Synthesis of 1β-hydroxydeoxycholic acid in H-2 and unlabeled forms

Article abstract of DOI:10.1002/jlcr.3495

1β–hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for use as a biomarker for Cytochrome P450 3A4/5 activity. A lengthy synthesis was undertaken to deliver the unlabeled compound and in the process, to develop a route to the deuterium labeled compound. The synthesis of the unlabeled compound was completed but in a very low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and this approach proved to be much more rapid and afforded the compound in both unlabeled and deuterium labeled forms in a 1-step oxidation from deoxycholic acid and [D₄]deoxycholic acid, respectively.

Full text of DOI:10.1002/jlcr.3495

Synthesis of 1β-hydroxydeoxycholic acid in H-2 and unlabeled forms
Martin A. Hayes,1 Ieuan Roberts,4 Gunnar Grönberg,2 Kexin Lv,3 Baorui Lin,3 Jonas
Bergare,5 and Charles S. Elmore5,*
1Cardiovascular and Metabolic Disease Innovative Medicines and Early Development
Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden
2Respiratory, Inflammation & Autoimmunity, Innovative Medicines and Early Development
Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden
3WuXi AppTec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
China
4Discovery Sciences, Innovative Medicines and Early Development Biotech Unit,
AstraZeneca, Cambridge Science Park, Cambridgeshire, CB4 0WG, UK
5Drug Safety & Metabolism, Innovative Medicines and Early Development Biotech Unit,
AstraZeneca, Pepparedsleden 1, Mölndal, 431 83, Sweden
Abstract
1β–hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for
use as a biomarker for Cytochrome P450 3A4/5. A lengthy synthesis was undertaken to
deliver the unlabeled compound and in the process, to develop a route to the deuterium
labeled compound. The synthesis of the unlabeled compound was completed, but in very
low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and
this approach proved to be much more rapid and afforded the compound in both unlabeled
and deuterium labeled forms in a one-step oxidation from deoxycholic acid and
[D4]deoxycholic acid respectively.
Introduction
Bile acids are steroids which are the major organic components of bile. In humans, cholic
acid and chenodeoxycholic acid are primary bile acids (figure 1); they are synthesized in the
liver from cholesterol in a multistep process.1 These compounds are conjugated to taurine
or glycine prior to secretion into the intestine and after conjugation are termed bile salts.2
Intestinal bacteria can then further metabolize these bile salts. One key pathway consists of
the hydrolysis of the bile salt to the bile acid and then dehydroxylation of the 7-hydroxyl of
cholic acid or chenodeoxycholic acid to give deoxycholic acid (DCA) or lithocholic acid
respectively.3 Bile acids and salts can be reabsorbed into the blood stream by enterohepatic
circulation and can undergo further biotransformation in the liver to give a plethora of
products among which are hydroxylated versions of DCA.1,4
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doi: 10.1002/jlcr.3495
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The formation of 1β –hydroxydeoxycholic acid was recently reported to be a biomarker for
cytochrome P-450 3A4 activity in vivo.5,6 To further develop this as a potential clinical
biomarker, both unlabeled and stable isotope labeled versions of the compound were
required.7,8
Results
The synthesis of 1β-hydroxydeoxycholic acid (1) in unlabeled form was reported
previously,7,8 and we did not anticipate any significant problems in replicating the reported
procedures (Scheme 1). Beta-hydroxy ketone 2 was proposed as a key intermediate to serve
as the starting material for the incorporation of the deuterium labels via a base catalyzed
exchange reaction. The protons on the carbon alpha to the ester could also potentially
exchange which was not viewed as problematic; however, the beta-ketoalcohol of 2 could
potentially eliminate to give enone 3 under the proposed exchange conditions as was
observed previously for a similar substrate (Scheme 2).9 Therefore, this reaction needed to
be probed as soon as possible. Assuming the elimination was minimal, once the deuterated
alcohol was obtained, it could then be converted to the final compound using the same
procedure used for the unlabeled compound.
