Enantioseparation of racemic bupivacaine via ultrasonic-assisted diastereomeric crystallization using 12,14-dinitrodehydroabietic acid

Article abstract of DOI:10.1016/j.ultsonch.2019.01.020

12,14-Dinitrodehydroabietic acid (12,14-dinitroDHAA), a chiral acid obtained by the nitration of optical dehydroabietic acid (DHAA), was successfully employed as resolving agent. The resolution of racemic bupivacaine by ultrasonic-assisted diastereomeric crystallization in ethanol was investigated. The results indicated that ultrasonic-assist can well facilitate resolution of (R,S)-bupivacaine and a higher enantiomeric excess (ee) and yield was obtained for (S)-bupivacaine, and while without ultrasound, the ee value decreases by increasing the crystallization time. A Box-Behnken experimental design with four factors (amount of 12,14-dinitroDHAA, ethanol amount, ultrasonic power and crystallization temperature) combined with response surface methodology (RSM) was applied to explore resolution effects. A second-order polynomial equation was adequate to model the relationship between the ee (or yield) and the dependent variables. When maintaining a lower limit of 90% for the yield of (S)-bupivacaine, the optimal resolution conditions by RSM were 12,14-dinitroDHAA/bupivacaine molar ratio of 1.6, solvent/propranolol ratio of 16.5 mL/g, 63.2 W ultrasonic power and crystallization temperature of 0 °C, respectively. Under the optimal conditions, the experimental ee and yield of (S)-bupivacaine were 69.8% and 87.5%.

Full text of DOI:10.1016/j.ultsonch.2019.01.020

Accepted Manuscript
Enantioseparation of racemic bupivacaine via ultrasonic-assisted diastereomer-
ic crystallization using 12, 14-dinitrodehydroabietic acid
Li Ge, Yi Zhu, Yonghui Qi, Yande Chen, Kedi Yang
PII:
S1350-4177(18)31300-2
DOI:
https://doi.org/10.1016/j.ultsonch.2019.01.020
ULTSON 4462
Reference:
To appear in:
Ultrasonics Sonochemistry
Received Date:
Revised Date:
Accepted Date:
26 August 2018
3 January 2019
17 January 2019
Please cite this article as: L. Ge, Y. Zhu, Y. Qi, Y. Chen, K. Yang, Enantioseparation of racemic bupivacaine via
ultrasonic-assisted diastereomeric crystallization using 12, 14-dinitrodehydroabietic acid, Ultrasonics
Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.2019.01.020
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Enantioseparation of racemic bupivacaine via
ultrasonic-assisted diastereomeric crystallization using 12,
1
4-dinitrodehydroabietic acid
a
b
b
b
a*
Li Ge , Yi Zhu , Yonghui Qi , Yande Chen , Kedi Yang
a
Department of Pharmaceutical Engineering, Medical College of Guangxi University, Nanning
30004, China
5
b
School of Chemistry & Chemical Engineering, Guangxi University, Nanning 530004, China
Corresponding Author. E-mail address: kdyang@163.com
*

ABSTRACT
12,14-Dinitrodehydroabietic acid (12,14-dinitroDHAA), a chiral acid obtained by the
nitration of optical dehydroabietic acid (DHAA), was successfully employed as resolving agent.
The resolution of racemic bupivacaine by ultrasonic-assisted diastereomeric crystallization in
ethanol was investigated. The results indicated that ultrasonic-assist can well facilitate resolution
of (R,S)-bupivacaine and a higher enantiomeric excess (ee) and yield was obtained for
(
S)-bupivacaine, and while without ultrasound, the ee value decreases by increasing the
crystallization time. A Box-Behnken experimental design with four factors (amount of
2,14-dinitroDHAA, ethanol amount, ultrasonic power and crystallization temperature) combined
1
with response surface methodology (RSM) was applied to explore resolution effects. A
second-order polynomial equation was adequate to model the relationship between the ee (or yield)
and the dependent variables. When maintaining a lower limit of 90% for the yield of
(S)-bupivacaine, the optimal resolution conditions by RSM were 12,14-dinitroDHAA/bupivacaine
molar ratio of 1.6, solvent/propranolol ratio of 16.5mL/g, 63.2W ultrasonic power and
crystallization temperature of 0℃, respectively. Under the optimal conditions, the experimental ee
and yield of (S)-bupivacaine were 69.8% and 87.5%.
Keywords: Bupivacaine, Resolution, Diastereomeric crystallization, Ultrasonic,
12,14-Dinitrodehydroabietic acid, Response surface methodology

