Multigram-scale HPLC enantioseparation as a rescue pathway for circumventing racemization problem during enantioselective synthesis of ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate

Article abstract of DOI:10.1002/chir.23313

Racemic ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate is a key synthon for the design of promising therapeutic drugs. It is mainly synthesized from racemic ethyl 2,3-dibromopropionate and 2-aminophenol in presence of K₂CO₃ in refluxed acetone. Unfortunately, synthesis of (R)- and (S)-enantiomers using the enantioselective version of this reaction, which should normally be performed with a double S_N2 mechanism, is unsuitable due to a racemization process involving the dehydrobromination of enantiopure ethyl 2,3-dibromopropionate into ethyl 2-bromoacrylate. For the first time, the enantioselective version is studied (ee ≈ 55–66%), and the percentage of racemization process has estimated to around 34–46% after determination of the optimal experimental conditions for analytical HPLC enantioseparation of racemate. The influence of the experimental and purification conditions on the racemization rate is also studied. The results indicate that racemization occurs faster at the beginning of the reaction but the initiation of the double S_N2 process takes place more faster to limit the racemization rate. The study of the influence of experimental conditions (reaction times, temperature, solvent or type of base, etc.) on the degree of racemization of the (R)- enantiomer is performed and shows that despite changes in the experimental conditions, the synthesis of the (R)- enantiomer is always accompanied by a racemization rate which is difficult in reducing. In parallel, (R)- and (S)-enantiomers are obtained in high enantiopurity (ee > 99.5%) by preparative HPLC enantioseparation of racemate on multigram scale and characterized by optical rotation measurements, ECD and UV spectra.

Full text of DOI:10.1002/chir.23313

Received: 2 December 2020
DOI: 10.1002/chir.23313
Revised: 23 March 2021
Accepted: 12 April 2021
S H O R T C O M M U N I C A T I O N
Multigram-scale HPLC enantioseparation as a rescue
pathway for circumventing racemization problem during
enantioselective synthesis of ethyl 3,4-dihydro-2H-
1,4-benzoxazine-2-carboxylate
1,2
1
3
Hong-Ngoc Pham
| Axelle Arrault
| Nicolas Vanthuyne
|
1
Samir Acherar
1
LCPM, CNRS, Université de Lorraine,
F-54000 Nancy, France
Abstract
2
Racemic ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate is a key synthon
for the design of promising therapeutic drugs. It is mainly synthesized from
racemic ethyl 2,3-dibromopropionate and 2-aminophenol in presence of
Vietnam Academy of Science and
Technology, University of Science and
Technology of Hanoi (USTH), Hanoi,
Vietnam
3
K CO in refluxed acetone. Unfortunately, synthesis of (R)- and (S)-
2 3
Centrale Marseille, iSm2, Aix Marseille
Université CNRS, Marseille, France
enantiomers using the enantioselective version of this reaction, which should
normally be performed with a double S 2 mechanism, is unsuitable due to a
N
Correspondence
racemization process involving the dehydrobromination of enantiopure ethyl
2,3-dibromopropionate into ethyl 2-bromoacrylate. For the first time, the
enantioselective version is studied (ee ≈ 55–66%), and the percentage of race-
mization process has estimated to around 34–46% after determination of the
optimal experimental conditions for analytical HPLC enantioseparation of
racemate. The influence of the experimental and purification conditions on
the racemization rate is also studied. The results indicate that racemization
occurs faster at the beginning of the reaction but the initiation of the double
SN2 process takes place more faster to limit the racemization rate. The study of
the influence of experimental conditions (reaction times, temperature, solvent
or type of base, etc.) on the degree of racemization of the (R)- enantiomer is
performed and shows that despite changes in the experimental conditions, the
synthesis of the (R)- enantiomer is always accompanied by a racemization rate
which is difficult in reducing. In parallel, (R)- and (S)-enantiomers are
obtained in high enantiopurity (ee > 99.5%) by preparative HPLC
enantioseparation of racemate on multigram scale and characterized by optical
rotation measurements, ECD and UV spectra.
Samir Acherar, Laboratoire de Chimie
Physique Macromoléculaire (LCPM),
Université de Lorraine, CNRS, 1 rue
Grandville BP 20451, Nancy Cedex 54001,
France.
Email: samir.acherar@univ-lorraine.fr
Funding information
Vietnam Ministry of Education and
Training (MOET); Vietnam International
Cooperation Department (ICD)
K E Y W O R D S
chiral stationary phases, enantioselective chromatography, ethyl 2,3-dibromopropionate,
ethyl 2-bromoacrylate, ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate, preparative
resolution, racemization
Chirality. 2021;1–13.
wileyonlinelibrary.com/journal/chir
© 2021 Wiley Periodicals LLC.
1

2
PHAM ET AL.