The synthesis started from the readily available DCA, and the first four steps – esterification
of the acid, oxidation of the C-3 hydroxyl to the ketone, acetylation of the C-12 hydroxyl, and
dibromination – rapidly delivered dibromoketone 4 for use in the Cr(OAc)2 mediated
debromination (see Scheme 3).10 Attemps to replicate the Cr(OAc)2 debromination failed to
give the target compound under a variety of conditions. Therefore, an alternate approach
was used in which the C-2 bromide was first eliminated with Li2CO3 to give
monobromoketone 5 followed by debromination with Zn to afford enone 6.11 Epoxidation
of 6 under basic conditions provided epoxide 7,8,11 but when 7 was reacted with Cr(OAc)2 in
EtOH, none of the target compound was observed under a variety of conditions.12 It has
been reported that Cr(OAc)2 is sensitive to oxygen;13 it is likely that the quality of the reagent
used in the debromination of 4 and in the epoxide opening of 6 was not sufficient to catalyze
these transformations since these reactions are well precedented.
Therefore, alternate epoxide opening conditions were investigated (Scheme 4). LiBr
opening of epoxide 7 followed by Pd-catalyzed hydrogenation gave a mixture of products
which were difficult to separate.14,15 SmI2 mediated epoxide ring opening gave a poor yield of
a low purity compound.16 Reduction with Na[PhSeB(OEt)3] in EtOH afforded the target
compound in 60% yield and good purity by NMR.17 This later route appeared promising,
but while this development work was proceeding, enough alcohol had been obtained from
these probe reactions to investigate the key step for deuterium incorporation.
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The reaction was probed with unlabeled materials first to simplify the analysis and under
several reaction conditions (LiOH or NaOH, room temp or 100 °C) complete elimination to
give the enone and either full or partial acetate hydrolysis (Scheme 5) was observed. This
result necessitated a redesign of the proposed route to the labeled compound.
A recent report used a silyl group as a surrogate for a hydroxy group in a similar compound;14
therefore, enone 6 was reacted with the cuprate formed from Me2PhSiCl to give 10 after
conjugate addition (Scheme 6). The silane was converted to alcohol 8 using a
Fleming–Tamao oxidation. Reduction of ketone 8 to alcohol 11 (as a mixture of
diastereomeric alcohols) and subsequent hydrolysis of the esters and purification by silica gel
chromatography gave an alcohol as the major product in 46% yield for the two steps. The
modest yield reflects the difficulty of the separation of the diastereotopic alcohols 1 and 12.
Unfortunately, on carefully analysis by NMR spectroscopy, the isolated alcohol was
determined to be the incorrect diastereomer, alcohol 12. A second preparation was
performed in which the diastereomeric alcohols were separated to give 13 in a 20% yield.
However, the final purification again proved difficult, and the final compound (1) was
isolated in only a 14% yield. A detailed NMR analysis was used to determine the structures
and is detailed in the supplementary material and summarized in the table.
Insert table and caption here.
The use of enzymes for the functionalization of inert C-H bonds has recently been reviewed.18
During this work, parallel efforts were ongoing to find a biocatalyst which converted DCA to
1 (Scheme 7).6 A library of commercially available Bacillus megaterium P450 mutants
from Codexis was investigated for their ability to convert DCA to 1 in a selective manner and
in high conversion. These results were compared with a human liver microsomal incubation
of DCA which was thought to produce 1 as the major mono-oxygenated metabolite.19 The
comparison allowed the selection of an engineered P450, MicroCYP0029, as a suitable
biocatalyst. A scale up reaction was performed according to the vendors protocol and 1 was
formed from DCA as the sole major product and was isolated in 27% chemical yield (10.5
mg from 39 mg) by mass directed preparative HPLC. The use of [D4]DCA afforded [D4]-1
with a high, uniform, isotopic incorporation (>95% D4 which reflected the isotopic
distribution of the starting material) in 33% yield (13 mg from 39 mg). This biosynthetic
route was competitively priced to the synthetic route so work was halted on the purely
synthetic route. For a small scale preparation (<100mg), it was clear the enzymatic method
was superior in price and speed. For larger amounts, a close comparison of the costs of the
two routes would be required to discern the less expensive option. Although not
investigated, it is likely that a purely synthetic method would provide a much less
homogenous isotopomer pattern than that of the enzymatic method.
