Cationic amphiphiles of methylphosphonates and cholesterol phosphocholines and their homo derivatives

Article abstract of DOI:10.1023/B:RUGC.0000016015.55496.b9

A method of synthesis of (P → O) and (P → N)-choline cholesteryl methylphosphonates and their homo derivatives of the cationic type is developed. These compounds were obtained by the Arbuzov reaction between bromomethane and cyclic cholesteryl phosphorous esters and amidoesters and subsequent aminolysis of the intermediate bromoalkyl phosphonates. Alkylation of trimethylamine with cholesteryl ethanediyl and propanediyl phosphates gives cholesterol phosphocholines and their homo derivatives.

Full text of DOI:10.1023/B:RUGC.0000016015.55496.b9

Russian Journal of General Chemistry, Vol. 73, No. 10, 2003, pp. 1530 1534. Translated from Zhurnal Obshchei Khimii, Vol. 73, No. 10, 2003,
pp. 1620 1625.
Original Russian Text Copyright
2003 by Predvoditelev, Malenkovskaya, Nifant’ev.
Cationic Amphiphiles of Methylphosphonates
and Cholesterol Phosphocholines and Their Homo Derivatives
D. A. Predvoditelev, M. A. Malenkovskaya, and E. E. Nifant’ev
Moscow State Pedagogical University, Moscow, Russia
Received April 24, 2002
Abstract A method of synthesis of (P O) and (P N)-choline cholesteryl methylphosphonates and their
homo derivatives of the cationic type is developed. These compounds were obtained by the Arbuzov reaction
between bromomethane and cyclic cholesteryl phosphorous esters and amidoesters and subsequent aminolysis
of the intermediate bromoalkyl phosphonates. Alkylation of trimethylamine with cholesteryl ethanediyl and
propanediyl phosphates gives cholesterol phosphocholines and their homo derivatives.
In the last ten years there has been considerable
O
synthetic work on cholesterol-containing phospohorus-
free cationic amphiphiles [1, 2]. Such lipids are being
actively studied as components of cationic liposoms
for transport of genetic material into the cell, as well
as medicinals [3, 4]. At the same time, we have been
the only who synthesized phosphorus lipids with
cholesterol as structural domain. Hence, the synthesis
of cholesterol-containing thiophospholipids, such as
R O P
(CH2)n
O
I, II
CH3
CH Br
3
R O P O (CH ) Br,
2
n
O
III, IV
phosphocholines,
phosphomethylcolamines,
and
phosphodiols [5], as well as of amidodiester (P N)-
cholesterol phospholipids of the cationic type [6] has
been accompished. Cholesteryl phospholipids of the
cationic type, owing to specific properties of chole-
sterol, may present considerable interest in terms of
transfection effectiveness [7].
R =
n = 2 (I, III), 2 (II, IV).
Proceeding with our research, we set ourselves the
task to develop synthetic approaches to formerly
unknown cationic methylphosphonate analogs of
cholesterol, as well as to cholesterol phosphocholines
and their homo derivatives.
1
30.34 and 30.02 ppm, respectively. The H NMR
spectra of these compounds show expected signals of
all proton groups.1 Hence, the PCH protons, due to
3
coupling with phosphorus, appear as a doublet at
.47 ppm ( J 17.04 17.50 Hz). The CH Br me-
The first line of out work was based on the use of
alkanediyl cholesteryl phosphites in the synthesis of
cationic cholesteryl methylphosphonates. To this end,
we performed Arbuzov alkylation of the available
ethanediyl and propanediyl phosphites (I, II) with
bromomethane.
2
1
PH
2
thylene protons give characteristic triplet signals at
3
3
.48 3.49 ppm, the cholesteryl C HOP proton, a
6
multiplet at
4.38 ppm, and the cholesteryl C H=
7
proton, due to coupling with the C H methylene
2
protons, a doublet at 5.38 ppm. The rest signals in
the spectra of the acyclic cholesteryl phosphonates
were roughly the same as in the spectra of the starting
phosphites I, II (see Experimental).
