Synthesis of cationic amphiphiles on the basis of deoxycholic acid

Article abstract of DOI:10.1023/B:RUBI.0000043792.13604.b3

Cationic derivatives of deoxycholic acid with N,N-dimethylenediamine, ε-aminocaproic acid, and pyridine as polar heads were synthesized. The cationic groups were linked to 3α- and 12α-hydroxy groups of the steroid moiety through ester or urethane bonds. Liposomal formulations of the compounds synthesized may be used for gene delivery in cells.

Full text of DOI:10.1023/B:RUBI.0000043792.13604.b3

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 5, 2004, pp. 477–481. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 5, 2004, pp. 531–536.
Original Russian Text Copyright © 2004 by Sokolova, Maslov, Serebrennikova.
Synthesis of Cationic Amphiphiles on the Basis
of Deoxycholic Acid
1
T. V. Sokolova , M. A. Maslov, and G. A. Serebrennikova
Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 119571 Russia
Received July 10, 2003; in final form, August 15, 2003
Abstract—Cationic derivatives of deoxycholic acid with N,N-dimethylenediamine, ε-aminocaproic acid, and
pyridine as polar heads were synthesized. The cationic groups were linked to 3α- and 12α-hydroxy groups of
the steroid moiety through ester or urethane bonds. Liposomal formulations of the compounds synthesized may
be used for gene delivery in cells.
Key words: cationic amphiphiles, deoxycholic acid, transfection
1
INTRODUCTION
lead to the creation of efficient systems for the introduc-
tion in cell of various biologically active substances:
nucleosides, oligo- and polynucleotides, hormones,
proteins, and other natural and synthetic macromole-
cules with negatively charged moiety.
An interest in basically new technologies that allow
the address delivery of new blocks of genetic informa-
tion to defect cells for the subsequent expression
increased in recent years. The contemporary medicine
knows several hundreds of diseases that are directly
connected with disturbances in gene functioning [1, 2].
Such defects could be repaired by the direct introduc-
tion into the cells of the corresponding organs and tis-
sues of the genetic material that would specially be con-
structed and would provide the synthesis of missing
protein. The gene therapy that is devoted to the intro-
duction of DNA, mRNA, or oligonucleotides into
organism with the aim to treat it is one of the directions
of modern medicine [3, 4].
The delivery of genetic material in cell (transfec-
tion) is a necessary stage of genetic therapy. Various
molecular constructs of viral and nonviral origin are
used for its realization. One of the modern methods
with a high therapeutic potential is the method of lipo-
fection, which is based on the use of positively charged
liposomes. It is promising, because liposomes are bio-
degradable and there is a minimal probability of the ini-
tiation of immune response or inflammation [5–7].
Metabolizable lipids with a minimal cytotoxicity are
the most promising for solving applied problems, and
the search for them is expedient to carry out among the
modified natural lipids [8]. The compounds whose
RESULTS AND DISCUSSION
We develop studies of the synthesis of cationic
amphiphiles on the basis of cholesterol [3] and describe
in this paper the synthesis of new representatives of the
class of cationic lipids on the basis of deoxycholic acid
(I). The use of polyfunctional deoxycholic acid as the
hydrophobic part of cationic amphiphile allows the
preparation of the compounds with several positively
charged groups. Such an approach could affect the
transfection efficiency, since the stability of liposome–
DNA (genosome) complexes depend on the density of
positive charge on the liposome surface [6, 7].
We synthesized cationic derivatives of deoxycholic
acid with various modes of linkage of polar grouping to
the steroid part of molecule. Octadecyl deoxycholate
(IIa) and methyl deoxycholate (IIb) were used as start-
ing compounds, which provided for the protection of
carboxyl group, necessary in subsequent conversions,
and helped change the total hydrophobicity of mole-
cule, which seems to affect the transfection efficiency.
We synthesized cationic lipids (Va) and (Vb)
hydrophobic part is represented by the derivatives of (Scheme 1) in which nitrogen base is attached to the
steroid series are now rather attractive among the cat- deoxycholate molecules with urethane bond, more sta-
ionic lipids of various types. The steroid structure was ble in biological media, in order to reveal the relation
found to significantly influence the DNA transfection between the structure and biological activity. The inter-
efficiency [9, 10].
