Enantioselective synthesis of (S)-salmeterol via asymmetric reduction of azidoketone by Pichia angusta

Article abstract of DOI:10.1016/S0957-4166(01)00350-0

An efficient enantioselective route to (S)-salmeterol involving asymmetric reduction of an azidoketone intermediate to an azido alcohol by Pichia angusta is described.

Full text of DOI:10.1016/S0957-4166(01)00350-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2005–2008
Enantioselective synthesis of (S)-salmeterol via asymmetric
reduction of azidoketone by Pichia angusta
Panayiotis A. Procopiou,* Gillian E. Morton, Martin Todd and Graham Webb
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
Received 19 June 2001; accepted 6 August 2001
Abstract—An efficient enantioselective route to (S)-salmeterol involving asymmetric reduction of an azidoketone intermediate to
an azido alcohol by Pichia angusta is described. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
toluolyl tartaric acid3 and the second patent described
the use of phenylglycinol as a chiral auxiliary, followed
by chromatographic separation.4 The Helquist synthesis
involved the enantioselective reduction of the phenacyl
bromide 3 to the corresponding bromohydrin using
Corey’s CBS-oxazaborolidine catalyst.5
Salmeterol (Serevent®) 1 is a potent, long acting b2-
adrenoreceptor agonist used as a bronchodilator for the
prevention of bronchospasm in patients with asthma
and chronic obstructive pulmonary disease.1 Interest in
the (S)-enantiomer of salmeterol had recently been
stimulated by the publication of a patent by Sepracor
which claimed that the (S)-enantiomer of salmeterol
had a higher selectivity for b2 receptors and that it did
not precipitate certain adverse effects associated with
the administration of ( )- or (R)-salmeterol.2
2. Results and discussion
We envisaged an enantioselective reduction of an aro-
matic a-azidoketone to provide an azido alcohol, using
a procedure similar to that described by Yadav et al.6
Thus, the salmeterol intermediate 4 was first protected
as the acetonide 5 and then converted to the azidoke-
tone 6 (95% yield for the two steps) (Scheme 1). Reduc-
tion of 6 with borane in the presence of 7.5%
(S)-2-methyl-CBS-oxazaborolidine provided the (S)-
azido alcohol 7 but with an unexpectedly low enantio-
We were interested in a practical and efficient synthesis
of (S)-salmeterol having >95% enantiomeric excess
(e.e.) for further pharmacological screening. Surpris-
ingly, the synthesis of non-racemic salmeterol has been
described in only two Glaxo patents and also in a
communication by Helquist. The first patent described
the resolution of the ethanolamine 2 using (−)-di-p-
* Corresponding author. Tel.: 01438-763423; fax: 01438-763438; e-mail: pap1746@gsk.com
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00350-0

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P. A. Procopiou et al. / Tetrahedron: Asymmetry 12 (2001) 2005–2008
Scheme 1. Reagents and conditions: (i) 2-methoxypropene, dichloromethane, TsOH; (ii) NaN3, DMF; (iii) Pichia angusta; (iv) H2,
10% Pd/C, EtOH; (v) Br(CH2)6O(CH2)4Ph 9, DMF; (iv) AcOH, water.
meric ratio (5:1). Since (R)-salmeterol is more potent
than the (S)-enantiomer even a small amount of the
(R)-isomer in the product could produce misleading
pharmacological results. Rather than attempting
lengthy and expensive purification, we investigated an
alternative procedure for the reduction of 6. The bio-
transformation of ketones to alcohols can be performed
with a variety of enzymes or whole cell preparations.
The latter are usually preferred on an industrial scale in
order to avoid the problem of cofactor regeneration.7
Moreover, although the prevalent enantiopreference in
the reduction is for alcohols with the (R)-configuration
a number of micro-organisms provide alcohols with the
(S)-configuration.8,9 Forty two micro-organisms were
screened for their ability to reduce the ketone group of
6 and five were identified that gave selective reduction.
Mucor racemosus C142, Curvularia lunata NRRL2434
and Aureobasidium pullulans ATCC16623 preferentially
converted the azidoketone 6 to the (R)-enantiomer of 7,
whereas Zygosaccharomyces rouxii NCYC381 and
Pichia angusta ATCC16623 preferentially converted 6
to the (S)-enantiomer of 7 (Table 1). On scale-up it was
found that the yeast Pichia angusta produced 7 with
excellent enantioselectivity (e.e. >98%) and 94% yield.
