A Facile Route to Ursodeoxycholic Acid Based on Stereocontrolled Conversion and Aggregation Behavior Research

Article abstract of DOI:10.1055/s-0035-1560388

A facile route to ursodeoxycholic acid (UDCA) and its aggregation behavior in aqueous phase solution, which is rarely known, are reported. The starting material, hyodeoxycholic acid (HDCA), is a relatively less expensive material and more easily obtained compared with chenodeoxycholic acid (CDCA). A facile route was developed to synthesize UDCA from HDCA with a Shapiro reaction as the key step and in 26% overall yield. A new strategy using organosilane reagent considering its stability, nontoxicity, and abundance in nature was carried out for a more rapid route and higher yield. It was found that the critical micelle concentration value, which is a critical value for surfactants of bile salts, was influenced by the number of hydroxyl groups.

Full text of DOI:10.1055/s-0035-1560388

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
©
Georg Thieme Verlag Stuttgart · New York
2016, 48, 588–594
588
paper
Synthesis
Q. Dou, Z. Jiang
Paper
A Facile Route to Ursodeoxycholic Acid Based on Stereocontrolled
Conversion and Aggregation Behavior Research
Qian Dou
COOH
NBS or TCCA
COOMe
Zhongliang Jiang*
acetone, r.t., 1 h
Department of Chemistry, Shanghai Key Laboratory
of Chemical Assessment and Sustainability, Tongji
University, Shanghai 200092, P. R. of China
zl_jiang@tongji.edu.cn
selective oxidation
RO
HO
R = H, Ac
OH
O
COOMe
total up to 28.8% yield
COOH
AcO
AcO
OSiMe3
key step
UDCA
6
,7-OH shift
COOMe
HO
OH
4
steps
total up to 25.8% yield
Received: 03.09.2015
Accepted after revision: 13.11.2015
Published online: 15.12.2015
et al.9,10 reported the synthesis of UDCA with cholic acid
and CDCA as the starting materials using an enzyme as
catalyst, which is also applied in the synthesis of UCA and
DOI: 10.1055/s-0035-1560388; Art ID: ss-2015-h0518-op
6
-FUDCA.11
Abstract A facile route to ursodeoxycholic acid (UDCA) and its aggre-
gation behavior in aqueous phase solution, which is rarely known, are
reported. The starting material, hyodeoxycholic acid (HDCA), is a rela-
tively less expensive material and more easily obtained compared with
chenodeoxycholic acid (CDCA). A facile route was developed to synthe-
size UDCA from HDCA with a Shapiro reaction as the key step and in
COOH
COOH
2
6% overall yield. A new strategy using organosilane reagent consider-
HO
HO
OH
1b
ing its stability, nontoxicity, and abundance in nature was carried out
for a more rapid route and higher yield. It was found that the critical
micelle concentration value, which is a critical value for surfactants of
bile salts, was influenced by the number of hydroxyl groups.
OH
1a
hyodeoxycholic acid
ursodeoxycholic acid
OH
Key words ursodeoxycholic acid, hyodeoxycholic acid, critical micelle
concentration, facile route, Shapiro reaction, Clemmensen reaction
COOH
COOH
Among the bile acids (BAs, Figure 1), which are natural
products and the fundamental component of bile, ursode-
HO
OH
HO
OH
1
1c
1d
chenodeoxycholic acid
cholic acid
oxycholic acid (UDCA) has an important medical applica-
tion due to its ability to resolve cholesterol gallstones.2
UDCA plays an important role in immunoregulation, pro-
motes the secretion of endogenous BAs, and protect the ep-
icytes of liver cells. Most of UDCA is prepared on a large
scale from raw materials with high bile content, such as
goose and bovine bile, of which the major component is
cholic acid that is frequently used as the starting material to
synthesize chenodeoxycholic acid (CDCA).4–7 Recently, nu-
merous routes have been developed for the synthesis of
UDCA by employing CDCA as the starting material, but
HDCA was not frequently used because a few extra steps
would be required for the conversion between 6-OH and 7-
OH. The transformation from other BAs to UDCA has been
,3
Figure 1 Different types of bile acids
Computation chemistry has been developed for many
years to predict the potential property and reactivity of
compounds based on the density functional theory.
12–16
Ac-
curate quantum chemical calculation was performed to
predict the enthalpy change between various products in
selective reactions. Moreover, according to a previous study
on avicholic acid,17 a major constituent of the bile of several
avian species, research on critical micelle concentration
(CMC) as a reflection of aggregation behavior, which indi-
cates the capability of surfactants, has also been covered in
8
18,19
reported. Zhou et al. described a 7-step sequence to
this paper.
synthesize UDCA in 15% total yield. More recently, Pedrini
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Paper
Path Finding Studies
was washed with cold ethanol and the yield was >95% over
all the operations. From the literature23 we discovered that
the base most frequently used in the Shapiro reaction was
n-BuLi or LDA. The first example of Shapiro reaction with
LiH as the base and toluene as solvent was developed in our
present route providing a yield of 74% of the olefin 6. NaH
could be used in this reaction as well, but the yield was low-
er at 37%. TMEDA played a very important role in this reac-
tion. With its unique coordination with Li, the C=C bond
was more easily formed. The ratio of 6/6a was 15:1
(Scheme 2).