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Experimental:
General: All solvents and reagents were obtained from Aldrich or Alfa or other local
sources and were used without further purification except for anhydrous solvents which were
pre-treated by passing through a 3A MS column before use.20 [D4]Deoxycholic acid was
obtained from C/D/N. Chromatographic purifications were conducted using flash
chromatography on 100~200 mesh silica gel.
LC/MS were acquired on a Shimadzu LC/MS-2010 (Column: Sepax ODS 50×2.0 mm, 5 um)
or Agilent 1200 HPLC-1956 MSD (Column: Shim-pack XR-ODS 30×3.0 mm，2.2 um). 1H
NMR spectra were recorded on Bruker Avance 400 MHz for 1H NMR or Bruker Avance 600
1
MHz instruments for H, 13C and 2D-experiments. Samples were dissolved in CDCl3,
1
DMSO-d6 or Methanol-d4. NMR experiments were recorded at 25 degrees C. H, 13C
chemical shifts were referenced via the residual solvent signal, set to 7.27/77.0 (CDCl3),
2.5/39.5 (DMSO-d6) or 3.31/49.0 ppm (Methanol-d4). 1, 12 and DCA were assigned using
COSY, ROESY, CH correlation and CH long-range correlation experiments using standard
Bruker pulse sequences. Methyl Deoxycholate, Methyl 3-oxo-5β-12ɑ -hydroxy-cholanoate,
Methyl 3-oxo-5β -12ɑ -acetoxy-cholanoate, 4, 6, 7, and 8 were characterized by 1H NMR and
LC/MS, and the spectra were consistant with previously reported data.8,21
(R)-methyl
4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanoate (Methyl Deoxycholate)
The procedure of Májer was modified as follows.22 A mixture of deoxycholic acid (50.0 g,
130 mmol) in 4 M HCl in MeOH (2 mol, 500 mL) was stirred at 20 °C for 12 h at which time
TLC indicated the reaction to be complete; therefore, the solvent was evaporated. The
resulting crude material was dissolved in EtOAc (300 mL), and the solution was washed with
sat. aq. NaHCO3 (3x100 mL) and brine (2x100 mL), and was then dried over MgSO4. After
filtering, the solution was concentrated to give methyl deoxycholate (45.0 g, 111 mmol, 85%)
as a colorless oil.
(R)-methyl
4-((5R,8R,9S,10S,12S,13R,14S,17R)-12-hydroxy-10,13-dimethyl-3-oxohexadecahydro-1H-c
yclopenta[a]phenanthren-17-yl)pentanoate (Methyl 3-oxo-5β-12ɑ -hydroxy-cholanoate)
A modification of the procedure of Aher was used.23 A flask containing a suspension of
methyl deoxycholate (25.0 g, 61.5 mmol) and Ag2CO3 (35 g, 170 mmol) in toluene (300 mL)
was fitted with a Dean-Stark apparatus, and the solution heated at reflux for 48 hrs at which
time analysis by TLC showed the reaction to be complete. The mixture was filtered through a
pad of celite, and the celite pad was washed with CH2Cl2 (3x500 mL). The combined
organic layers were concentrated under reduced pressure to give a residue which was purified
by silica gel chromatography (3:1, pet ether:EtOAc) to give methyl 3-oxo-5β -12ɑ -acetoxy-
cholanoate (21.0 g, 51.9 mmol, 84% yield) as a yellow oil.
