Process Research and Impurity Control Strategy for Obeticholic Acid, a Farnesoid X Receptor Agonist

Article abstract of DOI:10.1021/acs.oprd.9b00252

The process to obtain ICH-grade quality obeticholic acid (OCA) was improved, and the overall yield was 25.9%. The critical process parameters were established to reduce or avoid process-related impurities. The formation mechanisms, purge pathways, and control strategies for these impurities were also discussed for the first time. An high-performance liquid chromatography instrument utilizing the charged aerosol detection technique was applied for an impurity content assay in OCA for the first time. The developed process was robust and suitable for manufacturing scale-up.

Full text of DOI:10.1021/acs.oprd.9b00252

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Full Paper
Process Research and Impurity Control Strategy for
Obeticholic Acid, a Farnesoid X Receptor Agonist
Weidong Feng, Songming Zhuo, and Fu-Li Zhang
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00252 • Publication Date (Web): 04 Sep 2019
Downloaded from pubs.acs.org on September 4, 2019
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Process Research and Impurity Control Strategy for Obeticholic Acid,
a Farnesoid X Receptor Agonist
Wei-Dong Feng†, Song-Ming Zhuo‡, Fu-Li Zhang†§*
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†
Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang
University of Technology, 18 Chaowang Road, Hangzhou 310014, China
‡
Department of Pharmaceutial Engineering, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, China
§Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmacetical Industry, 285
Gebaini Road, Pudong, Shanghai 201203, China
*
E-mail: zhangfuli1@sinopharm.com
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TOC graphic
Quality Controllable and Robust Process for Obeticholic Acid
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steps
OH
OH
Overall yield: 25.9%
H
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HO
HO
OH
H
H
KLCA
1) Establish critical process parameters
OCA
Content assay 99.0%
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) Identify 24 process-related substances
) Formation reason and control strategy of impurities
) Apply HPLC analysis technique with charged aerosol detector
) ICH-grade quality
Max. single impurity content <0.1%
Total impurities content <0.5%
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ABSTRACT
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The process to obtain ICH-grade quality obeticholic acid (OCA) was improved, and
the overall yield was 25.9%. The critical process parameters were established to
reduce or avoid process-related impurities. The formation reasons, purge pathways
and control strategies for these impurities were also discussed for the first time. An
HPLC instrument with utilizing the charged aerosol detection (CAD) technique was
applied for an impurity content assay in OCA for the first time. The robust process
was suitable for manufacturing scale-up.
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KEYWORDS
Obeticholic acid, Process, Primary Biliary Cholangitis, Nonalcoholic Steatohepatitis
INTRODUCTION
OCA, which has the brand name Ocaliva, is a first-in-class farnesoid X receptor
(FXR) agonist that acts as a synthetic chenodeoxycholic acid (CDCA) analogue;
Ocaliva was approved by the FDA in May 2016 for the treatment of primary biliary
1
cholangitis (PBC). The new use of OCA for the treatment of patients with
noncirrhotic nonalcoholic steatohepatitis (NASH) should be approved in 2020.
2
The original route for the synthesis of OCA was proposed for the first time by
Pellicciari in 2001, as shown in Scheme 1. Although this process comprises only 4
steps, it presents a series of drawbacks, such as: (a) in all of the steps, the reaction
products are purified on a chromatographic column; (b) the reaction yield for the
preparation of 4 is extremely low (12%), while the overall yield is 3.1%; and (c)
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hexamethylenphosphonamide (HMPA) is used as a reactant, which is a known
carcinogen.
Scheme 1. Original Synthetic Route for OCA
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O
OH
DHP, TsOH
O
OH
n-BuLi, HMPA, diisopropylamine, EtBr
H
H
o
THF, -78 o
C
Dioxane, 25 C
74%
O
H
H
O
H
H
O
HO
12%
O
H
H
2
3
O
O
O
O
O
OH
H
H
H
NaBH4
NaOH, H O
2
H
H
OH
H
H
H
H
H
o
EtOH, 25 C
EtOH, reflux
43%
HO
HO
OH
HO
O
H
H
81%
4
5
1
The original commercial synthetic route for OCA was also developed by Pellicciari
3
4
in 2006, which was further developed by Steiner in 2013. The synthetic process of
OCA based on the reported commercial route is further researched by our group, as
5
shown in Scheme 2. Some routes similar to the commercial route have been reported
as well, and the ethyl of the 6-postion of OCA is constructed by a method similar to
the Mukaiyama reaction, which does not have obvious advantages over the above
6
route. The use of different starting materials to prepare OCA, such as cholic acid or
7
hyodeoxycholic acid (HDCA), have also been reported; however, the intermediates in
the novel synthetic routes need to be purified by a chromatographic column. Although
Scheme 2 utilizes more steps than Scheme 1, the total yield is satisfactory, and the
intermediates do not need to be purified by a chromatographic column as previously
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-4
reported, however, the drug substance OCA with ICH-grade quality is not obtained,
and the total yield is only 10%, even though we tried our best to follow the reported
procedures.
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Scheme 2. Improved Commercial Synthetic Route for OCAa
O
O
O
OH
O
O
H
H
H
Step 1
a
Step 2
b
H
H
O
H
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O
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HO
HO
TMSO
OTMS
H
H
H
6
7
2
KLCA
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OH
Step 3
c
H
Step 4
H
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d
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H
8
9
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OH
OH
H
H
Step 5
Step 6
f
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O
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e
HO
HO
OH
H
H
10
1
a
Reagents and conditions: (a) H SO , MeOH, 50~55 °C, 3 h, 90%; (b) LDA, TMSCl, THF,
2
4
-
60~-65 °C, 5 h, colored substance treated with silica gel adsorbent; (c) BF ·MeCN, CH CHO,
3 3
DCM, -60~-65 °C, 2.5 h, and then warmed to -5~-10 °C, 2 h; (d) NaOH, H O, EtOH, 20~25 °C, 1 h,
2
recrystallized twice with EtOH and H O, 52% yield from 6 to 9; (e) 10 wt% Pd/C, H , NaOH, H O,
2
2
2
20~25 °C, 10 h, and then heated to 95~100 °C, 2 h, recrystallized twice with EtOH and H O, 79%;
2
(
f) NaBH , NaOH, H O, 70~75 °C, 6 h, recrystallized twice with n-butyl acetate and n-heptane, 70%.