The reaction was carried out at 110 120 C. The
yield of bromoalkyl phosphonates III, IV reached
4
0 65%. The products are colorless oils. Importantly,
these compounds are stable and can be handled in
dark closed vessels at 5 C for several months. Reac-
3
1
1
1
tion progress was controlled by P NMR spectro-
Analysis of the H NMR spectra was carried out with account
scopy. The 31P { H} NMR spectra of cholesteryl
1
for data of Facke and Berger [8] who performed detailed
assignment of the H NMR spectra of cholesterol.
1
methylphosphonates III, IV contain singlets at
P
1
070-3632/03/7310-1530$25.00 2003 MAIK Nauka/Interperiodica

CATIONIC AMPHIPHILES OF METHYLPHOSPHONATES
1531
Bromoalkyl phosphonates III, IV were used for pre-
paring cationic amphiphiles V, VI. To this end, phos-
phonates III, IV were reacted with ith trimethylamine.
Propanediyl phosphorochloridate proved to be
much less reactive compared to propanediyl phos-
phorochloridite toward cholesterol (VIII) and could
only be reacted at 80 C within 8 h. The yield of the
target product IX isolated on a silica gel column was
as low as 35%.
CH3
+
N(CH3)3
III, IV
R O P O(CH ) N(CH ) Br ,
2
n
3 3
O
Cholesteryl alkanediyl phosphates VII, IX, unlike
the corresponding thiophosphates [5], are unstable
hygroscopic crystalline substances. They can be
stored without decomposition in closed vials only
for several days at 5 C.
V, VI
Quarternization of trimethylamine occurred at room
temperature (about 22 C) within 12 h. Earlier quarter-
nization of glycerol bromoalkyl phosphonates in the
synthesis of cationic amphiphiles of phosphoglycerols
was also performed at room temperature, but for a
longer time (70 72 h) [9]. Reaction progress was
controlled by TLC. The yields of chromatographically
pure ammonium compounds V, VI were 52 65%.
They are solid amorphous substances.
The purity and structure of phosphates VII, IX
31
1
was proved by TLC and P and H NMR spectro-
31
scopy. The P NMR spectra of compounds VII, IX
contained singlets at
15.98 and 8.76 ppm, res-
P
1
pectively. The H NMR spectra of phosphates VII,
IX showed signals characteristic of all proton groups.
Thus, the spectrum of ethanediyl phosphate VII
contained a multiplet of methylene protons of the
phospholane ring at 4.40 ppm, whereas the spectrum
of propanediyl phosphate IX, characteristic multiplets
of protons of the phosphorinane ring at 2.20 and
4.46 ppm. The other signals in the spectra of phos-
phates VII, IX were consistent with the proposed
structures (see Experimental).
1
The H NMR spectra of phosphonates V, VI
+
contain singlets of the N (CH ) group at
3.32
3
3
3
.40 ppm. The triplets of the CH Br methylene
protons have disappeared, and, instead of them,
multiplet signals of the CH N (CH ) methylene
protons have appeared. The other signals in the spec-
tra of compounds V, VI practically coincide with
those of the starting bromoalkanols (see Experimental).
2
+
2
3 3
To pass to cholesterol phosphatide structures of the
betain type, we performed alkylation of trimethyl-
amine with phosphates VII, IX.
Cholesteryl alkanediyl phosphates were also used
for preparing cholesterol phosphocholine and its homo
derivative. As the starting material for preparing
cholesterol phosphocholine we used cholesteryl
ethanediyl phosphite (I) which was oxidized to
cholesteryl ethanediyl phosphate (VII) with urea
hydrogen peroxide adduct (20 C, 5 h). The product
was isolated by column chromatograpghy in 40% yield.
N(CH3)3
+
VII, IX
R O P O(CH ) N(CH ) ,
2
n
3 3
O
O
X, XI
n = 2 (III, V), 3 (IV, VI).