1
. Introduction
Enantiomers of chiral molecules are a specific class of stereoisomers that can exhibit very
different biological activities in human body, such as pharmacology, toxicology, pharmacokinetics
and metabolism[1,2]. Following this reason, the single enantiomers as chiral pharmaceuticals are
required for the pharmaceutical industry, where a number of drugs sold nowadays are enantiopure
chiral compounds. So, economically effective route to optically pure enantiomers is becoming
increasingly important.
There are two main ways to obtain high-pure chiral substances up to now including
asymmetric synthesis[3-5] and resolution or separation of the racemate (a mixture with equal
amounts of enantiomers) using chiral chromatography[6,7], diastereomeric crystallization[8-10],
kinetic resolution[11,12], enantioselective extraction[13,14], chiral inclusion[15,16], and
adsorption resolution via chiral MOFs or MIPs[17-21]. However, the asymmetric synthesis of
enantiomers with high purities and yields remains a challenge. The separation of enantiomers is a
key strategy to produce optically chiral drugs. Among various resolution methods of the racemate,
diastereomer crystallization has been the most useful method to obtain pure chiral compounds,
especially at an industry scale. It is estimated that more than half of the optical chiral drugs on the
market were produced by this method[22].
As a means of enhancing mass transfer, the ultrasonic can be applied to crystallization
processes because of its several advantages, such as decrease of the induction time, narrowing of
the metastable zone width, accelerating the primary nucleation and so on[23-27]. Many
investigations have reported these effects of the ultrasound to crystallization of achiral compounds,
although the detailed mechanism of how the ultrasound facilitates crystallization is poorly
understood, which is generally considered that most effects are due to the cavitation phenomenon
and the acoustic streaming caused by the ultrasound in liquid[28]. Also, several published
literatures involved the application of ultrasound in asymmetric synthesis[29,30], chiral
amplification and symmetry breaking during the crystallization process[31,32], kinetic resolution
and kinetic control of sonocrystallization in some enantiomeric mixtures[33]. Recently, Stefanidis
summarized the application of ultrasound to chiral crystallization processes that separate

enantiomers, ranging from diastereomeric resolution and preferential crystallization to new and
emerging processes such as attrition-enhanced deracemization[34,35]. Only two previous studies
have reported the effects of the ultrasound on diastereomeric crystallization. Barve et al.[36]
reported the diastereomeric salt resolution of an intermediate of silodosin using (S)-mandelic acid
as chiral resolving agent, and a yield increase of the diastereomeric salt was observed from 10% to
34% by 30 min of ultrasonic irradiation. In another recent report, Szeleczky et al.[37] explored the
effect of ultrasound-assisted diastereomeric crystallization on the resolution of tetramisole
enantiomers, a faster crystallization and a higher enantiomeric excess of the desired
diastereomeric salt was achieved using 4.3W power of ultrasound irradiation for 15 min.
In the present work, our aim is to investigate the ultrasonic-facilitated enantioseparation
effect of racemic bupivacaine, currently used as an epidural anaesthetic during labour, and
(S)-isomer (levobupivacaine) was confirmed less cardiotoxic in man and significantly safer than
racemic bupivacaine[38]. For this purpose, 12,14-dinitrodehydroabietic acid (12,14-dinitroDHAA)
is employed as chiral resolving agent, which was synthesized from dehydroabietic acid (DHAA),
a largely and inexpensively obtained natural chiral carboxylic acid from the disproportionated
rosin. The successful resolution of (R,S)-bupivacaine via ultrasound-assisted diastereomeric
crystallization, and optimization of the resolution process parameters by response surface
methodology (RSM) for the better resolution efficiency of (S)-bupivacaine were reported.
2
. Materials and methods
2.1. Materials
The racemic bupivacaine hydrochloride was provided by Zhuhai Rundu Pharmaceutical Co.,
Ltd (Zhuhai, China), which was dissolved in 20% NaOH aqueous solution, followed by
recrystallization of the precipitate to obtain base form. Optically pure (R)- and (S)-bupivacaine
were purchased from Sigma-Aldrich Co. (Shanghai, China). The resolving agent,
1
1
2,14-dinitroDHAA was synthesized by the introduction of two nitro groups at the 12- and
4-positions of dehydroabietic acid according to the literature[39], and after purified on a silica
gel column, its purity (mass fraction) was higher than 99.5% as determined by LabAlliance Model
00 high performance liquid chromatography system (Scientific System Inc., United States). All
2

