Baker's yeast catalyzed preparation of a new enantiomerically pure synthon of (S)-pramipexole and its enantiomer (dexpramipexole) This work is dedicated to Professor Enzo Santaniello in the year of his 70th birthday

Article abstract of DOI:10.1016/j.tetasy.2014.07.011

A biocatalyzed reduction of a prochiral bicyclic ketone afforded enantiomerically pure (R)-2-acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothiazole, a synthon of the anti-Parkinson (S)-pramipexole and its (R)-isomer, which is currently under investigation for the treatment of amyotrophic lateral sclerosis (ALS).

Full text of DOI:10.1016/j.tetasy.2014.07.011

Tetrahedron: Asymmetry 25 (2014) 1239–1245
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Baker’s yeast catalyzed preparation of a new enantiomerically pure
synthon of (S)-pramipexole and its enantiomer (dexpramipexole)
a
b
a,
Patrizia Ferraboschi a, , Samuele Ciceri , Pierangela Ciuffreda , Maria De Mieri , Diego Romano c,
⇑
Paride Grisenti d
a Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università di Milano, Via Saldini 50, 20133 Milano, Italy
b Dipartimento di Scienze Biomediche e Cliniche ‘L. Sacco’, Università di Milano, Via Grassi 74, 20157 Milano, Italy
c Department of Food, Enviromental and Nutritional Sciences, Università di Milano, Via Mangiagalli 25, 20133 Milano, Italy
d EUTICALS SpA, Via Volturno 41/43, 20089 Rozzano (MI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2014
Accepted 23 July 2014
A biocatalyzed reduction of a prochiral bicyclic ketone afforded enantiomerically pure (R)-2-acetylamino-
6-hydroxy-4,5,6,7-tetrahydrobenzothiazole, a synthon of the anti-Parkinson (S)-pramipexole and its
(R)-isomer, which is currently under investigation for the treatment of amyotrophic lateral sclerosis
(ALS).
Ó 2014 Elsevier Ltd. All rights reserved.
This work is dedicated to Professor Enzo
Santaniello in the year of his 70th birthday
1. Introduction
as the dihydrochloride monohydrate (Mirapex brand name) for
Parkinson disease treatment. The (R)-isomer (dexpramipexole) is
Parkinson disease is a neurodegenerative disorder characterized
by the progressive loss of dopaminergic neurons of substantia nigra
in the brain basal glia accompanied by motor symptoms (bradyki-
nesia, muscle rigidity, resting tremor, and equilibrium impair-
ment). Parkinson disease is treated with dopamine replacement
agents, such as levodopa or dopaminergic agonists. Pramipexole
1, approved in 1997 in the USA and in 1998 in most European
countries, is the most prescribed dopamine agonist, both as mono-
therapy and as adjunctive therapy with levodopa.1–3
currently in clinical development for the treatment of amyotrophic
lateral sclerosis (ALS), a rapidly progressive and lethal neurodegen-
erative disorder of motor neurons. The enantiomeric purity of the
(R)-isomer drug substance is a critical parameter for accurately
interpreting studies conducted with dexpramipexole.4 The pres-
ence of a trace amount of (S)-pramipexole has a significant phar-
macological effect due to its extremely high dopamine agonist
affinity.4 Therefore the synthesis of both enantiomerically pure
(R)- and (S)-pramipexole is an engaging goal not only in laboratory
amounts but also for large scale preparative purposes.
Usually the synthesis of (S)-pramipexole is realized via the res-
olution of racemic pramipexole5 or of an intermediate6 by frac-
tional crystallization of a suitable diastereomeric salt. Preparative
chiral chromatography has also been applied to the separation of
(R)- and (S)-pramipexole7,8 or of a racemic precursor.8
In order to avoid the long and tedious processes that character-
ize fractional crystallization and preparative HPLC, an asymmetric
synthesis should be performed; we planned to take advantage of
the high stereoselectivity of biocatalysts in order to prepare an
enantiomerically pure intermediate, that could then be easily con-
verted into (S)-pramipexole or dexpramipexole.
R1
R2
7
1
S
2
6
NH2
5
N
3
4
1
R1 = H, R2 = NHCH2CH2CH3
(R)-pramipexole
(S)-pramipexole
R1 = NHCH2CH2CH3, R2 = H
Pramipexole
1
(2-amino-6-propylamino-4,5,6,7-tetra-
hydrobenzothiazole) bears a stereocenter at the 6-position. Only
the (S)-isomer is active as a dopamine agonist and is marketed,
2. Results and discussion
⇑
Corresponding author. Tel.: +39 02 50316052; fax: +39 02 50316036.
E-mail address: patrizia.ferraboschi@unimi.it (P. Ferraboschi).
Two distinct biocatalytic approaches to the synthesis of
(S)-pramipexole have been already described starting from
(RS)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole 29 or from the
Present address: Division of Pharmaceutical Biology, Department of Pharmaceu-
tical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
http://dx.doi.org/10.1016/j.tetasy.2014.07.011
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

1240
P. Ferraboschi et al. / Tetrahedron: Asymmetry 25 (2014) 1239–1245
ester of (RS)-6-carboxylic acid 3.10 In both cases, the (R)- and (S)-
intermediates were obtained via a lipase-catalyzed transformation.
pounds.23 However, with regard to the complete conversion of
ketone 4 into alcohol 8, the results were not satisfactory in terms
of the ee (see Table 2) and the low recovery.
