New chemical and chemo-enzymatic routes for the synthesis of (RS)- and (S)-enciprazine

Article abstract of DOI:10.1016/j.tetasy.2012.08.002

The chemo-enzymatic synthesis of racemic and enantiopure (RS)- and (S)-enciprazine 1, a non-benzodiazepine anxiolytic drug, is described herein. The synthesis started from 1-(2-methoxyphenyl) piperazine 3, which was treated with 2-(chloromethyl) oxirane (RS)-4 using lithium bromide to afford a racemic alcohol, 1-chloro-3-(4-(2-methoxyphenyl) piperazin-1-yl) propan-2-ol (RS)-6 in 85% yield. Intermediate (S)-6 was synthesized from racemic alcohol (RS)-6 using Candida rugosa lipase (CRL) with vinyl acetate as the acyl donor. Various reaction parameters such as temperature, time, substrate, enzyme concentration, and the effect of the reaction medium on the conversion and enantiomeric excess for the transesterification of (RS)-6 by CRL were optimized. It was observed that 10 mM of (RS)-6, 50 mg/mL of CRL in 4.0 mL of toluene with vinyl acetate (5.4 mmol) as acyl donor at 30 °C gave good conversion (C = 49.4%) and enantiomeric excess (ee_P = 98.4% and ee_S = 96%) after 9 h of reaction. Compound (S)-6 is a key intermediate for the synthesis of enantiopure (S)-1. The (RS)- and (S)-enciprazine drug 1 was synthesized by treating (RS)- and (S)-6 with 3,4,5-trimethoxyphenol 5 using MeCN as a solvent and K₂CO₃ as a base.

Full text of DOI:10.1016/j.tetasy.2012.08.002

Tetrahedron: Asymmetry 23 (2012) 1272–1278
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
New chemical and chemo-enzymatic routes for the synthesis of (RS)- and
(S)-enciprazine
⇑
Linga Banoth, Thete Karuna Narayan, Uttam C. Banerjee
Biocatalysis Laboratory, Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar 160062, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2012
Accepted 2 August 2012
Available online 14 September 2012
The chemo-enzymatic synthesis of racemic and enantiopure (RS)- and (S)-enciprazine 1, a non-benzodi-
azepine anxiolytic drug, is described herein. The synthesis started from 1-(2-methoxyphenyl) piperazine
3, which was treated with 2-(chloromethyl) oxirane (RS)-4 using lithium bromide to afford a racemic
alcohol, 1-chloro-3-(4-(2-methoxyphenyl) piperazin-1-yl) propan-2-ol (RS)-6 in 85% yield. Intermediate
(S)-6 was synthesized from racemic alcohol (RS)-6 using Candida rugosa lipase (CRL) with vinyl acetate as
the acyl donor. Various reaction parameters such as temperature, time, substrate, enzyme concentration,
and the effect of the reaction medium on the conversion and enantiomeric excess for the transesterifica-
tion of (RS)-6 by CRL were optimized. It was observed that 10 mM of (RS)-6, 50 mg/mL of CRL in 4.0 mL of
toluene with vinyl acetate (5.4 mmol) as acyl donor at 30 °C gave good conversion (C = 49.4%) and enan-
tiomeric excess (eeP = 98.4% and eeS = 96%) after 9 h of reaction. Compound (S)-6 is a key intermediate for
the synthesis of enantiopure (S)-1. The (RS)- and (S)-enciprazine drug 1 was synthesized by treating (RS)-
and (S)-6 with 3,4,5-trimethoxyphenol 5 using MeCN as a solvent and K2CO3 as a base.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
In general, the synthesis of the 1,2-aminoalcohol class of b-
adrenergic blocking agents involves the opening of an epoxide ring
Optically active aryloxypropanols have been widely employed
as starting materials for the synthesis of b-blockers, which are used
in the management of sympathetic nervous system associated car-
diovascular1 disorders such as hypertension,2 angina pectoris, car-
diac arrhythmias, and other disorders3 such as depression, loss of
appetite, asthma, migraine, glaucoma, and so on. All b-blockers
have at least one stereogenic center in their structure and so, have
two enantiomers.4 However, some b-adrenergic blocking agents
cross the blood–brain barrier because of their lipophilicity and
act on central nervous system functions.5 Propranolol has long
been used as a drug for the treatment of prophylaxis of migraine
headaches, anxiety syndromes, alcohol withdrawal, schizophrenia,
