Synthesis of polycationic lipids based on cholesterol and spermine

Article abstract of DOI:10.1007/s11172-010-0071-x

The synthesis of cholesterol-based cationic lipids in which the hydrophobic steroidal fragment is linked to a spermine residue as the hydrophilic domain by ester or carbamate linkage is described.

Full text of DOI:10.1007/s11172-010-0071-x

Russian Chemical Bulletin, International Edition, Vol. 59, No. 1, pp. 260—268, January, 2010
260
Synthesis of polycationic lipids based on cholesterol and spermine
I. A. Petukhov, M. A. Maslov, N. G. Morozova, and G. A. Serebrennikova
M. V. Lomonosov Moscow State Academy of Fine Chemical Tehnology,
86 Prospect Vernadskogo, 119571 Moscow, Russian Federation.
Fax: +7 (495) 936 8901. Eꢀmail: mamaslov@mail.ru
The synthesis of cholesterolꢀbased cationic lipids in which the hydrophobic steroidal fragment
is linked to a spermine residue as the hydrophilic domain by ester or carbamate linkage is described.
Key words: cholesterol, spermine, cationic lipids
In gene therapy, any curative action is achieved through
to eukaryotic cells more efficiently than lipophilic derivaꢀ
tives of spermine.17,18 On the basis of spermine, commerꢀ
cial products for transfection were developed.19—22
A number of approaches to the preparation of polycaꢀ
tionic amphiphiles whose hydrophobic parts are bound
to the polyamine by spacers of different length have
been reported. Most of the methods include initial inꢀ
troduction of the spacer group to the polyamine maꢀ
trix and subsequent linking of the obtained fragment to
the activated hydrophobic component.17,23—26 However,
the overall yields of the target compounds in these synꢀ
theses are 14—25% (see Refs 17, 23, 27). A solidꢀstate
method was proposed15,27 for the synthesis of cholesꢀ
terol polycationic amphiphiles, which increased the
yield to 87%.
This communication presents a new method for the
synthesis of unsymmetrical and symmetrical polycationic
amphiphiles based on molecular cell components: cholesꢀ
terol and spermine. The effect of the amphiphile structure
on the cytotoxicity and DNA transfer properties was studꢀ
ied by varying the length of the spacer group separating
spermine and cholesterol and the type of linkage (ester
and carbamate linkages). Our synthetic scheme was based
on the Fukuyama reaction: Nꢀalkylation of 2ꢀnitrobenzeꢀ
nesulfonamides derived from primary amines with alkyl
halides and subsequent removal of 2ꢀnitrobenzenesulfonyl
group to give secondary amines.28,29
introduction, into the organism, of DNA, mRNA, or oliꢀ
gonucleotides that reach the cells by special transport sysꢀ
tems.1 Cationic lipids and liposomes based on them are
attractive systems for the delivery owing to their biodeꢀ
gradability and low probability of eliciting immune and
inflammatory reactions.2—5 A cationic lipid molecule is a
combination of two structural domains, hydrophobic and
hydrophilic ones, which are connected by linkers. Longꢀ
chain hydrocarbons, steroids, and diglycerides are used as
the hydrophobic domains. The hydrophilic domain can
contain one (monocationic lipids) or more (polycationic
lipids) positively charged groups. Monocationic lipids are
most often tertiary or quaternary derivatives of aliphatic or
heterocyclic nitrogen bases. In polycationic lipids, natural
or synthetic polyamines or amino acids are used as the
hydrophilic domains.3,6,7 The type of linking of the hyꢀ
drophobic and hydrophilic domains determines the stabilꢀ
ity and toxicity of cationic amphiphiles in biological sysꢀ
tems.3,8 Stable lipids with ether linkage are more toxic to
cells than acyl lipids, which are readily hydrolyzed in the
cell by endogenous esterases. A carbamoyl linkage proꢀ
vides a more favorable compromise between the amꢀ
phiphile stability and toxicity.
Natural polyamines, spermine and spermidine, play
an important role in the vital activity of cells.9 Polyamines
can pack DNA to toroidal and rodꢀlike structures10 and
the methylene fragments separating the nitrogen atoms
play an important role in the interaction of polyamine
with the DNA double helix.11,12 The structureꢀfunctional
studies have shown that lipophilic derivatives of polyꢀ
amines condense DNA more efficiently than natural
polyamines13,14 and can be used as nonꢀviral systems for
the delivery of genetic material. Among lipophilic polyꢀ
amines, spermine derivatives bind and condense DNA
most efficiently15,16 and, therefore, they better transport it
to the cells. However, some researchers note that amꢀ
phiphiles based on synthetic polyamines can deliver DNA
For implementing this approach, in the first stage we
prepared bromo derivatives of cholesterol. Treatment of
cholesterol (1) with an excess of 5ꢀbromopentanoyl chloꢀ
ride gave cholesterol brominated derivative 2 with an ester
bond (Scheme 1). The yield of bromide 2 was 96%. The
structure of compound 2 was supported by 1H NMR specꢀ
troscopy data, in particular, the presence of a triplet for
the CH2Br group at δ 3.40 and spinꢀspin coupling conꢀ
stant of 6.7 Hz.
Compounds with carbamoyl group were obtained by
treatment of cholesterol (1) with N,N´ꢀcarbonyldiimidꢀ
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 254—261, January, 2010.
1066ꢀ5285/10/5901ꢀ0260 © 2010 Springer Science+Business Media, Inc.

Synthesis of polycationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
261
Scheme 1
azole (CDI) to give imidazolide 3 (see Scheme 1), which
was then made to react with amino alcohols to give hyꢀ
droxy derivatives 4a and 4b. The hydroxy group in alcoꢀ
hols 4a,b was replaced by bromine by treatment of 4a,b
with CBr4 in the presence of Ph3P; this gave bromides
5a,b in 72 and 75% yields (over three steps).
The reaction of Воcꢀprotected derivative 6, obtained
from spermine by regioselective protection/deprotection,24
with 2ꢀnitrobenzenesulfonyl chloride yielded derivative 7,
which was isolated in 88% yield by column chromatograꢀ
phy on silica gel (Scheme 2). The structure of compound
7 was confirmed by NMR spectra exhibiting proton
(δH 7.59—8.11) and carbon (δC 125.25—148.29) signals of
the aromatic ring.
The key step of the synthesis of unsymmetrical polycaꢀ
tionic lipids 10a—c was the Fukuyama Nꢀalkylation of
substituted sulfonamide 7 with cholesterol brominated deꢀ
rivatives 2, 5a,b. Compounds 8a—c were isolated in
86—94% yields and characterized by mass spectrometry
1
1
and H and 13C NMR spectroscopy data. The H NMR
spectra show the set of proton signals for the polyamine
and cholesterol components. In the final step, the amino
groups in lipoconjugates 8a—c were deprotected. First,
the 2ꢀnitrobenzenesulfonyl group was removed by treatꢀ
ment with benzenethiol in the presence of K2CO3. The
removal of the tertꢀbutoxycarbonyl protection was accomꢀ
plished by treating compounds 9a—c with 4 M HCl in
n = 1 (a), 2 (b)
Reagents and conditions: (a) Br(CH2)4COCl, Py, 24 °C;
(b) CDI, Et3N, 40 °C; (c) HO(CH2)mNH2, m = 4, 6, 40 °C;
(d) CBr4, Ph3P, 24 °C.