(R)-methyl
4-((5R,8R,9S,10S,12S,13R,14S,17R)-12-acetoxy-10,13-dimethyl-3-oxohexadecahydro-1H-c
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yclopenta[a]phenanthren-17-yl)pentanoate (Methyl 3-oxo-5β-12ɑ -acetoxy-cholanoate)
A modification of the procedure of Bonar-Law was used.24 A mixture of methyl
3-oxo-5β-12ɑ -hydroxy-cholanoate (50.0 g, 124 mmol), Ac2O (125 mL, 1.33 mol),
dimethylaminopyridine (1.5 g, 12 mmol) and pyridine (500 mL) was stirred for 1 hr at 20 °C
at which time analysis by TLC showed the reaction to be complete. The solvent was
evaporated, and the crude residue was taken up in EtOAc (800 mL). The solution was
washed with 1M HCl (2x300 mL), sat. aq. NaHCO3 (2x300 mL), and brine (2x300 mL) and
was dried over MgSO4. After filtration, the organic layer was concentrated to dryness to
give methyl 3-oxo-5β-12ɑ -acetoxy-cholanoate (42.0 g, 94.0 mmol, 76% yield) as a yellow
solid.
(R)-methyl
4-((2R,4S,5S,8R,9S,10R,12S,13R,14S,17R)-12-acetoxy-2,4-dibromo-10,13-dimethyl-3-oxoh
exadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (4)
A modification of the procedure of Tohma was used.8
A solution of methyl
3-oxo-5β-12ɑ -acetoxy-cholanoate (44.0 g, 98.5 mmol) in HOAc (160 mL) and CHCl3 (80
mL) was stirred as Br2 (30.7 g, 192 mmol) was added, and the resulting reaction mixture was
stirred for 1 hr at 20 °C at which time assay by LC/MS showed the reaction to be
complete. The reaction was diluted with sat aq NaHCO3 (300 mL), and the resulting biphasic
mixture was extracted with EtOAc (2x300 mL). The combined organic layers were washed
with sat aq NaHCO3 and dried over Na2SO4. After filtration, the solution was concentrated
to dryness under reduced pressure. The resulting residue was washed three times with
MeOH to give compound 4 (38.0 g, 62.8 mmol, 64% yield) as an off-white solid (>90% UV
purity at 220 nm by HPLC).
(R)-methyl
4-((4S,5S,8R,9S,10R,12S,13R,14S,17R)-12-acetoxy-4-bromo-10,13-dimethyl-3-oxo-4,5,6,7,
8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-17-yl)pentanoate
(5)
A modification of the procedure of Joly was used.11 A solution of compound 4 (49.0 g, 81.1
mmol) in DMF (400 mL) was stirred as Li2CO3 (8.8 g, 120 mmol) and LiBr (9.72 g, 112
mmol) were added, and the reaction was warmed to 80 °C for 4 hrs. The reaction mixture was
diluted with EtOAc (500 mL) and washed with 1 N HCl. The combined organic layers were
washed with brine, dried and filtered, and the resulting solution was concentrated to dryness
under reduced pressure to give compound 5 (40.0 g, HPLC purity of approximately 80%, 75%
yield) as a yellow solid, and 5 was used in the next step without further purification.
(R)-methyl
4-((5R,8R,9S,10R,12S,13R,14S,17R)-12-acetoxy-10,13-dimethyl-3-oxo-4,5,6,7,8,9,10,11,12,
13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-17-yl)pentanoate (6)
A modification of the procedure of Tohma was used.8 A solution of compound 5 (40.0 g, 80%
purity by HPLC, 61.1 mmol) in HOAc (300 mL) was stirred as Zn (16.0 g, 245 mmol) was
added, and the reaction was stirred at room temperature for 0.5 h at which time assay by
LC/MS showed the reaction to be complete. The mixture was filtered and concentrated under
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reduced pressure. The resulting residue was purified by column chromatography on silica
gel (80:1, Pet ether:EtOAc) to give compound 6 (22.0 g, 65% yield) as a yellow solid.