other reagents and solvents were of analytical grade.
.2. Resolution of (R, S)-bupivacaine using ultrasound-assist
.76g (20 mmol) racemic bupivacaine base and 1.0 equivalent of 12,14-dinitroDHAA were
2
5
dissolved in 30 mL ethanol, respectively. The two solutions were mixed and reacted in a jacked
glass vessel connected with thermostatic water circulator at 50℃for 30 min. Then the mixture was
exerted ultrasonic irradiation, and sequentially cooled to 5℃straightaway by using a cold bath
(Tokyo Rikakikai CO., Ltd. Japan). The power ultrasonic was supplied by one model of
XODL-1000N ultrasound apparatus (Nanjing Xianou Instruments Manufacture Co., Ltd., China)
equipped with a φ15 mm acoustic horn at a frequency of 25ꢀKHz. The ultrasonic irradiation was
operated with a work mode of radiating continuously for 4 s then followed an interval of 2 s. The
crystallized out (S)-bupivacaine diastereomeric salt was filtered and treated with 20% NaOH to
yield (S)-bupivacaine. The resolution efficiency was described by the enantiomeric excess (ee)
(Eq.1) and the yield was calculated based on (S)-bupivacaine (Eq.2), respectively.
ee % = ([S] - [R]) / ([R] + [S]) ×100
(1)
(2)
*
Yield %= [S] × m / [S ] × m ×100
2
1
*
where [S] and [R] are the purity of (S)- and (R)-bupivacaine in the product, [S ] is the purity of (S)-
bupivacaine in the racemic bupivacaine base, m and m are the mass of the racemic base and
1
2
product, respectively.
In order to compare the impact of ultrasonic on resolution, the reference experiments without
ultrasonic were performed. The resolution results were also evaluated by ee and yield of
(S)-bupivacaine.
2.3 Chromatographic Conditions
The analysis of (R)- and (S)-bupivacaine was carried out on a LabAlliance HPLC system
equipped with a CHROMTECH AGP chiral column (100 mm×4.0 mm, 5.0 μm). A mobile phase
composed of buffer solution of potassium dihydrogen phosphate (pH=7) and isopropanol in a
-1
volume ratio fraction of 90:10, was used to run the separation at a flow rate of 0.8 mL.min .
Detective wavelength was set at 215 nm. All chromatograph procedures were performed at room
temperature.

2.4. Experimental design
3
A 4 Box-Behnken factorial design was used to investigate factors (independent variables)
having significant effect on the ee and yield of (S)-bupivacaine (dependent variables) by
ultrasonic-assisted diastereomeric crystallization. The factor selection and its range assignment
were based on our previous screening experiments. Four factors, i.e. resolving agent-bupivacaine
molar ratio (n /n ), liquid-solid ratio (V /g, ethanol volume per gram of bupivacaine), ultrasonic
r
rp
s
power (P, electrical power) and crystallization temperature (T) were chosen. Each independent
variable was coded at three levels between −1 and +1. The factors with their variation levels are
shown in Table1. The ee and yield of (S)-bupivacaine influenced by four independent variables
are set as the desired goal (response) for fitting a second-order polynomial regression model
(Eq.3):
(3)
where Y is the predicted response of ee or yield, β is a constant, β , β and β are constant
0
i
ii
ij
coefficients for the linear, quadratic and interaction effects, respectively, x and x are coded
i
j
independent variables. The Design-Expert V10.0 was used for analysis of variance (ANOVA),
determination of coefficient as well as estimation of goodness of model fitting.
3. Results and discussion
3.1. Resolution of (R,S)-bupivacaine using 12,14-dinitroDHAA
Bupivacaine is a chiral secondary amine (Fig.1a), the resolution of this compound by
diastereomeric crystallization using optical tartaric acid as the resolving agent had previously been
documented in the literature[40,41], and however optical tartaric acid is relatively expensive.
Cheung et al.[42] reported that racemic mixtures of bupivacaine base can crystallize in the chiral
space P212121, suggesting a direct preferential crystallization may be used to resolve this racemic
mixture, however, bupivacaine base is a white powder, during their sample preparation, for which
handling is rendered difficult due to static charges. In addition, preferential crystallization requires
the racemic mixture exists as a conglomerate[43]. Therefore, the conglomerate and pure
enantiomers of bupivacaine base were obtained tediously in general. Dehydroabietic acid (DHAA,
Fig.1b) is a natural chiral acid, and can be largely and inexpensively obtained using fractional