The further study of positively charged lipids, in
particular, cholesterol and bile acid derivatives, could
action of (IIa) and (IIb) with 1,1'-carbonyldiimidazole
in dichloromethane catalyzed with triethylamine
resulted in octadecyl and methyl 3α,12α-bis(1-imida-
zolylcarbonyloxy)-5β-cholan-24-oates (IIIa) and
(IIIb) in 93–97% yields. These compounds were then
treated with N,N-dimethylethylenediamine in dichlo-
1
Corresponding author; phone: +7 (095) 434-8544; e-mail:
httos.mitht@g23.relcom.ru
1068-1620/04/3005-0477 © 2004 MAIK “Nauka /Interperiodica”

478
SOKOLOVA et al.
OH
OH
COOH
COOR
‡: C18H37OH, p-TsOH
·: CH3OH, H2SO4
HO
HO
H
H
(I)
(II‡, IIb)
Im2CO
BocHN(CH2)5COOH,
DCC
N COO
COOR
N
BocHN(CH2)5COO
COOR
N COO
H
(III‡, IIIb)
N
BocHN(CH2)5COO
COOR
H
(VI‡, VIb)
H2N(CH2)2N(CH3)2
CF3COOH
(CH3)2N(CH2)2NHCOO
+
H3 N(CH2)5COO
2CF COO–
COOR
(CH3)2N(CH2)2NHCOO
3
H
(IV‡, IVb)
+
H3 N(CH2)5COO
H
CH3I
(VII‡, VIIb)
+
(CH3)3 N(CH2)2NHCOO
COOR
2I–
‡: R = C18H37
b: R = CH3
+
(CH3)3 N(CH2)2NHCOO
H
(V‡, Vb)
Scheme 1.
romethane, which resulted in tertiary amines (IVa) and by the DCC-catalyzed acylation of starting esters (IIa)
and (IIb) with N-ÇÓÒ-ε-aminocaproic acid in dichlo-
romethane to (VIa) and (VIb) (yield 58 and 64%,
respectively) and subsequent removal of Boc protective
group with trifluoroacetic acid in chloroform; octadecyl
and methyl 3α,12α-bis(ε-ammoniocaproyloxy)-5β-
cholan-24-oate bistrifluoroacetates (VIIa) and (VIIb)
were obtained in 74 and 90% yields.
(IVb) in 62 and 66% yields, respectively. The interac-
tion of (IVa) and (IVb) with methyl iodide and the sub-
sequent chromatographic purification led to octadecyl
and methyl 3α,12α-bis(N,N,N-trimethylammonioethyl-
carbamoyloxy)-5β-cholan-24-oate diiodides (Va) and
(Vb) in 70 and 88% yields.
The synthesis of cationic lipids (VIIa) and (VIIb)
containing NH+3 group in their polar head was achieved
In addition to the compounds with aliphatic polar
groups, we synthesized a cationic lipid (IX) with pyri-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

SYNTHESIS OF CATIONIC AMPHIPHILES ON THE BASIS OF DEOXYCHOLIC ACID
479
N+
(CH2)4COO
Br(CH2)4COO
COOCH3
COOCH3
2Br–
Br(CH2)4COCl
Py
(IIb)
N+
(CH2)4COO
Br(CH2)4COO
H
H
(VIII)
(IX)
Scheme 2.
dinium residue (Scheme 2). To this end, a spacer group 3.62 (1 H, m, H3), 3.98 (1 H, m, H12), and 4.06 (2 H,
was introduced into (IIb) by the reaction with 5-bro- t, J 6.9, COOëç2).
movaleric acid chloride. The reaction product (VIII)
Metyl deoxycholate (IIb). Conc. ç2SO4 (0.09 ml)
(81% yield) was heated in anhydrous pyridine, which
was added to a solution of sodium deoxycholate
resulted in cationic lipid (IX) in 90% yield.
