Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid)

Article abstract of DOI:10.1016/j.steroids.2010.03.007

Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3α,12α,16α-trihydroxy-5β-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16β-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3α,7α,16α-trihydroxy-5β-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties.

Full text of DOI:10.1016/j.steroids.2010.03.007

Steroids 75 (2010) 506–512
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis, aggregation behavior and cholesterol solubilization studies of
16-epi-pythocholic acid (3␣,12␣,16␤-trihydroxy-5␤-cholan-24-oic acid)
Nonappa∗, Uday Maitra
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic
acid (3␣,12␣,16␤-trihydroxy-5␤-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnat-
ural epimer of pythocholic acid (3␣,12␣,16␣-trihydroxy-5␤-cholan-24-oic acid, PCA) involves a series of
simple and selective chemical transformations with an overall yield of 21% starting from readily available
cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was
determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated
using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy
bile acids. These studies showed that 16-epi-pythocholic acid (16␤-hydroxy-deoxycholic acid) behaves
similar to cholic acid (CA) and avicholic acid (3␣,7␣,16␣-trihydroxy-5␤-cholan-24-oic acid, ACA) in its
aggregation behavior and cholesterol dissolution properties.
Received 27 January 2010
Received in revised form 19 March 2010
Accepted 23 March 2010
Available online 30 March 2010
Keywords:
Bile acid
16-Epi-pythocholic acid
Cholesterol dissolution
CMC
© 2010 Elsevier Inc. All rights reserved.
Allylic oxidation
1. Introduction
The presence of 15␣- and 15␤-hydroxy derivatives have been
found to be the major bile acids in certain marsupials [12], whereas,
There has been considerable interest towards the synthesis of
natural and unnatural analogues of bile acids and their derivatives
in recent years [1,2]. Recent advances in the field of molecular biol-
ogy and genetics have greatly accelerated the knowledge related
to the role of bile acids and its derivatives in a number of physio-
logically important processes [3]. The studies on new roles of bile
acids as potential ligands for certain nuclear hormone receptors
[4,5], and as pheromones [6,7] have emerged as an attractive area
in steroid research. The importance of 15- and 16-hydroxy deriva-
tives of chenodeoxycholic acid (CDCA) and 16-hydroxy derivatives
deoxycholic acid (DCA) has recently been discussed in the literature
[8,9]. In the majority of vertebrates, the hydroxylation of primary
bile acids like chenodeoxycholic acid and certain secondary bile
acids (deoxycholic acid and lithocholic acid) occurs at an unusual
site on the steroid nucleus [10,11]. It has been suggested that such
unusual trihydroxy bile acids are formed due to the involvement
of hepatic enzymes (in CDCA) or intestinal microbial flora (in sec-
ondary bile acids) which mediates dehydroxylation at C-7 followed
by absorption of the 7-deoxy bile acids and subsequent hepatic
hydroxylation at additional sites.
the 16-hydroxy derivatives are major bile acids in snake and avian
bile. Kakiyama et al. [13] reported the presence of 15␣-hydroxy
derivative of lithocholic acid as a major biliary bile acid in the wom-
bat (Vombatus ursinus), a common Australian marsupial. Studies
on microbial transformation of lithocholic acid (LCA) into 3␣,15␤-
dihydroxy-5␤-cholan-24-oic acid by a fungus Cunninghamella
blakesleeana ST-22 was reported in 1985 [14]. In 2002 Iida et al. pub-
lished the synthesis of 3␣,7␣,15␣-trihydroxy-5␤-cholan-24-oic
acid and also its 15␤-epimer (3␣,7␣,15␤-trihydroxy-5␤-cholan-
24-oic acid) as a byproduct upon the synthesis of avicholic
acid (3␣,7␣,16␣-trihydroxy-5␤-cholan-24-oic acid). It is inter-
esting to note that four years later the same group identified
and structurally characterized 3␣,7␣,15␣-trihydroxy-5␤-cholan-
24-oic acid as a major biliary bile acid in swans, tree ducks and
geese as its taurine conjugates [11]. There are several exam-
ples where unusual trihydroxy bile acids like 3␣,7␣,14␣- or
3␣,7␣,16␣-trihydroxy-5␤-cholan-24-oic acid have been isolated
and structurally characterized [15,16]. Chemical synthesis of such
unusual bile acids is of considerable interest because the avail-
ability of synthetic samples provides useful reference towards a
definitive structural determination of the 15- and 16-hydroxy bile
acids. Unambiguous proof of many naturally isolated bile acids and
their structural characterization requires chemical synthesis and
demonstration of identity of the isolated compounds with syn-
thetic ones. The chemical synthesis of these uncommon bile acids
also provides a unique opportunity to study the chemical, biologi-
∗
Corresponding author. Current address: Department of Chemistry, University of
Jyväskylä, Survontie 9, P.O. Box 35, FI-40014 Jyväskylä, Finland.