and tremors.6 The newly developed aryloxypropanol derivatives
with various heterocycles in the hydrophilic moiety of the mole-
cule have shown anticonvulsive activity. Enciprazine 1 (Scheme 1)
is a non-benzodiazepine anxiolytic drug.7 It has very little sedative/
hypnotic side effects and its interaction with alcohol is far less than
that of the benzodiazepines.8 Enciprazine exhibits a variety of bio-
logical activities, such as cardiovascular, hypotensive, and local
anesthetic properties.9 The different approaches for the synthesis
of 1 are summarized in Scheme 1.10
with amines.11 The synthesis of enciprazine 1 involves the epoxide
ring opening reaction of rac-2-[(3,4,5,-trimethoxyphenoxy)methyl]
oxirane 2 with 1-(2-methoxyphenyl)piperazine 3. The methodol-
ogy for the synthesis of enantiopure/enantio-enriched 1 involves
the epoxide ring opening reaction of the glycidic ether (S)-2, pre-
pared by the hydrolytic kinetic resolution of (RS)-2 using an enan-
tioenriched (R,R)-salen–Cobalt(III) complex. The glycidic ether
(RS)-2 was synthesized by treatment of epichlorohydrin 4 with
3,4,5-trimethoxy phenol 5 in the presence of a base. The phenol
5 itself was synthesized by a two step procedure starting from
3,4,5-trimethoxybenzaldehyde.10 This reported synthesis has
drawbacks, such as it being a multistep procedure for the prepara-
tion of 2, which requires an expensive and toxic (R,R)-salen–
Co(OAc) complex which is carcinogenic and flammable.10 New
chemical and chemo-enzymatic routes for the synthesis of (RS)-
and (S)-enciprazine (Scheme 1) are discussed herein.
2. Results and discussion
The increasing influence of green chemistry tools on the synthe-
sis of new chemical entities and for the improvement of existing
chemical synthesis12–13 is now widely recognized. Integrating bio-
catalysis in chemical syntheses is an elegant approach in expand-
ing the organic synthesis toolbox.14 N-Heterocyclic amines are
very much in demand for the synthesis of various agro- and fine
⇑
Corresponding author. Tel.: +91 172 2214682 85x2142; fax: +91 172 2214692.
E-mail address: ucbanerjee@niper.ac.in (U.C. Banerjee).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.08.002

L. Banoth et al. / Tetrahedron: Asymmetry 23 (2012) 1272–1278
1273
MeO
MeO
OH
2.1. Synthesis of (RS)-1-chloro-3-(4-(2-methoxyphenyl)pip-
erazin-1-yl) propan-2-ol 6
O
+
+
Cl
As shown in the retrosynthetic analysis (Scheme 2), the synthe-
sis started from the commercially available 1-(2-methoxyphenyl)
piperazine 3, which was treated with commercially available 2-
(chloromethyl) oxirane (RS)-4 using lithium bromide to afford
racemic alcohol (RS)-6 in 85% yield (Scheme 3) as the substrate re-
quired for enzymatic kinetic resolution.
For the kinetic resolution of (RS)-6, authentic samples of (R)-
and (S)-6 and the corresponding O-acylated derivatives (RS), (R)-,
and (S)-7 were required.
OMe
O
4
(RS)-
5
O
MeO
N
HN
OMe
MeO
OMe
3
2
(RS)-
earlier work
2.2. Synthesis of authentic samples of (R)- and (S)-1-chloro-3-
(4-(2-methoxyphenyl)piperazin-1-yl)propan-2-ol 6 and (RS)-,
(R)- and (S)-1-chloro-3-(4-(2-methoxyphenyl) piperazin-1-yl)
propan-2-yl acetate 7
OH
N
N
MeO
MeO
O
OMe
The authentic samples of (R)- and (S)-6 were synthesized by the
reaction of 3 with (R)- and (S)-4, respectively. The enantiomeric
purity was determined by chiral HPLC and by the specific rotation
value. The ring opening of (S)-4 by treatment with 3 following the
same procedure as for (RS)-6 afforded (S)-6 in 83% yield and with
92% ee. Similarly, (R)-6 was obtained from (R)-4 in 84% yield and
with 91% ee. The treatment of (RS)-6 with acetic anhydride at
4 °C in the presence of pyridine afforded (RS)-7 in 90% yield. Acet-
ylation of (R)- and (S)-6 following a similar procedure resulted in
the formation of (R)- and (S)-7 in 92 and 91% yields with 87%
and 89% ee, respectively.
(RS)-enciprazine 1
OMe
Cl
present work
OH
N
5
+
N
(RS)-6
OMe
2.3. Lipase-catalyzed kinetic resolution of (RS)-6
4
(RS)-
+
3
Scheme 1. Various synthetic strategies for 1.
