Chemical Synthesis of the Epimeric (23R)- and (23S)-Fluoro Derivatives of Bile Acids via Horner-Wadsworth-Emmons Reaction

Article abstract of DOI:10.1007/s11745-015-4050-8

A method for the synthesis of two (23R)- and (23S)-epimeric pairs of 23-fluoro-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid and 23-fluoro-3α,7α-dihydroxy-5β-cholan-24-oic acid is described. The key intermediates, 23,24-dinor-22-aldehyde peracetates were prepared from cholic and chenodeoxycholic acids via the 24-nor-22-ene, 24-nor-22ξ,23-epoxy, and 23,24-dinor-22-aldehyde derivatives. The Horner-Wadsworth-Emmons reaction of the 23,24-dinor-22-aldehydes using triethyl 2-fluoro-2-phosphonoacetate in the presence of LiCl and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and subsequent hydrogenation of the resulting 23ξ-fluoro-22-ene ethyl esters, followed by hydrolysis, gave a mixture of the epimeric (23R)- and (23S)-fluorinated bile acids which were resolved efficiently by preparative RP-HPLC. The stereochemical configuration of the fluorine atom at C-23 in the newly synthesized compounds was confirmed directly by the X-ray crystallographic data. The ¹H and ¹³C NMR spectral differences between the (23R)- and (23S)-epimers were also discussed.

Full text of DOI:10.1007/s11745-015-4050-8

Lipids
DOI 10.1007/s11745-015-4050-8
METHODS
Chemical Synthesis of the Epimeric (23R)‑ and (23S)‑Fluoro
Derivatives of Bile Acids via Horner–Wadsworth–Emmons
Reaction
1
1
1
1
Kaoru Omura · Yuuki Adachi · Yuuki Kobayashi · Shoutaro Sekiguchi ·
1
1
Biao Zhou · Takashi Iida
Received: 7 May 2015 / Accepted: 8 July 2015
AOCS 2015
©
Abstract A method for the synthesis of two (23R)- and
23S)-epimeric pairs of 23-fluoro-3α,7α,12α-trihydroxy-
β-cholan-24-oic acid and 23-fluoro-3α,7α-dihydroxy-5β-
cholan-24-oic acid is described. The key intermediates,
3,24-dinor-22-aldehyde peracetates were prepared from
NMR Nuclear magnetic resonance
HPLC High-performance liquid chromatography
(
5
2
Introduction
cholic and chenodeoxycholic acids via the 24-nor-22-
ene, 24-nor-22ξ,23-epoxy, and 23,24-dinor-22-aldehyde
derivatives. The Horner–Wadsworth–Emmons reaction
of the 23,24-dinor-22-aldehydes using triethyl 2-fluoro-
The incorporation of a fluorine atom into an advanced drug
candidate is a common strategy in synthetic organic chem-
istry as well as medicinal chemistry. Extensive research has
established that introducing fluorine into a target molecule
can effectively modify its physicochemical and drug-like
properties by blocking undesired metabolism at a specific
site, increasing lipophilicity or binding affinity, or altering
drug absorption, distribution, or excretion [1–4].
Considerable effort has been devoted to the development
of methods for the introduction of a fluorine atom into a
steroid molecule. It has been reported that a 9α-fluoro sub-
stituent adjacent to the 11-oxygen function in the adrenal
cortical hormones markedly increases biological activity
such as a topical anti-inflammatory one [5]. For example,
the commercially available triamcinolone, dexamethasone,
and betamethasone are the 9α-fluoro derivatives of corti-
sol (cortisone and hydrocortisone) that have potent topical
anti-inflammatory properties. This finding substantiated the
concept that a fluorine substituent could strongly influence
the biological properties of a steroid. Therefore, the syn-
thesis of fluorinated steroids with stereo control is of major
importance in many areas including synthetic organic
chemistry, pharmaceuticals, and biochemistry.
2
-phosphonoacetate in the presence of LiCl and 1,8-diaz-
abicyclo[5,4,0]undec-7-ene (DBU), and subsequent
hydrogenation of the resulting 23ξ-fluoro-22-ene ethyl
esters, followed by hydrolysis, gave a mixture of the epi-
meric (23R)- and (23S)-fluorinated bile acids which were
resolved efficiently by preparative RP-HPLC. The ste-
reochemical configuration of the fluorine atom at C-23 in
the newly synthesized compounds was confirmed directly
1
13
by the X-ray crystallographic data. The H and C NMR
spectral differences between the (23R)- and (23S)-epimers
were also discussed.
