A convenient synthesis of dinorbile acids: Oxidative hydrolysis of norbile acid nitriles

Article abstract of DOI:10.1016/S0039-128X(99)00064-1

We report a convenient method for the synthesis of dinorbile acids (23,24-dinor-5β-cholan-22-oic acids, pregnane-20-carboxylic acids) in fair to good yields from norbile acid nitriles in one step by oxidative hydrolysis with oxygen in the presence of potassium-t-butoxide. The method results in stepwise overall removal of two carbon atoms in bile acid side chains in two steps. Dinorbile acids corresponding to several common bile acids have been prepared and their structures confirmed by spectroscopic methods. This simple method for synthesis of dinorbile acids may facilitate their study metabolically.

Full text of DOI:10.1016/S0039-128X(99)00064-1

Steroids 64 (1999) 780–784
A convenient synthesis of dinorbile acids:
Oxidative hydrolysis of norbile acid nitriles૾
Ashok K. Battaa,*, Subhash C. Dattaa, G. Stephen Tinta,b, David S. Albertsc,
David L. Earnestc, Gerald Salena,b
aDepartment of Medicine, University of Medicine and Dentistry-New Jersey Medical School, Newark, NJ 07103, USA
bDepartment of Medicine, GI Section (15A), Veterans Administration Medical Center, 185 Tremont Avenue, East Orange, NJ 07019, USA
cDepartment of Medicine, University of Arizona, Tuscon, AZ 85727, USA
Received 5 April 1999; accepted 5 May 1999
Abstract
We report a convenient method for the synthesis of dinorbile acids (23,24-dinor-5␤-cholan-22-oic acids, pregnane-20-carboxylic acids)
in fair to good yields from norbile acid nitriles in one step by oxidative hydrolysis with oxygen in the presence of potassium-t-butoxide.
The method results in stepwise overall removal of two carbon atoms in bile acid side chains in two steps. Dinorbile acids corresponding
to several common bile acids have been prepared and their structures confirmed by spectroscopic methods. This simple method for synthesis
of dinorbile acids may facilitate their study metabolically. © 1999 Elsevier Science Inc. All rights reserved.
Keywords: Dinorbile acids; Norbile acids; Bile acids; Synthesis; Nuclear magnetic resonance; Mass spectrometry
1. Introduction
cholic acid has been shown to be secreted into bile in the
unconjugated form. Dinorbile acids have not been detected
in bile from healthy humans, although dinorcholic acid has
been shown to be present in unconjugated form in bile from
patients with cerebrotendinous xanthomatosis [4] and dinot-
lithocholic acid was reported in human meconium [5] and
serum [6]. Dinorcholic acid, and its 7- and 12-oxo and 1␤-
and 2␤-hydroxy derivatives, were all detected in small
amounts in the urine of patients with cerebrotendinous xan-
thomatosis [7]. Dinorbile acids are also formed by microbial
degradation of bile acids [8,9]. Yeh et al. [3] have shown
that dinorchenodeoxycholic acid is metabolized in bile fis-
tula rat or hamster similarly to norchenodeoxycholic acid (a
greater proportion of unconjugated dinorbile acid was se-
creted into the bile than the norbile acid), but quite differ-
ently from chenodeoxycholic acid. In the view of renewed
interest in dinorbile acids and to learn more about their
metabolism, we needed to have a convenient method for the
synthesis of these short-chain bile acid derivatives.
Bile acids (hydroxy derivatives of 5␤-cholan-24-oic acid
[1]) are the end products of cholesterol biosynthesis in the
liver and play a major role in cholesterol and fat absorption.
Although the naturally occurring C24 bile acids are conju-
gated with glycine and taurine by hepatic enzymes and are
well absorbed from the intestine, the short-chain C23 and
C22 bile acids are choleretic in animals and are poorly
conjugated and largely excreted into the urine [1–3]. Be-
cause hepatocyte bile acid concentrations are abnormally
high in cholestatic liver disease, bile acids that cause
choleresis may be potentially useful in patients with cho-
lestasis. Norcholic acid and norchenodeoxycholic acid have
been shown to be highly choleretic, but they do not accu-
mulate in the bile. On the other hand, dinorchenodeoxy-
* Corresponding author. Tel.: ϩ1-201-676-1000/2289; fax: ϩ1-201-
676-2991.