The best operative enzymatic kinetic resolution procedure
using various lipases was followed with substrates (RS)-6 and the
authentic samples of (R)- and (S)-6 and (RS)-, (R)-, and (S)-7.
chemicals and pharmaceuticals.15 They also have remarkable prop-
erties in organocatalytic reactions. It has been reported in the liter-
ature15 that highly valuable, optically active cyclic amines, such as
piperidines, pyrrolidines, azetidines, aziridines, and biologically ac-
tive compounds such as antibiotics, antihypertensive drugs, and
alkaloids can be synthesized by lipase-catalyzed stereoselective
acylations. The enzymatic kinetic resolution of a racemic second-
ary alcohol provides16 a new chemo-enzymatic route for the syn-
thesis of (S)-enciprazine (Scheme 2).
2.3.1. Screening of lipases
Commercially available lipases, such as Pseudomonas cepacia
immobilized on sol-gel-Ak (PS-C), immobilized lipozyme Mucor
miehei, Candida antarctica immobilized on acrylic resin (CAL-B), li-
pase AY ‘Amano’30, lipase A from Candida antarctica (CAL-A) and
lipase from Candida rugosa (L8525), Candida cylindracea, Aspergillus
niger, and porcine pancreas lipase were screened for the transeste-
rification of (RS)-6 with vinyl acetate in toluene (Scheme 4).
OH
N
MeO
MeO
O
N
OMe
OH
N
Cl
N
OMe
OMe
(S)-1
6
(S)-
OH
N
N
O
Cl
Cl
N
OMe
HN
OMe
3
4
(RS)-6
(RS)-
Scheme 2. New chemo-enzymatic route for (RS)- and (S)-enciprazine.

1274
L. Banoth et al. / Tetrahedron: Asymmetry 23 (2012) 1272–1278
OH
N
N
O
LiBr
RT
Cl
Cl
N
OMe
HN
OMe
3
4
(RS)-6
(RS)-
Scheme 3. Synthesis of (RS)-6.
O
lipase,
O
N
OH
N
vinyl acetate
OH
N
Cl
N
OMe
Cl
N
OMe
Cl
N
OMe
(R)
(S)
RT, toluene
7
(R)-
(RS)-6
(S)-6
Scheme 4. Enzymatic kinetic resolution of (RS)-6.
Table 2
Lipases CRL, CAL, and PCL exhibited the best activity for the con-
version of (RS)-6 to (R)-7 and (S)-6. However, CRL was found to be
better than CAL and PCL in terms of conversion and enantiomeric
excess (Table 1).
The effect of the organic solvent on the enantioselectivity in the resolution of (RS)-6
with lipase CRLa
Cb (%)
ees (%)
eep (%)
Ee
9.4
c
d
Solvent
Acetone
4.0
29.5
17.1
26.1
32.5
39.0
41.2
21.5
27.8
30.3
30.1
29.4
3.4
35.1
19.7
31.6
43.9
59.4
62.7
25.4
35.1
38.9
38.5
37.6
80.2
83.7
95.5
89.5
91.1
92.8
89.7
92.7
90.9
89.7
89.5
90.5
Diethyl ether
Dichloromethane
Diisopropyl ether
Benzene
Chloroform
Toluene
Carbon tetrachloride
Cyclo hexane
Hexane
15.8
52.5
24.5
32.9
48.64
34.9
33.6
29.6
26.9
26.4
28.0
Table 1
Lipase-catalyzed transesterification of (RS)-6 with vinyl acetatea
Lipaseb
Time (h)
Cc (%)
ees (%)
eep (%)
Ef
d
e
CAL
PCL
CRL
24
24
24
18.7
26.4
29.7
21.2
32.5
37.8
91.9
90.6
89.5
29.4
27.9
26.2
Heptane
iso-Octane
a
Conditions: (RS)-6 (20 mM) in 4 mL of toluene was treated with vinyl acetate
(5.4 mmol) at 25 °C in the presence of the enzyme (25 mg/mL of CAL, PCL, and CRL).
b
a
PCL (immobilized lipase in sol-gel-Ak from Pseudomonas cepacia), CAL (lipase
Conditions: (RS)-6 (20 mM) in toluene (4 mL) was treated with vinyl acetate
acrylic resin from Candida antarctica), and CRL (lipase from Candida rugosa).
(5.4 mmol) at 25 °C in the presence of lipase CRL (25 mg/mL).
c
b
Conversions were calculated from the enantiomeric excess (ee) of (S)-6 (sub-
Conversions were calculated from the enantiomeric excess (ee) of (S)-6 (sub-
strate S) and (R)-7 (product P) using the formula: conversion (C) = ees/(ees + eep).
strate S) and (R)-7 (product P) using the formula: conversion (C) = ees/(ees + eep).
d
c
Enantiomeric excess of (S)-6 determined by HPLC analysis (Daicel Chiralcel OJ-
Enantiomeric excess of (S)-6 determined by HPLC analysis (Daicel Chiralcel OJ-
H column) 95:5:0.05; hexane/2-propanol/triethyl amine, 0.8 mL/min flow rate at
261 nm.