Chiral HPLC analysis indicated no detectable (R)-iso-
mer or residual ketone. The azido alcohol 7 was then
hydrogenated over palladium on charcoal in ethanol to
provide the amino alcohol 8 in 86% yield. The enan-
tiomeric purity of 8 was assessed by 750 MHz 1H NMR
using the chiral complexing agent (R)-(−)-2,2,2-tri-
fluoro-1-(9-anthryl)ethanol. The benzylic CHOH of
the (R)-isomer appears as a dd at 4.29, whereas the
analogous proton in the (S)-isomer appears as a dd at
Table 1. Reduction of ketone 6 by a selection of micro-
organisms
Organism
Hours
E.e. %
post-ketone
addition
(config.)
Mucor racemosus C142
18
42
18
42
18
42
6
24
18
42
85 (R)
88 (R)
80 (R)
83 (R)
85 (R)
83 (R)
94 (S)
94 (S)
94 (S)
93 (S)
Cur6ularia lunata
NRRL2434
Aureobasidium pullulans
ATCC16623
Zygosaccharomyces rouxii
NCYC381
Pichia angusta ATCC16623

P. A. Procopiou et al. / Tetrahedron: Asymmetry 12 (2001) 2005–2008
2007
1
4.33. The higher field dd was not detected indicating
that compound 8 was >95% pure. Reaction of 8 with
the bromide 9 (prepared from 4-phenyl-1-butanol and
1,6-dibromohexane under phase transfer conditions)10
gave the secondary amine 10 together with a small
amount of dialkylated product 11 which was removed
by filtration on silica gel. Deprotection of 10 with
aqueous acetic acid gave (S)-salmeterol acetate salt in
quantitative yield and 98.6% e.e. (chiral HPLC).
95%): H NMR (CDCl3; 400 MHz): l 7.72 (1H, dd, J
8, 2 Hz), 7.62 (1H, br s), 6.88 (2H, d, J 8 Hz), 4.89 (2H,
s), 4.49 (2H, s), 1.57 (6H, s); 13C NMR (CDCl3; 100
MHz): l 191.7, 156.5, 128.5, 126.8, 125.5, 119.6, 117.5,
100.8, 60.6, 54.5, 24.8; MS (TSP+ve) m/z 248 (M+H)+.
Found: C, 58.40; H, 5.22; N, 16.78. C12H13N3O3
requires C, 58.29; H, 5.30; N, 17.00%.
4.3. Biotransformations
Organisms were grown in 10 mL YEPD medium (yeast
extract 2%, peptone 2% and dextrose 2%) at 28°C with
shaking at 250 rpm. azidoketone 6 (2 mg) in acetonitrile
(200 mL) was added after 2 days growth, and incuba-
tion continued. The results are shown in the Table 1.
3. Conclusion
In conclusion, we have described an efficient and prac-
tical synthesis of (S)-salmeterol in very high e.e. and an
overall yield of 54%.
4.4. (1S)-2 Azido-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-
yl)ethanol 7
4. Experimental
4.4.1. Biotransformation method. The yeast Pichia
angusta ATCC16623 was grown in 5.2 L of YEPD
medium in 13 Florence flasks. The flasks were incu-
bated at 28°C with shaking until the organism had
reached late growth stage. After 16 h growth, a solution
of ketone 6 (5.2 g, 21 mmol) in acetonitrile (52 mL) was
added (4 mL to each flask) and incubation continued at
28°C for 48 h. At this stage all the ketone was con-
sumed and the contents of the flasks were combined.
The broth was centrifuged (4200 rpm for 30 min) and
the supernatant was decanted and filtered. The filtrate
(4.9 L) was extracted with tert-butyl methyl ether (4.9
L), the organic phase was separated, dried (Na2SO4),
filtered and concentrated to a pale yellow oil (5.35 g).
HPLC (Kromasil C8, 15 cm×4.6 mm, eluting with 49%
MeCN in 25 mM aq. NH4OAc isocratically at a flow
rate of 1 mL/min) showed no detectable residual ketone
6. HPLC (ChromTech Chiral AGP, 15 cm×4.6 mm,
eluting with 17.5% MeCN in 10 mM aq. KH2PO4 pH 7
isocratically with a flow rate of 0.5 mL/min) rt=4.39
min; rt [(R)-isomer]=3.71 min; rt [(S)-isomer]=4.49
min. The crude product was purified by chromatogra-
phy on silica gel (90 g Biotage cartridge) eluting with
15% diethyl ether–light petroleum (40–60°C) (1 L),
followed by 20% diethyl ether–light petroleum (1.5 L)
to give 7 as a colorless oil (4.92 g, 94%): NMR (CDCl3;
400 MHz): l 7.12 (1H, dd, J 8, 2 Hz), 6.99 (1H, d, J 2
Hz), 6.80 (1H, d, J 8 Hz), 4.81 (2H, s), 4.78 (1H, dd, J
8, 4 Hz), 3.43 (1H, dd, J 12, 8 Hz), 3.37 (1H, dd, J 12,
4 Hz), 2.64 (1H, br s), 1.53 (6H, s), 13C NMR (CDCl3;
100 MHz): l 151.2, 132.5, 125.8, 122.3, 119.6, 117.3,
99.7, 73.0, 60.8, 58.0, 24.7, 24.6; LCMS rt=2.82 min,
ES+ve m/z 250 [(M+H)+, 14%], ES-ve m/z 294 [(M+
HCO2)−, 24%]. Found: C, 57.89; H, 6.01; N, 16.46.