Scheme 2
X = CO (a), NHCO (b, c); n = 3 (a, b), 5 (c)
Reagents and conditions: (a) 2ꢀNO2C6H4SO2Cl, Et3N, 24 °C; (b) 2 or 5a,b, Cs2CO3, DMF, 60 °C; (c) PhSH, K2CO3, 24 °C;
(d) 4 M HCl—dioxane, 24 °C.

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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Petukhov et al.
dioxane for 2 h. The unsymmetrical lipophilic polyamines
10a—c were obtained in 89, 86, and 79% yields (over two
steps), respectively. The structures of the products were
confirmed by data from NMR spectroscopy and mass specꢀ
trometry.
In order to find new transfection agents, we syntheꢀ
sized symmetrical cationic polyamines with different spacꢀ
er lengths between the hydrophobic and polyamine parts
and different types of spacer binding to the hydrophobic
domain (Scheme 3).
presence of benzenesulfonyl groups. The condensation of
cholesterol brominated derivatives 2, 5a,b with sulfonaꢀ
mide 12 was carried out according to the Fukuyama reacꢀ
tion, the yields of derivatives 13a—c were 51—60%. The
final step of the synthesis was deprotection of amino groups
as in the case of compounds 10a—c. First, the 2ꢀnitrobenꢀ
zenesulfonyl (compounds 14a—c) and then tertꢀbutoxyꢀ
carbonyl protective groups were removed to give the target
symmetric lipopolyamines 15a—c in 54, 76, and 67% yields
over two steps.
DiꢀBocꢀprotected spermine 11 was prepared using
a known approach.30 Spermine was treated with a twofold
excess of ethyl trifluoroacetate to protect the primary amiꢀ
no groups. The secondary amino groups were protected by
reaction with diꢀtertꢀbutyl dicarbonate to afford a fully
protected spermine derivative. The removal of the trifluoꢀ
roacetyl groups on treatment with NaOH furnished parꢀ
tially protected polyamine 11 containing two free primary
amino groups. NꢀSulfonylation of this product with an
excess of 2ꢀnitrobenzenesulfonyl chloride in the presence
of Et3N gave diamide 12, which was isolated in 63% yield.
The structure of compound 12 was confirmed by NMR
spectroscopy. The signals of aromatic protons (δH 7.59—8.11)
appearing in the 1H NMR spectrum and a downfield shift
of the methylene proton signals of the CH2NH group
(δH 2.81—3.15 for 11, δH 3.10—3.38 for 12) attest to the
Cationic lipids 15a—c can be referred to the class
of gemini surfactants, i.e., compounds in which two
hydrophobic domains are symmetrically linked through
rigid or flexible spacers with a hydrophilic cationic doꢀ
main. They have a very high surface activity, which makes
them interesting for biological and biomedical studies.
During the last several years, about 250 geminiꢀsurꢀ
factants have been synthesized most of which showed
medium or high level of transfection activity in standard
tests in vitro.31
Thus, we prepared unsymmetrical and symmetriꢀ
cal polycationic amphiphiles based on cholesterol
and spermine differing in the mode of attachment of
the spacer to the hydrophobic domain (ester and carꢀ
bamate bonds) and in the spacer length (4 and 6 methꢀ
ylene units).
Scheme 3
X = CO (a), NHCO (b, c); n = 3 (a, b), 5 (c)
Reagents and conditions: (a) 2ꢀNO2C6H4SO2Cl, Et3N, 24 °C;
(b) 2 or 5a,b, Cs2CO3, DMF, 60 °C; (c) PhSH, K2CO3, 24 °C;
(d) 4 M HCl—dioxane, 24 °C.

Synthesis of polycationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
263
ed (%): C, 76.03; H, 10.08; N, 5.72; O, 8.17. IR, ν/cm–1: 2840,
1730, 1640, 1520, 1460, 1380. 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); 0.84 (d, 3 H, C(20)Me, J = 6.5 Hz); 0.99
(s, 3 H, C(10)Me); 0.90—1.58 (m, 19 H, Chol); 1.58—2.02
(m, 7 H, Chol); 2.38—2.47 (m, 2 H, H2C(4)); 4.69—4.83 (m, 1 H,
H(3)); 5.34—5.40 (m, 1 H, H(6)); 7.00, 7.36, 8.09 (all s, 1 H each,
Im). 13C NMR, δ: 11.88, 18.88, 19.35, 21.21, 22.63, 22.87, 23.98,
24.44, 27.81, 28.19, 28.38, 31.98, 35.96, 36.33, 36.71, 36.92,
38.03, 39.67, 39.83, 42.47, 50.11, 56.27, 56.81, 79.20, 117.44,
123.95, 130.05, 137.14, 138.74, 148.05.
Experimental
Distilled solvents, Russian (Khimmed) and foreign (Merck,
Fluka, Aldrich, Acros) commercial reagents were used. CH2Cl2
and Et3N were refluxed with CaH2 and distilled prior to the
reaction, DMF was kept over calcined molecular sieves 4 Å.
N1,N4,N9ꢀTriꢀtertꢀbutoxycarbonylꢀ1,12ꢀdiaminoꢀ4,9ꢀdiazaꢀ
dodecane (6) and N4,N9ꢀdiꢀtertꢀbutoxycarbonylꢀ1,12ꢀdiamiꢀ
noꢀ4,9ꢀdiazadodecane (11) were prepared by known proꢀ
cedures.24,30
Thinꢀlayer chromatography was carried out on Kieselgel 60
F254 plates (Merck) in solvent systems petroleum ether—EtOAc,
4 : 1 (A); CHCl3—MeOH, 40 : 1 (B); 36 : 1 (C); 25 : 1 (D); 20 : 1 (E);
13 : 1 (F); CHCl3—MeOH—PriNH2, 4 : 1 : 2 (G), 3 : 1 : 2 (H).
The compounds were detected by treatment with chlorine folꢀ
lowed by visualization using a benzidine solution,32 the Dragenꢀ
dorff reagent,32 phosphomolybdic acid—cerium(IV) sulfate sysꢀ
tem followed by heating,33 or UV light (254 nm). Column
chromatography was carried out on silica gel Kieselgel 60
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ(4ꢀhydroxybutyl)carbamate (4a).
4ꢀAminobutanꢀ1ꢀol (0.287 g, 2.78 mmol) was added to a soluꢀ
tion of compound 3 (1.00 g, 2.08 mmol) in anhydrous CH2Cl2
(10 mL). The reaction mixture was stirred under reflux for 18 h,
washed with 3% HCl (8 mL) and water (5×5 mL), and the orꢀ
ganic solvent was evaporated. The residue was chromatographed
on a column with silica gel using a CHCl3—MeOH mixture
(30 : 1) as the eluent to give compound 4a as white crystals (0.895 g,
87%), Rf 0.45 (E), m.p. 154—156 °C. MS, m/z (Irel (%)):
524.314 [M + Na]+ (100). Calculated for C32H56NO3: 501.418
[M]+. 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, 23 H, Chol, NHCH2(CH2)2); 1.68—2.02 (m, 7 H, Chol);
2.14—2.38 (m, 2 H, H2C(4)); 3.05—3.20 (m, 2 H, CH2N); 3.59
(t, 2 H, CH2OH, J = 5.9 Hz); 4.36—4.48 (m, 1 H, H(3));
4.48—4.62 (m, 1 H, NH); 5.27—5.35 (m, 1 H, H(6)). 13C NMR,
δ: 12.10, 18.99, 19.56, 21.14, 22.79, 23.07, 23.93, 24.38, 26.54,
28.12, 28.26, 29.64, 31.96, 35.91, 36.27, 36.65, 37.06, 38.65,
39.61, 39.82, 40.70, 42.40, 49.94, 56.21, 56.77, 62.10, 74.57,
122.63, 139.89, 156.72.