Keywords Bile acid · Epimeric (23R)-/(23S)-fluoro-bile
acids · Horner–Wadsworth–Emmons reaction · α-Fluoro-
α,β-unsaturated esterification
Abbreviations
MMO 4-Methylmorpholine N-oxide
DBU
1,8-Diazabicyclo[5,4,0]undec-7-ene
Although there have been many reports on the introduc-
tion of a fluorine atom in 5α- and 5β-steroid nuclei [6–13],
that into the aliphatic cholestane (iso-octane) and cholane
*
Takashi Iida
takaiida@chs.nihon-u.ac.jp
(
iso-pentane) side chain has not yet been reported. During
1
Department of Chemistry, College of Humanities
and Sciences, Nihon University, Sakurajousui, Setagaya,
Tokyo 156-8550, Japan
the course of our synthetic study of new and scarce ster-
oids, we herein report the preparation of two (23R)- and
1
3

Lipids
C F as an internal reference compound; chemical shifts
6
6
were expressed in δ (ppm) relative to C F (−162.9 ppm).
6
6
1
3
The C distortionless enhancement by polarization trans-
fer (DEPT; 135°, 90°, and 45°) spectra were measured to
determine the exact C signal multiplicity and to differen-
tiate among CH , CH , CH, and C based on their proton
1
3
3
2
1
13
environments. In order to further confirm the H and
C
signal assignments for some of compounds, two-dimen-
1
sional (2D) H detected heteronuclear multiple quantum
Fig. 1 Structure of epimeric (23R)- and (23S)-fluoro derivatives of
CA and CDCA
1
13
1
(
HMQC; H– C coupling) and H detected heteronuclear
1
13
multiple bond correlation (HMBC; long-range H– C cou-
pling) experiments were also performed. High-resolution
mass spectra by electrospray ionization (HR-ESI–MS)
were obtained using a JEOL AccuTOF JMS-T100LC liq-
uid chromatography-mass spectrometer equipped with an
ESI source and coupled to an Agilent 1200 series binary
pump (Agilent Technologies Inc., Santa Clara, CA, USA)
operated in the negative ion mode or positive ion mode.
Normal-phase TLC was performed on pre-coated Kie-
selgel 60F254 plates (E. Merck, Darmstadt, Germany)
using EtOAc-hexane mixtures as the developing solvent.
Reversed-phase (RP)-TLC was performed on pre-coated
RP-18F254s plates (E. Merck) using methanol–water mix-
tures as the developing solvents.
The analytical RP-HPLC apparatus used was a Jasco
LC-2000 plus HPLC system, which consisted of two
PU-2085 high-pressure pumps, an MX-2080-32 solvent
mixing module, and a CO-2060 column heater equipped
with a Chrom NAV data processing system (Tokyo, Japan).
A Capcell pack type AQ RP-C18 column (3.0 × 150 mm
I.D.; particle size, 3 μm; Shiseido, Tokyo, Japan) was
employed and kept at 37 °C. An Alltech 2000ES evapora-
tive light-scattering detector (ELSD; Deerfield, IL, USA)
was used under the following conditions: the flow rate of
purified compressed air used as the nebulizing gas was 2.2
L/min, and the temperature of the heated drift was 79.3 °C.
The mobile phase used was a mixture of 15 mM ammo-
nium acetate/acetic acid buffer solution (pH 5.4) and meth-
anol (35:65 or 33:67, v/v); the flow rate was kept at 400
μL/min during the analysis.
(
23S)-epimeric pairs of 23-fluorinated bile acids, starting
from primary bile acids, chenodeoxycholic acid (CDCA;
b) and cholic acid (CA; 2a), which are common in mam-
2
mals. Thus, the root bile acid should be 2b, as every bile
acid must a hydroxyl group at C-3 and a hydroxyl group
at C-7, as 7α-hydroxylation of cholesterol is the rate lim-
iting step in the pathway of bile acid synthesis. The most
frequent site of additional hydroxylation of 2b is at C-12
in 2a.
We applied herein the Horner-Wadsworth-Emmons reac-
tion of triethyl 2-fluoro-2-phosphonoacetate in the presence
of LiCl and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU)
as base sensitive reagents on the 23,24-dinor-22-aldehyde
derivatives (6a and 6b) of bile acids as the key intermedi-
ates. The novel 23-fluorinated bile acids prepared by the
method are shown in Fig. 1.
Experimental
Materials
CA (2a) was obtained from Wako Pure Chemical Indus-
tries, Ltd. (Osaka, Japan). CDCA (2b) was supplied from
Tanabe Mitsubishi Pharmaceutical Co. (Tokyo, Japan). All
other chemicals and solvents were of analytical reagent
grade and available from commercial sources. All com-
pounds were dried by azeotropic distillation before use in
reactions.
