Synthesis of conformationally restricted acetylcholine analogues. Comparing lipase-mediated resolution with simulated moving bed chromatography of arylated β-hydroxy-pyrrolidines

Article abstract of DOI:10.1016/j.tetasy.2007.01.030

The enantiodivergent synthesis of new, conformationally restricted acetylcholine analogues was accomplished using arylated endocyclic enecarbamates as key intermediates. Stereoselective hydroboration of the aryl enecarbamates provided the corresponding ar

Full text of DOI:10.1016/j.tetasy.2007.01.030

Tetrahedron: Asymmetry 18 (2007) 435–442
Synthesis of conformationally restricted acetylcholine
analogues. Comparing lipase-mediated resolution
with simulated moving bed chromatography
of arylated b-hydroxy-pyrrolidines
a
a
b
a,
*
´
Ricardo de L. Barreto, Marcos J. S. Carpes, Cesar C. Santana and Carlos R. D. Correia
aChemistry Institute, State University of Campinas—UNICAMP, C.P. 6154, CEP 13084-971, Campinas, Sa˜o Paulo, Brazil
bSchool of Chemical Engineering, State University of Campinas—UNICAMP, C.P. 6066, CEP 13093-970, Campinas, Sa˜o Paulo, Brazil
Received 2 January 2007; accepted 29 January 2007
Abstract—The enantiodivergent synthesis of new, conformationally restricted acetylcholine analogues was accomplished using arylated
endocyclic enecarbamates as key intermediates. Stereoselective hydroboration of the aryl enecarbamates provided the corresponding
aryl-b-hydroxy-pyrrolidines. Acetylation, deprotection, followed by N-bismethylation, led to the desired betaine products. The kinetic
resolution of the intermediate alcohols was performed by lipase-mediated hydrolysis of its acetate derivatives, resulting in excellent
enantioselectivities (E > 100). An enzymatic enantiopreference predicted by the Kazlauskas’s model was confirmed following Riguera’s
protocol. Finally, chromatographic resolution of racemic alcohol 5a was evaluated by semi-preparative scale chiral simulated moving
bed chromatography and its performance compared with biocatalysis.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Me
O
O
NH2
Acetylcholine (AcCh, Fig. 1) is an important neurotrans-
mitter acting at the central nervous system and the neuro-
muscular junction. Its deficiency in the cholinergic system1
is thought to be one of the causes of Alzheimer’s disease
(AD). AD is a severe neurodegenerative disorder leading
to a characteristic deterioration of intellectual capacity,
executive functions and personality changes.2
O
N
N
Me
Tacrine
Donepezil
CH3
H3C N
O
O
CH3
H3C
OH
AcCh
Alzheimer’s disease is the most common cause of dementia
in the elderly and experts estimate that 22 million people
around the world will be afflicted with AD by 2025.3 In
the United States, the annual direct and indirect costs
involved in caring for people with AD approached 100
billion dollars in 1991.4 One of the current therapeutic
treatments consists of the use of acetylcholinesterase
(AcChE) inhibitors,5 which increase the efficiency of
cholinergic transmission by preventing the hydrolysis of
O
Me
Me
O
Me
O
N
Me
N
Et
Me
O
N
Rivastigmine
Me
Galanthamine
Figure 1. Some examples of AcChE inhibitors.
released AcCh, thus raising its level at the cholinergic
synapse.
*
The cholinergic concept has led to the development
and commercialization of several drugs (Fig. 1) for the
Corresponding author. Tel.: +55 19 3788 3114; fax: +55 19 3788 3023;
e-mail: roque@iqm.unicamp.br
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.01.030

436
R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
symptomatic treatment of AD: tacrine (CognexÒ, Warner–
Lambert), donepezil (AriceptÒ, Eisai/Pfizer), rivastigmine
(ExelonÒ, Novartis) and galanthamine (Reminyl Ò, Shire/
Johnson & Johnson).
arylation of N-Cbz-3-pyrroline 2 with arenediazonium
tetrafluoroborates, and dehydration of the intermediate
lactamol 3 to give the arylated endocyclic enecarbamates
4a–d (Scheme 2).
Based on these considerations, we envisaged the synthesis
of new conformationally restricted AcCh analogues as
potential AcChE inhibitors. The synthetic route involved
an arylated endocyclic enecarbamate as a key intermediate,
which can be obtained in a few steps from commercial di-
allylamine, followed by the Heck arylation of 3-pyrroline, a
protocol previously developed in our research group6
(Scheme 1).
Ar
Ar
b
a
c
OH
N
N
N
N
Cbz
4a-d
Cbz
2
Cbz
3a-d
Cbz
1
30-75%
(over 3 steps)
Ar = a:
b:
p
p
p
-ClC6H4
-MeOC6H4
-NO2C6H4
-Naphthyl
c:
d: β
Ar
OAc
I -
Ar
Scheme 2. Reagents and conditions: (a) [Ru], CH2Cl2, rt, 5 h; (b)
Pd(OAc)2, ArN2BF4, CH3CN/H2O, rt, 3 h; (c) (CF3CO)2O, 2,6-lutidine,
0 °C to rt, 2 h, reflux, 30 min.
N
N
N
H
Me Me
( )
Cbz
Enecarbamates 4a–d were then subjected to hydrobora-
tion–oxidation, providing exclusively ( )-(trans)-alcohols
5a–d. As described in the literature,12 hydroboration of
substituted enecarbamates occurs in the b position and in
a stereoselective manner, resulting in the preferential attack
of the borane on the less esterically hindered side of the
double bond. Acetylation of alcohols 5a–d resulted in the
racemic acetate derivatives ( )-trans-6a–d (Scheme 3).
