Chemoenzymatic synthesis of the enantiomerically pure 1,2,3,4- tetrahydroquinoline moiety of the antithrombotic (21R)- and (21S)-argatroban

Article abstract of DOI:10.1016/j.tetasy.2013.07.008

The synthetic antithrombotic argatroban is a dipeptide between the nonproteogenic (2R,4R)-4-methyl-2-piperidine carboxylic acid and l-arginine, in turn bonded to a 3-methyltetrahydroquinoline sulfonyl group; the drug is usually prepared and administered as a mixture of C-21-diastereoisomers. By means of a biocatalytic transformation enantiomerically pure (R)- and (S)-synthons, suitable for the synthesis of separate (21R)- and (21S)- argatroban, were obtained.

Full text of DOI:10.1016/j.tetasy.2013.07.008

Tetrahedron: Asymmetry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemoenzymatic synthesis of the enantiomerically pure
1,2,3,4-tetrahydroquinoline moiety of the antithrombotic
(
21R)- and (21S)-argatroban
Patrizia Ferraboschi , Samuele Ciceri , Paride Grisenti b
a,
⇑
a
a
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università Statale di Milano, Via Saldini 50, 20133 Milano, Italy
EUTICALS SpA, Via Volturno 41/43, 20089 Rozzano (Mi), Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthetic antithrombotic argatroban is a dipeptide between the nonproteogenic (2R,4R)-4-methyl-2-
Received 4 July 2013
Accepted 10 July 2013
Available online xxxx
piperidine carboxylic acid and L-arginine, in turn bonded to a 3-methyltetrahydroquinoline sulfonyl
group; the drug is usually prepared and administered as a mixture of C-21-diastereoisomers. By means
of a biocatalytic transformation enantiomerically pure (R)- and (S)-synthons, suitable for the synthesis of
separate (21R)- and (21S)- argatroban, were obtained.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
the (4R)-stereocenter is subsequently obtained after hydrogena-
1
0,11
tion of the 4,5-double bond.
Recently we have identified a
Thrombin is a serine protease that plays a key role in the blood
complex between a rhodium compound and the Mandiphos ligand
coagulation and fibrinolysis that induces platelet aggregation and
that catalyzes the diastereoselective [98:2, (4R)/(4S)] hydrogena-
tion of the double bond. The use of L-arginine as a starting mate-
1
–3
12
secretion. The central role of thrombin in the coagulation cascade
has made it a target for antithrombotic agents in the management of
cardiovascular diseases.4 Heparin, a sulfated glycosaminoglycan,
has historically been used as the anticoagulant of choice in the man-
agement of thrombotic diseases: however limitations due to its
chemical heterogeneity, a high variability in dose efficacy, and sev-
eral adverse events, such as the heparin induced thrombocytopenia
rial leads to the introduction of a third stereocenter with the
13
desired configuration. An additional stereocenter is formed dur-
ing the last step of the synthesis, that is, the reduction of the het-
eroaromatic ring of a quinoline that affords the (21R)- and (21S)-
1
3
diastereoisomers. The diastereoisomeric mixture is applied as
the antithrombotic drug without separation of the (21R)- and
(21S)-epimers, 1a and 1b, respectively, provided that the diaste-
reomeric ratio (dr) is 64:36 ± 2. The (21R)- and (21S)-configura-
tions have been assigned in 1993 by an X-ray crystallographic
(
HIT) prompted the development of low molecular weight selective
4
and orally bioavailable direct inhibitors of thrombin.
Argatroban 1 is a synthetic, peptidomimetic, small molecule
that binds selectively and reversibly to the catalytic site of
1
4
study after HPLC separation. Compound (21S)-1b is twice as
5
14
thrombin, serving as a competitive inhibitor. It was identified by
potent as (21R)-1a and about five times less soluble in water.
6
–8
Ò
Okamoto et al.
and used in Japan (Novastan , MD-805) since
Recently, in order to fully characterize the two C-21-epimers of
argatroban 1 through crystallography, NMR, and computational
techniques, we accomplished their separation by means of
fractional crystallization. While the (21R)-diastereoisomer 1a
(96.8:3.2 dr, 19% yield) was easily obtained by two crystallizations,
the preparation of (21S)-1b (0.5:99.5 dr, 4.8% yield) required ten
the early 1980s; it was later approved in Europe and in the US
for patients with HIT.9 Three moieties are easily recognized as
constituents of 1, the 4-methyl-2-piperidine carboxylic acid
bonded to arginine, in turn bearing a 3-methyl-1,2,3,4-tetrahydro-
quinoline sulfonyl group. Four stereogenic centers are present in
the structure. The (2R)-stereocenter of the piperidine moiety is
usually introduced over the course of a Diels–Alder reaction, with
1
5
crystallizations. These results, in addition to the current regula-
tory guidelines that for all active pharmaceutical ingredients
(API), presenting one or more stereocenters, require that all of
the possible isomers undergo a full study from a chemical–physical
and pharmacological point of view, prompted us to investigate the
synthesis of the (R)- and (S)-isomers of 3-methyl-1,2,3,4-tetrahy-
droquinoline 2, that is, the suitable synthons for the preparation
of (21R)- and (21S)-1, giving priority to an enzymatic approach.