H column) 95:5:0.05; hexane/2-propanol/triethyl amine, 0.8 mL/min flow rate at
261 nm.
e
d
Enantiomeric excess of (R)-7 determined by HPLC analysis (Daicel Chiralcel OJ-
Enantiomeric excess of (R)-7 determined by HPLC analysis (Daicel Chiralcel OJ-
H column) 95:5:0.05; hexane/2-propanol/triethyl amine, 0.8 mL/min flow rate at
261 nm.
H column) 95:5:0.05; hexane/2-propanol/triethyl amine, 0.8 mL/min flow rate at
261 nm.
f
e
E values were calculated using the formula: E = [ln (1 ꢁ C (1 + eeP)]/[ln (1 ꢁ C
E values were calculated using the formula: E = [ln (1 ꢁ C (1 + eeP)]/[ln (1 ꢁ C
(1 ꢁ eeP)].17
(1ꢁeeP)].17
conversion or ees was observed. On the other hand, the eep of the
product decreased with time (eep = 80.6% at 6 h to 79.6% at 9 h)
(Fig. 1). Thus, 9 h was chosen as the optimal reaction time.
2.3.2. Selection of the organic solvent
The effect of various solvents (varying logP values such as tolu-
ene, iso-octane, chloroform, dichloromethane, etc) on the enanti-
oselectivity of enzymatic reaction,18 was investigated by
transesterification of (RS)-6 with vinyl acetate in the presence of
CRL (Table 2). The maximum enantiomeric excess of substrate
was observed in toluene.
2.3.4. Effect of acyl donors
The effect of acyl donors on the conversion rate and ee of the
CRL-catalyzed kinetic resolution of (RS)-6 was studied in toluene
(Table 3). The best results were obtained using vinyl acetate com-
pared to other acyl donors. The superiority of vinyl acetate as the
acyl donor for the enzymatic reaction is because of the in situ tau-
tomerization ability of vinyl alcohol to acetaldehyde, and so it does
not act as a competitive substrate and shifts the equilibrium to-
ward the product.19
2.3.3. Effect of reaction time
A time dependent CRL catalyzed transesterification reaction of
(RS)-6 with vinyl acetate was performed in toluene. Samples were
collected periodically from the reaction mixture and centrifuged at
10,000ꢀg for 5 min to remove the enzyme. The conversion and
enantiomeric excess were determined using chiral HPLC. It can
be seen from Table 1 that the conversion increased with time
and the maximum conversion (C = 45.9%) and ees (67.8%) were
achieved at 9 h, after which no significant change in the rate of
2.3.5. Effect of temperature
The influence of temperature on the activity and enantioselec-
tivity of the CRL-catalyzed kinetic resolution of (RS)-6 using vinyl
acetate as the acyl donor in toluene was determined (Fig. 2). The

L. Banoth et al. / Tetrahedron: Asymmetry 23 (2012) 1272–1278
1275
2.3.6. Effect of enzyme concentration
20
It is known
that the enzyme concentration has a significant
effect on the rate of conversion and ee. The reaction of (RS)-6 with
vinyl acetate was carried out using different concentrations of CRL
enzyme (25, 50, 75, and 150 mg/mL) in toluene. Samples were col-
lected from the reaction mixture at 9 h and the enzyme was re-
moved by centrifugation. The percentage of conversion and
enantiomeric excess were determined using chiral HPLC as de-
scribed in Section 4.1.1. It was observed that when the enzyme
concentration was increased, the conversion increased up to a cer-
tain level after which there was not any significant change in the
conversion rate or ee. The maximum conversion (47.5%) was ob-
tained at 50 mg/mL enzyme concentration with the highest ees val-
ues (Fig. 3).
Figure 1. Course of CRL catalyzed transesterification of (RS)-6 in toluene.
Table 3
The effect of acyl donors on the CRL catalyzed transesterification of (RS)-6 in toluenea
Acyl donorsa
Cb (%)
ees (%)
eep (%)
c
d
Phenylethylacetate
Ethyl acetate
Methylchloroacetate
Acetic anhydride
Vinyl acetate
9
8
100
100
100
100
80.2
3.3
5.4
0.6
34.2
3.4
5.8
0.6
41.7
a
Conditions: (RS)-6 (20 mM) in toluene (4 mL) was treated with the acyl donor
(5.4 mmol) at 25 °C in the presence of lipase CRL (25 mg/mL).
b
Conversions were calculated from the enantiomeric excess (ee) of (S)-6 (sub-
strate S) and (R)-7 (product P) using the formula: conversion (C) = ees/(ees + eep).
c
Enantiomeric excess of (S)-6 determined by HPLC analysis (Daicel Chiralcel OJ-
Figure 3. Effect of enzyme concentration on the CRL catalyzed transesterification of
(RS)-6 with vinyl acetate in toluene.
