Facile Synthesis, Aggregation Behavior, and Cholesterol Solubilization Ability of Avicholic Acid

Article abstract of DOI:10.1021/ol036073f

(Equation presented) Avicholic acid, a major constituent of the bile of several avian species, was synthesized in eight steps from readily available chenodeoxycholic acid in 9% overall yield using Breslow's remote functionalization strategy in a key step. Micelle formation and equilibrium cholesterol solubilization properties were studied for avicholate in aqueous solution.

Full text of DOI:10.1021/ol036073f

ORGANIC
LETTERS
2004
Vol. 6, No. 1
31-34
Facile Synthesis, Aggregation Behavior,
and Cholesterol Solubilization Ability of
Avicholic Acid
,†
Samrat Mukhopadhyay and Uday Maitra*
Department of Organic Chemistry, Indian Institute of Science,
Bangalore 560012, India
maitra@orgchem.iisc.ernet.in
Received October 24, 2003
ABSTRACT
Avicholic acid, a major constituent of the bile of several avian species, was synthesized in eight steps from readily available chenodeoxycholic
acid in 9% overall yield using Breslow’s remote functionalization strategy in a key step. Micelle formation and equilibrium cholesterol solubilization
properties were studied for avicholate in aqueous solution.
Bile acid science (cholanology) continues to have importance
in physiology and medicine.1 Bile acids conjugated with
taurine and/or glycine form mixed micelles with fatty acids
and cholesterol in the intestine.2 Bile acids from different
species chemically differ in two respects: (i) the side-chain
structure and (ii) the distribution of the number, position,
and stereochemistry of the hydroxyl groups in the steroid
nucleus. Several decades ago, Haslewood addressed the issue
of considering the bile acid structure as an aid to the
understanding of the evolutionary process.3 It has been noted
that the bile acid structure shows a pattern of progressive
molecular development along the line of vertebrate evolution.
It has been suggested that the most evolved mammalian bile
acids have a 5â configuration with hydroxyl groups at 3R,
7R, and 12R.4 An unusual 3R,7R,16R-trihydroxy bile acid
was recently isolated from storks and herons by Hagey et
al.5 It was called avicholic acid to signify that it is a class
that has to date been identified only in avian species. This
bile acid was a major constituent (>90%) of biliary bile acids
in the Shoebill stork and several herons. It was also suggested
that 16R-hydroxy is a primitive bile acid, whereas 12R-
hydroxy is a more evolved bile acid. The first and so far the
only chemical synthesis of avicholic acid was achieved by
Iida et al. from chenodeoxycholic acid.6 This synthesis
utilized a somewhat selective 17R-hydroxylation (ca. 15%)
of acetylated methyl chenodeoxycholate by dimethyldi-
oxirane. The overall yield of avicholic acid was <1%, clearly
suggesting the need for an improved synthetic route. Herein,
† Also at the Chemical Biology Unit, Jawaharlal Nehru Center for
Advanced Scientific Research (JNCASR), Bangalore, India.
(1) Hofmann, A. F. In Bile Acids and Hepatobiliary Disease; Northfield,
T., Zentler-Munro, P. L., Jazrawi, R. P., Eds.; Kluwer Academic Publish-
ers: Boston, 1999; p 303.
(2) (a) Hofmann, A. F. In The LiVer: Biology and Pathology, 3rd ed.;
Arias, I. M., Boyer, J. L., Fausto, N., Jakoby, W. B., Schachtetr, D. A.,
Shafritz, D. A., Eds.; Raven Press, Ltd.: New York, 1994; p 677. (b) The
Bile Acids: Chemistry, Physiology and Metabolism; Nair, P. P., Kritchevsky,
D., Eds.; Plenum Press: New York, 1973; Vols. 1-3.
(3) (a) Haslewood, G. A. D. Biol. ReV. 1964, 39, 537. (b) Haslewood,
G. A. D. The Biological Importance of Bile Salts; North-Holland Publishing
Co.: Amsterdam, 1978.
(4) Hofmann, A. F.; Schteingart, C. D.; Hagey, L. R. In Bile Acids and
LiVer Diseases (International Falk Workshop); Paumgartner, G., Beuers,
U., Eds.; Kluwer Academic Publishers: Boston, 1995; pp 3-30.
(5) Hagey, L. R.; Schteingart, C. D.; Ton-Nu, H.-T.; Hofmann, A. F. J.
Lipid Res. 2002, 43, 685 and references therein.
(6) Iida, T.; Hikosaka, M.; Kakiyama, G.; Shirashi, K.; Schteingart, C.
D.; Hagey, L. R.; Ton-Nu, H.-T.; Hofmann, A. F.; Mano, N.; Goto, J.;
Nambara, T. Chem. Pharm. Bull, 2002, 50, 1327.
10.1021/ol036073f CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/05/2003

