Enantioselective synthesis of (2S)-5-{4-[2-(4-fluorophenoxy)ethyl]piperazin-1-yl}-2-isopropyl-2-phenyl- pentanenitrile dihydrochloride (E2050) using enzyme-catalyzed kinetic resolution

Article abstract of DOI:10.1016/S0957-4166(02)00364-6

Immobilized lipase (Pseudomonas sp.)-catalyzed transesterification of (RS)-4-cyano-4-isopropyl-4-phenyl-1-butanol 4 in vinyl acetate gave (S)-4-cyano-4-isopropyl-4-phenyl-1-butyl acetate 3a with high enantiomeric excess values and conversions (95-97% ee,

Full text of DOI:10.1016/S0957-4166(02)00364-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 1493–1502
Enantioselective synthesis of
(2S)-5-{4-[2-(4-fluorophenoxy)ethyl]piperazin-1-yl}-2-isopropyl-2-
phenyl-pentanenitrile dihydrochloride (E2050) using
enzyme-catalyzed kinetic resolution
Yoshihiko Norimine,* Noboru Yamamoto, Yuichi Suzuki, Teiji Kimura, Koki Kawano, Koichi Ito,
Satoshi Nagato, Yoichi Iimura and Masahiro Yonaga
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
Received 10 May 2002; accepted 27 June 2002
Abstract—Immobilized lipase (Pseudomonas sp.)-catalyzed transesterification of (RS)-4-cyano-4-isopropyl-4-phenyl-1-butanol 4 in
vinyl acetate gave (S)-4-cyano-4-isopropyl-4-phenyl-1-butyl acetate 3a with high enantiomeric excess values and conversions
(95–97% ee, 30–47%, E=90–93), leaving the enantiomerically pure (R)-alcohol 4 (>99% ee) unreacted. As 3a can be efficiently
converted to E2050, use of the immobilized lipase should provide an economical route for large-scale synthesis of E2050. © 2002
Elsevier Science Ltd. All rights reserved.
1. Introduction
in their report, a second enzymatic resolution is neces-
sary to obtain the enantiomerically pure (S)-enantiomer
(E value4=49). Our method affords pure (S)- and
(R)-enantiomers by single-step enzymatic kinetic reso-
lution without using additives (E value=93).
The neuron-selective Ca2+ channel blocker E2050 ((2S)-
5-{4-[2-(4-fluorophenoxy)ethyl]piperazin-1-yl}-2-isopro-
pyl-2-phenylpentanenitrile
dihydrochloride)
was
discovered in our research program directed towards
novel neuroprotective agents.1 It possesses the same
moiety (with a quaternary stereogenic carbon) as the
well-known L-type calcium antagonist, emopamil. Sev-
eral reports2 have described the synthesis of the chiral
moiety 4 and its congeners, but these approaches are
limited to small-scale preparation because of the need
for many reaction steps or time-consuming chromatog-
raphy. Although the kinetic resolution of racemic 2-
phenyl-1-propanol using enzymatic transesterification
was previously reported by Kawasaki et al.,3a the
enzyme-catalyzed kinetic resolution of racemic 4-cyano-
4-isopropyl-4-phenyl-1-butanol, which has a longer
alkyl chain, has not been reported. Herein, we report a
practical enantioselective synthesis of chiral 4-cyano-4-
isopropyl-4-phenyl-1-butanol as a basis for practical
asymmetric synthesis of E2050. Recently, Im et al.
reported an improved enzyme-catalyzed resolution of
(RS)-4-cyano-4-(3,4-dimethoxyphenyl)-4-isopropyl-1-
butanol using enzymatic transesterification.3b However,
2. Results and discussion
Our strategy to prepare enantiomerically pure E2050 is
outlined in Fig. 1. Substrate types I and II were chosen
as key intermediates and subjected to enzymatic
resolution.
(RS)-4-Cyano-4-isopropyl-4-phenyl-1-butanoates (sub-
strate type I) and (RS)-4-cyano-4-isopropyl-4-phenyl-1-
butanol derivatives (substrate type II) were prepared as
shown in Scheme 1.
First, we examined the kinetic resolution of the car-
boxylates 1a,b,c (substrate type I) using enzymatic
hydrolysis. Since the resultant carboxylic acid was
obtained as a crystalline solid, its enantiomeric purity
might be easily improved by recrystallization. However,
preliminary studies gave disappointing results with
many kinds of lipase and esterase, such as Lipase AK,
Lipase PSA-30, porcine pancreatic lipase (PPL), Rhizo-
pus delemar lipase (RDL), immobilized lipase (Pseu-
* Corresponding author. Tel.: +81-298-47-5840; fax: +81-298-47-2037;
e-mail: y-norimine@hhc.eisai.co.jp
0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00364-6

1494
Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
O
N
CN
CN
CN
CN
OR
N
OR
OR
2HCl
F
S
S
S
O
E2050
CN
OR
Kinetic Resolution
Using Enzymatic
Hydrolysis and Transesterification
O
O
Type II
Type I
R
R
R
F
OR
N
OR
CN
CN
N
CN
O
Figure 1. Enantioselective synthetic route to E2050.