1
0,11
the stereochemical outcome determined by a chiral auxiliary;
⇑
Corresponding author. Tel.: +39 02 50316052; fax: +39 02 50316040.
E-mail address: patrizia.ferraboschi@unimi.it (P. Ferraboschi).
0
957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.07.008
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2
P. Ferraboschi et al. / Tetrahedron: Asymmetry xxx (2013) xxx–xxx
of commercially available 3-quinoline carboxylic acid we prepared
3-carboxymethyl-1,2,3,4-tetrahydroquinoline 5 (78% from 3-car-
12
H
N
COOH O
NH
8
5
4
2
1
7
5
7
2
8
9
1
boxylic acid) by selective reduction with sodium cyanoborohy-
N
6
N
H
NH2
3
22
3
4
dride, as described by Gotor et al. The LiAlH reduction of 5
6
HN
1
1
afforded the 3-hydroxymethyl derivative 3 (89%) (Scheme 1).
After protection of the amino group (80% yield), the obtained
tert-butyl carbamate 6 was used for screening with lipases, se-
lected for their well known stability in organic solvents, and capa-
bility of selectively transforming a wide spectrum of substrates,
SO2
H
N
4
13
10
1
4
1
9
2
20
1
5
H
N
R
1
6
1
8
21
1
7
22
R'
2
3
including many 2-substituted primary alcohols. Among the li-
pases tested under irreversible transesterification conditions,2
the highest but not satisfactory enantiomeric excess (ee) was ob-
tained by Pseudomonas fluorescens lipase (PFL); in fact at 32% con-
version 76% ee was determined by HPLC analysis on a chiral
stationary phase of the obtained (+)-acetate 7 (Scheme 2).
Other lipases, even at a very low extent of conversion, showed
lower ee values (Table 1). Also different solvents from the initially
employed toluene did not enhance the PFL stereoselectivity
(Table 2).
1
OH
4,25
a R = CH ; R' = H (21R)-argatroban
3
3
b R = H; R' = CH (21S)-argatroban
3
2
. Results and discussion
Two syntheses of optically active 3-methyl-1,2,3,4-tetrahydro-
quinoline 2, both aimed to the preparation of argatroban 1 or its
analogues have been reported in the literature: in one case the
starting material is Oppolzer’s sultam that, after acylation, in five
steps leads to the enantiomerically pure (3R)-methyl-6-bromo-
Table 1
Lipase-catalyzed irreversible transesterification of alcohol 6 in toluene
Entry Enzyme
Time
Conv.a
(%)
ee b (%)
(config)
E
1
,2,3,4-tetrahydroquinoline.16 The synthesis of (3R)- and (3S)-
(
h)
methyl-1,2,3,4-tetrahydroquinoline is described in a Synthelabo
patent: the starting material, in this case, is the enantiomerically
pure methyl (S)- and (R)-3-iodo-2-methyl-propanoate, respec-
a
b
c
Candida cylindracea lipase
CCL)
Candida antarctica lipase A 36
2.5
39
33 (S) c
2.4
2
(
23
5
30 (S) c
30 (R) c
1
7
tively. The use of a chiral hydrogenation catalyst is a well known
(CAL A)
1
8,19
Candida antarctica lipase B
CAL B)
2.5
1.9
method
for the preparation of enantiomerically pure tetrahy-
(
droquinolines starting from variously substituted quinolines; nev-
ertheless a chiral rhodium complex, giving optimal results (98% ee
and quantitative yield) in the case of 2-substituted quinolines,
e
d
d
e
CAL B CLEA
PFL
1
24
68
32
45 (S)
2.3
10.4
76 (S) c
a
b
c
20
From GLC analysis.
From HPLC on a chiral stationary phase.
Product ee and configuration.
Substrate ee and configuration.
Cross linked enzyme aggregate.
failed when applied to 3-methylquinoline.
