Asymmetric synthesis of an enantiomerically pure rivastigmine intermediate using ketoreductase

Article abstract of DOI:10.1016/j.tetasy.2013.01.023

A novel chemo-enzymatic synthesis of (S)-rivastigmine using ketoreductases with NADH/NADPH as the proton donor has been demonstrated. An exclusive enzymatic process has been developed by exploring the possible ketoreductases by screening to identify enzymes useful for the preparation of the (S)-isomer intermediate, and scaling up of the enzymatic process for the production of an adequate, enantiomerically pure precursor of rivastigmine and for the total synthesis of (S)-rivastigmine.

Full text of DOI:10.1016/j.tetasy.2013.01.023

Tetrahedron: Asymmetry 24 (2013) 374–379
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Asymmetric synthesis of an enantiomerically pure rivastigmine
intermediate using ketoreductase
⇑
⇑
Madhuresh K. Sethi , Somashekar R. Bhandya , Nagaraj Maddur, Rohit Shukla, Anish Kumar,
V. S. N. Jayalakshmi Mittapalli
R&D, Mylan Laboratories Ltd, Plot No. 31, 32, 33 and 34 A ANRICH Industrial Estate, Bollaram (Village), Jinnaram (Mandal), Medak (Dt) 502325, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel chemo-enzymatic synthesis of (S)-rivastigmine using ketoreductases with NADH/NADPH as the
proton donor has been demonstrated. An exclusive enzymatic process has been developed by exploring
the possible ketoreductases by screening to identify enzymes useful for the preparation of the (S)-isomer
intermediate, and scaling up of the enzymatic process for the production of an adequate, enantiomeri-
cally pure precursor of rivastigmine and for the total synthesis of (S)-rivastigmine.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 28 December 2012
Accepted 29 January 2013
Available online 23 March 2013
1. Introduction
NADH/NADPH from D-glucose to gluconic acid, thus shifting the
equilibrium of the reaction to the formation of the alcohol. This
process breaks the limitation of obtaining 50% maximum yield of
(S)-rivastigmine.
Rivastigmine {(S)-3-[1-(dimethylamino)-ethyl]phenyl ethyl-
(methyl)carbamate}1 is an active pharmaceutical ingredient which
inhibits acetylcholinesterase enzymes present in the nervous sys-
tem. It also helps in improving cognition, coordination for routine
functions for patients with mild to moderate Alzheimer disease2
and treatment of dementia caused by Parkinson’s disease3 and
Lewy body.4
2. Result and discussion
2.1. Synthesis
Early researchers synthesized (S)-rivastigmine by the resolution
of racemic rivastigmine or an intermediate by a catalytic asymmet-
ric hydrogen transfer based reaction using transition metals and
chiral organic ligands.5–18 Recently, researchers have reported on
the synthesis of rivastigmine via a chemoenzymatic route using li-
pases19–21 as well as transaminases.22 However, there are various
drawbacks for these methods such as complex operations, loss of
yield, metal impurities in final API, multiple purification proce-
dures in the lipase catalyzed resolution, three enzyme systems in
the transaminase reactions, and the need for multiple crystalliza-
tion steps with chiral acids.
Herein we report a practical and industrially scalable chemoen-
zymatic approach using ketoreductases toward the synthesis of
(S)-rivastigmine.23 Various references are available for stereoselec-
tive reduction reactions catalyzed by ketoreductases or alcohol
dehydrogenases.24–39 This procedure includes an enzyme cata-
lyzed stereoselective reduction as the key step where ketoreducta-
ses undergo stereoselective reduction by utilizing the proton from
NADH/NADPH while glucose dehydrogenase regenerates the
Our synthetic route (Scheme 1) involved the condensation of 3-
hydroxy acetopheneone with ethyl methyl carbomyl chloride to
give 1 which in turn was reduced stereoselectively to 2 using KRED
which was then followed by bromination to give 3; finally conden-
sation with dimethylamine gave (S)-rivastigmine free base 4.
2.2. Screening for the keto reductase
For the initial screening of the reduction of N-ethyl methyl
carbomoyl acetophenone 1, an ES-KRED 8000 sceening kit con-
taining 80 enzymes (Table 1) was used. The screening medium
containing 5 mg of each Keto reductase enzyme in 2 mL of phos-
phate buffer pH 7.0 (containing 5 mg NADPH/NADH, 330 mM D-
glucose, 2 U/mL glucose dehydrogenase CDX901) in a 2 mL screw
capped vial. A solution of N-ethyl methyl carbomyl acetophenone
in dimethyl sulfoxide (10 mg in 0.1 mL) was then added. The mix-
ture was incubated over a eppendorf thermomixture comfort at
30 °C at 1000 rpm for 24 h. The progress of the reaction was mon-
itored by TLC. After completion of the reaction, the product was
subjected to chiral HPLC analysis of the alcohol enantiomer. All
ketoreductases showed 100% conversion as indicated by RP-HPLC
with the formation of the (S)-alcohol (99% ee) as indicated by chi-
ral HPLC.