R = Me 1a or Et 1b
b)
CN
ref 2
CN
a)
R = H
2
OR
c)
CN
R = Pr 1c
O
d)
CN
O
CN
e)
R
R = Me 3a
OH
R = Et 3b
O
4
3
Scheme 1. Synthesis of substrates. Reagents and conditions: (a) methyl acrylate (for 1a) or ethyl acrylate (for 1b), KOBut, THF;
(b) 5N NaOH, aq. THF; (c) 1-propanol, 2-chloro-3,5-dinitropyridine, pyridine; (d) LiAlH4; (e) Ac2O, pyridine (for acetate (3a))
or propionyl chloride, Et3N, THF (for propionate (3b)).
domonas sp.), and pig liver esterase (PLE).† Therefore,
enzyme-catalyzed kinetic resolution of (RS)-4-cyano-4-
isopropyl-4-phenyl-1-butanol derivatives (substrate type
II) was investigated.
Next, we tried the enzymatic hydrolysis of the acylated
alcohols 3a,b in tert-butyl methyl ether (tBuOMe) or
diisopropyl ether saturated with water in the presence
of immobilized lipase (Table 2).
In these cases, the enzymatic hydrolyses of 3a,b
afforded (S)-4 with improved enantiomeric purity com-
pared with the reaction in phosphate buffer. The abso-
lute configurations of the hydrolyzed products were
determined by comparison with authentic samples
derived from the reported2a chiral aldehyde 6 by HPLC
using a chiral column (Scheme 2).
2.1. Kinetic resolution of the acylates 3a,b using enzy-
matic hydrolysis in phosphate buffer or organic solvent
Enzymatic hydrolysis of 3a,b was performed in phos-
phate buffer (pH 6.89) at room temperature using
several lipases (Lipase AK, immobilized lipase, PPL,
RDL, Lipase PSA-30) and PLE. The results are sum-
marized in Table 1. PLE did not significantly hydrolyze
the acetate 3a and propionate 3b. Lipase PSA-30 pre-
dominantly hydrolyzed (R)-3a,b, while other lipases
favored (S)-3a,b. Immobilized lipase gave the best
result ((S)-4, 85% ee, 21% yield; (R)-3a, 30% ee, 78%).
2.2. Kinetic resolution of the alcohol 4 using enzymatic
transesterification
The substrate 4 was subjected to enzymatic transester-
ification in vinyl acetate or vinyl propionate at room
temperature using several lipases (lipase AK, immobi-
lized lipase, PPL, RDL, lipase PSA-30) and PLE. The
ratio of substrate (alcohol 4), enzyme, and vinyl acetate
was ca. 100 mg:10 mg:10 ml. The results are summa-
rized in Table 3. Immobilized lipase gave superior
enantiomeric excesses and yields. The transesterification
of 4 in vinyl acetate using immobilized lipase gave the
corresponding (S)-3a (43% ee, 70%) and (R)-4 (99.3%
ee, 30%), while the same process in vinyl propionate
gave (S)-3b (99% ee, 3%) and (R)-4 (16% ee, 83%).
† When lipases other than RDL were used, the reaction did not
proceed within 180 h. With PLE, the reaction proceeded within 24
h
in all cases. The (S)-carboxylates 1 were predominantly
hydrolyzed to the (S)-carboxylic acid 2, but these enzymatic
hydrolyses showed low enantioselectivity. Although PLE-catalysed
hydrolysis of the methyl ester 1a gave the (S)-carboxylic acid 2 in
high conversion yield (51%), the enantiomeric excess of the obtained
(S)-2 was only 24% ee.

Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
Table 1. Enzymatic hydrolysis in phosphate buffer
1495
Type II
CN
Enzyme
CN
CN
O
R
O
R
OH
+
R
S
phosphate buffer
r,t,
O
O
R=Me 3a
Et 3b
R=Me 3a
Et 3b
4
Yield of (S)-4
Yield of (R)-3
Reaction
Time (h)
Enzymea
Entry
E
R
% eeb
% eeb
c.y.%
c.y.%
Lipase AK
1
2
3
4
18
48
78
70
30
52
21
72
Me
Me
Me
13
17
1
Immobilized lipase 168
85
13
39
PPL
RDL
24
9
67
70
8.1
11
23
21
Me
3
Lipase AK
5
6
8
24
1
Et
Et
26
48
70
57
40
23
64
90
Immobilized lipase 143
24.4
Et
Et
7
8
PPL
RDL
8
9
65
64
4.7
12
11
20
0.2
31
2
Type II
CN
Enzyme
CN
CN
O
R
O
R
OH
+
S
R
phosphate buffer
r,t,
O
O
R=Me 3a
Et 3b
R=Me 3a
Et 3b
4
Yield of (R)-4
Yield of (S)-3
c.y.%
50
54
Reaction
Time (h)
Enzymea
Entry
R
E
% eeb
23
13
% eeb
21
15
c.y.%
Lipase PSA-30
Lipase PSA-30
9
18
49
46
Me
Et
2
2
10
119
a) Lipase AK (Pseudomonas fluorescens), Immobilized lipase (Pseudomonas sp.), PPL; porcine pancreatic
lipase, RDL; Rhizopus delemar lipase, Lipase PSA-30 (Burkholderia cepacia)
b) Enantiomeric excess (% ee) was determined by HPLC (CHIRALCEL OJ, hexane:IPA:EtOH= 85:10:5)
Table 2. Enzymatic hydrolysis in organic solvent
Type II
CN
Immobilized lipase
R
CN
CN
O
OH
O
R
S
+
R
Solvent
(H2O saturated)
r.t.