We planned to explore an enzymatic approach to the synthesis
of both enantiomers of compound 2 by the resolution of a suitable
racemic substrate. It is known21 that despite the many enzymatic
methods available for the preparation of non-racemic chiral pri-
mary amines, only a few examples have been reported for the res-
olution of secondary amines, maybe due to steric hindrance. For
this reason our attention was focused on the identification of a
compound bearing a functional group convertible by a hydrolytic
enzyme and an immediate precursor of the 3-methyl group. The
d
e
The enantiomeric ratio (E)26,27 of the PFL-catalyzed transesteri-
fication was moderate (about 10) but adequate, according to the
observations of Sih et al., to achieve a satisfactory final ee after
a second enzymatic resolution of the enantiomerically enriched
compound. Taking into consideration the results of Davies and
Kanerva we chose to submit enantiomerically enriched (+)-ace-
2
6
2
8
3
-hydroxymethyl group of compound 3 was evaluated as being
2
9
the most suitable for our purposes. Starting from methyl ester 4
H
N
N
ii
8%
8
COOCH3
COOCH3
i
4
5
N
8
9%
iii 89%
H
COOH
Boc
N
N
iv
OH 80%
OH
6
3
2 3 4 2
Scheme 1. Reagents and conditions: (i) SOCl , MeOH; (ii) NaBH CN, THF, and MeOH; (iii) LiAlH , THF; (iv) (Boc) O, dioxane, and NaOH, water.
The best separation of (R)- and (S)-7 was obtained with a Phenomenex Lux 3
l
Cellulose-1; (R)- and (S)-alcohols 6 were not resolved. The Chiralpak IA (Daicell)
afforded only a partial separation of the acetates and did not resolve alcohols.
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P. Ferraboschi et al. / Tetrahedron: Asymmetry xxx (2013) xxx–xxx
3
Boc
N
Boc
Boc
N
N
OAc, PFL
+
OH
PhCH
3
OH
OAc
5
8% conv
(RS)-6
(
S)-7
(
R)-6, 81% ee
OAc, PFL
PhCH3
3
2% conv
OAc, PFL
PhCH3
41% conv
Boc
(
R)-6, >98% ee
N
PFL
buffer pH 7
(
S)-6, >98% ee
OAc
6
1% conv
(S)-7, 76% ee
Scheme 2. Double enzymatic resolutions of alcohol 6.
Table 2
PFL-catalyzed irreversible transesterification of alcohol 6 in different solvents
second PFL-catalyzed transesterification, a ‘continuation’29 of the
first resolution. The desired >98% ee of (R)-6 was achieved by stop-
ping the second reaction at 41% conversion (Scheme 2). Following
the same synthetic pathway previously optimized for (S)-2, com-
pound (R)-2 (Scheme 3) was also available as a chiral building
block for the synthesis of (R)- and (S)-1,2,3,4-tetrahydroquino-
line-8-sulfonic acid chloride according to the reported method.17
Enantiomerically pure (R)-1,2,3,4-tetrahydroquinoline-8-sul-
fonic acid chloride is the starting material for several analogues
Entry
Solvent
Time (h)
Conv.a (%)
eeb (%) (config)
E
a
b
c
d
e
f
Chloroform
Heptane
Tetrahydrofuran
Hexane
Cyclohexane
Vinyl acetate
36
6
24
158
6
/
/
/
33
16
64
26
58
61 (S) c
67 (S) c
5.5
5.7
6.5
6.6
9.2
d
82 (R)
c
68 (S)
66
81 (R) d
a
b
c
From GLC analysis.
From HPLC on a chiral stationary phase.
Product ee and configuration.
1
6
modified in the arginine or in the piperidyl moiety, prepared in
attempts to develop drugs with increased efficacy and
bioavailability.
d
Substrate ee and configuration.
3
. Conclusion
tate 7 to PFL-catalyzed hydrolysis. The reaction was monitored by
GLC and stopped at 61% conversion. Alcohol 6 showed >98% ee (by
chiral HPLC). In order to establish the stereochemical outcome of
the double resolution, alcohol 6, reported in the literature only as
Four stereocenters are present in argatroban 1, three of which
must present a well defined configuration; the fourth is introduced
over the course of the hydrogenation of a quinoline ring that leads
to a mixture of diastereoisomers.
Compound 1 is endowed with antithrombotic activity and
administered in the treatment of cardiovascular diseases as a
mixture of (21R)-1a and (21S)-1b diastereoisomers (in a ratio
3
0
a racemic mixture, was transformed into a known chiral synthon
by esterification to tosylate 8, lithium aluminum hydride reduc-
2
tion, and removal of the protecting group (59% yield from 6) (in
Scheme 3 the synthetic sequence is described for the (R)-enantio-
mer). By comparison of the specific rotation of 2 with the reported
6
4:36 ± 2), the (S)-isomer being twice as potent as the (R)-isomer
1
7
one the (S)-configuration was assigned to the 3-methyl-1,2,3,4-
tetrahydroquinoline obtained starting from (+)-acetate 7. The
enantiomeric purity of 2, determined by chiral HPLC, showed that
the ee of alcohol 6 remained unaltered over the course of the
synthesis.