H column) 95:5:0.05; hexane/2-propanol/triethyl amine, 0.8 mL/min flow rate at
261 nm.
Enantiomeric excess of (R)-7 determined by HPLC analysis (Daicel Chiralcel OJ-
H column) 95:5:0.05; hexane/2-propanol/triethyl amine, 0.8 mL/min flow rate at
261 nm.
d
2.3.7. Effect of substrate concentration
It was necessary to study the effect of the substrate concentra-
tion on the activity and ee of the CRL for the resolution of (RS)-6.
Various concentrations of the substrate were used, that is, 10, 20,
60, 120, and 180 mM. Samples were collected from the reaction
mixture after 9 h and the enzyme was removed by centrifugation.
The percentage conversion and enantiomeric excess were deter-
mined using chiral HPLC as described before. It was found that
the maximum conversion was obtained with a substrate concen-
tration of 10 mM (conversion 49.3%) and enantiomeric excess of
Figure 2. The effect of temperature on the CRL catalyzed transesterification of (RS)-
6 with vinyl acetate in toluene.
resolution was carried out at 15, 30, 45, and 60 °C and the conver-
sion and the enantiomeric excess were determined using chiral
HPLC after 9 h of reaction (Fig. 3). It was found that the conversion
increased from 5.1% at 15 °C to 45.9% at 30 °C and then decreased
to 25.3% at 60 °C. The ee of the substrate followed the same pat-
tern, however the enantiomeric excess (ee) of the product had in-
creased from 58.5% at 15 °C to 94.3% at 60 °C (Fig. 2).
Figure 4. Effect of substrate concentration on the CRL catalyzed transesterification
of (RS)-6 with vinyl acetate in toluene.

1276
L. Banoth et al. / Tetrahedron: Asymmetry 23 (2012) 1272–1278
OH
H3CO
H3CO
H3CO
OCH3
N
HO
Cl
OH
K2CO3
O
N
N
N
MeCN, Reflux
O
O
H3CO
O
6
1
(RS)- and (S)-
(RS)- and (S)-
5
Scheme 5. Synthesis of (RS)- and (S)-1.
eeP = 98.3% and eeS = 96% (Fig. 4). Therefore, a substrate concentra-
tion of 10 mM was used for further studies.
4.1.2. Reagents
(RS)-Epichlorohydrin, (S)-epichlorohydrin, (R)-epichlorohydrin,
1-(2-methoxyphenyl) piperazine, 3,4,5-trimethoxy phenol, Can-
dida antarctica, Candida rugosa L8525, Candida cylindracea, Aspergil-
lus niger, and porcine pancreas lipase and chemicals were
purchased from SIGMA (St. Louis, Missouri, USA). Solvents required
for the synthesis and extraction were acquired from commercial
sources and were either of analytical or commercial grade. HPLC
grade solvents used for HPLC analysis were obtained from J.T. Ba-
ker, Rankem and Merck Ltd. Immobilized lipase in sol-gel-Ak from
Pseudomonas cepacia, immobilized lipozyme from Mucor miehei, li-
pase A, Candida antarctica lipase were purchased from Fluka™ and
lipase AY ‘Amano’ 30 was purchased from Amano Chem Ltd. These
were used without any further treatment.
2.4. Synthesis of (RS)- and (S)-eciprazine 1
The (RS)- and (S)-enciprazine 1 drugs were synthesized by
treatment of (RS)- and (S)-6 with 5 using MeCN as the solvent
and K2CO3 as the base. The reaction was carried out under reflux
condition for 6 h at room temperature. The progress of the reaction
was monitored by TLC. After the complete consumption of (RS)-
and (S)-6, the product was extracted with ethyl acetate. The com-
bined organic layers were washed with water and brine, dried over
anhydrous sodium sulfate and evaporated under vacuum and puri-
fied by flash chromatography to yield (RS)- and (S)-1 (Scheme 5).
4.2. Synthesis of (RS)-, (R)- and (S)-1-chloro-3-(4-(2-methoxy
phenyl) piperazin-1-yl) propan-2-ol 6
3. Conclusion
From our results, we can conclude that enantio-enriched (S)-
enciprazine can be synthesized by a chemo-enzymatic route. The
kinetic resolution of racemic alcohol (RS)-6 to (S)-6 by Candida rug-
osa lipase is a good example of the enantiomeric synthesis of an
enantiomerically pure compound. Lipases from various sources
were screened and the reaction conditions for the enantioselective
resolution of (RS)-6 in organic solvents were optimized. On a pre-
parative scale, (RS)-6 was transesterified quantitatively and both
enantiomers were separated; (S)-6 was used for the synthesis of
(S)-1 enciprazine. A new chemical method has also been developed
for the synthesis of (RS)-enciprazine.