Diallylamine
Target-molecule
Enecarbamate
Scheme 1. The synthetic strategy.
Due to the increasing interest of the pharmaceutical indus-
tries in single enantiomers of chiral drugs,7 there has also
been special interest in the association of biocatalysis and
simulated moving bed (SMB) technology to organic syn-
thesis as an alternative method to asymmetric synthesis.
Ar
Ar
OAc
Ar
OH
Over the last two decades, biocatalytic systems have gained
growing importance in organic synthesis for the prepara-
tion of chiral compounds.7,8 A great number of enzymes
are commercially available for a variety of biotransforma-
tions. In particular, lipases9 (triacylglycerol hydrolases,
E.C. 3.1.1.3) are often used for the kinetic resolution of
racemates.
a
b
N
N
N
Cbz
4a-d
Cbz
( )-6a-d
Cbz
( )-5a-d
Ar = a:
b:
p
-ClC6H4
-MeOC6H4
-NO2C6H4
p
c:
p
β
d: -Naphthyl
The SMB chromatography is a large-scale version of tradi-
tional high-performance liquid chromatography (HPLC),
but, unlike normal HPLC, it operates continuously in an
automated multicolumn chromatographic system, with
reduced solvent consumption and purification costs.10 This
process consists of simulating the countercurrent move-
ment of adsorbent, producing two outlet streams, one rich
in the more adsorbable component (extract stream) and the
other rich in the less adsorbable one (raffinate stream),
which is particularly appropriate for binary separations
such as racemates.11
Scheme 3. Reagents and conditions: (a) (1) BH3ÆSMe2, 0 °C to rt, 3 h, (2)
H2O2, NaOH, 0 °C (45 min), rt (45 min), 90%; (b) (CH3CO)2O, DMAP,
Pyr. CH2Cl2, rt, 2 h, 95%.
Acetate derivatives ( )-trans-6a–d were then submitted to
enzymatic hydrolysis (Scheme 4) using lipase Pseudomonas
cepacia13 (lipase PS from Amano Pharmaceutical Co.) to
furnish alcohols (3S,4R)-trans-5a–d and acetates (3R,4S)-
trans-6a–d with excellent enantioselectivities (Table 1).
The enzymatic enantiopreference was predicted to be (S)
by the Kaslauskas’s empiric model.14
Herein we report the synthesis of new conformationally
restricted AcCh analogues and compare the resolution
power of a lipase with that of the SMB in the separation
of alcohol intermediates employed in the synthesis of the
new AcCh analogues.
In most experiments, enantioselectivities were excellent
(E > 100), especially for the alcohol bearing the large b-
naphthyl substituent (entry 4), in accordance with the
Kazlauskas’s model. Conversions (C) below 50% (entry
1) lead to considerable losses in the ees values, while C val-
ues slightly above 50% raise ees and diminishes eep values
(entry 4).
2. Results and discussion
In order to confirm the foreseen enzymatic enantioprefer-
ence employing Kazlauskas’s model, we determined the
The synthesis features a ring-closing metathesis of the
N-Cbz-protected diallylamine 1, followed by the Heck

R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
437
stereomeric esters (RSS)-11 and (SRS)-12, it was possible
to calculate DdRS values of +0.30 for L1 and ꢀ0.26 for
L2 (Scheme 6), which is in good agreement with the (S)
enantiopreference as predicted by the Kazlaukas’s model.
Ar
OAc
Ar
OAc
Ar
OH
3
3
3
a
+
N
N
N
Cbz
( )-trans-6a-d
Cbz
(3R,4S)-6a-d
Cbz
)-5a-d
(3S,
4
R
We also compared the resolution performance of the lipase
with the SMB technology. For this purpose we ran a chro-
matographic resolution of racemic alcohol 5a (Ar = p-Cl–
C6H4) in a semi-preparative SMB equipment composed
of eight columns having cellulose tris(3,5-dimethylphenyl-
carbamate) as the chiral stationary phase. The SMB run
was performed with a mixture of hexane and isopropyl
alcohol (85:15, v/v) as eluent, a feed concentration of
2 g/L, and a flow rate of 0.1 mL/min. Under these opera-
tional conditions, we obtained the raffinate stream with
a:
b:
c:
Ar =
p
p
p
-ClC6H4
-MeOC6H4
-NO2C6H4
β
d: -Naphthyl
Scheme 4. Reagents and conditions: (a) Lipase PS-AK, phosphate buffer
(10% toluene), pH 7.0.
Table 1. Enzymatic hydrolysis of acetate derivatives 6a–da
the enantiomerically enriched alcohol (R)-(ꢀ)-5a in 96.9%
b
20
Entry
Ar
eep
eesc
Cd
Ee
ee and ½aꢁD = ꢀ19.0 (c 3.0, CH2Cl2). On the other hand,
1
2
3
4
p-ClC6H4
98.8
96.3
92.9
97.2
58.0
97.1
93.0
99.4
37.0
50.2
50.0
50.6
288
228
94
alcohol (S)-(+)-5a was obtained in the extract stream in
20
only 83.5% ee and ½aꢁD = +15.0 (c 3.0, CH2Cl2).17
p-MeOC6H4
p-NO2C6H4
b-Naphthyl
386
Comparing the results of the chiral SMB and biocatalysis,
in terms of productivity (grams of material processed per
day), for this particular molecule, SMB was a more effec-
tive methodology for the resolution of the enantiomers.