We discuss here the oxidative reaction of hyodeoxycho-
lic acid methyl ester (2) with three potential oxidizing
agents to develop a facile route to synthesize UDCA by em-
ploying environment-friendly reagents and HDCA. Selective
oxidation of 6-OH could be achieved with NBS, NCS, or
TCCA (trichloroisocyanuric acid) as the oxidant. The poten-
tial result of oxidation was predicted by computations (Ta-
ble 1) that were accomplished using the density functional
theory method on potential products 3 and 3a performed
20–22
with the Gaussian 09 program,
the B3LYP functional
and the 6-31+G(d,p) basis for carbon and oxygen atoms.
The reaction with NBS was carried out to obtain the best re-
sult; the ratio of 3/3a was 8:1 and the yield 85% (Scheme 1).
The oxidation reaction of C=C bond to give 7 with m-
CPBA is a traditional method with mild conditions and high
yield (Scheme 3). Enantioselective ring opening of epoxides
required Pd/C and H to achieve the selective reduction of 7
2
Table 1 Calculated Energy of 2, 3, 3a
based on the higher stability of 7α-OH. N-Chlorosuccinim-
ide was used to oxidize 8, and the last step of subsequent
radical reduction to form 7β-OH was accomplished using
24
sodium in n-butanol or lithium in liquid ammonia. With
compound 9 available, the stereochemical course of nucleo-
philic attack at C=O bond was controlled by the solvent and
reductant.2
5,26
Considering the lower total yield of this route, another
route with higher yield was developed using organosilane
reagents. Because carbonyl group could be transformed to
enol with a highly reactive OH, 10 was obtained using 4 as
the substrate and LiH as the base at reflux in redistilled THF
with TMSCl overnight. Versatile oxidation of C=C bond re-
quired the combined use of m-CPBA and CH Cl to obtain
2
3
3a
–75.55
Relative energy (kcal/mol)
0
–27.41
2
2
After the protection of 3-OH using a traditional reaction
product 11. The key step in this route is the selective reduc-
tion of 11 with two ester groups and one carbonyl group,
under precise control of the conditions (Schemes 4 and 5).
However, zinc amalgam, which is mostly used in
Clemmensen reduction, was not used in our total synthesis
of UDCA. We first developed a new method without using
with Ac O and Na CO , the substrate 4 was used in the
2
2
3
Shapiro reaction as the key step of this route. The reaction
between p-toluenesulfonyl hydrazide and 4 was carried out
in refluxing ethanol for one hour and the product 5 was iso-
lated without further purification. The crucial product 5
COOMe
COOMe
COOMe
NBS
+
AcOH (cat.), acetone
HO
HO
O
OH
O
OH
2
3, major
3a, minor
Scheme 1 Selective oxidation of 2 with NBS
COOMe
COOMe
COOMe
LiH, TMEDA
toluene, reflux
+
AcO
NNHTs
5
AcO
AcO
6, major
6a, minor
Scheme 2 Shapiro reaction of 5
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Synthesis
Q. Dou, Z. Jiang
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COOH
COOMe
b
COOMe
a
HO
HO
RO
c
OH
OH
O
1a
2
3: R = H
: R = Ac
d
4
COOMe
f
COOMe
e
COOMe
AcO
AcO
AcO
O
NNHTs
7
6
5
g
COOMe
h
COOMe
i
COOH
AcO
OH
AcO
O
HO
OH
8
9
1b
Scheme 3 Path I. Reagents and conditions: a) MeOH, H SO , r.t., 8 h, 99%; b) NBS, AcOH, acetone, H O, r.t., 1 h, 85%; c) Ac O, Na CO , CH Cl , r.t., 8 h,
2
4
2
2
2
3
2
2
9
6%; d) TsNHNH , EtOH, reflux, 1 h, 87%; e) LiH, TMEDA, toluene, reflux, 18 h, 74%; f) m-CPBA, CH Cl , 0 °C, 1 h, 83%; g) Pd/C (10 mol%), H , 50 psi,
2
2
2
2
40 °C, 24 h, 62%; h) NCS, AcOH, acetone, r.t., 40 min, 96%; i) Na, n-BuOH, 60 °C, 2 h, 62%.
zinc amalgam such that zinc powder was added directly
into the reaction mixture, and the reaction time and tem-
perature were accurately controlled to produce 8 in 74%
yield for this single step (Scheme 4).
on the fluorescence absorption behavior in aqueous medi-
um are scarce, thus, we decided to research on the micelli-
zation in aqueous solution with pyrene as a fluorescent
probe to measure the CMC index of bile salts. The signal in-
tensity of two vibronic bands III/I of fluorescence spectrum
indicate the amount of fluorescence absorption, namely,
the amount of pyrene that dissolved in water together with
the surfactants (see Supporting Information).
