Lipase-mediated kinetic resolution of rigid clofibrate analogues with lipid-modifying activity

Article abstract of DOI:10.1016/S0957-4166(01)00119-7

The lipase-catalysed kinetic resolution of methyl esters of (±)-5-chloro-2,3-dihydro-1-benzofuran-2-carboxylic acid, (±)-6-chloro-2,3-dihydro-4H-1-benzopyran-2-carboxylic acid, and (±)-6-chloro-2,3-dihydro-4H-1-benzopyran-3-carboxylic acid, rigid analogues of clofibrate, was effected with fair to moderate enantioselectivities (E = 1.0-4.8), enantiomeric excesses of up to 86% and workable reaction rates. Enantiomerically pure (R)- and (S)-5-chloro-2,3-dihydro-1-benzofuran-2-carboxylic acids were obtained by fractional crystallisation of the diastereomeric salts of the corresponding racemic acid with (+)- and (-)-amphetamine from ethanol; the absolute configuration of the products were established by chemical correlation.

Full text of DOI:10.1016/S0957-4166(01)00119-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 853–862
Lipase-mediated kinetic resolution of rigid clofibrate analogues
with lipid-modifying activity
a
a
a
a,
Savina Ferorelli, Carlo Franchini, Fulvio Loiodice, Maria Grazia Perrone, Antonio Scilimati, *
b
c
Maria Stefania Sinicropi and Paolo Tortorella
a
Dipartimento Farmaco-Chimico, Universit a` di Bari, Via E. Orabona, no. 4, 70125 Bari, Italy
Dipartimento di Scienze Farmaceutiche, Universit a` della Calabria, 87030 Arcavacata di Rende (CS), Italy
Dipartimento di Scienze del Farmaco, Universit a` degli Studi ‘G. D’Annunzio’, Via dei Vestini, 66100 Chieti, Italy
b
c
Received 7 February 2001; accepted 8 March 2001
Abstract—The lipase-catalysed kinetic resolution of methyl esters of (±)-5-chloro-2,3-dihydro-1-benzofuran-2-carboxylic acid,
±)-6-chloro-2,3-dihydro-4H-1-benzopyran-2-carboxylic acid, and (±)-6-chloro-2,3-dihydro-4H-1-benzopyran-3-carboxylic acid,
rigid analogues of clofibrate, was effected with fair to moderate enantioselectivities (E=1.0–4.8), enantiomeric excesses of up to
6% and workable reaction rates. Enantiomerically pure (R)- and (S)-5-chloro-2,3-dihydro-1-benzofuran-2-carboxylic acids were
(
8
obtained by fractional crystallisation of the diastereomeric salts of the corresponding racemic acid with (+)- and (−)-amphetamine
from ethanol; the absolute configuration of the products were established by chemical correlation. © 2001 Elsevier Science Ltd.
All rights reserved.
1
. Introduction
2,2-Dialkyl-2-(4-chlorophenoxy)ethanoic acids 2 (R=
H) are analogues of the clofibric acid 1b, the active
metabolite of clofibrate 1a which is still on the market
in spite of side adverse effects on skeletal muscle (myal-
Fibrates (Fig. 1, structures 1 and 2) constitute a widely
used class of lipid-modifying agents. Treatment with
1
3–7
fibrates results in a substantial decrease in plasma
triglycerides and is usually associated with a moderate
decrease in LDL cholesterol and an increase in HDL
cholesterol concentration. Recent studies indicate that
the effects of fibrates are mediated, at least in part,
through alterations in the transcription of genes encod-
ing for proteins that control lipoprotein metabolism.
Fibrates activate specific transcription factors belonging
to the nuclear hormone receptor superfamily, termed2
peroxisome proliferator-activated receptors (PPARs).
gias and myotonias) and on the liver (carcinogenic-
8,9
ity). The two enantiomers of 2 in which R =H and
1
R =alkyl [e.g. (R)- and (S)-2-(4-chlorophenoxy)pro-
2
panoic acids, R =CH ] exhibit different pharmacologi-
2
3
cal profiles. In particular, the (R)-enantiomers have
10
11
higher anti-aggregatory activity, lower myotonic and
hepatocarcinogenicity
12–14
than the (S)-enantiomers,
whilst both have the same antilipidemic effect as clofi-
bric acid.
Rigid analogues of clofibric acid (Fig. 2) show an
antilipolytic activity comparable to that of 2-alkyl-2-(4-
15,16
chlorophenoxy)ethanoic acids.
These investigations
were performed using the racemic compounds 3–5, but
nothing is known, so far, about the pharmacological
behaviour of their pure enantiomers. This prompted us
to investigate the preparation of homochiral 3–5.
Several methods have already been reported for the
resolution of racemic phenoxyalkanoic acid derivatives
Figure 1.
17
including chemical kinetic resolution, crystallisation
1
8–28
of diastereomeric salts,
and biological methods
2
9,30
involving enantioselective microbial 3 reduction,
1,32
*
0
Corresponding author.
enantioselective esterification of acids
and enan-
957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00119-7

8
54
S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
Figure 2.
tioselective hydrolysis and/or transesterification of their
In this paper the results of this investigation are reported,
focusing on the e.e.s of the products and the enantiose-
lectivity factor (E) values of the lipase-catalysed hydrol-
ysis, esterification, and transesterification reactions.
3
3–36
esters.
In contrast, rigid analogues of clofibric acid
have received less attention.
In the course of our previous investigations we accom-
plished the resolution of 4b and 5b by crystallisation of
37
their diastereomeric salts with homochiral amines, and
the lipase-mediated kinetic resolution of 2-monoalkyl-2-
aryloxyethanoic acids and 2-methyl-2-(n-propyl)-2-(4-
2. Results and discussion
38
chlorophenoxy)ethanoic acid. Lipases, beyond their
biological function, are widely used as chiral catalysts for
asymmetric acyl transfer reactions between a number of
acyl acceptors and donors. The broad range of accepted
substrates (and consequently the broad range of possible
reaction types including hydrolysis, esterification, trans-
esterification, etc.) together with the high stability of
most lipases in aqueous solutions and in organic solvents,
has made lipases important catalysts for the preparation
of highly enantiopure building blocks and intermediates.