It is well known that substrate modification can reverse the bio-
transformation stereochemical outcome and also increase the
enantiomeric purity.11,24 In our case, the functionalization of the
2-amino group could improve the recovery of the alcohol from
the aqueous media. We therefore carried out Baker’s yeast cata-
lyzed reduction on N-acetyl derivative 9 (Scheme 2). In this case,
the addition of n-heptane improved the enantiomeric excess in
pH 7 buffer alone (92%). If the reaction was stopped at complete
conversion a 94% ee was obtained; lowering the conversion to
87%, >98% ee was achieved. The easier recovery of the less polar
alcohol 10 (compared with 8) from the reaction medium, by means
of a continuous extraction, led to 75–80% yields. The ee was deter-
mined by HPLC analysis on a chiral stationary phase (Chiralpak IA)
by comparison with a racemic sample. The formation over the
course of the bioreduction of a by-product (5%), with a retention
time very similar to that of the enantiomer later eluted, required
careful purification of the crude alcohol 10. The enantiomeric pur-
ity was unequivocally ascertained through high field 1H NMR anal-
ysis of the diastereomeric esters 11, which were obtained by
R
S
NH2
N
2
3
R = NH2
R = COOR
The well documented capability of microorganisms11,12 to per-
form asymmetric reductions of prochiral carbonyl groups to
alcohols prompted us to study this approach. Initially, 2-amino-
6-oxo-4,5,6,7-tetrahydrobenzothiazole 4 seemed to be the best
substrate for preliminary microorganism screening. Starting from
the commercially available monoprotected cyclohexandione 5,
we prepared enolsilyl ether 6,13 which was transformed into the
6-acetal of 2-amino-6-oxo-4,5,6,7-tetrahydrobenzothiazole 7 by
treatment with N-bromosuccinimide14 followed by cyclization
with thiourea. Hydrolysis of acetal with 5% hydrochloric acid affor-
ded the desired ketone 4 in 64% yields from 5 (Scheme 1).
O
reaction with the (S)-a-methoxy-a-trifluoromethylphenylacetic
O
O
O
O
O
O
acid (MTPA) chloride25 of alcohols (R)- and (R,S)-10. The same
analysis also allowed us to assign, according to the modified
method of Mosher,26 the (R)-configuration to alcohol 10, not previ-
ously described in the literature (see the NMR section below).
The knowledge of the stereocenter configuration was necessary
in order to plan how to accomplish the synthesis of (S)-pramipex-
ole 1. Therefore, (R)-10 was transformed into the corresponding
tosylate 12 (74%), which upon treatment with sodium azide in
dimethylformamide afforded azide 13 with the desired (S)-config-
uration. Reduction with polymer bonded triphenylphosphine gave
i
ii
iii
77%
83%
S
S
from 5
N
N
O
OSi(CH3)3
NH2
NH2
7
4
5
6
Scheme 1. Reagents and conditions: (i) (CH3)3SiOSO2CF3, (CH3CH2)3N, CH2Cl2;
(ii) NBS, AcONa, thiourea, THF/H2O; (iii) 5% HCl, THF.
(S)-2-acetylamino-6-amino-4,5,6,7-tetrahydrobenzothiazole
14
The choice of Saccharomyces cerevisiae (the common Baker’s
yeast), as the first microorganism to be screened was due to its
well-known advantages such as simple experimental conditions,
easy availability, and efficiency in relation to the yields and stere-
oselectivity.11,15–17 The bioreduction was carried out under differ-
ent conditions as summarized in Table 1, in order to achieve the
highest ee.
(76% from tosylate 12). By removing the acetyl group, (S)-2,6-dia-
mine 2, an advanced known intermediate of (S)-pramipexole 1,
was obtained [in 51% overall yield from (R)-alcohol 10] (Scheme 3).
The configuration and enantiomeric purity were determined by
comparison with obtained 2 with a sample of (S)-2, [previously
prepared by fractional crystallization6 of (R,S)-2] via HPLC analysis
on chiral stationary phase [Crownpak CR (+)]; the (S)-configuration
of 2 and the (R)-configuration of biosynthesized 10 were con-
firmed. In addition, the observed >98% ee allowed us to conclude
that the enantiomeric purity remained the same over the course
of the transformation of 10 into 2.
Commercial lyophilized Baker’s yeast18 catalyzed the complete
conversion of ketone 4 into alcohol 8 but with moderate ee (84%)
(Scheme 2). The best results, even if not yet completely satisfactory
(94% ee and low recovery), were observed by addition to the bio-
transformation medium of an organic solvent, according to a
reported procedure.19–22 Multiple reducing enzymes are present
in yeast with different enantioselectivities. The role of an organic
solvent can be explained either by a change in the substrate aque-
ous concentration, which should favor a single reducing enzyme, or
by possible inhibition of one or more reducing enzymes.11
In order to obtain enantiomerically pure alcohol 8, we focused
our attention on other microorganisms, listed in Table 2, that are
already used for stereoselective reduction of carbonyl com-
Finally, according to the literature,27 (S)-2,6-diamine 2 was
treated with 1-propanol tosylate 15 to afford after suitable work-
up the dihydrochloride monohydrate of (S)-pramipexole 1 in 42%
yields.
Enantiomerically pure alcohol (R)-10 was chosen as the starting
material for the synthesis of the intermediate of the (R)-isomer of
pramipexole 1, the dexpramipexole, which is undesired in anti-
Parkinson preparations, but is currently under investigation for
ALS treatment. A Mitsunobu reaction28 starting from alcohol (R)-
Table 1
Baker’s yeast-catalyzed reduction of ketones 4 and 9
Ketone
Alcohol
Experimental conditions
Time (h)
Conversiona (%)
eea (%)
4
4
4
9
9
9
8
8
8
10
10
10
Buffer pH 7
15
10
10
18
18
16
100
100
100
100
100
87
84
94
93
92
94
Buffer pH 7/n-heptane 1:1
Buffer pH 7/toluene 1:1
Buffer pH 7
Buffer pH 7/n-heptane 1:1
Buffer pH 7/n-heptane 1:1
>98
a
Determined by HPLC on chiral stationary phase.

P. Ferraboschi et al. / Tetrahedron: Asymmetry 25 (2014) 1239–1245
1241
OH
O
i
S
NH2
N
S
8
N
OMTPA
OH
N
O
NH2
4
ii
67%
iv
iii
79%
>98% ee
S
S
S
N
N
NHCOCH3
NHCOCH3
NHCOCH3
11
10
9
Scheme 2. Reagents: (i) Bioreduction; (ii) Ac2O; (iii) Baker’s yeast; (iv) MTPA-Cl.