4 2
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The safety of a drug is dependent not only on the toxicological properties of the
bulk drug itself but also on the impurities in the bulk drug, and the presence of these
unwanted impurities, even in small amounts, may influence the efficacy and safety of
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the pharmaceutical product. Therefore, identification, quantification, and control of
impurities in the drug substance and drug product are important steps in drug
9
development and regulatory assessment. These unknown impurities (>0.10%) in the
drug substance must be identified and characterized according to ICH guidelines.10
Therefore, developing a practicable process to obtain OCA with ICH-grade quality for
commercialization is highly desirable.
RESULTS AND DISCUSSION
We herein report the quality controllable and robust process for the synthesis of
OCA, as shown in Scheme 2. The process-related impurity profiles for the preparation
of OCA are shown in Table 1.
Table 1. Impurity Profiles for 1 (OCA)
Step
0
1
2 and 3
4
5
6
R1
R2
R2
R2
R1
R1
9
R1
1 (OCA)
2
(KLCA)
6
6a
9a
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OH
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TMSO
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R1
10
7
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TMSO
OTMS
R2
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R2
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H
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H
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H
R1
R2
R2
R2
R1
R1
Imp-1
Imp-4
Imp-7
Imp-8
H
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OH
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Imp-2
R1 Imp-5
Imp-9
R3
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H
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HO
O
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R1
R1 Imp-6
Imp-10
Imp-3
H
H
H
H
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H
HO
OH
O
OH
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OH
H
H
R1
Imp-11
H
H
H
HO
OH
H
R1
Imp-12
H
H
H
HO
OH
H
R3
Imp-13
H
H
H
HO
OH
H
H
HO
H
OH
R1
H
OH
O
O
Imp-14
H
O
O
OH
O
OH
Note: R1 =
; R2 =
; R3 =
.
Preparation of Methyl 3α-Hydroxy-7-keto-5β-cholanate (6, Step 1)
The starting material 2 (KLCA, purity>98.5%) was purchased from the supplier,
which was synthesized from chenodeoxycholic acid (CDCA), and Imp-1 (CDCA),
Imp-2 and Imp-3 (Table 1) were detected at approximately 0.46%, 0.44% and 0.36%
by HPLC, respectively. The esterification of 2 was carried out smoothly in the
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presence of H SO and MeOH, and no more than 0.5% of 2 remained, as determined
2
4
by HPLC. Imp-4 (0.48%), Imp-5 (0.40%) and Imp-6 (0.31%) (Table 1) were
generated from the esterification of Imp-1, Imp-2 and Imp-3, respectively. These
impurities were removed successfully, and no more than 0.1% was found by HPLC
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through recrystallization using MeOH and H O (1.5:1); the purity of 6 was greater
2
than 99.5%.
Preparation of 3α-Hydroxy-6-ethylidene-7-keto-5β-cholanic Acid (9, Steps 2, 3
and 4)
The alkenyl of the 6-position of 9 was constructed with the Mukaiyama reaction,
which was the most critical step and the most difficult to control in the process. The
Mukaiyama aldol addition is a type of aldol reaction between a silyl enol ether and an
aldehyde or formate, and the reactants allow for a crossed aldol reaction between an
aldehyde and a ketone or a different aldehyde without self-condensation of the
aldehyde.11
Obviously, the formation of silyl enol ethers 7 required low temperature to improve
the regioselectivity, and the more reasonable order of addition was that 6 was added
slowly to a mixture of TMSCl and LDA at -60~-65 °C. The crude 7 was a dark brown
oil that could not be purified by recrystallization, and the colored substance and the
impurities with high polarity were brought into the subsequent steps, which led to the
recovery rate of the purification of 9 being significantly decreased. Furthermore, the
hydrogenation of 9 did not occur completely because the Pd/C catalyst could be
poisoned by the colored substance. The colored substance was not adsorbed onto
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activated carbon at all. However, it adsorbed well with silica gel in n-heptane, and
then, it was filtered and washed with a mixed solvent of n-heptane and ethyl acetate
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6
7
8
9
1
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(20:1, v/v). The weights of the silica gel and solvent were only 2 times and 20 times
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that of 7, respectively. The in process control (IPC) for preparing 7 was monitored by
thin-layer chromatography (TLC), and TMSCl and LDA were added once more in
proportion. In this case, the spots of 6 or 6a (Table 1) were still observed by TLC.
Since 7 was not stable in acidic conditions and acetaldehyde (bp: 20.8 °C) could
easily volatilize at room temperature, the DCM solution of 7 and acetaldehyde were
4
poured into the mixture of BF and DCM, as described in the patent. Typically,
3
however, the solution of BF would be added slowly to the DCM solution containing 7
3
and acetaldehyde at -60~-65 °C. 7a (Table 1) was generated from the aldol addition
reaction of 7 and acetaldehyde, and 8 was obtained from the elimination of water from
7
a. After the reaction, 6 and 7a were detected at approximately 3.5% and 0.2% by
HPLC, respectively, and 6 was further hydrolyzed to 2 in step 4, while 2 and 7a would
be reduced or removed in the subsequent steps.
Initially, the hydrolyzation of 8 was carried out in a solution of NaOH, H O and
2
4
MeOH at 50 °C for 2 h, and the amounts of 2 increased continually with prolonged
time. Consequently, it is indicated that trans-9 was degraded to 2 and cis-9 under
alkaline conditions, and the formation mechanism was proposed, as shown in Scheme
3
. Impurity 2 was likely formed via the Michael addition of trans-9 followed by the
retro-aldol addition of 9c in NaOH aqueous solution. 9e was the rotational
conformational isomer of 9b, which upon retro-Michael addition with hydroxide
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would lead to cis-9 and would also produce 10 in a subsequent hydrogenation
reaction. There were three sources of impurity 2 in the preparation of 9; the first
source was from the hydrolyzation of impurity 6, the second source was from the
hydrolyzation of 7a to 9c followed by the retro-aldol addition of 9c, and the third
source was from the degradation of 9. Obviously, the degree of degradation of 9 was
related to the hydrolytic conditions. The hydrolyzation of high purity 8 was carried out
in various conditions to investigate the content changes of impurity 2, and the results
are summarized in Table 2. The hydrolyzation of 8 was carried out at 50 °C for 1~2 h,
and 2 was greater than 0.2% whether 1.2 equiv. or 1.5 equiv. of NaOH were used
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(entries 1 and 2), while 2 increased continually with prolonged time (entries 3 and 4).
However, 2 was present at no more than 0.2% if the hydrolyzation of 8 was performed
at 25 °C for 1 h (entry 5), and product 9 was relatively stable at 25 °C for 8 h (entry 6).