we report a facile synthesis of avicholic acid from readily
available chenodeoxycholic acid in about 9% overall yield
(Scheme 1). The aggregation behavior and the equilibrium
cholesterol solubilizing ability of avicholate in aqueous media
are also reported.
that the calculated distance from C-7 oxygen to the attached
chlorine in 2 (the reactive species for the template directed
chlorination) is 4.96 Å, and the C-7 oxygen to C-17 hydrogen
distance is 4.91 Å (Figure 2).8
Scheme 1. Synthesis of 10 from 3a
a (a) (i) MeOH/HCl, rt, 10 h, (ii) MeCOCl/pyridine/CH2Cl2, rt,
5 h (78%); (b) 3-iodobenzoyl chloride/CaH2/BnNEt3+Cl-/PhMe,
reflux, 13 h (50%); (c) PhICl2 (2.5 equiv)/tBuOH (0.3 M)/CH2Cl2/
hν; (d) pyridine, reflux, 14 h (65% from 3); (e) (i) BH3‚THF, (ii)
NaOH-H2O2, (iii) 5% KOH in MeOH, rt, 12 h (63%); (f) CH2Cl2-
H2O (carbonate buffer, pH 8.6) NCS-TEMPO/BnNEt3+Cl-, rt, 4 h
(63%); (g) (i) 5% KOH in MeOH, rt, 13 h, (ii) H3O+ (90%). Overall
yield: 9%.
Figure 2. (A) Perspective representation of the reactive species
2. (B) Optimized structure of 2 using semiempirical AM1 method.
(C) A view from the R-face of 2. Cl, 9-H, 14-H and 17-H are
labeled.
Breslow has pioneered (covalently linked) iodoaryl tem-
plate directed chlorination of cholestanol derivatives.7 Com-
pound 1a and 1b were shown to selectively chlorinate C-9
and C-17, respectively, under radical relay conditions (Figure
1).7 Despite the 5â-configuration, we envisioned that 3-iodo-
benzoate attached at C-7 (e.g., compound 2) would selec-
tively chlorinate C-17. Our modeling studies (AM1) indicated
Chenodeoxycholic acid (3) was converted to ester 5 in
two steps (Scheme 1). Photolysis was performed on 5 (10
mM) in 0.3 M tBuOH7d/dichloromethane (deoxygenated) in
the presence of dichloroiodobenzene to yield 17-Cl-steroid
(6). Regioselective introduction of the ∆16 double bond was
achieved by refluxing 6 in pyridine.7e Differences in the
chemical shift value of angular methyl groups were in
accordance with the literature data for other ∆16-steroids
(Table 1).9
Hydroboration-oxidation of
7 (BH3‚THF, followed
by H2O2/NaOH) yielded side chain reduced product 8
(7) (a) Breslow, R. Acc. Chem. Res. 1980, 13, 170 and references therein.
(b) Breslow, R.; Corcoran, R. J.; Snider, B. B.; Doll, R. J.; Khanna, P. L.;
Kaleya, R. J. Am. Chem. Soc. 1977, 99, 905. (c) White, P.; Breslow, R. J.
Am. Chem. Soc. 1990, 112, 6842. (d) Maitra, U.; Breslow, R. Tetrahedron
Lett. 1986, 27, 3087. (e) Breslow, R.; Maitra, U. Tetrahedron Lett. 1984,
25, 5843. (f) Breslow, R. In Templated Organic Synthesis; Diederich, F.,
Stang, P. J., Eds.; Wiley-VCH: Weinheim, 2000; pp 159-188.
(8) Spartan ′04; Wavefunction, Inc.: Irvine, CA.
Figure 1. Template-directed radical-relay chlorination developed
by Breslow.7
(9) Cragg, G. M. L.; Davey, C. W.; Hall, D. N.; Meakins, G. D.; Richards,
E. E.; Whateley, T. L. J. Chem Soc. C 1966, 1266.
32
Org. Lett., Vol. 6, No. 1, 2004