Tel.: +358 14 2602655.
E-mail addresses: nonappa@jyu.fi, nonappa@gmail.com ( Nonappa).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.03.007

Nonappa, U. Maitra / Steroids 75 (2010) 506–512
507
cal and physicochemical properties of rare and unusual natural bile
acids.
The first chemical synthesis, aggregation behavior and choles-
terol solubilization studies of pythocholic acid (3␣,12␣,16␣-
+53 (c 2.0, EtOH). HRMS: calculated for C25H40O4 + Na: 427.2824;
found: 427.2824. Analysis: calculated for C25H40O4·1/2H2O: C 72.6,
H 9.99; found: C 72.5, H 9.95.
trihydroxy-5␤-cholan-24-oic acid, PCA)
a
major component
2.1.2. Methyl 3˛,12˛-dihydroxy-5ˇ-chol-14-en-24-oate (3)
in python’s bile and 16␣-hydroxycholic acid (3␣,7␣,12␣,16␣-
tetrahydroxy-5␤-cholan-24-oic acid) a minor component in avian
bile was reported from our laboratory [17]. Pythocholic acid (PCA)
displayed unusual aggregation behavior and high cholesterol sol-
ubilization ability. A biomimetic remote functionalization strategy
utilized towards the first chemical synthesis of PCA resulted in 5.5%
overall yield [17]. Therefore, we decided to carry out the synthesis
of PCA via an alternative synthetic route. Final characterization of
the product revealed that it is the 16␤-epimer of pythocholic acid.
Herein we report the synthesis, aggregation properties and pre-
liminary in vitro cholesterol dissolution studies of this unnatural
epimer of PCA.
A solution of compound 2 (1.0 g, 2.5 mmol) in chloroform
(20 mL) was cooled to −78 ◦C with dry ice/acetone. A dry stream of
hydrogen chloride gas was passed through the solution for 4 h fol-
lowed by a stream of nitrogen to remove excess hydrogen chloride
from the reaction vessel. Then, 0.5 M aqueous sodium hydrogen
carbonate (10 mL) was added at low temperature (0 ◦C) and the
mixture was allowed to attain room temperature. The organic layer
was separated, washed with water, dried with MgSO4 and evapora-
tion of the volatiles under reduced pressure resulted in yellow solid.
Column chromatography on silica gel using 50–75% ethyl acetate
in petroleum ether furnished 0.68 g (68%) of 3 as white solid. Mp
92–95 ◦C. IR, v¯max : 3420, 2925, 2804, 1735 cm−1 1H NMR (CDCl3,
.
400 MHz) ı: 5.29 (d, J = 1.6 Hz, 1H, 15-H), 3.80 (s, 1H, 12␤-H), 3.67 (s,
3H, –CO2CH3), 3.65–3.60 (m, 1H, 3␤-H), 2.5–1.1 (m, steroidal –CH
and –CH2), 0.95 (d, J = 6.8 Hz, 3H, 21-CH3), 0.93 (s, 3H, 19-CH3), 0.92
(s, 3H, 18-CH3). 13C NMR (CDCl3, 75 MHz) ı: 174.70 (C-24), 151.40
(C-14), 120.10 (C-15), 73.22 (C-3), 71.67 (C-12), 51.68 (C-13), 51.47
(–CO2CH3), 46.91 (C-17), 41.90 (C-5), 36.24 (C-4), 34.87 (C-1), 34.69,
34.18, 33.49, 31.94, 31.06 (C-23), 30.86 (C-2), 30.51 (C-22), 29.20,
26.80, 23.93, 22.83 (C-19), 17.73 (C-21), 16.68 (C-18). [␣]D23 +54 (c
2.0, EtOH). HRMS: calculated for C25H40O4 + Na: 427.2824; found:
427.2824. Analysis: calculated for C25H40O4: C 74.21, H 9.96; found:
C 74.22, H 10.12.