⇑
Corresponding authors. Tel.: +91 4030492031; fax: +91 8458279305.
E-mail addresses: madhuresh.sethi@mylan.in (M.K. Sethi), somashekar.br@my-
lan.in (S.R. Bhandya).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.01.023

M. K. Sethi et al. / Tetrahedron: Asymmetry 24 (2013) 374–379
375
O
O
Et3N, CH2Cl2
+
N
O
HO
N
Cl
95 oC, 15h, yield = 99 %
O
1
O
ethyl methyl
3-hydroxy acetophenone
carbamoyl chloride
ketoreductase
30 oC, 24h
NAD(P)H
glucose dehydrogenase
glucose
yield = 95 %
phosphate buffer
PBr3, CH2Cl2
O
O
(R)
(S)
Na2CO3, 2h, rt
yield = 90 %
N
N
O
O
OH
Br
3
2
dimethyl amine
25 oC, 7h
hydrochloride
yield = 85 %
O
N
(S)
O
N
4
Scheme 1. Synthesis of (S)-rivastigmine from 3-hydroxy acetopheneone.
2.3. Reaction profile
dehydrogenase CDX901, 20% DMSO (Table 3). In all cases, the ee
remained constantly perfect (>99%).
In the presence of ES-KRED 119, the conversion showed an in-
crease in the formation of 2 from 1 over 0–24 h (Fig. 1). The rate
2.7. Effect of the co-solvents
of reduction was found to be 0.0021 mol hꢀ1 gꢀ1
.
A perfect stereoselectivity of more than >99% ee was observed
with or without DMSO as the co-solvent. DMF showed 97.97% ee
while methanol showed 84.58% conversion with 86.32% ee of the
(S)-isomer. The formation of more of the (R)-isomer (85.5% ee)
was observed when ethanol was used as the co-solvent (Table 5).
2.4. Effect of the enzyme concentration
A maximum conversion was achieved at 10% of ES-KRED-119 at
0.045 mol of N-ethyl methyl carbomyl acetophenone, 10% NADPH,
10% glucose dehydrogenase CDX901 and 119% D-glucose for incu-
bations up to 24 h (Table 2). A 1–5% of ES-KRED 119 took more
than 48 h to complete the reaction. In the case of glucose dehydro-
genase CDX 901, maximum conversion was achieved at 10% of en-
zyme concentration.
2.8. Effect of temperature
A decrease in stereoselectivity (96.3% ee) and % conversion
(70.0%) was observed when the temperature was increased from
30 to 40 °C (Table 6). This decrease in activity was due to the dena-
turation of the enzymes40 as well as the degradation of NADPH.41
The stereoselectivity can in theory, either increase or decrease
when the temperature is varied. The decrease in stereoselectivity
of the enzyme may be due to a change in the steric requirement
and Van der Waals interactions of the substrate binding at the ac-
tive site.42–44
2.5. Effect of the NADPH concentration
A maximum conversion was achieved at 10% of NADPH at
0.045 mol N-ethyl methyl carbomyl acetophenone, 10% ES-KRED-
119, 10% glucose dehydrogenase CDX901 and 119%
incubations up to 24 h (Table 2).
D-glucose for
2.6. Effect of the buffer
3. Conclusion
For the optimization of the reaction with respect to buffer pH
and molar concentration (Table 3 and 4), the complete reaction
was achieved at 20 mL of 0.25 M phosphate buffer pH 6.8–7.0 con-
taining N-ethyl methyl carbomyl acetophenone (0.0045 mol), 0.1 g
N-Ethyl methyl carbomyl acetophenone, synthesized by the
condensation of N-ethyl methyl carbomyl chloride with 3-hydroxy
acetophenone, has been screened for stereoselective reduction by
the enzymes ES-KRED, NAD/NADPH,
D-glucose and glucose dehy-
of ES-KRED 119, 0.1 g of NAD, 1.19 g of D-glucose, 0.1 g of glucose
drogenase. ES-KRED 119 taken for optimization used 10% of

376
M. K. Sethi et al. / Tetrahedron: Asymmetry 24 (2013) 374–379
Table 1 (continued)
Table 1
Screening for ketoreductases:a
S. No.
Enzyme
Conversion (%)
(S) (%)
(R) (%)
Cofactor
S. No.
Enzyme
Conversion (%)
(S) (%)
(R) (%)
Cofactor
76.