While the enzyme resolution required 30 days to process
0.5 g of racemic alcohol, the laboratory-scale SMB unit
was able to process the same amount in 2 days. Neverthe-
less, it is important to point out that there was a decrease in
enantioselectivity for the (S)-enantiomer (98.8–83.5% ee)
but a considerable increase for the (R)-enantiomer (58.0–
96.9% ee).
a Quantitative treatment of enzymatic kinetic resolutions developed by Sih
et al.15
b Enantiomeric excess (%) of the product.
c Enantiomeric excess (%) of the substrate.
d Extent of conversion (C), where C = ees/(ees + eep).
e Enantiomeric ratio (E), where E = ln[(1 ꢀ c)Æ(1 ꢀ ees)]/ln[(1 ꢀ c)Æ
(1 + ees)].
absolute configuration of the resolved alcohol 5b (p-MeO–
Ph) using Riguera’s protocol,16 after reacting it with (S)-
1
methoxyphenylacetic acid (MPA). The H NMR spectra
of the corresponding diastereomeric esters (RSS)-9 and
(SRS)-10 were rather complex, probably because of the
presence of rotamers. Therefore, we then removed the
Cbz protecting groups and compared the spectra of free
amines (RSS)-11 and (SRS)-12 (Scheme 5).
Despite the advantages of SMB chromatography, we found
that some separations can be excessively laborious in terms
of optimizing chromatographic parameters. This was the
case for the simpler alcohol ( )-N-Cbz-3-hydroxy-pyrrol-
idine 13. Various separation conditions were tested in
order to obtain acceptable resolutions (changes in eluent
polarity, flow rates, use of additives and different chiral
stationary phases), but all attempts were fruitless. On the
other hand, enzymatic resolution of pyrrolidine 13, via
Evaluating the differences in chemical shifts (Dd) between
the signals for hydrogens H-2 (L1 neighbourhood), H-4
and H-5 (L2 neighbourhood) from the corresponding dia-
Ph
Ph
Ar
Ar
OH
+
Ar
O
O
Ph
OMe
OMe
H
H
OH
a
H
O
O
b
MeO
N
N
N
H
O
Cbz
(3R,4S)-5b
Cbz
(RSS)-9
(RSS)-11
(S)-MPA
Ph
Ph
O
Ar
Ar
O
Ar
OH
OMe
Ph
OMe
H
H
OH
a
b
O
H
O
+
MeO
N
N
Cbz
N
H
O
Cbz
(SRS)-10
(SRS)-12
(S)-MPA
(3S,4R)-5b
Ar = p-CH3OC6H4
Scheme 5. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, 75%; (b) Pd(OH)2/C, H2, MeOH, rt, 2 h, 40%.

438
R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
L2
Ph
H
O
H
H
H
OMe
L1
H
4
H
Ar
L
MeO
Ar
5
s
3
H
O
1
N
Ar
L2
H
2
H
OMe 1
L2
H
H
H
sp
ap
L1
1
(R,S,S)-11
H
ΔδRS
ΔδRS
<
0
L2
RO
L
L1
1
>
0
L
L
1
1
H
H
O
H
H
Ph
OMe
L2
H
3
N1
H
Ar
MeO
Ar
2
s
H
O
H
4
Ar
L1
H
5
L
OMe 2
L1
H
H
H
ap
sp
L2
(S,R,S)-12
ΔδRS L1 = ΔδRS H-2 + ΔδRS H-2' = 0.22 + 0.08 = + 0.30
ΔδRS L2 = ΔδRS H-4 + ΔδRS H-5 + ΔδRS H-5'= - 0.17 + (+ 0.01) + (-0.09) = - 0.26
Scheme 6. Conformational models of the diastereomeric esters.
demanding (10 days to achieve 45% conversion). The
resolved acetylated alcohol (3R)-14 was obtained in
>99% ee and the remaining alcohol (3S)-13 in 81.3% ee.
OH
OAc
OH
3
3
3
a
+
N
N
N
Cbz
Cbz
14
Cbz
)-13
The absolute configuration was attributed by comparing
(+)-13
(3
R
)-
(3
S
the specific rotation obtained for acetate (3R)-14
-
20
½aꢁD = ꢀ13.5 (c 1.48, CH3OH) with that reported in the lit-
Scheme 7. Reagents and conditions: (a) lipase PS in sol gel AK, vinyl
acetate, CH2Cl2, 10 days.
erature.19 The Kazlauskas’s model was not applicable in
this case due to the similar sizes of the substituents.
Having enough quantity of the resolved alcohols (+)-
20
transesterification in organic solvent18 (Scheme 7), was very
effective in terms of enantioselectivity, but still time
(3S,4R)-5b (½aꢁD = +6.4 (c 1.6, CH2Cl2)) and (ꢀ)-(3R,
20
4S)-5b (½aꢁD = ꢀ6.7 (c 1.2, CH2Cl2)), we then synthesized
Ar
OAc
Ar
OAc
Ar
OH
Ar
OAc
I-
3
a
b
c
N
N
N
H
N
Me Me
Cbz
Cbz
(3S,
4
R
)-7b
8b
R)-
6b
(3S,4R)-
(-)-(3S,
Ar
4
5b
R)-
(+)-(3S,
4
Ar
OH
Ar
OAc
Ar
Ac
OAc
I-
3
b
c
a
N
N
N
H
4
N
Me Me
Cbz
)-6b
Cbz
(-)-(3R,
8b
(+)-(3R,4S)-
(3R,
S
)-7b
4
S
)-5b
(3R,4S
Ar = p-CH3OC6H4
Scheme 8. Reagents and conditions: (a) (CH3CO)2O, DMAP, Pyr., CH2Cl2, rt, 2 h, 95%; (b) Pd(OH)2/C, H2, MeOH, rt, 2 h, 88%; (c) MeI (excess),
NaHCO3, CH2Cl2, reflux, 4 days, 100%.

