Synthesis of lipid mediators based on 1,2-dialkylglycerol and cholesterol for targeted delivery of oligo-and polynucleotides into hepatocytes

Article abstract of DOI:10.1007/s11172-010-0070-y

The synthesis of cationic glycolipids containing galactose or lactose residue as hepatocytetargeted markers is described.

Full text of DOI:10.1007/s11172-010-0070-y

Russian Chemical Bulletin, International Edition, Vol. 59, No. 1, pp. 251—259, January, 2010
251
Synthesis of lipid mediators
based on 1,2ꢀdialkylglycerol and cholesterol
for targeted delivery of oligoꢀ and polynucleotides into hepatocytes
N. G. Morozova, M. A. Maslov, O. A. Petukhova, S. V. Andronova, A. O. Grishaeva, and G. A. Serebrennikova
M. V. Lomonosov Moscow State Academy of Fine Chemical Technology,
86 prosp. Vernadskogo, 119571 Moscow, Russian Federation.
Fax: +7 (495) 936 8901. Eꢀmail: mamaslov@mail.ru
The synthesis of cationic glycolipids containing galactose or lactose residue as hepatocyteꢀ
targeted markers is described.
Key words: cationic lipids, cholesterol, dialkylglycerol, galactose, lactose, lipofection, heꢀ
patocytes.
Among the methods of delivery of genetic material
into eukaryoic cells, considerable place belongs to lipofecꢀ
tion, i.e., transfer by means of cationic amphiphiles or
liposomes.1 The positively charged liposomes form elecꢀ
trostatic complexes with nucleic acid molecules that have
been called lipoplexes. In these complexes, nucleic acids
are protected from degradation under the action of cell
enzymes, they penetrate into the cell by the endocytosis
mechanism. The gene delivery in vitro by lipid transꢀ
fer agents is a routine procedure; however, the use of
this method in vivo is restricted by low efficiency of the
transfer, which is due to the presence of various types of
biological barriers (for example, to the interaction of
DNA—lipid complexes with blood plasma proteins and their
capture by the reticuloendothelial system, and to low specꢀ
ificity of targeting organs and tissues).2 For increasing the
efficiency of the delivery of genetic material, special eleꢀ
ments that help overcoming the biological barriers are
introduced in the structure of cationic amphiphiles.3 For
example, for addressing specific cells, the molecule of
a cationic lipid is modified by targeting peptide or carboꢀ
hydrate ligands.4
Cationic lipids comprise an extensive range of comꢀ
pounds with common structural features such as the presꢀ
ence of positively charged and hydrophobic domains conꢀ
nected by a spacer.5—7 The hydrophilic positively charged
domain is needed for binding nucleic acid and the hydroꢀ
phobic domain is needed for its encapsulation. In recent
years, carbohydrate residues have been incorporated more
and more often in cationic amphiphile structures; they
acts as targeting markers for the delivery of nucleic acids
to hepatocytes,8—11 macrophages,12,13 and the nucleus of
the HeLa cells.14 It is known that on the surface of hepaꢀ
tocytes, glycoprotein receptors are exposed, by means of
which cell selectively recognize, bind, and internalize gaꢀ
lactoseꢀcontaining molecular assemblies. This feature can
be used for targeted delivery of medical drugs by means of
liposomes incorporating synthetic galactolipids. In addiꢀ
tion, the carbohydrate residue can serve as the basis for
construction of one or more cationic groups.15—18 Amꢀ
phiphiles containing carbohydrate residues are able to
change the supramolecular structure of lipoplexes, inducꢀ
ing a рHꢀdependent transition of lipids from the lamellar
to micellar phase and thus increasing the efficiency of the
delivery of genetic material.19,20 In addition, carbohydrate
fragments increase the colloid stability of lipoplexes in
blood serum21,22 and decrease their toxicity.23
Here we report the synthesis of monocationic galactoꢀ
lipids, which are potential transfer agents of polyꢀ and
oligonucleotides into eukaryotic cells. We assume that synꢀ
thesized glycolipids would be able to specifically interact
with hepatocyte receptors owing to the presence of targetꢀ
ing carbohydrate residues.
As the hydrophobic domain needed for incorporation
into a lipid bilayer, we used cholesterol and 1,2ꢀdiꢀOꢀ
tetradecylꢀracꢀglycerol residues. In the series of glycerol
derivatives with saturated longꢀchain hydrocarbon resiꢀ
dues, lipids containing tetradecyl substituents favor the
best penetration of lipoplexes into the cells.5,24 The bindꢀ
ing of hydrophobic, hydrophilic, and targeting domains
was performed by a spacer based on 6ꢀaminohexanol,
which is a suitable bifunctional molecule for the assembly
of domains into a common structure of cationic glycoꢀ
lipid. The amino group in the 6ꢀaminohexanol molecule
can serve for the formation of a positively charged ammoꢀ
nium group, while the hydroxyl group can serve as the
acceptor for the introduction of carbohydrate residues. In
the designed glycolipid molecules, the cationic head is
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 245—253, January, 2010.
1066ꢀ5285/10/5901ꢀ0251 © 2010 Springer Science+Business Media, Inc.

252
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Morozova et al.
attached to the hydrophobic fragment either directly or
through a hexamethylene spacer. In the latter case, the
spacer was linked to the hydrophobic domain by an ureꢀ
thane bond.
group is easily removed under the action of benzenethiol
or thioglycolic acid. This approach was implemented as
the reaction between 6ꢀaminohexanol (4) and 2ꢀnitrobenꢀ
zenesulfonyl chloride in the presence of Et3N. This gave
sulfonamide 7 (Scheme 2), which was alkylated with broꢀ
mo derivatives 2, 6a, and 6b in the presence of Cs2CO3 in
DMF at 80 °C to give amides 8a—с in 64—86% yields.
Note that in the case of bromides 6a,b with a spacer group,
the reaction time was 3 h, whereas alkylation with broꢀ
mide 2 required 48 h, apparently, due to steric hindrance.
The next important stage of the synthesis was the
introduction of the carbohydrate markers by the formaꢀ
tion of a glycosidic bond. Glycosylation of compounds
8a—с with 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀgalactosyl and
2,2´,3,3´,4´,6,6´ꢀheptaꢀOꢀacetylꢀαꢀlactosyl bromides was
carried out by one of the variants of the Königs—Knorr
method in a Soxhlet apparatus using CdCO3 as the proꢀ
moter (see Ref. 28). The yields of the βꢀglycosylation prodꢀ
ucts 9a—c and 10 were 45% (for lactose) and 69% (for
According to the developed synthetic route, first, we
prepared bromo derivatives of 1,2ꢀdiꢀOꢀtetradecylꢀracꢀ
glycerol and cholesterol (Scheme 1). The starting 1,2ꢀdiꢀ
Oꢀtetradecylglycerol (1a) was made to react with CBr4 in
the presence of Ph3P to give bromide 2 in 90% yield. For
the synthesis of cationic lipids where the positively charged
group is remote from the hydrophobic residue, 1,2ꢀdiꢀOꢀ
tetradecylglycerol (1a) and cholesterol (1b) were treated,
in the first step, with an excess of carbonyldiimidazole
(CDI) in the presence of Et3N to give stable imidazolides
3a,b. The second step was addition of the spacer group to
the activated hydrophobic component. The optimal yields
of diglyceride 5a (80%) and cholesterol 5b (96%) derivaꢀ
tives were attained using a 1.5ꢀfold excess of 6ꢀaminohexꢀ
anol (4). Then the hydroxyl group was replaced by broꢀ
mine under the same conditions as for compound 2. For
both the diglyceride and the cholesterol derivatives, the
bromination proceeded smoothly and rapidly, resulting in
compounds 6a and 6b in 96 and 83% yields, respectively.
1
galactose). The H NMR spectra of these compounds
showed doublet signals for protons at the anomeric cenꢀ
ters with spinꢀspin coupling constants J1,2 = 7.8—8.0 Hz,
indicating the βꢀconfiguration of the anomeric center. In
the 13C NMR spectra, the signals for anomeric carbon
atoms occur at δ 101.20—101.53, thus supporting the
βꢀconfiguration of the anomeric center. The concomitant
αꢀanomers were isolated in 3 to 8% yields. The acetyl
derivative of alcohol 8a was formed as a glycosylation byꢀ
product (yield 10%). Glycosylation of alcohols is rather
often accompanied by the formation of their acetyl deꢀ
rivatives.29
Scheme 1
The subsequent stages of the synthesis were to be
Nꢀdesulfonylation and deacetylation of compounds 9a—с
and 10 and completion of the cationic head. Two sequencꢀ
es of deprotection reactions were tested. In the first of
them, 2ꢀnitrobenzenesulfonyl group was the first to be
removed (treatment with benzenethiol in the presence of
K2CO3). The secondary amines thus formed were isolated
in low yields, which was caused by the difficulty of their
preparative purification. In addition, we observed partial
deacetylation of the carbohydrate component, probably,
due to the basicity of the secondary amino group. The
second approach according to which compounds 9a—с
and 10 were first deacetylated and then the 2ꢀnitrobenzeꢀ
nesulfonyl group was removed was free from these drawꢀ
backs. In addition, in this case, the useful UVꢀdetectꢀ
able label was longer retained in the lipid molecule. To
remove the acetyl protection, compounds 9a—с and 10
were treated with a freshly prepared methanolic sodium
methoxide. After neutralization of the reaction mixture
with an ionꢀexchange resin, products 11a—с, 12 were isoꢀ
lated by column chromatography on silica gel in 61—98%
yields. The removal of the 2ꢀnitrobenzenesulfonyl protecꢀ
tion in compounds 11a—с and 12 was carried out by treatꢀ
ment with a tenfold excess of benzenethiol and potassium
Reagents and conditions: (a) CBr4, Ph3P, 24 °C; (b) CDI, Et3N,
40 °C; (c) HO(CH2)6NH2 (4), 40 °C.