2. Experimental
2.1. General methods
All reactions were carried out in oven-dried glassware. TLC was
checked on pre-coated glass plates (0.25 mm silica gel with fluores-
cent UV254) purchased from Aldrich. After elution the plates were
developed using the Liebermann-Burchard reagent. Commercial
grade solvents were distilled prior to use in column chromatogra-
phy. Silica gel (100–200 mesh) columns were run under gravity.
1
1H and 13C{ H} NMR spectra were recorded on a JEOL Lambda
2.1.3. Methyl 3˛,12˛-diacetoxy-5ˇ-chol-14-en-24-oate (4)
300 MHz spectrometer in deuterated solvents as indicated with
TMS or residual solvent signals as the internal standards. Infrared
spectra were recorded on a JASCO-70 FT-IR spectrophotometer, by
either making a film of the compounds on a NaCl plate from a chlo-
roform solution or using KBr pellets. ESI-QTOF MS was recorded
on a Micromass Q-TOF mass spectrometer. Optical rotations were
recorded on a DIP-370 digital polarimeter at 589 nm. Elemen-
tal analyses were done on a Carlo Erba Strumentazione CHNS
Analyzer-model 1106 and Flash EA 1112. Double distilled water
was used in fluorescence and absorption spectral measurements.
Fluorescence experiments were performed using a Perkin-Elmer
LS-50 B luminescence spectrometer, while absorption spectra were
recorded on a Shimadzu UV200 spectrometer.
To
a stirred solution of 3 (160 mg, 0.39 mmol) in pyri-
dine (3 mL), acetic anhydride (1 mL, 10.58 mmol) and
4-dimethylaminopyridine (20 mg, 0.16 mmol) were added and the
reaction mixture was stirred at 25 ◦C for 16 h. It was then neutral-
ized with 1 M HCl (10 mL), extracted with ethyl acetate (3 × 5 mL)
and washed with aqueous sodium bicarbonate solution. After
drying the organic layer over anhydrous. Na2SO4, the volatiles
were removed under reduced pressure. Column chromatographic
purification of crude product using 15–30% ethyl acetate in n-
hexane furnished 170 mg (90%) of 4 as a foamy solid. Mp 75–77 ◦C.
IR, v¯max : 2935, 1735, 1241, 1025 cm−1 1H NMR (CDCl3, 300 MHz)
.
ı: 5.28 (s, 1H, 15-H), 5.00 (br t, J = 3.0 Hz, 1H, 12␤-H), 4.75–4.65 (m,
1H, 3␤-H), 3.67 (s, 3H, –CO2CH3), 2.10 (s, 3H, CH3COO-12), 2.00
(s, 3H, CH3COO-3), 2.0–1.1 (m, steroidal –CH and –CH2), 0.96 (s,
3H, 19-CH3), 0.91 (s, 3H, 18-CH3), 0.81 (d, J = 6.6 Hz, 3H, 21-CH3).
13C NMR (CDCl3, 75 MHz) ı: 174.43 (C-24), 170.45 (CH3COO-12),
170.09 (CH3COO-3), 151.63 (C-14), 119.63 (C-15), 75.90 (C-12),
73.94 (C-3), 51.40 (CO2CH3), 49.66 (C-13), 47.65 (C-17), 41.50
(C-5), 34.74, 34.59, 34.35, 33.92, 32.91, 32.42, 31.86, 30.87, 30.74,
29.94, 26.55, 26.48, 26.38, 23.55, 22.57, 21.30 (C-19), 17.76 (C-21),
16.76 (C-18). HRMS: calculated for C29H44O6 + Na: 511.3036:
found: 511.3036. Analysis: calculated for C29H44O6: C 71.27, H
9.07; found: C 71.20, H 9.08.