R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
439
the desired quaternary ammonium salts (ꢀ)-(3S,4R)-
To a solution of lactamol 3a–d (5.2 mmol) in 52 mL of
anhydrous toluene was added 2,6-lutidine (3.0 mL;
26 mmol). To this mixture, at 0 °C, was slowly added
7.2 mL of a 0.7 M solution of trifluoroacetic anhydride
(5.2 mmol). The ice bath was removed and the reaction
mixture left under vigorous stirring for 2 h. The mixture
was then heated at reflux for 30 min. After cooling to room
temperature, the content was transferred to a separatory
funnel containing 50 mL of ethyl acetate. The organic
phase was washed with water and then with saturated
solutions of NaHCO3 and NaCl. The combined organic
phases were dried over anhydrous sodium sulfate, filtered,
and the solvent removed in vacuo to furnish a crude oil,
which was purified by flash chromatography (eluent:
hexane/ethyl acetate; 90:10; v/v), to provide the enecarb-
amates 4a–d with yields ranging from 35% to 85% (over
2 steps).
20
20
8b (½aꢁD = ꢀ13.5 (c 1.3, H2O)) and (+)-(3R,4S)-8b (½aꢁD
=
+17.5 (c 2.0, H2O)), by the sequence shown in Scheme 8
encompassing acetylation, removal of the Cbz protecting
group and bis methylation.
3. Conclusion
In conclusion, new and conformationally constrained
acetylcholine derivatives were synthesized in an enantio-
selective manner in nine steps from diallylamine in overall
yields of ꢂ15%. The key step in the synthesis involved a
kinetic resolution performed by lipase PS or by chiral
simulated moving bed chromatography. A comparison of
the two technologies was performed using key intermediate
5a. The absolute stereochemistry of the alcohol intermedi-
ates was determined using the procedure of Riguera’s.
Evaluation of the acetylcholinesterase inhibition capability
of these new acetylcholine derivatives are in progress.
Compond 4a IR (cmꢀ1): 3115, 3093, 3066, 3039, 2963,
2897, 1719, 1627, 1496, 1420, 1339, 1219, 1132, 1089,
1
1024, 964, 915, 861 and 828. H NMR (300 MHz, CCl4/
D2O): d = 7.3 (m, 5H), 7.21 (d, J = 8.4 Hz, 2H), 7.08 (d,
J = 8.1 Hz, 2H), 6.67 and 6.78 (s, 1H), 5.1 (m, 3H), 4.1
(m, 2H), 3.6 (m, 1H). 13C NMR (75 MHz, CCl4/D2O):
d = 150.9 and 150.2, 141.6, 135.8, 132.4, 131.0 and 129.8,
128.3, 127.5, 110.4 and 110.1, 66.6, 53.6, 47.6 and 46.3.
Anal. Calcd for C18H16ClNO2: C, 68.90; H, 5.14; N,
4.46. Found: C, 68.87; H, 5.16; N, 4.45.
4. Experimental
4.1. General
Reagents and solvents are of commercial grade and were
used as supplied, except when specified in the experimental
procedure. In cases where dry solvents were employed, they
were distilled from calcium hydride or Na. 1H NMR and 13
C
Compond 4b IR (cmꢀ1): 3111, 3091, 3072, 3043, 3008,
NMR data were recorded on a Varian Gemini 2000 (7.0 T)
or Varian Inova (11.7 T) spectrometer with tetramethyl-
silane as internal standard (1H NMR). Data are reported
as follows: chemical shift in ppm (d), multiplicity (s = sin-
glet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad signal), coupling constant (Hz), integration.
High-resolution mass spectra (HRMS) were measured on
a VG AutoSpec-Micromass spectrometer. Infrared spectra
(IR) were obtained on a Thermo-Nicolet IR-200 spectrom-
eter and absorptions are reported in reciprocal centimeters.
HPLC analyses were performed with a Hewlett Packard
HP-1100/HP-3395 equipment and optical rotations were
measured with a Perkin–Elmer 341 polarimeter.
2955, 2906, 2832, 1714, 1626, 1523, 1430, 1352, 1245,
1181, 1123, 1095, 1034, 966, 839, 766 and 703. H NMR
1
(300 MHz, CCl4/D2O): d = 7.22 (m, 5H), 7.01 (d,
J = 8.1 Hz, 2H), 6,71 (d, J = 8.4 Hz, 2H), 6.64 (s, 1H),
5.05 (m, 3H), 4.1 (m, 2H), 3.72 (s, 3H), 3.58 (m, 1H). 13C
NMR (75 MHz, CCl4/D2O): d = 158.2, 151.2 and 150.5,
136.3, 135.4, 130.4 and 129.3, 128.1–113.7, 111.7 and
111.3, 66.7 and 66.6, 54.6, 54.2 and 54.1, 47.7 and 46.4.
Anal. Calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53.
Found: C, 73.75; H, 6.23; N, 4.54.