and five times less soluble in aqueous solution. A double PFL-cata-
lyzed resolution of a 2-substituted primary alcohol, easily prepared
from commercially available quinoline 3-carboxylic acid, allowed
suitable chiral synthons to be obtained for both (21R)-1a and
(
21S)-1b argatroban, required, for example, for deeper pharmaco-
logical activity studies. The apparently low yields of synthons 2
approximately 10% from (±)-6 for either (R)- or (S)-enantiomer]
In order to accomplish our goal we also needed (R)-3-methyl-
1
,2,3,4-tetrahydroquinoline 2, corresponding to (R)-6, that is, the
[
slower reacting enantiomer in the course of the PFL-catalyzed
transformation. The PFL-catalyzed transesterification was stopped,
in this case, at 58% conversion and the ee of obtained (+)-6 was
determined, after acetylation, by HPLC on a chiral column. The ex-
pected moderate ee (81%) suggested submitting enriched (R)-6 to a
obtained by the double enzymatic resolution, necessary in order
to reach the desired >98% ee, represents 20% of the theoretical yield
that can be achieved by working on a racemic substrate.
The availability of the enantiomerically pure 1,2,3,4-tetrahydro-
quinoline moiety allows the separate C-21-diastereoisomers to be
Boc
N
Boc
R
N
N
i
ii
86%
OH
R)-6, >98% ee
82%
OTs
(
(R)-8
(R)-9 R = Boc
R)-2 R = H
, THF; (iii) TFA, CH
iii 83%
(
Scheme 3. Reagents and conditions: (i) TsCl, py; (ii) LiAlH
4
2 2
Cl .
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4
P. Ferraboschi et al. / Tetrahedron: Asymmetry xxx (2013) xxx–xxx
obtained in a more convenient way than the previous fractional
duced pressure. To the residue water (400 mL) and 1 M sodium
hydroxide (until pH 8) were added; the mixture was extracted
with dichloromethane (4 ꢀ 400 mL). The collected organic phases
were dried over sodium sulfate; after filtration, the solvent was re-
moved at reduced pressure, to afford title compound 4 (7.78 g,
89%), which was used directly in the next step without any further
purification. The chemical and physical properties were in agree-
1
5
crystallization of a diastereomeric mixture of argatroban [19%
and 4.8% yields for (21R)-1a and (21S)-1b, respectively]. Indeed
over the course of the fractional crystallization of a diastereomeric
mixture of 1, a significant amount of argatroban, obtained from
L-arginine and the precious (2R,4R)-piperidyl moiety, has to be dis-
carded, while 10% yields of the enzymatic method are related to a
less expensive intermediate.
2
2
ment with the reported ones.
The present synthesis is suitable for gram-scale preparations
and furnishes the enantiomerically pure compounds that are use-
4.2.2. (RS)-Methyl 1,2,3,4-tetrahydroquinoline-3-carboxylate 5
To a solution of methyl ester 4 (7.68 g, 41 mmol) in dry tetrahy-
drofuran (150 mL) and methanol (70 mL) sodium cyanoborohy-
ful not only for (21R)-1a and (21S)-1b argatroban synthesis, but
also for other analogues1
6,17
still under investigation. The devel-
oped analytical GLC and HPLC methods allow a careful control of
the outcome of the enzymatic reactions (rate of conversion and ee).
Moreover (R)- and (S)-6 could be useful in order to obtain the
farnesyl protein transferase inhibitors reported in a 2002 patent;
indeed, the use of (RS)-6 as the starting material for the prepara-
dride (10.8 g, 172 mmol) was added under
a
nitrogen
atmosphere. The pH was adjusted to 4, by the addition of 4 M
hydrogen chloride in dioxane and kept at this value over the course
of the reaction (10 h), by the addition of the same hydrogen chlo-
ride solution. The reaction progress was monitored by TLC (dichlo-
romethane/acetone 9:1) until the starting material disappeared.
The reaction mixture was cooled in an ice bath, after which water
(200 mL) and a saturated sodium hydrogen carbonate aqueous
solution (until neutral pH) were added. The organic solvents were
removed at reduced pressure. The aqueous phase was extracted
with ethyl acetate (3 ꢀ 200 mL). The collected organic phases were
dried over sodium sulfate and after the usual work-up an oily res-
idue (8.84 g) was obtained; the residue was purified by silica gel
column chromatography by elution with hexane/ethyl acetate
(9:1) to give pure 5 (6.88 g, 88%). The chemical and physical prop-
3
0
tion of a racemic imidazol-derivative of 1,2,3,4-tetrahydroquino-
line, requires separation by preparative HPLC on
stationary phase.
a chiral
Since argatroban is used for designing antithrombotic surfaces
in angioplasty devices,31 the availability of the separated
C-21-diastereoisomers could be very helpful in the enhancement
of its water-solubility, required in order to improve its efficiency
in coating hemocompatible polymers.