(0.50 g, 1.2 mmol) in methanol (6 ml). The reaction
mixture was refluxed for 3 h, neutralized with methan-
olic solution of KOH to pH 6, and evaporated. The res-
idue was dissolved in chloroform; the solution was
washed with water (2 × 15 ml), dried with Na2SO4, and
evaporated. The residue was chromatographed on a col-
umn eluted with chloroform. The yield of (IIb) was
The homogeneity and structure of the compounds
synthesized were confirmed by H NMR and mass
spectra.
1
EXPERIMENTAL
1
0.450 g (91%); Rf 0.68 (C); H NMR: 0.65 (3 H, s,
We used in this work the following solvents and
reagents: DMSO, N,N-dimethylethylenediamine, and
ε-aminocaproic acid (Sigma, United States); carbonyl-
diimidazole (Acros, Belgium); and triethylamine (Vek-
18-CH3), 0.86 (3 H, s, 19-CH3), 0.90 (3 H, d, J 6.9,
ëç3-21), 0.99–1.89 (24 H, steroid CH and ëç2), 2.22
(1 H, ddd, J 6.8, 9.0, and 15.4, H23a), 2.35 (1 H, ddd, J
5.1, 9.8, and 15.4, H23b), 3.59 (1 H, m, H3), 3.63 (3 H,
s, OCH3), and 3.92 (1 H, m, H12).
1
ton, Russia). H NMR spectra were registered on a
pulse Fourier-transform Bruker MSL-200 spectrometer
(200.13 MHz) and Bruker MSL-300 spectrometer
(300.13 MHz) in deuterochloroform with tetramethyl-
silane as an internal standard; chemical shifts are given
in ppm and spin coupling constants, J, in Hz. Mass
spectra were obtained on a time-of-flight Finnigan
MAT 900XL-TRAP mass spectrometer (San Jose, CA,
United States) with electrospray ionization (ESI MS).
Silufol UV-254 (Chemapol, Czech Republic) was used
for TLC; substance spot were detected on chromato-
grams by spraying with 10% phosphomolybdic acid
solution followed by heating. The following solvent
systems were used for TLC: (A) 10 : 1 chloroform–
methanol, (B) 7 : 1 chloroform–methanol, (C) 5 : 1
chloroform–methanol, and (D) 65 : 25 : 4 chloroform–
methanol–water. Silica gel L 40/100 µm (Chemapol,
Czech Republic) was used for column chromatography.
Octadecyl
3a,12a-bis(1-imidazolylcarbony-
loxy)-5b-cholan-24-oate (IIIa). A solution of (IIa)
(0.300 g, 0.5 mmol), 1,1'-carbonyldiimidazol (0.300 g,
1.8 mmol), and triethylamine (0.19 ml) in dichlo-
romethane (2 ml) was stirred for 3 h at 40°ë and evap-
orated. The residue was dissolved in chloroform
(15 ml), washed with 3% HCl (2 × 5 ml) and water,
dried with Na2SO4, and chromatographed on a column
eluted with 30 : 1 chloroform–methanol to give (IIIa);
yield 0.360 g (93%); Rf 0.81 (B); 1H NMR: 0.79 (3 H,
s, 18-CH3), 0.84 (3 H, t, J 6.7, ëç3-21), 0.87 [3 H, t, J
6.8, (ëç2)16ëç3), 0.90 (3 H, s, 19-CH3), 1.15–2.00
[58 H, steroid CH and CH2 and COOCH2(ëç2)16ëç3],
2.15 (1 H, ddd, J 7.3, 9.0, and 15.4, H23a), 2.27 (1 H,
ddd, J 5.5, 9.8, and 15.4, H23b), 4.00 (2 H, t, J 6.9,
COOëç2), 4.85 (1 H, m, H3), 5.32 (1 H, m, H12),
7.05–7.12 (2 H, m, Im), 7.32–7.47 (2 H, m, Im), and
8.05–8.19 (1 H, m, Im).