Table 2
OH
N
OCOPh
OH
N
Bioreduction of ketone 4 to alcohol 8
Microorganisma
Enantiopurity of alcoholb 8 (%)
ii
i
(R)-1
Torulopsis magnoliae IMAP 4425
Torulopsis molischiana CBS 837
Sporobolomyces salmonicolor
Pichia etchelsii
Kluyveromyces marxianus CBS 1553
Hansenula polymorpha CBS 4732
0
0
0
50
80
50
S
S
S
N
NHCOCH3
NHCOCH3
NHCOCH3
10
(R)-
16
(S)-10
Scheme 4. Reagents and conditions: (i) PhCOOH, DEAD, Ph3P, DMF; (ii) 1% NaOH,
MeOH.
a
Resting cells resuspended in 0.1 M phosphate buffer pH 7 containing 50 g/L of
glucose. The biotransformations were stopped after 48 h at 100% conversion (by
TLC).
b
Determined by HPLC on chiral stationary phase.
tons due to the presence of p-electron system. On the basis of these
observations, the spectra of other compounds were assigned.
In order to assign the configuration of the C(6) of secondary
alcohol 10, we used a modified Mosher’s method26 to establish
the absolute configuration of the secondary alcohols. Treatment
of 10 with (S)- and (R)-2-methoxy-2-(trifluoromethyl)-phenylace-
tyl chloride (MTPA-Cl)25 gave the 6-O-(R)-MTPA and 6-O-(S)-MTPA
esters, respectively, whose 1H NMR spectra at 500 MHz were
10 afforded enantiomerically pure (by HPLC) benzoate 16; alcohol
10 was obtained by ester hydrolysis and its the (S)-configuration
was assigned by comparison with the HPLC chromatograms of
(RS)- and (R)-10 (Scheme 4).
2.1. NMR analysis
acquired and assigned. The
D
d values (dS ꢀ dR) are expressed in
In spite of the narrow dimensions of the studied molecules, in
order to unequivocally assign the NMR signals, careful analysis
was required. Proton and carbon 1D NMR spectra, as well as 2D
NMR homocorrelation (COSY) and heterocorrelation (HMQC and
HMBC) spectra were employed for complete structural assign-
ments. In compound 4, the assignment of the signals of the protons
at C-4 and at C-5 was made on the basis of the long-range coupling
constants and splitting patterns observed in the 1H NMR. The res-
onance of the protons at d 2.95 was a triple triplet (J = 6.9 and
J = 1.8 Hz) which was correlated in the COSY experiment with both
protons at d 2.71 (t, J = 6.9 Hz) and d 3.43 (7-CH2, t, J = 1.8 Hz).
These data revealed that these hydrogens belonged to 4-CH2 since
they have a homoallylic coupling constant 5J with the 7-CH2 pro-
Hertz and are shown in Scheme 5. The chemical shifts of the pro-
tons at the 5-position of the (R)-MTPA esters appear shielded with
respect to those of the (S)-MTPA diastereomer. Conversely, the
chemical shifts of the protons at the 7-position appear deshielded
H3CO Ph
-15, -60 Hz
O
S
N
7
F3C
H
N
5
O
30 Hz
O
Scheme 5. Configuration assignment to alcohol 10 based on the lH NMR
obtained for their (S)- and (R)-MTPA esters.
(500 MHz).
D
d values
D
d values (dS ꢀ dR) are expressed in Hz
NH2
NH2
N3
OTs
OH
iv
ii
iii
i
57%
90%
S
from 10
S
S
S
S
N
N
N
N
N
NH2
NHCOCH3
2HCl.H2O
NHCOCH3
NHCOCH3
14
2
NHCOCH3
13
12
10
HN
S
v
OTs
NH2
+
2
42%
N
15
Scheme 3. Reagents and conditions: (i) TsCl, py; (ii) NaN3, DMF; (iii) Ph3P polymer bonded, THF/H2O; (iv) 5% HCl, THF; (v) NEt(iPr)2, DMF, 1 M NaOH, 12 M HCl.

1242
P. Ferraboschi et al. / Tetrahedron: Asymmetry 25 (2014) 1239–1245
in (R)-MTPA esters relative to the (S)-MPTA ones, thus allowing us
to assign the (R)-configuration at C-6.
Optical rotations were registered on a Perkin–Elmer (mod. 241)
polarimeter in a 1 dm cell at 20 °C, setting the wavelength at
589 nm or at 546 nm. Mass spectra were recorded on a Agilent
instrument (mod 6339 Ion trap LC/MS) using the ESI source with
positive and/or negative ion polarity; the samples were dissolved
The NMR analysis of (R)-MTPA esters 11 of (R)- and (R,S)-10 also
allowed us to confirm the ee of alcohol 10 previously determined
by HPLC. One of protons at the 7-position showed two double dou-
blets centered at 3.16 and 3.14 ppm in the case of (R,S)-10. The sig-
nal centered at 3.14 ppm was not detectable in the spectrum of
MTPA ester of (R)-10.
in methanol (0.05 lg/lL) and were examined utilizing the direct
inlet probe technique at an infusion rate of about 0.6 mL/min; data
acquisition and analysis were accomplished with Bruker Daltonics
Data Analysis 3.3 software. The infrared spectra were registered on
a Perkin Elmer instrument (mod. FT-IR spectrum one) equipped
with universal attenuated total reflection (ATR) sampling. Differen-
tial scanning calorimetry (DSC) analyses were performed on a Per-
kin Elmer DSC-7 instrument, heating 10 °C/min. Melting points
were performed on SMP3 Stuart Scientific apparatus, 2.5 °C/min.
3. Conclusion
The synthesis of alcohols via Baker’s yeast-catalyzed reduction
of carbonyl compounds represents a classic approach of organic
synthesis;15–17 when a prochiral ketone is reduced a chiral alcohol
is formed. Nevertheless several reductases with different stereose-
lectivities are present in the Saccharomyces cerevisiae, but enantio-
merically pure compounds are obtained in most cases. These
considerations prompted us to plan a biocatalyzed reduction of a
suitable ketone in order to obtain an enantiomerically pure
advanced intermediate of the synthesis of (S)-pramipexole, a
known anti-Parkinson drug. Modification of the starting ketone
and of the biotransformation medium allowed us to obtain enanti-
omerically pure (R)-alcohol 10, thus showing the Baker’s yeast effi-
ciency. This synthon, not yet described in the literature, was
transformed through very simple steps into the desired dihydro-
chloride monohydrate derivative of (S)-pramipexole 1 in 21% over-
all yield.