COOMe
COOMe
Zn
HCl in MeOH
AcO
OH
AcO
OH
O
11
8
Scheme 4 Clemmensen reduction without zinc amalgam
The 7-OH inversion was chemically accomplished
through the regioselective oxidation of the 7α-OH function
and reduction with Na in n-BuOH (Scheme 5). The overall
yield of this route is 29%.
Research on Aggregation Behavior of Bile Salts
Bile salts are a type of surfactants containing a hydro-
phobic group and a hydrophilic group. Critical micelle con-
centration (CMC) as a reflection of aggregation behavior in-
dicates the surfactant capability, which is mainly related to
the number of hydroxyl groups in steroid compounds.
Therefore, our research was focused on determining the
CMC index by detecting the fluorescence spectrum with
different types of steroid compounds, such as lithocholic
acid, HDCA, CDCA, UDCA, and cholic acid (Figure 2). Reports
Figure 2 Intensity ratios (III/I) of vibronic bands of pyrene fluorescence
as a function of bile salt concentration at room temperature with dis-
tilled water as solvent
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COOMe
COOMe
b
COOMe
a
AcO
AcO
AcO
OH
O
OSiMe3
O
4
10
11
c
COOH
e
COOMe
d
COOMe
HO
OH
AcO
O
AcO
OH
1b
9
8
Scheme 5 Path II. Reagents and conditions: a) LiH, TMSCl, THF, 60 °C, 6 h, 89%; b) m-CPBA, CH Cl , r.t., 4 h, 91%; c) Zn, HCl, –20 °C, 45 min, 74%;
2
2
d) NCS, AcOH, acetone, r.t., 1 h, 96%; e) Na, n-BuOH, 60 °C, 2 h, 62%.
The data in the diagram indicate that the value of CMC
correlated with the number of hydroxyl groups in bile salts.
Compound with fewer hydroxyl groups would have a lower
CMC value, which indicates the lower capacity of surfac-
tants. Moreover, the position and space configuration
would have tremendous influence on the value of III/I rath-
er than the CMC value, implying that the selective reduc-
tion of 11 and the transformation from 6-OH to 7-OH are
related to the reactivity of substrates but not to their aggre-
gation behavior.
In summary, a novel, facile, and efficient route to syn-
thesize UDCA was developed with the Shapiro reaction as
the key step for the 6,7-OH transformation, and the final
product was obtained through the 7-OH inversion employ-
ing regioselective oxidation and subsequent radical reduc-
tion. Moreover, another route with higher total yield was
discovered using organosilane reagents, which was proven
stable, nontoxic, and high yielding. In this route, the first
Clemmensen reduction performed without zinc amalgam
became the yield-controlling step, with precisely controlled
conditions and reaction time. Further studies on improved
routes and higher yield are underway in our laboratory.
spectra were recorded using KBr discs on Nicolet FT-IR spectrometer,
and fluorescence spectra were detected on Hitachi F-7000. Optional
rotations were recorded on SG WXG-4 automatic polarimeter.
Methyl 3α,6α-Dihydroxycholan-24-oate (2)
To a solution of hyodeoxycholic acid (1a; 6.00 g, 15.3 mmol) in MeOH
(
50 mL) at 5 °C was added H SO (5 drops) dropwise over 10 min with
2 4
stirring until all solids were dissolved completely. The mixture was
then allowed to warm to r.t. and stirred overnight. The reaction was
quenched with sat. aq NaHCO (10 mL) and evaporated under reduced
3
pressure. After extraction with EtOAc (50 mL), the combined organic
layers were washed with sat. aq NaHCO
(2 × 35 mL) and brine (3 × 50
3
mL), and dried (Na SO ). Evaporation of the solvent under reduced
2
4
pressure afforded 2 (6.22 g, 99%) as a white solid, which was recrys-
tallized from toluene; yield: 6.22 g (99%); mp 165–167 °C; [α]D25
+
15.7 (c 1 g/100 mL EtOH).
IR (KBr): 3350 (O–H), 1743 (C=O), 1123 cm–1 (C–O).
1
H NMR (400 MHz, CDCl /TMS): δ = 0.66 (3 H, s, 18-H), 0.94 (6 H, t,
3
J = 1.4 Hz), 3.63 (1 H, m, 6α-H), 3.69 (3 H, s), 4.07 (1 H, m, 3α-H).
13
C NMR (100 MHz, CDCl /TMS): δ = 12.05, 18.28, 20.79, 23.56, 24.24,
3
2
3
8.16, 29.27, 30.15, 30.98, 31.10, 34.85, 34.89, 35.39, 35.63, 35.97,
9.89, 39.99, 42.87, 48.47, 51.54, 55.96, 56.21, 68.05, 71.54, 174.79.
