Synthesis of ester-linked lithocholic acid dimers

Article abstract of DOI:10.1016/j.steroids.2003.08.015

Four lithocholic acid dimers were synthesised via esterification. The ester-linked dimer, 3-oxo-5β-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3α,5β), was obtained by condensation of methyl lithocholate with 3-oxo-5β-cholan-24-oic acid. Borohydride reduction of this ester-linked dimer gave 3α-hydroxy-5β-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3α,5β), which was acetylated to 3α-acetoxy-5β-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3α,5β). Reaction of methyl lithocholate with oxalyl chloride yielded the oxalate dimer, bis(5β-cholan-24-oic acid methyl ester)-3α-yl oxalate.

Full text of DOI:10.1016/j.steroids.2003.08.015

Steroids 68 (2003) 1157–1161
Synthesis of ester-linked lithocholic acid dimers
Lutfun Nahar∗, Alan B. Turner
Japp Laboratory, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, Scotland, UK
Received 30 May 2003; accepted 22 August 2003
Abstract
Four lithocholic acid dimers were synthesised via esterification. The ester-linked dimer, 3-oxo-5␤-cholan-24-oic acid (cholan-24-oic
acid methyl ester)-3-yl ester, (3␣,5␤), was obtained by condensation of methyl lithocholate with 3-oxo-5␤-cholan-24-oic acid. Borohydride
reduction of this ester-linked dimer gave 3␣-hydroxy-5␤-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3␣,5␤), which
was acetylated to 3␣-acetoxy-5␤-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3␣,5␤). Reaction of methyl lithocholate
with oxalyl chloride yielded the oxalate dimer, bis(5␤-cholan-24-oic acid methyl ester)-3␣-yl oxalate.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Lithocholic acid; Steroid dimers; Esterification; Hydrolysis; 13C NMR
1. Introduction
dures. The purity of the products and the reaction time were
supported by thin-layer chromatography (TLC) performed
on silica gel (Merck type 60) and visualised under UV il-
lumination and/or by I2 vapour. Melting points of the prod-
ucts were determined on a Gallenkamp melting point appa-
ratus and are uncorrected. Infrared spectra (wave numbers
in cm−1) were recorded on an ATI Mattson Genesis FTIR
spectrophotometer as KBr pellets or in CHCl3. Nuclear mag-
netic resonance (NMR) spectra were recorded on a Varian
Unity INOVA 400 MHz NMR spectrometer. Chemical shifts
(δ) are reported in ppm downfield from TMS, using the mid-
Steroid dimers were initially isolated as synthetic
by-products [1,2] and are also found in nature [3,4]. Later,
steroid dimers were chemically synthesised via an ester
[5–7] and an ether [7] linkages. Dimeric steroids exhibit
micellar, detergent, and liquid crystal behaviour [8] and are
pharmaceutically important [9]. They have been utilised as
catalysts for certain organic reactions [10] and can be used
to develop new pharmacologically active steroids [9].
We report here the synthesis of new lithocholic acid
dimers, which contain either a simple ester linkage between
the C24 position of 3-oxo-5␤-cholanoic acid (2) and the
C3 position of methyl 3␣-hydroxy-5␤-cholanate (3), or
an oxalate ester linkage between the C3 positions of two
molecules of 3.
1
dle resonance of CDCl3 (7.25 ppm for H and 77.23 ppm
for 13C) as an internal standard and coupling constants (J)
in Hz. Data are reported as follows: chemical shift, multi-
plicity (s: singlet, d: doublet, m: multiplet) coupling con-
stant(s), integration and peak assignment. The MS analyses
were carried out at the EPSRC Central Mass Spectrometry
Service at Swansea.
2. Experimental
2.1. 3-Oxo-5β-cholan-24-oic acid (2)
Lithocholic acid (1), 4-dimethylaminopyridine (DMAP),
1,3-dicyclohexyl carbodiimide (DDC), oxalyl chloride and
NaBH4 were purchased from Aldrich and used as received.