(R)-methyl
4-[(1S,2S,5R,8S,9S,10S,12S,13R,14S,17R)-12-acetoxy-1,2-epoxy-10,13-dimethyl-3-oxo-1,2,
4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]pentanoate (7)
A modification of the procedure of Tohma was used.8 A solution of compound 6 (5.00 g,
11.3 mmol) in dioxane (80 mL) was stirred at 0 °C as a solution of 30% aq. H2O2 (20.00 mL,
196 mmol) in 2 N NaOH (10.00 mL) was added dropwise. The solution was stirred for 12 h
at 25 °C at which point TLC showed the reaction to be completed. The reaction mixture was
diluted with brine (100 mL) and was extracted with ethyl acetate (2x150 mL). The
combined organic layers were washed with sat aq Na2S2O3 (2x100 mL) and were then dried
over Na2SO4. After filtering, the solution was concentrated to dryness and then purified by
column chromatography (80:1, CH2Cl2:MeOH) to afford compound 7 (3 g, 57% yield) as a
colorless oil.
(R)-methyl
4-((1R,5R,8S,9S,10S,12S,13R,14S,17R)-12-acetoxy-1-(dimethyl(phenyl)silyl)-10,13-dimeth
yl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (10)
A modification of the procedure of Wolfram was used.14 A solution of Li (780.0 mg, 112.4
mmol) in 50 mL THF was stirred at 0 °C as dimethylphenylsilyl chloride (9.60 g, 56.3 mmol)
was added. After stirring for 6 h at 0 °C, the resulting mixture was added to a suspension of
CuCN (1.6 g, 17.9 mmol) in 50 mL THF at -30 °C, and the slurry was stirred at -30 °C
for 0.5 hr. A solution of 7 (5.0 g, 11.2 mmol) in 50 mL of THF was then added, and the
resulting slurry was stirred at -30 °C for 2 hr. The mixture was diluted with 0.1 N HCl (200
mL) and was extracted with EtOAc (2x300 mL). The combined organic extracts were dried
over Na2SO4 and filtered, and the organic solution was concentrated to dryness under reduced
pressure. The residue was purified by column chromatography on silica gel (8:1 pet
ether:EtOAc) to give silane 10 (4.4 g, 67% yield) as a yellow oil. 1H NMR (400 MHz CDCl3)
δ 7.45-7.50 (m 2H), 7.37 - 7.32 (m, 3H), 5.13 (t, J=2.9 Hz, 1H), 3.66 (s, 3H), 2.64 (dd,
J=17.0, 14.0 Hz, 1H), 2.15 – 2.44 (m, 5H), 2.08 (s, 3H), 1.97-2.05 (m, 2H), 1.74-1.96 (m,
5H), 1.55-1.73 (m, 4H), 1.44-1.54 (m, 3H), 1.24-1.43 (m, 4H),0.8-0.95 (m, 1H), 0.98 (s,
3H), 0.81 (d, J=6.0 Hz, 3H), 0.72 (s, 3H), 0.38 (s, 3H), 0.33 (s, 3H). The spectrum shows
an approximately 1:1.4 molar ratio of 10 to EtOAc.
(R)-methyl
4-((1R,5R,8S,9S,10S,12S,13R,14S,17R)-12-acetoxy-1-hydroxy-10,13-dimethyl-3-oxohexade
cahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (8)
A modification of the procedure of Wolfram was used.14 A solution of 10 (3.0 g, 5.2 mmol)
in AcOH (10 mL) was stirred at room temperature as a solution of Hg(OAc)2 (2.1 g, 6.6
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mmol) in 20% aq AcOOH (5 mL, 5.2 mmol) were added, and the resulting mixture was
stirred for 3h. The mixture was diluted with sat aq NaHSO3 (100 mL) and was extracted
with EtOAc (2x200 mL). The combined organic layers were washed with sat. aq. NaHCO3
and were dried over Na2SO4. After filtration, the solution was concentrated under reduced
pressure, and the resulting residue was purified by column chromatography (4:1 Pet ether;
EtOAc) to give 8 (1.6 g, 67% yield) as a white solid.
