Identification, characterization, synthesis and strategy for minimization of potential impurities observed in the synthesis of brivaracetam

Article abstract of DOI:10.1016/j.tet.2020.131273

A first systematic impurity profile research concerning nine observed and potential process related impurities of antiepileptic drug brivaracetam is reported. Among which three (impurity G/H/I) have not been discovered or reported before, these nine impurities were monitored by HPLC, and their structures were identified on the basis of MS and NMR spectroscopy. In addition to the formation, synthesis, and characterization, strategies for minimizing these impurities to the levels accepted by ICH are also described in this report.

Full text of DOI:10.1016/j.tet.2020.131273

Journal Pre-proof
Identification, characterization, synthesis and strategy for minimization of potential
impurities observed in the synthesis of brivaracetam
Shouzhu Liao, Hongjun Chen, Guifei Wang, Shuming Wu, Zaiyou Yang, Weihe Luo,
Zhuanfeng Liu, Xun Gao, Junhai Qin, Chuan-hua Li, Zhongqing Wang
PII:
S0040-4020(20)30419-1
https://doi.org/10.1016/j.tet.2020.131273
TET 131273
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 20 March 2020
Revised Date: 20 April 2020
Accepted Date: 11 May 2020
Please cite this article as: Liao S, Chen H, Wang G, Wu S, Yang Z, Luo W, Liu Z, Gao X, Qin J, Li
C-h, Wang Z, Identification, characterization, synthesis and strategy for minimization of potential
impurities observed in the synthesis of brivaracetam, Tetrahedron (2020), doi: https://doi.org/10.1016/
j.tet.2020.131273.
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Identification, Characterization, Synthesis
and Strategy for Minimization of Potential
Impurities Observed in the Synthesis of
Brivaracetam
Leave this area blank for abstract info.
‖
‖
Shouzhu Liaob,c, , Hongjun Chenb,c, , Guifei Wangb, Shuming Wub, Zaiyou Yangb, Weihe Luob, Zhuanfeng
Liub, Xun Gaob, Junhai Qinb, Chuan-hua Lia, Zhongqing Wang*,a,b,c
a Hunan Provincial Key Laboratory of Xiangnan Rare-Precious Metals Compounds Research and Application,
Xiangnan University, Chenzhou 423000, China
b HEC Research and Development Center, HEC Pharm Group, Dongguan 523871, P. R. China
c State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co.,Ltd, Dongguan 523871,
P. R. China

1
Tetrahedron
journal homepage: www.elsevier.com
Identification, Characterization, Synthesis and Strategy for Minimization of Potential
Impurities Observed in the Synthesis of Brivaracetam
‖
‖
Shouzhu Liaob,c, , Hongjun Chenb,c, , Guifei Wangb, Shuming Wub, Zaiyou Yangb, Weihe Luob,
Zhuanfeng Liub, Xun Gaob, Junhai Qinb, Chuan-hua Lia, Zhongqing Wang*,a,b,c
a Hunan Provincial Key Laboratory of Xiangnan Rare-Precious Metals Compounds Research and Application, Xiangnan University, Chenzhou 423000, China
b HEC Research and Development Center, HEC Pharm Group, Dongguan 523871, P. R. China
c State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co.,Ltd, Dongguan 523871, P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A first systematic impurity profile research concerning nine observed and potential process
related impurities of antiepileptic drug brivaracetam is reported. Among which three (impurity
G/H/I) have not been discovered or reported before, these nine impurities were monitored by
HPLC, and their structures were identified on the basis of MS and NMR spectroscopy. In
addition to the formation, synthesis, and characterization, strategies for minimizing these
impurities to the levels accepted by ICH are also described in this report.
Available online
2009 Elsevier Ltd. All rights reserved
.
Keywords:
Brivaracetam
Process
impurity profile
impurity control
———
‖ These authors contributed equally.
* Corresponding author. e-mail: wangzhongqing@hecpharm.com.

2
Tetrahedron
1. Introduction
not to mention the synthetic methods (only impurity A/B/D were
reported considering this issue).3-6
Brivaracetam (Briviact®; BRIVLERATM), a 4-n-propyl
Therefore, we present herein a research on identification,
characterization and synthesis for nine observed and potential
impurities in 1. The corresponding possible formation pathways
were also discussed and the strategy for minimization of nine
impurities to the levels accepted by International Conference on
Harmonization (ICH)7 has also been demonstrated. We hope that
our report will provide useful information for other organic
chemists working on this field.
analogue of levetiracetam developed by UCB Pharma, which is a
high affinity synaptic vesicle protein 2A (SV2A) ligand, has been
approved as an adjunctive therapy for the treatment of partial-
onset seizures and epilepsy in Europe, the US, and many other
countries.1 In addition, Briviact was further approved for the
application on adolescents and children from 4 years of age by
FDA and EMA in 2018. Thus there has been a great interest on
the synthesis of brivaracetam (1).
2. Result and discussion
Scheme 1. Retrosynthetic Analysis for Reported Routes
towards Brivaracetam
The identification and possible pathways for the formation of
impurities are elaborated at first, and then the strategies we
adapted for assessing and minimizing theses impurities to the
level accepted by ICH are described as follows.
As the isomeric impurity control following ICH requirements
was a critical part of process research, the isomers of 1 were
initially taken into account during the process development
(Scheme 3). However, there has been little information on the
synthetic details of the stereoisomers of 1,3 except for some being
mentioned as side products toward the synthesis of the drug
substance.2b,8 Thus, the isomers should be synthesized at first to
help us better study their formation and removal, and the
plausible formation mechanism and characterization are
presented in the following context.