O
O
The reactions with trimethylamine were carried out
in benzene at 90 100 C for 30 35 h. Then benzene
was removed in a vacuum, and the remaining target
products were washed with hexane. Cholesterol
phosphocholine X and phosphohomocholine XI were
obtained as colorless crystals in 30 and 42% yields,
respectively. Note that in the case of cholesterol thio-
phosphocholines prepared by the analogous procedure
from cholesteryl thiophosphates the yields of the
H NCNH H O
2 2
2
2
I
R O P
O
O
VII
To extend the scope of the method of synthesis
of cholesterol phosphocholines, we made an at-
tempt to oxidize cholesteryl propanediyl phosphite
(
II). Unfortunately, this oxidation failed. Therefore,
the target product was prepared by reacting chole-
sterol (VIII) with propanediyl phosphorochloridate in
the presence of triethylamine.
target products were higher, 40 and 80%, respectively
31
[
5]. The P NMR spectra of phosphocholines X, XI
contained singlet signals at
0.82 and 0.27 ppm,
P
1
O
O
respectively. The H NMR spectra of these com-
pounds displayed singlets of ammonium methyl
protons at 3.32 3.40 ppm. In addition, the spectrum
of betain X contained multiplets of -methylene
O
Cl P
O
R OH
R O P
O
O
VIII
+
protons of the CH N (CH ) group at 3.65 ppm,
2
3 3
IX
that of betaine XI, multiplets of - and -methylene
+
protons of the CH CH N (CH ) group at 2.38 ppm
2
2
3 3
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 73 No. 10 2003

1532
PREDVODITELEV et al.
1
and 2.85 ppm. The other signals in the H NMR
spectra had only slightly different chemical shifts
compared to the respective signals of the starting
cyclic phosphates VII, IX (see Experimental).
proton signals of compounds XIII, XIV almost did
not differ from each other (see Experimental).
The present work resulted in the synthesis of a
group of original phosphocholesterol systems belong-
ing to cationic phospholipids. The synthesized com-
pounds offer interest as objects for research into lipid
exchange. Moreover, they can be used for studying
the behavior of a polar head incorporated into the
rigid lipid matrix.
In the final stage of this work we applied the
proposed synthetic scheme for preparing cholesteryl
ethylamidophosphonate of the cationic type. Such
(
P
N) phospholipids, unlike (P O) phospholipids,
have a more polar phosphoryl group due to the pre-
sence of a phosphamide fragment in its structure,
and this may influence the properties of membrane
constructions on their basis.
EXPERIMENTAL
1
The H NMR spectra were obtained on a Bruker
CH3
O
WM-250 (250 MHz) spectrometer against internal
HMDS. Signal assignment was made on the basis of
double-resonance data. The 31P { H} spectra were
measured on a Bruker WP-80 SY spectrometer
CH Br
3
R O P
R O P NCH Br
2
1
N
O CH3
CH3
XII
XIII
(
32.4 MHz) against external 85% phosphoric acid.
Column chromatography was performed on a column
5 mm in diameter, filled on Silica gel L (100
CH3
1
N(CH3)3
+
R O P NCH CH N(CH ) Br
160 m). Thin-layer chromatography was performed
on Silufol UV-254 plates, eluents 3:1 benzene di-
oxane (A), 3:1 hexane dioxane (B), and 65:25:4
chloroform methanol water (C). The melting points
were measured in a sealed capillary at a heating rate
of 1 deg/min. The urea hydrogen peroxide adduct was
prepared according to [10], 2-chloro1,3,2-dioxaphos-
pholane, according to [11], 2-chloro-1,3,2-dioxaphos-
phorinane, according to [12], and propanediyl pros-
phorochloridite, according to [13].
2
2
3 3
O CH3
XIV
The synthesis of lipid aminophosphonate amphi-
philes was begun from alkylation of the previously
prepared by us [5] cholesteryl phosphoramidite XII
with bromomethane (90 C, 8 h). Then cholesteryl
phosphonamidate XIII was purified by column
chromatography on silica gel and reacted with tri-
methylamine to obtain cationic ammonium phospho-
lipid XIV (22 C, 12 h). This compound was preci-
pitated from benzene as a white powder. The yield of
chromatographically pure product XIV was 42%.
The 31P NMR spectra of compounds XIII, XIV
2-Bromoethyl cholesteryl methylphosphonate
(III). A sealed ampule with a solution of ethanediyl
phosphite (I) (prepared according to [5] from
2 g of cholesterol, 0.64 g of ethanediyl phosphorochlo-
ridite, and 0.5 g of triethylamine) and 2.95 g of bro-
momethane in 20 ml of anhydrous benzene was kept
for 12 h at 110 C. The solvent was removed in a
vacuum, and ethanediyl phosphonate III was isolated
on a column of silica gel (20 g), filled with hexane.