Herein, we report the resolution of (±)-3b by the classical
method of crystallisation of the diastereomeric salts, the
establishment of the absolute configuration of (+)-3b and
(−)-3b, and the lipase-catalysed kinetic resolution results
of (±)-3, (±)-4 and (±)-5. (±)-5-Chloro-2,3-dihydro-1-
benzofuran-2-carboxylic acid 3b, prepared by the litera-
ture method, was resolved by fractional crystallisation
of the diastereomeric salts with (−)- and (+)-
amphetamine from ethanol. Interestingly, the crystallisa-
tion of a partially resolved mixture of the antipodal forms
led to the separation of the racemate similarly to our
15
3
7,39
We have investigated the pharmacological activity of
clofibric acid derivatives [such as 2-alkyl-2-(4-chlorophe-
noxy)ethanoic acids] in order to find and, possibly,
dissociate the structural determinants of the different
effects. Thus, we decided to synthesise optically active
rigid analogues of aryloxyalkanoic acids by chemical and
chemoenzymatic routes and compare the two methods.
previous report on other 2-aryloxyalkanoic acids.
The absolute configuration was established by chemical
correlation as depicted in Scheme 1. For this purpose, the
dextro-acid was converted, by catalytic hydrogenation,
into the corresponding dehalogenated compound (−)-
2,3-dihydro-1-benzofuran-2-carboxylic acid of known
40
(S)-configuration.
Scheme 1.
Scheme 2.

S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
855
Table 1. Hydrolysis of (9)-5-chloro-2,3-dihydro-1-benzofuran-2-carboxylic acid methyl ester 3a, (9)-6-chloro-2,3-dihydro-
4
H-1-benzopyran-2-carboxylic acid methyl ester 4a, and (9)-6-chloro-2,3-dihydro-4H-1-benzopyran-3-carboxylic acid methyl
a
ester 5a in the presence of lipase
Comp.
Lipase (source)
Reaction time (h)
Cb (%)
E.e.sc (%)
E.e.pc (%)
Ed
(
absolute configuration)
3a
3a
3a
3a
3a
3a
3a
4a
5a
5a
5a
5a
5a
5a
Candida cylindracea (AY-Amano)
Candida cylindracea (AY-Amano)
Humicola sp. (CE-10)
2.5
7
3
37
77
79
85
91
44
43
93
94
51
73
6
27 (R)
60 (R)
45 (R)
78 (R)
27 (R)
17 (R)
35 (R)
3
39 (S)
35 (S)
75 (S)
27 (S)
46 (S)
40 (S)
46 (S)
19 (S)
12 (S)
14 (S)
3 (S)
3.5
2.4
1.8
2.6
1.3
1.8
3.8
1.0
1.3
2.8
3.6
1.0
1.5
1.8
Mucor ja6anicus (MAP)
9
Humicola lanuginosa (R-10)
Humicola lanuginosa (R-10)
30
51
56
24
72
72
72
72
72
72
e
22 (S)
46 (S)
Rhizopus delemar
f
g
Candida cylindracea (AY-Amano)
Candida cylindracea (OF-MeitoSanjo)
Humicola sp. (CE-10)
Mucor ja6anicus (MAP)
Humicola lanuginosa (R-10)
Rhizopus ni6eus (N-conc)
Rhizopus delemar
–
3
h
(R)
h
34 (R)
h
28 (R)
f
–
h
91
30
5
(R)
h
86 (R)
a
b
c
d
e
f
Unless otherwise indicated the substrate:lipase ratio=1:5 (w/w).
C=conversion determined by HPLC.
Unless otherwise indicated enantiomeric excess of unreacted substrate (e.e. ) and product (e.e. ) was determined by HPLC (see Section 3).
E=enantioselectivity factor.
Substrate:lipase ratio=1:1 (w/w).
The product was recovered as a racemate.
s
p
g
The same result was obtained performing the reaction in the presence of other lipases such as those ones from Humicola sp. (CE-10), Mucor
javanicus (MAP), Humicola lanuginosa (R-10), Rhizopus delemar.
h
Calculated according to the following equation: C=e.e. /(e.e. +e.e. ), where e.e. is the enantiomeric excess of remaining unreacted ester substrate
s
s
p
s
and e.e.p is the enantiomeric excess of the acid product.
Compounds 4b–5b were synthesised as previously
Humicola lanuginosa) and 78% (using the lipase from
1
5,16
reported.
Their resolution was accomplished by
Mucor javanicus). The e.e. of the product (e.e. ) varied
p
crystallisation of their diastereomeric salts obtained by
reaction with optically active amines, and the enan-
tiomers used as standards for the present investigation.
between 3% (lipase from Humicola lanuginosa) and 46%
(lipase from Rhizopus delemar and Candida cylin-
dracea). No enantioselectivity was found in the hydrol-
ysis of substrate 4a, indicating that both (R)- and
(S)-enantiomers of 4a react at very similar rates irre-
spective of the lipase chosen to catalyse the hydrolysis,
so racemic unreacted substrate 4a and racemic product
4b are always isolated. The E value is approximately 1.0
with all the lipases examined (see also footnote g of
Table 1).
3
7
As far as the lipase-mediated kinetic resolution of com-
pounds 3–5 is concerned, a variety of experimental
conditions were investigated. Initially, hydrolysis of the
methyl esters 3a–5a was performed in the presence of
several lipases (Scheme 2). A summary of the results
obtained is listed in Table 1. It seems that reactions of
the three compounds 3a, 4a and 5a are strongly depen-
dent upon the lipase used.