As depicted in Scheme 1, various routes to synthesize
brivaracetam with different retrosynthetic disconnecting patterns
have been reported since 2001.2 These synthetic strategies can be
divided into three categories according to the origins of the two
chiral centers in brivaracetam: by chiral chromatography2a, by
chiral starting material and chiral seperation2b-d and all by chiral
starting materials.2e-k
Scheme 3. Brivaracetam and Its Isomers
In view of that procedures with chiral chromatography as a
separation method were undoubtedly lack of practicality and
viability in large-scale production and effective fine control over
isomeric impurities with aspect to manipulation, we considered
utilizing the synthetic method that starts with two enantiospecific
compounds as starting materials for process development
(Scheme 2). Meanwhile, the corresponding impurity profile of
this procedure was thus studied in detail.
Based on our initial studies, impurity A was believed to spring
from enantiomer of the starting material (R)-2-(2-(tert-butoxy)-2-
oxoethyl)pentanoic acid 2. For impurity B, it might mostly come
from the racemization during the reaction process and partly
originate from enantiomer of the other starting material (S)-2-
aminobutanamide hydrochloride 6. Comparatively, impurity C as
the enantiomer of 1 was considered as a result of joint effects
which might lead to the formation of impurity A and B
respectively.
Scheme 2. Our Route towards Brivaracetam (1)
Considering that impurity A and B were reported to be
achieved by resolution or chiral chromatography from mixtures,3
which was neither atom economic nor convenient for
manipulation, other alternative synthetic methods were
attempted. And as a result, impurity A, B and C were prepared
in analogy to the synthetic procedure of 1 by employing
corresponding chiral starting materials for oriented synthesis.
These stereoisomers were characterized by Nuclear Magnetic
Resonance (NMR), High Resolution Mass Spectrometry
(HRMS) and optical rotation (see experimental section and
supporting information) which matched with literatures.4 Aside
from this, for a better validation of the synthetic strategy and the
structure of the impurities, HSQC, HMBC and 1H-1H COSY
data were recorded and herein we provide assignment of impurity
C for illustration (Table 1). For example, location of ethyl group
in impurity C was proven by 1H-1H COSY NMR showing spin
interaction between 8-H (4.46 ppm) and 10-H (1.94 ppm and
1.69 ppm) while 3-H (1.42 ppm) showed interaction with 2-H
(1.33 ppm) and 4-H (2.33 ppm).
Impurities are inevitable during the process of drug
development. For the purpose of ensuring safety and efficacy of
the drug substance, impurity profile research which is the basis of
specification setting for active pharmaceutical ingredient (API) is
of great significance. To the best of our knowledge, no
systematic study of quality research involved in the preparation
of brivaracetam has been reported. Besides, three among them
(impurity G/H/I) have not been discovered or reported before,
Table 1. NMR Correlation Spectroscopy of Impurity C

3
Impurity C
23.61
2.33
a Agilent HPLC. Column: Chiralpak IC (4.6 mm × 250 mm), 5 μm. Flow rate:
1.0 mL/min. λ: 210 nm. Injection vol: 10 μL. Mobile phase A: hexane.
Mobile phase B: isopropanol. Run time: 30 min. Temperature: 30 °C.
Gradient (A:B = 45:55, v/v).
HSQC
1H-1H
COSY
No.
HMBC(1H→13C)
δH
δC
Except for stereoisomers, LC-MS was used as a common
approach to identify impurities in 1, by which the molecular
weights for impurities D-I were observed (Table 3, relative
retention time of impurity A\B in this method was 1.08). Their
structures were later confirmed by HRMS, NMR and HPLC co-
injection. These impurities are formed due to degradation
(impurities D), contaminants in starting materials (impurity E, I),
incomplete reactions (impurities F, H), or overreaction
(impurities G). During the study we realized that almost all the
impurities listed in Table 3 (except for impurity D) could not be
completely removed from 1 due to solubility issues. Therefore,
understanding how these impurities were formed could be of
great value to late stage specification establishment and control
strategy development.
1
2
3
4
0.92 (3H, overlap)
1.33 (2H, m)
14.0
20.6
36.6
31.9
C-2, 3
H-2
H-1, 3
H-2, 4
H-3, 5, 7
C-1, 3, 4
1.42 (2H, m)
C-1, 2, 4, 5, 7
C-2, 3, 5, 6, 7
2.33 (1H, m)
2.57 (1H, dd, J=16.9,
8.6 Hz)
2.08 (1H, dd, J=16.9,
8.1 Hz)
5
6
7
37.9
175.7
49.6
C-3, 4, 6, 7
N/A
H-4
N/A
H-4
N/A
3.49 (1H, dd, J=9.8,
7.9 Hz)
3.04 (1H, dd, J=9.8,
7.1 Hz)
4.46 (1H, dd, J=8.9,
6.9 Hz)
Table 3. Potential Impurities in Brivaracetam (reverse phase)a
C-3, 4, 5, 6
relative
retention time
Name
Structure
8
56.0
172.1
N/A
20.9
10.5
C-6, 7, 9, 10, 11
N/A
H-10
N/A
9
N/A
Impurity D
0.44
6.35 (1H, s); 5.64
(1H, s)
1.94(1H, m); 1.69
(1H, m)
9a
10
11
N/A
N/A
C-8, 9, 11
C-8, 10
H-8, 11
H-10
0.91 (3H, overlap)
Impurity E
Impurity F
0.89
With all the stereoisomers as reference compounds in hand,
we developed a normal-phase HPLC analytical method for a
simultaneous detection of the four isomers to verify their
0.91
1.08
1.29
presence or absence and thereafter provide
a
more
comprehensive understanding and a better control. The retention
time of 1 and impurity A, B and C in this method were 10.13
min, 15.04 min, 17.33 min and 23.61 min respectively (Table 2).