1
Table 1. Differences in H NMR Chemical Shift Value for
Table 2. 1H NMR Chemical Shift Data for
Methyl-3R,7R,16R-triacetoxy-5â-cholane-24-oate in CDCl3
Steroid 5 and ∆16-Steroid 7 (in CDCl3)
18-CH3
19-CH3
21-CH3
5
7
0.68
0.76
0.99
1.02
0.92
1.01
∆δ
-0.08
-0.03
-0.09
(3R,7R,16R, 24-tetrol), as observed earlier by Iida et al.6
Spectral data of 8 matched with the data published in ref 6,
confirming the site-specific introduction of the hydroxyl
group at the 16R-position. Our next aim was to oxidize the
primary alcohol (24-OH) in the presence of three secondary
alcohols. In the earlier work by Iida et al., the tetrol was
oxidized to the triketo carboxylic acid,6 but the selective
reduction of the oxo groups of the triketo methyl ester did
not lead to satisfactory selectivity in favor of R-OH groups
(yield 16%). Therefore, we felt the need for an efficient
method of oxidation of 8 to the final product. Initially, the
24-OH was selectively protected (trityl), followed by the
protection of the secondary alcohols (acetate) and subsequent
deprotection/oxidation of the CH2OTr under acidic condition
(Jones reagent).10 The final product (10) was then obtained
by cleaving the acetate groups (not shown).
derived from synthetic
avicholic acida
derived from naturally
occurring avicholic acidb
18-CH3
19-CH3
21-CH3
OAc’s
CO2Me
3â-H
0.702
0.928
0.960 (d, 6.6 Hz)
1.992, 2.037, 2.043
3.650
4.591
4.817
4.938 (br t, 6.6 Hz)
0.702
0.928
0.960 (d, 6.5 Hz)
1.997, 2.035, 2.049
3.649
4.591
4.813
4.939 (br t, 6.5 Hz)
7â-H
16â-H
a This work. b Reference 5.
vibronic bands (I3/I1) in the fluorescence spectrum of pyrene
is indicative of the polarity experienced by the probe
solubilized in the micellar aggregates.13 Using this technique,
we have measured the CMC values of dihydroxy (cheno-
deoxycholate, and 7-deoxycholate) and trihydroxy (avicholate
and cholate) bile salts. The CMCs of avicholate and cholate
at pH 9 were found to be ca. 15 mM (Figure 3), whereas
Unsatisfactory overall yield and long reaction times (2 days
for 24-O-tritylation) compelled us to go for an alternative
oxidation methodology. Einhorn et al.11 had successfully
performed the oxidation of a primary diol to the correspond-
ing lactone using TEMPO-mediated N-chlorosuccinimide
oxidation in a biphasic mixture using a phase transfer catalyst
+
such as BnNEt3 Cl-. By employing the same methodology
(with slight variation in the condition; see Scheme 1 and
Supporting Information) a one-step oxidation of 8 to avi-
cholic lactone (9) has been accomplished. Spectral data for
9 matched with the data reported for 3R,7R-dihydroxy-5â-
cholane O-24,16R-lactone.6 Cleavage of the lactone (5%
KOH/MeOH), acidification (cold 1 M HCl), and quick
extraction yielded 10. Longer exposer of 10 to an acidic
aqueous medium always led to partial lactonization. This
facile lactonization of 16R-hydroxy bile acid was also
reported for pythocholic (3R,12R,16R-trihydroxy) acid.12 It
was suggested that the 16-hydroxyl group is in a geo-
metrically favorable position to form an ꢀ-lactone. After the
avicholic acid was obtained, the triacetoxy methyl ester
derivative was prepared to compare with the data published
for the same compound from avicholic acid Hagey et al.
isolated from the avian bile (Table 2).
Figure 3. The ratio of vibronic bands (III/I) of pyrene fluorescence
as a function of bile salt concentration at pH 9 (TRIS buffer) at 25
°C: (O) chenodeoxycholate, (9) 7-deoxycholate, (]) cholate, (b)
avicholate.
Since there was no report on the aggregation behavior of
avicholate in aqueous medium, we decided to study the
micellization of avicholate in aqueous solution. Pyrene was
used as a fluorescent probe to measure the CMC (critical
micellar concentration) of avicholate. The ratio of the two
chenodeoxycholate (3R,7R-dihydroxy) and 7-deoxycholate
(3R,12R-dihydroxy) had CMCs below 5 mM.14 The intro-
duction of the third hydroxyl group increased the aqueous
(10) Matsuoka, K.; Kurosawa, H.; Esumi, Y.; Terunuma, D.; Kuzuhara,
H. Carbohydr. Res. 2000, 329, 765.
(11) Einhorn, J.; Einhorn, C.; Ratajczak, F. Pierre, J. L. J. Org. Chem.
1996, 61, 7452.
(12) (a) Haslewood, G. A. D.; Wootton, V. Biochem. J. 1950, 47, 584.
(b) Haslewood, G. A. D.; Wootton, V. Biochem. J. 1951, 49, 67.
(13) Kalyansundaram. K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99,
2039.
(14) (a) Coello, A.; Meijide, F.; Nu´n˜ez, E. R.; Tato, J. V. J. Pharm. Sci.
1996, 85, 9. (b) Gouin, S.; Zhu, X. X. Langmuir 1998, 14, 4025.
Org. Lett., Vol. 6, No. 1, 2004
33

