Synthesis and characterization of new impurities in obeticholic acid

Article abstract of DOI:10.1177/1747519819875858

Novel and efficient synthetic strategies are developed for the first synthesis of two new impurities found in obeticholic acid. The synthetic routes to the impurities are designed without column purification using 4-nitrobenzoyl chloride as a selective pr

Full text of DOI:10.1177/1747519819875858

875858CHL0010.1177/1747519819875858Journal of Chemical ResearchFeng et al.
research-article2019
Research Paper
Journal of Chemical Research
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Synthesis and characterization of new
impurities in obeticholic acid
https://doi.org/10.1177/1747519819875858
DOI: 10.1177/1747519819875858
journals.sagepub.com/home/chl
Wei-Dong Feng1, Song-Ming Zhuo2 and Fu-Li Zhang1,3
Abstract
Novel and efficient synthetic strategies are developed for the first synthesis of two new impurities found in obeticholic
acid. The synthetic routes to the impurities are designed without column purification using 4-nitrobenzoyl chloride
as a selective protecting group. The impurities, which are obtained in good yields and high purity, are identified and
characterized using high-resolution mass spectrometry, Fourier transform infrared, one-dimensional nuclear magnetic
resonance (1H, 13C, distortionless enhancement by polarization transfer), and two-dimensional nuclear magnetic
resonance (Correlated Spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple bond correlation,
and rotating-frame Overhauser effect spectroscopy) techniques.
Keywords
bile acids, farnesoid X receptor, impurities, nuclear magnetic resonance, obeticholic acid
Date received: 26 March 2019; accepted: 21 August 2019
1Collaborative Innovation Center of Yangtze River Delta Region Green
Pharmaceuticals, Zhejiang University of Technology, Hangzhou, P.R. China
2Department of Pharmaceutical Engineering, China Pharmaceutical
University, Nanjing, P.R. China
3Shanghai Institute of Pharmaceutical Industry, China State Institute of
Pharmaceutical Industry, Shanghai, P.R. China
Corresponding author:
Fu-Li Zhang, Collaborative Innovation Center of Yangtze River Delta
Region Green Pharmaceuticals, Zhejiang University of Technology, 18
Chaowang Road, Hangzhou 310032, P.R. China.
Email: zhangfuli01@126.com

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Journal of Chemical Research 00(0)
(HPLC) using an evaporative light-scattering detector
(ELSD). The impurities were isolated from the crystal
mother liquor of OCA and identified using high-resolution
mass spectrometry (HRMS) and nuclear magnetic reso-
nance (NMR) techniques (Figure 1). To the best of our
knowledge, impurities 1 and 2 have not been previously
reported. We have successfully synthesized these impuri-
ties without column purification for the first time.
Introduction
Obeticholic acid (OCA) is a farnesoid X receptor (FXR)
agonist for the treatment of primary sclerosing cholangitis
(PSC), primary biliary cholangitis (PBC), and non-alco-
holic steatohepatitis (NASH).1,2 OCA was approved by the
Food and Drug Administration (FDA) under the brand
name Ocaliva in 2016 for patients not responding to or who
are intolerant to ursodeoxycholic acid (UDCA).3
The safety of a drug is dependent not only on the toxi-
cological 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 quantities, may influ-
ence the efficacy and safety of the pharmaceutical prod-
uct.4 Thus, the detection, identification, synthesis,
quantification, and control of these impurities, which orig-
inate in the manufacturing process, have become impor-
tant elements of drug development to ensure product
quality and ultimately patient safety.5 According to the
international conference on harmonization (ICH) guide-
lines, impurities in drug substances should be identified
and controlled when present at a level of ⩾0.1% by isolat-
ing or synthesizing them in their pure form for analytical
method development.6 The process-related impurities of
OCAhave been previously reported in Douša et al.7 During
the analysis of laboratory batches of OCA, two unknown
impurities of OCA at 0.13% and 0.10%, respectively, were
detected by high-performance liquid chromatography
Results and discussion
Formation of impurities 1 and 2
The synthetic route to OCA is shown in Scheme 1.8,9 OCA
is synthesized using a sequence of reactions involving oxi-
dation, esterification, Mukaiyama aldol addition, hydroly-
sis, Pd/C catalytic reduction, and NaBH4 reduction. The
mechanisms of formation of impurities 1 and 2 involve the
carboxyl moieties of unreacted intermediates 7 and 6,
respectively, being partially reduced by NaBH4 in the last
step.
Design of synthetic routes to impurities 1
and 2
The synthetic routes to impurities 1 and 2 are shown in
Schemes 2 and 3, respectively. The reduction of a carbonyl
is far easier than that of a carboxyl under typical reaction
Figure 1. Structures of OCA, impurity 1, and impurity 2.
Scheme 1. Commercial process route to OCA (1).

Feng et al.
3
Scheme 2. Synthetic route to impurity 1.