Octadecyl deoxycholate (IIa). Deoxycholic acid
(I) (0.70 g, 1.7 mmol) was fused together with octade-
canol (0.72 g, 2.6 mmol) in the presence of p-toluene-
sulfonic acid (0.37 g, 2.1 mmol) for 2 h at 100°ë. The
residue was chromatographed on a column eluted with
chloroform to give (IIa); yield 0.76 g (70%); Rf 0.77
Methyl
3a,12a-bis(1-imidazolylcarbonyloxy)-
5b-cholan-24-oate (IIIb). A solution of (IIb) (0.300 g,
0.7 mmol), 1,1'-carbonyldiimidazol (0.479 g,
2.9 mmol), and triethylamine (0.2 ml) in dichlo-
romethane (2 ml) was stirred for 5 h at 40°ë and evap-
orated. The residue was dissolved in chloroform (2 ml),
washed with 3% HCl (2 × 5 ml) and water, dried with
(A); 1H NMR: 0.65 (3 H, s, 18-CH3), 0.84 [3 H, t, J 6.8,
(ëç2)16ëç3), 0.87 (3 H, s, 19-ëç3), 0.90 (3 H, d, J 6.7,
ëç3-21), 1.05–1.89 [58 H, steroid CH and ëç2 and
COOCH2(ëç2)16ëç3), 2.25 (1 H, ddd, J 7.0, 9.3, and Na2SO4, and chromatographed on a column eluted with
15.1, H23a), 2.38 (1 H, ddd, J 5.2, 9.8, and 15.1, H23b), 30 : 1 chloroform–methanol to give (IIIb); yield 0.428 g
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

480
SOKOLOVA et al.
(97%); mp 55–56°ë; Rf 0.56 (Ä); 1ç NMR: 0.80 (3 H,
s, 18-CH3), 0.84 (3 H, d, J 6.7, 21-CH3), 0.95 (3 H, s,
(ëç2)16ëç3], 0.89 (3 H, s, 19-CH3), 0.92–1.95 [56 H,
m, COOCH2(ëç2)16ëç3 and steroid CH and ëç2],
19-CH3), 1.01–1.99 (26 H, steroid CH and ëç2), 2.22 2.14–2.42 (2 H, m, H23), 3.44 [18 H, s, 2N+(CH3)3],
(1 H, ddd, J 6.9, 9.0, and 15.4, H23a), 2.35 (1 H, ddd, 3.65–3.89 [8 H, m, 2ëç2N(CH3)2 and 2 2NHCH2],
J 5.3, 9.8, and 15.4, H23b), 3.63 (1 H, s, OCH3), 4.83
(1 H, m, H3), 5.35 (1 H, m, H12), 7.01–7.12 (2 H, m,
Im), 7.32–7.46 (2 H, m Im), and 8.05–8.19 (1 H, m,
Im).
4.01 (2 H, t, J 6.9, COOëç2), 4.54 (1 H, m, H3), 4.92
(1 H, m, H12), 6.09 (1 H, m, NH), and 6.44 (1 H, m,
NH).
Methyl 3a,12a-bis(N,N,N-trimethylammonioet-
hylcarbamoyloxy)-5b-cholan-24-oate diiodide (Vb).
Methyl iodide (0.08 ml, 0.5 mmol) was added to a solu-
tion of tertiary amine (IVb) (0.035 g (0.05 mmol) in
methylethylketone (1 ml); the reaction mixture was
stirred for 4 h at 60°ë and evaporated. The residue was
chromatographed on a column successively eluted
with10 : 1 and 5 : 1 chloroform–methanol mixtures to
give quaternary iodide (Vb); yield 0.045 g (88%); Rf
Octadecyl 3a,12a-bis(N,N-dimethylaminoethyl-
carbamoyloxy)-5b-cholan-24-oate (IVa). A solution
of (IIIa) (0.4 g, 0.5 mmol) and N,N-dimethylethylene-
diamine (0.2 ml) in dichloromethane (3 ml) was stirred
for 24 h at 15–20°ë, diluted with dichloromethane
(15 ml), washed with 5% KOH (2 × 8 ml) and water
(2 × 15 ml), dried with Na2SO4, and evaporated. The
residue was chromatographed on a column succes-
sively eluted with 50 : 1, 25 : 1, and 10 : 1 chloroform–
methanol mixtures to give (IVa); yield 0.267 g (62%);
Rf 0.33 (D); MS (m/z): 876.2 [M + H]+; 1ç NMR: 0.68
(3 H, s, 18-CH3), 0.81–2.0 (67 H, m, 19-CH3, 21-CH3,
COOCH2(ëç2)16ëç3, and steroid CH and ëç2), 2.07–
2.38 (2 H, m, H23), 2.58 [12 H, s, 2 N(CH3)2], 2.75–
2.88 [4 H, m, 2CH2N(CH3)2), 3.40–3.52 (4 H, m,
2NHCH2), 4.05 (2 H, t, J 6.9, COOëç2), 4.6 (1 H, m,
H3), 4.95 (1 H, m, H12), 5.85 (1 H, m), and 6.01 (1 H,
m, 2 NH).