Compound (RS)-6 was synthesized by adding (RS)-epichloro-
hydrin 4 (0.93 g, 0.01 mol), 1-(2-methoxyphenyl) piperazine 3
(1.92 g, 0.01 mol) and LiBr (0.86 g, 0.01 mol) in 10 ml of water.
The reaction was then allowed to stir at room temperature.
The progression of the reaction was monitored by TLC. After
completion of the reaction, the product was extracted with ethyl
acetate and washed with a brine solution. The organic layer was
separated, dehydrated using anhydrous Na2SO4, and concentrated
under vacuum. The residue was purified by column chromatogra-
phy on silica gel (60–120 mesh), eluting with ethyl acetate:hex-
ane (15:85) to afford (RS)-6, which was then subjected to chiral
HPLC analysis using a Chiralcel OJ-H column and the two enan-
tiomers were eluted after 25.42 and 28.28 min (95:5:0.05; hex-
ane/2-propanol/triethyl amine), respectively and in a ratio of
50.25:49.74.
4. Experimental
4.1. General experimental
(RS)-6: White solid, yield 2.41 g (85%); 1H NMR (400 MHz,
CDCl3): d (ppm): 7.00–7.02 (m, 1H), 6.91–6.94 (m, 2H), 6.86–6.88
(m, 1H), 3.98–4.00 (m,1H), 3.87 (s, 3H), 3.56–3.65 (m, 2H), 3.11
(s, 4H), 2.86–2.88 (m, 2H), 2.66 (s, 2H), 2.56 (d, J = 5.6 Hz, 2H),
1.68 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3): d (ppm): 152.2,
140.9, 123.1, 120.9, 118.2, 111.1, 66.4, 61.0, 55.3, 53.5, 50.6, 47.0;
FTIR (neat cmꢁ1) ꢁ3400 cmꢁ1 (OH); LC–MS (m/z): 285.24, identical
with an authentic sample.21
4.1.1. Analysis
Enzymatic reactions were carried out on a ‘stackable Kuhner-
shaker’ at 200 rpm. 1H NMR and 13C NMR spectra were obtained
with Bruker DPX 400 (1H 400 MHz and 13C 100 MHz), chemical
shifts are expressed in d units relative to the tetramethylsilane
(TMS) signal as an internal reference in CDCl3. IR spectra (wave
number in cmꢁ1) were recorded on Nicolet FT-IR impact 400
instruments as either neat for liquid or KBr pellets for solid sam-
ples. Analytical TLC of all reactions was performed on Merck pre-
pared plates. Column chromatography was performed using SRL
silica gel (60–120 mesh). LC–MS analysis was carried out on Fin-
ninganmat LCQ instrument (USA) using a C-18 hypersil ODS
(4.6 ꢀ 15 mm, 5 m) column. Optical rotations were measured in a
Rudolph, AutopolR IV polarimeter. The enantiomeric excesses (ee)
were determined by HPLC performed on Shimadzu LC-10AT ‘pump,
(R)-6: A white solid, yield 0.238 g (84%), 91% ee. ½a D20
¼ ꢁ28:6 (c
ꢂ
1.0, EtOH). The product was then subjected to chiral HPLC analysis
using chiral OJ-H column, the two enantiomers were eluted at
tS = 25.42 min and tR = 28.28 min (95:5:0.05; hexane/2-propanol/
triethyl amine) with peak areas of 4.5% and 95.5%, respectively
(91% ee).
(S)-6: White solid, yield 0.235 g (83%), 92% ee. ½a D20
¼ þ28:9 (c
ꢂ
1.0, EtOH). The product was then subjected to chiral HPLC analysis
using chiral OJ-H column, the two enantiomers were eluted at
tS = 25.42 min and tR = 28.28 min (95:5:0.05; hexane/2-propanol/
triethyl amine) with peak areas of 96% and 4%, respectively (92%
ee).
SPD-10A UV–vis detector using
a Chiralcel OJ-H column
m, Daicel Chemical Industries, Japan) under
(0.46 ꢀ 250 mm; 5
l
the following conditions: mobile phase, 95:5:0.05; hexane/2-pro-
panol/triethyl amine, 0.8 mL/min flow rate at column temperature,
25 °C at 262 nm.

L. Banoth et al. / Tetrahedron: Asymmetry 23 (2012) 1272–1278
1277
4.3. Synthesis of (RS)-, (R)- and (S)-1-chloro-3-(4-(2-
methoxyphenyl)piperazin-1-yl) propan-2-yl acetate 7
zyme concentrations (25, 50, 75, and 150 mg/mL) were used.