HRMS (ESI): m/z [M + Na]+ calcd for C25H42O Na: 429.2975; found:
4
429.2965.
Methyl 3α-Hydroxy-6-oxocholan-24-oate (3)
The silica gel used for column chromatography was 300–400 mesh in
size. TLC was carried out using commercial silica gel GF254, and
plates were developed with CMC (carboxymethylcellulose sodium)
The reagents used were of analytical grade, all of the solvents were
redistilled before use, and all of the reactions were performed in
Method 1: To a stirred solution of 2 (2.40 g, 5.91 mmol) in acetone
(100 mL) containing AcOH (0.40 mL, 7.09 mmol) and H O (5 mL) was
2
added NBS (2.10 g, 11.8 mmol) in ten portions at 0 °C. The mixture
was then allowed to warm to r.t. after stirring for 10 min. The course
of the reaction was monitored by TLC. After 1 h, the reaction was then
quenched with sat. aq Na SO (20 mL) and the mixture was evaporat-
oven-dried three-necked flasks. Compounds were detected with 10%
2
3
1
ethanolic phosphomolybdic acid solution as chromogenic agent.
H
ed under reduced pressure. EtOAc (25 mL) was added to the residue
and filtered. The filtrate was washed with sat. aq Na SO (2 × 25 mL),
13
and C NMR spectra were recorded on a Bruker ARX 400 spectrome-
2
3
ter in CDCl3 or DMSO-d6 with reference to the residual CHCl at
3
sat. aq NaHCO3 (2 × 25 mL), and brine (3 × 15 mL). The organic layer
was dried (Na SO ), filtered, and concentrated in vacuo to give the
1
7
.26 ppm and DMSO at 1.56 ppm for H NMR, and 77.0 ppm for CHCl
and 39.52 ppm for DMSO for C NMR. High-resolution mass spectra
were obtained on a Bruker MicroTOF II ESI-TOF mass spectrometer. IR
3
2
4
13
crude product, which was chromatographed on a silica gel column
eluting with EtOAc–PE (1:5) to afford 3 as white crystals; yield: 2.0 g
(85%).
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Method 2: TCCA (225 mg, 0.95 mmol) dissolved in acetone (10 mL)
was added dropwise to a stirred solution of 2 (1.00 g, 2.45 mmol) in
freshly distilled pyridine (0.225 mL, 2.85 mmol) and acetone (15 mL)
at 0 °C, followed by gentle heating to r.t. over 40 min. The reaction
was then quenched with sat. aq Na SO (15 mL) and the volatiles were
evaporated under reduced pressure. After extraction of the residue
with EtOAc (15 mL), the separated aqueous layer was extracted with
EtOAc (10 mL). The combined organic solutions were washed with aq
min under N2 atmosphere and then allowed to stir at r.t. for 15 min
prior to dilution with Et O (10 mL). The course of the reaction was
2
monitored by TLC. The mixture was evaporated under reduced pres-
sure to leave a light yellow solid, which was dissolved in EtOAc (15
mL) and the EtOAc layer was washed with brine (2 × 20 mL). The
EtOAc solution was dried (Na SO ) and concentrated. After the crude
2
3
2
4
product was recrystallized from ethanol twice, chromatography of
the residue on silica gel (eluent: EtOAc–CH Cl –PE, 1:1:6) gave 5 as a
2
2
1
M HCl (2 × 10 mL), sat. aq NaHCO (3 × 25 mL) and brine (2 × 20 mL),
white solid; yield: 0.96 g (87%); mp 210–211 °C (EtOH); [α]D25 –18.3
3
dried (Na SO ), and concentrated. The product was purified by silica
(c 1 g/100 mL CH Cl ).
2
4
2
2
gel column chromatography (eluent: EtOAc–PE, 1:5) to afford 3 as
IR (KBr): 3389 (N–H), 1743 and 1690 (C=O), 1127 (C–O), 710 and 650
cm (C–H).
2
5
white crystals; yield: 753 mg (76%); mp 133–135 °C (EtOH); [α]D
7.1 (c 1 g/100 mL CH Cl ).
–1
–
2
2
1
H NMR (400 MHz, CDCl /TMS): δ = 0.64 (3 H, s, 18-H), 0.90 (3 H, d,
3
IR (KBr): 3330 (O–H), 1743 and 1690 (C=O), 1123 cm–1 (C–O).
J = 6.4 Hz, 21-H), 0.96 (3 H, d, J = 6.4 Hz, 19-H), 2.04 (3 H, s), 2.43 (3 H,
s), 3.66 (3 H, s), 5.14 (1 H, m, 3α-H), 7.31 (2 H, d, J = 7.8 Hz), 7.84 (2 H,
1
H NMR (400 MHz, CDCl /TMS): δ = 0.70 (3 H, s, 18-H), 0.94 (3 H, d,
3
J = 6.4 Hz, 21-H), 1.03 (3 H, s, 19-H), 3.69 (3 H, s), 4.13 (1 H, m, 3α-H).
d, J = 7.8 Hz).
