D. Yu et al. / Steroids 77 (2012) 1335–1338
1337
under argon or nitrogen. Melting points were determined and re-
ported automatically by an optoelectronic sensor in open capillary
tubes and were uncorrected. 1H NMR and 13C NMR spectra were
measured at 500 and 125 MHz, respectively, and using CDCl3 or
CD3OD as the solvents and tetramethylsilane (Me4Si) as the inter-
nal standard. Flash column chromatography was performed using
Sigma–Aldrich silica gel 60 (200–400 mesh), carried out under
moderate pressure by using columns of an appropriate size packed
and eluted with appropriate eluents. Silica gel chromatography
was performed on a Biotage flash column gradient pump system
using 15 cm long columns. All reactions were monitored by TLC
on precoated plates (silica gel HLF). TLC spots were visualized
either by exposure to iodine vapors or by irradiation with UV light.
7α-OH (axial)
3α-OH (equatorial)
Fig. 3. MM2 structure of CDCA (Chem3D). Ovals indicate pertinent cyclohexane
chairs with the equatorial and axial hydroxyls.
2.1. 3a-Hydroxy-7-keto-5b-cholan-24-oic acid (1)
To a suspension of chenodeoxycholic acid (CDCA, Sigma–
Aldrich, 1.0 g, 2.5 mmol) and silica gel (4 g, 200–400 mesh, Aldrich)
in anhydrous CHCl3 (2 mL) was added, portionwise, pyridinium
chlorochromate (PCC, 0.61 g, 2.8 mmol) in 25 mL of CH2Cl2 and
the reaction mixture was stirred at room temperature for 15 min.
The mixture was filtered and the filtrate was washed with water
(20 mL) and brine (20 mL). The organic layer was dried over Na2-
SO4 and concentrated. The resulting crude oil was purified by flash
column chromatography (CH2Cl2: MeOH 95:5) to afford 1 as a solid
(0.79 g, 82% yield), mp 201.1–201.7 oC (lit [11]. mp 201–203 oC). 1H
NMR (500 MHz, CD3OD) d 3.50 (m, 1H), 2.94 (m, 1H), 2.52 (t, 1H),
2.30 (m, 2H), 2.19 (m, 6H), 1.70 (m, 2H), 1.43 (m, 4H), 1.31 (m, 6H),
1.19 (s, 3H), 1.12 (m, 4H), 0.92 (d, 3H), 0.67 (s, 3H). 13C NMR
(125 MHz,CD3OD) d 213.7, 176.8, 70.1, 54.8, 49.2, 48.9, 47.7, 46.0,
44.9, 43.0, 42.4, 38.9, 36.8, 35.1, 34.9, 33.7, 31.0, 30.6, 29.2, 27.8,
24.3, 22.0, 21.4, 17.3, 10.5. Anal. Calcd for C24H38O4: C, 73.81; H,
9.81. Found: C, 73.50; H, 9.63.
CH3
CH3
H
COOH
14
H
9
H
H
H
H
CH
O
2
4
O
Cr
O
O
Cl
H
CH3
CH3
H
COOH
2.2. 3a-Tetrahydropyranyloxy-7-keto-5b-cholan-24-oic acid (2)
H
To a solution of 1 (0.50 g, 1.3 mmol) in 16 mL of CHCl3:Cl2CH2:-
Et2O (1:1:2) were added p-toluensulfonic acid (0.06 g, 0.3 mmol)
and 3,4-dihydro-2H-pyrane (0.41 g, 4.9 mmol). The reaction mix-
ture was stirred at room temperature for 60 min. Water (10 mL)
was added and the mixture was extracted with EtOAc
(3 Â 30 ml); the combined organic layers were washed with satu-
rated NaHCO3 and brine and concentrated. The resulting crude oil
was purified by flash column chromatography (CH2Cl2:Et2O 1:2) to
afford 2 as a white solid (0.51 g, 82% yield), mp 160.0–160.8 oC (lit
[9]. mp 157–159 oC). 1H NMR (500 MHz, CDCl3) d 4.73 (d, 1H), 3.86
(m, 1H), 3.59 (m, 1H), 3.46 (m, 1H), 2.82 (m, 1H), 1.17 (s, 3H), 0.92
(d, 3H), 0.63 (s, 3H). 13C NMR (125 MHz, CDCl3) d 212.3, 179.8, 96.4,
62.8, 62.1, 19.8, 18.1, 11.4. Anal. Calcd for C29H46O5: C, 73.38; H,
9.77. Found: C, 73.30; H, 9.76.
H
H
O
H
CH
2
O
H
Fig. 4. Oxidation of CDCA with PCC.
The identity of 6ECDCA we synthesized in improved way was
secured by comparison of its 1H NMR and 13C NMR with those pre-
viously reported. In a reporter assay we demonstrated that the so
obtained 6ECDCA is a very potent FXR agonist with EC50 values
around 100 nM. This result is consistent with previous observation
[12].
In summary, we have modified several critical steps in the syn-
thesis of 6ECDCA, resulting in an economical and efficient strategy.
The two key synthetic improvements that allow a productive yield
are the use of PCC in the oxidation step and the use of HMPA/ethyl
iodide in the stereo-selective alkylation step that provides a simple
and cost-effective procedure for potential large-scale production of
this promising FXR agonist, which is a research tool and potential
drug substance of current interest.
2.3. 3a-Hydroxy-6a-ethyl-7-keto-5b-cholan-24-oic acid (3)
To a solution of 2 (0.30 g, 0.63 mmol) in dry THF (20 mL) at
À78 oC were added dropwise n-butyllithium (1.0 mL, 1.6 M
solution in hexane, 1.6 mmol), HMPA (0.7 g, 4 mmoL) and LDA
(2.0 mL, 1.8 M in THF/heptane/ethylbenzene, 3.6 mmol). The reac-
tion mixture was stirred for 30 min. Iodoethane (2.0 g, 13 mmol)
was slowly added and the reaction mixture was allowed to warm
overnight to room temperature. After rotary evaporation, water
and ether were added and the aqueous layer was acidified with
10% HCl and extracted with EtOAc (5 Â 20 mL). The organic layers
were washed with brine, dried over Na2SO4, and concentrated to
give a yellow oil. After a short column (CH2Cl2:Et2O 1:2), the
crude semi-solid was dissolved in ethanol (5 mL) and pyridinium
p-toluenesulfonate (15 mg, 0.06 mmol) was added. The reaction
2. Experimental section
General procedures: Organic reagents were purchased from
commercial suppliers unless otherwise noted and were used with-
out further purification. All solvents were analytical or reagent
grade. All reactions were carried out in flame-dried glassware