1
| INTRODUCTION
starting from our targeted ethyl 3,4-dihydro-2H-
1,4-benzoxazine-2-carboxylate scaffold in a racemic form
Since essential biomolecules needed for human body
appeared as enantiomerically pure, chiral drugs have
been and being a vital topic in the past decades as
approximately 56% of marketed medicines are used in
rac-1 (Figure 1). In 2005, an overview of different strate-
gies to provide racemic 1,4-benzoxazine analogs has been
22
published, but only few reports brought up procedures
to obtain enantiomerically pure analogs. These proce-
1,2
23–27
28
one of their enantiomeric form. Chiral molecules have
the same chemical behavior in an achiral environment
but may containing different biological activities such in
dures use metal
or phase transfer
catalyzed
2
9,30
asymmetric reactions, kinetic resolution with enzyme
or chiral resolving agents,
addition, asymmetric baker's yeast reduction, and
chiral 1,2-cyclic sulfamidates nucleophilic cleavage.
31–33
stereoselective aryl
3
4,5
34
35
the special case of as ibuprofen or thalidomide. Those
compounds have been reported that only one optically
active isomer can exhibit beneficial pharmacological
activities than the other.
3
6
It is interesting to note that, while our targeted scaf-
fold 1 is a key synthon for the design of promising
therapeutic drugs (Figure 1), its synthesis and character-
izations (optical rotation and electronic circular
dichroism [ECD]) as enantiomers were never described
in the literature. Rac-1 is mainly synthesized by
reacting 2-aminophenol and racemic ethyl 2,3
dibromopropionate rac-2 with anhydrous K CO in
Due to the extraordinary diversity and multi-
functions of heterocycles, heterocycle-based molecules
are becoming key structural in development of new drugs
with two third of marketed drugs containing heterocy-
6
cles. 1,4-Benzoxazine derivatives are a fascinating class
of heterocyclic compounds which can exhibit biological
2
3
7,8
9,11,37–40
properties such as antibacterial, treatment against car-
refluxing dry acetone,
but to date, its
9
,10
11
diovascular diseases,
anti-hypertensive, angiogenesis
enantioselective version has never been reported for two
reasons. First, both enantiomers of 2 have been just
recently isolated by our team through a preparative
HPLC enantioseparation of racemate on multigram
12–14
15,16
inhibitor,
anti-thrombiotic,
Among them, various promising therapeutic
drugs (e.g., anti-tumour, procognitive, and EGFR
kinase inhibitor ) were described in the literature
or neuroprotective
17,18
factor.
19
20
21
41
scale. Second, this reaction should be proceeded via a
FIGURE 1 Examples of designed
therapeutic drugs starting from rac-1

PHAM ET AL.
3
double S 2 reaction, but a racemization is proposed to
occur through a relay mechanism involving (i) the
dehydrobromination of 2, achieving the ethyl
other reagent-grade solvents were used as received.
2-Aminophenol (99%, 109.13 g/mol) and ethyl
2,3-dibromopropionate racemate rac-2 (≥ 98%,
259.92 g/mol) were purchased from Sigma-Aldrich. (R)-
N
2
-bromoacrylate 3; (ii) the Michael addition of the amine
group of 2-aminophenol on the primary carbon; and
iii) the S 2 reaction of the phenolate anion on the
2
5
Ethyl 2,3-dibromopropionate (R)-2 (ee > 99.5%, [α]D
=
(
+ 12 (CH Cl , c = 3.29)) was obtained previously by our
N
2
2
41
α-bromoester group to afford rac-1 (Figure 2).
The goal of this work is to use analytical and prepara-
tive HPLC enantioseparation of rac-1 for the first time
team through preparative HPLC enantioseparation of
commercially available rac-2 on multigram scale.
Ethanol, heptane, and acetonitrile were HPLC grade and
were purchased from VWR.
(i) to investigate the enantioselective version of this
reaction with (R)-2 and to evaluate the percentage of
racemization that occurred during the synthesis of (R)-1
and (ii) to offer a cost-efficient alternative solution for the
obtainment of both pure enantiomers of 1 in multigram
scale. This work shows that (i) a minor amount of
racemization (≈ 34–46%) occurred during the synthesis
of (R)-1 (ee ≈ 55–66%) and (ii) preparative HPLC
enantioseparation of rac-1 on multigram scale afforded
both enantiomers with high enantiomeric excess
2.2 | Characterization
Reactions were monitored by thin-layer chromatography
(TLC) using aluminum-backed silica gel plates
®
(Macherey-Nagel ALUGRAM SIL G/UV254). TLC spots
were viewed under ultraviolet light or/and by heating
the plate after treatment with a staining solution of
phosphomolybdic acid or ninhydrin. Product purifica-
tions were performed using Geduran 60 H Silica Gel
(ee > 99.5%).