Dinorbile acids have been synthesized by the Barbier–
Wieland degradation [10] or oxidation [11] of 24-nor-5␤-
cholan-22-enes obtainable by lead tetraacetate oxidation of
the C24 bile acids [12,13] or of the noraldehydes [14].
However, the methods involve several steps and the inter-
૾ This work was supported by U.S. Public Health Service Grant HL-
17818, a grant from the Veterans Affairs Research Service, Washington,
D.C., and National Cancer Institute Grant PO1-CA-41108. The contents of
this publication are solely the responsibility of the authors and do not
necessarily represent the official views of the National Cancer Institute.
0039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(99)00064-1

A.K. Batta et al. / Steroids 64 (1999) 780–784
781
10% sulfuric acid and subsequent heating at 110°C for 2
min.
2.3. NMR spectroscopy
The proton NMR spectra of the dinorbile acids were
obtained at 200 MHz in deutereochloroform on a Varian
Associates XL-200 spectrometer (Palo Alto, CA, USA) and
tetramethylsilane was used as the internal standard.
2.4. GLC
A Hewlett–Packard model 5890A gas chromatograph
(Palo Alto, CA, USA) equipped with a flame ionization
detector and an injector with a split/splitless device for
capillary columns was used for GLC. The chromatographic
column consisted of a chemically bonded fused silica CP-
Sil-5 CB (stationary phase, 100% dimethylsiloxane) capil-
lary column (20 m ϫ 0.22 mm inside diameter; Chrompack,
Inc., Raritan, NJ, USA) and helium was used as the carrier
gas at a flow rate of 1 ml/min. The GLC operating condi-
tions were as follows. Injector and detector temperatures
were 260°C and 290°C, respectively. After injection, oven
temperature was kept at 100°C for 2 min, then programmed
at a rate of 20°C/min to a final temperature of 260°C.
Aliquots of dinorbile acids (10–20 ␮g) were treated with
3% anhydrous methanolic hydrochloric acid to obtain the
methyl esters, which were then reacted with 100 ␮l of
Sil-Prep for 20 min at 55°C. Solvents were evaporated at
55°C under N2 and the TMS ether derivative formed was
taken in 100 ␮l of hexane. One to 2 ␮l was injected into the
GLC column simultaneously with nordeoxycholic acid used
as the internal standard.
Fig. 1. Structures of dinorbile acids.
mediate 24-nor-5␤-cholan-22-enes are obtained in rela-
tively low yields. Schteingart and Hofmann have reported
the synthesis of 24-nor-23-nitriles of bile acids in Ͼ90%
yields [15] via ␣-nitrosation-fragmentation with sodium ni-
trite in trifluoroacetic acid/trifluoroacetic anhydride [16].
We report a convenient synthesis of dinorbile acids (Fig. 1,
1a–e) by oxidative hydrolysis of the 24-nor-23-nitriles of
bile acids, and thus, the synthesis of C22 bile acids in two
steps from C24 bile acids.
RCH2CH2COOH 3 RCH2CN 3 RCOOH.
2. Experimental
Ursodeoxycholic acid was a gift from Tokyo Tanabe,
Tokyo, Japan. All other bile acids were purchased from
Aldrich (Milwaukee, WI, USA). Sil-Prep (hexamethyldisi-
lazane:trimethylchlorosilane:pyridine, 3:1:9) used for mak-
ing trimethylsilyl ether derivatives was purchased from All-
tech Associates, Inc. (Deerfield, IL, USA). All reagents and
solvents used were reagent grade and were purchased from
Aldrich.