It is noteworthy that the hydrolysis of 3a occurred
almost always faster than for 4a, for the reaction
executed in the presence of the same lipase. The hydrol-
ysis of (±)-6-chloro-2,3-dihydro-4H-1-benzopyran-3-
carboxylic acid methyl ester 5a gave an E of 1.0–3.6,
The enantioselectivity factor E value for 3a ranges from
1
.3 (lipase from Humicola lanuginosa) to 3.8 (lipase
from Rhizopus delemar), whereas the e.e. of the unre-
acted substrate (e.e. ) ranges between 17% (lipase from
with e.e. from 27% (using the lipase from Humicola
s
s
Scheme 3.

8
56
S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
Scheme 4.
lanuginosa) to 75% (using lipase from Mucor javanicus)
occurred and methyl ester 3a (or 5a, respectively)
formed.
and e.e. from 3% (lipase from Candida cylindracea) to
p
86% (lipase from Rhizopus delemar).
In a further attempt to develop a more enantioselective
method for the preparation of this class of compounds,
it was decided to investigate the kinetic resolution of
the alcohols 6–8, which can be considered as precursors
of 3–5. (±)-6–8 were prepared in 93–96% yield, by
reducing the corresponding esters 3a–5a (Scheme 3).
These were incubated in hexane, in the presence of a
lipase and vinyl acetate acyl donor (Scheme 4 and
Table 2).
It is known that the enantioselectivity of the lipase-
catalysed kinetic resolution of racemic acids (and their
derivatives) can be enhanced by substrate modifica-
tion. Hence, the allyl esters 3c and 5c were prepared
and incubated with the lipase from Pseudomonas sp.
4
1
(
AK-Amano) in phosphate buffer. The lipase from
Pseudomonas sp. (AK-Amano) was chosen for further
investigation because in the preliminary screening its
reaction proceeded at an appreciable rate. No marked
improvement was observed. Similar results were
obtained when 3c (or 5c) was incubated in hexane, in
the presence of lipase from Pseudomonas sp. (AK-
Amano) and methanol; a transesterification reaction
An enzymatic screening was performed and the best
results obtained in the case of compound 6 were found
to be as follows: e.e.s of the unreacted substrate (e.e.s)
Table 2. Transesterification reaction of (9)-2-hydroxymethyl-5-chloro-2,3-dihydro-1-benzofuran 6 and (9)-2-hydroxymethyl-
a
6-chloro-2,3-dihydro-4H-1-benzopyran 8 with vinyl acetate in hexane in the presence of lipase
Comp.
Lipase (source)
Reaction time (h)
Cb (%)
E.e.sc (%)
E.e.pd (%)
Ee
(
absolute configuration)
6
6
6
6
6
6
6
6
6
6
6
8
8
8
8
8
8
8
8
Candida cylindracea (AY)
Rhizopus ni6eus (N-conc)
Mucor ja6anicus (MAP)
Pseudomonas sp. (K-10)
Aspergillus niger (AP)
Geotrichum candidum (GC)
Penicillium cyclopium (G)
Candida antarctica (S 435 L)
Candida lipolytica (L)
Pseudomonas sp. (AK)
Porcine pancreas (PPL)
Rhizopus ni6eus (N-conc)
Humicola sp. (CE-10)
Mucor meihei (M)
1
30
23
7
47
8
6 (S)
–
20 (S)
14 (S)
17 (S)
5 (S)
3 (R)
–
1.2
–
f
f
50
30
43
10
33
71
19
51
22
14
43
25
52
26
40
43
35
20 (R)
10 (R)
22 (R)
45 (R)
32 (R)
9 (R)
1.8
2.2
1.8
2.8
2.3
1.5
4.5
1.4
1.8
–
2.1
4.4
1.1
2.0
1.3
1.7
4.4
8
8.5
8.5
0.25
32
1
1.5
7.5
2
6
24
5
5
3
16 (S)
23 (S)
14 (S)
11 (S)
7 (S)
60 (R)
11 (R)
25 (R)
g
g
–
–
20 (R)
19 (R)
7 (R)
10 (R)
6 (R)
26 (S)
57 (S)
6 (S)
28 (S)
9 (S)
Humicola sp. (R-10)
Mucor ja6anicus (MAP)
Penicillium cyclopium (G)
Pseudomonas sp. (K-10)
Pseudomonas sp. (AK)
14 (R)
29 (R)
18 (S)
54 (S)
1.5
a
Substrate:lipase=1:5 (w/w).
C=conversion determined by HPLC.
E.e. of the unreacted substrate determined by HPLC.
b
c
d
E.e. of the product calculated according to the following equation: C=e.e. /(e.e. +e.e. ), where e.e. is the enantiomeric excess of remaining
s
s
p
s
unreacted alcohol and e.e.p is the enantiomeric excess of the produced ester.
E=enantioselectivity factor.
Both unreacted substrate and product were recovered as a racemate. Humicola sp. (CE-10), Pseudomonas fluorescens (Sigma), and Candida
cylindracea (Meito-Sanjo) afforded the same results.