Impurity A/B
(Diastereoisomers)
-
Table 2. HPLC Chromatogram of Brivaracetam and Its
Impurity G
Stereoisomers (Normal Phase)a
Impurity H
Impurity I
1.76
1.99
Relative
Retention
Time
Retention
time (min)
Name
Structure
a Agilent HPLC. Column: Waters BEH shield RP18 (4.6 mm × 100 mm), 2.5
μm. Flow rate: 1.0 mL/min. λ: 210 nm. Injection vol: 5 μL. Mobile phase A:
the phosphate buffer solution (dissolved 1.36 g potassium dihydrogen
phosphate of in water and diluted to 1 L, adjusted to pH = 7. 0 with 1N
potassium hydroxide aqueous solution) / acetonitrile (95:5, v/v). Mobile
phase B: acetonitrile. Run time: 40 min. Brivaracetam retention time: about
9.49 min. Temperature: 25 °C. Gradient (A:B, 0-12 min: 90:10; 12-22 min:
82:18; 22-31 min: 25:75; 31-40 min: 90:10).
Impurity A
15.04
1.49
1.71
Impurity B
17.23
(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]-butanoic
acid
(Impurity D), resulting from hydrolysis of the amide moiety, was
not only a degradation product but also a metabolite of 1.5 There
are several literature reports, predominantly focusing on the
synthesis of 1, in which impurity D was mentioned as one

4
Tetrahedron
intermediate.6 In our process, impurity D, which occurred from
Impurity F, which arose from incomplete reaction, had a
slightly complicated origin. Residual 2 which was still retained in
intermediate 4 after several steps of preparation esterificated
the hydrolysis of compound 1 (Scheme 4), was an unwanted
compound in API that needed to be controlled under certain
limit.
under the condition to produce
5 (TMSBr/EtOH/DCM),
providing (R)-diethyl 2-propylsuccinate (F-1), which further
underwent a double amidation with the same condition to
synthesize compound 1 to afford impurity F (Scheme 7). Also
outlined in Scheme 7, incomplete reaction of starting material 6
with 5 was the causation of impurity H. The reaction stopped
after an SN2 substitution to provide impurity H instead of further
latamization to furnish 1. These two impurities were henceforth
synthesized as proposed.
Scheme 4. Possible Pathway to Impurity D
Scheme 7. Possible Pathway to Impurity F\H
Thus, we developed a much simpler method different from the
known procedures, using D-1 instead of 6 to generate the methyl
ester analogue of 1 (D-2), followed by a basic hydrolysis with
LiOH to furnish impurity D (see scheme 5).
Scheme 5. Synthesis of Impurity D
Impurity G appeared as a notable overreaction impurity during
the process development. And it might emerge in the final step
while the newly formed target compound 1 continued to
condense with another molecule of (S)-2-aminobutanamide (B-2)
(scheme 8). According to our study, the amount of impurity G
had a positive correlation of the reaction temperature and the
amount of starting material 6 employed.
Scheme 8. Possible Pathway to Impurity G
According to the synthetic route, 2 and 6 were not only starting
materials but also critical components as they have contained all
atoms required in brivaracetam molecule. Hence, impurities in 2
and 6 were easy to be brought in and transferred to the
corresponding byproducts in API. As depicted in Scheme 6,
impurity E was also believed to be introduced from (S)-4-(tert-
butoxy)-2-isopropyl-4-oxobutanoic acid (E-2), structural isomer
of 2 and impurity I was believed to originate from 2-
(methylamino)butanamide (I-1), an impurity in 6. These two
impurities were synthesized as proposed.
Scheme 6. Possible Pathway to Impurity E\I
Br
OEt
O
O
O
O
O
R
1.ClCO2Et,
TEA
O
Conc.HCl
TMSBr
O
To the best of our knowledge, there has been no report
regarding impurity G. Consequently, we designed a route to
synthesize impurity G instead of the aforementioned pathway by
which the target impurity could not be prepared in sufficient
quantity. After benzyloxycarbonyl (Cbz) group protection of (S)-
2-aminobutanoic acid (G-1), amidation reaction with (S)-2-
aminobutanamide (B-2) under condition of ethyl chloroformate
was performed followed by a Cbz group deprotection to provide
G-4, dimer derivative of B-2. Final amidation with compound 5
in ethanol furnished impurity G in an overall yield of 35% from
G-1 (see scheme 9).
HO
2. NaBH4,
EtOH, DCM
HO
EtOH
DCM
R
R
R
O
R = iPr
R = nPr
E-5
5
R = iPr
R = nPr
E-2
2
R = iPr
R = nPr
E-3
3
R = iPr
R = nPr
E-4
4
(S)
H2N
NH2.HCl
(S)
Br
OEt
O
R
6
O
N
(S)
NH2
Na2CO3 / EtOH
O
O
R = iPr
E-5
Impurity E
Scheme 9. Synthesis of Impurity G
H2N
N
H
Br
OEt
O
R
I-1
N
(R)
Na2CO3 / EtOH
NH2
O
OEt
O
O
R = nPr
5
Impurity I

5
Table 5. Effect of Recrystallization Solvents of 1 for
Isomersa
Impurity (HPLC area%)
Yield
Solvent
(%)
93
A
B
C
Isopropyl ether
Methyl tert-butyl
ether
0.09
0.12
0.03
0.09
0.08
0.14
0.02
0.02
0.01
91
88
Isopropyl acetate
a
All recrystallization procedures performed with 10.00g crude 1 (impurity A
0.09%, impurity B 0.35%, impurity C 0.03%) and solvent (30.0 ml).