1
(0.040—0.063 mm, Merck). H and 13C NMR spectra were reꢀ
corded on a Bruker Avance DPXꢀ300 pulse Fourier transform
spectrometer (Germany) in CDCl3 unless otherwise stated
(internal SiMe4). Mass spectra were run on a Bruker Ultraflex
timeꢀofꢀflight mass spectrometer (Germany) with laser desorpꢀ
tion ionization using 2,5ꢀdihydroxybenzoic and cyanohydroxyꢀ
cinnamic acids as the matrices. For compounds 9a,c, 10a—c,
and 15b,c, the mass spectra were measured on an Agilent
1100 GC/MS spectrometer (Agilent Technologies, USA) by
chemical ionization under atmospheric pressure. IR spectra were
recorded on a Shimadzu IRꢀ435 spectrometer (Japan). The
melting points were determined on a Boetius hot stage (Gerꢀ
many). Compounds 10a—c и 15a—c decompose without meltꢀ
ing above 270 °C.
(5ꢀBromopentanoyl)cholesterol (2). Pyridine (3 mL) and then
a solution of 5ꢀbromopentanoyl chloride (2.25 g, 11.3 mmol) in
CH2Cl2 (2.5 mL) were added to a cooled (0 °C) solution of
cholesterol (1) (2.18 g, 5.6 mmol) in anhydrous CH2Cl2 (15 mL).
The reaction mixture was stirred for 1 h at 24 °C, washed with
10% HCl (3×5 mL) and water (5 mL), and dried with Na2SO4,
and the solvent was evaporated. The residue was chromatoꢀ
graphed on a column with silica gel using a CHCl3—MeOH
mixture (40 : 1) as the eluent to give compound 2 as white crysꢀ
tals (2.955 g, 96%), Rf 0.48 (А), m.p. 112—114 °C. Found (%):
C, 70.30; H, 10.00. C32H53BrO2. Calculated (%): C, 69.92;
H, 9.72; Br, 14.54; O, 5.82. IR, ν/cm–1: 2830, 1736, 1460, 1370,
1240, 670. 1H NMR, δ: 0.68 (s, 3 H, C(13)Me); 0.79, 0.80 (both d,
3 H each, C(25)Me, J = 6.5 Hz); 0.86 (d, 3 H, C(20)Me,
J = 6.5 Hz); 1.02 (s, 3 H, C(10)Me); 0.88—1.65 (m, 23 H, Chol,
OC(O)CH2(CH2)2); 1.68—2.00 (m, 7 H, Chol); 2.13—2.34
(m, 2 H, H2C(4)), 3.06—3.19 (m, 2 H, OC(O)CH2); 5.27—5.34
(m, 1 H, H(6)).
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ(6ꢀhydroxyhexyl)carbamate (4b) was
prepared as compound 4a from compound 3 (1.00 g, 2.08 mmol)
and 6ꢀaminohexanꢀ1ꢀol (0.366 g, 3.12 mmol). Chromatography
on silica gel using CHCl3—MeOH (50 : 1) gave compound 4b as
white crystals (0.992 g, 90%), Rf 0.48 (E), m.p. 186—188 °C.
Found (%): C, 76.96; H, 11.22; N, 2.57. C35H61NO3. Calculatꢀ
1
ed (%): C, 77.29; H, 11.30; N, 2.58. H 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, 27 H, Chol, NHCH2(CH2)4);
1.68—2.00 (m, 7 H, Chol); 2.13—2.35 (m, 2 H, H2C(4)); 3.03—3.16
(m, 2 H, CH2N); 3.57 (t, 2 H, CH2OH, J = 6.4 Hz); 4.34—4.49
(m, 1 H, H(3)); 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.
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ(4ꢀbromobutyl)carbamate (5a). Triꢀ
phenylphosphine (0.793 g, 3.02 mmol) was added to a cooled
(0 °C) solution of compound 4a (0.843 g, 1.68 mmol) in anhyꢀ
drous CH2Cl2 (10 mL). Then CBr4 (1.0 g, 3.02 mmol) was added
in portions and the mixture was stirred for 1 h at 24 °C. Methanol
(5 mL) was added and the solvent was evaporated. The residue
was chromatographed on a column with silica gel with CHCl3 as
the eluent to give compound 5a as white crystals (0.400 g, 84%),
Rf 0.8 (E), m.p. 92—94 °C. Found (%): C, 68.10; H, 9.90; N, 2.48.
C32H54BrNO2. Calculated (%): C, 68.06; H, 9.64; N, 2.48.
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,
(Cholestꢀ5ꢀenꢀ3βꢀyl) imidazoleꢀ1ꢀcarboxylate (3). N,N´ꢀCarꢀ
bonyldiimidazole (1.678 g, 10.35 mmol) and triethylamine (2 mL,
3.74 mmol) were added to a solution of cholesterol (1) (4.00 g,
10.35 mmol) in anhydrous CH2Cl2 (20 mL). The reaction mixꢀ
ture was stirred under reflux for 16 h, washed with 10% HCl
(3×5 mL) and water (5 mL), and dried with Na2SO4, and the
solvent was evaporated to give compound 3 as white crystals
(4.904 g, 99%), Rf 0.7 (B), m.p. 124—126 °C. Found (%):
C, 76.26; H, 10.40; N, 5.56. 2 C35H61NO3•H2O. Calculatꢀ

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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Petukhov et al.
C(20)Me, J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.98—1.57
(m, 23 H, Chol, NHCH2(CH2)2); 1.70—2.00 (m, 7 H, Chol);
2.15—2.38 (m, 2 H, H2C(4)); 3.05—3.16 (m, 2 H, CH2N); 3.35
(t, 2 H, CH2Br, J = 6.5 Hz); 4.36—4.48 (m, 1 H, H(3));
4.48—4.62 (m, 1 H, NH); 5.27—5.34 (m, 1 H, H(6)). 13C NMR,
δ: 12.17, 18.74, 19.37, 21.20, 22.87, 23.14, 24.00, 24.44, 28.18,
28.33, 28.90, 30.00, 32.02, 33.43, 35.97, 36.33, 36.71, 37.12,
38.72, 39.67, 39.88, 40.12, 42.46, 50.02, 56.27, 56.83, 74.48,
122.69, 139.93, 156.34.
(Cholestꢀ5ꢀenꢀ3βꢀyl) Nꢀ(6ꢀbromohexyl)carbamate (5b) was
prepared in the same way as compound 5a from compound 4b
(0.585 g, 1.1 mmol), Ph3P (0.579 g, 2.2 mmol), and CBr4 (0.945 g,
2.89 mmol). Compound 5b was obtained as white crystals (0.548 g,
84%), Rf 0.85 (E), m.p. 106—108 °C. Found (%): C, 69.24;
H, 10.15; N, 2.31. C35H60BrNO2. Calculated (%): C, 69.28;
H, 9.97; N, 2.31. 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, 27 H, Chol, NHCH2(CH2)4); 1.68—1.98 (m, 7 H,
Chol); 2.12—2.34 (m, 2 H, H2C(4)); 3.03—3.15 (m, 2 H, CH2N);
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.