2.1.1. Methyl 3˛,12˛-dihydroxy-5ˇ-chol-8(14)-en-24-oate (2)
A solution containing cholic acid 1 (3.0 g, 7.34 mmol) and anhy-
drous ZnCl2 (3.0 g, 22.06 mmol) in acetone (30 mL) was refluxed
at 80 ◦C for 2 h and the solvent was slowly distilled off until TLC
showed conversion of the starting material. The solution was then
cooled to room temperature and aqueous acetic acid (30 mL of
0.5%) was added. The precipitate was collected by filtration and
dried in vacuum. The solid obtained was dissolved in cold methanol
(15 mL) containing anhydrous HCl (generated in situ by the drop-
wise addition of AcCl into methanol). After stirring for 12 h at room
temperature (∼28 ◦C), the volatiles were removed under reduced
pressure. The crude product obtained after aqueous work up was
column purified on silica gel using 50–70% ethyl acetate in n-
2.1.4. Methyl
3˛,12˛-diacetoxy-16-keto-5ˇ-chol-14(15)-en-24-oate (5)
A
mixture of olefin
4
(162 mg, 0.33 mmol), N-
hexane to afford 2 in 78% yield as white solid. Mp 84–86 ◦C. IR, v¯max
:
hydroxysuccinimide (150 mg, 1.30 mmol) and Na2Cr2O7·2H2O
(250 mg, 0.83 mmol) was stirred in acetone (8 mL) at 40 ◦C for 48 h.
A saturated solution of solution of Na2SO3 (12 mL) was added and
the contents were extracted with ethyl acetate (2 × 10 mL). The
organic layer was washed with saturated solution of NaCl (15 mL)
and dried over anhydrous Na2SO4. The crude product obtained
after the removal of volatiles was purified by column chromatog-
raphy using silica gel in 20–30% ethyl acetate in petroleum ether
to yield 120 mg (72%) of 5. Mp 120–121 ◦C. IR v¯max : 2933, 1737,
3420, 2925, 2804, 1740, 1630 cm−1 1H NMR (CDCl3, 300 MHz) ı
.
3.90 (br s, 1H, 12␤-H), 3.66 (s, 4H, 3␤-H and –CO2CH3), 2.2–1.2 (br
m, steroidal –CH and –CH2), 1.00 (d, J = 6.0 Hz, 3H, 21-CH3), 0.865
(s, 3H, 19-CH3), 0.81 (s, 3H, 18-CH3). 13C NMR (CDCl3, 75 MHz) ı:
174.74(C-24), 137.88(C-8), 127.40(C-14), 72.16 (C-12), 71.85(C-3),
51.49 (CO2CH3), 47.19 (C-17), 42.08 (C-5), 36.02 (C-4), 35.63 (C-1),
34.44, 33.75, 31.17, 30.94 (C-23), 30.76 (C-22), 30.51, 27.24, 26.60,
23
26.37, 26.25, 24.56, 23.77 (C-19), 19.24 (C-21), 17.62 (C-18). [␣]D

508
Nonappa, U. Maitra / Steroids 75 (2010) 506–512
1241, 1025 cm−1
.
1H NMR (CDCl3, 300 MHz) ı: 5.82 (d, J = 1.5 Hz,
1H NMR (CDCl3, 300 MHz) ı: 4.15 (m, 1H, 16␣-H), 3.66–3.55 (m,
2H, 12␤-H + 3␤-H), 1.07 (d, J = 6.5 Hz, 3H, 21-CH3), 0.96 (s, 3H, 19-
CH3), 0.90 (s, 3H, 18-CH3). 1H NMR (acetone-d6, 400 MHz) ı: 4.06
(q, J = 7.5 Hz, 1H, 16␣-H), 3.57 (br s, 1H, 12␤-H), 3.55–3.45 (m,
1H, 3␤-H), 2.0–1.1 (m, steroidal –CH and –CH2), 1.04 (d, J = 7.2 Hz,
3H, 21-CH3), 1.00 (s, 3H, 19-CH3), 0.91 (s, 3H, 18-CH3). 13C NMR
(acetone-d6, 100 MHz) ı: 174.71 (C-24), 76.47 (C-16), 73.34 (C-12),
71.47 (C-3), 61.17 (C-17), 50.42 (C-14), 46.32 (C-13), 43.09 (C-5),
38.13 (C-8), 37.17 (C-4), 35.92 (C-1), 35.59 (C-10), 34.72 (C-15),
34.35 (C-23), 33.45 (C-22), 27.98, 26.97, 26.53, 26.05, 25.72, 25.00,
23.53, 22.73 (C-19), 20.92, 20.62 (C-21), 19.53 (C-18). HRMS: cal-
culated for C24H40O5 + Na: 431.2773: found: 431.2772. Analysis:
calculated for C24H40O5: C 70.55, H 9.87; found: C 70.56, H 10.0.