(
63–200 mesh). NMR spectra were recorded on a Brucker
Advance 300 spectrophotometer in CDCl3 solvent at
98 K. CDCl (δ = 7.26 and 77.0 ppm) residual peaks
2
2
| MATERIALS AND METHODS
.1 | Chemicals
2
3
1
13
were used as internal references for H and C NMR,
respectively. Chemical shifts (δ) were reported in parts
per million (ppm), and coupling constants (J) were given
in hertz (Hz). Multiplicity was defined as Ar for aromatic,
s for singlet, d for doublet, t for triplet, q for quadruplet,
m for multiplet, and br for broad or combinations
Unless otherwise stated, all chemicals were purchased as
the highest purity commercially available and were used
without further purification. Dry acetone was obtained
by distillation over P O under an argon atmosphere, and
2
5
FIGURE 2 Proposed mechanism for the synthesis of rac-1 from 2-aminophenol and 2

4
PHAM ET AL.
thereof. Electrospray mass spectroscopy (ESI-MS) was
performed on a ToF-Q HR spectrometer. HRMS spectra
were obtained by the ESI method.
2.5 | Analytical achiral HPLC
monitoring of the dehydrobromination of
(R)-2
Analytical achiral HPLC monitoring of reaction progress
of synthesis of 3 through dehydrobromination of (R)-2 at
intervals of 0, 10, 60, 120, 180, 240, 300, 360, 420, 480,
540, and 960 min was performed using a Shimadzu LC-
2.3 | Synthesis of racemic and (R)-ethyl
3,4-dihydro-2H-1,4-benzoxazine-
2-carboxylate, rac-1 and (R)-1
20A HPLC system, consisting of a degasser (DGU-20A3),
To
a
stirring solution of 2-aminophenol (2.00 g,
two pumps (LC-20AT), an UV photo-diode array detector
(SPD-M20A), and an oven (CTO-20A). Separations of
peaks for (R)-2 and 3 were achieved using a Macherey-
Nagel Nucleosil 300-7 C4 column (250 Â 4 mm). The
mobile phase was consisted of CH CN/H O (v/v, con-
1
8.3 mmol, 1.0 equiv.) and anhydrous potassium carbon-
ate (5.57 g, 40.3 mmol, 2.2 equiv.) in dry acetone (30 mL),
was added rac-2 or (R)-2 (5.24 g, 20.2 mmol, 1.1 equiv.).
The reaction was refluxed at 60 C and allowed to stir for
ꢀ
3
2
1
6 h. After filtration on celite to remove carbonate salts,
taining 0.1% of TFA) from 5/95 to 100/0 in 20 min, then
100/0 to 5/95 in 5 min. The flow rate and the UV detec-
tion were 1.0 mL/min and 254 nm, respectively.
the resulting filtrate was concentrated in vacuo, diluted
in CH Cl (30 mL) and washed with water (10 mL). The
2
2
organic layer was dried over MgSO and concentrated in
4
vacuo. The resulting crude material was purified by flash
chromatography (Petroleum ether/EtOAc, 30/70, v/v) to
2.6 | Enantioselective HPLC
afford rac-1 or (R)-1 in good yields (83–85%) as yellow
1
oil. H NMR (300 MHz, CDCl ) δ 1.27 (t, 3H, J = 6.9 Hz,
Analytical and preparative HPLC experiments were per-
formed on an Agilent 1260 Infinity unit (pump G1311B,
autosampler G1329B, DAD G1315D), with Jasco OR-1590
online polarimeter and UV (254 nm) detectors. The
analytical (250 Â 4.6 nm) and preparative columns were
purchased from Chiral Technologies Europe (Illkirch,
France), Phenomenex (Le Pecq, France) or Regis
Technologies (Morton Grove, USA) (see Appendix S2 for
more details). HPLC grade heptane, hexane, and ethanol
were degassed and filtered on a 45 μm Millipore mem-
brane before use. Retention times Rt in minutes, reten-
tion factors k = (Rt ÀRt )/Rt and enantioselectivity
3
CH ), 3.50–3.65 (m, 2H, N-CH ), 3.77 (br s, 1H, NH), 4.25
3
2
(
q, 2H, J = 6.9 Hz, O-CH ), 4.80 (dd, 1H, J = 6.9 and
2
3
.3 Hz, CH [CO]), 6.63 (dd, 1H, J = 7.8 and 1.8 Hz,
CH ), 6.72 (td, 1H, J = 7.5 and 1.8 Hz, CH ), 6.78 (td,
Ar
Ar
1
1
4
H, J = 7.5 and 1.8 Hz, CH ), 6.92 (dd, 1H, J = 7.8 and
Ar
13
.8 Hz, CHAr); C NMR (75 MHz, CDCl ) δ 14.80 (CH ),
3
3
3.27 (N CH ), 62.31 (O CH ), 73.39 (CH [CO]), 116.74
2
2
(
CH ), 117.70 (CH ), 120.64 (CH ), 122.35 (CH ),
Ar Ar Ar Ar
1
32.77 (N C ), 143.83 (O C ), 170.00 (C O); HRMS
Ar Ar
+
(ESI) calculated for C H NO [M + Na] m/z 230.0793,
11 13 3
found 230.0799 (see Appendix S1 for a copy of 1D and 2D
NMR spectra).
i
i
0
0
factor α = k /k and resolution Rs = 1.18 (Rt ÀRt )/
2
1
2
1
(
w + w ) are given. Rt was determined by injection of
1 2 0
1
,3,5-tri-tert-butylbenzene, and w was the peak width at
i
2
2
.4 | Synthesis of ethyl
-bromoacrylate, 3
half-height.