All chemicals and solvents were used throughout without
further purification; solvents were dried by standard proce-
Jones’ reagent (1 ml) was added dropwise to a solution
of lithocholic acid (100 mg, 0.27 mmol) in acetone (10 ml)
and stirred at 4 ◦C for 30 min. Then the reaction mixture
was concentrated under reduced pressure, diluted with ether
(15 ml), washed with saturated NaHCO3 (2×20 ml) solution
and H2O (2 × 20 ml), and dried (MgSO4). Evaporation of
the solvent yielded the title compound as a colourless solid
(94 mg, 93%), mp: 122–123 ◦C (Lit. mp: 121–122 ◦C) [11];
∗
Corresponding author. Tel.: +44-1224-272919;
fax: +44-1224-272921.
E-mail address: l.nahar@abdn.ac.uk (L. Nahar).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2003.08.015

1158
L. Nahar, A.B. Turner / Steroids 68 (2003) 1157–1161
Table 1
O
O
1H NMR (coupling constant J in Hz in parentheses) data of dimers 4
and 5
OH
OH
i
No.
δ (ppm)
No.
δ (ppm)
4
5
4
5
HO
O
1a
2a
3␤
4
5␤ 1.35
6
7
8␤ 1.38
9␣ 1.41
11
12
14␣ 1.05
15
16
1.75, 0.95
1.29, 1.60
−
1.76, 0.96
1.30, 1.62
4.67m (1H)
1.72, 1.48
1.36
1.25, 1.84
1.10, 1.42
1.40
1ꢀ
2ꢀ
1.75, 0.95
1.29, 1.60
4.72m (1H) 4.67m (1H)
1.71, 1.45
1.35
1.23, 1.83
1.09, 1.39
1.38
1.76, 0.96
1.30, 1.62
H
H
1
2
3ꢀ␤
1.71, 1.45
4ꢀ
1.72, 1.48
1.36
1.25, 1.84
1.10, 1.42
1.40
5ꢀ␤
6ꢀ
ii
1.23, 1.83
1.09, 1.39
7ꢀ
8ꢀ
O
1.42
9ꢀ
1.41
1.42
OMe
1.38, 1.23
1.14, 1.96
1.39, 1.25
1.15, 1.98
1.07
1.05, 1.56
1.87, 1.29
1.09
0.60s (3H)
0.86s (3H)
1.43
11ꢀ
1.38, 1.23
1.14, 1.96
1.05
1.04, 1.56
1.85, 1.27
1.10
0.65s (3H)
0.93s (3H)
1.41
0.91d (6.2)
1.75, 1.29
2.25, 2.25
1.39, 1.25
1.15, 1.98
1.07
1.05, 1.56
1.87, 1.29
1.09
0.60s (3H)
0.86s (3H)
1.43
0.87d (6.2)
1.76, 1.32
2.25, 2.25
3.62s (3H)
12ꢀ
14ꢀ
15ꢀ
16ꢀ
17ꢀ
18ꢀ
19ꢀ
20ꢀ
21ꢀ
22ꢀ
23ꢀ
1.04, 1.56
1.85, 1.27
HO
H
17␣ 1.10
3
18␤ 0.69s (3H)
19␤ 1.02s (3H)
20␤ 1.41
Scheme 1. (i) Jones’ reagents, 4 ◦C, 30 min. (ii) MeOH, AcCl, RT, 24 h.
21␣ 0.93d (6.2) 0.87d (6.2)
1
22
23
–
1.75, 1.29
2.32, 2.12
–
1.76, 1.32
2.25, 2.25
–
IR (KBr) [12]; H NMR (CDCl3): δ 0.68 (s, 3H, 18-Me),
0.92 (d, J = 6.1 Hz, 3H, 21-Me), 1.01 (s, 3H, 19-Me); 13
C
MeO 3.66s (3H)
NMR (CDCl3): δ 37.5 (C-1), 37.3 (C-2), 203.8 (C-3), 42.6
(C-4), 44.6 (C-5), 26.1 (C-6), 26.9 (C-7), 35.6 (C-8), 41.1
(C-9), 35.2 (C-10), 21.5 (C-11), 40.3 (C-12), 43.1 (C-13),
56.7 (C-14), 24.4 (C-15), 28.4 (C-16), 56.3 (C-17), 12.4
(C-18), 22.9 (C-19), 35.8 (C-20), 18.5 (C-21), 31.2 (C-22),
31.0 (C-23), 180.1 (C-24); ESIMS m/z: 397 [M + Na]+
(Scheme 1).