15-H), 5.08 (t, J = 2.7 Hz, 12␤-H), 4.75–4.68 (m, 1H, 3␤-H), 2.10
(s, 3H, CH3COO-12), 2.00 (s, 3H, CH3COO-3), 2.0–1.1 (m, steroidal
–CH and –CH2), 1.28 (S, 3H, 19-CH3), 1.06 (d, J = 6.6 Hz, 3H, 21-
CH3), 1.00 (s, 3H, 18-CH3). 13C NMR (CDCl3, 75 MHz) ı: 207.89
(C-16), 184.98 (C-14), 173.99 (C-24), 170.48 (CH3COO-12), 170.04
(CH3COO-3), 126.07 (C-15), 75.49 (C-12), 73.58 (C-3), 55.89 (C-17),
51.45 (–CO2CH3), 49.89 (C-13), 41.25, 36.04, 34.59, 34.33, 32.06,
31.96, 30.87, 28.65, 26.55, 26.10, 25.43, 23.46, 22.58 (C-19), 21.35
(CH3COO-12), 21.14 (CH3COO-3), 20.48 (C-21), 19.13 (C-18).
23
[␣]D +115 (c 2.0, EtOH). HRMS: calculated for C29H42O7 + Na:
525.2828; found: 525.2828. Analysis: calculated for C29H42O7: C
69.29, H 8.42; found: C 69.27, H 8.46.
2.1.5. Methyl 3˛,12˛-diacetoxy-16-keto-5ˇ-cholan-24-oate (7)
To a solution containing olefin 5 (180 mg, 0.35 mmol) in ethyl
acetate (3 mL), 10% Pd–C (12 mg) was added and the vessel was
evacuated and flushed with hydrogen. The reaction mixture was
stirred under H2 atmosphere for 1 h. The reaction mixture was
filtered using celite over a sintered funnel. The crude product
obtained after the removal of volatiles was purified by column chro-
matography in 20–30% ethyl acetate in petroleum ether to yield
162 mg (90%) of 7 as a white solid. IR v¯max : 2933, 1737, 1241,
2.2. Determination of critical micellar concentration (CMC)
Pyrene (Fluka, 99%, recrystallized from hot EtOH) was added to
aqueous TRIS buffer (0.1 M, pH 9). The mixture was sonicated for
½ h and filtered through a 0.4 ␮m filter. Samples were made by
diluting the stock solutions of bile acids with the saturated pyrene
solution. The concentration of pyrene was ∼0.5 ␮M. Samples were
excited at 336 nm and the I3/I1 ratios were calculated by taking
the ratio of maximum peak intensity at 384 nm to that at 373 nm.
All spectra were recorded on a Perkin-Elmer LS 50B luminescence
spectrometer.
1025 cm−1 1H NMR (CDCl3, 300 MHz) ı: 5.07 (br s, 1H, 12␤-H),
.
4.77–4.64 (br m, 1H, 3␤-H), 3.66 (s, 3H, –CO2CH3), 2.10 (s, 3H,
CH3COO-12), 2.00 (s, 3H, CH3COO-3), 2.0–1.1 (m, steroidal –CH and
–CH2), 0.90 (s, 3H, 19-CH3), 0.80 (d, J = 6.3 Hz, 3H, 21-CH3), 0.72 (s,
3H, 18-CH3). 13C NMR (CDCl3, 75 MHz) ı: 220.20 (C-16), 173.79 (C-
24), 170.62 (CH3COO-12), 170.07 (CH3COO-3), 78.38 (C-12), 73.84
(C-3), 61.67 (C-17), 51.59 (CO2CH3), 45.88 (C-13), 44.23, 41.81 (C-5),
39.97, 34.18, 33.67, 33.19, 32.34, 32.04, 31.98, 28.18, 26.80, 26.60,
26.35, 25.91, 25.81, 23.0, 21.43 (C-19), 21.40 (CH3COO-12), 21.07
2.3. In vitro cholesterol solubilization studies
A commercial ‘cholesterol reagent set’ based on enzymatic
method (using cholesterol esterase, cholesterol oxidase and perox-
idase) was used for the determination of the amount of cholesterol
solubilized by bile salts [17]. Solid anhydrous cholesterol was added
to bile salt solutions prepared by dissolving bile acids in carbonate
buffer of pH 10. The mixtures were stirred at 37 ◦C for one day.