A solution of (R)-2 (500 mg, 1.92 mmol, 1.0 equiv.) and
2.6.1 | Analytical HPLC
anhydrous potassium carbonate (532 mg, 3.85 mmol, 2.0
ꢀ
equiv.) in dry acetone (5 mL) was refluxed at 60 C and
Twenty microliter of a 3 mg/mL solution in ethanol was
injected on the chiral column and detected with an
UV (254 nm) and polarimeter detectors. A mixture of
heptane/EtOH (70/30, v/v) as mobile phase with a flow-
rate of 1 mL/min was used.
allowed to stir for 16 h. The reaction progress and the
conversion yield (%) were estimated using analytical
HPLC analysis (see Section 2.5) by comparing the ratio
of peak areas of 3 and (R)-2 (retention times of 7.8 and
9
.9 min, respectively). At the end of the reaction, the
crude reaction product comprising 3 and residual (R)-2
1
was concentrated in vacuo. H NMR of 3 (300 MHz,
2.6.2 | Preparative HPLC
CDCl ) δ 1.29 (t, 3H, J = 6.9 Hz, CH ), 4.24 (q, 2H,
3
3
J = 6.9 Hz, O CH ), 6.21 (s, 1H, =CH), 6.90 (s, 1H,
The preparative HPLC on multigram scale was per-
formed starting from a solution of ꢁ12 g of synthesized
rac-1 in 180 mL of hexane/EtOH (50/50, v/v). Every
2
1
=
CH) (see Appendix S1 for a copy of H NMR
spectrum).

PHAM ET AL.
5
8
min, 200 μL of this solution (900 injections) were
The baseline was always measured for the same solvent
and in the same cell as the samples. The spectra are pres-
ented without smoothing and further data processing;
0.1 nm as intervals, scanning speed 50 nm/min, band
width 2 nm, and 3 accumulations per sample were used
as acquisition parameters.
injected (stacked method) in the preparative Lux-
®
Cellulose-2 (250 Â 10 mm, 5 μm) column using a
mobile phase of hexane/EtOH (70/30, v/v), a flow-rate of
5
mL/min, and UV (254 nm) detection. Both enantiomers
of 1 were separated and collected in two fractions of
around 5.7 g with high purity (ee > 99.5%).
3
3
| RESULTS AND DISCUSSION
.1 | Racemic and enantioselective
2
2
.7 | Chiroptical characterization
.7.1 | Optical rotation measurements
synthesis of (R,S)- and (R)-ethyl
,4-dihydro-2H-1,4-benzoxazine-
3
Optical rotations of both enantiomers of 1 (CH Cl ,
c = 0.2) were measured on a JASCO P-2000 polarimeter
2-carboxylate, rac-1 and (R)-1
2
2
with a sodium lamp (λ = 589 nm) or a halogen lamp
The racemic synthesis of rac-1 on multigram scale (83%
yield) is carried out by a mainly used procedure using
2-aminophenol and rac-2 with anhydrous K CO in
(
λ = 578, 546, 436, and 405 nm) in a 10 cm cell
ꢀ
thermostated at 25 C with
a
Peltier controlled
2
3
cell holder.
refluxed dry acetone during 16 h (Scheme 1). The first
enantioselective version of this reaction (85% yield,
synthesis performed twice) is also achieved using (R)-2
25
2.7.2 | ECD and UV spectra
([α]D = + 12 (CH Cl , c = 3.29), which was recently
2
2
41
isolated for the first time by our team (Scheme 1). This
reaction should normally be performed through a double
SN2 process and afforded (R)-1 from (R)-2. For this type
of reaction, it is generally postulated, however, that a
racemization process occurs during the synthesis, as
already described above, in Figure 2. The racemic synthe-
sis of rac-1 allowed us to investigate the analytical and
preparative HPLC enantioseparation of rac-1 in
ECD and UV spectra of both enantiomers of 1 in acetoni-
trile (c = 0.61 mM) were measured on a JASCO J-815
spectrometer equipped with a JASCO Peltier cell holder
ꢀ
PTC-423 to maintain the temperature at 25.0 ± 0.2 C. A
CD quartz cell of 1 mm of optical pathlength was used.
The CD spectrometer was purged with nitrogen before
recording each spectrum, which was baseline subtracted.