1
0.23 (D); MS, m/z: 649.8 [M – ëç3]+; H NMR: 0.69
(3 H, s, 18-CH3), 0.81 (3 H, d, J 6.7, 21-CH3), 0.88
(3 H, s, 19-CH3), 0.92–2.00 (24 H, m, steroid CH and
CH2), 2.10–2.35 (2 H, m, H23), 3.48 [18 H, s,
2N+(CH3)3], 3.61 (3 H, s, Oëç3), 3.68–3.90 [8 H, m,
2NçCH2ëç2N), 4.52 (1 H, m, H3), 4.92 (1 H, m, H12),
6.01 (1 H, m, NH), and 6.70 (1 H, m, NH).
Octadecyl 3a,12a-bis(N-tert-butyloxycarbonyl-
amidocapronoyloxy)-5b-cholan-24-oate (VIa). Octa-
decyl deoxycholate (IIa) (0.100 g, 0.2 mmol) was
added to a solution of N-Boc-aminocaproic acid (0.143 g,
0.6 mmol), DCC (0.250 g, 1.2 mmol), and a catalytic
amount of DMAP in dichloromethane (2 ml). The reac-
tion mixture was stirred for 3 h at 15–20°ë and evapo-
rated. The residue was treated with ether, and the dicy-
clohexylurea precipitate was filtered off. The filtrate
was evaporated and chromatographed on a column
eluted with 60 : 1 dichloromethane–methanol mixture.
The yield of (VIa) was 0.134 g (58%); Rf 0.39 (A); MS,
Methyl 3a,12a-bis(N,N-dimethylaminoethylcar-
bamoyloxy)-5b-cholan-24-oate (IVb). A solution of
(IIIb) (0.400 g, 0.67 mmol) and N,N-dimethylethylene-
diamine (0.23 ml, 2.0 mmol) in dichloromethane
(2.5 ml) was stirred for 8 h at 25°ë, diluted with dichlo-
romethane (6 ml), washed with 5% KOH (2 × 8 ml),
dried with Na2SO4, and evaporated. The residue was
chromatographed on a column successively eluted with
30 : 1, 10 : 1 and 5 : 1 chloroform–methanol mixtures
to give (IVb); yield 0.280 g (66%); Rf 0.28 (C); MS
m/z: 1073.9 [M]+; 1H NMR: 0.70 (3 H, s, 18-CH3), 0.78
(3 H, d, J 6.7, ëç3-21), 0.86 [3 H, t, J 6.8,
(ëç2)16ëç3), 0.88 (3 H, s, 19-CH3), 1.01–2.0 [88 H, m,
COOCH2(ëç2)16ëç3, steroid CH and CH2, and
2NHCH2(CH2)3CH2CO], 2.05–2.40 (6 H, m,
2ééëëç2 and H23), 3.05–3.15 (4 H, m, 2CH2NH),
4.05 (2 H, t, J 6.9, COOëç2), 4.72 (1 H, m, H3), and
5.01 (1 H, m, H12).
(m/z): 634.9 [M + ç]+; 1ç NMR: 0.65 (3 H, s, 18-CH3),
0.84 (3 H, d, J 6.7, 21-CH3), 0.88 (3 H, s, 19-CHJ 6.7,
ëç3-21), 0.88), 0.92–1.99 (26 H, m, steroid CH and
CH2), 2.20 [6 H, s, N(CH3)2], 2.24 [6 H, s, N(CH3)2],
2.30–2.48 [6 H, m, 23-CH2 and 2ëç2N(CH3)2], 3.18–
3.30 (4 H, m, 2NHCH2), 3.64 (3 H, s, OCH3), 4.55
(1 H, m, H3), 4.92 (1 H, m, H12), and 5.05–5.20 (1 H,
m, NH).
