Efficient chemoenzymatic synthesis of (RS)-, (R)-, and (S)-bunitrolol

Article abstract of DOI:10.1055/s-0033-1340465

A new chemical and the first chemoenzymatic synthesis of β-adrenergic receptor blocking agent bunitrolol is reported in racemic (RS) and enantioenriched forms (R and S). The intermediates (R)- and (S)-1-chloro-3-(2- cyanophenoxy)propan-2-ol intermediates were synthesized from the corresponding racemic alcohol through enzymatic kinetic resolution. The commercial available lipases PS-C and CCL exhibited complementary enantioselectivity during transesterification of the racemic alcohol with vinyl acetate affording the (R)-alcohol along with (S)-acetate and the (S)-alcohol along with (R)-acetate, respectively, and represent an example of enzymatic switch for reversal of enantioselectivity. The effects of various reaction parameters, such as temperature, time, substrate and enzyme concentration, and reaction medium, on the activity and enantioselectivity were optimized. The (R)- and (S)-alcohols were converted into (S)-and and (R)-bunitrolol, respectively, by treatment with tert-butylamine. The (R)- and (S)-acetates, obtained enzymatically were deacetylated to the corresponding alcohol by chemical hydrolysis and further converted into (S)-and and (R)-bunitrolol by chemical means. This is the first chemoenzymatic synthesis of both of the enantiomers of the drug. (RS)-, (R)-, and (S)-Bunitrolol were also synthesized following the 'all chemical' routes from (RS)-, (R)-, and (S)-epichlorohydrin via the corresponding (RS)-, (S)-, and (R)-2-cyanoglycidyl ether and the (RS)-, (R)-, and (S)-1-chloro-3-(2- cyanophenoxy)propan-2-ol intermediates with improved overall yields and better enantiomeric excesses compared to the reported processes.

Full text of DOI:10.1055/s-0033-1340465

PAPER
▌479
paper
Efficient Chemoenzymatic Synthesis of (RS)-, (R)-, and (S)-Bunitrolol
Chemoenzymatic Synthesis of (RS)-, (R)-, and (S)-Bunitrolol
Linga Banoth,a Bhukya Chandarrao,a Brahmam Pujala,b Asit K. Chakraborti,*b U. C. Banerjee*a
a
Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, S. A. S. Nagar-160062, Punjab, India
E-mail: ucbanerjee@niper.ac.in
b
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S. A. S. Nagar-160062,
Punjab, India
E-mail: akchakraborti@niper.ac.in
Received: 10.09.2013; Accepted after revision: 25.11.2013
ments for therapeutic advantage in the acute vasodilatory
Abstract: A new chemical and the first chemoenzymatic synthesis
effect as its α-adrenoreceptor blocking action may con-
of β-adrenergic receptor blocking agent bunitrolol is reported in ra-
tribute to the decrease in total peripheral resistance when
hypertensive patients are treated chronically with bunitro-
cemic (RS) and enantioenriched forms (R and S). The intermediates
(R)- and (S)-1-chloro-3-(2-cyanophenoxy)propan-2-ol intermedi-
ates were synthesized from the corresponding racemic alcohol lol.8
through enzymatic kinetic resolution. The commercial available li-
The several approaches to the synthesis of 1 are summa-
pases PS-C and CCL exhibited complementary enantioselectivity
during transesterification of the racemic alcohol with vinyl acetate
affording the (R)-alcohol along with (S)-acetate and the (S)-alcohol
along with (R)-acetate, respectively, and represent an example of
enzymatic switch for reversal of enantioselectivity. The effects of
various reaction parameters, such as temperature, time, substrate
and enzyme concentration, and reaction medium, on the activity and
enantioselectivity were optimized. The (R)- and (S)-alcohols were
converted into (S)-and and (R)-bunitrolol, respectively, by treat-
ment with tert-butylamine. The (R)- and (S)-acetates, obtained en-
zymatically were deacetylated to the corresponding alcohol by
rized in Scheme 1.9 The most common strategy for the
synthesis of the 1,2-amino alcohol class of β-adrenergic
blocking agents involves the opening of the epoxide ring
by an amine.10 Efforts in this direction in the synthesis of
1 involve the reaction of tert-butylamine with the requisite
epoxide, 2-cyanophenyl glycidyl ether 2.9 However, most
of these have one or more drawbacks such as a multistep
procedure for the preparation of 2 that requires a costly
and toxic osmium catalyst, corrosive reagents, or long re-
chemical hydrolysis and further converted into (S)-and and (R)- action times.9a,c
bunitrolol by chemical means. This is the first chemoenzymatic
Although currently 1 is marketed in its racemic form, the
synthesis of both of the enantiomers of the drug. (RS)-, (R)-, and (S)-
Bunitrolol were also synthesized following the ‘all chemical’ routes
from (RS)-, (R)-, and (S)-epichlorohydrin via the corresponding
(RS)-, (S)-, and (R)-2-cyanoglycidyl ether and the (RS)-, (R)-, and
(S)-1-chloro-3-(2-cyanophenoxy)propan-2-ol intermediates with
improved overall yields and better enantiomeric excesses compared
to the reported processes.
S-enantiomer is the eutomer with 20 times higher
potency11 than that of the distomeric R-form, which has
undesirable effects,11,12 and the two enantiomers have dif-
ferent metabolic profiles.13 This has generated an interest
in preparing the enantioenriched/enantiopure forms of 1.
The reported methodologies (routes A and B) for the syn-
thesis of enantiopure/enantioenriched 1 involves the ep-
oxide ring-opening reaction of the glycidic ethers (R)- and
(S)-2, prepared from enantioenriched epichlorohydrin
(R)-4,9b multistep reaction of (R)- and (S)-6 obtained
through spontaneous resolution of the alkylated product
of 2-cyanophenol 3 with (RS)-8,9c and a multistep proce-
dure involving toxic osmium tetroxide mediated asym-
metric dihydroxylation of 7.9a These reported procedures
afford overall 26–37% chemical yields with 60–88% ee.
Key words: bunitrolol, biocatalysis, lipase, drug, overall enantio-
selectivity synthesis
Cardiovascular diseases (CVDs) account for one third
(16.6 million) of global deaths in 2001 and they are the
leading cause of death worldwide.1 Effective life-saving
medicines for the management of cardiovascular disor-
ders,2 including hypertension,3 angina pectoris, cardiac
arrhythmias, and other disorders4 related to the sympa-
thetic nervous system, are β-adrenergic blocking agents
that have been in use for more than 25 years. However, the
increase in total peripheral resistance by β-adrenergic
blocking agents is thought to oppose their antihyperten-
sive effect5 and this has led to the development of drugs
with dual α- and β-adrenergic blocking effects.6 Bunitro-
lol (1), a vasodilatory drug known for its β-adrenergic
blocking properties,7 is expected to meet new require-
Herein we describe (route C) concise new chemical and
novel chemoenzymatic synthetic routes for (RS)-, (R)-,
and (S)-bunitrolols with much improved overall chemical
yields (59–61%) with higher (80–94%) enantiomeric ex-
cesses.
While designing a new synthetic route, we took into con-
sideration the increasing influence of green chemistry
tools on medicinal chemistry and chemistry research
based organization14 and the necessity to improve existing
transformations to enrich the medicinal chemist’s tool box
for more general and amenable applications.15 Integrating
biocatalysis in synthesis is an elegant approach to expand
the organic toolbox and it adds and additional dimension
SYNTHESIS 2014, 46, 0479–0488
Advanced online publication: 11.12.2013
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0033-1340465; Art ID: SS-2013-Z0620-OP
© Georg Thieme Verlag Stuttgart · New York

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L. Banoth et al.
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CN
O
7
CN
OH
OH
O
O
OH
O
Cl
OH
3
+
(RS)-8
6
O
S
CN
CN
O
O
(RS)-5
B
OH
H
O
N
C (this work)
A
(RS)-bunitrolol (1)
CN
CN
O
OH
O
O
Cl
(RS)-5
(RS)-9
(RS)-2
CN
(RS)-2
OH
+
O
Cl
(RS)-4
3
Scheme 1 Various synthetic strategies for 1
in greener chemistry.16 In this context, the enzymatic ki- The starting racemic epoxide (RS)-2 was prepared in 80%
netic resolution of a racemic secondary alcohol provides yield by the reaction of 3 with (RS)-4 in the presence of
the desired scope17 and encouraged us to design a new potassium carbonate in acetonitrile under reflux following
chemoenzymatic route for (R)- and (S)-bunitrolols the reported procedure.10b The treatment of (RS)-2 with
(Scheme 2).
lithium chloride in tetrahydrofuran and acetic acid afford-
ed the desired substrate (RS)-9 required for enzymatic ki-
netic resolution18 (Scheme 3).
OH
OH
CN
CN
H
Cl
Cl
O
N
O
CN
CN
O
OH
O
(S)-9
K2CO3, MeCN
reflux, 16 h
(S)-1
O
Cl
+
3
(RS)-4
(RS)-2
CN
OH
CN
O
LiCl, AcOH
THF, r.t., 12 h
O
O
CN
OH
(RS)-2
(RS)-9
O
Cl
Scheme 2 New chemoenzymatic route for (R)- and (S)-bunitrolols
(RS)-9
Scheme 3 Synthesis of (RS)-9
Synthesis 2014, 46, 479–488
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Chemoenzymatic Synthesis of (RS)-, (R)-, and (S)-Bunitrolol
481
To assess the catalytic efficiency of the enzyme and to de- mol%)19 afforded (RS)-10 in 95% yield. Acetylation of
rive the best operative experimental conditions for kinetic (R)- and (S)-9 following a similar procedure resulted in
resolution, it is necessary to have authentic samples of the formation of (R)- and (S)-10 in 89% and 87% yields
(R)- and (S)-9 and the corresponding O-acylated deriva- with 82% and 81% ee, respectively.
tives (R)- and (S)-10, respectively.
With the substrate (RS)-9 and authentic samples of (R)-
The requisite starting materials (S)- and (R)-2 for the au- and (S)-9 and (R)- and (S)-10 in hand, it was planned to
thentic samples of (R)- and (S)-9 were obtained by reac- derive the best operative enzymatic kinetic resolution pro-
tion of 3 with (R)- and (S)-4, respectively, following the cedure using various lipases.
modified procedure.10b The enantiomeric excess/optical
Initially, commercially available lipases: Pseudomonas
cepacia immobilized on sol-gel-Ak (PS-C), immobilized
lipozyme Mucor miehei (MML), Candida antarctica im-
mobilized on acrylic resin (CAL), lipase A Candida ant-
purity was determined by chiral HPLC and from the opti-
cal rotation value. As observed in a previous report,10b al-
kylation using (R)-4 resulted in (S)-2 (80% yield, 82% ee)
and (S)-4 afforded (R)-2 (78% yield, 81% ee) (Scheme 4).
arctica (CAL-A), Candida rugosa (CRL L8525),
The formation of (S)-2 from (R)-4 can be attributed due to
Candida rugosa (CRL L-1754), Candida cylindracea
the nucleophilic ring opening of the epoxide ring at the
(CCL), Aspergillus niger, porcine pancreas lipase, lipase
AY ‘Amano’ 30 were screened for the transesterification
of (RS)-9 with vinyl acetate in toluene (Scheme 5).
least substituted carbon atom of the epoxide followed by
nucleophilic displacement of the chlorine atom (Scheme
4, path b), rather than direct nucleophilic substitution of
The lipases PS-C exhibited best activity for conversion of
the chlorine atom (Scheme 4, path a). This, accounts for
(RS)-9 into (R)-9 and (S)-10, respectively. The lipases
CCL exhibited best activity for conversion of (RS)-9 into
(S)-9 and (R)-10, respectively. Thus, the enzymes PS-C
and CCL showed complementary action with respect to
enantioselectivity. These enzymes were better in terms of
conversion and different enantioselectivity compared to
the observed inversion of configuration.
Ring opening of (S)-2 by lithium chloride following the
same procedure as used for (RS)-2 afforded (R)-9 in 84%
yield with 82% ee. Similarly, (S)-9 was obtained from
(R)-2 in 82% yield with 81% ee. The treatment of (RS)-9
with acetic anhydride at room temperature under neat con-
other lipases (Table 1).
ditions in the presence of zirconium(IV) chloride (2
O
K+O–
Cl
OH
O–K+
O
O
H
(R)-4
NC
NC
H
CN
CN
K2CO3, MeCN
reflux, 16 h
Cl
path 'b'
3
(R)-4
path 'a'
NC
O
O
O
H
H
(R)-2
(80%, 82% ee)
Scheme 4 Formation of (S)-2 from (R)-4
CN
OAc
O
CN
OH
CN
OH
lipase PS-C
vinyl acetate
Cl
O
Cl
O
Cl
+
r.t., PhMe
(RS)-9
(R)-9
(S)-10
Scheme 5 Enzymatic kinetic resolution of (RS)-9
© Georg Thieme Verlag Stuttgart · New York
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L. Banoth et al.
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Table 1 Lipase-Catalyzed Transesterification of (RS)-9 with Vinyl
Table 2 The Effect of Organic Solvent on Enantioselectivity in the
Resolution of (RS)-9 with Lipase PS-Ca
Acetatea
Lipase
Time
(h)
Cb
(%)
eec (%) eec (%) Eb
of 10 of 9
Config
of 10
Solvent
Log P
Cb
(%)
eec (%) eec (%) Eb
of 10
of 9
CRL L1754
CRL L8525
CCL
48
48
48
48
48
48
18
76
32
30
2
55
11
59
79
16
4
12
33
27
34
1
R
S-Selective with PS-C
MeCN
1.61
R
–0.33
–1.1
1.35
1.9
2
5
2
70
92
37
90
92
79
95
4
4
1
1
2
2
1
1
R
1,4-dioxane
t-BuOMe
i-Pr2O
PS-C
S
18
7
20
3
2
CAL-A
MML
0.3
0.1
RS
RS
1
3
benzene
2.0
4
4
1
a Conditions: (RS)-9 (1 mmol), toluene (3.5 mL) treated with vinyl
acetate (5.4 mmol) at 25 °C in the presence of the enzyme (15 mg/mL).
b For the method of calculation see the experimental section.
c Enantiomeric excess of the substrate 9 or product 10 was determined
by HPLC analysis (Daicel Chiralcel OD-H column, hexane–i-PrOH,
80:20, 0.5 mL/min flow rate, 254 nm).
heptane
4.0
17
30
43
19
34
72
1
toluene
2.5
1
isooctane
R-Selective with CCL
benzene
4.5
11
2.0
1.25
1.9
23
3
73
68
56
70
55
86
45
46
22
2
2
1
To evaluate the effect of the solvent on the enantioselec-
tivity of the enzymatic reaction,21 various other organic
solvents with varying log P values, such tert-butyl methyl
ether, isooctane, chloroform, dichloromethane etc, were
investigated for the transesterification of (RS)-9 with vi-
nyl acetate in the presence of PS-C and CCL (Table 2).
The maximum enantioselectivity was observed in tert-bu-
tyl methyl ether and isooctane for the PS-C-catalyzed re-
actions. On the other hand, for reactions using CCL,
maximum enantioselectivity was observed in toluene. It
has been reported22 that in polar solvents (log P < 2), the
rate of biocatalytic reactions is lower compared to that in
nonpolar solvents (log P > 4). Moderate reactions rates are
observed in organic solvents with log P value between 2
and 4.23a It has been observed that hydrophobic solvents
are unable to strip away the water molecules associated
with the enzymes and in this process enzymes retain the
required degree of hydration to remain catalytically ac-
tive, whereas hydrophilic solvents, due to their water lov-
ing nature, strip away water molecules from the enzyme
complex, which leads to catalytic deactivation.23b,c In the
case of isooctane, a hydrophobic solvent, a positive corre-
lation between the activity of lipase and increased log P
value of the solvent could be seen. Thus isooctane and
tert-butyl methyl ether were used for subsequent enzy-
matic reactions with PS-C and toluene was used for CCL.
CH2Cl2
i-Pr2O
59
41
3
81
49
1
–
Et2O
0.85
–1.1
2.5
4
1,4-dioxane
toluene
1
46
66
42
74
88
33
13
–
hexane
isooctane
4.5
2
a Conditions: (RS)-9 (1 mmol), toluene (3.5 mL) treated with vinyl
acetate (5.4 mmol) at 25 °C in the presence of the lipase (15 mg/mL)
for 48 h.
b For the method of calculation see the experimental section.
c Enantiomeric excess of the substrate 9 or product 10 was determined
by HPLC analysis (Daicel Chiralcel OD-H column, hexane–i-PrOH,
80:20, 0.5 mL/min flow rate, 254 nm).
product decreased with time (E = 47.1 at 96 h to E = 25.5
at 168 h and eeP = 94.3% at 96 h to 89.4% at 168 h) (Fig-
ure 1).
A time dependent PS-C-catalyzed transesterification reac-
tion of (RS)-9 was performed with vinyl acetate separately
in tert-butyl methyl ether and isooctane and in toluene for
CCL-catalyzed reactions. The conversion and enantio-
meric excess were determined using chiral HPLC. In the
case of tert-butyl methyl ether the conversion increased
with time. Maximum conversion (C = 25.3%) was
achieved after 96 hours and thereafter no significant
change in the rate of conversion was observed. On the
other hand, the enantiomeric excess and E values of the
Figure 1 Course of PS-C-catalyzed transesterification of (RS)-9 in
tert-butyl methyl ether
Synthesis 2014, 46, 479–488
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Chemoenzymatic Synthesis of (RS)-, (R)-, and (S)-Bunitrolol
483
Maximum conversion (C = 46.2%) was achieved after 48 ascribed to the ability of the in situ generated vinyl alcohol
hours of reaction and thereafter no significant change in to tautomerize to acetaldehyde, there by not acting as a
the rate of conversion was observed when the reaction competitive substrate and shifting the equilibrium to the
medium was isooctane. On the other hand, the enantio- product.24b
meric ratio of the product decreased with time (E = 84.5
at 48 h to E = 63.67 at 168 h) (Figure 2). Thus, 96 and 48
hours were taken as the optimum time for further study in
Table 3 Effect of Acyl Donors on the PS-C-Catalyzed Transesteri-
fication of (RS)-9 in tert-Butyl Methyl Ethera
tert-butyl methyl ether and isooctane, respectively.
Acyl donorsa
Cb
(%)
eec (%)
of 10
eec (%) Eb
of 9
BnOAc
–
4
–
87
86
79
96
–
3
–
1
1
1
2
EtOAc
butanoic anhydride
isobutanoic anhydride
vinyl acetate
4
4
3
3
26
33
a Conditions: (RS)-9 (1 mmol) in t-BuOMe (3.5 mL) treated with acyl
donor (5.4 mmol) at 25 °C in the presence of the lipase PS-C (15
mg/mL) for 96 h.
b For the method of calculation see the experimental section.
c Enantiomeric excess of (R)-9 or (S)-10 determined by HPLC analy-
sis (Daicel Chiralcel OD-H column, hexane–i-PrOH, 80:20, 0.5
mL/min flow rate, 254 nm).
Figure 2 Course of PS-C-catalyzed transesterification of (RS)-9 in
isooctane
Table 4 Effect of Acyl Donors on the PS-C-Catalyzed Transesteri-
fication of (RS)-9 in Isooctanea
For the CCL-catalyzed reaction, maximum conversion
(C = 48%) was achieved after 36 hours and there after no
significant change in the rate of conversion was observed
when the reaction medium was toluene. The enantiomeric
ratio of the product increased with time (Figure 3). Thus,
36 hours was taken as an optimum time for further study
in toluene.
Acyl donorsa
Cb (%)
eec (%)
eec (%)
Eb
of 10
of 9
BnOAc
–
4
–
51
64
37
95
–
2
–
1
EtOAc
butanoic anhydride
isobutanoic anhydride
vinyl acetate
4
3
1
4
2
1
43
72
11
a Conditions: (RS)-9 (1 mmol) in isooctane (3.5 mL) treated with acyl
donor (5.4 mmol) at 25 °C in the presence of the lipase PS-C (15
mg/mL) for 48 h.
b For the method of calculation see the experimental section.
c Enantiomeric excess of (R)-9 or (S)-10 determined by HPLC analy-
sis (Daicel Chiralcel OD-H column, hexane–i-PrOH, 80:20, 0.5
mL/min flow rate, 254 nm).
The influence of temperature on activity and enantioselec-
tivity of the PS-C-catalyzed kinetic resolution of (RS)-9
using vinyl acetate as the acyl donor in tert-butyl methyl
ether (Figure 4) and isooctane (Figure 5) was also deter-
mined. The resolution was carried out at 15, 25, 30, 45,
and 60 °C and the conversion and the enantiomeric excess
were determined using chiral HPLC after 96 hours in tert-
butyl methyl ether (Figure 4) and after 48 hours in isooc-
tane (Figure 5). It was found that in tert-butyl methyl ether
the conversion had increased from 5% at 15 °C to 46% at
45 °C and then decreased to 25% at 60 °C. On the other
hand, the enantiomeric excess of the product decreased
from 94% at 25 °C to 90% at 60 °C [i.e. enantiomeric ratio
(E) decreased from E = 47 to E = 26] (Figure 4). In the
Figure 3 Course of CCL-catalyzed transesterification of (RS)-9 in
toluene
The acyl donor has significance influence on enzymatic
acylation24 on various aspects such as conversion, enan-
tioselectivity, and greenness of the process.24a Therefore,
the effect of the acyl donor on the rate of conversion and
enantioselectivity of the PS-C-catalyzed kinetic resolu-
tion of (RS)-9 was studied separately in tert-butyl methyl
ether (Table 3) and isooctane (Table 4) and the best results
were obtained in using vinyl acetate and poor results were
obtained with other acyl donors. The superiority of vinyl
acetate as the acyl donor for enzymatic reactions has been
© Georg Thieme Verlag Stuttgart · New York
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484
L. Banoth et al.
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case of isooctane, it was observed that the conversion in- 7). Thus, for all the subsequent experiments, an enzyme
creased from 22% at 15 °C to 52% at 45 °C and then de- concentration of 20 mg/mL was used.
creased to 35% at 60 °C. On the other hand, the
enantiomeric ratio (E) decreased from E = 85 to E = 48)
(Figure 5). Thus, 25 °C was used as the optimum temper-
ature for PS-C-catalyzed kinetic resolution to achieve
good enantiomeric ratio and enantiomeric excess both in
tert-butyl methyl ether and isooctane.
Figure 6 Effect of enzyme concentration on the PS-C-catalyzed
transesterification of (RS)-9 with vinyl acetate in tert-butyl methyl
ether
Figure 4 The effect of temperature on the PS-C-catalyzed trans-
esterification of (RS)-9 with vinyl acetate in tert-butyl methyl ether
Figure 7 Effect of enzyme concentration on the PS-C-catalyzed
transesterification of (RS)-9 with vinyl acetate in isooctane
As the enzyme concentration often exhibits a significant
effect on the rate of conversion as well as the enantiomeric
excess, the reaction of (RS)-9 with vinyl acetate was car-
Figure 5 The effect of temperature on the PS-C-catalyzed trans-
esterification of (RS)-9 with vinyl acetate in isooctane
ried out using different concentrations of CCL (10, 50, 75,
and 150 mg/mL) in toluene. The optimum enzyme con-
centration was 50 mg/mL with conversion (46%), eeS =
82%, eeP = 95%, and E = 100 (Figure 8).