crystallization from the disproportionated rosin. Some base enantiomers can be seperated using
DHAA[44,45], but not effective for the resolution of racemic bupivacaine demonstrated by our
previous experimentals. Based on three-point interactions model in enantiomer separation
invented by Dalgliesh[46], we synthesised 12,14-dinitroDHAA (Fig. 1b) from DHAA via
electrophilic aromatic substitution, intrducing a dipole interaction of nitro group to improve its
chiral recognition selectivity to (R)- and (S)-bupivacaine. The structure of 12,14-dinitroDHAA
1
13
was identified by NMR ananysis, and its chemical shifts of H and C in DMSO-d are as follows:
6
1
H NMR: δ 11.89 (s, 1H), 7.49 (s, 1H), 2.27 (dd, J = 18.1, 6.6 Hz, 1H), 2.16 (ddd, J = 18.2, 10.8,
7.7 Hz, 1H), 2.07 (s, 1H), 1.93 (d, J = 12.6 Hz, 1H), 1.65 (s, 1H), 1.57 (d, J = 12.5 Hz, 1H), 1.25
(m, 3H）,1.05 (dd, J = 13.4, 7.8 Hz, 1H), 0.90 (t, J = 11.8 Hz, 2H), 0.79 (t, J = 7.4 Hz, 6H), 0.74 (s,
1
3
3
3
H), 0.72 (s, 3H) and C NMR: δ 178.9, 151.6, 151.0, 149.1, 130.0, 126.9, 122.0, 46.1, 43.3,
7.49, 36.9, 36.0, 28.4, 24.3, 24.1, 20.5, 20.4, 19.5, 17.8, 16.3. The resolution of
(R,S)-bupivacaine using DHAA and 12,14-dinitroDHAA were performanced in several solvents.
As showed in Table 2, the chiral discrimination ability for bupivacaine enantiomers increases with
adding polar nitro on the aromatic ring of DHAA, and consequently, a successful resolution of the
racemic bupivacaine are obtained using 12,14-dinitroDHAA as the chiral resolving agent.
3.2. Effect of ultrasound on the crystallization process
When crystalline phases begin to precipitate, ee and yield of the obtained (S)-bupivacaine
product at different crystallization periods were determined (Fig.2). It was found, at the same
cooling rate, that ultrasound caused crystals to form faster than crystallization process without
ultrasound, probably due to the narrowing of the metastable zone width. As seen From Fig.2，at a
short time period of irradiation, the ultrasonic-assisted crystallization does not produce expected
results on resolution, and its ee and yield are even lower than those of crystallization without
ultrasonic. However, a higher ee and yield can be achieved along with a prolonged acting period
of ultrasound, but a decline in ee with yield increasing for crystallization without ultrasonic. By
comparing resolution results between ultrasonic and reference experiments, the ultrasonic can
enhance liquid-solid mass transfer, and facilitate (S)-diastereomeric salt crystallization. Moreover,
by using ultrasound, precipitation of the later less-stable crystallizing can be reduced and