Scheme 3. Synthetic route to impurity 2.
conditions, and therefore, impurity 1 is difficult to obtain chloride to produce 9, which was oxidized with sodium
directly by reduction of 7. In Scheme 2, impurity 1 can be hypochlorite and acetic acid to afford 10. This was fol-
obtained directly by oxidation of 8, but purification by lowed by hydrolysis to yield impurity 1. Intermediates 9
recrystallization or column chromatography is challenging and 10 and impurity 1 are easily crystallized from appropri-
due to the production of byproducts resulting from the ate solvents. The synthetic route to impurity 2 was designed
overoxidation of 8. Hence, the primary hydroxy and as shown in Scheme 3. The hydroxy groups of intermediate
hydroxy at C-3 of 8 should be protected to avoid oxidation. 11 must also be selectively protected using p-nitrobenzoyl
Furthermore, the protecting group should be chemoselec- chloride to produce 12, followed by oxidation to yield 13,
tive and not react with the C-7 hydroxy group. To our which is then hydrolyzed to give compound 14.
delight, p-nitrobenzoyl chloride is an appropriate hydroxy Subsequently, intermediate 14 was protected with tri-
protecting group. The primary hydroxy and C-3 hydroxy methylsilyl chloride to afford 15. Finally, impurity 2 was
groups of 8 were, thus, protected with p-nitrobenzoyl obtained from 15 using the Mukaiyama aldol addition

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Journal of Chemical Research 00(0)
1
Table 1. H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) assignments for impurities 1 and 2.
Position
1H
13C
DEPT
1H
13C
35.41
DEPT
1.
2.
3.
4.
5.
6.
7.
8.
1.21 (m), 1.85 (m)
1.22 (m), 1.63 (m)
3.46 (sept, J = 4.8 Hz)
0.79 (m), 1.64 (m)
1.86 (m)
2.82 (q, J = 5.4 Hz)
–
2.49 (t, J = 11.4 Hz)
1.79 (td, J = 12, 4.8 Hz)
–
35.25
CH2
CH2
CH
CH2
CH
CH
C
CH
CH
C
1.93 (m), 1.20 (m)
1.69 (m), 1.29 (m)
3.59 (sept, J = 4.8 Hz)
1.55 (m), 1.26 (m)
2.68 (dd, J = 13.2, 4.2 Hz)
–
CH2
CH2
CH
CH2
CH
C
30.53
71.66
32.57
52.16
53.29
215.48
51.19
45.36
36.82
22.97
40.43
43.76
50.49
25.62
29.42
56.51
12.52
23.92
36.85
19.28
33.21
30.26
63.54
20.07
12.29
30.46
70.96
38.37
46.88
145.38
207.64
50.18
40.74
35.77
22.45
40.32
44.66
52.03
27.03
29.53
56.18
12.49
23.24
36.87
19.35
33.24
30.28
63.55
130.34
12.75
–
C
2.32 (t, J = 11.4 Hz)
1.99 (m)
–
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
CH2
CH
CH3
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
1.53 (m)
CH2
CH2
C
1.49 (m)
2.04 (dt, J = 13.2, 3 Hz), 1.14 (m)
–
1.44 (m)
0.95 (m), 2.12 (m)
1.30 (m), 1.92 (m)
1.15 (m)
0.71 (s)
1.25 (s)
1.42 (m)
0.96 (d, J = 6.6 Hz)
1.09 (m), 1.48 (m)
1.41 (m), 1.63 (m)
3.51 (m)
1.11 (m), 1.70 (m)
0.81 (t, J = 7.2 Hz)
2.08 (dt, J = 13.2, 2.4 Hz), 1.16 (m)
–
1.42 (m)
1.44 (m), 2.34 (m)
1.28 (m), 1.89 (m)
1.12 (m)
0.69 (s)
1.04 (s)
1.41 (m)
0.96 (d, J = 6.6 Hz)
1.07 (m), 1.48 (m)
1.39 (m), 1.59 (m)
3.51 (m)
6.08 (q, J = 7.2 Hz)
1.71 (d, J = 7.2 Hz)
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
CH2
CH2
CH3
NMR: nuclear magnetic resonance; DEPT: distortionless enhancement by polarization transfer.
reaction because this impurity could not be directly obtained impurity 2. Therefore, impurity 1 contains hydroxy and
from 13 via this reaction. Intermediates 12, 13, and 14 and carbonyl functional groups, and impurity 2 contains an alk-
impurity 2 can be purified by recrystallization. Finally, the enyl group as well as hydroxy and carbonyl groups.
synthetic routes to impurities 1 and 2 were successfully
A series of one-dimensional (1D) and two-dimensional
designed without the use of column chromatography purifi- (2D) NMR experiments were performed on impurities 1
cation and can be employed in the development of an HPLC and 2, including distortionless enhancement by polarization
method for OCA and as external standards with high purity transfer (DEPT), H-H Correlated Spectroscopy (COSY),
to analyze the content of these impurities in OCA.
heteronuclear single quantum coherence (HSQC), and het-
eronuclear multiple bond correlation (HMBC) to assign all
1
the H and 13C signals (Table 1). The H-H COSY and key
Structural elucidation of impurities 1 and 2
HMBC data of impurities 1 and 2 are described in Figure 2.