Methyl 3a,12a-bis(N-tert-butyloxycarbonylami-
docapronoyloxy)-5b-cholan-24-oate (VIb). A solu-
tion of methyl deoxycholate (IIb) (0.250 g, 0.6 mmol),
N-Boc-aminocaproic acid (0.355 g (1.5 mmol), DCC
(0.475 g, 2.3 mmol) and a catalytic amount of DMAP
in dichloromethane (3 ml) was stirred for 23 h at 25°ë
and evaporated. The residue was treated with ether, and
dicyclohexylurea was filtered off. The filtrate was evap-
Octadecyl 3a,12a-bis(N,N,N-trimethylammonio-
ethylcarbamoyloxy)-5b-cholan-24-oate
diiodide
(Va). Methyl iodide (0.12 ml) was added to a solution
of tertiary amine (IVa) (0.065 g (0.07 mmol) in DMSO
(2 ml); the reaction mixture was stirred for 8 h at 70°ë,
diluted with chloroform (15 ml), washed with water
(2 × 15 ml), dried with Na2SO4, and evaporated. The
residue was chromatographed on a column succes- orated and chromatographed on a column successively
sively eluted with10 : 1 and 5 : 1 chloroform–methanol eluted with chloroform and 30 : 1 chloroform–metha-
mixtures to give quaternary iodide (Va); yield 0.060 g nol mixture. Ester (VIb) was obtained; yield 0.312 g
1
(70%); Rf 0.32 (D); H NMR: 0.65 (3 H, s, 18-CH3), (64%); Rf 0.85 (B); MS, m/z: 874.8 [M + K]+; 1ç NMR:
0.68 (3 H, d, J 6.7, 21-CH3), 0.85 [3 H, t, J 6.8, 0.70 (3 H, s, 18-CH3), 0.78 (3 H, d, J 6.7, 21-CH3), 0.88
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004

SYNTHESIS OF CATIONIC AMPHIPHILES ON THE BASIS OF DEOXYCHOLIC ACID
481
[3 H, s, 19-CH(3 ç, Ò, ëç3-19), 0.92–2.01), 0.92–2.01
[56 H, m, 26 steroid CH and CH2, 2(ëç2)3ëç2ëéé,
and 2ë(ëç3)3], 2.15–2.38 (6 H, m, H23 and
2ééëëç2), 3.01–3.18 (4 H, m, 2NHCH2), 3.62 (3 ç,
Ò, Oëç3), 4.63 (1 H, m, H3), and 5.08 (1 H, m, H12).
Methyl 3a,12a-bis[5-(pyridinio)pentanoyloxy]-
5b-cholan-24-oate dibromide (IX). A solution of
(VIII) (0.110 g, 0.15 mmol) in pyridine (2 ml) was
heated for 6 h at 60°ë and evaporated. The residue was
chromatographed on a column successively eluted with
30 : 1 and 6 : 1 chloroform–methanol mixtures to give
(IX); yield 0.131 g (90%); Rf 0.37 (B); MS, m/z: 765.1
Octadecyl
3a,12a-bis(e-ammmoniocapronoy-
loxy)-5b-cholan-24-oate bistrifluoroacetate (VIIa).
A solution of (VIa) (0.050 g) and trifluoroacetic acid
(0.08 ml) in chloroform (0.5 ml) was stirred for 2 h at
40°ë and evaporated. The residue was chromato-
graphed on a column eluted with 30 : 1 chloroform–
methanol mixture to get (VIIa); yield 0.035 g (90%); Rf
[M + Na]+; 1ç NMR: 0.69 (3 H, s, 18-CH3), 0.74 (3 H,
d, J 6.7, ëç3-21), 0.87 (3 H, s, 19-CH3), 1.05–2.65 [38
H, m, steroid CH and ëç2, 2ëééëç2(ëç2)3ëç2N+,
and H23], 3.63 (3 H, s, OCH3), 4.75 (1 H, m, H3), 4.98–
5.12 (5 H, m, 2ëç2N+ and H12), 8.03–8.19 (4 H, m),
8.40–8.55 (2 H, m), and 9.45–9.66 (4 H, m, 2ë5ç5N+).