SCHEME 1 Synthesis of rac-1 and (R)-1

6
PHAM ET AL.
multigram scale. The enantioselective synthesis of (R)-1,
for its part, allowed us to determine, for the first time, the
percentage of racemization in this reaction by measure-
ment of the enantiomeric excess (ee).
(Rs < 1.5, entries 1, 12, and 16, Table 1). Good baseline
separation is observed for all other CSPs (Rs > 2.3) with
®
the best values for Lux-Cellulose-2 and Lux-Cellulose-
®
3
(Rs > 10, entries 3 and 4, Table 1). The polarimeter
detector reveals an elution order of (À) and (+). Among
®
these two CSPs, Lux-Cellulose-2 is chosen because of its
3
.2 | Analytical and preparative HPLC
better separation factor (α = 2.16) and lower retention
enantioseparation of (R,S)-ethyl
times for both eluted enantiomers (R = 5.70 and
t
3
2
,4-dihydro-2H-1,4-benzoxazine-
-carboxylate, rac-1
8.87 minutes for (À)-1 and (+)-1, respectively (Figure 3A
and entry 3, Table 1).
Based on the analytical HPLC results, preparative
HPLC enantioseparation of rac-1 on multigram scale is
successfully applied to access both enantiomers of our
targeted scaffold 1 with high enantiopurity and their
characterizations (optical rotation and electronic circular
To determine the ee of the synthesized (R)-1 and, at the
same time, to estimate the percentage of racemization
occurring during the synthesis, analytical chiral HPLC
separation of rac-1 is critical. Therefore, a screening
of 16 different chiral stationary phases (CSPs,
dichroism [ECD] are also investigated). This cost-efficient
®
2
50 Â 4.6 mm) using a mixture of heptane/EtOH (70/30,
strategy uses
a
preparative Lux-Cellulose-2
CSP
v/v) as mobile phase with a flow rate of 1 mL/min under
UV polarimetric detection is conducted to find the best
suitable phase for the separation of both enantiomers of
(250 Â 10 mm, 5 μm), hexane/EtOH (70/30, v/v) as
mobile phase with a flow rate of 5 mL/min under UV
detection at 254 nm. About 12 g of rac-1 is diluted in
hexane/EtOH (50/50, v/v, 180 mL), and enantiomers are
separated using a stacked injection technique (i.e., 900
1. The screening results are reported in Table 1 (see
Appendix S2 for HPLC chromatograms). These results
show that a poorly baseline separation is obtained only
for Chiralpak AS-H, Chiralpak ID and (S,S)-Whelk-O1
injections of 200 μL every
8
min). Two highly
enantiopure eluted fractions (5.7 g, ee > 99.5%) are
TABLE 1 Screening results for analytical HPLC enantioseparation of rac-1
First eluted enantiomer
Second eluted enantiomer
a
b
a
b
αc
Rsd
0.82
Entry
Column
Heptane/EtOH (v/v)
70/30
Rt
1
k
1
Rt
2
k
2
1
2
3
4
5
6
7
8
9
Chiralpak AS-H
Chiralcel OD-3
Lux-Cellulose-2
Lux-Cellulose-3
Lux-Cellulose-4
Lux-Amylose-1
Lux-Amylose-2
Chiralpak AZ-H
Chiralpak IA
Chiralpak IB
Chiralpak IC
Chiralpak ID
Chiralpak IE
Chiralpak IF
4.43(À)
6.32(À)
5.70(À)
14.56(À)
5.23(À)
6.79(+)
5.94(À)
5.31(À)
6.13(+)
5.39(À)
5.09(À)
5.13(À)
5.49(À)
5.45(À)
6.66(À)
6.90(À)
0.50
1.14
0.93
3.94
0.77
1.30
1.01
0.80
1.08
0.83
0.72
0.74
0.86
0.85
1.26
1.34
4.66(+)
7.99(+)
8.87(+)
22.01(+)
7.17(+)
8.23(À)
6.79(+)
7.02(+)
7.25(À)
6.00(+)
5.77(+)
5.44(+)
6.00(+)
7.21(+)
7.36(+)
7.33(+)
0.58
1.71
2.01
6.46
1.43
1.79
1.30
1.38
1.46
1.03
0.95
0.84
1.04
1.45
1.49
1.49
1.15
1.50
2.16
1.64
1.85
1.37
1.29
1.72
1.35
1.25
1.32
1.14
1.20
1.70
1.19
1.11
70/30
6.08
10.09
10.06
8.43
5.23
3.89
6.38
2.85
2.60
2.90
0.98
2.30
5.60
2.46
1.20
70/30
70/30
70/30
70/30
70/30
70/30
70/30
1
1
1
1
1
1
1
0
1
2
3
4
5
6
70/30
70/30
70/30
70/30
70/30
Chiralpak IG
(S,S)-Whelk-O1
70/30
70/30
a
Retention time (min).
b
Retention factor.
c
Enantioselectivity factor.
d
Resolution.

PHAM ET AL.