The limitation usually ascribed to the biocatalyzed reduction of
a ketone, namely that only one enantiomer is achievable, was over-
come by the complete inversion of the configuration realized under
the Mitsunobu conditions.28 The (S)-alcohol 10 obtained is a suit-
able substrate for dexpramipexole, the (R)-isomer of pramipexole,
which is currently under investigation in the treatment of ALS.
The use of readily available and inexpensive Saccharomyces
cerevisiae, the easy preparation of the biotransformation substrate,
and the simple steps required to accomplish the synthesis, make
the method applicable to a preparative scale. The optimized HPLC
analysis conditions (on a chiral stationary phase) and careful NMR
studies allow us to monitor the identity of the compounds and the
stereochemical outcome of the whole process.
4.1. 1,4-Cyclohexanedione-1-ethyleneacetal-4-trimethylsilyl-
enolether 6
To a solution of 1,4-cyclohexanedione monoethylene acetal 5
(7 g, 44.8 mmol) in dichloromethane (400 mL) and triethylamine
(18.5 mL, 0.133 mol) at 0 °C, a solution of trimethylsilyl trifluoro-
methanesulfonate (10 mL, 0.055 mol) in dichloromethane
(120 mL) was added dropwise. The reaction mixture was kept
under stirring at 0 °C under an N2 atmosphere until the starting
material disappeared (1 h), monitoring the reaction progress by
TLC (hexane/ethyl acetate/triethyl amine 9.5:0.45:0.05). Saturated
sodium hydrogen carbonate aqueous solution (30 mL) and water
(200 mL) were then added. The organic phase was washed with
brine (4 ꢁ 100 mL). After drying over sodium sulfate, filtration,
and removal of the solvent under reduced pressure, enol ether 6
(9.9 g, 96%) was recovered as an oil, which was used in the next
step, without further purification. The chemical and physical prop-
erties were in agreement with the literature values.13
4.2. 2-Amino-6-(ethylenedioxy)-4,5,6,7-tetrahydrobenzothiazole 7
To a solution of silyl enol ether 6 (9.9 g, 43.4 mmol) in tetrahy-
drofuran/water 1:1 (380 mL), N-bromosuccinimide (9.6 g,
53.9 mmol) and sodium acetate (0.500 g, 6.1 mmol) were added.
The reaction mixture was kept at room temperature with stirring
until the starting material disappeared (2 h) (TLC hexane/ethyl
acetate 9:1). Thiourea (3.46 g, 45.5 mmol) was added and the reac-
tion mixture was kept at 80 °C (6 h) and then at room temperature
overnight, under stirring. The reaction was monitored by TLC
(dichloromethane/methanol 9:1). Tetrahydrofuran was removed
at reduced pressure and the aqueous phase was extracted with
dichloromethane (4 ꢁ 50 mL). To the aqueous phase, 2 M sodium
hydroxide was added until pH 9. Extraction with dichloromethane
(5 ꢁ 50 mL), and usual work-up afforded 7 as a solid (7.9 g, 83%
from 5), which was used in the next step without further purifica-
tion. The chemical and physical properties are in agreement with
the literature values.29
4. Experimental
General: The reagents were purchased from Sigma–Aldrich
(Italy). The solvents were purchased from Panreac (Novachimica,
Italy). Dry Baker’s yeast was supplied by Paneangeli, Italy. Racemic
2,6-diamino-4,5,6,7-tetrahydrobenzothiazole was from Pen Tsao
Chemical & Pharmaceutical Industry Co., Ltd (China). All reactions
were monitored by TLC on silica gel 60 F254 precoated plates with a
fluorescent indicator (Merck) with detection by spraying with a
10% phosphomolybdic acid ethanol solution and heating at
110 °C. Column chromatography was performed on silica gel 60
(70–230 mesh) (Merck). HPLC analyses were performed with a
4.3. 2-Amino-6-oxo-4,5,6,7-tetrahydrobenzothiazole 4
Merck-Hitachi L-6200; RP column: Waters
(0.39 cm ꢁ 30 cm, 10 m); chiral columns: Daicel Crownpak
CR(+) m); Daicel Chiralpak IA
(0.4 cm ꢁ 15 cm,
(0.46 cm ꢁ 25 cm, m); UV detector wavelength 254 nm.
lBondapak C18
l
To a solution of acetal 7 (6.6 g, 0.031 mol) in tetrahydrofuran
(120 mL), 5% hydrogen chloride aqueous solution (120 mL) was
added. The reaction mixture was kept at reflux with stirring while
5
l
5
l
Nuclear magnetic resonance (NMR) spectra were recorded at
300 K on a Bruker-Avance 500 MHz spectrometer operating at
500.13 and 125.76 MHz for 1H and 13C acquisitions, respectively.
Chemical shifts (d) of the 1H NMR and 13C NMR spectra are
reported in ppm using the signal for residual solvent proton reso-
nance as the internal standard (1H NMR: CDCl3 7.26, DMSO-d6
2.49, CD3OD 3.31 ppm; 13C NMR: CDCl3 77.0 (central line),
DMSO-d6 39.50 (central line), CD3OD 49.00 (central line) ppm).
monitoring the reaction progress by HPLC (C18
lBondapak col-
umn, water/acetonitrile 7:3 as eluent, flow rate: 0.5 mL/min). After
the disappearance of the starting material (4 h), the work-up was
carried out as reported in the literature.30 A saturated aqueous
potassium carbonate solution (75 mL) was then added until pH 7.