Spectra obtained in CDCl3, 400 MHz.
product was accomplished by preparative TLC and eluted
with 15% EtOAc in pet-ether (40–60 ◦C). The title com-
pound 4 was found as a colourless solid (99 mg, 51%), mp:
201–202 ◦C; IR (CHCl3): νmax (cm−1) 2933s (O–H), 2860s
=
=
(O–H), 1736s (ester C O), 1715m (ketonic C O), 1453m,
1384m, 1257s (C–O), 1222m, 1175s (C–O), 1118m, 840m,
779m; 1H NMR (Table 1) and 13C NMR (Table 2); FABMS
m/z: 747 [M +H]+, 769 [M +Na]+; HRFABMS calculated
for C49H79O5: 747.5927; found: 747.5929.
2.2. 3α-Hydroxy-5β-cholan-24-oic acid methyl ester (3)
Esterification of lithocholic acid (100 mg, 0.27 mmol)
was carried out according to the method described in the lit-
erature [13]. The title compound 3 was obtained as a white
solid (104 mg, 99%), mp: 116–117 ◦C (Lit. mp: 115–117 ◦C)
[14]; IR (KBr) [13]; 1H NMR [15]; 13C NMR [16] and
MS [17].
2.4. 3α-Hydroxy-5β-cholan-24-oic acid (cholan-24-oic
acid methyl ester)-3-yl ester, (3α,5β) (5)
To a stirred solution of 4 (89 mg, 0.12 mmol) in EtOH
(10 ml) at RT was added NaBH4 (9 mg, 2 equivalent) and
stirring was continued for 2 h. A pale yellow solution was
found. Then, H2O (15 ml) was added to the solution, and
extracted with EtOAc (3 × 15 ml). The organic extracts
were separated and dried (MgSO4). Evaporation of the
solvent gave oily product. The compound was purified by
preparative TLC and eluted with 18% EtOAc in pet-ether
(40–60 ◦C). The title compound 5 was found as a white solid
2.3. 3-Oxo-5β-cholan-24-oic acid (cholan-24-oic acid
methyl ester)-3-yl ester, (3α,5β) (4)
To a stirred solution of 2 (97 mg, 0.26 mmol) in dry DCM
(5 ml) was added DMAP (1.6 mg, 0.027 mmol) and then 3
(102 mg, 0.26 mmol) at RT. After cooling to 4 ◦C and a solu-
tion of DCC (54 mg, 0.27 mmol) in DCM (1 ml) was added
slowly to the reaction mixture and stirred continued for an-
other 20 min at 4 ◦C, then 72 h at RT. Finally, a precipitate
(dicyclohexylurea) was formed. The precipitate was filtered
off and the filtrate evaporated down in vacuo. The residue
was taken up in DCM and washed subsequently twice with
0.5N HCl and saturated NaHCO3 solution and then dried
over MgSO4. The solvent was removed by evaporation and
the product was found as a crude solid. Purification of the
(69 mg, 77%), mp: 203–204 ◦C; IR (CHCl3): νmax (cm−1
3342br (O–H), 2931vs (O–H), 2864s (O–H), 1735vs (ester
)
=
C O), 1450m, 1379m, 1271s (C–O), 1168s (C–O), 1125m,
1
1070m (alcoholic C–O), 892m, 778m; H NMR (Table 1)
and 13C NMR (Table 2); FABMS m/z: 749 [M + H]+,
771 [M + Na]+; HRFABMS calculated for C49H81O5:
749.6084; found: 749.6087.

L. Nahar, A.B. Turner / Steroids 68 (2003) 1157–1161
1159
Table 2
tallisation from a mixture (2:1) of CHCl3 and EtOAc. The ti-
tle compound 7 was obtained as a colourless solid (1.20 mg,
56%), mp: 195–196 ◦C; IR (CHCl3): νmax (cm−1) 2934s
13C NMR data of dimers 4 and 5
No.