(R)-methyl
4-((1R,3R/S,5R,8S,9S,10S,12S,13R,14S,17R)-12-acetoxy-1,3-dihydroxy-10,13-dimethylhex
adecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (11)
A modification of the procedure of Tohma was used.8 A solution of compound 8 (1.5 g, 3.2
mmol) in CH2Cl2 (5 mL) and THF (10 mL) was stirred as NaBH4 (750. mg, 19.8 mmol) was
added and the resulting reaction mixture was stirred at room temperature for 1 hr. TLC
showed the reaction was completed; therefore, the reaction mixture was diluted with sat.
NH4Cl (100 mL) and was extracted with EtOAc (300 mL). The organic layer was washed
with brine and dried over Na2SO4, and after filtration, the organic layer was concentrated
under reduced pressure to dryness to give compound 11 (1.3 g) as a colorless oil.
(R)-4-((1R,3R,5R,8S,9S,10S,12S,13R,14S,17R)-1,3,12-trihydroxy-10,13-dimethylhexadecah
ydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (12)
A modification of the procedure of Tohma was used.8 A solution of compound 11 (1.3 g, 2.8
mmol) in MeOH (30 mL) and H2O (20 mL) was stirred as NaOH (1.1 g, 27.1 mmol) was
added. The reaction was warmed to 110 °C and was stirred at that temperature for 12 hr at
which time LC-MS showed the reaction to be complete. The mixture was cooled to room
temperature, and 0.5 N HCl (10 mL) was added. The reaction mixture was extracted with
EtOAc (2x20 mL), and the combined organic layers were dried over Na2SO4.
After filtration, the solution was concentrated under reduced pressure, and the resulting
residue was purified by column chromatography on silica gel (20:1, CH2Cl2:MeOH) to give
12 (0.6 g, 46% yield from 8) as a white solid (96.5% purity by 1H NMR25 using fumaric acid
as the internal standard). LC/MS [M-H] 407.4. NMR data is compiled in the table.
(R)-methyl
4-((1R,3S,5R,8S,9S,10S,12S,13R,14S,17R)-12-acetoxy-1,3-dihydroxy-10,13-dimethylhexad
ecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (13)
A modification of the procedure of Tohma was used.8 A solution of compound 8 (1.5 g, 3.2
mmol) in in CH2Cl2 (5 mL) was stirred at 0°C as NaBH4 (600. mg, 15.9 mmol) was added,
and the resulting reaction mixture was stirred at at 0 °C for 0.5 hr. TLC showed the reaction
to be complete; therefore, it was diluted with sat. NH4Cl (30 mL) and was extracted
with EtOAc (2x30 mL). The combined organic layers were washed with brine and dried over
Na2SO4, and after filtration, the organic layer was concentrated under reduced pressure to
dryness to give compound 11. Purification by column chromatography on silica gel (3:1 to
1:1, Hexane:EtOAc = 3:1, 1:1) afforded compound 13 (0.30 g, 20%yield) as a white solid.
1H NMR (400 MHz, CDCl3) δ 0.75 (s, 3H), 0.82 (d, J = 6.4 Hz, 3H), 1.03 – 1.19 (m, 1H),
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1.04 (s, 3H), 1.18 – 1.78 (m, 20H), 1.81 – 1.97 (m, 4H), 2.10 (s, 3H), 2.22 (ddd, J = 15.6, 9.3,
6.7 Hz, 1H), 2.36 (ddd, J = 15.1, 9.9, 5.0 Hz, 1H), 3.69 (s, 3H), 3.85 (s, 1H), 4.14 (dq, J =
12.1, 6.0, 5.9, 5.9 Hz, 1H), 5.08 (d, J = 2.8 Hz, 1H).
(R)-4-((1R,3S,5R,8S,9S,10S,12S,13R,14S,17R)-1,3,12-trihydroxy-10,13-dimethylhexadecah
ydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (1)
A modification of the procedure of Tohma was used.8 A solution of compound 13 (0.30 g,
0.65 mmol) in MeOH (3 mL) and H2O (2 mL) was stirred as NaOH (120 mg, 3.0 mmol) was
added. The reaction was warmed to 100 °C and was stirred at that temperature for 12 hr at
which time TLC showed the reaction to be complete. The mixture was cooled to room
temperature, and the pH was adjusted to 4 with 1 N HCl. The reaction mixture was then
extracted with EtOAc (2x80 mL), and the combined organic layers were dried over Na2SO4.