Solvents were removed by evaporation at reduced pressure. To
the obtained solid residue, diethyl ether (60 mL) and saturated
aqueous potassium carbonate solution were added. The red precip-

P. Ferraboschi et al. / Tetrahedron: Asymmetry 25 (2014) 1239–1245
1243
itate was recovered by suction and washed with diethyl ether
(3 ꢁ 20 mL) to afford a crude product (6.2 g) which was purified
by silica gel column chromatography (1:10); elution with ethyl
acetate/methanol/triethylamine (84:8:8) to give pure 4 (4.0 g,
77%). Rt compound 4 8.96 min; compound 7 15.44 min. TLC
(dichloromethane/methanol/triethylamine 95:4:1) Rf 0.38. Mp
175 °C (ethyl acetate). DSC endothermic peak at 194 °C. IR mmax
3368.74, 3280.27, 3087.31, 2904.41, 1693.43, 1637.37, 1595.85,
on sodium sulfate and, after filtration, the solvent was removed
under reduced pressure to give an oily residue (80 mg). The aque-
ous phase was extracted in a continuous extraction apparatus with
the ethyl acetate previously used in order to wash the Celite pad
(5 ꢁ 200 mL). The organic phase was separated, dried over sodium
sulfate, and filtered. Evaporation of the solvent under reduced
pressure afforded a solid residue (2.2 g) which was purified by sil-
ica gel column chromatography (1:20). Pure 10 (1.99 g, 79%) was
recovered by elution with ethyl acetate. The ee (>98%) was estab-
lished by HPLC on chiral stationary phase by comparison with the
previously prepared (R,S)-10. Rt (R)-isomer 10.90 min; (S)-isomer
14.85 min. TLC (dichloromethane/methanol 95:5) Rf 0.25.
1540.22, 1493.29 cmꢀ1 1H NMR (CDCl3): d 3.43 (2H, t, J = 1.8 Hz,
.
7-H), 2.95 (2H, tt, J = 6.9, 1.8 Hz, 4-H), 2.71 (2H, t, J = 6.9 Hz, 5-H).
13C NMR (CDCl3): d 207.2 (6-C), 167.4 (2-C), 144.3 (N-C), 113.7
(S-C), 38.7 (5-C), 37.4 (7-C), 25.3 (4-C). 1H NMR (DMSO-d6): d
3.36 (2H, t, J = 0.9 Hz, 7-H), 2.77 (2H, tt, J = 7.0, 0.9 Hz, 4-H), 2.60
(2H, t, J = 7.0 Hz, 5-H). 13C NMR (DMSO-d6): d 207.6 (6-C), 167.6
(2-C), 144.6 (N-C), 111.5 (S-C), 38.6 (4-C), 37.6 (7-C), 25.6 (5-C).
MS (m/z) (ESI-) 167.3 [Mꢀ1]+. (ESI+) 201.1 [M+CH3OH]+ 223
[M+Na+CH3OH]+.
[a
]
D
20 = +25.5 (c 1, methanol); [
a
]
546
20 = +30.7 (c 1, methanol).
Mp 179 °C (ethyl acetate). DSC endothermic peak 187 °C. IR mmax
3421.46, 3234.12, 3151.18, 3031.33, 2930.41, 2853.17, 2767.79,
1687.99, 1667.98, 1537.67, 1435.72, 1361.70 cmꢀ1 1H NMR (CD3-
.
OD): d 4.15 (1H, dddd, J = 8.6, 6.9, 4.7, 2.9 Hz, 6-H), 2.99 (1H, ddd,
J = 15.7, 4.7, 1.8 Hz, 7a-H), 2.79 (1H, dddd, J = 16.5, 5.9, 5.9,
1.8 Hz, 4a-H), 2.71–2.61 (2H, m, 4b-H and 7b-H), 2.26 (3H, s,
CH3), 2.04 (1H, dddd, J = 14.2, 6.2, 5.9, 2.9 Hz, 5a-H), 1.91 (1H,
dddd, J = 14.2, 8.6, 7.7, 5.9 Hz, 5b-H), 13C NMR (CD3OD): d 169.1
(NHCOCH3), 156.4 (2-C), 143.2 (N-C), 120.9 (S-C), 66.2 (6-C), 30.7
(7-C), 30.5 (5-C), 23.2 (4-C), 21.2 (CH3). MS (m/z) (ESIꢀ) 212.1
[M]+. (ESI+) 235.0 [M+Na]+.
4.4. 2-Acetylamino-6-oxo-4,5,6,7-tetrahydrobenzothiazole 9
A solution of 4 (3.12 g, 18.6 mmol) in acetic anhydride (7 mL)
was kept under stirring at 100 °C.31 Monitoring the reaction by
TLC (dichloromethane/methanol 95:5), complete conversion was
observed after 30 min. The reaction mixture, after cooling to room
temperature, was poured into water (130 mL). The aqueous phase
was extracted with ethyl acetate (5 ꢁ 50 mL). The organic phases
were washed with water (3 ꢁ 50 mL), dried over sodium sulfate
and, after filtration, evaporated at reduced pressure. The crude
product (5.6 g) was purified by silica gel column chromatography
(1:10). Elution with ethyl acetate/hexane 9:1 afforded pure 9
(2.6 g, 67%). TLC Rf 0.38. Mp 220 °C (ethyl acetate). DSC endother-
mic peak 235.83 °C. IR mmax 3371.99, 3234.12, 3148.90, 3023.23,
4.7. General procedure for the preparation of Mosher’s ester 11
To a stirred solution of alcohol (R)-10 or (R,S)-10 (10 mg,
0.047 mmol) in carbon tetrachloride (0.2 mL) and pyridine
(0.2 mL), (S)-MTPA-chloride (12 lL, 0.061 mmol) was added. The
reaction mixture was allowed to stand at room temperature for
12 h. Next, dichloromethane (3 mL) and 3-dimethylaminopropan-
1-amine (13 lL) were added, the organic phase was washed with
2935.27, 2894.40, 2765.87, 1705.59, 1682.65, 1539.23 cmꢀ1
.
1H
NMR (CDCl3): d 3.59 (2H, br s, 7-H), 3.12 (2H, br t, J = 7.0 Hz,
4-H), 2.79 (2H, t, J = 7.0 Hz, 5-H), 2.32 (3H, s, CH3).13C NMR (CDCl3):
d 205.5 (6-C), 167.7 (NHCOCH3), 158.1 (2-C), 141.1 (N-C), 119.4 (S-
C), 38.4 (4-C), 37.0 (7-C), 24.7 (5-C), 23.3 (CH3). MS (m/z) (ESIꢀ)
209.7 [Mꢀ1]+. (ESI+) 211.0 [M+1]+, 233.0 [M+Na]+, 265.1
[M+Na+CH3OH]+.
1 M hydrogen chloride aqueous solution (2 ꢁ 5 mL), a saturated
sodium hydrogen carbonate aqueous solution (2 ꢁ 5 mL) and with
brine (5 mL). After drying over anhydrous sodium sulfate and
filtration, the solvents were evaporated under reduced pressure.