δ (ppm)
No.
δ (ppm)
=
(O–H), 2859s (O–H), 1762s (oxalate C O), 1739vs (ox-
4
5
4
5
=
alate and ethyl ester C O), 1449m, 1376w, 1175s (C–O) and
1
2
3
4
5
6
7
8
9
37.2
37.1
213.4
42.4
44.4
25.8
26.7
35.8
41.9
34.9
21.2
40.2
42.8
56.6
24.2
28.2
56.1
12.1
22.7
35.4
18.3
31.8
31.0
173.8
−
35.3
30.5
71.8
36.5
42.1
27.2
26.4
35.8
40.4
34.6
20.8
40.2
42.7
56.5
24.2
28.2
56.0
12.0
23.3
35.8
18.3
31.7
31.0
173.8
−
1ꢀ
2ꢀ
35.1
26.4
74.1
32.3
40.5
27.1
26.7
35.6
40.8
34.6
20.9
40.1
42.8
56.5
24.2
28.2
56.1
12.1
23.4
35.3
18.3
31.1
31.0
174.8
51.5
35.0
26.3
74.1
32.3
41.9
27.0
26.7
35.4
40.4
34.6
20.8
40.2
42.7
56.5
24.2
28.2
56.1
12.1
23.4
35.4
18.3
31.1
31.0
758w; 1H NMR (CDCl3): δ 0.60 (s, 6H, 18-Me and 18ꢀ-Me),
0.86 (d, J = 6.2 Hz, 6H, 21-Me and 21ꢀ-Me), 0.89 (s, 6H,
19-Me and 19ꢀ-Me), 3.62 (s, 6H, 2×24-OMe), 4.85 (m, 2H,
3-CH–O and 3ꢀ-CH–O) and 13C NMR (CDCl3): δ 34.9 (C-1
and C-1ꢀ), 26.2 (C-2 and C-2ꢀ), 77.8 (C-3 and C-3ꢀ), 31.8
(C-4 and C-4ꢀ), 41.9 (C-5 and C-5ꢀ), 27.0 (C-6 and C-6ꢀ),
26.3 (C-7 and C-7ꢀ), 35.8 (C-8 and C-8ꢀ), 40.3 (C-9 and
C-9ꢀ), 34.6 (C-10 and C-10ꢀ), 20.8 (C-11 and C-11ꢀ), 40.1
(C-12 and C-12ꢀ), 42.7 (C-13 and C-13ꢀ), 56.5 (C-14 and
C-14ꢀ), 24.2 (C-15 and C-15ꢀ), 28.2 (C-16 and C-16ꢀ), 56.0
(C-17 and C-17ꢀ), 12.0 (C-18 and C-18ꢀ), 23.2 (C-19 and
C-19ꢀ), 35.4 (C-20 and C-20ꢀ), 18.3 (C-21 and C-21ꢀ), 31.1
(C-22 and C-22ꢀ), 31.0 (C-23 and C-23ꢀ), 174.7 (C-24 and
C-24ꢀ), 51.5 (2 × 24-OMe), 157.8 (2 × O–CO); HRFABMS
calculated for C52H86NO8 [M + NH4]+: 852.6353; found:
852.6355.
3ꢀ
4ꢀ
5ꢀ
6ꢀ
7ꢀ
8ꢀ
9ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
MeO
10ꢀ
11ꢀ
12ꢀ
13ꢀ
14ꢀ
15ꢀ
16ꢀ
17ꢀ
18ꢀ
19ꢀ
20ꢀ
21ꢀ
22ꢀ
23ꢀ
24ꢀ
MeO
2.7. Hydrolysis of dimer 6
174.8
51.5
2.7.1. Acid hydrolysis
To a solution of the 3␣-acetoxy dimer (6; 30 mg,
0.038 mmol) in MeCN (3 ml) at RT was added conc. HCl
(1 ml). The resulting suspension was stirred at RT for 48 h
and no change was found. Finally, the dimer was refluxed at
80 ◦C for 24 h and only 10% lithocholic acid was obtained.