The preparative RP-HPLC consisted of a Hitachi
L-7100 pump (Tokyo, Japan) and was carried out by iso-
cratic elution using a Capcell Pack type MG RP-C18 col-
umn (20 × 250 mm I.D.; particle size, 5 μm). A mixture of
15 mM ammonium acetate/acetic acid buffer solution (pH
5.4) and methanol (35:65 or 33:67, v/v) was used as the
mobile phase; the flow rate was kept at 9.99 mL/min.
Instruments
All melting points (mp) were determined on a micro hot
stage apparatus and are uncorrected. H and C NMR
spectra were obtained with a JEOL ECA 500 FT instrument
1
13
operated at 500 and 125 MHz, respectively, with CDCl or
3
CD OD containing 0.1 % Me Si as the solvent; chemical
X‑Ray Crystal Structure Determination of 1b‑S
3
4
shifts were expressed in δ (ppm) relative to Me Si (0 ppm).
4
1
9
F NMR was measured on the same instrument oper-
ated at 470 MHz with CDCl or CD OD containing 0.1 %
A high quality single crystal of 1b‑S suitable for X-ray dif-
fraction studies was grown by evaporation of a solution of
3
3
1
3

Lipids
methanol at room temperature. The X-ray diffraction data
were collected on a Rigaku AFC-8 diffractometer equipped
with a Mercury CCD detector using monochromated Mo
Chemical Synthesis of 23‑Fluorinated Bile Acids
Ethyl (22E)‑ 23‑Fluoro‑3α,7α,12α‑triacetoxy‑5β‑chol‑22‑e
n‑24‑oate (7a)
(
Kα) radiation (λ = 0.71075 Å) at 173 K. The structure
was solved using direct methods (SHELXS-97) and refined
by a full-matrix least-squares procedure based on F using
2
Triethyl 2-fluoro-2-phosphonoacetate (800 mg) and
1,8-diazabicyclo[5,4,0]-7-undecene (DBU, 350 mg) were
added gradually to a stirred solution of LiCl (120 mg)
the CrystalStructure crystallographic software package.
The crystal structures were refined with anisotropic tem-
perature factors for all non-hydrogen atoms. The positions
of hydrogen atoms were calculated geometrically, and were
refined using the riding model. The crystal data and refine-
ment details for 1b‑S are given in Tables 1 and 2.
in anhydrous CH CN (40 mL). To the solution, the
3
23,24-dinor-22-aldehyde triacetate 6a (1.2 g, 2.4 mmol)
was then added, and the mixture was further stirred under
N at room temperature for 3 h. After adding an ice-cold
2
ammonium chloride solution, the reaction product was
extracted with EtOAc. The combined extract was washed
with saturated brine, dried over Drierite, and evaporated
under reduced pressure. The oily residue was chromato-
graphed on a column of silica gel (40 g) and elution with
Table 1 Crystallographic details for 1b‑S
Formula
M
C H FO ·1.5H O
24 39 4 2
2
2
hexane–EtOAc (80:20, v/v) afforded the desired Δ -
3-fluoro-24-carboxylate (7a) which crystallized from
434.57
2
Crystal system
Space group
a (Å)
Monoclinic
P21
acetone-Et O as colorless prisms: yield, 990 mg (70 %);
2
1
mp, 98–99 °C. H NMR (CDCl ) δ: 0.80 (3H, s, 18-CH ),
3
3
10.482(3)
7.582(2)
15.134(4)
90
0
1
3
1
.93 (3H, s, 19-CH ), 0.96 (3H, d, J = 6.3 Hz, 21-CH ),
3
3
b (Å)
.26 (3H, t, J = 6.9 Hz, –COOCH CH ), 2.05 (3H, s,
2
3
c (Å)
α-OCOCH ), 2.08 (3H, s, 7α-OCOCH ), 2.16 (3H, s,
3
3
α (°)
2α-OCOCH ), 4.13 (2H, m, –COOCH CH ), 4.58 (1H,
3
2
3
β (°)
100.563(6)
90
brm, 3β-H), 4.91 (1H, brs, 7β-H), 5.10 (1H, brs, 12β-H),
γ (°)
V (Å3)
1
3
5
.68 (1H, m, 22-H). C NMR (CDCl ) δ: 34.5 (C-1), 25.4
3
1182.3(6)
2
(
(
C-2), 73.8 (C-3), 34.5 (C-4), 40.7 (C-5), 31.0 (C-6), 70.4
C-7), 37.6 (C-8), 28.8 (C-9), 34.2 (C-10), 26.4 (C-11),
Z value
−3
D /g cm
1.221
c
7
5.0 (C-12), 45.0 (C-13), 43.3 (C-14), 22.6 (C-15), 26.7
F(000)
472
(
(
(
C-16), 47.5 (C-17), 12.4 (C-18), 22.4 (C-19), 31.8
C-20), 19.5 (C-21), 128.5 (d, J = 15.5 Hz, C-22), 145.4
d, J = 250.4 Hz, C-23), 160.9 (d, J = 36.0 Hz, C-24),
Radiation type
Mo Kα
0.090
−
1
μ(Mo-K )/cm
α
Crystal description
Crystal size (mm)
No of reflns measured
No of unique reflns
No. observed (I > 2σ)
R1 (I > 2σ)
Block
1
70.3, 170.5, 170.5 (each OCOCH ), 21.2, 21.3, 21.4 (each
3
0.20 × 0.20 × 0.20
19
OCOCH ), 13.9 (CH CH ), 61.2 (CH CH ). F NMR
3
2
3
2
3
25770
5405
(
CDCl ) δ: −131.8 (1F, t, J = 29.3 Hz, 23-F). LC–ESI–
3
+
MS: Calcd. for C H FNaO [M+Na] , 601.3153; found,
3
2
47
8
4530
m/z 601.3151.
