First synthesis and characterization of key stereoisomers related to Ezetimibe

Article abstract of DOI:10.1016/j.cclet.2014.03.035

During the laboratory optimization and the late phase manufacturing studies of the cholesterol absorption inhibitor Ezetimibe 1, the formation of several stereoisomers was observed. To study the complete stereoisomer profile of Ezetimibe 1, we have synthesized and completely characterized several key stereoisomers of Ezetimibe 1 for the first time. This study will provide an access to the reference standard of these stereoisomers and may have some implications in the development of new medicines.

Full text of DOI:10.1016/j.cclet.2014.03.035

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CCLET-2921; No. of Pages 4
Chinese Chemical Letters xxx (2014) xxx–xxx
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
First synthesis and characterization of key stereoisomers related to
Ezetimibe
*
*
Yun Ren, Yan-Jun Duan, Ren-Jun Li, Yong Deng, Li Hai , Yong Wu
Key Laboratory of Drug Targeting and Drug Delivery System of the Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy,
Sichuan University, Chengdu 610041, China
A R T I C L E I N F O
A B S T R A C T
Article history:
During the laboratory optimization and the late phase manufacturing studies of the cholesterol
absorption inhibitor Ezetimibe 1, the formation of several stereoisomers was observed. To study the
complete stereoisomer profile of Ezetimibe 1, we have synthesized and completely characterized several
key stereoisomers of Ezetimibe 1 for the first time. This study will provide an access to the reference
standard of these stereoisomers and may have some implications in the development of new medicines.
ß 2014 Li Hai and Yong Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Received 9 January 2014
Received in revised form 3 March 2014
Accepted 12 March 2014
Available online xxx
Keywords:
Ezetimibe
Related substances
Stereoisomers profile
Synthesis
Characterization
1. Introduction
stereoisomers, the synthesis of the final products with the required
stereochemistry is a significant challenge [10]. However, stereo-
The safety of a drug product is not only dependent on the
toxicological properties of the active drug substance (or API), but
also on the impact of impurities formed during the various
chemical transformations. Therefore, the development of a drug
substance, especially for a chiral drug, is incomplete without the
identification and characterization of its stereoisomer profile [1–
3]. Strict regulatory guidelines of the International Conference on
Harmonization (ICH) have led to an increasing need for identifica-
tion, quantification and control of trace impurities in the drug
substances and drug products to obtain marketing approval [4–6].
However, it is more challenging to identify the impurities that are
formed in very small quantities. Since frequently it is very difficult
to identify and control impurities within acceptable levels in the
process, extra purification steps may then be necessary thereby
making the process less competitive. More often than not, the
syntheses of impurities are not described in the literature, which
posts a greater challenge [1].
isomers of chiral drugs often show different behaviors in
pharmacological action and metabolic process. It is definitely
common that one stereoisomer is active while other stereoisomers
are toxic in biological systems. The pharmaceutical industry has
recently raised its emphasis on the generation of single optical
isomer before undertaking pharmacokinetic, metabolic, physio-
logical, and toxicological evaluation in the search for drugs with
greater therapeutic benefits and lower toxicity [11].
During the laboratory optimization and the late phase
manufacturing studies of Ezetimibe 1, low levels (<0.1%) of SRR-
Ezetimibe 2 and RRS-Ezetimibe 4 were discovered as shown in
Fig. 2. In our previous research [12], we have successfully designed
an unambiguous synthetic approach to prepare SRR-Ezetimibe 2
and its enantiomer RSS-Ezetimibe 3. Single-crystal X-ray analysis
of SRR-Ezetimibe 2 [13] provided the final verification of the
structural assignments. Considering that the fact that the synthesis
and characterization of stereoisomers 4–8 is almost completely
overlooked, we explored the synthesis and characterization of
stereoisomers 4–8 of Ezetimibe 1 for the first time in this study. It is
essential to characterize the complete stereoisomer profile of
Ezetimibe 1 and meet the stringent regulatory requirements of
ICH. These results will be of immense help for organic chemists to
understand the potential stereoisomers in Ezetimibe 1 API and
may have some implications in the development of new medicines
[12].
Ezetimibe 1 shown in Fig. 1 has been commercialized as an
effective inhibitor of intestinal cholesterol and related phytosterol
absorption for lowering cholesterol levels [7–9]. As three
asymmetric carbons in the Ezetimibe molecule give rise to eight
*
Corresponding authors.
E-mail addresses: smile@scu.edu.cn (L. Hai), wyong@scu.edu.cn (Y. Wu).
http://dx.doi.org/10.1016/j.cclet.2014.03.035
1001-8417/ß 2014 Li Hai and Yong Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: Y. Ren, et al., First synthesis and characterization of key stereoisomers related to Ezetimibe, Chin.
Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.035