3. Impurity Control
Subsequently, the depletion of other process related impurities
were also evaluated (Table 6). Except for impurity D, which has
an excellent solubility in water and was not detected in final
product 1 due to effective elimination through acidic and basic
work-up conditions, impurity E-I exhibited better purging results
in isopropyl acetate compared to other solvents.
As mentioned in previous sections, the configuration
inversions of the two chiral centers had different explanations.
Epimerization at 4’-position led to the formation of impurity A,
which originated from the enantiomer impurity in starting
material 2, while inversion of the configuration at 2-position
mostly arose from racemization during the reaction process,
delivering impurity B (see Table 4). And it could be inferred that
impurity C was generated as an outcome of these joint effects.
Thus for the control of impurity A, it was confirmed that the
concentration of impurity A in 1 could be under 0.15% by
demonstrating an epimerization level below 0.15% at the source
in starting material 2, which also helped to control the level of
impurity C accordingly.
Table 6. Screening Recrystallization Solvents of 1a
Impurity (HPLC area%)
Recrystallization solvent
E
F
G
H
I
Isopropyl ether
Methyl tert-butyl ether
Isopropyl acetate
Heptaneb
0.09
0.09
0.08
0.10
ND
0.01
ND
0.20
0.13
0.10
ND
0.01
ND
ND
0.50
ND
0.01
ND
0.09
In comparison, the formation of impurity B was much more
process-related and after detailed optimization, it was found that
the content of impurity B increased as the reaction temperature
escalated (Table 4). Hence, it could be concluded that precise
control on reaction temperature would certainly play an
important role in the suppression of impurity B.
0.48
a All recrystallization procedures performed with 10.00 g crude 1 (impurity E
0.10%, impurity F 0.21%, impurity G 0.48%, impurity H 0.65%, impurity I
0.11%) and solvent (30.0 ml).
b Crude 1 was not soluble in heptane but just heated at 50.0 °C.
Impurity E and I were both closely related to the starting
materials. However, impurity E could merely get purged during
the recrystallization process whilst impurity I could be controlled
easily to the limit required according to ICH guidelines. Hence,
E-2 needed to be controlled to <0.10% in the starting material 2
to ensure the level of impurity E in 1 below 0.10%. In addition,
impurity F and H could also be well removed by recrystallization
in isopropyl acetate (0.21% to ND for impurity F and 0.65% to
ND for impurity H). Impurity G was an ineluctable overreaction
byproduct which correlated with the reaction conditions of
preparing target compound 1. To our delight, impurity G could
also be purged almost completely in isopropyl acetate and
therefore controlled within specification.
Table 4. Effect of Temperature in The Reaction of Preparing
1 for Isomersa
Entry
1
Temp
40℃
45℃
50℃
55℃
60℃
65℃
70℃
80℃
Impurity A
Impurity B
Impurity C
0.09%
0.04%
0.01%
2
3
4
5
6
7
8
0.08%
0.09%
0.09%
0.09%
0.08%
0.09%
0.09%
0.06%
0.06%
0.08%
0.15%
0.35%
0.68%
1.05%
0.02%
0.01%
0.01%
0.02%
0.03%
0.03%
0.03%
4. Conclusion
For better understanding of the synthetic pathway of an active
pharmaceutical ingredient (API), it is necessary to identify all the
impurities formed/anticipated. In this regard, nine observed and
potential impurities of brivaracetam have been identified,
synthesized, and characterized while three (impurity G/H/I)
among which have not been discovered or reported before, not to
mention the synthetic methods. The corresponding possible
formation pathways were also discussed and the strategy for
minimization of impurities A−I to the levels accepted by ICH has
also been demonstrated. We envision this to be the first report on
impurity profile research of brivaracetam and from which we
hope other process chemists on drug development will benefit.
a
All reactions performed at EtOH as the solvent, with 1.0 eq 5 (99.90% ee),
1.0 eq Na2CO3 and 2.0 eq 6 (99.99% ee). The reaction time was about 24h.
Furthermore, for a double security on the quality control of 1,
recrystallization was applied and several solvents were
henceforth screened (Table 5). Except for impurity A, which
could not be completely eliminated during the recrystallization,
the removal effect of impurity B and C in isopropyl acetate was
acceptable. Based on that, controlling the presence of enantiomer
within the specification of the starting materials was thus of
paramount significance to limit impurity A below permitted
levels.
5. Experimental Section
If no specially indicated, all reagents and solvents were used
as commercially available without further purification. NMR
spectra were measured on a Bruker Avance 400 spectrometer in
the solvents indicated; chemical shifts are reported in units (ppm)

6
Tetrahedron
by assigning TMS resonance in the 1H spectrum as 0.00ppm.
overlap); 13C NMR (151 MHz, CDCl3) δ 175.51 (s), 172.32 (s),
55.92 (s), 49.67 (s), 37.68 (s), 36.86 (s), 31.79 (s), 21.11 (s),
20.60 (s), 14.00 (s), 10.56 (s); NMR were in accordance with
Coupling constants are reported in Hz with multiplicities denoted
as singlet (s), doublet (d), triplet (t), quartet (q), dd (doublet of
doublets); m (multiplets), and etc. HRMS were performed on
Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.