O
O
R=Me 3a
Et 3b
R=Me 3a
Et 3b
4
Entry
R
Solvent
Reaction time (h)
Yield of (S)-4
% eea
Yield of (R)-3
E
c.y. (%)
c.y. (%)
% eea
1
2
3
4
Me
Et
Me
Et
t-BuOMe
t-BuOMe
Diisopropyl ether
Diisopropyl ether
24
144
163
22
20
20
–
95
94
93
–
66
74
75
–
35
–
–
55
–
–
\163
–
–
a Enantiomeric excess (% ee) was determined by HPLC (CHIRALCEL OJ, hexane:IPA:EtOH=85:10:5).
To optimize the reaction conditions, we varied the
ratio of substrate, enzyme and vinyl acetate (entries
7–9). Decreasing the amount of enzyme or vinyl
acetate resulted in high enantiomeric purity of (S)-3a,
probably because of the prolonged reaction time (94–
96% ee and 17–44%). The reaction conditions
detailed in entry 9 of Table 3 were selected as the
optimum.

1496
Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
NaBH4
CN
CN
CN
H
OH
OH
S
S
MeOH
O
4
4
(S)-6
product
Analysis by Chiral HPLC
reported by Gilmore, et al. 2)
K2CO3
NaBH4
MeOH
CN
CN
CN
H
OH
O
R
R
R
MeOH
O
O
4
3a,3b
recovered form
(R)-6
Scheme 2. Determination of absolute configuration.
Table 3. Enzymatic transesterification of (9)-4
Type II
Enzyme
O
CN
CN
CN
OH
OH
O
R
+
R
S
O
r.t.
4
4
R=Me 3a
Et 3b
O
R
Entry
R
Enzyme
Reaction time (h)
Yield of (S)-3
Yield of (R)-4
E
c.y. (%)
% ee
c.y. (%)
% ee
1
2
3
4
5
6
7
8
9
Me
Me
Me
Me
Et
Lipase Ak
Immobilized lipase
PPL
RDL
PPL
Immobilized lipase
Immobilized lipase
Immobilized lipase
Immobilized lipase
45
0.3
168
\168
24
52
70
29
–
48
3
17
18
44
64
43
26
–
9.8
99
95
96
94
48
30
70
–
52
83
83
79
54
57
99.3
9.2
–
7.1
16
19
26
71
8
12
2
–
1
230
47
63
69
Et
15
27
6
1.5
Me
Me
Me
Entries 1–6: Substrate (20 mg), lipase (2 mg added all at once), vinyl acetate or vinyl propionate (excess).
Entry 7: Substrate (200 mg), lipase (4×0.2 mg added portionwise), vinyl acetate (excess).
Entry 8: Substrate (20 mg), lipase (2 mg added all at once), vinyl acetate (1.2 equiv.), EtOAc (excess).
Entry 9: Substrate (200 mg), lipase (2 mg added all at once), vinyl acetate (excess).
2.3. Enzymatic transesterification on a large scale
NMR to avoid undesired over-reaction. The best result
was obtained when the enzymatic transesterification
was terminated at the point of 60–70% conversion.
Isolation of the product was carried out by silica gel
chromatography. These results suggest that this process
should be feasible for larger scale synthesis on kilogram
scale (Table 4).
Based on the above results, the ratio of substrate 4,
immobilized lipase and vinyl acetate was fixed at 1 g:2
mg:1 ml. A preparative-scale transesterification was
performed in a Taitec Personal-10 Incubator® (111
min−1, at 30°C). The reaction was monitored by 1H
Table 4. Enzymatic transesterification on a large scale
Immobilized lipase
CN
OH
CN
CN
OAc
+
OH
R
S
OAc
3a
4
4
111 min-1
30 °C
Entry
Substrate (g)
Reaction time (h)
Yield of (S)-3a
Yield of (R)-4
E
c.y. (%)
% ee
c.y. (%)
% ee
1
2
18
35
3.7
7.2
30
32
97
94.7
67
68
33
39
91
54

Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
1497
2.4. Large-scale double-step resolution of ( )-4 by
been due to acetaldehyde produced during the transes-
terification in vinyl acetate. Utilization of an alternative
acetylating agent or reaction in a column loaded with
lipase is under investigation.
transesterification using immobilized lipase
In order to improve the enantiomeric purity of (S)-3a
obtained through enzymatic esterification, subsequent
enzymatic resolution was investigated. Ideally, direct
enzymatic hydrolysis by switching the solvent from
vinyl acetate to buffer solution would give the desired
(S)-4, which can be used in the next process to synthe-
size E2050. However, the enzymatic hydrolysis required
a long reaction time on large scale. Thus, re-transester-
ification was investigated. Termination of the reaction
after 80–90% conversion of the substrate gave (S)-3a in
>99% ee (11.5 g) and 29% yield from ( )-4 (35 g)
(theoretical maximum 50%), as shown in Scheme 3.
2.6. Asymmetric synthesis of E2050 from (S)-3a
The enantiomerically pure (S)-3a (99% ee) was then
used for the synthesis of E2050. Hydrolysis of (S)-3a
afforded the corresponding alcohol (S)-4 in 95% iso-
lated yield. (S)-4 was converted to the corresponding
mesylate, followed by coupling with p-fluorophe-
noxyethylpiperazine to afford the free form of E2050 in
84% yield (two steps from (S)-4). The HCl salt of
E2050 was prepared by a standard procedure and had
99.9% ee after one recrystallization (Scheme 4).
2.5. Reuse of immobilized lipase
The feasibility of reusing the immobilized lipase was
investigated. As shown in Table 5, transesterification
using fresh immobilized lipase afforded (S)-3a in 95%
ee and 47% yield in 23 h (E=93).