These mixtures were filtered using 0.2 ␮m membrane filter. The
filtrate (10 ␮L) was added to 1 mL reagent, and then it was incu-
bated at 37 ◦C for 10 min. Absorbance was checked at 510 nm. The
absorbance of the standard cholesterol solution (200 mg %, 5.2 mM)
at 510 nm was 0.364. Conc. of cholesterol (in mg %) = [absorbance of
the sample/absorbance of the standard] × 200 conc. of cholesterol
(in mM) = [conc. of cholesterol (in mg %) × 1000]/[386.7 × 100].
23
(CH3COO-3), 19.03 (C-21), 19.00 (C-18). [␣]D +112 (c 2.0, EtOH).
HRMS: calculated for C29H44O7 + Na: 527.2984: found: 527.2982.
2.1.6. Methyl 3˛,12˛,16ˇ-triacetoxy-5ˇ-cholan-24-oate (8)
To a stirred solution of 7 (62 mg, 0.12 mmol) in MeOH/THF
1:2 (v/v) (2 mL) at 0 ◦C was added CeCl3·7H2O (45 mg, 0.12 mmol)
followed by NaBH4 (7 mg, 0.185 mmol) and stirred at rt for 2 h.
After aqueous work up the crude product was dried under vacuum
and dissolved in pyridine (500 ␮L). Anhydrous acetic anhydride
(500 ␮L) was added and the reaction mixture was stirred at room
temperature. After stirring for 12 h, the reaction mixture was neu-
tralized with 1 M HCl and extracted using CH2Cl2 (2 × 4 mL). The
crude product was purified by column chromatography over sil-
ica using 15–20% ethyl acetate in n-hexane to yield 49 mg (72%)
3. Results and discussions
3.1. Retrosynthetic analysis
of 8 as gummy solid. IR v¯max : 2940, 1735 cm−1 1H NMR (CDCl3,
.
We envisioned intermediate 1a (Scheme 1) as a suitable pre-
cursor towards the synthesis of 16-hydroxy bile acid. Selective
chemical transformation of 1a into 16-keto derivative utilizing
allylic oxidation followed by stereoselective reduction will result
in the formation of 16␣/␤-hydroxy bile acid. During the course of
our investigation, we noticed that the intermediate could easily
be prepared starting from cholic acid 1. These intermediates have
been reported in the literature during the synthesis of cephalostatin
[18]. Careful analysis of the structure and synthetic procedure indi-
cated that there are two modified bile acid units where one of the
intermediates used was olefin 1a.
300 MHz) ı: 5.08 (q, J = 7.5 Hz, 1H, 16␣-H), 4.77 (br s, 1H, 12␤-
H), 4.75–4.66 (m, 1H, 3␤-H), 3.66 (s, 3H, –CO2CH3), 2.12 (s, 3H,
CH3COO-16), 2.05 (s, 6H, CH3COO-12 and CH3COO-3), 2.0–1.1 (m,
steroidal –CH and –CH2), 1.04 (s, 3H, 19-CH3), 0.90 (t, 6H, 21-CH3
and 18-CH3). 13C NMR (CDCl3, 75 MHz) ı: 174.38 (C-24), 171.21
(CH3COO-16), 170.98 (CH3COO-12), 170.91 (CH3COO-3), 78.45 (C-
12), 76.61 (C-16), 74.60 (C-3), 54.48 (C-17), 51.96 (CO2CH3), 49.28
(C-15), 44.68 (C-14), 42.27 (C-13), 34.72 (C-1), 34.61, 33.59, 32.96,
32.87, 32.64, 29.21, 27.54, 27.42, 27.35, 27.31, 26.83, 23.41, 22.09,
23
21.88 (C-19), 20.50 (C-21), 20.05 (C-18). [␣]D +132 (c 0.5, EtOH).
HRMS: calculated for C31H48O8 + Na: 571.3247: found: 571.3247.