2.5. GC-MS
Mass spectra of the dinorbile acids were obtained on a
Hewlett–Packard model 5988 gas chromatograph–mass
spectrometer (Palo Alto, CA, USA) by using a 25-m
CP-Sil-5 CB capillary column [17].
2.1. Methods
2.6. Synthesis of dinorursodeoxycholic acid (1d)
The elemental analysis of the synthesized compounds
was performed at the Spang Microanalytical Laboratory
(Eagle Harbor, MI, USA). Melting points were determined
on a Thermolyne 12 000 apparatus (Dubuque, Iowa, USA)
and are uncorrected.
The nornitrile (Fig. 2, 3; 2 g) synthesized from ursode-
oxycholic acid [2], according to the method of Schteingart
and Hofmann [15], was dissolved in anhydrous tetrahydro-
furan (20 ml) and cooled at 0°C. Potassium t-butoxide (1 g)
was added and the resulting solution was stirred for 30 min
at 0°C. 18-Crown-6 ether (1 g) was then added to the
reaction mixture and a slow stream of oxygen was passed
while the temperature was allowed to rise to room temper-
ature [18]. After stirring for 18 h in oxygen atmosphere,
ethyl acetate (100 ml) was added and the contents were
extracted with water (4 ϫ 20 ml). The combined water
extract was acidified to pH 1 and the liberated dinorursode-
oxycholic acid was extracted with ethyl acetate (4 ϫ 50 ml),
2.2. TLC
TLC of the dinorbile acids was performed on precoated
silica-gel plates (0.25 mm thickness; Analabs, New Haven,
CT, USA). Plates were developed in a solvent system of
chloroform/methanol (90:10 v/v). After development, spots
were visualized by spraying the plates with phosphomolyb-
dic acid (3.5% in isopropanol) followed by a spray with

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A.K. Batta et al. / Steroids 64 (1999) 780–784
bile acids via ␣-nitrosation fragmentation with sodium ni-
trite in trifluoroacetic acid/trifluoroacetic anhydride [16].
Alkaline hydrolysis of the nornitrile then produces the nor-
bile acid in very good yields and the method may be a
preferred alternative to Barbier–Wieland degradation for
synthesis of dinorbile acids. However, nitriles are highly
resistant to alkaline hydrolysis and 24-nor-23-nitriles of bile
acids are hydrolyzed with 10% sodium hydroxide at reflux
temperature for 96 h—conditions that result in partial dis-
solution of silicates from the glass reaction vessel [15]. We
considered the penultimate oxidative hydrolysis of norni-
triles as a means of converting the nitrile into carboxyl
group under milder conditions [18] and thereby providing
us with a method to synthesize a C22 bile acid in two steps
from a C24 bile acid. The reaction would involve creation of
a carbanion ␣- to the nitrile, followed by oxidative hydro-
lysis with O2 in presence of a strong base. Treatment of the
24-nor-23-nitrile with a slight excess of lithium diisopro-
pylamide at low temperatures (Ϫ20°C) in oxygen atmo-
sphere and in the presence of 18-crown-6 ether for 4 h
produced the required dinorbile acid, albeit in only 10–15%
yield, and the starting nor-nitrile was largely recovered.
Prolonged reaction time (up to 48 h) did not significantly
improve the yield. In an earlier publication, DiBiase et al.
[18] have reported that treatment of nitriles from several
long-chain aliphatic acids with potassium-t-butoxide in the
presence of 18-crown-6 ether at 65°C in oxygen atmosphere
resulted in 60–90% yields of the lower homologs. We used
this method to make dinorbile acids from bile acid norni-
triles and obtained 33–75% yield of the products. The iso-
lation procedure was simple; the unreacted nornitrile could
be removed by simple solvent extraction of the basic aque-
ous reaction product and the dinor bile acid was then iso-
lated after acidification of the aqueous layer, followed by
direct crystallization or a simple column chromatography. A
typical procedure for the oxidative hydrolysis is described
in the experimental section for the synthesis of dinorursode-
oxycholic acid. Apparently, the addition of oxygen to the
generated anion or the hydrolysis of the acyl nitrile (A,
below) requires more rigorous conditions [18]; thus, lithium
Fig. 2. Synthesis of dinorursodeoxycholic acid. a, Trifluoroacetic acid/
trifluoroacetic anhydride/sodium nitrite on diformate. b, Potassium t-bu-
toxide/18-crown-6 ether/O2.