consequently higher ee and yield are available at a sufficient period of time. Based on our
experiments, the potential positive impact of ultrasound for discrimination of (S)- and
(R)-diastereomeric salt in ethanol solution was observed. We speculated the mechanism is that
ultrasonic possibly reduce the induction time of nucleation of (S)-diastereomeric salt, and when
more (S)-diastereomeric salt crystals are formed initially, the cavitation-induced secondary
nucleation of ultrasound presumably prompt the formation of much more secondary nuclei of (S)-
than (R)-diastereomeric salt, then deplete of more mass from the solution, resulting in precipitate
and enrichment of (S)-diastereomeric salt with higher yield and purity in smaller size. Therefore,
ultrasonic treatment can be beneficial for the enantiomeric separation of (R,S)-bupivacaine via
diastereomeric salt crystallization.
3.3. Response surface methodology (RSM) results
A total of 29 experiments (Table 3) by RSM were designed to optimize the resolution of
(R,S)-bupivacaine. The design of experiments matrix, resolution conditions and the results
obtained are also listed in Table 3. The The experimental data were fitted to the second-order
polynomial equation (Eq.3) and only the significant terms were maintained in the models. The
quadratic equations (in coded factors) for ee value and yield of (S)-bupivacaine can be expressed
as Eq.4 and Eq.5, respectively.
The quality of the adjusted models was estimated with determination coefficients, residues and
lack-of-fit tests from the analysis of variance (ANOVA) (Table 4), indicating the quadratic model
is significant for ee and yield. The model can be used to navigate the design space and predict the
responses. Among the four factors considered, the amount of resolving agent and solvent are
significant for resolution process. Also, based on the obtained models, it can be seen that
ultrasonic power has the important linear and quadratic effect on ee and yield, and crystallization
temperature has relatively little influence. The effect of four factors on the resolution of
(
R,S)-bupivacaine via ultrasound-assisted diastereomeric crystallization is x >x >x >x .
2 1 3 4
2
2
2
2
ee % =70.74-5.10x +6.42x +4.12x -4.20 x -4.47x -11.42x -2.55x
4
(4)
(5)
1
2
3
1
2
3
2
2
2
2
2
Yield %=89.06+20.18x -4.07x -5.12x +6.67x x -11.96x -12.54x -7.50x -5.68x
4
1
2
3
1
3
1
3
3.4. Response surface analysis

In the above second-order models (Eq.4 and 5), we ignored some less prominent factors and
interactions between the various factors influencing resolution effects. Three process parameters
(
x , x and x ), i.e. amount of resolving agent, solvent amount and ultrasonic power, have the most
1 2 3
critical impact on ee and yield for resolution of (R,S)-bupivacaine. To attain a better understanding
of the three key factors as well as their interactions and to determine the optimized process
parameters for a high ee and yield, the three-dimensional response surfaces and contour plots of
the relationship between x and x , x and x , x and x are constructed in Fig.3 while the other
1
2
1
3
2
3
variables remain unchanged at the central level. Fig.3A and 3B depict the effects of amount of
2,14-dinitroDHAA and ethanol amount under ultrasound irradiation. It is observed that ee
1
reaches a high level of about 70% with ethanol amount increasing from 10.0 to 20.0 mL/g at a
wide range of amount of resolving agent. The yield rises with increasing amount of resolving
agent, but reaches a maximum followed by an obvious decline with the solvent amount further
increased due to the soluble ability enhancing for desired (S)-form diastereomeric salts in ethanol.
Fig.3C and 3D show the effects of the ultrasonic power and amount of 12,14-dinitroDHAA on ee
and yield, indicating that ultrasonic power has the remarkable effect on ee, and a relatively slight a
slight improvement in yield. A similar influence of ultrasonic power on ee and yield is also
observed in Fig.3E and 3F. This behavior may be the result of differences in the crystallization
rates between (R)- and (S)-propranolol diastereomeric salts in ethanol, which is analogous to
ultrasonic-facilitated preferential crystallization as described in literatures[33,47]. It’s important to
observe from Fig.3C to 3F, on the other hand, that yield does not necessarily correlate with ee
(purity). Often a process with a high yield will provide a low level of purity and vice versa. This
indicates that a high ee and a good yield may be obtained simultaneously by optimizing resolution
conditions.
3.5. Resolution process optimization
The purpose of this resarch is to optimize the four operating parameters (resolving agent
amount, solvent amount, ultrsonic power and crystallization temperature) to achieve the best
resolution results for prescribed amount of (R,S)-bupivacaine. Based on the discusion above, in
this work, an optimization function that maximizes ee while maintaining a lower limit of 90% for