7
fraction (see Appendix S1 for more details about the
absolute configuration determination by comparison of
calculated and experimental ECD and UV spectra).
Thanks to this assignment and ECD spectra, we can
conclude that (R)-1 has a positive and a negative
maximum of same intensity at 205 and 219 nm, respec-
tively, with an additional negative maximum of lower
25
intensity at 254 nm, and a specific rotation [α]D = + 37
CH Cl , c = 0.19) (see Appendix S1 for characterization
(
2
2
data).
3.3 | Determination of the percentage of
racemization accompanying the synthesis
of (R)-ethyl 3,4-dihydro-2H-
1,4-benzoxazine-2-carboxylate, (R)-1
The ee value of the synthesized (R)-1 is determined by
analytical chiral HPLC with the best chromatographic
conditions obtained in Section 3.2 (entry 3, Table 1). The
analytical HPLC chromatograms of (R)-1 obtained from
the two repeated enantioselective synthesis is given in
Figure 4A,B. Ee values of ≈ 55% and ≈ 66% are obtained
for the synthesized (R)-1 highlighting the formation of
the undesired (S)-enantiomer in ≈ 23% and ≈ 17%,
respectively. These data are used to estimate the percent-
age of the racemization process compared to that of dou-
ble S 2 process. It is interesting to note that the
N
percentage of racemization is evaluated at around
34–46% (Figure 4A,B) indicating that the racemization is
not the major mechanism involved in this reaction.
To confirm that the racemization is only due to the
formation of the ethyl 2-bromoacrylate 3 intermediate as
mentioned in Figure 2, the influence of the experimental
(
i.e., anhydrous K CO , refluxed dry acetone, 16 h), and
2 3
FIGURE 3 (A) Analytical HPLC enantioseparation of rac-1
and (B,C) preparative HPLC chromatograms of the separated
enantiomers from rac-1 under UV (254 nm) detection
purification (i.e. flash chromatography (Petroleum ether/
EtOAc, 30/70, v/v)) conditions on (R)-1 and/or (R)-2 is
investigated (Table 2 and Figure 4C). First, to be sure that
experimental conditions and purification step do not
induce a racemization of (R)-1, pure enantiomer of (R)-1
isolated by preparative chiral HPLC (ee > 99.5%) is
placed in dry acetone under reflux for 16 h in presence of
anhydrous K CO (entry 1, Table 2) or passed through a
silica gel column chromatography using Petroleum
ether/EtOAc (30/70, v/v) as eluent (entry 2, Table 2). In
both cases, analytical chiral HPLC chromatogram of the
recovered (R)-1 shows no racemization (ee > 99.5%),
indicating that the racemization only occurs before the
formation of (R)-1. Second, to confirm that racemization
of 1 is proceeding by the postulated mechanism involving
ethyl 2-bromoacrylate 3 as intermediate, enantiopure
(R)-2 (ee > 99.5%) is brought into solution in dry acetone
obtained at a retention time of 5.59 and 8.53 min,
respectively (Figure 3B,C). The two separated enantio-
mers of 1 are characterized by optical rotation measure-
ments and ECD and UV spectra (see Appendix S1 for
characterization data). Two highly enantiopure eluted
fractions show opposite values in optical rotation and
ECD spectra, and same UV spectra. The assignment of
the absolute configuration of both eluted fraction
2
3
(
i.e., the (S)-enantiomer is the first eluted) is based on the
analytical HPLC chromatogram of the synthesized (R)-1
see Section 3.3) and the comparison of calculated UV
(
and ECD spectra for (S)-enantiomer (DFT and TD-DFT
calculations) with experimental ones for the first eluted
with anhydrous K CO3 and refluxed for 16 h (entry
2

8
PHAM ET AL.
3, Table 2). The reaction progress and the conversion
yield (%) of (R)-2 into 3 are estimated using analytical
achiral HPLC analysis (see Section 2.5) by comparing the
ratio of peak areas of 3 and (R)-2 (retention times of 7.8
and 9.9 min, respectively) (Figure 4C). The monitoring of
the reaction indicates a fast dehydrobromination of (R)-2
into 3 during the first 2 h (i.e., conversion of
about 57% of (R)-2 to 3), followed by a slower increase
of dehydrobromination during the rest of the reaction,
reaching a maximum of around 93% within 16 h
(
Figure 4C). Given that percentage of racemization in
the enantioselective synthesis of (R)-2 does not exceed
6% (Figure 4A,B), this means that the first step in the
4
double S 2 process (i.e., substitution of primary alkyl
N
bromide by the amine group) proceeds very quickly in less
than 1–2 h to limit the dehydrobromination reaction
(Scheme 2).
Finally, we decided to study the influence of experi-
mental conditions on the degree of racemization of (R)-1.
To achieve this, we resynthesized (R)-1 at a 50-mg scale
of 2-aminophenol by changing the reaction conditions
(
reaction times, temperature, solvent or type of base, etc.)
under the same experimental procedure described in Sec-
tion 2.3 (Table 3). All the results are compared to the ini-
tial conditions used to synthesize (R)-1 (entry 1, Table 3).