The reactions were monitored from time to time by TLC
(CHCl3:EtOH = 99:1). Both the starting material and the
product were identified by comparison of their mp, TLC
Spectra obtained in CDCl3, 100 MHz.
2.5. 3α-Acetoxy-5β-cholan-24-oic acid (cholan-24-oic
acid methyl ester)-3-yl ester, (3α,5β) (6)
Acetic anhydride (1 ml) was added to a solution of dimer
5 (90 mg, 0.12 mmol) in pyridine (0.5 ml), and the mixture
was left at RT for 36 h. To this mixture H2O (10 ml) was
added to quench the excess acetic anhydride, and the mix-
ture was subsequently extracted with CHCl3 (3 × 15 ml).
The combined organic layer was washed successively with
H2O, 1 M HCl, saturated NaHCO3 solutions and brine, and
then dried (MgSO4). The solvent was removed by evap-
oration and the product was found as a solid. The com-
pound was purified by preparative TLC and eluted with 20%
EtOAc in pet-ether (40–60 ◦C) and the title compound 6 as
a white powder (68 mg, 72%), mp: 210–212 ◦C (Lit. mp:
1
and H NMR data with those of the authentic samples.
2.7.2. Alkaline hydrolysis
To a solution of the 3␣-acetoxy dimer (6; 30 mg,
0.038 mmol) in MeCN (3 ml) at RT was added methano-
lic KOH (1 M; 1 ml). The resulting suspension was stirred
at RT for 48 h and it was found that the dimer was con-
verted almost 50% to lithocholic acid. Then, the mixture
was refluxed at 80 ◦C for 18 h and 90% lithocholic acid
was obtained. Both reactions were monitored from time to
time by TLC (CHCl3:EtOH = 99:1). The final product was
identified by comparison of its mp, TLC and 1H NMR data
with those of the authentic sample.
212–214 ◦C; MS; IR; H NMR and 13C NMR) [5].
1
2.6. bis(5β-Cholan-24-oic acid methyl ester)-3α-yl
oxalate (7)
3. Results and discussion
A stirred solution of 3␣-hydroxy-5␤-cholan-24-oic acid
methyl ester 3 (2.0 g, 5.12 mmol) in a mixture of toluene
(3 ml) and dry pyridine (2 ml) was treated dropwise with oxa-
lyl chloride (325 mg, 2.56 mmol in 10 ml toluene) over 5 min
under N2. After 48 h, the resulting off-white suspension was
quenched cautiously with H2O (20 ml). The toluene layer
was washed with H2O and extracted with EtOAc (3×20 ml),
dried over MgSO4. The crude solid was purified by recrys-
Lithocholic acid (3␣-hydroxy-5␤-cholan-24-oic acid),
which has an alcohol and an acid group at opposite ends of
the molecule, can easily be dimerised after protecting one
of the functional groups. This interesting structural feature
of lithocholic acid is ideal for the synthesis of symmetri-
cal dimers with distinct architectures. This feature may be

1160
L. Nahar, A.B. Turner / Steroids 68 (2003) 1157–1161
O
OMe
O
i
O
ii
+
2
3
H
4
O
H
iii
AcO
HO
H
H
6
5
Scheme 2. (i) DCM, DMAP, DCC, 72 h, RT (ii) NaBH4, EtOH, 2 h, RT (iii) Ac2O, pyridine, 24 h, RT.
useful in the synthesis of pharmaceuticals. The ␣-dimer
is capable of acquiring a folded conformation as demon-
strated by the formation of dimeric cyclocholates [18]. In
order to prepare ester-type dimers from 1, it was necessary
to use carboxylic acid and alcohol precursors. The chosen
acid component, 3-oxo-5␤-cholan-24-oic acid (2), was pre-
pared by oxidation of the secondary 3␣-hydroxyl group
with Cr(VI) and the alcohol component was obtained by
esterification of the carboxyl group with MeOH, to give
3␣-hydroxy-5␤-cholan-24-oic acid methyl ester.