Both unreacted substrate and product were recovered as a racemate. Aspergillus niger (AP), Geotrichum candidum (GC), Rhizopus delemar, and
Pseudomonas fluorescens (Sigma) afforded the same results.
e
f
g

S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
857
Scheme 5.
ranged between 5% (lipase from Geotrichum candidum)
and 23% (lipase from Candida antartica), while the e.e.p
varied between 3% (using lipase from Candida cylin-
dracea) and 60% (with lipase from Candida lipolytica),
ously (Tables 1 and 2) and no marked difference was
seen in the reaction performed with both anhydrides in
the presence or absence of KHCO
. E.e. values of
3
s
3
–76% for 6 and 23–78% for 8 were obtained.
and E=1.2–4.5; whereas for 8: e.e. ranges between 6%
s
In summary, using the methods presented, 5a can be
prepared with an e.e. of up to 86%, for 4a and 7 the e.e.
was close to zero, while for 3a, 6 and 8 the e.e.s ranged
from 10 to 80%. Such results could be improved by
recycling the reaction product, as has been done
already for other substrates and also for analogues of
compound 4, which is an intermediate for the synthesis
(
lipase from Penicillium cyclopium) and 29% (lipase
from Pseudomonas sp.-AK), e.e. =between 6% (lipase
p
from Humicola sp.) and 57% (lipase from Mucor mei-
hei), and E=1.1–4.4. The reaction with 7 was unselec-
tive and always afforded the racemic unreacted
substrate and a racemic product. By comparing the
results in Tables 1 and 2, it can be seen that the
hydrolysis of substrates 3a–5a gave higher e.e. values
than the transesterification reactions of 6–8 with vinyl
acetate.
43
of natural tocols.
The enzymatic hydrolyses (Scheme 2 and Table 1) are
complementary to the enzymatic transesterifications
(
Schemes 4 and 5, Tables 2 and 3), in terms of stereo-
chemical preference. In fact, all of the lipases examined
preferentially promoted the hydrolysis of (S)-5-chloro-
2
ester 3a. The same hydrolysis reactions performed on
the benzopyran derivative (±)-6-chloro-2,3-dihydro-4H-
1-benzopyran-3-carboxylic acid methyl ester 5a pro-
ceeded with an (R)-preference.
,3-dihydro-1-benzofuran-2-carboxylic acid methyl
Table 3. Enzymatic acylation of (9)-2-hydroxymethyl-5-chloro-2,3-dihydro-1-benzofuran 6 and (9)-2-hydroxymethyl-6-
a
chloro-2,3-dihydro-4H-1-benzopyran 8 using succinic or acetic anhydride in TBME in the presence of lipase
Comp.
Lipase (source)
Reaction time (h)
Cb (%)
E.e.sc (%)
E.e.pc (%)
Ed
(
absolute configuration)
6
6
6
6
6
6
8
8
8
Candida cylindracea (AY)
Pseudomonas sp. (K-10)
24
24
24
24
25
48
24
48
48
37
67
34
54
55
26
37
70
70
11 (S)
66 (S)
38 (S)
76 (S)
31 (S)
3
23 (R)
78 (R)
74 (R)
19 (R)
32 (R)
74 (R)
65 (R)
25 (R)
8 (R)
39 (S)
34 (S)
60 (S)
1.6
3.6
4.8
2.4
2.2
1.2
1.8
4.3
3.9
e
Pseudomonas sp. (K-10)
Pseudomonas sp. (K-10)
e,f
Pseudomonas sp. (AK)
Rhizopus delemar
Candida cylindracea (AY)
Pseudomonas sp. (AK)
Pseudomonas sp. (K=10)
a
Substrate:lipase=1:1 (w:w). Unless otherwise indicated the acyl donor used was the succinic anhydride.
C=conversion determined by HPLC.
b
c
E.e. is the enantiomeric excess of remaining unreacted alcohol and e.e. is the enantiomeric excess of the produced ester, both determined by
s
p
HPLC.
E=enantioselectivity factor.
KHCO3 was added to the medium in ratio 1:1 (w/w) with the substrate.
Acetic anhydride was used.
d
e
f

8
58
S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
a
Table 4. Chromatographic data
Comp.
Abs. conf.
tR
k%
k%%
h
Mobile phase, flow rate, UV-u
3
3
3
4
4
5
5
5
6
8
1
1
1
a
b
c
(S)-(+)
20.05
23.22
18.51
14.18
21.28
23.83
9.33
13.07
12.07
10.51
14.54
15.33
9.61
10.00
14.00
14.97
23.02
18.19
34.55
31.61
13.30
14.12
40.81
32.71
39.01
34.84
2.13
0.92
3.46
0.44
1.38
0.95
1.40
2.50
4.05
3.04
1.42
1.35
4.01
2.62
1.50
3.99
1.02
1.74
1.06
1.50
2.74
5.40
3.42
1.57
1.93
4.61
1.23
1.63
1.15
2.31
1.26
1.18
1.07
1.09
1.33
1.12
1.10
1.43
1.15
Hexane-iso-propanol (98:2), flow 0.8 mL/min, u=230 nm
Hexane-iso-propanol-trifluoroacetic acid (90:10:0.75), flow 0.7 mL/min, u=280 nmb
Hexane-iso-propanol (99:1), flow 0.8 mL/min, u=280 nm
Hexane-iso-propanol (90:10), flow 0.8 mL/min; u=230 nm
Hexane-iso-propanol-trifluoroacetic acid (90:10:0.5), flow 1 mL/min, u=230 nm
Hexane-iso-propanol (98:2), flow 0.8 mL/min, u=230 nmc
Hexane-iso-propanol-trifluoroacetic acid (99:1:0.1), flow 0.5 mL/min, u=254 nm
Hexane-iso-propanol (99:1), flow 1 mL/min, u=280 nm
Hexane-iso-propanol (95:5), flow 1 mL/min, u=230 nmd
Hexane-iso-propanol (95:5), flow 0.5 mL/min, u=230 nmd
Hexane-iso-propanol (98:2), flow 1 mL/min, u=254 nm
Hexane-iso-propanol (95:5), flow 0.7 mL/min, u=230 nmf
Hexane-iso-propanol (90:10), flow 0.5 mL/min, u=230 nmg
(
R)-(−)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(S)-(−)
R)-(+)
(S)-(−)
R)-(+)
(S)-(−)
R)-(+)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(S)-(+)
R)-(−)
(
(
a
b
a
b
c
(
(
(
(
(
(
(
0
1
2
(
e
e
(
(
a
b
c
Stationary phase=Chiralcel OD (Daicel), t =retention time (min), k% and k%%=capacity factors of the first and second eluted enantiomer,
respectively, h=separation factor.