4.7.1. (R)-Mosher’s ester 11 of alcohol (R)-10
Colorless oil; 1H NMR (CDCl3) representative signals d 5.60 (1H,
m, 6-H), 3.17 (1H, dd, J = 16.5, 4.5, 7a-H), 3.01 (1H, dd, J = 16.5,
6.1 Hz, 7b-H), 2.73 (2H, m, 4-CH2).
4.5. (RS)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothi
azole 10
To a solution of ketone 9 (0.365 g, 1.74 mmol) in methanol
(20 mL), sodium borohydride (0.250 g, 6.60 mmol) was slowly
added. The solution was kept under stirring at room temperature
while monitoring the reaction progress until the starting material
disappeared (1 h) (TLC dichloromethane/methanol 95:5). Next,
3 M hydrochloric acid was added until pH 7; the solvents were
removed at reduced pressure and the solid residue was washed
with ethyl acetate (2 ꢁ 10 mL). After filtration, the filtrate was
dried over sodium sulfate and the usual work-up afforded pure
(RS)-10 (0.353 g, 95%).
4.7.2. (R)-Mosher’s ester 11 of alcohol (RS)-10
Colorless oil; 1H NMR (CDCl3) representative signals d 5.60 (1H,
m, 6-H), 5.55 (1H, m, 6-H), 3.17 (1H, dd, J = 16.5, 4.5, 7a-H), 3.13
(1H, dd, J = 16.5, 4.5, 7a-H), 3.01 (1H, dd, J = 16.5, 6.1 Hz, 7b-H),
2.90 (1H, dd, J = 16.5, 6.1 Hz, 7b-H), 2.78 (2H, m, 4-CH2), 2.73
(2H, m, 4-CH2).
4.8. (R)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothia
zole, 6-tosylate 12
To a solution of (R)-10 (1.5 g, 7.1 mmol) in dry pyridine (5 mL)
cooled at 0 °C, tosyl chloride (1.9 g, 9.9 mmol) was added. The solu-
tion was kept under stirring at room temperature until TLC analy-
sis (dichloromethane/methanol 95:5) showed complete conversion
(2 h). The reaction mixture was then poured into water and ice
(10 mL). The aqueous phase was extracted with dichloromethane
(3 ꢁ 10 mL). The organic phase was washed with water
(3 ꢁ 10 mL). The oily residue (1.92 g) was used in the next step
without further purification. TLC Rf 0.49. 1H NMR (CDCl3): d 7.82
(2H, d, J = 8.0 Hz, 20 and 60), 7.38 (2H, d, J = 8.0 Hz, 30 and 50), 5.04
(1H, dddd, J = 7.2, 5.2, 4.2, 2.4 Hz, 6-H), 2.98 (1H, ddd, J = 16.7,
4.2, 1.6 Hz, 7a-H), 2.92 (1H, dd, J = 16.7, 5.2 Hz, 7b-H), 2.82 (1H,
ddd, J = 16.5, 8.4, 5.7 Hz, 4a-H), 2.73 (1H, dddd, J = 16.5, 6.2, 5.7,
4.6. (R)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothia
zole 10
To a solution of sucrose (55 g) in a phosphate buffer (pH 7, 1 L)
dry Baker’s yeast (16 g) was added. The suspension was kept for
1 h at 30 °C under vigorous mechanical stirring. n-Heptane (1 L)
and ketone 9 (2.5 g, 11.9 mmol) were then added and the mixture
was kept at 30 °C, under vigorous stirring. The reaction progress
and the ee of the alcohol were monitored by HPLC on chiral sta-
tionary phase (Chiralpak IA, hexane/2-propanol 8:2 as eluent, flow
rate: 0.7 mL/min). At 87% conversion (16 h), the suspension was fil-
tered through a Celite pad. The organic phase was separated, dried

1244
P. Ferraboschi et al. / Tetrahedron: Asymmetry 25 (2014) 1239–1245
1.6 Hz, 4b-H), 2.49 (3H, s, PhCH3), 2.32 (3H, s, CH3CO), 2.18 (1H,
dddd, J = 14.0, 7.2, 5.7, 5.7 Hz, 5a-H), 2.00 (1H, dddd, J = 14.0, 8.4,
6.2, 2.4 Hz, 5b-H). 13C NMR (CDCl3): d = 167.9 (NHCOCH3), 158.1
(2-C), 145.1 (N-C), 138.8 (10-C), 133.9 (40-C), 130.0 (30C and 50-C),
127.7 (20-C and 60-C), 118.4 (S-C), 75.7 (6-C), 29.1 (7-C), 27.6
(5-C), 23.3 (COCH3), 21.7 (4-C), 21.3 (Ph-CH3).
(7-C), 24.2 (4-C), 21.2 (CH3). MS (m/z) (ESIꢀ) 210.1 [Mꢀ1]+.
(ESI+) 212.0 [M+1]+, 445.0 [2M+Na]+.
4.10. (S)-2,6-Diamino-4,5,6,7-tetrahydrobenzothiazole 2
To a solution of 14 (0.520 g, 2.47 mmol) in tetrahydrofuran
(10 mL), 5% hydrogen chloride aqueous solution (10 mL) was
added. The solution was kept under stirring at 80 °C (12 h) until
the starting material disappeared (TLC dichloromethane/metha-
nol/triethylamine 8:1.5:0.5). Saturated potassium carbonate aque-
ous solution was then added until pH 7. The solvents were
removed under reduced pressure and the solid brown residue
was suspended in dichloromethane/methanol 7:3 (10 mL). After
filtration, the solvents were removed under reduced pressure.