Finally, in order to optimize the substrate concentration with re-
spect to constant enzyme concentration (50 mg/ml), various sub-
strate concentrations (10 20, 60, 120, and180 mM) were used.
The samples were taken at regular time intervals and analyzed
for the enantioselectivity of the transesterification reaction.
Compound (RS)-7 was synthesized chemically via treatment of
the corresponding alcohol of (RS)-6 (0.142 g, 0.0005 mol) and ace-
tic anhydride (2 ml) in the presence of pyridine [0.395 mg,
0.05 mmol] as a catalyst. The reaction mixture was stirred at 4 °C
until the complete consumption of (RS)-6, as monitored by TLC.
After the consumption of (RS)-6, the reaction mixture was poured
into ice water (25 ml), which was acidified with HCl (3.0 M) to pH
1–2 and then extracted three times with ethyl acetate. The com-
bined organic layers were washed with water and brine, dried over
anhydrous sodium sulfate, and evaporated to yield (RS)-7.
4.6. Preparative-scale transesterification reaction
The resolution of (RS)-6 was carried out on a preparative scale
under optimized condition. The reaction was performed by sub-
jecting 45 mL (10 mM substrate) of the reaction mixture to resolu-
tion by CRL at 30 °C using vinyl acetate as the acyl donor in
toluene. After 9 h (49.3% conversion), the reaction mixture was fil-
tered off and the enzyme preparation was washed with a solvent.
The solvent was evaporated under reduced pressure and the
resulting dried residue was subjected to flash chromatography
using ethyl acetate: hexane (15:85 v/v) as the mobile phase. We
observed that after 9 h the isolated yield of (S)-6 was 41.8% with
enantiomeric excess eeS = 96%, (Chiralcel OJ-H) and that of (R)-7
was 44.7% with enantiomeric excess eeP = 98.3% (Chiralcel OJ-H).
(RS)-7: Yellow semisolid, yield 0.147 g (90%); 1H NMR
(400 MHz, CDCl3): d (ppm): 6.97–6.99 (m, 1H), 6.89–6.92 (m,
2H), 6.85–6.86 (m, 1H), 3.86 (m 3H), 3.79 (s, 1H), 3.73 (s, 1H)
3.05 (s, 4H), 2.73 (s, 4H),2.66 (s, 2H), 2.11 (s, 3H); 13C NMR
(100 MHz, CDCl3): d (ppm): 170.3, 141.1, 122.9, 120.9, 118.2,
111.1, 70.3, 58.4, 55.3, 53.9, 50.6, 44.7, 21.0; FTIR (neat cmꢁ1):
ꢁ1
1741 cm
(C@O); LC–MS (m/z): 327.18. Next, (RS)-7 was sub-
jected to chiral HPLC analysis using a Chiralcel OJ-H column and
the two enantiomers were eluted after 17.95 min and 23.57 min
(95:5:0.05; hexane/2-propanol/triethyl amine), respectively and
were present in a ratio of 49.89:50.11.
(S)-6: White solid, yield 0.105 g (41.8%), 96% ee. ½a D20
¼ þ30:15 (c
ꢂ
1.0, EtOH). The product was then subjected to chiral HPLC analysis
using chiral OJ-H column, the two enantiomers were eluted at
tS = 25.42 min and tR = 28.28 min (95:5:0.05; hexane/2-propanol/
triethyl amine) with peak areas of 98% and 2%, respectively (96% ee).
(R)-7: Yellow colored semisolid, yield 0.115 g (44.7%);
(R)-7: Yellow semisolid, yield 0.15 g (92%); ½a D20
¼ ꢁ32:8 (c 1.0,
ꢂ
EtOH). The product was then subjected to chiral HPLC analysis
using chiral OJ-H column, the two enantiomers were eluted at
tR = 23.57 min and tS = 17.95 min (95:5:0.05; hexane/2-propanol/
triethyl amine) with peak areas of 93.5% and 6.5%, respectively
(87% ee).
½
a 2D0
ꢂ
¼ ꢁ36:2 (c 1.0, EtOH). The product was then subjected to chi-
ral HPLC analysis using chiral OJ-H column, the two enantiomers
were eluted at tR = 23.57 min and tS = 17.95 min (95:5:0.05; hex-
ane/2-propanol/triethyl amine) with peak areas of 99.15% and
0.85%, respectively (98.3% ee).
(S)-7: Yellow semisolid, yield 0.148 g (91%); ½a D20
¼ þ33:5 (c 1.0,
ꢂ
EtOH). The product was then subjected to chiral HPLC analysis
using chiral OJ-H column, the two enantiomers were eluted at
tR = 23.57 min and tS = 17.95 min (95:5:0.05; hexane/2-propanol/
triethyl amine) with peak areas of 5.5% and 94.5%, respectively
(89% ee).