R
By using hexane–isopropanol–trifluoroacetic acid (95:5:0.75), flow 1.0 mL/min, u=280 nm, 3a and 3b were eluted in the same analysis: (S)-3a
t =9.35 min, (R)-3a t =10.19 min, (S)-3b t =18.75 min, (R)-3b t =13.61 min.
R
R
R
R
By using hexane–isopropanol–trifluoroacetic acid (90:10:5.0), flow 0.4 mL/min, u=230 nm, 5a and 5b were eluted in the same analysis: (S)-5a
t =21.45 min, (R)-5a t =23.95 min, (S)-5b t =17.91 min, (R)-5b t =22.95 min.
R
R
R
R
d
Chromatogram of the transesterification reaction crude with vinyl acetate (Scheme 4) or acetic anhydride (Scheme 5) contained also 9 or 10 not
resolved with t =10.29 min and t =11.50 min, respectively.
R
R
e
f
Analysed after its conversion into methyl ester by CH N .
2
2
The analysis could be performed directly on the reaction crude (Scheme 5) after its treatment with CH N [hexane–isopropanol (90:10), flow 1.0
2
2
mL/min, u=230 nm]: (S)-6 t =42.67 min, (R)-6 t =34.29 min, (S)-11 methyl ester t =44.04 min, (R)-11 methyl ester t =52.23 min.
R
R
R
R
g
The analysis could be performed directly on the reaction crude (Scheme 5) after its treatment with CH N [hexane–isopropanol (90:10), flow 1.0
2
2
mL/min, u=230 nm]: (S)-8 t =39.01 min, (R)-8 t =34.84 min, (S)-12 methyl ester t =20.57 min, (R)-12 methyl ester t =18.98 min.
R
R
R
R
In contrast, in the case of the transesterification
reactions, (R)-2-hydroxymethyl-5-chloro-2,3-dihydro-
per million (l ppm). Absolute values of the coupling
constant (J) are reported. IR spectra were recorded on
a Perkin–Elmer 681 spectrometer. GC analyses were
performed by using a HP1 column (methyl silicone
gum; 5 m×0.53 mm×2.65 mm film thickness) on a HP
1
-benzofuran 6 and (S)-2-hydroxymethyl-6-chloro-2,
3-dihydro-4H-1-benzopyran 8 were preferred, respec-
tively.
5
890 model, Series II. HPLC analyses (Table 4) for the
determination of e.e.s were carried out using a
DAICEL Chiralcel OD column (tris-3,5-di-
The pharmacological activity of compounds 3–5 and
6–8 is currently under evaluation and will be reported
methylphenylcarbamate, derivatised cellulose film) on a
HP series 1050 instrument. Optical rotation measure-
ments were obtained using a Perkin–Elmer digital
polarimeter, model 241 MC. Thin-layer chromatogra-
phy (TLC) was performed on silica gel sheets with
fluorescent indicator (Statocrom SIF, Carlo Erba), the
spots on the TLC were observed under ultraviolet light
separately.
3. Experimental
3.1. General methods
or were visualised with I vapour. Flash chromatogra-
2
phy was conducted by using silica gel with an average
particle size of 60 mm, a particle size distribution of
40–63 mm and 230–400 ASTM. GC–MS analyses were
performed on a HP 5995C model and microanalyses on
an elemental analyser 1106-Carlo Erba-instrument.
Melting points were taken on electrothermal apparatus
and are uncorrected. H NMR spectra were recorded in
1
CDCl on a Varian EM 390 or Mercury 300 MHz
3
spectrometer and chemical shifts are reported in parts

S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
859
3
.2. Materials
30 min and evaporated under reduced pressure. The
resulting oil was dissolved in chloroform and washed
with 5% aqueous NaHCO3 to remove trace of the
unreacted acid. The chloroform solution was dried over
anhydrous Na SO and then evaporated, affording the
The enzymes used in this paper were obtained from
either Amano Enzyme Co., Meito Sangyo, or Sigma
Chemical Co. All other chemicals and solvents were of
the highest quality grade available and purchased from
Aldrich Chemical Co. or Sigma Chemical Co.
2
4
ester as an oil (yield 93–98%).
3
.5.1. (±)-5-Chloro-2,3-dihydro-1-benzofuran-2-carbox-
1
ylic acid methyl ester 3a. H NMR (CDCl , l): 3.35–
3
3
.3. Resolution of (±)-5-chloro-2,3-dihydro-1-benzofuran-
3.60 (m, 2H, CH ); 3.80 (s, 3H, CH ); 5.15–5.38 (m,
2 3
2
-carboxylic acid 3b
1
H, CH); 6.81–7.30 (m, 3H, aromatic protons). GC–
+
MS (70 eV) m/z (rel. int.): 212 (M , 79), 125 (100).
9
The racemic acid (11.2 g, 0.056 mol) and (−)-
amphetamine (7.6 g, 0.056 mol) were mixed and crys-
tallised from EtOH. After three crystallisations the salt
had [h]D −17.0 (c 1.0, MeOH), which remained con-
stant after two more crystallisations. This salt was
3
.5.2. (±)-6-Chloro-2,3-dihydro-4H-1-benzopyran-2-car-
1
boxylic acid methyl ester 4a. H NMR (CDCl , l):
3
2
2
1
.05–2.35 (m, 2H, CH CHCOOCH ), 2.60–2.91 (m,
2 3
H, ArCH CH ), 3.81 (s, 3H, COOCH ), 4.65–4.85 (m,
2
2
3
treated with 5% H SO4 and extracted with CHCl3
2
H, CHCOOCH ), 6.80–7.21 (m, 3H, aromatic pro-
tons). GC–MS (70 eV) m/z (rel. int.): 226 (M , 64), 167
100).