The HRMS spectrum of impurity 1 provides a mass ion
The 13C NMR and DEPT spectra of impurity 1 indicate
signal at m/z 405.3368 ([M + H]+, Calcd. for C26H45O3: the presence of 26 carbon atoms including 4 methyl carbon
405.3369) in positive ion mode. This result indicates that atoms, 11 methylene carbon atoms, 8 methine carbon
the molecular formula of impurity 1 is C26H44O3 and that atoms, and 3 quaternary carbon atoms. The quaternary car-
the molecular weight is 404, which is 16 Da (O) less than bon signal at 215.48 ppm indicates that C7 is a carbonyl
that of OCA (C26H44O4). The HRMS spectrum of impurity carbon atom. The chemical shifts of H3 and H24 should be
2 shows a mass ion at m/z 403.3208 ([M + H]+, Calcd. for located in a lower field region (>3.0 ppm) in the 1H NMR
C26H43O3: 403.3212) in positive ion mode. This result indi- spectrum because the positions of 3 and 24 are adjacent to
cates that the molecular formula of impurity 1 is C26H42O3, oxygen atoms. The chemical shifts of C3 and C24 were
and the molecular weight is 402, which is 18 Da (H2O) less determined by the HSQC spectrum. The H18 and H19
than that of OCA. The infrared (IR) absorptions of impurity methyl signals are single peaks, and the methyl signals of
1 were observed for hydroxy (3269 cm−1) and carbonyl H21 and H26 appear as a double and triple, respectively. In
(1705 cm−1) functional groups. The IR spectrum of impu- addition, the chemical shift of H18 is lower than that of
rity 2 indicates the presence of hydroxy (3337 cm−1), car- H19 in various bile acid compounds,10 and the chemical
bonyl (1690 cm−1), and alkenyl (1636 cm−1) functional shifts of C18, C19, C21, and C26 were determined from the
groups, and the characteristic absorption of the carbonyl HSQC spectrum. The chemical shift of the quaternary car-
shifts to a lower number (<1700 cm−1) due to the conju- bon C10 was determined from the HMBC spectrum via the
gated structure of the α,β-unsaturated ketone moiety of correlations of C10 and H19. Similarly, the chemical shift

Feng et al.
5
Figure 2. H-H COSY and key HMBCs of impurity 1 (left) and impurity 2 (right) (CD3OD, 600 MHz).
Figure 3. Key ROESY correlations for impurity 1 (CD3OD, 600 MHz).
of the quaternary carbon C13 was also determined from the HMBC spectrum, which correlates with long-range cou-
HMBC spectrum due to the correlations of C13 and H18. pling of a propylene. The chemical shifts of impurity 2
The chemical shift of H23 was determined from the H-H were determined using NMR analysis analogous to that
COSY spectrum due to the correlations of H23 and H24, employed in the analysis of impurity 1.
and the corresponding chemical shift of C23 was deter-
As shown in Scheme 1, intermediate 7 was synthesized
mined by analysis of the HSQC spectrum. In addition, the from 6 in the presence of Pd/C/H2 and sodium hydroxide in
chemical shifts of H25 and C25 were determined by the water and was obtained via tautomerism of the α-ketone of
correlations between positions 25 and 26. H6 correlates the 6β-ethyl intermediate under strong alkaline conditions.
with H5 and H25 in the H-H COSY spectrum and with A small amount of the 6β-ethyl intermediate most likely
positions 7 and 26 in the HMBC spectrum. Moreover, the remained in intermediate 7. Therefore, the relative configu-
chemical shifts of H6, H5, and C5 were deduced based on ration of the ethyl moiety of impurity 1 was confirmed by
the H-H COSY and HSQC spectra. The methylene at C-1 the rotating-frame Overhauser effect spectroscopy
correlates with positions 3, 5, 10, and 19 in the HMBC (ROESY) experiment. The key ROESY correlation of
spectrum, and thus, the chemical shifts of H1 and C1 were impurity 1 is shown in Figure 3. The 6α-orientation of the
determined. The chemical shifts of H4, C4, H2, and C2 ethyl moiety of impurity 1 was determined based on H6
were easily deduced based on the 2D NMR spectrum. The being correlated with H5, H8, and H19, but not correlated
assignments of the 1H and 13C resonance signals of the B-, with H9, as shown in Figure 4. In addition, the ROESY cor-
C-, and D-rings were determined based on an analogous relations of H9 and H25 or H26 also provided support for
analysis.
the relative configuration. In the ROESY spectrum, H5 is
1
In the H NMR spectrum of impurity 2, the resonance correlated with H3, H6, and H19, and H8 is correlated with
signal of the alkenyl group was observed at 6.08 ppm, and H6, H18, and H19. Furthermore, H14 is correlated with H9
the characteristic signals of the carbon atoms of the alkenyl and H17, which indicates that the relative configurations of
group were observed at 145.38 and 130.34 ppm. These H3, H5, H6, H8, H18, and H19 have a β orientation.
results indicate that impurity 2 contains an ethylene group. However, the relative configurations of H9, H14, and H17
Interestingly, C5 and C7 were correlated to H26 in the have an α orientation.

6
Journal of Chemical Research 00(0)
Figure 4. ROESY spectrum for impurity 1 (CD3OD, 600 MHz).
melting points were measured on a WRS-1B apparatus
(YiCe, Shanghai, China). HRMS measurements were per-
formed on a Bruker Daltonics Solarix 7.0T (Bruker,
Karlsruhe, DE, Germany). The IR spectra were recorded in
the solid state on a Tracer-100 FTIR spectrometer
(Shimadzu, Kyoto, Japan). The NMR spectra were recorded
on a Bruker Avance III instrument (Bruker) operating at
Conclusion
As per the stringent regulatory requirements that have been
recommended by the ICH, we have developed efficient and
practical approaches to deliver new impurities 1 and 2 that
are found in OCA. In general, many bile acids and their
derivatives are oils or foamy solids, which are difficult to
purify via recrystallization. However, using the synthetic
strategy outlined in this study, the crystallizability can be
improved significantly by the formation of a nitrobenzoic
ester at the 3-position hydroxy groups in these compounds.
In addition, these compounds can be detected by HPLC
with a typical UV–Vis detector rather than with ELSD or
CAD (charged aerosol detector). The new impurities were
characterized using HRMS, FTIR, 1D NMR and 2D NMR
techniques. The relative configuration of impurity 1 was
determined from the ROESY spectrum. This study pro-
vides useful reference information for organic, process, and
analytical chemists in pharmaceutical companies and drug
regulatory authorities.