After filtration, the solution was concentrated under reduced pressure, and the resulting
residue was purified twice by column chromatography on silica gel (3:1, CH2Cl2: MeOH) to
give 1 (38 mg, 14% yield) as a white solid (79% purity by NMR25 using
2,3,5,6-tetrachloronitrobenzene as an internal standard). LC/MS [M-H] 407.4. NMR data is
compiled in the table.
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Table. NMR spectra were recorded in D6-DMSO, and chemical shifts are reported relative to the residual solvent signal set to 2.5 ppm(1H) and 39.5 ppm (13C). Chemical shifts for
overlapping 1H signals are taken from relevant 2D experiments with the chemical shift taken at the center of the cross-peak. 1H-Coupling patterns and J values are reported from
1
1D H- spectra for selected, non overlapping, signals (Multipicity, J Hz). For multiplets, the center of the signal is taken as the chemical shift. Abbreviations; s(singlet),
d(doublet), t(triplet), dt(doublet of triplet), tt(triplet of triplet), qt(quartet of triplet), m(multiplet), br.(broad), nr. (narrow), n.a.(not applicable), n.d. (not determined).
(1) 1-OH deoxycholic acid
13C ppm 1H ppm (multiplicity, J)
(12) 1--OH deoxycholic acid
13C ppm 1H ppm (multiplicity, J)
(Ref) deoxycholic acid
1H ppm (multiplicity, J)
Position
1
13C ppm
71.4
3.59
72.1
3.53 (br.s)
35.2
1.62, 0.88
1-OH
2
n.a.
4.16 (d, J=4.6)
1.64,1.46
n.a.
4.77 (br.d,6.0)
1.68, 1.68
n.a.
n.a.
37.4
31.8
30.2
1.45, 1.27
3
64.6
3.82 (tq, J=9.9,9.9,4.8,4.8,4.8)
66.8
3.99 (br.s)
69.9
3.36 (tt, J10.9,10.9,4.4,4.4)
3-OH
n.a.
36.0
35.1
26.6
4.28 (d, J=4.8)
1.62, 1.29
1.69
n.a.
33.2
30.3
25.9
5.14
1.93, 1.30
1.93
n.a.
36.3
41.6
27.0
4.45 (br.s)
1.63, 1.33
1.29
4
5
6
1.64, 1.17
1.75, 1.16
1.76, 1.17
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7
8
25.7
35.7
34.2
38.0
28.7
70.9
n.a.
1.32, 1.08
1.35
25.6
35.6
34.1
38.8
28.6
71.0
n.a.
1.34, 1.07
1.38
26.1
35.6
32.9
33.8
28.6
71.0
n.a.
1.33, 1.05
1.33
9
1.77
1.62
1.80
10
11
12
12OH
13
14
15
16
17
18
19
20
21
22
23
n.a.
n.a
n.a.
1.35,1.30
3.76 (br.m)
4.19 (d, J=4.0)
n.a.
1.39, 1.25
3.75
1.36, 1.36
3.78
4.14 (br.s)
n.a
4.2 (br.d, J=3.2)
n.a.