77.
78.
79.
80.
KRED-176
KRED-177
KRED-178
KRED-179
KRED-180
100
100
100
100
100
100
NADPH
NADPH
NADPH
NADH
1.
2.
3.
4.
5.
6.
7.
8.
KRED-101
KRED-102
KRED-103
KRED-104
KRED-105
KRED-106
KRED-107
KRED-108
KRED-109
KRED-110
KRED-111
KRED-112
KRED-113
KRED-114
KRED-115
KRED-116
KRED-117
KRED-118
KRED-119
KRED-120
KRED-121
KRED-122
KRED-123
KRED-124
KRED-125
KRED-126
KRED-127
KRED-128
KRED-129
KRED-130
KRED-131
KRED-132
KRED-133
KRED-134
KRED-135
KRED-136
KRED-137
KRED-138
KRED-139
KRED-140
KRED-141
KRED-142
KRED-143
KRED-144
KRED-145
KRED-146
KRED-147
KRED-148
KRED-149
KRED-150
KRED-151
KRED-152
KRED-153
KRED-154
KRED-155
KRED-156
KRED-157
KRED-158
KRED-159
KRED-160
KRED-161
KRED-162
KRED-163
KRED-164
KRED-165
KRED-166
KRED-167
KRED-168
KRED-169
KRED-170
KRED-171
KRED-172
KRED-173
KRED-174
KRED-175
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
>99.0
>99.0
100
100
100
100
100
100
100
100
100
100
100
>99.0
100
100
>99.0
100
100
100
100
100
100
100
100
97.5
100
100
96.1
95.5
99.4
100
100
100
100
100
100
100
100
100
100
71.0
100
100
100
97.50
>99.0
>99.0
100
100
100
100
100
>99.0
100
100
100
100
>99.0
>99.0
>99.0
>99.0
100
100
100
100
100
100
100
>99.0
>99.0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
>99.0
>99.0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
<1.0
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADH
61.4
>99.0
100
>99.0
NADPH
a
N-Ethyl methyl carbomyl acetophenone in dimethyl sulfoxide (10mg in 0.1ml),
5mg of each keto reductase enzyme in 2m1 of phosphate buffer pH 7.0 (containing
5 mg of NADP/NAD, 330 mM of -glucose, 5 mg of glucose dehydrogenase CDX901)
in a 2 mL screw capped vial. Temperature ꢀ30°C, Incubation period; 24hrs.
<1.0
<1.0
<1.0
<1.0
D
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
enzyme with 10% of NADPH, 0.55 M D-glucose, 10% glucose dehy-
drogenase CDX901 with 20% DMSO was able to complete the
reduction of N-ethyl methyl carbomyl acetophenone to give the
(S)-alcohol with >99% ee.
<1.0
<1.0
4. Experimental
4.1. General methods
NADPH
NADPH
NADPH
NADH
NADH
NADPH
NADH
Reverse phase-high performance liquid chromatography analy-
sis (RP-HPLC) of N-ethyl methyl carbomyl acetophenone 1 and N-
ethyl-methyl-carbamic acid-3-[(1S)-hydroxy-ethyl]-phenyl ester
2 was performed on Agilent Technologies high performance liquid
chromatography instrument connected with a UV detector at
NADH
NADH
NADPH
NADH
NADH
NADH
NADPH
NADPH
NADH
210 nm using Inertsil ODS 3V column (5 lm particle size,
150 ꢁ 4.6 mm length) eluted with 20 mM of phosphate buffer pH
3.0:acetonitrile (70:30 v/v) at a flow rate of 1 mL/min. The reten-
tion times were found to be 4.0, 14.0 and 9.0 min for 3-hydroxy
acetophenone, N-ethyl methyl carbomyl acetophenone 1 and N-
ethyl-methyl-carbamic acid-3-[(1S)-hydroxy-ethyl]-phenyl ester
2, respectively. Chiral HPLC of N-ethyl-methyl-carbamic acid-3-
[(1S)-hydroxy-ethyl]-phenyl ester 2 was performed on Agilent
Technologies High performance liquid chromatography instrument
connected with a UV detector at 215 nm using Chiralpak AD-
H(250 ꢁ 4.6 mm length) column eluted with n-hexane/ethanol/tri-
fluroacetic acid (95:0.5:0.1 v/v/v) at a flow rate of 1 mL/min. The
retention time were found to be 13.8 and 15.5 min for the (R)-iso-
mer and the (S)-isomer respectively of N-ethyl-methyl-carbamic
acid-3-[(1S)-hydroxy-ethyl]-phenyl ester 2.45
<1.0
<1.0
NADH
NADPH
NADPH
NADH
NADH
NADH