2.1.7. 3˛,12˛,16ˇ-Trihydroxy-5ˇ-cholan-24-oic acid (9)
3.2. Synthesis
A solution of 8 (45 mg, 0.08 mmol) in 5% KOH–MeOH (2.5 mL)
was stirred at 65 ◦C. After stirring for 16 h the volatiles were
removed under reduced pressure. The residue was dissolved in
ice-cold water (5 mL) and neutralized with 1 M HCl solution. The
precipitate was filtered and dried to yield in 31 mg (92%) of 9 as
Selective dehydration of cholic acid 1 was performed using
anhydrous ZnCl2 in refluxing acetone to furnish the olefin and the
crude product was stirred in methanol containing anhydrous HCl to
yield the methyl ester 2 [18]. Compound 2 was subjected to isomer-
ization using dry HCl in chloroform at −78 ◦C to yield the desired
a white solid. Mp: 192–193 ◦C. IR v¯max : 3410, 2939, 1705 cm−1
.

Nonappa, U. Maitra / Steroids 75 (2010) 506–512
509
Scheme 1. Retrosynthetic approach.
Table 1
Comparison of 1H NMR (in CDCl3, 300 MHz) chemical shift values (in ppm) of pythocholic acid [Ref. [17]] and its 16␤-epimer 9.
ꢀı
18-H
19-H
21-H
3␤-H
12␤-H
16␤/␣-H
0.70 (s)
0.89 (s)
1.00 (d)
3.71–3.61 (br m)
3.88 (br s)
4.0 (t, 6.6 Hz)
0.90 (s)
0.96 (s)
1.07 (d)
0.20
0.07
0.07
–
–
0.15
3.66–3.55 (merged with 12␤-H)
3.66–3.55 (merged with 3␤-H)
4.15 (br m)
precursor 3 [18]. Our next aim was to carry out selective functional-
ization at C-16. In order to achieve that, the olefin 3 was converted
into its diacetyl derivative 4 under pyridine-acetic anhydride con-
dition. The selective oxidation of 4 was obtained using a mixture
sodium dichromate dihydrate and N-hydroxysuccinimide (NHS) in
acetone at 40 ◦C for 48 h to obtain the enone 5 [19]. Compound
5 upon reduction using NaBH4–CeCl3·7H2O in methanol followed
by hydrogenation resulted in dehydroxylation and furnished the
undesired diacetyl derivative of deoxycholic acid 6. The structure
of 6 was confirmed by comparing the spectral data of authentic
diacetyl derivative of methyl deoxycholate prepared from commer-
cial deoxycholic acid.
gave the 16-hydroxy bile acid 9 with an overall yield of 21%. On the
other hand NaBH4 reduction of the 16-ketosteroid 7, followed by
peracetylation in pyridine/Ac2O furnishedthetriacetyl derivative 8.
After successful synthesis we compared the NMR spectral data
of compound 9 with that of pythocholic acid. Interestingly 1H NMR
spectral data of 9 showed a considerable difference in the chemical
shift values of angular methyl protons and in the splitting pat-
tern of 16-H compared to that of pythocholic acid [17]. Careful
analysis of spectral data with pythocholic acid revealed a signifi-
cant difference in the chemical shift values (0.20 ppm) of angular
methyl 18-CH3. However, the difference in the chemical shift val-
ues of other angular methyl protons (19-CH3 and 21-CH3) was
much smaller. A similar difference was also observed in the chem-
ical shift values of 16-H (0.15 ppm) compared to other ␤-protons
(Tables 1 and 2). It clearly suggests that the stereochemistry at C16
might be different. A detailed analysis of avicholic acid and its 16␤-
In order to overcome this problem the enone 5 was selectively
reduced using Pd–C/H2 condition to yield the 16-ketosteroid 7. The
ketosteroid 7 was then subjected for reduction under NaBH4/MeOH
followed by hydrolysis using 5% KOH in methanol. This procedure
Table 2
Comparison of 1H NMR (in CDCl3) chemical shift values (in ppm) of avicholic acid and its 16␤-epimer [Ref. [8]].
ꢀı
18-H
19-H
21-H
3␤-H
7␤-H
16␤/␣-H
0.67 (s)
0.89 (s)
0.95 (d)
3.43 (br m)
3.84 (br s)
4.01 (t, 6.6 Hz)
0.87 (s)
0.91 (s)
1.07 (d)
3.46
3.88
4.50 (br m)
0.20
0.02
0.02
0.03
0.04
0.49

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Nonappa, U. Maitra / Steroids 75 (2010) 506–512
Scheme 2. Synthesis of 16-epi-pythocholic acid from 1.
epimer reported in the literature also revealed similar difference
in the chemical shift values [8]. On the basis of the above results
conclude that compound 9 is the 16␤-epimer of pythocholic acid.