Therefore, the hydrolyzation of 8 should be carried out at 25 °C for 1 h. Although
crude 9 was recrystallized with EtOH to give high purity 9, the yield was only 31%
from 6 to 9, even though the weight of the recrystallization solvent was only 2~3 times
than that of 9; furthermore, the recrystallization mixture was too sticky to filter. As
expected, 9 was recrystallized twice with a mixture of EtOH and H O (1:1), and the
2
yield was 52% from 6 to 9. Finally, the key impurity 2 was found in quantities of no
more than 1.0%, and the purity of 9 was greater than 98.0% by HPLC. Furthermore,
the recrystallization mixture was easily filter.
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Scheme 3. Proposed Degradation Mechanism of trans-9 in NaOH Aqueous Solution
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
OH
OH
OH
OH
OH
H
H
H
O
H
H
OH, H2O
OH, H O
- CH3CHO
- OH
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
O
Michael addition
H
H
O
H
H
O
H
Retro-Aldol
HO
H
H
O
H
H
O
HO
HO
HO
HO
H
H
H
H
H
OH
H
OH
OH
OH
trans-9
9b
9c
9d
H
OH
2
O
O
O
OH
OH
OH
H
H
H
OH
HO
- OH
H
H
H
H
O
H
H
O
H
H
Retro-Michael addition
HO
O
HO
H
H
H
OH
HO
HO
9e
cis-9
9e
Table 2. Hydrolyzation of High Purity 8 under Various Conditions
O
O
O
O
OH
OH
Step 5
H
H
H
NaOH, H O
2
H
H
O
+
H
H
O
H
H
O
MeOH
HO
HO
HO
H
H
H
8
9
2
HPLC (%)b
2
entrya NaOH (equiv.) T/t (°C/h)
8
9 (cis /trans)
97.13
1c
2d
3
1.2
1.5
1.5
1.5
1.5
1.5
50/2
50/1
50/2
50/8
25/1
25/8
2.60
0.12
0.11
0.12
0.12
0.10
0.27
0.25
99.63
0.61
99.28
4
1.97
97.91
5
0.05
99.83
6
0.15
99.75
a
Reaction solvent: EtOH (6 v/w) and H O (1 v/w); the purity of 8 is over 99.0%, and impurity 6 is
2
b
present at quantities of no more than 0.02% by HPLC. Only peak areas (%) of 8, 2 and 9 were
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c,d
integrated by HPLC. The reaction conditions followed references 4 and 3, respectively.
Preparation of 3α-Hydroxy-6α-ethyl-7-keto-5β-cholanic Acid (10, Step 5)
The reaction mechanism from 9 to 10 was proposed as shown in Scheme 4, and 9a
was the main hydrogenated product of 9 due to steric hindrance effect. Compound 10,
with a smaller steric hindrance, was obtained via the tautomerism of the α-ketone of
the 6β-ethyl intermediate (9a) under strong alkaline conditions. In the spatial stereo
structure of 9a, the conformation of 5β-H, 6β-ethyl and 7-ketone in B-ring was at the
equatorial (e), axial (a) and equatorial (e) bond orientation, respectively. However, the
conformation of 5β-H, 6α-ethyl and 7-ketone in B-ring was all at the equatorial (e)
bond orientation. Obviously, the conformation of 10 was more stable than that of 9a,
and more than 97% 9a was finally transformed into 10 in NaOH aqueous solution.
Scheme 4. Proposed Mechanism for the Preparation of 10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
OH
OH
OH
H
H
H
Pd/C, H2
OH, H O
2
+
H
H
O
H
H
H
O
H
H
O
HO
HO
HO
H
H
H
H
9
9a
10
O
O
O
OH
OH
a
H
OH
e
H
H
H
6
Oe
7
5
Tautomerism
Tautomerism e
HO
H
H
H
H
O
e
HO
O
HO
H
H
H
H
O
OH
H
H
9a
9f
10
The hydrogenation reduction and configuration inversion from 9 to 10 was
performed in NaOH aqueous solution, which was environmentally friendly and safety
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without any organic solvent. As mentioned above, 9 was partially degraded in NaOH
aqueous solution at high temperature. The hydrogenation of 9 under various
conditions was screened, and the results are shown in Table 3. The reduction was
initially carried out at 25 °C/1 atm with 5% (w/w) Pd/C for 8 h, and 9.93% 9 remained
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(entry 1). When the reaction time and pressure were increased to 16 h and 3 atm,
respectively, and 3.74% 9 still remained (entry 2). When the reaction was carried out
at 25 °C/3 atm with 10% (w/w) Pd/C for 10 h, only 0.42% 9 remained (entry 3).
Fortunately, impurity 2 from the degradation of 9 was increased to less than 0.1% at
25 °C in 8 h; however, impurity 2 increased over 0.5% by HPLC at 50 °C in 8 h
(
entries 4 and 5). Although the hydrogenation of 9 was rapid at 75 °C, impurity 2 was
increased over 0.7% in 2 h (entry 6). Therefore, the hydrogenation of 9 was performed
with 10 wt% Pd/C (10% w/w), NaOH (2 equiv.), H O and H (pressure: 3 atm) at
2
2
2
5 °C for 10 h, and the ratio of 9a to 10 was approximately 50:50, as determined by
HPLC.
Table 3. Hydrogenation of 9 under Various Conditions
O
O
O
O
OH
OH
OH
OH
Step 6
H
H
H
H
Pd/C, H2
H
H
O
H
H
O
+
H
H
O
+
H
H
O
HO
NaOH, H O HO
HO
HO
2
H
H
H
H
9
9a
10
2
HPLC (%)c
entrya T/t (°C/h) P (atm) Pd/C (%w/w)
b
9
2
9a&10
1
25/8
1
5
9.93
0.77
89.30
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3d
4
25/16
25/10
50/8
3
3
3
3
3
5
3.74
0.42
0.30
0.28
0.20
0.80
0.85
1.16
1.67
1.59
95.46
98.73
98.54
98.05
98.21
10
10
10
10
5
50/16
75/2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
a
Reaction conditions: NaOH (2 equiv.), H O (5 v/w); the purity of 9 is 98.5%, and impurity 2 is
2
b
c
0
.82% by HPLC. 10 wt% palladium on carbon (wetted with ca. 55% water). Only the peak areas
d
(%) of 9, 2 and 9a & 10 were integrated by HPLC. The peak areas (%) of 9a and 10 were 50.02 and
4
8.71 by HPLC, respectively.