The residue (0.580 g) was suspended twice in 0.9 M hydrogen chlo-
ride dioxane solution (10 mL) and recovered by filtration. The res-
idue (0.650 g) was dissolved in water (2 mL); the precipitate
obtained after addition of 85% potassium hydroxide aqueous solu-
tion (3.4 mL) was recovered by suction and washed with cool
water (2 ꢁ 1 mL) to afford pure 2 (0.375 g, 90%). TLC Rf 0.37. The
ee (>98%) and the (S)-configuration were determined by HPLC
(Crownpak CR(+)). Eluant: perchloric acid aqueous solution at pH
2; flow rate 0.3 mL/min). Rt (R)-isomer 11.97 (S)-isomer
14.87 min. DSC endothermic peak at 240.00 °C. IR mmax 3369.03,
3353.54, 3255.58, 3085.39, 3007.93, 2911.77, 2835.29, 2757.84,
4.9. (S)-2-Acetylamino-6-amino-4,5,6,7-tetrahydrobenzothiazole
14
4.9.1. (S)-2-Acetylamino-6-azido-4,5,6,7-tetrahydrobenzothiazole
13
To a solution of (R)-tosylate 12 (1.9 g, 5.2 mmol) in dimethyl-
formamide (75 mL) under a nitrogen atmosphere at room temper-
ature, sodium azide (1.35 g, 20.8 mmol) was added. The reaction
mixture was heated at 70 °C until the starting material disap-
peared (TLC toluene/ethanol 9:1, two elutions) (6 h). After cooling
at room temperature, the reaction mixture was poured into water
(350 mL). The aqueous phase was extracted with ethyl acetate
(5 ꢁ 100 mL); the collected organic phases were washed with
brine (2 ꢁ 100 mL) and water (100 mL). The usual work-up affor-
ded crude azide 13 (1.07 g), which was directly used in the next
step. For analytical purposes, a sample (0.2 g) was purified by silica
gel column chromatography (1:10); elution with hexane/ethyl ace-
tate 3:7 afforded pure 13. TLC Rf (TLC toluene/ethanol 9:1, two elu-
2240.12, 1632.72, 1587.66, 1573.68, 1532.80, 1440.83 cmꢀ1
.
tions) 0.46. [
a
]
20 = ꢀ18.0 (c 1, CHCl3). [
a
]
20 = ꢀ21.9 (c 1, CHCl3).
[a]
]
20 = ꢀ91.0 (c 1, methanol) (lit.6 ꢀ94.2, c 1 in methanol).
D
546
D
DSC endothermic peak at 193.17 °C. IR mmax 3242.06, 3158.10,
[a
20 = ꢀ110.1 (c 1, methanol). 1H NMR (CD3OD): d 3.15 (1H,
546
3031.88, 2946.32, 2923.19, 2895.91, 2836.91, 2761.90, 2534.94,
dddd, J = 8.5, 8.5, 5.0, 3.0 Hz, 6-H), 2.81 (1H, dd, J = 15.6, 6.2 Hz,
7a-H), 2.62–2.52 (2H, m, 4a-H and 4b-H), 2.35 (1H, ddd, J = 15.6,
8.8, 2.3 Hz, 7b-H), 2.02 (1H, m, 5a-H), 1.68 (1H, dddd, J = 13.9,
8.5, 6.1, 2.4 Hz, 5b-H). 13C NMR (CD3OD): d 168.2 (2-C), 143.3 (N-
C), 113.9 (S-C), 47.4 (6-C), 31.4 (7-C), 31.3 (5-C), 24.3 (4-C). MS
(m/z) (ESI+) 118 [MꢀSC(NH2)N+Na]+, 170.1 [M+1]+, 361.1
[2M+Na]+, 401.1 [2M+2CH3OH]+.
2108.71, 2079.59, 1688.98, 1576.56, 1533.22 cmꢀ1
.
1H NMR
(CDCl3): d 4.02 (1H, m, 6-H), 3.06 (1H, ddd, J = 16.0, 4.7, <1 Hz,
7a-H), 2.89–2.72 (3H, m, 4a-H, 4b-H and 7b-H), 2.30 (3H, s, CH3),
2.15 (1H, dddd, J = 13.0, 6.2, 5.9, 2.9 Hz, 5a-H), 2.06 (1H, dddd,
J = 13.0, 10.2, 6.7, 4.9 Hz, 5b-H). 13C NMR (CDCl3):
d 167.9
(NHCOCH3), 157.9 (2-C), 140.1 (N-C), 119.3 (S-C), 56.2 (6-C), 28.3
(5-C), 27.4 (7-C), 23.3 (4-C), 22.8 (CH3). MS (m/z) (ESIꢀ) 235.9
[Mꢀ1]+. (ESI+) 238.1 [M+1]+, 260.1 [M+Na]+.
4.11. (S)-Pramipexole 1 dihydrochloride monohydrate
4.9.2. (S)-2-Acetylamino-6-amino-4,5,6,7-tetrahydrobenzothia
zole 14
The p-toluenesulfonic acid salt of (S)-pramipexole 1 was pre-
pared in 54% yield, according to the literature,27 by reaction of dia-
mine (S)-2 with n-propyl tosylate 15, which was also prepared
according to the literature.32 1H NMR (CD3OD): d 7.72 (2H, d,
J = 8.0 Hz, 20 and 60), 7.25 (2H, d, J = 8.0 Hz, 30 and 50), 3.55 (1H,
dddd, J = 11.6, 8.8, 5.3, 2.90 Hz, 6-H), 3.09–3.05 (3H, m, 7a-H and
CH2CH2CH3), 2.71–2.60 (3H, m, 4a-H, 4b-H and 7b-H), 2.39 (3H,
s, PhCH3), 2.27 (1H, m, 5a-H), 1.93 (1H, dddd, J = 12.9, 10.6, 9.6,
6.2 Hz, 5b-H), 1.75 (2H, tq, J = 8.0, 7.4 Hz, CH2CH2CH3), 1.06 (3H,
t, J = 7.4 Hz, CH2CH2CH3). (S)-Pramipexole p-toluenesulfonic salt
(0.350 g, 0.914 mmol) was suspended in water (1.5 mL) and, after
Crude azide 13 (0.8 g) was dissolved in tetrahydrofuran (30 mL),
after which water (1.5 mL) and polymer bonded triphenyl phos-
phine (2.3 g, 3 mmol/g) were sequentially added. The reaction mix-
ture was kept under stirring at 40 °C for 12 h. The reaction progress
was monitored by TLC until complete conversion. The TLC eluant
was prepared by mixing water, n-butanol, and triethylamine
(5:4:1), and separating the organic phase, after vigorous stirring.