(Tables 1 and 2) of 4 showed signals similar to that of its
component steroid monomers 2 and 3 with the exception
that the signals arising from the C-3 oxymethine proton
(δH 4.72) and carbon (δC 74.1) were much deshielded
compared to those of 3 (δH 3.65 and δC 71.9). This down-
field shift was due to dimersiation of 3 at C-3 with the
side-chain carbonyl of 2. This fact was confirmed further
1
by a H–13C long-range correlation (3J) observed between
the C-3 oxymethine proton (δH 4.72) of 3 and the C-24
carbonyl carbon of 2 (δC 173.8) in its HMBC spectrum.
3-Oxo dimer (4) was selectively reduced to 3␣-hydroxy
dimer (5) which on acetylation produced 3␣-acetate dimer
(6) (Scheme 2). The dimer 6 contains three different types
of ester linkages. A high yielding synthesis of the dimer 6
was reported by Li and Dias [5], but the dimer was pre-
pared by direct coupling of the acetate of lithocholic acid
with its methyl ester. The FABMS spectrum of 5 revealed
the [M + H]+ and [M + Na]+ ions, respectively, at m/z 749
and 771 confirming the molecular formula C49H80O5. The
The lithocholic acid dimer 4 was synthesised from
these two components via esterification using DCC as the
acid-activating agent, leading to the linkage of the acid
group on C24 position of 2 with the hydroxyl group on C3
position of 3 (Scheme 2). The IR spectrum of 4, showed
an absorption band at 1736 cm−1, charact+eristic for an ester
C O. The FABMS of 4 showed [M + H] and [M + Na]+
=
ions, respectively, at m/z 747 and 769 and thus confirmed
the formation of dimer. The 1H and 13C NMR spectra
O
OMe
O
OMe
i
O
H
O
O
H
O
HO
H
MeO
3
7
O
Scheme 3. (i) Pyridine, oxalyl chloride in toluene, 48 h, RT.

L. Nahar, A.B. Turner / Steroids 68 (2003) 1157–1161
1161
1H and 13C NMR spectra (Tables 1 and 2) were similar to
References
those of 4 with the exception that the signals supporting the
presence of an oxymethine, instead of the ketonic carbonyl,
at C-3 were observed. The oxymethine proton and carbon
(C-3) showed signals, respectively, at δH 3.58 and δC 71.8.
Dimer 6 was readily identified by comparison of its mp, IR,
1H NMR and 13C NMR data with the published data [5].
The dimer 6 was subjected to both acid and base hydrol-
ysis. While at RT the dimer was significantly stable in acid,
it was unstable under basic condition. The acid hydrolysis
of the dimer (refluxing at 80 ◦C for 24 h) resulted in small
amounts (∼10%) of lithocholic acid. The basic hydrolysis
of 6 (refluxing at 80 ◦C for 18 h) provided about 90% of
lithocholic acid.
Methyl lithocholate (3) was treated with oxalyl chloride
in presence of pyridine for 48 h to give bis(5␤-cholan-24-oic
acid methyl ester)-3␣-yl oxalate (7) in a yield of 56%
(Scheme 3). The dimer 7 was purified by recrystallisation
from a mixture (2:1) of CHCl3 and EtOAc. In the 1H NMR
of 7, the down field shift (δ 4.85) of the signal for the C-3
and C-3ꢀ oxymethines compared to those of the starting ma-
terial (δ 3.60) confirmed the oxalate ester formation at C-3
and C-3ꢀ. This fact was corroborated further from the 13C
NMR spectrum where the chemical shift of C-3 and C-3ꢀ
oxymethine carbons was also deshielded (δ 77.8 as opposed
to δ 71.9 of the starting material). The HRFABMS spectrum
of 7 confirmed the structure by exhibiting the [M + NH4]+
ion at m/z 852.6353, calculated 852.6358 for C52H86NO8.
This report demonstrates the convenient routes for the
synthesis of ester-linked dimers either by simple ester (4–6)
or via oxalate ester (7) formation. The dimers were obtained
with high yields. We have shown the use of these dimerisa-
tion methods on lithocholic acid derivatives. Therefore, it is
reasonable to assume that such methods could also be ap-
plied for the dimerization of other bile acids.
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The authors thank the funding body the Aberdeen Univer-
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