The configuration inversion from 9a to 10 was carried out in NaOH aqueous
solution, and the conversion rate was related to the temperature, as shown in Table 4.
a, and was greater than 3.0% by HPLC when the temperature was below 50 °C for 16
9
h (entries 1 and 2); 2.87% 9a remained at 75 °C for 5 h (entry 3). The amount of 9a
remaining was 2.78% at 95 °C after only for 2 h (entry 4), and 2.75% 9a still remained
even though the time was prolonged to 8 h (entry 5). Interestingly, approximately
2
.7% 9a was detected by HPLC even though the high purity (>99.5%) 10 was used in
the presence of NaOH (2, 3 or 4 equiv.) aqueous solution at 95 °C for 2 h, and this
result indicated that the configuration inversion from 9a to 10 was an equilibrium
reaction. Initially, the emission of hydrogen was performed immediately after the
hydrogenation of 9 considering safety; then, the configuration inversion of 9a was
carried out at 95 °C for 2 h. Approximately 10% of an unknown impurity was detected
by HPLC, and it increased with prolonged time. The impurity was identified by MS
and NMR, and the structure of the impurity is shown in Table 1 (Imp-7). To the best
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of our knowledge, it has not yet been reported that the oxidation of a hydroxyl group
to a ketone carbonyl was carried out in the presence of Pd/C, air and NaOH aqueous
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
solution. Despite that, the reduction of Imp-7 with NaBH would produce the final
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
product 1 in the next step, and the recovery yield of 10 was decreased because Imp-7
was easily removed in the crystallization process of crude 10. Therefore, the
coexistence of Pd/C with air should be avoided for the configuration inversion from 9a
to 10 after completing hydrogenation. To prevent the coexistence of Pd/C and air, two
approaches could be used: first, the configuration inversion process could be
performed in a hydrogen atmosphere, and second, the Pd/C filtered off prior to the
reaction. Obviously, the former was performed easily in a pilot plant.
Table 4. Screening of the Temperature and Time for Configuration Inversion from 9a
to 10
O
O
O
OH
OH
OH
H
H
H
NaOH, H O
2
+
H
H
O
H
H
O
H
H
O
HO
HO
HO
H
H
H
9a
10
10
HPLC (%)b
entrya T (°C) t (h)
9a
10
1
2
3
4
5
25
50
75
95
95
16 12.43
16 3.01
87.57
96.99
97.13
97.22
97.25
5
2
8
2.87
2.78
2.75
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reaction conditions: NaOH (2 equiv.), H O (5 v/w); the initial ratio of 9a to 10 was approximately
2
b
5
0:50 by HPLC. Only the peak areas (%) of 9a and 10 were integrated on the HPLC.
Approximately 0.8% 2, 0.5% 9 and 2.8% 9a were detected by HPLC in crude 10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(HPLC purity: 91.2%), and these impurities would lead to the generation of new
impurities (Table 1: Imp-1, Imp-8, Imp-9, Imp-10 and Imp-11) in step 6. The above
impurities in crude 10 have already been controlled well in the hydrogenation reaction,
and 10 was further recrystallized twice using a mixture solution of EtOH and H O
2
(
1:1). Approximately 0.3% 2, 0.3% 9 and 0.5% 9a were detected in intermediate 10
(HPLC purity: 97.3%).
Preparation of 3α,7α-Dihydroxy-6α-ethyl-5β-cholanic Acid (1, Step 6)
OCA (1) was obtained by the reduction of 10 with NaBH in NaOH aqueous
4
4
solution. The reaction mechanism from 10 to 1 was proposed as shown in Scheme 5,
and the 7-ketone of 10 was reduced by hydride ion more easily from β-plane to
α-plane due to the 6α-ethyl of 10 with high steric hindrance in α-plane. The reduction
reaction of 10 with NaBH in various conditions is shown in Table 5, and a large
4
amount of the starting material 10 remained when it was carried out at 25 or 50 °C for
2
4 h (entries 1 and 2), while 0.68% of the starting material remained with increasing
the reaction temperature to 75 °C (entry 3). Despite the reduction reaction being
performed at 95 °C for 3 h (entry 4), Imp-12 was produced, resulting in a content
greater than 3.0%, as determined by HPLC. The amounts of Imp-12, Imp-13 (Table 1)
and 10 were almost unchanged despite the different stoichiometry of NaOH in the
reduction reaction (entries 4, 5 and 6). To summarize the above results, the optimized
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reaction conditions were carried out with NaBH (1.1 equiv.), NaOH (2 equiv.) and
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H O at 75 °C for 6 h.
2
Scheme 5. Proposed Mechanism for the Preparation of 1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
OH

plane
H
a
H
OH
H
OH
H
H
H
H
6
O
O
7
e
H
NaBH , NaOH
H
H
H
H
4
H
B
H
H
HO
HO
HO
OH
e
H O
2
H
H
H
H
H

plane
1
0
1
Table 5. Reduction Reaction of 10 with NaBH under Various Conditions
4
O
O
O
OH
OH
OH
OH
H
H
H
H
NaBH4
HO
+
H
H
+
H
H
O
H
H
H
H
HO
OH
HO
OH
HO
OH
H
H
H
H
10
Imp-12
Imp-13
1
HPLC (%)b
entrya NaOH (equiv.) T/t (°C/h) Imp-12
Imp-13
ND
10
1
1
2
3
4
5
6
2
2
2
2
3
4
25/24
50/24
75/6
95/3
95/3
95/3
0.75
2.24
2.97
3.61
3.53
3.65
80.69 18.56
12.96 84.80
0.68 96.30
0.62 95.71
0.57 95.85
0.72 95.56
ND
0.05
0.06
0.05
0.07
a
b
Reaction conditions: NaBH (1.1 equiv.), H O (8 v/w). Only the peak areas (%) of Imp-12, Imp-13,
4
2
1
0 and 1 were integrated on the HPLC.
The dimeric impurity Imp-14 (Table 1) was generated from the self-esterification of
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0 in acidic conditions, which was removed by usual recrystallization with difficulty.
The level of Imp-14 had been controlled well and was no more than 0.1% in the final
product, and HOAc aqueous solution, rather than diluted hydrochloric acid, was used
to adjust the alkaline reaction mixture until the pH=3~4. Imp-1 was almost not
removed at all by recrystallization using the usual solvents except for n-butyl acetate.