Compound III was eluted with 50 ml of a 5:1
hexane dioxane mixture. The solvents were removed
contained two singlet signals at
33.12 and 33.42
P
(
XIII) and 34.39 and 34.69 ppm (XIV). Such a
complification of the spectra is connected with the
fact that these compounds are present as diastereo-
meric pairs due to the cholesteryl radicals and racemic
1
phosphorus atom. The H NMR spectra of phos-
in a vacuum, and the residue was kept for 2 h at 40 C
phonates XIII, XIV contained signals characteristic
of all proton groups, sometimes complicated by
20
(
1 mm). Yield 1.2 g (40%), n 1.5057, R 0.55 (A),
D
f
1
0
2
1
.27 (B). H NMR spectrum (CDCl ), , ppm: 0.68
3
stereomeric anisochronicity. Hence, the PCH protons
.47 (cholesterol H),2 1.47 d (3H, PCH3, 2JHP
3
appear as doublets at
1.45 ppm, and the NCH3
7.50 Hz), 3.48 t (2H, CH Br), 4.24 m (3H, POCH ,
cholesterol OPC H), 5.33 d (1H, cholesterol C H=,
JHH 3.40 Hz). P NMR spectrum (chloroform), P,
2
2
protons, as a doublet at 2.75 3.40 ppm. In addition,
3
6
1
the H NMR spectrum of compound XIII showed a
3
31
triplet at 3.02 ppm, characteristic of CH Br groups.
2
ppm, P: 30.34 s. Found,%: C 63.10; H 9.25; P 5.50.
C H BrO P. Calculated,%: C 63.03, H 9.17, P 5.42.
The spectrum of choline phosphonate XIV differed
from the spectrum of the acyclic compound XIII by
3
0
52
3
the presence of a singlet at
3.50 ppm from the
+
+
2
N (CH ) methyl protons, a multiplet of the CH N
Here and hereafter, the region at 0.68 2.47 ppm, characte-
3
3
2
1
,2
4,5
7 27
(
CH ) methylene protons at 3.56 ppm, as well as
ristic of protons on the C , C , and C
atoms of the
3
3
by the absence of the CH Br triplet. All the other
phosphocholesterol moiety [8], is labeled (cholesterol H).
3
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 73 No. 10 2003

CATIONIC AMPHIPHILES OF METHYLPHOSPHONATES
1533
3
-Bromopropyl cholesteryl methylphosphonate
according to [5] from 1 g of cholesterol and 0.32 g of
ethanediyl phosphorochloridite in the presence of
0.3 g of triethylamine) in 20 ml of anhydrous benzene,
0.2 g of urea hydrogen peroxide adduct was added at
room temperature, and the reaction mixture left to
stand at that temperature for 5 h. Excess adduct was
filtered off, and the solvent was removed in a vacuum.
Compound VII was purified on a column of silica gel
(10 g), filled with hexane, eluent 5:1 hexane dioxane
(40 ml). The solvents were removed in a vacuum, and
the residue was kept for 2 h at 40 C (1 mm). Yield
(
IV). A sealed ampule with a solution of propanediyl
phosphite (II) (prepared according to [5] from 2.5 g
of cholesterol, 0.73 g of propanediyl phosphorochlori-
dite, and 0.62 g of triethylamine), and 3 g of bromo-
methane in 20 ml of anhydrous benzene was kept at
1
20 C for 15 h. The solvent was removed in a
vacuum, and phosphonate IV was isolated on a
column of silica gel (30 g), filled with hexane. Com-
pound IV was eluted with 50 ml of a 3:1 hexane
dioxane mixture. The solvents were removed in a
vacuum, and the residue was kept for 2 h at 40 C
1 mm). Yield 2.5 g (65%), n 1.4981. R 0.60 (A),
.30 (B). H NMR spectrum (CDCl ), , ppm: 0.67
.47 (cholesterol H), 1.47 d (3H, PCH3, JPH
7.04 Hz), 2.61 m (2H, POCH CH CH , JHH
.15 Hz), 3.49 t (2H, CH Br, J 6.6 Hz), 4.14 m
2H, POCH ), 4.38 m (chlesterol OPC H), 5.38 d
0.38 g (40%), mp 145 148 C, R 0.70 (A), 0.45 (B).
f
2
0
1
(
H NMR spectrum (CDCl3), , ppm: 0.68-2.47
D
f
1
0
2
1
7
(
(
(cholesterol H), 4.40 m (4H, OCH CH O), 4.47 m
3
2
2
2
3
(1H, cholesterol OPC H), 5.39 d (1H, cholesterol
3
6
3
31
C H=,
JHH 4.30 Hz).
P
NMR spectrum
2
2
2
3
(chloroform), P, ppm: 15.98 s. Found, %: C 36.91,
H 9.89, P 6.03. C H O P. Calculated, %: C 36.56,
2
HH
3
2
29 49
4
6
3
31
1H, cholesterol C H=, J 3.80 Hz). P NMR spec-
rum (chloroform), P, ppm: 30.02 s. Found,%: C
3.61; H 9.51; P 5.43. C H BrO P. Calculated,%:
H 10.02, P 6.29.
HH
5
2-Cholesteryloxy-1,3,2 -dioxaphosphorinane
2-oxide (IX). To a solition of a mixture of 1 g of
cholesterol (VIII) and 0.25 g of triethylamine in
6
3
1
54
3
C 63.57; H 9.29, P 5.29.
3
0 ml of anhydrous benzene, a solution of 0.4 g of
Cholesteryl 2-(dimethylamino)ethyl methyl-
phosphonate bromomethylate (V). A sealed ampule
with a solution of 1 g of phosphonate III and 0.36 g
of trimethylamine in 10 ml of anhydrous benzene was
kept at room temperature for 12 h. To remove the
solvent, the gel-like reaction mixture was kept for
propanediyl phosphorochloridate in 5 ml of the same
solvent was added dropwise with stirring at 0 C. The
temperature of the reaction mixture was gradually
raised to 80 C and maintained for 8 h. Then triethyl-
amine hydrochloride was filtered off, and benzene was
removed in a vacuum. Compound IX was purified on
a column of silica gel (15 g), filled with hexane,
eluent 3:1 hexane dioxane (50 ml). The solvent was
removed in a vacuum, and the residue was kept for
2 h at 40 C (1 mm). Yield 0.45 g (35%), mp 190
192 C, R 0.70 (A), 0.50 (B). H NMR spectrum
(CDCl ), , ppm: 0.67-2.46 (cholesterol H), 2.20 m
(2H, OCH CH CH O), 4.46 m (5H, POCH CH
CH O, cholesterol OPC H), 5.41 d (1H, cholesterol
C H=, J
form), P, ppm: 8.76 s. Found,%: C 71.38,H 10.31,
P 6.23. C H O P. Calculated,%: C 71.11; H 10.14;
P 6.11.
0
2
.5 h at 40 C (1 mm). Yield 0.6 g (55%), mp 210
1
12 C (begins to melt at 185 C), R 0.60 (C). H
f
NMR spectrum (CDCl ), , ppm: 0.67-2.38 (chole-
sterol H), 1.56 d (3H, PCH , J 17.05 Hz), 3.54 s
3
2
3
PH
+
+
(9H, N Me ), 4.22 br.m (2H, POCH CH N ),
3 2 2
1
+
f
4
.44 br.m (2H, POCH CH N ), 4.54 br.m (1H,
2 2
3
3
cholesterol OPC H), 5.39 br.m (1H, cholesterol
6
31
2
2
2
2
2
C H=). P NMR spectrum (chloroform), P, ppm:
1.00 s. Found, %: C 63.01; H 9.98; P 5.01. C H
3
2
3
3
3
61
6
3
31
4.85 Hz). P NMR spectrum (chloro-
BrNO P. Calculated, %: C 62.84; H 9.75; P 4.91.