1
0.42 (D); MS, m/z: 873.9 [M – ëF3COO]+; H NMR:
0.71 (3 H, s, 18-CH3), 0.77 (3 H, d, J 6.7, 21-CH3), 0.86
[3 H, t, J 6.8, (ëç2)16ëç3], 0.88 (3 H, s, 19-CH3),
1.01–2.51 [70 H, m, COOCH2(ëç2)16ëç3, steroid CH
and ëç2, and 2 2HNCH2(CH2)3CH2CO], 2.02–2.41
(6 H, m, 2ééëëç2 and H23), 2.81–3.00 (4 H, m,
2CH2N+), 4.05 (2 H, t, J 6.9, COOëç2), 4.72 (1 H, m,
H3), 5.1 (1 H, m, H12), and 7.85 (6 H, br s, 2N+H3).
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research, project nos. 01-03-33234, 00-15-
866, and 03-03-32482, and the grants: Scientific
Research of High Schools on the Priority Directions of
Science and Technology, subsection Medicinal and
Biologically Active Substances no. 203.05.04.005, and
the President’s Grant for the Support of Leading Scien-
tific Schools of Russia no. NSh-2013.2003.3.
Methyl 3a,12a-bis(e-ammmoniocapronoyloxy)-
5b-cholan-24-oate bistrifluoroacetate (VIIb). A
solution of (VIb) (0.170 g, 0.2 mmol) and trifluoroace-
tic acid (0.2 ml, 2.4 mmol) in chloroform (2 ml) was
stirred for 2 h at 50°ëand evaporated. The residue was
chromatographed on a column successively eluted with
chloroform and 30 : 1 and 10 : 1 chloroform–methanol
mixtures to get (VIIb); yield 0.124 g (74%); Rf 0.36
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1
(B); MS, m/z: 636.1 [M]+; ç NMR: 0.68 (3 H, s,
18-CH3), 0.76 (3 H, d, J 6.7, ëç3-21), 0.88 (3 H, s,
19-CH3), 0.92–2.02 [38 H, m, 26 steroid CH and ëç2
and 2HNCH2(ëç2)3ëç2ëé], 2.15–2.38 (6 H, m,
2ééëëç2 and H23), 2.70–2.79 (4 H, m, 2CH2N+),
3.62 (3 H, s, OCH(3 ç, Ò, Oëç3), 4.61 (1 H, m, H3),
5.00 (1 H, m, H12), and 7.98 (6 H, m, 2N+H3).
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Methyl 3a,12a-bis(5-bromopentanoyloxy)-5b-
cholan-24-oate (VIII). Pyridine (0.1 ml) and 5-bro-
moisovaleric acid chloride (0.780 g (3.9 mmol) were
added to a stirred solution of (IIb) (0.220 g, 0.5 mmol)
in chloroform (2 ml) at 0°ë. The reaction mixture was
kept for 1 h at 25°ë and evaporated. The residue was
chromatographed on a column eluted with chloroform
to give (VIII); yield 0.322 g (81%); Rf 0.86 (B); MS,
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m/z: 754.9 [M + Na]; 1ç NMR: 0.73 (3 H, s, 18-CH3),
0.81 (3 H, d, J 6.7, ëç3-21), 0.91 (3 H, s, 19-CH(3 ç,
Ò, ëç3-19), 1.05–2.01 [32 H, m, steroid CH and CH2
and 2(ëç2)2ëç2Br], 2.18–2.45 (6 H, m, 2 OOCCH2
and H23), 3.44 (4 H, q, J 6.4, 2ëç2Br), 3.67 (3 ç, Ò,
Oëç3), 4.75 (1 H, m, H3), and 5.10 (1 H, m, H12).
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 5 2004