The recrystallization recovery yield of crude 1 was only 60% even though the weight
of n-butyl acetate was only 2~3 times than that of 1, and the amount of Imp-1 was
only reduced by half at every recrystallization according to the spiking/purge studies
of the impurity. Furthermore, the recrystallization mixture was too sticky to filter, and
the drawback was that some of the mother liquor containing the impurities remained in
the crystals. The crude 1 was recrystallized twice with n-butyl acetate and n-heptane
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(1:1, v/v), which provided a good yield (~70%) of OCA with ICH-grade quality, and
the recrystallization mixture was filtered smoothly.
Control Strategy and Impurity Limit of Process Related Impurity in OCA
Drug Substance
Our primary goal was not only to obtain the high quality drug substance but also to
develop a quality controllable and robust process. The process parameters were
optimized and improved well as above described. The structures and physical
properties of process-related impurities, intermediates and 1 are very similar to each
other; therefore, the purification of crude 1 to obtain high-purity OCA was a highly
challenging task. The control strategy and impurity limit of process related impurities
were proposed, as shown in Table 6.
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Table 6. Control Strategy and Impurity Limit of Process Related Impurity
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Intermediate
Crude product
Control strategy
Impurity limit
9a
2 (3.5%)
Recrystallized twice with EtOH/H O:
2≤1.0%
2
(
1) 2≤1.5%;
2) 2≤1.0%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
10a
2 (0.8%)
Recrystallized twice with EtOH/H O:
2≤0.3%
2
9
9
(0.5%)
(1) 2≤0.6%; 9≤0.4%; 9a≤1.5%;
(2) 2≤0.3%; 9≤0.3%; 9a≤0.5%.
Recrystallized twice with n-butyl acetate/
n-heptane:
9≤0.5%
a (2.8%)
9a≤0.5%
2<0.10%
9<0.10%
9a<0.10%
1b
2 (ND)c
(ND)
9a (ND)
0 (0.61)
9
1
(1) 10≤0.20%; Imp-1≤0.15%; Imp-10≤0.25%; 10<0.10%
Imp-1 (0.32%)
Imp-8 (0.23%)
Imp-9 (0.04%)
Imp-12≤0.10%; Imp-8(ND); Imp-9(ND);
Imp-1<0.10%
Imp-11(ND); Imp-13(ND); Imp-14(ND);
Imp-8<0.10%
Imp-9<0.10%
Imp-10<0.10%
Imp-11<0.10%
Imp-12<0.10%
Imp-13<0.10%
Imp-10 (0.43%) (2) 10(ND); Imp-1<0.10%; Imp-10<0.10%;
Imp-11 (0.27%)
Imp-12 (2.82%)
Imp-13 (0.02%)
Imp-14 (0.04%)
Imp-12 (ND).
Imp-14<0.10%
b
a
Content assay of impurities in crude 9 and 10 using peak area normalization method. Content assay
c
of impurities in crude 1 using the main component self-compare method. No detected on HPLC.
As discussed above, Imp-1, Imp-8, Imp-9, Imp-10 and Imp-11 were generated from
the NaBH reduction of 2, 9 and 9a in intermediate 10, and the key of quality control
4
of these impurities was to control the contents of 2, 9 and 9a. Imp-1 mainly originated
from steps 1, 2, 3 and 4, and impurity 2 was no more than 3.5% in intermediate 9 on
HPLC, and the impurity limits of 2 must be set in a strict quality specification so as to
ensure that the content of Imp-1 no more than 0.1%. Imp-8 and Imp-9 were generated
from 9, and therefore the control strategy of Imp-8 and Imp-9 was that the content of 9
should be controlled no more than 0.5% in step 5. Impurity 9a is a diastereomer of 10,
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7
8
9
1
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and Imp-10 and Imp-11 are diastereomers of OCA which generated from 9a, and
therefore the control strategy of Imp-10 and Imp-11 was that the content of 9a should
be controlled no more than 0.5% in step 5. In step 6, Imp-1, Imp-10 and 10 should be
controlled no more than 0.15%, 0.25% and 0.20% after the first recrystallization,
respectively, and the content of all these impurities was no more than 0.1% after the
second recrystallization. Imp-12, Imp-13 (<0.05%) and Imp-14 (<0.05%) were the
byproducts of the preparation of 10, Imp-12 is a diastereomer of OCA, which removed
easily by recrystallization twice.
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
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9
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1
2
3
4
5
6
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8
9
0
As shown in the supporting information, the content assay of the process-related
impurities of OCA calculated them to be no more than 0.1% using the main
component self-compare HPLC method, and the drug substance of OCA was prepared
by our improved process. However, the content assay of the three known impurities in
the original drugs (from Intercept Pharmaceuticals Inc.) calculated content of these
impurities to be over 0.1%.