4.7. Synthesis of (RS)- and (S)-enciprazine 1
The (RS)- and (S)-enciprazine 1 was synthesized by reaction of
(RS)- and (S)-6 (0.0569 g, 0.0002 mol) and trimethoxyphenol 5
(0.0272 g, 0.0002 mol) using MeCN as the solvent and K2CO3
(0.0552 g, 0.0004 mol) as the catalyst. The reaction was carried
out under reflux conditions for 6 h. Progress of the reaction was
monitored by TLC. After the complete consumption of (RS)- and
(S)-6, the mixture was diluted with ethyl acetate (15 mL) and
washed with water. The organic layer was separated, dried over
anhydrous Na2SO4, and concentrated under vacuum. The residue
was purified by passing through a column of silica (60–12 mesh)
and eluting with ethyl acetate/hexane (15:85) to obtain (RS)- and
(S)-enciprazine 1.
4.4. Enantioselective transesterification of (RS)-6
In a 10 mL round bottom flask containing magnetic beads, a
mixture of (RS)-6 (0.022 g, 0.08 mmol) in 4 mL of toluene and vinyl
acetate (0.464 g, 5.40 mmol) was placed. Lipases from different
sources (commercial lipase from lipase A, Candida antarctica, Can-
dida rugosa L8525, Candida cylindracea, Aspergillus niger, porcine
pancreas, and AY ‘Amano’ 30) were used to carry out the reaction.
The round bottom flask was capped and placed on a magnetic stir-
rer which was maintained at room temperature. Immobilized li-
pase in sol-gel-Ak from Pseudomonas cepacia, immobilized
lipozyme from Mucor miehei, and lipase acrylic resin from Candida
antarctica, were individually taken into separate 10 mL conical
flasks. The flasks were then capped and placed in a shaker, which
(RS)-Enciprazine 1: Yellow viscous liquid yield 0.082 g (95%); 1H
NMR (400 MHz, CDCl3): d (ppm): 6.99–7.04 (m, 1H), 6.93–6.96 (m,
2H) 6.86–6.91 (m, 1H), 6.20 (s, 2H), 4.12–4.15 (m, 1H), 3.98–3.99
(d, J = 4.8 Hz, 2H), 3.84–3.87 (m, 9H), 3.79 (s, 3H), 3.11 (s, 4H),
2.89–2.92 (m, 2H), 2.62–2.69 (m, 4H); 13C NMR (100 MHz, CDCl3):
d (ppm): 155.4, 153.6, 152.2, 141.2, 132.4, 123.0, 122.9, 121.0,
118.2, 111.1, 92.3, 70.6, 65.4, 61.0, 60.5, 56.0, 55.3, 53.5, 50.7; FTIR
(neat cmꢁ1): 3394 cmꢁ1(OH); LC–MS (m/z): 433.37. identical with
an authentic sample.10 Next, (RS)-1 was subjected to chiral HPLC
analysis using a Chiralcel OJ-H column and the two enantiomers
were eluted after 29.43 min and 30.98 min (95:5:0.05; hexane/2-
propanol/triethyl amine), respectively and were present in a ratio
of 49.9:50.1.
was maintained at 30 °C (200 rpm). Samples (400 lL) were with-
drawn from reaction mixture and the conversion and enantiomeric
excess of the reaction were monitored by HPLC.
4.5. Optimization of the transesterification reaction
The effect of different organic solvents such as diisopropyl
ether, diethyl ether, dichloromethane, benzene, heptane, iso-oc-
tane, and toluene on the transesterification of (RS)-6 was studied.
The optimum temperature was determined by carrying out the
reaction at various temperatures in the range of 15–60 °C. In order
to determine the effect of different acyl donors, various acyl donors
such as ethyl acetate, acetic anhydride, and vinyl acetate
(5.40 mmol) were used. In order to optimize the enzyme concen-
tration with respect to a constant amount of substrate, various en-
(S)-1: Yield .0812 g (94%).½a D20
ꢂ
¼ þ29 (c 0.5, EtOH) {lit22
½
a 2D0
ꢂ
¼ þ3:0 (c 0.5, EtOH)}. The product was then subjected to chi-
ral HPLC analysis using chiral OJ-H column, the two enantiomers
were eluted at tR = 30.98 min and tS = 29.43 min (95:5:0.05; hex-
ane/2-propanol/triethyl amine) with peak areas of 2.5% and
97.5%, respectively (95% ee).

1278
L. Banoth et al. / Tetrahedron: Asymmetry 23 (2012) 1272–1278
Chakraborti, A. K.; Kondaskar, A.; Rudrawar, S. Tetrahedron 2004, 60, 9085–
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