yield was selected. By employing the Design-Expert software, an optimization response result was
generated. The numerical solution suggested that an optimum exists at 1.6, 16.5mL/g, 63.2W and
0℃corresponding to molar ratio of resolving agent to (R,S)-bupivacaine, liquid-solid ratio of
solvent volumeto (R,S)-bupivacaine amount, ultrasonic power and temperature, respectively. It
predicted that these resolution conditions will provide a ee of 71.5% and a yield of 90% for
(S)-bupivacaine.
3.6. Verification experiments
Five repeated experiments were performed to validate the optimal conditions. 50g of racemic
bupivacaine base and an prescribed amount of 12,14-dinitroDHAA were loaded into a reactor
followed by the prescribed amount of ethanol, and to reacted at 50℃for 30 min, then exerted
ultrasonic irradiation for 50 min, and sequentially cooled to 0℃over 1h. The crystalline solid was
filtrated and dried, and treated with 20% NaOH aqueous solution to yield the desired white
(
S)-bupivacaine. These experiments provided a ee of 69.8% (corresponding purity of 84.9%) and a
yield of 87.5% very close to the 71.5% and 90.0% predicted by RSM.
. Conclusion
Racemic bupivacaine base was successfully enantioseparated via ultrasonic-assisted
4
diastereomeric crystallization in ethanol using 12, 14-dinitroDHAA as resolving agent, a optically
chiral acid prepared by the nitration of dehydroabietic acid (DHAA). The results indicated that
ultrasonic-assist can well intensify resolution of (R,S)-bupivacaine and a higher ee and yield was
obtained for (S)-bupivacaine, and while without ultrasound, the ee value decreases by increasing
the crystallization time. In this work, amount of 12, 14-dinitroDHAA, ethanol amount, ultrasonic
power and crystallization temperature, which may play a key role for resolution of
(R,S)-bupivacaine, were evaluated and optimized by employing a Box-Behnken design with
response surface method. The predicted optimal resolution parameters within the experimental
ranges were: 1.6 molar ratio of 12,14-dinitroDHAA to bupivacaine, 16.5mL/g of solvent to
bupivacaine, 63.2W ultrsonic and 0℃. Under the optimal conditions, the experimental ee and yield
of (S)-bupivacaine were 69.8% and 87.5%, very close to the predicted 71.5% ee and 90.0% yield.

Acknowledgements
This work was financially supported by the National Natural Science Foundation of China
(No.21666003)
Conflict of Interest
The authors declare that they have no conflict of interest.

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[
2
[
[

Figures
Fig.1 Chemical structures of bupivacaine, DHAA and 12,14-dinitroDHAA
Fig.2 Enantiomeric excess and yield of (S)-bupivacaine product obtained at different
crystallization time with or without ultrasonic assist.
Fig.3 Response surface plots show the correlative influence of ultrsound power, the usage of
resolving agent and solvent on ee and yield of (S)-bupivacaine.
Fig.1 Chemical structures of bupivacaine, DHAA and 12,14-dinitroDHAA
1
00
100
80
60
40
20
0
1
0W ultrsonic assist
1
0W ultrsonic assist
without ultrsonic assist
Without ultrsonic assist
8
6
4
2
0
0
0
0
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
t/min
t/min
Fig.2 Enantiomeric excess and yield of (S)-bupivacaine product obtained at different
crystallization time with or without ultrasonic assist.

100
80
90
80
70
Yield 70
60
60
ee
(%)
50
(%) 50
40
30
40
B
A
20
2
20
2
1
8
1.8
1
8
1.8
16
1.6
16
1.6
14
1.4
14
1.4
12
1.2
V /g (mL/g) 12
s
1.2 nr/nrp
rp
V /g (mL/g)
s
10
1
nr/nrp
rp
10
1
80
100
90
70
80
Yield 70
ee
6
0
60
(%)
(%)
50
5
0
40
40
30
C
D
100
2
100
2
1.8
80
1.8
8
0
1
.6
1.6
60
60
1
.4
1.4
1.2 nr/nrp
P (W)
40
nr/nrp
P (W) 40
1.2
20
1
20
1
100
80
90
70
80
Yield 70
ee
6
0
6
0
(%)
(%)
5
0
5
0
40
30
40
E
F
100
20
100
20
1
8
80
18
8
0
16
16
60
60
40
14
14
40
P (W)
12V /g (mL/g)
P (W)
12
s
rp
V /g (mL/g)
s rp
20
10
20 10
Fig.3 Response surface plots show the correlative influence of ultrsound power, the usage of
resolving agent and solvent on ee and yield of (S)-bupivacaine.