3. Summary
An asymmetric synthesis of the neuron-selective cal-
cium channel blocker E2050 was established by using
immobilized lipase-catalyzed transesterification to pre-
pare the key intermediate. This method is expected to
be suitable for kilogram-scale synthesis.
On the other hand, reuse of immobilized lipase gave
almost the same result ((S)-3a >94% ee and 38%), but
the reaction rate decreased ten-fold. This may have
Immobilized lipase
CN
OH
CN
CN
OAc
OH
+
+
R
S
3a
4
4
OAc
32%
68%
111 min-1
(35 g)
30°C
94% ee
39% ee
Lipase, immobilized
t-BuOMe
K2CO3
MeOH
Immobilized lipase
OH
CN
CN
CN
OAc
OH
4
S
S
S
4
3a
OAc
87%
13%
111 min-1
(11.4 g)
30°C
99% ee
73% ee
(11.5 g)
(1.4 g)
Scheme 3. Double-step resolution using transesterification.
Table 5. Recovery and re-use of immobilized lipase
Immobilized lipase
CN
CN
CN
OH
OAc
OH
+
R
S
OAc
3a
4
4
30 °C 111 min-1
Entry
Immobilized lipase
Substrate (g)
Reaction time (h)
Yield of (S)-3a
Yield of (R)-4
E
c.y. (%)
% ee
c.y. (%)
% ee
1
2
Fresh
Recovered
1
0.6
23
257
47
38
95
\94
53
62
78
\57
93
58
Entry 1: substrate:lipase:vinyl acetate=1 g:5 mg:1 ml.
Entry 2: substrate:lipase:vinyl acetate=0.6 g:3 mg:1 ml.

1498
Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
F
HN
1)
2)
K2CO3
MeOH
N
O
CN
CN
OAc
OMs
S
S
NaI, Et3N,
MeCN
MsCl
Et3N,
MeCN
3a
(11.5 g)
99.15% ee
(15.2 g, 87% c.y.)
O
1) 4 N HCl/EtOAc
2) Recrystallization
N
E2050.2HCl salt of 5
CN
N
F
S
5
(10.8 g)
99.6% ee
ee% was improved by recrystallization
Scheme 4. Enantioselective synthesis of E2050.
4. Experimental
2H), 3.60 (s, 3H), 7.29–7.41 (m, 5H); EI(+)HRMS calcd
for C15H19O2N (M+) 245.1416, found 245.1444.
1H NMR spectra were measured at 400 MHz on a
Varian UNITY400 spectrometer. Chemical shifts (ppm)
were reported relative to internal CDCl3 (residual
CHCl3, 1H, 7.26 ppm). EI(+)HRMS spectra were
recorded on a Thermoquest Finnigan Mat SSQ7000.
High-performance liquid chromatography for the deter-
mination of enantiomeric purity was performed on a
Shimadzu SIL-10AD VP with a Shimadzu SPD-10A
VP UV–vis detector set at 200 nm and Chiralcel OJ
4.6×250 mm (eluent, hexane:ethanol:isopropanol
(850:100:50), flow rate; 0.7 ml/min). Specific rotations
were measured on a JASCO DIP-1000 digital polarime-
ter. Vinyl acetate (monomer) and vinyl propionate were
purchased from Tokyo Kasei Corp. (Japan), Immobi-
lized lipase (EC 3.1.1.3) was purchased from Wako
Pure Chemical industries Ltd. (Japan), PPL (type II)
was purchased from Sigma Corp., RDL (EC 3.1.1.3)
was purchased from Seikagaku Kogyo Corp. (Japan).
Pig liver esterase (EC 3.1.1.1) was purchased from
Sigma Corp., and Lipase AK (Amano 20) and lipase
PSA-30 (Amano 20) were given courtesy of Amano
Pharmaceutical Corp. (Japan). All materials were used
as received.
4.2. ( )-Ethyl 4-cyano-5-methyl-4-phenylhexanoate, 1b
1b was prepared from acrylic acid ethyl ester in a
similar manner to that described above (75%). 1H
NMR (400 MHz, CDCl3); l (ppm) 0.79 (d, J=6.8 Hz,
3H), 1.20 (t, J=7.1 Hz, 3H), 1.23 (d, J=6.6 Hz, 3H),
1.89–2.00 (m, 1H), 2.10–2.26 (m, 2H), 2.34–2.56 (m,
2H), 3.98–4.13 (m, 2H), 7.29–7.42 (m, 5H); EI(+)-
HRMS calcd for C16H21O2N (M+) 259.1572, found
259.1601.
4.3. ( )-4-Cyano-5-methyl-4-phenylhexanoic acid, 2
( )-2 was prepared from ( )-1a or 1b by non-enzymatic
hydrolysis (76 or 78% yield, respectively). 1H NMR
(400 MHz, CDCl3); l (ppm) 0.79 (d, J=6.8 Hz, 3H),
1.23 (d, J=6.6 Hz, 3H), 1.94–2.06 (m, 1H), 2.08–2.23
(m, 2H), 2.40–2.55 (m, 2H), 7.29–7.42 (m, 5H); EI(+)-
HRMS calcd for C14H17O2N (M+) 231.1259, found
231.1266.
4.4. ( )-Propyl 4-cyano-5-methyl-4-phenylhexanoate, 1c
4.1. ( )-Methyl 4-cyano-5-methyl-4-phenylhexanoate,
1a
2-Chloro-3,5-dinitropyridine (278.4 mg, 0.14 mmol)
was added to a stirred solution of ( )-4-cyano-5-
methyl-4-phenylhexanoic acid 2 (316 mg, 0.14 mmol)
and 1-propanol (0.2 ml, 2.68 mmol) in pyridine (1.4 ml)
under an N2 atmosphere at room temperature. The
mixture was stirred at room temperature overnight,
then the reaction was quenched with saturated
NaHCO3 and the whole mixture was extracted with
EtOAc. The combined extracts were dried over MgSO4.