C12H15N3O3 requires C, 57.83; H, 6.07; N, 16.86%.
LCMS analysis was conducted on
a Supelcosil
LCABZ+PLUS column (3.3 cm×4.6 mm) eluting with
0.1% HCO2H and 0.01 M NH4OAc in water (solvent
A), and 0.05% HCO2H and 5% water in MeCN (sol-
vent B), using the following elution gradient 0–0.7 min
0% B, 0.7–4.2 min 100% B, 4.2–5.3 min 0% B, 5.3–5.5
min 0% B at a flow rate of 3 mL/min. The mass spectra
were recorded on a Fisons VG Platform spectrometer
using electrospray positive and negative mode (ES+ve
and ES-ve).
4.1. 2-Bromo-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-
yl)ethanone 5
2-Methoxypropene (24 mL, 250 mL) was added to an
ice-cooled mixture of bromide 4 (50 g, 204 mmol)
(Caution: potent alkylating agent) and p-toluenesul-
fonic acid monohydrate (230 mg, 1.2 mmol) in
dichloromethane (500 mL). After the addition was
complete, the mixture was stirred for 30 min and then
more 2-methoxypropene (7.5 mL, 78 mmol) was added
and the mixture was stirred for an additional 10 min.
Aqueous sodium bicarbonate solution was added and
the two phases were separated. The organic phase was
washed with aqueous sodium bicarbonate, dried
(MgSO4) and evaporated to dryness to give 5 as a beige
1
solid (58.3 g, 100%). H NMR (CDCl3; 400 MHz): l
7.82 (1H, dd, J 8, 2 Hz), 7.70 (1H, d, J 2 Hz), 6.88 (1H,
d, J 8 Hz), 4.90 (2H, s), 4.39 (2H, s), 1.58 (6H, s); 13C
NMR (CDCl3; 100 MHz): l 188.9, 155.3, 128.6, 125.7,
125.5, 118.5, 116.3, 99.8, 59.6, 29.7, 23.8.
4.2. 2-Azido-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-
yl)ethanone 6
A suspension of bromoketone 5 (52 g, 181 mmol)
(Caution: potent alkylating agent) in DMF (300 mL)
was treated with sodium azide (12.24 g, 188 mmol) and
the mixture was stirred at 20°C for 2 h, by which time
the mixture became homogeneous and red colored. The
mixture was diluted with ethyl acetate and washed with
water, brine and dried (MgSO4). The filtrate was evapo-
rated to dryness to give 6 as a pale yellow solid (42.5 g,
4.4.2. Chemical method. (S)-2-Methyl-CBS-oxazaboroli-
dinine (1 M solution in toluene, 4.4 mL) was diluted
with THF (43.6 mL), cooled to 0°C and then treated
with a solution of BH3·THF (1 M, 72.6 mL) under
nitrogen. After 15 min at 0°C, a solution of 6 (14.37 g,
58.1 mmol) in THF (145 mL) was added dropwise over
1.5 h at 5°C. The reaction mixture was stirred for a
further hour and then quenched by the addition of

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P. A. Procopiou et al. / Tetrahedron: Asymmetry 12 (2001) 2005–2008
aqueous HCl (2 M, 58.1 mL) and extracted with ethyl
acetate. The organic layer was washed with aqueous
HCl (2 M), aq. NaHCO3, brine and dried (MgSO4).
The residue was purified by chromatography on silica
gel (90 g Biotage cartridge) eluting with light petroleum
(40–60°C) dichloromethane (2:3) to give 7 as a yellow
liquid (3.0 g, 21%). The enantiomeric purity of this
material was not determined at this stage, but was
examined after conversion to the amine 8.