Compond 4c IR (cmꢀ1): 3115, 3071, 3039, 2957, 2903,
1730, 1627, 1529, 1431, 1360, 1219, 1127, 986, 861, 763
and 703. 1H NMR (300 MHz, CCl4/D2O): d = 8.10 (d,
J = 8.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.27 (m, 5H),
6.83 and 6.75 (s, 1H), 5.10 (m, 3H), 4.2 (m, 2H), 3.6 (m,
1H). 13C NMR (75 MHz, CCl4/D2O): d = 151.1, 150.3,
147.0, 135.9, 132.0 and 130.9, 128.2–127.6, 123.7, 109.7
and 109.4, 67.1, 53.6, 48.1 and 46.8. Anal. Calcd for
C18H16N2O4: C, 66.66; H, 4.97; N, 8.64. Found: C, 66.62;
H, 4.95; N, 8.61.
Compounds 1,20 2,21 1319 and 1419 have been described in
the literature.
4.2. N-(Cbz)-4-Aryl-2-pyrroline 4a–d
To a solution of N-Cbz-3-pyrroline 2 (1.061 g; 5.2 mmol)
in 36 mL of CH3CN/H2O (1:1; v/v) was added the
arenediazonium salt (7.8 mmol) and 22 mg of Pd(OAc)2-
(0.1 mmol). The reaction mixture was stirred for 3 h at
room temperature, when the total consumption of the sub-
strate was observed by TLC analysis. The reaction mixture
was then diluted with ethylacetate (ꢂ50 mL), transferred to
a separatory funnel and extracted with saturated solutions
of NaHCO3 and NaCl. The organic layer was collected,
dried over anhydrous Na2SO4, filtered and the solvent
evaporated in vacuo, to give a dark brown oil (lactamol
3a–d), which was submitted to the next reaction without
further purification.
Compond 4d IR (cmꢀ1): 3047, 2955, 2891, 1719, 1616,
1499, 1426, 1347, 1216, 1132, 1098, 961, 898, 864, 825,
1
756 and 707. H NMR (300 MHz, CCl4/D2O): d = 7.8–
7.1 (m, 12H), 6.81 and 6.71 (s, 1H), 5.1 (m, 3H), 4.2 (m,
2H), 3.7 (m, 1H). 13C NMR (75 MHz, CCl4/D2O):
d = 151.3 and 150.6, 140.6, 136.2, 133.2, 132.3, 131.0 and
129.8, 128.5–125.1, 111.2 and 110.8, 66.80 and 66.65,
53.9, 48.6 and 47.3. Anal. Calcd for C22H19NO2: C,
80.22; H, 5.81; N, 4.25. Found: C, 80.17; H, 5.79; N,
4.24.

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R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
4.3. ( )-trans-N-(Cbz)-3-Hydroxy-4-arylpyrrolidine 5a–d
952, 918, 869, 825, 747 and 698. 1H NMR (500 MHz,
CDCl3): d = 7.8–7.2 (m, 12H), 5.15 (s, 2H), 4.31 (m, 1H),
3.95 (m, 1H), 3.78 (m, 1H), 3.65 (m, 1H), 3.35 (m, 2H).
13C NMR (125 MHz, CDCl3): d = 154.7 and 154.6, 136.5
and 136.4, 136.2 and 136.1, 133.2, 132.3, 128.4–125.1,
76.2 and 75.3, 66.8, 52.2, 51.7 and 51.1, 49.8. Anal. Calcd
for C22H21NO3: C, 76.06; H, 6.09; N, 4.03. Found: C,
76.11; H, 6.20; N, 4.10.
To a solution of enecarbamate 4a–d (1.1 mmol) in 11.5 mL
of dry THF, under an inert atmosphere, at 0 °C, was added
slowly BH3ÆSMe2 (0.167 g; 0.21 mL; 2.2 mmol). The reac-
tion mixture was then maintained at room temperature
for 3 h. After total consumption of the substrate, the sys-
tem was cooled again to 0 °C when 8.0 mL of a 3 M NaOH
solution was added dropwise, followed by 8.0 mL of 30%
H2O2. The mixture was stirred for 45 min in an ice bath fol-
lowed by another 45 min at room temperature. After that,
the mixture was transferred to a separatory funnel with
30 mL of ethyl acetate, the organic layer was washed with
water, and then with saturated solutions of NaHCO3 and
NaCl. Finally, the combined organic layers were dried over
anhydrous Na2SO4, filtered, and the solvent evaporated in
vacuo. The resultant oil was purified by flash chromatogra-
phy (hexane/ethyl acetate; 60:40; v/v), to give alcohols 5a–d
with yields of 94%, 81%, 90% and 85%, respectively.
4.4. ( )-trans-N-(Cbz)-3-Acetyl-4-aryl-pyrrolidine 6a–d
Alcohol substrate 5a–d (1.0 mmol) was dissolved in
21.0 mL of dichloromethane. To this solution was added
acetic anhydride (2.55 g; 2.3 mL; 25 mmol), pyridine
(0.989 g; 1 mL; 12,5 mmol) and DMAP (12.2 mg;
0.1 mmol), and the reaction mixture magnetically stirred
at room temperature for 2 h. Next, the solvent was re-
moved in vacuo, and the residue was purified by flash chro-
matography to provide compounds 6a–d in yields of 98%,
89%, 82% and 91%, respectively.
Compound 5a IR (cmꢀ1): 3419, 3062, 3038, 2945, 2891,
1704, 1421, 1357, 1211, 1093, 1025, 971, 918, 830, 737
Compound 6a IR (cmꢀ1): 3062, 3043, 2964, 2886, 1743,
20
and 698. (3S,4R)-5a; ½aꢁD = +15.0 (c 3.0, CH2Cl2)
1709, 1499, 1416, 1357, 1240, 1172, 1108, 1093, 1059,
20
1
ee = 83.5%; (3R,4S)-5a; ½aꢁD = ꢀ19.0 (c 3.0, CH2Cl2)
1015, 986, 913, 825, 766 and 693. H NMR (500 MHz,
1
ee = 96.9%; H NMR (500 MHz, CDCl3): d = 7.5–7.1 (m,
CDCl3): d = 7.5–7.1 (m, 9H), 5.19 (d, J = 7.6 Hz, 2H),
5.1 (m, 1H), 3.88 (m, 1H), 3.77 (m, 1H), 3.71 (dd,
J = 11.3 and 3.9 Hz, 1H), 3.53 (dd, J = 12.9 and 2.5 Hz,
1H), 3.46 (m, 1H), 2.1 (s, 3H). 13C NMR (125 MHz,
CDCl3): d = 170.4 and 170.3, 154.6, 137.17 and 137.03,
136.53 and 136.47, 133.2, 129.1–127.9, 78.5 and 77.6,
67.1, 49.9 and 49.6, 49.15 and 49.11, 48.4 and 47.4, 21.0.
HRMS (ESI): m/z calcd for C20H20ClNO4+[H+]:
374.1159; found: 374.1137.
9H), 5.1 (s, 2H), 4.27 (m, 1H), 3.93 (dd, J = 11.3 and
7.9 Hz, 1H), 3.77 (m, 1H), 3.55 (m, 1H), 3.37 (m, 1H),
3.24 (m, 1H). 13C NMR (125 MHz, CDCl3): d = 154.8,
137.5, 136.6, 133.1, 129.0–127.9, 76.5 and 75.6, 67.1, 52.4
and 52.0, 51.2 and 50.6, 49.9. HRMS (ESI): m/z calcd for
C18H18ClNO3+[H+]: 332.1019; found: 332.1053. Anal.
Calcd for C18H18ClNO3: C, 65.15; H, 5.47; Cl, 10.69; N,
4.22. Found: C, 65.09; H, 5.35; Cl, 10.49; N, 4.19.
Compound 5b IR (cmꢀ1): 3412, 3063, 3032, 2950, 2894,
2835, 1705, 1612, 1584, 1514, 1498, 1428, 1359, 1324,
Compound 6b IR (cmꢀ1): 3071, 3033, 2957, 2897, 2843,
1746, 1708, 1621, 1588, 1523, 1426, 1355, 1241, 1181,
1111, 1062, 839, 768 and 698. 1H NMR (300 MHz,
CDCl3): d = 7.5–6.8 (m, 9H), 5.20 (d, J = 4.0 Hz, 2H),
5.13 (m, 1H), 3.8 (m, 6H), 3.5 (m, 2H), 2.1 (s, 3H); 13C
NMR (75 MHz, CDCl3): d = 170.2, 158.6, 154.5, 136.5,
130.5 and 130.4, 128.3–127.7, 114.1, 78.7 and 77.9, 67.0,
55.2, 50.1 and 4.7, 49.4, 48.2 and 47.2, 21.1. HRMS
(ESI): m/z calcd for C21H23NO5+[H+]: 370.1654; found:
370.1601.
1306, 1283, 1249, 1210, 1179, 1114, 1101, 1076, 1033,
20
960, 913, 831, 768, 738 and 698. (3S,4R)-5b; ½aꢁD = +6.4
20
(c 1.6, CH2Cl2) ee = 96.3%; (3R,4S)-5b; ½aꢁD = ꢀ6.7 (c
1.2; CH2Cl2) ee = 97.1%; 1H NMR (300 MHz, CDCl3):
d = 7.4–6.8 (m, 9H), 5.1 (s, 2H), 4.22 (m, 1H), 3.89 (dd,
J = 11.3 and 7.7 Hz, 1H), 3.75 (s, 3H), 3.70 (m, 1H), 3.53
(m, 1H), 3.33 (dd, J = 11.3 and 5.5 Hz, 1H), 3.2 (m, 1H).
13C NMR (75 MHz, CDCl3): d = 158.5, 154.7, 136.6,
130.5, 130–127, 114.0, 76.5 and 75.7, 66.9, 55.2, 52.3 and
51.9, 51.0 and 50.4, 50.2. Anal. Calcd for C19H21NO4: C,
69.71; H, 6.47; N, 4.28. Found: C, 69.53; H, 6.54; N, 4.20.
Compound 6c IR (cmꢀ1): 3119, 3074, 3035, 2956, 2895,
1745, 1711, 1616, 1543, 1419, 1363, 1234, 1111, 1066,
1
869, 757 and 707. H NMR (300 MHz, CDCl3): d = 8.19
Compound 5c IR (cmꢀ1): 3426, 3112, 3072, 3031, 2950,
2886, 1697, 1604, 1523, 1441, 1348, 1203, 1174, 1116,
971, 919, 861, 774, 751 and 704. 1H NMR (300 MHz,
CDCl3): d = 8.16 (d, J = 8.5 Hz, 2H), 7.5–7.3 (m, 7H),
5.15 (s, 2H), 4.35 (m, 1H), 3.99 (dd, J = 11.4 and 7.7 Hz,
1H), 3.80 (dd, J = 11.3 and 6.2 Hz, 1H), 3.62 (m, 1H),
3.4 (dd, J = 11.4 and 5.5 Hz, 2H). 13C NMR (75 MHz,
CDCl3): d = 154.4, 146.9, 146.5, 135.1, 128.3–127.7,
123.8, 76.2 and 75.3, 67.1, 52.4 and 52.1, 51.5 and 50.9,
49.56. Anal. Calcd for C18H18N2O5: C, 63.15; H, 5.30; N,
8.18. Found: C, 63.07; H, 5.43; N, 8.08.
(d, J = 8.5 Hz, 2H), 7.4 (m, 7H), 5.20 (d, J = 3.3 Hz,
2H), 5.14 (m, 1H), 3.94 (m, 1H), 3.8 (m, 2H), 3,56 (m,
2H), 2.1 (s, 3H). 13C NMR (75 MHz, CDCl3): d = 170.1,
154.3, 147.1, 145.8, 136.2, 128.3–127.8, 124.0, 78.0 and
77.1, 67.2, 49.9 and 49.6, 48.9, 48.9 and 48.0, 20.9. HRMS
(ESI): m/z calcd for C20H20N2O5+[H+]: 384.13214; found:
384.1336.