400 or 600 MHz for 1H NMR and 100 or 150 MHz for 13
C
NMR with Me4Si (tetramethylsilane (TMS)) as the internal
standard. DEPT and 2D NMR (H-H COSY, HSQC, HMBC,
and ROESY) experiments were also performed on the same
instrument to assign the correlations.
Synthesis of impurity 1 (Scheme 2)
(3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-17-((R)-5-hydrox-
ypentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]
phenanthrene-3,7-diol (8). To a mixture of 1 (10.0 g, 23.8
mmol) in dry tetrahydrofuran (100 mL), lithium aluminum
hydride (1.8 g, 47.6 mmol) was added in batches at 25 °C
under nitrogen followed by stirring at 65 °C for 4 h. The
reaction mixture was cooled to 0–5 °C, and then, water (1.8
mL) followed by aqueous 10% sodium hydroxide solution
(1.8 mL) were slowly added to the reaction mixture. After
stirring for 30 min, the mixture was filtered and the filter
cake was washed with tetrahydrofuran (50 mL), and the fil-
trate was dried using anhydrous sodium sulfate. The solvent
was concentrated under reduced pressure to afford com-
pound 8 as a white foamy solid (8.9 g, yield: 92%, purity:
98.3%). 1H NMR (400 MHz, CD3OD): δ 3.65 (m, 1H), 3.48–
3.52 (m, 2H), 3.28–3.33 (m, 1H), 1.98–2.02 (m, 1H),
Experimental
Materials and instruments
All solvents and reagents were purchased from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China). OCA and
its intermediates were prepared in-house. HPLC analyses
were performed on a Dionex UltiMate 3000 HPLC (Thermo
Fisher, Massachusetts, USA) instrument using a reverse-
phase Agilent ZOBAX XDB-C18, 250 mm × 4.6 mm, and
5-μm particle size column (Agilent, California, USA). The

Feng et al.
7
1.79–1.94 (m, 4H), 1.70–1.77 (m, 2H), 1.57–1.64 (m, 2H),
1.27–1.56 (m, 13H), 1.16–1.21 (m, 2H), 1.05–1.14 (m, 2H),
0.98–1.03 (m, 1H), 0.96 (d, J = 4.4 Hz, 3H), 0.91 (s, 3H),
0.90 (t, J = 4.8 Hz, 3H), 0.69 (s, 3H) (Supplemental mate-
rial, Figure S2);13C NMR (100 MHz, DMSO-d6): δ 73.18,
71.19, 63.58, 57.56, 51.67, 46.95, 43.69, 43.16, 41.55, 41.08,
37.06, 36.79, 36.65, 34.52, 34.42, 33.23, 31.26, 30.28, 29.39,
24.59, 23.79, 23.50, 21.99, 19.25, 12.27, 12.06 (Supplemen-
tal material, Figure S3). HRMS: m/z [M + Na]+ Calcd for
C26H46NaO3: 429.3345; found: 429.3336 (Supplemental
material, Figure S1).
acetate (50 mL × 2). The organic layer was dried over
anhydrous sodium sulfate and concentrated under reduced
pressure to afford a residue, which was recrystallized
from methyl tert-butyl ether (50 mL) and heptane (50
mL) to yield compound 10 as a white solid (9.1 g, yield:
91%, purity: 97.8%). IR (KBr) νmax/cm−1: 3426 (OH),
2957, 2874, 1722 (C=O), 1607, 1530 (N=O), 1350
(N=O), 1277, 1119, 1103, 1015, 874, 719 (Supplemental
material, Figure S7). 1H NMR (400 MHz, CDCl3): δ 8.29
(dd, J = 10.8 Hz, 6 Hz, 4H), 8.19 (dd, J = 16 Hz, 6 Hz,
4H), 4.94 (hept, J = 3.2 Hz, 1H), 4.31–4.39 (m, 2H), 2.77
(q, J = 8.8 Hz, 4 Hz, 1H), 2.42 (t, J = 7.6 Hz, 1H),
2.17–2.22 (m, 1H), 2.03 (dt, J = 8.8 Hz, 2.4 Hz, 1H),
1.91–1.98 (m, 3H), 1.83–1.88 (m, 4H), 1.66–1.77 (m,
2H), 1.46–1.55 (m, 6H), 1.30–1.34 (m, 1H), 1.28 (s, 3H),
1.09–1.27 (m, 6H), 0.98 (d, J = 4.4 Hz, 3H), 0.91–0.96
(m, 1H), 0.82 (t, J = 4.8 Hz, 3H), 0.69 (s, 3H) (Supple-
mental material, Figure S8). 13C NMR (100 MHz,
CDCl3): δ 212.83, 164.76, 164.07, 150.50, 135.92,
135.85, 130.69, 130.65, 123.56, 123.48, 75.05, 66.51,
54.94, 52.05, 50.63, 50.01, 48.99, 43.92, 42.66, 38.98,
35.78, 35.29, 33.84, 32.05, 28.44, 27.81, 26.08, 25.21,
24.57, 23.53, 21.98, 18.88, 18.71, 12.11, 12.06 (Supple-
mental material, Figure S9). Anal. cacld for C40H50N2O9
(702.35): C, 68.36; H, 7.17; N, 3.99; found: C, 68.11; H,
6.97; N, 4.24.
(3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-7-hydroxy-
10,13-dimethyl-17-((R)-5-((4-nitrobenzoyl)oxy)pentan-2-yl)
hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-nitro-
benzoate (9). To a mixture of 8 (8.0 g, 19.7 mmol), N,N-
diisopropylethylamine (7.6 g, 59.0 mmol) and
4-dimethylaminopyridine (0.24 g, 2.0 mmol) in dry tet-
rahydrofuran (40 mL) was added to 4-nitrobenzoyl chlo-
ride (7.7 g, 41.4 mmol) in batches at 0–5 °C followed by
stirring at 0 °C for 2 h. The reaction mixture was
quenched with an aqueous solution of 20% citric acid
(100 mL) and extracted with ethyl acetate (50 mL × 2).