4
4
. Experimental
2
2
erties were in agreement with the reported ones.
.1. General
0
4
.2.3. (RS)-3-(1 -Hydroxymethyl)-1,2,3,4-tetrahydroquinoline 3
To a suspension of lithium aluminum hydride (5.3 g, 140 mmol)
in dry tetrahydrofuran (125 mL), cooled at 0-5 °C, ester 5 (6.68 g,
5 mmol) dissolved in tetrahydrofuran (125 mL) was added drop-
All of the reagents and enzymes were purchased by Sigma–Al-
drich. CAL B CLEA was purchased by CLEA Technologies (The Neth-
erlands). All reactions were monitored by TLC on silica gel 60 F254
precoated plates with a fluorescent indicator (Merck) with detec-
tion by spraying with 10% phosphomolybdic acid ethanol solution
and heating at 110 °C. Column chromatography was performed on
silica gel 60 (70–230 mesh) (Merck) with a substrate/silica gel ratio
3
wise. The ice bath was then removed and the reaction mixture was
kept at room temperature (4 h), with the reaction progress moni-
tored by TLC (dichloromethane/acetone 9:1) until the starting
material disappeared. To the reaction mixture, cooled at 0–5 °C,
water (5.3 mL), 15% sodium hydroxide aqueous solution (5.3 mL),
and water (16 mL) were sequentially added. The white precipitate
was removed by suction through a Celite pad. The solvent was
evaporated at reduced pressure and the recovered oily 3 (5.02 g,
1
:20. HPLC analyses were performed with a Merck–Hitachi L-6200;
chiral column: Phenomenex Lux 3 Cellulose-1, 250 ꢀ 4.6 mm; UV
detector wavelength 254 nm). GLC analyses were performed with
1
a Hewlett–Packard 5890-series II. H NMR spectra were recorded
on a Bruker-Avance 500 MHz spectrometer. 13C NMR spectra were
collected at 125.76 MHz. The values of the optical rotations were
registered on a Perkin–Elmer (mod. 343) polarimeter in a 1 dm cell
at 20 °C, setting the wavelength at 589 nm. Mass spectra were re-
corded on a Agilent instrument (mod 6339 Ion trap LC/MS) using
the ESI source with positive ion polarity; the samples were dis-
8
9%) was used in the next step without further purification. 1
NMR (CDCl ) d 2.23 (m, 1H, H-3); 2.56 (dd, 1H, J = 16.17 and
.40 Hz, H-4); 2.88 (dd, 1H, J = 16.17 and 5.18 Hz, H-4); 2.62–
.82 (m, 2H, exchange with D O); 3.15 (dd, 1H, J = 8.09 and
H
3
8
2
1
(
5
2
1.14 Hz, H-2), 3.46 (ddd, 1H, J = 11.14, 3.51 and 1.52, H-2); 3.64
0
dd, 1H, J = 11.14 and 7.62 Hz, H-1 ); 3.72 (dd, 1H, 11.14 and
ꢁ1
0
.95 Hz, H-1 ); 6.53 (d, 1H, J = 7.78 Hz, H-5); 6.67 (dd, 1H, J = 7.78
solved in methanol (0.02
lg lL ) and were examined utilizing
13
and 8.24 Hz, H-6); 6.97–7.05 (m, 2H, H-7 and H-8). C NMR
the direct inlet probe technique at an infusion rate of about
ꢁ1
0
(
1
(
2
1
CDCl
3
) d 29.56 (C-4); 34.91 (C-3), 44.03 (C-2); 65.23 (C-1 );
0
.6 mL min ; data acquisition and analysis were accomplished
14.16 (C-5); 117.36 (C-6); 120.21 (C-10); 126.84 (C-8); 129.82
C-7); 144.48 (C-9). IR max 3380.86, 3238.16, 2918.32, 2864.52,
837.38, 1602.37, 1582.56, 1494.88, 1471.35, 1368.95, 1323.05,
with BRUKER DALTONICS DATA ANALYSIS 3.3 software. The infrared spectra
were registered on a Perkin Elmer instrument (mod. FT-IR spec-
trum one) equipped with universal attenuated total reflection
m
ꢁ1
293.83, 1266.81, 1071.36, 1022.97 cm ; MS (ESI+) m/z 164.1
(
ATR) sampling.
+
+
[
M+1] , 186.0 [M+Na] .