Analytical HPLC for liquid phase was carried out on an Agilent
HPLC workstation. Optical rotation was recorded on a Rudolph
Autopol IV polarimeter. The angle of rotation was recorded at 20
°C on 1% solutions in MeOH.
literature.4b HRMS [M+H]+ for C11H21N2O2 , calculated:
213.1598; found 213.1608.
+
Impurity C. According to the synthesis of impurity A and
using (S)-2 and (R)-6 as starting materials, impurity C is
provided as a white solid (50.12 g, 51%) with a purity of 98.9%
20
1
by HPLC and 99.5% ee. [α]D =56.69° (c=1.01, CH3OH); H
NMR (600 MHz, CDCl3) δ 6.35 (1H, s), 5.64 (1H, s), 4.46 (1H,
dd, J=8.9, 6.9 Hz), 3.49 (1H, dd, J=9.8, 7.9 Hz), 3.04 (1H, dd,
J=9.8, 7.1 Hz), 2.57 (1H, dd, J=16.9, 8.6 Hz), 2.33 (1H, m), 2.08
(1H, dd, J=16.9, 8.1 Hz), 1.94(1H, m), 1.69 (1H, m), 1.42 (2H,
m), 1.33 (2H, m), 0.92 (3H, overlap), 0.91 (3H, overlap); 13C
NMR (151 MHz, CDCl3) δ 175.67 (s), 172.13 (s), 55.98 (s),
49.56 (s), 37.89 (s), 36.60 (s), 31.92 (s), 20.92 (s), 20.55 (s),
14.00 (s), 10.46 (s); NMR were in accordance with literature.4b
Impurity A. A 2.0 L double jacketed reactor equipped with a
mechanical stirrer and a condenser is charged with (S)-2-(2-(tert-
butoxy)-2-oxoethyl)pentanoic acid ((S)-2) (100.00 g, 0.46 mol)
and dichloromethane (1.0 L). The solution is cooled to −10.0 °C
under nitrogen flow. Triethylamine (60.83 g, 0.60 mol) is added,
and then ethyl chloroformate (73.53 g, 0.60 mol) is added
dropwise maintaining the temperature between −10.0 °C and
−5.0 °C over a period of 30 min. The heterogeneous reaction
mixture is stirred for 0.5 h at −10 °C, diluted with water (0.40 L),
followed by standing for 20 min for organic layer separation. The
organic layer is cooled to −15.0 °C. Under nitrogen flow,
powdered sodium borohydride (43.73 g, 1.16 mol) is added.
Then ethanol (0.2 L) is transferred dropwise in order to maintain
the temperature below −5 °C. The reaction mixture is stirred for
minimum 3 h at −15 °C, and then quenched with HCl aqueous
solution (1.2 mol/L, 0.6 L). The mixture is warmed to 25 °C, and
the aqueous layer is discarded. Hydrochloric acid (14.05 g, 0.14
mol) is added one pot and the reaction mixture is heated at 30 °C
for 6 h. After cooled to room temperature, the dichloromethane
solution is washed with water (0.4 L) then evaporated to give (S)-
4 (59.25 g, 100%). To the oil of compound (S)-4, CH2Cl2 (0.4 L)
and EtOH (53.27 g, 1.16 mol) are added. Under a nitrogen flow,
TMSBr (53.27 g, 1.16 mol) is added dropwise at 20 °C, and then
the reaction mixture is stirred for 6 h. After quenched with the
aqueous solution (0.4 L) of sodium carbonate (35.00 g), the
mixture is separated and the organic layer is evaporated under
reduced pressure to obtain the crude oil. Then S-2-
aminobutanamide (prepared from 6 according to the patent
WO2017076738, 94.48 g, 0.92 mol) and Na2CO3 (48.76g,
0.46mol) in EtOH (0.5 L) is added and the mixture is heated at
50 °C for 16 h. After removal of solvent EtOH by evaporation,
water (0.2 L) is added and the aqueous solution is extracted with
dichloromethane (3 × 0.4 L). The combined organic phase is
evaporated under reduced pressure to obtain the crude solid. The
residue is purified by recrystallization from isopropyl acetate
(IPAC) to afford impurity A as a white solid (50.03 g, 51%) with
+
HRMS [M+H]+ for C11H21N2O2 , calculated: 213.1598; found
213.1609.