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CCLET-2921; No. of Pages 4
2
Y. Ren et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Fig. 1. Ezetimibe 1, process related stereoisomers 2–8.
reduction of ketone 11 with borane dimethyl-sulfide at À5 8C
delivered the pure RRS-Ezetimibe 4 after recrystallization from
i-PrOH and water.
The same strategy to synthesize the stereoisomer 4 can be used
to prepare stereoisomer 5 when we employed compound 9b as our
starting material as shown in Scheme 2.
As mentioned in Scheme 3, our synthesis approach to prepare
stereoisomers 6–8 firstly began with the coupling of 4-(2-(4-
fluorophenyl)-5,5-dimethyl-1,3-dioxan-2-yl)butanoic acid with
(R)-4-phenyl-2-oxazolidinone which was done by the mixed
anhydride method using pivaloyl chloride and triethylamine to
obtain oxazolidinone 14. The crucial enolate condensation of
oxazolidinone 14 with N-(4-((tert-butyldimethylsilyl)oxy)benzy-
lidene)-4-fluoroaniline in the presence of TiCl4 and Ti(OiPr)4 at
À40 8C delivered the key intermediates 15a and 15b with the ratio
of two stereoisomers (15a:15b) about 1:8.
Fig. 2. HPLC chromatogram of the crude Ezetimibe 1.
2. Experimental
Based on the mechanism of Ti(IV)-catalyzed Mannich-type
equilibrium reaction and the fact that all the physical properties
(melting point, ESI-MS, NMR) of the intermediates 15a and 9b are
identical except for the optical rotation values, which are of
opposite sign, it can be concluded that the intermediates 15a and
9b are a pair of enantiomers. Same is true for the intermediates 15b
and 9a. Having established the absolute configuration of inter-
mediates 15a and 15b, we were able to obtain stereoisomers 6–8
using a similar sequence as shown in Scheme 4.
Stereoisomers 4–8 are major byproducts in the preparation of
the crude Ezetimibe 1 API, and difficult to be purified by column
chromatography. Herein, we design an efficient route to synthesize
the stereoisomers 4–8. As shown in Scheme 1, compound 9a on
reaction with N,O-bis(trimethylsilyl)acetamide and tetrabutyl-
ammonium fluoride trihydrate in acetonitrile at 25 8C gave the
cyclized
b-lactam product 10. b-Lactam product 10 was treated
with 2 mol/L H2SO4 in i-PrOH to get the ketone 11 which is purified
by column chromatography. S-Methyloxazaborolidine-mediated
(S)
(S)
C6H5
O
C6H5
O
O
O
O
O
O
O
TBSO
TBSO
N
(R)
O
N
(R)
O
O
N
O
O
O
OH
(R)
F
(S)
(S)
C6H5
+
O
O
F
NH
NH
O
O
F
F
OTBS
F
F
F
N
9a
9b
a
OH
O
OTBS
O
O
N(S)
c
(R)
b
4
N(S)
(R)
F
O
F
O
F
11
10
F
Scheme 1. Reagents and conditions: (a) BSA, TBAFÁ3H2O, CH3CN, 25 8C, 86.7%; (b) 2 mol/L H2SO4, i-PrOH, 50 8C, 79.6%; (c) S-CBS, 94% BH3-DMS, DCM, À5 8C, 82.3%.
Please cite this article in press as: Y. Ren, et al., First synthesis and characterization of key stereoisomers related to Ezetimibe, Chin.
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3
Scheme 2. Reagents and conditions: (a) BSA, TBAFÁ3H2O, CH3CN, 25 8C, 92.8; (b) 2 mol/L H2SO4, i-PrOH, 50 8C, 80.3%; (c) S-CBS, 94% BH3-DMS, DCM, À5 8C, 84.9%.
Scheme 3. Reagents and conditions: (a) pivaloyl chloride, TEA, DCM, r.t.; (R)-4-phenyloxazolidin-2-one, LiCl, 45 8C, 78.5%; (b) TiCl4, Ti(OiPr)4, DIEA, DCM, À40 8C.
and practical method to access stereoisomers 4–8 of Ezetimibe 1 as
shown in Schemes 1–4.
The RRS-Ezetimibe 4 was a main by-product when we used R-
methyloxazaborolidine-mediated reduction of ketone 11 with
borane dimethyl-sulfide to prepare the crude Ezetimibe 1 API [14–
16]. The polarity of RRS-Ezetimibe 4 and Ezetimibe 1 is so close that
they are difficult to be separated completely even using HPLC as
shown in Fig. 2. High levels (>3%) of RRS-Ezetimibe 4 may be
monitored by HPLC when the temperature was unsatisfactory. The
RRS-Ezetimibe 4 with high purity can be prepared and recrys-
tallized from i-PrOH and water when we employed S-methylox-
azaborolidine to replace the R-methyloxazaborolidine as shown in
Scheme 1. Thus, we can make full use of the RRS-Ezetimibe 4 to
establish an HPLC method for the analysis of Ezetimibe 1 API.
In our previous work [12], we have confirmed the absolute
configuration of intermediate 9b using single-crystal X-ray
analysis of the final product SRR-Ezetimibe 2 derived from 9b.
In our large scale preparation of Ezetimibe 1 API, we can