The use of lower reaction times (4 and 10 h, entries 2 and
3, Table 3) reduced the isolated yield of (R)-1 while
maintaining a similar degree of racemization (≈ 30–40%),
and it is also noteworthy that the TLC spots of the
starting materials disappear after approximately 2 h.
These findings confirm that the racemization and the
first S 2 reaction in the double S 2 process occur quickly
N
N
FIGURE 4 (A,B) Analytical HPLC chromatograms of the
synthesized (R)-1 (synthesis performed twice) and (C) progress of
the synthesis of 3 from (R)-2 monitored by achiral HPLC under UV
detection (254 nm)
(less than 2 h) as postulated in Scheme 2 and that the sec-
ond S 2 reaction in the double S 2 process yielding (R)-1
N
N
takes more time to proceed. Decreasing the temperature
ꢀ
ꢀ
ꢀ
from 60 C (entry 1, Table 3) to 40 C and 20 C (entries
TABLE 2 Study of the racemization process of (R)-1
a
Entry
Compound
Conditions
Results
b
1
(R)-1
Experimental
The resulted mixture was extracted with CH
and concentrated in vacuo.
2 2
Cl
No racemization was observed by analytical chiral
®
HPLC analysis using Lux-Cellulose-2 CSP.
c
2
3
(R)-1
(R)-2
Purification
The collected fraction was concentrated in vacuo.
No racemization was observed by analytical chiral
HPLC analysis using Lux-Cellulose-2 CSP.
®
b
Experimental
The reaction progress (i.e., formation of 3 from
(
R)-2) was monitored by achiral HPLC using
Nucleosil 300–7 C4 column (see Figure 4C).
a
ee > 99.5%.
b
anhydrous K
2
CO
3
(2.2 equiv.), refluxed dry acetone, 16 h.
c
flash chromatography (Petroleum ether/EtOAc, 30/70, v/v).

PHAM ET AL.
9
SCHEME 2 Racemization
and double S
involving in the synthesis of
R)-1
N
2 processes
(
4
and 5, Table 3) does not drastically reduce the degree of
dehydrobromination of (R)-2 (i.e., deprotonation of the
racemization (≈ 22–28%) but does negatively affect the
isolated yield of (R)-1 by lowering it to 20–45%. Aware
that the use of K CO with (R)-2 is responsible for the
H-atom alpha to the ester group). The replacement of
K CO3 with weaker bases such as 2,6-lutidine
2
(pKa ≈ 6,7) or NaHCO3 (pKa ≈ 6,4) (entries 13 and
2
3
formation of 3 and so the racemization of (R)-1, we inves-
14, Table 3) is unsuccessful, while the use of NEt (entry
3
tigated the influence of K CO addition and equivalent
15, Table 3) having a similar basicity (pKa ≈ 10.8) led to
a high degree of racemization (90%). This high racemiza-
tion can be explained by the fact that the synthesis of
3 through dehydrobromination of (R)-2 in the presence
of NEt3 (1.1 eq.) in acetone at room temperature is
achieved with 97% yield in 30 min. Finally, a stronger
base (KOH, pKa ≈ 13.5) is also tried (entry 15, Table 3)
after necessary modifications in the experimental condi-
tions (i.e., refluxed acetone is replaced by refluxed tolu-
ene to avoid the formation of enolate from acetone and a
Dean-Stark is also used to remove water from the reac-
tion medium). This last attempt gives (R)-1 in 9% isolated
yield with high degree of racemization (70%). In
summary, this study revealed that notwithstanding the
changes in the experimental conditions, the synthesis of
(R)-1 is always accompanied by a racemization rate
which is difficult in reducing.
2
3
(entries 6–8, Table 3) and also the influence of (R)-2 addi-
tion (entries 9 and 10, Table 3). No reaction is observed
by using one equivalent of K CO or with addition of
2
3
K CO or (R)-2 after 1 h, emphasizing the importance
2
3
42
of the presence of K CO and (R)-2 from the beginning of
2
3
the reaction. However, only a moderate yield of 20–34%
is achieved with addition of K CO or (R)-2 after 30 min
2
3
without significant improvement in degree of racemiza-
tion. The solvent's change from refluxed acetone (entry
1
, Table 3) to refluxed acetonitrile (MeCN) or dic-
hloromethane (DCM) (entries 11 and 12, Table 3) has a
negative impact on the isolated yield of (R)-1 without any
improvement in the degree of racemization. In the initial
conditions (entry 1, Table 3), K CO3 (pKa ≈ 10.3) is
2
used to deprotonate 2-aminophenol (pKa ≈ 9.7), but
these basic conditions are also sufficient for the

10
PHAM ET AL.