solubility of the bile salt. Hence, CMC was higher for
avicholate, as observed for cholate.14
In conclusion, we have developed a good synthetic route
for chemical synthesis of avicholic acid. To the best of our
knowledge this is the first demonstration of “Breslow remote
funtionalization” on a bile acid backbone. This strategy may
hold the promise to prepare other natural/unnatural bile acids
using different combinations of substrates and templates. For
the first time, we have studied the properties of avicholate
in aqueous solutions. Our data show the similarity among
avicholate and cholate in aqueous media. It would be useful
to take up further studies on this unusual and rare bile acid.
Because the cholesterol solubilization ability is one of the
significant properties of bile salts,15 cholesterol solubilization
ability of avicholate was also evaluated. For this study, a
mixture of bile salt and solid anhydrous cholesterol was
stirred at 37 °C for 1 day. After filtration, the solubilized
cholesterol was assayed using a commercially available
enzymatic assay (Supporting Information). Maximum aque-
ous solubility of cholesterol was found to be much lower
with avicholate (0.8 mM, bile salt:cholesterol ∼63:1) com-
pared to that in chenodeoxycholate (2.5 mM, bile salt:
cholesterol ∼20:1) in 50 mM bile salt concentration at pH
10. This is not surprising because avicholate, with three
hydroxyl groups, is less hydrophobic compared to cheno-
deoxycholate (which was also clear from the CMC data).
Our data are consistent with the hypothesis that the
more hydrophilic bile salts are poorer solubilizers of
cholesterol.15
Acknowledgment. We thank JNCASR, Bangalore and
IFCPAR, New Delhi for financial assistance, Prof. Alan
Hofmann (at UCSD, San Diego) for sending us several
reprints, and Prof. Scott Rychnovsky (UC, Irvine) for
discussions.
Supporting Information Available: Synthetic proce-
dures, characterization data, details of the calculation, and
materials and methods for the determination of CMC and
cholesterol solubilization. This material is available free of
charge via the Internet at http://pubs.acs.org.
(15) (a) Carey, M. C.; Montet, J.-C.; Phillips, M. C.; Armstrong, M. J.;
Mazer, N. A. Biochemistry 1981, 20, 3637. (b) Matsuoka, K.; Kuranaga,
Y.; Moroi, Y. Biochim. Biophys. Acta. 2002, 1580, 200.
OL036073F
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Org. Lett., Vol. 6, No. 1, 2004