13
13
C NMR (100 MHz, CDCl /TMS): δ = 12.04, 18.26, 21.09, 22.84, 24.15,
C NMR (100 MHz, CDCl /TMS): δ = 12.01, 14.22, 18.25, 21.07, 21.44,
3
3
2
3
8.06, 30.94, 31.04, 34.36, 34.56, 35.31, 36.04, 36.21, 37.05, 37.09,
9.83, 40.27, 42.84, 50.18, 51.48, 55.94, 56.12, 67.62, 174.66, 212.71.
21.64, 22.81, 22.96, 28.06, 30.93, 30.97, 31.05, 34.39, 34.45, 35.31,
36.50, 39.76, 42.85, 45.42, 46.70, 51.53, 55.91, 56.05, 56.13, 70.75,
128.00, 128.10, 129.52, 129.57, 135.46, 143.93, 161.99, 170.48,
HRMS (ESI): m/z [M + Na]+ calcd for C25H40O Na: 427.2819; found:
4
174.70.
427.2814.
+
HRMS (ESI): m/z [M + Na] calcd for C34H50N O SNa: 637.3282; found:
2
6
637.3273.
Methyl 3α-Acetoxy-6-oxocholan-24-oate (4)
Method 1: Compound 3 (1.50 g, 3.71 mmol) dissolved in anhydrous
Methyl 3α-Acetoxychol-6-en-24-oate (6)
CH Cl (50 mL) was reacted with Ac O (0.42 mL, 4.45 mmol) in the
2
2
2
presence of Na CO (39.21 mg, 0.37 mmol). The mixture was allowed
A magnetically stirred solution of 5 (1.30 g, 2.12 mmol) in freshly dis-
tilled toluene (100 mL) was treated dropwise with a solution of LiH in
freshly distilled toluene (20 mL) over 10 min at 60 °C, and TMEDA
(0.22 mL) was then added dropwise. The suspension was warmed to
2
3
to stand for 8 h at r.t. and diluted with sat. aq NaHCO3 (10 mL). The
organic layer was then separated and the aqueous phase was extract-
ed with an additional amount of CH Cl (15 mL). The combined ex-
2
2
tracts were successively washed with aq 1 M HCl (2 × 15 mL), sat. aq
NaHCO3 (3 × 25 mL), and brine (2 × 25 mL). The organic layer was
dried (Na SO ) and filtered. The filtrate was evaporated under re-
90 °C at reflux under N atmosphere. The process of the reaction was
monitored by TLC. After stirring for 18 h, the reaction was quenched
2
with sat. aq NH Cl (12 mL) and filtered through a Celite pad. The fil-
2
4
4
duced pressure to leave 4 as a pale yellow solid, which was used in the
next step without purification; yield: 1.59 g (96%).
trate was evaporated under reduced pressure and extracted with
EtOAc (25 mL). The combined organic extracts were washed with sat.
aq NaHCO (2 × 25 mL) and brine (20 mL), dried (Na SO ) and filtered.
Method 2: To a stirred solution of 3 (300 mg, 0.74 mmol) in anhydrous
3
2
4
Evaporation of the solvent gave a brown oil, which was purified chro-
matographically on a silica gel column (eluent: EtOAc–CH Cl –PE,
CH Cl (15 mL) was added sequentially dropwise AcCl (0.06 mL, 0.81
2
2
mmol) and Et N (0.10 mL, 0.72 mmol). The mixture was stirred at r.t.
2
2
3
1
:5:50) to give the pure product 6 as a white solid; yield: 528 mg
overnight. After removal of volatiles, the mixture was acidified with
aq 1 M HCl (2 × 15 mL), basified with sat. aq NaHCO3 (2 × 10 mL),
washed with brine (20 mL), and filtered. The solid residue was puri-
fied on a silica gel column (4 cm × 5 cm) using EtOAc–PE (1:3) to af-
ford 4 as a pale yellow solid; yield: 231.0 mg (86%); mp 183–185 °C
25
(74%); mp 120–123 °C (EtOH); [α]D +3.1 (c, 1 g/100 mL CH Cl ).
2 2
IR (KBr): 3299 (C=C), 1743 and 1700 (C=O), 1127 cm–1 (C–O).
1
H NMR (400 MHz, CDCl /TMS): δ = 0.67 (3 H, s, 18-H), 0.92 (3 H, d,
3
J = 6.4 Hz, 21-H), 1.02 (3 H, s, 19-H), 2.07 (3 H, s), 3.67 (3 H, s), 5.14 (1
H, m, 3α-H), 5.68 (1 H, m, 6-H), 5.78 (1 H, m, 7-H).
25
(
EtOH); [α]D –9.4 (c 1 g/100 mL CH Cl ).
2 2
IR (KBr): 1770, 1743 and 1690 (C=O), 1123 cm–1 (C–O).