4.7. (S)-Salmeterol acetate 1
The acetonide 10 (59 mg, 0.13 mmol) was heated in a
mixture of acetic acid (5 mL) and water (2 mL) at 71°C
for 0.5 h. The mixture was concentrated under reduced
pressure, the residue was dissolved in methanol and
evaporated to dryness to give the acetate salt of 1 as a
1
colorless gum (62 mg, 100%). H NMR (CD3OD; 400
MHz): l 7.34 (1H, d, J 2 Hz), 7.23 (1H, t, J 8 Hz), 7.21
(1H, s), 7.20–7.10 (4H, m), 6.79 (1H, d, J 8 Hz), 4.86
(1H, dd, J 9, 4 Hz), 4.65 (2H, s), 3.43 (2H, t, J 7 Hz),
3.41 (2H, t, J 7 Hz), 3.15–2.95 (4H, m), 2.61 (2H, t, J
7 Hz), 1.92 (3H, s), 1.75–1.53 (8H, m), 1.45–1.35 (4H,
m). Chiral HPLC on a Sumichiral OA-4100 column (25
cm×4.6 mm) eluting with hexane:ethanol:dichloro-
methane:TFA (240:30:130:1), at a flow rate of 1 mL/
min, detecting at 276 nm: rt=17.45 min, 99.3% [(S)–
isomer]; rt=21.15 min, 0.7% [(R)-isomer].
4.5. (1S)-2-Amino-1-(2,2-dimethyl-4H-1,3-benzodioxin-
6-yl)ethanol 8
The azide 7 (4.92 g, 19.7 mmol) was hydrogenated over
10% Pd/C (0.5 g) in ethanol (150 mL) over 3 h. The
catalyst was removed by filtration and washed with
ethanol. The combined filtrate and washings were evap-
orated to dryness. The residue was filtered on silica gel
(40 g Biotage cartridge) eluting with toluene:ethanol:aq.
880 ammonia (85:14:1) to give 8 as a white solid (3.77
1
g, 86%): [h]2D0 +17 (c 0.83 in MeOH); H NMR (CDCl3;
400 MHz): l 7.12 (1H, dd, J 8, 2 Hz), 7.00 (1H, d, J 2
Hz), 6.80 (1H, d, J 8 Hz), 4.84 (2H, s), 4.54 (1H, dd, J
8, 4 Hz), 2.98 (1H, dd, J 13, 4 Hz), 2.77 (1H, dd, J 13,
8 Hz), 2.05–1.70 (3H, br), 1.54 (6H, s); 13C NMR
(CDCl3; 100 MHz): l 150.6, 134.4, 125.8, 122.2, 119.3,
117.0, 99.5, 74.0, 61.0, 49.2, 24.8, 24.6; LCMS rt=1.75
min, ES+ve m/z 206 [(MH−H2O)+, 100%]; 224 [(M+H)+,
10%]. Found: C, 64.58; H, 7.58; N, 6.20. C12H17NO3
requires C, 64.54; H, 7.67; N, 6.27%. The enantiomeric
excess of 8 was determined by 750 MHz 1H NMR using
8 (1 mg) in CDCl3 (0.8 mL) and (R)-(−)-2,2,2-trifluoro-
1-(9-anthryl)ethanol (18 mg).
Acknowledgements
We thank Mr. E. G. Hortense for the determination of
the ee of 1 by chiral HPLC and Dr. R. J. Upton for the
determination of the e.e. of 8 by NMR.
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{[6-(4-phenylbutoxy)hexyl]amino}ethanol 10
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A mixture of the amino alcohol 8 (223 mg, 1 mmol)
and bromide 9 (178 mg, 0.57 mmol) in DMF (2 mL)
was stirred at 20°C for 67 h. The reaction mixture was
concentrated under reduced pressure, the residue was
dissolved in ethyl acetate and washed with water and
brine. The organic solution was dried (MgSO4) concen-
trated to a colorless oil, and purified by chromatogra-
phy (10 g silica Bond Elut cartridge) eluting with 1, 2
and 5% 2 M ammonia in methanol–dichloromethane to
give 10 as a colorless oil (181 mg, 70%). 1H NMR
(CDCl3; 400 MHz): l 7.3–7.25 (2H, m), 7.2–7.1 (4H,
m), 7.00 (1H, d, J 2 Hz), 6.78 (1H, d, J 8 Hz), 4.83 (2H,
s), 4.60 (1H, dd, J 9, 4 Hz), 3.41 (2H, t, J 7 Hz), 3.38
(2H, t, J 7 Hz), 2.83 (1H, dd, J 12, 4 Hz), 2.70–2.55
(5H, m), 2.55–2.30 (2H, br), 1.7–1.4 (8H, m), 1.54 (6H,
s), 1.4–1.28 (4H, m); LCMS rt=3.21 min, ES+ve m/z
456 (M+H)+, ES-ve m/z 500 (M+HCO2)−