4
.2. (RS)-3-Hydroxymethyl-1-tertbutyloxycarbonyl-1,2,3,4-tet-
0
4
4
.2.4. (RS)-3-(1 -Hydroxymethyl)-1-tert-butyloxycarbonyl- 1,2,3,
-tetrahydroquinoline 6
rahydroquinoline 6
To a solution of 3 (4.90 g, 30 mmol) in dioxane (230 mL) and
4
.2.1. Methyl quinoline-3-carboxylate 4
water (290 mL), sodium hydroxide (14 g, 0.35 mol) and di-tertbutyl
carbonate (72 mL, 313 mmol) were added sequentially. The reac-
tion mixture was kept, under stirring, at room temperature
Quinoline-3-carboxylic acid (8 g, 46.2 mmol) was dissolved in
methanol (900 mL), after which thionyl chloride (5 mL, 68.5 mmol)
was added at 0 °C. The solution was kept at reflux, under stirring
10 h), monitoring the reaction progress by TLC (dichlorometh-
ane/methanol 9:1). An additional amount of thionyl chloride
5 mL) was added and the solution was kept at reflux (20 h). After
cooling at room temperature, the solvent was evaporated at re-
(
24 h). The reaction progress was monitored by TLC (dichlorometh-
(
ane/methanol 9:1). Dioxane was removed under reduced pressure
and the remaining aqueous phase was extracted with dichloro-
methane (4 ꢀ 70 mL). The collected organic phases were washed
(
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P. Ferraboschi et al. / Tetrahedron: Asymmetry xxx (2013) xxx–xxx
5
with water (2 ꢀ 100 mL) until pH 7. After the usual work-up, a yel-
(R)- and (S)-isomers (see footnote in the Results and discussion
section).
low oil was obtained, which was purified by silica gel column chro-
matography. The desired Boc derivative
recovered by elution with hexane/ethyl acetate 8:2. 1H NMR
CDCl ) d 1.54 (s, 9H, CH ); 1.89 (br s, 1H, exchange with D O);
.31 (m, 1H, H-3); 2.53 (dd, 1H, J = 16.9 and 5.6 Hz, H-4); 2.99
6 (6.3 g, 80%) was
4
.3.2. Second resolution
To a solution of 6 (81% ee, 0.600 g, 2.28 mmol) in toluene
(
2
3
3
2
(
60 mL), vinyl acetate (0.9 mL, 9.65 mmol) and PFL (13 mg) were
(
8
dd, 1H, J = 16.9 and 6.99 Hz, H-4); 3.50 (dd, 1H, J = 11.7 and
added. The mixture was kept under stirring at room temperature
until 41% conversion. The residue, obtained after filtration and
evaporation of the solvent, was purified by silica gel column chro-
matography. By elution with hexane/ethyl acetate 9:1, pure ace-
tate 7 (0.278 g, 40%) was obtained. (R)-Alcohol 6 (0.300 g, 50%)
0
.9 Hz, H-2); 3.57–3.65 (m, 2H, H-2 and H-1 ); 3.92 (dd, 1H,
0
J = 13.5 and 5.9 Hz, H-1 ); 7.04 (dd, 1H, J = 7.87 and 7.35 Hz, H-6);
7
.11 (d, 1H, J = 7.35 Hz, H-5); 7.16 (dd, 1H, J = 8.22 and 7.87 Hz,
13
H-7); 7.59 (d, 1H, J = 8.22 Hz, H-8). C NMR d 28.35 ((CH
2
3
)
3
C);
0
9.50 (C-4); 36.15 (C-3); 45.19 (C-1 ); 63.67 (C-2); 81.42
was obtained by elution with hexane/ethyl acetate 7:3. (R)-6
(
(CH
3
)
3
C); 123.91 (C-6); 124.57 (C-8); 125.58 (C-7); 128.28 (C-
0); 129.13 (C-5); 138.68 (C-9); 154.63 (N-COO). IR max 3430.76,
975.98, 2929.84, 1690.36, 1673.95, 1492.20, 1367.09,
20
D
½
a
ꢂ
¼ þ11:8 (c 1, chloroform). Ee >98% (from HPLC); (R)-7 ob-
1
2
1
m
20
D
tained by acetylation of (R)-6 ½
aꢂ
¼ ꢁ28:7 (c 1, chloroform).
ꢁ
1
+
+
159.36 cm ; MS (ESI+) m/z 286.1 [M+Na] , 549.1 [2M+Na] .