13
C NMR (100 MHz, CDCl /TMS): δ = 11.96, 18.28, 21.40, 21.45, 22.25,
3
1
H NMR (400 MHz, CDCl /TMS): δ = 0.70 (3 H, s, 18-H), 0.94 (3 H, d,
22.83, 24.15, 28.07, 31.00, 31.05, 32.79, 33.81, 34.63, 35.26, 35.32,
39.99, 40.25, 42.85, 46.98, 51.49, 55.87, 55.91, 71.86, 125.11, 128.13,
170.58, 174.76.
3
J = 6.4 Hz, 21-H), 1.09 (3 H, s, 19-H), 2.03 (3 H, s), 3.69 (3 H, s), 5.19 (1
H, m, 3α-H).
13
HRMS (ESI): m/z [M + Na]+ calcd for C27H42O Na: 453.2975; found:
C NMR (100 MHz, CDCl /TMS): δ = 12.02, 18.26, 21.03, 21.20, 22.63,
3
4
2
3
1
4.05, 28.04, 30.91, 30.93, 31.02, 34.43, 35.29, 36.31, 36.77, 36.85,
6.89, 39.78, 40.32, 42.88, 47.09, 51.45, 55.92, 56.12, 70.56, 170.24,
74.58, 211.85.
453.2946.
Methyl 3α-Acetoxy-6,7-epoxycholan-24-oate (7)
HRMS (ESI): m/z [M + Na]+ calcd for C27H42O : 469.2924; found:
5
m-CPBA (3-chloroperbenzoic acid, 324 mg, 1.88 mmol) dissolved in
469.2915.
anhydrous CH Cl (10 mL) was added dropwise at 0 °C to a stirred
2
2
solution of 6 (675 mg, 1.57 mmol) in anhydrous CH Cl (15 mL) over
2
2
Methyl 3α-Acetoxy-6-[2-[(4-methylphenyl)sulfonyl]hydrazinyl-
idene]cholan-24-oate (5)
15 min. The reaction mixture was allowed to reach 35 °C and stirred
for 1 h and the course of reaction was monitored by TLC. The reaction
was quenched with sat. aq Na SO (20 mL) and evaporated under re-
p-Toluenesulfonyl hydrazone (0.40 g, 2.15 mmol) was added in five
portions to a stirred solution of 4 (0.80 g, 1.79 mmol) in anhydrous
EtOH (15 mL) over 10 min. The mixture was heated at reflux for 45
2
3
duced pressure prior to extraction with Et O (20 mL). The combined
2
organic phases were washed with sat. aq Na SO (20 mL), sat. aq
2
3
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Synthesis
Q. Dou, Z. Jiang
Paper
NaHCO3 (2 × 25 mL), and brine (2 × 20 mL), dried (Na SO ), and con-
centrated. Purification by chromatography on a silica gel column (elu-
min and the reaction mixture was then allowed to rise to r.t. during
40 min with stirring before quenching with sat. aq Na SO (15 mL).
2
4
2
3
ent: EtOAc–CH Cl –PE, 1:4:60) resulted in pure 7 as a white solid;
yield: 581 mg (83%); mp 158–160 °C (EtOH); [α]D +13.1 (c, 1 g/100
The course of the reaction was monitored by TLC. The mixture was
evaporated under reduced pressure and extracted with EtOAc (20
mL). The combined organic layers were washed with sat. aq NaHCO3
2
2
25
mL CH Cl ).
2
2
IR (KBr): 1749 and 1700 (C=O), 1127 and 1121 cm–1 (C–O).
(25 × 2 mL) and brine (25 mL), dried (Na SO ), and concentrated to
2 4
give 9 as a white solid, which was pure enough to be used directly in
1
H NMR (400 MHz, CDCl /TMS): δ = 0.66 (3 H, s, 18-H), 0.91 (3 H, s,
25
3
the next step; yield: 440 mg (96%); mp 144–147 °C (EtOH); [α]D –1
19-H), 0.93 (3 H, d, J = 1.5 Hz, 21-H), 2.08 (3 H, s), 3.20 (1 H, d, J = 4.0
(
c 1 g/100 mL CH Cl ).
2 2
Hz, 6-H), 3.22 (1 H, d, J = 3.6 Hz, 7-H), 3.67 (3 H, s), 5.17 (1 H, m, 3α-
H).
IR (KBr): 3330 (O–H), 1743 and 1720 (C=O), 1123 cm–1 (C–O).
1H NMR (400 MHz, CDCl
/TMS): δ = 0.72 (3 H, s, 18-H), 0.97 (3 H, d,
13
3
C NMR (100 MHz, CDCl /TMS): δ = 11.92, 18.31, 20.30, 20.75, 21.52,
3
J = 6.4 Hz, 21-H), 1.07 (3 H, s, 19-H), 2.08 (3 H, s), 3.03 (1 H, t, J = 14.4
Hz), 3.70 (3 H, s), 5.00 (1 H, q, J = 3.1 Hz, 3α-H).