3
affording the dextro-acid, which was recrystallised from
CHCl :petroleum ether; mp 123–125°C; [h] =+14.7 (c
+
3
D
(
1.0, MeOH). The mother liquors from the first crystalli-
sation of the (−)-amphetamine salt were evaporated to
3
.5.3. (±)-6-Chloro-2,3-dihydro-4H-1-benzopyran-3-car-
1
dryness; the residue was treated with 5% H SO and
2
4
boxylic acid methyl ester 5a. H NMR (CDCl , l): 3.05
3
extracted with CHCl obtaining a partially enriched
3
(
(
(
d, 2H, ArCH CH), 3.71 (s, 3H, COOCH ), 4.10–4.61
m, 3H, OCH CH and, CH CHCOOCH ), 6.75–7.40
m, 3H, aromatic protons). GC–MS (70 eV) m/z (rel.
2
3
mixture of the levo-acid, which was mixed with an
equivalent amount of (+)-amphetamine and crystallised
from EtOH. After two crystallisations, the salt had
2
2
3
+
int.): 226 (M , 83), 166 (100).
[
5
h] =+17.0 (c 1.0, MeOH). This salt was treated with
D
% H SO and extracted with CHCl3 affording the
2 4
3
.6. General procedure for the esterification of (±)-5-
levo-acid which was crystallised from CHCl :petroleum
3
chloro-2,3-dihydro-1-benzofuran-2-carboxylic acid 3b,
1
ether; mp 123–125°C; [h] =−14.5 (c 1.0, MeOH). H
D
(
±)-6-chloro-2,3-dihydro-(4H)-1-benzopyran-3-carboxylic
NMR (CDCl , l): 3.33–3.66 (m, 2H, CH ); 5.25 (dd,
3
2
acid 5b with allyl alcohol
1
6
H, CH); 5.84 (bs, 1H, COOH: exchanged with D O);
.77–7.20 (m, 3H, aromatic protons).
2
A solution of the acid (1.4 mmol) and SOCl2 (1.6
mmol) was stirred under reflux for 5 h. After cooling at
room temperature, the reaction mixture was concen-
trated and the crude acyl chloride was dissolved in allyl
alcohol (4.2 mmol). The solution was stirred for 1 h at
room temperature. Then, the solution was concentrated
under reduced pressure. The resulting oil was dissolved
in chloroform, washed with 10% aqueous NaHCO and
then with H O. The organic phase was dried over
anhydrous Na SO and concentrated affording the ester
as a colourless oil (yield 80–85%).
3
.4. Assignment of the absolute configuration to 3b
(
Scheme 1)
The dextro-acid (430 mg, 2.17 mmol) with [h] +12.3 (c
D
1
.5, MeOH) was dissolved in EtOH (15 mL) and hydro-
3
genated at atmospheric pressure overnight in the pres-
ence of catalytic 5% Pd/C at room temperature. The
solution was filtered through Celite and the solvent
removed under reduced pressure. The residue was dis-
2
2
4
solved in Et O and extracted with 10% NaHCO3
2
aqueous solution. The aqueous phase was acidified with
3
.6.1. (±)-5-Chloro-2,3-dihydro-1-benzofuran-2-carbox-
1
2
N HCl and extracted with Et O. The organic layer
2
ylic acid, allyl ester 3c. H NMR (CDCl , l): 3.32 (dd,
3
was washed with water, dried over anhydrous Na SO4
2
1
H, J=10.5 Hz and 16.0 Hz, benzylic CH ); 3.50 (dd,
2
and evaporated to dryness affording a solid (100 mg)
4
0 1
1H, J=9.6 Hz and 16.0 Hz, benzylic CH ); 4.65 (d, 2H,
2
with [h] =−16.0 (c 1.1, EtOH). H NMR (CDCl , l):
D
3
CH CHꢀCH2); 5.10–5.38 (m, 3H, CHꢀCH and OCH-
2
2
3
.35–3.69 (m, 2H, CH ); 5.24 (dd, 1H, CH); 5.65 (bs,
2
COO); 5.78–5.98 (m, 1H, CHꢀCH ), 6.70–7.15 (m, 3H,
2
1H, COOH); 6.85–7.22 (m, 4H, aromatic protons).
aromatic protons). GC–MS (70 eV) m/z (rel. int.): 238
+
(
M , 50), 125 (100).
3
.5. General procedure for the esterification of (±)-5-
chloro-2,3-dihydro-1-benzofuran-2-carboxylic acid 3b,
(
±)-6-chloro-2,3-dihydro-4H-1-benzopyran-2-carboxylic
acid 4b and (±)-6-chloro-2,3-dihydro-4H-1-benzopyran-
-carboxylic acid 5b with CH N2
3.6.2. (±)-6-Chloro-2,3-dihydro-4H-1-benzopyran-3-car-
1
boxylic acid, allyl ester 5c. H NMR (CDCl , l): 2.90–
3
3
3.25 (m, 2H, benzylic CH ); 3.95–4.92 (m, 3H,
2
2
COOCH and CHCOO); 4.50–4.72 (m, 2H, OCH );
2
2
A slight excess of diazomethane in ethereal solution,
was added to a solution of the acid in ethyl ether. The
resulting solution was stirred at room temperature for
5.18–5.50 (m, 2H, CHꢀCH ); 5.68–6.25 (m, 1H,
2
CHꢀCH ), 6.65–7.25 (m, 3H, aromatic protons). GC–
2
+
MS (70 eV) m/z (rel. int.): 252 (M , 2), 167 (100).