4,9,N12ꢀTri(tertꢀbutoxycarbonyl)ꢀN1ꢀ(2ꢀnitrophenylsulfoꢀ
nyl)ꢀ1,12ꢀdiaminoꢀ4,9ꢀdiazadodecane (7). Molecular sieves 4 Å
(1.0 g), Et3N (0.239 mL, 1.716 mmol), and 2ꢀnitrobenzeneꢀ
sulfonyl chloride (0.228 g, 1.02 mmol) were added to a cooled
(0 °C) solution of compound 6 (0.431 g, 0.858 mmol) in anhyꢀ
drous CH2Cl2 (5 mL). The reaction mixture was stirred for 1 h at
24 °C. The molecular sieves were filtered off and washed with
CH2Cl2, and the solvent was removed in vacuo. The resiꢀ
due was chromatographed on a column with silica gel using
a CHCl3—MeOH mixture (50 : 1) as the eluent to give comꢀ
pound 7 as a crystallizing oil (0.507 g, 88%), Rf 0.40 (D). MS,
m/z (Irel (%)): 710.341 [M + Na]+ (100). Calculated for
C31H53N5O10S: 687.351 [M]+. 1H NMR, δ: 1.27 (br.s,
31 H, 3 C(CH3)3 and NCH2(CH2)2CH2N); 1.52—1.71 (m, 4 H,
(s, 3 H, C(10)Me), 0.90—2.00 (m, 38 H, Chol, 2 CH2(CH2)2CH2,
2 NCH2CH2CH2N); 1.36 (br.s, 18 H) and 1.39 (br.s, 9 H,
3 C(CH3)3); 2.11—2.33 (m, 4 H, H2C(4), OC(O)CH2);
2.95—3.12 (m, 8 H, 4 NCH2); 3.12—3.30 (m, 6 H, CH2NH,
2 NCH2); 4.43—4.59 (m, 1 H, H(3)); 5.01—5.26 (m, 1 H, NH);
5.26—5.34 (m, 1 H, H(6)); 7.50—7.59 (m, 1 H); 7.59—7.70
(m, 2 H) and 7.87—8.00 (m, 1 H, C6H4). 13C NMR, δ: 11.94,
18.80, 19.39, 21.11, 22.03, 22.64, 22.89, 23.90, 24.36, 25.61,
25.79, 27.87, 28.07, 28.29, 28.53, 31.94, 31.98, 34.00, 35.86,
36.26, 36.68, 37.07, 37.67, 38.22, 39.60, 39.82, 42.40, 43.94,
44.54, 45.19, 46.77, 47.07, 50.12, 56.23, 56.78, 74.02, 79.58,
122.73, 124.24, 130.80, 131.73, 133.50, 133.58 139.71, 148.13,
155.50, 156.10, 172.51.
9,14,N17ꢀTri(tertꢀbutoxycarbonyl)ꢀ5ꢀ(2ꢀnitrophenylsulfonyl)ꢀ
N1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ1,17ꢀdiaminoꢀ5,9,14ꢀtriꢀ
azaheptadecane (8b) was prepared in the same way as compound
8a from compound 7 (0.098 g, 0.142 mmol) and bromide 5a
(0.096 g, 0.170 mmol) in the presence of Cs2CO3 (0.046 g, 0.142
mmol) to give compound 8b as a crystallizing oil (0.157 g, 94%),
Rf 0.4 (В). MS, m/z (Irel (%)): 1193.762 [M + Na]+ (100). Calꢀ
culated for C63H106N6O12S: 1170.759 [M]+. 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); 0.84 (d, 3 H, C(20)Me,
J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.90—2.00 (m, 38 H, Chol,
2 CH2(CH2)2CH2, 2 NCH2CH2CH2N); 1.36 (br.s, 18 H) and
1.39 (br.s, 9 H, 3 C(CH3)3); 2.11—2.33 (m, 2 H, H2C(4));
2.91—3.12 (m, 10 H, CH2NH, 4 NCH2); 3.12—3.30 (m, 6 H,
CH2NH, 2 NCH2); 4.32—4.47 (m, 1 H, H(3)); 4.55—4.96
(m, 1 H, NH); 4.96—5.25 (m, 1 H, NH); 5.25—5.33 (m, 1 H,
H(6)); 7.50—7.59 (m, 1 H); 7.59—7.70 (m, 2 H) and 7.87—8.00
(m, 1 H, C6H4). 13C NMR, δ: 11.97, 18.84, 19.44, 21.17,
22.67, 22.92, 23.95, 24.40, 25.61, 26.01, 27.20, 27.57,
28.11, 28.30, 28.58, 32.01, 35.90, 36.31, 36.54, 36.69,
37.13, 37.80, 38.70, 39.63, 39.87, 40.36, 42.44, 44.16,
44.70, 45.57, 46.70, 50.15, 56.27, 56.82, 74.36, 79.66, 122.56,
124.27, 130.83, 131.77, 133.48, 133.61, 139.98, 148.18, 155.56,
156.16, 156.35.
11,16,N19ꢀTri(tertꢀbutoxycarbonyl)ꢀ7ꢀ(2ꢀnitrophenylsulfonyl)ꢀ
N1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ1,19ꢀdiaminoꢀ7,11,16ꢀ
triazanonadecane (8c) was prepared in the same way as comꢀ
pound 8a from compound 7 (0.203 g, 0.302 mmol) and bromide
5b (0.215 g, 0.362 mmol) in the presence of Cs2CO3 (0.098 g,
0.302 mmol). Compound 8c was obtained as a crystallizing oil
(0.382 g, 88%), Rf 0.45 (C). MS, m/z (Irel (%)): 1221.424
[M + Na]+(100). Calculated for C65H110N6O12S: 1198.790 [M]+.
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); 0.84 (d, 3 H,
C(20)Me, J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.90—2.00 (m, 42 H,
Chol protons, NHCH2(CH2)2CH2N, 2 NCH2CH2CH2N,
CH2(CH2)4CH2); 1.37 (br.s, 18 H) and 1.39 (br.s, 9 H, 3 C(CH3)3);
2.11—2.35 (m, 2 H, H2C(4)); 2.92—3.13 (m, 10 H, CH2NH,
4 NCH2); 3.13—3.33 (m, 6 H, CH2NH, 2 NCH2); 4.33—4.47
(m, 1 H, H(3)); 4.50—4.85 (m, 1 H, NH); 4.96—5.25 (m, 1 H,
NH); 5.26—5.35 (m, 1 H, H(6)); 7.51—7.59 (m, 1 H); 7.59—7.70
(m, 2 H) and 7.87—8.00 (m, 1 H, C6H4). 13C NMR, δ: 12.00,
18.87, 19.48, 21.20, 22.70, 23.00, 23.98, 24.43, 25.91, 26.31,
26.37, 27.60, 28.09, 28.14, 28.36, 28.60, 30.30, 32.05, 35.93,
36.34, 36.72, 37.16, 37.81, 38.74, 39.66, 39.90, 40.88, 42.47,
44.13, 44.67, 45.28, 46.82, 47.48, 50.19, 56.30, 56.85, 74.36,
79.65, 122.57, 124.29, 130.87, 131.73, 133.57, 133.68, 140.03,
148.20, 155.59, 156.18, 156.33.
2
NCH2CH2CH2N); 2.92—3.11 (m, 8 H, 4 NCH2);
3.11—3.30 (m, 4 H, 2 NHCH2); 7.59—7.71 (m, 2 H); 7.71—8.89
(m, 1 H); 8.00—8.11 (m, 1 H, C6H4). 13C NMR (δ: 25.96,
28.54, 28.59, 28.93, 37.91, 41.03, 43.78, 44.23, 46.82,
47.05, 79.16, 79.74, 80.02, 125.25, 130.93, 132.68, 133.51,
148.29, 156.17.