recrystallize the pure compound 9b from the concentrated mother
liquor after removing the main product 9a. Thus, the same strategy
Scheme 4. Reagents and conditions: (a) BSA, TBAFÁ3H2O, CH3CN, 25 8C; (b) 2 mol/L
H2SO4, i-PrOH, 50 8C; (c) R-CBS, 94%BH3-DMS, DCM, À5 8C; (d) S-CBS, 94% BH3-DMS,
DCM, 0 8C.
3. Results and discussion
Based on a comprehensive review of the literature, we found
little research on the synthesis of the stereoisomers 4–8 related to
Ezetimibe, which represents a defect and deficiency in the quality
analysis and quality control of Ezetimibe API. Katarzyna Filip and
his co-workers [10] indicated that the formation of RRS-Ezetimibe
4 is inconvenient because this impurity is difficult to remove from
the final product Ezetimibe 1. Compound EZ-6, which is a key
intermediate to prepare RRR-Ezetimibe 5 as shown in Scheme 5,
was not isolated in Filip’s work. We also failed to observe the
desired RRS-Ezetimibe 4 and RRR-Ezetimibe 5 under the reported
reaction conditions or their slight variations. Gratifyingly, the
disappointing results have prompted us to identify an alternative
to synthesize the stereoisomer 2 can be used to prepare
stereoisomer 5 when we employed S-methyloxazaborolidine to
replace the R-methyloxazaborolidine in the reduction of ketone 13
with borane dimethyl-sulfide as shown in Scheme 2.
The impressive stereodirecting power of (S)-4-phenyl-2-oxa-
zolidinone in the crucial enolate condensation suggests that its
enantiomer (R)-4-phenyl-2-oxazolidinone can be used to generate
the key chiral intermediates 15a and 15b as shown in Scheme 3
[12]. In some of commercial samples, (S)-4-phenyl-2-oxazolidi-
none was found to be contaminated with trace amount of (R)-4-
phenyl-2-oxazolidinone, which is why low levels (<1%) of
intermediates 15a and 15b may be detected in the key
Scheme 5. The route to prepare RRR-Ezetimibe 5 of Katarzyna Filip and his co-workers.
Please cite this article in press as: Y. Ren, et al., First synthesis and characterization of key stereoisomers related to Ezetimibe, Chin.
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detected in the crude Ezetimibe 1.
[4] International Conference on Harmonization (ICH) Guidelines, Q3A (R) Impurities
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application on the content and qualification of impurities in new drug substances
produced by the chemical synthesis).
4. Conclusion
[5] International Conference on Harmonization (ICH) guidelines, Q3B (R) Impurities
in New Drug Substances, 2002 (Guidance for registration or marketing application
on the content and qualification of impurities in new drug product).
In summary, to better understand the synthetic pathway of an
active pharmaceutical ingredient (API), we have synthesized and
completely characterized several key stereoisomers of Ezetimibe 1
for the first time. This research, together with previous work [12],
completed the synthesis and characterization of all stereoisomers
related to Ezetimibe 1, which is essential to establish the complete
´
´
ˇ
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stereoisomer profile of Ezetimibe
1 to meet the stringent
regulatory requirements of ICH. Our research will provide an
access to the reference standard of these stereoisomers and may
have some implications in the development of new medicines.
[10] K. Filip, K. Bankowski, K. Sidoryk, et al., Physicochemical characterization of
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Acknowledgments
[12] Y. Ren, R.J. Li, Y. Deng, et al., First synthesis and characterization of SRR/RSS-
Ezetimibe, Tetrahedron Lett. 54 (2013) 6443–6446.
We would like to express our gratitude to Sichuan Provincial
Science and Technology Support Program (No. 2011SZ0014) and
Sichuan Bali Pharmaceutical Co., Ltd. for financial support.
[13] Crystallographic data (excluding structure factors) for the structures in this paper
have been deposited with the Cambridge Crystallographic Data Centre as sup-
plementary publication nos. CCDC 944997. Copies of the data can be obtained,
free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(fax: +44 01223 336033 or email:deposit@ccdc.cam.ac.uk).
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Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.035