22.15, 24.15, 28.08, 29.10, 30.98, 31.06, 34.15, 34.23, 35.34, 39.76,
42.04, 42.81, 47.11, 51.10, 51.56, 52.78, 55.94, 55.97, 70.47, 77.25,
170.49, 174.73.
13
C NMR (100 MHz, CDCl /TMS): δ = 11.75, 18.32, 21.06, 21.49, 21.89,
3
HRMS (ESI): m/z [M]+ calcd for C27H42O Na: 469.2924; found:
23.57, 28.01, 30.95, 31.00, 31.05, 34.93, 35.01, 35.29, 36.58, 36.91,
5
37.93, 39.41, 42.60, 42.77, 44.76, 50.34, 51.53, 55.74, 71.14, 170.26,
74.67, 212.50.
469.2929.
1
HRMS (ESI): m/z [M + Na]+ calcd for C27H42O Na: 469.2924; found:
Methyl 3α-Acetoxy-7α-hydroxycholan-24-oate (8)
5
469.2917.
Path I: To a pressure bottle containing anhydrous CH Cl (15 mL) was
2
2
added a solution of 7 (2.1 g, 4.71 mmol) in freshly distilled CH Cl (60
mL) and anhydrous pyridine (1 mL, 12.44 mmol) in the presence of
Pd/C (200 mg, 10 mol%) at r.t. The reaction mixture was heated to
2
2
Ursodeoxycholic Acid (UDCA, 1b)
Na (86.85 mg, 3.78 mmol), cut into pieces was added in portions to a
stirred solution of 9 (281 mg, 0.63 mmol) in freshly distilled n-BuOH
(15 mL) at r.t. within 15 min. After refluxing the reaction mixture at
60 °C for 2 h under N2 atmosphere, the mixture was quenched with
4
0 °C for 24 h under 50 psi of H . H O (30 mL) was then added and the
2 2
catalyst was removed by filtration through a Celite pad. The filtrate
was concentrated and the residue was dissolved in EtOAc (30 mL). The
organic layer was washed with aq 1 M HCl (15 × 2 mL), sat. NaHCO3
sat. aq NH Cl (15 mL) and evaporated under reduced pressure. The
4
(
50 × 3 mL) and brine (25 × 2 mL), and dried (Na SO ). The mixture
residue was dissolved in CH Cl (12 mL) and the organic layer was
2
4
2
2
was concentrated to yield an 8:1 mixture of hydroxy epimers (α/β) as
washed with sat. aq NaHCO3 (15 mL) and brine (15 × 2 mL), dried
(Na SO ), and concentrated to obtain an orange oil. The oil was puri-
fied by chromatography on a silica gel column (eluent: MeOH–
shown by the integration of methine signals connected to hydroxy
2
4
1
epimer in the H NMR spectrum. Chromatography of the residue on
silica gel column (eluent: EtOAc–CH Cl –hexane, 1:4:45) afforded
pure 8 as a white solid; yield: 1.31 g (62%).
CH Cl –PE, 11:4) to furnish 1b as a white solid; yield: 153 mg (62%);
mp 195–197 °C (EtOH); [α]D +54 (c 1 g/100 mL EtOH).
2
2
2
2
25
Path II: Compound 11 (0.12 g, 0.26 mmol) was dissolved in MeOH (10
mL) at –20 °C and stirred for 30 min. To the mixture was added concd
HCl (5 mol/L, 2.0 mL) dropwise and activated Zn powder (0.12 g, 1.82
mmol) in five portions at intervals of 1 min until the solution began
to turn yellow. The reaction was terminated by the addition of sat. aq
NaHCO3 (15 mL), filtered, and evaporated under reduced pressure.
The residue was dissolved in EtOAc (15 mL) and washed with sat. aq
NaHCO3 (25 × 3 mL) and brine (20 × 2 mL), and dried (Na SO ). The
IR (KBr): 3330 cm–1 (O–H).
1
H NMR (400 MHz, DMSO-d /TMS): δ = 0.62 (3 H, s, 18-H), 0.90 (6 H,
6
m, 19,21-H), 3.29 (1 H, m, 7β-H), 3.90 (1 H, d, J = 8.0 Hz), 4.47 (1 H, d,
J = 4.5 Hz), 11.97 (1 H, s).
13
C NMR (100 MHz, DMSO-d /TMS): δ = 12.50, 14.55, 18.77, 19.02,
6
2
3
1.32, 23.78, 27.18, 28.64, 30.72, 31.24, 34.23, 35.31, 37.74, 38.19,
9.19, 42.65, 43.48, 43.56, 55.15, 56.31, 56.50, 69.92, 70.19, 175.35.
2
4
HRMS (ESI): m/z [M + Na]+ calcd for C24
415.2811.