8
60
S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
3.7. Enzymatic enantioselective hydrolysis of 3a–5a
3.9. Reduction of (±)-6-chloro-2,3-dihydro-4H-1-ben-
zopyran-2-carboxylic acid methyl ester 4a with BMS
The reaction mixture containing the powdered enzyme
1 g) in phosphate buffer, (0.2N, 20 mL, pH 7.0), the
racemic substrate (200 mg), CaCl (10 mg) and NaCl
(
A solution of methyl ester (2.74 mmol) and BMS (5.48
mmol) in anhydrous THF (10 mL) was stirred under
2
(
30 mg) was placed in a screw-cap bottle and shaken on
reflux for 5 h. After cooling to room temperature H O
2
an orbit shaker at 250 rpm and 37°C. After the time
specified in Table 1, the reaction mixture was extracted
(0.57 mL) was added followed by 3N aqueous NaOH
(0.4 mL) and 30% aqueous H O2 (0.4 mL). After
2
with CHCl . The organic phase was washed with 10%
stirring at room temperature for 0.5 h, the reaction
mixture was extracted with ethyl acetate. The organic
layer was dried over anhydrous Na SO and the solvent
3
aqueous NaHCO , dried over anhydrous Na SO and
3
2
4
the solvent evaporated under reduced pressure. The
residue consisted of the remaining unreacted ester. The
aqueous phase was acidified with 6N HCl and then
2
4
evaporated under reduced pressure to afford a colour-
less oil (yield 96%).
extracted three times with CHCl . The chloroform solu-
3
tion was dried over anhydrous Na SO and concen-
2
4
3
.9.1. (±)-2-Hydroxymethyl-6-chloro-2,3-dihydro-4H-1-
trated under reduced pressure to afford a white solid
residue. The recovered substrates and products were
then analysed by HPLC (Table 4) for the determination
of the e.e. and e.e. (the product was methylated by
1
benzopyran 8. H NMR (CDCl , l): 1.73–1.99 (m, 2H,
3
CH CHCH OH); 2.00–2.25 (bs, 1H, OH, D O
exchanged); 2.66–2.91 (m, 2H, benzylic CH ); 3.74 (dd,
2
2
2
2
s
p
1
H, J=12.0 Hz and 6.0 Hz, CH OH); 3.83 (dd, 1H,
2
CH N treatment at 0°C and then used for the determi-
2
2
1
J=12.0 Hz and 3.5 Hz, CH OH), 4.03–4.13 (m, 1H,
2
nation of e.e. values), and by H NMR to compare
p
CHCH OH), 6.70–7.05 (m, 3H, aromatic protons).
GC–MS (70 eV) m/z (rel. int.): 198 (M , 78), 167 (100).
2
their spectra with those of authentic samples. Reaction
times, e.e. and E values for the three compounds are
reported in Table 1.
+
3
.10. Lipase-catalysed acylation of 6–8 using vinyl ace-
Similar conditions were used to hydrolyse allyl esters 3c
and 5c. In these reactions 15% of methanol was added
as co-solvent. Enantiomeric excesses were determined
as reported in Table 4.
tate in hexane
Crude lipase (500 mg) and vinyl acetate (1 mL) were
added to a solution of racemic alcohol (100 mg) in
hexane (10 mL). The heterogeneous mixture was incu-
bated at 37°C with stirring at 250 rpm. The reaction
was followed by TLC and stopped at the time indicated
in the Table 2. The mixture was filtered through a
sintered glass funnel to recover the enzyme extract. The
hexane was removed under reduced pressure. Product
and remaining unreacted substrate were separated by
chromatography (silica gel, eluent: petroleum
ether:ethyl acetate=8:2). The enantiomeric excesses
were determined by chiral HPLC (Table 4) on a column
supplied from DAICEL (Chiralcel OD) on the mixture
containing the remaining substrate and product, just
after the filtration. The extent of the conversion was
also determined from the same HPLC chromatogram.
3.8. General procedure for the reduction of (±)-6-
chloro-2,3-dihydro-1-benzofuran-2-carboxylic acid
methyl ester 3a and (±)-6-chloro-2,3-dihydro-4H-1-ben-
zopyran-3-carboxylic acid methyl ester 5a with LiAlH4
A suspension of the methyl ester (2.35 mmol) and
LiAlH (3.06 mmol) in benzene (14 mL) was stirred
4
under reflux and N atmosphere for 5 h. After cooling
2
at room temperature, H O was added to the reaction
2
mixture which was then extracted with ethyl acetate.
The organic phase was dried over anhydrous Na SO4
2
and the solvent was evaporated under reduced pressure
affording a colourless oil (yield 93%).
The e.e. of the remaining alcohol (e.e. ) and the extent
s
of the conversion (C) were used to calculate the enan-
tioselectivity factor E (Table 2).
3
.8.1. (±)-2-Hydroxymethyl-5-chloro-2,3-dihydro-1-ben-
1
zofuran 6. H NMR (CDCl , l): 1.79 (bs, 1H, OH, D O
3
2
exchanged); 3.01 (dd, 1H, J=16.0 Hz and 7.6 Hz,
benzylic CH ); 3.22 (dd, 1H, J=16.0 Hz and 9.5 Hz,
2
3
.11. Enzymatic acylation using succinic (or acetic)
benzylic CH ); 3.70 (dd, 1H, J=12.0 Hz and 6.0 Hz, 1
2
anhydride in TBME
of CH OH); 3.84 (dd, 1H, J=12.0 Hz and 3.3 Hz, 1 of
2
CH OH); 4.86–4.97 (m, 1H, CH OCH); 6.65–7.13 (m,
2
2
To a TBME (10 mL) solution of the substrate (±)-6
3
H, aromatic protons). GC–MS (70 eV) m/z (rel. int.):
84 (M , 100), 165 (85), 125 (99).