NADH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADH
NADPH
NADPH
NADPH
NADH
NADPH
NADPH
NADPH
NADPH
NADPH
NADH
RP-HPLC analysis of N-ethyl-methyl-carbamic acid-3-[1R-bro-
mo-ethyl]-phenyl ester 3 was performed on an Agilent Technolo-
gies high performance liquid chromatography instrument
connected with a UV detector at 210 nm using an Inertsil ODS 3V
column (5
l
m particle size, 150 ꢁ 4.6 mm length) eluted with
20 mM phosphate buffer pH 3.0:acetonitrile (60:40 v/v) at a flow
rate of 1 mL/min. The retention times were found to be 4.0 and
15.0 min for N-ethyl-methyl-carbamic acid-3-[(1S)-hydroxy-
ethyl]-phenyl ester 2 and N-ethyl-methyl-carbamic acid-3-[(1R)-
bromo-ethyl]-phenyl ester 3, respectively. Chiral HPLC of N-
ethyl-methyl-carbamic acid-3-[(1R)-bromo-ethyl]-phenyl ester 3
was performed on an Agilent Technologies high performance liquid
chromatography instrument connected with a UV detector at
260 nm using Chiralpak AD-3 (3
lm particle size, 250 ꢁ 4.6 mm
length) column eluted with n-hexane/ethanol/methanol/trifluro
acetic acid (85:10:5:0.1 v/v/v/v) at a flow rate of 1 mL/min. The
retention times were found to be 5.7 and 6.7 min for the (R)-isomer
and the (S)-isomer, respectively of N-ethyl-methyl-carbamic acid-
3-[(1R)-bromo-ethyl]-phenyl ester 3.
RP-HPLC analysis of rivastigmine base 4 was performed on an
Agilent Technologies high performance liquid chromatography
instrument connected with
NADH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
a UV detector at 210 nm using
Lichro CART RP-18 eluted with 20 mM of phosphate buffer pH

M. K. Sethi et al. / Tetrahedron: Asymmetry 24 (2013) 374–379
377
0.05
0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
0
5
10
15
20
25
30
Reaction time in hours
Figure 1. Reaction profile: Reagents and conditions: N-ethyl methyl carbomyl acetophenone (0.045 mol), 1 g of ES-KRED 119, 0.25 M phosphate buffer pH 6.8, 1 g of NAD,
10 g of glucose, 1 g of glucose dehydrogenase CDX901, 20% DMSO, 200 mL of 0.25 M phosphate buffer, with shaking at 30 °C for 24 h.
Table 2
Effect of enzyme and cofactor concentration.a
% conversion
% concentration (with respect to N-ethyl methyl carbomyl acetophenone)
ES-KRED-119b
Glucose dehydrogenase CDX901c
NADPd
1
5
10
20
45.8
69.4
99.1
99.3
30.2
45.8
99.1
99.21
54.1
74.2
99.1
99.5
a
b
% conversion from HPLC determined with respect to N-ethyl methyl carbomyl acetophenone with shaking at 30 oC for 24 h.
N-Ethyl methyl carbomyl acetophenone (0.045 mol), 0.25 M phosphate buffer pH 6.8, 1 g of NADPH, 11.9 g of glucose, 1 g of glucose dehydrogenase CDX901, 20% DMSO,
200 mL of 0.25 M phosphate buffer.
c
N-Ethyl methyl carbomyl acetophenone (0.045 mol), 1 g of ES-KRED 119, 0.25M phosphate buffer pH 6.8, 1 g of NADPH, 11.9g of glucose, 20% DMSO, 200mL of 0.25M
phosphate buffer.
d
N-Ethyl methyl carbomyl acetophenone (0.045 mol), 1 g of ES-KRED 119, 0.25 M phosphate buffer pH 6.8, 11.9 g of glucose, 20% DMSO, 200mL of 0.25M phosphate buffer.
Table 3
Table 5
Effect of buffer.a
Effect of co-solvent.a
pH
% conversion
Solvent
% conversion
(S)-isomer (%ee)
6.0
6.8
7.0
7.5
8.0
51.6
>99.0
>99.0
61.6
MeOH
DMF
EtOH
DMSO
No solvent
84.6
96.5
87.4
>99.0
90.6
86.3
98.0
14.5
>99.0
>99.0
63.2
a
a
N-Ethyl methyl carbomyl acetophenone (0.0045mol), 0.1 g of ES-KRED 119, 0.1 g
of NADPH, 1.19 g of glucose, 0.1 g of glucose dehydrogenase CDX901, 20% DMSO,
200 mL of 0.25M phosphate buffer with shaking at 30 oC for 24 h.