For the formation of the "procationic" center, the reꢀ
action of nitrobenzenesulfonamides with alkyl halides in
the presence of cesium carbonate was used. The introducꢀ
tion of 2ꢀnitrobenzenesulfonyl protection provides conꢀ
trol over the degree of the Nꢀalkylation. This reaction (the
Fukuyama reaction25—27) is widely used for the synthesis
of secondary amines, because the nitrobenzenesulfonyl

Synthesis of cationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
253
Scheme 2
Comꢀ
pound
R2
R3
9a—c
10
11a—c, 13a—c, 15a—c
12, 14, 16
OAc
H
H
OH
H
H
OꢀβꢀGal(OAc)4
OꢀβꢀGal
Reagents and conditions: (a) 2ꢀNO2C6H4SO2Cl, Et3N, 24 °C; (b) 2 or 6a,b, Cs2CO3, DMF, 80 °C; (c) Gal(OAc)4Br or Lac(OAc)7Br,
CdCO3, C6H6, 80 °C; (d) MeONa/MeOH, 24 °C; (e) PhSH, K2CO3, 24 °C; (f) MeI, K2CO3, MeCOEt, 50 °C.
carbonate in DMF. Due to the high polarity of secondary
amines 13a—с, 14, they were isolated by reversed phase
chromatography on silica gel LiChroprep® RPꢀ18.
The synthesis of monocationic lipids was completed
by quaternization of secondary amines 13a—с, 14, which
was performed by treatment with an excess of MeI. Afꢀ
ter reversed phase chromatography, compounds 15a—с,
16 were isolated in 69—87% yields and their strucꢀ
tures were confirmed by NMR spectroscopy and mass
spectrometry.
Thus, we developed an approach to the preparation
of monocationic lipids with a marker group based
on galactose and lactose. The obtained glycolipids
are meant for targeted delivery of the genetic material to
hepatocytes.
Experimental
Distilled solvents, Russian (Khimmed, Reachim) and forꢀ
eign (Merck, Fluka, Aldrich, Acros) commercial reagents were
used. Benzene was refluxed with Na metal and distilled immediꢀ
ately prior to the reaction; CH2Cl2 and Et3N were refluxed with
CaH2 and distilled; DMF (Merck) was kept over calcined moꢀ
lecular sieves 4 Å.
Thinꢀlayer chromatography was carried out on Kieselgel
60 F254 and Kieselgel RPꢀ18 F254S plates (Merck). The spots
were visualized by the Dragendorff reagent,30 phosphomolybdic
acid—cerium(IV) sulfate system followed by heating,31 or under
UV light (254 nm). Column chromatography was carried out on
silica gel Kieselgel 60 (40—63 μm, Merck) and LiChroprep®
RPꢀ18 (40—63 μm, Merck). The 1H and 13C NMR spectra were
recorded on a Bruker DPXꢀ300 pulse Fourier transform specꢀ

254
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Morozova et al.
trometer in CDCl3 unless otherwise stated (internal SiMe4). Mass
spectra were run on Bruker Ultraflex timeꢀofꢀflight mass specꢀ
trometer (Germany) with laser desorption ionization using
2,5ꢀdihydroxybenzoic acid as the matrix. IR spectra were reꢀ
corded on a Shimadzu IRꢀ435 spectrophotometer (Japan). Opꢀ
tical rotation was measured on a Digytor Jasco DIP 360 photoꢀ
electrical spectropolarimeter (Japan). The melting points were
determined on a Boetius hot stage (Germany).
(2×5 mL), dried with Na2SO4, and the solvent was evaporated.
The residue was chromatographed on a column with silica gel
using the CHCl3—MeOH mixture (40 : 1) as the eluent to give
20
1.39 g (96%) of compound 5b, m.p. 186—188 °C, [α]D –12
(c 0.5, CHCl3). Found (%): C, 76.96; H, 11.22; N, 2.57.
C34H59NO3. Calculated (%): C, 77.07; H, 11.22; N, 2.64.
1H NMR, δ: 0.61 (s, 3 H, C(13)Me); 0.79 (d, 3 H, C(25)Me,
J = 6.5 Hz); 0.80 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.85 (d, 3 H,
C(20)Me, J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.98—1.57
(m, 29 H, Chol, NHCH2(CH2)4); 1.68—2.00 (m, 5 H, Chol);
2.13—2.35 (m, 2 H, H2C(4)); 3.03—3.16 (m, 2 H, CH2NH);
3.57 (t, 2 H, CH2OH, J = 6.4 Hz); 4.34—4.49 (m, 1 H, H(3));
4.50—4.61 (m, 1 H, NH); 5.27—5.34 (m, 1 H, H(6)). 13C NMR,
δ: 12.03, 18.89, 19.51, 21.21, 22.73, 22.99, 24.00, 24.46, 25.48,
26.57, 28.19, 28.35, 28.41, 30.19 , 32.06, 35.97, 36.36, 36.73,
37.17, 38.76, 39.69, 39.91, 40.87, 42.49, 50.18, 56.30, 56.86,
62.90, 74.39, 122.64, 140.03, 156.40.
[racꢀ2,3ꢀBis(tetradecyloxy)propyl] Nꢀ(6ꢀbromohexyl)carbꢀ
amate (6a). Triphenylphosphine (1.34 g, 5.12 mmol) was added
to a cooled (0 °C) solution of compound 5a (2.14 g, 3.41 mmol)
in anhydrous CH2Cl2 (10 mL). Then CBr4 (1.69 g, 5.12 mmol)
was added in portions, and the mixture was stirred for 1 h at
25 °C. Methanol (5 mL) was added and after 10 min, the solvents
were removed in vacuo. The residue was chromatographed on
a column with silica gel using a petroleum ether—EtOAc mixꢀ
ture (10 : 1) as the eluent to give 2.25 g (96%) of compound 6a,
m.p. 32—34 °C. Found (%): C, 65.95; H, 11.14; N, 1.91.
C38H76BrNO4. Calculated (%): C, 66.06; H, 11.09; N, 2.03.
1H NMR, δ: 0.85 (t, 6 H, 2 CH2Me, J = 6.9 Hz); 1.15—1.58
(m, 54 H, 2 (CH2)11, 2 OCH2CH2, NHCH2(CH2)3(CH2)2Br);
1.78—1.88 (m, 2 H, CH2CH2Br); 3.14 (dt, 2 H, NHCH2,
J = 6.2 Hz, J = 6.9 Hz); 3.36 (t, 2 H, CH2Br, J = 6.8 Hz);
3.38—3.63 (m, 7 H, OCH2CHCH2, 2 OCH2CH2); 4.06 (dd, 1 H,
CH2OC(O), J = 5.3 Hz, J = 11.4 Hz); 4.18 (dd, 1 H, CH2OC(O),
J = 3.8 Hz, J = 11.4 Hz); 4.64—4.73 (m, 1 H, NH). 13C NMR,
δ: 14.50, 23.07, 26.26, 26.42, 26.47, 28.17, 29.75, 29.87, 30.03,
30.07, 30.20, 30.39, 32.30, 32.99, 34.06, 41.26, 64.57, 70.75,
70.95, 72.15, 77.21, 156.77.
racꢀ1ꢀBromoꢀ1ꢀdeoxyꢀ2,3ꢀdiꢀOꢀtetradecylglycerol (2). A soꢀ
lution of CBr4 (8.257 g, 24.90 mmol) in CH2Cl2 (10 mL) was
added with stirring to a cooled (4 °C) solution of 1,2ꢀdiꢀOꢀtetꢀ
radecylꢀracꢀglycerol (1a)32 (6.036 g, 12.45 mmol) and Ph3P
(6.531 g, 24.90 mmol) in anhydrous CH2Cl2 (30 mL). After
50 min, MeOH (20 mL) was added, the mixture was stirred for
10 min, and the solvents were removed in vacuo. The solid resiꢀ
due was chromatographed on a column with silica gel using the
petroleum ether—EtOAc mixture (50 : 1) as the eluent to give
6.571 g (96%) of compound 2. Found (%): C, 67.97; H 11.65.
1
C31H63BrO2. Calculated (%): C, 67.98; H, 11.59. H NMR, δ:
0.85 (t, 6 H, 2 CH2Me, J = 6.4 Hz); 1.24 (br.s, 44 H, 2 (CH2)11);
1.49—1.62 (m, 4 H, 2 OCH2CH2); 3.43 (t, 2 H, CH2Br, J = 6.1 Hz);
3.46—3.63 (m, 7 H, OCH2CHCH2, 2 OCH2CH2).
[racꢀ2,3ꢀBis(tetradecyloxy)propyl]imidazoleꢀ1ꢀcarboxylate
(3a). N,N´ꢀCarbonyldiimidazole (0.7 g, 4.32 mmol) and Et3N
(0.43 mL, 3.1 mmol) were added to a solution of 1,2ꢀdiꢀOꢀtetꢀ
radecylꢀracꢀglycerol (1a) (1.0 g, 2.06 mmol) in anhydrous
CH2Cl2 (17 mL). The reaction mixture was refluxed for 2 h,
cooled, washed with 3% HCl (3×5 mL) and water (2×5 mL), and
dried with Na2SO4, and the solvent was evaporated to give 1.19 g
of product 3a, which was used without further purification.
(Cholestꢀ5ꢀenꢀ3βꢀyl) imidazoleꢀ1ꢀcarboxylate (3b) was preꢀ
pared in the same way as compound 3a from cholesterol (1b)
(1.0 g, 2.59 mmol) and CDI (0.81 g, 4.99 mmol) in the presence
of Et3N (0.52 mL, 3.74 mmol). This gave 1.17 g of compound
3b, which was used without further purification.
[racꢀ2,3ꢀBis(tetradecyloxy)propyl] Nꢀ(6ꢀhydroxyhexyl)ꢀ
carbamate (5a). 6ꢀAminohexanol (4) (0.75 g, 6.40 mmol) was
added to a solution of compound 3a (2.47 g, 4.27 mmol) in
anhydrous CH2Cl2 (10 mL), and the mixture was stirred under
reflux for 2.5 h. The reaction mixture was washed with 3% HCl
(5 mL) and water (2×5 mL), and dried with Na2SO4, and the
solvent was evaporated. The residue was chromatographed on
a column with silica gel, the target compound being eluted with
a petroleum ether—EtOAc mixture (2 : 1) to give 2.14 g (80%) of
compound 5a, m.p. 54—56 °C. Found (%): C, 72.60; H, 12.17;
N, 2.13. C38H77NO5. Calculated (%): C, 72.67; H, 12.36;
N, 2.23. 1H NMR, δ: 0.85 (t, 6 H, 2 CH2Me, J = 6.6 Hz);
1.20—1.40 (m, 48 H, 2 (CH2)11, N(CH2)2(CH2)2(CH2)2OH);
1.42—1.58 (m, 8 H, 3 OCH2CH2, NHCH2CH2); 3.15 (dt, 2 H,
NHCH2, J = 6.3 Hz, J = 6.7 Hz); 3.37—3.64 (m, 9 H,
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ(6ꢀbromohexyl)carbamate (6b) was
prepared in the same way as compound 6a from 5b (1.39 g,
2.57 mmol), Ph3P (1.35 g, 5.13 mmol), and CBr4 (1.70 g,
5.13 mmol). The product was isolated by chromatography on
silica gel (petroleum ether—EtOAc (15 : 1)) giving 1.29 g (83%)
20
of compound 6b, m.p. 106—108 °C, [α]D –21 (c 1, CHCl3).
Found (%): C, 69.24; H, 10.15; N, 2.31. C34H58BrNO2. Calꢀ
culated (%): C, 68.90; H, 9.86; N, 2.36. 1H NMR, δ: 0.61
(s, 3 H, C(13)Me); 0.79 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.80
(d, 3 H, C(25)Me, J = 6.5); 0.85 (d, 3 H, C(20)Me, J =
= 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.98—1.57 (m, 27 H, Chol,
NHCH2(CH2)3(CH2)2Br); 1.68—1.98 (m,
7 H, Chol,
OCH2CHCH2,
2
OCH2CH2, CH2OH); 4.06 (dd,
1
H,
CH2CH2Br); 2.12—2.34 (m, 2 H, H2C(4)); 3.03—3.15 (m, 2 H,
NHCH2); 3.34 (t, 2 H, CH2Br, J = 6.8 Hz); 4.35—4.48 (m, 1 H,
H(3)); 4.48—4.62 (m, 1 H, NH); 5.28—5.33 (m, 1 H, H(6)).
13C NMR, δ: 12.04, 18.90, 19.52, 21.23, 22.74, 23.00, 24.01,
24.47, 26.08, 27.99, 28.19, 28.37, 28.42, 30.08, 32.07 32.81, 33.91,
35.98, 36.37, 36.75, 37.18, 38.76, 39.70, 39.92, 40.94, 42.50,
50.20, 56.32, 56.87, 74.40, 122.66, 140.03, 156.35.
Nꢀ(6ꢀHydroxyhexyl)ꢀ2ꢀnitrobenzenesulfonamide (7). Molecꢀ
ular sieves 4 Å and Et3N (6 mL) were added with stirring to a
cooled (4 °C) solution of 6ꢀaminohexanol (4) (2.00 g, 17.07 mmol)
in anhydrous CH2Cl2 (30 mL). Then a solution of 2ꢀnitrobenzeꢀ
CH2OC(O), J = 5.4 Hz, J = 11.5 Hz); 4.17 (dd, 1 H, CH2OC(O),
J = 4.0 Hz, J = 11.5 Hz); 4.62—4.77 (m, 1 H, NH). 13C (δ:
14.49, 23.07, 25.69, 26.42, 26.48, 26.78, 29.75, 29.88, 30.03,
30.08, 30.35, 30.39, 32.30, 32.96, 41.24, 63.12, 64.58, 70.79,
70.97, 72.16, 77.24, 156.84.
Cholestꢀ5ꢀenꢀ3βꢀylꢀNꢀ(6ꢀhydroxyhexyl)carbamate (5b).
6ꢀAminohexanol (4) (0.47 g, 4.01 mmol) was added to a solution
of compound 3b (1.29 g, 2.68 mmol) in anhydrous CH2Cl2
(10 mL), and the mixture was stirred under reflux for 2.5 h. The
reaction mixture was washed with 3% HCl (5 mL) and water

Synthesis of cationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
255
nesulfonyl chloride (4.51 g, 20.35 mmol) in anhydrous CH2Cl2
(10 mL) was added dropwise. After 24 h, the reaction mixture
was filtered through Celite® 545 and washed with CH2Cl2, and
the solvent was removed in vacuo. The residue was chromatoꢀ
graphed on a column with silica gel using a CHCl3—MeOH
mixture (100 : 1) as the eluent to give 3.97 g (77%) of compound
7, m.p. 77—78 °C. Found (%): C, 47.77; H, 6.15; N, 9.28.
C12H18N2O5S. Calculated (%): C, 47.67; H, 6.00; N, 9.27.
1H NMR, δ: 1.21—1.59 (m, 8 H, NCH2(CH2)4CH2OH); 3.07
(dt, 2 H, CH2NH, J = 6.4 Hz); 3.58 (t, 2 H, CH2OH, J = 6.4 Hz);
5.23—5.38 (m, 1 H, NH); 7.68—7.78 (m, 2 H, Ar); 7.80—7.88
and 8.06—8.15 (both m, 1 H each, Ar).
Nꢀ(6ꢀHydroxyhexyl)ꢀNꢀ[racꢀ2,3ꢀbis(tetradecyloxy)propyl]ꢀ
2ꢀnitrobenzenesulfonamide (8a). Cesium carbonate (0.63 g,
1.94 mmol) was added to a solution of compound 7 (0.50 g,
1.66 mmol) in anhydrous DMF (10 mL). Then a solution of
compound 2 (0.73 g, 1.33 mmol) in anhydrous DMF (3 mL) was
added in portions. The reaction mixture was kept for 48 h at
80 °C and cooled to 24 °C, petroleum ether (15 mL) was added,
and the mixture was washed with water (3×5 mL). The organic
products were extracted from the aqueous phase with petroleum
ether (5×10 mL). The combined organic extract was dried with
Na2SO4 and filtered, and the solvent was removed in vacuo.
The residue was chromatographed on a column with silica
gel using a petroleum ether—EtOAc mixture (2.5 : 1) as the eluꢀ
ent to give 0.66 g (64%) of compound 8a as a crystallizing oil.
Found (%): C, 67.14; H, 10.48; N, 3.73. C43H80N2O7S. Calꢀ
culated (%): C, 67.15; H, 10.48; N, 3.64. 1H NMR, δ: 0.81
28.21, 29.53, 29.67, 29.82, 29.86, 30.17, 32.09, 32.66, 41.00,
47.33, 62.80, 64.36, 70.55, 70.76, 71.95, 77.00, 124.27, 130.87,
131.68, 133.47, 133.89, 148.18, 156.59.
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ[13ꢀhydroxyꢀ7ꢀ(2ꢀnitrophenylsulfoꢀ
nyl)ꢀ7ꢀazatridecyl]carbamate (8с) was prepared in the same way
as compound 8b from 7 (0.13 g, 0.43 mmol) and 6b (0.26 g,
0.43 mmol) in the presence of Cs2CO3 (0.84 g, 2.58 mmol).
Compound 8с was formed (0.301 g, 86%) as a crystallizing oil,
[α]D20 –15 (c 1, CHCl3). Found (%): C, 68.01; H, 10.21; N, 5.51.
C46H75N3O7S. Calculated (%): C, 67.86; H, 9.28; N, 5.16.
1H NMR, δ: 0.65 (s, 3 H, C(13)Me); 0.73 (d, 3 H, C(25)Me,
J = 6.5 Hz); 0.84 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.89 (d, 3 H,
C(20)Me, J = 6.5 Hz); 0.98 (s, 3 H, C(10)Me); 0.99—1.57
(m, 37 H, Chol, NHCH2(CH2)4CH2NCH2(CH2)4CH2OH);
1.68—1.98 (m, 5 H, Chol); 2.15—2.38 (m, 2 H, H2C(4));
3.05—3.19 (m, 2 H, NHCH2); 3.20—3.28 (m, 4 H, CH2NCH2);
3.60 (t, 2 H, CH2OH, J = 6.5 Hz); 4.38—4.53 (m, 1 H, H(3));
4.54—4.64 (m, 1 H, NH); 5.32—5.37 (m, 1 H, H(6)); 7.56—7.68
(m, 3 H, Ar); 7.96—8.00 (m, 1 H, Ar). 13C NMR, δ: 12.24, 19.09,
19.71, 21.42, 22.94, 23.20, 24.21, 24.67, 25.64, 26.55, 26.65,
28.39, 28.44, 28.56, 28.61, 29.74, 29.87, 30.07, 30.19, 30.27,
32.90, 36.17, 36.56, 36.94, 37.38, 38.96, 39.90, 40.12, 41.07,
42.69, 46.03, 47.52, 50.39, 56.51, 57.07, 63.06, 64.72, 122.84,
124.48, 131.11, 131.88, 133.65, 135.22, 140.25, 148.01, 156.53.
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(2,3,4,6ꢀtetraꢀ
OꢀacetylꢀβꢀDꢀgalactopyranosyloxy)hexyl]ꢀ2ꢀnitrobenzenesulfonꢀ
amide (9a). Calcined CdCO3 (0.230 g, 1.333 mmol) was added
to a solution of compound 8a (0.343 g, 0.446 mmol) in anhyꢀ
drous benzene (40 mL), and the mixture was stirred under reflux
in a Soxhlet apparatus with calcined granulated silica gel. After
six cycles, a solution of 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀgalactopyrꢀ
anosyl bromide33 (0.549 g, 1.333 mmol) in anhydrous benzene
(2 mL) was added dropwise to the reaction mixture. After 7 h,
the precipitate was filtered off and washed with CHCl3, and the
solvents were removed in vacuo. The residue was chromatoꢀ
graphed on a column with silica gel using a toluene—EtOAc
mixture (10 : 1 → 2 : 1) as the eluent to give 0.338 g (69%) of
(t, 6 H, 2 CH2Me, J = 6.5 Hz); 1.15—1.29 (m, 48 H, 2 (CH2)11
,
N(CH2)2(CH2)2(CH2)2OH); 1.34—1.54 (m, 8 H, 2 OCH2CH2,
NCH2CH2(CH2)2CH2CH2OH); 3.21—3.50 (m, 11 H,
OCH2CHCH2NCH2, 2 OCH2CH2); 3.54 (t, 2 H, CH2OH,
J = 6.5 Hz); 7.52—7.64 (m, 3 H, Ar); 7.93—7.99 (m, 1 H, Ar).
13C NMR, δ: 14.49, 23.06, 25.64, 26.45, 26.48, 26.61, 27.95,
29.74, 29.87, 30.02, 30.08, 30.42, 32.29, 32.93, 49.06, 49.10,
63.08, 70.71, 70.87, 72.12, 78.09, 124.40, 131.35, 131.81, 133.56,
134.36, 148.47.
20
[racꢀ2,3ꢀBis(tetradecyloxy)propyl] Nꢀ[13ꢀhydroxyꢀ7ꢀ(2ꢀ
nitrophenylsulfonyl)ꢀ7ꢀazatridecyl]carbamate (8b). Cesium carꢀ
bonate (0.23 g, 0.70 mmol) and then a solution of compound 6a
(0.36 g, 0.52 mmol) in anhydrous DMF (5 mL) were added with
stirring to a solution of compound 7 (0.11 g, 0.364 mmol) in
anhydrous DMF (10 mL). The reaction mixture was kept for 3 h
at 80 °C, cooled to 24 °C, diluted with CHCl3 (10 mL), and
washed with water (10 mL), the aqueous phase was extracted
with CHCl3 (3×5 mL), the combined organic extract was washed
with water (5 mL), dried with Na2SO4, and filtered, and the
solvents were removed in vacuo. The residue was chromatoꢀ
graphed on a column with silica gel using a petroleum ether—
EtOAc mixture (1.5 : 1) as the eluent to give 0.285 g (86%) of
compound 8b as a crystallizing oil. Found (%): C, 64.83;
H, 10.89; N, 4.49. C50H93N3O9S•H2O. Calculated (%): C, 64.55;
H, 10.59; N, 4.52. 1H NMR, δ: 0.83 (t, 6 H, 2 CH2Me,
J = 6.8 Hz); 1.17—1.33 (m, 52 H, 2 (CH2)11, 2 N(CH2)2(CH2)2);
1.35—1.55 (m, 12 H, 2 OCH2CH2, 3 NCH2CH2, CH2CH2OH);
3.03—3.13 (m, 2 H, NHCH2); 3.18—3.26 (m, 4 H, CH2NCH2);
3.35—3.55 (m, 7 H, OCH2CHCH2, 2 OCH2CH2); 3.56 (t, 2 H,
CH2OH, J = 6.4 Hz); 4.03 (dd, 1 H, CH2OC(O), J = 5.3 Hz,
J = 11.3 Hz); 4.14 (dd, 1 H, CH2OC(O), J = 4.3 Hz, J = 11.3 Hz);
4.67—4.78 (m, 1 H, NH); 7.55—7.69 (m, 3 H, Ar); 7.92—8.05
(m, 1 H, Ar). 13C NMR, δ: 14.29, 22.85, 25.42, 26.27, 26.42,
compound 9a as a crystallizing oil, [α]D –2 (c 1.9, CHCl3).
Found (%): C, 62.39; H, 9.17; N, 2.57. C57H98N2O16S. Calcuꢀ
lated (%): C, 62.27; H, 8.98; N, 2.55. 1H NMR, δ: 0.81 (t, 6 H,
2 CH2Me, J = 6.5 Hz); 1.16—1.25 (m, 48 H, 2 (CH2)11
,
N(CH2)2(CH2)2(CH2)2O); 1.31—1.57 (m, 8 H, 2 CH2CH2O,
NCH2CH2(CH2)2CH2CH2O); 1.91 (s, 3 H), 1.98 (s, 6 H), 2.08
(s, 3 H) (4 MeCO); 3.16—3.56 (m, 12 H, OCH2CHCH2NCH2,
2 CH2CH2O, CHHaO); 3.70—3.88 (m, 2 H, CHHbO, H(5)
Gal); 4.07—4.11 (m, 2 H, H(6) Gal); 4.38 (d, 1 H, H(1) Gal,
J1,2 = 7.8 Hz); 4.94 (dd, 1 H, H(3) Gal, J3,4 = 3.4 Hz, J3,2
=
= 10.3 Hz); 5.12 (dd, 1 H, H(2) Gal, J2,1 = 7.8 Hz, J2,3 = 10.3 Hz);
5.30—5.35 (m, 1 H, H(4) Gal); 7.50—7.64 (m, 3 H, Ar);
7.93—8.00 (m, 1 H, Ar). 13C NMR, δ: 14.20, 20.67, 20.76, 20.83,
22.75, 25.45, 26.14, 26.27, 27.65, 29.35, 29.43, 29.56, 29.72,
29.77, 30.11, 32.00, 48.76, 61.31, 67.12, 68.95, 70.08, 70.40,
70.56, 70.64, 71.03, 71.80, 76.98, 101.34, 124.10, 131.06, 131.54,
133.28, 134.05, 148.15, 169.48, 170.27, 170.38, 170.49.
[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[13ꢀ(2,3,4,6ꢀtetraꢀOꢀ
acetylꢀβꢀDꢀgalactopyranosyloxy)ꢀ7ꢀ(2ꢀnitrophenylsulfonyl)ꢀ7ꢀ
azatridecyl]carbamate (9b). Calcined CdCO3 (0.78 g, 4.52 mmol)
was added to a solution of compound 8b (1.37 g, 1.50 mmol) in
anhydrous benzene (20 mL), and the mixture was refluxed with
stirring in a Soxhlet apparatus with calcined granulated silica
gel. After five cycles, 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀgalactopyraꢀ

256
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Morozova et al.
nosyl bromide (0.93 g, 2.26 mmol) was added in portions. The
reaction mixture was stirred under reflux for 15 h, the precipitate
was filtered off, and the solvents were removed in vacuo.
The residue was chromatographed on a column using a toluꢀ
ene—EtOAc mixture (4.5 : 1) as the eluent to give 1.13 g (60%)
of compound 9b as a crystallizing oil, [α]D20 +3.5 (c 1, CHCl3).
Found (%): C, 61.76; H, 9.30; N, 3.36. C64H111N3O18S. Calcuꢀ
lated (%): C, 61.86; H, 9.00; N, 3.38. 1H NMR, δ: 0.81 (t, 6 H,
[α]D23 –8.15 (c 1, CHCl3). MS, m/z (Irel (%)): 1387.935 [M + H]+
(100). Calculated for C69H114N2O24S: 1386.748 [M]+. 1H NMR,
δ: 0.84 (t, 6 H, 2 CH2Me, J = 6.8 Hz); 1.20—1.30 (m, 48 H,
2 (CH2)11, N(CH2)2(CH2)2(CH2)2O); 1.32—1.55 (m, 8 H,
2 CH2CH2O, NCH2CH2(CH2)2CH2CH2O); 1.91, 1.96, 1.98,
1.99, 2.06, 2.08 and 2.10 (all s, 3 H each, 7 COMe); 3.18—3.57
(m, 12 H, OCH2CHCH2NCH2, 2 CH2CH2O, CHHaO);
3.65—3.75 (m, 2 H, CHHbO, H(5) Lac); 3.78—3.86 (m, 1 H,
H(5´) Lac); 3.91—4.12 (m, 4 H, H(6), H(6´) Lac); 4.37 (d, 1 H,
H(1´) Lac, J1´,2´ = 7.8 Hz); 4.41 (m, 1 H, H(4) Lac); 4.43 (d, 1 H,
H(1) Lac, J1,2 = 7.8 Hz); 4.79 (dd, 1 H, H(2) Lac, J2,1= 7.8 Hz,
2 CH2Me, J = 6.5 Hz); 1.14—1.31 (m, 52 H, 2 (CH2)11
,
2 N(CH2)2(CH2)2); 1.34—1.56 (m, 12 H, 3 OCH2CH2,
3 NCH2CH2); 1.91 (s, 3 H), 1.98 (s, 6 H), 2.08 (s, 3 H)
(4 MeCO); 3.01—3.10 (m, 2 H, NHCH2); 3.15—3.24 (m, 4 H,
CH2NCH2); 3.34—3.58 (m, 8 H, OCH2CHCH2, 2 OCH2CH2,
CHHaO); 3.74—3.87 (m, 2 H, CHHbO, H(5) Gal); 3.97—4.17
J2,3 = 9.4 Hz); 4.89 (dd, 1 H, H(3´) Lac, J3´,4´ = 3.4 Hz, J3´,2´
= 10.3 Hz); 5.03 (dd, 1 H, H(2´) Lac, J2´,1´ = 7.8 Hz, J2´,3´
=
=
= 10.3 Hz); 5.12 (t, 1 H, H(3) Lac, J3,2 = J3,4 = 9.4 Hz); 5.29
(m, 1 H, H(4´) Lac); 7.50—7.62 (m, 3 H, Ar); 7.90—8.03 (m, 1 H,
Ar). 13C NMR, δ: 14.19, 20.57, 20.70, 20.92, 22.79, 25.53, 26.22,
26.24, 26.36, 27.75, 29.43, 29.46, 29.62, 29.77, 29.81, 30.21,
32.04, 48.89, 48.96, 61.00, 62.27, 66.86, 69.39, 69.99, 70.64,
70.91, 71.19, 71.90, 71.98, 72.85, 73.09, 76.49, 77.90, 100.75,
101.20, 124.17, 131.16, 131.53, 133.30, 148.15, 169.16, 169.63,
169.83, 170.12, 170.19, 170.42.
(m, 4 H, CH2OC(O), H(6) Gal); 4.38 (d, 1 H, H(1) Gal, J1,2
=
= 8.0 Hz); 4.62—4.70 (m, 1 H, NH); 4.94 (dd, 1 H, H(3) Gal,
J3,4 = 3.4 Hz, J3,2 = 10.6 Hz); 5.13 (dd, 1 H, H(2) Gal, J2,1 = 8.0 Hz,
J2,3 = 10.6 Hz); 5.31—5.34 (m, 1 H, H(4) Gal); 7.52—7.66
(m, 3 H, Ar); 7.91—8.26 (m, 1 H, Ar). 13C NMR, δ: 14.29,
20.77, 20.86, 20.95, 22.86, 25.55, 26.22, 26.27, 26.42, 28.20,
29.53, 29.67, 29.83, 30.18, 32.10, 41.02, 47.18, 61.42, 64.39,
67.23, 69.08, 70.18, 70.56, 70.76, 71.12, 71.95, 77.03, 101.53,
124.29, 130.90, 131.71, 133.50, 133.95, 148.18, 156.57, 169.60,
170.35, 170.46.
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ[13ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀDꢀ
galactopyranosyloxy)ꢀ7ꢀ(2ꢀnitrophenylsulfonyl)ꢀ7ꢀazatridecyl]ꢀ
carbamate (9с) was prepared in the same way as compound 9b
from 8с (0.98 g, 1.20 mmol), CdCO3 (0.617 g, 3.58 mmol), and
2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀgalactopyranosyl bromide (0.736 g,
1.79 mmol). Chromatography on silica gel (toluene—EtOAc,
3 : 1) gave 0.882 g (64%) of compound 9с as a crystallizing oil,
[α]D20 –1.5 (c 1, CHCl3). Found (%): C, 63.33; H, 8.19; N, 3.22.
C60H93N3O16S. Calculated (%): C, 62.97; H, 8.19; N, 3.67.
1H NMR, δ: 0.65 (s, 3 H, C(13)Me); 0.73 (d, 3 H, C(25)Me,
J = 6.5 Hz); 0.84 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.89 (d, 3 H,
C(20)Me, J = 6.5 Hz); 0.98 (s, 3 H, C(10)Me); 0.99—1.57
(m, 37 H, Chol, NHCH2(CH2)4CH2NCH2(CH2)4CH2O);
1.68—1.95 (m, 5 H, Chol); 1.92 (s, 3 H), 1.98 (s, 6 H), 2.08 (s, 3 H)
(4 MeCO); 2.16—2.34 (m, 2 H, H2C(4)); 3.00—3.10 (m, 2 H,
NHCH2); 3.14—3.25 (m, 4 H, CH2NCH2); 3.39 (dt, 1 H,
CHHaO, J = 6.9 Hz, J = 9.5 Hz); 3.75—3.87 (m, 2 H,
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(βꢀDꢀgalactoꢀ
pyranosyloxy)hexyl]ꢀ2ꢀnitrobenzenesulfonamide (11a). A 1 M soꢀ
lution of MeONa in MeOH (0.5 mL) was added to a solution of
compound 9a (0.973 g, 0.885 mmol) in a CHCl3 : MeOH mixꢀ
ture (1 : 1). After 30 min, the reaction mixture was neutralized
with the ionꢀexchange resin Amberlite IRꢀ120 (H+). The resin
was filtered off and washed with MeOH, the solvents were reꢀ
moved in vacuo, and the residue was chromatographed on
a column with silica gel using a toluene—acetone mixture
(2 : 1 → 1 : 2) as the eluent to give 0.501 g (61%) of comꢀ
20
pound 11a as a crystallizing oil, [α]D –2.5 (c 3, CHCl3).
Found (%): C, 63.00; H, 10.07; N, 2.95. C49H90N2O12S. Calꢀ
culated (%): C, 63.19; H, 9.74; N, 3.01. 1H NMR, δ: 0.82 (t, 6 H,
2 CH2Me, J = 6.8 Hz); 1.16—1.28 (m, 48 H, 2 (CH2)11
,
N(CH2)2(CH2)2(CH2)2O); 1.32—1.56 (m, 8 H, 2 CH2CH2O,
NCH2CH2(CH2)2CH2CH2O); 3.19—3.64 (m, 15 H,
OCH2CHCH2NCH2, 2 CH2CH2O, CHHaO, H(6) Gal, H(3)
Gal); 3.72—3.87 (m, 3 H, CHHbO, H(5) Gal, H(2) Gal);
3.95—3.99 (m, 1 H, H(4) Gal); 4.17 (d, 1 H, H(1) Gal, J1,2
=
= 7.4 Hz); 7.53—7.65 (m, 3 H, Ar); 7.94—87.98 (m, 1 H, Ar).
13C NMR, δ: 14.17, 22.75, 25.41, 26.11, 26.16, 27.47, 29.43,
29.56, 29.72, 29.77, 30.09, 31.98, 48.58, 61.85, 69.23, 70.07,
70.55, 71.49, 71.80, 73.67, 74.21, 77.58, 103.27, 124.15, 130.98,
131.68, 133.46, 133.88, 148.09.
CHHbO, H(5) Gal); 4.01—4.16 (m,
2 H, H(6) Gal);
4.33—4.48 (m, 1 H, H(3)); 4.38 (d, 1 H, H(1) Gal, J1,2 = 7.9 Hz);
4.50—4.59 (m, 1 H, NH); 4.95 (dd, 1 H, H(3) Gal, J3,4 = 3.5 Hz,
J3,2 = 10.5 Hz); 5.13 (dd, 1 H, H(2) Gal, J2,1 = 7.9 Hz, J2,3
=
= 10.5 Hz); 5.26—5.33 (m, 2 H, H(6) Chol, H(4) Gal);
7.52—7.65 (m, 3 H, Ar); 7.91—7.96 (m, 1 H, Ar). 13C NMR, δ:
12.01, 18.86, 19.49, 20.79, 20.87, 20.96, 21.19, 22.72, 22.99,
23.97, 24.44, 25.53, 26.36, 26.42, 28.17, 28.33, 28.38, 29.44,
29.85, 30.06, 32.02, 32.06, 35.95, 36.33, 36.71, 37.15, 38.73,
39.67, 39.87, 40.85, 42.45, 47.21, 47.27, 50.15, 56.26, 56.83,
61.40, 67.21, 68.90, 69.05, 70.19, 70.73, 71.10, 71.84, 74.35,
101.50, 122.63, 124.28, 130.87, 131.71, 133.50, 133.89, 140.00,
148.16, 156.34, 169.60, 170.37, 170.47, 170.61.
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(2,2´,3,3´,4´,
6,6´ꢀheptaꢀOꢀacetylꢀβꢀlactosyloxy)hexyl]ꢀ2ꢀnitrobenzenesulfonꢀ
amide (10) was prepared in the same way as compound 9a from 8
(0.760 g, 0.988 mmol), CdCO3 (0.341 g, 1.978 mmol), and
2,2´,3,3´,4´,6,6´ꢀheptaꢀOꢀacetylꢀαꢀlactosyl bromide34 (1.411 g,
2.017 mmol). Chromatography on silica gel (toluene—EtOAc,
12 : 1) gave 0.740 g (54%) of compound 10 as a crystallizing oil,
[racꢀ2,3ꢀBis(tetradecyloxy)propyl] Nꢀ[13ꢀ(βꢀDꢀgalactopyrꢀ
anosyloxy)ꢀ7ꢀ(2ꢀnitrophenylsulfonyl)ꢀ7ꢀazatridecyl]carbamate
(11b). A 0.5 M solution of MeONa in MeOH (0.2 mL) was
added to a solution of compound 9b (0.13 g, 0.11 mmol) in
MeOH (10 mL). After 1 h, the reaction mixture was neutralized
with the ionꢀexchange resin Dowex 50W×8 (H+). The resin was
filtered off and washed with MeOH, and the solvent was reꢀ
moved in vacuo. The residue was chromatographed on a column
with silica gel using a toluene—acetone mixture (1 : 1) as the
eluent to give 0.094 g (84%) of compound 11b as a crystallizing
20
oil, [α]D +4 (c 1, CHCl3—MeOH, 1 : 1). MS, m/z (Irel (%)):
1096.576 [M + Na]+ (100), 1112.578 [M + K]+ (100). Calculated
1
for C56H103N3O14S: 1073.716 [M]+. H NMR, δ: 0.80 (t, 6 H,
2 CH2Me, J = 6.5 Hz); 1.18—1.35 (m, 52 H, 2 (CH2)11
,
2 N(CH2)2(CH2)2); 1.36—1.61 (m, 12 H, 2 OCH2CH2,
3 NCH2CH2, CH2CH2O); 3.04—3.13 (m, 2 H, NHCH2);

Synthesis of cationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
257
3.20—3.28 (m, 4 H, CH2NCH2); 3.38—3.90 (m, 14 H,
OCH2CHCH2OC(O), 2 OCH2CH2, CH2O, H(2) Gal, H(3)
Gal, H(5) Gal, H(6) Gal); 3.99—4.04 (m, 1 H, H(4) Gal); 4.05
(dd, 1 H, CHHaOC(O), J = 5.3 Hz, J = 11.2 Hz); 4.15 (dd, 1 H,
CHHbOC(O), J = 4.1 Hz, J = 11.2 Hz); 4.20—4.25 (m, 1 H,
H(1) Gal); 4.76—4.90 (m, 1 H, NH); 7.57—7.72 (m, 3 H, Ar);
7.93—8.00 (m, 1 H, Ar). 13C NMR, δ: 14.29, 22.85, 25.50, 26.20,
26.26, 26.32, 26.41, 28.09, 29.52, 29.67, 29.81, 29.86, 29.98,
30.17, 32.08, 41.03, 47.18, 61.97, 64.39, 69.32, 70.12, 70.58,
70.77, 71.64, 71.96, 73.71, 74.34, 77.01, 103.34, 124.33, 130.76,
131.87, 133.68, 148.13, 156.63.
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ[13ꢀ(βꢀDꢀgalactopyranosyloxy)ꢀ7ꢀ
(2ꢀnitrophenylsulfonyl)ꢀ7ꢀazatridecyl]carbamate (11с) was preꢀ
pared in the same way as compound 11b from 9с (0.350 g). This
gave 0.26 g (87%) of compound 11c, m.p. 78—80 °C, [α]D20 –18
(c 1, CHCl3—MeOH, 1 : 1). MS, m/z (Irel (%)): 998.597 [M + Na]+
(100), 1014.588 [M + K]+ (100). Calculated for C52H85N3O12S:
975.585 [M]+. 1H NMR, δ: 0.60 (s, 3 H, C(13)Me); 0.70 (d, 3 H,
C(25)Me, J = 6.5 Hz); 0.80 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.86
(d, 3 H, C(20)Me, J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.98—1.54
(m, 37 H, Chol, NHCH2(CH2)4CH2NCH2(CH2)4CH2O);
1.70—1.99 (m, 5 H, Chol); 2.13—2.31 (m, 2 H, H2C(4));
2.95—3.06 (m, 2 H, NHCH2); 3.13—3.26 (m, 4 H, CH2NCH2);
3.35—4.00 (m, 8 H, CH2O, H(2) Gal, H(3) Gal, H(4) Gal, H(5)
Gal, H(6) Gal); 4.13—4.22 (m, 1 H, H(1) Gal, J1,2 = 7.9 Hz);
4.34—4.45 (m, 1 H, H(3)); 4.68—4.71 (m, 1 H, NH); 5.26—5.31
(m, 1 H, H(6)); 7.52—7.68 (m, 3 H, Ar); 7.86—7.94 (m, 1 H,
Ar). 13C NMR, δ: 12.03, 18.88, 19.51, 21.20, 22.73, 22.99, 24.02,
24.45, 25.48, 26.34, 26.44, 28.17, 28.35, 28.40, 29.49, 30.02,
32.02, 35.97, 36.35, 36.72, 37.15, 38.76, 39.68, 39.90, 40.90,
42.47, 47.25, 50.15, 56.30, 56.84, 61.60, 69.12, 70.17, 71.51,
73.70, 74.36, 103.38, 122.62, 124.34, 130.70, 131.95, 133.66,
133.74, 140.02, 148.12.