Removal of the solvent in vacuo gave an oily residue,
which was purified by column chromatography on sil-
ica gel. The fraction eluted with 20% EtOAc in hexane
Acrylic acid methyl ester (16.7 g, 96.9 mmol), a solution
of potassium tert-butoxide in 2-methyl-2-propanol (1.0
M, 19.4 ml, 19.4 mmol) and 18-crown-6 (512 mg, 1.94
mmol) were added to a stirred solution of 3-methyl-2-
phenylbutyronitrile (15.4 g, 96.9 mmol) in THF (150
ml). The reaction mixture was heated under reflux
overnight, then cooled to room temperature, and ice-
water was added. This mixture was extracted with
EtOAc. The combined extracts were dried over
Na2SO4. Removal of the solvent in vacuo gave an oily
residue, which was purified by column chromatography
on silica gel. The fraction eluted with 50% EtOAc in
hexane afforded ( )-1a as a colorless oil (21.7 g, 91%).
1H NMR (400 MHz, CDCl3); l (ppm) 0.79 (d, 6.8 Hz,
3H), 1.23 (d, J=6.8 Hz, 3H), 1.96 (ddd, J=4.8 Hz,
12.0 Hz, 16.4 Hz, 1H), 2.10–2.24 (m, 2H), 2.37–2.61 (m,
1
afforded ( )-1c (283 mg, 76%) as a colorless oil. H
NMR (400 MHz, CDCl3); l (ppm) 0.79 (d, J=6.6 Hz,
3H), 0.89 (t, J=7.4 Hz, 3H), 1.23 (d, J=6.6 Hz, 3H),
1.52–1.63 (m, 2H), 1.95 (ddd, J=7.2 Hz, 12.0 Hz, 16.4
Hz, 1H), 2.01–2.24 (m, 2H), 2.35–2.53 (m, 2H), 3.89–
4.02 (m, 2H), 7.28–7.41 (m, 5H); EI(+)HRMS calcd for
C17H23O2N (M+) 273.1729, found 273.1740.

Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
1499
4.5. ( )-4-Cyano-5-methyl-4-phenylhexanol, 4
4.8. General method for enzymatic hydrolysis of the
( )-acyl alcohol 3a,b (substrate type II) in phosphate
buffer
A solution of ( )-ethyl 4-cyano-5-methyl-4-phenylhex-
anoate 1a (9.4 g, 36.4 mmol) in THF (60 ml) was added
dropwise to a solution of LiAlH4 (1.03 g, 27.3 mmol) in
THF (60 ml) at 0°C, and the mixture was stirred at the
same temperature for 1 h. After stirring at room tem-
perature overnight, the mixture was cooled to 0°C, then
the reaction was quenched with saturated NaHCO3 (10
ml) and 1N NaOH (12 ml). After removal of the
precipitate, the organic layer was dried over MgSO4.
Removal of the solvent in vacuo gave an oily residue,
which was purified by column chromatography on sil-
ica gel (50% EtOAc in hexane) to afford ( )-4 as a
colorless oil (5.01 g, 64%). 1H NMR (400 MHz,
CDCl3); l (ppm) 0.79 (d, J=6.6 Hz, 3H), 1.22 (d,
J=6.6 Hz, 3H), 1.17–1.28 (m, 1H), 1.62–1.66 (m, 1H),
1.98 (td, J=4.4 Hz, 12.5 Hz, 1H), 2.14 (qu, J=6.8 Hz,
1H), 2.25 (ddd, J=4.4 Hz, 12.0 Hz, 13.6 Hz, 1H), 3.59
(m, 2H), 7.26–7.40 (m, 5H); EI(+)HRMS calcd for
C14H19ON (M+) 217.1467, found 217.1470.
A suspension of the ( )-acetete 3a (or ( )-propionate
3b) (100 mg) and enzyme (10 mg) in acetone (0.1 ml) (in
the case of long reaction times, acetonitrile (10 ml) was
added instead of acetone (0.1 ml)) and phosphate buffer
(10 ml, pH 6.8) was stirred at 23°C, and the reaction
was monitored by TLC. After the period of time noted
in Table 1, hydrolysis was terminated by extracting the
mixture with CHCl3. The combined extracts were dried
over MgSO4 and then concentrated in vacuo to leave
an oily residue, which was purified by column chro-
matography on silica gel (hexane:ethyl acetate=4:1) to
give the hydrolyzed alcohol 4 and recovered acetate 3a
(or propionate 3b) as colorless oils.
4.8.1. (S)-(−)-4-Cyano-5-methyl-4-phenylhexanol, 4. 52%
yield, 72% ee, by Lipase AK hydrolysis of 4-cyano-5-
methyl-4-phenylhexyl acetate in phosphate buffer, [h]D27
−5.3 (c 0.8, CHCl3).
4.6. ( )-4-Cyano-5-methyl-4-phenylhexyl acetate, 3a
4.8.2. (R)-(+)-4-Cyano-5-methyl-4-phenylhexanol, 4.
49% yield, 21% ee, by Lipase PSA-30 hydrolysis of
4-cyano-5-methyl-4-phenylhexyl acetate in phosphate
buffer, [h]2D7 +3.9 (c 0.9, CHCl3).