the ethyl acetate extract was washed with water to neutral-
ity, dried over anhydrous sodium sulfate, and concentrated
to a small volume. On keeping overnight in a refrigerator,
colorless microscopic crystals of dinorursodeoxycholic acid
(1d) were separated (yield, 1.05 gm); crystals from acetone,
melting point, 177–180°C; TLC, Rf, 0.45 (Table 1); GLC
retention time of the methyl ester-TMS ether derivative,
19.333 min (Table 1). Found: C, 72.47; H, 9.92%; calcu-
lated for C22H36O4: C, 72.53; H, 9.89. The proton signals in
1
the H-NMR spectrum of the methyl ester and the major
mass ion fragments in the mass spectrum of the methyl
ester-TMS ether derivatives are given in Tables 2 and 3.
Silica gel column chromatography of the mother liquor after
filtration of 1d yielded another 310 mg of the compound
(total yield, 68% from 2).
3. Results and discussion
The recently reported synthesis of norbile acids [15]
involves the formation of 24-nor-23-nitriles from the C24
Table 1
Physical characteristics of dinorbile acids
Dinorbile acid
m.p.
TLC Rf
GLC rt (min)
C,H analysis
Found
Calculated
Dinorlithocholic
Dinordeoxycholic
Dinorcholic
207–209°C
(Lit. 210°C [21])
236–239°C
(Lit. 238°C [22])
285–288°C
(Lit. 285–286°C [23])
177–180°C
0.79
0.42
0.19
0.45
0.20
16.342
17.483
18.206
19.333
19.806
C, 75.80%
H, 10.31%
C, 72.59%
H, 9.82%
C, 69.56%
H, 9.43%
C, 72.47%
H, 9.92%
C, 69.41%
H, 9.41%
C, 75.86%
H, 10.34%
C, 72.53%
H, 9.89%
C, 69.47%
H, 9.47%
C, 72.53%
H, 9.89%
C, 69.47%
H, 9.47%
Dinorursodeoxycholic
Dinorursocholic
217–218°C

A.K. Batta et al. / Steroids 64 (1999) 780–784
783
Table 2
Proton signals in the 1H-NMR spectra of the methyl esters of dinorbile acids
Dinorbile acid
1H signal
CH3
CHOH
C-3
COOCH3
C-18
C-19
C-21
C-7
C-12
Dinorlithocholic
Dinordeoxycholic
Dinorcholic
0.674
(s)
0.687
(s)
0.677
(s)
0.686
(s)
0.925
(s)
0.910
(s)
0.878
(s)
0.949
(s)
1.231
(d, J ϭ 6.8 Hz)
1.228
(d, J ϭ 6.8 Hz)
1.232
(d, J ϭ 6.8 Hz)
1.190
(d, J ϭ 6.8 Hz)
1.244
(d, J ϭ 6.8 Hz)
3.630
(m)
3.630
(m)
3.480
(m)
3.620
(m)
3.647
(s)
3.650
(s)
3.643
(s)
3.648
(s)
3.920
(bs)
3.820
(bs)
3.620
(m)
3.620
(m)
3.920
(bs)
Dinorursodeoxycholic
Dinorursocholic
0.717
(s)
0.941
(s)
3.620
(m)
3.920
(bs)
3.658
(s)
diisopropylamide at the low temperatures yielded poor
yields of the dinorbile acids.
tained 20–30% of norcholic acid in addition to dinor-
cholic acid, thus suggesting competing hydrolysis of the
nornitrile at higher temperatures.