H O Na: 415.2819; found:
40 4
residue obtained after evaporation of the solvent was chromato-
graphed on a silica gel column (eluent: EtOAc–CH Cl –PE, 1:1:25) to
2
2
afford 8 as a white solid; yield: 86.1 mg (74%); mp 173–176 °C
25
(
EtOH); [α]D +27 (c 1 g/100 mL CH Cl ).
Methyl 3α-Acetoxy-6-trimethylsilyloxychol-6-en-24-oate (10)
2
2
IR (KBr): 3330 (O–H), 1743 and 1720 (C=O), 1123 cm–1 (C–O).
A Schlenk tube containing a suspension of LiH (0.27 g, 33.61 mmol) in
1
freshly distilled THF (10 mL) was stirred at 30 °C for 5 min under N
2
H NMR (400 MHz, CDCl /TMS): δ = 0.68 (3 H, s, 18-H), 0.95 (3 H, d,
3
atmosphere. To this was added dropwise a solution of TMSCl (5.25
mL, 42.01 mmol) in freshly distilled THF (10 mL). After stirring the
mixture for 5 min, a solution of 4 (1.5 g, 3.36 mmol) in freshly dis-
tilled THF (15 mL) was added dropwise and the mixture was stirred
for 30 min until the solution turned yellow. The mixture was heated
J = 3.1 Hz, 21-H), 0.97 (3 H, s, 19-H), 2.09 (3 H, s), 3.54 (1 H, m, 7α-H),
3.70 (3 H, s), 4.91 (1 H, q, J = 3.1 Hz, 3α-H).
13
C NMR (100 MHz, CDCl /TMS): δ = 11.72, 18.30, 20.67, 21.65, 22.75,
3
23.58, 28.05, 29.72, 30.98, 31.02, 31.46, 34.14, 34.77, 35.22, 35.31,
37.96, 38.94, 39.54, 41.14, 42.72, 50.43, 51.52, 55.73, 71.40, 71.83,
170.67, 174.74.
to 60 °C and treated dropwise with Et N (10.51 mL, 33.61 mmol). Af-
3
ter 6 h, sat. aq NH Cl (20 mL) was poured into the mixture to quench
4
HRMS (ESI): m/z [M + Na]+ calcd for C27H44O Na: 471.3081; found:
the reaction, followed by evaporation under reduced pressure. The
mixture was extracted with EtOAc (25 mL). The combined organic
phases were washed with sat. aq NaHCO3 (2 × 30 mL) and brine (20
5
471.3083.
Methyl 3α-Acetoxy-7-oxocholan-24-oate (9)
mL), dried (Na SO ), and concentrated. The residue was purified by
2 4
silica gel column chromatography (eluent: EtOAc–PE, 1:50) to afford
1
To a stirred solution of 8 (460 mg, 1.03 mmol) in a mixture of acetone
0 as a colorless oil; yield: 1.55 g (89%); mp 167–170 °C (EtOH); [α]D25
(30 mL) and AcOH (0.07 mL, 1.24 mmol) was added dropwise a solu-
+
12.5 (c 1 g/100 mL CH Cl ).
tion of NCS (205 mg, 1.54 mmol) in acetone (10 mL) at 0 °C over 10
2 2
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 588–594

5
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Synthesis
Q. Dou, Z. Jiang
Paper
IR (KBr): 1743 and 1720 (C=O), 1123 cm–1 (C–O).
References
1
H NMR (400 MHz, CDCl /TMS): δ = 0.24 (9 H, s), 0.67 (3 H, s, 18-H),
3
(
(
(
(
1) Kritchevsky, D. The Bile Acids - Chemistry, Physiology and Metab-
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HRMS (ESI): m/z [M + Na] calcd for C30H50O SiNa: 541.3320; found:
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541.3323.
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2
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2
3
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10) Giovannini, P. P.; Grandini, A.; Perrone, D.; Pedrini, P.; Fantin,
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(
Na SO ). The solvent was evaporated under vacuum and the residue
2 4
was chromatographed on a silica gel column (eluent: EtOAc–PE, 1:12)
(
to afford pure 11 as a white solid; yield: 0.62 g (91%); mp 151–153 °C
25
(
EtOH); [α]D +51.3 (c 1 g/100 mL CH Cl ).
2 2
(
(
IR (KBr): 1743, 1720, and 1690 (C=O), 1123 cm–1 (C–O).
1
H NMR (400 MHz, CDCl /TMS): δ = 0.71 (3 H, s, 18-H), 0.94 (3 H, d,
3
J = 6.4 Hz, 21-H), 1.12 (3 H, s, 19-H), 2.04 (3 H, s), 3.70 (3 H, s), 4.49 (1
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3
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4
1
2.98, 24.04, 28.08, 30.94, 31.04, 31.97, 35.05, 35.31, 38.65, 39.67,
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(
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HRMS (ESI): m/z [M + Na]+ calcd for C27H42O Na: 485.2874; found:
4
6
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The research was supported by Shanghai Key Laboratory of Chemical
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his help in the preparation of this manuscript.
(
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(
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©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 588–594