N-Ethyl methyl carbomyl acetophenone (0.0045 mol), 0.1 g of ES-KRED 119, 0.1 g
of NADP, 1.19g of glucose, 0.1 g of glucose dehydrogenase CDX901, 20% solvent,
200 mL of 0.25M phosphate buffer with shaking at 30oC for 24h.
Table 4
Table 6
Effect of Molar concentration.a
Effect of Temperature.a
Molar concentration pH 7.0
% conversion
Temperature (oC)
% conversion
(S)-isomer (%ee)
0.10
0.20
0.25
0.30
0.40
88.0
89.4
99.8
65.6
82.2
30
40
70.0%
99.1
96.3
99.4
a
N-Ethyl methyl carbomyl acetophenone (0.0045mol), 0.1 g of ES-KRED 119, 0.1 g
of NADP, 1 g of glucose, 0.1 g of glucose dehydrogenase CDX901, 20% DMSO, 200mL
of 0.25 M phosphate buffer with shaking 24 h.
a
N-Ethyl methyl carbomyl acetophenone (0.0045mol), 0.1 g of ES-KRED 119, 0.1 g
of NADP, 1.19 g of glucose, 0.1 g of glucose dehydrogenase CDX901, 20% DMSO,
200mL of phosphate buffer pH 7.0 with shaking at 30 oC for 24 h.
4 and N-ethyl-methyl-carbamic acid-3-[(1R)-bromo-ethyl]-phenyl
ester 3, respectively. Chiral HPLC of rivastigmine base 4 was per-
formed on an Agilent Technologies high performance liquid chro-
2.5:acetonitrile (60:40 v/v) at a flow rate of 1 mL/min. The reten-
tion times were found to be 2.5 and 36.5 min for rivastigmine base

378
M. K. Sethi et al. / Tetrahedron: Asymmetry 24 (2013) 374–379
matography instrument connected with a UV detector at 260 nm
using a Chiralpak OD-H (5
m particle size, 250 ꢁ 4.6 mm length)
(m, 1H), 3.07–2.89 (m, 3H), 1.32–1.30 (d, J = 6.3 Hz, 3H), 1.20–
l
1.06 (m, 3H). 1H NMR data are in good agreement with those re-
column eluted with n-hexane/ethanol/trifluroacetic acid/diethyl-
amine (90:10:5:0.1:0.1 v/v/v/v) at a flow rate of 1 mL/min. The
retention times were found to be 10.1 and 13.4 min for the (R)-iso-
mer and the (S)-isomer, respectively, of rivastigmine base 4.
1H NMR spectra were recorded on a Bruker Avance 300 spec-
trometer. The spectra were recorded with DMSO-d6/CDCl3 as the
internal standard for measuring chemical shifts with 0.01 ppm.
The region of 0–10 ppm was scanned for all the samples. The spe-
cific rotations of the isolated compounds were measured using Per-
kin–Elmer 243 polarimeter (Uberlingen, Germany).
ported in the literature.20
½
a 2D5
ꢂ
¼ ꢀ25:8 (c 1.1, CHCl3) 99.5% ee.
4.6. Preparation of 3-[(1R)-bromoethyl)phenyl ethyl (methyl)
carbamate 3
Phosphorus tribromide was added dropwise to a solution of 2
(10 g) in 50 mL of CH2Cl2 at 0–5 °C and stirred for 30 min. The tem-
perature was then gradually increased to room temperature and
stirred for 2 h. The progress of the reaction was monitored by
TLC. After completion of the reaction, the reaction mass was
poured into 50 mL of ice water and extracted with 100 mL of meth-
ylene dichloride. The organic layer was separated, dried over so-
dium sulfate, and distilled off to obtain a brownish oily product.
The product obtained was monitored by Chiral HPLC, ESI-MS and
1H NMR (11.54 g, 90% isolated yield, ee: >90%): ESI-MS 287.1
(M+); 1H NMR (DMSO, 300 MHz, ppm) d 7.40–7.34 (m, 2H), 7.30–
7.25 (m, 1H), 7.08–7.04 (m, 1H), 5.70–5.46 (m, 1H), 3.46–3.28
(m, 2H), 3.07–2.90 (d, J = 37.2 Hz, 3H), 1.98–1.91 (s, 3H), 1.31–
1.01 (m, 3H).
4.2. Enzymes
ES-KRED-8000, a 80 ketoreductase containing kit was purchased
from Syncore Laboratories (Shanghai) Co Ltd, Shanghai, China and
Glucose dehydrogenase CDX901 from Codexis Inc. California, USA.