HH
3
Cholesteryl 3-(dimethylamino)propyl methyl-
phosphonate bromomethylate (VI) was obtained
analogously to phosphonate V from 1.5 g of com-
pound IV and 0.62 g of trimethylamine in 15 ml of
3
0
51
4
Cholesterol phosphocholine X. A sealed ampule
with a solution of 0.4 g of phosphate VII and 0.15 g
of trimethylamine in 5 ml of anhydrous benzene was
heated at 90 95 C for 30 h. The solvent was removed
in a vacuum, and the oily residue was washed with
hexane (2 5 ml) and dried for 2 h at 40 C (1 mm).
anhydrous benzene. Yield 1.02 g (62%), mp 255
1
2
57 C (begins to melt at 210 C), R 0.63 (C). H
f
NMR spectrum (CDCl ), , ppm: 0.63 2.40 (chole-
3
3
sterol H), 1.49 d (3H, PCH , J 17.18 Hz), 2.23 br.s
3
PH
+
(
2H, POCH CH CH N ), 4.17 br.m (1H, choleste-
2 2 2
3
6
31
Yield 0.3 g (30%), mp 285 286 C (begins to melt at
2
rol OPC H), 5.35 br.m (1H, cholesterol C H=).
NMR spectrum (chloroform), P, ppm: 30.69 s. Found,
: C 63.51; H 10.01; P 5.05. C H BrNO P. Cal-
P
1
50 C), R 0.00 (A), 0.30 (C). H NMR spectrum
f
(
CDCl ), , ppm: 0.67 2.42 (cholesterol H), 3.40 s
%
3
3
4
63
3
+
+
(9H, N Me ), 3.65 br.s (2H, POCH CH N ),
culated, %: C 63.33; H 9.85; P 4.80.
3 2 2
3
4
.38 br.m (1H, cholesterol OPC H), 5.40 br.m (1H,
2
-Cholesteryloxy-1,3,2 5-dioxaphospholane
cholesterol C H=). P NMR spectrum (chloroform),
P, ppm: 0.2 s. Found, %: C 69.72; H 10.49, P 5.43.
6
31
2
-oxide (VII). To a solution of ester I (prepared
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 73 No. 10 2003

1534
PREDVODITELEV et al.
2
3
C H NO P. Calculated, %: C 69.65; H 10.60,
P 5.61.
(3H, PCH , J 15.79 Hz), 2.75 d (3H, PNCH , J
PH
3
2
58
4
3
PH
3
+
9.81 Hz), 3.50 s (9H, N Me ), 3.56 br.m (2H, PNCH
3 2
CH ), 4.03 br,m (3H, PNCH CH and cholesterol
2
2
2
Cholesterol phosphohomocholine (XI) was pre-
pared analogously from 0.5 g of phosphate IX and
3
6
31
OPC H), 5.36 br.m (1H, cholesterol C H=). P NMR
spectrum (chloroform), P, ppm: 34.39 and 34.69,
intensity ratio 10:8. Found, %: C 63.53; H 10.21,
P 4.95. C H BrN O P. Calculated, %: C 63.43;
0
.25 g of trimethylamine in 10 ml of anhydrous ben-
zene at 90 100 C for 35 h. Yield 0.3 g (42%), mp
3
4
64
2
2
3
0
00 301 C (decomp.) (begins to melt at 285 C), R
f
H 10.02, P 4.81.
1
.00 (A), 0.25 (B). H NMR spectrum (CDCl ), ,
3
ppm: 0.67 2.48 (cholesterol H), 2.38 br.m (2H,
ACKNOWLEDGMENTS
+
POCH CH CH N ), 2.85 br.m (2H, POCH CH
2
2
2
2
2
+
+
CH N ), 3.32 s (9H, N Me ), 3.75 br.m (2H, POCH
The work was financially supported by the Russian
Foundation for Basic Research (project no. 01-03-
32459).
2
3
2
+
3
CH CH N ) 4.20 br.m (1H, cholesterol OPC H),
2
2
6
31
5
.35 br.m (1H, cholesterol C H=). P NMR spectrum
chloroform), P, ppm: 0.27 s. Found, %: C 70.15; H
0.81, P 5.58. C H NO P. Calculated, %: C 70.04;
(
1
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3
60
4
H 10.69; P 5.47.