9,14,N17ꢀTri(tertꢀbutoxycarbonyl)ꢀ5ꢀ(2ꢀnitrophenylsulfonyl)ꢀ
1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ17ꢀaminoꢀ5,9,14ꢀtriazaꢀ
heptadecane (8a). Cesium carbonate (0.083 g, 0.254 mmol) and
bromide 2 (0.168 g, 0.306 mmol) were added successively to
a solution of compound 7 (0.175 g, 0.254 mmol) in anhydrous
DMF (3 mL). The reaction mixture was stirred for 1 h at 60 °C.
The precipitate was filtered off through Celite® 545 and washed
with CH2Cl2. After removal of the solvents in vacuo, the resiꢀ
due was chromatographed on a column with silica gel using
a CHCl3—MeOH mixture (100 : 1) as the eluent to give comꢀ
pound 8a as a crystallizing oil (0.253 g, 86%), Rf 0.38 (C). MS,
m/z (Irel (%)): 1178.741 [M + Na]+ (100). Calculated for
C63H105N5O12S: 1155.748 [M]+. 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); 0.83 (d, 3 H, C(20)Me, J = 6.5 Hz); 0.94

Synthesis of polycationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
265
9,14,N17ꢀTri(tertꢀbutoxycarbonyl)ꢀ1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)ꢀ
oxycarbonyl]ꢀ17ꢀaminoꢀ5,9,14ꢀtriazaheptadecane (9a). Potassiꢀ
um carbonate (0.072 g, 0.520 mmol) and then PhSH (0.13 mL,
1.310 mmol) were added with stirring to a solution of compound
8a (0.150 g, 0.130 mmol) in DMF (3 mL). After 1 h, the reaction
mixture was filtered through Celite® 545, the precipitate was
washed with MeOH, and the solvent was evaporated in vacuo.
Chromatography on silica gel (CHCl3—MeOH—25% aq. NH3,
25 : 1 : 0.1) gave compound 9a as a yellowish crystallizing oil
(0.079 g, 83%), Rf 0.33 (F). MS, m/z (Irel (%)): 971.7 [M + H]+
(100). Calculated for C57H102N4O8: 970.7 [M]+. 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); 0.84 (d, 3 H, C(20)Me,
J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.90—2.00 (m, 38 H, Chol,
2 CH2(CH2)2CH2, 2 NCH2CH2CH2N); 1.36 (br.s, 18 H) and
1.39 (br. s, 9 H, 3 C(CH3)3); 2.15—2.33 (m, 4 H, OC(O)CH2,
H2C(4)); 2.60—2.87 (m, 4 H, CH2NHCH2); 2.96—3.35 (m, 10 H,
CH2NH, 4 NCH2); 4.44—4.59 (m, 1 H, H(3)); 5.01—5.26
(m, 1 H, NHBoc); 5.26—5.33 (m, 1 H, H(6)). 13C NMR, δ:
11.94, 18.81, 19.40, 21.12, 22.29, 22.66, 22.91, 23.91, 24.37,
25.82, 27.88, 28.09, 28.31, 28.54, 31.94, 31.99, 34.02, 35.87,
36.27, 36.68, 37.07, 37.53, 38.22, 39.60, 39.82, 42.39, 43.42,
43.99, 44.35, 45.45, 46.88, 47.25, 50.12, 56.22, 56.78, 74.12,
79.70, 122.74, 139.71, 156.14, 172.53.
19.43, 21.13, 22.66, 22.92, 23.92, 24.38, 25.59, 25.96,
26.31, 26.37, 27.60, 28.09, 28.29, 28.32, 28.54, 29.93, 31.97, 35.89, 36.27,
36.65, 37.10, 37.55, 38.69, 39.60, 39.83, 40.79, 42.40,
43.73, 44.20, 44.30, 45.83, 45.90, 46.47, 46.60, 46.84,
47.08, 50.10, 56.22, 56.78, 74.22, 79.56, 122.50, 139.97, 156.13,
156.18, 156.33.
17ꢀAminoꢀ1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ5,9,14ꢀtriꢀ
azaheptadecane tetrahydrochloride (10a). A 4 M solution of HCl
in dioxane (4 mL) was added to a solution of compound 9a
(0.164 g, 0.169 mmol) in 4 mL of MeOH, and the mixture was
stirred for 2 h at 24 °C. The solvents were removed in vacuo, the
residue was recrystallized from a CHCl3—EtOH mixture (1 : 1)
to give compound 10a as beigeꢀcolored crystals (0.136 g, 99%),
Rf 0.35 (G). MS, m/z (Irel (%)): 671.5 [M – 4 HCl + H]+ (100).
Calculated for C42H82Cl4N4O2: 670.61 [M – 4 HCl]+. 1H NMR
(D2O), δ: 0.61 (s, 3 H, C(13)Me); 0.77 (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); 0.94 (s, 3 H, C(10)Me); 0.90—2.00
(m, 38 H, Chol, NHCH2(CH2)2CH2N, 2 NCH2CH2CH2N,
CH2(CH2)2CH2); 2.11—2.32 (m, 2 H, H2C(4)); 2.90—3.15
(m, 16 H, OC(O)CH2, 7 CH2N); 4.20—4.40 (m, 1 H, H(3));
5.20—5.35 (m, 1 H, H(6)).
1,17ꢀDiaminoꢀN1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ5,9,14ꢀ
triazaheptadecane tetrahydrochloride (10b) was prepared in the
same way as compound 10a from 9b (0.120 g, 0.122 mmol).
Compound 10b was obtained as beigeꢀcolored crystals (0.098 g,
97%), Rf 0.38 (G). MS, m/z (Irel (%)): 686.6 [M – 4 HCl + H]+
(100). Calculated for C42H83Cl4N5O2: 685.63 [M – 4 HCl]+.
1H NMR (D2O), δ: 0.60 (s, 3 H, C(13)Me); 0.76 (d, 3 H,
C(25)Me, J = 6.5 Hz); 0.79 (d, 3 H, C(25)Me, J = 6.5 Hz); 0.85
(d, 3 H, C(20)Me, J = 6.5 Hz); 0.95 (s, 3 H, C(10)Me); 0.90—2.00
(m, 38 H, Chol, NHCH2(CH2)2CH2N, 2 NCH2CH2CH2N,
CH2(CH2)2CH2); 2.10—2.33 (m, 2 H, H2C(4)); 2.91—3.15
(m, 16 H, 8 CH2N); 4.13—4.39 (m, 1 H, H(3)); 5.17—5.38
(m, 1 H, H(6)).
1,19ꢀDiaminoꢀN1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ7,11,16ꢀ
triazanonadecane tetrahydrochloride (10c) was prepared in the
same way as compound 10a from 9c (0.144 g, 0.142 mmol).
Compound 10c was obtained as white crystals (0.116 g, 95%),
Rf 0.40 (G). MS, m/z (Irel (%)): 714.6 [M – 4 HCl + H]+(100).
Calculated for C44H87Cl4N5O2: 713.65 [M – 4 HCl]+. 1H NMR
(D2O), δ: 0.61 (s, 3 H, C(13)Me); 0.77 (d, 3 H, C(25)Me,
J = 6.5 Hz); 0.79 (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); 0.91—2.05
(m, 42 H, Chol, NHCH2(CH2)2CH2N, 2 NCH2CH2CH2N,
CH2(CH2)4CH2); 2.12—2.35 (m, 2 H, H2C(4)); 2.91—3.14
(m, 16 H, 8 CH2N); 4.13—4.40 (m, 1 H, H(3)); 5.17—5.38
(m, 1 H, H(6)).