As the enzyme concentration often exhibits a significant
effect on the rate of conversion as well as the enantiomeric
excess,25 the reaction of (RS)-9 with vinyl acetate was car-
ried out using different concentrations of PS-C (10, 15,
20, 23, 25, 30, 60, and 120 mg/mL) separately in tert-bu-
tyl methyl ether and isooctane. It was observed that with
the increase in enzyme concentration, the conversion in-
creased up to a certain level after which there was no sig-
nificant change in the conversion. Although the
conversion obtained at 20 mg/mL (46%) was slightly less
than at 25 mg/mL (50%) of the enzyme, the enantiomeric
excess and E values were better at 20 mg/mL (Figure 6).
In the case of isooctane, the conversion (46%) obtained at
20 mg/mL enzyme concentration was slightly less (48%)
than that of at 25 mg/mL but the enantiomeric excess and
E values were better at 20 mg/mL enzyme level (Figure
Figure 8 Effect of enzyme concentration on the CCL-catalyzed
transesterification of (RS)-9 with vinyl acetate in toluene
Synthesis 2014, 46, 479–488
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Chemoenzymatic Synthesis of (RS)-, (R)-, and (S)-Bunitrolol
485
The liberation of the parent alcohol from the acetylated in- The present study describes an efficient chemical synthe-
termediate (RS)-, (R)-, and (S)-10 was achieved by deacet- sis, and the first chemoenzymatic synthesis, of the enan-
ylation in aqueous potassium carbonate at room tiomerically enriched cardiovascular drug bunitrolol in
temperature for two hours26 (Scheme 6).
improved overall yields (59–61% as compared to reported
yields of 26–37%) and higher enantiomeric access (80–
94% as compared to the reported of 60–88% ee). This
commercially available lipases PS-C and CCL offered
complementary activity during the transesterification of
(RS)-9 with vinyl acetate to afford the key intermediates
(R)- and (S)-9 for (R)- and (S)-10 required for the synthe-
sis of enantioenriched (R)- and (S)-bunitrolol. This offers
a handle for an enzymatic switch towards the production
of bunitrolol in an enantiodivergent fashion. These enan-
tiomeric intermediates are also used for the synthesis of
other chiral drugs such as epanolol and bucindolol. An
‘all-chemical’ synthetic route was also achieved for effi-
cient synthesis of (RS)-, (R)-, and (S)-bunitrolol.
CN
OH
CN
OAc
K2CO3
Cl
O
Cl
O
MeOH, H2O
(RS)-10
(RS)-9
Scheme 6 Synthesis of (RS)-, (R)-, and (S)-9
The treatment of (R)-9 [obtained from (i) the enzymatic
kinetic resolution of (RS)-9 with vinyl acetate by lipase
PS-C, (ii) the enzymatic kinetic resolution of (RS)-9 with
vinyl acetate by lipase CCL to form (R)-10 followed by
deacetylation, and (iii) epoxide ring opening of (S)-2 with
lithium chloride] with tert-butylamine in ethanol under re-
flux for overnight27 afforded (S)-bunitrolol [(S)-1]
(Scheme 7).
Enzymatic reactions were carried out on a ‘stackable Kuhner-shak-
er’ at 200 rpm. 1H and 13C NMR spectra were obtained with a Bruk-
er DPX 400 (1H 400 MHz and 13C 100 MHz) relative to TMS as an
internal reference in CDCl3. IR spectra were recorded on Nicolet
FT-IR impact 400 instrument as either neat for liquid or KBr pellets
for solid samples. Analytical TLC of all the reactions was per-
formed on Merck prepared plates. Column chromatography was
performed using SRL silica gel (60–120 mesh). MS analysis was
carried out on Finninganmat LCQ instrument (USA). Optical rota-
tion was measurement in a Rudolph, Autopol IV polarimeter. Enan-
Similarly, the treatment of (S)-9 [obtained from (i) the en-
zymatic kinetic resolution of (RS)-9 with vinyl acetate by
lipase CCL, (ii) the enzymatic kinetic resolution of (RS)-
9 with vinyl acetate by lipase PS-C to form (S)-10 fol-
lowed by deacetylation, and (iii) epoxide ring opening of
(R)-2 with lithium chloride] with tert-butylamine in etha-
nol under reflux for overnight27 afforded (R)-bunitrolol tiomeric excesses were determined by HPLC performed on
Shimadzu LC-10AT pump, SPD-10A UV-VIS detector using a
Chiralcel OD-H column (0.46 mm × 250 mm; 5 μm, Daicel Chem-
[(R)-1].
ical Industries, Japan) under the following conditions: mobile
CN
OAc
CN
OH
CN
OH
lipase CCL
vinyl acetate
Cl
O
Cl
O
Cl
O
+
r.t., PhMe
(R)-10
(S)-9
(RS)-9
lipase PS-C
vinyl acetate
r.t., isooctane
K2CO3
MeOH–H2O
CN
OH
CN
CN
OAc
O
O
Cl
O
O
Cl
LiCl, AcOH
THF, r.t.
+
(R)-9
(R)-2
(S)-10
Et3N
EtOH
reflux
t-BuNH2
CN
OH
H
N
O
(S)-1
Scheme 7 Chemical and chemoenzymatic synthesis of (S)-bunitrolol [(S)-1]
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 479–488

486
L. Banoth et al.
PAPER
phase, hexane–i-PrOH, 80:20; flow rate, 0.5 mL/min; column tem-
perature, 25 °C at 254 nm.
(RS)-9
White solid; yield: 173 mg (82%).
IR (neat): 3466 cm–1 (OH).