4.3. Chemicals and reagents
Nicotinamide adenine dinucleotide and nicotinamide adenine
dinucleotide phosphate were from SISCO Research Laboratories
Pvt. Ltd, Mumbai, India, potassium dihydrogen phosphate, dipotas-
sium hydrogen phosphate, acetonitrile, ethanol, methanol and di-
methyl sulfoxide were from Spectrochem Pvt. Ltd, India.
Triethylamine, 3-hydroxy acetophenone, methylene dichloride, so-
dium sulfate anhydrous, phosphorus tribromide, trifluroacetic acid,
diethylamine, dimethyl amine hydrochloride and sodium hydrox-
ide were from Avra Laboratories Pvt. Ltd, Hyderabad, India. N-Ethyl
methyl carbomyl chloride was from Chemrich Fine Chemicals Pvt.
Ltd, Hyderabad India.
4.7. Preparation of rivastigmine base 4
At first, 41.5 g of dimethyl amine hydrochloride in 50 mL of
methanol was taken and stirred for 60 min at 0–5 °C. The reaction
mass was neutralized using methanol/sodium hydroxide (20 g in
85 mL methanol) and stirred for 60 min at 0–5 °C. The reaction
mixture was filtered under nitrogen. To the filtrate were added
10 g of ethyl-methyl-carbamic acid-3-[(1R)-bromo-ethyl]-phenyl
ester 3 in 10 mL of methanol. The reaction mixture was then stir-
red at 25 °C for 5–7 h. The progress of the reaction was monitored
by TLC. After completion, the reaction mass was distilled off to re-
move methanol under reduced vacuum. The crude oil was acidified
with 100 mL of 10% hydrochloric acid solution. The product was
then extracted into methylene dichloride. The organic layer was
separated, basified with a saturated sodium carbonate solution,
dried over sodium sulfate, and distilled off to obtain a colorless oily
product. The product was analyzed by chiral HPLC, ESI-MS and 1H
NMR. (7.43 g, 85% isolated yield, ee: >99%) ESI-MS was found to be
250.1 (M+). 1H NMR (CDCl3, 300 MHz, ppm) d 7.33–7.26 (m, 1H),
7.14–7.11 (d, J = 7.2 Hz, 1H), 7.07–7.02 (m, 2H), 3.48–3.34 (m,
3H), 3.06–2.99 (d, J = 22.5 Hz, 3H), 2.26 (s, 6H), 1.41–1.39 (d,
J = 6.9 Hz, 3H), 1.27–1.17 (m, 3H).
4.4. Synthesis of N-ethyl methyl carbomyl acetophenone 1
To a solution of triethyl amine (910 mL, 4.55 vol) were added N-
ethyl methyl carbomyl chloride (182 g, 1.5 mol) and 3-hydroxy
acetophenone (200 g, 1.47 mol). The mixture was then stirred at
95 °C for 10–15 h. The reaction mixture was extracted with CH2Cl2
(1000 mL), washed with 0.5 M NaOH solution (2 ꢁ 2000 mL), dried
over Na2SO4, and distilled under vacuum to obtain a brown oil
(321.74 g 99% isolated yield) ESI-MS found 221.1 (M+). 1H NMR
(CDCl3, 300 MHz, ppm) d 7.80–7.67 (m, 1H), 7.69 (s, 1H), 7.48–
7.42 (m, 1H), 7.35–7.27 (m, 1H), 3.52–3.38 (m, 2H), 3.08–3.00 (d,
J = 25.5 Hz, 3H), 2.60 (s, 3H), 1.28–1.13 (m, 3H). 1H NMR data are
in good agreement with those reported in literature.20
1H NMR data are in good agreement with those reported in lit-
erature.20,46
½
a 2D5
ꢂ
¼ ꢀ32:3 (c 1.3, EtOH) 99% ee.11
4.5. Preparation of ethyl-methyl-carbamic acid-3-[(1S)-
hydroxy-ethyl]-phenyl ester 2
Acknowledgments
Our group thanks the Department of Scientific and Industrial
Research, India, Dr. Hari Babu (CEO Mylan Laboratories Ltd, India);
Mr. Sanjeev Sethi (Head Mylan Global R&D); Dr. Ramesh Dandala
(Head MLL R&D); Dr. Abhay Gaitonde (Head Manufacturing
MLL); Dr. Suryanarayana Mulukutla (Head Analytical Dept MLL
R&D) as well as the analytical development team of Mylan Labora-
tories Ltd for their encouragement and support. We would also like
to thank Dr. Narahari Ambati (Head IPR MLL R&D) and his Intellec-
tual property team for their support.