Tables
Table 1 Independent variable levels and codified values for the Box-Behnken experimental design.
Level
Factors
Coded factor
-
1
0
1
nr/nrp
x1
x2
1.0
10
1.5
15
2.0
20
Vs/g (mL/g)
P (W)
T (℃)
x3
x4
20
-5
60
0
100
5
Table 2 Resolution of (R,S)-bupivacaine with DHAA and 12,14-dinitroDHAA in various solvents.
1
Resolving solvents
DHAA
12,14-dinitroDHAA
2
ee (%)
Yield (%)
ee (%)
Yield (%)
Methanol
Ethanol
A little crystal
9.8
3.4
10.3
44.7
28.2
39.4
11.5
8.6
39.0
9
2
0% Methanol
-Propanol
4.7
16.8
1.8
26.5
2.3
Ethyl acetate
Not crystallized
Not crystallized
Not crystallized
Not crystallized
Acetone
1
Solvent volume was 15 mL per gram of (R,S)-bupivacaine, molar ratio of resolving agent to (R,S)-bupivacaine
was 1.0 and crystallization temperature was set at 5℃.
2
Yield was calculated based on (S)-bupivacaine.
Table 3 Experimental Box-Behnken design matrix, the measured ee value and yield of
S)-bupivacaine.
(
Factors
Response
Run
x1
x2 (mL/g)
x3 (W)
x4 (℃)
ee (%)
Yield (%)
1
2
3
4
5
6
7
8
9
1.5
1.5
2
15
15
15
15
20
15
15
20
15
10
15
100
60
20
100
60
60
20
60
60
60
60
5
58.6
72.1
45.8
63.8
70.4
69.2
53.0
70.8
70.8
56.6
70.6
67.2
86.8
88.0
33.4
41.0
90.6
85.2
66.4
91.3
69.1
51.2
0
0
1
0
1
0
1.5
1.5
1.5
1.5
1.5
1
0
5
-5
0
1
1
0
1
5
-5

1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
2
15
20
20
20
10
10
15
10
10
15
15
15
20
15
15
15
10
15
60
-5
0
58.6
59.2
65.6
64.6
60.2
44.8
71.0
48.4
55.1
59.8
54.6
60.6
69.2
72.2
55.8
70.6
57.8
50.2
89.2
71.6
55.4
85.4
57.8
70.2
86.2
89.8
72.2
90.2
89.6
80.7
64.6
48.2
58.5
90.4
74.4
86.2
1.5
1.5
2
20
100
60
0
0
1
60
0
1.5
1.5
2
20
0
60
0
60
0
1.5
2
100
60
0
5
2
100
100
60
0
1.5
1.5
1
-5
5
60
5
1
20
0
1.5
1.5
1.5
60
0
60
-5
-5
20
Table 4 Fitted quadratic polynomial models.
ee
Yield
Coefficient
70.74
-5.10
p-Value
< 0.0001
< 0.0001
< 0.0001
< 0.0001
Coefficient
89.06
20.18
-4.07
-5.12
n.s.
p-Value
< 0.0001
< 0.0001
0.0058
Β0
β1
β2
6.42
β3
4.12
0.0011
1
β4
n.s.
β12
β13
β14
β23
β24
β34
β11
β22
β33
β44
n.s.
.
n.s.
n.s.
6.67
0.0083
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
-3.04
-3.29
-7.62
-1.77
0.9798
< 0.0001
< 0.0001
< 0.0001
0.0018
-11.96
-12.54
-7.50
-5.68
0.9661
< 0.0001
< 0.0001
0.0006
0.0050
2
R
Lack-of-fit test
0.14
0.086
1
Not significant (95%).

Highlights




12,14-Dinitrodehydroabietic acid was synthesized as a chiral resolving agent.
(R,S)-bupivacaine was separated successfully via diastereomeric crystallization.
Ultrasonic-assisted was used for intensifying racemate resolution.
The parameters were optimized by employing a Box-Behnken design with RSM.