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(βꢀlactosyloxy)ꢀ
hexyl]ꢀ2ꢀnitrobenzenesulfonamide (12) was prepared in the same
way as compound 11a from 10 (0.376 g) and a 0.1 M solution of
MeONa in MeOH (2 mL). Chromatography on silica gel (toluꢀ
ene—acetone—water, 10 : 50 : 1) gave 0.295 g (98%) of comꢀ
pound 12 as a crystallizing oil, [α]D23 –4 (c 1, CHCl3). MS, m/z
(Irel (%)): 1131.960 [M + K]+ (100). Calculated for C55H100N2O17S:
1092.674 [M]+. 1H NMR, δ: 0.80 (t, 6 H, 2 CH2Me, J = 6.8 Hz);
1.21—1.31 (m, 48 H, 2 (CH2)11, N(CH2)2(CH2)2(CH2)2O);
1.32—1.55 (m, 8 H, 2 CH2CH2O, NCH2CH2(CH2)2CH2CH2O);
3.05—4.50 (m, 27 H, OCH2CHCH2NCH2, 3 CH2CH2O, Lac);
7.52—7.67 (m, 3 H, Ar); 7.91—7.98 (m, 1 H, Ar). 13C NMR, δ:
14.27, 22.86, 25.55, 26.26, 26.31, 26.37, 27.60, 28.40,
29.54, 30.26, 32.11, 48.67, 61.89, 70.24, 70.69, 70.90, 71.93,
73.58, 76.88, 77.68, 102.81, 103.70, 124.33, 131.09, 131.89,
133.73, 134.00.
for C43H87NO8: 745.643 [M]+. 1H NMR, δ: 0.79 (t, 6 H,
2 CH2Me, J = 6.8 Hz); 1.13—1.33 (m, 48 H, 2 (CH2)11
,
N(CH2)2(CH2)2(CH2)2O); 1.35—1.60 (m, 8 H, 2 CH2CH2O,
NCH2CH2(CH2)2CH2CH2O); 2.50—2.69 (m, 4 H, CH2NCH2);
3.26—3.59 (m, 11 H, OCH2CHCH2N, 2 CH2CH2O, CHHaO,
H(6) Gal, H(3) Gal); 3.72—3.85 (m, 3 H, CHHbO, H(5) Gal,
H(2) Gal); 3.91—3.95 (m, 1 H, H(4) Gal); 4.13 (d, 1 H, H(1)
Gal, J1,2 = 7.4 Hz). 13C NMR, δ: 14.18, 22.78, 25.82, 26.24,
26.75, 29.16, 29.46, 29.62, 29.79, 30.31, 32.03, 49.55, 51.24,
61.97, 69.40, 70.15, 70.51, 71.52, 71.86, 73.75, 74.42, 103.53.
[racꢀ2,3ꢀBis(tetradecyloxy)propyl] Nꢀ[13ꢀ(βꢀDꢀgalactopyrꢀ
anosyloxy)ꢀ7ꢀazatridecyl]carbamate (13b) was prepared in the
same way as compound 13a from 11b (0.07 g, 0.06 mmol), K2CO3
(0.08 g, 0.5 mmol), and PhSH (0.06 mL, 0.59 mmol). Reversꢀ
edꢀphase chromatography (CHCl3—MeOH—25% aq. NH3
(20 : 100 : 1)) gave 0.05 g (87%) of compound 13b, m.p. 40—42 °C,
[α]D20 –4 (c 1, CHCl3—MeOH, 1 : 1). MS, m/z (Irel (%)): 889.6
[M + H]+ (100), 911.6 [M + Na]+ (50), 927.6 [M + K]+ (90).
Calculated for C50H100N2O10: 888.74 [M]+. 1H NMR (400 MHz,
CDCl3—CD3OD, 3 : 1), δ: 0.80 (t, 6 H, 2 CH2Me, J = 7.0 Hz);
1.13—1.32 (m, 52 H, 2 (CH2)11, 2 N(CH2)2(CH2)2); 1.36—1.59
(m, 12 H, 2 OCH2CH2, 3 NCH2CH2, CH2CH2O); 2.45 (br.t, 4 H,
CH2NCH2, J = 7.6 Hz); 3.03 (br.t, 2 H, NHCH2, J = 7.0 Hz);
3.33—3.80 (m, 14 H, OCH2CHCH2OC(O), 2 OCH2CH2,
CH2O, H(2) Gal, H(3) Gal, H(5) Gal, H(6) Gal); 3.98 (dd,
1 H, CHHaOC(O), J = 5.0 Hz, J = 11.5 Hz); 4.07 (dd, 1 H,
CHHbOC(O), J = 4.2 Hz, J = 11.5 Hz); 4.13 (d, 1 H, H(1) Gal,
J1,2 = 6.9). 13C NMR (100 MHz, CDCl3—CD3OD, 3 : 1), δ:
13.99, 22.64, 25.70, 25.97, 26.55, 26.92, 29.32, 29.46, 29.61,
29.66, 29.90, 31.89, 49.51, 61.30, 64.01, 68.88, 69.96, 70.31,
70.62, 71.33, 71.82, 73.52, 74.61, 103.39, 156.91.
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ[13ꢀ(βꢀDꢀgalactopyranosyloxy)ꢀ7ꢀ
azatridecyl]carbamate (13с) was prepared in the same way as
compound 13b from 11с (0.07 g, 0.07 mmol), K2CO3 (0.08 g,
0.61 mmol), and PhSH (0.06 mL, 0.61 mmol). This gave 0.05 g
20
(88%) of compound 13c, m.p. 63—65 °C, [α]D –11 (c 1,
CHCl3—MeOH, 1 : 1). MS, m/z (Irel (%)): 791.751 [M + H]+
(100). Calculated for C46H82N2O8: 790.607 [M]+. 1H NMR
(400 MHz, CDCl3—CD3OD, 5 : 1), δ: 0.61 (s,
3 H,
C(13)Me); 0.79 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.80 (d, 3 H,
C(25)Me, J = 6.5 Hz); 0.84 (d, 3 H, C(20)Me, J = 6.5 Hz);
0.93 (s,
3 H, C(10)Me); 0.90—1.60 (m, 37 H, Chol,
NHCH2(CH2)4CH2NCH2(CH2)4CH2O); 1.72—1.98 (m, 5 H,
Chol); 2.14—2.32 (m, 2 H, H2C(4)); 2.48—2.61 (m, 4 H,
CH2NCH2); 2.98—3.14 (m, 2 H, NHCH2); 3.40—4.18 (m, 9 H,
CH2O, Gal); 4.35—4.46 (m, 1 H, H(3)); 4.90—4.97 (m, 1 H,
NH); 5.24—5.32 (m, 1 H, H(6)). 13C NMR (100 MHz,
CDCl3—CD3OD, 5 : 1), δ: 11.89, 18.75, 19.33, 21.15, 22.54,
22.79, 23.94, 24.34, 28.04, 28.26, 29.72, 31.97, 35.85, 36.27,
36.77, 37.37, 38.62, 39.58, 39.84, 42.40, 56.31, 56.83, 60.62,
60.79, 61.41, 65.19, 69.72, 71.19, 73.46, 75.68, 79.20, 100.29,
122.35, 140.28.
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(βꢀDꢀgalactoꢀ
pyranosyloxy)hexyl]amine (13a). Potassium carbonate (0.742 g,
5.369 mmol) and PhSH (0.550 mL, 5.369 mmol) were added to
a solution of compound 11a (0.500 g, 0.537 mmol) in anhydrous
DMF (5 mL), and the mixture was stirred for 2 h at 24 °C. The
reaction mixture was filtered through Celite® 545 and washed
with MeOH, and the solvents were removed in vacuo. The resiꢀ
due was chromatographed on a column with reversedꢀphase silꢀ
ica gel LiChroprep RPꢀ18 using a CHCl3—MeOH—25% aq.
NH3 mixture (10 : 100 : 1) as the eluent to give 0.334 g (83%) of
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(βꢀlactosyloxy)ꢀ
hexyl]amine (14) was prepared in the same way as compound
13b from 12 (0.231 g, 0.211 mol), K2CO3 (0.301 g, 2.201 mmol),
and PhSH (0.21 mL, 2.110 mmol). This gave 0.126 g (66%)
23
of compound 14 as a crystallizing oil, [α]D –6.23 (c 1,
CHCl3—CH3OH, 4 : 1). MS, m/z (Irel (%)): 908.889 [M + H]+
(100), 930.904 [M + Na]+ (25), 946.892 [M + K]+ (50). Calcuꢀ
lated for C49H97NO13: 907.696 [M]+. 1H NMR (400 MHz,
Pyꢀd5), δ: 0.74 (t, 6 H, 2 CH2Me, J = 7.0 Hz); 1.03—1.34 (m, 50 H,
20
compound 13a as a crystallizing oil, [α]D –4 (c 1.6, CHCl3).
MS, m/z (Irel (%)): 746.683 [M + H]+ (100). Calculated

258
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Morozova et al.
2 (CH2)11, NCH2(CH2)3(CH2)2O); 1.42—1.53 (m, 6 H,
3 CH2CH2O); 2.37—2.48 and 2.59—2.71 (both m, 2 H each,
CH2NCH2); 3.28—4.23 (m, 21 H, OCH2CHCH2N, 3 CH2CH2O,
Lac); 4.44 (d, 1 H, H(1´) Lac, J1´,2´ = 7.8 Hz); 4.71 (d, 1 H, H(1)
Lac, J1,2 = 8.1 Hz). 13C NMR (100 MHz, Pyꢀd5), δ: 14.78,
23.40, 26.82, 27.04, 27.98, 30.08, 30.27, 30.44, 30.63, 30.99,
31.15, 32.63, 50.88, 52.34, 62.36, 62.80, 70.34, 70.88, 72.20,
72.59, 73.12, 74.87, 75.35, 76.53, 76.83, 77.31, 79.12, 82.57,
104.62, 105.97.
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(βꢀDꢀgalactoꢀ
pyranosyloxy)hexyl]ꢀN,Nꢀdimethylammonium iodide (15a). Poꢀ
tassium carbonate (7.4 mg, 0.0533 mmol) and MeI (1 mL,
16.07 mmol) were added successively to a solution of compound
13a (39.8 mg, 0.0533 mmol) in anhydrous ethyl methyl ketone
(1.5 mL). The reaction mixture was stirred for 2.5 h at 80 °C and
the solvent was removed in vacuo. The residue was chromatoꢀ
graphed on a column with silica gel LiChroprep®RPꢀ18 using
a MeOH—water mixture (100 : 1 → 100 : 0) as the eluent to give
0.0364 g (76%) of compound 15a as a crystallizing oil. MS, m/z
(Irel (%)): 774.840 [M – I]+ (100). Calculated for C45H92NO8:
774.682 [M – I]+. 1H NMR (400 MHz, CDCl3—Pyꢀd5, 2.5 : 1),
1H NMR (400 MHz, CDCl3—CD3OD, 5 : 1), δ: 0.61 (s, 3 H,
C(13)Me); 0.78 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.79 (d, 3 H,
C(25)Me, J = 6.5 Hz); 0.84 (d, 3 H, C(20)Me, J = 6.5 Hz);
0.93 (s,
3 H, C(10)Me); 0.90—1.98 (m, 42 H, Chol,
NHCH2(CH2)4CH2NCH2(CH2)4CH2O); 2.14—2.30 (m, 2 H,
H2C(4)); 3.00 and 3.03 (both s, 3 H each, N+Me2); 3.40—4.18
(m, 15 H, NHCH2, CH2NCH2, CH2O, Gal); 4.31—4.45
(m, 1 H, H(3)); 5.27—5.35 (m, 1 H, H(6)). 13C NMR (100 MHz,
CDCl3—CD3OD, 2 : 1), δ: 11.87, 18.66, 19.24, 21.04, 22.40,
22.53, 22.66, 23.83, 24.24, 25.18, 25.57, 25.76, 26.05, 27.95,
28.17, 28.94, 29.47, 31.91, 35.76, 36.20, 36.58, 37.01,
38.58, 39.50, 39.78, 40.37, 42.34, 50.12, 51.09, 56.23, 56.75,
61.28, 64.56, 64.69, 68.97, 69.56, 71.35, 73.48, 74.62, 103.31,
122.44, 139.90.
Nꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyl]ꢀNꢀ[6ꢀ(βꢀlactosyloxy)ꢀ
hexyl]ꢀN,Nꢀdimethylammonium iodide (16). Potassium carbonꢀ
ate (8.5 mg, 0.0616 mmol) and MeI (0.0766 mL, 0.123 mmol)
were added with stirring to a solution of amine 14 (0.056 g,
0.0616 mmol) in ethyl methyl ketone (9 mL). The reaction mixꢀ
ture was kept for 3 h at 50 °C, the solvent was removed in vacuo,
and the residue was chromatographed on a column with the silica
gel LiChroprep®RPꢀ18 using a toluene—CHCl3—MeOH—
—AcOH mixture (2 : 2 : 5 : 0.02 → 1 : 1 : 4 : 0.01) as the eluent
to give 0.0458 g (69%) of compound 16 as a crystallizing oil,
δ: 0.80 (t, 6 H, 2 CH2Me, J = 6.8 Hz); 1.21 (br.s, 48 H, 2 (CH2)11
,
N(CH2)2(CH2)2(CH2)2O); 1.48—1.62 (m, 8 H, 2 CH2CH2O,
NCH2CH2(CH2)2CH2CH2O); 3.48 and 3.50 (both s, 3 H each,
N+Me2); 3.39—3.87 (m, 12 H, OCH2CHCH2N, 2 CH2CH2O,
CH2N+CH2, CHHaO); 3.96—4.14 (m, 4 H, H(2) Gal, H(3)
Gal, H(5) Gal, CHHbO); 4.33—4.44 (m, 2 H, H(6) Gal);
23
[α]D –3.1 (c 1, CHCl3—CH3OH, 1 : 1). MS, m/z (Irel (%)):
936.759 [M – I]+ (100). Calculated for C51H102NO13: 936.735
[M – I]+. 1H NMR (CDCl3—CD3OD, 3 : 1), δ: 0.84 (t, 6 H,
4.51—4.54 (m, 1 H, H(4) Gal); 4.67 (d, 1 H, H(1) Gal, J1,2
=
2 CH2Me, J = 6.8 Hz); 1.21 (br.s, 48 H, 2 (CH2)11,
= 7.5 Hz). 13C NMR (100 MHz, CDCl3—CD3OD, 2 : 1), δ:
14.05, 22.40, 22.70, 25.29, 25.49, 26.10, 26.24, 28.85, 29.39,
29.52, 29.72, 30.11, 31.97, 52.23, 52.50, 61.42, 65.66, 66.49,
68.86, 69.00, 71.45, 72.18, 73.26, 73.70, 74.71, 77.40, 103.48.
Nꢀ{6ꢀ[racꢀ2,3ꢀBis(tetradecyloxy)propyloxycarbonylamino]ꢀ
hexyl}ꢀNꢀ[6ꢀ(βꢀDꢀgalactopyranosyloxy)hexyl]ꢀN,Nꢀdimethylamꢀ
monium iodide (15b). Methyl iodide (0.01 mL, 0.12 mmol) was
added to a solution of compound 13b (0.03 g, 0.03 mmol) in
anhydrous ethyl methyl ketone (1.5 mL). The reaction mixture
was stirred for 2.5 h at 80 °C and the solvent was removed in vacuo.
The residue was chromatographed on a column with silica gel
LiChroprep®RPꢀ18 using a MeOH—water mixture (100 : 1) as
the eluent to give 0.029 g (81%) of compound 15b as a crystallizꢀ
ing oil. MS, m/z (Irel (%)): 917.999 [M – I]+ (100). Calculated
for C52H105N2O10: 917.777 [M – I]+. 1H NMR (400 MHz,
CDCl3—CD3OD, 4 : 1): 0.80 (t, 6 H, 2 CH2Me, J = 7.0 Hz);
1.13—1.33 (m, 52 H, 2 (CH2)11, 2 N(CH2)2(CH2)2), 1.36—1.71
(m, 12 H, 2 OCH2CH2, 3 NCH2CH2, CH2CH2O); 3.00
(s, 6 H, N+Me2); 3.02—3.87 (m, 20 H, NHCH2, CH2N+CH2,
OCH2CHCH2OC(O), 2 OCH2CH2, CH2O, Gal); 3.95—4.06
(m, 2 H, CH2OC(O)); 4.12 (d, 1 H, H(1) Gal, J1,2 = 8.4 Hz).
13C NMR (100 MHz, CDCl3—CD3OD, 2 : 1), δ: 14.19, 22.55,
22.77, 22.94, 25.53, 25.85, 26.15, 26.36, 29.16, 29.63, 29.79,
29.97, 30.32, 32.23, 33.75, 40.91, 51.04, 61.76, 64.62, 64.81,
64.96, 69.37, 69.72, 70.83, 71.02, 71.81, 72.23, 74.16, 75.26,
77.39, 77.86, 103.87.
N(CH2)2(CH2)2(CH2)2OH); 1.45—1.65 (m, 8 H, 2 CH2CH2O,
NCH2CH2(CH2)2CH2CH2O); 3.31—4.49 (m, 33 H,
OCH2CHCH2N, 3 CH2CH2O, CH2N+CH2, N+Me2, Gal).
13C NMR (CDCl3—Pyꢀd5, 2 : 1), δ: 13.24, 21.80, 25.25, 25.37,
27.09, 28.49, 28.85, 29.28, 29.46, 31.03, 32.26, 32.67, 34.88,
35.28, 36.13, 37.83, 38.60, 38.82, 49.09, 51.17, 55.74, 60.04,
60.82, 64.16, 65.03, 67.08, 68.28, 70.71, 70.94, 72.45, 72.86,
74.45, 76.56, 102.31, 103.05.
This work was supported by the Russian Foundation
for Basic Research (Projects No. 09ꢀ03ꢀ00874a and
No. 07ꢀ03ꢀ00632a).
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