A mixture of ( )-4-cyano-5-methyl-4-phenylhexanol 4
(1.32 g, 6.12 mmol), acetic anhydride (1 ml, 9.05 mmol)
and pyridine (2 ml) was stirred at room temperature
overnight. After having been cooled to 0°C, the reac-
tion mixture was neutralized with saturated NaHCO3,
then extracted with EtOAc. The combined extracts
were dried over MgSO4. Removal of the solvent in
vacuo gave an oily residue, which was purified by
column chromatography on silica gel. The fraction
eluted with 10% EtOAc in hexane afforded ( )-3a as a
4.8.3. (R)-(+)-4-Cyano-5-methyl-4-phenylhexyl acetate,
3a. 48% yield (recovery), 70% ee, by Lipase AK hydrol-
ysis of 4-cyano-5-methyl-4-phenylhexyl acetate in phos-
phate buffer, [h]2D7 +19.1 (c 0.9, CHCl3).
4.8.4. (S)-(−)-4-Cyano-5-methyl-4-phenylhexyl acetate,
3a. 50% yield (recovery), 23% ee, by Lipase PSA-30
hydrolysis of 4-cyano-5-methyl-4-phenylhexyl acetate in
phosphate buffer, [h]2D7 −3.8 (c 0.8, CHCl3).
1
colorless oil (1.3 g, 80%). H NMR (400 MHz, CDCl3);
l (ppm) 0.79 (d, J=6.8 Hz, 3H), 1.21 (d, J=6.8 Hz,
3H), 1.28 (m, 1H), 1.70 (m, 1H), 1.90 (ddd, J=4.4 Hz,
12.4 Hz, 13.6 Hz, 1H), 2.02 (s, 3H), 2.13 (qu, J=6.8
Hz, 1H), 2.21 (ddd, J=4.4 Hz, 12.0 Hz, 13.6 Hz, 1H),
3.99 (t, J=6.2 Hz, 2H), 7.28–7.41 (m, 5H); EI(+)-
HRMS calcd for C16H21O2N (M+) 259.1572, found
259.1575.
4.8.5. (R)-(+)-4-Cyano-5-methyl-4-phenylhexyl propi-
onate, 3b. 48% yield (recovery), 57% ee, by Lipase AK
hydrolysis of 4-cyano-5-methyl-4-phenylhexyl propi-
onate in phosphate buffer, [h]2D8 +12.0 (c 0.88, CHCl3).
4.7. ( )-4-Cyano-5-methyl-4-phenylhexyl propionate, 3b
4.8.6. (S)-(−)-4-Cyano-5-methyl-4-phenylhexyl propi-
onate, 3b. 54% yield (recovery), 13% ee, by Lipase
PSA-30 hydrolysis of 4-cyano-5-methyl-4-phenylhexyl
propionate in phosphate buffer, [h]2D8 −2.2 (c 0.88,
CHCl3).
Propionyl chloride (773 mg, 8.35 mmol) was added to a
stirred solution of ( )-4-cyano-5-methyl-4-phenylhex-
anol 4 (1.21 g, 5.57 mmol) and Et3N (1.6 ml, 11.1
mmol) in THF (10 ml). The reaction mixture was
stirred at room temperature overnight, then the reac-
tion was quenched with brine, and extracted with
EtOAc. The combined extracts were dried over MgSO4.
Removal of the solvent in vacuo gave an oily residue,
which was purified by column chromatography on sil-
ica gel. The fraction eluted with 10% EtOAc in hexane
4.9. General method of enzymatic hydrolysis of the ( )-
acyl alcohol 3a,b (substrate type II) in organic solvent
A suspension of ( )-acetete 3a (or ( )-propionate 3b)
(100 mg) and immobilized lipase (10 mg) in water-satu-
rated tert-butyl methyl ether (10 ml) or water-saturated
diisopropyl ether (10 ml) was stirred at 23°C, and the
reaction was monitored by TLC. After the time period
noted in Table 2, immobilized lipase was removed by
filtration, and the filtrate was concentrated in vacuo to
leave an oily residue, which was purified by column
chromatography on silica gel (hexane:ethyl acetate=
4:1) to give the hydrolyzed alcohol 4 and recovered
acetate 3a (or propionate 3b) as colorless oils.
1
afforded ( )-3b as a colorless oil (1.3 g, 87%). H NMR
(400 MHz, CDCl3); l (ppm) 0.78 (d, J=4.8 Hz, 3H),
1.12 (d, J=7.6 Hz, 3H), 1.20 (d, J=4.8 Hz, 3H),
1.24–1.34 (m, 1H), 1.65–1.76 (m, 1H), 1.91 (td, J=4.4
Hz, 12.8 Hz, 1H), 2.13 (qu, J=6.8 Hz, 1H), 2.20 (td,
J=4.4 Hz, 13.0 Hz, 1H), 2.30 (q, J=7.6 Hz, 2H), 3.99
(t, J=6.0 Hz, 2H), 7.29–7.41 (m, 5H); EI(+)HRMS
calcd for C17H23O2N (M+) 273.1729, found 273.1737.

1500
Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
4.10. Determination of enantiomeric excess of
hydrolyzed products 4 and 3a or 3b
4.15. General method of enzymatic transesterification
of the ( )-alcohol 4 (substrate type II)
The enantiomeric excess (% ee) of the hydrolyzed alco-
hol 4 was determined by HPLC (CHIRALCEL OJ,
eluent, hexane:IPA:EtOH=850:100:50, flow rate; 0.7
ml/min, retention time (min) 9.5 for (R)-alcohol 4 and
7.5 for (S)-alcohol 4).