0
4
.4. (S)-3-(1 -Hydroxy-methyl)-1-tert-butyloxycarbonyl- 1,2,3,4-
tetrahydroquinoline 6
0
4
4
.2.5. (RS)-3-(1 -Hydroxymethyl)-1-tert-butyloxycarbonyl-1,2,3,
0
-tetrahydroquinoline, 1 -acetate 7
To a solution of (RS)-6 (0.200 g, 0.76 mmol) in dry pyridine
4.4.1. First resolution
The irreversible transterification of (RS)-6 (1.21 g, 4.6 mmol)
was carried out under the same conditions as described for the
preparation of (R)-6, but the reaction was stopped at 32% conver-
sion. The residue (1.29 g), obtained after the usual work-up, was
purified by silica gel column chromatography. By elution with hex-
ane/ethyl acetate 9:1, (S)-acetate 7 was recovered as an oil (0.40 g,
29%, 76% ee from HPLC); unreacted (R)-6 was recovered by elution
with hexane/ethyl acetate 8:2 as an oil (0.80 g, 65%).
(
2 mL) acetic anhydride (0.25 mL, 2.64 mmol) was added. The reac-
tion mixture was kept at room temperature overnight, after which
TLC analysis (hexane/ethyl acetate 7:3) showed complete conver-
sion. The solution was poured in ice cooled water (40 mL) and
the product was recovered by extraction with dichloromethane
(
(
3 ꢀ 40 mL). The collected organic phases were washed with water
3 ꢀ 40 mL). After the usual work-up crude acetate 7 was recov-
ered. Purification on silica gel column chromatography (hexane/
1
ethyl acetate 9:1 as eluant) afforded pure 7 (0.204 g, 88%).
NMR (CDCl ) d 1.55 (s, 9H, (CH C); 2.11 (s, 3H, CH CO); 2.37
m, 1H, H-3); 2.58 (dd, 1H, J = 16.02 and 8.70 Hz, H-4); 2.93 (dd,
H
4.4.2. Second resolution
3
)
3 3
3
To a suspension of acetate 7 (76% ee, 0.310 g, 1 mmol) in phos-
phate buffer (pH 7, 18 mL), PFL (6 mg) was added. The pH of the
mixture was kept at 7 over the course of the reaction (5 h) by
the addition of 0.1 M sodium hydroxide aqueous solution until a
calculated 70% conversion. The reaction progress (61%) was veri-
fied by GLC (see above for analysis conditions); the aqueous phase
was extracted with dichloromethane (3 ꢀ 15 mL). The collected or-
ganic phases were washed with water (2 ꢀ 50 mL) and after the
usual work-up, an oily residue was recovered (0.28 g); purification
by silica gel column chromatography afforded pure acetate 7
(0.085 g, 28%, hexane/ethyl acetate 9:1 as eluant) and (S)-alcohol
6 (0.153 g, 58%, hexane/ethyl acetate 8:2 as eluant. Compound
(
1
H, J = 16.02 and 5.65 Hz, H-4); 3.37 (dd, 1H, J = 12.8 and 8.85 Hz,
0
H-2); 3.96-4.16 (m, 3H, 2H-1 and H-2); 7.02 (dd, 1H, J = 7.56 and
7
and 7.56 Hz, H-7); 7.65 (d, 1H, J = 7.95 Hz, H-8). C NMR (CDCl )
3
d 20.90 (CH
(
1
(
1
3
.02 Hz, H-6); 7.11 (d, 1H, J = 7.02 Hz, H-5); 7.17 (dd, 1H, J = 7.95
13
3
CO); 28.37 ((CH
C-2); 65.71 (C-1 ); 81.06 ((CH
3
)
3
C); 30.09 (C-4); 33.77 (C-3); 46.28
C); 123.61 (C-6); 124.08 (C-8);
0
3
)
3
25.95 (C-7); 127.87 (C-10); 128.88 (C-5); 138.44 (C-9); 153.75
); IR max 2976.11, 2932.21, 1741.73,
697.36, 1492.67, 1367.59, 1239.61, 1161.70 cm ; MS (ESI+) m/z
NCOO); 171.03 (COCH
3
m
ꢁ1
+
+
28.2 [M+Na] , 344 [M+K] .
(
S)-6 showed a >98% ee (by HPLC, eluant: n-hexane/2-propanol
ꢁ1
20
D
0
100:2; flow rate 0.250 mL min ). ½
aꢂ
¼ ꢁ12:4 (c 1, chloroform).