The combined organic layer was washed with 10%
sodium bicarbonate solution (50 mL) and then dried over
anhydrous sodium sulfate and concentrated under
reduced pressure. The residue was recrystallized from
ethyl acetate (40 mL) and heptane (80 mL) to yield com-
pound 9 as a white solid (11.4 g, yield: 82%, purity:
98.7%). IR (KBr) νmax (cm−1): 3559 (OH), 3431, 2941,
2903, 2870, 1722 (C=O), 1607, 1528 (N=O), 1350
(N=O), 1277, 1119, 1103, 1015, 874, 719 (Supplemental
material, Figure S4). 1H NMR (400 MHz, CDCl3): δ 8.30
(dt, J = 6 Hz, 1.6 Hz, 2H), 8.27 (dt, J = 6 Hz, 1.2 Hz,
2H), 8.20–8.22 (m, 4H), 4.83–4.87 (m, 1H), 4.31–4.39
(m, 2H), 3.75 (s, 1H), 2.13 (q, J = 8.8 Hz, 1H), 2.01 (dt,
J = 8 Hz, 2 Hz, 1H), 1.82–1.95 (m, 6H), 1.13–1.73 (m,
20H), 0.99 (d, J = 4.4 Hz, 3H), 0.96 (s, 3H), 0.92 (t, J =
4.8 Hz, 3H), 0.69 (s, 3H) (Supplemental material, Figure
S5); 13C NMR (100 MHz, CDCl3): δ 164.91, 164.38,
150.64, 150.52, 136.52, 135.99, 130.83, 130.79, 123.71,
123.55, 76.75, 71.00, 66.69, 56.04, 50.71, 45.25, 42.92,
41.29, 40.12, 39.73, 35.76, 35.58, 35.28, 33.46, 32.15,
29.78, 28.44, 26.85, 25.34, 23.86, 23.27, 22.36, 20.95,
18.76, 11.97, 11.80 (Supplemental material, Figure S6).
Anal. cacld for C40H52N2O9 (704.37): C, 68.16; H, 7.44;
N, 3.97; found: C, 68.01; H, 7.28; N, 4.14.
(3R,5S,6R,8S,9S,10S,13R,14S,17R)-6-ethyl-3-hydroxy-17-((R)-
5-hydroxypentan-2-yl)-10,13-dimethylhexadecahydro-7H-
cyclopenta[a]phenanthren-7-one (impurity 1). To a mixture
of 10 (9.0 g, 12.8 mmol) in a solution consisting of water
(6 mL), tetrahydrofuran (30 mL), and methanol (15 mL),
sodium hydroxide (1.3 g, 32 mmol) was added at 25 °C
followed by stirring at 25 °C for 1 h. The reaction mixture
was diluted with water (50 mL) and extracted with ethyl
acetate (50 mL × 2). The organic layer was dried over
anhydrous sodium sulfate and concentrated under reduced
pressure to afford a residue, which was recrystallized from
dichloromethane (90 mL) and heptane (45 mL) to yield
impurity 1 as a white solid (4.5 g, yield: 87%, purity:
99.3%). m.p.: 108–110 °C. IR (KBr) νmax/cm−1: 3269
(OH), 2945, 2874, 1705 (C=O), 1464, 1452, 1377, 1061,
1
1013, 735 (Supplemental material, Figure S10). H NMR
(600 MHz, CD3OD): δ 3.49–3.52 (m, 2H), 3.46 (sept, J =
4.8 Hz, 1H), 2.82 (q, J = 12.6 Hz, 5.4 Hz, 1H), 2.49 (t, J
= 11.4 Hz, 1H), 2.10–2.16 (m, 1H), 2.04 (dt, J = 13.2 Hz,
3 Hz, 1H), 1.89–1.95 (m, 1H), 1.83–1.88 (m, 2H), 1.79 (td,
J = 12.0 Hz, 4.8 Hz, 1H), 1.70 (quin, J = 7.2 Hz, 1H),
1.56–1.66 (m, 3H), 1.53 (dd, J = 12.6 Hz, 3.6 Hz, 1H),
1.46–1.51 (m, 2H), 1.38–1.45 (m, 3H), 1.26–1.33 (m, 1H),
1.25 (s, 3H), 1.06–1.23 (m, 6H), 0.96 (d, J = 6.6 Hz, 3H),
0.92–0.95 (m, 1H), 0.81 (t, J = 7.2 Hz, 3H), 0.77–0.80 (m,
(3R,5S,6R,8S,9S,10S,13R,14S,17R)-6-ethyl-10,13-dimethyl-
17-((R)-5-((4-nitrobenzoyl)oxy)pentan-2-yl)-7-oxohexadeca-
hydro-1H-cyclopenta[a]phenanthren-3-yl 4-nitrobenzoate
(10). To a mixture of 9 (10.0 g, 14.2 mmol), sodium bro-
mide (73 mg, 0.71 mmol), and tetrabutylammonium bro-
mide (229 mg, 0.71 mmol) in a mixture of water (5 mL),
tetrahydrofuran (20 mL), isopropanol (30 mL), and acetic
acid (10 mL), was added slowly to an aqueous solution
consisting of 5% sodium hypochlorite (42.3 g, 28.4
mmol) at −15 °C followed by stirring at −15 °C for 2 h.