0.058
0.164
1.123
wR (I > 2σ)
2
Ethyl (22E)‑23‑Fluoro‑3α,7α‑diacetoxy‑5β‑chol‑22‑en‑
4‑oate (7b)
Goodness of fit
2
The 23,24-dinor-22-aldehyde diacetate 6b (1.2 g,
.3 mmol) was subjected to the Horner-Wadsworth-
Table 2 Selected bond lengths (Å) and angles (°) for 1b‑S
2
Emmons reaction with triethyl 2-fluoro-2-phosphonoac-
etate and DBU and LiCl processed as described for the
preparation of 7a. Recrystallization of the product from
O1–C3
1.415
1.450
1.397
1.302
1.208
110.2
107.0
111.5
O2–C7
F1–C23
2
2
methanol-CHCl3 gave the desired Δ -23-fluoro-24-
carboxylate (7b) as colorless prisms: yield, 870 mg (72 %);
O3–C24
O4–C24
1
mp, 96–98 °C. H NMR (CDCl ) δ: 0.73 (3H, s, 18-CH ),
3
3
F1–C23–C22
F1–C23–C24
C22–C23–C24
0
1
.94 (3H, s, 19-CH ), 1.04 (3H, d, J = 6.8 Hz, 21-CH ),
3
3
.20 (3H, t, J = 6.9 Hz, –COOCH CH ), 2.05 (6H, s, 3α-
2
3
and 7α-OCOCH ), 4.13 (2H, m, –COOCH CH ), 4.58 (1H,
3
2
3
1
3

Lipids
brm, 3β-H), 4.91 (1H, brs, 7β-H), 5.73 (1H, m, 22-H). 13C
(23R)‑ and (23S)‑23‑Fluoro‑3α,7α,12α‑trihydroxy‑5β‑chol
an‑24‑oic acids (1a‑R and 1a‑S)
NMR (CDCl ) δ: 34.6 (C-1), 26.8 (C-2), 74.1 (C-3), 34.9
3
(
(
5
C-4), 40.9 (C-5), 31.3 (C-6), 71.2 (C-7), 37.9 (C-8), 34.1
C-9), 34.8 (C-10), 20.6 (C-11), 39.4 (C-12), 42.9 (C-13),
0.4 (C-14), 23.6 (C-15), 27.4 (C-16), 56.0 (C-17), 12.0
A solution of a mixture (8a) of the epimeric 23-fluoro-
triacetoxy esters (100 mg, 0.17 mmol) dissolved in meth-
anol (1 mL) and 20 % NaOH (1.5 mL) was left stand at
room temperature for 12 h. After evaporation of the sol-
vents under reduced pressure, the residual oily product
was dissolved in water and then acidified with 10 % H SO
(
C-18), 22.7 (C-19), 34.1 (C-20), 20.2 (C-21), 129.0 (d,
J = 14.8 Hz, C-22), 145.6 (d, J = 250.4 Hz, C-23), 161.0
(
2
d, J = 35.8 Hz, C-24), 170.4, 170.6 (each OCOCH ), 21.5,
3
1
9
1.6 (each OCOCH ), 14.1 (CH CH ), 61.3 (CH CH ). F
3 2 3 2 3
2
4
NMR (CDCl ) δ: −131.0 (1F, t, J = 29.3 Hz, 23-F). LC–
under ice-bath cooling. The precipitated solid was filtered,
washed with water, and dried: yield, 70 mg (96 %). The
crude product consisted of an epimeric mixture of (23R)-
and (23S)-23-fluorinated epimers (ratio of 9:1) of CA (2a)
by RP-HPLC. Preparative RP-HPLC of a part of the mix-
ture resulted in separation of the two epimers.