CONCLUSION
In conclusion, we have developed a quality controllable, reproducible, and
industrially scalable synthetic process for OCA. The high purity intermediate 6 was
obtained smoothly, and the maximum impurity in 6 was less than 0.1%. The
Mukaiyama reaction from 6 to 8 was performed well in a reasonable order of addition
at the given temperature. The hydrolyzation of intermediate 8 was carried out under
the optimized reaction conditions to reduce the generation of 2. Impurity 2 was also
controlled well in the hydrogenation reduction of intermediate 9, and diketone
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impurity Imp-7 content was no more than 1.0% in the improved configuration
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
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5
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5
5
6
inversion conditions from 9a to 10. The NaBH reduction of 10 was carried out in an
4
appropriate temperature to reduce the generation of Imp-12 and Imp-13. Three
intermediates with stable transient states (6a, 7a and 9a) and fourteen process-related
impurities were confirmed and characterized by HRMS and NMR. The formation
mechanisms/reasons, purge pathways and control strategies for the process-related
substances were discussed in detail. The purification processes of 7, 9, 10 and 1 were
efficient, which were reported for the first time. The limits and control strategy for
these impurities were set strictly according to their origination along with the
spiking/purge studies of the impurities. Finally, OCA with ICH-grade quality was
successfully obtained, and the overall process yield was 25.9%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
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5
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9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
All solvents and reagents were purchased from suppliers and were used without
further purification. NMR spectra were recorded on a Bruker Avance III instrument
1
13
operated at 400 MHz or 600 MHz for H NMR and 100 MHz or 150 MHz for C
NMR with Me Si (TMS) as an internal standard. High-resolution mass spectrometry
4
(HRMS) measurements were recorded on a Bruker Daltonics Solarix 7.0T. Melting
points (MP values) were measured on a WRS-1B apparatus. The TLC method was
carried out to monitor steps 2 and 3 using the ethanol solution of 10%
phosphomolybdic acid as the chromogenic agent and n-heptane and ethyl acetate (1:1
or 8:1, v/v) as the developing solvent. Three HPLC methods were developed to
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8
9
1
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2
2
2
2
2
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3
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5
5
6
monitor the reactions and to detect the impurities. (a) The HPLC method was carried
out to monitor steps 1 and 6 and to detect the purity of 6, which was performed on a
Dionex UltiMate 3000 HPLC instrument using a Waters XBridge C18 column (250
mm × 4.6 mm, 3.5 μm) at 30 °C with a flow rate of 1 mL/min and detection at 192 nm
for 20 min or 50 min. The mobile phase involved a mixture of 50% aqueous solution
(pH adjusted to 2.6 by using phosphoric acid) and 50% MeCN with isocratic elution.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
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9
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2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
(b) The HPLC method was carried out to monitor steps 4 and 5 and to detect the
purities of 8, 9 and 10, which was performed on a Dionex UltiMate 3000 HPLC
instrument using a Waters XBridge C18 column (250 mm × 4.6 mm, 3.5 μm) at 30 °C
with a flow rate of 1 mL/min and detection at 200 nm for 35 min. The mobile phase
involved an aqueous solution (pH adjusted to 2.6 by using phosphoric acid) as phase A
and MeCN as phase B. The HPLC analyses were accomplished with a gradient elution
program (time (min)/% B: 0/40, 25/60, 35/90, 35/40, and 40/40). (c) The content assay
of the related impurities of OCA was performed using the main component
self-compare HPLC method. The HPLC method was carried out on a Dionex UltiMate
3
000 HPLC instrument with Corona CAD using a Waters Cortecs Shield RP18
column (150 mm × 4.6 mm, 2.7 μm) at 35 °C with a flow rate of 1 mL/min for 60 min.
The mobile phase involved an aqueous solution (0.1% formic acid) as phase A and
MeCN as phase B. The HPLC analyses were accomplished with a gradient elution
program (time (min)/% B: 0/35, 35/95, 50/95, 50.1/35, and 60/35).
Methyl 3α-Hydroxy-7-keto-5β-cholanate (6) A 3-L reactor was charged with 2
(
300.0 g, 0.77 mol) and MeOH (1.5 L) under an atmosphere of nitrogen. H SO (2 g,
2
4
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0.4 mmol) was added slowly to the stirring mixture at 20~25°C. The reaction mixture
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was then heated to 50~55 °C for 3 h. The reaction was monitored by HPLC and
deemed complete when 2 was less than 0.5%. NaHCO (3.4 g, 40.8 mmol) aqueous
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solution (0.5 L) was added slowly to the reaction mixture at 20~25 °C over 30 min.
The mixture was stirred at 5~10 °C for 1 h until good precipitation appeared. Another
portion of water (0.5 L) was added slowly at 5~10 °C over 30 min. After stirring for 1
h, the solid product was isolated by filtration, washed twice with a mixture of MeOH
and H O (1:2, 0.5 L × 2) and then dried in an air-drying oven at 55~60 °C to yield the
2
title compound as a white solid (279.7 g, yield: 90%, HPLC purity>99.5%, water
1
content<1.0%). Mp 88.8 ~ 99.1 °C. H NMR (400 MHz, DMSO-d ) δ 4.49 (d, J = 3.2
6
Hz, 1H), 3.57 (s, 3H), 2.90 (dd, J = 4 Hz, 8.4 Hz, 1H), 2.44 (t, J = 7.6 Hz, 1H),
2.31-2.35 (m, 1H), 2.18-2.23 (m, 1H), 2.03-2.09 (m, 1H), 1.92 (dt, J = 2.4 Hz, 6.4 Hz,
1
0
H), 1.78-1.84 (m, 2H), 1.66-1.73 (m, 4H), 1.00-1.51 (m, 14 H), 1.14 (s, 3H),
13
.89-0.94 (m, 1H), 0.87 (d, J = 4.4 Hz, 3H), 0.61 (s, 3H); C NMR (100 MHz,
DMSO-d ) δ 211.94, 174.21, 69.55, 54.69, 51.69, 49.26, 49.05, 45.85, 45.54, 42.65,
6
3
1
9.03, 37.89, 35.24, 35.19, 34.33, 31.11, 30.83, 30.30, 28.31, 24.87, 23.27, 21.68,
+
8.66, 12.35. HRMS m/z [M+Na] Calcd for C H NaO : 427.2824; found: 427.2822.
2
5
40
4
Methyl 3α-Trimethylsiloxy-7-keto-5β-cholanate (7) The water and MeOH in
intermediate 6 (250.0 g, 0.62 mol) were removed by azeotropic distillation with THF
at normal pressure until the water content was no more than 0.05% (Karl Fischer
titration). A 10-L reactor was charged with LDA (1.86 L, 3.72 mol, 2 mol/L) and THF
(1 L) at -60~-65 °C under an atmosphere of nitrogen. TMSCl (336.8 g, 3.10 mol) was
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9
1
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2
2
2
2
3
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5
5
5
5
6
added slowly to the stirring mixture at -60~-65 °C over 20 min. The mixed solution of
and THF (total volume of approximately 1.2 L) was added slowly to the 10-L
6
reactor at -60~-65 °C over 1.5 h, and then, the mixture was stirred at -60~-65 °C for
another 3 h. The reaction was monitored by TLC and deemed complete when the spots
of 6 and 6a disappeared when using n-heptane and EA (1:1 and 8:1, v/v) as the
developing solvent. The reaction mixture was warmed to -5~0 °C and diluted with EA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2 L), and then, citric acid aqueous solution (260 g citric acid in 2 L water) was added
slowly to the mixture over 1 h at -5~0 °C. After the addition, the aqueous phase was
separated and discarded, the organic solvent was evaporated, and it was replaced twice
with n-heptane (1 L × 2) under reduced pressure at 40~45 °C. The oily residue was
added to n-heptane (2.5 L) and 300~400 mesh silica gel (500 g) and then stirred for
another 30 min. The mixture was filtered and then washed with a mixture of n-heptane
and EA (20:1, v/v, 2.5 L), and the organic solvent in the filtrate was evaporated and
replaced twice with DCM (1 L × 2) under reduced pressure at 35~40 °C. Water in
intermediate 7 was removed by azeotropic distillation with DCM at normal pressure
until the water content was no more than 0.05% (Karl Fischer titration). The mixed
solution of 7 and DCM (total volume of approximately 2.5 L) was directly used for the
next step. A small analytical sample of 7 was purified by a chromatographic column.
1
H NMR (400 MHz, CDCl ) δ 4.72 (dd, J = 1.6 Hz, 6 Hz, 1H), 3.66 (s, 3H), 3.47-3.55
3
(m, 1H), 2.31-2.39 (m, 1H), 2.18-2.26 (m, 1H), 1.51-2.00 (m, 10H), 0.99-1.44 (m, 12
1
3
H), 0.92 (d, J = 6.4 Hz, 3H), 0.61 (s, 3H), 0.68 (s, 3H), 0.16 (s, 9H), 0.10 (s, 9H). C
NMR (100 MHz, CDCl ) δ 174.76, 151.80, 108.90, 71.63, 54.95, 54.20, 51.53, 44.49,
3
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2.77, 41.10, 41.05, 40.45, 40.23, 35.39, 34.76, 33.08, 31.18, 31.14, 30.78, 28.79,
7.19, 22.65, 21.05, 18.55, 12.37, 0.47, 0.35.
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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3
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3
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4
4
4
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5
5
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5
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5
5
6
Methyl 3α-Hydroxy-6-ethylidene-7-keto-5β-cholanate (8) A 5-L reactor was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
charged with the mixed solution of 7, DCM (2.5 L) and acetaldehyde (68.3 g) at
-60~-65 °C under an atmosphere of nitrogen. BF (663.4 g, 1.86 mol, 19 wt% solution
3
in MeCN) was added slowly to the stirring mixture at -60~-65 °C over 1.5 h, and then,
the mixture was stirred at -60~-65 °C for another 1 h. The reaction was monitored by
TLC and deemed complete when the spot of 7 disappeared when using n-heptane and
EA (8:1, v/v) as the developing solvent. The reaction mixture was warmed to
-5~-10 °C for 1 h and stirred at -5~-10 °C for another 1 h. The reaction was monitored
by TLC and deemed complete when the spot of 7a disappeared when using n-heptane
and EA (1:1, v/v) as the developing solvent. The reaction mixture was added slowly to
NaOH aqueous solution (75 g of NaOH in 1 L of water) over 30 min at -5~0 °C, and
then, the aqueous phase was separated and discarded; the organic solvent was
evaporated and replaced twice with EtOH (1 L × 2) under reduced pressure at
3
5~40 °C. The mixed solution of 8 (HPLC purity > 90.0%) and EtOH (total volume of
approximately 1.7 L) was directly used for the next step. A small analytical sample of
1
8 was purified by a chromatographic column. H NMR (400 MHz, CD OD) δ 6.11 (q,
3
J = 6.8 Hz, 14 Hz, 1H), 3.67 (s, 3H), 3.58-3.66 (m, 1H), 2.70 (dd, J = 4 Hz, 12.8 Hz,
1
H), 2.23-2.43 (m, 4H), 1.10-2.11 (m, 24H), 1.06 (s, 3H), 0.98 (d, J = 6.4 Hz, 3H),
1
3
0.71 (s, 3H); C NMR (100 MHz, CD OD) δ 205.63, 174.49, 143.51, 128.53, 69.10,
3
5
4.07, 50.17, 50.15, 48.30, 45.01, 42.85, 38.87, 38.41, 36.53, 34.70, 33.93, 33.57,
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
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5
5
5
5
5
5
5
5
6
3
0.41, 30.01, 28.63, 27.57, 25.17, 21.41, 20.60, 17.08, 10.92, 10.65. HRMS m/z
+
[M+H] Calcd for C H O : 431.3136; found: 431.3161.
2
7
43
4
3α-Hydroxy-6-ethylidene-7-keto-5β-cholanic Acid (9) A 5-L reactor was charged
with the mixed solution of 8 and EtOH (1.7 L) under an atmosphere of nitrogen.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NaOH (37.2 g, 0.93 mol) aqueous solution (0.25 L of H O) was added and stirred at
2
20~25 °C. After stirring for 1 h, the reaction was monitored by HPLC and deemed
complete when 8 was less than 0.5%. The reaction mixture was diluted with water
(1.17 L), and then, hydrochloric acid (37%, 80 mL) was added slowly to the mixture
until the pH = 3~4. The mixture was refluxed to form a clear solution within 30 min
and then cooled naturally to 60~62 °C before the crystal seeds (0.2 g) were added to
the solution. After adding the crystal seeds, the mixture was stirred at 60~62 °C for 1
h. The mixture was cooled naturally to 20~25 °C within 1.5 h and stirred for another 2
h. The solid was isolated by filtration, washed twice with a mixture of EtOH and H O
2
(1:2, 0.5 L × 2), and dried in an air-drying oven at 55~60 °C to yield crude 9 (157.5 g).
A 3-L reactor was charged with the crude product (157.5 g), EtOH (1.57 L), and H O
2
(1.57 L). The mixture was refluxed to form a clear solution within 30 min and then
cooled naturally to 20~25 °C within 1.5 h, followed by stirring for another 2 h. The
solid was isolated by filtration and washed twice with a mixture of EtOH and H O
2
(1:2, 0.3 L × 2) and then dried in an air-drying oven at 55~60 °C to give the title
compound as an off-white solid (134.3 g, 52% yield from 6 to 9, HPLC purity >
1
9
8.0%). Mp 187.8 ~ 190.7 °C. H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 5.97
(q, J = 4.8 Hz, 9.6 Hz, 1H), 4.54 (d, J = 2.8 Hz, 1H), 3.42-3.47 (m, 1H), 2.58 (dd, J =
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.8 Hz, 8.4 Hz, 1H), 2.17-2.30 (m, 3H), 2.08-2.13 (m, 1H), 1.95 (dt, J = 2 Hz, 8.4 Hz,
H), 1.86-1.91 (m, 1H), 1.78-1.83 (m, 2H), 1.66-1.71 (m, 1H), 1.65 (d, J = 4.8 Hz,
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3H), 1.55-1.57 (m, 1H), 1.32-1.45 (m, 5H), 1.04-1.27 (m, 8H), 0.94 (s, 3H), 0.89 (d, J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
=
4 Hz, 3H), 0.60 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 204.16, 175.35, 144.15,
1
28.88, 69.04, 54.49, 50.71, 48.64, 45.35, 43.53, 39.06, 38.94, 38.02, 35.20, 34.67,
34.50, 31.20, 30.06, 28.46, 26.13, 23.07, 21.33, 18.77, 12.80, 12.33. HRMS m/z
+
[M+H] Calcd for C H O : 417.3005; found: 417.3004.
2
6
41
4
3
α-Hydroxy-6α-ethyl-7-keto-5β-cholanic Acid (10) A 2-L hydrogenation reactor
was charged with NaOH (24.8 g, 0.62 mol) and H O (0.65 L) and cooled to 20~25 °C.
2
9
(130 g, 0.31 mol) and Pd/C (10 wt%, 13 g) were added to the stirring mixture at
0~25 °C, and then, the reactor was flushed three times with nitrogen (2 atm) and
2
three times with hydrogen (2 atm). A hydrogen pressure of 3 atm was applied for 10 h
until hydrogen absorption was no longer noted. The reaction was monitored by HPLC
and deemed complete when 9 was less than 0.5%. The hydrogen pressure was adjusted
to 1 atm, and the reaction mixture was heated up to 95~100 °C for 2 h. The reaction
was monitored by HPLC and deemed complete when 9a was less than 3.0%. The
reaction mixture was cooled to 40~50 °C, Pd/C was filtered off, and it was washed
with water twice (65 mL × 2). The filtrate (approximately 0.95 L) and EtOH (0.78 L)
were transferred into a 2-L reactor, and citric acid monohydrate (52 g, 0.25 mol) was
added in batches to the mixture over 10 min. The mixture was refluxed to form a clear
solution within 30 min and then cooled naturally to 58~61 °C before the crystal seeds
(0.1 g) were added to the solution. After adding the crystal seeds, the mixture was
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1
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9
1
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stirred at 58~61 °C for 1 h. The mixture was cooled naturally to 20~25 °C within 1.5 h
and stirred for another 2 h. The solid was isolated by filtration, washed twice with a
mixture of EtOH and H O (1:2, 0.3 L × 2), and dried in an air-drying oven at
2
0
1
2
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9
0
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0
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0
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5~60 °C to yield crude 10 (113.9 g). A 2-L reactor was charged with the crude
product (113.9 g), EtOH (0.68 L) and H O (0.68 L). The mixture was refluxed to form
2
a clear solution within 30 min and then cooled naturally to 60~63 °C before the crystal
seeds (0.1 g) were added to the solution. After adding the crystal seeds, the mixture
was stirred at 60~63 °C for 1 h. The mixture was cooled naturally to 20~25 °C within
1.5 h and stirred for another 2 h. The solid was isolated by filtration and washed twice
with a mixture of EtOH and H O (1:2, 0.2 L × 2) and then dried in an air-drying oven
2
at 55~60 °C to give the title compound as an off-white solid (103.5 g, yield: 79%,
1
HPLC purity >97.0%). Mp 189.3 ~ 193.7 °C. H NMR (400 MHz, DMSO-d6) δ
1
1.95 (br, 1H), 4.47 (d, J = 3.2 Hz, 1H), 3.25-3.32 (m, 1H), 2.74 (q, J = 4.0 Hz, 8.8
Hz, 1H), 2.42 (t, J = 7.6 Hz, 1H), 2.21-2.26 (m, 1H), 2.02-2.13 (m, 2H), 1.92 (dt, J =
2.0 Hz, 6.4 Hz, 1H), 1.78-1.85 (m, 1H), 1.73-1.77 (m, 1H), 1.65-1.71 (m, 3H),
1
8
.53-1.60 (m, 1H), 1.46-1.49 (m, 2H), 1.31-1.43 (m, 4H), 1.17 (s, 3H), 0.98-1.27 (m,
H), 0.90-0.92 (m, 1H), 0.88 (d, J = 4.4 Hz, 3H), 0.74 (t, J = 4.8 Hz, 3H), 0.62 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 212.69, 175.35, 69.73, 54.83, 51.54, 50.41, 49.48,
4
2
4
3
9.34, 43.66, 42.67, 39.09, 35.80, 35.21, 34.42, 32.16, 31.18, 30.20, 28.35, 24.70,
+
3.72, 21.88, 19.08, 18.70, 12.40, 12.33. HRMS m/z [M+H] Calcd for C H O :
2
6
43
4
19.3161; found: 419.3157.
α,7α-Dihydroxy-6α-ethyl-5β-cholanic Acid (Obeticholic acid, 1) A 2-L reactor
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Organic Process Research & Development
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was charged with 10 (100 g, 0.24 mol), NaOH (19.2 g, 0.48 mol) and H O (0.7 L)
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under an atmosphere of nitrogen. The mixture of NaBH (9.8 g, 0.26 mol), NaOH (1
4
g) and H O (0.1 L) was added to the stirring mixture at 20~25 °C over 10 min, and
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0
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then, the mixture was stirred at 70~75 °C for another 6 h. The reaction was monitored
by HPLC and deemed complete when 10 was less than 0.5%. The reaction mixture
was cooled to 35~40 °C. Acetic acid aqueous solution (53.3 g of HOAc and 0.2 L of
H O) was added slowly to the reaction mixture over 30 min. The mixture was
2
combined with n-butyl acetate (0.6 L). The aqueous layer was discarded, and the
organic layer was washed with NaCl aqueous solution (5% of NaCl, 0.1 L). The
organic layer was transferred into a 2-L reactor and heated to 70~75 °C, and n-heptane
(0.6 L) was added slowly to the mixture over 30 min to form a clear solution; then, the
solution was cooled naturally to 58~60 °C before the crystal seeds (0.1 g) were added
to the solution. After adding the crystal seeds, the mixture was stirred at 58~60 °C for
1
h. The mixture was cooled naturally to 20~25 °C within 1.5 h and stirred for another
2 h. The solid was isolated by filtration, washed twice with a mixture of n-butyl
acetate and n-heptane (1:2, 0.2 L × 2), and dried in an air-drying oven at 55~60 °C to
yield crude 1 (81.4 g). A 2-L reactor was charged with the crude product (81.4 g),
n-butyl acetate (0.49 L) and n-heptane (0.49 L). The mixture was heated to 70~75 °C
and formed a clear solution within 30 min and then cooled naturally to 60~62 °C
before the crystal seeds (0.1 g) were added to the solution. After adding the crystal
seeds, the mixture was stirred at 60~62 °C for 1 h. The mixture was cooled naturally
to 20~25 °C within 1.5 h and stirred for another 2 h. The solid was isolated by
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