(RS)-Epichlorohydrin, (R)-epichlorohydrin, 2-hydroxybenzonitrile,
Candida antarctica (5.72 U/mg), Candida rugosa L8525 (13060
U/mg), Candida rugosa L-1754 (742 U/mg), Candida cylindracea
(≥2 U/mg), Aspergillus niger (184 U/g), and porcine pancreas lipase
(54 U/mg) and chemicals were purchased from Sigma. Solvents re-
quired for the synthesis and extraction were acquired from commer-
cial sources and they were either of analytical or commercial
grades. HPLC grade solvents used for HPLC analysis were obtained
from J. T. Baker, Rankem, and Merck Ltd. immobilized lipase in
sol-gel-Ak from Pseudomonas cepacia (186 U/g), immobilized li-
pozyme from Mucor miehei (140 U/g), lipase A, Candida antarcti-
ca lipase (5.72 U/mg) were purchased from Fluka and lipase AY
‘Amano’30 was purchased from Amano Chem Ltd. They were used
without any further treatment. Although the definition of enzyme
activity depends on enzyme preparation, in most of the cases 1 unit
of enzyme corresponds to the amount of the enzyme that frees 1
μmol fatty acid per min at the specified pH and temperature with the
corresponding glyceryl esters as the substrates. The strains 5b1,
5b2, 5a1, 1b1 (N), 5d1, 1b1 used were previously isolated from soil
in our laboratory for the resolution of (RS)-3-[5-(4-fluorophenyl)-5-
hydroxypentanoyl]-4-phenyloxazolidin-2-one, an intermediate for
ezitimibe synthesis. These isolates were maintained on selective
media at 4 °C.
1H NMR (400 MHz, CDCl3): δ = 2.85 (s, 1 H, OH), 3.79–3.87 (m,
2 H), 4.18–4.25 (m, 2 H), 4.28–4.36 (m, 1 H), 7.0–7.08 (m, 2 H),
7.53–7.58 (m, 2 H).
13C NMR (100 MHz, CDCl3): δ = 42.0, 45.6, 69.4, 102.2, 112.60,
116.2, 121.6, 133.7, 134.5, 159.81.
MS (+ESI): m/z = 212.18.
Following a similar procedure (R)-9 and (S)-9 were prepared from
(S)-2 and (R)-2, respectively.
(R)-9
20
White solid; yield: 177 mg (84%); 82% ee; [α]D –3.0 (c 1.0,
CHCl3). Enantiomeric excess was determined by chiral HPLC anal-
ysis (chiral OD-H column, hexane–i-PrOH, 80:20): tR = 21.45 (S),
23.6 min (R), peak areas of 9% and 91%, respectively.
(S)-9
20
White solid; yield: 173 mg (82%); 81% ee; [α]D +2.9 (c 1.0,
CHCl3). Enantiomeric excess was determined by chiral HPLC anal-
ysis (chiral OD-H column, hexane–i-PrOH, 80:20): tR = 21.45 (S),
23.6 min (R), peak areas of 90.5% and 9.5% respectively.
(RS)-, (R)-, and (S)-1-Chloro-3-(2-cyanophenoxy)propan-2-yl
Acetate [(RS)-10, (R)-10, and (S)-10]
Conversions were calculated from the enantiomeric excess using
the formula: Conversion (C) = eeS/(eeS + eeP)20a–e [for Tables 3 and
4 (R)-9 (substrate S) and (S)-10 (product P)]; E values were
calculated using the formula: E = [ln (1 – C (1 + eeP)]/[ln (1 – C (1
– eeP)].20e,f
Compound (RS)-10 was synthesized chemically through the treat-
ment of (RS)-9 (106 mg, 0.5 mmol) with Ac2O (0.065 mL, 0.6
mmol) in the presence of ZrCl4 (5 mg, 2 mol%) in MeCN at r.t. un-
der magnetic stirring. After disappearance of (RS)-9 (TLC, 2 h),
H2O was added to the mixture and it was washed with NaHCO3.
The organic layer was then separated and concentrated under vacu-
um to afford (RS)-10 (115 mg, 91%) as a white solid. (RS)-10 was
subjected to chiral HPLC analysis (Chiralcel OD-H column, hex-
ane–i-PrOH, 80:20): tR = 26.2 (R), 28.0 min (S), ratio 49.9:50.1.
(RS)-, (R)-, and (S)-2-(Oxiranylmethoxy)benzonitrile [(RS)-2,
(R)-2, and (S)-2]
To a mixture of 3 (2.38 g, 20 mmol) and K2CO3 (11.04 g, 40 mmol)
in anhyd MeCN (100 mL) was added (RS)-4 (3.5 mL, 30 mmol),
and the mixture was heated under reflux for 16 h.16 The mixture was
cooled to r.t., filtered, and washed with MeCN, and the combined
organic layers were concentrated under vacuum. The residue was
purified by column chromatography (silica gel, 60–120 mesh,
EtOAc–hexane, 15:85) to afford (RS)-2 (2.8 g, 80%) as a white solid.
IR (KBr): 1745 cm–1 (ester).
1H NMR (400 MHz, CDCl3): δ = 2.12–2.16 (s, 3 H), 3.84–3.95 (m,
2 H), 4.32 (d, J = 4.9 Hz, 2 H), 5.34–5.4 (m, 1 H), 6.99–7.09 (m, 2
H), 7.53–7.58 (m, 2 H).
13C NMR (100 MHz, CDCl3): δ = 42.02, 66.6, 70.5, 77.0, 102.58,
112.49, 115.8, 121.69, 133.8, 134.3, 159.7.
(R)-2
20
White solid; yield: 680 mg (78% ); 81% ee; [α]D –14.8 (c 1.0,
EtOH) [Lit.13 [α]D20 –16.9 (c 1.0, EtOH), 94% ee].
(S)-2
MS (+ESI): m/z = 253.92.
Following a similar procedure (R)-10 and (S)-10 were prepared
from (R)-9 and (S)-9, respectively.
20
White solid; yield: 700 mg (80%); 82% ee; [α]D +15.0 (c 1.0,
EtOH) [Lit.13 [α]D20 +10.7 (c 1.0, EtOH), 59% ee].