3
+
ESI–MS: Calcd. for C H FNaO [M+Na] , 543.3098;
3
0
42
6
found, m/z 543.3110.
An Epimeric Mixture of Ethyl (23R)/(23S)‑23‑Fluoro‑3α,7
α,12α‑triacetoxy‑5β‑cholan‑24‑oates (8a)
The more polar, major fraction (90 % by RP-HPLC)
eluted with a mixture of 15 mM ammonium acetate/ace-
tic acid buffer solution (pH 5.4) and methanol (65:35,
v/v) and gave a homogeneous oil which crystallized from
methanol as a colorless amorphous solid and was identi-
The 23-fluoro-22-ene ethyl ester 7a (1.0 g, 1.7 mmol) dis-
solved in dry THF (15 mL) was catalytically hydrogen-
ated with 10 % Pd/C (0.6 g) at a slight positive pressure
of H . After stirring at room temperature overnight, the
2
1
catalyst was filtered off, and the solvent was evaporated
under reduced pressure. The oily residue was chromato-
graphed on a silica gel column (30 g) and eluted with hex-
ane–EtOAc (50:50, v/v). The combined homogeneous frac-
tion showed a single spot on TLC, but it was found to be an
fied as the (23R)-fluorinated epimer (1a‑R) of 2a. H NMR
(CDCl containing 20 % CD OD) δ: 0.71 (3H, s, 18-CH ),
3
3
3
0.89 (3H, s, 19-CH ), 1.12 (3H, d, J = 5.2 Hz, 21-CH ),
3
3
3.38 (1H, brm, 3β-H), 3.83 (1H, brs, 7β-H), 3.97 (1H, brs,
1
3
12β-H), 4.91 (1H, brd, J = 52.7 Hz, 23-H). C NMR:
1
9
epimeric mixture (8a) of (23R)- and (23S)-23-fluoro esters
see Table 3. F NMR (CDCl containing 20 % CD OD)
3
3
1
by RP-HPLC: yield, 910 mg (91 %). H NMR (CDCl ) δ:
δ: −185.22 (1F, brs, 23-F). LC–ESI–MS: Calcd. for
3
−
0
.75 (3H, s, 18-CH ), 0.92 (3H, s, 19-CH ), 0.94 (3H, d,
C H FO [M-H] , 425.2703; found, m/z 425.2684.
3
3
24 38
5
J = 6.3 Hz, 21-CH ), 1.29 (3H, t, J = 7.4 Hz, –OCH CH ),
The less polar, minor fraction (10 % by RP-HPLC),
which was eluted with the same solvent system, gave a
homogeneous oil which crystallized from methanol as a
3
2
3
2
2
4
.05 (3H, s, 3α-OCOCH ), 2.09 (3H, s, 7α-OCOCH ),
3
3
.14 (3H, s, 12α-OCOCH ), 4.13 (2H, m, –OCH CH ),
3
2
3
.58 (1H, brm, 3β-H), 4.91 (1H, brs, 7β-H), 4.92 (1H,
colorless amorphous solid and was identified as the (23S)-
1
9
1
d, J = 49.2 Hz, 23-H), 5.10 (1H, brs, 12β-H). F NMR
fluorinated epimer (1a‑S) of 2a. H NMR (CDCl contain-
3
(
(
[
CDCl ) δ: −188.9 (1F, t, J = 29.3 Hz, 23-F) and −192.9
ing 20 % CD OD) δ: 0.72 (3H, s, 18-CH ), 0.90 (3H, s,
3
3
3
1F, m, 23-F). LC–ESI–MS: Calcd. for C H FNaO
19-CH ), 1.10 (3H, d, J = 6.3 Hz, 21-CH ), 3.38 (1H, brm,
3
2
49
8
3
3
+
M+Na] , 603.3309; found, m/z 603.3311.
3β-H), 3.84 (1H, brs, 7β-H), 3.98 (1H, brs, 12β-H), 4.96
1
3
(
1H, dd, J = 50.6, 10.9 Hz, 23-H). C NMR: see Table 3.