45.7
47.4
23.5
27.0
46.2
12.5
18.2
35.0
16.9
30.8
45.8
47.4
23.5
27.0
46.2
12.4
18.5
34.9
16.9
30.8
46.2
47.5
23.5
27.2
46.2
12.4
23.1
35.0
16.9
30.8
1.55
1.52
1.57
1.51, 0.98
1.73, 1.18
1.74
1.50, 1.00
1.72, 1.16
1.73
1.50, 0.98,
1.75, 1.17
1.76
0.59 (s)
0.89 (s)
1.28
0.60 (s)
0.96, (s)
1.28
0.59 (s)
0.84 (s)
1.29
0.9 (d, J=6.6Hz)
1.64, 1.18
0.9 (d, J=6.5)
1.64, 1.18
0.91 (d, J=6.5Hz)
1.64, 1.18
2.09 (ddd, J=15.5,9.1,7.1), 2.21
(ddd, J=15.5,9.6,5.2)
2.09 (ddd, J=15.6,9.2,6.9),
2.21 (ddd, J=15.6,9.6,5.2)
2.09 (ddd, J=15.6,9.3,6.9), 2.22
(ddd, J=15.6,9.7,5.2)
31.0
30.8
30.8
24
175.3
n.a
n.a.
174.9
n.a.
n.a.
174.9
n.a
n.a.
24OH
11.91 (br.s)
11.92 (br.s)
11.93 (br.s)
Chemical shifts are reported relative to the residual solvent signal set to 2.5 ppm(1H) and 39.5 ppm (13C).
Chemical shifts for overlapping 1H signals are taken from relevant 2D experiments with the chemical shift taken at the center of the cross-peak
1H-Coupling patterns and J values are reported from 1D 1H- spectra for selected, non overlapping, signals (Multipicity, J Hz). For multiplets the center of the signal is taken as the chemical shift
Abbreviations; s(singlet), d(doublet), t(triplet), dt(doublet of triplet), tt(triplet of triplet), qt(quartet of triplet), m(multiplet), br.(broad), nr. narrow, n.a.(not applicable), n.d. (not determined)
Numbering follows the IUPAC approved atom numbering of steroids.
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Bile Acids
Bile Salts
18
12
17
15
11
13
14
19
16
1
4
9
2
10
8
3
5
7
6
R = OH, R' = OH cholic acid
R = OH, R' = OH, R''=NHCH2CO2H
Glycocholoic acid
R = H, R'= OH
R = OH, R' = H
R = H, R' = H
chenodeoxycholoic acid
deoxycholic acid (DCA)
lithocholic acid
R = H, R'= OH, R''=NHCH2CH2SO3H Taurocholoic acid
R = H, R'= OH, R''=NHCH2CO2H
Glycochenodeoxycholoic acid
R = OH, R' = H, R''=NHCH2CH2SO3H Taurodeoxycholic acid
Figure 1. Structures of some common bile acids and bile salts.
Scheme 1. Previously reported synthesis of 1-beta-hydroxydeoxycholic acid by Tohma.7,8
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D2O, base
2
[D6]-2
Scheme 2. Proposed key exchange reaction.
1. MeOH, HCl (85%)
2. Ag2CO3, PhCH3 (84%)
Li 2CO 3, LiBr
3. Ac 2O, DMAP (76%)
DMF, 80 °C (75%)
4. Br2, HOAc, CHCl3 (64%)
deoxycholic acid
4
1. Zn, HOAc (65%)
2. H2O2, NaOH (57%)
5
7
Scheme 3. Synthesis of epoxide 7 from deoxycholic acid.
Scheme 4. Attempted ring opening of epoxide 7.
5-10 eq of LiOH or NaOH
Roomtemp or 100 °C
+
3
9
8
Scheme 5. Probing the stability of the 1β- hydroxy of 8 to the proposed exchange reaction
conditions.
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PhMe2SiCl,
Li, CuCN
Hg(OAc)2
THF, 30 oC
67%
AcOOH, AcOH
67%
6
10
NaBH4
CH2Cl2, THF
8
11, 3 OH = R/S mix
13, 3 OH = R (20%)
NaOH,
MeOH, H2O
1
12
14% from 13
46% from 8
Scheme 6. Conversion of 7 to 1 and 12.
1. MicroCYP0029
2. mass directed
prep HPLC
R=H, deoxycholic acid
R=D, [2,2',4,4'-D4]deoxycholic acid
R=H, 1 (27%)
R=D, [2,2',4,4'-D4]-1 (33%)
Scheme 7. Biocatalytic route from DCA to 1.
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