A suspension of the ( )-alcohol 4 (100 mg, 0.46 mmol)
and enzyme (10 mg) in vinyl acetate (or vinyl propi-
onate) (10 ml) was stirred at 23°C. The reaction was
monitored by TLC. After the time period noted in
Table 3, the enzyme was removed by filtration, and the
filtrate was concentrated in vacuo to leave an oily
residue, which was purified by column chromatography
on silica gel. The fraction eluted with 25% EtOAc in
hexane afforded the acetate 3a (or propionate 3b) and
the recovered alcohol 4 as colorless oils.
The enantiomeric excess (% ee) of the recovered acetate
3a or propionate 3b was determined after conversion
into the corresponding alcohol 4, which was prepared
by the method described below.
4.11. Conversion of recovered acetate 3a (or propionate
3b) into the corresponding alcohol 4
4.15.1. (S)-(−)-4-Cyano-5-methyl-4-phenylhexyl acetate,
3a. 18% yield, 96% ee, by immobilized lipase (Pseu-
domonas sp.)-catalysed transesterification of 4-cyano-5-
methyl-4-phenylhexanol, [h]2D9 −25 (c 0.558, CHCl3).
A mixture of the recovered (R)- or (S)-acetate 3a (or
propionate 3b) (14 mg) and K2CO3 (3 mg) in methanol
(10 ml) was stirred at room temperature for 3 h. It was
neutralized with 5N aqueous HCl, then concentrated in
vacuo to give an oily residue, to which water was
added, and the mixture was extracted with EtOAc. The
combined extracts were dried over Na2SO4. After
removal of the solvent in vacuo, the residue was
purified by short column chromatography on silica gel
(50% EtOAc in hexane) to give (R)- or (S)-alcohol 4 as
a colorless oil for HPLC analysis.
4.15.2. (S)-(−)-4-Cyano-5-methyl-4-phenylhexyl propi-
onate, 3b. 48% yield, 9.8% ee, by PPL-catalysed transes-
terification of 4-cyano-5-methyl-4-phenylhexanol, [h]D30
−2.7 (c 1.2, CHCl3).
4.15.3. (R)-(+)-4-Cyano-5-methyl-4-phenylhexanol, 4.
30% yield (recovery), 99.3% ee, by immobilized lipase
(Pseudomonas sp.) transesterification of 4-cyano-5-
methyl-4-phenylhexanol in vinyl acetate, [h]2D9 +19.4 (c
0.42, CHCl3).
4.12. Determination of absolute configuration of the
hydrolyzed products 3a or 3b and 4
The enantiomeric excess of the resolved products 3a or
3b and 4 was determined in a similar manner to that
described for the determination of the enantiomeric
excess of hydrolyzed products 4 and 3a or 3b.
The absolute configuration of the hydrolyzed products
was determined by comparison of the retention time on
chiral HPLC with the value for the (R)-alcohol 4 (or
(S)-alcohol 4) derived from the reported (R)-aldehyde 6
(or (S)-aldehyde 6). The conversion of the reported
(R)-aldehyde 6 (or (S)-aldehyde 6)2a into the corre-
sponding (R)-alcohol 4 (or (S)-alcohol 4) is described
below.
4.16. Large-scale enzymatic transesterification of
alcohol ( )-4 using immobilized lipase
A suspension of ( )-4-cyano-5-methyl-4-phenylhexanol
4 (18 g) and immobilized lipase (35 mg) in vinyl acetate
(20 ml) was stirred at 30°C in Taitec Personal-10 Incu-
1
4.13. (S)-4-Cyano-5-methyl-4-phenylhexnol, 4
bator® (111 min−1). The reaction was monitored by H
NMR at intervals of 1 h. Immobilized lipase was
removed by filtration at the point of 60% conversion,
and the filtrate was concentrated in vacuo to leave an
oily residue, which was purified by column chromatog-
raphy on silica gel. The fraction eluted with 25%
EtOAc in hexane afforded the (S)-acetate 3a of 97% ee
(6.4 g, 30%) and recovered (R)-alcohol 4 of 33% ee (12
g, 67%) as colorless oils.
NaBH4 (20 mg, 0.53 mmol) was added to a stirred
solution of (S)-4-cyano-5-methyl-4-phenylhexanal 6 (18
mg, 0.08 mmol) prepared by the reported method.2a
After stirring at room temperature for 1 h, the reaction
mixture was concentrated in vacuo to give an oily
residue, to which water was added, and the mixture was
extracted with EtOAc. The combined extracts were
dried over Na2SO4. After removal of the solvent in
vacuo, the residue was purified by short column chro-
matography on silica gel (50% EtOAc in hexane) to
give (S)-(−)-4-cyano-5-methyl-4-phenylhexanoic acid 4
as a colorless oil for HPLC analysis.
4.17. Re-resolution of (S)-acetate 3a of 94.7% ee by
means of immobilized lipase-catalyzed
transesterification
The (S)-acetate 3a of 94.7% ee (13.4 g), prepared from
the ( )-alcohol 4 (35 g) in a similar manner to that
described above, was converted into the corresponding
(S)-alcohol 4 in the same manner as described for the
conversion of the recovered acetate 3a (or propionate
3b) into the corresponding alcohol 4. The obtained
(S)-alcohol 4 (11.4 g) was submitted to the same enzy-
matic procedure as described above. After 80–90% con-
4.14. (R)-4-Cyano-5-methyl-4-phenylhexanol, 4
(R)-(+)-4-Cyano-5-methyl-4-phenylhexanol 4 was pre-
pared from (R)-4-cyano-5-methyl-4-phenylhexanal 6
(18 mg), which was prepared by the reported method in
the same manner as described above for the HPLC
analysis sample.