The reaction mixture was filtered and the solid was washed with
methanol (3 ꢁ 10 mL). The collected organic phases were evapo-
rated under reduced pressure. The solid residue (0.710 g) was puri-
fied by silica gel column chromatography (1:10). Elution with
dichloromethane/methanol 9:1 afforded (S)-14 (0.620 g, 76% from
tosylate 12). The ee (>98%) was determined by HPLC on chiral sta-
tionary phase (Chiralpak IA; eluant hexane/2-propanol/diethyl-
amine 7:2.95:0.05; flow rate 0.7 mL/min). Rt (R)-isomer
cooling at 0 °C, 12 M hydrogen chloride aqueous solution (76 lL)
was added. The mixture was kept under stirring for 15 min. after
which 1 M sodium hydroxide aqueous solution (0.914 mL) was
added while keeping the temperature at 0 °C over 3 h. The precip-
itate pramipexole 1 was recovered by filtration and following the
reported method,27 was transformed into the corresponding dihy-
drochloride monohydrate (0.245 g, 78%). The ee was established by
HPLC analysis (Chiralpak IA, hexane/ethanol/diethylamine
70:30:0.1) Rt (R)-isomer 5.53 min; (S)-isomer 7.39 min. 1H NMR
(CD3OD): d 3.69 (1H, dddd, J = 11.4, 8.6, 5.3, 3.0 Hz, 6-H), 3.17
(1H, dd, J = 16.0, 6.2 Hz, 7a-H), 3.12 (2H, t, J = 8.0 Hz, CH2CH2CH3),
2.85–2.72 (3H, m, 4a-H, 4b-H and 7b-H), 2.41 (1H, dddd, J = 13.9,
5.3, 3.0, 1.4 Hz, 5a-H), 2.08 (1H, dddd, J = 13.9, 8.6, 6.3, 2.4 Hz,
5b-H), 1.82 (2H, tq, J = 8.0, 7.4 Hz, CH2CH2CH3), 1.08 (3H, t,
J = 7.4 Hz, CH2CH2CH3). 13C NMR (CD3OD): d 170.1 (2-C), 133.0
(N-C), 111.6 (S-C), 53.2 (6-C), 46.9 (CH2CH2CH3), 24.9 (7-C), 23.9
(5-C), 20.6 (4-C), 19.5 (CH2CH2CH3), 9.9 (CH2CH2CH3). Other chem-
11.59 min; (S)-isomer 16.25 min. Rf 0.47. [
a
]
20 = ꢀ59.1 (c 1, meth-
D
anol). [
a]
20 = ꢀ70.4 (c 1, methanol). DSC endothermic peak at
546
192.50 °C. IR mmax 3334.13, 3272.90, 3165.16, 3050.47, 2921.44,
2843.81, 2634.92, 2504.97, 1667.67, 1567.19 cmꢀ1 1H NMR (CD3-
.
OD): d 3.20 (1H, dddd, J = 10.0, 8.3, 5.3, 2.9 Hz, 6-H), 2.97 (1H,
ddd, J = 15.7, 5.3, <1 Hz, 7a-H), 2.76 (1H, dddd, J = 16.5, 5.9, 4.9,
<1 Hz, 4a-H), 2.68 (1H, dddd, J = 16.5, 7.7, 6.2, 1.8 Hz, 4b-H), 2.49
(1H, ddd, J = 15.7, 8.3, 1.8 Hz, 7b-H), 2.20 (3H, s, CH3), 2.05 (1H,
dddd, J = 13.0, 6.2, 5.9, 2.9 Hz, 5a-H), 1.74 (1H, dddd, J = 13.0,
10.0, 7.7, 4.9 Hz, 5b-H).13C NMR (CDCl3): d 169.1 (NHCOCH3),
156.4 (2-C), 143.2 (N-C), 120.3 (S-C), 56.2 (6-C), 31.1 (5-C), 30.8

P. Ferraboschi et al. / Tetrahedron: Asymmetry 25 (2014) 1239–1245
1245
ical–physical properties are in agreement with the literature
values.6,27,33,34
Ricerca Applicata’ carried out in collaboration with Università degli
Studi di Milano. We thank Dr Valentina Vandoni for her technical
assistance and Mrs Riccardo Monti and Stefano Novelli for FT-IR
and MS spectra.
4.12. (S)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothia
zole 10
References
4.12.1. (S)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzo-
thiazole, 6-benzoate 16
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To a solution of benzoic acid (0.215 g, 1.76 mmol) and diethyl
azadicarboxylate (0.80 mL of a 40% solution in toluene, 1.66 mmol)
in dry dimethylformamide (4 mL), kept under nitrogen at 0 °C with
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4.12.2. (S)-2-Acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzo-
thiazole 10
(S)-Benzoate 16 (0.050 g, 0.158 mmol) was dissolved in a 1%
sodium hydroxide solution in methanol (6.2 mL). The solution
was kept at room temperature overnight. After complete conver-
sion (TLC dichloromethane/methanol 95:5) 1 M hydrochloric acid
was added until pH 7. The solvents were removed and to the res-
idue (0.115 g) dichloromethane (0.5 mL) was added. The obtained
suspension was purified by silica gel (1 g) column chromatogra-
phy. Elution with hexane/ethyl acetate 3:7 afforded pure (S)-10
27. Raje, P.; Gadikota, R. R.; Chen, J. -X.; Lapina, O.; McCall, J. M. WO 113056, 2008.
28. Mitsunobu, O. Synthesis 1981, 1–28.
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1994, 77, 1256–1266.
30. Erlenmeyer, H.; Schoenauer, W. Helv. Chim. Acta 1941, 24, 172–179 E.
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20 = ꢀ22.6 (c 1, methanol).
ˇ
(0.025 g, 75%, >98% ee by HPLC). [
a
]
ˇ
33. Zivec, M.; Anzic, B.; Gobec, S. Org. Process Res. Dev. 2010, 14, 1125–1129.
D
20
34. Ravinder, B.; Rajeswar Reddy, S.; Panasa Reddy, A.; Rakeshwar, B. Tetrahedron
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[
a]546 = ꢀ26.8. Other chemical–physical properties are in agree-
ment with those of (R)-10.
Acknowledgments
M.D.M. acknowledges the financial support provided by
Regione Lombardia and Euticals SpA under the program ‘Dote di