At first, 1 g of ES-KRED 119 was mixed with 200 mL of buffer pH
6.8 containing 0.25 mM potassium dihydrogen phosphate,
0.25 mM dipotassium hydrogen phosphate, 1 g of NAD, 10 g of D-
glucose, 1 g glucose dehydrogenase CDX901. Next, a solution of 1
in dimethyl sulfoxide (10 g in 40 mL) was added. A value of pH
6.8 was maintained by adding 1 M NaOH throughout the reaction.
The mixture was stirred at 30 °C for 16–24 h and monitored by
HPLC. After completion of the reaction, the reaction mass was fil-
tered through Celite and the filtrate was extracted with 200 mL
of CH2Cl2 (200 mL). The organic layer was separated, dried over so-
dium sulfate, and distilled off to obtain a colorless oil. The product
was analyzed by chiral HPLC, ESI-MS and 1H NMR (9.58 g, 95% iso-
lated yield, ee: >99%). ESI-MS was found to be 223.1 (M+). 1H NMR
(DMSO, 300 MHz, ppm) d 7.33–7.27 (t, J = 7.8 Hz, 1H), 7.17–7.15 (d,
J = 7.5 Hz, 1H), 7.06 (s, 1H), 6.96–6.93 (m, 1H), 5.23–5.21 (d,
J = 4.2 Hz, 1H), 4.75–4.67 (m, 1H), 3.49–3.38 (m, 1H), 3.34–3.27
References
1. Polinsky, R. J. Clin. Ther. 1998, 20, 634–647.
2. Rosler, M.; Anand, R.; Cicin-Sain, A.; Gauthier, S.; Agid, Y.; Dal-Bianco, P.;
Stahelin, H. B.; Hartman, R.; Gharabawi, M. Br. Med. J. 1999, 318, 633–638.
3. Reading, P. J.; Luce, A. K.; McKeith, I. G. Mov. Disord. 2001, 16, 1171–1174.
4. McKeith, I.; Del Ser, T.; Spano, P.; Emre, M.; Wesnes, K.; Anand, R.; Cicin-Sain,
A.; Ferrara, R.; Spiegel, R. Lancet 2000, 356, 2031–2036.

M. K. Sethi et al. / Tetrahedron: Asymmetry 24 (2013) 374–379
379
5. Enz, A. United Kingdom Patent GB 2,203,040A, CAN 111:187620, 1988.
6. Chen, W.; Wen, F. China Patent 200,710,030,658, CAN 148:379318, 2007.
7. Boezio, A. A.; Pytkowicz, J.; Cote, A.; Charette, A. B. J. Am. Chem. Soc. 2003, 125,
14260–14261.
8. Hu, M.; Zhang, F. L.; Xie, M. H. Synth. Commun. 2009, 39, 1527–1533.
9. Stepankova, N.; Hajicek, J. European Patent 1,556,338, CAN 142:6315, 2008.
10. Dubey, S. K.; Bansal, V.; Pannu, K.; Dubey, S. K. WO Patent 2,010,023,535, CAN
152:311264, 2010.
26. Davis, S. C.; Grate, J. H.; David, R. G.; Gruber, J. M.; Huisman, G. W.; Ma, S. K.;
Newman, L. M.; Sheldon, R.; Wang, L. A. U.S. Patent 7,125,693, CAN
141:378916, 2006.
27. Werner, H.; Bettina, R. U.S. Patent 6,413,750, CAN 131:239737, 2002.
28. Wong, C.-H.; Bradshaw, C. W. U.S. Patent 5,225,339, CAN 119:115519,
1993.
29. Patel, R. N.; Banerjee, A.; McNamee, C. G.; Szarka, L. J. U.S. Patent 5,391,495,
CAN 121:132396, 1995.
11. Stepankova, C. B. H.; Hajicek, P. J.; Simek, P. U.S. Patent 20,060,122,417, CAN
142:6315, 2006.
30. Moore, J. C.; Sturr, M. G.; McLaughlin, K.; Kim. J. U.S. Patent 7,109,004, CAN
141:5870, 2006.
12. Tian, G.; Zhu, Y.; Shen, J. WO Patent 2,008,124,969, CAN 149:448060, 2008.
13. Mandava, V. N. B. R.; Vajrala, V. R., Varanasi, G.; Adla, V. K.; Jambula, M. R.;
Reddy, V. R. K. U.S. Patent 20,080,255,383, CAN 149:471196, 2008.
14. Patel, H. V. U.S. Patent 20,060,293,518, CAN 140: 27668, 2006.
15. Jaweed, M. S. M.; Upadhye, B. K.; Rai, V. K.; Zia, H. WO Patent 2,007,026,373,
CAN 146:295628, 2007.