1
9
An Epimeric Mixture of Ethyl (23R)/(23S)‑23‑Fluoro‑3α,7
α‑diacetoxy‑5β‑cholan‑24‑oates (8b)
F NMR (CDCl containing 20 % CD OD) δ: −191.92
3
3
(1F, brs, 23-F). LC–ESI–MS: Calcd. for C H FO
2
4
38
5
−
[
M−H] , 425.2703; found, m/z 425.2691.
The 23-fluoro-22-ene ethyl ester 7b (1.0 g, 1.9 mmol),
hydrogenated with 10 % Pd/C and processed as described
for the preparation of 8a yielded an epimeric mixture (8b)
of (23R)- and (24S)-24-fluoro esters: yield, 920 mg (92 %).
(23R)‑ and (23S)‑23‑Fluoro‑3α,7α‑dihydroxy‑5β‑cholan‑2
4‑oic acids (1b‑R and 1b‑S)
1
H NMR (CDCl ) δ: 0.62 (3H, s, 18-CH ), 0.83 (3H,
The compounds were prepared from a mixture (8b) of the
epimeric 23-fluorodiacetoxy esters (100 mg, 0.19 mmol)
by the procedure described for the preparation of 1a. The
crude product (70 mg, 90 %) consisted of a 23R/23S ratio
of 6:4 by RP-HPLC.
The first fraction (60 % by RP-HPLC) which eluted with
a mixture of 15 mM ammonium acetate/acetic acid buffer
solution (pH 5.4) and methanol (67:33, v/v) consisted of a
3
3
s, 19-CH ), 1.05 (3H, d, J = 6.3 Hz, 21-CH ), 1.27 (3H,
3
3
m, –OCH CH ), 1.98 (3H, s, 3α-OCOCH ), 2.00 (3H, s,
2
3
3
7
3
2
2
α-OCOCH ), 4.20 (2H, m, –OCH CH ), 4.53 (1H, brm,
3 2 3
β-H), 4.83 (1H, brs, 7β-H), 4.91 (1H, d, J = 50.4 Hz,
1
9
3-H). F NMR (CDCl ) δ: -188.2 (1F, t, J = 29.3 Hz,
3
3-F) and −192.9 (1F, m, 23-F). LC–ESI–MS: Calcd. for
+
C H FNaO [M+Na] , 545.3254; found, m/z 545.3253.
3
0
47
6
1
3

Lipids
Table 3 13C NMR spectral
data for the epimeric 23-fluoro
derivatives of bile acids
Carbon
(23R)-23-Fluoro-
α,7α,12α-
trihydroxy-5β-
(23S)-23-Fluoro-
3α,7α,12α-
trihydroxy-5β-
cholan-24-oic acid
(1a‑S)
(23R)-23-Fluoro-
3α,7α-
dihydroxy-5β-
cholan-24-oic acid
(1b‑R)
(23S)-23-Fluoro-
3α,7α-
dihydroxy-5β-
cholan-24-oic acid
(1b‑S)
3
cholan-24-oic acid
(
1a‑R)
1
2
3
4
5
6
7
8
9
35.1
35.0
35.2
35.1
29.7
29.6
30.1
30.1
71.3
71.3
71.5
71.5
39.0
39.0
39.1
39.1
41.2
41.2
41.4
41.3
34.3
34.3
34.4
34.4
68.1
68.1
68.1
68.1
39.2
39.2
39.1
39.1
26.1
26.1
32.6
32.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
34.5
34.5
34.9
34.8
27.8
27.8
20.4
20.4
72.7
72.7
39.4
39.4
46.3
46.3
42.6
42.6
41.5
41.5
50.2
50.2
23.0
23.0
23.4
23.4
27.5
27.3
28.2
28.0
47.0
47.1
56.1
56.0
12.1
12.2
11.4
11.5
22.2
22.2
22.6
22.6
33.9
32.0
34.0
32.1
18.0
16.7
19.1
17.8
38.6 (J, 19.2 Hz)
89.2 (J, 180.0 Hz)
174.1 (J, 25.2 Hz)
38.5 (J, 20.4 Hz)
87.2 (J, 182.4 Hz)
173.2 (J, 25.2 Hz)
38.7 (J, 19.2 Hz)
89.2 (J, 183.6 Hz)
174.0 (J, 20.3 Hz)
38.6 (J, 20.4 Hz)
86.7 (J, 182.4 Hz)
173.2 (J, 22.8 Hz)
Measured in CDCl containing 20 % of CD OD
3
3
homogenous oil that crystallized from methanol as a color-
Results and Discussion
less amorphous solid and was characterized as the (23R)-
1
fluorinated epimer (1b-R) of CDCA (2b). H NMR (CDCl
As illustrated in Fig. 2, the synthetic routes to key inter-
mediates (6a and 6b) from primary bile acids (2a and
2b) are essentially the same as those reported previously
[14]. Thus, treatment of 2a and 2b with acetic anhydride
and 4-dimethylaminopyridine resulted in the formation of
the peracetate derivatives (3a and 3b), which in turn were
transformed into the C23 24-nor-22-enes (4a and 4b) by the
3
containing 20 % CD OD) δ: 0.68 (3H, s, 18-CH ), 0.91 (3H,
3
3
s, 19-CH ), 1.06 (3H, d, J = 6.3 Hz, 21-CH ), 3.41 (1H, brm,
3
3
3
β-H), 3.83 (1H, brs, 7β-H), 4.90 (1H, brd, J = 49.0 Hz, 23-H).