Compound 6d IR (cmꢀ1): 3066, 3035, 2959, 2887, 1744,
1708, 1606, 1422, 1361, 1320, 1243, 1177, 1116, 1059,
1024, 968, 922, 865, 830, 768, 753 and 707. 1H NMR
(300 MHz, CDCl3): d = 7.9–7.2 (m, 12H), 5.22 (m, 3H),
3.89 (m, 3H), 3.58 (m, 2H), 2.1 (s, 3H). 13C NMR
(75 MHz, CDCl3): d = 170.2, 154.4, 136.4, 135.8 and
Compound 5d IR (cmꢀ1): 3414, 3062, 2950, 2886, 1704,
1680, 1601, 1430, 1367, 1323, 1220, 1181, 1098, 1074,

R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
441
135.7, 133.1, 132.3, 128.5–125.3, 78.5 and 77.6, 67.0, 50.1
and 49.7, 49.2, 49.0 and 48.1, 21.1. HRMS (ESI): m/z calcd
for C24H23NO4+[H+]: 389.16271; found: 389.16319.
2.0 (s, 3H). 13C NMR (75 MHz, D2O/CCl4): d = 172.5,
158.14, 128,6–128.3, 114.3, 77.9, 69.6 55.3, 53.4, 52.5,
47.9, 20.1. (ESI): m/z calcd for C15H22NO3+[H+]:
264.1600; found: 264.1574.
4.5. Resolution of ( )-trans-N-(Cbz)-3-acetyl-4-aryl-pyrrol-
idine 6a–d (enzymatic hydrolysis)
4.8. Pyrrolidine-(3R,4S)-methoxyphenylacetate 11 and
pyrrolidine-(3S,4S)-methoxyphenylacetate 12
To a round-bottomed reaction flask was added 50 mL of
potassium phosphate buffer (pH 7.0), 5.0 mL of toluene,
acetate 6a–d (1.5 mmol) and 5.42 g of Pseudomonas cepacia
AY lipase. The reaction was maintained at room tempera-
ture for 30 days under rigorous stirring. After this period,
the flask contents were filtered through Celite. The organic
phase was separated and washed with water and with sat-
urated solutions of NaHCO3 and NaCl. The solvent was
removed in vacuo and the crude product purified by flash
chromatography (hexane:ethyl acetate, 6:4; v/v), furnishing
alcohols 5a–d (98.8%, 96.3%, 92.9% and 97.2% ee, respec-
tively) and the remaining acetates 6a–d (58.0%, 97.1%,
93.0% and 99.4% ee, respectively).
To a solution of the resolved alcohols (ꢀ)-(3R,4S)-5b
(0.060 g; 0.2 mmol) or (+)-(3S,4R)-5b (0.046 g; 0.1 mmol)
in 5.0 mL of dichloromethane was added methoxyphenyl-
acetic acid (MPA; 1 equiv) and catalytic amounts of
DMAP (2 mol %), and the mixture was left stirring for
3 h. The reaction mixture was then filtered to remove solid
side products (mostly dicyclohexylurea). The filtrate was
evaporated in vacuo and the residue purified by flash chro-
matography (hexane:ethyl acetate; 6:4; v/v), furnishing the
respective esters (75% yield). Deprotection of the esters
were conducted as described in item 4.5 to give diastereo-
meric free-amines (RSS)-11 and (SRS)-12 in 40% yield.
4.6. ( )-trans-3-Acetyl-4-p-MeO-phenyl-pyrrolidine 7b
(RSS)-11 IR (cmꢀ1): 3362, 3059, 3046, 2932, 2844, 1756,
1621, 1514, 1460, 1252, 1191, 1124, 1030, 828, 734 and
1
To a solution of acetate 6b (0.75 mmol) in 22.5 mL of
methanol was added palladium hydroxide dispersed over
activated carbon (26.3 mg; 0.19 mmol). Hydrogen was
purged through the resulting suspension for about 10 min
under vigorous stirring, and then left under a balloon of
hydrogen for 2 h. Next, the reaction was filtered through
Celite, and the solvent evaporated in vacuo. The crude
oil was then purified by flash chromatography (CHCl3/
MeOH, 9:1, v/v), to yield free amine 7b in 88%. Compound
7b IR (cmꢀ1): 3321, 2959, 2939, 2865, 2830, 1740, 1646,
1621, 1517, 1423, 1378, 1249, 1190, 1036, 992 and 833.
1H NMR (300 MHz, CDCl3): d = 7.15 (d, J = 8.7, 2H),
6.85 (d, J = 8.4 Hz, 2H), 5.14 (ddd, J = 5.9, 3.7 and
2.6 Hz, 1H), 3.78 (s, 3H), 3.50 (dd, J = 11.3 and 7.7 Hz,
1H), 3.27 (m, 2H), 3.08 (dd, J = 12.8 and 2.2 Hz, 1H),
2.91 (dd, J = 11.3 and 7.7 Hz, 1H); 2.53 (s, 1H), 2.05 (s,
3H). 13C NMR (75 MHz, CDCl3): d = 170.5, 158.2,
132.9, 128.1, 113.9, 82.3, 55.2, 53.9, 53.4, 51.2, 21.2. Anal.
Calcd for C13H17NO3: C, 66.36; H, 7.28; N, 5.95. Found:
C, 66.32; H, 7.25; N, 5.93.
701. H NMR (500 MHz, CDCl3): d = 7.5–7.3 (m, 5H),
6.97 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 5.14
(ddd, J = 5.8, 3.3 and 2.1 Hz, 1H), 3.8 (s, 3H), 3.4 (s,
3H), 3.35 (dd, J = 11.3 and 7.6 Hz, 1H), 3.26 (dd,
J = 12.8 and 5.8 Hz, 1H), 3.08 (td, J = 7.6 and 3.9 Hz,
1H), 3.06 (dd, J = 12.8 and 2.1 Hz, 1H), 2.9 (dd, J = 11.3
and 7.3 Hz, 1H), 1.9 (s, 1H). 13C NMR (75 MHz, CDCl3):
d = 170.0, 158.1, 135.8, 132.4, 128.5–126.8, 113.8, 83.1,
82.3, 57.2, 55.1, 53.6, 53.0, 51.1. HRMS (ESI): m/z calcd
for C20H23NO4+[H+]: 342.1705; found: 342.1696.
(SRS)-12 IR (cmꢀ1): 3381, 3069, 3051, 2943, 2847, 1759,
1615, 1519, 1453, 1249, 1183, 1111, 1033, 828, 738 and
702. 1H NMR (500 MHz CDCl3): d = 7.4–7.3 (m, 5H),
7.11 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 5.181
(ddd, J = 5.5, 3.4 and 2.1 Hz, 1H), 3.44 (dd, J = 11.3 and
7.9 Hz, 1H), 3.25 (td, J = 7.3 and 3.3 Hz, 1H), 3.18 (dd,
J = 13.2 and 5.5 Hz, 1H), 2.89 (dd, J = 11.6 and 7.3 Hz,
1H), 2.84 (dd, J = 13.2 and 1.9 Hz, 1H), 1.9 (1H). 13C
NMR (75 MHz, CDCl3): d = 170.4, 158.4, 136.1, 132.7,
128.9–127.1, 114.1, 83.3, 82.5, 57.3, 55.3, 53.8, 53.2, 51.2.
HRMS (ESI): m/z calcd for C20H23NO4+[H+]: 342.1705;
found: 342.1698.
4.7. ( )-trans-3-Acetyl-4-p-MeO-phenyl-pyrrolidinium
iodide 8b
A mixture of free amine 7b (0.24 mmol), methyl iodide
(1.7 g; 0.75 mL; 12 mmol), sodium bicarbonate (0.101 g;
1.2 mmol) in 10.0 mL of anhydrous dichloromethane was
heated at reflux under inert atmosphere for 4 days. After
the consumption of amine 7b (TLC), the solvent was
removed in vacuo to give a crude oil, which was purified
by flash chromatography (CHCl3/MeOH, 8:2, v/v), provid-
ing the quaternary ammonium salt 8b in quantitative yield.
4.9. Resolution of ( )-N-(Cbz)-3-hydroxy-pyrrolidine 13
(enzymatic transesterification)
To a solution of racemic alcohol ( )-13 (0.518 g; 2.3 mmol)
in 25 mL of dichloromethane it was added vinyl acetate
(0.198 g; 2.1 mL; 23 mmol) and the immobilized lipase
(Pseudomonas cepacia AK). The resulting suspension was
maintained under vigorous stirring at room temperature
for 10 days. After this period, the mixture was filtered
through a synterized funnel and the solvent evaporated in
vacuo. The crude oil was then purified by flash chromato-
graphy (hexane:ethyl acetate; 6:4; v/v), providing 0.262 g of
Compound 8b IR (cmꢀ1): 3425, 2924, 1738, 1614, 1517,
20
1458, 1253, 1181, 1026. (3S,4R)-8b; ½aꢁD = ꢀ13.5 (c 1.3,
20
H2O) ee = 96.3%; (3R,4S)-8b; ½aꢁD = +17.5 (c 2.0, H2O)
ee = 97.1%; 1H NMR (300 MHz, D2O): d = 7.25 (d,
J = 9.2 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 5.40 (m, 1H),
3.7 (s, 3H), 4.2–3.5 (m, 5H), 3.29 (s, 3H), 3.26 (s, 3H),
the acetylated compound (3R)-14 (>99% ee) and 0.280 g of
20
alcohol (3S)-13 (81.3% ee). (3R)-14; ½aꢁD = ꢀ13.5 (c 1.48;
CH3OH) ee >99%.

442
R. de L. Barreto et al. / Tetrahedron: Asymmetry 18 (2007) 435–442
8. Santaniello, E.; Ferraboshi, P.; Grisenti, P.; Manzocchi, A.
Acknowledgments
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D.; Furlan, L. T.; Santana, C. C. Braz. J. Chem. Eng. 2004,
21, 127.
This work was supported by a grant from the Research
Supporting Foundation of the State of Sao Paulo (FA-
˜
PESP, 01-07232-7). We also thank CNPq and CAPES for
´
fellowships and Professor Fabio Gozzo (Unicamp) for
the ESI-MS analyses.
12. (a) Shono, T.; Matsumura, Y.; Tsubata, K.; Sugihara, Y.;
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