(R)-10
1H NMR (400 MHz, CDCl3): δ = 2.85–2.87 (m, 1 H), 2.93–2.96 (m,
1 H), 3.39–3.43 (m, 1 H), 4.13 (dd, J = 5.3, 11.4 Hz, 1 H), 4.38 (dd,
J = 2.9, 11 Hz, 1 H), 7.01–7.06 (m, 2 H), 7.51–7.59 (m, 2 H).
20
White solid; yield: 113 mg (89%); 82% ee; [α]D –26.02 (c 1.0,
CHCl3). Enantiomeric excess was determined by chiral HPLC anal-
ysis (chiral OD-H column, hexane–i-PrOH, 80:20): tR = 26.2 (R), 28
min (S), peak areas of 91% and 9%, respectively.
13C NMR (100 MHz, CDCl3): δ = 44.5, 49.8, 69.3, 102.3, 112.68,
116.2, 121.3, 133.8, 134.4, 160.0.
MS (+ESI): m/z = 176.34 (identical with an authentic sample12).
(S)-10
20
White solid; yield: 110 mg (87%); 81% ee; [α]D +25.89 (c 1.0,
CHCl3). Enantiomeric excess was determined by chiral HPLC anal-
ysis (chiral OD-H column, hexane–i-PrOH, 80:20): tR = 26.2 (R), 28
min (S), peak areas of 9.5% and 90.5%, respectively.
(RS)-, (R)-, and (S)-1-Chloro-3-(2-cyanophenoxy)propan-2-ol
[(RS)-9, (R)-9, and (S)-9]
To a stirred solution of (RS)-2 (175 mg, 1 mmol) in THF (10 mL)
containing AcOH (0.2 mL) was added LiCl (85 mg, 2 mmol).17 The
resultant mixture was stirred at r.t. for 12 h and on completion of the
reaction (TLC), the mixture was diluted with EtOAc (15 mL) and
washed with aq NaHCO3 to remove excess AcOH. The organic lay-
er was separated, dried (Na2SO4), and concentrated under vacuum.
The residue was purified by column chromatography (silica gel,
60–12 mesh, EtOAc–hexane, 15:85) to obtain (RS)-9 which was
then subjected to chiral HPLC analysis (Chiralcel OD-H column,
hexane–i-PrOH, 80:20): tR = 21.45 (S), 23.6 min (R), ratio 49:51.
Enantioselective Transesterification of (RS)-9
To a round-bottomed flask (10 mL) containing a magnetic bead
were added a mixture of (RS)-9 (1 mmol) in t-BuOMe (3.5 mL) and
vinyl acetate (5.40 mmol). Lipases from different sources [commer-
cial lipase from lipase A, Candida Antarctica, Candida rugosa
L8525, Candida rugosa L-1754, Candida cylindracea, Aspergillus
niger, porcine pancreas, and AY ‘Amano’30] and crude lipase
[from strains 5b1, 5b2, 5a1, 1b1 (N), 5d1, 1b1 laboratory strains]
were used to carry out the reaction. The round-bottomed flask was
capped and placed on a magnetic stirrer, which was maintained at
Synthesis 2014, 46, 479–488
© Georg Thieme Verlag Stuttgart · New York

PAPER
Chemoenzymatic Synthesis of (RS)-, (R)-, and (S)-Bunitrolol
Deacylation of (RS)-, (R)-, and (S)-10
487
r.t. Immobilized lipase in sol-gel-Ak from Pseudomonas cepacia,
immobilized lipozyme from Mucor miehei, lipase acrylic resin from
Candida antarctica, were individually taken into a separate 10-mL
conical flask, the flasks were capped and placed in shaker which
was maintained at 25 °C (200 rpm). Samples (300 μL) were with-
drawn from mixture and conversion and the enantiomeric excess of
the reaction were monitored by HPLC.
A solution of K2CO3 (270 mg, 2 mmol) in distilled H2O (1 mL) was
added to a solution of 10 (250 mg, 1 mmol) in MeOH (5 mL) and
the resultant mixture was allowed to stir at r.t. for 2 h. After com-
pletion of the reaction, the mixture was extracted with EtOAc (3 ×
15 mL) and H2O (10 mL). The combined organic extracts were
dried (Na2SO4) and concentrated under vacuum to obtain the crude
product, which was purified by column chromatography (silica gel,
100–200 mesh) to obtain the corresponding alcohol.
Optimization of Transesterification Reaction
The effect of different organic solvents (MeCN, 1,4-dioxane,
t-BuOMe, i-Pr2O, Et2O, CH2Cl2, benzene, heptanes, isooctane, and
toluene) was determined on the transesterification of rac-CCPP.
The optimum temperature was determined by carrying out reaction
at different temperatures in the range 15–60 °C. To find out the ef-
fect of different acyl donors, different acyl donors such as EtOAc,
BnOAc, butanoic anhydride, and isobutanoic anhydride (5.40
mmol) were used. Finally in order to optimize the enzyme concen-
tration with respect to constant substrate concentration (1 mmol),
various enzyme concentrations (10, 15, 20, 23, 25, 30, 60, and 120
mg/mL) were used. The samples were taken at regular time inter-
vals and analyzed for enantioselectivity of transesterification reac-
tion.
(RS)-9
White solid; yield: 170 mg (82%).
(R)-9
20
White solid; yield: 170 mg (81%); 95% ee; [α]D –3.46 (c 1.0,
CHCl3).
(S)-9
20
White solid; yield: 173 mg (82%); 94% ee; [α]D +3.43 (c 1.0,
CHCl3).
Synthesis of (RS)-1
Compound (RS)-9 (100 mg, 1 mmol) was treated with t-BuNH2
(110 mg, 0.16 mL, 1 mmol) in EtOH (10 mL) in the presence of
Et3N (100 mg, 0.35 mL, 1 mmol) for 10 h. On completion of the re-
action (TLC), the mixture was diluted with EtOAc (15 mL) and
washed with H2O. The organic layer was separated and dried
(Na2SO4), and concentrated under vacuum. The residue was puri-
fied by column chromatography (silica gel, 60–12 mesh, EtOAc–
hexane, 15:85) to give (RS)-1 (114 mg, 92%) as a white solid.