Since the ␤-face of the steroid is sterically more hindered,
the hydride attack occurs predominantly from the ␣-face. Spar-
tan’ 06 energy minimized structure of compound 7 also showed
that the ␣-face of the steroidal skeleton is sterically less hindered
(Fig. 1). Therefore, the reduction of 16-ketosteroid 7 results in the
formation of 3␣,12␣,16␤-trihydroxy-5␤-cholan-24-oic acid 9, an
unnatural epimer of pythocholic acid.
In order to ensure that the steroidal skeleton remained
unchanged during the course of our synthesis, some of the
intermediates were converted into known bile acid deriva-
tives (Scheme 2). The olefin 3 upon hydrogenation furnished
the methyl ester of deoxycholic acid 10. Wolf-Kishner reduc-
tion of ketone
acid 11 [20]. The structure of compounds 10, 11 (Scheme 2)
and (Scheme 3) was confirmed by comparing the spec-
8 resulted in the formation of deoxycholic
6
tral data of standard derivatives obtained from deoxycholic
acid.
Scheme 3. Conversion of intermediates into known bile acid derivatives.

Nonappa, U. Maitra / Steroids 75 (2010) 506–512
511
Fig. 1. Spartan’ 06 energy minimized structure of 7.
Fig. 3. Solubilization of cholesterol by bile salts (50 mM bile salt, pH 10.0 at 37 ◦C).
3.3. Determination of critical micellar concentration (CMC)
Being facially amphiphilic, bile salts show unique aggrega-
tion behavior compared to conventional surfactants. Bile salts
carry extensive hydrophobic segments, which are responsible for
reduced contact with water. Bile salts undergo rapid, dynamic
association–dissociation equilibrium to form self-aggregates or
micelles as the total concentration of the bile salt are increased [21].
The aggregation properties of bile salts have been studied exten-
sively by various methods and this property of 16-epi-pythocholate
in aqueous medium was studied using pyrene as the fluores-
cent probe. The ratio of the two vibronic bands (I3/I1) in the
fluorescence spectrum is indicative of the polarity experienced
by the probe solubilized in the micellar aggregates [22]. Using
this technique we have measured the CMC values of cholate,
deoxycholate, and pythocholate. The CMC of 16-epi-pythocholate
was 14 mM, whereas pythocholate and cholate showed CMC
of 3–4 mM and 15 mM respectively under similar experimen-
tal conditions (Fig. 2). It is interesting to note that pythocholate
shows very low CMC [17], whereas its epimer behaves similar
to other trihydroxy bile acids. These results are consistent with
the hypothesis that aggregation behavior of bile salts depends on
the number, position and the stereochemistry of hydroxyl groups
[23].
3.4. In vitro cholesterol solubilization studies
Mixed micelle formation leading to solubilization of lipolysis
products (fatty acids and monoglycerides) is one of the significant
properties of bile salts. Bile salt micelles increase the solubility of
cholesterol (aqueous solubility is 1 nM) by more than a million
fold. The solubility of cholesterol is better in dihydroxy bile salts
compared to trihydroxy bile salts. In vitro cholesterol solubilization
study of epi-pythocholate was evaluated using anhydrous choles-
terol with bile salt solution in carbonate-bicarbonate buffer (pH
10.0) Maximum aqueous solubility of cholesterol was found to be
much less with epi-pythocholate (0.35 mM, bile salt: cholesterol
∼114:1) as compared to cholic acid (0.8 mM, bile salt: cholesterol
∼63:1) (Fig. 3).
4. Conclusions
In summary, we have developed a synthetic route for the
chemical synthesis of 16-epi-pythocholic acid, an unnatural ana-
logue of pythocholic acid starting from readily available cholic
acid. The aggregation behavior in aqueous media and preliminary
cholesterol dissolution studies revealed high CMC values and low
cholesterol solubilization ability of 16-epi-pythocholate compared
to pythocholate. Synthesis and characterization of unusual bile
acids will provide an easy access towards the identification and
characterization of bile acids from biological sources.
Acknowledgements
We thank the Jawaharlal Nehru Center for Advanced Scientific
Research, Bangalore, for a research grant. We also thank the Coun-
cil of Scientific and Industrial Research (CSIR) New Delhi, India for
senior research fellowship (SRF to N).
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