TABLE 3 Study of experimental conditions influence on the degree of racemization of (R)-1
Yield of (R)-1
T ( C) Time (h) (%)
Ee of (R)-1
(%)
Racemization
(%)
ꢀ
a
Entry Base
Dry solvent
b
Initial conditions
1
K
2
CO
3
(2.2 eq.)
Acetone refluxed 60
16
85
66–55
34–46
b
Influence of reaction time
2
3
K
2
2
CO
CO
3
3
(2.2 eq.)
(2.2 eq.)
Acetone refluxed 60
Acetone refluxed 60
10
4
46
32
60
70
40
30
K
Influence of temperature
4
5
K
2
2
CO
CO
3
(2.2 eq.)
(2.2 eq.)
Acetone
Acetone
40
20
16
16
40
25
78
72
22
28
K
3
Influence of K
2
CO
3
addition and equivalent
c
6
7
8
K
2
K
2
K
2
CO
CO
CO
3
3
3
(2.2 eq.)
Acetone refluxed 60
Acetone refluxed 60
Acetone refluxed 60
16
16
16
34
80
―
―
20
―
―
d
e
(2.2 eq.)
(1.0 eq.)
0
e
0
Influence of (R)-2 addition
f
9
1
K
2
2
CO
CO
3
3
(2.2 eq.)
(2.2 eq.)
Acetone refluxed 60
Acetone refluxed 60
16
16
20
73
27
g
e
0
K
0
―
―
Influence of solvent
1
1
2
K
2
2
CO
CO
3
3
(2.2 eq.)
(2.2 eq.)
DCM refluxed
MeCN refluxed
40
80
16
16
13
46
62
68
38
32
1
K
Influence of type of base
e
1
1
1
3
4
5
NaHCO
2,6-lutidine (2.2 eq.) Acetone refluxed 60
Et N (2.2 eq.) Acetone refluxed 60
3
(2.2 eq.)
Acetone refluxed 60
16
16
16
0
―
―
10
―
―
90
e
0
3
78
9
Influence of type of base and solvent
h
1
6
KOH (2.2 eq.)
Toluene refluxed 112
16
30
70
a
Isolated yield after silica gel chromatography purification.
b
The TLC spots of the starting materials disappear after a reaction time of around 2 h.
c
Addition of K
2
CO
3
after 30 min.
3
after 1 h.
d
Addition of K
2
CO
e
No conversion was observed on TLC.
f
Addition of (R)-2 after 30 min.
g
Addition of (R)-2 after 1 h.
h
ꢀ
ꢀ
With KOH, refluxed dry acetone (60 C) was replaced by refluxed dry toluene (112 C) to avoid the formation of enolate from acetone and a Dean-Stark was
also used to remove water from the reaction medium.

PHAM ET AL.
11
4
| CONCLUSIONS
broadly varied new enantiopure heterocyclic therapeutic
drugs or as β-amino acid analogs for designing new
pseudopeptides and foldamers.
In this work, the enantioselective version of the known
ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate syn-
thesis from (R)-ethyl 2,3-dibromopropionate and
ACKNOWLEDGMENTS
2
-aminophenol in the presence of K CO in refluxing
The authors acknowledge Mr. O. Fabre for running NMR
experiments. The authors would like to thank Vietnam
International Cooperation Department (ICD) and
Vietnam Ministry of Education and Training (MOET) for
financial support as a part of the 911 Project (PhD fellow-
ship). DFT and TD-DFT calculations were supported by
the computing facilities of the CRMMM, “Centre
Régional de Compétences en Modélisation Moléculaire
de Marseille.”
2
3
acetone was investigated for the first time. This reaction
normally should be proceeded via a double S 2 reaction,
N
but a racemization is postulated to occur through a
dehydrobromination of (R)-ethyl 2,3-dibromopropionate,
yielding the ethyl 2-bromoacrylate. The objectives of this
study were to quantify the racemization rate on carrying
out this enantioselective synthesis and to offer a cost-
efficient alternative for the obtainment of both pure
enantiomers. To achieve these goals, analytical HPLC
enantioseparation of the synthesized racemic form was
investigated and successfully run. Based on the best ana-
DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no
datasets were generated or analysed during the
current study.
®
lytical enantioseparation results (Lux-Cellulose-2 CSP,
α = 1.70 and Rs = 10.09), the synthesized (R)-enantiomer
indicated an ee around 55–66%, highlighting a racemiza-
tion rate of ≈ 34–46%. The influence study of the experi-
mental and purification conditions on the racemization
was performed and revealed that (i) racemization only
occurred before the formation of the (R)-enantiomer and
ORCID
Axelle Arrault https://orcid.org/0000-0003-3410-7183
Nicolas Vanthuyne https://orcid.org/0000-0003-2598-
7940
(ii) despite a very fast dehydrobromination of (R)-ethyl
Samir Acherar https://orcid.org/0000-0002-8776-1632
2
,3-dibromopropionate with K CO in refluxing acetone,
2
3
the initiation of the double S 2 process took place more
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