4,9ꢀDi(tertꢀbutoxycarbonyl)ꢀ1,12ꢀbis(2ꢀnitrophenylsulfonyꢀ
lamino)ꢀ4,9ꢀdiazadodecane (12). Molecular sieves 4 Å (0.5 g),
Et3N (1.0 mL, 6.72 mmol) and 2ꢀnitrobenzenesulfonyl chloride
(0.893 g, 4.03 mmol) were added successively to a cooled (0 °C)
solution of compound 11 (0.676 g, 1.68 mmol) in anhydrous
CH2Cl2 (5 mL). The reaction mixture was stirred for 5 h at
24 °C. The molecular sieves were filtered off and
washed with CH2Cl2, and the solvents were removed in vacuo.
The residue was chromatographed on a column with silica gel
(CHCl3 → CHCl3—MeOH, 50 : 1) to give compound 12 as
a yellowish crystallizing oil (0.818 g, 63%), Rf 0.35 (D). 1H NMR,
δ: 1.35 (br.s, 22 H, 2 C(CH3)3, CH2(CH2)2CH2); 1.48—1.70
(m, 4 H, 2 CH2CH2CH2); 2.86—3.10 (m, 8 H, 4 NCH2); 3.10—3.38
9,14,N17ꢀTri(tertꢀbutoxycarbonyl)ꢀN1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀ
yl)oxycarbonyl]ꢀ1,17ꢀdiaminoꢀ5,9,14ꢀtriazaheptadecane (9b) was
prepared in the same way as compound 9a from 8b (0.130 g,
0.110 mmol), K2CO3 (0.050 g, 0.360 mmol), and PhSH (0.1 mL,
1.10 mmol). Compound 9b was obtained as a yellowish amorꢀ
phous solid (0.107 g, 99%). Rf 0.35 (Е). MS, m/z (Irel (%)):
986.796 [M + H]+ (100). Calculated for C57H103N5O8: 985.781
[M]+. 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);
0.84 (d,
3 H, C(20)Me, J = 6.5 Hz); 0.94 (s, 3 H,
C(10)Me); 0.90—2.00 (m, 38 H, Chol, 2 CH2(CH2)2CH2,
2 NCH2CH2CH2N); 1.36 (br.s, 18 H, 2 C(CH3)3); 1.39 (br.s,
9 H, C(CH3)3); 2.11—2.33 (m, 2 H, H2C(4)); 2.53—2.82
(m, 4 H, CH2NHCH2); 2.95—3.33 (m, 12 H, 2 CH2NH,
4 NCH2); 4.32—4.47 (m, 1 H, H(3)); 4.55—4.96 (m, 1 H, NH);
4.96—5.25 (m, 1 H, NH); 5.25—5.33 (m, 1 H, H(6)). 13C NMR,
δ: 11.92, 18.77, 19.40, 21.10, 22.62, 22.88, 23.88, 24.34, 26.01,
27.40, 28.05, 28.25, 28.28, 28.51, 31.93, 35.84, 36.23, 36.61,
37.06, 37.58, 38.66, 39.57, 39.79, 40.29, 42.36, 43.73, 44.21,
46.56, 46.84, 50.07, 56.19, 56.75, 74.21, 79.67, 122.46, 139.94,
156.15, 156.41.
11,16,N19ꢀTriꢀ(tertꢀbutoxycarbonyl)ꢀN1ꢀ[(cholestꢀ5ꢀenꢀ3βꢀ
yl)oxycarbonyl]ꢀ1,19ꢀdiaminoꢀ7,11,16ꢀtriazanonadecane (9c)
was prepared in the same way as compound 9a from 8c (0.106 g,
0.088 mmol), K2CO3 (0.053 g, 0.380 mmol), and PhSH (0.1 mL,
1.10 mmol).Compound 9c was obtained as a yellowish amorꢀ
phous solid (0.079 g, 89%), Rf 0.37 (F). MS, m/z (Irel (%)):
1014.7 [M + H]+ (100). Calculated for C59H107N5O8: 1013.8
[M]+. 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); 0.84
(d, 3 H, C(20)Me, J = 6.5 Hz); 0.94 (s, 3 H, C(10)Me); 0.90—2.00
(m, 42 H, Chol, NHCH2(CH2)2CH2N, 2 NCH2CH2CH2N,
CH2(CH2)4CH2); 1.37 (br.s, 18 H) and 1.39 (br. s, 9 H,
3 C(CH3)3); 2.11—2.35 (m, 2 H, H2C(4)); 2.47—2.76 (m, 4 H,
CH2NHCH2); 2.93—3.30 (m, 12 H, 2 CH2NH, 4 NCH2);
4.32—4.52 (m, 1 H, H(3)); 4.50—4.85 (m, 1 H, NH); 4.96—5.25
(m, 1 H, NH); 5.26—5.35 (m, 1 H, H(6)). 13C NMR, δ: 11.95, 18.81,

266
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Petukhov et al.
1
(m, 4 H, 2 NCH2); 7.50—7.75 (m, 4 H); 7.95—8.05 (m, 2 H)
and 8.05—8.20 (m, 2 H, 2 C6H4). 13C NMR, δ: 25.42,
25.83, 28.38, 28.86, 29.22, 40.66, 43.32, 44.25, 46.46,
46.90, 79.84, 124.47, 130.62, 132.41, 135.69, 148.02, 148.18,
155.42, 156.38.
56%), Rf 0.40 (C). H NMR, δ: 0.61 (s, 6 H, 2 C(13)Me); 0.79
(d, 6 H, 2 C(25)Me, J = 6.5 Hz); 0.80 (d, 6 H, 2 C(25)Me,
J = 6.5 Hz); 0.83 (d, 6 H, 2 C(20)Me, J = 6.5 Hz); 0.94 (s, 6 H,
2 C(10)Me); 0.90—2.00 (m, 76 H, Chol, NCH2(CH2)2CH2N,
2 NCH2CH2CH2N, 2 CH2(CH2)4CH2); 1.37 (br.s, 18 H,
2 C(CH3)3); 2.11—2.37 (m, 4 H, 2 H2C(4)); 2.96—3.14 (m, 12 H,
2 CH2NH, 4 NCH2); 3.14—3.30 (m, 8 H, 4 NCH2); 4.33—4.50
(m, 2 H, 2 H(3)); 4.55—4.78 (m, 2 H, 2 NH); 5.26—5.32
(m, 2 H, 2 H(6)); 7.51—7.58 (m, 2 H); 7.59—7.68 (m, 4 H)
and 7.88—7.97 (m, 2 H, 2 C6H4). 13C NMR, δ: 11.97,
18.83, 19.45, 21.14, 22.68, 22.94, 23.93, 24.40, 25.68, 26.25,
26.38, 27.50, 28.03, 28.11, 28.30, 28.34, 28.57, 29.97,
31.97, 35.90, 36.28, 36.66, 37.10, 38.70, 39.61, 39.83, 40.79,
42.41, 44.65, 45.14, 47.20, 50.10, 56.22, 56.78, 74.25, 79.61,
122.53, 124.26, 130.77, 131.78, 133.54, 133.62, 139.98, 148.11,
155.54, 156.29.