RCH2CN 3 RCHϪCN 3
The structures of the dinorbile acids were confirmed by
1
RCH(OOϪ)CN 3 RCOCN 3 RCOOH(A)
their H-NMR and MS analyses. The proton signals in the
1H-NMR spectra of the methyl esters of compounds 1a–e
are given in Table 2. As expected, both C-18 and C-19
proton signals were not influenced by the length of the side
chain and appeared at approximately the same positions, as
in case of the corresponding C23 and C24 bile acids [3,19].
Thus, the C-18 proton signals appeared as a singlet at
␦0.674–0.717 ppm in all spectra, and the C-19 protons
appeared as singlet at ␦0.878–0.949 ppm. However, the
shortened side chain showed a strong deshielding effect of
approximately ␦0.2 ppm on the C-21 proton signals, which
appeared as a doublet (J ϭ 6.8 Hz) at ␦1.190–1.244 ppm in
the spectra of these compounds [8]. The mass spectral frag-
mentation pattern of the TMS ether methyl esters of the dinor-
bile acids [4,6,8] was essentially identical to that reported for
the corresponding C24 bile acids [20] and norbile acids [15]
with the ion fragments with intact side chains being 28 and 14
mass units less, respectively. The relative intensities of the ion
fragments varied sometimes; e.g. in the mass spectrum of
dinordeoxycholic acid derivative, the ion fragment at m/z 180
The dinorbile acids, 1a and 1d (Fig. 1), were isolated in
pure form after crystallization from acetone, whereas the
compounds 1b, 1c, and 1e needed initial purification by
column chromatography. Care was taken in the isolation of
the more water-soluble dinorcholic acid (1c) and, in partic-
ular, dinorursocholic acid (1e) to exhaustively extract these
compounds from the aqueous solutions after acidification.
Further, whereas the 23-nornitriles of ursodeoxycholic acid
and lithocholic acid yielded 68% and 73% of the corre-
sponding dinorbile acids, the nornitriles from cholic acid,
deoxycholic acid, and ursocholic acid, the 12␣-hydroxy-
lated bile acids, produced the dinor acids in 33%, 40%,
and 50% yields, respectively, even when the reaction was
performed at 65°C [18], suggesting steric hindrance
caused by the 12␣-hydroxyl group. Elevated tempera-
tures resulted in undesirable side products. Thus, during
the preparation of dinorcholic acid, 20–25% of norcholic
acid amide was formed in the neutral fraction in addition
to the unreacted nornitrile, and the acidic fraction con-
Table 3
Major ion fragments in the mass spectra of methyl ester-trimethylsilyl ester derivatives of dinorbile acidsa
Dinorbile acid
Ion fragment (m/z)
Dinorlithocholic
Dinordeoxycholic
434 (0.1), 419 (8), 344 (35), 329 (20), 257 (12), 215 (34), 75 (100)
522 (0.1), 507 (38), 432 (1), 342 (10), 327 (6), 255 (60), 180 (63),
73 (100)
Dinorcholic
595 (7), 505 (1), 430 (13), 415 (3), 340 (13), 325 (5), 281 (6), 253
(37), 73 (100)
Dinorursodeoxycholic
Dinorursocholic
522 (0.1), 507 (4), 432 (32), 342 (12), 327 (11), 283 (4), 255 (10),
73 (100)
595 (9), 520 (9), 433 (5), 430 (7), 343 (10), 340 (12), 281 (3), 253
(45), 73 (100)
a Major on fragments above m/z 200 are reported.

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In summary, we have described a method for the synthesis
of dinorbile acids from bile acids via penultimate oxidative
hydrolysis of norbile acid nitriles. The reaction involves mild
hydrolysis conditions and good yields are obtained for dinor-
bile acids without a 12␣-hydroxyl group. The 12␣-hydroxyl
group interferes with the oxidative hydrolysis with competing
hydrolysis of the nornitrile. It is hoped that this facile synthesis
of dinorbile acids will generate a renewed interest in their
metabolism and physiological properties.
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