1
3
19
C NMR: see Table 3. F NMR (CDCl containing 20 %
3
CD OD) δ: −185.85 (1F, brs, 23-F). LC–ESI–MS: Calcd. for
3
−
C H FO [M-H] , 409.2754; Found, m/z 409.2800.
24
38
4
The second fraction (40 % by RP-HPLC), which eluted
side chain degradation with lead tetraacetate [(AcO) Pb]
4
with the same solvent system, gave an oil that crystallized
from methanol as colorless prisms and was characterized
as the (23S)‑fluorinated epimer (1b‑S) of 2b. H NMR
and cuprous acetate [(AcO) Cu] in boiling pyridine [15,
16]. Epoxidation of 4a and 4b with m-chloroperbenzoic
2
1
acid (m-CPBA) in the presence of NaHCO at room tem-
3
(
0
3
CDCl containing 20 % CD OD) δ: 0.69 (3H, s, 18-CH ),
perature [16] gave the 24-nor-22,23-epoxides (5a and 5b)
in good isolated yields (85 and 83 %). Subsequent oxida-
tive-cleavage of the 22,23-epoxide ring in 5a and 5b with
3
3
3
.91 (3H, s, 19-CH ), 1.04 (3H, d, J = 6.3 Hz, 21-CH ),
3
3
.41 (1H, brm, 3β-H), 3.83 (1H, brs, 7β-H), 4.96 (1H,
1
3
19
dd, J = 51.0, 9.8 Hz, 23-H). C NMR: see Table 3.
F
orthoperiodic acid (HIO ) in THF at room temperature [16]
4
NMR (CDCl containing 20 % CD OD) δ: −191.89 (1F,
afforded the corresponding C22 23,24-dinor-22-aldehyde
derivatives (6a and 6b) in isolated yields of 43 and 45 %,
respectively.
3
3
−
brs, 23-F). LC–ESI–MS: Calcd. for C H FO [M−H] ,
2
4
38
4
4
09.2754; Found, m/z 409.2798.
1
3

Lipids
Fig. 2 Synthetic route to epimeric (23R)- and (23S)-fluoro-bile acids from CA and CDCA
When 6a and 6b were subjected to the Horner-Wads-
worth-Emmons reaction with triethyl 2-fluoro-2-phos-
phonoacetate in the presence of LiCl and 1,8-diazabicy-
clo[5,4,0]undec-7-ene (DBU) as base-sensitive reagents
probably due to steric hindrance of the hydroxyl group.
Careful separation of each of the epimeric mixtures by pre-
parative RP-HPLC provided two well-separated fractions.
The more polar fractions of each of the epimeric mixtures
eluted with a mixture of 15 mM ammonium acetate/acetic
acid buffer solution (pH 5.4) and methanol (35:65 ~ 33:67,
v/v) were characterized as the desired (23R)-23-fluoro-
3α,7α,12α-trihydroxy acid (1a‑R) and (23R)-23-fluoro-
3α,7α-dihydroxy analog (1b‑R) and less polar fractions
as the corresponding (23S)-epimers (1a‑S and 1b‑S) as
described below.