16. Deshpande, P. B.; Boda, B. B.; Acharya, H. H.; Luthra, P. K. U.S. Patent
20,080,045,743, CAN 148:284915, 2008.
17. Deshpande, P. B.; Khemani, K.; Boda, B. B.; Shah, T. R.; Acharya, H. H.; Luthra, P.
K. U.S. Patent 7,683,205, CAN 148:284915, 2010.
18. Deshpande, P. B.; Khemani, K.; Boda, B. B.; Shah, T. R.; Acharya. H. H.; Luthra, P.
K. WO Patent 2,008,020,452, CAN 148:284915, 2008.
19. Mangas-Sanchez, J.; Rodrıguez-Mata; Busto, E.; Gotor-Fernandez, V.; Gotor, V.
J. Org. Chem. 2009, 74, 5304–5310.
20. Han, K.; Kim, C.; Park, J.; Kim, M. J. J. Org. Chem. 2010, 75, 3105–3108.
21. Arunkumar, K.; AppiReddy, M.; SravanKumar, T.; VijayaKumar, B.;
Chandrasekhar, K. B.; RajenderKumar, P.; Pal, M. Beilstein J. Org. Chem. 2010,
6, 1174–1179.
31. Valerie, N. D. WO Patent 2,009,045,507, CAN 150:396693, 2009.
32. Keinan, E.; Sinha, S. C.; Sinha-Bagchi, A. J. Org. Chem. 1992, 57, 3631–3636.
33. Patel, R. N.; Chu, L.; Mueller, R. Tetrahedron: Asymmetry 2003, 14, 3105–3109.
34. Zhou, B. N.; Gopalan, A. S.; Van-Middlesworth, F.; Shieh, W. R.; Sih, C. J. J. Am.
Chem. Soc. 1983, 105, 5925–5926.
35. Gröger, H.; Hummel, W.; Buchholz, S.; Drauz, K.; Nguyen, T. V.; Rollmann, C.;
Hüsken, H.; Abokitse, K. Org. Lett. 2003, 5, 173–176.
36. Truppo, M. D.; Pollard, D.; Devine, P. Org. Lett. 2007, 9, 335–338.
37. Kim, M. J.; Ahn, Y.; Park, J. Curr. Opin. Biotechnol. 2002, 13, 578–587.
38. Pamies, O.; Beackvall, J.-E. Chem. Rev. 2003, 103, 3247–3262.
39. Kim, M. J.; Park, J.; Ahn, Y. In Biocatalysis in the Pharmaceutical and
Biotechnology Industries; Patel, R. N., Ed.; CRC Press: Boca Raton, 2007; pp
249–272.
40. Calvin, S. J.; Mangan, D.; Miskelly, I.; Moody, T. S.; Stevenson, P. J. Org. Process
Res. Dev. 2012, 16, 82–86.
41. Wu, J. T.; Wu, L. H.; Knight, J. A. Clin. Chem. 1986, 32, 314–319.
42. Phillips, R. S.; Zheng, C.; Pham, V. T.; Andrade, F. A. C.; Andrade, M. A. C. Biocatal.
Biotrans. 1994, 10, 77–86.
22. Fuchs, M.; Koszelewski, D.; Tauber, K.; Kroutil, W.; Faber, K. Chem. Commun.
2010, 46, 5500–5502.
23. Sethi, M. K.; Bhandya, S. R.; Maddur, N.; Potluri, S.; Dutta, D.; Dandala, R. India
Patent Office 2681/CHE/2010, 2012.
43. Pham, V. T.; Phillips, R. S. J. Am. Chem. Soc. 1990, 112, 3629–3632.
44. Tripp, A. E.; Burdette, D. S.; Zeikus, J. G.; Phillips, R. S. J. Am. Chem. Soc. 1998,
120, 5137–5141.
45. Sushmitha, P.; Narasu, M. L.; Suryanarayana, M. V. Rasayan J. Chem. 2012, 5, 5–
9.
24. Prelog, V. Pure Appl. Chem. 1964, 9, 119–123.
25. Davis, S. C.; Grate, J. H.; Gray, D. R.; Gruber, J. H.; Huisman, G. W.; Ma, S. K.;
Newman, L. M.; Sheldon, R.; Wang, L. A. U.S. Patent 7,132,267, CAN
141:378916, 2006.
46. Srinivasu, M. K.; Rao, B. M.; Reddy, B. S.; Kumar, P. R.; Chandrasekhar, K. B.;
Mohakhud, P. K. J. Pharm. Biomed. Anal. 2005, 38, 320–325.