4
.3. (R)-3-(1 -Hydroxymethyl)-1-tert-butyloxycarbonyl-1,2,3,4-
tetrahydroquinoline 6
4
.5. (R)-3-Methyl-1,2,3,4-tetrahydroquinoline 2
4
.3.1. First resolution
To a solution of (RS)-6 (1.95 g, 7.4 mmol) in toluene (168 mL),
0
4.5.1. (R)-3-(1 -Hydroxymethyl)-1-tert-butyloxycarbonyl-1,2,3,
0
vinyl acetate (2.93 mL, 31.4 mmol) and PFL (36 mg, 40.2 U/mg)
were added sequentially. The reaction mixture was kept at room
temperature with vigorous stirring in a screw cap flask. The reac-
tion progress was monitored by GLC (column: HP-5 WB, 30 m,
4-tetrahydroquinoline, 1 -tosylate 8
To a solution of (R)-6 (>98% ee, 0.27 g, 1.03 mmol) in pyridine
(0.7 mL) cooled in an ice bath, tosyl chloride (0.38 g, 2 mmol)
was added slowly. The reaction mixture was kept at room temper-
ature until the starting material disappeared (4 h, by TLC hexane/
ethyl acetate 7:3). The solution was then poured into ice cooled
water (5 mL). The precipitate was recovered by suction, washed
with water (3 ꢀ 5 mL), and dried under reduced pressure. The
0
.88
l
2
m, ID 0.53 mm; oven temperature: 160 °C, isothermal; car-
; 140 kPa). R Alcohol 6 9.8 min; acetate 7 15.5 min. The
rier N
t
reaction was stopped at 58% conversion; the enzyme was removed
by filtration and the solvent was evaporated at reduced pressure.
The residue (1.91 g) was purified by silica gel column chromatog-
raphy. By elution with hexane/ethyl acetate 9:1, acetate 7 was
recovered (1.02 g, 45%). Elution with hexane/ethyl acetate 7:3
afforded alcohol 6 (0.69 g, 35%). The ee of (R)-6 (81%) was deter-
mined after acetylation (acetic anhydride in pyridine) by HPLC
analysis on a chiral stationary phase (eluant: n-hexane/2-propanol
recovered tosylate 8 (0.35 g, 82%) was used in the next step with-
out further purification. H NMR (CDCl
(m, 1H, H-3), 2.48 (s, 3H, CH
8.74 Hz, H-4), 2.89 (dd, 1H, J = 16.7 and 5.96 Hz, H-4); 3.34 (dd,
1H, J = 12.9 and 8.54 Hz, H-2); 3.94 (dd, 1H, J = 12.9 and 4.17 Hz,
H-2); 4.01 (m, 2H, H-4); 7.00 (dd, 1H, J = 8.34 and 6.95 Hz, H-6);
7.05 (d, 1H, J = 6.95 Hz; H-5); 7.16 (dd, 1H, J = 8.34 and 7.75 Hz,
H-7); 7.37 (d, 2H, J = 8.15 Hz, Ar-CH
8); 7.81 (d, 2H, J = 8.15 Hz, ArSO ).
1
3
3
) d 1.53 (s, 9H, CH ); 2.39
3
Ar); 2.56 (dd, 1H, J = 16.7 and
ꢁ1
1
00:2; flow rate 0.250 mL min ), by comparison with the
chromatogram of racemic acetate 7. R (S)-7 48.72; (R)-7 52.34.
The acetylation was required in order to suitably separate
3
); 7.63 (d, 1H, J = 8.34 Hz, H-
t
2
Please cite this article in press as: Ferraboschi, P.; et al. Tetrahedron: Asymmetry (2013), http://dx.doi.org/10.1016/j.tetasy.2013.07.008

6
P. Ferraboschi et al. / Tetrahedron: Asymmetry xxx (2013) xxx–xxx
4
.5.2. (R)-3-Methyl-1-tert-butyloxycarbonyl-1,2,3,4-tetrahydro-
Acknowledgements
quinoline 9
To a solution of tosylate 8 (0.35 g, 0.84 mmol) in dry tetrahydro-
furan (7 mL) lithium aluminum hydride (0.143 g, 3.77 mmol) was
added. The reaction was kept under stirring at room temperature
We thank Dr. Maria De Mieri for her technical assistance at the
early stage of the work and Mr. Riccardo Monti and Mr. Stefano
Novelli for IR and MS spectra.
(
2 h) until the starting material disappeared (by TLC hexane/ethyl
acetate 9:1). Water (0.14 mL), 15% sodium hydroxide aqueous
solution (0.14 mL) and water (0.42 mL) were added sequentially.
The white precipitate was removed by suction through a Celite
pad and the filtrate evaporated at reduced pressure affording an
oily residue (0.178 g, 86%), which was used in the next step with-
out further purification. For analytical purposes a sample (50 mg)
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.5.3. (R)-3-Methyl-1,2,3,4-tetrahydroquinoline 2
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1
flow rate 0.5 mL min ). R
(
(
t
(R)-2 15.33, (S)-2 12.99 min. H NMR
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m
max 3318.07, 2943.52, 2831.70, 1448.90,
ꢁ1
+
+
20
D
17
ms/ms 106.0 [M-CH
3
CHCH
2
] ; ½
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¼ ꢁ73:4 (c 3, methanol) (lit.
(S)-2 = +79).
Please cite this article in press as: Ferraboschi, P.; et al. Tetrahedron: Asymmetry (2013), http://dx.doi.org/10.1016/j.tetasy.2013.07.008