Impurity D. A 1.0 L double jacketed reactor equipped with a
mechanical stirrer and a condenser is charged with compound 4
(50.00 g, 0.39 mol), CH2Cl2 (0.4 L) and EtOH (44.93 g, 0.98
mol). Under a nitrogen flow, TMSBr (119.45 g, 0.78 mol) is
added dropwise at 20 °C, and then the reaction mixture is stirred
for 6 h. After quenched with the aqueous solution (0.4 L) of
sodium carbonate (35.00 g), the mixture is separated and the
organic layer is evaporated under reduced pressure to obtain the
crude oil. Then (S)-methyl-2-aminobutanoate hydrochloride
(94.48 g, 0.92 mol) and Na2CO3 (97.52 g, 0.92 mol) in EtOH (0.5
L) is added and the mixture is heated at 50 °C for 48 h. After
removal of solvent EtOH by evaporation, water (0.2 L) is added
and the aqueous solution is extracted with dichloromethane (3 ×
0.4 L). The combined organic phases are evaporated under
reduced pressure to obtain the crude oil (D-2). The residue is
added lithium hydroxide monohydrate (15.55 g, 0.37 mol),
dichloromethane (0.2 L) and water (0.4 L), then the mixture is
stirred at room temperature for 6 h. The reaction mixture is added
water (0.2 L) and exacted with CH2Cl2 (3 × 0.2 L). The combined
organic phase is evaporated under reduced pressure to afford
impurity D as a yellow solid (43.25 g, 52%) with a purity of
1
95.2% by HPLC. H NMR (600 MHz, CDCl3) δ 9.91 (1H, brs),
4.65 (1H, dd, J=10.9, 5.0 Hz), 3.43 (1H, dd, J=9.3, 8.0 Hz), 3.19
(1H, dd, J=9.4, 7.3 Hz), 2.61 (1H, dd, J=16.9, 8.5 Hz), 2.34 (1H,
m), 2.17 (1H, dd, J=16.9, 8.3 Hz), 2.05 (1H, m), 1.71 (1H, m),
1.47 (2H, m), 1.34 (2H, m), 0.93 (3H, overlap), 0.92 (3H,
overlap); 13C NMR (151 MHz, CDCl3) δ 176.78 (s), 174.33 (s),
55.41 (s), 49.57 (s), 37.65 (s), 36.42 (s), 32.17 (s), 21.86 (s),
20.65 (s), 14.01 (s), 10.84 (s); NMR were in accordance with
20
a purity of 99.1% by HPLC and 99.2% ee. [α]D =-46.46°
1
(c=1.00, CH3OH); H NMR (600 MHz, CDCl3) δ 6.31 (1H, s),
5.56 (1H, s), 4.46 (1H, t, J=7.8 Hz), 3.56 (1H, t, J=8.8 Hz), 3.01
(1H, dd, J=9.6, 6.8 Hz), 2.53 (1H, dd, J=16.7, 8.6 Hz), 2.36 (1H,
m), 2.13 (1H, dd, J=16.7, 7.9 Hz), 1.97(1H, m), 1.68 (1H, m),
1.43 (2H, m), 1.34 (2H, m), 0.93 (3H, overlap), 0.91 (3H,
overlap); 13C NMR (151 MHz, CDCl3) δ 175.53 (s), 172.32 (s),
55.97 (s), 49.68 (s), 37.66 (s), 36.86 (s), 31.81 (s), 21.01 (s),
20.59 (s), 14.00 (s), 10.55 (s); NMR were in accordance with
+
literature.6a HRMS [M+H]+ for C11H20NO3 , calculated:
214.1438; found 214.1448.
Impurity E. According to the synthesis of impurity A,
impurity E is synthesized using the starting materials E-2 and 6
as white solid (48.32 g, 49%) with a purity of 98.6% by HPLC.
1H NMR (400 MHz, CDCl3) δ 6.47 (1H, s), 5.78 (1H, s), 4.46
(1H, dd, J = 8.7, 7.0 Hz), 3.45 (1H, dd, J = 9.7, 8.2 Hz), 3.17 –
3.03 (1H, m), 2.52 (1H, dd, J = 16.5, 8.4 Hz), 2.19 – 1.85 (3H,
m), 1.77 – 1.50 (2H, m), 0.94 – 0.86 (9H, m); 13C NMR (151
MHz, CDCl3) δ 175.77 (s), 172.17 (s), 55.84 (s), 48.02 (s), 39.37
(s), 36.32 (s), 32.43 (s), 20.94 (s), 20.45 (s), 20.03 (s), 10.41 (s);
+
literature.4b HRMS [M+H]+ for C11H21N2O2 , calculated:
213.1598; found 213.1609.
Impurity B. According to the synthesis of impurity A and
using 2 and R-2-aminobutanamide hydrochloride ((R)-6) as
starting materials, impurity B is provided as a white solid (50.00
20
g, 51%) with a purity of 99.5% by HPLC and 99.6% ee. [α]D
+
HRMS [M+H]+ for C11H21N2O2 , calculated: 213.1603; found
1
=48.32° (c=1.01, CH3OH); H NMR (600 MHz, CDCl3) δ 6.47
213.1603.
(1H, s), 5.81 (1H, s), 4.48 (1H, t, J=7.8 Hz), 3.58 (1H, t, J=8.8
Hz), 3.00 (1H, t, J=8.2 Hz), 2.52 (1H, dd, J=16.7, 8.6 Hz), 2.36
(1H, m), 2.12 (1H, dd, J=16.7, 7.9 Hz), 1.96(1H, m), 1.66 (1H,
m), 1.43 (2H, m), 1.35 (2H, m), 0.93 (3H, overlap), 0.91 (3H,
Impurity F. A 1.0 L double jacketed reactor equipped with a
mechanical stirrer and a condenser is charged with compound 2
(50.00 g, 0.23 mol), CH2Cl2 (0.4 L) and EtOH (26.63 g, 0.58

7
+
mol). Under a nitrogen flow, TMSBr (70.79 g, 0.46 mol) is added
dropwise at 20 °C, and then the reaction mixture is stirred for 66
h. After quenched with the aqueous solution (0.4 L) of sodium
carbonate (17.15 g), the mixture is separated and the organic
layer is evaporated under reduced pressure to obtain the crude oil.