The reaction mixture was quenched with 10% sodium
bisulfite solution (50 mL) and extracted with ethyl
1H), 0.71 (s, 3H) (Supplemental material, Figure S12); 13
C
NMR (150 MHz, CD3OD) δ 215.48, 71.66, 63.54, 56.51,
53.29, 52.16, 51.19, 50.49, 45.36, 43.76, 40.43, 36.85,
36.82, 35.25, 33.21, 32.57, 30.53, 30.26, 29.42, 25.62,
23.92, 22.97, 20.07, 19.28, 12.52, 12.29 (Supplemental
material, Figure S13). HRMS: m/z [M + H]+ Calcd for
C26H45O3: 405.3369; found: 405.3368 (Supplemental
material, Figure S11).

8
Journal of Chemical Research 00(0)
(Supplemental material, Figure S25). Anal. cacld for
C38H48N2O9 (676.34): C, 67.44; H, 7.15; N, 4.14; found: C,
67.21; H, 7.07; N, 4.31.
Synthesis of impurity 2 (Scheme 3)
(3R,5S,7R,8R,9S,10S,13R,14S,17R)-17-((R)-5-hydroxypentan-
2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan-
threne-3,7-diol (11). To a mixture of 2 (20.0 g, 50.9 mmol)
in dry tetrahydrofuran (200 mL), lithium aluminum hydride
(3.9 g, 101.8 mmol) was added in batches at 25 °C under
nitrogen followed by stirring at 65 °C for 3 h. The reaction
mixture was cooled to 0–5 °C, and then, water (3.9 mL)
followed by 10% sodium hydroxide solution (3.9 mL) were
slowly added to the reaction mixture. After 30 min, the
mixture was filtered, and the filter cake was washed with
tetrahydrofuran (100 mL), and the filtrate was dried over
anhydrous sodium sulfate. The solvent was concentrated
under reduced pressure to afford compound 11 as a white
(3R,5S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-((R)-5-((4-
nitrobenzoyl)oxy)pentan-2-yl)-7-oxohexadecahydro-
1H-cyclopenta[a]phenanthren-3-yl 4-nitrobenzoate (13). To a
mixture of 12 (20.0 g, 29.6 mmol), sodium bromide (152
mg, 1.48 mmol), and tetrabutylammonium bromide (477
mg, 1.48 mmol) in a solution consisting of water (10 mL),
tetrahydrofuran (40 mL), isopropanol (60 mL), and acetic
acid (20 mL), a 5% sodium hypochlorite solution (88.1 g,
59.2 mmol) was slowly added at −15 °C followed by stir-
ring at −15 °C for 2 h. The reaction mixture was quenched
with aqueous 10% sodium bisulfite (100 mL) and extracted
with ethyl acetate (100 mL × 2). The organic layer was
dried using anhydrous sodium sulfate and concentrated
under reduced pressure to afford a residue, which was
recrystallized from methyl tert-butyl ether (100 mL) and
heptane (100 mL) to yield compound 13 as a white solid
(18.8 g, yield: 94%, purity: 98.2%). IR (KBr) νmax/cm−1:
2949, 2874, 1717 (C=O), 1607, 1526 (N=O), 1348(N=O),
1287, 1117, 1105, 1015, 872, 716 (Supplemental material,
Figure S26). 1H NMR (400 MHz, CDCl3): δ 8.25–8.30 (m,
4H), 8.15–8.21 (m, 4H), 4.98 (sept, J = 4.8 Hz, 1H), 4.29–
4.39 (m, 2H), 2.91 (dd, J = 12.4 Hz, 5.6 Hz, 1H), 2.44 (t, J
= 11.2 Hz, 1H), 2.15–2.23 (m, 1H), 1.46–2.05 (m, 16H),
1.14–1.34 (m, 6H), 1.25 (s, 3H), 0.91–1.04 (m, 1H), 0.97
(d, J = 6.8 Hz, 3H), 0.68 (s, 3H) (Supplemental material,
Figure S27); 13C NMR (100 MHz, CDCl3): δ 212.04,
164.88, 164.19, 150.62, 136.02, 135.98, 130.78, 123.68,
123.61, 74.85, 66.62, 55.03, 49.68, 49.04, 46.09, 45.39,
43.12, 42.75, 39.07, 35.40, 35.37, 33.88, 33.22, 32.16,
28.53, 26.25, 25.36, 24.88, 23.18, 21.93, 18.84, 12.21
(Supplemental material, Figure S28). Anal. cacld for
C38H46N2O9 (674.32): C, 67.64; H, 6.87; N, 4.15; found: C,
67.46; H, 6.58; N, 4.38.
1
foamy solid (17.3 g, yield: 90%, purity: 98.6%). H NMR
(400 MHz, CD3OD): δ 3.82 (q, J = 1.6 Hz, 1H), 3.49–3.56
(m, 2H), 3.37–3.42 (m, 1H), 2.28 (q, J = 8.4 Hz, 1H), 2.03
(dt, J = 6.4 Hz, 2.4 Hz, 1H), 1.97–2.01 (m, 1H), 1.86–1.95
(m, 3H), 1.73–1.78 (m, 1H), 1.60–1.69 (m, 3H), 1.29–1.56
(m, 11H), 1.18–1.24 (m, 2H), 1.07–1.16 (m, 2H), 1.00–1.03
(m, 1H), 0.99 (d, J = 4.4 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H)
(Supplemental material, Figure S21); 13C NMR (100 MHz,
CD3OD): δ 72.86, 69.08, 63.59, 57.54, 51.54, 43.64, 43.18,
41.08, 40.77, 40.46, 37.06, 36.57, 36.23, 35.90, 34.05,
33.23, 31.36, 30.28, 29.36, 24.65, 23.44, 21.81, 19.26,
12.21 (Supplemental material, Figure S22). HRMS: m/z [M
+ H]+ Calcd for C24H43O3: 379.3212; found: 379.3207
(Supplemental material, Figure S20).
(3R,5R,7R,8R,9S,10S,13R,14S,17R)-7-hydroxy-10,13-di-
methyl-17-((R)-5-((4-nitrobenzoyl)oxy)pentan-2-yl)hexadeca-
hydro-1H-cyclopenta[a]phenanthren-3-yl 4-nitrobenzoate
(12). To a mixture of 11 (16 g, 42.3 mmol), N,N-diisopro-
pylethylamine (16.4 g, 126.8 mmol), and 4-dimethylami-
nopyridine (0.51 g, 4.2 mmol) in dry tetrahydrofuran (100
mL), 4-nitrobenzoyl chloride (16.5 g, 88.8 mmol) was
added in batches at 0–5 °C followed by stirring at 0 °C for
2 h. The reaction mixture was quenched with aqueous 20%
citric acid (200 mL) and extracted with ethyl acetate (100
mL × 2). The combined organic layer was washed with
10% sodium bicarbonate solution (100 mL). The organic
layer was dried using anhydrous sodium sulfate and con-
centrated under reduced pressure to afford a residue, which
was recrystallized from ethyl acetate (80 mL) and heptane
(160 mL) to yield compound 12 as a white solid (22.3 g,
yield: 78%, purity: 98.0%). IR (KBr) νmax/cm−1: 3564 (OH),
2940, 2868, 1722 (C=O), 1607, 1528 (N=O), 1350 (N=O),
1277, 1119, 1103, 1015, 874, 719 (Supplemental material,
Figure S23). 1H NMR (400 MHz, CDCl3): δ 8.28 (dd, J =
12.4 Hz, 6 Hz, 4H), 8.20–8.22 (m, 4H), 4.88 (sept, J = 4.8
Hz, 1H), 4.33–4.39 (m, 2H), 3.89 (s, 1H), 2.49 (t, J = 8.8
Hz, 1H), 2.00–2.05 (m, 2H), 1.83–1.95 (m, 6H), 1.60–1.72
(m, 3H), 1.11–1.55 (m, 15H), 0.99 (d, J = 4.4 Hz, 3H), 0.97
(s, 3H), 0.69 (s, 3H) (Supplemental material, Figure S24);
13C NMR (100 MHz, CDCl3): δ 164.75, 164.24, 150.49,
150.37, 136.35, 135.85, 130.67, 130.63, 123.56, 123.41,
76.19, 68.52, 66.52, 55.90, 50.50, 42.72, 41.24, 39.59,
39.36, 35.43, 35.25, 35.13, 34.94, 34.46, 32.91, 32.00,
28.26, 26.75, 25.19, 23.72, 22.74, 20.64, 18.59, 11.81
(3R,5S,8R,9S,10S,13R,14S,17R)-3-hydroxy-17-((R)-5-hydroxy-
pentan-2-yl)-10,13-dimethylhexadecahydro-7H-cyclopenta[a]
phenanthren-7-one (14). To a mixture of 13 (18.0 g, 26.7
mmol) in a solution consisting of water (13 mL), tetrahydro-
furan (60 mL), and methanol (30 mL), sodium hydroxide
(2.7 g, 66.8 mmol) was added at 25 °C followed by stirring
at 25 °C for 1 h. The reaction mixture was diluted with water
(100 mL) and extracted with ethyl acetate (100 mL × 2).
The organic layer was dried over anhydrous sodium sulfate
and concentrated under reduced pressure to afford a residue,
which was recrystallized from ethyl acetate (180 mL) and
heptane (110 mL) to yield compound 14 as a white solid
1
(8.4 g, yield: 84%, purity: 96.2%). H NMR (400 MHz,
CDCl3): δ 3.56–3.64 (m, 3H), 2.85 (dd, J = 12.4 Hz, 6 Hz,
1H), 2.38 (t, J = 11.2 Hz, 1H), 1.77–2.02 (m, 6H), 1.60–
1.72 (m, 3H), 1.35–1.51 (m, 7H), 1.05–1.33 (m, 9H), 1.19
(s, 3H), 0.88–0.99 (m, 1H), 0.93 (d, J = 6 Hz, 3H), 0.65 (s,
3H) (Supplemental material, Figure S30); 13C NMR (100
MHz, CDCl3): δ 212.16, 71.10, 63.73, 55.11, 49.69, 49.06,
46.24, 45.56, 42.90, 42.77, 39.13, 37.57, 35.65, 35.31,
34.30, 32.00, 30.06, 29.59, 28.57, 25.00, 23.21, 21.86,
18.89, 12.21 (Supplemental material, Figure S31). HRMS:

Feng et al.
9
then warming to −10 °C for 1 h. The reaction mixture was
diluted with dichloromethane (50 mL) and quenched with
50 mL of aqueous sodium hydroxide solution (2.6 g, 65.8
mmol). The organic layer was separated, dried over anhy-
drous sodium sulfate, and concentrated under reduced pres-
sure to afford a residue, which was recrystallized from
dichloromethane (80 mL) to yield impurity 2 (4.5 g, 78%
yield from 14, purity: 98.8%). m.p.: 106–107 °C. IR (KBr)
m/z [M + H]+ Calcd for C24H41O3: 377.3056; found:
377.3050 (Supplemental material, Figure S29).