1
2
. Okayama, R., FEBS Lett., 1997, vol. 408, no. 2,
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phospholane (XIII) (prepared according to [5] from
. Konstantinova, T.V., Klykov, V.N., and Serebrenni-
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2
g of cholesterol and 2.56 g of hexaethylphosphorous
3
4
. Miller, A.D., Angew. Chem. Int. Ed. Engl., 1998,
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cholesterol tetraethylphosophorodiamidite with 0.4 g
of N-methylethanolamine) and 3.5 g of bromometha-
ne in 25 ml of anhydrous benzene was kept at 90 C
for 8 h. The solvent was removed in a vacuum, and
phosphonate XIII was isolated on a column of silica
gel (15 g), filled with hexane, eluent 1:1 hexane di-
oxane (50 ml). The solvent was removed in a vacuum,
and the residue was kept for 2 h at 40 C (1 mm).
. Maslov, M.A., Sycheva, E.V., Morozova, H.G., and
Serebrennikova, G.A., Izv. Ross. Akad. Nauk, 2000,
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fant’ev, E.E., Trudy 13 mezhdunarodnoi konferentsii
po khimii soedinenii fosfora (ICCPC-XII) (Proc. 13-th
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pounds), St. Petersburg, 2002, p. 130.
2
0
Yield 1.4 g (45%), n 1.4785, R 0.45 (A), 0.19 (B).
D
f
1
H NMR spectrum (CDCl3), , ppm: 0.65 2.47
6. Predvoditelev, D.A., Kosarev, G.V., Suvorkin, S.V.,
and Nifant’ev, E.E., Zh. Org. Khim., 2002, vol. 38,
no. 121, p. 1671.
2
(cholesterol H), 1.45 d (3H, PCH , J 12.02 Hz),
3 PH
3
2
2
.06 m and 2.66 m (2H, PNCH CH , J 9.35 Hz),
2 2 PH
3
7
. Tarakhovskii, Yu.S. and Ivanitskii, T.V., Biokhimiya,
998, vol. 63, no. 6, p. 723 725.
.99 t and 3.02 t (2H, CH Br, J 7.14 Hz), 3.43 d
2
HH
3
1
(
3H, PNCH , J 2.20 Hz), 4.30 m (1H, cholesterol
3 HP
3
6
3
OPC H), 5.36 d (1H, cholesterol C H=, J 5.50 Hz).
8. Facke, T. and Berger, S., Tetrahedron, 1995, vol. 51,
HH
3
1
P (chloroform), P, ppm: 33.12 s and 33.42 s,
intensity ratio 10:8. Found, %: C 63.72; H 9.63.
C H BrNO P. Calculated, %: C 63.68, H 9.48,
no. 12, pp. 3521 3524.
9
. Alarkon, Kh. Kh., Predvoditelev, D.A., and Nifan-
t’ev, E.E., Bioorg. Khim., 1978, vol. 4, no. 11,
pp. 1513 1519.
3
1
55
2
P 5.30.
1
0. Nifant’ev, E.E., Rasadkina, E.N., and Yankovich, I.V.,
Cholesteryl N-(2-bromoethyl)-N,P-dimethyl-
phosphonamidate bromomethylate (XIV) was ob-
tained similarly to phosphonate V from 1 g of com-
pound XIII and 0.45 g of triethylamine in 10 ml of
Zh. Obshch. Khim., 1999, vol. 69, no. 1, pp. 36 42.
11. Gefter, E.L., Zh. Obshch. Khim., 1956, vol. 26, no. 5,
pp. 1440 1443.
anhydrous benzene. Yield 0.46 g (42 C), mp 225
1
2. Zwierzak A., Can. J. Chem., 1967, vol. 45, no. 21,
1
2
27 C (begins to melt at 205 C), R 0.69 (C). H
f
pp. 2501 2512.
NMR spectrum (CDCl ), , ppm: 0.66 2.37 (chole-
3
13. US Patent 2892862, Chem. Abstr., 1960, vol. 54,
2
sterol H), 1.46 d (3H, PCH , J 15.79 Hz), 1.46 d
15740.
3
PH
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 73 No. 10 2003