Then (S)-2-aminobutanamide (prepared from 6 according to the
patent WO2017076738, 47.23 g, 0.46 mol ) and Na2CO3 (24.50
g, 0.46 mol) in EtOH (0.3 L) are added and the mixture is heated
at 50 °C for 16 h. After removal of solvent EtOH by evaporation,
water (0.2 L) is added and the aqueous solution is extracted with
dichloromethane (3 × 0.3 L). The combined organic phases are
evaporated under reduced pressure to obtain the crude solid. The
residue is purified by recrystallization from IPAC to provide
impurity F as white solid (20.02 g, 38%) with a purity of 95.1%
by HPLC. 1H NMR (400 MHz, CDCl3) δ 7.49 (1H, s), 7.06 (1H,
s), 4.28 (1H, dd, J = 11.0, 5.0 Hz), 2.96 – 2.73 (2H, m), 2.39 (1H,
dd, J = 17.3, 3.5 Hz), 2.10 – 1.79 (2H, m), 1.74 – 1.55 (1H, m),
1.43 (1H, dt, J = 21.2, 7.9 Hz), 1.35 – 1.24 (2H, m), 0.95 – 0.74
(4H, m), 0.73 – 0.52 (3H, m); 13C NMR (151 MHz, D2O) δ
183.58 (s), 180.17 (s), 173.60 (s), 55.07 (s), 39.56 (s), 33.69 (s),
32.45 (s), 20.80 (s), 19.18 (s), 13.02 (s), 9.89 (s); HRMS [M+H]+
13.99 (s), 10.51 (s), 9.97 (s); HRMS [M+H]+ for C15H28N3O3 ,
calculated: 298.2125; found 298.2134.
Impurity H. A 1.0 L double jacketed reactor equipped with a
mechanical stirrer and a condenser is charged with compound 4
(50.00 g, 0.39 mol), CH2Cl2 (0.4 L) and EtOH (44.93 g, 0.98
mol). Under a nitrogen flow, TMSBr (119.45 g, 0.78 mol) is
added dropwise at 20 °C, and then the reaction mixture is stirred
for 6 h. After quenched with the aqueous solution (0.4 L) of
sodium carbonate (35.00 g), the mixture is separated and the
organic layer is evaporated under reduced pressure to obtain the
crude oil. Then (S)-2-aminobutanamide (39.84 g, 0.39 mol) and
Na2CO3 (8.27 g, 0.08 mol) in EtOH (0.5 L) are added and the
mixture is heated at 50 °C for 12 h. After removal of solvent
EtOH by evaporation, water (0.2 L) is added and the aqueous
solution is extracted with dichloromethane (2 × 0.4 L). The
combined organic phase is evaporated under reduced pressure to
obtain the crude oil. After preparative chromatography, impurity
H is obtained as a light yellow oil (15.22 g, 15%) with a purity of
95.6% by HPLC. For the convenience in storage, Impurity H was
prepared as a hydrochloride salt before NMR detection. 1H NMR
(600 MHz, DMSO) δ 9.48 (1H, brs), 8.62 (1H, m), 8.16 (1H, s),
7.73 (1H, s), 4.07 (2H, q, J=7.0 Hz), 3.67 (1H, m), 2.86 (1H, m),
2.71 (1H, m), 2.63 (1H, dd, J=16.4, 5.4 Hz), 2.33 (1H, dd,
J=16.4, 7.5 Hz), 2.20 (1H, m), 1.97(1H, m), 1.81 (1H, m), 1.38
(1H, m), 1.25 (3H, m), 1.19 (3H, t, J=7.1 Hz), 0.90 (3H, t, J=7.5
Hz), 0.86 (3H, t, J=7.1 Hz); 13C NMR(151 MHz, DMSO) δ
171.70 (s), 169.06 (s), 61.42 (s), 59.89 (s), 49.28 (s), 35.98 (s),
33.13 (s), 31.88 (s), 22.77 (s), 18.79 (s), 14.09 (s), 13.88 (s), 9.20
+
for C11H19N2O3 , calculated: 227.1396; found 227.1395.
Impurity G. A 1.0 L double jacketed reactor equipped with a
mechanical stirrer and a condenser is charged with NaOH (38.79
g, 0.92 mol) and water (0.5 L). The solution is cooled to −5.0 °C
and then (S)-2-aminobutyric acid (50.00 g, 0.49 mol) is added.
Benzyl chloroformate (102.50 g, 0.63 mol) in toluene (0.3 L) is
added dropwise and the reaction mixture is stirred at room
temperature for 6 h. The mixture is added Na2CO3 until the pH of
aqueous layer is about 9, and the organic layer is discarded. The
aqueous layer is extracted with toluene (2 × 0.2 L) and then is
added hydrochloric acid until the pH of aqueous layer is about 3.
It is extracted with toluene (2 × 0.3 L) and the combined organic
phases are evaporated under reduced pressure to obtain the crude
colorless liquid. N-methyl morphofine (46.90 g, 0.46 mol) and
CH2Cl2 (1.0 L) are added and the mixture is cooled to −15.0 °C.
Ethyl chloroformate is added dropwise maintaining the
temperature between −15.0 °C and −5.0 °C over a period of 30
min, then the precipitated solid is filtered and the filtrate is still
stirred at −15.0 °C. (S)-2-aminobutanamide (46.98 g, 0.46 mol) is
added. Followed by standing for 30 min, the reaction mixture is
warmed to room temperature for 2 h, and then the precipitated
solid is filtered, washed with water, dried at 50 °C under reduced
pressure to provide G-3 as a white solid. To the solution of G-3
in isopropyl alcohol (1.2 L) 10% Pd/C (9.00 g) is added, and the
mixture is stirred under H2 balloon at room temperature
overnight. After filtration, the filtrate is evaporated to provide G-
4 (78.96 g, 0.42 mol, 86% from G-1) as a white solid. 5 (50.00 g,
0.21 mol) and Na2CO3 (22.35 g, 0.21 mol) are added to the
solution of G-4 in EtOH (0.3 L) and the mixture is heated at 50
°C for 16 h. After removal of solvent EtOH by evaporation,
water (0.2 L) is added and the aqueous solution is extracted with
dichloromethane (3 × 0.3 L). The combined organic phase is
evaporated under reduced pressure to obtain the crude solid. The
residue is purified by recrystallization from IPAC to provided
impurity G as a white solid (36.52 g, 58%) with a purity of
(s); HRMS [M+H]+ for C13H27N2O3 , calculated: 259.2016; found
+
259.2040.