9,14ꢀDi(tertꢀbutoxycarbonyl)ꢀ5,18ꢀbis(2ꢀnitrophenylsulfoꢀ
nyl)ꢀ1,22ꢀdi[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ5,9,14,18ꢀtetꢀ
raazadocosane (13a). Cesium carbonate (0.084 g, 0.259 mmol)
and bromide 2 (0.300 g, 0.546 mmol) were added successively to
a solution of compound 12 (0.200 g, 0.259 mmol) in anhydrous
DMF (7 mL). The reaction mixture was stirred for 1 h at 60 °C.
The precipitate was filtered off through Celite® 545 and washed
with CH2Cl2. After removal of the solvents in vacuo, the resiꢀ
due was chromatographed on a column with silica gel using
a CHCl3—MeOH—25% aq. NH3 mixture (60 : 2 : 0.1 →
→ 60 : 4 : 0.1) as the eluent to give compound 13a as a lightꢀ
yellow crystallizing oil (0.240 g, 61%). Rf 0.38 (В). 1H NMR, δ:
0.61 (s, 6 H, 2 C(13)Me); 0.79 (d, 6 H, 2 C(25)Me, J = 6.5 Hz);
0.80 (d, 6 H, 2 C(25)Me, J = 6.5 Hz); 0.83 (d, 6 H, 2 C(20)Me,
J = 6.5 Hz); 0.94 (s, 6 H, 2 C(10)Me); 0.90—2.07 (m, 68 H,
Chol, 3 CH2(CH2)2CH2, 2 NCH2CH2CH2N); 1.36 (br.s, 18 H,
2 C(CH3)3); 2.20—2.38 (m, 8 H, 2 H2C(4); 2 OC(O)CH2);
3.00—3.17 (m, 8 H, 4 NCH2); 3.17—3.31 (m, 8 H, 4 NCH2);
4.33—4.49 (m, 2 H, 2 H(3)); 5.25—5.34 (m, 2 H, 2 H(6));
7.49—7.60 (m, 2 H); 7.58—7.69 (m, 4 H) and 7.88—7.98 (m, 2 H,
2 C6H4). 13C NMR, δ: 11.95, 18.64, 19.21, 20.96, 21.88, 22.46,
22.69, 23.75, 24.19, 25.58, 25.74, 27.30, 27.46, 27.72, 27.89,
28.11, 28.37, 31.81, 31.82, 33.84, 35.68, 36.12, 36.52, 36.93,
38.07, 39.44, 39.68, 42.25, 44.48, 45.07, 46.99, 47.02, 50.01,
56.12, 56.64, 73.85, 79.37, 122.2, 124.06, 130.60, 131.54, 133.38,
139.58, 147.00, 155.32, 172.30.
9,14ꢀDi(tertꢀbutoxycarbonyl)ꢀ5,18ꢀbis(2ꢀnitrophenylsulfoꢀ
nyl)ꢀ1,22ꢀdi[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonylamino]ꢀ5,9,14,18ꢀ
tetraazadocosane (13b) was prepared in the same way as comꢀ
pound 13a from 12 (0.290 g, 0.375 mmol) and 5a (0.509 g,
0.901 mmol) in the presence of Cs2CO3 (0.244 g, 0.750 mmol).
Chromatography (CHCl3—MeOH, 100 : 0.5 → 100 : 1) gave
compound 13b as a lightꢀyellow crystallizing oil (0.390 g, 60%),
Rf 0.38 (C). 1H NMR, δ: 0.61 (s, 6 H, 2 C(13)Me); 0.79 (d, 6 H,
2 C(25)Me, J = 6.5 Hz); 0.80 (d, 6 H, 2 C(25)Me, J =
= 6.5 Hz); 0.83 (d, 6 H, 2 C(20)Me, J = 6.5 Hz); 0.94 (s, 6 H,
2 C(10)Me); 0.90—2.00 (m, 68 H, Chol, 3 CH2(CH2)2CH2,
2 NCH2CH2CH2N); 1.36 (br.s, 18 H, 2 C(CH3)3); 2.11—2.38
(m, 4 H, 2 H2C(4)); 2.96—3.14 (m, 12 H, 2 CH2NH, 4 NCH2);
3.14—3.29 (m, 8 H, 4 NCH2); 4.33—4.49 (m, 2 H, 2 H(3));
4.58—4.75 (m, 2 H, 2 NH); 5.26—5.33 (m, 2 H, 2 H(6));
7.51—7.58 (m, 2 H); 7.58—7.69 (m, 4 H) and 7.88—7.98
(m, 2 H, 2 C6H4). 13C NMR, δ: 11.95, 18.81, 19.43,
21.13, 22.66, 22.92, 23.92, 24.37, 25.52, 26.02, 27.13,
27.54, 28.09, 28.27, 28.32, 28.56, 31.96, 31.98, 35.88,
36.27, 36.64, 37.08, 38.68, 39.60, 39.81, 40.30, 42.39, 44.71,
45.20, 45.67, 47.28, 47.64, 50.09, 56.22, 56.77, 74.27, 79.62,
122.53, 124.25, 130.72, 131.81, 133.36, 133.65, 139.93, 148.11,
155.53, 156.33.
9,14ꢀDi(tertꢀbutoxycarbonyl)ꢀ1,22ꢀdi[(cholestꢀ5ꢀenꢀ3βꢀyl)ꢀ
oxycarbonyl]ꢀ5,9,14,18ꢀtetraazadocosane (14a). Potassium
carbonate (0.035 g, 0.252 mmol) and then PhSH (0.13 mL,
1.13 mmol) were added with stirring to a solution of compound
13a (0.215 g, 0.126 mmol) in DMF (2 mL). After 1 h, the reacꢀ
tion mixture was filtered through Celite® 545, the precipitate
was washed with MeOH, and the solvent was evaporated in vacuo.
The residue was chromatographed on a column with silica gel
using a CHCl3—MeOH—25% aq. NH3 mixture (100 : 10 : 1) as
the eluent to give compound 14a as a yellow crystallizing oil
(0.100 g, 60%). Rf 0.32 (Е). MS, m/z (Irel (%)): 1339.642 [M]+
(100). Calculated for C84H146N4O8: 1339.114 [M]+. 1H NMR,
δ: 0.61 (s, 6 H, 2 C(13)Me); 0.79 (d, 6 H, 2 C(25)Me, J = 6.5 Hz);
0.80 (d, 6 H, 2 C(25)Me, J = 6.5 Hz); 0.83 (d, 6 H, 2 C(20)Me,
J = 6.5 Hz); 0.94 (s, 6 H, 2 C(10)Me); 0.90—2.03 (m, 68 H,
Chol, 3 CH2(CH2)2CH2, 2 NCH2CH2CH2N); 1.37 (br.s, 18 H,
2 C(CH3)3); 2.30—2.35 (m, 8 H, 2 H2C(4), 2 OC(O)CH2);
2.50—2.70 (m, 8 H, 2 CH2NHCH2); 3.00—3.30 (m, 8 H,
4 NCH2); 4.40—4.50 (m, 2 H, 2 H(3)); 5.25—5.33 (m, 2 H,
2 H(6)). 13C NMR, δ: 12.03, 18.91, 19.47, 21.24, 22.71, 22.94,
24.03, 24.46, 26.07, 28.03, 28.16, 28.38, 28.66, 29.20, 32.10,
34.60, 35.95, 36.40, 36.80, 37.21, 38.37, 39.72, 39.97, 42.54,
45.03, 47.03, 49.55, 50.30, 56.41, 56.93, 74.04, 79.59, 122.76,
139.90, 155.97, 172.99.
9,14ꢀDi(tertꢀbutoxycarbonyl)ꢀ1,22ꢀdi[(cholestꢀ5ꢀenꢀ3βꢀyl)ꢀ
oxycarbonylamino]ꢀ5,9,14,18ꢀtetraazadocosane (14b) was preꢀ
pared in the same way as compound 14a from 13b (0.200 g,
0.115 mmol), K2CO3 (0.048 g, 0.345 mmol), and PhSH (0.14 mL,
1.15 mmol). Chromatography (CHCl3—MeOH—25% aq. NH3,
120 : 7.6 : 1) gave compound 14b as a lightꢀyellow crystallizing
oil (0.121 g, 77%). Rf 0.30 (Е). MS, m/z (Irel (%)): 1370.339
[M + H]+ (100). Calculated for C84H148N6O8: 1369.136 [M]+.
1H NMR, δ: 0.61 (s, 6 H, 2 C(13)Me); 0.79 (d, 6 H, 2 C(25)Me,
J = 6.5 Hz); 0.80 (d, 6 H, 2 C(25)Me, J = 6.5 Hz); 0.83 (d, 6 H,
2 C(20)Me, J = 6.5 Hz); 0.94 (s, 6 H, 2 C(10)Me); 0.90—2.00
(m, 68 H, Chol, 3 CH2(CH2)2CH2, 2 NCH2CH2CH2N); 1.37
(br.s, 18 H, 2 C(CH3)3); 2.10—2.35 (m, 4 H, 2 H2C(4));
2.47—2.62 (m, 8 H, 2 CH2NHCH2); 2.98—3.28 (m, 12 H,
2 CH2NH, 4 NCH2); 4.34—4.49 (m, 2 H, 2 H(3)); 4.58—4.70
(m, 2 H, 2 NH); 4.92—5.11 (m, 2 H, 2 NH); 5.25—5.33 (m, 2 H,
2 H(6)). 13C NMR, δ: 12.00, 18.86, 19.49, 21.19, 22.70, 22.96,
23.97, 24.43, 25.86, 26.14, 27.13, 27.97, 28.15, 28.37, 28.62,
32.02, 35.94, 36.33, 36.70, 37.15, 38.76, 39.66, 39.88, 40.86,
42.45, 44.45, 45.17, 46.81, 47.41, 50.15, 56.28, 56.83, 74.25,
79.54, 122.55, 140.02, 155.53, 156.38.
11,16ꢀDi(tertꢀbutoxycarbonyl)ꢀ7,20ꢀbis(2ꢀnitrophenylsulfoꢀ
nyl)ꢀ1,26ꢀdi[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonylamino]ꢀ7,11,16,20ꢀ
tetraazahexacosane (13c) was prepared in the same way as comꢀ
pound 13a from 12 (0.200 g, 0.259 mmol) and 5b (0.338 g,
0.569 mmol) in the presence of Cs2CO3 (0.084 g, 0.259 mmol).
Chromatography (CHCl3—MeOH, 100 : 0.5 → 100 : 1) affordꢀ
ed compound 13c as a lightꢀyellow crystallizing oil (0.262 g,

Synthesis of polycationic lipids
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
267
11,16ꢀDi(tertꢀbutoxycarbonyl)ꢀ1,26ꢀdi(cholestꢀ5ꢀenꢀ3βꢀ
yloxycarbonylamino)ꢀ7,11,16,20ꢀtetraazahexacosane (14c) was
prepared in the same way as compound 14a from 13c (0.330 g,
0.184 mmol), K2CO3 (0.076 g, 0.552 mmol), and PhSH (0.19 mL,
1.83 mmol). Compound 14c was obtained as a lightꢀyellow crysꢀ
tallizing oil (0.185 g, 71%), Rf 0.33 (F). MS, m/z (Irel (%)):
1426.238 [M + H]+ (100). Calculated for C88H156N6O8:
1425.198 [M]+. 1H NMR, δ: 0.61 (s, 6 H, 2 C(13)Me); 0.79
(d, 6 H, 2 C(25)Me, J = 6.5 Hz); 0.80 (d, 6 H, 2 C(25)Me,
J = 6.5 Hz); 0.83 (d, 6 H, 2 C(20)Me, J = 6.5 Hz); 0.94 (s, 6 H,
2 C(10)Me); 0.90—2.00 (m, 76 H, Chol, NCH2(CH2)2CH2N,
2 NCH2CH2CH2N, 2 CH2(CH2)4CH2); 1.37 (br.s, 18 H,
2 C(CH3)3); 2.12—2.35 (m, 4 H, 2 H2C(4)); 2.47—2.61 (m, 8 H,
2 CH2NHCH2); 2.99—3.27 (m, 12 H, 2 CH2NH, 4 NCH2);
4.34—4.49 (m, 2 H, 2 H(3)); 4.57—4.70 (m, 2 H, 2 NH);
5.26—5.33 (m, 2 H, 2 H(6)). 13C NMR, δ: 11.97, 18.83,
19.45, 21.14, 22.68, 22.94, 23.93, 24.40, 25.68, 26.25, 26.38,
27.50, 28.03, 28.11, 28.30, 28.34, 28.57, 29.97, 31.97, 35.90,
36.28, 36.66, 37.10, 38.70, 39.61, 39.83, 40.79, 42.41, 44.65,
45.14, 47.20, 50.10, 56.22, 56.78, 74.25, 79.61, 122.53, 139.98,
155.54, 156.29.
1,22ꢀDi[(cholestꢀ5ꢀenꢀ3βꢀyl)oxycarbonyl]ꢀ5,9,14,18ꢀtetꢀ
raazacosane tetrahydrochloride (15a). A 4 M solution of HCl in
dioxane (4 mL) was added to a solution of compound 14a (0.100 g,
0.075 mmol) in MeOH (3 mL) and the mixture was stirred for
2 h at 24 °C. The solvent was removed in vacuo to give compound
15a as beigeꢀcolored crystals (79 mg, 82%), Rf 0.40 (G). MS,
m/z (Irel (%)): 1139.784 [M – 4 HCl]+ (100). Calculated
for C74H134Cl4N4O4: 1139.009 [M – 4 HCl]+. 1H NMR
(DMSOꢀd6 — CDCl3, 1 : 3), δ: 0.61 (s, 6 H, 2 C(13)Me); 0.77
(d, 6 H, 2 C(25)Me, J = 6.5 Hz); 0.79 (d, 6 H, 2 C(25)Me,
J = 6.5 Hz); 0.84 (d, 6 H, 2 C(20)Me, J = 6.5 Hz); 0.94 (s, 6 H,
2 C(10)Me); 0.90—2.03 (m, 68 H, Chol, NHCH2(CH2)2CH2N,
2 NCH2CH2CH2N, 2 CH2(CH2)4CH2); 2.12—2.35 (m, 4 H,
2 H2C(4)); 2.40—2.61 (m, 8 H, 2 CH2NHCH2); 2.99—3.27
(m, 12 H, 2 CH2NH, 4 NCH2); 4.34—4.49 (m, 2 H, 2 H(3));
4.57—4.70 (m, 2 H, 2 NH); 5.26—5.33 (m, 2 H, 2 H(6)).
This work was supported by the Russian Foundation
for Basic Research (Project No. 07ꢀ03ꢀ00632a) within
a Federal target program (contract No. 02.512.11.2200).
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Received May 15, 2009;
in revised form October 26, 2009