Y. Norimine et al. / Tetrahedron: Asymmetry 13 (2002) 1493–1502
1501
version of the substrate, immobilized lipase was
removed by filtration, and the filtrate was concentrated
in vacuo to leave an oily residue, which was purified by
column chromatography on silica gel. The fraction
eluted with 25% EtOAc in hexane afforded the (S)-ace-
tate 3a of 99.15% ee (11.5 g, 87%) and the recovered
(S)-alcohol 4 of 73% ee (1.4 g, 13%) as colorless oils.
cipitated colorless crystals were collected by filtration
and dried in an oven at 60°C for 9 days to give E2050
as colorless crystals (10.77 g, 61%), >99.9% ee, [h]D29
1
−5.2 (c 0.73, EtOH), H NMR (400 MHz, DMSO-d6);
l (ppm) 0.68 (d, J=6.6 Hz, 3H), 1.11 (d, J=6.6 Hz,
3H), 1.22–1.34 (m, 1H), 1.58–1.72 (m, 1H), 2.06–2.30
(m, 3H), 3.00–3.25 (m, 2H), 3.30–3.80 (m, 10H), 4.36
(brs, 2H), 6.98–7.07 (m, 2H), 7.11–7.20 (m, 2H), 7.32–
7.40 (m, 1H), 7.40–7.50 (m, 4H), ESI-Mass; 424 (MH+),
mp 183–187°C. HPLC impurities: 0.68%, residual sol-
vent: 0.09%, water content: 0.30%, chloride ion content:
14.56% (% anhydrous/desolvated theoretical value=
14.28%)).
4.18. (2S)-5-{4-[2-(4-Fluorophenoxy)ethyl]piperazin-1-
yl}-2-isopropyl-2-phenylpentanenitrile dihydrochloride
(E2050)
To a solution of the (S)-acetate 3a of 99.15% ee (11.51
g, 44.5 mmol) in methanol (500 ml) was added K2CO3
(11 g, 79.6 mmol). The mixture was stirred at room
temperature overnight, then neutralized with 5N
aqueous HCl, and concentrated in vacuo to give an oily
residue, to which water was added. The mixture was
extracted with EtOAc. The combined extracts were
dried over Na2SO4. Removal of the solvent in vacuo
gave the (S)-alcohol 4 as a colorless oily residue (9.16
g), which was dissolved in acetonitrile (70 ml). To this
solution, Et3N (12.8 ml, 91.8 mmol) was added. The
mixture was cooled to 0°C, and a solution of methane-
sulfonyl chloride (3.66 ml, 46.3 mmol) in acetonitrile
(70 ml) was added dropwise. The reaction mixture was
stirred at 0°C for 1 h, then at room temperature
overnight, and concentrated in vacuo to give an oily
residue, which was dissolved in ether (300 ml). This
solution was washed with brine. The organic layer was
dried over Na2SO4. Removal of the solvent in vacuo
gave 12.13 g of the mesylate as a dark reddish oil,
which was dissolved in acetonitrile (113 ml). To this
solution, sodium iodide (6.97 g, 46.5 mmol), Et3N (6.4
ml, 45.9 mmol) and p-fluorophenoxyethylpiperazine
(10.41 g, 46.4 mmol) were added. The mixture was
stirred at 70°C for 3 h. The reaction mixture was cooled
to room temperature and concentrated in vacuo to give
an oily residue, which was dissolved in EtOAc (400 ml).
This solution was washed with water (400 ml), and the
organic layer was dried over Na2SO4. Removal of the
solvent in vacuo left an oily residue, which was purified
by column chromatography on Cromatorex NH silica
gel. The fraction eluted with 17% EtOAc in hexane
afforded the free form of E2050 as a colorless oil (15.22
g, 80.2% yield from compound 4). 99.6% ee, [h]2D0 −3.9
A second crop of crystals of E2050 was obtained; 1.19
g (6.7%), 99.05% ee. Filtrate; 2.58 g (14.5%), 99.47% ee.
Acknowledgements
We wish to thank members of the Analytical Chemistry
Section of Eisai Tsukuba Research Laboratories for
interpretation of NMR and mass spectra and for
obtaining elemental analysis data.
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1
(c 1.46, CHCl3), H NMR (400 MHz, CDCl3); l (ppm)
0.77 (d, J=6.8 Hz, 3H), 1.05–1.17 (m, 1H), 1.20 (d,
J=6.8 Hz, 3H), 1.50–1.60 (m, 1H), 1.88 (dt, J=4.4 Hz,
12.4 Hz, 1H), 2.06–2.19 (m, 2H), 2.24–2.30 (m, 2H),
2.30–2.43 (m, 4H), 2.46–2.62 (m, 4H), 2.77 (t, J=5.8
Hz, 2H), 4.04 (t, J=5.8 Hz, 2H), 6.80–6.85 (m, 2H),
6.91–6.99 (m, 2H), 7.25–7.32 (m, 1H), 7.32–7.40 (m,
4H).
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(150 ml), and the mixture was stirred at room tempera-
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white precipitate was dissolved in 1-propanol (88.5 ml)
under reflux. The solution was stirred while being grad-
ually cooled to room temperature overnight. The pre-
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