[
17], the reaction proceeded smoothly to give the more
thermodynamically stable (E)-isomer of the C24 α-fluoro-
α,β-unsaturated esters (7a and 7b) in reasonable isolated
yields of 70 and 72 %. The stereochemical configuration
2
2
of the Δ -bond in 7a and 7b was conclusively deter-
1
mined as the E-form, on the basis of a multiplet H signal
1
at 22-H appearing at 5.68-5.73 ppm in the H NMR spec-
1
3
13
tra [18–20]. The characteristic C doublet signals occur-
Table 3 shows the C NMR chemical shifts for the iso-
1
ring at 128.5–129.0 (J, 14.8-15.5 Hz), 145.4–145.6 (J,
lated 1a‑R, 1a‑S, 1b‑R, and 1b‑S. In the H NMR spectra,
2
49.0–250.4 Hz), and 160.9–161.0 (J, 35.8–36.8 Hz) ppm
the two epimeric pairs showed essentially identical spectral
1
3
were also confirmed as C-22, C-23, and C-24, respectively,
with a fluoro atom at the C-23 position. Furthermore, the
patterns and chemical shift values. In the C NMR spec-
tra, however, a doublet signal (J, 180.0–183.6 Hz) arising
from the C-23 (89.2 ppm) in the (23R)-epimers (1a‑R and
1b‑R) resonated at a lower field than the corresponding
signal (87.2 and 86.7 ppm) in the (23S)-epimers (1a‑S and
1b‑S), strongly suggesting the presence of a fluoro atom at
C-23 and the stereochemical difference. A minor, but con-
sistent chemical shift difference between the two epimeric
pairs was observed for the C-20 and C-21 signals. A sig-
nificantly large, distinct difference was also observed in the
1
9
F signal was observed as the triplet signal (J, 29.3 Hz) at
−
131.8 ppm in 7a and at −131.0 ppm in 7b.
Catalytic hydrogenation of the α-fluoro-α,β-unsaturated
esters (7a and 7b) in the presence of Pd/C as a catalyst
yielded an epimeric mixture of the (23R)- and (23S)-
2
3-fluoro-peracetate esters (8a and 8b) nearly quantita-
tively. Without isolating each of the epimeric 23ξ-fluoro
acetate-ethyl esters, the mixtures were hydrolyzed with
methanolic NaOH to afford epimeric mixtures of the free
1
9
F NMR spectra, in which the 23-F signals (−185.22 and
2
3ξ-fluoro acids (1a and 1b). Figure 3 shows the RP-HPLC
of 1a and 1b, in which the presence of an axially-oriented
2α-hydroxy group in 1a appreciably decreased the for-
mation of one (1a‑S) of the two epimers (ratio of 9:1),
−185.85 ppm) in the (23R)-epimers (1a‑R and 1b‑R) reso-
nated at an appreciably higher field than the corresponding
signals (−191.92 and −191.89 ppm) in the (23S)-epimers
(1a‑S and 1b‑S).
1
1
3

Lipids
Fig. 3 RP-HPLC profile of the epimeric mixtures of the 23ξ-fluoro derivatives of (1) 3α,7α,12α-trihydroxy-5β-cholanoic acid (1a), and (2)
3
α,7α-dihydroxy-5β-cholanoic acid (1b)
Fig. 4 The molecular and crystal structures of compound 1b‑S
Fractional crystallization of 1b‑S from methanol gave
single-crystals suitable for X-ray measurement. Figure 4
shows the molecular and crystal structures of 1b‑S deter-
mined by X-ray analysis, respectively. Table 1 shows the
crystallographic details for 1b‑S. Selected bond lengths
and angles for 1b‑S are also given in Table 2. 1b‑S crys-
tallizes in the monoclinic system with space groups P21,
with one independent molecule in an unit cell. The crystals
contain about 1.5 molecule of water per molecule of 1b‑
S, likely from methanol used for recrystallization that had
not been dried prior to use. Thus, the X-ray analysis pro-
vided directly the absolute configuration at C-23, in anal-
ogy with hydroxylated derivatives epimeric at C-23, C-25,
C-22/C-25 or C-24/C-25 of bile acids and bile alcohols
[14, 21–24], and conclusive evidence for the stereochemi-
cal assignment of 1b‑S as the (23S)-configuration, which
was the secondary-eluted peak in Fig. 3 (2); 1b‑R was
therefore assigned as (23R). Based on the above finding
and the C and F NMR characteristics, analogous 1a‑R
and 1a‑S were assigned as (23R)- and (23S)-configuration,
respectively.
The biological evaluation of the synthesized 23-fluoro-
bile acids is now progressing in our laboratory, and the
result will be reported elsewhere.
1
3
19
Acknowledgments This work was supported in part by a Grant-in-
Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Sciences and Technology of Japan (to T.I., 21550091) for
2
012-2014.
1
3

Lipids
1
3. Acebedo SL, Alonso F, Galagovsky LR, Ramírez JA (2011) Syn-
thesis and biological activity of ring-A difluorinated brassinos-
teroids. Steroids 76:1016–1020
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A, Hagey LR, Hofmann AF, Iida T (2014) Improved chemical
synthesis, X-ray crystallographic analysis, and NMR charac-
terization of (22R)-/(22S)-hydroxy epimers of bile acids. Lipids
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