(3R,5S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-3-
((trimethylsilyl)oxy)-17-((R)-5-((trimethylsilyl)oxy)pentan-2-yl)
hexadecahydro-7H-cyclopenta[a]phenanthren-7-one (15). To a
mixture of 14 (8 g, 21.2 mmol) and triethylamine (6.4 g, 63.6
mmol) in dichloromethane (80 mL), trimethylsilyl chloride
(6.9 g, 63.6 mmol) was added slowly at 0 °C followed by
stirring at 25 °C for 2 h. The reaction mixture was diluted
with water (50 mL). The organic layer was separated, dried
over anhydrous sodium sulfate, and concentrated under
reduced pressure to afford 15 as yellow oil (14.2 g), which
was not purified prior to use in the next step. A small analyti-
cal sample of 15 was purified by column chromatography.
IR (KBr) νmax/cm−1: 2955, 2866, 1707 (C=O), 1466, 1381,
1252, 1098, 1078(Si–O), 1015, 885, 841(Si–C), 756, 746
ν
max/cm−1: 3337 (OH), 2940, 2872, 1690 (C=O), 1636
(C=C), 1458, 1375, 1065, 1011, 735, 669 (Supplemental
material, Figure S35). 1H NMR (600 MHz, CD3OD): δ 6.08
(q, J = 7.2 Hz, 1H), 3.59 (sept, J = 4.8 Hz, 1H), 3.48–3.54
(m, 2H), 2.68 (dd, J = 13.2 Hz, 4.2 Hz, 1H), 2.35–2.38 (m,
1H), 2.32 (t, J = 11.4 Hz, 1H), 2.08 (dt, J = 13.2 Hz, 3 Hz,
1H), 1.89–2.00 (m, 3H), 1.71 (d, J = 7.2 Hz, 3H), 1.69 (m,
1H), 1.46–1.65 (m, 5H), 1.39–1.45 (m, 3H), 1.27–1.35 (m,
3H), 1.06–1.24 (m, 5H), 1.04 (s, 3H), 0.96 (d, J = 6.6 Hz,
1
3H), 0.69 (s, 3H) (Supplemental material, Figure S37); 13
C
(Supplemental material, Figure S32). H NMR (600 MHz,
NMR (150 MHz, CD3OD): δ 207.64, 145.38, 130.34,
70.96, 63.55, 56.18, 52.03, 50.18, 46.88, 44.66, 40.74,
40.32, 38.37, 36.87, 35.77, 35.41, 33.24, 30.46, 30.28,
29.53, 27.03, 23.24, 22.45, 19.35, 12.75, 12.49 (Supple-
mental material, Figure S38). HRMS: m/z [M + H]+ Calcd
for C26H43O3: 403.3212; found: 403.3208 (Supplemental
material, Figure S36).
CDCl3): δ 3.51–3.57 (m, 3H), 2.81 (dd, J = 12.6 Hz, 6 Hz,
1H), 2.34 (t, J = 11.4 Hz, 1H), 2.15–2.22 (m, 1H), 1.98 (dt,
J = 12.6 Hz, 3 Hz, 1H), 1.78–1.92 (m, 5H), 1.55–1.61 (m,
3H), 1.35–1.46 (m, 7H), 1.19–1.26 (m, 2H), 1.17 (s, 3H),
1.03–1.16 (m, 4H), 0.87–0.95 (m, 4H), 0.63 (s, 3H), 0.11 (s,
9H), 0.09 (s, 9H) (Supplemental material, Figure S33); 13C
NMR (100 MHz, CDCl3): δ 211.92, 71.45, 63.41, 55.12,
49.65, 49.04, 46.25, 45.60, 42.73, 42.47, 39.10, 37.76, 35.66,
35.26, 34.49, 32.04, 30.46, 29.54, 28.54, 25.05, 23.19, 21.77,
18.84, 12.19, 0.30, -0.27 (Supplemental material, Figure
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
S34). Anal. cacld for C30H56O3Si2 (520.38): C, 69.17; H, respect to the research, authorship, and/or publication of this
article.
10.84; found: C, 68.89; H, 10.61.
Funding
(((3R,5S,8S,9S,10S,13R,14S,17R)-10,13-dimethyl-17-((R)-5-
( ( t r i m e t h y l s i l y l ) o x y ) p e n t a n - 2 - y l ) -
The author(s) received the following financial support for the
2,3,4,5,8,9,10,11,12,13,14,15,16,17-tetradecahydro- research, authorship, and/or publication of this article: Financial
support was provided by the Zhejiang University of Technology
and the China State Institute of Pharmaceutical Industry.
1H-cyclopenta[a]phenanthrene-3,7-diyl)bis(oxy))
bis(trimethylsilane) (16). To a mixture of lithium diisopro-
pylamide (28.8 mL, 57.6 mmol, 2 mol L−1) and trimethylsi-
lyl chloride (5.2 g, 48.0 mmol) in tetrahydrofuran (50 mL),
a tetrahydrofuran (30 mL) solution containing 15 (10 g,
19.2 mmol) was added slowly at −70 °C followed by stir-
ring for 2 h. The reaction mixture was diluted with ethyl
acetate (100 mL) and quenched with an aqueous solution
(50 mL) of citric acid monohydrate (4 g, 19.2 mmol). The
organic layer was separated, dried over anhydrous sodium
sulfate, and concentrated under reduced pressure to afford
16 as yellow oil (13.5 g), which was not purified prior to
use in the next step. Intermediate 16 was not analyzed due
to its inherent instability.
Supplemental material
Supplemental material for this article is available online.
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