Preparative-Scale Transesterification Reaction
The resolution of (RS)-9 was carried out on a preparative scale un-
der optimized conditions. The reaction was performed by subjecting
the substrate mixture (45 mL, 1 mmol) to resolution by PS-C and
CCL lipase at 25 °C and 30 C using vinyl acetate as the acyl donor
in isooctane and with toluene as the solvent. When the transforma-
tion was ca. 50% (48 h, 46.2% conversion and 36 h, 46.32% con-
version) the mixture was filtered off and enzyme preparation was
washed with solvent. The solvent was evaporated under reduced
pressure and the resulting dried residue was subjected to flash chro-
matography (EtOAc–hexane, 15:85). Using PS-C after 48 h were
isolated (R)-9; yield: 146 mg (46%); 81% ee (Chiralcel OD-H), and
(S)-10; yield: 139 mg (37%); 94% ee (Chiralcel OD-H). Using CCL
after 36 h were isolated (S)-9; yield: 148 mg (47%); 82% ee (Chi-
ralcel OD-H), and (R)-10; yield: 170 mg (45%); 95% ee (Chiralcel
OD-H).
1H NMR (400 MHz, CDCl3): δ = 1.14 (s, 9 H), 2.76–2.81 (m, 2 H),
3.64–3.68 (m, 3 H), 4.11 (d, J = 4.8 Hz, 1 H), 6.99–7.03 (m, 2 H),
7.50–7.56 (m, 2 H).
13C NMR (100 MHz, CDCl3): δ = 28.9, 44.3, 50.6, 67.8, 72.7, 102.1,
112.5, 121.0, 133.6, 134.3, 160.5. MS (+ESI): m/z = 248.15.
Synthesis of (S)- and (R)-1
Compound (R)- and (S)-9 (100 mg, 1 mmol), obtained either by
chemical synthesis from (R)- and (S)-4 or kinetic resolution of (RS)-
9 by the enzyme PS-C/CCL, was treated with t-BuNH2 (110 mg,
0.16 mL, 1 mmol) in EtOH (10 mL) in the presence of Et3N (100
mg, 0.35 mL, 1 mmol) for 10 h. On completion of the reaction
(TLC), the mixture was diluted with EtOAc and washed with H2O.
The EtOAc layer was separated and dried (Na2SO4), and it was con-
centrated under vacuum. The residue was purified by column chro-
matography (silica gel, 60–12 mesh, EtOAc–hexane, 15:85) to
obtain (S)- and (R)-1. In all cases optical rotation can be compared
to Lit.9a [α]D20 –10.0 (c 1.4, H2O), 60% ee.
PS-C-Catalyzed Reactions
(R)-9
White solid; yield: 146 mg (46%); [α]D20 –3.0 (c 1.0, CHCl3).
IR (neat): 3466 cm–1 (OH).
1H NMR (400 MHz, CDCl3): δ = 2.85 (s, 1 H, OH), 3.79–3.87 (m,
2 H), 4.18–4.25 (m, 2 H), 4.28–4.36 (m, 1 H), 7.0–7.08 (m, 2 H),
7.53–7.58 (m, 2 H).
13C NMR (100 MHz, CDCl3): δ = 42.0, 45.6, 69.4, 102.2, 112.60,
116.2, 121.6, 133.7, 134.5, 159.8.
For Chemoenzymatic Routes
Using Enzymatically Prepared (R)- and (S)-9
(S)-10
White solid; yield: 139 mg (37%); [α]D20 +30.36 (c 1.0, CHCl3).
(S)-1
Yield: 114 mg (92%); 80% ee; [α]D20 –13.4 (c 1.4, H2O).
IR (KBr): 1745 cm–1 (ester).
1H NMR (300 MHz, CDCl3): δ = 2.12–2.16 (s, 3 H), 3.84–3.95 (m,
2 H), 4.32 (d, J = 5 Hz, 2 H), 5.34–5.4 (m, 1 H), 6.99–7.09 (m, 2 H),
7.53–7.58 (m, 2 H).
(R)-1
Yield: 115 mg (93%); 81% ee; [α]D20 +13.5 (c 1.4, H2O).
By Deacylation of Enzymatically Prepared (R)- and (S)-10
13C NMR (100 MHz, CDCl3): δ = 42.02, 66.6, 70.5, 77.0, 102.58,
112.49, 115.8, 121.69, 133.8, 134.3, 159.7.
(S)-1
Yield: 114 mg (92%); 94% ee; [α]D20 –15.7 (c 1.4, H2O).
CCL-Catalyzed Reactions
(R)-1
Yield: 112 mg (91%); 94% ee; [α]D20 +15.58 (c 1.4, H2O).
(S)-9
White solid; yield: 148 mg (47%); [α]D20 +3.0 (c 1.0, CH2Cl2).
For All-Chemical Route
(R)-10
White solid; yield: 1.70 g (45%); [α]D20 –30.14 (c 1.0, CH2Cl2).
(S)-1
Yield: 112 mg (91%); 80% ee; [α]D20 –13.4 (c 1.4, H2O).
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 479–488

488
L. Banoth et al.
PAPER
(f) Chakraborti, A. K.; Kondaskar, A. Tetrahedron Lett.
2003, 44, 8315. (g) For a review: Schneider, C. Synthesis
2006, 3919.
(R)-1
Yield: 114 mg (92%); 80% ee; [α]D20 +13.3 (c 1.4, H2O).
(11) Narimatsu, S.; Kato, R.; Horie, T.; Ono, S.; Tsutsui, M.;
Yabusaki, Y.; Ohmori, S.; Kitada, M.; Ichioka, T.; Shimada,
N. Chirality 1999, 11, 1.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
n
nfomartit
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