(
100 mg) or (±)-8 (100 mg) and acid anhydride (100 mg)
+
1
was added lipase (100 mg). When KHCO was added
3
(
100 mg), a finely powdered anhydrous compound was
3
.8.2. (±)-3-Hydroxymethyl-6-chloro-2,3-dihydro-4H-1-
used. The mixture was shaken at 250 rpm on a rotatory
shaker at 35°C for the time indicated in Table 3. After
filtration of the solid (lipase powder and KHCO ), H O
1
benzopyran 7. H NMR (CDCl , l): 2.18–2.40 (bs, 1H,
3
OH, D O exchanged); 2.45–2.76 (m, 1H, benzylic CH );
2
2
3
2
2
.78–3.10 (m, 1H, benzylic CH ), 3.50–3.88 (m, 3H,
and ethyl acetate were added to the organic solution.
The organic layer was extracted with 10% NaHCO3
aqueous solution, dried over anhydrous Na SO and
evaporated under reduced pressure affording the unre-
acted alcohol. The alkaline solution was acidified with
2
OCH CH); 3.90–4.15 (m, 1H, 1 of CH OH); 4.18–4.45
2
2
(
m, 1H, 1 of CH OH); 6.60–7.20 (m, 3H, aromatic
2
+
2
4
protons). GC–MS (70 eV) m/z (rel. int.): 198 (M , 65),
67 (100).
1

S. Ferorelli et al. / Tetrahedron: Asymmetry 12 (2001) 853–862
861
6
N HCl and extracted three times with ethyl acetate.
3.13.1. (±)-Mono (5-chloro-2,3-dihydro-1-benzofuran-2-
1
The organic layers were dried and the solvent was
evaporated, affording the acid as a white solid. For the
reaction performed with acetic anhydride, TBME was
removed under reduced pressure after filtration of the
lipase powder. Product and unreacted substrate were
separated by chromatography (silica gel, eluent:
petroleum ether:ethyl acetate=8:2). Reaction times and
e.e.s are reported in Table 3 (see Table 4 in which are
listed the HPLC conditions for e.e. determination).
methyl)butanedioate 11. H NMR (CDCl , l): 2.55–2.75
3
(m, 4H, CH CH COOH); 2.97 (dd, 1H, J=16.0 Hz and
2
2
7.4 Hz, benzylic CH ); 3.20–3.35 (dd, 1H, J=16.0 Hz
2
and 9.6 Hz, benzylic CH ); 4.18–4.26 (dd, 1H, J=12.0
2
Hz and 6.4 Hz, CH OCO); 4.28–4.38 (dd, 1H, J=12.0
2
Hz and 3.6 Hz, CH OCO); 4.92–5.05 (m, 1H, CH);
2
6.65–7.25 (m, 3H, aromatic protons), 7.5–9.4 (bs,
COOH, D O exchanged). GC–MS (70 eV) m/z (rel.
2
+
int.): 284 (M , 2), 166 (100), 153 (15), 131 (25), 101 (27);
+
(
methyl ester) 298 (M , 34), 166 (100).
3
.12. General procedure for the esterification of
3
.13.2. (±)-Mono (6-chloro-2,3-dihydro-4H-1-benzopy-
1
racemic alcohols (±)-6–8 with acetic anhydride
ran-2-methyl)butanedioate 12. H NMR (CDCl , l):
3
1
.62–1.90 (m, 1H, 1 of CH CH Ar); 1.93–2.06 (m, 1H,
2 2
A slight excess of acetic anhydride (10 mL) was added
to a solution of racemic alcohol (100 mg) in pyridine
CH CH Ar); 2.58–2.94 (m, 6H, CH CH COOH and
2
2
2
2
benzylic CH ); 4.15–4.27 (m, 1H, CH); 4.29–4.35 (m,
2
2
(
10 mL). The resulting solution was stirred at room
H, CH OCO); 6.69–7.08 (m, 3H, aromatic protons);
2
temperature for 1 h. Then, CHCl (10 mL) was added.
3
8
.50–9.95 (bs, COOH, D O exchanged). GC–MS (70
2
The organic solution was washed three times with H O,
dried over anhydrous Na SO and concentrated afford-
ing an oil. Chromatography of the residue using silica
gel and ethyl acetate:petroleum ether (1:4) afforded the
ester as a colourless oil (yield 95–98%).
2
+
eV) m/z (rel. int.): (methyl ester) 312 (M , 34), 180
2
4
(
100).
Acknowledgements
3
.12.1. (±)-2-Methylacetoxy-5-chloro-2,3-dihydro-1-ben-
1
zofuran 9. H NMR (CDCl , l): 2.11 (s, 3H, CH ); 2.96
3
3
This work was supported by the Ministero dell’Univer-
sit a` e della Ricerca Scientifica e Tecnologica (MURST,
Rome), and by the University of Bari and CNR-MISO
(Bari). The authors wish to express their cordial thanks
to Amano Pharm. Co. Ltd. (Japan) for the generous
donation of lipases.
(
(
(
dd, 1H, J=16.0 Hz and 7.4 Hz, benzylic CH ); 3.32
dd, 1H, J=16.0 Hz and 9.0 Hz, benzylic CH ); 4.23
dd, 1H, J=12.0 Hz and 7.0 Hz, CH OCO); 4.37 (dd,
2
2
2
1
H, J=12.0 Hz and 3.5 Hz, CH OCO); 4.92–5.12 (m,
2
1
H, OCH); 6.68–7.22 (m, 3H, aromatic protons). GC–
+
MS (70 eV) m/z (rel. int.): 226 (M , 27), 165 (100).
References
3
.12.2. (±)-2-Methylacetoxy-6-chloro-2,3-dihydro-4H-1-
1
benzopyran 10. H NMR (CDCl , l): 1.15–1.40 (m, 2H,
3
1
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3
2
2
2
+
(
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(
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8
1
3
.13. General procedure for the esterification of
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added. The organic solution was washed three times
with H O, dried over anhydrous Na SO and concen-
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residue using silica gel and ethyl acetate:petroleum
ether:acetic acid (1:1:0.1) afforded the ester as a colour-
less oil (yield 90–95%).
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