solved in chloroform and reprecipitated with ethyl
ether. The thick oily substance was washed with ether
O-(1,2-Distearoyl-3-glycerol)-O-(3-menthyl)-[N-
(2-bromoethyl-N-methyl]amidophosphate
(IX).
Similarly to the synthesis of glyceroamidophosphate
VIII from 0.42 g of bromide IV, 0.62 g of distearo-
ylglycerol (VI), and 0.08 g of pyridine in 10 ml of
anhydrous dioxane by heating to 80 C for 26 h was
obtained compound IX. Glycerophosphate IX was
isolated as an individual compound by chromato-
graphy on a column charged with silica gel (10 g) and
filled with benzene. A mixture of benzene dioxane,
8: 1 (250 ml) was used as eluent. The solvents were
removed in a vacuum, and the compound obtained
was kept at 1 mm Hg and 40 C for 3 h. Yield 0.54 g
(58%), mp 64 67 C, Rf (elution system): 0.71 (A),
0.59 (B), 0.94 (C). 1H NMR spectrum, , ppm:
0.85 m [9H, CH3 (menthyl) and 6H, CH2C_H3 (di-
stearoylglycerol)], 1.05 2.29 m [9H, CH2 and CH
(menthyl)], 1.26 m [56H, CH3(C_H2)14], 1.64 m [4H,
4
(2 2 ml) and was kept in a vacuum (1 10 mm Hg)
over P2O5 at 40 C for 3 h. Yield 0.41 g (73%). Rf
1
(elution system): 0.24 (C), 0.61 (D). H NMR spec-
trum, , ppm: 0.72 d (3H), 0.83 d [6H, CH3
3
(menthyl), JHH 6.8 Hz], 1.06 2.28 m [9H, CH2 and
CH (menthyl)], 1.24 s (3H), 1.32 s [3H, [C(CH3)2],
3
2.64 d (3H, PNCH3, JPH 9.94 Hz), 3.55 s [9H,
N+ (CH3)3], 3.76 m [4H, PN(CH3)C_H2C_H2], 3.82
4.08 m (5H, C_H2CHCH_ 2OPOC_H), 4.31 mm(1H,
*
Here and hereinafter the region of 0.67 2.47 ppm character-
27
istic of protons attached to atoms C1,2, C4,5 and C7 of the
phosphocholesterol part of the molecule [8] will be designated
as (H, cholesterol).
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 38 No. 11 2002

SYNTHESIS OF DIESTERAMIDE PHOSPHOLIPIDS OF CATIONIC TYPE
1619
3
CH2C_HCH2OP, JHH 5.6 Hz). 31P NMR spectrum,
, ppm: 9.84 br.s. Found, %: C 49.98; H 8.67; P
5.88. C22H46BrN2O5P. Calculated, %: C 49.90;
H 8.76; P 5.85.
PNC_H2CH2), 3.80 4.16 m [2H, POCH (menthyl) and
C3H (cholesterol)], 5.21 m [1H, HC6 (cholesterol)].
31P NMR spectrum, , ppm: 9.96 br.s. Found, %:
C 65.73; H 10.22; P 3.91. C43H80BrN2O3P. Cal-
culated, %: C 65.88; H 10.29; P 3.95.
O-(1,2-Distearoyl-3-glycerol)-O-(3-menthyl)[N-
methyl-N-(2-trimethylammonioethyl)]amidophos-
phate bromide (XII) was prepared in a similar way
as compound XI from 0.3 g of amidophosphate IX
and 0.17 g of trimethylamine at 80 C within 16 h.
Yield 0.24 g (75%), mp 142 147 C. Rf (elution
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1
system): 0.09 (A), 0.27 (C). H NMR spectrum, ,
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Khimiya, 1983.
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(distearoylglycerol)], 0.89 2.32 m [9H, CH2 and CH
(menthyl)], 1.29 m [56H, CH3(C_H2)14], 1.68 · [4H,
C_H2CH2C(O)], 1.98 m [4H, CH2C_H2C(O)], 2.77 d
(3H, PNCH3), 3.16 s [9H, N+ (CH3)3], 3.28 m (2H,
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(2H, CH2CHCH_ 2OP), 5.14
m
,
(1H,
ppm:
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P 3.03.
O-(3-Menthyl)-O-(3-cholesteryl)-[N-methyl-N-(2-
trimethylammonioethyl)]amidophosphate bromide
(XIII). Likewise from 0.2 g of amidophosphate X
and 0.16 g of trimethylamine at 80 C within 16 h was
prepared compound XIII. Yield 0.15 g (69%), mp
136 138 C. Rf (elution system): 0.04 (A), 0.21 (C).
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3
2.62 d (3H, PNCH3, JPH 9.52 Hz), 3.07 s [9H,
N+ (CH3)3], 3.36 m (2H, PNCH2C_H2), 3.68 m (2H,
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 38 No. 11 2002