Impurities I. A 1.0 L double jacketed reactor equipped with a
mechanical stirrer and
a condenser is charged with 2-
bromobutylamide (50.00 g, 0.31 mol), 30% methylamine ethanol
solution (93.54 g, 0.90 mol) and ethanol (0.2 L). The mixture is
heated at 30.0 ℃ for 24 h and then evaporated to remove
methylamine and ethanol. The residue is added 30% hydrogen
chloride ethanol solution (96.60 g) and ethanol (0.6 L). After
being stirred for 2.0 h, the precipitated solid is filtered, washed
with ethanol (0.1 L). The wet solid is added isopropyl alcohol
(0.3 L) and treated with ammonia for 2 h. The precipitated solid
is filtered and the filtrate is evaporated to provide I-1 (30.01 g,
86% from 2-bromobutylamide) as colorless oil. To the oil of I-1,
5 (50.00 g, 0.21 mol) and Na2CO3 (22.35 g, 0.21 mol) in EtOH
(0.3 L) are added and the mixture is heated at 50 °C for 16 h.
After removal of solvent EtOH by evaporation, water (0.4 L) and
hydrochloric acid (0.1 L) are added and the aqueous solution is
extracted with dichloromethane (2 × 0.2 L). The retained aqueous
layer is added sodium carbonate until the pH is about 9. The
aqueous solution is extracted with dichloromethane (2 × 0.3 L)
and the combined organic phases are evaporated under reduced
pressure to obtain impurity I as a light yellow oil (16.87 g, 48%)
1
with a purity of 94.9% by HPLC. H NMR (400 MHz, CDCl3) δ
6.71 (1H, d, J = 48.4 Hz), 6.51 (1H, d, J = 12.3 Hz), 4.02 – 3.85
(2H, m), 2.73 – 2.56 (1H, m), 2.25 – 1.97 (8H, m), 1.50 (2H, pd,
J = 13.5, 7.4 Hz), 1.19 – 1.02 (7H, m), 0.81 – 0.69 (6H, m); 13C
NMR (151 MHz, CDCl3) δ 176.44 (s), 176.17 (s), 173.79 (s),
173.65 (s), 71.42 (s), 69.74 (s), 61.59 (s), 60.06 (*2), 59.08 (s),
38.65 (*2), 38.06 (s), 37.38 (s), 35.15 (*2), 33.32 (*2), 20.85 (s),
19.78 (*2), 19.72 (s), 14.07 (s), 14.03 (s), 14.01 (s), 13.99 (s),
1
97.5% HPLC. H NMR (600 MHz, CDCl3) δ 7.02 (1H, d, J=7.9
Hz), 6.37 (1H, s), 5.95 (1H, s), 4.48 (1H, dd, J=8.9, 6.8 Hz), 4.34
(1H, m), 3.51 (1H, dd, J=9.8, 7.9 Hz), 3.07 (1H, dd, J=9.8, 6.8
Hz), 2.58 (1H, dd, J=16.9, 8.6 Hz), 2.33 (1H, m), 2.07 (1H, dd,
J=16.9, 7.8 Hz), 1.93 (1H, m), 1.89 (1H, m), 1.71(1H, m), 1.65
(1H, m), 1.40 (2H, m), 1.32 (2H, m), 0.92 (3H, overlap), 0.91
(3H, overlap), 0.89 (3H, overlap); 13C NMR (151 MHz, CDCl3) δ
175.67 (s), 173.77 (s), 170.35 (s), 56.52 (s), 54.16 (s), 49.77 (s),
37.87 (s), 36.70 (s), 31.90 (s), 25.01 (s), 21.25 (s), 20.52 (s),
11.71 (s), 11.09 (s); HRMS [M+H]+ for C14H29N2O3 , calculated:
+
273.2178; found 273.2212.
Acknowledgement
This work was financially supported by the State Key
Laboratory of Anti-Infective Drug Development (Sunshine Lake

8
Tetrahedron
8
Norrant, E.; Nuel, D.; Giordano, L.; Leclaire, J.; Septavaux, J.
Continuous Process for Preparing Brivaracetam. PCT Int. Patent
Application WO2017/076737 A1, May 11, 2017.
Pharma Co.,Ltd), (N0. 2015DQ780357). We also gratefully
acknowledge financial support from the Key research and
development program of Guangdong Province, China. (Grant No.
2019B02021002) and the Hunan Provincial Natural Science
Foundation of China (No. 2017JJ2240).
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5
6
7
ICH Q3A (R2) Impurities in New Drug Substances, International
Conference on Harmonisation ofTechnical Requirements for
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Switzerland, October 2006 ICH Q3A Impurities in New Drug
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Human Use (ICH): Geneva, Switzerland, October,2006.

1. In this work, nine potential process related impurities detected during the impurity profile study of antiepileptic drug,
brivaracetam, have been given.
2. A systematic study of quality research involved in the preparation of brivaracetam has been reported, describing the
possible formation pathways, synthesis, and characterization, strategies for minimizing these impurities to the levels
accepted by ICH.
3. It is the first report on impurity profile research of brivaracetam from which we hope other chemists on drug
development will benefit.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: